MXPA01006496A - Microbial transformation method for the preparation of an epothilone - Google Patents

Abstract

A microbial method for the preparation of an epothilone containing a terminal hydroxyalkyl group, comprising contacting at least one epothilone having a terminal alkyl group with an enzyme or microorganism capable of catalyzing the selective hydroxylation of said alkyl group to a hydroxyalkyl group, and effecting said hydroxylation.

Description

METHOD OF MICROBIAL TRANSFORMATION FOR THE PREPARATION OF AN EPOTILONE FIELD OF THE INVENTION The present invention is concerned with a microbial method for the preparation of an epothilone.

BACKGROUND OF THE INVENTION Epothilones are macrolide compounds that find utility in the pharmaceutical field. For example, it has been found that epothilones A and B that have the structures: Epothilone AR = H Epothilone BR = I exert microtubule stabilizing effects similar to paclitaxel (TAXOL®) and hence cytotoxic activity against rapidly proliferating cells, such as tumor cells or other hyperproliferative cell disease, see Bollag et al, Cancer Res., Vol. 55, No. 11, 2325-2333 (1995). Epothilones A and B are natural anticancer agents produced by Sorangium cellulosum that were isolated and characterized for the first time by Hofle et al., REF: 128776 DE 4138042; WO 93/10121; Angew, Chem. Int. Ed. Engl. Vol 35, No 13/14 1567-1569 (1996), and J. Antibiot, Vol 49, No. 6, 560-563 (1996) Subsequently, the total syntheses of the epothilones A and B have been published. by Balog et al, Angew, Chem. Int. Ed. Engl., Vol. 35, No. 23/24, 2801-2803, 1996; Meng, et al, J. Am. Chem. Soc, Vol. 119, No 42, 10073-10092 (1997), Nicolaou et al., J. Am. Chem. Soc., Vol 119, No. 34, 7974-7991 (1997), Schinzer et al., Angew, Chem. Int. Ed. Eng., Vol 36, No. 5, 523-524 (1997), and Yang et al, Angew, Chem. Int. Ed. Engl., Vol. 36 No. 1/2, 166-168, 1997. PCT document W098 / 25929 describes the methods for the chemical synthesis of epothilone A, epothilone B, epothilone analogs and epothilone analog libraries The structure and production of Sorangium cellulosum DSM 6773 of epothilones C, D, E and F is revealed in W098 / 22461.

BRIEF DESCRIPTION OF THE INVENTION The present invention is concerned with a method for obtaining epothilones with desired substituents in a terminal carbon position. In particular, the present invention provides a method for the preparation of hydroxyalkyl-bearing epothilones, such compounds find utility as antitumor agents and as starting materials in the preparation of other epothilone analogues.

One embodiment of the invention provides a method for the preparation of at least one epothilone of the following formula I H0-CH2- (A?) N- (Q) m- (A2) or E (I) wherein: Ai and A2 are independently selected from the group of alkyl and alkenyl of 1 to 3 carbon atoms optionally substituted; Q is an optionally substituted ring system containing one to three rings and at least one carbon-carbon double bond in at least one ring; n, m and o are integers selected from the group consisting of zero and 1, where at least one of m or n or is zero is 1; and E is a nucleus of epothilone; comprising the steps of contacting at least one epothilone of the following formula II CH3- (A?) n- (Q) m- (A2) or E (II) wherein Ai, Q, A2, E, n, myo are as defined above, with a microorganism or an enzyme derived therefrom, which is capable of selectively catalyzing the hydroxylation of formula II and effecting such hydroxylation.

In another embodiment, the present invention provides a method for the preparation of at least one epothilone of the following formula III: where: Q is selected from the group consisting of Gi is the following formula V HO-CH2- (A?) N- (Q) m- (A2) or (V) wherein: Ai and A2 are independently selected from the alkyl and alkenyl group of 1 to 3 carbon atoms optionally substituted; Q is an optionally substituted ring system containing one to three rings and at least one carbon-carbon double bond in at least one ring; n, m and o are integers selected independently of the group consisting of zero and 1, where at least one of m or n or is l; W is O or NR6; X the group consisting of 0 is selected; H, 0R7; M is O, S, NR8, CR9R? 0; Bi and B2 are selected from the group consisting of ORn, 0C0Ri2; R? -R5 and? 2-7 are selected from the group consisting of H, alkyl, substituted alkyl, aryl and heterocycle and wherein, when Ri and R2 are alkyl, they can be joined to form a cycloalkyl; Re is selected from the group consisting of H, alkyl and substituted alkyl; R7 and Rii are selected from the group consisting of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl and dialkylarylsilyl; Re is selected from the group consisting of H, alkyl, substituted alkyl, R? 3C = 0, R? 0C = 0 and R? 5S02; R-9 and Rio are selected from the group consisting of H, halogen, alkyl, substituted alkyl, aryl, heterocycle, hydroxy, R? 6C = 0 and R? 70C = 0, the pharmaceutically acceptable salts thereof and any hydrates, solvates or geometric, optical and stereoisomeric isomers thereof; comprising the steps of contacting at least one epothilone of the following formula IV: wherein Q, W, X, M, Bi, B2 and R? -R17 are as defined above; G2 is the following formula VI CH3- (A?) N- (Q) m- (A2) or (VI) wherein Ai, Q, A2, n, m and o are as defined above; the pharmaceutically acceptable salts thereof and any hydrates, solvates or geometrical, optical and stereoisomeric isomers thereof; with a microorganism or enzyme derived therefrom capable of selectively catalyzing the hydroxylation of G2 to Gi and effecting such hydroxylation.

DETAILED DESCRIPTION OF THE 3: NVENTION The present invention provides an efficient process for the preparation of epothilones having a terminal substituted hydroxyalkyl or hydroxyalkyl group from epothilones having an alkyl or substituted alkyl group in a terminal position. A single epothilone can be hydroxylated or a mixture of different epothilones can be hydroxylated sequentially or simultaneously according to the present invention. All stereoconfigurations of the unspecified chiral centers of the compounds of formulas I to VI are contemplated in the hydroxylation method of the present invention either alone (i.e., substantially free of other stereoisomers) or in admixture with other stereoisomeric forms. In the method of the present invention, the stereoconfiguration of the terminal alkyl or substituted alkyl group of the starting epothilone is preferably retained in the epothilone product.

