387268a0Nature3876630199705152682720028-0836199710.1038/387268a01476-4679199717 April 199715 May 1997ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v387/n6630issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupSupplementsCurrent issue387268a0Synthesis of epothilones A and B in solid and solution phase
AU  - Nicolaou, K. C.
AU  - Winssinger, N.
AU  - Pastor, J.
AU  - Ninkovic, S.
AU  - Sarabia, F.
AU  - He, Y.
AU  - Vourloumis, D.
AU  - Yang, Z.
AU  - Li, T.
AU  - Giannakakou, P.
AU  - Hamel, E.[ast] Department ofChemsitry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA, and Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA[dagger] Medicine Branch, DCS, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA[Dagger] Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, DCTDC, NCI, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USAEpothilones A and B, two compounds that have been recently isolated1 from myxobacterium Sorangium cellulosum strain 90, have generated intense interest2-16 among chemists, biologists and clinicians owing to the structural complexity, unusual mechanism of interaction with micro tubules and anticancer potential of these molecules. Like taxol[ast] (refs 17, 18), they exhibit cytotoxicity against tumour cells by inducing microtubule assembly and stabilization3,4, even in taxol-resistant cell lines. Following the structural elucidation of these molecules by X-ray crystallography in 19961, several syntheses of epothilones A (refs 12-16) and B (ref. 19) have been reported, indicative of the potential importance of these molecules in the cancer field. Here we report the first solid-phase synthesis of epothilone A, the total synthesis of epothilone B, and the generation of a small epothilone library. The solid-phase synthesis applied here to epothilone A could open up new possibilities in natural-product synthesis and, together with solution-phase synthesis of other epothilones, paves the way for the generation of large combinatorial libraries of these important molecules for biological screening.The strategy for the solid-phase synthesis of epothilone A (1) was based on the retro synthetic analysis indicated in Fig. 1 (refs 5, 13). Thus, it was anticipated that the three requisite fragments (5-7), one on a solid support (7), would be coupled together sequentially through an aldol reaction, an esterification reaction, and an olefin metathesis reaction20'25, the latter simultaneously cyclizing and liberating the product from the solid support (6 + 7 + 5-> 4 -> 3). A simple desilylation and epoxidation reaction would then complete the total synthesis of epothilone A (1) and analogues thereof (3 -*o!). The outlook for obtaining two products at each of the aldol, metathesis and epoxidation steps was considered advantageous for the purposes of library generation.







Merrifield resin (8, Fig. 2) was converted to phosphonium salt 9 in >90% yield by sequential reaction with: (1) 1,4-butanediol-NaH-n-Bu4NI cat.: (2) Ph3P-iodine-imidazole; and (3) Ph3P (for abbreviations see figure legends). Ylide 1026, generated from 9 by the action of NaHMDS in THF: DMSO at 25 [deg]C, reacted with aldehyde







11 (K.C.N. et a/., unpublished results) at 0[deg]C to form olefinic compound 12 in >70% yield. The geometry of the double bond in 12 was tentatively14 assigned as Z, but its geometry was neither rigorously determined nor did it matter for our purposes. Desilylation of 12 with HF-pyridine, followed by Swern oxidation of the resulting primary alcohol furnished aldehyde 7 in high yield (>95%). The aldol condensation of the polymer-bound aldehyde 7 with the dianion derived from keto acid 6 in the presence of ZnCl2 in tetrahydrofuran (THF) gave a mixture of diastereoisomers  







[ast] Bristol-Myers Squibb has registered Taxol as a trademark and wishes the sceintific community to use the name paclitaxel. 







