WO2004014919A1 - Epothilone derivatives - Google Patents

Abstract

This invention relates Epothilone derivatives of formula (I), wherein the symbols and substituents are as defined in the description, to processes for the preparation thereof, to pharmaceutical compositions comprising such derivatives and to the use of such derivatives , for the preparation of pharmaceutical compositions for the treatment especially of a proliferative disease, such as a tumour.

Description

EPOTHILONE DERIVATIVES

Description

Technical Field:

The invention relates to antitumor agents. More particularly, the invention related to analogs of epothilone B, cis and trans-12,13-cyclopropyl epothilone B and cis and trans 12, 13-epoxide Epothilone B as antitumor agents.

Summary:

The invention is directed to analogs of epothilone B cis and trans-12,13- cyclopropyl epothilone B and cis and trans 12, 13-epoxide Epothilone B having potent cytotoxic active against a variety of cell lines, including Taxol^@-resistant tumor cells. Exemplary embodiments of the invention include compounds 3, 6, and 8-14, as illustrated in Figure 1 and compounds 3, 49, and 50-60, as illustrated in figure 11. Another aspect of the invention is directed to the use of such compounds as cytotoxic agents.

One aspect of the invention is directed to a compound represented by formula

wherein, wherein,

when a compound of formula I is a cis- isomer X is oxygen or CH₂ and when a compound of formula I is a trans- isomer X is CH₂;

when compound of formula I is a cis- isomer and X is oxygen then R1 is methyl and R is a radical selected from the group consisting of

₂Me

wherein R2 is a radical selected from the group consisting of -Me, -CI, -Br, -SMe and -CF₃;

when a compound of formula I is a cis isomer and X is CH₂then R1 is methyl and R is a radical selected from the group consisting of

wherein R2 is a radical selected from the group consisting of -Me, -CI, -Br, -SMe and -CF₃; when a compound of formula I is a trans isomer and X is CH₂ then R1 is methyl or Hydrogen;

wherein, when R1 is methyl R is a radical selected from the group consisting of

and, when R1 is hydrogen,

R is a radical selected from the group consisting of

or a salt of a compound of formula I where a salt-forming group is present.

Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like ("a" as an indefinite article or as a numeral meaning "one").

Salts are primarily the pharmaceutically acceptable salts of compounds of formula I.

Such salts are formed, for example, as acid addition salts, preferably with organic or inorganic acids, from compounds of formula I with a basic nitrogen atom, especially the pharmaceutically acceptable salts. Suitable inorganic acids are, for example, hydrohalic acids, such as hydrochloric acid, sulphuric acid, or phosphoric acid. Suitable organic acids are, for example, carboxylic, phosphonic, sulphonic or sulphamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, 2-hydroxybutyric acid, gluconic acid, glucosemonocarboxylic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, glucaric acid, galactaric acid, amino acids, such as glutamic acid, aspartic acid, N-methylglycine, acetylaminoacetic acid, N-acetylasparagine or N-acetylcysteine, pyruvic acid, aceto- acetic acid, phosphoserine, 2- or 3-glycerophosphoric acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, benzoic acid, salicylic acid, 1- or 3-hydroxynaphthyl-2-carboxylic acid, 3,4,5-trimethoxybenzoic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, glucuronic acid, galacturonic acid, methane- or ethane- sulphonic acid, 2-hydroxyethanesulfonic acid, ethane-1 ,2-disulphonic acid, benzene- sulphonic acid, 2-naphthalenesulphonic acid, 1,5-naphthalene-disulphonic acid, N- cyclohexylsulphamic acid, N-methyl-, N-ethyl- or N-propyl-sulphamic acid, or other organic protonic acids, such as ascorbic acid.

Salts of compounds of formula I with a salt-forming group may be prepared in a manner known per se. Acid addition salts of compounds of formula I may thus be obtained e.g. by treatment with an acid or with a suitable anion exchange reagent.

Salts can usually be converted to free compounds, e.g. by treating with suitable basic agents, for example with alkali metal carbonates, -hydrogencarbonates, or - hydroxides, typically potassium carbonate or sodium hydroxide.

For isolation or purification purposes it is also possible to use pharmaceutically unacceptable salts, for example picrates or perchlorates. Only the pharmaceutically acceptable salts or free compounds (if the occasion arises, in the form of pharmaceutical preparations) attain therapeutic use, and these are therefore preferred.

In view of the close relationship between the novel compounds in free form and in the form of their salts, including those salts that can be used as intermediates, for example in the purification or identification of the novel compounds, hereinbefore and hereinafter any reference to the free compounds is to be understood as referring also to the corresponding salts, as appropriate and expedient.

Another aspect of the invention is directed to a compound represented by formula II:

In Formula II, R is a radical selected from the group consisting of the following structures:

Preferred embodiments of this aspect of the invention include compounds represented by the following formulae:

Another aspect of the invention is directed to a compound represented by formula III:

In formula III, R is a radical selected from the group consisting of the following structures:

Preferred embodiments of this aspect of the invention are represented by the following formula:

Another aspect of the invention is directed to a compound represented by the following structure:

.Another aspect of the invention is directed to a compound represented by the following structure:

Another aspect of the invention is directed to a compound represented by formula IV:

wherein R is a radical selected from the group consisting of radicals represented by the following structures:

Preferred embodiments of this aspect of the invention include compound represented by the following structures:

Another aspect of the invention is directed to a compound represented by the formula V:

wherein, R is a radical selected from group consisting of -Me, -CI, -Br, -SMe, and -CF₃. Preferred embodiments of this aspect of the invention include compounds represented by the following structures:

Other aspects of the invention are directed to compounds represented by the following structures:

Another aspect of the invention is a process for synthesizing any of the compounds described above or intermediates thereof, as described in the specification, in particular a process for the preparation of a compound of formula I, II, III, IV or V wherein a compound of the formula VI

wherein X and R have any of the meanings as defined herein before and PG is a protecting group for a hydroxy function, in a first step is condensed by a esterification reaction, optionally in the presence of a catalyst, and in a second step the protecting group is detached thus furnishing a lacton of formula I.

The term "protecting groups for a hydroxy group" as used herein refers to acid labile protecting groups for a hydroxy group, which groups are known as such. It is a characteristic of protecting groups that they lend themselves readily, i.e. without undesired secondary reactions, to removal, typically by solvolysis, reduction, photolysis or also by enzyme activity, for example under conditions analogous to physiological conditions, and that they are not present in the end-products.The specialist knows, or can easily establish, which protecting groups are suitable with the reactions mentioned hereinabove and hereinafter.

The protection of hydroxy groups by protecting groups, the protecting groups themselves, and their cleavage reactions are described for example in standard reference works, such as J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973, in T. W. Greene, "Protective Groups in Organic Synthesis", Wiley, New York 1981, in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in "Methoden der organischen Chemie" (Methods of organic chemistry), Houben Weyl, 4th edition, Volume 15/1, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Proteine" (Amino acids, peptides, proteins), Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and in Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide und Derivate" (Chemistry of carbohydrates: monosaccharides and derivatives), Georg Thieme Verlag, Stuttgart 1974.

Preferred protecting groups are silyl ethers which are acid labile like terf-butyl- dimethyl-silyl (TBS) ether, triethylsilyl (TES) ether, triisopropylsilyl (TIPS) ether, diethylisopropylsilyl (DEIPS) ether, isopropyldimethylsilyl (IPDMS) ether or thexyldimethylsilyl (TDS) ether.

Brief Description of Drawings:

Figure 1 illustrates the structures of selected natural and designed epothilones. Grey boxes indicate compounds synthesized in this study.

Figure 2 illustrates a chart disclosing the displacement of the fluorescent taxoid Flutax-2 (50 nM) from microtubule binding sites (50 nM) by competing ligands at 37°C.

Figure 3 illustrates the synthesis of 2-(thiomethyl)thiazole epothilone B (3) via Stille coupling.

Figure 4 illustrates a retrosynthetic analysis of .rans-cyclopropyl epothilone B analogues (1 - 6, 8, 10, and 12-14).

Figure 5 illustrates the construction of aldehyde 32.

Figure 6 illustrates the construction of vinyl iodides 20c-g.

Figure 7 illustrates the synthesis of epothilone analogues 8-14. Figure 8 illustrates a table disclosing the cytotoxicity of epothilones 1 through 14 and paclitaxel against 1A9 human ovarian carcinoma cells and D-tubulin mutant cell lines selected with paclitaxel or epothilone A.

Figure 9 illustrates a table disclosing the tubulin polymerization potency and cytotoxicity of epothilones 1-8, 10-14, and paclitaxel against human epidermoid cancer cell lines.

Figure 10 illustrates a table disclosing binding affinities of epothilone analogues to the taxoid binding site of microtubules.

Figure 11 shows a series of structures of the various designed analogs of epothilones A and B along with the structures of epothilone A and B.

Figure 12 is a scheme showing the last step in the synthesis of many of the analogs from the vinyl iodide 15.

Figure 13 is a scheme showing the steps required to synthesize the stannanes used in the scheme in Figure 12.

Figure 14 is scheme illustrating the synthetic route taken to build the skeleton of the cyclopropyl analogs of epothilone B.

Figure 15 is a scheme showing the final steps used in the synthesis of cyclopropyl analogs 48 and 50.

Figure 16 is a table with the cytotoxicities of epothilones 48, 50 and 51-60 against human carcinoma cells and β-tubulin mutant cell lines selected with paclitaxel or epothilone A.

Figure 17 is a table with the cytotoxicities (IC50's in nM) of selected epothilones against the human epidermoid cell lines KB-3 and KB-8511.

Another aspect of the invention is directed to a pharmaceutical composition containing a therapeutic dose of a compound within either formula I, II, III, IV or formula V, represented above, for the treatment of a proliferative disease in a mammal. In a preferred mode, the mammal is a human.

Detailed Description

The construction of a series of epoxide and cyclopropane epothilones with varying side chains by chemical synthesis and biologically evaluated is disclosed. The design of the present focused epothilone library was based on the current knowledge of structure activity relationships (SAR), specifically the facts that: (1) epothilone B (2) is considerably more potent than epothilone A (1); (2) a thiomethyl replacement for the methyl group on the thiazole moiety enhances the potency (Nicolaou, K. O; et al. Angew. Chem. 1998, 110, 2120-2153; Angew. Chem. Int. Ed. 1998, 37, 2014-2045. Nicolaou, K. O; et al. Tetrahedron 2002, 58, 6413-6432; Nicolaou, K. O; et al. Angew. Chem. 1998, 110, 89-92; Angew. Chem. Int. Ed. 1998, 37, 84-87.); (3) a heterocycle such as pyridine (Nicolaou, K. O; et al. Chem. Biol. 2000, 7, 593-599.) replacement for the thiazole ring needs to maintain the proper position for the nitrogen for biological activity; and (4) a cyclopropane ring can replace the epoxide moiety without loss of activity (Nicolaou, K. C; et al. J. Am. Chem. Soc. 2001, 23, 9313-9323; Nicolaou, K. C; et al. ChemBioChem. 2001 , 2, 69-75; Johnson, J. A.; et al. Org. Lett. 2000, 2, 1537-1540.). From these considerations, epothilones 3, 6 and 8-14 (figure 1) and 48, 50 and 51-60 (Figure 11 ) were considered as prime candidates for chemical synthesis and biological evaluation.The biological evaluation of these compounds led to the identification of the thiomethylthiazole side chain as a desirable pharmacophoric group improving the biological activity of the epothilones with regard to cytotoxicity and tubulin polymerizing properties. The enhanced activity was confirmed by three distinct biological assays where the effects of the compounds tested were determined both in cells and in vitro.

Design and chemical synthesis of epothilone analogues: As an initial foray, we decided to confirm the potency enhancement bestowed on the epothilone scaffold by the methylthio group as compared to the methyl substituent in the epothilone B series. The methylthiothiazole epothilone B (3) was thus synthesized by Stille coupling of stannane 16 (Nicolaou, K. O; et al. Bioorg. Med. Chem. 1999, 7, 665-697) with vinyl iodide 15 (Nicolaou, K. C; et al. Chem. Eur. J. 2000, 6, 2783-2800) (80% yield) as shown in Figure 3. The observed high potency of analogue 3 against a series of tumor cell lines (see Table 1 ) encouraged us to proceed with the design and synthesis of an entire family of methylthio analogues as well as a number of new pyridine-containing epothilones.

The designed epothilone analogs (51-60) were synthesized in a convergent manner from vinyl iodide 15 (Nicolaou, K. O; et al. Chem. Eur. J. 2000, 6, 2783- 2800.) and the corresponding aromatic stannanes as shown in Figure 12 (for abbreviations of reagents and protective groups, see the detailed description of figures). Thus, a Stille-type coupling of 15 with appropriate stannanes (64a-d, 66a- d, 67 and 68) was carried out in the presence of PdCI₂(MeCN)₂, Cul and AsPh₃ in DMF at ambient temperature, leading directly to the desired epothilones (51-60) in the indicated yields. The required aromatic stannanes were prepared as summarized in Figure 13. Thus, for the thiazole compounds 64a-64d, the commercially available 2,4-dibromothiazole (62) was reacted with the corresponding thiol in the presence of NaH leading first to the intermediate sulfides (63a-63d) through replacement of the more reactive 2-bromide substituent. Subsequent coupling of these substrates with Me₃SnSnMe₃ in the presence of Pd(PPh₃) in toluene at 100 _C then gave the desired products 64a-64d via reaction of the second bromide residue. The pyridyl stannanes 66a-66d were similarly synthesized from the readily available 2-bromopyridines 65a, 65b (Virgilio, N. J. Org. Chem. 1973, 38, 2660-2664), 65c (Nicolaou, K. O; et al. Tetrahedron 2002, 58, 6413- 6432) and 65d (Testaferri, L.; et al. Tetrahedron 1985, 41, 1373-1384) via metal- halogen exchange (πBuLi) followed by quenching of the resulting 2-lithioderivatives (Gilman, H.; et al. J. Org. Chem. 1951, 16, 1788-1791) with r/Bu₃SnCI. Stannanes 67 (Dinnell, K.; et al. Bioorg. Med. Chem. Lett. 2001, 11, 1237-1240) and 68 (Jessie, S.; Kjell, U. Tetrahedron 1994, 50, 275-284) were prepared according to the corresponding literature procedures from the respective halides.

