Tetrahedron Vol. 48. No. 34. pp. 6985-7012 1W2 0040-4020/92 85.00+.00 Printed in Chat Britain Porg- Ress Lrd NEW AND EFFICIENT APPROACHES TO THE SEMISYNTHESIS OF TAXOL AND ITS C-13 SIDE CHAIN ANALOGS BY MEANS OF f+LACTAM SYNTHON METHOD IWAO OIIMA*, IVAN HABUS, MANGZHU ZHAO, MARTINE ZUCCO, YOUNG HOON PARR, CHUNG MING SUN and THIERRY BRIGAUD Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, U. S. A. (Received in USA 20 April 1992) Summary: Highly efficient chiral ester enolate-imine condensation giving 3-hydroxy+aryl- g-lactams with excellent enantlomeric purity is successfully applied to the asymmetric synthesis of the enantiomerically pure tax01 C-13 side chain, N-benxoyl-(2f?,3S>3-phenyl-isoserine and its analogs. (3R,4S)-N-benzoyl-3-(1-ethoxyethoxy)-4-phenyl-2-azetidinone readily derived from the 3-hydroxy-4-phenyl-g-lactam is coupled with protected baccatin IRS, followed by deprotection to give optically pure tax01 and lO-deacetyl-7,10-bis(Troc)-taxol in good yields. Fully assigned IH, 1%. and 2D (CGSY and HETCOR) NMR spectra of tax01 thus synthesized are shown and discussed. Taxol, a complex diterpene,l is currently considered the most exciting lead in cancer chemotherapy. Tax01 possesses high cytotoxicity and strong antitumor activity against different cancers which have not been effectively treated by existing antitumor drugs. For example, tax01 is currently in phase III clinical trial for advanced overian cancer, phase II for breast cancer, and phase I for lung cancers, colon cancer and acute leukemia.2 At present, the supply of tax01 is solely dependent on the extraction from the bark of Tams brevifoliu (Pacific yew), which is a very slowly growing tree in old growth forests in the Northwest of the United States and the total number is estimated to one million. For the set of clinical trials only, more than 25,000 trees are required because of the low concentration of taxol in the bark. The harvest of those trees endangers not only the old growth forest in the Northwest of the United States, but also the future supply of taxol. Taxol lo-Deacetylbaccatin III 1) 6985
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`6986 I.
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`OJIMA et al.
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`Accordingly, it is obviously an absolute necessity to develop practical cell culture or synthesis for this extremely promising anticancer drug. A couple of reports have appeared for the cell culture approach,3 but the efficiency and practicality of those reported methods are still unknown. The total synthesis of tax01 has already been attempted by a number of synthetic chemists without success so far, and requires much further elaborations.4 However, it has recently been found that the most complicated tetracyclic diterpene moiety of taxol, i.e., 10deacetylbaccatin III
`(l), which is the most demanding in total synthesis, is more readily available from the leaves of Taxus baccuta (European yew).5 The extraction of the fresh leaves yields IO-deacetylbaccatin III in a very good yield, i.e., lg/lKg. The leaves are reproduced quickly, and thus it is unnecessary to cut down the trees to obtain the bark, which makes a sharp contrast to the case of taxol. With the availability of IO-deacetylbaccatin III
`(l), it appears that sufficient supplies of tax01 can now be produced in a semisynthetic fashion. Namely, if the C-13 side chain can be synthesized effectively and coupled to lo-deacetylbaccatin III
`
`(1) with proper protective groups, the semisynthetic process would be the most practical approach to the production of taxol and sufficient supplies of tax01 may well be secured. Although tax01 is an extremely important “lead” in cancer chemotherapy, tax01 has a problem in solubility in aqueous media, which may impose some serious limitation in its use. Also, it is common that better drugs can be derived from naturally occurring lead compounds. In fact, French researchers, Potier, Gu&itte-Voegelein, Guenard et al. have discovered that a modification of the C-13 side chain of tax01 brought about a new anticancer agent which seems to have antitumor activity superior to tax01 with better bioavailability. This unnatural compound was named “taxoti?te”, which has t-butoxycarbonyl instead of benzoyl on the amino group of (ZR,X)- phenylisoserine moiety at the C-13 position and a hydroxyl group instead of acetoxy group at c-10.6 Taxotke is currently in phase II clinical trial in both United States and Europe. Taxotere has been synthesized by a semisynthetic process, including a coupling of N-tert-butoxycarbonyl-(2R,3S)-3-phenylisoserine (2) with lo- deacetylbaccatin III