(-90% yield, -1:1 ratio). Finally, introduction of the heterocyclic segment 513 onto the growing substrate was achieved by esterifica-tion, leading to the required precursor 14 in -80% yield. Exposure of 14 to RuCl2(= CHPh)(PCy3)2, where Cy is cyclohexyl, catalyst (15) in CH2C12 at 25 [deg]C released from the resin olefinic compounds 16-18 and 3 (52% total yield, 16 : 17 : 18 : 3 " 3 : 3 : 1 : 3 as determined by high pressure liquid chromatography (HPLC)). Compounds 16-18 and 3 could be separated either by HPLC or by preparative layer silica gel chromatography, and the two with the correct C6-C7 stereochemistry (that is, 17 and 3) were desilylated by exposure to TFA (see Fig. 2 legend) to afford epothilone precursors 19 (92%) and 20 (90%), respectively. Epoxidation of 19 and 20 with methyl(trifluoromethyl)dioxirane27 then furnished epothilone A (1, 70%) and its diastereoisomer 21 (45%), respectively. The a-epoxy isomers of 1 and 21 were also obtained in these epoxidation reactions. Pure synthetic epothilone A (1) exhibited identical properties (as determined by thin layer chromatography, [a]D (optical rotation), 1H and 13C NMR, infrared and HRMS (high resolution mass spectrum)) to those of an authentic sample.







Figure 1 Retrosynthetic analysis of epothilone A (1) by a solid-phase olefin metathesis strategy. IBS, f-BuMe2Si; the shaded circle indicates polystyrene.







Figure 2 (a) 1,4-butanediol (5.0 equiv.), NaH (5.0 equiv.), />Bu4NI (0.1 equiv.), DMF, 25 [deg]C, 12 h; (b) Ph3P (4.0 equiv.), I2 (4.0 equiv.), imidazole (4.0 equiv.), CH2CI2, 25[deg]C, 3h; (c) Ph3P (10 equiv.), 90[deg]C, 12h (>90% for 3 steps based on mass gain of polymer); (d) NaHMDS (3.0 equiv.), THF: DMSO (1 :1), 25 [deg]C, 12 h; (e) 11 (2.0 equiv.), THF, 0[deg]C, 3h (>70% based on aldehyde recovered from ozonolysis); (f) 10% HF-pyridine in THF, 25[deg]C,12 h; (g) (COCI)2 (4.0equiv.), DMSO (8.0 equiv.), Et3N (12.5 equiv.), -78-* -25[deg]C (estimated yield -95% for 2 step; the reaction was monitored by IR analysis of polymer-bound material and by TLC analysis of the products obtained by ozonolysis); (h) 6 (2.0 equiv.), LDA (2.2 equiv), THF, -78-" -40[deg]C, 1 h; then add resulting enolate to the resin suspended in a ZnCI2 (2.0 equiv.) solution in THF, -78-* -40[deg]C, 2h (-90%; estimated yield, as step g); (i) 5, (5.0 equiv.), DCC (5.0 equiv.), 4-DMAP (5.0 equiv.), 25 [deg]C, 15 h (80% yield as determined by recovered heterocycle fragments obtained by treatment with NaOMe); (j) 15 (0.75 equiv.), CH2CI2, 25 [deg]C, 48 h (52%; 16 :17 :18 : 3 = ˜3 : 3 : 1 : 3); (k) 20% TFA in CH2CI2 (v/v), 92% for 19 and 90% for 20; (I) 22 [methyl(trifluoromethyl)dioxirane, acetonitrile], 0[deg]C, 2h (70% for 1, 45% for 21; in addition to these products, the corresponding a-epoxides were also obtained). NaHMDS, sodium bis(trimethylsilyl)amide; DMSO, dimethyl sulphoxide; LDA, lithium diisopropylamide; TBS, f-BuMe2Si; 4-DMAP, 4-dimethyl-aminopyridine: TFA, trifluoroacetic acid. Selected physical data for compound 20; 1H NMR (400MHz, CDCI3) 6 6.95 (s, 1 H, ArH), 6.59 (s, 1 H, ArCH = C(CH3)), 5.44 (ddd,7 = 10.5,10.5, 4.5 Hz, 1 H, CH = CHCH2), 5.36 (ddd,7 = 10.5,10.5, 5.0 Hz. 1 H, CH = CHCH2), 5.28 (d,7 - 9.4 Hz, 1 H, CO2CH), 4.23 (d,7 = 11.1 Hz, 1 H, (CH3)2CC\-H(OH)), 3.72 (m, CAYOH(CHCH3)), 3.43-3.37 (m, 1 H, OH), 3.14 (q, J = 6.7 Hz, 1 H, CH3CH(C = 0)), 3.05 (bs, 1 H, OH), 2.72-2.63 (m,1 H), 2.69 (s, 3 H, CH3Ar), 2.48 (dd, J = 14.8,11.3 Hz, 1 H, CH2COO), 2.33 (dd,7 = 14.8, 2.0 Hz, 1 H, CH2COO), 2.30-2.13 (m, 2 H) 2.07 (s, 3 H, ArCH = CCH3), 2.07-1.98 (m, 1 H), 1.80-1.60 (m, 2 H), 1.32 (s, 3 H, C(CH3)2), 1.36-1.13 (m, 3 H), 1.17 (d, J = 6.8 Hz, 3 H, Cf/3CH(C = 0)), 1.06 (s, 3 H, C(CH3)2), 0.99 (d,y = 7.0 Hz, 3 H, CH3CHCH2); 13C NMR (150.9 MHz, CDCI3) d 220.6, 170.4,165.0,151.9,138.7,133.4,125.0,119.4,115.8, 78.4, 74.1, 72.3, 53.3, 41.7, 39.2, 38.5, 32.4,31.7,27.6,27.4,22.7,19.0,18.6,15.9,15.5,13.5; infrared (thin film) j>max 3,453,2,929, 1,733,1,686,1,506,1,464,1,250, 978cm'1; [a]2D2 - 80.2 (c 1.36, CHCI3); HRMS (FAB), calc. for C26H39CsN05S (M + Cs+) 610.1603, found 610.1580.