Figure 4 outlines, in retrosynthetic format, the pathway that was followed for the construction of the cyclopropyl epothilone B analogues. Based on our previously reported strategy, the adopted sequence required a Charette cyclopropanation reaction (Nicolaou, K. C; et al. J. Am. Chem. Soc. 2001 , 123, 9313-9323; Charette, A. B.; et al. J. Am. Chem. Soc. 1998, 720, 11943-11952) to establish early on in the synthesis the 12,13-cyclopropyl site, an aldol reaction according to our optimized procedure (Nicolaou, K. C; et al. Chem. Eur. J. 2000, 6, 2783-2800) to construct the C6-C7 bond with its two stereocenters, a Nozaki-Hiyama-Kishi coupling (Nicolaou, K. O; et al. J. Am. Chem. Soc. 2001, 723, 9313-9323; Takai, K.; et al. Tetrahedron Lett. 1983, 24, 5281-5284; Jin, H.; et al. J. Am. Chem. Soc. 1986, 708, 5644-5646) to introduce the side chain, and a Yamaguchi macrolactonizaion (Inanaga, J.; et al. Bull. Chem. Soc. Jpn. 1979, 52, 1989-1993; Mulzer, J.; et al. Synthesis 1992, 215- 228; Nicolaou, K. C; et al. J. Am. Chem. Soc. 1997, 779, 7974-7991) to complete the macrocyclic structure. Key building blocks 18 or 69 and 19, and 20 were thus defined as the starting points for these constructions. Construction of the corresponding epothilone A analogues was envisaged to be carried out in the same manner as previously reported by us (Nicolaou, K. O; et al. J. Am. Chem. Soc. 2001, 723, 9313-9323).

Figure 5 and figure 14 outline the synthesis of the required aldehyde 32 or 82 from the readily available geraniol (18 or 69). Thus, Charette cyclopropanation of 18 (Et₂Zn-CH₂l₂, in the presence of chiral ligand 21) (Charette, A. B.; et al. J. Am. Chem. Soc. 1998, 720, 11943-11952) furnished cyclopropyl alcohol 22 in 87% yield and 93% ee or 71 in 80% yield, 95%ee. Protection of the hydroxy group in 22 or 71 (NaH-BnBr) (for abbreviations of reagents and protecting groups, see legends in schemes) followed by ozonolysis (O₃; NaBH₄) of the remaining double bond led to compound 23 or 72 in 89% or 83% overall yield respectively. Conversion of alcohol 23 or 72 to the corresponding iodide (24, 95% yield or 73, 91 %) was accomplished upon mesylation and subsequent reaction with Nal. Alkylation of (-)- propionaldehyde SAMP hydrazone (25) (Nicolaou, K. C; et al. J. Am. Chem. Soc. 1997, 779, 7974-7991 ; Enders, D. Aymmetric Synth. 1984, 3, 275-339; Enders, D.; Klatt, M. Synthesis 1996, 1403-1418) with iodide 24 or 73 under the influence of LDA gave compound 26 or 75 (84% yield, 87% yield respectively), whose cleavage (Mel; HCIg_q) led to aldehyde 17 in 86% yield or 76 in 91% yield. The ratio of the resulting C-8 epimers was determined to be ca. 97:3 by ¹H NMR analysis of the MTPA esters derived from aldehyde 17 (Tsuda, M.; Endo, T.; Kobayashi, J. J. Org. Chem. 2000, 65, 1349-1352 and references cited therein). The aldol condensation between ketone 19 and aldehyde 17 or 76 under the previously defined conditions [LDA (2.4 equiv), ketone 19 (2.3 equiv), -78 to -40 °C, 30 min; then aldehyde 17 or 76, -78 °C, 5 min] (Nicolaou, K. O; et al. Chem. Eur. J. 2000, 6, 2783-2800) afforded aldol product 27 or 78 which was isolated in a diastereomerically pure form (81 % yield). Subsequent protection of the secondary alcohol in 27 or 78 as a TBS ether (TBSOTf, 2,6-lutidine) followed by selective cleavage of the primary TBS group (HF^»py) afforded, in 88% overall yield, alcohol 28 or 86% overall yield, alcohol 79. The compound was stepwise oxidized to the carboxylic acid (DMP; then NaClO₂) which was then protected as the TMSE ester 29 or 80(TMSE-OH, EDC, 4-DMAP) in 75% or 73% overall yield. Hydrogenolysis of the benzyl ether in 29 or 80 followed by oxidation with DMP led to aldehyde 30 or 82 (84% yield, 87% yield) whose homologation (NaHMDS-MeOCH₂PPh₃CI; then PPTS) to the coveted higher aldehyde 32 or 83 proceeded smoothly, and via vinyl ether 31 (ca. 1 :1 £:Z ratio), with 82% overall yield.

The side chains (20a-g, Scheme 4 and 64a-d, 66a-d, 84a and 84b, scheme 15) were synthesized either as previously reported (20a, 20b and 84a) (Nicolaou, K. C; et al. J. Am. Chem. Soc. 2001 , 723, 9313-9323), (84b) (Nicolaou, K. C; et al. J. Tetrahedron 2002, 58, 6413-6432J, or from the corresponding aryl halides (33 (Ellingboe, J. W.; et al. J. Med. Chem. 1994, 37, 542-550), 37, 38, 39) as shown in Scheme 4. Protection of 4-hydroxymethyl-2-pyridyl bromide 33 as a trityl ether (TrCI, 4-DMAP, 100%) followed by Sonogashira coupling (Arcadi, A.; et al. Tetrahedron 1994, 50, 437-452) of the resulting aryl bromide 34 with propyne [Pd(PPh₃)₂CI₂-Cul, 96%] led to acetylenic compound 35 which served as a precursor to vinyl iodide 20c (n-BuLi; then (n-Bu₃Sn)₂, CuCN, MeOH; then l₂, 80% yield). Exchange of the trityl for a MOM group within 35 [HCI(g), CHCI₃; then NaH, MOM-CI, 34% overall yield] (Betzer, J.-F.; et al. Tetrahedron Lett. 1997, 38, 2279-2282) allowed access to vinyl iodide 20d (67% yield) by exposure of the resulting intermediate 36 to the same conditions described above for the 35 to 20c conversion. Similar chemistry was employed to construct vinyl iodides 20e-20g from 37-39, respectively, as shown in Scheme 4.

Two crucial bond formations and two accompanying deprotections separated key building blocks 32 (prepared in this study for epothilone B analogues), 40 (prepared as previously described for epothilone A analogues) (Nicolaou, K. O; et al. J. Am. Chem. Soc. 2001, 723, 9313-9323), and 20a-g (for side chains) from the targeted epothilone analogues. The first operation was the Nozaki-Hiyama-Kishi coupling (Takai, K.; et al. Tetrahedron Lett. 1983, 24, 5281-5284; Jin, H.; et al. J. Am. Chem. Soc. 1986, 708, 5644-5646) of aldehydes 32 and 40 with vinyl iodides 20a-g. This carbon-carbon bond forming reaction worked admirably in this instance (CrCI₂, NiCI₂, 4-f-BuPy, DMSO), furnishing, after TBAF-induced carboxylic acid generation, coupling products (41a, 41b, 41d-g, 42c, 42e, 85 and 88) in yields indicated in Schemes 5 and 15 (as ca. 1:1 mixtures of C-15 diastereomers). Each mixture of hydroxy acid diastereomers (41a, 41b, 41d-g, 42c, 42e, 85 and 88) was then subjected to Yamaguchi macrocyclization (2,4,6-trichlorobenzoyl chloride, 4- DMAP) (Inanaga, J.; et al. Bull. Chem. Soc. Jpn. 1979, 52, 1989-1993; Mulzer, J.; et al. Synthesis 1992, 215-228) to afford the desired 15(S) lactone in the indicated (unoptimized) yields together with its 5(R) epimer. The separation of the two epimers at this juncture was facilitated by their rather drastically different R_f values on silica gel. Final deprotection of protected derivatives either with 20% TFA in CH₂CI₂ (43a, 43b, 43e-g, 44c, 44e, 86 and 89) or with TMSBr-4A MS in CH₂CI₂, followed by 20% TFA in CH₂CI₂ (43d), led to epothilones 6, 8-14 in the indicated (unoptimized) yields (Schemes 5 and 15). Chromatographically and spectroscopically pure compounds were subjected to biological evaluations as described below. Chemical biology:

The biological activities of the synthesized epothilones were evaluated through cytotoxicity, in vitro tubulin polymerization, and tubulin binding assays. Cytotoxicity was first evaluated in a set of ovarian carcinoma cell lines, including a parental cell tine (IA9) and three drug-resistant cell lines, namely the paclitaxel- resistant strains (Giannakakou, P.; et al. J. Biol. Chem. 1997, 272, 17118-17125) IA9/PTX10 and IA9/PTX22 and the epothilone-resistant strain (Giannakakou, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2904-2909) 1A9/A8. These resistant cell lines harbor distinct acquired β-tubulin mutations which affect drug-tubulin interaction and result in impaired taxane and epothilone-driven tubulin polymerization. The results of these biological investigations are summarized in Table 1 (Skehan, P.; et al. J. Natl. Cancer Inst. 1990, 82, 1107-1112). Further cytotoxicity and in vitro tubulin polymerization assays were carried out using a set of human epidermoid cancer cell lines, including a parent cell line (KB-31) and a paclitaxel-resistant (due to Pgp overexpression) cell line (KB-8511). The results of these studies are summarized in Table 2 (Nicolaou, K. O; et al. Chem. Biol. 2000, 7, 593-599; Meyer, T.; et al. Int. J. Cancer 1989, 43, 851-856).

In general, there is good agreement between the in vitro tubulin polymerization potency and the cytotoxicity profile of the tested compounds against both the 1A9 human ovarian carcinoma cells and the KB-31 human epidermoid carcinoma cells. In agreement with original observations with the naturally occurring epothilones A and B, none of the epothilone A or B analogues tested herein appears to be a good substrate for the drug-efflux pump P-glycoprotein (Pgp). This is evident by the lack of cross-resistance of each of these analogues to the Pgp expressing cell line KB-8511, in contrast to paclitaxel-a known Pgp substrate- which is 214-fold less active against KB-8511 cells (see figure 9 and figure 17). It is noteworthy that all the epothilone analogues appear more active against the β-tubulin mutants compared to epothilone A (1) and epothilone B (2) (see figure 8 and figure 16, RR values). This is more pronounced with compounds 10-14, 52, 54, 58 and 60 for which the relative resistance values (RR) range from 1.6-9.3 against PTX10 (β270) and A8 (β274) cells compared with 9.4-24.9 RR values for Epo A (1) and Epo B (2) (figure 8). Furthermore, in the current study, and in agreement with previous reports (Nicolaou, K. C; et al. ChemBioChem 2001, 2, 69-75; Giannakakou, P.; et al. J. Biol. Chem. 1997, 272, 17118-17125; Giannakakou, P.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2904-2909), we found that the paclitaxel-selected mutant PTX22 (β364) retains almost full sensitivity to the epothilones, and to all epothilone analogues tested in this report (RR values 33 3.3).

There is a general agreement in the relative potency of the substituted epothilone B analogs against the 1A9 human ovarian and the KB-31 human epidermoid cancer cells. Collectively, the results of these cytotoxicity assays revealed interesting information in terms of structure-activity relationships within the epothilone family. First, compounds 4 and 6 in which the C12-C13 epoxide moiety is replaced by a cyclopropane ring are the two most potent compounds among all the epothilone B analogs presented here. This result reaffirms that the C12-C13 epoxide moiety is not necessary for biological activity as previously noted (Nicolaou, K. O; et al. J. Am. Chem. Soc. 2001, 123, 9313-9323; Nicolaou, K. O; et al. ChemBioChem. 2001 , 2, 69-75; Johnson, J. A.; et al. Org. Lett. 2000, 2, 1537-1540.). Compound 4 is 6-fold more active than the parent epothilone B (2) against the 1A9 human ovarian carcinoma cells (Figure 6) further confirming that the replacement of the methyl group on the thiazole side-chain with a thiomethyl group leads to increased activity. This result is in agreement with previous data on a similar substitution in epothilone B without replacement of the C12-C13 epoxide (i.e. compound 3) (Nicolaou, K. O; et al. Tetrahedron 2002, 58, 6413-6432). The latter compound (3) was about 2-fold more active than the parent epothilone B, while compound 4 is 6- fold more potent than epothilone B. This result makes compound 4, the most active epothilone B analog against the 1 A9 cell line synthesized to date and suggests that replacement of the epoxide by a cyclopropane moiety together with the replacement of the methyl substituent on the thiazole moiety with a thiomethyl group act synergistically, leading to the observed enhancement of biological activity. Interestingly, substitution of the methyl group of the thiazole ring with larger moieties (compounds 7-10) (The IC50 value for compound 7 was found to be 2.5 nM against the 1A9 cell line.) led to diminished biological activity as compared to epothilone B (Figures 6 and 7).

Among the epothilone B analogs with substituted pyridine side-chains at the 5-position of the pyridine ring (compounds 55-57 and 59), the thiomethyl analog (compound 57) is the most potent followed by the bromo-substituted derivative (compound 55) followed by the chloro-substituted system (compound 56). When the thiomethyl group is relocated from the 5-position of the pyridine ring (compound 57) to the 6-position (compound 58) loss of activity occurs as the IC50 value drops from 0.4 nM (compound 57) to 3.3 nM (compound 58) (Figure 16). Furthermore, replacement of the thiomethyl group at the 5-position of the pyridine ring (compound 57) with a trifluoromethyl group (compound 59) results in loss of activity by 10-fold. Finally, the least active of the synthesized epothilone B analogs is compound 60 where a pyrimidine side-chain with a thiomethyl substitution has replaced the thiazole side-chain of the parent compound.

