Received 29th October 2012,Accepted 11th January 2013
DOI: 10.1039/c3ob27111k
www.rsc.org/obc
Synthesis and structure–activity relationship studies ofnovel tubulysin U analogues – effect on cytotoxicity ofstructural variations in the tubuvaline fragment†
Sreejith P. Shankar,a Monika Jagodzinska,a,b Luciana Malpezzi,a Paolo Lazzari,c
Ilaria Manca,d Iain R. Greig,e Monica Sani*c,f and Matteo Zanda*c,e,f
Tubulysins are cytotoxic natural products with promising anti-cancer properties, originally isolated from
myxobacterial cultures. Structurally, tubulysins are tetrapeptides, incorporating three unusual (Mep, Tuv
and Tup) and one proteinogenic amino acid (Ile). Here we describe the synthesis and structure–activity
relationship studies of novel tubulysin U and V analogues, with variations in the central Tuv fragment,
which is known to be of paramount importance for tubulysins’ potency and hence cytotoxicity, but has
seldom been modified in previous studies. Specifically, we replaced the natural iso-propyl and acetoxy
functionalities with other structurally related groups. In general, the new analogues showed much lower
potency relative to native tubulysin U. However, one of the synthetic analogues (1f ) having a MOM func-
tion replacing the acetyl group exhibited a 22 nM IC50 on the HT-29 cell line which is comparable to the
IC50 displayed by tubulysin U (3.8 nM). Furthermore, the synthetic methodology reported herein was
found to be flexible enough to deliver different core-modified tubulysin analogues and hence may be
regarded as a scalable and convenient strategy for the chemical generation of novel tubulysin analogues.
Introduction
Tubulysins 1 (Fig. 1) are a family of tetrapeptides produced inrather small quantities (<4 mg L−1 culture broth) by twodifferent species of Myxobacteria.1 First isolated from theculture supernatant of an Archangium gephyra strain, tubuly-sins were later found in the fermentation broth of Angiococcusdisciformis, which preferentially produced tubulysinD. Tubulysins are extremely toxic to mammalian cells, includ-ing multidrug-resistant cell lines, with IC50 values between0.01 and 10 nM.2 The cytotoxic activity of the tubulysins stems
from their ability to bind tubulin and disintegrate micro-tubules of dividing cells, thus inducing apoptosis and hencethe name.3
From a structural point of view, tubulysins are linear tetra-peptides incorporating L-isoleucine and three unnatural aminoacids. At the N-terminus, all the family members haveN-methyl pipecolic acid (Mep) and isoleucine (Ile, the onlyproteinogenic amino acid). The central position is occupied bythe unusual amino acid tubuvaline (Tuv) containing a thiazoleheterocycle. The tetrapeptide is completed at the C-terminusby either tubuphenylalanine (Tup, present in tubulysins D, E,F, H, U and V) or tubutyrosine (Tut, present in tubulysins A, B,C, G, I, Y and Z), which are γ-amino acid homologues of
Fig. 1 Structure of the tubulysins.
†CCDC 853619 for 8a and 853618 for 15b. For crystallographic data in CIF orother electronic format see DOI: 10.1039/c3ob27111k
aDipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”,
Politecnico di Milano, Via Mancinelli 7, 20131 Milan, ItalybDepartment of Pharmaceutical Chemistry, Jagiellonian University,
Collegium Medicum, Medyczna 9 30-688 Krakow, PolandcKemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3,
Loc. Piscinamanna, 09010 Pula, CA, ItalydC.N.R. Istituto di Farmacologia Traslazionale, UOS Cagliari, Edificio 5,
Loc. Piscinamanna, 09010 Pula, CA, ItalyeKosterlitz Centre for Therapeutics, Institute of Medical Sciences,
University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK.
http://www.abdn.ac.uk/kosterlitz/fC.N.R. – Istituto di Chimica del Riconoscimento Molecolare, Sezione ‘A. Quilico’,
phenylalanine and tyrosine, respectively. Additionally, theN-terminal moiety of Tuv may be further functionalized withan unusual N,O-acetal substituent having different ester func-tionalities (tubulysins A–I) (Table 1). Tubulysins U–Z aredevoid of such an N-Tuv substituent and as a consequenceshow lower cytotoxicity, albeit for tubulysin U this is still in thenanomolar range.
Although at first sight the tubulysins present a relativelysimple linear tetrapeptide structure, the presence of six stereo-genic centres and several chemically and configurationally-sensitive functionalities render their total synthesis a challen-ging endeavour. Due to their considerable interest and potentialas powerful anticancer agents, several reports on the total syn-thesis of natural or modified tubulysins have been published.4
Among them, one should mention the structurally simplified“Tubugis” compounds,4m “pre-tubulysins”,4i,n–p oxo-tubuly-sins,4c N-methyl-tubulysins,4d,f and tubulysins U and V.5,6
These modified tubulysin analogues are generally less potentthan the more synthetically challenging natural tubulysins Aand D, although they mostly display sub-nanomolar cyto-toxicity on several different cancer cell lines. Remarkably, onlya few papers describing scalable syntheses of the tubulysinshave been published.5 Last but not least, little information isavailable on the structural modification of the central Tuv frag-ment, which would be essential for drawing a reliable pictureof the structure–activity relationship (SAR) features of the tubu-lysins, with the view of developing more synthetically accessi-ble and chemically/metabolically stable tubulysin analoguesfor pre-clinical studies.
