MXPA01001459A - Process for the preparation of c-4 deacetyltaxanes - Google Patents

Abstract

A process useful for the preparation of intermediates in synthesis or semi-synthesis of paclitaxel analogs wherein a starting taxane such as 10-deacetylbaccatin III is deacetylated at the C-4 position using a microorganism or an enzyme derived therefrom to provide 4-deacetyltaxanes, such as 4,10-dideacetylbaccatin III.

Description

PROCEDURE FOR THE PREPARATION OF DEACETILTAXANES WITH FOUR CARBON ATOMS Field of the Invention The present invention is directed to a process using microorganisms or enzymes derived therefrom for the deacetylation of the taxanes at C-4 to give the 4-deacetyltaxanes, which are useful intermediates for the synthesis of new anti-cancer agents.

Background of the Invention The taxanes are diterpene compounds which find utility in the pharmaceutical field. For example, taxanes containing aryl heterocyclic or cycloalkyl groups on the side chain with C-13 find utility as anticancer agents. The taxanes include paclitaxel, cephalomannin, taxol c, 10-desacetylpaclitaxel, 10-desacetyl-cefalomanin, 7-β-xylosylpaclitaxel, baccatine-III, 10-desacetylbaccatine III, 7-β-xylosyl-10-deacetyl cephalomannin, 7-β-xylosyl-10-deacetylbaccatin III, 7-β-xylosylbaccatine III, and -desacetyl-taxol c. Ref.126985 Paclitaxel (Taxol), a diterpene-taxane compound, is a natural product extracted from the bark of the Pacific yew tree, Taxus Brevifolia. It has been shown to have excellent anti-tumor activity in animal models in vivo, and recent studies have clarified its unique mode of action, which involves the abnormal polymerization of tubulin and the alteration of mitosis during the cell cycle. Taxol has recently been approved for the treatment of advanced refractory ovarian cancer, breast cancer, non-small cell lung cancer, and more recently, Kaposi's sarcoma related to AIDS. The results of clinical trials of paclitaxel are replete in scientific journals and have been reviewed by numerous authors, such as Rowins and Donehower in "The Clinical Pharmacology and Use of Antimicrotubule Agents in Cancer Chemotherapeutics," Phamac. Ther., 52, pp. 35-84 (1991); Spencer and Faulds, Paclitaxel, A Review of its Pharmacodynamic and Pharmacokinetic Properties and Therapeutic Potential in the Treatment of Cancer, Drugs, 48 (5), p. 794-847 (1994); K. C. Nicolau et al., Chemistry and Biology of Taxol, Angew. Chem., Int. Ed. Eng., 33, pp. 15-44 (1994); FA Holmes, AP Kudelka, JJ Kavanaugh, MH Huber, JA Ajani, and V. Valero, "Taxane Anticancer Agents - Basic Science and Current Status", edited by Gunda I. Georg, Thomas C. Chen, Iwao Ojima, and Dolotrai M Vyas, pp. 31-57 American Chemical Society, Wash., D.C. (nineteen ninety five); Susan G. Arbuck and Barbara Blaylock, "Taxol (Science and Applications)", edited by Matthew Suffness, pp. 379-416, CRC Press, Boca Raton, FL (1995) and the references cited there. The structure of Taxol® is subsequently shown in the company of the conventional numbering system for the molecules belonging to the Taxano class; such a numbering system is also used in this application.

With reference to the taxane numbering, the reference to a particular carbon on the structure of the taxane will be indicated throughout this application by a "C number", which means the carbon on the taxane according to the previous numbering system. For example, "C-13" refers to the carbon at position 13 on the taxane ring as shown above, which has a side chain coupled thereto. The structural unit of the central skeleton of paclitaxel is baccatin III, a diterpenoid that has the chemical structure: It is also very similar in structure to 10-desacetylbaccatine III ("10-DAB III"), which has the chemical structure: but which lacks an acetate ester in the alcohol of position 10. The chemical modification of the structure of paclitaxel in C-4 and other positions has been explored by many groups to determine the relationships of structure / activity and to try to obtain compounds with efficacy superior to taxol for development as second generation drugs. See Pat. U.S. No. 5,773,461; Gunda I. George, Syed M. Ali, Thomas C. Boge, Apurba Datta, and Lise Falborg, "Selective C-2 and C-4 Deacilation of Taxol: The First Synthesis of a C-4 Substituted Taxol Analogue", Tetrahedron Let ., 35:48, pp. 8931-8934 (1994); Shu-Hui Chen. John F. Kadow, Vittorio Fariña, Craig R. Fairchild and Kathy A. Johnston, "First Synthesis of Novel Paclitaxel (Taxol) Analogs Modified at the C-4 Position", J. Org. Chem. 59, pp. 61-56-6158 (1994); S. Py, and F. Khuong-Huu, "A Novel Rearrangement of The Taxane Skeleton", Bull. Soc. Chim. Fr., 130, pp. 189-191 (1993). The replacement of the C-4-acetyl group of paclitaxel with other substituents has led to compounds with improved potency in the activity assays (S. Chen et al., Biorganix and Medicinal Chemistry Letters, 5: 2741-2748 (1995)). ). An enzyme capable of specifically removing the C-4-acetyl group of the taxanes will be useful in the synthesis of paclitaxel analogs modified with C-4 to provide a starting material to allow the incorporation of other groups in this position, by Examples are esters of butyrate with C-4, esters of cyclobutyl with C-4, esters of propyl with C-4, esters of cyclopropyl with C-4 and carbonates of methyl and ethyl with C-4.