In addition to the above biological assays, the relative potency of each epothilone analogue was measured by the fluorescent taxoid displacement assay (Andreu, J. M.; Barasoain, I. Biochemistry 2001, 40, 11975-11984). The purpose of these experiments was to compare the equilibrium constants with which microtubules bind at their taxane site the epothilone analogues investigated. The inhibition of the binding of the well-characterized fluorescent taxoid Flutax-2 (Souto, A. A.; et al. Angew. Chem. Int. Ed. Engl. 1995, 34, 2710-2712; Diaz, J. F.; et al. J. Biol. Chem. 2000, 275, 26265-26276; Abal, M.; et al. Cell. Motil. Cytoskeleton 2001, 49, 1-15) to microtubules by each of the epothilone analogues was measured at 37 °C (Figure 2). The resulting equilibrium dissociation constants shown in Table 3 indicate that epothilone A (1) has the lowest binding affinity among the epothilone analogues tested (Kd = 34A4). The most powerful ligand among those measured in this assay is compound 3, with a Kd value of 0.64A0.24 nM, followed by compounds 8, 11-13, with similar Kd values comprised between 1.6 and 1.9 nM. With the possible exception of compound 13, the binding affinities of the analogues tested mirror their respective activities in both cell growth inhibition and in vitro tubulin polymerization assays.

Collectively from all three biological assays employed herein, a number of conclusions can be drawn in terms of structure-activity relationships within the epothilone family. First, the addition of the C12 methyl group does not enhance the activity in the trans- cyclopropyl series (compound 5 vs 6, 7 vs 8, 9 vs 10), contrary to the result in the cis epoxide series, where epothilone B (2) is at least 10-fold more active than epothilone A (1). This could be due to the different orientation of the C12 methyl group in the cis and trans compounds or to overall differences in conformation between the cis and trans compounds, although the details remain to be elucidated. Second, the introduction of the 2- thiomethylthiazole side chain enhances the activity compared with the natural 2- methylthiazole side chain (compounds 2 vs 3, 5 vs 11, and 6 vs 12). This effect was previously observed for epothilone C and D analogues (Nicolaou, K. O; et al. Angew. Chem. 1997, 709, 2181-2187; Angew. Chem. Int. Ed. Engl. 1997, 36, 2097-2103; see also: Sinha, S. C; et al. ChemBioChem 2001, 2, 656-665). Third, the replacement of a methyl group with a thiomethyl group in the pyridine side chain series (compounds 8 vs 13) reduces potency, contrary to the results obtained for the thiazole side chains above. This conclusion was based on the cell cytotoxicity and in vitro tubulin polymerization data, while in the fluorescent taxoid displacement assay the replacement of the methyl group with a thiomethyl moiety in the pyridine side chain is indifferent in terms of binding affinity. This discrepancy may simply reflect differences in cell uptake and permeability of the compounds tested or differences in the sensitivity of the two tubulin assays. Despite this discrepancy, it is clear from these data that the introduction of a thiomethyl group at the thiazole side chain is a more favorable modification than the introduction of a thiomethyl group at the pyridine side chain, which may be due to differing steric requirements by the two side chain scaffolds. In agreement with previous data obtained with cis pyridine epothilone analogues (Nicolaou, K. C; et al. Chem. Biol. 2000, 7, 593-599), relocation of the thiomethyl group of the pyridine side chain from the position 5 (compound 13) to position 6 (compound 14) resulted in significant loss of activity. Fourth, mixed results are obtained with compounds 7 vs 9 and 8 vs 10 in which the 5-methylpyridine side chain (compounds 7 and 8) is substituted by the 5- hydroxymethylpyridine side chain (compounds 9 and 10). This substitution appears indifferent in cytotoxicity assays against the 1A9 human ovarian carcinoma cells (Table 1) where very similar IC₅₀ values are obtained for each pair (e.g. 0.6 and 0.7 nM for compounds 7 and 9, respectively; 1.7 nM for compounds 8 and 10). On the other hand, in the human epidermoid carcinoma cells KB-31, compound 10 is 2-fold more active than its counterpart compound 8 with IC₅₀s at 0.44 vs 0.9 nM, respectively. Given the small differences in the growth rate of the two human cancer cell lines that could account for the differential results, we could conclude that the introduction of the 5-hydroxymethylpyridine side chain is not likely to enhance activity in, at least, frans-12,13-cyclopropyl analogues of the epothilone family.

Owing to these properties, the compounds are suitable for the treatment of proliferative diseases, especially tumour diseases, including metastases; for example solid tumours such as lung tumours, breast tumours, colorectal tumours, prostate tumours, melanomas, brain tumours, pancreas tumours, neck tumours, bladder tumours, neuroblastomas, throat tumours, but also proliferative diseases of blood cells, such as leukaemia; also for the treatment of other diseases which respond to treatment with microtubule depolymerisation inhibitors, such as psoriasis.

A compound of formula I can be administered alone or in combination with one or more other therapeutic agents, possible combination therapy taking the form of fixed combinations or the administration of a compound of the invention and one or more other therapeutic agents being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic agents. A compound of formula I can besides or in addition be administered for tumour therapy in combination with chemotherapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumour regression, or even chemopreventive therapy, for example in patients at risk.

Therapeutic agents for possible combination are especially one or more antiproliferative, cytostatic or cytotoxic compounds, for example one or more chemotherapeutic agent(s) selected from the group comprising the classical chemotherapeutic agents, an inhibitor of polyamine biosynthesis, an inhibitor of protein kinase, especially of serine/threonine protein kinase, such as protein kinase C, or of tyrosine protein kinase, such as epidermal growth factor receptor protein tyrosine kinase, a cytokine, a negative growth regulator, such as TGF-β or IFN-β, an aromatase inhibitor, and a classical cytostatic.

Compounds according to the invention are not only for the (prophylactic and preferably therapeutic) treatment of humans, but also for the treatment of other warm-blooded animals, for example of commercially useful animals, for example rodents, such as mice, rabbits or rats, or guinea-pigs. They may also be used as a reference standard in the test systems described above to permit a comparison with other compounds.

A compound of formula I may also be used for diagnostic purposes, for example with tumours that have been obtained from warm-blooded animal "hosts", especially humans, and implanted into mice to test them for decreases in growth after treatment with such a compound, in order to investigate their sensitivity to the said compound and thus to improve the detection and determination of possible therapeutic methods for neoplastic diseases in the original host.

Stereoisomeric mixtures, e.g. mixtures of diastereoisomers, can be separated into their corresponding isomers in a manner known per se by means of suitable separation methods. Diastereoisomeric mixtures may thus be separated into their individual diastereoisomers by means of fractionated crystallization, chromatography, solvent distribution, and similar procedures. This separation may take place either at the stage of one of the starting compounds or in a compound of formula I itself. Enantiomers may be separated through the formation of diastereoisomeric salts, for example by salt formation with an enantiomer-pure chiral acid, or by means of chromatography, for example by HPLC, using chromatographic substrates with chiral ligands. (Enantiomer separation is normally effected at the intermediate stage).

Pharmaceutical preparations, methods, and uses

The present invention relates also to pharmaceutical preparations that contain a compound of formula I as active ingredient and that can be used especially in the treatment of the diseases mentioned above. Preparations for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for parenteral administration, such as intravenous, intramuscular or subcutaneous administration, to warm-blooded animals, especially humans, are especially preferred. The preparations contain the active ingredient alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration.

The precise dosage of the compounds of formula I to be employed for inhibiting proliferative disease, preferably tumors, depends upon several factors including the host, the nature and the severity of the condition being treated, the mode of administration and the particular compound employed. However, in general, satisfactory inhibition of tumors is achieved when a compound of formula I is administered parenterally, e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally, or enterally, e.g., orally, preferably intravenously or orally, more preferably intravenously at a single dosage of 1-300 mg/kg body weight per cycle (cycle = 3-6 weeks) or, for most larger primates, a single dosage of 50-5000 mg per treatment cycle. A preferred intravenous single dosage per 3-6 week treatment cycle is 1-75 mg/kg body weight or, for most larger primates, a daily dosage of 50-1500 mg. A typical intravenous dosage is 45 mg/kg, once every three weeks.

Usually, a small dose is administered initially and the dosage is gradually increased until the optimal dosage for the host under treatment is determined. The upper limit of dosage is that imposed by side effects and can be determined by trial for the host being treated.

The invention relates also to pharmaceutical preparations for use in a method for the prophylactic or especially therapeutic treatment of the human or animal body, to a process for the preparation thereof (especially in the form of compositions for the treatment of tumours) and to a method of treating the above-mentioned diseases, primarily neoplastic diseases, especially those mentioned above. The invention relates also to processes and to the use of compounds of formula I for the preparation of pharmaceutical preparations which contain compounds of formula I as active component (active ingredient).

Preference is given to a pharmaceutical composition that is suitable for administration to a warm-blooded animal, especially a human or commercially useful mammal, suffering from a disease that is responsive to the inhibition of microtubule depolymerisation, for example psoriasis or especially a neoplastic disease, comprising a correspondingly effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof when salt-forming groups are present, together with at least one pharmaceutically acceptable carrier.

A pharmaceutical composition for the prophylactic or especially therapeutic treatment of neoplastic and other proliferative diseases of a warm-blooded animal, especially a human or a commercially useful mammal requiring such treatment, especially suffering from such a disease, comprising a new compound of formula I, or a pharmaceutically acceptable salt thereof, as active ingredient in a quantity that is prophylactically or especially therapeutically active against said diseases, is likewise preferred.

Pharmaceutical preparations contain from about 0.000001 % to 95 % of the active ingredient, whereby single-dose forms of administration preferably have from approximately 0.00001 % to 90 % and multiple-dose forms of administration preferably have from approximately 0.0001 to 0.5 % in the case of preparations for parenteral administration or 1 % to 20 % active ingredient in the case of preparations for enteral administration. Unit dose forms are, for example, coated and uncoated tablets, ampoules, vials, suppositories or capsules. Further dosage forms are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, dispersions, etc. Examples are capsules containing from about 0.0002 g to about 1.0 g active ingredient.

The pharmaceutical preparations of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilising processes. The formulations suitable for parenteral administration are primarily aqueous solutions ([or example in physiological saline, obtainable by diluting solutions in polyethylene glycol, such as polyethylene glycol (PEG) 300 or PEG 400] of an active ingredient in water-soluble form, e.g. a water-soluble salt, or aqueous injectable suspensions containing viscosity-increasing agents, e.g. sodium carboxymethyl cellulose, sorbitol and/or dextran, and where appropriate stabilisers. The active ingredient, if need be together with excipients, can also be in the form of a lyophilisate and can be made into a solution before parenteral administration by the addition of suitable solvents.

Solutions such as those used, for example, for parenteral administration can also be employed as infusion solutions.

The invention similarly relates to a process or a method for the treatment of one of the above-mentioned pathological conditions, especially a disease which responds to an inhibition of microtubule depolymerisation, especially a corresponding neoplastic disease. A compound of formula I can be administered as such or in the form of pharmaceutical compositions, prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment, the compounds especially being used in the form of pharmaceutical compositions. In the case of an individual having a bodyweight of about 70 kg the dose administered is from approximately 0.1 mg to approximately 1 g, preferably from approximately 0.5 mg to approximately 200 mg, of a compound of the present invention. Administration is preferably effected e.g. every 1 to 4 weeks, for example weekly, every two weeks, every three weeks or every 4 weeks.

The present invention also relates in particular to the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, especially a compound of formula I named as a preferred compound, or a pharmaceutically acceptable salt thereof, as such or in the form of a pharmaceutical formulation containing at least one pharmaceutically employable carrier, for the therapeutical and also prophylactic treatment of one or more of the above diseases. The present invention also relates in particular to the use of a compound of formula I, or a pharmaceutically acceptable salt thereof, especially a compound of formula I named as a preferred compound, or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical formulation for the therapeutical and also prophylactic treatment of one or more of the above diseases.

Experimental

General

All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Anhydrous solvents were obtained by passing them through commercially available activated alumina columns. All reagents were purchased at highest commercial quality and used without further purification. Reactions were generally monitored by thin-layer chromatography carried out on 0.25 mm E. Merck silica gel plates (60F-254). E. Merck silica gel (60, particle size 0.040-0.063 mm) was used for flash column chromatography. Preparative thin-layer chromatography (PTLC) separations were carried out on 0.25, 0.50 or 1 mm E. Merck silica gel plates (60F-254). Melting points (mp) are uncorrected and were recorded on a Thomas-Hoover Unimelt capillary melting point apparatus. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter. NMR spectra were recorded on Bruker DRX-600, DRX-500, AMX-400 or AC-250 instruments and calibrated using residual undeuterated solvents as an internal reference. All labeling of carbon atoms, e.g. C15, refers to epothilone A (1) numbering (see Figure 1). IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR spectrometer. High resolution mass spectra were recorded on a PerSeptive Biosystems Voyager™ lonSpec mass spectrometer (MALDI-FTMS) or on an API 100 Perkin-Elmer mass spectrometer (ESI).

Synthesis of epothilone 3

Stille coupling of vinyl iodide 15 with stannane 16. A solution of Pd₂(dba)₃(CHCI₃ (3.9 mg, 3.8 μmol), AsPh₃ (4.6 mg, 15 μmol), and Cul (7.2 mg, 38 μmol) in DMF (degassed, 0.5 mL) was added at 25 °C to a solution of iodide 15 (10 mg, 19 μmol) (Nicolaou, K. C, et al., Chem. Eur. J. 2000, 6, 2783-2800) and stannane 16 (11 mg, 38 μmol) (Nicolaou, K. C, et al., Bioorg. Med. Chem. 1999, 7, 665-697) in DMF (degassed, 0.5 mL), and the resulting solution was stirred for 2 hours. Water (10 mL) was added, and the mixture was extracted with EtOAc (3 ( 10 mL). The combined organic phase was washed with water (30 mL), brine (30 mL), and dried (Na₂SO ). After evaporation of the volatiles, the residue was purified by flash column chromatography (sillica, hexanes:EtOAc 2:1 ( 1 :1) to yield epothilone 3 as a white solid (7.2 mg, 72%); TLC R_f = 0.29 (silica, hexanes:EtOAc 1:1); [α]_D22 -53 (c 0.51, CH₂CI₂); IR (film) v_max 3472 (br), 2967, 2920, 1731, 1684, 1461, 1420, 1378, 1249, 1143, 1032, 973, 879, 732, 667 cm^-1; MALDI- FTMS m/z 562.2267 (MNa⁺), calcd for C₂₇H₄₁NO₆S₂Na 562.2267.

