indian Journal of Chemistry Vol 39B, November 2000, pp. 803 - 807

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Rhizophorin B: A novel beyerane diterpenoid from the Indian mangrove plant Rhizophora

mucronatat

AS R Anjaneyulu· , V Anjaneyulu & V Lakshmana Rao

School of Chemistry, Andhra University, Visak­hapatnam-530 003, India

Fax: 0891-555547 & 0891-570365 ; E-mail address: orgchem@satyasaionline.net.in

Received 21 June 2000; accepted 19 October 2000

A novel beyerane diterpenoid has been isolated from the ethyl acetate extract of the Indian mangrove plant Rhizophora mucro­nata and its structure established as ent-3P.20-epoxy-3a,l8-dihydroxy-15-beyerene 2 by physical and spectral e H, 13C, DEPT, 1H- 1H COSY, 1H- 1H NOESY, HMQC, HMBC & MASS) data and chemical reactions.

Rhizophora mucronata Lam (family: Rhizopho­raceae) is one of the two mangrove plants of the Rhizophora genus that occur along the tidal shores and creeks of India including the Andaman and Nicobar Islands1

• Besides being a source of tan­nins , R. mucronata is known to be powerful astrin­gent and is used in the treat ment of haemmorrhage, haematuria and anginaz. The bark of R. mucronata is also used as a cure for diabetesz. A variety of steroid and triterpenoid derivatives have been re­ported from the leaves3.4 and bark5 of thi s plant. In our continuing interest on the chemical constitu­ents of the Indian mangrove plants6

'7 we have ex­

amined the roots of R. mucronata collected from the Mangalore coast of India and reported recentl y the isolation of a novel seco labdane diterpenoid rhizophorin-A from the eth yl acetate extract8

.

Further, elution of the residue from the EtOAc ex­tract over a column of a silica gel with hex­ane:EtOAc (7 :3) furnished another interes tin g bey­erane diterpenoid , designated rhizophorin-B . Structural elucidation of rhizophorin-B using all spectral data and some chemical reactions is pre­sented here.

1Part- IV of the series: Chemical Constituents of Indi an Mangrove Plants

Rhizophorin-B was crystallised from chloro­form-methanol as colourless needles , yield 250

mg (0.00 I%), mp 220°C, [a]~ -59 .2° (c 1.9 in

CHCI 3). Its molecular formula was deduced as Cz0H 300 3 from elemental analysi s and the mass ion rn/z 300, (M+ -18) in its EI mass spectrum. A preliminary study of its physical and spectral data revealed that it is a new beyerane diterpenoid9

. In its 1H NMR spectrum it showed only two out of the four methyl groups of a beyarane skeleton [8H 0.97 (s), 8H 1.10 (s)] suggesting that the other two methyl groups might be functionalised . In support of this two sets of oxymethylene protons were noticed, one set at 3 .79 (2H, ABq, J = 9 Hz, 20-Hz) and the other set at 3 .24 (1 H, d , J = 10.5 Hz), 3.55 (I H, d , J = I 0 .5 Hz). The diagnostic olefinic protons 15-H and 16-H of beyerene skeleton were noticed at 8 5 .57 and 5.45 each as a doublet (J = 5.5 Hzt Its IR spectrum showed strong hy­droxylic absorption (3390 cm- 1

), cis-disubstituted olefinic band (720 cm-1

) besides multiple ether absorption bands between I 050-1100 cm·1

, but no carbonyl absorption. Rhizophorin B on acetyla­tion furni shed a monoacetate 3, C22H3204, mp 142°C, rn/z 360 (M +), and a monotosylate 4,

C27 H360 5S, mp 190°, which still showed ~1tron g hydroxylic absorptions (3420 and 3400 em ) be­sides the other absorptions e .g., aceta te absorp­tion ( 1710, 1225 cm-1

) in the acetate and tosylate absorption ( 1380, 1150 cn,- 1

) in the tosylate 4 suggesting that the other oxygens of the molecul e might be present as tertiary hydrox yls or involved in ether linkages . The 1 H NMR spectrum of the acetate showed the acetyl protons at 8 2.05 (3H , s) and the acetoxy methylene protons at 8 3.95 (2H, s) besides the 20-Hz at 8 3.85 (2H, ABq , J = 8 Hz) accounting for the presence of one hy­droxymethyl group in the parent compound 2. Similarl y, in the 1H NMR spectrum of the tosylate th e oxymethylene protons over tosylate appeared at 8 3.80 (2H, br s, 18-H2) and the 20-H2 at 8 3.90 (2H, ABq , J =9Hz).