Definitions Definitions of various terms used to describe this invention are listed below. These definitions apply to terms as used throughout this specification, unless otherwise limited in specific instances either individually or as part of a larger group. The terms "microbial processes" or "microbial methods" as used herein, mean a process or method of the present invention that employs a microorganism or an enzyme derived therefrom. The term "hydroxylation" as used herein means the formation of a substituted hydroxyalkyl or hydroxyalkyl group of the corresponding alkyl or substituted alkyl group and can be obtained, for example, by contact with a microorganism or an appropriate enzyme. The term "epothilone" as used herein means compounds that contain an epothilone core and a side chain group as defined herein. The term "epothilone core", as used herein, means a portion containing the core structure (with the numbering of the ring system positions used shown herein): wherein the substituents are as defined hereinbefore. The term "side chain group" refers to substituent G as defined by Gi and G2 herein above. The term "terminal carbon" or "terminal alkyl group" refers to the terminal carbon or terminal methyl group of the portion either directly attached to the epothilone nucleus at position 15 or to the terminal carbon or terminal alkyl group of the attached side chain group at position 15. It is understood that the term "alkyl group" includes alkyl and substituted alkyl as defined herein. The term "pharmaceutically active agent" or "pharmaceutically active epothilone" refers to an epothilone that is pharmacologically active in the treatment of cancer or other diseases described herein. The term "alkyl" refers to straight or branched chain saturated, optionally substituted, hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms. The term "lower alkyl" refers to optionally substituted alkyl groups of 1 to 4 carbon atoms. The term "substituted alkyl" refers to an alkyl group substituted by, for example, 1 to 4 substituents, such as halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, aralkylamino, cycloalkylamino, heterocycloamino, disubstituted amines in which the two amino substituents are selected from alkyl, aryl or aralkyl, alkanoylamino, aralylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, aralkylthio, cycloalkylthio, heterocyclic, alkylthion, arylthione, aralkylthion, alkylsulfonyl, arylsufonyl, aralkylsulfonyl, sulfonamido (for example S02NH2), substituted sulfonamido, nitro, cyano, carboxy, carbamyl (for example C0NH2), substituted carbamyl (for example CONH alkyl, CONH aryl, CONH aralkyl or cases where there are two substituents on the nitrogen selected from alkyl, aryl or aralkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino and heterocycles, such as indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where indicated above, where the substituent is further substituted, it will be with halogen, alkyl, alkoxy, aryl or aralkyl. The term "ring system" refers to an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring. Exemplary ring systems include, but are not limited to, an aryl or a partially or completely unsaturated heterocyclic ring system, which may be optionally substituted. The term "aryl" refers to monocyclic or bicyclic aromatic hydrocarbon groups having from 6 to 12 carbon atoms in the ring portion, such as phenyl, naphthyl, biphenyl or diphenyl groups, each of which may be optionally substituted .

The term "substituted aryl" refers to an aryl group substituted by, for example, one to four substituents such as alkyl; substituted alkyl, halo, trifluoromethoxy, trifluoromethyl, hydroxy, alkoxy, cycloalkyloxy, heterocycloxy, alkanoyl, alkyloxy, amino, alkylamino, aralkylamino, cycloalkylamino, heterocycloamino, dialkylamino, alkanoylamino, thiol, alkylthio, cycloalkylthio, heterocyclic, ureido, nitro, cyano, carboxy , carboxyalkyl, carbamyl, alkoxycarbonyl, alkylthion, arylthion, alkylsulfonyl, sulfonamido, - aryloxy and the like. The substituent may be further substituted by halo, hydroxy, alkyl, alkoxy, aryl, substituted aryl, substituted alkyl or aralkyl. The term "aralkyl" refers to an aryl group directly ched through an alkyl group, such as benzyl. The term "substituted alkene" and "substituted alkenyl" refers to a portion having a carbon-carbon double bond, which may be part of a ring system, with at least one substituent that is a lower alkyl or lower alkyl replaced. Other substituents are as defined for the substituted alkyl.

The term "cycloalkyl" refers to saturated, optionally substituted cyclic hydrocarbon ring systems, preferably containing from 1 to 3 rings and from 3 to 7 carbon atoms per ring, which may be further fused with a carbocyclic ring of 3. at 7 unsaturated carbon atoms. Exemplary groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl and adamantyl. Exemplary substituents include one or more alkyl groups as described above or one or more groups described above as alkyl substituents. The terms "heterocycle", "heterocyclic" and "heterocycle" refer to an unsaturated, partially saturated or fully saturated, optionally substituted, aromatic or unsaturated cyclic group, for example, which is a monocyclic ring system of 4 to 7 members , 7 to 11 membered bicyclic or 10 to 15 membered tricyclic, having at least one heteroatom in at least one ring containing carbon atoms. Each ring of the heterocyclic group containing a heteroatom may have 1, 2 or 3 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms, where the nitrogen and sulfur heteroatoms may also be optionally oxidized and the nitrogen heteroatoms may also be be optionally quaternized. The heterocyclic group may be ched to any heteroatom or carbon atom. Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolidyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxazepinyl, azepinyl, 4-piperidonyl, pyridyl, N-oxo-pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl, sulfoxide of thiomorpholinyl, thiomorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothienyl, dioxanyl, isothiazolidinyl, thietanyl, tyranyl, triazinyl and triazolyl and the like. Exemplary bicyclic heterocyclic groups include benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, quinolinyl-N-oxide, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinilyl (such as furo [2, 3-c] pyridinyl, furo [3, 1-b] pyridinyl] or furo [2,3-b] pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as, 4-dihydro-4-oxo-quinazolinyl), benzyl-thiazolyl, benzisoxazolyl, benzodiazinyl, benzofurazanyl, benzothiopyranyl, benzotriazolyl, benzopyrazolyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothio-pyranyl, dihydrobenzothiopyranyl sulfone, dihydrobenzo-pyranyl, indolinyl, isochromanyl, isoindolinyl, naphthyridinyl , phthalazinyl, piperonyl, purinyl, pyridopyridyl, quinazolinyl, tetrahydroquinolinyl, thienofuryl, thienopyridyl, thienothienyl and the like. Exemplary substituents include one or more alkyl groups as described above or one or more groups described above as alkyl substituents. Also included are small heterocycles, such as epoxides and aziridines. The term "alkanoyl" refers to -C (O) -alkyl. The term "substituted alkanoyl" refers to -C (O) -substituted alkyl. The term "aroyl" refers to -C (O) -aryl. The term "substituted aroyl" refers to -C (O) -substituted aryl. The term "trialkylsilyl" refers to -Si (alkyl) 3.

The term "aryl dialkylsilyl" refers to -Si (alkyl) 2 (aryl). The term "diarylkylsilyl" refers to -Si (aryl) 2 (alkyl). The term "heteroatoms" will include oxygen, sulfur and nitrogen. The term "halogen" or "halo" refers to fluorine, chlorine, bromine and iodine. The compounds of formulas I to IV can form salts with alkali metals such as sodium, potassium and lithium, with alkaline earth metals such as calcium and magnesium, with organic bases such as dicyclohexylamine and tributylamine, with pyridine and amino acids such as arginine, lysine and the similar. Such salts can be obtained, for example, by exchanging the protons of the carboxylic acid, if they contain a carboxylic acid, of compounds of formulas I and II with the desired ion, in a medium in which the salt precipitates or in an aqueous medium followed by evaporation. Other salts can be formed as is known to those skilled in the art. The compounds of formula I to IV form salts with a variety of organic and inorganic acids.

Such salts include those formed with hydrogen chloride, hydrogen bromide, methanesulfonic acid, hydroethanesulfonic acid, sulfuric acid, acetic acid, trifluoroacetic acid, maleic acid, benzenesulfonic acid, toluene sulfonic acid and various other (eg, nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates, salicylates and the like). Such salts are formed by reacting a compound of formula I to IV in an equivalent amount of acid in a medium in which the salt precipitates or in an aqueous medium followed by evaporation. In addition, amphoteric ions ("inner salts") can be formed and are included within the term salts as used herein. The prodrugs and solvates of the compounds of formula I to IV are also contemplated herein. The term "prodrug" as used herein means a compound that, upon administration to a subject, is subjected to chemical conversion by metabolic or chemical processes to produce a compound of formulas I through IV or a salt and / or solvate thereof. For example, the compounds of formulas I to IV can form a carboxylate ester moiety. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid functionalities found in the described ring structure (s). The solvates of the compounds of formula I to IV are preferably hydrates.

Various forms of prodrugs are well known in the art. For examples of such prodrug delivery derivatives, see: (a) Design of Prodrugs, H. Bundgaard (editor), Elsevier (1985); (b) Methods in Enzymology, K. Widdler et al (editors), Academic Press, Vol. 42, 309-396 (1985); (c) A Textbook of Drug Design and Development, Krosgaard-Larsen and H. Bundgaard (editors), Chapter 5, "Design and Application of Prodrugs", 113-191 (1991); (d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992); (e) H. Bundgaard, J. of Pharm. Sciences, 77, 285 (1988); and (f) N. Kakeya et al., Chem. Pharm. Bull., 32 692 (1984) . The compounds of the invention can exist as multiple optical, geometric isomers and stereoisomers. While the compounds shown herein are depicted for an optical orientation, included within the present invention are all isomers and mixtures thereof.