`(1) with proper protecting groups.7 It is known that the C-13 side chain, i.e., N-benxoyl-(2R,3S)-3-phenylisoserine (3) moiety, is crucial for the strong antitumor activity of taxol. 8 Moreover, some modification of the C-13 side chain can provide a new series of taxol analogs which may have higher potency, better bioavailability and less unwanted toxicity, as exemplified by the discovery of taxotere. Accordingly, it is quite promising to investigate the structure-activity relationship (SAR) for the C-13 side chain analogs of tax01 with some modification of the baccatin III moiety in order to find more effective anticancer agents with better pharmacological property.9 Taxol C-13
`
`side chain
`
`(3)
`
`Tax&&e
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`Semisynthesis of tax01 and its C-13 side chain analogs 6987 Accordingly, the development of an efficient method which can be applied not only to taxol, but also to taxoti% and other analogs, i.e, the method having flexibility and wide applicability is extremely important and of current demand to promote the research in this area. We describe here an efficient and practical approach to the semisynthesis of the C-13 side chain analogs of tax01 on the basis of the “p-Lactam Synthon Metbod”,lO which has the desired flexibility and wide applicability. The first enantioselective synthesis of the important side chain 3 was obtained in 8 steps and 23% yield via a Sharpless epoxidation from cis-cinnamyl alcohol with au enantiomeric excess of X-80%.11 The obtained 3 was coupled with 7-triethylsilylbaccatin BI (4a) by esterification. 12 A recent publication describes the chemo- enzymatic synthesis of a derivative of 3, in which the racemic mixture was resolved by enzymatic hydrolysis with lipases.13 We successfully applied lithium chiral ester enolate - imine cyclocondensation strategy14 to the asymmetric synthesis of 3 via a (3R,4S)-3-hydroxy-4-phenylazetidin-2-one
`
`@a) as the key-intermediate.15 Based on this protocol, 3 can be obtained in 3 steps in high yield with virtually 100% e.e. Quite recently, it was found that l-benzoyl-(3R,4S)-3-( l-ethoxyethoxy)-4-phenyl-2-azetidinone
`(6a), readily derived from the hydroxy-a-lactam
`(Sa), served as the key-intermediate for the synthesis of taxol.16 Therefore, our fi-lactam intermediate
`5a serves as the key-intermediate for both coupling methods.
`
`6a
`
`(Chart 1)
`
`10 (q.
`
`RESULTS AND DISCUSSION Synthesis of C-13 side chain of tax01 and its analogs First, we carried out the reactions of chiral lithium ester enolates (8) generated in situ from silyloxyacetates (7)
`1). Results am summarized in Table 1. OR’
`R’-0 LDA )_ (1) 0 O-LI R-CH=N-TMS 7 8 9
`
`OR’
`
`W-O*
`R 0.
`l
`
`9a:
`9b:
`R = 4-(MeO)CsH, 9c: R = 3,4-(MeO&H,
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`with N-trimethylsilylimines (9). which gave the corresponding chiral p-lactams
`R=Ph
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`Chart1 c Ph 0 L 0 0 -d, 7a Table 1. Asymmetric synthesis of ~lactams (10) through chiral enolate - imine cyclocondensation Entry Ester Imine PLactam Isolated Configuration Enantionmic Yield (%) purity (96 e.e.)O 1 7a 2 7b 3 7c 4 7d 5 (-)7e 6 (+)7e 7 (-)7e 8 (-)7e 9a (-)10-A 18 3R,4S 15 9a (+)10-A 20 3S,4R 67 9a 10-B 52 3R,4S 50 9a 10-B 90 3R,4S 76 9a (+)10-C 85 3R,4S 96 9a (-)10-C 80 3S,4R 97 9b (+)10-D 80 3R,4S 96 9c (+)10-E 80 3R,4S 98 ~Detined by 1H NMR analysis using a chiral shift reagent, (+)-Eu(hfc)g. (Entries l-3) and by HF’LC analysis on a chiral column - DAICEL CHIRACEL OD using n-hexane - 2-propanol as the solvent (Entries 4-8).