We have very recently reported the synthesis and cyto-toxicity evaluation of an oxazole analogue of tubulysin U thatshowed slightly improved activity towards human promyelo-genous leukaemic cells (HL-60),4l and this prompted us toevaluate the significance of the Tuv moiety in deciding thecytotoxicity of tubulysins. In this article we describe the totalsynthesis of tubulysin U and V analogues bearing structuralmodifications in the Tuv fragment, with the aim of carryingout an SAR study designed to shed light on the role and impor-tance of the Tuv amino acid on the cytotoxic activity of thetubulysins.
Results and discussion
Back in 2007 we reported a scalable synthesis of tubulysins Uand V.6 With a reliable synthetic strategy in hand, we decidedto explore whether the same could be extended to analogues oftubulysin U, preferably incorporating modified Tuv fragments.We decided to explore two different structural variations,namely (1) the replacement of the iso-propyl group by otheralkyl and aryl groups and (2) the replacement of the O-acetylmoiety with alkoxy or benzoyl groups. This endeavour wasundertaken in order to determine the importance and role ofthe central part of the tubulysins on their biological activity,with the view of using this knowledge in future studies on thekey binding interactions with tubulin. So far the only availableinformation on the role of the Tuv fragment was published byWipf and co-workers,7 who demonstrated that the natural(R)-configuration of the O-acetate is preferred and it influencestubulin assembly. Subsequently, Shibue et al. demonstratedthat tubulysin D stereoisomers having different configurationsof the Tuv stereogenic centres are equivalent to the naturalisomer in terms of inhibition of tubulin polymerisation, butare considerably less cytotoxic, generally displaying an IC50 atleast three orders of magnitude higher.8 To the best of ourknowledge, nothing has been published about the replace-ment of the Tuv iso-propyl group. However, a recent accurateNMR structural analysis of the tubulin-bound conformation oftubulysins suggested that the hydrophobic Tuv core of thetubulysins, which includes the iso-propyl group, plays an extre-mely important role in the binding process.9
Satisfactorily, we were able to extend our existing syntheticstrategy,6 with only slight modifications, to the generation ofTuv-analogues. Thus, the synthesis started from the conden-sation of L-cysteine hydrochloride ethyl ester 2 with methylglyoxal in the presence of a base affording the thiazoline 3,which was immediately oxidised using MnO2 to the corres-ponding 2-acetyl thiazole ethyl ester 4 in 52% overall yield(Scheme 1).
Meanwhile, a one-pot reaction involving Boc-carbamate,benzene sulfinic acid sodium salt and the corresponding alde-hyde in THF–H2O or MeOH–H2O afforded the α-amino sul-fones 5a–d (Scheme 2). Enolisation of the thiazole 4 usingNaH followed by the addition of amino sulfones 5a–d gave theβ-amino ketones 6a–d in satisfactory yields as racemic mix-tures. In order to obtain stereochemically pure Tuv precursors,we performed an oxazoborolidine (CBS) mediated reduction10
of 6a–d to 7a–d using the (S)-CBS catalyst in the presence of aBH3·Me2S complex at 0 °C. The required alcohols (R,R)-7a–d,
Scheme 1 Synthesis of 2-acetyl-thiazole ethyl ester 4. Reagents and con-ditions: (a) methyl glyoxal solution, NaHCO3, EtOH–H2O (1 : 1), overnight;(b) MnO2, MeCN, 65 °C, overnight.
Table 1 The tubulysins family
Tubulysin R1 R2 R3
A p-OH-Ph Ac –CH2OCO-i-butylB p-OH-Ph Ac –CH2OCO-n-propylC p-OH-Ph Ac –CH2OCOEtD Ph Ac –CH2OCO-i-butylE Ph Ac –CH2OCO-n-propylF Ph Ac –CH2OCOEtG p-OH-Ph Ac –CH2OCOCHvC(CH3)2H Ph Ac –CH2OCOMeI p-OH-Ph Ac –CH2OCOMeU Ph Ac HV Ph H HY p-OH-Ph Ac HZ p-OH-Ph H H
which could be obtained in pure form by flash chromato-graphy (FC), were produced along with the diastereoisomers8a–d in a ca. 1 : 1 ratio (Scheme 2), as determined by NMRanalysis of the crude reaction mixtures.
Diastereomers 8, with the exception of 8a, could not beobtained in pure form by FC because they were always recov-ered as mixtures either with 7 or with unidentified by-products, which were initially not observed in the reduction of6a. Diastereomers 7a–d were obtained in 88–90% ee, as deter-mined by chiral HPLC analysis, whereas among diastereomers8, only 8a could be submitted to chiral HPLC analysis,showing 96% ee.
The relative configuration of the two stereocenters in 8a wasconfirmed by X-ray diffraction analysis (Fig. 2) of a suitableracemic crystal and the absolute configuration was assessed bychemical correlation with the enantiopure O-Ac-Cbz-Tuv-OEtdescribed by Wipf et al.11
The next step consisted of the O-alkylation of the freehydroxy group in 7a, so as to obtain the O-alkylated N,C-pro-tected tubuvalines 7e,f. This was achieved by reaction ofmethyl iodide or MOM-Cl with the corresponding potassiumor sodium bases, respectively (Scheme 3).
The synthesis of the Tup unit is shown in Scheme 4. Thekey reaction for assembling the Tup fragment was a Wittigreaction between N-Boc protected phenylalanine aldehyde 95
and the ylide 13, which was chosen because we knew thatN-Boc(−)-menthyl esters 15a and 15b could eventually be sepa-rated by FC. Starting from (−)-menthol, acylation with
bromoacetyl bromide followed by reaction with triphenylphos-phine gave rise to the ylide 12, which was reacted with methyliodide to yield 13, which was subjected to Wittig olefinationwith the aldehyde 9. The latter was obtained by Dess–Martinoxidation of Boc-phenylalaninol 9, affording the α,β unsatu-rated compound 14 in good yield. Hydrogenolysis of 14 withPd/C provided a 2 : 1 diastereomeric mixture of Tup-derivatives15, separable by FC. One-pot acid hydrolysis of all the protect-ing groups gave stereochemically pure H-Tup-OH·HCl 16,which was then esterified to yield H-Tup-OMe·HCl 17, readyfor subsequent couplings.