General Methods of Preparation In general, the hydroxyalkyl-bearing epothilone product of the invention can be produced by culturing a microorganism or enzyme capable of selectively hydroxylating a terminal carbon or alkyl, in the presence of a suitable epothilone substrate in a medium of aqueous nutrient that contains sources of carbon and nitrogen assimilable under submerged aerobic conditions.

Starting materials The epothilones used as starting materials for the present invention can be any compound such that it has a terminal carbon or terminal alkyl group capable of undergoing enzymatic hydroxylation of the present invention. The starting material or substrate can be isolated from natural sources, such as Sorangium cellulosum or can be synthetically formed epothilones. In a preferred embodiment, the starting material is epothilone B. Epothilone B can be obtained from the fermentation of Sorangium cellulosum So ce90, as described in DE 41 38 042 / WO 93/10121. The strain has been deposited in the Deutsche Sammlung von Mikroorganismen (German Collection of Microorganisms) (DSM) under No. 6773. The fermentation process is also described in Hofle, G., et al, Angew, Chem, Int. Ed. Engl., Vol. 35, No. 13/14, 1567-1569 (1996). Epothilone B can also be obtained by chemical means, such as those described by Meng, D., et al., J. Am. Chem. Soc., Vol. 119, No. 42, 10073-10092 (1996); Nicolaou, K., et al., J. Am. Chem. Soc, Vol. 119, No. 34, 7974-7991 (1997) and Schinzer, D. et al., Chem. Eur. J., Vol. 5, No. 9, 2483-2491 (1999).

Enzymes and Microorganisms The enzyme or microorganism employed in the present invention can be any enzyme or microorganism capable of selectively catalyzing the enzymatic hydroxylation described herein. Specifically, the microorganism or any enzyme derived from the microorganism employed in the present invention can be any microorganisms capable of selectively converting the terminal carbon or the terminal alkyl group to a hydroxymethyl or hydroxyalkyl group. The microorganism, regardless of origin or purity, can be used in a free state or immobilized on a support such as by physical absorption or entrapment. Suitable microorganisms for the selective hydroxylation process of the invention can be selected from the genera including, but not limited to, Actinomycete, Amycolata, Amycolatopsis, Beauveria, Candida, Gilbertella, Nocardia, Pseudomonas, Saccharopolyspora, Saccharothrix and Streptomyces. Examples of species of microorganisms known to hydroxylate the terminal portions include Beauveria basiana, Candida rugosa and Pseudomonas putida. Examples of microorganisms that have been shown to selectively hydroxylate a terminal alkyl or substituted alkyl of an epothilone include Amycolata autotrophica ATCC 35203 and Actinomycete Sp. Cepa SC 15847 PTA-1043. In a preferred embodiment of the invention, the microorganism is Actinomycete sp. Cepa SC 15847 PTA-1043. The actinomycete SP strain SC 15847 was isolated from the Bristol-Myers Squibb soil collection. The term "PTA-1043" as used herein refers to the access number of the American type culture collection, 10801 üniversity Blv, Manassas, VA, the depository for the referred organism. The microorganism Actinomycete SP was deposited with the ATCC under the deposit number PTA-1043. The term "SC" means the designation given to the microorganism as part of the Squibb culture collection. The microorganism Amycolata autotrophica was purchased from ATCC. The taxonomic analysis of Amycolata autotrophica has been described by Okazaki, T., Serizawa, N., Enokita, R., Torikata, A. and Terahara, A., J. Antibiot., 36, 1176-1183 (1983) and Lechevalier , MP, Prauser, H., Labeda, DP and Rúa, J.-S., Int. J. Systemic Bacteriol. 36, 29-37 (1986).

The biologically pure microorganisms Amycolata autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043 are novel microorganisms capable of carrying out the process of selective hydroxylation. It should be understood that mutants of these organisms are also contemplated by the present invention, for use in the hydroxylation method described herein, such as those modified by chemical, physical (e.g., X-ray) or biological (e.g. , using molecular biology techniques). Those skilled in the art can select other microorganisms for use in the present invention by using the following protocol.

Test for the Selection of the Microorganism: To a 25 ml flask containing 2 ml of transformation medium with the same composition as described in Example 1, a small aliquot (approximately 0.1 ml) of microbial culture is inoculated in this flask . The culture is incubated at 28 ° C and 250 r.p.m. on a rotary shaker for 24 hours. To the culture, 0.2 mg of an epothilone substrate is added and the culture is returned to the agitator for further incubation. At 45 and 60 hours, an aliquot of 0.5 ml is removed and the test for the formation of the epothilone carrying hydroxyalkyl is carried out by the HPLC analysis that follows. The microorganisms found capable of carrying out the inventive process are then selected for further analysis. Exemplary enzymes for use in the present hydroxylation are monooxygenases dependent on cytochrome P-450 isolated from microbial, mammal and plant systems. See, H.L. Holland, Organic Synthesis With Oxidative Enzymes, VCH Publishers, Inc., New York, NY, 5-12 (1991). Enzymes can be isolated, for example, by extraction and purification methods such as by the use of hydrophobic interaction chromatography, gel filtration, followed by an anion exchange column. The present invention further provides that enzymes capable of the selective hydroxylation method, present, can be isolated from the genera listed above, including, but not limited to, Amycolata autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043 by the above techniques. Where microorganisms are employed, the cells can be used in the form of intact moist cells or dry cells, such as lyophilized cells dried by heat or dried thermally or in the form of treated cellular material such as broken or broken cells or cell extracts. The use of genetically designed organisms is also contemplated. The host cell can be any cell, for example Escherichia coli, modified to contain a gene or genes to express one or more enzymes capable of catalysis as described herein. Where one or more microorganisms are employed, the enzymatic hydroxylation method of the present invention can be carried out subsequent to the fermentation of microorganisms (fermentation and two-step hydroxylation) or concurrently with it, that is, in the latter case, by in situ fermentation and hydroxylation (single stage fermentation and hydroxylation). The microorganisms and enzymes used herein can be prepared by known means. See, for example, J.C. Hunter-Cevera, M.E. Fonda and A. Belt, Chapter 1: "Isolation of Cultures", Manual of Industrial Microbiology and Biotechnology, edited by A.L. Demain and N.A. Solomon, American Society for Microbiology, Washington, D.C., 3-23 (1986). The size of the inoculum, i.e. the amount of microorganism employed in relation to the volume of the reaction mixture, is selected to allow the catalysis of the enzymatic hydroxylation of the present invention. It is preferred to obtain yields above 20%. Typically, to carry out the process, the size of the inoculum used varies from 1% to 20% of the reaction mixture. Preferably, the inoculum size is 2%.