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`Semisynthesis of tax01 and its C-13 side chain analogs 6989 As Table 1 shows, the chiral auxiliary and the O-protecting group exert marked effects on the enantioselectivity as well as on the chemical yield of the reaction. l7 For example, the reactions of 7e, beating (-)- or (+)-trans-2phenyl-l-cyclohexyl as the chiral auxiliary19 and ttiisopmpylsilyl (TfPS) as the O-protecting group, with 9a-c give exclusively the corresponding cis-p-lactams
`
`10-B and
`
`(+)10-C
`
`(+)10-D and
`
`(+)10-E, absolute configurations were assigned by analogy with
`
`(+)lO-
`
`10 in high yields with extremely high enantiomeric purity (9698% e.e.) (Entries 5-8). However, the reaction of
`7d bearing t-butyldimethylsilyl (TBDMS) as the O- protecting group with 9a gives
`10-B in 90% yield, but with 76% e.e. (Entry 4). When (-)-menthyl is used as the chiral auxiliary and t-butyldimethylsilyl as the O-protecting group (7~). the reaction with
`9a gives 10-B in 52% yield with only 50% enantiomeric purity (Entry 3). The use of benxyl as the O-protecting group and (-)- menthyl or ephedrinyl,
`7a and 7b, gives (-)10-A (18% e.e.) or (+)10-A (67% e.e.) in low yield (1520%) (Entries 1,2). Absolute configurations of p-lactams
`(10) were determined by chemical correlation with authentic samples:
`were converted to (R)-3-phenyllactic acid via hydrogenolysis on 10% Pd-C followed by hydrolysislw-f and to (2R,3S)-3-phenylisoserine by hydrolysis with 6N hydrochloric acid (vi& infra), respectively. For
`C based on specific rotations and retention times on HPLC analyses on a chiral column (see Experimental Section). The absolute configuration of
`(+)10-B (3S,4R). The chiral auxiliaries, (+)- and (-)-uans-2-phenyl-1-cyclohexanol were recovered >90% yield in the Entries 5-8. The exclusive formation of cis-p-lactams
`(+)lO-C,D,E with 96-988 e.e. is rationalized by taking into account the highly selective generation of @)-lithium enolates, (-)-E-8e, and the transition state
`A depicted in Scheme 1 on the basis of analysis discussed below.
`
`bearing (-)-menthyl group gives (3R,4S)-p-lactam
`
`(-)10-A and
`
`(+)10-A were determined on the basis of the fact that
`(10-B). i.e,
`(-)10-A should have (3R,4S) configuration and
`
`7d
`
`Scheme 1
`
`H O-TIPS ‘TMS (-)-E-8e O-TIPS 5 1 TIPS-O,, R’ ‘._ p N-TMS -
`
`lo-C,D,E;
`
`96-98%
`
`e.e.
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`6990 I.
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`OlBlA
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`63 al.
`
`TIPS
`
`I &A R’ I’ A , W-0 :x
`
`There are two possible mechanistic pathways, i.e., (a) E-enolate formation followed by a chair-like transition state A and (b) Z-enolate formation followed by a boat-like transition state B, which can ammmdate the observed stereochemical outcome. Since these are early transition states, the boat-like transition state B is not necessarily unfavorable. However, the chlral auxiliary is in an exo position in B, whereas it is located in au endo position in A. Accordingly, it is reasonable to assume that the transition state A would bring about much better asymmetric induction than B. For the formation of E- or Z-enolate, E-enolate formation should be kinetically favorable.20 In order to examine thermodynamic preference, we carried out MM2 calculations for both (-)-E&.3THF and (-)-Z- &.3THF using MACROMODEL program. Then, it was found that (-)-E-&.3THF is more favorable than (-)- 2-&.3THF by 2.5 kcaI/mol. Therefore, the formation of E-enolate is clearly preferred in this case. Z-enolate
`Li' 0 B E-enolate, (-)-E-Se.3THF Z-enolate, (-)-Z-8e.3THF It is apparent that the chiral auxiliary, (-)-trans-2-phenyl-1-cyclohexyl, directs the approach of the N- TMS-imines @a-c) extremely effectively from the G-face of (-)-E-&z to give N-lithiated-P-amino esters (ll), which then cyclize to afford the corresponding cis-p-lactams lo-C,D.E (Scheme 1). In the same manner, the enantiomeric (-)10-C is yielded from (+)-E-t& with 97% e.e. Until now, the asymmetric synthesis of 3- hydroxy-p-lactam has been limited by low stereoselectivity and often low chemical yield.21 Thus, our method provides the first efficient and practical route to 3-hydroxy-plactams with extremely high enantiomeric purity.
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`Semisynthesis of tax01 and its C-13 side chain analogs 6991 Next,
`(+)10-C thus obtained was converted to the desired N-benzoyl-(2R,3S)-phenylisoserine (3) through the procedure illustrated in Scheme 2. As Scheme 2 shows, (+)10-C ‘was hydrolyzed with 6N hydrochloric acid at 2YJC for 3-4 h to give (2R,3S)-phenylisoserine hydrochloride (1211) in quantitative yield, which was benwylated by the usual Schotten-Baumann procedure followed by purification on a short silica gel column to give enantiomerically pure N-benzoyl-(2R,3S)-phenylisoserine (3) in 72% yield. Alternatively, (+)10X was deprotected by reacting with tetra-n-butylammonium fluoride in e at 25oC to give the (3R,4S)-3-hydroxy-j3-lactam
`Sa was hydrolyzed with 6N hydrochloric acid at 25oC for 3 h to afford 12a in quantitative yield. Other 3-silyloxy-4-aryl-~-lactams, (+)lO. D and (+)10-E, can be converted to the corresponding substituted N-benxoylphenylisoserines in the same manner. Scheme 2 b
`
`Sa in 97% yield (Scheme 2). Then,
`
`12a
`
`a: 6N HCI, 25%. b: n-&NF, THF. c: PhCOCI, NaHCQ, CHg& - Hfl. 1. LDA TlPS-0,. ,,R’ . TIPS-O-CH2-COOR* (2) (-Ye R’O- = 6N HCI t- YH2.HCI RI+ COOH
`
`6H The substituent Rt other than phenyl is important for the synthesis of tax01 analogs which may possess better bioavailability as well as cytotoxicity and/or stronger binding ability to micrombles. Therefore, we synthe-
`
`12
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`6992 I.