The 13C NMR spectrum of rhizophorin-B showed all the 20 carbon signal s which were analysed to be 2

804 INDIAN J CHEM, SEC 8 , NOVEMBER 2000

methyls, 9 methylenes, 4 methines and 5 quaternary carbons from its DEPT spectrum. The assignment of the chemical shifts of the respective carbons was made with the help of HMQC, 1H- 1H COSY data of the compound and also by comparative literature val-

9 ues . The spectrum showed three oxygenated carbons at 5 70.5 (t), 67 .3 (t) and 98.0(s), the last of which accounted for the presence of a hemiketal carbon which might be linked to a hydroxymethylene carbon. The molecular formula requiring six double bond equivalence of which only one being accounted for in the C-15-C-16 olefinic bond [8 132.8 (d), 137.4 (d)] indicates that the molecule is pentacyclic. The prob­lems to be addressed now are: (i) which two of the four methyls of the beyarane skeleton are functional­ised, (ii) which of these two again is present as hy­droxymethyl while the other being involved in the hemiketalic linkage and (iii) location of the hemiketalic carbon in one of the rings. All these points were settled by the help of HMBC correla-

tions. That carbon-3 is involved in hemiketalic link­age was indicated by the HMBC correlations noticed between C-3 (8c 98.0) with 19-H3 (8H 1.1), 18-H2 (8H 3.55 & 3.44), 20-H2 (8H 3.79), 2-H2 (~ 3.12, 1.8) and I-H2 (8H 2.15 & 1.12). These correlations also con­firmed the presence of 18-H3 as hydroxymethyJ. and 20-H3 as oxymethylene group linked with 3-keto car­bon as a hemiketal. These conclusions. were also sup­ported by the HMBC correlations of C-18 (8c 70.5) and C-20 (8c 67.3) with the respective protons as noted in the Table I. The structure of rhizophorin B could thus be established as ent-3~ ,20-epoxy-3a, 18-dihydroxy-15-beyerene 2.

The relative stereochemistry of the respective chi­raJ centers was revealed from the NOESY correla­tions (Table I). The 5-H (8 0.89) as well as 9-H (8 1.05) showed NOESY correlation with one of the protons at C-7 (o 1.62) showing their cis relationship. The 5-H also showed correlation with the hy-

T able I-1H NMR and 13C NMR assignments and HMBC, NOESY and COSY correlations of rhizophorin-B (2)

Carbon No. 1H (0) 13C (li) HMBC NOESY COSY

C-1 2.15 m 32.3 2-H2, 5-H, 20-H2,9-H 2-He, 11-H, 1-H. 1.12 m

C-2 2.12 m 30.2 l-H2 1-H. 2-H. 1.80 m 19-H3 1-H.

C-3 98 .0 2-H2, l-H2, l8-H2, 19-HJ,20-H2 C-4 43 .8 5-H , 18-H3, 19-H3 C-5 0.89 m 47. 1 6-H2, 7-H2, 9-H, J -H2, 20-Hz, 18-H3, 19-HJ C-6 1.65 m 22.4 5-H,7-H2 19-H3 5-H

1.53 m C-7 1.62 m 35.4 6-H2, 5-H, 14-H2,9-H,l5-H 5-H,7-He 7-H.

!.24m C-8 48 .3 6-Hz, ll-H2, 7-H2, 14-H2,9-H, 15-H, 16-H C-9 1.05 m 45 .5 12-H2, 11-Hz, l-H2, 7-H2, 15-H, 14-H2 C-10 34.6 1-Hz, 2-Hz, 5-H, 9-H, 20-Hz, 6-Hz C- 11 1.63 m 21.0 9-H, l2-H2 9-H, 7-H.

1.08 m C-12 1.22 m 32.4 ll-H2,9-H,l4-H2,16-H,l7-H3

1.12 m C- 13 43 .8 17-H3,14-H2,12-H2,15-H,l6-H,ll-H2 C-14 1.48 m 60.1 15-H, 16-H, 12-H2,7-Hz,I7-H3.,9-H 17-HJ 17-HJ

0 .98 m 0.98 m

C-15 5.57 (d, J = 5.5 ) 132.8 9-H,7-H2, 14-H2,16-H 20-Hz 16-H,l7-H3

C-16 5.45 (d. J = 5.5 ) 137.4 · J5-H,I4-Hz, 17-HJ 17-H3 C-17 0.97 s 24.6 12-Hz,l4-H2, 16-H C- 18 3.55 (d. J = I 0.5) 70.5 19-H3 5-H 18-H.