Fermentation Medium The growth of the microorganism selected for use in the process can be achieved by one of ordinary skill in the art through the use of the appropriate nutrient medium. Suitable means for the growth of microorganisms, include those that provide the nutrients necessary for the growth of microbial cells. See, for example, T. Nagodawithana and J. M. Wasileski, Chapter 2: "Media Design for Industrial Fermentation," Nutritional Requirements of Commercially Important Microorganism, edited by T.W. Nagodawithana and G. Reed, Esteekay Associates, Inc. Milwaukee, Wl, 18-45 (1998); T.L. Miller and B.W. Churchill, Chapter 10: "Substrates for Large Scale Fermentations", Manual of Industrial Microbiology and Biotechnology, edited by A. L. Demain and N.A. Solomon, American Society for Microbiology, Washington D.C., 122-136 (1986). A typical medium for growth includes necessary carbon sources, nitrogen sources and minor elements. You can also add inductors to the medium. The term "inducer" as used herein includes any compound that increases the formation of the desired enzymatic activity within the microbial cell. Typical inducers as used herein may include solvents used to dissolve substrates, such as dimethyl sulfoxide, dimethyl formamide, dioxane, ethanol and acetone. In addition, some substrates such as epothilone B may also be considered to be inducers. Carbon sources may include sugars such as glucose, fructose, galactose, maltose, sucrose, mannitol, sorbitol, glycerol starch and the like.; organic acids such as sodium acetate, sodium citrate and the like; alcohols such as ethanol, propanol and the like, preferred carbon sources include, but are not limited to, glucose, fructose, sucrose, glycerol and starch. The nitrogen sources may include a NZ amine A, starch soaked with liquor, soybean meal, beef extract, yeast extract, tryptone, peptone, cottonseed meal, peanut flour, amino acids such as sodium glutamate. and the like, sodium nitrate, ammonium sulfate and the like. The minor elements may include salts of magnesium, manganese, calcium, cobalt, nickel, iron, sodium and potassium. Phosphates can also be added in small amounts or, preferably, greater than small amounts. The medium used for the fermentation may include more than one source of carbon or nitrogen or another nutrient. The preferred means for growth includes aqueous media, particularly those described in the examples herein. Preferably, the hydroxylation process of the present invention is carried out under submerged aerobic conditions. For the growth of the microorganisms and / or hydroxylation according to the method of the present invention, the pH of the medium is preferably from about 5 to about 8 and the temperature is from about 24 ° C to about 37 ° C. preference the temperature is 28 ° C. The aqueous medium is incubated for a period of time necessary to complete the biotransformation, it is verified by liquid chromatography at high pressure (HPLC). Typically, the period of time necessary to complete the transformation is 12 to 60 hours and preferably about 45 hours after the addition of the substrate. The medium is placed in a rotary agitator (New Brunswick Scientific Innova 5000) which operates at 150 to 300 r.p.m. and preferably at 250 r.p.m. with a 5.08 cm (2 inch) stroke.

Separation and Isolation The hydroxyalkyl bearing product can be recovered from the fermentation broth by conventional means, which are commonly used for the recovery of other known biologically active substances.

Examples of such recovery means include, but are not limited to, isolation and purification by extraction with a conventional solvent, such as ethyl acetate and the like; by adjusting the pH, by treatment with a conventional resin, for example, by treating an anionic or cation exchange resin or a nonionic absorption resin; by treatment with a conventional absorbent, for example, by distillation, by crystallization or by recrystallization and the like. The extract obtained above from the biotransformation reaction mixture can be further isolated and purified by gradient elution column chromatography and analytical thin layer chromatography. The protocol for the extraction of the product from the following examples is described below.

Gradient Elution Chromatography All column chromatography was carried out using a 1.5 cm column (internal diameter) per 20 cm length Spectra / Chrom ™ purchased from Spectrum Medical Industries, Los Angeles, CA. The column was packed in water paste for each experiment. About 16 grams of silica gel (63-200 μm, 70-230 mesh, purchased from EM Separations, New Jersey) was made watery in 75 to 100 ml of hexane or toluene and added to the column in a single pour. The bed was allowed to form and pack under maximum gravity flow. Samples were absorbed into the silica gel before application to the packed column. Linear gradients were formed using a Spectrum gradient elution apparatus consisting of two 500 ml chambers.

Thin Analytical Layer Chromatography (TLC): Aliquots (9 μl) of column fractions were deposited onto pre-coated thin layer chromatography plates Uniplate Silica Gel GHLF (labeled 10 x cm, thickness of 250 microns, purchased from Analtech, Inc., Newark, DE) using disposable 3 μl Nicrocaps pipettes (again, trade name or trademark or the name of a type?). Stained plates were deployed in tanks coated with filter paper equilibrated with the specific eluents. After spraying the plates spread with vanillin (99 parts of 2% vanillin) (weight / volume) / ethanol - 1 part of concentrated sulfuric acid), the compounds were visualized with gentle heating.