Construction of aldehyde 32

Alcohol 23. To a solution of cyclopropyl alcohol 22 (4.08 g, 24 mmol) (Charette, A. B.; et al. J. Am. Chem. Soc. 1998, 720, 11943-11952) in DMF (40 mL) was added sodium hydride (1.45 g, 36 mmol, 60% in mineral oil) portionwise with stirring at 0 °C. After stirring for 0.5 h at 25 °C, the mixture was cooled to 0 °C, benzyl bromide (4.3 mL, 36 mmol) was added over 2 min, and stirring was continued for 12 h at 25 °C The reaction was quenched with NH CI (sat., 50 mL), the mixture was extracted with EtOAc (3 ( 50 mL) and the combined extract was washed with brine (2 ( 100 mL), dried (Na₂SO ) and evaporated. The residue was dissolved in CH₂CI₂:MeOH 4:1 (60 mL), and the solution was ozonized (100 L/h, ca. 5 g O₃/h) at -78 °C for 21 min. (NOTE: Longer reaction times must be avoided to prevent oxidation of the benzyl ether to the corresponding benzoate.) Excess ozone was removed by flushing with N₂ for 1 min, and then NaBH₄ (2.75 g, 73 mmol) was added in small portions (CAUTION! Exothermic!) followed by methanol (20 mL). The mixture was warmed to 25 °C over 1 hour, and the reaction was quenched by the addition of NH₄CI (sat., 20 mL). The mixture was extracted with CH₂CI₂ (2 ( 50 mL), and the combined extract was washed with brine (100 mL), dried (Na₂SO ) and evaporated. The residue was purified by flash chromatography (silica, hexanes:EtOAc 5:2) to yield 23 as a yellow oil (5.07 g, 89%). TLC R_f = 0.20 (silica, hexanes:EtOAc 3:1); [α]_D22 -7.5 (c 1.76, CHCI₃); IR (film) v_max 3390 (br), 2933, 2859, 1452, 1070, 739, 698 cm^-1; MALDI- FTMS m/z 257.1519 (MNa⁺), calcd for C₁₅H₂₂O₂Na 257.1512.

Iodide 24. To a solution of cyclopropyl alcohol 23 (10.08 g, 43.0 mmol) in dry CH₂CI₂ (100 mL) at 0 °C was added methanesulfonyl chloride (4.2 mL, 54 mmol) followed by triethylamine (9.0 mL, 65 mmol) dropwise. A white precipitate started to form immediately. The mixture was stirred at 25 °C for 1 hour, then NH₄CI (sat., 50 mL) and water (50 mL) were added and the phases were separated. The aqueous phase was extracted with EtOAc (100 mL), and the combined organic phase was washed with brine, dried (Na₂SO₄) and evaporated. The residue was dissolved in dry acetone (200 mL), and sodium iodide (19.3 g, 129 mmol) was added. The initially almost clear solution was refluxed for 40 min, during which time a white precipitate formed. Water (100 mL) was added and the mixture was extracted with ether (500 + 250 mL). The combined extract was dried and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 5:1) to yield 24 as a colorless oil (14.16 g, 95%). TLC R_f = 0.66 (silica, hexanes:EtOAc 5:1); [α]_D22 -16 (c 2.05, CHCI₃); IR (film) I 2916, 2848, 1453, 1217, 1098, 1073, 735, 697 cm^"1; ESI-MS m/z 367 (MNa⁺), calcd for Cι₅H₂ιIONa 367.

Hydrazone 26. A solution of LDA was prepared by adding n-BuLi (13.1 mL, 21.0 mmol, 1.6 M in hexanes) to diisopropylamine (2.94 mL, 21.0 mmol) in THF (10 mL) at -78 ^CC, then warming the solution to 0 °C, and stirring for 10 min. To this LDA solution was added propionaldehyde SAMP hydrazone 25 (3.32 g, 19.5 mmol) (Nicolaou, K. O, et a!., J. Am. Chem. Soc. 1997, 779, 7974-7991 ; Enders, D. Aymmetric Synth. 1984, 3, 275-339; and Enders, D., et al., Synthesis 1996, 1403- 1418), and the mixture was stirred for 6 h at 0 °C, during which time a white precipitate formed. The mixture was cooled to -98 °C (MeOH/N₂(l) bath) and a solution of iodide 24 (5.16 g, 15.0 mmol) in THF (20 mL) was added over 0.5 hour. The reaction mixture was then allowed to warm to -10 °C over 14 hours, and then the reaction was quenched with NH₄CI (sat., 10 mL). The mixture was extracted with EtOAc (100 mL + 2 ( 50 mL), the combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 6:1 ( 4:1) to yield hydrazone 26 as a yellow oil (4.88 g, 84%). TLC R_f = 0.38 (silica, hexanes:EtOAc 5:1); [α]_D22 -61 (c 1.45, CHCI₃); IR (film) v_max 2926, 1454, 1097, 736, 697 cm^-1; MALDI-FTMS m/z 387.3008 (MH⁺), calcd for C₂₄H₃₉N₂O₂ 387.3006.

Aldehyde 17. A solution of hydrazone 26 (3.82 g, 9.9 mmol) in iodomethane ( 10 mL) was heated at 60 °C (reflux condenser) for 3 hours, and was then cooled to 25 °C. Excess iodomethane was evaporated and traces removed under oil pump vacuum. The residual yellow syrup was vigorously stirred with 3 N HCl (190 mL) and pentane (190 mL) for 3 h at 25 °C, the phases were separated, and the aqueous phase was extracted with pentane (100 mL). The combined organic phase was dried (Na₂SO₄, NaHCO₃) and evaporated to yield aldehyde 17 as a yellow oil (2.38 g, 88%). [α]_D22 +2 (c 1.3, CHCI₃); IR (film) v_max 2931 , 2856, 1724, 1454, 1095, 1074, 736, 698 cm^-1; MALDI-FTMS m/z 297.1830 (MNa⁺), calcd for Cι₈H₂₆O₂Na 297.1825.

Due to the configurational lability at C8 (epothilone numbering), the aldehyde should be used immediately in the next step. The dr at C8 was estimated as follows: A sample of 17 was treated with excess NaBH in methanol for 10 min. The reaction was quenched with NH₄CI (sat.), the mixture was extracted with EtOAc, and the extract was dried (Na₂SO₄) and evaporated. The residue was treated with (R)-(-)- MTPACI (2-3 equiv.), excess triethylamine and 4-DMAP in CH₂CI₂ for 3 hours. Purification by preparative TLC yielded a sample of the (S)-MTPA ester, which by ¹H NMR analysis showed a dr = 97:3, with the correct absolute stereochemistry at C8 as the major isomer (Tsuda, M., et al., J. Org. Chem. 2000, 65, 1349-1352). Analogous results were obtained by using (S)-(+)-MTPACI.

Aldol product 27. A solution of LDA was prepared by adding ti-BuLi (7.5 mL, 12 mmol, 1.6 M in hexanes) to diisopropylamine (1.68 mL, 12 mmol) in THF (12 mL) at -78 °C, then warming the solution briefly to 0 °C, and finally cooling back to -78 °C. A solution of ketone 19 (4.63 g, 11.5 mmol) (Nicolaou, K. O, et al., J. Am. Chem. Soc. 1997, 779, 7974-7991) in THF (12 mL) was added dropwise over 2 min, and the mixture was stirred for 1 h at -78 °C and then for 0.5 h at -40 °C. It was again cooled to -78 °C, and a solution of aldehyde 17 (1.37 g, 5.0 mmol) in THF (25 mL), pre-cooled to -78 °C, was added via cannula over 1 min, taking care to ensure minimal warming during transfer. The mixture was stirred for 5 min, and the reaction was then quenched by rapid injection of a solution of AcOH (1.4 mL) in THF (4.2 mL). After 5 min at -78 °C, the mixture was warmed to 25 °C and partitioned between NH₄CI (sat, 50 mL) and ether (50 mL). The aqueous phase was extracted with ether (2 ( 50 mL), the combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:ether 20:1 ( 6:1) to yield recovered ketone 19 (1.71 g, 4.25 mmol) followed by the aldol product 27 in diastereomerically pure form (2.73 g, 81 %). TLC R_f = 0.34 (silica, hexanes:EtOAc 5:1 ); [α]_D22 -40 (c 1.0, CHCI₃); IR (film) v_max 3502 (br), 2954, 2928, 2856, 1681 , 1472, 1255, 1098, 836, 776 cm^-1; MALDI-FTMS m/z 699.4796 (MNa⁺), calcd for C₃₉H₇₂O₅Si₂Na 699.4816.

Alcohol 28. A solution of. aldol product 27 (2.71 g, 4.0 mmol) and 2,6-lutidine (1.40 mL, 12 mmol) in CH₂CI₂ (25 mL) was cooled to -20 °C and then TBSOTf (1.84 mL, 8.0 mmol) was added dropwise. The mixture was stirred for 1 h at -20 °C and the reaction was then quenched by the addition of NH₄CI (sat, 25 mL). The mixture was warmed to 25 °C, the phases were separated and the aqueous phase was extracted with CH₂CI₂ (25 mL) and ether (25 mL). The combined organic phase was dried (Na₂SO ) and evaporated, and the residue was filtered through a plug of silica eluting with hexane:ether 10:1. The filtrate was evaporated and the resulting crude silyl ether (3.14 g, 4.0 mmol, 99%) was dissolved in THF (40 mL). To this was added a cold (0 °C) solution of HF-pyridine complex (6.4 mL) and pyridine (18 mL) in THF (32 mL) at 0 °C (this solution was prepared by slowly adding the HF-pyridine complex to a solution of pyridine in THF at 0 °C; CAUTION! HF-pyridine is highly corrosive. The addition of HF-pyridine to the pyridine-THF solution is highly exothermic, and must be done with stirring and cooling in ice bath to prevent splashing), and the resulting solution was stirred at 25 °C for 4 hours. The mixture was diluted with EtOAc (100 mL), placed in an ice bath, and quenched by the careful addition of NaHCO₃ (sat, 100 mL) and as much solid NaHCO₃ as needed to ensure complete neutralization (CAUTION! Foaming!). The mixture was extracted with EtOAc (3 ( 100 mL), and the combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 5:1) to yield 28 as a colorless oil (2.40 g, 89%). TLC R_f = 0.39 (silica, hexanesiEtOAc 5:1); [α]_D22 -26 (c 1.1 , CHCI₃); IR (film) v_mex 3458 (br), 2929, 2856, 1693, 1472, 1462, 1255, 1093, 986, 836, 775 cm^-1; MALDI-FTMS m/z 699.4807 (MNa⁺), calcd for C39H₇₂O₅Si₂Na 699.4816.

Ester 29. The alcohol 28 (2.40 g, 3.5 mmol), Dess-Martin periodinane (3.75 g, 8.8 mmol), NaHCO₃ (0.74 g, 8.8 mmol) and water (76 μL, 4.2 mmol) were mixed in CH₂CI₂ (80 mL), and the resulting suspension was stirred for 1 hour. The mixture was diluted with ether (200 mL), water (100 mL) and NaHCO₃ (sat., 100 mL), and was then filtered. The phases were separated and the aqueous phase was extracted with ether (2 ( 100 mL). The combined extract was dried (Na₂SO₄) and evaporated, and the residue was filtered through a plug of silica eluting with hexanes:EtOAc 6:1. The filtrate was evaporated and the resulting crude aldehyde (2.15 g, 3.2 mmol, 90%) was dissolved in a mixture of THF (80 mL), .-BuOH (145 mL) and 2-methyl-2- butene (25 mL). To this solution was added a solution of NaH₂PO₄ (0.95 g, 6.7 mmol) and NaCIO₂ (1.14 g, 10 mmol) in water (31 mL), and the resulting mixture was stirred vigorously for 1 hour. The volatiles were removed by evaporation, and the residue was partitioned between EtOAc (100 mL) and brine (100 mL). The phases were separated and the aqueous phase was extracted with EtOAc (3 ( 100 mL). The combined extract was dried (Na₂SO₄) and evaporated, and the residue was dissolved in DMF (5 mL) and evaporated again to remove traces of .-BuOH. The so obtained crude acid (2.4 g, ca. 3.2 mmol >100%,) was again dissolved in DMF (10 mL), to which 2-(trimethylsilyl)ethanol (1.83 mL, 12.7 mmol), EDC (0.92 g, 4.8 mmol), and 4-DMAP (40 mg, 0.33 mmol) were added. The resulting suspension was stirred for 14 hours, after which time a clear solution was obtained. Water (10 mL) was added and the mixture was extracted with ether (3 ( 50 mL). The combined extract was washed with water-brine mixture (100 + 100 mL), dried (Na₂SO ) and evaporated. The residue was purified by flash chromatography (silica, hexanes:EtOAc 10:1) to yield ester 29 as a viscous, pale yellow oil (2.08 g, 74%). TLC R_f = 0.57 (silica, hexanes:EtOAc 10:1); [α]_D22 -33 (c 1.2, CHCI₃); IR (film) v_max 2954, 2930, 2856, 1735, 1695, 1472, 1385, 1252, 1090, 988, 836, 776 cm^"1; MALDI- FTMS m/z 813.5315 (MNa⁺), calcd for C^HsaOeSisNa 813.5311.