Figure 3 Retrosynthetic analysis of epothilone B (2) by a solution-phase strategy. TBS,f-BuMe2Si.







The total synthesis of epothilone B (2) followed a strategy derived from the retrosynthetic analysis shown in Fig. 314. This strategy called for coupling of intermediates 6, 25 and 26 via a Wittig olefination, an aldol reaction, and a macrolactonization, followed by epoxidation, and was expected to proceed via intermediates 24 and 23. This plan was deliberately chosen for its potential to deliver both diastereoisomers at C6-C7 (aldol reaction) and both geometrical isomers at C12-C13 (Wittig reaction) for molecular diversity and biological screening purposes.







The phosphonium salt 25 (K.C.N. etal, unpublished results) was converted to the corresponding ylide by treatment with NaHMDS which reacted with ketone 26 (K.C.N. etal, unpublished results) to afford a mixture of Z- and E-olefins 27 in 73% yield and -1:1 ratio (by XH NMR) (Fig. 4). The primary hydroxyl group in 27 was selectively liberated by exposure to CSA (97% yield) and oxidized with SO3-pyridine-Et3N-DMSO to afford aldehyde 28 in 95% yield. Treatment of keto acid 613 with excess LDA in THF, followed by reaction with aldehyde 28, furnished a mixture of four compounds corresponding to the two geometrical isomers of the C12-C13 double bond and the two diastereomeric isomers at C6-C7 in high yield. This mixture was persilylated by exposure to excess TBSOTf and 2,6-lutidine, and then selectively deprotected at the carboxylic acid site (K2CO3-MeOH) to afford chromatographically separable (silica gel) carboxylic acids 29 (31% yield from 28) and its 6S,7[pound]-diastereoisomer 29a (30% yield from 28).