Use and Utility The invention is a process by which compounds that are stabilizing agents of microtubules are produced. The compounds and thus the process are useful in the treatment of a variety of cancers and other proliferative diseases including, but not limited to, the following: - carcinoma, including that of the bladder, breast, colon, kidney, liver, lung , ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; - Haematopoietic tumors of the lineage or lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B cell lymphoma, T cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hair cell lymphoma and lymphoma from Burketts; - hematopoietic tumors of the lineage or myeloid line, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; - other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, and osteosarcoma; and - other tumors, including melanoma, xenoderma pigmentosum, keratoactantoma, seminoma, follicular thyroid cancer and teratocarcinoma. The compounds produced by the invention will also inhibit angiogenesis, thereby affecting the growth of tumors and providing treatment of tumors and disorders related to tumors. Such anti-angiogenesis properties of the compounds of formulas I and II, will also be useful in the treatment of other conditions sensitive to antiangiogenesis agents including, but not limited to, certain forms of blindness related to retinal vascularization, arthritis, especially inflammatory arthritis, multiple sclerosis , resinosis and psoriasis. The compounds produced by the invention will induce or inhibit apoptosis, a critical physiological cell death process for normal development and homeostasis. Alterations of apoptotic trajectories contribute to the pathogenesis of a variety of human diseases. The compounds of formulas I and II, as modulators of apoptosis, will be useful in the treatment of a variety of human diseases with aberrations in apoptosis, including, but not limited to, cancer and precancerous lesions, diseases related to the immune response, infections viral diseases, degenerative diseases of the musculoskeletal system and. kidney disease. Without wishing to be limited to any mechanism or morphology, the compounds produced by the invention can be used to treat conditions other than cancer or other proliferative diseases. Such conditions include, but are not limited to, viral infections such as herpes viruses, poxviruses, Epstein-Barr virus, Sindbis virus and adenovirus.; autoimmune diseases such as systemic lupus erythematosus, immune-mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases and autoimmune diabetes mellitus; neurodegenerative disorders such as Alzheimer's disease, dementia related to AIDS, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration; AIDS, myelodysplastic syndromes, aplastic anemia; myocardial infarctions associated with ischemic injury; apoplexy and reperfusion injury; stenosis, arrhythmia; atherosclerosis; toxin-induced or alcohol-induced liver diseases; hematological diseases such as chronic anemia and aplastic anemia; degenerative diseases of the musculoskeletal system such as osteoporosis and arthritis; rhinosinusitis sensitive to aspirin; cystic fibrosis; multiple sclerosis; kidney diseases; and pain from cancer. Thus, the present invention provides a method for treating a subject, preferably mammals and especially humans, in need of treatment for any of the conditions mentioned above, especially cancer or other proliferative diseases, comprising the step of administering to a subject in need thereof, at least one compound of formulas I and II in an amount effective therefor. Other therapeutic agents such as those described below can be employed with the compounds inventive in the present method. In the method of the present invention, other therapeutic agent (s) such as can be administered before, concurrent with, or after administration of the compound (s) of the present invention. The effective amount of a compound produced by the present invention can be determined by one of ordinary skill in the art and includes exemplary dosages for a human from about 0.05 to 200 mg / Kg / day, which can be administered in a single dose or in form of divided individual doses, such as 1 to 4 times per day. Preferably, the compounds are administered in a dose of 100 mg / Kg / day, in a single dose or in 2 to 4 divided doses. It will be understood that the specific dose level and frequency of dosing for any particular subject can be varied and will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and the duration of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, the rate of excretion, the combination of drugs and the severity of the particular condition. Preferred subjects for treatment include animals, preferably mammalian species such as humans and domestic animals such as dogs, cats and the like, which suffer from the disorders mentioned above. The present invention also provides compounds for a pharmaceutical composition, comprising at least one of the compounds produced by the invention, capable of treating cancer or other proliferative diseases in an effective amount therefor and a pharmaceutically acceptable carrier or diluent. The compositions of the present invention may contain other therapeutic agents as described below and may be formulated, for example by using conventional solid or liquid carriers or diluents, as well as pharmaceutical additives of a type appropriate to the desired mode of administration (eg, excipients). , binders, preservatives, stabilizers, flavorings, etc.) according to the techniques, such as those well known in the pharmaceutical formulating art or necessary or required by accepted pharmaceutical practice. The compounds produced by the invention can be administered by any suitable means, for example, in oral form, such as in the form of tablets, capsules, granules or powders.; in sublingual form; oral; parenteral, such as by subcutaneous, intravenous, intramuscular or intrasternal injection or infusion techniques (e.g., as aqueous or non-aqueous, injectable, sterile solutions or suspensions); nasal, such as by inhalation spray; in topical form, such as the form of a cream or ointment or rectally such as in the form of suppositories; in unit dosage formulations containing pharmaceutically acceptable, non-toxic carriers or diluents. The present compounds, for example, can be administered in a form suitable for immediate release or prolonged release. Immediate release or prolonged release can be achieved by the use of suitable pharmaceutical compositions comprising the present compounds or in particular in the case of prolonged release, by the use of devices such as subcutaneous implants or osmotic pumps. The present compounds can also be administered liposomally. For example, the active substance can be used in a composition such as a tablet, capsule, solution or suspension containing from about 5 to about 500 mg per unit dose of a compound or mixture of compounds, produced by the invention or in a form topical (0.01 to 5% by weight of the compound, from 1 to 5 treatments per day). These can be combined or compounded in a conventional manner with a physiologically acceptable carrier or carrier, excipient, binder, preservative, stabilizer, flavoring, etc., or with a topical carrier. The compounds of the invention can also be formulated into compositions such as sterile solutions or suspensions for parenteral administration. From about 0.1 to 500 mg of a compound produced by the invention, it can be combined with a physiologically acceptable carrier, carrier, excipient, binder, preservative, stabilizer, etc., in a unit dosage form as required by accepted pharmaceutical practice. . The amount of active substance in these compositions or preparations is preferably such that a suitable dosage is obtained in the range or range indicated. Exemplary compositions for oral administration include suspensions which may contain, for example, microcrystalline cellulose to bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity booster, buffering agents, solubilizers and sweetening and flavoring agents. such as those known in the art and immediate release tablets which may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and / or lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. Molded tablets, compressed tablets or freeze-dried tablets are exemplary forms that can be used. Exemplary compositions include those which formulate the present compound (s) with fast dissolving diluents such as mannitol, lactose, sucrose and / or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG). Such formulations may also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC), maleic anhydride copolymer (e.g. Gantrez) and agents to control the release such as the polyacrylic copolymer (for example, Carbopol 934). Lubricants, glidants, flavorings, colorants and stabilizers can also be added for ease of manufacture and use. Exemplary compositions for administration of nasal spray or inhalation include solutions in saline solution which may contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to increase bioavailability and / or other solubilizing or dispersing agents such as those known in the art. Exemplary compositions for parenteral administration include injectable solutions or suspensions which may contain, for example, non-toxic parenterally acceptable diluents or solvents, such as cremophor, mannitol, 1,3-butanediol, water, Ringer's solution, a solution of isotonic sodium or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides and fatty acids, including oleic acid. Exemplary compositions for rectal administration include suppositories which may contain, for example, a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at normal temperature but are liquified and / or dissolved in the cavity rectal to release the drug. Exemplary compositions for topical administration include a topical carrier such as Plastibase (mineral oil gelled with polyethylene). For example, the compounds of the invention can be administered topically to treat plaques associated with psoriasis and as such, can be formulated as a cream or ointment. The compounds of the invention can be administered alone or in combination with other anticancer and cytotoxic agents and treatments useful in the treatment of cancer or other proliferative diseases. Especially useful are combinations of anticancer and cytotoxic drugs wherein the second selected drug acts in a different manner or different phase of the cell cycle, for example the S phase, than the present compounds of formulas I and II which exert their effects in the G2-M phase. Typical examples of anticancer and cytotoxic agents include, but are not limited to: alkylating agents, such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylene imines and triazenes, antimetabolites, such as folate antagonists, purine analogs and analogues of pyrimidine; antibiotics, such as anthracyclines, bleomycins, mitomycin, dactinomycin and plicamycin; cyclin-dependent kinase inhibitors, such as flavopiridol; enzymes, such as L-asparaginase; farnesyl protein transferase inhibitors; hormonal agents such as glucocorticoids, estrogens / antiestrogens, androgens / antiandrogens, progestins and hormone antagonists, which release luteinizing hormone, ocherotide acetate; microtubule-dissociating agents, such as ecteinascidins or other analogs and derivatives; microtubule stabilizing agents such as paclitaxel (Taxol®), docetaxel (Taxotere®); plant derived products, such as vinca alkaloids, epipodophyllotoxins, taxanes and topoisomerase inhibitors, prenyl protein transferase inhibitors and various agents such as hydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinum coordination complexes such as cisplatin and carboplatin and other agents such as cytotoxic anticancer agents such as biological response modifiers, growth factors; immune modulators and monoclonal antibodies. The compounds of the invention can also be used in conjunction with radiation therapy.

Representative examples of these classes of anticancer and cytotoxic agents include, but are not limited to, mechlorethamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan, carmustin, lomustin, semustin, steptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribin, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, safracin, saframycin, quinocarcin, discodermolide, vincristine, vinblastine, vinorelbine tartrate, etoposide, teniposide, paclitaxel, tamoxifen, estramustine, estramustine sodium phosphate, flutamide, buserelin, leuprolide, pteridines, diinesis, levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride, betamethasone, gemcitabine hydrochloride, altretamine and a library and some analogs or derivatives of the m isms. Preferred members of these classes include, but are not limited to paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, metopterin, mitomycin C, ecteinascidin 743, porphyromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podophyllotoxin derivatives such as etoposide, etoposide or teniposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine and leurosin. Examples of anticancer agents and other cytotoxic agents include the following: epothilone derivatives as found in German Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890 and WO 99/28324; the cyclin-dependent kinase inhibitors as found in WO 99/24416; and prenyl protein transferase inhibitors as found in WO 97/30992 and WO 98/54966. The combinations of the present invention may also be formulated or co-administered with other therapeutic agents that are selected for their particular utility in administration to therapies associated with the conditions mentioned above. For example, the compounds of the invention can be formulated with agents to prevent nausea, hypersensitivity and gastric irritation, such as antihistamines and antihistamines Hi and H2. The above therapeutic agents when used in combination with the compounds of the present invention can be used in those amounts indicated in the Physicians' Desk Reference (PDR) or as otherwise determined by one of ordinary skill in the art.

Preferred Compounds A preferred embodiment of the present invention, epothilone F is obtained by fermentation of epothilone B with the microorganism Amicolata autotrophica ATCC 35203. The epothilones B and F are antitumor agents useful in treatment of cancers in humans and have the following structures Chemicals: Epothilone B Epothilone F In another preferred embodiment of the invention, epothilone F is obtained by fermentation of epothilone B with the microorganism Actinomycete sp. strain SC15847 PTA-1043.