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`OJIMA Of d.
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`sired new p-lactams beating aryl, heteroaryl, and alkyl substituents at C-4, which can be converted to the C-13
`side chain analogs of taxoL In these syntheses, N-PMP-imines (PMP = 4-methoxyphenyl)
`(13) were used for
`the chiral enolate - imine cyclocondensation (eq. 2). These imines 13 are much more stable than IV-TMS-imines
`9, especially for alkylaldimks.
`It is worthy of note that alkylaldumnes can be used for this reaction in spite of
`the acidity of hydrogen at the u-carbon. Results are listed in Table 2. As Table 2 shows, the reactions achieve
`
`extremely high stereoselectlvity (9899% e.e.) for phenyl-, 4-fluorophenyl- and 4-(trifltmromethyl)phenylaldimine
`(13a-c) (Entries l-3). The reactions also give excellent stereoselectivity for phenylethenyl-, 2-tkylethenyl,
`isobutyl, and cyclohexylmethylaldimines
`(13e-h) (Entries J-8).
`7 and ,I?
`first
`give the
`provide the
`corresponding /Hactanrs in chiral
`(14) with (3R,4S) configuration exclusively except for the case of
`All reactions give cis-p-lactams
`furfurylaldimine
`(14d), which gives a small amount of trans-isomer (cis/trans = 91/9); Nevertheless, the
`stereoselectivity for the formation of &-isomer
`(14d) is excellent (92% e.e.) @my 4).
`
`Table 2. Asymmetric synthesis of p-lactams (14)
`
`Rl
`
`-0 \-/
`
`m
`
`I\
`0
`
`Entry
`
`1
`
`2
`
`3
`
`4
`
`8
`
`14a
`
`14b
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`14c
`
`14d
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`14e
`
`89
`
`81
`
`84
`
`78C
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`85
`
`98
`
`98
`
`99
`
`92d
`
`96
`
`14h
`
`85
`
`90
`
`a All &lactams are cis and (3&W) configuration unless otherwise noted. @zermined by
`chiral HPLC on a chiral column - DAICRL CHIRACRL OD using n-hexane - 2-pmpanol as
`a mixture of cis- and tram-isomers (cis/trans = 9 l/9).
`the solvent . C Obtained as
`d Enantiomeric excess for the cis-isomer.
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`NEPTUNE GENERICS EX. 1052 00008
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`To the best of our knowledge, Entries
`exmqles in which aldimines of em&able aikylaldehydes are successfully used to
`ester - e&ate condensation process.=
`

`

`Semisynthesis of tax01 and its C-13 side chain analogs 6993 The p-lactams 14a, Me, 140, and 14h thus obtained were treated with cerium ammonium nitrate (CAN) in water at ODC for 1 h to give p-lactams (+)10-C, 1Oe. log. and 10h. respectively, in 7585% yields (eq. 2). The hydrolysis of 10e. log, and 10h with 6N hydrochloric acid at 25eC for 3-4 h gave the corresponding (2R,3S)-isoserines in quantitative yields (see Experimental Section). The j3-lactam 14e was further hydrogenated on 10% W-C to give the 4-phenylethyl-P-lactam 14i (96% yield), followed by reacting with CAN at OW in water to afford the j3-lactam 1Oi in 85% yield. The p-lactam 1Oi was further hydrogenated on 5% Rh-C at 50X and 800 psi of hydrogen to give the 4-cyclohexylethyl-p-lactam 1Oj in 93% yield. The j3-lactams 1Oi and 1Oj were hydrolyzed with 6N hydrochloric acid in the same manner to that described above to afford the corresponding isoserine hydrochlorides, 12i and 12j, respectively, in quantitative yields. These transformations are illustrated in Scheme 3. Scheme 3 14e w OBk 14i o+,, “->[- qz NHI.HCI 0 12j 1Oj 12i a: WPd-C, MeOH-AtSEt, 25’C. b: CAN, CH$N-Hfi. c: 6N HCI, 2!j°C. cl: H2/Rh-Ci (NMl psi), M&H, got2 Coupling of the C-13 side chain precursors with baccatin III derivatives - semisynthesis of tax01 The N-benzoylphenylisoserine (3) with O-( 1-ethoxyethyl) (GEE) protecting group has already been coupled with 7-TES-baccatin III (4a) (TES = triethylsilyl) in the presence of dipyridylcarbonate @PC!) and 4- dimethylaminopyridine @MAP) by Greene et al. (eq. 3). lZa The best result so far reported for the synthesis of 7-TES-2’-EE-taxol (15) is 80% yield at 50% conversion by using ca. 6 equiv. of O-EE-N-benzoyl- phenylisoserine (3-EE) at 73oC in toluene.lh
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`I. OJIMA er al.
