8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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Original article

Synthesis and antitumor activities of naturally occurring oleanolic acidtriterpenoid saponins and their derivatives

Qingchao L iu a Hongchun Liu d Lei Zhang c Tiantian Guo c Peng Wang c Meiyu Geng dYingxia Li b

a Department of Pharmaceutical Engineering Northwest University Xirsquo an 710069 Chinab School of Pharmacy Fudan University Shanghai 201203 Chinac School of Medicine and Pharmacy Ocean University of China Qingdao 266003 Chinad Division of Antitumor Pharmacology Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai 201203 China

a r t i c l e i n f o

Article history

Received 4 March 2013Received in revised form4 April 2013Accepted 6 April 2013Available online 15 April 2013

Keywords

Oleanolic acid triterpenoid saponinsCytotoxic activityGlycosylationLog P

IC50 value

a b s t r a c t

Twenty-six naturally occurring oleanolic acid saponins and their derivatives 16 of which were synthe-sized in this study were preliminarily evaluated against human cancer cells From SAR studies thepresence of a-L -rhamnosyl residue at the terminal of both C-3 and C-28 position for oleanolic acidbidesmosides was important to enhance cytotoxicity and introducing more sugar residues at C3eOH of compound 12 with C-28 carboxylic acid is a favorable modi1047297cation to ameliorate the anticancer activityFurthermore a-L -rhamnosyl moiety linked to C2eOH of the 1047297rst monosaccharide (a-L -alabinose b-D-xylose b-D-galactose or b-D-glucose) in C3eOH of oleanolic acid was helpful to improve the cytotoxicityAccording to the predicted log P values lipophilicity of the synthesized saponins was not an importantfactor for cytotoxicity

2013 Elsevier Masson SAS All rights reserved

1 Introduction

Triterpenoid saponins glycosylated plant secondary metabolites[1] which exist widely in many signi1047297cant foods forage crops andherbal medicinal plants with high contents [2] act as naturalchemical barriers to fungal attack because of their remarkableantifungal properties [3] Apart from the natural protective activitymany of them are also discovered as resources for drugs at thefolkloric usage such as the saponin extracts from ginseng licoricesenega roots and ivy leaves [45] or food crops such as oats andlegumes [6] However the occurrence of triterpenoid saponins

with prominent structural microheterogeneity makes separation of the homogeneous component especially in a suitable amountcomparatively dif 1047297cultwhich results in the hysteretic developmenton elucidating the structureeactivity relationships and the bio-logical mechanisms of action of triterpenoid saponins components[78] Therefore chemical synthesis would provide a feasible accessto these biologically triterpenoid components for further under-standing and application of this type of natural products [7910]

As we all known the most predominant member of this familyof triterpenoids is probably oleanolic acid [1112] which has beenclinically used as a hepatoprotectiveantihepatitis drug in China forseveral decades However its low water solubility results in itsunfavorably absolute oral bioavailability [1314] Therefore com-pounds derived from chemical modi1047297cation of oleanolic acid withprodrug strategy or isolation of oleanolic acid saponins from nat-ural resources for improving pharmacokinetics was researched[1516] In previous communications [17e20] we reported that thenaturally occurring oleanolic acid saponins (1e10) with signi1047297cantbiological activities were synthesized via concise and ef 1047297cient

strategies With the continuous interest in the oleanolic acid sa-ponins on the biological activity and in order to search for potentialnew antitumor agents we decided to investigate the effect of modi1047297cation of sugar moiety of oleanolic acid saponins on the tu-mor cell growth inhibitory activity Herein we disclose the ef 1047297cientsynthesis of some natural (12 14e16) [21e25] (Fig 1) and non-natural sugar-modi1047297ed oleanolic acid saponins (11 13 17e26)(Fig 2) Furthermore structureeactivity relationships analysis of this series of natural and non-natural oleanolic acid saponinsagainst tumor cells including human promyelotic leukemia cancer(HL-60) non-small-cell lung cancer (A549) and human melanomacancer (A375) is discussed These results obtained have provided

Corresponding author Telfax thorn86 21 51980127E-mail address liyx417fudaneducn (Y Li)

Contents lists available at SciVerse ScienceDirect

European Journal of Medicinal Chemistry

j o u r n a l h o m e p a g e h t t p w w w e l se v i e r c o m l o c a t e ej m e c h

0223-5234$ e see front matter 2013 Elsevier Masson SAS All rights reserved

httpdxdoiorg101016jejmech201304016

European Journal of Medicinal Chemistry 64 (2013) 1e15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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glycosylation with the acceptor 48 [3233] in the presence of NISand AgOTf stereoselectively affording the fully protected interme-diate 49 in 35 yield The diisopropylidene was achieved bytreatment of compound 49 with p-TsOH in CH2Cl2eMeOH solutionto obtain 50 Subsequent deprotection of the benzyl ether with 10

PdeC and the benzoyl ester with NaOMe in CH2Cl2eMeOH wasachieved to afford the target compound 20The synthesis of non-natural saponins 21e26 was achieved in a

straight-forward manner As shown in Scheme 8 the oleanolic acidsaponin acceptor 51 (or 53) was condensed with the known donor40 in the presence of TMSOTf to obtain the intermediates 52aec (or54aec) Subsequent removal of benzyl and acetyl groups providedthe desired saponins 21e23 (or 24e26)

22 Cytotoxic activity

To examine the potential ability of the natural and non-naturaloleanolic acid saponins 1e26 the in vitro cytotoxic activity againsthuman promyelotic leukemia cancer (HL-60) non-small-cell lung

cancer (A549) and human melanoma cancer (A375) was assessedusing methyl-thiazol-tetrazolium (MTT) reduction test and sulfo-rhodamine B (SRB) staining methods for protein The cytotoxicityresults presented in Table 1 are shown as the IC50 values They aresubdivided into two groups Oleanolic acid bidesmosides (1e4 11)and Oleanolic acid monodesmosides (5e10 12e26) In the 1047297rstgroup saponin 4 exhibited the most cytotoxic activity against thethree tumor cells (with IC50 ranging from 32 to 105 mM) followed

by saponin 11 (IC50 47e103 mM) In contrast saponins 1e3 did notshow any cytotoxic activity (IC50 gt 20 mM) against HL-60 A549 andA375 cell lines These results proved that the presence of a-L -rhamnosyl residue at the terminal of both C-3 and C-28 positionwas critical to enhance cytotoxicity

In the second group most of compounds except 20 22e

23 and25e26 exerted better anticancer activity When compared com-pounds 6 16e17 with 1e3 the in1047298uence of free C-28 carboxylicacid on enhancing the cytotoxicity was clear As for compound 12we anticipated that changing the spatial con1047297guration of L -alabi-nosyl moiety at the C-3 position of oleanolic acid should improveanticancer activity Actually the changed compound 20 exhibitedno cytotoxicity against all tested cell lines (IC50 gt 20 mM) Howeverwhen a-L -rhamnosyl residue of compound 12 was converted tob-L -rhamnosyl residue the derived compound 13 exerted good cyto-toxicity especially enhanced the cytotoxicity about 2-fold againstHL-60 cell line (IC50 42 mM) Meanwhile compounds 5 (IC50

61 mM) 9 (IC50 35 mM) 10 (IC50 31 mM) 14 (IC50 34 mM) and 15

(IC50 42 mM) were 15- to 3-fold more potent against HL-60 cell

line comparing with compound 12 (IC50 92 mM) which suggestedthat introducing more sugar residues at C3eOH of compound 12 is afavorable modi1047297cation to ameliorating the anticancer activityReplacement of a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose at the C3eOH of 12 afforded compound 19 21 or 24 whichhas no signi1047297cant increase in cytotoxicity However adding a-L -rhamnosyl moiety at the C2eOH and C3eOH (or C3eOH solely) of b-D-galactose or b-D-glucose respectively obtained compounds

OOO

O

OAcOLevO OAc

OBn

O

O

OOO

HO

OBn

O

O

OAcOLevO

OAc

STol

29

30a

31a

AgOTf NIS

CH Cl 76

(1) p -TsOH CH2Cl2-CH3OH

(2)10Pd-C H2 NaOMe12

53 for 3 steps

OBnOBnO OBn

STol

30b

AgOTf NIS

CH Cl 43

OOO

OOBnOBnO

OBn

OBn

O

O

31b

(1) p -TsOH CH2Cl2-CH3OH

(2)10Pd-C H2

65 for 2 steps

13

Scheme 2 Synthesis of oleanolic acid saponins 12 and 13

Scheme 3 Synthesis of disaccharide donor 33

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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22e23 or 25e26 which did not exhibit desirable antitumor activ-ity These results demonstrated that the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obviousin1047298uence on improving the cytotoxicity and a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) favored thecytotoxic activity

The logarithm of a partition coef 1047297cient (log P ) is a parameterwhich re1047298ects a drug equilibrium partition ratio between polar(water) and non-polar (octanol) phases and it has been proved todetermine the absorption the distribution the biological avail-ability and pharmacological activity of drugs In this study we have

employed the ACD lab program to predict the values of log P foreach oleanolic acid saponin 1e26 The results are arranged inTable 2 comparing the log P values of all synthesized compoundswe found that the order of antitumor activity was not consistentwith the order of lipophilicity which indicated that lipophilicity of the compounds had no important effect on cytotoxicity

3 Conclusions

A series of natural and non-natural sugar-modi1047297ed oleanolicacid saponins was synthesized in a concise and practical way andtheir cytotoxicity was evaluated in vitro In terms of structureeac-tivity relationships we can conclude that (i) with regard to ole-anolic acid bidesmosides the presence of a-L -rhamnosyl residue atthe terminal of both C-3 and C-28 position was critical to enhancecytotoxicity (ii) for oleanolic acid monodesmosides free C-28carboxylic acid favors the cytotoxicity (iii) changing the spatialcon1047297guration of L -alabinosyl moiety at the C-3 position of oleanolicacid disfavor anticancer activity (iv) introducing more sugar resi-

dues at C3e

OH of compound 12 is a favorable modi1047297

cation toameliorate the anticancer activity (v) the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obvious in-1047298uence on improving the cytotoxicity (vi) a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) was helpful

Scheme 4 Synthesis of target compounds 14e15

Scheme 5 Synthesis of target compounds 16e

18

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 5

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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to improve the cytotoxic activity Meanwhile lipophilicity of thecompounds was not an important factor for cytotoxicity

4 Experimental section

41 Chemistry

Thin-layer chromatography (TLC) was performed on precoatedE Merck Silica Gel 60 F254 plates Flash column chromatographywas performed on silica gel (200e300 mesh Qingdao China)Optical rotations were determined with a PerkineElmer Model 241MC polarimeter 1HNMRand 13C NMR spectra were taken on a JEOL

JNM-ECP 600 spectrometer with tetramethylsilane as the internalstandard and chemical shifts are recorded in d values Mass spectrawere recorded on a Q-TOF Global mass spectrometer

Commercial reagents were all analytically or chemically pure andused without further puri1047297cation unless speci1047297ed All anhydroussolvents were dried and redistilled prior to use in the usual way

411 28-O-a-L-Rhamnopyranosyl-(1 4)-O-b-D-glucopyranosyl-

(16)-O-b-D-glucopyranosyl oleanate 3-O-b-D-xylopyranoside (11)

To a solution of compound 28 (100 mg) in dry CH2Cl2eMeOH(12 12 mL) was added a newly prepared NaOMe in MeOH solution(10 molL 020 mL) The mixture was stirred at room temperaturefor 5 h and neutralized with Dowex Hthorn resin to pH 7 andthen 1047297ltered The 1047297ltrate was concentrated and the resultingresidue was subjected to a silica gel column chromatography

(MeOHeCHCl3eH2O 12501) to give 11 (40 mg 87) as a whiteamorphous solid frac12a25

D 133 (c 025 CH3OH) IR (KBr) nmax 34992938 1731 1645 1453 1073 cm1 1H NMR (500 MHz C5D5N) d

623 (d J frac14 81 Hz1H H-100

)585 (br s1H H-1000

) 539 (t J frac14 36 Hz1H H-12) 498 (d J frac14 78 Hz 1H H-10) 496 (qd J frac14 91 60 Hz 1HH-5000 ) 482 (d J frac14 75 Hz 1H H-10000) 468 (dd J frac14 3217 Hz1H H-2000 ) 465 (m 1H H-40) 454 (dd J frac14 93 32 Hz 1H H-3000 ) 441 (t J frac14 96 Hz 1H H-300) 431e437 (m H-30000 H-4000 H-50000-1 H-60-1)409e423 (m 8H H-200 H-30 H-400 H-40000 H-50 H-500 H-600-1 H-60-2) 402 (t-like J frac14 88 79 Hz 1H H-20000) 393 (t-like J frac14 87 83 Hz1H H-20) 378 (t J frac14 110 Hz 1H H-50000-2) 364 (m 1H H-600-2)333 (dd J frac14 117 43 Hz 1H H-3) 315 (dd J frac14 137 37 Hz 1H H-18) 169 (d J frac14 60 Hz 3H H-6000 ) 129 124 109 099 089 089089 (s each 3H each CH3 7) 13C NMR (125 MHz C5D5N) d 1765(C-28) 1440 (C-13) 1229 (C-12) 1076 (C-10000) 1049 (C-100) 1028(C-1000 ) 956 (C-10) 887 (C-3) 788 786 784 781 772 766 755754 740 739 728 726 713 710 703 693 671 620 560 497482 471 463 422 418 400 396 389 371 341 332 326 308284 283 268 261 239 238 234 185 176 170 157 HR-MS(ESI) m z calcd for C53H86O21Na [M thorn Na]thorn 10815554 found10815579

412 General procedure for synthesizing compounds 31ae 31b

A mixture of compound 29 (0139 mmol) powdered 4 A mo-lecular sieves and compound 30a or 30b (0209 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (0221 mmol) and AgOTf (0016 mmol) was

OOBzO

O

OBzOBzO OBz

OBn

O

BzO

OOBzO

HO

OBn

O

BzO

41

10Pd-C H2

TMSOTf CH2Cl2

NaOMe CH2Cl2-CH3OHOO

HOO

OHOHO OH

OH

O

HO

OBzOBzO OBz

O

40

CCl3

NH

45

19

Scheme 6 Synthesis of target compound 19

Scheme 7 Synthesis of target compound 20

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e156

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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added and the reaction mixture was stirred for 30 min and thenwarmed to room temperature The product was detected on TLC(21 petroleum ethereEtOAc) After completion of the reaction thereaction mixture was quenched by addition of Et3N and then

1047297ltered The 1047297ltrate was concentrated and puri1047297ed by a silica gelcolumn chromatography to afford the products

4121 Benzyl oleanolate 24-di-O-acetyl-3-O-levulinoyl-a-L-rham-

nopyranosyl-(1 2)-34-O-isopropylid-ene-a-L-arabinopyranoside

( 31a) Yield 76 frac12a25D thorn213 (c 08 CHCl3) IR (KBr) vmax 2929

1731 1447 1356 1123 1023 707 673 cm1 1H NMR (CDCl3600 MHz) d 707e735 (m 5H PheH) 534 (s 1H H-100 ) 529e532(m 3H H-30 H-40 H-12) 503e511 (m 3H H-200 PhCH2) 434(d J frac14 77 Hz1H H-10) 410e421(m 4H H-300 H-400 H-500 H-50-1)373e378 (m 2H H-20 H-50-2) 308 (dd J frac14 118 44 Hz 1H H-3)290 (dd J frac14 139 40 Hz 1H H-18) 272e276 (m 1H LeveCH H)261e266 (m 1H LeveCH H) 250e255 (m 1H LeveCHH ) 239e244 (m1H LeveCHH ) 216 215 205 (s each 3H each Ac2Leve

CH3) 153 133 (s each 3H each Oe

(CH3)2Ce

O) 120 (d J frac14 62 Hz

3H H-600 ) 112 104 092 089 088 081 060 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 2062 1775 (C-28) 17141702 1437 (C-13) 1364 1284 1280 1279 1225 (C-12) 1104((CH3)2C) 1031 (C-10) 952 (C-100 ) 890 (C-3) 791 751 733 709

705 697 696 693 662 659 626 558 476 467 459 417 414393 390 378 377 376 367 339 331 327 324 309 307 297280 278 276 261 259 237 234 230 210 208 182173 169164 154 153 HR-MS (ESI) m z calcd for C60H86O15Na [M thorn Na]thorn

10695859 found 10695883

4122 Benzyl oleanolate 234-tri-O-benzyl-b-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-a-L-arabin-opyranoside ( 31b)

Yield 43 frac12a23D thorn592 (c 118 CHCl3) IR (KBr) vmax 2939 1733

1454 1368 1116 1023 738 685 cm1 1H NMR (CDCl3 600 MHz)d 727e734 (m 20H PheH) 533 (d J frac14 11 Hz 1H H-100 ) 529(t J frac14 36 Hz 1H H-12) 511 (d J frac14 127 Hz 1H PhCH H) 505(d J frac14 127 Hz 1H PhCHH ) 501 (d J frac14 154 Hz 1H PhCH H) 495(d J frac14 135 Hz 1H PhCHH ) 476 (d J frac14 127 Hz 1H PhCH H) 467

(d J frac14 110 Hz 1H PhCHH ) 464 (d J frac14 104 Hz 1H PhCH H) 456

Scheme 8 Synthesis of target compounds 21e26

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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glycosylation with the acceptor 48 [3233] in the presence of NISand AgOTf stereoselectively affording the fully protected interme-diate 49 in 35 yield The diisopropylidene was achieved bytreatment of compound 49 with p-TsOH in CH2Cl2eMeOH solutionto obtain 50 Subsequent deprotection of the benzyl ether with 10

PdeC and the benzoyl ester with NaOMe in CH2Cl2eMeOH wasachieved to afford the target compound 20The synthesis of non-natural saponins 21e26 was achieved in a

straight-forward manner As shown in Scheme 8 the oleanolic acidsaponin acceptor 51 (or 53) was condensed with the known donor40 in the presence of TMSOTf to obtain the intermediates 52aec (or54aec) Subsequent removal of benzyl and acetyl groups providedthe desired saponins 21e23 (or 24e26)