The relative stereochemistry of the two isomers wasassigned based on X-ray analysis of the undesired diastereo-isomer 15b (Fig. 3).
Final assembly of tubulysins 1a–f was achieved by a stan-dard stepwise peptide synthesis protocol, as shown inScheme 5. Boc deprotection of 7a–f with 20% TFA in CH2Cl2and subsequent coupling of the resulting amines 18a–f withBoc-Ile-OH afforded the corresponding dipeptides 19a–f in
Scheme 2 Synthesis of stereopure (R,R)-Tuv precursor. Reagents and con-ditions: (a) formic acid, THF–H2O or MeOH–H2O, 24 h; (b) NaH, THF, 2–3 h;(c) (S)-(−)-2-methyl-CBS-oxazaborolidine, BH3·Me2S, THF, 0 °C, 2–3 h.
Fig. 2 X-ray structure of racemic 8a.
Scheme 3 Synthesis of O-alkylated Tuv precursors. Reagents and conditions:(a) MeI, tBuOK, THF, −78 °C; (b) MOMCl, NaH, THF, 0 °C.
Scheme 4 Synthesis of stereopure Tup-OMe. Reagents and conditions:(a) Et3N, dry THF, 0 °C to rt, 2 h; (b) PPh3, THF, 2 h, 0.38 N NaOH, toluene, 3 h;(c) MeI, CH2Cl2, 0 °C to rt, overnight; (d) Dess–Martin periodinane, CH2Cl2, 6 h;(e) CH2Cl2, 0 °C to rt, 8 h; (f ) H2, Pd/C, EtOAc, overnight; (g) 6 N HCl, 130 °C,1.5 h; (h) 2,2-dimethoxypropane, conc. HCl, MeOH, 60 °C, overnight.
satisfactory yields. Hydrolytic cleavage of ethyl ester undermild alkaline conditions delivered the acids 20a–f, which, onsubsequent coupling with H-Tup-OMe, 17, afforded the tripep-tides 21a–f. Coupling between N-methyl pipecolic acid 23 andthe tripeptides 22a–f, obtained by treatment of 21a–f with TFA,proceeded smoothly in the presence of HATU, HOAt and Et3N,affording the tetrapeptides 24a–f in good yields and withoutany detectable loss of stereochemical purity. Saponification ofthe methyl ester function using 1 M aqueous LiOH in THFgave the final carboxylic acid tubulysin U analogues 1e,f and25a–d in good yields. The latter compounds were eventuallyacetylated by treatment with acetic anhydride in pyridine,affording the corresponding tubulysin U derivatives 1a–d instereopure form.
Eventually we accomplished the synthesis of the O-benzoylderivative of tubulysin U, which turned out to be more challen-ging than expected. In fact, attempts to achieve direct benzoy-lation of the Tup hydroxy group were unsuccessful, possibly
because of steric reasons. Furthermore the benzoate moietyproved to be labile even to the mild basic reaction conditionsused for hydrolysing the C-terminal ethyl ester. Thus, wedecided to modify the synthetic sequence starting from theTuv stereoisomer ent-8a, as portrayed in Scheme 6. Theracemic β-amino ketone 6a was subjected to reduction using(R)-CBS catalyst, yielding a 1 : 1 diastereomeric mixture of alco-hols ent-7,8a. Saponification of ent-8a with LiOH to 26 andsubsequent coupling with Tup-OMe 17 afforded the corres-ponding dipeptide 27 in good yield. Next, a Mitsunobu reac-tion using PPh3, benzoic acid and a 40% toluene solution ofDEAD afforded the O-benzoyl derivative 28, having the desiredstereochemistry, resulting from inversion of configuration ofthe starting (S)-Tuv stereocenter. TFA mediated Boc deprotec-tion of 28 to 29, and subsequent coupling with Boc-Ile-OHgave the tripeptide 30. After further N-deprotection, 31 wascoupled with N-methyl pipecolic acid (23) affording the tetra-peptide 32. Eventually, selective cleavage of the C-terminalmethyl ester was accomplished using Me3SnOH which was pre-viously shown to be effective for a highly selective hydrolysis oftubulysin methyl esters.8 Thus treatment of 32 with Me3SnOHat 70 °C for 32 h delivered 1g, albeit in modest yields, due tothe formation of several unidentified by-products.
The tubulysin analogues 1a–f were tested for their anti-tumour activity on the human colon cancer cell line HT-29 andthe results are summarised in Table 2.
These data show that replacement of the acetyl group Tuvfragment of tubulysin U by a methyl group (compound 1e)caused a two-fold loss of potency, in line with the previouslyobserved decreased cytotoxicity of O-deacetylated tubulysin V,5
whereas the methoxymethyl group produced a minimal dropof cytotoxicity and the resulting compound 1f showed remark-able potency. These findings are very important because theacetyl group of tubulysins is hydrolytically- and metabolically-labile, and its replacement with a functionality less susceptibleto hydrolysis could produce more stable and easier-to-handletubulysin derivatives. Finally, replacement of the acetyl withthe benzoyl group in compound 1g also produced a drop ofpotency.