The tris(silylether) 29 was then selectively desilylated at C15 (TBAF, 75% yield) to produce hydroxy acid 24 as a mixture of 12Z- and 12E-isomers. Macrolactonization14 of 24 by the Yama-guchi method (2,4,6-trichlorobenzoylchloride, Et3N, 4-DMAP) resulted in the formation of macrocyclic olefins 30 (40%) and 31 (37%), which were chromatographically separated (silica gel). Exposure of 30 and 31 to TFA led to dihydroxy lactones 32 (89%) and 23 (91%), respectively. Finally, epoxidation of 23 with methyl(trifluoromethyl)dioxirane27 furnished epothilone B (2) together with its a-epoxide epimer 35 in 85% yield and -5:1 ratio in favour of 2. Pure synthetic epothilone B (2) was obtained by preparative layer silica gel chromatography (Rf = 0.24, 4% MeOH in CH2C12) and exhibited identical properties (thin layer chromatography, [a]D, 1H and 13C NMR, infrared and HRMS) to those of an authentic sample of epothilone B (2). Similar treatment of 32 resulted in the formation of epothilones 33 and 34 in 86% yield and -4:1 ratio. The use of raCPBA for these epoxidations gave slightly different results leading to 2 and 35 in 66% total yield and -5:1 ratio, and 33 and 34 in 73% total yield and -4:1 ratio.







Table 1 Relative activities of epothilones A (1) and B (2) as compared with synthetic analogues 23, 20, 32, 34 and taxol







Compound







Induction of tubulin assembly*







Parental







Inhibition of human ovarian carcinoma cell growtht Taxol-resistant







p-tubulin mutants







   MDR-line A2780AD















   EC50 (fxM) + s.d.







   1A9







   1A9PTX10







   1A9PTX22 IC50nM (relative resistance) 4.2(2.1)







   















1







   14 [plusmn]0.4







   2.0







   19(9.5)







   







   2.4(1.2)















2







   4.0 [plusmn]0.1







   0.040







   0.035 (0.88)







   0.045(1.1)







   0.040(1.0)















23







   3.3 [plusmn] 0.2







   2.0







   33(17)







   3.5(1.8)







   1.5 (0.80)















20







   25 [plusmn] 1







   25 48







   >100(>4) >100(>2)







   75 (3.0)







   22 (0.88)















32







   39 [plusmn]2







   







   







   75(1.6)







   24 (0.50)















34







   22 [plusmn] 0.9 15[plusmn]2







   3.5 2.0







   30 (8.6) '50(25)







   5.5(1.6)







   3.0 (0.86) >100(>50)















Taxol







   







   







   







   43 (22)







   















*See Fig. 5.







t The growth of all cell lines was evaluated by quantitation of the protein in microtitre plates4. The parental cell line 1A9, a clone of the A2780 cell line, was used to select two taxol resistant sublines(1A9PTX10and 1A9PTX22)29. These sublines were selected by growth in the presence of taxol and verapamil, a P-glycoprotein modulator. Two distinct point mutations in the p-tubulin isotype M40 gene were identified. In 1A9PTX10 amino acid residue 270 was changed from Phe (TTT) to Val (GTT), and in 1A9PTX22 residue 364 was changed from Ala (GCA) to Thr (ACA). The A2780AD line is a multi-drug resistant (MDR) line expressing high levels of P-glycoprotein30. Relative resistance refers to the ratio of the IC50 value obtained with a resistant cell line to that obtained with the parental cell line.







The synthesized epothilones were tested for their action on tubulin assembly using purified tubulin with an assay28 developed to amplify differences between compounds more active than taxol. As demonstrated in Fig. 5, both epothilone B (2) (EC50 = 4.0 [plusmn] 1 |JiM; defined in Fig. 5 legend) and its progenitor 23 (EC50 = 3.3 [plusmn] 0.2 jjiM) were significantly more active than taxol (EC50 = 15.0[plusmn]2|xM) and epothilone A (1) (EC50 = 14.0 [plusmn] 0.4 |xM), whereas compounds 34, 20 and 32 were less effective than taxol.