22 Cytotoxic activity

To examine the potential ability of the natural and non-naturaloleanolic acid saponins 1e26 the in vitro cytotoxic activity againsthuman promyelotic leukemia cancer (HL-60) non-small-cell lung

cancer (A549) and human melanoma cancer (A375) was assessedusing methyl-thiazol-tetrazolium (MTT) reduction test and sulfo-rhodamine B (SRB) staining methods for protein The cytotoxicityresults presented in Table 1 are shown as the IC50 values They aresubdivided into two groups Oleanolic acid bidesmosides (1e4 11)and Oleanolic acid monodesmosides (5e10 12e26) In the 1047297rstgroup saponin 4 exhibited the most cytotoxic activity against thethree tumor cells (with IC50 ranging from 32 to 105 mM) followed

by saponin 11 (IC50 47e103 mM) In contrast saponins 1e3 did notshow any cytotoxic activity (IC50 gt 20 mM) against HL-60 A549 andA375 cell lines These results proved that the presence of a-L -rhamnosyl residue at the terminal of both C-3 and C-28 positionwas critical to enhance cytotoxicity

In the second group most of compounds except 20 22e

23 and25e26 exerted better anticancer activity When compared com-pounds 6 16e17 with 1e3 the in1047298uence of free C-28 carboxylicacid on enhancing the cytotoxicity was clear As for compound 12we anticipated that changing the spatial con1047297guration of L -alabi-nosyl moiety at the C-3 position of oleanolic acid should improveanticancer activity Actually the changed compound 20 exhibitedno cytotoxicity against all tested cell lines (IC50 gt 20 mM) Howeverwhen a-L -rhamnosyl residue of compound 12 was converted tob-L -rhamnosyl residue the derived compound 13 exerted good cyto-toxicity especially enhanced the cytotoxicity about 2-fold againstHL-60 cell line (IC50 42 mM) Meanwhile compounds 5 (IC50

61 mM) 9 (IC50 35 mM) 10 (IC50 31 mM) 14 (IC50 34 mM) and 15

(IC50 42 mM) were 15- to 3-fold more potent against HL-60 cell

line comparing with compound 12 (IC50 92 mM) which suggestedthat introducing more sugar residues at C3eOH of compound 12 is afavorable modi1047297cation to ameliorating the anticancer activityReplacement of a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose at the C3eOH of 12 afforded compound 19 21 or 24 whichhas no signi1047297cant increase in cytotoxicity However adding a-L -rhamnosyl moiety at the C2eOH and C3eOH (or C3eOH solely) of b-D-galactose or b-D-glucose respectively obtained compounds

OOO

O

OAcOLevO OAc

OBn

O

O

OOO

HO

OBn

O

O

OAcOLevO

OAc

STol

29

30a

31a

AgOTf NIS

CH Cl 76

(1) p -TsOH CH2Cl2-CH3OH

(2)10Pd-C H2 NaOMe12

53 for 3 steps

OBnOBnO OBn

STol

30b

AgOTf NIS

CH Cl 43

OOO

OOBnOBnO

OBn

OBn

O

O

31b

(1) p -TsOH CH2Cl2-CH3OH

(2)10Pd-C H2

65 for 2 steps

13

Scheme 2 Synthesis of oleanolic acid saponins 12 and 13

Scheme 3 Synthesis of disaccharide donor 33

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22e23 or 25e26 which did not exhibit desirable antitumor activ-ity These results demonstrated that the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obviousin1047298uence on improving the cytotoxicity and a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) favored thecytotoxic activity

The logarithm of a partition coef 1047297cient (log P ) is a parameterwhich re1047298ects a drug equilibrium partition ratio between polar(water) and non-polar (octanol) phases and it has been proved todetermine the absorption the distribution the biological avail-ability and pharmacological activity of drugs In this study we have

employed the ACD lab program to predict the values of log P foreach oleanolic acid saponin 1e26 The results are arranged inTable 2 comparing the log P values of all synthesized compoundswe found that the order of antitumor activity was not consistentwith the order of lipophilicity which indicated that lipophilicity of the compounds had no important effect on cytotoxicity

3 Conclusions

A series of natural and non-natural sugar-modi1047297ed oleanolicacid saponins was synthesized in a concise and practical way andtheir cytotoxicity was evaluated in vitro In terms of structureeac-tivity relationships we can conclude that (i) with regard to ole-anolic acid bidesmosides the presence of a-L -rhamnosyl residue atthe terminal of both C-3 and C-28 position was critical to enhancecytotoxicity (ii) for oleanolic acid monodesmosides free C-28carboxylic acid favors the cytotoxicity (iii) changing the spatialcon1047297guration of L -alabinosyl moiety at the C-3 position of oleanolicacid disfavor anticancer activity (iv) introducing more sugar resi-

dues at C3e

OH of compound 12 is a favorable modi1047297

cation toameliorate the anticancer activity (v) the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obvious in-1047298uence on improving the cytotoxicity (vi) a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) was helpful

Scheme 4 Synthesis of target compounds 14e15

Scheme 5 Synthesis of target compounds 16e

18

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 5

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to improve the cytotoxic activity Meanwhile lipophilicity of thecompounds was not an important factor for cytotoxicity

4 Experimental section

41 Chemistry

Thin-layer chromatography (TLC) was performed on precoatedE Merck Silica Gel 60 F254 plates Flash column chromatographywas performed on silica gel (200e300 mesh Qingdao China)Optical rotations were determined with a PerkineElmer Model 241MC polarimeter 1HNMRand 13C NMR spectra were taken on a JEOL

JNM-ECP 600 spectrometer with tetramethylsilane as the internalstandard and chemical shifts are recorded in d values Mass spectrawere recorded on a Q-TOF Global mass spectrometer

Commercial reagents were all analytically or chemically pure andused without further puri1047297cation unless speci1047297ed All anhydroussolvents were dried and redistilled prior to use in the usual way

411 28-O-a-L-Rhamnopyranosyl-(1 4)-O-b-D-glucopyranosyl-

(16)-O-b-D-glucopyranosyl oleanate 3-O-b-D-xylopyranoside (11)

To a solution of compound 28 (100 mg) in dry CH2Cl2eMeOH(12 12 mL) was added a newly prepared NaOMe in MeOH solution(10 molL 020 mL) The mixture was stirred at room temperaturefor 5 h and neutralized with Dowex Hthorn resin to pH 7 andthen 1047297ltered The 1047297ltrate was concentrated and the resultingresidue was subjected to a silica gel column chromatography

(MeOHeCHCl3eH2O 12501) to give 11 (40 mg 87) as a whiteamorphous solid frac12a25

D 133 (c 025 CH3OH) IR (KBr) nmax 34992938 1731 1645 1453 1073 cm1 1H NMR (500 MHz C5D5N) d

623 (d J frac14 81 Hz1H H-100

)585 (br s1H H-1000

) 539 (t J frac14 36 Hz1H H-12) 498 (d J frac14 78 Hz 1H H-10) 496 (qd J frac14 91 60 Hz 1HH-5000 ) 482 (d J frac14 75 Hz 1H H-10000) 468 (dd J frac14 3217 Hz1H H-2000 ) 465 (m 1H H-40) 454 (dd J frac14 93 32 Hz 1H H-3000 ) 441 (t J frac14 96 Hz 1H H-300) 431e437 (m H-30000 H-4000 H-50000-1 H-60-1)409e423 (m 8H H-200 H-30 H-400 H-40000 H-50 H-500 H-600-1 H-60-2) 402 (t-like J frac14 88 79 Hz 1H H-20000) 393 (t-like J frac14 87 83 Hz1H H-20) 378 (t J frac14 110 Hz 1H H-50000-2) 364 (m 1H H-600-2)333 (dd J frac14 117 43 Hz 1H H-3) 315 (dd J frac14 137 37 Hz 1H H-18) 169 (d J frac14 60 Hz 3H H-6000 ) 129 124 109 099 089 089089 (s each 3H each CH3 7) 13C NMR (125 MHz C5D5N) d 1765(C-28) 1440 (C-13) 1229 (C-12) 1076 (C-10000) 1049 (C-100) 1028(C-1000 ) 956 (C-10) 887 (C-3) 788 786 784 781 772 766 755754 740 739 728 726 713 710 703 693 671 620 560 497482 471 463 422 418 400 396 389 371 341 332 326 308284 283 268 261 239 238 234 185 176 170 157 HR-MS(ESI) m z calcd for C53H86O21Na [M thorn Na]thorn 10815554 found10815579

412 General procedure for synthesizing compounds 31ae 31b

A mixture of compound 29 (0139 mmol) powdered 4 A mo-lecular sieves and compound 30a or 30b (0209 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (0221 mmol) and AgOTf (0016 mmol) was

OOBzO

O

OBzOBzO OBz

OBn

O

BzO

OOBzO

HO

OBn

O

BzO

41

10Pd-C H2

TMSOTf CH2Cl2

NaOMe CH2Cl2-CH3OHOO

HOO

OHOHO OH

OH

O

HO

OBzOBzO OBz

O

40

CCl3

NH

45

19

Scheme 6 Synthesis of target compound 19

Scheme 7 Synthesis of target compound 20

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e156

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added and the reaction mixture was stirred for 30 min and thenwarmed to room temperature The product was detected on TLC(21 petroleum ethereEtOAc) After completion of the reaction thereaction mixture was quenched by addition of Et3N and then

1047297ltered The 1047297ltrate was concentrated and puri1047297ed by a silica gelcolumn chromatography to afford the products

4121 Benzyl oleanolate 24-di-O-acetyl-3-O-levulinoyl-a-L-rham-

nopyranosyl-(1 2)-34-O-isopropylid-ene-a-L-arabinopyranoside

( 31a) Yield 76 frac12a25D thorn213 (c 08 CHCl3) IR (KBr) vmax 2929

1731 1447 1356 1123 1023 707 673 cm1 1H NMR (CDCl3600 MHz) d 707e735 (m 5H PheH) 534 (s 1H H-100 ) 529e532(m 3H H-30 H-40 H-12) 503e511 (m 3H H-200 PhCH2) 434(d J frac14 77 Hz1H H-10) 410e421(m 4H H-300 H-400 H-500 H-50-1)373e378 (m 2H H-20 H-50-2) 308 (dd J frac14 118 44 Hz 1H H-3)290 (dd J frac14 139 40 Hz 1H H-18) 272e276 (m 1H LeveCH H)261e266 (m 1H LeveCH H) 250e255 (m 1H LeveCHH ) 239e244 (m1H LeveCHH ) 216 215 205 (s each 3H each Ac2Leve

CH3) 153 133 (s each 3H each Oe

(CH3)2Ce

O) 120 (d J frac14 62 Hz

3H H-600 ) 112 104 092 089 088 081 060 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 2062 1775 (C-28) 17141702 1437 (C-13) 1364 1284 1280 1279 1225 (C-12) 1104((CH3)2C) 1031 (C-10) 952 (C-100 ) 890 (C-3) 791 751 733 709

705 697 696 693 662 659 626 558 476 467 459 417 414393 390 378 377 376 367 339 331 327 324 309 307 297280 278 276 261 259 237 234 230 210 208 182173 169164 154 153 HR-MS (ESI) m z calcd for C60H86O15Na [M thorn Na]thorn

10695859 found 10695883

4122 Benzyl oleanolate 234-tri-O-benzyl-b-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-a-L-arabin-opyranoside ( 31b)

Yield 43 frac12a23D thorn592 (c 118 CHCl3) IR (KBr) vmax 2939 1733

1454 1368 1116 1023 738 685 cm1 1H NMR (CDCl3 600 MHz)d 727e734 (m 20H PheH) 533 (d J frac14 11 Hz 1H H-100 ) 529(t J frac14 36 Hz 1H H-12) 511 (d J frac14 127 Hz 1H PhCH H) 505(d J frac14 127 Hz 1H PhCHH ) 501 (d J frac14 154 Hz 1H PhCH H) 495(d J frac14 135 Hz 1H PhCHH ) 476 (d J frac14 127 Hz 1H PhCH H) 467

(d J frac14 110 Hz 1H PhCHH ) 464 (d J frac14 104 Hz 1H PhCH H) 456

Scheme 8 Synthesis of target compounds 21e26

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1315

(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1415

(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

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glycosylation with the acceptor 48 [3233] in the presence of NISand AgOTf stereoselectively affording the fully protected interme-diate 49 in 35 yield The diisopropylidene was achieved bytreatment of compound 49 with p-TsOH in CH2Cl2eMeOH solutionto obtain 50 Subsequent deprotection of the benzyl ether with 10

PdeC and the benzoyl ester with NaOMe in CH2Cl2eMeOH wasachieved to afford the target compound 20The synthesis of non-natural saponins 21e26 was achieved in a

straight-forward manner As shown in Scheme 8 the oleanolic acidsaponin acceptor 51 (or 53) was condensed with the known donor40 in the presence of TMSOTf to obtain the intermediates 52aec (or54aec) Subsequent removal of benzyl and acetyl groups providedthe desired saponins 21e23 (or 24e26)

22 Cytotoxic activity

To examine the potential ability of the natural and non-naturaloleanolic acid saponins 1e26 the in vitro cytotoxic activity againsthuman promyelotic leukemia cancer (HL-60) non-small-cell lung

cancer (A549) and human melanoma cancer (A375) was assessedusing methyl-thiazol-tetrazolium (MTT) reduction test and sulfo-rhodamine B (SRB) staining methods for protein The cytotoxicityresults presented in Table 1 are shown as the IC50 values They aresubdivided into two groups Oleanolic acid bidesmosides (1e4 11)and Oleanolic acid monodesmosides (5e10 12e26) In the 1047297rstgroup saponin 4 exhibited the most cytotoxic activity against thethree tumor cells (with IC50 ranging from 32 to 105 mM) followed

by saponin 11 (IC50 47e103 mM) In contrast saponins 1e3 did notshow any cytotoxic activity (IC50 gt 20 mM) against HL-60 A549 andA375 cell lines These results proved that the presence of a-L -rhamnosyl residue at the terminal of both C-3 and C-28 positionwas critical to enhance cytotoxicity

In the second group most of compounds except 20 22e

23 and25e26 exerted better anticancer activity When compared com-pounds 6 16e17 with 1e3 the in1047298uence of free C-28 carboxylicacid on enhancing the cytotoxicity was clear As for compound 12we anticipated that changing the spatial con1047297guration of L -alabi-nosyl moiety at the C-3 position of oleanolic acid should improveanticancer activity Actually the changed compound 20 exhibitedno cytotoxicity against all tested cell lines (IC50 gt 20 mM) Howeverwhen a-L -rhamnosyl residue of compound 12 was converted tob-L -rhamnosyl residue the derived compound 13 exerted good cyto-toxicity especially enhanced the cytotoxicity about 2-fold againstHL-60 cell line (IC50 42 mM) Meanwhile compounds 5 (IC50

61 mM) 9 (IC50 35 mM) 10 (IC50 31 mM) 14 (IC50 34 mM) and 15

(IC50 42 mM) were 15- to 3-fold more potent against HL-60 cell

line comparing with compound 12 (IC50 92 mM) which suggestedthat introducing more sugar residues at C3eOH of compound 12 is afavorable modi1047297cation to ameliorating the anticancer activityReplacement of a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose at the C3eOH of 12 afforded compound 19 21 or 24 whichhas no signi1047297cant increase in cytotoxicity However adding a-L -rhamnosyl moiety at the C2eOH and C3eOH (or C3eOH solely) of b-D-galactose or b-D-glucose respectively obtained compounds

OOO

O

OAcOLevO OAc

OBn

O

O

OOO

HO

OBn

O

O

OAcOLevO

OAc

STol

29

30a

31a

AgOTf NIS

CH Cl 76

(1) p -TsOH CH2Cl2-CH3OH

(2)10Pd-C H2 NaOMe12

53 for 3 steps

OBnOBnO OBn

STol

30b

AgOTf NIS

CH Cl 43

OOO

OOBnOBnO

OBn

OBn

O

O

31b

(1) p -TsOH CH2Cl2-CH3OH

(2)10Pd-C H2

65 for 2 steps

13

Scheme 2 Synthesis of oleanolic acid saponins 12 and 13

Scheme 3 Synthesis of disaccharide donor 33

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22e23 or 25e26 which did not exhibit desirable antitumor activ-ity These results demonstrated that the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obviousin1047298uence on improving the cytotoxicity and a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) favored thecytotoxic activity

The logarithm of a partition coef 1047297cient (log P ) is a parameterwhich re1047298ects a drug equilibrium partition ratio between polar(water) and non-polar (octanol) phases and it has been proved todetermine the absorption the distribution the biological avail-ability and pharmacological activity of drugs In this study we have

employed the ACD lab program to predict the values of log P foreach oleanolic acid saponin 1e26 The results are arranged inTable 2 comparing the log P values of all synthesized compoundswe found that the order of antitumor activity was not consistentwith the order of lipophilicity which indicated that lipophilicity of the compounds had no important effect on cytotoxicity

3 Conclusions

A series of natural and non-natural sugar-modi1047297ed oleanolicacid saponins was synthesized in a concise and practical way andtheir cytotoxicity was evaluated in vitro In terms of structureeac-tivity relationships we can conclude that (i) with regard to ole-anolic acid bidesmosides the presence of a-L -rhamnosyl residue atthe terminal of both C-3 and C-28 position was critical to enhancecytotoxicity (ii) for oleanolic acid monodesmosides free C-28carboxylic acid favors the cytotoxicity (iii) changing the spatialcon1047297guration of L -alabinosyl moiety at the C-3 position of oleanolicacid disfavor anticancer activity (iv) introducing more sugar resi-

dues at C3e

OH of compound 12 is a favorable modi1047297

cation toameliorate the anticancer activity (v) the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obvious in-1047298uence on improving the cytotoxicity (vi) a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) was helpful