The inactivity of analogues 1b–d demonstrates the impor-tance of the iso-propyl group on the Tuv fragment – replacingthe iPr group with other alkyl and aryl groups causes a dra-matic drop of cytotoxicity. These finding are in line with therecently published SAR data analysis based on the tubulin-bound structure of the tubulysins determined by NMR struc-tural analysis. Indeed, the Tuv iso-propyl belongs to the“hydrophobic core” of the tubulysins, that extends from the Ileside chain to the thiazole ring, which was correctly deemed tobe essential by Carlomagno et al.9
Conclusions
We developed a scalable and efficient total synthesis of tubuly-sins U and V,6 which is flexible enough to be used for the syn-thesis of analogues incorporating different tubuvaline
Fig. 3 X-ray structure of the N-boc-(−)-menthyl ester 15b.
Scheme 5 Final assembling of fragments. Reagents and conditions: (a) TFA–CH2Cl2 (1 : 4), 0 °C to rt, 1 h; (b) HOBt, EDC·HCl, then Boc-Ile-OH, DIPEA, CH2Cl2,0 °C to rt, 3 h; (c) LiOH·H2O, THF–H2O (4 : 1), 0 °C to rt, 5 h; (d) HOAt, HATU,then H-Tup-OMe (17), Et3N, CH2Cl2, 0 °C to rt, 3 h; (e) TFA–CH2Cl2 (1 : 4), 0 °C tort, 1 h; (f ) HOAt, HATU, then Mep-OH (23), Et3N, 0 °C to rt, 3 h; (g) 1 N LiOH,THF, 0 °C to rt, 2–3 days; (h) Ac2O, pyridine, overnight.
fragments in quantities and purities sufficient to allow in vitrobiological screenings for cytotoxicity. An SAR study on a small
set of novel tubulysin U analogues was performed. One of thesynthesised analogues 1f, containing an O-MOM replacementof the natural O-acetyl group on the central Tuv fragment,essentially retained the activity of the parent analogue 1a. Allthe other replacements rendered the molecule biologicallyinactive or remarkably less active than the natural analogue,demonstrating the importance of the Tuv iso-propyl side-chainfor the biological activity of the tubulysins.
Experimental sectionGeneral methods
Commercially-available reagent-grade solvents were employedwithout purification. All reactions where an organic solventwas employed were performed under a nitrogen atmosphere,after flame-drying of the glass apparatus. Melting points (m.p.)are uncorrected and were obtained on a capillary apparatus.TLCs were run on silica gel 60 F254 Merck. Flash
Scheme 6 Synthesis of O-benzoyl tubulysin U analogue 1g. Reagents and conditions: (a) (R)-(+)-2-methyl-CBS-oxazaborolidine, BH3·Me2S, THF, 0 °C, 2–3 h; (b)LiOH·H2O, THF–H2O (4 : 1), 0 °C to rt; (c) HOAt, HATU, then H-Tup-OMe 17, Et3N, CH2Cl2, 0 °C to rt; (d) PPh3, PhCOOH, DEAD, benzene; (e) TFA–CH2Cl2 (1 : 4), 0 °C tort; (f ) HOBt, EDC·HCl, then Boc-Ile, sym-collidine, CH2Cl2, 0 °C to rt; (g) TFA–CH2Cl2 (1 : 4), 0 °C to rt, 1 h; (h) HOAt, HATU, then Mep 23, Et3N, 0 °C to rt, 3 h; (i)Me3SnOH, DCE, 70 °C, 32 h.
Table 2 Biological tests of the tubulysin analogues
Tubulysin analogue IC50 (nM)
1a R1 = iPr, R2 = Ac 3.81b R1 = Ph, R2 = Ac >10001c R1 = PMP, R2 = Ac >10001d R1 = c-hex, R2 = Ac >10001e R1 = iPr, R2 = Me 2401f R1 = iPr, R2 = MOM 221g R1 = iPr, R2 = COPh >1000
Chromatography (FC) purifications were performed with silicagel 60 (60–200 μm, Merck). 1H-, 13C-, and 19F-NMR spectrawere run at 250, 400 or 500 MHz. Chemical shifts areexpressed in ppm (δ), using tetramethylsilane (TMS) as theinternal standard for 1H and 13C nuclei (δH and δC = 0.00),while C6F6 was used as the external standard (δF −162.90)for 19F.
Ethyl 2-acetylthiazole-4-carboxylate, 4. To a solution ofcysteine hydrochloride ethyl ester 2 (15 g, 80.79 mmol) in a1 : 1 EtOH–H2O mixture (1.5 L) NaHCO3 (6.786 g, 80.79 mmol)and pyruvic aldehyde (35% w in H2O, 17.5 mL, 114 mmol)were added. The reaction mixture was stirred at rt for 18 h,then concentrated to half of its original volume (no heating).NaCl was added to saturate the aqueous phase. The aqueouslayer was extracted with CHCl3 (2 × 300 mL). The combinedorganic phase was dried over Na2SO4, concentrated in vacuoand the crude was used in the next step without any furtherpurification.
To a solution of compound 3 (16.31 g, 80.74 mmol) inMeCN (500 mL) MnO2 (140 g, 1.615 mol) was added. The reac-tion mixture was heated at 65 °C overnight, then filtered over acelite pad and the residue washed with AcOEt (2 × 200 mL).The filtrate was concentrated in vacuo. The crude was purifiedby FC (1 : 3 AcOEt–hexane) to give the acyl thiazole 4 (8.321 g,52% in two steps) as a yellow solid. Rf = 0.33 (1 : 3 AcOEt–hexane); 1H NMR (400 MHz, CDCl3) δ: 8.40 (s, 1H), 4.43 (q, J =7.1 Hz, 3H), 2.75 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). LC/MS (ESI)m/z 199.8 [M + H]+, 221.8 [M + Na]+. For a complete set of spec-troscopic data, see ref. 12.