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`DPC, DMAP 7-TES-2’-EE-taxol (1% (3) Recently, Holton developed a newer coupling method (eq. 4), 16 directly from l+enzoyl-(3R,4s)-3-(1- ethoxy)ethoxy-4-phenyl-2-azetidinone (6a), which was derived from (3&U)-3-hydroxy-j3-lactam 5a. The required 5a was obtained through tedious optical resolution of racemic cis-3-hydroxy-elactam.16 As shown in Scheme 2, Sa can be obtained in two steps in excellent yield through our method. According to Holton’s procedure in his patent application, 16 the coupling of 6a (5 equiv.) with 4a proceeds at 25oC in the presence of DMAP and pyridine for 12 h to give 7-TES-2’-EE-taxol (U) in 92% yield, which was deprotected with 0.5% hydrochloric acid in ethanol at OT to afford tax01 in ca.90% yield. We carried out the coupling following the Holton procedure and found that the reported result was reproducible except for the reaction time, i.e., the reaction proceeded much slower than reported and thus 24-36 h were necessary for completion. Since Holton’s protocol requires 5 equiv. of 6a and the reaction is very slow even under almost neat conditions, we looked for a better coupling procedure. Thus, we investigated the coupling of the sodium salt of 4a, which should have higher nucleophilicity than neutral 4a, with 6a in ‘II-IF. The sodium salt of 4a was generated by reacting 4a with NaH in THF (eq. 4). Although the reaction conditions have not been optimized yet, some encouraging result has been obtained. 4a + DMAP, Pyridine ) ‘I-TES-2’-EE-taxol (4) or NaH (15) 1 0.5% HCVEtOH 6a Taxol A mixture of 7-TES-baccatin III (4a) (0.10 mmol) and 6a (0.15 mmol, 1.5 equiv.) in THF was added to NaH (large excess) in THF suspension at BC. The suspension was stirred at 35oC for 3 h and quenched with brine at Ooc. Conversion was 50% on the basis of 1H NMR analysis. After column chromatography on silica gel, 7-TES-2’-EE-tax01 (15) was isolated in 40% yield (80% yield based on 4a reacted) and unreacted 4a was recovered (37%). The 7-TES-2’-EE-taxol (lS) thus obtained was a 1: 1 mixture of diastereomers due to the chiral center at 2’-EE (Note: Holton reported in his patent application 16 that a 2:l diastereomer mixture of 15 was obtained. It is possible to assume that one of the two diastereomers was enriched through kinetic resolution since
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`Semisynthesis
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`of taxol and its C-13 side chain analogs
`
`6995 5 equiv. of B-lactam (racda) were employed.). In order to examine the stereochemical integrity during the coupling, 15 was deprotected by 0.5% hydrochloric acid in ethanol at Ooc. following Greene’s procedure,t2a to give tax01 in 88% yield after column chromatography on silica gel. It was then confirmed by 1H and 13C NMR analyses that no racemization took place during the coupling process and optically pure tax01 was obtained. We also carried out the coupling of 6a with 7,10_bis(Troc)-baccatin III (4b)lzh (Tmc = trichloroethoxy- carbonyl) in the same manner as that described above. In this coupling, the sodium salt of 4b was generated first at -15nC, followed by the addition of 6a at the same temperature. The suspension was stirred for 1 h and quenched with .brine. lo-Deacetyl-7,10-bis(Troc)-2’-EE-taxol (16) was obtained in 88% yield based on 4b reacted (43% conversion). It is noteworthy that 4b showed substantially higher reactivity than 4a in this coupling process in spite of the fact that the substituents at C-7 and C-10 do not seem to be in a proximity of C- 13 hydroxyl group. After deprotecting 2’-EE with 0.5% hydrochloric acid in THE, lO-deacetyl-7,1O-bis(Troc)-taxol(17)t2h was obtained in 90% yield after column chromatography on silica gel (eq. 5). No racemization was observed during the coupling on the basis of tH and t3C NMR analyses of 17.