Scheme 4 Synthesis of target compounds 14e15

Scheme 5 Synthesis of target compounds 16e

18

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to improve the cytotoxic activity Meanwhile lipophilicity of thecompounds was not an important factor for cytotoxicity

4 Experimental section

41 Chemistry

Thin-layer chromatography (TLC) was performed on precoatedE Merck Silica Gel 60 F254 plates Flash column chromatographywas performed on silica gel (200e300 mesh Qingdao China)Optical rotations were determined with a PerkineElmer Model 241MC polarimeter 1HNMRand 13C NMR spectra were taken on a JEOL

JNM-ECP 600 spectrometer with tetramethylsilane as the internalstandard and chemical shifts are recorded in d values Mass spectrawere recorded on a Q-TOF Global mass spectrometer

Commercial reagents were all analytically or chemically pure andused without further puri1047297cation unless speci1047297ed All anhydroussolvents were dried and redistilled prior to use in the usual way

411 28-O-a-L-Rhamnopyranosyl-(1 4)-O-b-D-glucopyranosyl-

(16)-O-b-D-glucopyranosyl oleanate 3-O-b-D-xylopyranoside (11)

To a solution of compound 28 (100 mg) in dry CH2Cl2eMeOH(12 12 mL) was added a newly prepared NaOMe in MeOH solution(10 molL 020 mL) The mixture was stirred at room temperaturefor 5 h and neutralized with Dowex Hthorn resin to pH 7 andthen 1047297ltered The 1047297ltrate was concentrated and the resultingresidue was subjected to a silica gel column chromatography

(MeOHeCHCl3eH2O 12501) to give 11 (40 mg 87) as a whiteamorphous solid frac12a25

D 133 (c 025 CH3OH) IR (KBr) nmax 34992938 1731 1645 1453 1073 cm1 1H NMR (500 MHz C5D5N) d

623 (d J frac14 81 Hz1H H-100

)585 (br s1H H-1000

) 539 (t J frac14 36 Hz1H H-12) 498 (d J frac14 78 Hz 1H H-10) 496 (qd J frac14 91 60 Hz 1HH-5000 ) 482 (d J frac14 75 Hz 1H H-10000) 468 (dd J frac14 3217 Hz1H H-2000 ) 465 (m 1H H-40) 454 (dd J frac14 93 32 Hz 1H H-3000 ) 441 (t J frac14 96 Hz 1H H-300) 431e437 (m H-30000 H-4000 H-50000-1 H-60-1)409e423 (m 8H H-200 H-30 H-400 H-40000 H-50 H-500 H-600-1 H-60-2) 402 (t-like J frac14 88 79 Hz 1H H-20000) 393 (t-like J frac14 87 83 Hz1H H-20) 378 (t J frac14 110 Hz 1H H-50000-2) 364 (m 1H H-600-2)333 (dd J frac14 117 43 Hz 1H H-3) 315 (dd J frac14 137 37 Hz 1H H-18) 169 (d J frac14 60 Hz 3H H-6000 ) 129 124 109 099 089 089089 (s each 3H each CH3 7) 13C NMR (125 MHz C5D5N) d 1765(C-28) 1440 (C-13) 1229 (C-12) 1076 (C-10000) 1049 (C-100) 1028(C-1000 ) 956 (C-10) 887 (C-3) 788 786 784 781 772 766 755754 740 739 728 726 713 710 703 693 671 620 560 497482 471 463 422 418 400 396 389 371 341 332 326 308284 283 268 261 239 238 234 185 176 170 157 HR-MS(ESI) m z calcd for C53H86O21Na [M thorn Na]thorn 10815554 found10815579

412 General procedure for synthesizing compounds 31ae 31b

A mixture of compound 29 (0139 mmol) powdered 4 A mo-lecular sieves and compound 30a or 30b (0209 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (0221 mmol) and AgOTf (0016 mmol) was

OOBzO

O

OBzOBzO OBz

OBn

O

BzO

OOBzO

HO

OBn

O

BzO

41

10Pd-C H2

TMSOTf CH2Cl2

NaOMe CH2Cl2-CH3OHOO

HOO

OHOHO OH

OH

O

HO

OBzOBzO OBz

O

40

CCl3

NH

45

19

Scheme 6 Synthesis of target compound 19

Scheme 7 Synthesis of target compound 20

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added and the reaction mixture was stirred for 30 min and thenwarmed to room temperature The product was detected on TLC(21 petroleum ethereEtOAc) After completion of the reaction thereaction mixture was quenched by addition of Et3N and then

1047297ltered The 1047297ltrate was concentrated and puri1047297ed by a silica gelcolumn chromatography to afford the products

4121 Benzyl oleanolate 24-di-O-acetyl-3-O-levulinoyl-a-L-rham-

nopyranosyl-(1 2)-34-O-isopropylid-ene-a-L-arabinopyranoside

( 31a) Yield 76 frac12a25D thorn213 (c 08 CHCl3) IR (KBr) vmax 2929

1731 1447 1356 1123 1023 707 673 cm1 1H NMR (CDCl3600 MHz) d 707e735 (m 5H PheH) 534 (s 1H H-100 ) 529e532(m 3H H-30 H-40 H-12) 503e511 (m 3H H-200 PhCH2) 434(d J frac14 77 Hz1H H-10) 410e421(m 4H H-300 H-400 H-500 H-50-1)373e378 (m 2H H-20 H-50-2) 308 (dd J frac14 118 44 Hz 1H H-3)290 (dd J frac14 139 40 Hz 1H H-18) 272e276 (m 1H LeveCH H)261e266 (m 1H LeveCH H) 250e255 (m 1H LeveCHH ) 239e244 (m1H LeveCHH ) 216 215 205 (s each 3H each Ac2Leve

CH3) 153 133 (s each 3H each Oe

(CH3)2Ce

O) 120 (d J frac14 62 Hz

3H H-600 ) 112 104 092 089 088 081 060 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 2062 1775 (C-28) 17141702 1437 (C-13) 1364 1284 1280 1279 1225 (C-12) 1104((CH3)2C) 1031 (C-10) 952 (C-100 ) 890 (C-3) 791 751 733 709

705 697 696 693 662 659 626 558 476 467 459 417 414393 390 378 377 376 367 339 331 327 324 309 307 297280 278 276 261 259 237 234 230 210 208 182173 169164 154 153 HR-MS (ESI) m z calcd for C60H86O15Na [M thorn Na]thorn

10695859 found 10695883

4122 Benzyl oleanolate 234-tri-O-benzyl-b-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-a-L-arabin-opyranoside ( 31b)

Yield 43 frac12a23D thorn592 (c 118 CHCl3) IR (KBr) vmax 2939 1733

1454 1368 1116 1023 738 685 cm1 1H NMR (CDCl3 600 MHz)d 727e734 (m 20H PheH) 533 (d J frac14 11 Hz 1H H-100 ) 529(t J frac14 36 Hz 1H H-12) 511 (d J frac14 127 Hz 1H PhCH H) 505(d J frac14 127 Hz 1H PhCHH ) 501 (d J frac14 154 Hz 1H PhCH H) 495(d J frac14 135 Hz 1H PhCHH ) 476 (d J frac14 127 Hz 1H PhCH H) 467

(d J frac14 110 Hz 1H PhCHH ) 464 (d J frac14 104 Hz 1H PhCH H) 456

Scheme 8 Synthesis of target compounds 21e26

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e158

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 9

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1510

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1415

(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

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[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

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22e23 or 25e26 which did not exhibit desirable antitumor activ-ity These results demonstrated that the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obviousin1047298uence on improving the cytotoxicity and a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) favored thecytotoxic activity

The logarithm of a partition coef 1047297cient (log P ) is a parameterwhich re1047298ects a drug equilibrium partition ratio between polar(water) and non-polar (octanol) phases and it has been proved todetermine the absorption the distribution the biological avail-ability and pharmacological activity of drugs In this study we have

employed the ACD lab program to predict the values of log P foreach oleanolic acid saponin 1e26 The results are arranged inTable 2 comparing the log P values of all synthesized compoundswe found that the order of antitumor activity was not consistentwith the order of lipophilicity which indicated that lipophilicity of the compounds had no important effect on cytotoxicity

3 Conclusions

A series of natural and non-natural sugar-modi1047297ed oleanolicacid saponins was synthesized in a concise and practical way andtheir cytotoxicity was evaluated in vitro In terms of structureeac-tivity relationships we can conclude that (i) with regard to ole-anolic acid bidesmosides the presence of a-L -rhamnosyl residue atthe terminal of both C-3 and C-28 position was critical to enhancecytotoxicity (ii) for oleanolic acid monodesmosides free C-28carboxylic acid favors the cytotoxicity (iii) changing the spatialcon1047297guration of L -alabinosyl moiety at the C-3 position of oleanolicacid disfavor anticancer activity (iv) introducing more sugar resi-

dues at C3e

OH of compound 12 is a favorable modi1047297

cation toameliorate the anticancer activity (v) the type of the 1047297rst mono-saccharide linked to C3eOH of oleanolic acid has no obvious in-1047298uence on improving the cytotoxicity (vi) a-L -rhamnose moietylinked to C2eOH with free C3eOH of the 1047297rst monosaccharide (a-L -alabinose by b-D-xylose b-D-galactose or b-D-glucose) was helpful

Scheme 4 Synthesis of target compounds 14e15

Scheme 5 Synthesis of target compounds 16e

18

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to improve the cytotoxic activity Meanwhile lipophilicity of thecompounds was not an important factor for cytotoxicity

4 Experimental section

41 Chemistry

Thin-layer chromatography (TLC) was performed on precoatedE Merck Silica Gel 60 F254 plates Flash column chromatographywas performed on silica gel (200e300 mesh Qingdao China)Optical rotations were determined with a PerkineElmer Model 241MC polarimeter 1HNMRand 13C NMR spectra were taken on a JEOL

JNM-ECP 600 spectrometer with tetramethylsilane as the internalstandard and chemical shifts are recorded in d values Mass spectrawere recorded on a Q-TOF Global mass spectrometer

Commercial reagents were all analytically or chemically pure andused without further puri1047297cation unless speci1047297ed All anhydroussolvents were dried and redistilled prior to use in the usual way

411 28-O-a-L-Rhamnopyranosyl-(1 4)-O-b-D-glucopyranosyl-

(16)-O-b-D-glucopyranosyl oleanate 3-O-b-D-xylopyranoside (11)

To a solution of compound 28 (100 mg) in dry CH2Cl2eMeOH(12 12 mL) was added a newly prepared NaOMe in MeOH solution(10 molL 020 mL) The mixture was stirred at room temperaturefor 5 h and neutralized with Dowex Hthorn resin to pH 7 andthen 1047297ltered The 1047297ltrate was concentrated and the resultingresidue was subjected to a silica gel column chromatography

(MeOHeCHCl3eH2O 12501) to give 11 (40 mg 87) as a whiteamorphous solid frac12a25

D 133 (c 025 CH3OH) IR (KBr) nmax 34992938 1731 1645 1453 1073 cm1 1H NMR (500 MHz C5D5N) d

623 (d J frac14 81 Hz1H H-100

)585 (br s1H H-1000

) 539 (t J frac14 36 Hz1H H-12) 498 (d J frac14 78 Hz 1H H-10) 496 (qd J frac14 91 60 Hz 1HH-5000 ) 482 (d J frac14 75 Hz 1H H-10000) 468 (dd J frac14 3217 Hz1H H-2000 ) 465 (m 1H H-40) 454 (dd J frac14 93 32 Hz 1H H-3000 ) 441 (t J frac14 96 Hz 1H H-300) 431e437 (m H-30000 H-4000 H-50000-1 H-60-1)409e423 (m 8H H-200 H-30 H-400 H-40000 H-50 H-500 H-600-1 H-60-2) 402 (t-like J frac14 88 79 Hz 1H H-20000) 393 (t-like J frac14 87 83 Hz1H H-20) 378 (t J frac14 110 Hz 1H H-50000-2) 364 (m 1H H-600-2)333 (dd J frac14 117 43 Hz 1H H-3) 315 (dd J frac14 137 37 Hz 1H H-18) 169 (d J frac14 60 Hz 3H H-6000 ) 129 124 109 099 089 089089 (s each 3H each CH3 7) 13C NMR (125 MHz C5D5N) d 1765(C-28) 1440 (C-13) 1229 (C-12) 1076 (C-10000) 1049 (C-100) 1028(C-1000 ) 956 (C-10) 887 (C-3) 788 786 784 781 772 766 755754 740 739 728 726 713 710 703 693 671 620 560 497482 471 463 422 418 400 396 389 371 341 332 326 308284 283 268 261 239 238 234 185 176 170 157 HR-MS(ESI) m z calcd for C53H86O21Na [M thorn Na]thorn 10815554 found10815579

412 General procedure for synthesizing compounds 31ae 31b

A mixture of compound 29 (0139 mmol) powdered 4 A mo-lecular sieves and compound 30a or 30b (0209 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (0221 mmol) and AgOTf (0016 mmol) was

OOBzO

O

OBzOBzO OBz

OBn

O

BzO

OOBzO

HO

OBn

O

BzO

41

10Pd-C H2

TMSOTf CH2Cl2

NaOMe CH2Cl2-CH3OHOO

HOO

OHOHO OH

OH

O

HO

OBzOBzO OBz

O

40

CCl3

NH

45

19

Scheme 6 Synthesis of target compound 19

Scheme 7 Synthesis of target compound 20

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added and the reaction mixture was stirred for 30 min and thenwarmed to room temperature The product was detected on TLC(21 petroleum ethereEtOAc) After completion of the reaction thereaction mixture was quenched by addition of Et3N and then

1047297ltered The 1047297ltrate was concentrated and puri1047297ed by a silica gelcolumn chromatography to afford the products

4121 Benzyl oleanolate 24-di-O-acetyl-3-O-levulinoyl-a-L-rham-

nopyranosyl-(1 2)-34-O-isopropylid-ene-a-L-arabinopyranoside

( 31a) Yield 76 frac12a25D thorn213 (c 08 CHCl3) IR (KBr) vmax 2929

1731 1447 1356 1123 1023 707 673 cm1 1H NMR (CDCl3600 MHz) d 707e735 (m 5H PheH) 534 (s 1H H-100 ) 529e532(m 3H H-30 H-40 H-12) 503e511 (m 3H H-200 PhCH2) 434(d J frac14 77 Hz1H H-10) 410e421(m 4H H-300 H-400 H-500 H-50-1)373e378 (m 2H H-20 H-50-2) 308 (dd J frac14 118 44 Hz 1H H-3)290 (dd J frac14 139 40 Hz 1H H-18) 272e276 (m 1H LeveCH H)261e266 (m 1H LeveCH H) 250e255 (m 1H LeveCHH ) 239e244 (m1H LeveCHH ) 216 215 205 (s each 3H each Ac2Leve

CH3) 153 133 (s each 3H each Oe

(CH3)2Ce

O) 120 (d J frac14 62 Hz

3H H-600 ) 112 104 092 089 088 081 060 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 2062 1775 (C-28) 17141702 1437 (C-13) 1364 1284 1280 1279 1225 (C-12) 1104((CH3)2C) 1031 (C-10) 952 (C-100 ) 890 (C-3) 791 751 733 709

705 697 696 693 662 659 626 558 476 467 459 417 414393 390 378 377 376 367 339 331 327 324 309 307 297280 278 276 261 259 237 234 230 210 208 182173 169164 154 153 HR-MS (ESI) m z calcd for C60H86O15Na [M thorn Na]thorn

10695859 found 10695883

4122 Benzyl oleanolate 234-tri-O-benzyl-b-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-a-L-arabin-opyranoside ( 31b)

Yield 43 frac12a23D thorn592 (c 118 CHCl3) IR (KBr) vmax 2939 1733

1454 1368 1116 1023 738 685 cm1 1H NMR (CDCl3 600 MHz)d 727e734 (m 20H PheH) 533 (d J frac14 11 Hz 1H H-100 ) 529(t J frac14 36 Hz 1H H-12) 511 (d J frac14 127 Hz 1H PhCH H) 505(d J frac14 127 Hz 1H PhCHH ) 501 (d J frac14 154 Hz 1H PhCH H) 495(d J frac14 135 Hz 1H PhCHH ) 476 (d J frac14 127 Hz 1H PhCH H) 467

(d J frac14 110 Hz 1H PhCHH ) 464 (d J frac14 104 Hz 1H PhCH H) 456

Scheme 8 Synthesis of target compounds 21e26

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1315

(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1415

(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

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to improve the cytotoxic activity Meanwhile lipophilicity of thecompounds was not an important factor for cytotoxicity

4 Experimental section

41 Chemistry

Thin-layer chromatography (TLC) was performed on precoatedE Merck Silica Gel 60 F254 plates Flash column chromatographywas performed on silica gel (200e300 mesh Qingdao China)Optical rotations were determined with a PerkineElmer Model 241MC polarimeter 1HNMRand 13C NMR spectra were taken on a JEOL

JNM-ECP 600 spectrometer with tetramethylsilane as the internalstandard and chemical shifts are recorded in d values Mass spectrawere recorded on a Q-TOF Global mass spectrometer

Commercial reagents were all analytically or chemically pure andused without further puri1047297cation unless speci1047297ed All anhydroussolvents were dried and redistilled prior to use in the usual way

411 28-O-a-L-Rhamnopyranosyl-(1 4)-O-b-D-glucopyranosyl-

(16)-O-b-D-glucopyranosyl oleanate 3-O-b-D-xylopyranoside (11)

To a solution of compound 28 (100 mg) in dry CH2Cl2eMeOH(12 12 mL) was added a newly prepared NaOMe in MeOH solution(10 molL 020 mL) The mixture was stirred at room temperaturefor 5 h and neutralized with Dowex Hthorn resin to pH 7 andthen 1047297ltered The 1047297ltrate was concentrated and the resultingresidue was subjected to a silica gel column chromatography