While epothilone B and epothilone F can be used, in and of themselves, as the end-point product, ie, as pharmaceutically active agents, the invention contemplates that the epothilone product of the present process can be used to prepare other pharmaceutically active epothilones or analogous derivatives thereof. For example, the epothilone product of the present process can be used to prepare the epothilone analogues described in DE 199 07 588.3, the text of which is incorporated herein as being summarized in detail or the examples set forth herein, in which epothilone F is used as the starting material or intermediate. In a preferred embodiment of the invention, the process is used to prepare epothilones having lower hydroxyalkyl or substituted lower hydroxyalkyl substituents. In a preferred embodiment of the invention, in formulas I and III, n is zero and m is 1. In a more preferred embodiment, in formulas I and III, n is zero, m is 1 and A2 is alkenyl. Those skilled in the art will be able to optimize culture conditions, including inoculum size, composition of the medium, reaction conditions such as temperature, aeration, agitation, pH and time, the solvents used to dissolve the substrate and the concentrations using known methods and the general methods of preparation described herein. All references cited herein with respect to the synthesis, preparation and analytical procedures are incorporated by reference as set forth in detail herein. The following examples are provided for the purpose of illustrating the present invention and should not be construed as limiting the scope or spirit of the present invention.

EXAMPLE 1 Biotransformation of Epothilone B. to Epothilone F Microorganism and Culture Conditions A frozen flask (approximately 2 ml) of Amycolata autotrophica ATCC 35203 was used to inoculate a 500 ml flask containing 100 ml of the transformation medium. The transformation medium consisted of 10 g of dextrose (Em Science, Gibbstown, NJ), 5.0 g of polypeptone, 3.0 g of yeast extract (Difco, Detroit, MI) and 3.0 g of malt extract (Difco, Detroit, MI). ) in one liter of deionized water. Ten flasks were inoculated and the cultures were incubated at 28 ° C and 250 r.p.m. for 24 hours. A total of 840 ml of the resulting culture was combined in a 2 liter flask, to which 610 mg of the substrate, epothilone B, was added in 25 ml of ethanol. The culture was then divided into 42 250 ml flasks (approximately 20 ml of culture per flask) and incubated at 28 ° C and 250 rpm The conversion of epothilone B to epothilone F in the culture was verified by HPLC to determine the time in which no additional production of epothilone F was observed. The maximum conversion yield was obtained in approximately 45 hours after the addition of epothilone B to the culture.

Isolation and Purification of [1S- [1R *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-Dihydroxy-8, 8, 10, 12, 16- pentamethyl-3- [l-methyl-2- (2-hydroxymethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione) The reaction culture by biotransformation, above, it was rinsed in a 4 liter beaker with deionized water to produce approximately 2 liters of liquid. The rinsed reaction liquid was mixed vigorously with 2 liters of ethyl acetate using a magnetic stirrer. After 2 hours approximately 500 ml of Dicalite auxiliary filters (diatomaceous earth, Grefco Minerals, Torrance CA) were added and the resulting mixture was filtered. The filtrate was transferred to a 6-liter separatory funnel and in the separation phase, the lower aqueous layer was discarded. The upper organic phase was concentrated to dryness in a rotary evaporator to give a crude extract. The crude extract was then subjected to column chromatography using a linear gradient of 1 liter of hexane to 50% acetone in hexane. A total of 20 50 ml fractions was collected. After analysis with TLC, fractions 10 and 11 were accumulated and evaporated to yield 88 mg of epothilone B. Fraction 15 was evaporated to produce 71 mg of epothilone F. Fractions 16 and 17 were collected and evaporated to give a mixture of epothilone F and 26-hydroxyepotilone B (130.3 mg). The total synthesis of 26-hydroxyepothilone B has been previously reported (K.C. Nicolau et al, Tetrahedron, 54, 7127-7166 (1998)). The mixture of epothilone F and 26-hydroxyepothilone B was further purified by column chromatography using a linear gradient of one liter of hexane to 80% ethyl acetate in hexane. A total of 40 25 ml fractions were collected. After analysis with TLC, fractions 14 to 19 were accumulated and evaporated to give 55.4 mg of epothilone F. Fractions 20 to 25 were pooled and evaporated to give the 26-hydroxyepothilone B contaminated with a small amount of epothilone F.

The last mixture of epothilone F and 26-h droxiepotilone B was further refined by column chromatography using a gradient of 700 ml of toluene to 35% acetone in toluene. A total of 20 35 ml fractions were collected. Analysis with TLC showed that fractions 14 and 15 contained pure epothilone F, while fractions 18 to 20 contained pure 26-hydroxyepotilone B. The total yield of epothilone F, calculated from the amount of epothilone F recovered (126.4 mg) to the amount of starting substrate, epothilone B (610 mg), was 20.7%.

Example 2 Biotransformation of Epothilone B to Epothilone F Microorganism and Culture Conditions A frozen flask (approximately 2 ml) of Actinomycete sp. strain PTA-1043 to inoculate a 500 ml flask containing 100 ml of the medium. The vegetative medium consists of 20 g of dextrose (EM Science, Gibbstown, NJ), 10 g of malt extract (Difco, Detroit, MI), 10 g of yeast extract (Difco, Detroit, MI) and 1 g of peptone (Difco, Detroit, MI) in one liter of water deionized. The vegetative culture was incubated for 3 days at 28 ° C on a rotary shaker operating at 250 rpm, and 1 ml of the resulting culture was added to each of the 62 500 ml flasks containing the transformation medium, which has the same composition as the vegetative medium. The cultures were incubated at 28 ° C and 250 r.p.m. for 24 hours. Epothilone B (4.96 g) was dissolved in 155 ml of ethanol and the solution was distributed to the sixty-two flasks. The flasks were then returned to the agitator and incubated for an additional 43 hours at 28 ° C and 250 r.p.m. The reaction culture was then processed for the recovery of epothilone F.

Removal and Purification of [1S- [IR *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-Dihydroxy-8, 8, 10, 12, 16-pentamethyl-3- [l-methyl-2- (2-hydroxymethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadesan-5, 9-dione) The biotransformation reaction culture The previous one was rinsed in a 10-liter polypropylene bucket with deionized water to produce approximately four liters of liquid. The rinsed reaction liquid was vigorously stirred with four liters of ethyl acetate. After one hour of mixing, approximately one liter of Dicalite auxiliary filter is added (diatomaceous earth, Grefco Minerals, Torrance CA) and the resulting mixture was filtered on a bed of filter aid (Dicalite, Grefco Minerals, Torrance CA). The filtrate was transferred to a 20 liter separatory funnel and the phases allowed to separate. The solids were extracted once with four liters of acetone and filtered. The aqueous acetone filtrate was concentrated to approximately 1 liter and extracted three times with one liter aliquots of ethyl acetate. All ethyl acetate extracts were accumulated and concentrated to dryness under vacuum to yield 6.7 g of residue. This residue was preabsorbed on 6 g of silica gel and subjected to column chromatography (60 g of silica gel, column of 2.5 cm internal diameter x 30 cm in length Spectra / Chrom equipped with an adjustable end fitting). The column was eluted with a linear gradient of two liters of hexane to 50% acetone in hexane. A total of twenty 100 ml fractions were collected. After TLC, fractions 13 to 20 were accumulated and evaporated to yield 1.65 g of epothilone F. The total yield of epothilone F, calculated from the amount of epothilone F recovered (1.65 g) to the amount of starting substrate , epothilone B (4.96 g) was 33.3%.