3.24 (d, J = I 0.5) 5-H

C-19 1.10 s 13.3 18-Hz, 5-H C-20 3.79 (ABq, J = 9) 67 .3 5-H,9-H 6-H.

Chemical shifts in li from TMS (multiplicity, J in Hz) in CDCI3

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droxymethylene protons 18-H2; on the other hand 19-HJ (o 1.1) showed correlation with 20-H2 suggesting trans-trans relationship between C-5, C-10 and C-10, C-9. The presence of 20-methylene on the a side in­dicated the hemiketalic linkage also to be on the same side leaving the hydroxyl at C-3 with ~-configuration .

In view of the close chemical shifts of the carbons C-8, C-13, C-14 as well as other carbons with the values of beyrane carbons, and also from the NOESY corre­lation of 20-H with 15-H the carbon bridge at C-8 and C-13 was taken to be a to represent the stereochem­istry of rhizophorin-B as indicated in the structure 2. The structure was well supported by the 1H and 13C NMR spectral data (cf experimental) of the acetate 3 and the tosylate 4 of rhizophorin B.

In our attempt to provide some chemical proof to the structure of rhizophorin-B (2) it was refluxed with

\8

2 R= H

3 R= Ac

4 R= Ts

16

17

y-' HMBC

...r---- __. NOESY

methanolic KOH (5 %) for 3 hr. The compound ob­tained in the reaction on purification over Si-gel chromatography came as colourless needles from methanol. The reaction product 5 analysing for C1 9H2s02 and supported by mass ion rn/z 288 (M+) was found to be a nor diterpene resulting out of re­verse aldol reaction (Chart 1), as expected, and as noticed with 3-keto-24-hydroxy triterpenes 10

. Com­pound 5 showed in its IR spectrum hydroxylic (3400 cm.1) absorption . The 1H NMR spectrum showed two methyls as in 2, but only one set of oxymethylene protons at o 4.10 (2H, ABq, J = 8 Hz) instead of two sets in 2. Its 13C NMR spectrum showed only 19 car­bons of which only two are oxygenated carbons, one the oxymethylene o 67.6 (C-20) and the other hemiketalic o 97.2 (C-3) while the third present in 2 as hydroxymethyl at C-18 is lost in the reaction as

Char t l

806 INDIAN J CHEM, SEC B, NOVEMBER 2000

formaldehyde. Thus the structure of compound 5 was derived as ent-3~,20-epoxy-3a-hydroxy-18-nor-15-

beyerene.

Experimental Section Powdered roots (2.5 kg) were exhaustively ex­

tracted with CH2Ciz : MeOH (I : I) for six times. Removal of the solvent from the combined CHzCiz : MeOH extracts gave a residue (60 g) which was ex­tracted with EtOAc (4x500 mL) . Removal of the sol­vent from the EtOAc extract under reduced pressure gave a residue (45 g) . A part of this residue (30 g) was subjected to column chromatography over a col­umn of Si-gel using solvents of increasing polarity from n-hexane through EtOAc. Elution of column with hexane-EtOAc (7.0 : 3.0) gave a close running mixture of two compounds, which on rechromatogra­phy over a col umn of silver nitrate (20 %) impreg­nated Si-gel furnished rhizophorin-A (25 mg) and rhizophorin-B (250 mg) .

Rhizophorin-A, ( 6R, 11S,13S)-6,11,13-trihydroxy-2,3-seco-14-labden-2,8-olid-3-oic acid 1. Rhizo­phorin-A came as colourless needles from CHClr

MeOH, mp 242-43°C, [a]~ (+)128.0° (c 0.2,

CHCI3), IR (Nujol): 3450, 1765, 1720, 940, 985, 990 cm-1

; CIMS: m/z 349(M++ H-2H20). 1H, De and 20 NMR data are given in reference 8.

Rhizophorin-B, ent-3~, 20-epoxy-3a, 18-dihydr­oxy-15-beyerene 2. Rhizophorin-B came as colour-

less needles from CHCJ3-MeOH, mp 220°C, [a]~

-59.2° (c 1.6 in CHCI3). Anai.Calcd for C20H,o0,: C, 75.47; H, 9.43. Found: C, 75 .10; H, 9.12 %. IR (Nu­jol): 3390, 1050-1100, 720 cm-1

; 1H NMR (300 MHz,

CDCI3) and 13C NMR (75 MHz, CDCb) : see -Table I; ElMS : m/z 300 (M+-H20) , 285, 244, 229, 187, 159, 119.