(MeOHeCHCl3eH2O 12501) to give 11 (40 mg 87) as a whiteamorphous solid frac12a25

D 133 (c 025 CH3OH) IR (KBr) nmax 34992938 1731 1645 1453 1073 cm1 1H NMR (500 MHz C5D5N) d

623 (d J frac14 81 Hz1H H-100

)585 (br s1H H-1000

) 539 (t J frac14 36 Hz1H H-12) 498 (d J frac14 78 Hz 1H H-10) 496 (qd J frac14 91 60 Hz 1HH-5000 ) 482 (d J frac14 75 Hz 1H H-10000) 468 (dd J frac14 3217 Hz1H H-2000 ) 465 (m 1H H-40) 454 (dd J frac14 93 32 Hz 1H H-3000 ) 441 (t J frac14 96 Hz 1H H-300) 431e437 (m H-30000 H-4000 H-50000-1 H-60-1)409e423 (m 8H H-200 H-30 H-400 H-40000 H-50 H-500 H-600-1 H-60-2) 402 (t-like J frac14 88 79 Hz 1H H-20000) 393 (t-like J frac14 87 83 Hz1H H-20) 378 (t J frac14 110 Hz 1H H-50000-2) 364 (m 1H H-600-2)333 (dd J frac14 117 43 Hz 1H H-3) 315 (dd J frac14 137 37 Hz 1H H-18) 169 (d J frac14 60 Hz 3H H-6000 ) 129 124 109 099 089 089089 (s each 3H each CH3 7) 13C NMR (125 MHz C5D5N) d 1765(C-28) 1440 (C-13) 1229 (C-12) 1076 (C-10000) 1049 (C-100) 1028(C-1000 ) 956 (C-10) 887 (C-3) 788 786 784 781 772 766 755754 740 739 728 726 713 710 703 693 671 620 560 497482 471 463 422 418 400 396 389 371 341 332 326 308284 283 268 261 239 238 234 185 176 170 157 HR-MS(ESI) m z calcd for C53H86O21Na [M thorn Na]thorn 10815554 found10815579

412 General procedure for synthesizing compounds 31ae 31b

A mixture of compound 29 (0139 mmol) powdered 4 A mo-lecular sieves and compound 30a or 30b (0209 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (0221 mmol) and AgOTf (0016 mmol) was

OOBzO

O

OBzOBzO OBz

OBn

O

BzO

OOBzO

HO

OBn

O

BzO

41

10Pd-C H2

TMSOTf CH2Cl2

NaOMe CH2Cl2-CH3OHOO

HOO

OHOHO OH

OH

O

HO

OBzOBzO OBz

O

40

CCl3

NH

45

19

Scheme 6 Synthesis of target compound 19

Scheme 7 Synthesis of target compound 20

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added and the reaction mixture was stirred for 30 min and thenwarmed to room temperature The product was detected on TLC(21 petroleum ethereEtOAc) After completion of the reaction thereaction mixture was quenched by addition of Et3N and then

1047297ltered The 1047297ltrate was concentrated and puri1047297ed by a silica gelcolumn chromatography to afford the products

4121 Benzyl oleanolate 24-di-O-acetyl-3-O-levulinoyl-a-L-rham-

nopyranosyl-(1 2)-34-O-isopropylid-ene-a-L-arabinopyranoside

( 31a) Yield 76 frac12a25D thorn213 (c 08 CHCl3) IR (KBr) vmax 2929

1731 1447 1356 1123 1023 707 673 cm1 1H NMR (CDCl3600 MHz) d 707e735 (m 5H PheH) 534 (s 1H H-100 ) 529e532(m 3H H-30 H-40 H-12) 503e511 (m 3H H-200 PhCH2) 434(d J frac14 77 Hz1H H-10) 410e421(m 4H H-300 H-400 H-500 H-50-1)373e378 (m 2H H-20 H-50-2) 308 (dd J frac14 118 44 Hz 1H H-3)290 (dd J frac14 139 40 Hz 1H H-18) 272e276 (m 1H LeveCH H)261e266 (m 1H LeveCH H) 250e255 (m 1H LeveCHH ) 239e244 (m1H LeveCHH ) 216 215 205 (s each 3H each Ac2Leve

CH3) 153 133 (s each 3H each Oe

(CH3)2Ce

O) 120 (d J frac14 62 Hz

3H H-600 ) 112 104 092 089 088 081 060 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 2062 1775 (C-28) 17141702 1437 (C-13) 1364 1284 1280 1279 1225 (C-12) 1104((CH3)2C) 1031 (C-10) 952 (C-100 ) 890 (C-3) 791 751 733 709

705 697 696 693 662 659 626 558 476 467 459 417 414393 390 378 377 376 367 339 331 327 324 309 307 297280 278 276 261 259 237 234 230 210 208 182173 169164 154 153 HR-MS (ESI) m z calcd for C60H86O15Na [M thorn Na]thorn

10695859 found 10695883

4122 Benzyl oleanolate 234-tri-O-benzyl-b-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-a-L-arabin-opyranoside ( 31b)

Yield 43 frac12a23D thorn592 (c 118 CHCl3) IR (KBr) vmax 2939 1733

1454 1368 1116 1023 738 685 cm1 1H NMR (CDCl3 600 MHz)d 727e734 (m 20H PheH) 533 (d J frac14 11 Hz 1H H-100 ) 529(t J frac14 36 Hz 1H H-12) 511 (d J frac14 127 Hz 1H PhCH H) 505(d J frac14 127 Hz 1H PhCHH ) 501 (d J frac14 154 Hz 1H PhCH H) 495(d J frac14 135 Hz 1H PhCHH ) 476 (d J frac14 127 Hz 1H PhCH H) 467

(d J frac14 110 Hz 1H PhCHH ) 464 (d J frac14 104 Hz 1H PhCH H) 456

Scheme 8 Synthesis of target compounds 21e26

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e158

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

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PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

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[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

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5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

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Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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added and the reaction mixture was stirred for 30 min and thenwarmed to room temperature The product was detected on TLC(21 petroleum ethereEtOAc) After completion of the reaction thereaction mixture was quenched by addition of Et3N and then

1047297ltered The 1047297ltrate was concentrated and puri1047297ed by a silica gelcolumn chromatography to afford the products

4121 Benzyl oleanolate 24-di-O-acetyl-3-O-levulinoyl-a-L-rham-

nopyranosyl-(1 2)-34-O-isopropylid-ene-a-L-arabinopyranoside

( 31a) Yield 76 frac12a25D thorn213 (c 08 CHCl3) IR (KBr) vmax 2929

1731 1447 1356 1123 1023 707 673 cm1 1H NMR (CDCl3600 MHz) d 707e735 (m 5H PheH) 534 (s 1H H-100 ) 529e532(m 3H H-30 H-40 H-12) 503e511 (m 3H H-200 PhCH2) 434(d J frac14 77 Hz1H H-10) 410e421(m 4H H-300 H-400 H-500 H-50-1)373e378 (m 2H H-20 H-50-2) 308 (dd J frac14 118 44 Hz 1H H-3)290 (dd J frac14 139 40 Hz 1H H-18) 272e276 (m 1H LeveCH H)261e266 (m 1H LeveCH H) 250e255 (m 1H LeveCHH ) 239e244 (m1H LeveCHH ) 216 215 205 (s each 3H each Ac2Leve

CH3) 153 133 (s each 3H each Oe

(CH3)2Ce

O) 120 (d J frac14 62 Hz

3H H-600 ) 112 104 092 089 088 081 060 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 2062 1775 (C-28) 17141702 1437 (C-13) 1364 1284 1280 1279 1225 (C-12) 1104((CH3)2C) 1031 (C-10) 952 (C-100 ) 890 (C-3) 791 751 733 709

705 697 696 693 662 659 626 558 476 467 459 417 414393 390 378 377 376 367 339 331 327 324 309 307 297280 278 276 261 259 237 234 230 210 208 182173 169164 154 153 HR-MS (ESI) m z calcd for C60H86O15Na [M thorn Na]thorn

10695859 found 10695883

4122 Benzyl oleanolate 234-tri-O-benzyl-b-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-a-L-arabin-opyranoside ( 31b)

Yield 43 frac12a23D thorn592 (c 118 CHCl3) IR (KBr) vmax 2939 1733

1454 1368 1116 1023 738 685 cm1 1H NMR (CDCl3 600 MHz)d 727e734 (m 20H PheH) 533 (d J frac14 11 Hz 1H H-100 ) 529(t J frac14 36 Hz 1H H-12) 511 (d J frac14 127 Hz 1H PhCH H) 505(d J frac14 127 Hz 1H PhCHH ) 501 (d J frac14 154 Hz 1H PhCH H) 495(d J frac14 135 Hz 1H PhCHH ) 476 (d J frac14 127 Hz 1H PhCH H) 467

(d J frac14 110 Hz 1H PhCHH ) 464 (d J frac14 104 Hz 1H PhCH H) 456

Scheme 8 Synthesis of target compounds 21e26

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 7

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1510

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

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[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(d J frac14 127 Hz 1H PhCHH ) 431 (d J frac14 72 Hz 1H H-10) 421(dd J

frac14104 44 Hz 1H H-50-1) 399e402 (m 2H H-40 H-400 ) 395

(dq J frac14 91 66 Hz 1H H-500 ) 388 (dd J frac14 94 28 Hz 1H H-50-2)369e372 (m 2H H-20 H-300 ) 361 (dd J frac14 96 38 Hz 1H H-30)301 (dd J frac14 121 49 Hz 1H H-3) 291 (dd J frac14 137 38 Hz 1H H-18) 148 133 (s each 3H each Oe(CH3)2CeO) 128 (d J frac14 66 Hz3H H-600 ) 111 092 091 089 088 081 061 (s each 3H eachCH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 1439 (C-13)1389 1386 1366 1286 1284 1283 1282 1228 (C-12) 1104((CH3)2C) 1033 (C-10) 967 (C-100 ) 889 (C-3) 801 800 793 753749 735 723 687 685 661 559 478 469 462 417 415 400393 382 369 336 333 309 280 263 239 236 181171 159156 HRMALDIMS calcd for [M thorn Nathorn] C72H94O11Na 11576686found 11576688

4123 1-(4-Tolyl)thio-23-O-isopropylidene-4-O-(1-methoxy-1-

methylethyl)-b-D-xylopyranoside ( 33b) Compound 33a (160 g624 mmol) was dissolved in DMF (2 mL) and heated to 40 CTri1047298uoroacetic acid (100 mL 1 TFA in DMF) was added followed by2-methoxypropene (2 mL 2122 mmol) The reaction mixture wasstirred for 12 h at this temperature TLC (101 petroleum ethereEtOAc) indicated that the reaction was complete The reaction wasquenched with Et3N (20 mL) and the mixture was diluted withCH2Cl2 and sequentially washed with saturated NaHCO3 saturatedNaCl dried over Na2SO4 1047297ltered and concentrated The residue waspuri1047297ed by column chromatography (101 petroleum ethereEtOAc)affording compound 33b (150 g 65) as a white solid R f frac14 035(petroleum ethereEtOAc 101) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 82 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 469 (d J frac14 91 Hz 1H H-1) 409 (dd J frac14 115 50 Hz 1H H-5-1) 400 (ddd J frac14 96 92 50 Hz 1H H-4) 353 (t J frac14 91 Hz 1H H-3) 322 (s 3HOCH3) 318e321 (m 2H H-2 H-5-2) 235 (s 3H SPhCH3) 146141 137 133 (s each 3H each CH3 4) HRESIMS calcd for[M thorn Nathorn] C19H28O5SNa 3911550 found 3911576

4124 1-(4-Tolyl)thio-23-O-isopropylidene-b-D-xylopyranoside

( 33c ) Compound 33b (140 g 380 mmol) was dissolved in MeOH

(15 mL) and cooled to 0 C A solution of p-toluenesulfonic acid inMeOH (1 mgmL 200 mL) was added The reaction mixture wasstirred and monitored by TLC (61 petroleum ethereEtOAc) Whenthe starting material was disappeared the reaction was quenchedwith strongly basic ion exchange resin (OH form) The solutionwas 1047297ltrated and the 1047297ltrate was concentrated The residue waspuri1047297ed by column chromatography (61 petroleum ethereEtOAc)affording compound 33c as a white solid (100 g 88) R f frac14 031(petroleum ethereEtOAc 61) 1H NMR (CDCl3 600 MHz) d 747(d J frac14 78 Hz 2H PheH) 713 (d J frac14 78 Hz 2H PheH) 473(d J frac14 96 Hz 1H H-1) 412 (dd J frac14 115 50 Hz 1H H-5-1) 394e399 (m 1H H-4) 352 (t J frac14 92 Hz 1H H-3) 320e324 (m 2HH-2 H-5-2) 235 (s 3H SPhCH3) 149 144 (s each 3H eachCH3 2) ESI-MS (m z ) 2971 (M thorn Hthorn) 6152 (2M thorn Nathorn)

HRESIMS calcd for [M thorn Nathorn] C15H20O4SNa 3190975 found3190997

4125 1-(4-Tolyl)thio-4-O-acetyl-b-D-xylopyranoside ( 33e ) To asolution of compound 33c (140 mg 0472 mmol) in pyridineeCH2Cl2 (2 mL 11) Ac2O (120 mL) and DMAP (5 mg) was addedThe mixture was stirred for 2 h After completion of the reaction(monitored by TLC) the reaction mixture was quenched withCH3OH concentrated under reduced pressure The residue wasdiluted with EtOAc and sequentially washed with saturated 1 molL HCl saturated NaHCO3 saturated NaCl dried over Na2SO41047297ltered and concentrated The crude product 33d was dissolved inCH3OHeCH2Cl2 (5 mL 11) and then p-toluenesulfonic acid(24 mg) was added The mixture was stirred for 2 h The reaction

mixture was concentrated and puri1047297ed by column chromatog-raphy (petroleum ethereEtOAc 21) to afford 33e as a white solid(130 mg 92 for two steps) Rf frac14 031 (petroleum ethereEtOAc21) frac12a23

D 542 (c 170 CHCl3) IR (KBr) nmax 3403 3337 29722913 2860 1713 1487 1242 1070 950 824 804 592 cm1 1HNMR (DMSO-d6 600 MHz) d 735 (d J frac14 82 Hz 2H PheH) 715(d J frac14 82 Hz 2H PheH) 551 (d J frac14 59 Hz 1H C3eOH) 541(d J frac14 55 Hz 1H C2eOH) 461 (d J frac14 92 Hz 1H H-1) 450 (ddd J frac14 101 55 Hz 1H H-4) 384 (dd J frac14 110 50 Hz 1H H-5-1) 343(ddd J frac14 92 50 Hz 1H H-2) 326 (dd J frac14 110 101 Hz 1H H-5-2) 309 (ddd J frac14 92 59 Hz 1H H-3) 228 (s 3H SPhCH 3) 201(s 3H AceCH3) 13C NMR (DMSO-d6 150 MHz) d 1707 13751332 1301 1296 882 (C-1) 745 725 716 657 212 210 ESI-MS (m z ) 3211 (M thorn Nathorn) ESIHRMS m z calcd for C14H18O5NaS

[M thorn Na

thorn

] 3210773 found 3210780

Table 1

In vitro cytotoxicity of oleanolic acid bidesmosides (1e4 11) and oleanolic acidmonodesmosides (5e10 12e26) against cancer cell lines

Groupa Compound IC50 (mmolL)b

HL-60 A549 A375

I 1 gt20 gt20 gt202 gt20 gt20 gt203 gt20 gt20 gt204 32 10 105 23 73 1411 57 13 119 41 135 17

II 5 61 23 167 15 121 116 46 29 147 16 55 177 101 37 179 42 97 238 125 26 153 32 103 319 35 13 79 13 59 0910 31 18 62 11 35 0412 92 29 158 43 84 1813 42 15 175 34 77 3814 34 12 154 13 89 2415 42 03 gt20 221 6116 45 15 115 31 63 2917 25 09 78 44 61 1318 93 08 162 34 84 3319 36 01 107 42 50 1820 gt20 gt20 gt20

21 25 11 124 55 51 1222 gt20 152 07 154 0923 gt20 gt20 gt2024 129 27 235 27 116 1825 gt20 gt20 gt2026 gt20 gt20 gt20ADMc 090 03 056 02 075 01HCPTd 025 01 030 01 015 01

a (I) oleanolic acid bidesmosides (II) oleanolic acid monodesmosidesb Data represent mean values standard deviation for three independent ex-

periments made in triplicatec Adriamycin positive controld Hydroxycamptothecin positive control

Table 2

Lipophilicity (log P ) of oleanolic acid saponins 1e

26

Compound Log P a Compound Log P a

1 626 14 10022 406 15 7383 386 16 10024 611 17 7425 749 18 10516 749 19 9967 680 20 9968 680 21 10069 742 22 98710 478 23 103411 583 24 100612 996 25 98713 996 26 1034

a

Predicted octanolwater partition coef 1047297

cient

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e158

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

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8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1315

(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1415

(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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4126 1-(4-Tolyl)thio-23-di-O-benzoyl-4-O-acetyl-b-D-xylopyrano-

side ( 33f ) To a solution of 33e (100 mg 0335 mmol) and4-dimethylamino-pyridine (5 mg 0034 mmol) in pyridine (5 mL)and CH2Cl2 (10 mL) was added benzoyl chloride (156 mL101 mmol) dropwise at 0 C The reaction mixture was stirred for3 h and then quenched with CH3OH The solvents were evapo-rated in vacuo The resulting residue was diluted with EtOAc andsequentially washed with 1 N HCl saturated NaHCO3 and satu-rated NaCl dried over Na2SO4 1047297ltered and concentrated Theresidue was puri1047297ed by column chromatography (petroleumethereEtOAc 61) to afford 33f as a white solid (163 mg 94)Rf frac14 041 (petroleum ethereEtOAc 51) frac12a23