General procedure for the synthesis of the tert-butyl (phe-nylsulfonyl)methyl carbamates 5a–d. To a solution of tert-butyl carbamate (1 equiv.) in a H2O–MeOH 1 : 2 mixture orH2O–THF 1 : 2 were added the aldehyde (2 equiv.), benzenesulfinic acid sodium salt (2 equiv.) and formic acid (2 equiv.).The reaction mixture was stirred at rt for 24 h and cooled in anice-bath. The white precipitate was filtered off, washed withwater and hexane and dried to give the corresponding tert-butyl(phenylsulfonyl)methylcarbamate as a white solid andwas used in the next step without further purification orcharacterisation.
General procedure for the preparation of β-amino carbonylcompounds, 6a–d. To a suspension of NaH (60% dispersionin mineral oil, 995 mg, 24.87 mmol) in dry THF (60 mL) acylthiazole 4 (3 g, 15.07 mmol) was added. After stirring for15 min a solution of sulfone 5a (3.067 g, 9.8 mmol) in dryTHF (60 mL) was added over a period of 30 min. The reactionwas stirred for 2 h, quenched with an NH4Cl saturatedaqueous solution and extracted with AcOEt (2 × 50 mL). Thecollected organic phases were dried over anhydrous Na2SO4,filtered and concentrated in vacuo. The crude residue was puri-fied by FC.
General procedure for the stereoselective reduction of 6a–d. Toa solution of (S)-(−)-2-methyl-CBS-oxazaborolidine (152 mg,0.55 mmol) in dry THF (20 mL) cooled at 0 °C, BH3·Me2S(10 M solution in THF, 499 μL, 4.99 mmol) was added. Thesolution was stirred for 10 min at 0 °C and a solution of 6a(1.846 g, 4.99 mmol) in dry THF (10 mL) was added. The reac-tion was warmed to rt and stirred for 3 h. The reaction wasquenched with MeOH (2 mL), the solvent removed in vacuoand the crude purified by FC.
Ethyl 2-((1R,3S)-3-(tert-butoxycarbonylamino)-1-methoxy-4-methylpentyl)thiazole-4-carboxylate, 7e. To a 1 M solution oft-BuOK (0.54 mL, 0.54 mmol) in dry THF (5 mL), cooledat −78 °C, a solution of 7a (100 mg, 0.28 mmol) in dry THF(3 mL) was added. After 15 min, MeI (83 μL, 1.34 mmol) wasadded and the resulting mixture was stirred at −78 °C for 1 h.H2O (6 mL) was added and the aqueous layer was extractedwith EtOAc (3 × 10 mL). The solvent was removed in vacuo andthe crude was purified by FC (2 : 8 AcOEt–hexane) affording 7e(48 mg, 45%) as a yellowish foam. Rf = 0.54 (4 : 6 AcOEt–
Ethyl 2-((1R,3S)-3-(tert-butoxycarbonylamino)-1-methoxy-methyl-4-methylpentyl)thiazole-4-carboxylate, 7f. To a solu-tion of alcohol 7a (1.1 g, 2.96 mmol) in dry THF (50 mL)cooled at 0 °C, NaH (60% dispersion in mineral oil, 355 mg,8.87 mmol) was added. The resulting suspension was stirredat 0 °C for 15 min, then neat MOMCl (1.12 mL, 14.8 mmol)was added. After stirring at rt for 3 h, the reaction wasquenched by adding a 1 N HCl aqueous solution (20 mL). Thelayers were separated and the organic layer was washed withbrine, dried over anhydrous Na2SO4, filtered and concentratedin vacuo. The residue was purified by FC to give 7f (2 : 8 AcOEt–hexane) (428 mg, 35%) as a white foam. Rf = 0.43 (3 : 7 AcOEt–hexane); [α]23D = +12.6 (c = 0.75, CHCl3); FT-IR (film) νmax:3342.6, 2932.5, 2879.9, 1680.8, 1650.2, 1528.2, 1235.8,1171.1 cm−1; 1H NMR (400 MHz, CDCl3) δ: 7.97 (s, 1H), 4.84(br d, J = 9.0 Hz, 1H), 4.57 (d, J = 6.7 Hz, 1H), 4.55 (d, J =6.7 Hz, 1H), 4.48 (br d, J = 10.6 Hz, 1H), 4.22 (q, J = 7.06 Hz,2H), 3.63 (m, 1H), 3.20 (s, 3H), 1.74 (m, 1H), 1.64 (m, 1H), 1.56(m, 1H), 1.26 (s, 9H), 1.20 (t, J = 7.1 Hz, 3H), 0.72 (d, J = 6.7 Hz,3H), 0.70 (d, J = 7.1 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ:175.8, 161.5, 155.9, 147.2, 127.7, 97.6, 61.5, 56.9, 51.9, 40.9,33.2, 28.5, 19.0, 17.9, 14.6; LC/MS (ESI) m/z 417.1 [M + H]+,439.1 [M + Na]+.