Preliminary cytotoxicity experiments with 1A9, 1A9PTX10 ((3-tubulin mutant)29, 1A9PTX22 (p-tubulin mutant)29 and A2780AD cell lines revealed a number of interesting results (Table 1). Despite its high potency in the tubulin assembly assay, compound 23 did not display the potent cytotoxicity of 2 against 1A9 cells, being similar to 1 and taxol. These data suggest that whereas the C12-C13 epoxide is not required for the epothilone-tubulin interaction, it may play an important role in localizing the agent to its target within the cell. Like the naturally occurring epothilones 1 and 2, analogue 23 showed significant activity against the MDR line A2780AD and the altered p-tubulin-expressing cell lines 1A9PTX10 and 1A9PTX22, suggesting, perhaps, different contact points for the epothilones and taxol with tubulin (that is, stronger binding of epothilones around residue 364 than around 270 relative to taxoids).







Figure4(a) 25 (1.5 equiv.), NaHMDS (1.5 equiv.), THF, 0[deg]C,15 min; then add 26 (1.0 equiv.), - 20[deg]C, 12 h, 73% (Z: [pound]-1 : 1); (b) CSA (1.0 equiv.), CH2CI2: MeOH (1:1), 0[deg]C, 1 h; then 25 [deg]C, 0.5 h, 97%; (c) S03-pyr. (2.0 equiv.), DMSO (10 equiv.), Et3N (5 equiv.), CH2CI2, 25[deg]C, 0.5 h, 95%; (d) LDA (3.0 equiv.), THF, 0[deg]C, 15 min; then 6 (1.2 equiv. in THF), -78 -40[deg]C, 0.5h; then 28 (1.0 equiv. in THF), -78[deg]C; (e) TBSOTf (3.0 equiv.), 2.6-lutidine (5.0 equiv.), CH2CI2, 0[deg]C, 2 h; (f) K2C03 (2.0 equiv.), MeOH, 25[deg]C, 15 min, 31% of 29 from 28 and 30% of 29a from 28; (g) TBAF (6.0 equiv.), THF, 25[deg]C, 8 h, 75%; (h) 2,4,6-trichlorobenzoylchloride (2.0equiv.), Et3N (2.0 equiv.), THF, 0[deg]C, 1 h; then add to a solution of 4-DMAP (10.0 equiv. in toluene, 0.002 M), 25[deg]C, 12 h, 40% of 30 and 37% of 31; (i) 20% TFA (by volume) in CH2CI2, - 10-> -0[deg]C, 1 h, 89%; (j) same as/, 91%; (k) methyl(trifluoromethyl) dioxirane, acetonitrile, 0[deg]C, 86% (33:34 ˜1:1 diastereoisomers ) or /r[quest]cPBA (1.5 equiv.), benzene. 3[deg]C, 2 h, 73% (33:34 ˜4:1 ratio of stereoisomers); (I) methyl(trifluor-omethyl)dioxirane, acetonitrile, 0[deg]C, 85% (2:35 ˜5:1 ratio of diastereoisomers) or/7[quest]cPBA (1.5 equiv.), benzene, 3[deg]C, 2h, 66% (2 : 35 ˜5 : 1 ratio of diastereoisomers); NaHMDS, sodium bis(trimethylsilyl)amide; CSA, camphorsulphonic acid; DMSO, dimethyl sulphoxide; LDA, lithium diisopropylamide; TBS,f- BuMe2Si; TBSOTf, f-BuMe2SiOS02CF3; TBAF, tetra-n-butylammonium fluoride; 4-DMAP, 4-dimethylaminopyridine; mCPBA, 3-chloroperoxybenzoic acid; TFA, trifluoroacetic acid. Selected physical data for compound 23: 1H NMR (600 MHz, CDCI3) <5 6.94 (s, 1 H, SCH = C), 6.57 (s, 1 H, CH = CCH3), 5.20 (d, J = 9.7 Hz, 1 H, CH2COOCH), 5.13 (dd,7 = 9.6, 4.6 Hz, 1 H, CH3C = C/-/CH2), 4.28 (d,7 = 9.7 Hz, 1 H, (CH3)2CC/-/OH), 3.71 (s, 1 H, CAyOH), 3.47 (bs, 1 H, OH), 3.15 (q, J = 6.8 Hz, 1 H, C(O)CHCH3), 3.04 (bs, 1 H, OH), 2.68 (s, 3 H, N = C(CH3)S), 2.62 (ddd,7 = 15.0,10.2, 10.1 Hz,1 H,CAy2CH = CCH3),2.45(dd,7 = 14.7,11.1 Hz,1 H,CH2COOCH), 2.38-2.24 (m, 1H), 2.28 (dd, 7 = 14.8, 2.2Hz, CH2COOCH), 2.22 (d, 7 = 14.9Hz, 1 H, CH2C(CH3) = CHCH2), 2.06 (s, 3H, CH = CCH3), 1.90-1.84 (m, 1 H), 1.76-1.69 (m, 1 H), 1.65 (s, 3 H, CH2C(CA73) = CH), 1.33 (s, 3 H, C(CH3)2), 1.32-1.22 (m, 4 H), 1.19 (d, J = 6.8 Hz, 3 H, CH(Oy3)), 1.06 (s, 3 H, C(CH3)2), 1-00 (d,7 - 7.0 Hz, 3 H, CH(CH3)); 13C NMR(150.9 MHz, CDCI3)5220.4,170.2,164.9,151.8,139.1,138.3,120.8,119.1,115.5,78.9, 74.1, 72.3, 53.6, 41.7, 39.7, 32.6, 31.8, 31.7, 25.4, 23.0,19.1,18.1,16.0,15.8,13.5; infrared (thin film) max 3,460, 2,954, 2,919,1,725,1,684,1,455,1,379,1,290,1,249,1,184,1,143, 1,043,1,008,973,750cm ˜1; [o;]2D2 - 91.5 s, (c0.3, CHCI3); HRMS (FAB)m/e 492.2795, (M -f- H+) calc. for C27H4iN05S 492.2784.