D thorn212 (c 130CHCl3) IR (KBr) nmax 2946 2873 1739 1229 1123 1056 804705 cm1 1H NMR (CDCl3 600 MHz) d 711e802 (m 14H PheH)556 (t J frac14 71 Hz 1H H-3) 536 (t J frac14 71 Hz 1H H-2) 512 (td J frac14 77 44 Hz 1H H-4) 506 (d J frac14 71 Hz 1H H-1) 448 (dd J frac14 121 44 Hz 1H H-5-1) 364 (dd J frac14 121 71 Hz 1H H-5-2)234 (s 3H SPhCH3) 201 (s 3H AceCH3) 13C NMR (CDCl3150 MHz) d 1701 1655 1652 1388 1337 1336 1301 13001289 1287 1286 868 (C-1) 716 702 684 644 214 210 ESI-MS (m z ) 5291 (M thorn Nathorn) ESIHRMS m z calcd for C28H26O7NaS[M thorn Nathorn] 5291297 found 5291302

4127 1-(4-Tolyl)thio-23-di-O-benzoyl-4-b-D-xylopyranoside ( 33g )

To a solution of 33f (120 mg 0237 mmol) in CH3OH (10 mL) wasadded acetyl chloride (120 mL) dropwise at 0 C The reactionmixture was stirred for 5 h and then quenched with Et3N Thesolvent was evaporated in vacuo The resulting residue was puri1047297edby column chromatography (petroleum ethereEtOAc 41) to afford33g as a white solid (100 mg 90) Rf frac14 032 (petroleum ethereEtOAc 31) frac12a23

D thorn471 (c 088 CHCl3) IR (KBr) nmax 3456 30592846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3600 MHz) d 712e804 (m 14H PheH) 540 (t-like J frac14 78 73 Hz1H H-3) 532 (t-like J frac14 78 73 Hz 1H H-2) 501 (d J frac14 73 Hz 1HH-1) 442 (dd J frac14 119 46 Hz 1H H-5-1) 398 (td J frac14 77 46 Hz1H H-4) 357 (dd J frac14 119 78 Hz 1H H-5-2) 234 (s 3H SPhCH3)

13C NMR (CDCl3 150 MHz) d 1672 1653 1387 1339 1337 13351303 1290 1287 871 (C-1) 763 703 686 678 214 ESI-MS(m z ) 4871 (M thorn Nathorn) ESIHRMS m z calcd for C26H24O6NaS[M thorn Nathorn] 4871191 found 4871174

4128 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-1-(4-

tolyl)thio-23-di-O-benzoyl-b-D-xylopyranoside ( 33i) A mixture of compound 33g (100 mg 022 mmol) powdered 4 A molecularsieves and compound 33h (239 mg 032 mmol) in dry CH2Cl2

(3 mL) was stirred at rt for 30 min then cooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwise and the reaction mixturewas stirred for 30 min and then warmed to rt The product wasdetected on TLC (21 petroleum ethereEtOAc) After completion of the reaction the reaction mixture was quenched with Et3N

(005 mL) and1047297ltered The1047297ltrate was concentrated and puri1047297edbya silica gel column chromatography (31 petroleum ethereEtOAc)to afford 33i (204 mg 91) frac12a23

D 118 (c 092 CHCl3) IR (KBr) nmax

3456 3059 2846 1726 1487 1269 1063 804 705 cm1 1H NMR (CDCl3 600 MHz) d 708e803 (m 34H PheH) 581 (t J frac14 96 Hz1H H-30) 568 (t J frac14 74 Hz1H H-3) 544 (dd J frac14 97 78 Hz1H H-20) 536 (t J frac14 96 Hz1H H-40) 531 (t-like J frac14 87 70 Hz 1H H-4)530 (d J frac14 73 Hz 1H H-1) 499 (t J frac14 78 64 Hz 1H H-2) 497 (d J frac14 78 Hz 1H H-10) 425 (dd J frac14 124 46 Hz 1H H-60-1) 417 (dd J frac14 119 32 Hz 1H H-5-1) 403 (m 1H H-50) 398 (dd J frac14 11955 Hz 1H H-5-2) 345 (dd J frac14 124 77 Hz 1H H-60-2) 231 (s 3HSPhCH3) 13C NMR (CDCl3 150 MHz) d 1714 1661 1659 16531651 1650 1385 1336 1334 1332 1300 1299 1298 12961286 1285 1284 1019 (C-1) 868 (C-10) 730 728 721 705

696 652 631 606 299 229 214 213 144 ESI-MS (m z )

10656 (Mthorn Nathorn) ESIHRMS m z calcd for C60H50O15NaS [Mthorn Nathorn]10652768 found 10652798

4129 2346-Tetra-O-benzoyl-b-D-glucopyranosyle(1 4)-23-di-

O-benzoyl-b-D-xylopyranoside trichloroacetimidate ( 33) To a solu-tion of compound 33i (150 mg 0144 mmol) in 10 mL of acetone-H2O (91) NBS (67 mg 0374 mmol) were added at 20 C Themixture was stirred for 5 min quenched with satd aq NaHCO3 Thereaction mixture was concentrated and the residue was dilutedwith EtOAcwashed with satd aq NaHCO3 brine dried over Na2SO4and concentrated The residue was puri1047297ed by silica gel columnchromatography (31 to 11 petroleum ethereEtOAc) to give 33j

(115 mg 85) as a white solid A solution of 33j (100 mg0107 mmol) CNCCl3 (008 mL 0859 mmol) and DBU (002 mL0054 mmol) in dry CH2Cl2 (2 mL) was stirred for 3 h at roomtemperature then the solvent was evaporated in vacuo to give aresidue which was puri1047297ed by silica-gel 1047298ash column chromatog-raphy (petroleum ethereEtOAc 31) to afford 33 (97 mg 83) as awhite solid Rf frac14 036 (petroleum ethereEtOAc 11) 1H NMR (CDCl3) d 852 (s 1H N-H) 721e818 (m 30H PheH) 657 (d J frac14 37 Hz 1H H-1) 598 (t J frac14 96 Hz 1H H-3) 583 (t J frac14 96 Hz1HH-30) 552 (t J frac14 96 Hz1H H-40) 545 (dd J frac14 96 77 Hz1H H-

20) 535 (dd J frac14 101 37 Hz 1H H-2) 497 (d J frac14 82 Hz 1H H-10)426 (m 1H H-4) 409 (m 2H H-60-1 H-60-2) 397 (m 1H H-50)389 (dd J frac14 115 55 Hz 1H H-5-1) 377 (t J frac14 110 Hz 1H H-5-2)ESI-MS m z 10803 (M thorn Hthorn) HRESIMS calcd for C55H45O16NCl3[M thorn Hthorn] m z 10801804 found m z 10801849

413 General procedure for synthesizing compounds 35e 36

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperatureunder argon for 30 min and then cooled to 78 C At this tem-perature a solution of TMSOTf (02 equiv) in dry CH2Cl2 wasinjected and after 10 min a trichloroacetimidate 32 or 33

(21 equiv) in dry CH2Cl2 was added The mixture was stirred foradditional 30 min and then warmed up to 10 C To the above

mixture was added a solution of saponin acceptor 29 (79 mg0112 mmol 10 equiv) in CH2Cl2 (2 mL) followed by NIS (50 mg0112 mmol 20 equiv) After being stirred for 1 h the reactionmixture was quenched with Et3N and then 1047297ltered through a padof Celite The 1047297ltrate was concentrated The residue was puri1047297edsilica gel column chromatography (251 petroleum ethereEtOAc)to give the fully protected saponin The amounts of the reactantsand the yields of the saponin products were calculated based onsaponin accepter 29

4131 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-iso-

propylidene-a-L-arabinopyranoside ( 35) Yield 73 for two stepsfrac12a22

D thorn368 (c 288 CHCl3) Mp 137e139 C IR (KBr) nmax 2939

1719 1448 1255 1096 705 cm1

1

H NMR (CDCl3 600 MHz)d 725e803 (m 20H PheH) 572 (t-like J frac14 73 69 Hz 1H H-30)535 (dd J frac14 32 19 Hz 1H H-200) 531 (d J frac14 19 Hz 1H H-100)526e529 (m 3H H-12 H-20H-40) 511 (d J frac14 129 Hz1H PhCH H)508 (d J frac14 127 Hz 1H PhCHH ) 507 (t J frac14 101 Hz 1H H-400) 486(d J frac14 50 Hz 1H H-10) 436 (dd J frac14 124 41 Hz 1H H-50-1) 431(d J frac14 73 Hz 1H H-1000 ) 421 (dd J frac14 101 37 Hz 1H H-300) 415e419 (m 2H H-4000 H-5000-1) 410e414 (m 2H H-3000 H-5000-2) 401(dq J frac14 92 60 Hz 1H H-500) 372 (m1H H-2000 ) 365 (dd J frac14 12464 Hz 1H H-50-2) 305 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 134 41 Hz 1H H-18) 209 204 (s each 3H each Ac 2) 157131 (s each 3H each Oe(CH3)2eO) 112 (d J frac14 64 Hz 3H H-600)109 099 092 089 088 080 060 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1705 1697 1656 1655

1437 (C-13) 13651335 13001285 1226 (C-12)1104 ((CH3)2C)

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 9

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1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1510

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

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PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1015

1035 (C-1000 ) 1016 (C-10) 949 (C-100) 891 (C-3) 761 758 734717 705 660 628 613 609 559 477 468 463 418 394 368308 282 278 262 237 206 175 165 155 138 HRMALDIMScalcd for [M thorn Nathorn] C81H100O20Na 14156721 found m z

14156700

4132 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-34-O-isopropylidene-a-

L-arabinopyranoside ( 36 ) Yield 73 for two steps frac12a22D thorn120 (c

110 CHCl3) IR (KBr) nmax 2934 1731 1455 1369 1260 1097 10581027 707 cm1 1H NMR (CDCl3 600 MHz) d 727e798 (m 35HPheH) 582 (t J frac14 99 Hz 1H H-30000) 563 (t J frac14 71 Hz 1H H-30)547 (dd J frac14 99 77 Hz 1H H-20000) 541 (t J frac14 99 Hz 1H H-40000)529 (t J frac14 36 Hz 1H H-12) 527 (dd J frac14 3317 Hz1H H-200) 525(d J frac14 17 Hz 1H H-100) 513 (dd J frac14 71 55 Hz 1H H-20) 511(d J frac14 121 Hz 1H PhCH H) 507 (d J frac14 121 Hz 1H PhCHH ) 502(t J frac14 99 Hz 1H H-400) 497 (d J frac14 77 Hz 1H H-10000) 467(d J frac14 55 Hz 1H H-10) 427 (d J frac14 77 Hz 1H H-1000 ) 420 (dd J frac14 121 28 Hz 1H H-5000-1) 417 (m 1H H-4000 ) 409e413 (m 2HH-3000 H-60000-1) 408 (dd J frac14 99 33 Hz 1H H-600) 402e406 (m2H H-40 H-50000) 396e401 (m 3H H-50-1 H-500 H-60000-2) 368e

372 (m 2H H-2000 H-50-2) 336 (dd J frac14 127 71 Hz 1H H-5000-2)302 (dd J frac14 121 44 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 204 201 (s each 3H each Ac 2) 149 129 (s each 3H eachOe(CH3)2eO) 110 (d J frac14 60 Hz 3H H-600) 113 092 090 087086 075 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1706 1661 1659 1655 1653 16511439 (C-13) 1366 1336 1335 1334 1333 1299 1298 12971286128512821232 (C-12)1105 ((CH3)2C) 1035 (C-1000 )1020(C-10) 1018 (C-10000) 950 (C-100) 891 (C-3) 790 731 721 711694 666 661 631 606 560 478 470 461 420 416 393 389369 356 333 328 325 309 299 282 279 263 261 239233 213 206 184 176 171 166 156 144 HRMALDIMS calcdfor [M thorn Nathorn] C108H122O28Na 18898059 found m z 18898015

414 General procedure for synthesizing compounds 37 e 38

To a solution of compound 35 or 36 (0054 mmol) in CH2Cl2eMeOH (V V 12 5 mL) was added p-TsOH (0054 mmol) was stirredat room temperature When TLC (32 petroleum ethereEtOAc)showed that deprotection had completed Et3N (01 mL) was addedand the mixture was concentrated and puri1047297ed through a silica gelcolumn chromatography (petroleum ethereEtOAc) affording theproduct 37 or 38

4141 Benzyl oleanolate 234-tri-O-benzoyl-b-D-xylopyranosyl-

(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabi-

nopyranoside ( 37 ) Yield95 frac12a23D 176 (c 365 CHCl3) Mp 141e

143 CIR (KBr) nmax 29451731 1451 137312601097 707 cm1 1HNMR (CDCl3 600 MHz) d 727e804 (m 20H PheH) 570 (t-like

J frac14 71 66 Hz1H H-3000

) 527e530 (m 4H H-12 H-300

H-2000

H-4000

)510 (d J frac14 121 Hz1H PhCH H) 508 (s 1H H-200) 506 (d J frac14 127 Hz1H PhCHH ) 501 (s1H H-100) 497 (d J frac14 50 Hz1H H-1000) 474 (brs 1H H-10) 435 (dd J frac14 127 38 Hz 1H H-5000-1) 410 (dd J frac14 9938 Hz1H H-50-1) 388e392 (m 2H H-30 H-40) 383e387 (m 2HH-20 H-500) 371e375 (m 2H H-400H-50-2) 361 (dd J frac14 115 44 Hz1H H-5000-2) 310 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 13738 Hz 1H H-18) 209 180 (s each 3H each Ac 2) 112 (d J frac14 66 Hz 3H H-600) 111 094 092 089 088 078 060 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 17051698165616551650 1438 (C-13)136513351334130012991285 1284 1281 1225 (C-12)1017 (C-10)1013 (C-1000) 980 (C-100)905 (C-3) 758 756 719 718 702 701 699 689 673 660 649611 604 555 477 468 460 418 415 394 392 386 368 339

332 327 325 308 298 282 277 259 258 237 235 231 211

206 183 175 169 165 154 HRMALDIMS calcd for [M thorn Nathorn]C78H96O20Na 13756371 found m z 13756387

4142 Benzyl oleanolate 2346-tetra-O-benzoyl-b-D-glucopyr-

anosyl-(1 4)-23-di-O-benzoyl-b-D-xylopy-ranosyl-(1 3)-24-

di-O-acetyl-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyranoside

( 38 ) Yield 90 frac12a22D 06 (c 085 CHCl3) IR (KBr) nmax 3451

2922 1735 14471256 109310621023 711 cm1 1H NMR (CDCl3600 MHz) d 726e794 (m 35H PheH) 582 (t J frac14 99 Hz 1H H-30000) 561 (t-like J frac14 77 71 Hz 1H H-3000 ) 547 (dd J frac14 99 77 Hz1H H-20000) 541 (t J frac14 94 Hz1H H-40000) 528 (t-like J frac14 38 33 Hz1H H-12) 518 (dd J frac14 3411 Hz 1H H-200) 514 (dd J frac14 77 55 Hz1H H-2000 ) 510 (d J frac14 126 Hz 1H PhCH H) 506 (d J frac14 127 Hz 1HPhCHH ) 501 (t J frac14 99 Hz1H H-400) 497 (d J frac14 77 Hz 1H H-10000)493 (d J frac14 17 Hz 1H H-100) 474 (d J frac14 55 Hz1H H-1000) 470 (d J frac14 28 Hz 1H H-10) 422 (dd J frac14 116 16 Hz 1H H-60000-1) 403e408 (m 2H H-4000 H-50000) 396e402 (m 3H H-300 H-5000-1 H-60000-2) 382e386 (m 3H H-20 H-30 H-40) 379 (dq J frac14 99 66 Hz 1HH-500) 370 (dd J frac14 115 88 Hz1H H-50-1) 370 (dd J frac14 115 49 Hz1H H-50-2) 340 (dd J frac14 121 77 Hz 1H H-5000-2) 308 (dd J frac14 11544 Hz 1H H-3) 290 (dd J frac14 137 38 Hz 1H H-18) 204 201 (seach 3H each Ac 2) 108 (d J frac14 61 Hz 3H H-600) 110 091 090

089 087 076 059 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1774 (C-28) 1704 1696 1659 1658 1657 16511650 1648 1437 (C-13) 1364 1334 1332 1330 1297 12961295 1294 1292 1289 1285 1283 1279 1278 1224 (C-12)1016 (C-10) 1015 (C-10000) 1014 (C-1000 ) 980 (C-100) 904 (C-3) 757753 729 723 718 716 707 705 700 694 671 659 629 619601 554 476 467 459 417 413 392 390 384 367 338 331326 307 297 281 276 258 256 236 234 230 209 204182173 168 164 153 HRMALDIMS calcd for [M thorn Nathorn]C105H118O28Na 18497706 found m z 18497702

415 General procedure for synthesizing compounds 42 e 44

A solution of Mbp thioglycoside 34 (40 mg 0112 mmol) and 4 AMS (80 mg) in CH2Cl2 (5 mL) was stirred at room temperature under

argon for 30 min and then cooled to 78 C At this temperature asolution of TMSOTf (02 equiv) in dry CH2Cl2 was injected and after10 min a trichloroacetimidate 32 39 or 40 (21 equiv) in dry CH2Cl2was added The mixture was stirred for additional 30 min and thenwarmed up to 10 C To the above mixture was added a solution of saponin acceptor 41 (99 mg 0112 mmol 10 equiv) in CH2Cl2 (2 mL)followed by NIS (50 mg 0112 mmol 20 equiv) After being stirredfor 1 h the reaction mixture was quenched with Et3N and then1047297ltered through a pad of Celite The 1047297ltrate was concentrated Theresidue was puri1047297ed silica gel column chromatography (251 pe-troleum ethereEtOAc) to give the fully protected saponin Theamounts of the reactants and the yields of the saponin products werecalculated based on saponin accepter 41