Aldehyde 30. To a solution of benzyl ether 29 (2.08 g, 2.63 mmol) in EtOH:EtOAc 1 :1 (50 mL) was added 20% Pd(OH)₂ on carbon (2.1 g, 60% moisture), and the mixture was hydrogenated for 1 hour. It was then filtered through celite to remove the catalyst, the filtrate was evaporated, and the residue was co-evaporated with benzene to remove traces of EtOH. The resulting crude alcohol (1.89 g, ca. 2.6 mmol, >100%) was dissolved in CH₂CI₂ (60 mL), Dess-Martin periodinane (2.76 g, 6.5 mmol), NaHCO₃ (0.55 g, 6.5 mmol) and water (56 μL, 3.1 mmol) were added, and the resulting suspension was stirred for 1 hour. The mixture was diluted with ether (150 mL), water (75 mL) and NaHCO₃ (sat, 75 mL), and was then filtered. The phases were separated and the aqueous phase was extracted with ether (2 ( 75 mL). The combined extract was dried (Na₂SO ) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 15:1) to yield aldehyde 30 as a viscous oil (1.55 g, 84%). TLC R_f = 0.24 (silica, hexanes:EtOAc 15:1); [α]_D22 - 47 (c 1.3, CHCI₃); IR (film)

2954, 2856, 1734, 1703, 1251 , 1173, 1084, 988, 837, 776 cm^-1; MALDI-FTMS m/z 721.4671 (MNa⁺), calcd for C₃₇H₇₄O₆Si₃Na 721.4685.

Enol ether 31. To a suspension of MeOCH₂PPh₃CI (3.09 g, 9.0 mmol) in THF (20 mL) at 0 °C was added NaHMDS (8.5 mL, 8.5 mmol, 1 M in THF) dropwise. A red color developed. The mixture was stirred at 0 °C for 0.5 h and it was then cooled to - 40 °C. A solution of aldehyde 30 (2.12 g, 3.0 mmol) in THF (7 mL) was added, and the mixture was allowed to warm to -10 °C over 2 hours. The reaction was quenched with NH CI (sat., 15 mL), the phases were separated, and the aqueous phase was extracted with EtOAc (2 ( 75 mL). The combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 30:1) to yield enol ether 31 as a colorless, viscous oil (1.85 g, 84%, olefin cisdrans ca. 1 :1 by ¹H NMR). TLC R_f = 0.23 (silica, hexanes:EtOAc 30:1); [α]_D22 -36 (c 1.2, CHCI₃); IR (film) v_max 2954, 2930, 2856, 1735, 1695, 1251 , 1171, 1105, 988, 836, 776 cm^-1; MALDI-FTMS m/z 749.4996 (MNa⁺), calcd for C₃₉H₇₈θ₆Si₃Na 749.4998. Aldehyde 32. To a solution of enol ether 31 (847 mg, 1.16 mmol) in dioxane.water 9:1 (12 mL) was added pyridinium para-toluenesulfonate (2.34 g, 9.31 mmol) and the mixture was stirred at 70 °C until TLC indicated the completion of the reaction (6-10 h). The reaction was then quenched with NaHCO₃ (sat., 15 mL), and the mixture was extracted with EtOAc (3 ( 50 mL). The combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 15:1) to yield 32 as a colorless, viscous oil (681 mg, 82%). TLC R_f = 0.29 (silica, hexanes.EtOAc 15:1); [α]_D22 -34 (c 1.0, CHCI₃); IR (film) v_max 2954, 2856, 1731, 1695, 1251, 1086, 988, 836, 776 cm^"1; MALDI-FTMS m/z 735.4823 (MNa⁺), calcd for C₃₈H₇₆θ₆Si₃Na 735.4842.

Construction of vinyl iodides 20c-g

2-Bromo-5-[(trityloxy)methyl]pyridine 34. Trityl chloride (3.90 g, 14 mmol), 4- DMAP (2.08 g, 17 mmol) and 2-bromo-5-hydroxymethylpyridine 33 (1.88 g, 10 mmol) (Ellingboe, J. W., et al., J. Med. Chem. 1994, 37, 542-550) were dissolved in DMF (15 mL) and the solution was stirred at 80 °C for 48 hours. A white precipitate formed during this time. After cooling, the mixture was diluted with NaHCO3 (sat., 25 mL) and extracted with EtOAc (3 ( 50 mL). The combined extract was washed with brine, with a few drops of NaOH (1 M) added (2 ( 100 mL). After drying and evaporation, the solid residue was purified by flash chromatography (silica, hexanes:EtOAc 15:1) to yield 34 as a white solid (4.46 g, 100%). TLC R_f = 0.30 (silica, hexanes:EtOAc 15:1); IR (film) v_max 3057, 1448, 1086, 764, 700, 632 cm^"1; MALDI-FTMS m/z 430.0792 (MH⁺), calcd for C₂₅H₂₁BrNO 430.0801.

Sonogashira coupling of aryl bromdes (34, 37, 38, and 39) with propyne (general procedure). To a briefly deoxygenated (Ar bubbling) solution of the aryl bromide 34, 37, 38, or 39 (3.5 mmol) in DMF (3 mL) and diisopropyl amine (2.5 mL) were added Pd(PPh₃)₂CI₂ (25 mg, 36 μmol) and Cul (13 mg, 70 μmol) under Ar(g), and then the inert atmosphere was replaced by propyne (1 atm, balloon). The mixture was stirred at 25 °C for 3 hours. During this time, a precipitate formed, and the reaction mixture turned dark brown. Water (15 mL) was added, the mixture was extracted with EtOAc, and the combined extract was dried (Na₂SO₄) and evaporated. The pure 1-arylpropyne was obtained by flash chromatography (silica, hexane: EtOAc mixtures).

Propynylpyridine 35. Brown foam (96%); TLC R_f = 0.23 (silica, hexanes:EtOAc 5:1); IR (film) v_max 3057, 2229, 1594, 1560, 1478, 1448, 1075, 702 cm^"1; MALDI-FTMS m/z 390.1851 (MH⁺), calcd for C₂₈H₂₄NO 390.1852.

Pyridine 36. A solution of trityl ether 35 (1.38 g, 3.54 mmol) in CHCI₃ (15 mL) was cooled to 0 °C and then saturated with HCl (g). After 1 h at 0 °C, the reaction was quenched by the addition of NaHCO₃ (sat., 50 mL), and the phases were separated. The aqueous phase was extracted with CH₂CI₂ (50 mL) and the combined organic phase was dried (Na₂SO₄) and evaporated. Flash chromatography (silica, hexanes:EtOAc 1:2 + 5% MeOH) afforded 5-hydroxymethyl-2-prop-1-ynylpyridine as a yellow, viscous oil (0.36 g, 69%). TLC R_f = 0.29 (silica, hexanes:EtOAc 1:2 + 5% MeOH); IR (film) v_max 3262, 2916, 2230, 1596, 1561, 1023, 838 cm^"1; MALDI-FTMS m/z 148.0754 (MH⁺), calcd for C₉H₁₀NO 148.0757. To a solution of this alcohol (0.40 g, 2.7 mmol) in THF (10 mL) at 0 °C was added NaH (0.13 g, 3.3 mmol, 60% in oil). After stirring for 5 min, chloromethyl methyl ether (0.25 mL, 3.3 mmol) was added, and the mixture was stirred at 0 °C for 1 hour. The reaction was then quenched with NaCI (sat.), and a few drops of NaOH (1 M) were added. The mixture was extracted with EtOAc (3 ( 50 mL), the combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc 1:1) to yield 36 as a pale yellow oil (0.26 g, 50%). TLC R_f = 0.41 (silica, hexanes:EtOAc 1 :1); IR (film) v_max 2947, 2230, 1595, 1560, 1478, 1149, 1104, 1047, 919, 830 cm^"1; MALDI-FTMS m/z 192.1014 (MH⁺), calcd for CnH₁₄NO₂ 192.1019.

Sonogashira coupling product from 37. The reaction was very slow, probably due to Pd coordination to the thioether moiety; therefore, 10 mol% Pd(PPh₃)₂CI₂ and 20 mol% Cul were used. The product was obtained as a brown oil (42%). TLC R_f = 0.37 (silica, hexanes:EtOAc 15:1); IR (film) \ 3110, 2914, 2240, 1493, 1417, 1278, 1037, 966, 735 cm^"1; MALDI-FTMS m/z 170.0092 (MH⁺), calcd for C₇H₈NS₂ 170.0093. Sonogashira coupling product from 38. Brown oil (97%); TLC R_f = 0.21 (silica, hexanes:EtOAc 5:1); IR (film) v_max 2908, 2226, 1567, 1531, 1461, 1431, 1361, 1108, 1008, 832 cm^"1; MALDI-FTMS m/z 164.0527 (MH⁺), calcd for C₉H₁₀NS 164.0528.

Sonogashira coupling product from 39. Yellow oil (70%); TLC R_f = 0.36 (silica, hexanes:EtOAc 20:1); IR (film) v_max 2924, 2231 , 1566, 1554, 1431, 1156, 1140, 790 cm^"1; MALDI-FTMS m/z 164.0526 (MH⁺), calcd for C₉H₁₀NS 164.0528.

Hydrostannylation-iodination (general procedure). This is an adaption of the previously reported procedure (Betzer, J.-F., et al., Tetrahedron Lett. 1997, 38, 2279- 2282). To a solution of hexabutylditin (10.1 mL, 20 mmol) in dry THF (40 mL) at -78 °C was added n-BuLi (12.9 mL, 20 mmol, 1.55 M in hexanes), and the resulting clear solution was stirred at -40 °C for 30 min. It was then transferred via cannula to a suspension of CuCN (0.90 g, 10 mmol) in THF (2 mL) at -78 °C. A clear yellow solution formed, and it was stirred for 5 min at -40 °C before being re-cooled to -78 °C. Then dry methanol (23 mL, 0.57 mol) was added to yield a red solution, which was stirred at -40 °C for 15 min, after which a solution of the arylpropyne (5.0 mmol) in THF (5 mL) was added. The orange-red solution was stirred at -10 °C overnight (some Cu and/or Cu²⁺ salts precipitate), then cooled to -20 °C, followed by the addition of methanol (10 mL). After 15 min at -20 °C, water (10 mL) was added, and stirring was continued for another 15 min, while warming to 25 °C. The mixture was extracted with ether, and the organic phase was washed with brine, dried (Na₂SO₄) and evaporated. Flash chromatography (silica, hexanes:EtOAc mixtures) yielded the intermediate vinylstannane, which was dissolved in CH₂CI₂ (5 mL). A solution of iodine (1.05 equiv.) in CH₂CI₂ (40 mL per g l₂) was then added dropwise to this solution at 0 °C. After the last few drops, the color of l₂ persisted, and the reaction was allowed to continue for another 5 min at 0 °C. Then the solvent was evaporated and the residue was dissolved in ether. KF (1 M solution in water, 3 equiv.) and Na₂S₂O₃ (sat., 10 mL per mmol substrate) were added, and the mixture was stirred for 15 min at 25 °C during which time a white precipitate formed. The mixture was filtered through celite, and the organic phase was dried (Na₂SO₄) and evaporated. The residue was purified by flash chromatography (silica, hexanes:EtOAc mixtures) to yield the desired vinyl iodide.

Vinyl iodide 20c. White cloudy film (80%). TLC R_f - 0.25 (silica, hexanes:EtOAc 20:1); IR (film) v_max 3060, 2919, 1619, 1596, 1484, 1443, 1373, 1214, 1061 , 985, 873, 761, 703, 632 cm^"1; MALDI-FTMS m/z 518.0990 (MH⁺), calcd for C₂₈H₂₅INO 518.0975.

Vinyl iodide 20d. Yellow oil (67%). TLC R_f = 0.51 (silica, hexanes:EtOAc 4:1); IR (film) v_max 2924, 1716, 1619, 1596, 1481 , 1372, 1211 , 1 149, 1102, 1045, 918, 873, 609, 517 cm^"1; MALDI-FTMS m/z 320.0142 (MH⁺), calcd for CnH₁₅INO₂ 320.0142.

Vinyl iodide 20e. The intermediate vinyl stannane is readily protodestannylated; therefore, flash chromatography of this intermediate must be performed using hexanes:EtOAc:Et₃N 50:1 :1 as eluent, and the so obtained vinylstannane contained other butyl tin compounds. Following the general procedure, the mixture was treated with enough l₂ that the brown color persisted at the end of the addition (ca. 2 equiv. of l₂). After flash chromatography (hexanes:EtOAc 50:1), vinyl iodide 20e was obtained as a yellow oil (74%). TLC R_f = 0.41 (silica, hexanes:EtOAc 50:1); IR (film) v_max 3102, 2923, 1620, 1423, 1300, 1065, 1035, 964, 863, 723, 562 cm^"1; MALDI- FTMS m/z 297.9215 (MH⁺), calcd for C₇H₉INS₂ 297.9216.

Vinyl iodide 20f. Yellow solid (80%). TLC R_f = 0.19 (silica, hexanes:EtOAc 40:1); IR (film) v_max 2919, 1619, 1567, 1467, 1431 , 1373, 1108, 1067, 1014, 961 , 867, 820, 521 cm^"1; MALDI-FTMS m/z 291.9655 (MH⁺), calcd for CgHnlNS 291.9651.

Vinyl iodide 20g. Yellow oil (83%). TLC R_f = 0.28 (silica, hexanes:EtOAc 40:1); IR (film) v_max 2919, 1620, 1549, 1425, 1155, 1138, 1061, 991 , 961 , 861 , 785, 732, 550 cm^"1; MALDI-FTMS m/z 291.9653 (MH⁺), calcd for CgHnlNS 291.9651.

Synthesis of epothilone analogues 8-144.

Nozaki-Hiyama-Kishi coupling of aldehydes (34, 40) with vinyl stannanes (20a- g) (general procedure). To a briefly vacuum-degassed solution of aldehyde 32 (107 mg, 0.15 mmol), the requisite vinyl iodide 20 (0.45 mmol), and 4-.ert-butylpyridine (665 μL, 4.5 mmol) in DMSO (3 mL) were added anhydrous CrCI₂ (184 mg, 1.5 mmol) and anhydrous NiCI₂ (4 mg, 0.03 mmol). The mixture was stirred at 25 °C for 3 hours, after which another portion of vinyl iodide (0.45 mmol) was added, and stirring was continued for a further 3 hours. This was repeated one more time, after which stirring was continued overnight. The reaction was then quenched with water (5 mL), pyridine (1 mL) was added to prevent Cr-product complexes from being extracted into the water phase, and the mixture was extracted with EtOAc (3 ( 25 mL). The combined extract was washed with brine (2 ( 100 mL), dried (Na₂SO₄) and evaporated. Flash chromatography (silica, hexanes:EtOAc mixtures) yielded the coupling product, in most cases inseparable from excess 4-fer.-butylpyridin.