Example 3 The following syntheses provide examples wherein the hydroxyalkyl bearing product of the invention has been used as an intermediate or as the starting material for preparing other analogs or epothilone derivatives. Epothilone analogs or derivatives prepared from hydroxyalkyl-bearing epothilones are described in DE 19907588.3, the text of which is incorporated herein as being summarized in detail.

A. Synthesis of [ÍS- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-Dihydroxy-8, 8, 10, 12, 16- pentamethyl-3-- [l-methyl-2- (2-azi-domethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione To a stirred solution of epothilone ( 957 mg, 1828 mmol) in 20.0 ml of tetrahydrofuran at 0 ° C under argon atmosphere were added 0.47 ml of diphenylphosphoryl azide (604 mg, 2194 mmol, 1.2 equivalents). The mixture was stirred for about 3 minutes, then 1,8-diaza-bicyclo [5.4.0] undec-7-ene (0.27 ml, 278 mg, 1828 mmol, 1 equivalent) was added and the resulting mixture was stirred at 0 ° C. ° C. After two hours, the mixture was heated to 25 ° C and stirred for an additional twenty hours. The reaction mixture was diluted with 150 ml of ethyl acetate and washed with 50 ml of water. The aqueous layer was extracted with 35 ml of ethyl acetate. The combined organic layers were dried over Na2SO4 and concentrated under vacuum. The crude material was subjected to chromatography using silica gel eluted with 50% ethyl acetate in hexane to give 913 mg (91%) of 21-azido-epothilone B, as a clear, colorless oil. MS (ESI +): 549.3 (M + H) +.

B. Synthesis of [ÍS- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-dihydroxy-8, 8, 10, 12, 16- pentamethyl-3- [l-methyl-2- (2-ami-nomethyl-4-thiazolyl) ethenyl] -4,17-dioxabisyclo [14.1.0] hepta-decan-5, 9-dione To a stirred solution of the product of azide (from section A above) (1070 g, 1950 mmol) in 30.0 ml of tetrahydrofuran under argon atmosphere are added 0.22 ml of trimethylphosphine (0.163 g, 2145 mmol, 1.1 equivalents) and 5.5 ml of water. The mixture is allowed to stir at room temperature for 3 hours. The azide was completely consumed and 3 ml of NH4OH (aq) were added to 28% to complete the conversion of phosphorylamine to amine. After stirring at room temperature for one hour, the solvent (s) was (r) separated under vacuum. The crude material was subjected to chromatography using silica gel eluting with 1% Et3N, 2.5% MeOH in CHC13 to yield 924 mg (91%) of a white solid. In addition to the aforementioned epothilone analogs and derivatives, the following compounds can be made from the hydroxyalkyl-bearing epothilones of the present invention: [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [l-methyl-2- (2-n-pro? Ionyl-aminomethyl-4- thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] hepta-decan-5, 9-dione; [lS- [lR *, 3R * (E), 7R *, 10S *, llR *, 12R *, 16S *]] - 7, 11-di-hydroxy-8, 8,10, 12-tetramethyl-3- [L-methyl-2- (2-n-pentanoyl-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] -heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12, tetramethyl-3- [L-methyl-2- (2-chloromethyl-4-thiazolyl) ethenyl] -4,17-dioxoabicyl [14.1.0] heptadecan-5,9-dione; [l- [lR * f 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, ll-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [L-methyl-2- (2-aminomethyl-4-thiazoyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione. [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8,10, 12-tetramethyl-3- [l-methyl-2- (2-azidomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione and [1S- [IR *, 3R * (E)], 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [l-methyl-2- (2-hydroxymethyl-4 -thiazolyl) ethenyl-4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione.