4151 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-b-D-xylopyr-anosyl-(1 3)-24-di-O-acetyl-a-L-rhamno-pyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 42 ) Yield 67 Mp 140e142 Cfrac12a25

D 281 (c 204 CHCl3) IR (KBr) nmax 2945 1731 1603 14511256 1093 711 cm1 1H NMR (CDCl3 600 MHz) d 727e810 (m30H PheH) 559 (t J frac14 71 66 Hz 2H H-30 H-3000 ) 529 (t J frac14 38 Hz 1H H-12) 520e522 (m 2H H-200 H-2000 ) 514e517 (m2H H-40 H-4000) 510 (d J frac14 127 Hz 1H PhCH H) 505 (s 1H H-100)504 (d J frac14 127 Hz 1H PhCHH ) 503 (m 1H H-400) 478 (d J frac14 49 Hz1H H-10) 460 (d J frac14 50 Hz1H H-1000 ) 437 (dd J frac14 12139 Hz1H H-50-1) 411 (dd J frac14 121 38 Hz 1H H-5000-1) 401e404(m 2H H-300 H-500) 397 (t J frac14 55 Hz 1H H-20) 362 (dd J frac14 12166 Hz 1H H-50-2) 333 (dd J frac14 121 66 Hz 1H H-5000-2) 315 (dd J frac14 115 44 Hz 1H H-3) 291 (dd J frac14 132 44 Hz 1H H-18) 192

177 (s each 3H each Ac2) 113 (d J frac14 66 Hz 3H H-600

) 112 095

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1510

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092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

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[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1115

092 090 089 074 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1699 1695 1658 1656 16551654 1652 1438 (C-13) 1365 1335 1334 1299 1298 1286128512841226 (C-12)1031 (C-10) 1011 (C-1000 ) 976 (C-100) 894(C-3) 759 741 714 712 703 695 691 672 660 609 557 477468 460 418 415 394 392 388 368 339 332 328 325308 281 277 260 259 237 235 231 209 206 183 175 169164 155 HRMALDIMS calcd for [M thorn Nathorn] C92H104O22Na15836952 found m z 15836912

4152 Benzyl oleanolate 3b-O-23462030406 0-octa-O-benzoyl-b-D-

cellobicopyranosyl-(1 3)-24-di-O-acetyl-a-L-rhamnopyranosyl-

(1 2)-34-di-O-benzoyl-b-D-xylopyranoside ( 43) Yield 66 Mp144e146 C frac12a24

D thorn262 (c 192 CHCl3) IR (KBr) nmax 2945 17311599 1451 12641085 707 cm1 1H NMR (CDCl3 600 MHz) d 727e807 (m 50H PheH) 565 (t J frac14 94 Hz 1H H-3000) 551e554 (m 2HH-2000 H-30) 540(t-like J frac14 99 33 Hz1H H-30000) 527e531(m 2HH-12 H-300) 525 (dd J frac14 96 78 Hz 1H H-20000) 515 (m 1H H-40)510 (d J frac14 121 Hz 1H PhCH H) 506 (d J frac14 126 Hz 1H PhCHH )492e495 (m 3H H-100 H-200 H-400) 474 (d J frac14 77 Hz 1H H-1000)466 (d J frac14 49 Hz 1H H-10) 434 (dd J frac14 116 38 Hz 1H H-50-1)428 (dd J frac14 126 27 Hz 1H H-6000-1) 422 (t J frac14 99 Hz 1H H-4000)

405(d J frac14 78 Hz 1H H-10000) 403 (t J frac14 96 Hz 1H H-40000) 399 (dd J frac14 121 28 Hz1H H-60000-1) 389e392 (m 1H H-500) 385e387(m1H H-5000) 384 (t J frac14 66 Hz1H H-20) 367 (m1H H-50000) 357 (dd J frac14 121 55 Hz 1H H-60000-2) 351 (dd J frac14 121 56 Hz 1H H-50-2)312 (dd J frac14 115 44 Hz1H H-3) 291 (dd J frac14 137 38 Hz1H H-18)271 (t J frac14 105 Hz 1H H-6000-2) 174 174 (s each 3H each Ac 2)104 (d J frac14 61 Hz 3H H-600) 112 092 090 089 086 067 059 (seach 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d frac14 1775 (C-28)16981697 1657 16561654165316511438 (C-13)136513331298 1297 1285 1283 1281 1226 (C-12) 1034 (C-10) 1009 (C-1000) 1005 (C-10000) 979 (C-100) 889 (C-3) 764 754 730 724 723719 697 696 669 666 660 627 617 557 477 468 459 418415 394 392 388 368 332 328 308 280 260 237 231 207201 174 169 162 155 HRMALDIMS calcd for [M thorn Nathorn]

C127H132O32Na 21918557 found m z 21918594

4153 Benzyl oleanolate 3b-O-234-tri-O-benzoyl-a-L-rhamnopyr-

anosyl-(1 3)-24-di-O-acetyl-a-L-rha-mnopyranosyl-(1 2)-34-

di-O-benzoyl-b-D-xylopyranoside ( 44) Yield 70 Mp 136e138 Cfrac12a24

D thorn369 (c 210 CHCl3) IR (KBr) nmax 2945 1727 1603 1451 12641093 707 cm1 1H NMR (CDCl3 600 MHz) d 728e812 (m 30HPheH) 557e562 (m 3H H-30 H-3000 H-400) 538 (t-like J frac14 2319 Hz 1H H-2000) 529 (t J frac14 37 Hz 1H H-12) 524 (br s1H H-200)513e519 (m 2H H-40 H-400) 509 (d J frac14 124 Hz 1H PhCH H) 508(s 1H H-100) 506 (d J frac14 129 Hz 1H PhCHH ) 489 (s 1H H-1000)481 (d J frac14 46 Hz 1H H-10) 448 (dd J frac14 124 41 Hz 1H H-50-1)412e415 (m 3H H-300 H-500 H-5000) 400 (t-like J frac14 60 50 Hz 1HH-20) 363 (dd J frac14 119 64 Hz1H H-50-2) 318 (dd J frac14 119 46 Hz

1H H-3) 290 (dd J frac14 138 46 Hz 1H H-18) 222 210 (s each 3Heach Ac 2) 123 (d J frac14 59 Hz 3H H-600) 112 098 092 089 089077 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d frac14 1776 (C-28) 1704 1700 1658 1659 1658 1654 1438 (C-13)1365 1337 1334 1331 1299 1288 1285 1281 1226 (C-12) 1029(C-10) 987 (C-1000) 976 (C-100) 895 (C-3) 747 738 725 716 710695 675 672 660 608 557 477 468 460 418 415 394 393388 368 339 332 327 308 281 277 261 260 237 231 210209183177 174169165 155 HRMALDIMScalcd for [M thorn Nathorn]C93H106O22Na 15977089 found m z 15977068

416 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-34-di-O-benzoyl-b-D-xylo-pyranoside] ( 45)

A mixture of compound 41 (120 mg 014 mmol) powdered 4 A

molecular sieves and compound 40 (104 mg 017 mmol) in dry

CH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C TMSOTf (10 mL 002 mmol) was added dropwise andthe reaction mixture was stirred for 30 min and then warmed toroom temperature The product was detected on TLC (21 petro-leum ethereEtOAc) After completion of the reaction the reactionmixture was quenched with Et3N (005 mL) and 1047297ltered The 1047297ltratewas concentrated and puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 45 (171 mg 91)frac12a20

D thorn632 (c 052 CHCl3) 1H NMR (CDCl3 600 MHz) d 723e805(m 30H PheH) 582 (dd J frac14 101 32 Hz 1H H-300) 571 (t-like J frac14 73 69 Hz 1H H-30) 560 (t-like J frac14 101 96 Hz 1H H-400) 556(dd J frac14 32 14 Hz 1H H-200) 534 (d J frac14 18 Hz 1H H-100) 530(t-like J frac14 37 32 Hz 1H H-12) 521e524 (m 1H H-40) 509(dd J frac14 344 128 Hz 2H CH2ePh) 487 (d J frac14 55 Hz 1H H-10)448e452 (m 1H H-500) 440 (dd J frac14 124 46 Hz 1H H-50-1) 412(dd J frac14 69 60 Hz 1H H-20) 366 (dd J frac14 124 74 Hz 1H H-50-2)325 (dd J frac14 119 46 Hz 1H H-3) 291 (dd J frac14 142 46 Hz 1H H-18)199 (dt J frac14 132 41 Hz 1H H-16)131 (d J frac14 59 Hz 3H H-600)113 112 092 089 089 083 062 (s each 3H each CH3 7) 13CNMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1658 1652 1438(C-13) 1366 1334 1300 1284 1226 (C-12) 1035 (C-10) 977(C-100) 896 (C-3) 742 725 719 706 660 613 558 478 468

460 394 393 369 332 308 282 259 238 175 166 155ESI-MS (m z ) 13456 (M thorn Hthorn) HRMALDIMS calcd for [M thorn Nathorn]C83H92O16Na 13676309 found m z 13676278

417 234-Tri-O-benzoyl-a-L-rhamnopyranosyl-(1 2)-1-(4-

tolyl)thio-34-O-isopropylidene- a-L-arabino-pyranoside ( 47 )

A mixture of compound 46 (100 mg 034 mmol) powdered 4 Amolecular sieves and compound 39 (314 mg 051 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 78 C TMSOTf (10 mL 002 mmol) was added dropwiseand the reaction mixture was stirred for 30 min and then warmedto room temperature The product was detected on TLC (21 pe-troleum ethereEtOAc) After completion of the reaction the reac-tion mixture was quenched with Et3N (005 mL) and 1047297ltered The

1047297ltrate was concentrated and puri1047297ed by a silica gel column chro-matography (51 petroleum ethereEtOAc) to afford 47 (221 mg86) frac12a23

D thorn779 (c 095 CHCl3) IR (KBr) vmax 3364 3231 29791726 1448 1255 1109 831 705 cm1 1H NMR (CDCl3 600 MHz)d 713e809 (m 19H PheH) 581 (dd J frac14 99 33 Hz1H H-30) 574(dd J frac14 33 16 Hz 1H H-20) 566 (t-like J frac14 99 98 Hz 1H H-40)549 (d J frac14 17 Hz 1H H-10) 482 (d J frac14 83 Hz 1H H-1) 455 (dq J frac14 99 61 Hz 1H H-50) 432 (m 1H H-4) 429 (t J frac14 61 Hz 1H H-3) 420 (dd J frac14 132 38 Hz1H H-5-1) 398 (dd J frac14 83 61 Hz 1HH-2) 380 (dd J frac14 127 38 Hz 1H H-5-2) 233 (s 3H SToleCH3)152135 (s each 3H each Oe(CH3)2CeO)132 (d J frac14 66 Hz 3H H-60) 13C NMR (CDCl3 150 MHz) d 1660 1658 1657 1381 13371335 1333 1328 1302 1300 1299 1298 1297 1296 12881287 1286 1285 1107 (C-1) 968 (C-10) 869 (C-30) 785 760

728 719 709 703 677 651 280 263 214 177 HRMALDIMScalcd for [M thorn Nathorn] C42H42O11SNa m z 7772359 found m z

7772340

418 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-34-O-isopropylidene-b-L-arabinopyranoside ( 49)

A mixture of compound 48 (80 mg 015 mmol) powdered 4 Amolecular sieves and compound 47 (166 mg 022 mmol) in dryCH2Cl2 (5 mL) was stirred at room temperature for 30 min thencooled to 0 C NIS (54 mg 023 mmol) and AgOTf (5 mg002 mmol) was added the reaction mixture was stirred for 30 minand then warmed to room temperature The product was detectedon TLC (31 petroleum ethereEtOAc) After completion of the re-action the reaction mixture was quenched with Et3N (020 mL) and1047297

ltered The1047297

ltrate was diluted with CH2Cl2 and then washed with

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 11

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

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(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

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[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

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bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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saturated Na2S2O3 and brine respectively The organic layer wasdried over anhydrous Na2SO4 and then concentrated in vacuo Theresulting residue was puri1047297ed by a silica gel column chromatog-raphy (31 petroleum ethereEtOAc) to afford 49 (60 mg 35)frac12a23

D thorn1317 (c 050 CHCl3) IR (KBr) vmax 2919 1726 1441 12551096 705 cm1 1H NMR (CDCl3 600 MHz) d 710e810 (m 20HPheH) 584 (dd J frac14 99 33 Hz 1H H-300 ) 577 (dd J frac14 33 17 Hz1H H-200 ) 564 (t J frac14 99 Hz1H H-400 ) 534 (d J frac14 17 Hz1H H-100 )529 (t-like J frac14 38 33 Hz 1H H-12) 511 (d J frac14 127 Hz 1HPhCH H) 510 (d J frac14 33 Hz 1H H-10) 506 (d J frac14 127 Hz 1HPhCHH ) 444 (dd J frac14 77 60 Hz 1H H-30) 427e430 (m 2H H-40H-500 ) 411 (m 1H H-50-1) 397 (d J frac14 127 Hz 1H H-50-2) 390(dd J frac14 77 33 Hz 1H H-20) 329 (dd J frac14 115 38 Hz 1H H-3)290 (dd J frac14 143 38 Hz 1H H-18) 155 136 (s each 3H eachOe(CH3)2CeO) 135 (d J frac14 64 Hz 3H H-600 ) 113 105 095 092092 090 062 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1777 (C-28) 1658 1439 (C-13) 1366 1337 13351300 1295 1286 1285 1283 1282 1226 (C-12) 1092 993(C-100 ) 935 (C-10) 888 (C-3) 811 (C-300 ) 710 704 702 699 693672 661 627 557 478 469 419 417 395 389 381 378 333331 328 327 315 313 312 300 299 287 280 238 237 235233 232 180 179 165 155 142 116 HRMALDIMS calcd for

[M thorn Nathorn] C72H88O14Na m z 11996078 found m z 11996066

419 Benzyl oleanolate 234-tri-O-benzoyl-a-L-rhamnopyranosyl-

(1 2)-b-L-arabinopyranoside (50 )

A mixture of compound 49 (100 mg 009 mmol) and p-TsOH(15 mg 009 mmol) in CH2Cl2eMeOH (V V 12 6 mL) was stirred atrt When TLC (32 petroleum ethereEtOAc) showed that depro-tection had completed Et3N (01 mL) was added and the mixturewas concentrated and puri1047297ed through a silica gel column chro-matography (21 petroleum ethereEtOAc) to afford 50 (81 mg84) as a white solid frac12a23

D thorn1562 (c 040 CHCl3) IR (KBr) vmax

2938 1727 1455 1260 1108 1066 707 cm1 1H NMR (CDCl3600 MHz) d 727e807 (m 20H PheH) 580 (dd J frac14 99 33 Hz1HH-300 ) 575 (dd J frac14 28 16 Hz 1H H-200 ) 566 (t J frac14 99 Hz 1H

H-400 ) 528 (br s 2H H-100 H-12) 522 (d J frac14 38 Hz1H H-10) 512(d J frac14 127 Hz 1H PhCH H) 505 (d J frac14 127 Hz 1H PhCHH ) 438(dq J frac14 99 60 Hz 1H H-500 ) 420 (d J frac14 88 Hz 1H H-30) 409(m 1H H-40) 402 (dq J frac14 93 33 Hz1H H-20) 391 (d J frac14 126 Hz1H H-50-1) 375 (d J frac14 121 Hz 1H H-50-2) 331 (dd J frac14 11639 Hz 1H H-3) 289 (dd J frac14 137 44 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600 ) 113 107 097 091 089 087 061 (s each 3Heach CH3 7) 13C NMR (CDCl3 150 MHz) d 1777 (C-28) 17141658 1440 (C-13) 1367 1337 1336 1333 1301 1299 12881286 1285 1282 1226 (C-12) 993 (C-100 ) 935 (C-10) 887 (C-3)812 (C-300 ) 719 708 702 699 685 672 661 627 606 557478 469 461 419 416 395 389 383 371 341 333 329 325309 287 278 261 238 236 232 213 211 185 179 171 169155 144 ESI-MS m z 11597 (M thorn Nathorn)

4110 General procedure for synthesizing compounds 52aec and

54aec

Compound 51 or 53 (013 mmol) trichloroacetimidate 40

(013 mmol) and powdered 4 A molecular sieves (010 g) werestirred for 40 min at room temperature in dry CH2Cl2 (2 mL)TMSOTf (0002 mL 0013 mmol) was added dropwise The mixturewas stirred for 10 min followed by addition of Et3N and 1047297ltrationThe 1047297ltrate was concentrated and puri1047297ed by a silica gel columnchromatography (51 petroleum ethereEtOAc) to afford thesaponin products 52aec and 54aec

41101 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52a) Yield 51 frac12a20

D thorn708 (c 062 CHCl3)

1

H NMR (CDCl3

600 MHz) d 723e807 (m 25H PheH) 590 (dd J frac14 106 37 Hz1H H-300) 585 (d J frac14 18 Hz 1H H-30) 581 (m 1H H-40) 565(t J frac14 101 Hz 1H H-400) 555 (s 1H PhCHO) 530 (t J frac14 36 Hz 1HH-12) 509 (dd J frac14 408 128 Hz 2H CH2ePh) 461e464 (m 1HH-500) 452 (d J frac14 73 Hz 1H H-10) 432 (dd J frac14 102 18 Hz 1HH-60-1) 418 (d J frac14 28 Hz 1H H-100) 408e410 (m 2H H-50 H-60-2) 396 (m 2H H-20 H-200) 320 (dd J frac14 119 41 Hz 1H H-3) 291(dd J frac14 133 37 Hz 1H H-18) 1136 (d J frac14 64 Hz 3H H-600) 114097 093 092 090 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1659 1656 1438 (C-13) 13751365 1330 1283 1280 1227 (C-12) 1040 (C-10) 1013 (PhC(O))972 (C-1000 ) 899 (C-3) 721 707 701 665 561 478 469 460415 395 393 332 308 283 260 238 176 170 169HRMALDIMS calcd for [M thorn Nathorn] C77H90O15Na 12776202 foundm z 12776172