Product from 20a and 32. Yellow oil (85% as a ca. 1:1 mixture of C15 epimers). TLC R_f = 0.26 (silica, hexanes.EtOAc 4:1); [α]_D22 -25 (c 0.36, CH₂CI₂); IR (film) v_max 2943, 2860, 1731, 1696, 1467, 1384, 1290, 1249, 1173, 1079, 985, 832, 773 cm^"1; MALDI-FTMS m/z 860.5128 (MNa⁺), calcd for C₄₄H₈₃NO₆SSi₃Na 860.5141.

Product from 20b and 32. This coupling product was inseparable from 4-te/f-butyl pyridine, and was subjected to the TBAF deprotection conditions (vide infra) as a crude mixture.

Product from 20d and 32. This coupling product was inseparable from 4-ter.-butyI pyridine, and was subjected to the TBAF deprotection conditions (vide infra) as a crude mixture.

Product from 20e and 32. Yellow glass (78%, ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.40 (silica, hexanes: EtOAc 5:1); [α]_D22 -28 (c 2.0, CHCI₃); IR (film) V 3416 (br), 2929, 2856, 1732, 1694, 1472, 1251, 1037, 988, 836, 776 cm^"1; MALDI-FTMS m/z 906.5021 (MH⁺), calcd for C₄₅H₈₅NO₆S₂Si₃Na 906.5018.

Product from 20f and 32. This coupling product was inseparable from 4-.er.-butyl pyridine, and was subjected to the TBAF deprotection conditions (vide infra) as a crude mixture. Product from 20g and 32. This coupling product was inseparable from 4-terf-butyl pyridine, and was subjected to the TBAF deprotection conditions (vide infra) as a crude mixture.

Product from 20c and 40. Yellow glass (87% for two steps from aldehyde 40 as a ca. 1:1 mixture of C15 epimers). TLC R_f= 0.15 (silica, hexanes:EtOAc 4:1); [α]_D22 - 23 (c θ.19, CH₂CI₂); IR (film) v_max 2931 , 2861, 1731 , 1690, 1467, 1384, 1355, 1249, 1167, 1061 , 985, 832, 773, 703 cm^"1; MALDI-FTMS m/z 1112.6634 (MNa⁺), calcd for C₆₅H₉₉NO₇Si₃Na 1112.6621.

Product from 20e and 40. Colorless glass (59%, ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.27 (silica, hexanes.EtOAc 5:1); [α]_D22 -28 (c 2.0, CHCI₃); IR (film) v_max 3396 (br), 2928, 2855, 1734, 1693, 1472, 1251 , 1037, 988, 836, 775 cm^"1; MALDI- FTMS m/z 892.4861 (MNa⁺), calcd for C₄₄H₈₃NO₆S₂Si₃Na 892.4862.

TBAF deprotection (general procedure). The product mixture from the Nozaki- Hiyama-Kishi coupling was dissolved in THF (1.5 mL), and TBAF (1 M in THF, 0.30 mL, 0.30 mmol) was added at 0 °C. After 1 h at 0 °C, another portion of TBAF (0.30 mL, 0.30 mmol) was added, and the mixture was stirred at 25 °C for 1 hour. The reaction was quenched with NH₄CI (sat., 5 mL), and the mixture was extracted with EtOAc (4 ( 20 mL). The combined extract was dried (Na₂SO₄) and evaporated, and the residue was purified by flash chromatography (silica, hexanes:EtOAc mixtures) to yield the desired hydroxy acid as a ca. 1 :1 mixture of C15 epimers (inseparable at this stage).

Hydroxy acid 41a. The reaction mixture from the deprotection was quickly filtered through a plug of silica gel, and this crude product (73% yield from aldehyde 32) was subjected to the Yamaguchi macrolactonization (vide infra) without further purification.

Hydroxy acid 41b. Yellow solid (57%, ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.19 (silica, hexanes:EtOAc 2:1); [α]_D22 -6 (c 1.0, CHCI₃); IR (film) v_max 3369 (br), 2930, 2857, 1783, 1694, 1471 , 1251 , 1085, 1084, 988, 836, 775 cm^"1; MALDI-FTMS m/z 768.5028 (MNa⁺), calcd for C₄₂H₇₅NO₆Si₂Na 768.5025. Hydroxy acid 41 d. Yellow glass (49% for 2 steps from aldehyde 32 as a ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.20 (silica, hexanes: EtOAc 1 :1); [α]_D22 +1 (c 0.19, CH₂CI₂); IR (film) v_max 2933, 2858, 1694, 1600, 1563, 1463, 1382, 1357, 1251 , 1145, 1096, 1046, 989, 834, 772, 666 cm^"1; MALDI-FTMS m/z 806.5437 (MH⁺), calcd for C₄₄H₈₀NO₈Si₂ 806.5417.

Hydroxy acid 41e. Yellow solid (79%, ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.37 (silica, hexanes: EtOAc 2:1); [α]_D22 -23 (c 2.3, CHCI₃); IR (film) v_max 3356 (br), 2929, 2856, 1712, 1472, 1253, 1085, 1038, 988, 836, 776 crrf¹; MALDI-FTMS m/z 806.4282 (MNa⁺), calcd for C₄₀H₇₃NO₆S₂Si₂Na 806.4315.

Hydroxy acid 41f. Colorless glass (63%, ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.21 (silica, hexanes:EtOAc 2:1); [α]_D22 -3 (c 0.44, CH₂CI₂); IR (film) v_max 2933, 2858, 1693, 1467, 1253, 1086, 984, 833, 774 cm^"1; MALDI-FTMS m/z 800.4754 (MNa⁺), calcd for C₄₂H₇₅NO₆SSi₂Na 800.4746.

Hydroxy acid 41 g. Yellow glass (46% for 2 steps from aldehyde 32 as a ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.46 (silica, hexanes:EtOAc 2:1); [α]_D22 -11 (c 0.19, CH₂CI₂); IR (film) v_max 2933, 2858, 1706, 1557, 1463, 1426, 1364, 1251 , 1083, 989, 834, 772, 666 cm^"1; MALDI-FTMS m/z 800.4746 (MNa⁺), calcd for C₄₂H₇₅NO₆SSi₂Na 800.4746.

Hydroxy acid 42c. The reaction mixture from the deprotection was quickly filtered through a plug of silica gel, and this crude product (46% yield from aldehyde 40) was subjected to the Yamaguchi macrolactonization (vide infra) without further purification.

Hydroxy acid 42e. Pale yellow glass (66%, ca. 1 :1 mixture of C15 epimers). TLC R_f = 0.39 (silica, hexanes:EtOAc 2:1); [α]_D22 -20 (c 1.0, CHCI₃); IR (film) v_max 3354 (br), 2928, 2856, 1713, 1471 , 1253, 1087, 988, 836, 775 cm^"1; MALDI-FTMS m/z 792.4161 (MNa⁺), calcd for C₃₉H₇₁NO₆S₂Si₂Na 792.4153.

Yamaguchi macrolactonization (general procedure). To a solution of the hydroxy acid (95 μmol) in dry THF (8 ml) at 0 °C was added triethylamine (79 μl, 0.57 mmol) and 2,4,6-trichlorobenzoyl chloride (40 μl, 0.23 mmol). After stirring at 0 °C for 1 hour, the resulting solution was added over 2 h to a solution of 4-DMAP (26 mg, 0.21 mmol) in toluene (20 mL) at 75 °C using a syringe pump. Stirring was continued at 75 °C for another 1 h after which the toluene was evaporated under reduced pressure. The residue was directly subjected to flash chromatography (silica, hexanes:EtOAc mixtures) to yield the macrolactone and its (15R)-epimer, readily separable. In all cases the desired (15S)-epimer eluted after the less polar (15R)- epimer.

Macrolactone 43a. colorless glass (28% for two steps from the Nozaki-Hiyama-Kishi coupling product of aldehyde 32 and vinyl iodide 20a.); TLC R_f = 0.21 (silica, hexanes:EtOAc 20:1); [α]_D22 -33 (c 0.56, CH₂CI₂); IR (film)

2932, 2855, 1739, 1689, 1465, 1383, 1252, 1181 , 1153, 1099, 1066, 1017, 984, 869, 836, 776 cm^"1; MALDI-FTMS m/z 734.4639 (MH⁺), calcd for C₄₀H₇₂NO₅SSi₂ 734.4664.

Macrolactone 43b. Colorless glass (28%); TLC R_f = 0.27 (silica, hexanes:EtOAc 10:1); [α]_D22 -28 (c 1.0, CHCI₃); IR (film) 2929, 2856, 1740, 1695, 1472, 1384, 1253, 1100, 1020, 986, 836, 775 cm^"1; MALDI-FTMS m/z 728.5109 (MH⁺), calcd for C₄₂H₇₄NO₅Si₂ 728.5106.

Macrolactone 43d. Yellow glass (35%); TLC R_f = 0.14 (silica, hexanes: EtOAc 6:1); [α]_D22 -28 (c 0.12, CH₂CI₂); IR (film) v_max 2931 , 2861 , 1737, 1690, 1596, 1467, 1378, 1249, 1149, 1102, 1049, 985, 832, 773 cm^"1; MALDI-FTMS m/z 810.5116 (MNa⁺), calcd for C ₄H₇₇NO₇Si₂Na 810.5130.

Macrolactone 43e. This product was isolated as a crude mixture which was directly subjected to the global desilylation conditions (vide infra) without further purification.

Macrolactone 43f. Colorless glass (45%); TLC R_f = 0.20 (silica, hexanes:EtOAc 10:1); [α]_D22 -0.30 (c 0.10, CH₂CI₂); IR (film) v_max 2933, 285, 1737, 1668, 1463, 1382, 1357, 1251 , 1102, 1015, 983, 871 , 834, 772 cm^"1; MALDI-FTMS m/z 760.4799 (MH⁺), calcd for C₄₂H₇₄NO₅SSi₂ 760.4820. Macrolactone 43g Yellow glass (37%); TLC R_f = 0.47 (silica, hexanes: EtOAc 10:1); [α]_D22 -14 (c θ.31 , CHCI₃); IR (film) v_max 2929, 2856, 1740, 1696, 1557, 1461 , 1431 , 1379, 1250, 1099, 107, 979, 836, 774 cm^"1; MALDI-FTMS m/z 760.4802 (MH⁺), calcd for C₄₂H₇₄NO₅SSi₂ 760.4820.

Macrolactone 44c. Colorless glass (33% for 2 steps from the Nozaki-Hiyama-Kishi coupling product of aldehyde 40 and vinyl iodide 20c); TLC R = 0.46 (silica, hexanes: EtOAc 10:1); [α]_D22 -17 (c 0.56, CH₂CI₂); IR (film) v_max 2931 , 2861 , 1743, 1696, 1467, 1378, 1249, 1161, 1073, 1020, 985, 873, 833, 773, 703, 579 cm^"1; MALDI-FTMS m/z 972.5969 (MH⁺), calcd for C₆oH₈₆NO₆Si₂ 972.5988.

Macrolactone 44e. Colorless glass (47%); TLC R = 0.31 (silica, hexanes:EtOAc 15:1); [α]_D22 -19 (c 0.50, CHCI₃); IR (film) v_max 2929, 2855, 1741 , 1697, 1472, 1254, 1102, 1036, 986, 836, 775 cm^"1; MALDI-FTMS m/z 774.4056 (MNa⁺), calcd for C₃₉H₆₉NO₅S₂Si₂Na 774.4048.

Global desilylation (general procedure). The macrolactone was dissolved in 20% v/v TFA in CH₂CI₂, and the solution was kept at 25 °C for 3 hours, after which the volatiles were evaporated without heating. The residue was dissolved in EtOAc, and the solution was washed with NaHCO₃ (sat.), dried (Na₂SO₄) and evaporated. Flash chromatography (silica, hexanes:EtOAc mixtures) afforded the pure epothilone.

Epothilone 6. Colorless glass (73%); TLC R_f = 0.25 (silica, hexanes:EtOAc 2:1); [α]_D22 -34 (c 0.11 , CH₂CI₂); IR (film) v_max 3472 (br), 2931 , 1732, 1684, 1456, 1378,

1258, 1179, 1149, 1067, 1043, 1012, 973, 873, 732 cm^"1; MALDI-FTMS m/z 506.2931 (MH⁺), calcd for C₂₈H₄₄NO₅S 506.2935.

Epothilone 8. Colorless glass (48%); TLC R_f = 0.52 (silica, hexanes:EtOAc 1 :1); [α]_D22 -54 (c 0.30, CHCI₃); IR (film) v_max 3445 (br), 2936, 1732, 1682, 1454, 1383,

1259, 756 cm^"1; MALDI-FTMS m/z 500.3369 (MH⁺), calcd for C₃oH₄₆NO5 500.3376.

Epothilone 10. The general procedure failed to cleave the MOM protecting group cleanly. Therefore, this group was first removed using bromotrimethylsilane as follows: To a solution of protected epothilone 43d (11 mg, 14 μmol) in dry CH₂CI₂ (0.4 mL) was added powdered 4A MS (5 mg), and the resulting mixture was cooled to -30 °C. Bromotrimethylsilane (18.4 μL, 140 μmol) was added dropwise, and the mixture was stirred at -30 °C for 1 hour, after which the reaction was quenched with NaHCO₃ (sat.) and extracted five times with EtOAc. The combined extract was dried and evaporated, and the residue subjected to the general desilylation procedure to yield 10 as a colorless glass (56%); TLC R_f - 0.42 (silica, hexanes:EtOAc 1 :4); [α]_D22 -52 (c θ.12, CH₂CI₂); I R (film) v_max 3401 (br), 2931 , 1731 , 1684, 1596, 1561 , 1461 , 1378, 1331 , 1290, 1255, 1173, 1149, 1044, 1008, 979, 879, 732 cm^"1; MALDI- FTMS m/z 516.3330 (MH⁺), calcd for C₃oH ₆NO6 516.3319.