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-bromoacetate,11. To a solution of menthol (5 g, 32 mmol) in dry THF(30 mL) Et3N (4.8 mL, 35 mmol) was added. After cooling at0 °C, bromoacetyl bromide (3 mL, 35 mmol) was added drop-wise. The temperature was allowed to warm to rt and the reac-tion mixture was stirred for 2 h. After cooling at 0 °C, thereaction was quenched with a 1 N HCl aqueous solution(5 mL) and AcOEt was added (30 mL). The layers were sepa-rated and the organic phase was dried over anhydrous Na2SO4,filtered and concentrated in vacuo. The crude was purified byFC (3 : 97 AcOEt–hexane) to give 11 (6.4 g, 72%) as a colorlessoil. Rf = 0.5 (2 : 98 AcOEt–hexane); [α]23D = −64.1 (c = 1.68,CHCl3); spectral data matches with those reported in theliterature.13
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-(triphenylphos-phoranylidene) acetate, 12. To a solution of 11 (6.4 g,23 mmol) in dry THF (30 mL), under a nitrogen atmosphere,PPh3 (6 g, 23 mmol) was added. After refluxing for 2.5 h, thereaction mixture was concentrated in vacuo. The resulting solidwas washed with a 7 : 3 mixture of hexane–Et2O and filtered togive 11 g of phosphonium salt as a white solid, which wasused in the next step without further purification. To a suspen-sion of phosphonium salt (11 g, 23 mmol) in toluene (150 mL)
a 0.38 N NaOH aqueous solution (25 mL) was added dropwiseover a period of 5 min. The reaction mixture was stirred for 3 hand the layers were separated. The organic phase was driedover anhydrous Na2SO4, filtered and concentrated in vacuo togive 12 (11 g, quantitative yield) as a white foam. Rf = 0.87 (3 : 7MeOH–CHCl3); [α]
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-(triphenylphos-phoranylidene)propanoate, 13. To a suspension of phosphor-ane 12 (11 g, 23 mmol) in toluene (150 mL) a 0.38 N NaOHaqueous solution (25 mL) was added dropwise over a period of5 min. The reaction mixture was stirred for 3 h and the layerswere separated. The organic phase was dried over anhydrousNa2SO4, filtered and concentrated in vacuo to give 11 g of purephosphonium salt as a white foam. To a solution of phos-phonium salt (11 g, 23 mmol) in DCM (60 mL), cooled at 0 °C,MeI (2.1 mL, 34 mmol) was added dropwise. After stirring for10 min the temperature was allowed to warm to rt. The reac-tion mixture was stirred overnight and the solvent was evapor-ated. The crude was dissolved in toluene (100 mL) and a 0.38N NaOH aqueous solution (25 mL) was added. The mixturewas stirred for 2 h and the layers were separated. The organicphase was dried over anhydrous Na2SO4, filtered and concen-trated in vacuo to give pure 13 (10.4 g, 96%) as a yellow foam.Rf = 0.6 (2 : 8 MeOH–CHCl3); [α]
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 4-(tert-butoxycar-bonylamino)-2-methyl-5-phenylpent-2-enoate, 14. To a solu-tion of 13 (4.53 g, 9.6 mmol) in DCM (80 mL), cooled at 0 °C,aldehyde 95 (1.6 g, 6.4 mmol) was added. After stirring for15 min at 0 °C, the temperature was allowed to warm to rt andthe reaction mixture was stirred for 8 h. The reaction wasquenched with a 1 N NaHSO4 aqueous solution (50 mL) andextracted with DCM (2 × 50 mL). The organic layer was washedwith brine (1 × 50 mL), dried over anhydrous Na2SO4, filteredand concentrated in vacuo. The crude was purified by FC (2 : 8AcOEt–hexane) to give 14 (2.3 g, 78%) as a white foam; Rf = 0.5(3 : 7 AcOEt–hexane); [α]23D = −8.52 (c = 0.7, CHCl3); FT-IR (film)νmax: 3096.4, 2641.9, 1974.6, 1881.2, 1711.8, 1620.6,1459.8 cm−1; 1H NMR (400 MHz, CDCl3) δ: 7.25–7.18 (m, 3H),7.13–7.11 (m, 3H), 6.48–6.45 (m, 1H), 4.72–4.65 (m, 3H), 2.91
(2S,4R)-Methyl 4-amino-2-methyl-5-phenylpentanoate hydro-chloride, 17. A suspension of N-Boc menthyl ester 15a(950 mg, 2.13 mmol) in 6 N HCl (10 mL) was refluxed at145 °C for 3–5 h and then cooled to rt. AcOEt (10 mL) wasadded and the phases were separated. The aqueous layer wasconcentrated under reduced pressure to obtain Tup·HCl 16(422 mg, 95%) as a white solid and was used in the next stepwithout further purification. To a suspension of 16 (398 mg,1.64 mmol) in MeOH (15 mL), 2,2-dimethoxypropane (405 μL,3.28 mmol) followed by conc. HCl (4.8 μl, 0.015 mmol) wereadded. The reaction mixture was heated to 50 °C and stirredovernight. The solvent was removed in vacuo to give pureTup-OMe 17 (383 mg, 91%) as a white foam; Rf = 0.3 (1 : 9MeOH–CH2Cl2); [α]
General procedure for coupling Tuv 7a–f with Boc-Ile. Asolution of 7a (150 mg, 0.40 mmol) in a 20% mixture of TFA inDCM (5 mL) was stirred for 1 h. The solvent was removedunder reduced pressure to give 18a as the TFA salt and wasused in the next step without further purification. To a solu-tion of Boc-isoleucine (93 mg, 0.40 mmol) in DCM (5 mL),HOBt (60 mg, 0.44 mmol), EDC·HCl (84 mg, 0.44 mmol),DIPEA (153 μL, 0.88 mmol) and Tuv-TFA salt 18a (160 mg,0.40 mmol) were added. After stirring for 3 h water (10 mL)was added and the layers separated. The organic phase waswashed with a 1 N HCl aqueous solution (10 mL), a saturatedNaHCO3 aqueous solution (10 mL) and brine (10 mL). Afterdrying over anhydrous Na2SO4 and concentration in vacuo thecrude dipeptide was purified by FC.