`HCI (5) While our NMR study was in progress, complete assignments for the 1H and 13C NMR of tax01 with the aid of 2D NMR experiments were teported by Falzone.24a Chmumy, sh BakerDc and their coworkers which prompts us to report here lH andt3C NMR, HETCOR and COSY spectra of tax01 thus synthesized. The tH (600.139 MHz) and t3C (150.925 MHz) NMR spectra are shown in Figure 1, and Figures 2 and 3 illustrate the HETCOR and COSY spectra, respectively. The tH and *3C NMR data perfectly match with those reported for naturally occurring tax01.~ On the basis of lH, t3C, DEPT, COSY and lH - t3C HETCOR (CSCM) NMR spectra of tax01 as well as 1H and 13C NMR spectra of lo-deacetylbaccatin III (1). 7-TES-baccatin III (4a) and lo-deacetyl-‘l,lO-bis(Troc)- baccatin III (4b), all protons and all carbons were unambiguously assigned. The assignments of protons and carbons of three phenyl groups are not shown for simplicity. Our COSY and HETCOR analyses disclosed that the previously reported~s assignment of the metbylene protons at Cl4 (Cl4Hz) was incorrect, i.e., the Cl4-H2 protons were reported to appear at 2.5 ppm in CM313 as a multiplet, and the C&H2 methylene protons were not assigned. Although C-6 virtually overlaps with C-14 at 35.64 ppm, the expanded HETCOR spectrum at the C- 14 and C-6 region (Figure 2) clearly indicates the existence of diastereotopic protons. The COSY spectrum shows a cross peak due to the geminal coupling between the C6- Hz methylene protons. These protons also show two cross peaks arising from the vicinal couplings with the C7- H methine pmton.(4.40 ppm). A cross peak due to the vicinal coupling of Cl4-H2 with ClfCH (6.23 ppm) is
`
`1. NaH t 2. 6a
`
`3. 0.5%
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`6996 I. OIIMA et al. * - -
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`NEPTUNE GENERICS EX. 1052 00012
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`Semisynthesis of tax01 and its C-13 side chain analogs Figure 2. ‘H-13C HETCOR spectra (250 MHZ) of taxol in CDCl3 6997 ,“.‘,‘.-,....,....,....,....I....,....,....,....,.*.,,...,,,.,, ppI(
`
`-
`
`-
`
`-
`
`-
`
`_
`
`-
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`-
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`ii0
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`160
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`c;O
`
`PPM
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`$0
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`io
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`;0
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`2.0
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`3.0
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`4.0
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`5.0
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`6.0
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`7.0
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`6.0
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`6.0
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`69% I. OJMA et d, Figure 3. COSY spectra
`
`MHz)
`
`(250
`
`of tax01 in CDC& L PPY 6.0
`
`7.0
`
`6.0
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`5.0
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`PPU
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`4.0
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`3.0
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`2.0
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`2.0
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`3.0
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`4.0
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`5.0
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`6.0
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`7.0
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`6.0
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`2.0
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`3.0
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`4.0
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`5.0
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`6.0
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`Semisynthesis of taxol and its C-13 side chain analogs 6999 observed as well. Therefore, the signals at 1.86 (m) ppm and 2.53 (ddd) ppm in the *H NMR spectrum are unambiguously assigned to C6-Hz and the signals at 2.26 (dd) ppm and 2.34 (dd) ppm to Cl4-Hz. Our assignments are consistent with those mported by Falzone et al.% as well as Chmurny et alah In conclusion, it is demonstrated that 3-hydroxy4substit~lactams serve as the key-intermediates for the asymmetric synthesis of the tax01 C-13 side chain and its analogs, which are readily obtained through highly efficient chiral ester enolate - imine cyclocondensations with extremely high enantiomeric purity. These 3- hydroxy-4-substituted-B-lactams are readily converted to the corresponding 1-acyl-3-EEO-P-lactams in high yields, which can directly be coupled with protected baccatin BIs to give tax01 and its analogs after depmtection. The most efficient and crucial chiral auxiliary, (-)-trans-2-phenylcyclohexanol can readily be obtained in 100 g scale using the lipase-catalyzed kinetic resolution of its racemic acetate developed by Whitesell et al.,*9 which is fully recyclable after the reaction. Triisopropylsilyl protecting group (TIPS) can also be recycled and its recovery process is currently in active investigation. This synthetic method provides efficient and practical routes to a variety of new tax01 C-13 side chain analogs which may have better bioavailability and cytotoxicity with lower undesired toxicity as well as various photoaffinity and radio-labeled tax01 analogs which would play a key-role in biochemical study of taxol. EXPERIMENTAL SECTION
`
`General Method. Melting points were measured with a Thomas Hoover capillary melting point apparatus and are uncorrected. IR spectra were recorded with a Perkin-Elmer FTIR 1600 series spectrophotometer. 1H. l3C, and 2D NMR spectra were measured with a Bruker AMX 600, a Bruker AC 250 or a General Electric QE-300 spectrometer for solutions using tetramethylsilane as the internal standard. Optical rotations were measured with a Perkin-Elmer Model 241 polarimeter. Thin Layer chromatography was performed on Merck DC-alufolien with Kieselgel6OF-254. Column chromatography was carried out on silica gel (Silica gel 60,230-400
`mesh ASTM, Merck). Chiral HPLC analysis for the determination of enantiomeric excess, was carried out with a Waters HPLC assembly consisting of a Waters M45 solvent delivery system, a Waters Model 680 gradient controller, ‘and a Waters M440 detector (at 254 nm), equipped with a Spectra Physics Model SP4270 integrator using a chiral column J. T. Baker DAICEL - CHIRACEL OD employing hexane/;l-propanol(13/1) as the solvent system with a flow rate of 0.2~min. MACROMODEL program was run on a Vax Station 3100 (digital).