Acetylation of rhizophorin-B (2). Rhizophorin-B (35 mg) was acetylated with a mixture of acetic an­hydride (2 mL) and pyridine (2 mL) at room tem­perature for 24 hr. After usual work-up, it yielded a

monoacetyl derivative 3 as an oil yield, 30 mg, [a]~ -40.2° (c 0.8 in CHCI 3) . Anai.Calcd for C22H3z04: C, 73.33; H, 8.8. Found: C, 72.8; H, 8.32 %. IR (Nujol): 3420, 1710, 1225, 1060-1100, 730 cm- 1

; 1H NMR (90

MHz, CDCI3): 1.0 (3 H, s), 1.10 (3H, s), 2.05 (3H, s), 3.85 (2H, ABq, J = 9 Hz, 20-H2), 3.95 (2H, s, 18-H2),

5.50 (I H, d, 1 = 6 Hz), 5.65 (I H, d, 1 = 6 Hz) ; De NMR (22.5 MHz, CDCI3):171.2, 137.5, 133.0, 71.2 , 67.3, 60.2, 48.5, 47.6, 45.6, 43.8, 43 .7, 35.7, 34.9, 32.5, 32.3, 30.2, 29.6, 24.6, 22.4, 2 1.0, 21.0, 13 .3; ElMS: m/z 360 (M+), 342, 299, 284, 243, 184, I 04, 92.

Tosylation of rhizophorin B (2). To rhizophorin­B (30 mg) in dry pyridine (2 mL) .was added p­toulene sulphonyl chloride ( 40 mg) and the solution left for over night at room temperature. After usual work-up, it yielded a monotosylate 4 {35 mg); solid,

crystallised from CHClr MeOH, mp 190° C, [a] ~

+25 .6° (c 1.0 in CHCI 3). Anai.Calcd for C27H360 5S: C, 68.64; H, 7.62. Found: C, 68.14; H, 7.24 %. IR (Nujol): 3400, 1040-1100, 760 cm- 1

; 1H NMR (90

MHz, CDCI3) : 1.10 (6H, s), 2.46 (3H, s), 3.80 (2H, br s, 18-H2), 3.90 (2H, AB q, J = 9 Hz, 20-H2), 5.48 ( I H, d, J =5Hz), 5.58 (IH, d, J =5Hz), 7.40 (2H, d, J = 9 Hz, Ar-H), 7.85 (2H, d, J = 9 Hz, Ar-H); DC NMR (22.5 MHz, CDCI 3): 144.7, 137.5, 133.0, 132.9, 129.8, 127.9, 95.8, 77.2, 67 .3, 60.2, 48 .3, 46.9, 45.4 , 44.0, 43 .8, 35.4, 34.8, 32.4, 32.0, 30.1, 24.5 , 22.4, 21 .6, 20.9, 13.2.

Alkaline hydrolysis of rhizophorin-B 2: Isola­tion of ent-3~,20-epoxy-3a-hydroxy-18-nor-15-

beyerene 5. Rhizophorin-B (40 mg in 5 mL MeOH) was refluxed with methanolic KOH (5 %, 5 mL) 0n a water-bath for 3 hr. The reaction mixture was then neutralised with dil. HCI and extracted with EtOAc, the EtOAc layer on evaporating and purification of the residue over a small column of Si-gel furnished the compound 5, solid, colourless needles from

methanol, mp 198° C, [a]~~ -35.2° (c 0.8 in CHCb.

Anai.Calcd for C 19H280 2: C, 79.16; H, 9.72. Found: C, 78.74; H, 9.32 %. IR (Nujol): 3410, 1060-1120, 740 em·'; 1H NMR (90 MHz, d5-Pyridine): 1.10 (6H, s), 4.10 (2H, ABq, J =9Hz, 20-H2), 5.48 (IH, d, J = 5Hz), 5.70 (IH, d, J =5Hz); 13C NMR (22.5 MHz, ds-Pyridine): 137.1, 133.9, 97.2, 67.6, 60.5, 49.5, 49.2, 45.8 , 45.4, 43.9, 35.8, 35.0, 32.8, 32.7, 28.7, 27.6, 24.9, 21.2, 17.3.

Acknowledgement The authors are grateful to the Department of

Ocean Development, New Delhi for fin ancial support and to the CSIR, New Delhi for sancti oni ng Emeritus Scientist scheme to one of them (ASRA). We are also grateful to the Head, RSIC, Lucknow and to the Co­ordinator UGC-COSIST and SAP programmes,

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School of Chemistry for providing spectral facilities and to Dr H R V. Reddy, College of Fisheries, Man­galore for identification of the plant material.

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