General procedure for coupling Ile-Tuv-COOEt 19a–f withTup-OMe 17. To a solution of dipeptide 19a (210 mg,0.45 mmol) in a 4 : 1 THF–H2O mixture (5 mL), LiOH·H2O
(28 mg, 0.67 mmol) was added. The reaction mixture wasstirred for 5 h. H2O (5 mL) and AcOEt (10 mL) were added andthe layers were separated. The aqueous phase was acidified topH 1–2 with a 1 N HCl aqueous solution and was extractedwith AcOEt (2 × 10 mL). The combined organic extract wasdried over anhydrous Na2SO4, filtered and concentratedin vacuo to give the corresponding acid 20a, which was used inthe next step without further purification. To a solution of 20a(180 mg, 0.40 mmol) in DCM (5 mL), HOAt (60 mg,0.44 mmol), HATU (167 mg, 0.44 mmol), Et3N (123 μL,0.88 mmol) and Tup-OMe 17 (103 mg, 0.40 mmol) were added.After stirring for 3 h water (10 mL) was added and the layersseparated. The organic phase was washed with a 1 N HClaqueous solution (10 mL), a saturated NaHCO3 aqueous solu-tion (10 mL) and brine (10 mL). After drying over anhydrousNa2SO4 and concentration in vacuo the crude tripeptide waspurified by FC.
Ethyl 2-((1S,3S)-3-(tert-butoxycarbonylamino)-1-hydroxy-4-methylpentyl)thiazole-4-carboxylate, ent-7a. The reductionwas carried out with (R)-CBS catalyst according to the sameprocedure as described for the reduction of 6a–d to 7a–d(white solid, 35% yield); Rf 0.50 (2 : 3 AcOEt–hexane);[α]23D = −5.0 (c = 1.70, CHCl3);
4-{[2-(3-tert-Butoxycarbonylamino-1-hydroxy-4-methyl-pentyl)-thiazole-4-carbonyl]-amino}-2-methyl-5-phenylpentanoic acidmethyl ester, 27. To a solution of ent-8a (212 mg, 0.57 mmol)in a 4 : 1 THF–H2O mixture (5 mL), LiOH·H2O (34 mg,0.85 mmol) was added. The reaction mixture was stirred for5 h. H2O (5 mL) and AcOEt (10 mL) were added and the layerswere separated. The aqueous phase was acidified to pH 1–2with a 1 N HCl aqueous solution and was extracted with AcOEt(2 × 10 mL). The combined organic extract was dried overanhydrous Na2SO4, filtered and concentrated in vacuo to givethe corresponding acid 26, which was used in the next stepwithout further purification. To a solution of 26 (138 mg,0.40 mmol) in DCM (5 mL), HOAt (60 mg, 0.44 mmol), HATU(167 mg, 0.44 mmol), Et3N (123 μL, 0.88 mmol) and Tup-OMe17 (103 mg, 0.40 mmol) were added. After stirring for 3 hwater (10 mL) was added and the layers separated. The organicphase was washed with a 1 N HCl aqueous solution (10 mL), asaturated NaHCO3 aqueous solution (10 mL) and brine(10 mL). After drying over anhydrous Na2SO4 and concen-tration in vacuo the crude was purified by FC to give the dipep-tide 27 (186 mg, 85%) as a white foam. Rf = 0.35 (1 : 1 AcOEt–hexane); [α]23D = −32.8 (c = 0.51, CHCl3);
(1R,3R)-3-(tert-Butoxycarbonylamino)-1-(4-((2R,4S)-5-methoxy-4-methyl-5-oxo-1-phenylpentan-2-ylcarbamoyl)thiazol-2-yl)-4-methylpentyl benzoate, 28. To a solution of 27 (120 mg,0.21 mmol) in benzene (5 mL), PPh3 (144 mg, 0.54 mmol) andbenzoic acid (66 mg, 0.54 mmol) were added followed byDEAD (40% solution in toluene, 249 μL, 0.54 mmol). The reac-tion mixture was stirred at rt for 45 min and then a NaHCO3
saturated aqueous solution was added (10 mL). The layerswere separated and the organic layer was concentratedin vacuo. The crude was purified via FC (3 : 7 AcOEt–hexane)affording 28 (129 mg, 95%) as a white foam. Rf = 0.47 (2 : 3AcOEt–hexane); [α]23D = −3.84 (c = 0.26, CHCl3);
(1R,3R)-3-((2S,3S)-2-(tert-Butoxycarbonylamino)-3-methylpen-tanamido)-1-(4-((2R,4S)-5-methoxy-4-methyl-5-oxo-1-phenylpen-tan-2-ylcarbamoyl)thiazol-2-yl)-4-methylpentyl benzoate, 30. Asolution of 28 (120 mg, 0.18 mmol) in a 20% mixture of TFA inDCM (5 mL) was stirred for 1 h. The solvent was removedin vacuo to give 29, which was used in the next step withoutfurther purification. To a solution of 29 (120 mg, 0.18 mmol)in DCM (5 mL), HOAt (27 mg, 0.20 mmol), HATU (70 mg,0.20 mmol), Et3N (54 μL, 0.40 mmol) and Boc-Ile (46 mg,0.20 mmol) were added. After stirring for 3 h water (10 mL)was added and the layers separated. The organic phase waswashed with a 1 N HCl aqueous solution (10 mL), a saturatedNaHCO3 aqueous solution (10 mL) and brine (10 mL). Afterdrying over anhydrous Na2SO4 and concentration in vacuo, thecrude was purified by FC to give the tripeptide 30 (white foam,82%). Rf = 0.67 (1 : 1 AcOEt–hexane); [α]23D = −13.3 (c = 0.59,CHCl3);
General procedure for coupling the tripeptide Ile-Tuv-Tup-OMe with Mep. A solution of tripeptide 21a (150 mg,0.27 mmol ) in a 20% mixture of TFA in DCM (5 mL) wasstirred for 1 h. The solvent was removed in vacuo to give 22a,which was used in the next step without further purification.To a solution of 22a (100 mg, 0.18 mmol) in DCM (5 mL),HOAt (27 mg, 0.2 mmol), HATU (76 mg, 0.2 mmol), Mep(N-methyl-(R)-pipecolic acid (28 mg, 0.18 mmol) and Et3N(50 μL, 0.36 mmol) were added. The reaction mixture wasstirred for 3 h and then washed with a 1 N HCl aqueoussolution (10 mL), a saturated NaHCO3 aqueous solution(10 mL) and brine (10 mL). The combined organic extractswere dried over Na2SO4 and concentrated in vacuo. The crudetetrapeptide 35a–e thus obtained was purified using silica gelchromatography.