Example 4 Characterization of Epothilone F The epothilone F of this invention was identified by NMR spectroscopy and has the following characteristics: Description: White crystalline solid Melting Point: 141-143 ° C Molecular Formula: C27H4? N07S Molecular Weight: 523 NMR: observed chemical changes Solvent CDC13 (7.24, 77.0) Bruker DRX-500: proton 500.13 MHz, carbon 125.76 MHz Proton position Pattern C-l3 1 - - 170.5 2 2.45 m 39.19 2 2.30 m 3 4.12 d, J = 9.3 73.00 4 - - 52.95 5 - - 170.2 6 3.21 m 43.10 7 3.68 m 74.31 1.63 m 36.41 Proton position Pattern C-13 9 1.35 m 31.22 1.37 m 32.20 11 1.63 m 30.74 11 1.37 m 12 31.34 13 2.72 m 61.54 14 1.99 m 31.99 14 1.86 m 15 5.36 m 77 16 -. 16 - 137.72 17 6.52 s 119.50 18 -. 18 - 152.17 19 7.03 s 116.92 twenty - . 20 - 170.20 21 4.83 s 62.01 22 1.30 s 22.50 23 0.99 s 21.36 24 1.08 d, J = = 6. .90 13.80 0.92 d, J = = 6., 65 17.11 26 1.20 s 22.74 27 2.00 s 15.74 3-OH 3.98 s It is noted that, in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (6)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for the preparation of at least one epothilone of the following formula I H0-CH2 (A?) N- (Q) m- (A2) or E (I) wherein: and A2 are independently selected from the group of alkyl and alkenyl of 1 to 3 carbon atoms optionally substituted; Q is an optionally substituted ring system containing one to three rings and at least one carbon-carbon double bond in at least one ring; n, m and o are integers selected from the group consisting of 0 and 1, wherein at least one of m or n o is l and E is a nucleus of epothilone; characterized in that it comprises the steps of contacting at least one epothilone of the following formula II CH3- (A?) n- (Q) m- (A2) oE (ID wherein Ai, Q, A2, E, n, myo are as defined above, with a microorganism or an enzyme derived therefrom, which is capable of selectively catalyzing the hydroxylation of a compound of formula II to one of formula I and effecting such hydroxylation 2. The method according to claim 1 1, characterized in that n is zero and m is 1. 3. The method according to claim 1, characterized in that n is zero, m is 1 and A2 is alkenyl. The method according to claim 1, characterized in that such epothilone of formula I is epothione F and such epothilone of formula II is epothilone B. 5. The method according to claim 1, characterized in that the microorganism is selected from the group which consists of Actinomycete, Amycolata, Amycola topsis, Beauveria, Candida, Gilbertella, Nocardia, Pseudomonas, Saccharopolyspora, Saccharothrix and Streptomyces. 6. The method according to claim 1, characterized in that the microorganism is selected from the group consisting of Amycolata autotrophica, Beauveria basiana, Candida rugosa and Pseudomonas putida. 1 . The method according to claim 5, characterized in that the microorganism is selected from the group consisting of Amycola ta autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043. 8. The method according to claim 7, characterized in that the microorganism is Actinomycete sp. strain SC 15847 PTA-1043. The method according to claim 1, characterized in that said enzyme is derived from a microorganism selected from the group consisting of Actinomycete, Amycola ta, Amycola topsis, Beauveria, Candida, Gilbertella, Nocardia, Pseudomonas, Saccharopolyspora, Saccharothrix and Streptomyces. The method according to the claim 1, characterized in that said enzyme is derived from a microorganism selected from the group consisting of Amycola ta autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043. 11. The method according to the claim 1, characterized in that a pharmaceutically active epothilone or derivative or analogue thereof used to treat a mammal is finally prepared by such a method. The method according to claim 1, characterized in that the epothilone product derived therefrom is further reacted to prepare another pharmaceutically active epothilone. The method according to claim 1, characterized in that the epothilone product is used to prepare a pharmaceutically active epothilone selected from the group consisting of: [ÍS- [IR *, 3R * (E), 7R *, IOS * , 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12, 16-pentamethyl-3- [l-methyl-2- (2-azidomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] hepta-decan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12, 16-pentamethyl- 3- [1-methyl-2- (2-aminomethyl-4-thiazolyl) ethenyl] -A, 17-dioxabicyclo [14.1.0] heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] - 7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [l-methyl-2- (2-n-propionyl-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] he- ptadecan-5,9-dione; [1S- [IR *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [L-methyl-2- (2-n-pentanoyl-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] hep-tadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]], 7,11-di-hydroxy-8, 8,10, 12-tetramethyl-3- [L-methyl-2- (2-chloromethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [1.1.0] heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8,10, 12-tetramethyl-3- [L-methyl-2- (2-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] - 7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [l-methyl-2- (2-azidomethyl-4-thiazolyl) ethenyl-4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione and [1S- [1R *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [l-methyl-2- (2-hydroxymethyl-4- thiazolyl) ethenyl-4,17-dioxabicyclo- [14.1.0] heptadecan-5,9-dione. 14. A method for the preparation of at least one hydroxyalkyl-bearing epothilone of the following formula III: where: Q is selected from the group consisting of Gi is the following formula V HO-CH2- (A?) N- (Q) m- (A2) or (V) where: Ai and A2 are independently selected from the group of alkyl and alkenyl of 1 to 3 carbon atoms optionally substituted; Q is an optionally substituted ring system containing one to three rings and at least one carbon-carbon double bond in at least one ring; n, m and o are integers selected independently of the group consisting of zero and 1, where at least one of m or n or is 1; W is O or NR6; X the group consisting of O is selected; H, OR7; M is O, S, NR8, CR9R? 0; Bi and B2 are selected from the group consisting of ORn, 0C0R? 2; R1-R5 and? 2-7 are selected from the group consisting of H, alkyl, substituted alkyl, aryl and heterocycle and wherein, when Ri and R2 are alkyl, they can be joined to form a cycloalkyl; Re is selected from the group consisting of H, alkyl and substituted alkyl; R and Rn are selected from the group consisting of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl and dialkylarylsilyl; Rs is selected from the group consisting of H, alkyl, substituted alkyl, R? 3C = 0, R? 4OC = 0 and R? 5S02; R9 and Rio are selected from the group consisting of H, halogen, alkyl, substituted alkyl, aryl, heterocycle, hydroxy, Ri6C = 0 and R? 7OC = 0; the pharmaceutically acceptable salts thereof and any hydrates, solvates or geometrical, optical and stereoisomeric isomers thereof; characterized in that it comprises the steps of contacting at least one epothilone of the following formula IV: where: Q, W, X, M, Bi, B2 and R? ~ R? they are as defined above; G2 is the following formula VI CH3- (A?) N- (Q) m- (A2) 0 (VI) wherein Ai, Q, A2, n, m and o are as defined above; the pharmaceutically acceptable salts thereof and any hydrates, solvates or geometrical, optical and stereoisomeric isomers thereof; with a microorganism or enzyme derived therefrom capable of selectively catalyzing the hydroxylation of G2 to Gi and effecting such hydroxylation. The method according to claim 14, characterized in that the epothilone of formula III is epothilone F and the epothilone of formula IV is epothilone B. 16. The method according to claim 14, characterized in that n is zero and m is 1 . eleven .' The method according to claim 14, characterized in that n is zero, m is 1 and A2 is alkenyl. The method according to claim 14, characterized in that the microorganism is selected from the group consisting of Actinomycete, Amycola ta, Amycola topsis, Beauveria, Candida, Gilbertella, Nocardia, Pseudomonas, Saccharopolyspora, Saccharothrix and Streptomyces. The method according to claim 14, characterized in that the microorganism is selected from the group consisting of Amycola ta autotrophica, Beauveria basiana, Candida rugosa and Pseudomonas putida. The method according to claim 14, characterized in that the microorganism is selected from the group consisting of Amycolata autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043. 21. The method in accordance with the claim 14, characterized in that the microorganism is Actinomycete sp. strain SC 15847 PTA-1043. 22. The method according to claim 14, characterized in that it comprises a biotransformation under aerobic conditions immersed in an aqueous medium. 23. The method according to the claim 15, characterized in that the aqueous medium contains carbon and nitrogen nutrients at a pH of about 5-8. 24. A method for the hydroxylation of a terminal alkyl group of an epothilone, characterized in that a microorganism or a mutant thereof is used. 25. The method according to claim 24, characterized in that the microorganism is selected from the group consisting of Actinomycete, Amycola ta, Amycolatopsis, Beauveria, Candida, Gilbertella, Nocardia, Pseudomonas, Saccharopolyspora, Saccharothrix and Streptomyces. 26. The method according to claim 24, characterized in that the microorganism is selected from the group consisting of Amycola ta autotrophica, Beauveria basiana, Candida rugosa and Pseudomonas putida. 27. The method according to claim 24, characterized in that the microorganism is selected from the group consisting of Amycola ta autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043. 28. The method according to claim 24, characterized in that the microorganism is Actinomycete sp. strain SC 15847 PTA-1043. 29. A method for the preparation of at least one epothilone carrying hydroxyalkyl from an epothilone having a terminal alkyl group using a microorganism or an enzyme derived therefrom, characterized in that it comprises the steps of: (a) fermenting a microorganism or an enzyme derived therefrom grown in a medium in the presence of an epothilone having a terminal alkyl group; (b) isolating the resulting hydroxyalkyl-bearing epothilone product. 30. The method according to claim 30, characterized in that the microorganism is Amicola ta autotrophica ATCC 35203. 31. The method according to claim 30, characterized in that the microorganism is Actinomycete sp. strain SC 15847 PTA-1043. 32. The method according to claim 14, characterized in that the enzyme is derived from a microorganism selected from the group consisting of Actinomycete, Amycola ta, Amycola topsis, Beauveria, Candida, Gilbertella, Nocardia, Pseudomonas, Saccharopolyspora, Saccharothrix and Streptomyces. 33. The method according to claim 14, characterized in that the enzyme is derived from a microorganism selected from the group consisting of Amycola ta autotrophica ATCC 35203 and Actinomycete sp. strain SC 15847 PTA-1043. 34. The method according to claim 14, characterized in that a pharmaceutically active epothilone or derivative or analogue thereof used to treat a mammal is finally prepared by such a method. 35. The method according to claim 14, characterized in that the epothilone product derived therefrom is further reacted to prepare another pharmaceutically active epothilone. 36. The method according to claim 14, characterized in that the epothilone product is used to prepare a pharmaceutically active epothilone selected from the group consisting of: [1S- [1R *, 3R * (3), 7R *, IOS * , 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12, 16-pentamethyl-3- [l-methyl-2- (2-azidomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12, 16-pentamethyl- 3- [1-methyl-2- (2-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione; [1S- [IR *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [L-methyl-2- (2-n-propionyl-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] -heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [L-methyl-2- (2-n-pentanoyl-aminomethyl-4-thiazole) ethenyl] -4,17-dioxabicyclo [14.1.0] hepta-decan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [l-methyl-2- (2-chloromethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dicna; [1S- [1R *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8,10, 12-tetramethyl-3- [L-methyl-2- (2-aminomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione; [1S- [1R *, 3R * (E), 7R *, 10S *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8, 10, 12-tetramethyl-3- [L-methyl-2- (2-azidomethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione; and [ÍS- [IR *, 3R * (E), 7R *, IOS *, 11R *, 12R *, 16S *]] -7, 11-di-hydroxy-8, 8,10, 12-tetramethyl-3 - [L-methyl-2- (2-hydroxymethyl-4-thiazolyl) ethenyl] -4,17-dioxabicyclo [14.1.0] heptadecan-5,9-dione.