Epothilone 12. Viscous oil (17% from 41e); TLC R_f = 0.38 (silica, hexanes:EtOAc 2:1); [α]_D22 -52 (c 0.50, CHCI₃); IR (film) v_max 3490 (br), 2933, 1732, 1686, 1255, 1038, 756 cm^"1; MALDI-FTMS m/z 538.2666 (MH⁺), calcd for C₂₈H₄₄NO₅S₂ 538.2655.

Epothilone 13. Colorless glass (68%); TLC R_f = 0.57 (silica, hexanes:EtOAc 1 :1 ); [α]_D22 -46 (c 0.34, CH₂CI₂); IR (film) v_max 3484 (br), 2932, 1731 , 1684, 1469, 1367, 1255, 1150, 1044, 1009, 973, 879, 826, 732 cm^"1; MALDI-FTMS m/z 554.2915 (MNa⁺), calcd for C₃oH₄₅NO₅SNa 554.2910.

Epothilone 14. Colorless glass (48%); TLC R_f = 0.42 (silica, hexanes:EtOAc 2:1 ); [α]_D22 -38 (c 0.34, CH₂CI₂); IR (film) v_max 3478 (br), 2930, 1732, 1682, 1556, 1434, 1378, 1257, 1149, 1137, 1067, 1044, 1012, 979, 785, 732 cm^"1; MALDI-FTMS m/z 532.3078 (MH⁺), calcd for C₃oH₄₅NO₅S 532.3091.

Epothilone 9. Colorless glass (54%); TLC R_f = 0.13 (silica, hexanes: EtOAc 1 :2); [α]₀22 -24 (c 0.14, CH₂CI₂); IR (film) v_max 3379, 2920, 2857, 1725, 1688, 1600, 1459, 1370, 1255, 1151 , 1047, 1010, 979, 880, 734 cm^"1; MALDI-FTMS m/z 524.3004 (MNa⁺), calcd for C₂₉H₄₃NO₆Na 524.2982.

Epothilone 11. Colorless glass (68%); TLC R_f = 0.28 (silica, hexanes:EtOAc 2:1 ); [α]_D22 -26 (c 0.30, CHCI₃); IR (film) v_max 3444 (br), 2925, 1731 , 1693, 1454, 1258, 1037, 756 cm^"1; MALDI-FTMS m/z 546.2330 (MNa⁺), calcd for C₂₇H₄₁NO₅S₂Na 546.2318. Compound 48: R_f = 0.19 (silica gel, ethyl acetate/hexanes = 3/7); [α]_D ²⁰ -19.3 (c 0.14, CH₂CI₂); IR (film): v_max 3484 (br), 2932, 1729, 1459, 1375, 1249, 1043, 982, 733 cm^"1; ¹H NMR (400 MHz, CDCI₃): δ = 6.97 (s, 1 H), 6.47 (s, 1 H), 5.25 (dd, J = 5.7, 7.1 Hz, 1 H), 4.04 (dd, J = 3.0, 8.1 Hz, 1 H), 3.91 (dd, J = 4.1, 4.1 Hz, 1 H), 3.23 (m, 1 H), 2.69 (s, 3 H), 2.52 (dd, J = 8.4, 14.9 Hz, 1 H), 2.46 (dd, J = 2.6, 14.9 Hz, 1 H), 2.11 (s, 3 H), 2.04 (dd, J = 4.0, 14.5 Hz, 1 H), 1.66-1.72 (m, 1 H), 1.44-1.62 (m, 4 H), 1.36 (s, 3 H), 1.22-1.35 (m, 2 H), 1.17 (d, J = 7.5 Hz, 3 H), 1.16 (s, 3 H), 1.04-1.15 (m, 1 H), 0.99 (d, J = 7.0 Hz, 3 H), 0.97 (s, 3 H), 0.48 (m, 1 H), 0.40 (dd, J = 3.9, 8.8 Hz, 1 H), -0.11 ppm (br t, J = 4.6 Hz, 1 H); ¹³C NMR (100 MHz, CDCI₃): δ = 221.5, 171.1, 165.7, 152.9, 138.6, 120.1, 116.2, 82.0, 73.8, 73.2, 52.0, 42.9, 39.4, 36.5, 35.0, 33.2, 31.6, 24.6, 23.5, 22.54, 22.49, 21.1, 20.8, 19.4, 17.4, 16.8, 15.0, 13.2 ppm; MALDI-FTMS: m/z 538.2632 (MH⁺), calcd for C₂₈H₄₄NO₅S_ 538.2655.

Compound 50: R_f = 0.27 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -61 (c 0.12, CH₂CI₂);

MALDI-FTMS: m/z 500.3376 (MH⁺), calcd for C₃₀H₄₆NO5 500.3370.

Compound 51: R_f = 0.37 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -44 (c 0.14, CH₂CI₂);

MALDI-FTMS: m/z 554.2604 (MH⁺), calcd for C₂8H₄₄N0₆S₂ 554.2604.

Compound 52: R_t = 0.31 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -32 (c 0.33, CH₂CI₂);

MALDI-FTMS: m/z 608.2334 (MH⁺), calcd for C₂8H₄₁F₃N0₆S₂ 608.2322.

Compound 53: R_f = 0.38 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -43 (c 0.12, CH₂CI₂);

MALDI-FTMS: m/z 568.2777 (MH⁺), calcd for C₂₉H₄6N0₆S₂ 568.2761.

Compound 54: R_f = 0.27 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -28 (c 0.26, CH₂CI₂);

MALDI-FTMS: m/z 628.2376 (MNa⁺), calcd for C₃ιH₄3 0₇S₂Na 628.2373.

Compound 55: R_f = 0.24 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -49 (c 0.45, CH₂CI₂);

MALDI-FTMS: m/z 566.2116 (MH⁺), calcd for C₂₈H₄ιBrNO₆ 566.2112.

Compound 56: R_f = 0.36 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -27 (c 0.15, CH₂CI₂);

MALDI-FTMS: m/z 544.2419 (MNa⁺), calcd for C₂₈H₄₀CINO₆Na 544.2436. Compound 57: R_f = 0.28 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -49 (c 0.45, CH₂CI₂);

MALDI-FTMS: m/z 534.2907 (MH⁺), calcd for C₂₉H₄₄N0₆S 534.2884.

Compound 58: R_f = 0.35 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -50 (c 0.62, CH₂CI₂);

MALDI-FTMS: m/z 556.2724 (MNa⁺), calcd for C₂₉H₄₃NO₆SNa 556.2703.

Compound 59: R_f = 0.37 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -34 (c 0.24, CH₂CI₂);

MALDI-FTMS: m/z 556.2891 (MH⁺), calcd for C₂₉H₄iF₃NO₆ 556.2880.

Compound 60: R_f = 0.34 (silica gel, ethyl acetate/hexanes = 1/1); [α]_D ²⁰ -33 (c 0.80, CH₂CI₂);

MALDI-FTMS: m/z 535.2820 (MH⁺), calcd for Cz^NzOeS 535.2836.

Example : Soft capsules

5000 soft gelatin capsules, each containing as active ingredient 0.05 g of one of the compounds of formula I named in the preceding examples, e.g. the compounds of example 1, 2, 3 or 4, are prepared as follows:

composition

active ingredient 250 g

Lauroglycol 2 litres

Preparation process: The pulverised active ingredient is suspended in Lauroglykol^® (propylene glycol laurate, Gattefosse S.A., Saint Priest, France) and ground in a wet pulverizer to a grain size of approximately 1 to 3

Portions each containing 0.419 g of the mixture are then filled into soft gelatin capsules by a capsule filling machine.

Example : Infusion solution

The compound of example 1 , 2, 3 or 4 is dissolved at a concentration of 1 mg/ml in polyethylene glycol 300 (PEG 300) and filled into 2 ml vials. For infusion, this solution is diluted with 50 to 100 ml of 0.9% saline according to US Pharmacopoeia.

Detailed Description of Figures Figure 1 shows the structures of selected natural and designed epothilones. Grey boxes indicate compounds synthesized in this study.

Figure 2 is a chart illustrating the displacement of the fluorescent taxoid Flutax-2 (50 nM) from microtubule binding sites (50 nM) by competing ligands at 37 °C. The dots indicate acquired data points and the lines were generated so that they give the best fit value of the binding equilibrium constant of each competitor, assuming one-to-one binding to the same site. Ligands assayed are paclitaxel (Taxol^®) (dark blue), epothilone A (1) (red), epothilone B (2) (violet), compound 3 (yellow), compound 4 (light brown), and compound 8 (green). Representative curves for selected epothilone analogues (3, 4, and 8) are presented in this figure to exemplify how the binding affinities were measured for each compound in Table 3.

Figure 3 shows the synthesis of 2-(thiomethyl)thiazole epothilone B (3) via Stille coupling. Reagents and conditions: Pd₂(dba)₃-CHCI₃ (0.2 equiv), Cul (2.0 equiv), AsPh₃ (0.8 equiv), DMF, 25 °C, 80%. dba = dibenzylideneacetone.

Figure 4 shows the retrosynthetic analysis of .rans-cyclopropyl epothilone B analogues (1 - 6, 8, 10, 12-14).

Figure 5 shows the construction of aldehyde 32. Reagents and conditions: (a) See Nicolaou, K. O, et al. ChemBioChem 2001, 2, 69-75; Charette, A. B.; et al. J. Am. Chem. Soc. 1998, 120, 11943-11952; (b) NaH (1.5 equiv), BnBr (1.5 equiv), DMF, 0 → 25 °C, 12 h; (c) O₃, CH₂CI₂:MeOH 4:1, -78 °C, 21 min; then NaBH₄ (3.0 equiv), - 78 → 25 °C, 1 h, 89% for 2 steps; (d) MsCI (1.3 equiv), Et₃N (1.5 equiv), CH₂CI₂, 25 °C , 1 h; (e) Nal (3.0 equiv), acetone, reflux, 40 min, 95% for 2 steps; (f) LDA (1.4 equiv), 25 (1.3 equiv), THF, 0 °C, 6 h; then 24, -98 → -10 °C, 14 h, 84%; (g) Mel, 60 °C, 3 h; (h) 3 N HCIφentane 1:1, 25 °C, 3 h, 88% for 2 steps; (i) LDA (2.4 equiv), 19 (2.3 equiv), THF, -78 °C, 1 h; then -40 °C, 0.5 h; then 17 at -78 °C, 5 min, 81%; 0) TBSOTf (2.0 equiv), 2,6-lutidine (3.0 equiv), CH₂CI₂, -20 °C, 1 h; (k) HF-py, pyridine, THF, 25 °C, 4 h, 89% for 2 steps; (I) DMP (2.5 equiv), NaHCO₃ (2.5 equiv), H₂O, CH₂CI₂, 25 °C, 1 h; (m) NaCIO₂ (3.1 equiv), NaH₂PO₄ (2.1 equiv), 2-methyl-2-butene (74 equiv), .-BuOH, THF, H₂O, 25 °C, 1 h; (n) 2-(trimethylsilyl)ethanol (4.0 equiv), EDC (1.5 equiv), 4-DMAP (0.1 equiv), DMF, 25 °C, 14 h, 74% for 3 steps; (o) 20% Pd(OH)₂/C, H₂ (1 atm), EtOH:EtOAc 1:1, 25 °C, 1 h; (p) DMP (2.5 equiv), NaHCO₃ (2.5 equiv), H₂O, CH₂CI₂, 25 °C, 1 h, 84% for 2 steps; (q) MeOCH₂PPh₃CI (3.0 equiv), NaHMDS (2.8 equiv), THF, -40 → -10 °C, 2 h, 84%; (r) PPTS (8.0 equiv), dioxane:H₂O 9:1, 70 °C, 6 h, 82%. 4-DMAP = 4-(dimethylamino)pyridine; DME = 1,2-dimethoxy-ethane; DMP = Dess-Martin periodinane; EDC = 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HF-py = hydrogen fluoride- pyridine complex; NaHMDS = sodium hexamethyldisilaz.de; PPTS = pyridinium para- toluenesulfonate; TMSE = 2-trimethylsilylethyl.

Figure 6 shows the construction of vinyl iodides 20c-g. Reagents and conditions: (a) TrCI (1.4 equiv), 4-DMAP (1.7 equiv), DMF, 80 °C, 48 h, 100%; (b) Pd(PPh₃)₂CI₂ (0.01 equiv), Cul (0.02 equiv), HCeCCH₃ (1 atm), DMF, /-Pr₂NEt, 25 °C, 3 h, 35: 96%; (c) (i) π-BuLi (4.0 equiv), (π-Bu₃Sn)₂ (4.0 equiv), CuCN (2.0 equiv), MeOH, THF, -10 °C, 12 h; (ii) l₂ (1.05 equiv), CH₂CI₂, 0 °C, 5 min, 20c: 80% from 35; 20d: 67% from 36; 20e: 37% from 37; 20f: 97% from 38; 20g: 58% from 39; (d) (i) HCI(g), CHCI₃, 0 °C, 1 h, 69%; (ii) MOMCI (1.2 equiv), NaH (1.2 equiv), THF, 0 °C, 1 h, 50%. TrCI = triphenylmethyl chloride; 4-DMAP = 4-(dimethylamino)pyridine; MOMCI = chloromethyl methyl ether.

Figure 7 is the synthesis of epothilone analogues 8-14. Reagents and conditions: (a) CrCI₂ (10 equiv), NiCI₂ (0.2 equiv), 4-.-butylpyridine (30 equiv), 20 (3.0 equiv), DMSO, 25 °C, overnight; (b) TBAF (4.0 equiv), THF, 0 °C, 1 h; then 25 °C, 1 h; (c) Et₃N (6.0 equiv), 2,4,6-trichlorobenzoylchloride (2.4 equiv), 41 or 42, THF, 0 °C, 1 h; then 4- DMAP (2.2 equiv), toluene, 75 °C, 3 h; (d) 20 v/v% TFA in CH₂CI₂, 25 °C, 3 h (except 43d); (e) Estimated by ¹H NMR ; (f) Deprotection of 43d: TMSBr (10 equiv), 4A MS, CH₂CI₂, -30 °C, 1 h; then 20 v/v% TFA in CH₂CI₂, 25 °C, 3 h. TBAF = tetrabutylammonium fluoride; 4-DMAP = 4-(dimethylamino)pyridine; TFA = trifluoroacetic acid; TMSBr = trimethylsilyl bromide; MS = molecular sieves.