41102 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-galactopyranoside]

(52b) Yield 29 frac12a20D thorn808 (c 055 CHCl3) 1H NMR (CDCl3

600 MHz) d 704e811 (m 25H PheH) 596 (dd J frac14 105 32 Hz1H H-300) 583 (t J frac14 101 Hz 1H H-400) 576 (m 1H H-200) 558 (t J frac14 101 Hz1H H-30) 553 (s1H H-100) 538 (s 1H PhCHO) 530 (br

s1H H-12) 510 (dd J frac14 344125 Hz 2H CH2ePh) 491 (m 1H H-

500) 466 (d J frac14 73 Hz 1H H-10) 455 (m 1H H-40) 443 (t J frac14 87 Hz 1H H-20) 433 (dd J frac14 115 50 Hz 1H H-60-1) 411 (dd J frac14 92 68 Hz 1H H-60-2) 386 (m 1H H-50) 328 (dd J frac14 11444 Hz 1H H-3) 292 (dd J frac14 137 32 Hz 1H H-18) 140 (d J frac14 60 Hz 3H H-600) 114 093 093 092 092 090 063 (s each3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 16581655 1634 1438 (C-13) 1378 1365 1333 1298 1283 1227 (C-12) 1053 (C-10) 1012 (PhC(O)) 999 (C-1000 ) 899 (C-3) 818 709703 701 660 605 558 478468 460 418 392 369 332 308283 260 238 192 178 168 154 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776173 found m z 12776172

41103 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzyl-idene- b-D-galactopyranoside] (52c ) Yield 7 frac12a20

D thorn641 (c 065 CHCl3) 1H NMR (CDCl3600 MHz) d 715e811 (m40H PheH) 590 (dd J frac14 100 36 Hz1HH-3000 ) 583 (dd J frac14 101 37 Hz 1H H-300) 577 (m 1H H-40) 570(m 1H H-4000) 562 (t J frac14 101 Hz 1H H-400) 556 (s 1H PhCHO)532 (d J frac14 19 Hz 1H H-1000) 530 (t J frac14 36 Hz 1H H-12) 508 (dd J frac14 311 124 Hz 2H CH2ePh) 448e450 (m 1H H-500) 444e447(m 1H H-5000 ) 440 (d J frac14 73 Hz 1H H-10) 435 (d J frac14 32 Hz 1HH-100) 433 (dd J frac14 115 16 Hz 1H H-60-1) 408e412 (m 4H H-20H-30 H-50 H-60-2) 375 (dd J frac14 70 37 Hz 1H H-200) 317 (dd J frac14 119 46 Hz1H H-3) 291 (dd J frac14 128 41 Hz1H H-18)144 (d J frac14 50 Hz 3H H-6000 ) 134 (d J frac14 59 Hz 3H H-600) 112 105 091089 086 085 060 (s each 3H each CH3 7) 13C NMR (CDCl3150 MHz) d 1775 (C-28) 1655 1652 1649 1438 (C-13) 1376

1365 1298 1285 1283 1280 1227 1043 (C-10

) 1012 (C-100

)1002 (PhC(O)) 977 (C-1000 ) 896 (C-3) 858 725 716 709 660563 479 469 460 415 395 393 332 308 283 260 238175171 156 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357533 found m z 17357538

41104 Benzyl oleanolate 3b-O-[2-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54a) Yield 45 frac12a20D thorn495 (c 050 CHCl3) 1H NMR (CDCl3

600 MHz) d 729e809 (m 25H PheH) 591 (dd J frac14 101 32 Hz1H H-300) 583 (dd J frac14 50 18 Hz 1H H-200) 576 (br s 1H H-100)569 (t J frac14 101 Hz 1H H-400) 553 (s 1H PhCH(O)) 530 (t J frac14 36 Hz 1H H-12) 509 (dd J frac14 376 124 Hz 2H CH2ePh) 467(d J frac14 73 Hz 1H H-10) 452e457 (m 1H H-500) 435 (dd J frac14 105

55 Hz1H H-60

-1) 407 (m 1H H-30

) 383e

388 (m1H H-20

) 378

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1512

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1315

(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

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[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

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HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1315

(t J frac14 101 Hz 1H H-60-2) 356 (t J frac14 96 Hz 1H H-40) 345 (dt J frac14 101 50 Hz 1H H-50) 325 (dd J frac14 119 46 Hz 1H H-3) 291(dd J frac14 131 32 Hz 1H H-18) 135 (d J frac14 60 Hz 3H H-600) 114094 093 092 091 089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1776 (C-28) 1659 1657 1438 (C-13) 13711365 1334 1298 1284 1226 (C-12) 1047 (C-10) 1019 (PhC(O))977 (C-1000 ) 899 (C-3) 808 751 725 707 695 672 661 658560 478 468 460 418 415 394 385 341 332 308 281 260259 238 229 216 192 176 166 155 HRMALDIMS calcd for[M thorn Nathorn] C77H90O15Na 12776210 found m z 12776172

41105 Benzyl oleanolate 3b-O-[3-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylidene-b-D-glucopyranoside]

(54b) Yield 35 frac12a20D thorn359 (c 104 CHCl3) 1H NMR (CDCl3

600 MHz) d 720e808 (m 25H PheH) 583 (dd J frac14 101 37 Hz1H H-300) 572 (dd J frac14 37 19 Hz 1H H-200) 563 (s 1H PhCH(O))557 (t-like J frac14 101 96 Hz 1H H-400) 553 (d J frac14 14 Hz 1H H-100)529 (t J frac14 37 Hz1H H-12) 508 (dd J frac14 311 128 Hz 2H CH2ePh)453 (dt J frac14 124 64 Hz1H H-500) 447 (d J frac14 78 Hz1H H-10) 435(dd J frac14 106 50 Hz 1H H-60-1) 401 (t-like J frac14 92 91 Hz 1HH-30) 386 (t J frac14 96 Hz 1H H-60-2) 374e377 (m 1H H-20) 372(t-like J frac14 97 91 Hz 1H H-40) 349 (dt J frac14 96 50 Hz 1H H-50)

320 (dd J frac14 115 46 Hz1H H-3) 29 (dd J frac14 137 41 Hz1H H-18)200 (dt J frac14 137 41 Hz1H H-16)100 (d J frac14 60 Hz 3H H-600) 111099 091 089 089 083 060 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz) d 1775 (C-28) 1657 1438 (C-13) 1373 13651335 1285 1282 1226 (C-12) 1057 (C-10) 1018 (PhC(O)) 977(C-1000 ) 903 (C-3) 782 715 709 702 685 669 660 556 477468 463 418 394 368 332 308 284 276 259 238 237235 177 171 168 154 HRMALDIMS calcd for [M thorn Nathorn]C77H90O15Na 12776201 found m z 12776172

41106 Benzyl oleanolate 3b-O-[23-di-O-(234-tri-O-benzoyl-a-L-

rhamnopyranosyl)-46-di-O-benzylid-ene- b-D-glucopyranoside]

(54c ) Yield 20 frac12a20D thorn379 (c 064 CHCl3) 1H NMR (CDCl3

600 MHz) d 716e797 (m 40H PheH) 586 (dd J frac14 105 37 Hz

1H H-300) 579 (dd J frac14 101 32 Hz 1H H-3000 ) 575 (dd J frac14 3213 Hz 1H H-200) 568 (t J frac14 101 Hz 1H H-400) 563 (dd J frac14 3214 Hz 1H H-2000 ) 562 (s 1H H-1000) 558 (s 1H H-100) 550 (t J frac14 101 Hz 1H H-4000 ) 537 (s 1H PhCH(O)) 530 (t J frac14 32 Hz 1HH-12) 509 (dd J frac14 343 124 Hz 2H CH2ePh) 482 (d J frac14 64 Hz1H H-10) 464e469 (dt J frac14 124 60 Hz 1H H-500) 449 (dt J frac14 124 59 Hz 1H H-5000) 440 (dd J frac14 105 50 Hz 1H H-600-1)424 (dd J frac14 92 73 Hz 1H H-30) 410 (t-like J frac14 73 69 Hz 1HH-200) 396 (t-like J frac14 95 92 Hz 1H H-40) 382e388 (m 1HH-60-2) 364 (td J frac14 96 46 Hz1H H-50) 332 (dd J frac14 115 41 Hz1H H-3) 291 (dd J frac14 137 46 Hz 1H H-18) 137 (d J frac14 60 Hz3H H-600) 088 (d J frac14 59 Hz 3H H-6000 ) 118 114 097 092 090089 063 (s each 3H each CH3 7) 13C NMR (CDCl3 150 MHz)d 1776 (C-28) 1654 1653 1636 1438 (C-13) 1372 1365 1329

1297 1284 1226 (C-12) 1035 (C-10

) 1022 (C-100

) 974 (PhC(O))971 (C-1000 ) 919 (C-3) 891 787 775 718 700 689 671 660559 478 469 418 392 369 332 308 283 261 258 237175 171 155 HRMALDIMS calcd for [M thorn Nathorn] C104H112O22Na17357564 found m z 17357538

4111 General procedure for synthesizing compounds 14e 26

To a solution of 37 38 42e45 50 52aec or 54aec (50 mg) inCH2Cl2eMeOH (V V 11 8 mL) was added 10 PdeC (30 mg) andAcOH (2 drops) under 1 atm of H2 for 4 h The reaction mixture wasthen 1047297ltered and the 1047297ltrate was concentrated to dryness to give awhite solid The solid was dissolved in MeOHeCH2Cl2 (V V 218 mL) and then NaOMe (40 mg) was added After stirring at roomtemperature for 8 h the solution was neutralized with ion-

exchange resin (H

thorn

) then 1047297

ltered and concentrated The residue

was puri1047297ed by column chromatography on silica gel (31 CHCl3e

MeOH) to give the products 14e26

41111 Oleanolate 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rhamno-

pyranosyl-(1 2)-a-L-arabinopyranoside (14) Yield 74 for twosteps frac12a25

D 430 (c 080 CH3OH) Mp 219e221 C IR (KBr) nmax

3397 2941 1688 1455 1385 1046 cm1 1H NMR (C5D5N600 MHz) d 630 (br s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 539(d J frac14 74 Hz 1H H-1000 ) 495 (br s 1H H-200) 489 (d J frac14 59 Hz1HH-10) 477 (dd J frac14 96 32 Hz1H H-300) 467 (dq J frac14 96 59 Hz1HH-500) 462 (t-like J frac14 69 64 Hz 1H H-20) 452 (t-like J frac14 9791 Hz 1H H-400) 432e435 (m 2H H-40 H-5000-1) 426e430 (m2H H-30 H-50-1) 423 (m1H H-4000 ) 419 (t-like J frac14 87 83 Hz 1HH-3000) 411 (t J frac14 82 Hz1H H-2000 ) 384 (d J frac14 109 Hz1H H-50-2)372 (t J frac14 110 Hz 1H H-5000-2) 330e333 (m 3H H-3 H-18) 157(d J frac14 60 Hz 3H H-600) 134 133 116 103 100 098 085 (s each3H each CH3 7) 13C NMR (C5D5N 150 MHz) d 1808 (C-28)1453(C-13) 1225 (C-12) 1079 (C-1000 ) 1058 (C-10) 1019 (C-100) 893(C-3) 834 (C-300) 789 761 759 735 725 716 702 679 661565 486 472 470 427 425 403 401 394 376 348 338 337315 305 288 287 272 267 243 189 179 177161 ESI-HRMSm z calcd for C46H73O15 [M Hthorn] 8654949 found 8654961

41112 Oleanolate 3b-O-b-D-glucopyranosyl-(1 4)-b-D-xylopyr-

anosyl-(1 3)-a-L-rhamnopyranosyl-(1 2)-a-L-arabinopyrano-

side (15) Yield 75 for two steps frac12a25D 179 (c 080 CH3OH) IR

(KBr) nmax 3412 2945 1693 1499 1071 cm1 1H NMR (C5D5N500 MHz) d 619 (s 1H H-100) 549 (t J frac14 36 Hz 1H H-12) 523(d J frac14 70 Hz1H H-1000 ) 501(d J frac14 79 Hz1H H-10000) 487 (br s1HH-200) 485 (d J frac14 50 Hz 1H H-10) 467 (dd J frac14 95 30 Hz 1HH-300) 460 (dq J frac14 93 55 Hz 1H H-500) 453e456 (m 2H H-400H-60000-1) 447 (t J frac14 95 Hz 1H H-3000 ) 437 (dd J frac14 125 27 Hz1HH-60000-2) 424e433 (m 5H H-30 H-40 H-50-1 H-4000 H-30000) 418(t J frac14 96 Hz 1H H-40000) 397e406 (m 4H H-2000 H-20000 H-5000-1H-50000) 382 (dd J frac14 121 23 Hz 1H H-5000-2) 363 (m 1H H-50-2)328e331 (m 2H H-3 H-18) 155 (d J frac14 60 Hz 3H H-600) 129

126 110 099 096 094 082 (s each 3H each CH3 7) 13C NMR (C5D5N 125 MHz) d 1802 (C-28) 1450 (C-13) 1227 (C-12) 1068(C-1000 ) 1049 (C-10) 1035 (C-10000) 1014 (C-100) 887 (C-3) 829(C-300) 787 781 777 761 755 752 742 739 727 717 716696 647 643 626 566 480 466 464 421 419 397 395 369342 332 331 320 309 307 298 297 295 282 281 265 261237 236 228 184183 173 170 154141 ESI-HRMS m z calcdfor C52H83O20 [M Hthorn] 10275478 found 10275460

41113 Oleanolic acid 3b-O-b-D-xylopyranosyl-(1 3)-a-L-rham-

nopyranosyl-(1 2)-b-D-xylopyranoside (16 ) Yield78for2stepsfrac12a25

D 382(c 050CH3OH)IR(KBr) nmax 341229381696153614551385 1046 cm1 1H NMR (C5D5N 600 MHz) d 662 (br s 1H H-100)550(br s1H H-12)544(d J frac1478Hz1HH-1000)506(brs1HH-200)

488 (d J frac14 78 Hz 1H H-10

) 483e487 (m 2H H-20

H-500

) 458 (t J frac14 91 Hz1H H-30)436e439 (m 2H H-40H-4000)430(t J frac14 88Hz1H H-400) 421e427 (m 5H H-3000 H-400 H-50-1 H-5000-1 H-5000-2)414 (dd J frac148773Hz1HH-2000)374e379(m2HH-3H-50-2)339(dd J frac14 115 41 Hz1H H-18)169 (d J frac14 59 Hz 3H H-600)143135126105 099 099088 (s each 3H each CH3 7) 13CNMR(C5D5N150 MHz) d frac14 1769 (C-28) 1458 (C-13) 1238 (C-12) 1083 (C-1000)1069 (C-10)1024(C-100) 894 (C-3) 838 (C-300) 806 794 781 765737 727 724 720 705 683 679 570 489 430 405 379 351342 318 292 290 277 270 246 195 181 164 ESI-HRMS m z

calcd for C46H73O15 [M Hthorn] 8654949 found 8654980

41114 Oleanolic acid 3b-O-b-D-glucopyranosyl-(1 4)-b-D-glu-

copyranosyl (1 3)-a-L-rhamnopyranosyl-(1 2)-b-D-xylopyr-

anoside (17 ) Yield 78 for 2 steps frac12a25

D 586 (c 050 CH3OH) IR

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 13

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

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(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1415

(KBr) nmax 3397 2945 1692 1459 1389 1042 cm1 1H NMR (C5D5N 600 MHz) d 651 (br s 1H H-100) 549 (d J frac14 73 Hz 1H H-1000 ) 547 (br s 1H H-12) 505 (br s 1H H-200) 485 (dd J frac14 9635 Hz 1H H-300) 482 (d J frac14 73 Hz 1H H-10) 479 (dq J frac14 9355 Hz 1H H-500) 454e458 (m 3H H-20 H-400 H-6000-1) 442 (dd J frac14 121 52 Hz 1H H-60000-1) 433e437 (m 2H H-4000 H-6000-2)426e430 (m 2H H-30 H-40) 413e422 (m 5H H-3000 H-30000H-40000 H-50-1 H-60000-2) 410 (t J frac14 83 Hz 1H H-2000 ) 407 (t J frac14 83 Hz 1H H-20000) 410 (m 1H H-50000) 395 (m1H H-5000 ) 372(m 1H H-50-2) 332 (m 2H H-3 H-18) 167 (d J frac14 59 Hz 3HH-600) 139 132 122 103 098 098 086 (s each 3H eachCH3 7) 13C NMR (C5D5N 150 MHz) d 1789 (C-28) 1455 (C-13)1229 (C-12) 1070 (C-1000 ) 1067 (C-10) 1055 (C-10000) 1021 (C-100)890 (C-3) 839 (C-300) 816 799 790 788 773 759 753 735720 675 629 623 566 486 472 470 427 425 403 394 376338 315 289 287 272 267 243 192 190 179 178 161 ESI-HRMS m z calcd for C53H85O21 [M Hthorn] 10575583 found10575569