`
`Materials.
`
`(1) was a gift from Indena. SpA. lo-Deacetyl-7,10-bis(trichloroethoxy- carbonyl)baccatin III (4b) was a gift from Rhone-Poulenc Rarer. 7-Triethylsilylbaccatin III
`IO-Deacetylbaccatin III
`(4a) was prepared from IO-deacetylbaccatin III
`
`(1) by the literature method. 12a (-)- and (+)-trans-2-Phenylcyclohexanol were prepared by the literature method.19 Triisopropylsilyl chloride was obtained from Aldrich Chemical Co.
`A solution of (-)-(1R,2S)-Zphenyl-1-cyclohexyl hydroxyacetate (851 mg, 3.63 mmol) was prepared through esterification of benzyloxyacetyl chloride with (-)-(1R,2S)-2-phenyl-1-cyclohexanol followed by hydrogenolysis. Then, triiso-
`
`Preparation of (-)-(lR,2S)-2-phenyl-1-cyclohexyl
`
`triisopropylsilyloxyacetate
`
`(7e):
`
`NEPTUNE GENERICS EX. 1052 00015
`
`

`

`7Om
`
`I. OJIMA et al. propylsilyl chloride (840 mg, 4.36 mmol) and imidazole (618 mg, 9.08 mmol) in dimethylformamide (DMF) (1.7 mL) was stirred at room tempemtute for 12-20 hours. The mixture was poured into pentane (25 mL), and washed with water and brine. The combined organic layers were dried over anhydrous
`MgSO4 and concentrated in vacua. The crude product was subjected to a purhication on a short silica gel column using hexaneMloroform (3/l) as the eluant to give pure (-)7e (1.35 g, 95% yield) as a colorless oil.
`
`(-)7e:
`
`[ch20
`
`-17.1O (c 3.15. cI+213); IR
`
`(CDQ3) 6 0.93-0.99 (m. 21H), 1.30-1.62 (m, 4H), 1.72-2.0 (m. 3H), 2.10-2.19 (m, lH), 2.66 (dt, J = 11.5, 4.0 Hz, lH), 3.90 (d, J = 16.6 Hz, 1H). 4.07 (d, J = 16.6Hz, lH), 5.07 (dt, J = 10.6, 4.0 Hz, 1H). 7.16-7.30 (m, 5H). Anal. Calcd for Q&aO$Ii: C, 70.72; H, 9.81. Found: C, 70.79; H, 9.85. In the same manner, 7c, 7d. and (+)7e were prepared by the combinations of (-)-menthyl hydroxyacetate with tert-butyldimethylsilyl chloride, (-)-(ZR,B)-2-phenyl-l-cyclohexyl hydroxyacetate with tert-butyldimethyl- silyl chloride, and (+)-(Z&2@-2-phenyl-1-cyclohexyl hydroxyacetate with triisopropylsilyl chloride, respective- ly. in 90-955 yields. In a similar manner, 7a sad 7b were prepared by the reactions of benzyloxyacetyl chloride with (-)-menthol and (lR,2.S)-N-methylephedrine, respectively, in 92-95% yields. (-)Menthyl benzyloxyacetate (7a): Colorless oil; [a]D 2o -59.2“ (c 0.85. CHC13); 1H NMR (CDC13) 6 0.77 (d, J = 6.9 Hz, 3H). 0.89 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H). 0.92-1.14 (m, 3H). 1.34-1.56 (m, Z-I). 1.64-1.73 (m. W), 1.84 (m, lH), 2.02 (m, lH), 4.07 (s, 2H), 4.64 (s, 2H), 4.80 (dt, J = 10.9, 4.4 HZ, lH), 7.30-7.45 (m, 5H). (lR,2S)-N-Methylephedrinyl benzyloxyacetate (7b): Pale yellow oil; [u]um -25.780 (c 1.28, CHC13); lH NMR (CDC13) 6 1.05 (d, J = 6.7 Hz, 3H), 2.28 (s, 6H), 2.91 (m, lH), 4.15 (s, W), 4.62 (s, 2H), 6.04 (d, J = 5.4 Hz, IH), 7.25-7.35 (m, 10 H). (-)Menthyl t-butyldimethylsilyloxyacetate (7~): Colorless oil; [u]uzo -59.30 (c 1.00, CHC13); IH NMR (CDCI3) 6 0.11 (s. 6H). 0.76 (d, J = 7.0 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H), 0.92
`
`(s,
`