Figure 8 illustrates a table disclosing the cytotoxicity of epothilones 1 through 14 and paclitaxel against 1A9 human ovarian carcinoma cells and β-tubulin mutant cell lines selected with paclitaxel or epothilone A. The anti-proliferative effects of the tested compounds against the parental 1 A9 and the paclitaxel- and epothilone-selected drug-resistant clones (PTX10, PTX22 and A8, respectively) were assessed in a 72 h growth inhibition assay using the SRB (sulforhodamine-B) assay (Skehan, P.; et al. J. Natl. Cancer Inst. 1990, 82, 1107-1112). IC₅o values for each compound are given in nM and represent the mean of 3-9 independent experiments A standard error of the mean. Relative resistance (RR) is calculated as an IC₅₀ value for each resistant sub-line divided by that for the parental cell line (1 A9). CP = cyclopropyl; py = 5- methylpyridine side chain; pyOH = 5-hydroxymethylpyridine side chain; 5tmpy = 5- thiomethylpyridine side chain; 6tmpy = 6-thiomethylpyridine side chain; tmt = 2- thiomethyl thiazole side chain.

Figure 9 illustrates a table disclosing the tubulin polymerization potency and cytotoxicity of epothilones 1-8, 10-14, and paclitaxel against human epidermoid cancer cell lines, (a) The extent of porcine tubulin polymerization (TP) by 4 μM compound was quantified relative to the effect of 25 μM epothilone B (which was defined as 100%) as described (Nicolaou, K. O; et al. Chem. Biol. 2000, 7, 593- 599). (b) Drug concentration required for maximal inhibition of cell growth (IC₅₀ values given in nM) was assessed after a 96 hour drug exposure by quantification of cell mass using a protein dye method as described (Meyer, T.; et al. Int. J. Cancer 1989, 43, 851-856). KB-31 : epidermoid Taxol^®-sensitive cells, KB-8511 : epidermoid Taxol^®-resistant cells (due to Pgp overexpression). Relative resistance (RR) was calculated by dividing the IC₅₀ value for the resistant cell line by that of the sensitive cell line, (c) Data from ref. 3 (%TP values for Taxol^®, Epo A and Epo B were 49, 69 and 90, respectively). CP = cyclopropyl; py = 5-methylpyridine side chain; pyOH = 5- hydroxymethylpyridine side chain; 5tmpy = 5-thiomethylpyridine side chain; 6tmpy = 6-thiomethylpyridine side chain; tmt = 2-thiomethyl thiazole side chain.

Figure 10 illustrates a table disclosing binding affinities of epothilone analogues to the taxoid binding site of microtubules. (a) The binding of the different ligands to the taxoid site of microtubules was measured by the displacement of a fluorescent Taxol^® derivative (Flutax-2) from its binding site (Figure 2) (Diaz, J. F.; et al. J. Biol. Chem. 2000, 275, 26265-26276). The Flutax-2 displacement isotherm of each ligand was measured at least twice with a fluorescence polarization microplate reader in a modified procedure from the previous report (Andreu, J. M.; Barasoain, I. Biochemistry 2001 , 40, 11975-11984). Cross-linked stabilized microtubules which had been stored under liquid nitrogen were employed. The binding constant of the reference ligand Flutax-2 was measured by centrifugation and fluorescence anisotropy, at each temperature (Diaz, J. F.; et al. J. Biol. Chem. 2000, 275, 26265- 26276). The resulting reference value was 2.2 ( 10⁷ M^"1 at 37 °C. (b) The equilibrium dissociation constants (Kd) are given in nM. (c) The standard binding free energy changes (DG⁰ _app) are given in kJ mol^"1.

Figure 11 shows a series of structures of the various designed analogs of epothilones A and B along with the structures of epothilone A and B. The design of the present epothilone library is based on the current knowledge of structure activity relationships (SAR), specifically the facts that: epothilone B (2) is considerably more potent than epothilone A (1); a thiomethyl replacement for the methyl group on the thiazole moiety enhances the potency; and a heterocycle such as pyridine or pyrimidine replacement for the thiazole side chain is needed to maintain the proper position of the nitrogen for biological activity.

Figure 12 is a scheme showing the last step in the synthesis of many of the analogs from the vinyl iodide 15 and the corresponding aromatic stannanes. A Stille-type coupling of 15 with appropriate stannanes was carried out in the presence of PdCI₂(MeCN)₂, Cul and AsPh₃ in DMF at ambient temperature, leading directly to the analogs in the indicated yields. Reagents and conditions: a. PdCI₂(MeCN)₂ (0.5 eq), Cul (2.0 eq), AsPh₃ (1.0 eq), 64a-64d, 66a-66d, 67-68 (2.5 eq), DMF, 25 _C, 1-3 h, 41-80%.

Figure 13 is a scheme showing the steps required to synthesize the stannanes used in the scheme in Figure 12. The thiazole compounds (64a-64d) were synthesized from the commercially available 2,4-dibromothiazole (62) by reacting the corresponding thiol with NaH in the presence of the dibromothiazole. Coupling of the product with Me₃SnSnMe₃ in the presence of Pd(PPh₃) in toluene at 100 _C gave the desired products 64a-64d. Reagents and conditions: a) NaH (3 eq), RSH (3 eq), i-PrOH, 24 h, 70-81 %; b) (Me₃Sn)₂ (5-10 eq), Pd(PPh₃)₄ (5 mol%), toluene, 100 _C, 1-3 h, 71-88%; c) π-Buϋ (1.1 eq), ether, -78 _C, 1 h, then n-Bu₃SnCI (1.2 eq), -78 to 25 _C, 1 h, 49-62%.

Figure 14 is scheme illustrating the synthetic route taken to build the skeleton of the cyclopropyl analogs of epothilone B. Reagents and conditions: (a) Nicolaou, K. O; et al. J. Am. Chem. Soc. 2001, 723, 9313 and Jessie, S; Kjell, U. Tetrahedron 1994, 50, 275; (b) NaH (1.5 eq), BnBr (1.2 eq), DMF, 0 to rt, 12 h, 100%; (c) O₃, CH₂CI₂, MeOH (4:1), -78 _C, then NaBH₄ (3 eq), -78 _C to rt, 1 h, 83%; (d) MsCI (1.3 eq), Et₃N (1.5 eq), DCM, rt, 1 h; (e) Nal (3 eq), acetone, rt, 12 h, 91% (2 steps); (f) LDA (1.4 eq), 25 (1.3 eq), THF, 0 _C, 6 h, then 73, -98 to -10 _C, 14 h, 87%; (g) Mel, reflux, 3 h; (h) 3N HCI:pentane (1 :1), rt, 3 h, 91% (2 steps); (i) LDA (2.4 eq), 19 (2.3 eq), THF:ether (1 :1), -78 _C, 1 h, then -40 _C, 30 min, then 76 at -78 _C, 5 min, 80%; 0) TBSOTf (1.5 eq), 2,6-lutidine (2 eq), DCM, -20 _C, 1 h; (k) HF-py, pyridine, THF, 0 _C, 8 h, 86% (2 steps); (I) (COCI)₂ (1 .2 eq), DMSO (2.0 eq), DCM, -78 _C, 5 min, then 79 (1 eq), 20 min, then Et₃N (3 eq), -78 to 0 __C; (m) NaCIO₂ (5 eq), NaH₂PO₄ (3 eq), 2-methyl-2-butene (75 eq), f-BuOH, THF, H₂O, rt, 1 h; (n) 2- (trimethylsilyl)ethanol (4 eq), EDC (1.5 eq), DMAP (0.1 eq), DMF, rt, 12 h, 73% (3 steps); (o) 20% Pd(OH)₂/C, H₂, EtOH:EtOAc (1 :1 ), rt, 2 h, 89%; (p) (COCI)₂ (1.2 eq), DMSO (2.0 eq), DCM, -78 _C, 5 min, then 81 (1 eq), 20 min, then Et₃N (3 eq), -78 to 0 _C, 99%; (q) MeOCH₂PPh₃CI (3 eq), n-BuLi (2.8 eq), THF, 0 _C, 1 h, then 82, -78 to 0 _C, 2 h, 79%; (r) PPTS (10 eq), dioxane.water (9:1), 70 _C, 12 h, 81%.

Figure 15 is a scheme showing the final steps used in the synthesis of cyclopropyl analogs 48 and 50. Reagents and conditions: (a) CrCI₂ (10 eq), NiCI₂(0.2 eq), A-t- BuPy (30 eq), 84a or 84b (3 eq), DMSO, 25 _C, 24 h; (b) TBAF (2 eq), THF, rt, 2 h; (c) Et₃N (6 eq), 2,4,6-trichlorobenzoyl chloride (2.4 eq), 85 or 88, THF, 0 _C, 1 h, then DMAP (2.2 eq), toluene, 75 _C, 3 h; (d) 20% v/v TFA in CH₂CI₂, rt, 3 h.

Figure 16 is a table with the cytotoxicities of epothilones 48,50 and 51-60 against human carcinoma cells and b-tubulin mutant cell lines selected with paclitaxel or epothilone A. The anti-proliferative effects of the tested compounds against the parental 1A9 and the paclitaxel- and epothilone-selected drug resistant clones (PTX10, PTX22 and A8, respectively) were assessed in a 72 h growth inhibition assay using the SRB (sulforhodamine-B) assay (Skehan, P.; et al. J. Natl. Cancer. Inst. 1990, 82, 1107-1112.). IC₅₀ values for each compound are given in nM and represent the mean of 3 independent experiments A standard error of the mean. Relative resistance (RR) is calculated as an IC₅o value for each resistant sub-line divided by that for the parental cell line (1 A9). The results for compound 3 are taken from Nicolaou, K. O; et al. Tetrahedron 2002, 58, 6413-6432.

Figure 17 is a table with the cytotoxicities (IC₅₀'s in nM) of selected epothilones against the human epidermoid cell lines KB-3 and KB-8511. The antiproliferative effects of the tested compounds were assessed in two human epidermoid cancer cell lines, including a parent cell line (KB-31) and a Taxol™- resistant (due to Pgp- overexpression) cell line (KB-8511). The results for Epo B and 3 were taken from Nicolaou, K. O; et al. Tetrahedron 2002, 58, 6413-6432.

Claims

What is Claimed:

1. A compound represented by formula

wherein,

when a compound of formula I is a cis- isomer X is oxygen or CH₂ and when a compound of formula I is a trans- isomer X is CH₂;

when compound of formula I is a cis- isomer and X is oxygen then R1 is methyl and R is a radical selected from the group consisting of

₂M e

wherein R2 is a radical selected from the group consisting of -Me, -CI, -Br, -SMe and -CF₃;

when a compound of formula I is a cis isomer and X is CH₂then R1 is methyl and R is a radical selected from the group consisting of

wherein R2 is a radical selected from the group consisting of -Me, -CI, -Br, -SMe and -CF₃;

when a compound of formula I is a trans isomer and X is CH₂then R1 is methyl or Hydrogen;

wherein, when R1 is methyl R is a radical selected from the group consisting of

and, when R1 is hydrogen,

R is a radical selected from the group consisting of

or a salt of a compound of formula I where a salt-forming group is present.

2. A compound according to claim I represented by the formula 11:

wherein R is a radical selected from the group consisting of the following structures:

3. A compound according to claim 2 represented by the following formula:

4. A compound according to claim 2 represented by the following formula:

5. A compound according to claim 2 represented by the following formula:

6. A compound according to claim 2 represented by the following formula:

7. A compound according to claim 2 represented by the following formula:

8. A compound according to claim 2 represented by the following formula:

9. A compound represented by the following formula:

wherein R is a radical selected from the group consisting of the following structures:

10. A compound according to claim 9 represented by the following formula:

11. A compound according to claim 9 represented by the following formula:

12. A compound according to claim 1 represented by the following structure:

13. A compound according to claim 1 represented by the following structure:

14. A compound according to claim 1 represented by formula IV:

wherein R is a radical selected from the group consisting of radicals represented by the following structures:

15. A compound according to claim 14 represented by the following structure:

16. A compound according to claim 14 represented by the following structure:

17. A compound according to claim 14 represented by the following structure:

18. A compound according to claim 14 represented by the following structure:

19. A compound according to claim 14 represented by the following structure:

20. A compound according to claim 1 represented by the formula V:

wherein, R is a radical selected from group consisting of -Me, -CI, -Br, -SMe, and CF₃.

21. A compound according to claim 20 represented by the following structure:

22. A compound according to claim 20 represented by the following structure

23. A compound according to claim 20 represented by the following structure

24. A compound according to claim 20 represented by the following structure

25. A compound according to claim 20 represented by the following structure

26. A compound according to claim 1 represented by the following structure:

27. A compound according to claim 1 represented by the following structure:

28. A pharmaceutical composition containing a therapeutic dose of any one or more of the compounds of claims 1-27 together with a pharmaceutically acceptable carrier for the treatment of a proliferative disease in a mammal.

29. A pharmaceutical composition according to claim 28 where the mammal is a human.

30. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound according to any one of the claims 1 to 27, or a pharmaceutically acceptable acid or base addition salt thereof, where possible.

31. Use of a compound according to any one of the claims 1 to 27 for the preparation of a medicament for the treatment of a proliferative disease.

32. A compound according to any of the one claims 1 to 27 for use in a method for treatment of the human or animal body.

33. A method of treating a proliferative disease comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound according to any one of claims 1 to 27, or a pharmaceutically acceptable acid or base addition salt thereof, where possible.

34. A process for the preparation of a compound according to any one of the claims 1-27, wherein a compound of the formula VI

wherein X and R have any of the meanings as defined in claims 1 to 27 and PG is a protecting group for a hydroxy function, in a first step is condensed by a esterification reaction, optionally in the presence of a catalyst, and in a second step the protecting group is detached thus furnishing a lacton of formula I.