Figure 5 The tubulin assembly assay was performed essentially as described previously28. Reaction mixtures contained purified tubulin at LOmgml'1, 0.4 M monosodium glutamate, 5% dimethyl sulphoxide, and varying drug concentrations. Each compound was evaluated in three different experiments and average values are shown. The EC50 is defined as the drug concentration that causes 50% of the tubulin to assemble into polymer. In the absence of drug, <5% of the tubulin was removed by centrifugation, while with high concentrations of the most active drugs, >95% of the protein formed polymer. This suggests that at least 90% of the tubulin had the potential to interact with epothilones and taxoids. Although the EC50 value obtained for taxol was higher than that obtained in an alternate assay3, the agent's role in these experiments was only as a control. The numbers on the curves correspond to compound numbers in the text.







The solid-phase synthesis of epothilone A (1) described here represents a new concept for the total synthesis of natural products, traces a highly efficient pathway to the naturally occurring epothi-lones, and opens the way for the generation of large combinatorial epothilone libraries. The biological results demonstrate that more potent microtubule binding analogues than the parent epothilones can be obtained (for example, compound 23) by chemical synthesis. Furthermore, our findings point to lipophilic substituents rather than the epoxide moiety as important elements for binding activity. The role of the epoxide in the cytotoxicity of epothilones, however, still remains to be elucidated.







Acknowledgements. We thank G. Hofle and Merck Research Laboratories for gifts of epothilones A and B; and D. H. Huang and G. Siuzdak for NMR and mass spectroscopic assistance, respectively. This work was supported by the NIH, The Skaggs Institute for Chemical Biology and the CaP CURE Foundation, and fellowships from the Ministerio de Educacion y Ciencia (Spain) (J.P.), Fundacion Ramon Areces (Spain) (F.S.) and Novartis (D.V.), and grants from Merck, DuPont-Merck, Schering Plough, Hoffmann La Roche and Amgen.







Correspondence and requests for materials should be addressed to K.C.N. at Scripps Research Institute.







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