`9H), 0.90-1.13 (m, 3H), 1.32-1.43 (m, lH), 1.40-1.56 (m. lH), 1.63-1.72 (m, W), 1.80-1.91 (m, lH), 1.98-2.05 (m lH), 4.22 (s, W), 4.75 (ddd, J = 10.9, 10.9, 4.4 Hz, 1H). HRMS Calcd for Cl&&Sk C, 65.80; H, 11.05. Found: C, 65.60, H, 10.96. (l&ZS)-2-Phenyl-1-cyclohexyl t-butyldimethylsilyloxyacetate (7d): Colorless oil; [o]Dzo -18.70 (c 1.03. CHC13); lH NMR (CDC13) 6 -0.08 (s, 3H), -0.06 (s, 3H), 0.83 (s, 9H), 1.25-1.62 (m, 4H), 1.76-1.98 (m. 3H), 2.10-2.17 (m. lH), 2.66 (dt, J = 3.7, 11.5 Hz, lH), 3.83 (d, J = 16.8 Hz, lH), 3.99 (d, J = 16.8 HZ. lH), 5.06 (dt, J = 4.4, 10.5 Hz, 1H). 7.18-7.31 (m, 5H). Anal. Calcd for QoH3203Si: C, 68.92; H, 9.25. Found: C. 68.83; H, 9.18. (+)7e: Colorless oil; [a]D2’ +17.07O (c 3.29, CHCl3); lH NMR spectrum is identical to that of (-)7e.
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`NEPTUNE GENERICS EX. 1052 00016
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`(neat) 1759, 1730 (VCC) cm-l; 1H NMR
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`Semisynthesis of taxol and its C-13 side chain analogs 7001
`
`Preparation of N-trimethylsilylimines (9): N-Trimethylsilylaldimines used in these syntheses can readily be obtained by the reaction of lithium hexamethyldisilazide with aldehydes. Typical procedure is described for the preparation of N-trimethylsilylbenzaldimine @a): In 75 mL of anhydrous THP were added 17.29 mL (75 mmol) of hexamethyldisilazane and 30 mL (75 mmol) of n-butyllithium (2.5 M in hexane) at Ooc under nitrogen. After stirring for lh, 7.65 mL (75 mmol) of benzaldehyde was added at room temperature, and the mixture was refluxed for 3h. Then, 9.52 mL (75 mmol) of freshly distilled trimetbylsilyl chloride was added via a syringe. The mixture was refluxed for 2h. White precipitate came out during this process. The reaction mixture was then cooled to mom temperature and the liquid layer was transferred to a distillation flask under nitrogen via a syringe. The solvent was evaporated in vacua. and the oily residue was distilled under reduced pressure (68oc/lmm Hg) to give pure
`9a as a pale yellow oil (10.6 g, 80%): 1H NMR (CDC13) 6 0.18 (s, 9 H), 7.33-7.36 (m, 3H), 7.72- 7.75 (m, 2H), 8.89 (s, 1H); 13C NMR (CDC13) 6 -1.25, 128.34, 128.39, 131.96. 138.70, 168.32 In the same manner,
`9b and 9c were prepared from 4methoxybenzaldehyde and 3,4-dimethoxybenz- aldehyde, respectively, in 78-82% yields.
`9b: Pale yellow oil; bp 105WO.4 mmHg; 1H NMR (CDC13) 6 0.00 (s, 9H), 3.60 (s, 3H), 6.69 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 8.66 (s, 1H). 9c: Colorless oil; bp 140W0.2 mmHg; IH NMR 6 0.00 (s, 9H), 3.67 (s. 3H), 3.71 (s, 3H). 6.65 (d, J = 8.2 Hz, lH), 7.01 (dd, J = 8.2, 1.8 Hz, lH), 7.22 (d, J = 1.8 Hz. 1H). 8.63 (s, 1H).
`synthesis of 3-triisopropylsilyloxy-Carylazetidin-2-ones
`To a solution of diisopropylamine (223 mg, 2.20 mmol) in THP (2.0 mL) was added
`2.5 M solution of n-butyllithium (2.20 mmol) in THP (1.0 mL) at OK The solution was stirred for 30 min. at OOC and then cooled to 78oC. To the mixture was added a solution of (-)7e or (+)7e (781 mg,
`
`(2.0 mLJ. The solution was stirred for 2 h followed by addition of a solution of N-trimethylsilylal~ne 2.0 mmol) in TI-IP
`(9a-c) (2.0 mmol) in THP (2.0 mL). The mixture was stirred at -78OC for 4 h, and then slowly allowed to warm to room temperature, and further stirred overnight. The reaction was quenched with saturated aqueous solution of NH&l (50 mL), and the reaction mixture was extracte