41115 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 3)-a-L-

rhamnopyranosyl-(1 2)-b-D-xylopyranoside (18 ) Yield 73 for 2steps frac12a25

D 111 (c 065 CH3OH) IR (KBr) nmax 3350 2922 1595

1544 14241042 704 cm1 1HNMR(C5D5N 600 MHz) d 660 (br s1H H-100) 611 (br s 1H H-1000 ) 547 (br s 1H H-12) 499 (br s 1HH-200) 486 (d J frac14 68 Hz 1H H-10) 478e484 (m 3H H-2000 H-300H-5000 ) 474 (dq J frac14 91 64 Hz1H H-500) 467 (dd J frac14 91 32 Hz1HH-50-1) 449 (t J frac14 96 Hz 1H H-40) 430e436 (m 3H H-30 H-3000 H-400) 427 (dd J frac14 87 72 Hz 1H H-20) 419 (m 1H H-40) 374 (t J frac14 110 Hz 1H H-50-2) 330e336 (m 2H H-3 H-18) 169 (d J frac14 64 Hz 3H H-6000 ) 162 (d J frac14 64 Hz 3H H-600) 138 132 123102 097 097 085 (s each 3H each CH3 7) 13C NMR (C5D5N150 MHz) d 1764 (C-28) 1435 (C-13) 1237 (C-12) 1065 (C-1 0)1047 (C-1000 ) 1019 (C-100) 890 (C-3) 801 (C-300) 800 (C-3000 ) 787747 731 728 726 724 700 685 566 493 425 401 399 374337 313 303 287 265 242 190176 159 ESI-HRMS m z calcdfor C47H75O15 [M Hthorn] 8795106 found 8795103

41116 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-

xylopyranoside (19) Yield 65 for 2 steps frac12a20D thorn596 (c 062

CH3OH) 1H NMR (CD3OD 600 MHz) d 535 (s1H H-100) 527 (br s1H H-12) 441 (d J frac14 61 Hz 1H H-10) 395e398 (m 2H H-200 H-5000 ) 387 (dd J frac14 116 55 Hz 1H H-50-1) 325 (dd J frac14 99 33 Hz1H H-300) 348e351 (m 1H H-40) 339e346 (m 3H H-20 H-30 H-50-2) 320 (t J frac14 99 Hz 1H H-400) 314 (dd J frac14 121 44 Hz1H H-3)288 (dd J frac14 137 44 Hz 1H H-18) 125 (d J frac14 60 Hz 3H H-600)119 107 097 097 094 089 085 (s each 3H each CH 3 7) 13CNMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1049 (C-10)1006 (C-100) 888 (C-3) 775 773 727 708 703 687 559 416409 393 389 366 327 323 303 272 251 227 180167158147 ESI-HRMS m z calcd for [M thorn Na]thorn C41H66O11Na 7574503

found 7574509

41117 Oleanolic acid 3b-O-a-L-rhamnopyranosyl-(1 2)-b-L-

arabinopyranoside ( 20 ) Yield 74 for 2 steps frac12a20D thorn783 (c 084

CH3OH) IR (KBr) vmax 3420 2941 1688 1455 1139 1066 cm1 1HNMR (CD3OD 600 MHz) d 524 (t J frac14 33 Hz 1H H-12) 505 (d J frac14 33 Hz 1H H-10) 495 (d J frac14 11 Hz 1H H-100 ) 395 (dd J frac14 3316 Hz 1H H-200 ) 391 (dd J frac14 99 33 Hz1H H-30) 387 (m 2H H-20 H-40) 385 (dd J frac14 98 33 Hz 1H H-50-1) 375 (qd J frac14 9960 Hz 1H H-500 ) 366 (dd J frac14 94 33 Hz 1H H-300 ) 357 (dd J frac14 121 16 Hz 1H H-50-2) 340 (t-like J frac14 99 93 Hz 1H H-400 )320 (dd J frac14 121 44 Hz 1H H-3) 284 (dd J frac14 121 38 Hz 1H H-18) 126 (d J frac14 60 Hz 3H H-600 ) 116 103 096 094 091 085082 (s each 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1821

(C-28) 1454 (C-13) 1237 (C-12) 1038 (C-100

) 960 (C-10

) 833 (C-

3) 773 739 725 722 714 703 648 644 572 478 474 430429 407 398 395 383 351 342 340 337 318 294 290 266247 242 241 228 196 182 179 174 161 ESI-HRMS m z calcdfor [M Hthorn] C41H65O11 7334527 found 7334543

41118 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-gal-

actopyranoside ( 21) Yield 56 for two steps frac12a20D 116 (c 064

CH3OH) 1HNMR(CD3OD 600 MHz) d 524 (t J frac14 37 Hz1H H-12)505 (d J frac14 19 Hz 1H H-100) 431 (d J frac14 78 Hz 1H H-10) 396 (dd J frac14 38 14 Hz1H H-200) 392 (d J frac14 32 Hz 1H H-40) 375e379 (m2H H-300 H-500) 369e373 (m 2H H-60-1 H-60-2) 365 (dd J frac14 9778 Hz 1H H-20) 351 (dd J frac14 96 32 Hz 1H H-30) 349 (t-like J frac14 64 59 Hz 1H H-50) 339 (t-like J frac14 97 91 Hz 1H H-400) 318(dd J frac14 115 41 Hz 1H H-3) 286 (dd J frac14 133 32 Hz 1H H-18)201 (dt J frac14 142 37 Hz1H H-16)124 (d J frac14 64 Hz 3H H-600)116106 095 094 091 084 081 (s each 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1440 (C-13) 1223 (C-12) 1048 (C-1 0) 1007(C-100) 889 (C-3) 750 749 727 707 697 611 609 561 416393 366 327 323 303 272 251 232 227 180167159147ESI-HRMS m z calcd for [M thorn H]thorn C42H69O12 7654789 found7654783

41119 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-gal-actopyranoside ( 22 ) Yield 58 for two steps frac12a20

D thorn543 (c 056CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 527 (t J frac14 36 Hz 1H H-12) 440 (d J frac14 77 Hz1H H-10) 398e403 (m 2HH-200 H-500) 380 (d J frac14 33 Hz 1H H-40) 378 (dd J frac14 99 33 Hz1H H-30) 374 (dd J frac14 55 28 Hz 1H H-300) 370 (t-like J frac14 8877 Hz 1H H-20) 360e363 (m 2H H-60-1 H-60-2) 350 (t-like J frac14 66 61 Hz 1H H-50) 341 (t-like J frac14 99 93 Hz 1H H-400) 320(dd J frac14 121 44 Hz 1H H-3) 287 (dd J frac14 135 32 Hz 1H H-18)124 (d J frac14 66 Hz 3H H-600) 119 108 097 097 094 089 084 (seach 3H each CH3 7) 13C NMR (CD3OD 150 MHz) d 1439 (C-13)1223 (C-12) 1059 (C-10) 1026 (C-100) 895 (C-3) 805 748 728714 708 688 608 558 414 393 383 366 323 273 257 251232 227 167 164 157 146 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874595

411110 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-46-di-

O-benzylidene-b-D-galactopyranoside ( 23) Yield 51 for two stepsfrac12a20

D 272 (c 075 CH3OH) 1H NMR (CD3OD 600 MHz) d 556 (brs1H PhCHO) 524 (t J frac14 38 Hz1H H-12) 513 (d J frac14 11 Hz1H H-100) 490 (s 1H H-1000 ) 453 (d J frac14 77 Hz 1H H-10) 438 (d J frac14 33 Hz 1H H-40) 397 (dq J frac14 94 61 Hz 1H H-500) 391 (dd J frac14 3312 Hz1H H-2000 ) 388 (br s 1H H-200) 383e386 (m 2H H-20 H-5000 ) 377 (dd J frac14 99 33 Hz 1H H-30) 368 (dd J frac14 94 33 Hz1H H-300) 366 (dd J frac14 94 33 Hz1H H-3000 )354 (br s1H H-60-2)339 (t J frac14 99 Hz1H H-4000 ) 336 (t J frac14 93 Hz1H H-400) 320 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 137 38 Hz 1H H-18)121 (d J frac14 60 Hz 3H H-600) 119 (d J frac14 61 Hz 3H H-6000 ) 117 108 096

094 091 089 082 (s each 3H each CH3 7) 13

C NMR (CD3OD150 MHz) d 1826 (C-28) 1627 1454 (C-13) 1397 1300 12921276 1237 (C-12) 1060 (C-10) 1049 (C-1000 ) 1027 (C-100) 1024(PhCHO) 908 (C-3) 844 (C-300) 776 (C-3000 ) 756 740 737 725724 719 707 705 677 576 431 429 407 405 403 381 351342 338 318 290 286 274 266 247 242 195183 182180172 162 HRMALDIMS calcd for [M thorn Nathorn] C55H82O16Na10215507 found m z 10215495

411111 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 2)-b-D-glu-

copyranoside ( 24) Yield 55 for two steps frac12a20D thorn595 (c 100

CH3OH) 1H NMR (CD3OD 600 MHz) d 537 (s 1H H-100) 524 (t J frac14 36 Hz 1H H-12) 441 (d J frac14 77 Hz 1H H-10) 398 (td J frac14 9461 Hz 1H H-500) 395 (dd J frac14 34 16 Hz 1H H-200) 383 (dd

J frac14 115 16 Hz 1H H-60

-1) 374 (dd J frac14 93 33 Hz1H H-300

) 366

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e1514

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15

8102019 Synthesis and Antitumor Activities of Naturally Occurring Oleanolic Acid

httpslidepdfcomreaderfullsynthesis-and-antitumor-activities-of-naturally-occurring-oleanolic-acid 1515

(dd J frac14 121 55 Hz1H H-60-2) 346 (t J frac14 88 Hz 1H H-30) 341 (t-like J frac14 88 77 Hz 1H H-20) 338 (t-like J frac14 98 94 Hz 1H H-400)329 (t J frac14 88 Hz 1H H-1-40) 321e324 (m 1H H-50) 318 (dd J frac14 121 44 Hz1H H-3) 286 (dd J frac14 143 38 Hz1H H-18)121 (d J frac14 66 Hz 3H H-600)116 105 095 094 091 086 082 (s each 3Heach CH3 7) 13C NMR (CD3OD150 MHz) d 1439 (C-13)1223 (C-12) 1043 (C-10) 1005 (C-100) 889 (C-3) 782 776 763 708 707687 615 560 416 393 389 366 323 303 275 259 251 227167 164 159 147 ESI-HRMS m z calcd for [M thorn Na]thorn

C42H68O12Na 7874608 found 7874584

411112 Oleanolic acid 3-O-a-L-rhamnopyranosyl-(1 3)-b-D-

glucopyranoside ( 25) Yield 54 for two steps frac12a20D thorn290 (c 065

CH3OH) 1H NMR (CD3OD 600 MHz) d 527 (br s 1H H-12) 517 (s1H H-100) 435 (d J frac14 82 Hz 1H H-10) 401 (dq J frac14 94 66 Hz 1HH-500) 397 (br s 1H H-200) 386 (dd J frac14 109 35 Hz 1H H-60-1)371 (ddd J frac14 94 50 33 Hz1H H-50) 349 (t J frac14 88 Hz1H H-30)342 (t-like J frac14 99 93 Hz 1H H-400) 336 (t-like J frac14 94 93 Hz1HH-20) 328e332 (m 2H H-40 H-60-2) 321 (dd J frac14 116 44 Hz 1HH-3) 289 (dd J frac14 121 33 Hz 1H H-18) 127 (d J frac14 61 Hz 3H H-600) 119 108 098 097 093 087 084 (s each 3H each CH3 7)13C NMR (CD3OD 150 MHz) d 1439 (C-13) 1223 (C-12) 1052 (C-

10) 1016 (C-100) 896 (C-3) 837 748 729 711 709 690 688614 557 416 393 388 385 366 323 303 273 251 232 227180 166 157 146 ESI-HRMS m z calcd for [M thorn H]thorn

C42H69O12Na 7654789 found 7654806

411113 Oleanolic acid 3-O-23-di-O-(a-L-rhamnopyranosyl)-b-D-

glucopyranoside ( 26 ) Yield 57 for two steps frac12a20D 149 (c 075

CH3OH) 1H NMR (CD3OD 600 MHz) d 524 (br s 1H H-12) 513 (s1H H-100) 492 (s1H H-1000) 448 (d J frac14 71 Hz1H H-10) 399 (br s1H H-200) 390e396 (m 3H H-2000 H-50 H-5000) 384e386 (m 2HH-3000 H-40) 364e371 (m 3H H-400 H-50 H-60-1) 358 (t-like J frac14 88 83 Hz 1H H-300) 342e349 (m 3H H-20 H-4000 H-60-2)339 (t-like J frac14 99 94 Hz1H H-30) 322 (dd J frac14 121 44 Hz1H H-3) 287 (dd J frac14 132 33 Hz1H H-18)127 (d J frac14 66 Hz 3H H-600)

122 (d J frac14 61 Hz 3H H-6000 ) 116 105 095 094 091 087 082 (seach 3H each CH3 7) 13C NMR (CD3OD150 MHz) d 1442 (C-13)1222 (C-12) 1040 (C-10) 1022 (C-1000 ) 1007 (C-100) 888 (C-3)869 774761 722 719 708 705 698 689 630 613 560 416393 390 366 325 323 303 272 251 232 227 167 165158147 ESI-HRMS m z calcd for [M thorn Na]thorn C48H78O16Na 9335188found 9335175

42 Cytotoxic assay

The cytotoxicity of all synthesized oleanolic acid saponins wasexamined using a panel of human tumor cell lines including onehuman promyelotic leukemia cell line (HL-60) one human non-small-cell lung cancer cell line (A549) and one human melanoma

cancer cell lines (A375) Cells were seeded into 96-well plates andtreated in triplicate with gradient concentrations of tested com-pounds at 37 C for 72 h Cytotoxicity to HL-60 cellswas assessedbyMTT assay and cytotoxicity to A549 and A375 was assessed by SRBassay as previous described [3435] The cytotoxicity of testedcompounds was expressed as an IC50 determined by the Logitmethod from at least three independent experiments

Acknowledgments

This project was 1047297nancially supported by the National NaturalScience Foundation of China (No 30701046) and China Post-doctoral Science Foundation funded project (No 2012M512023)

Appendix A Supplementary data

Supplementary data related to this article can be found at httpdxdoiorg101016jejmech201304016

References

[1] KR Price IT Johnson GR Fenwick CRC Crit Rev Food Sci Nutr 26 (1987)27e133

[2] RA Hill JD Connolly Nat Prod Rep 28 (2011) 1087e1117[3] K Papadopoulou RE Melton M Legget MJ Daniels AE Osbourn Proc Natl

Acad Sci U S A 96 (1999) 12923e12928[4] K Hostettmann A Marston Saponins Cambridge University Press Cam-

bridge UK 1995[5] LP Christensen M Jensen U Kidmose J Agric Food Chem 54 (2006) 8995e

9003[6] K Haralampidis M Trojanowska AE Osbourn Adv Biochem Eng Bio-

technol 75 (2002) 31e49[7] B Yu JS Sun Chem J Asian 4 (2009) 642e654[8] B Yu JS Sun XY Yang Acc Chem Res 45 (2012) 1227e1236[9] B Yu YC Zhang PP Tang Eur J Org Chem 31 (2007) 5145e5161

[10] C Gauthier J Legault A Pichette Mini-Rev Org Chem 6 (2009) 321e344[11] J Liu J Ethnopharmacol 49 (1995) 57e68[12] Aacute Guinda M Rada T Delgado P Gutieacuterrez-Adaacutenez J Agric Food Chem 58

(2010) 9685e9691[13] HY Cheung QF Zhang J Chromatogr A 1213 (2008) 231e238[14] DW Jeong YH Kim HH Kim HY Ji SD Yoo WR Choi SM Lee CK Han

HS Lee Biopharm Drug Dispos 28 (2007) 51e57[15] F Cao JH Jia Z Yin YH Gao L Sha YS Lai QN Ping YH Zhang Mol

Pharm 9 (2012) 2127e2135[16] XA Wen HB Sun J Liu KG Cheng P Zhang LY Zhang J Hao LY Zhang

PZ Ni SE Zographos DD Leonidas KM Alexacou T Gimisis JM HayesNG Oikonomakos J Med Chem 51 (2008) 3540e3554

[17] TT Guo QC Liu P Wang L Zhang W Zhang YX Li Carbohydr Res 344(2009) 1167e1174

[18] QC Liu P Wang L Zhang TT Guo GK Lv YX Li Carbohydr Res 344 (2009)1176e1181

[19] QC Liu L Zhang XP Li TT Guo P Wang YX Li J Carbohydr Chem 28(2009) 506e519

[20] TTGuo QC LiuL Zhang PWang YX Li SynthCommun41 (2011) 357e371[21] MS ChengMCYanY Liu LGZheng J LiuCarbohydr Res 341 (2005) 60e67[22] Q Zheng K Koike T Nikaido J Nat Prod 67 (2004) 604e613[23] D Panov V Grishkovets V Kachala AS Shashkov Chem Nat Compd 42

(2006) 49e54[24] T Kanchanapoom R Kasai K Yamasaki Chem Pharm Bull 49 (2001) 1195e

1197[25] J Tian F Wu M Qiu R Nie Phytochemistry 32 (1993) 1539e1542[26] N Ding ZH Zhang W Zhang YX Chun P Wang HM Qi S Wang YX Li

Carbohydr Res 346 (2011) 2126e2135[27] GP Song HC Liu W Zhang MY Geng YX Li Bioorg Med Chem 18 (2010)

5183e5193[28] CS Yu HY Wang LW Chiang K Pei Synthesis 9 (2007) 1412e1420[29] RF Helm J Ralph L Anderson J Org Chem 56 (1991) 7015e7021[30] QC Liu Z Fan D Li WH Li TT Guo J Carbohydr Chem 29 (2010) 386e402

[31] D Zheng L Zhou YY Guan XZ Chen WQ Zhou XG Chen PS Lei BioorgMed Chem Lett 20 (2010) 5439e5442[32] CS Zhu PP Tang B Yu J Am Chem Soc 130 (2008) 5872e5873[33] MC Yan Y Liu H Chen Y Ke QC Xu MS Cheng Bioorg Med Chem Lett

16 (2006) 4200e4204[34] Z Tao Y Zhou J Lu W Duan Y Qin X He L Lin J Ding Cancer Biol Ther 6

(2007) 691e696[35] HR Lu H Zhu M Huang Y Chen YJ Cai ZH Miao JS Zhang J Ding Mol

Pharmacol 68 (2005) 983e994

Q Liu et al European Journal of Medicinal Chemistry 64 (2013) 1e15 15