General procedure for the hydrolysis of methyl ester 24a–fand acetylation of 25a–d. To a solution of compound 24a(120 mg, 0.17 mmol) in THF (5 mL), a 1 N aqueous solution ofLiOH (510 μL, 0.51 mmol) was added. After stirring for 3 days,TFA (53 μL, 0.68 mmol) was then added and the solvent wasremoved in vacuo to give 25a, which was used in the next stepwithout further purification. To a solution of 25a (126 mg,0.16 mmol) in pyridine (3 mL), Ac2O (1 mL) was added. Theresulting mixture was stirred overnight. The mixture wasconcentrated under reduced pressure and the crude purifiedby FC.
(2S,4R)-4-(2-((1R,3R)-1-Benzoyloxy-4-methyl-3-((2S,3S)-3-methyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)pentyl)-thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid,1g. To a solution of ester 32 (73 mg, 0.095 mmol) in DCE(3 mL), Me3SnOH (11 mg, 0.58 mmol) was added and the reac-tion mixture was stirred under reflux for 32 h. The solvent wasremoved in vacuo and the residue was purified by FC (5 : 95MeOH–CH2Cl2) to give 1g (15 mg, 20%) as a white foam. Rf =0.41 (1 : 9 MeOH–CH2Cl2); [α]23D = −26.6 (c = 0.33, CHCl3);1H NMR (400 MHz, CD3OD) δ: 8.11 (s, 1H), 8.09–7.68 (m, 5H),7.24–7.20 (m, 5H), 6.21–6.18 (m, 1H), 4.40–4.34 (m, 1H),4.09–4.06 (m, 1H), 3.16–3.10 (m, 1H), 2.98–2.91 (m, 3H),2.60–2.56 (m, 1H), 2.48–2.43 (m, 2H), 2.39 (s, 3H), 2.30–2.24(m, 1H), 2.05–1.98 (m, 1H), 1.93–1.85 (m, 3H), 1.75–1.53(m, 7H), 1.41–1.30 (m, 2H), 1.18 (d, J = 7.2 Hz, 3H), 1.00–0.91(m, 12H); 13C NMR (100.5 MHz, CD3OD) δ: 176.4, 174.5, 169.9,165.8, 153.9, 142.4, 137.2, 133.8, 133.7, 133.5, 133.3, 132.7,135.5, 132.2, 130.2, 127.8, 74.9, 72.8, 62.3, 59.3, 55.2, 53.9,46.9, 44.8, 42.1, 41.4, 40.5, 36.7, 34.0, 28.7, 28.4, 26.5, 22.3,21.6, 21.4, 19.2, 14.0; LC/MS (ESI) m/z 776.3 [M + H]+, 798.3[M + Na]+.
Biological tests
Potential anti-tumor activity was evaluated through in vitroassays based on the determination of cytotoxicity of the com-pounds towards the HT29 (Human Caucasian Colon Adeno-carcinoma) cell line. HT29 cells from ECACC (EuropeanCollection of Cell Cultures) were purchased from SigmaAldrich, Milan, Italy.
The assays were performed according to the previouslyreported procedure. The cell line was grown in 75 cm2 flaskswith culture medium DMEM (Dulbecco’s Medium Eagle Modi-fied, Sigma Aldrich) and the following additives (SigmaAldrich): L-glutamine 2 mM, 10% fetal bovine serum, penicil-lin/streptomycin, fungizone, gentamycin. The incubation wascarried out in a modified atmosphere incubator (37 °C, 5%CO2). When cell confluence was obtained, the cells were propa-gated by dilution in a ratio 1 : 3/1 : 10 by using a 0.25% trypsinsolution and then transferred into 96 well plates in the suit-able culture medium. The cells were then treated for 72 hourswith different doses of the compounds under examination. Forthese assays, the compounds were solubilised in DMSO. Allthe tests were carried out at a constant DMSO concentrationequal to 0.1% by weight.
To control the cellular viability, the ATPlite test (PerkinElmer) was used, based on the determination of the ATP pro-duction. ATP is in fact a marker of cellular viability as it ispresent in all the metabolically active cells. The test is basedon the chemiluminescence due to the ATP reaction with theluciferase and the D-luciferin. The emitted light is proportionalto the ATP concentration. The tests were carried out by a Victor3 instrument (Perkin Elmer).
IC50 values were obtained from four different experiments.
Notes and references
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