Description of the Drawings Figure 1 illustrates the effect of methanol concentration on deacetylation at C-4 by strain SC16249; Figure 2 illustrates the effect of methanol concentration on deacetylation with C-4 by strain SC 16250.

Description of the invention It is an object of the present invention to provide a new, useful and efficient protocol for the preparation of 4-deacetyltaxanes. Another object of the present invention is the provision of a method using microorganisms or enzyme derived therefrom for the deacetylation of the taxanes at the C-4 position to provide the 4-desacetyltaxanes. A further object of the present invention is the provision of a simple, efficient, and cost-effective protocol for the provision of the second generation taxol analogs having several substituents at the C-4 position. Accordingly, the present invention encompasses a novel method whereby 10-desacetyl baccatine III can be efficiently converted to 4,10-didesacetylbaccatin III using a microorganism or one or more enzymes derived from the microorganism. The resulting 4,10-didesacetylbaccatin III compound can then be used as part of the novel processes for the synthesis and semi-synthesis of paclitaxel analogues. The present disclosure is broadly directed to a process for efficient deacetylation of 10-DAB III at the C-4 position. Deacetylation at C-4 occurs as a result of biotransformation caused by a microorganism or an enzyme derived from said microorganism. More specifically, the present invention is directed to the use of an organism that transforms 10-DAB III into 4, 10-didesacetylbaccatine III. The terms "enzymatic process" or "enzymatic method", as used herein, denote a method or method of the present invention that employs an enzyme or a microorganism. The use of "an enzyme or a microorganism" in the present method includes the use of one, as well as two or more, enzymes or microorganisms.

The term "taxane", as used herein, denotes compounds having a taxane portion as described below. The term "taxane portion", as used herein, denotes portions containing the core structure (with the numbering of the ring system positions used herein, shown): such a structure of the core can be replaced and as such can contain an ethylenic unsaturation in the ring system thereof. Such portions having an oxetane ring fused at positions 4 and 5, such as those found in paclitaxel, are preferred. The enzyme or microorganism employed in the present invention can be an enzyme or microorganism capable of catalyzing the enzymatic hydrolysis described herein. Enzymatic or microbial materials, regardless of origin or purity, can be employed in the free state or immobilized on a support such as by adsorption or physical entrapment.

Exemplary microorganisms, which have been identified through a selection process, include Rhodococcus sp. ATCC 202192 (SC 16249) and Rhodococcus sp. ATCC (SC 16250). The term "ATCC" as used herein refers to the access number of the American Type Culture Collection, 10801 University Blvd., Manassas, VA, the depositor for the agency referred thereto. The above ATCC 202192 and ATCC 202191 microorganisms were deposited on January 14, 1999. The term "SC" denotes the designation given to the microorganism as part of the Squibb culture collection. It should be understood that the mutants of the biologically pure ATCC 202192 (SC 16249) and ATCC 202191 (SC 16250) microorganisms are also contemplated by the present invention for use in the biotransformation described herein, such as those modified by the use of chemical means, physical (for example, X-rays), or biological (for example, molecular biology techniques). Rhodococcus sp. ATCC 202192 (SC 16249) and Rhodococcus sp. ATCC 202191 (SC 16250) can be grown on medium 0.5% toasted nutrisoya, 2% glucose, 0.5% yeast extract, 0.5% K2HP04, 0.5% NaCl, adjusted to pH 7 with HCl. The organisms were isolated from the soil (from a sample of Parsippany, N.J.), and are non-mobile, gram-positive shoots with an aerobic requirement. 0 Preferred enzymes include those derived from microorganisms, particularly those microorganisms described above. Enzymes can be isolated, for example, by extraction and purification methods, such as ion exchange chromatography, followed by hydrophobic interaction chromatography and gel filtration. The present invention also provides enzymes capable of the present hydrolysis which can be isolated from Rhodococcus sp. ATCC 202192 (SC 16249) and Rhodococcus sp. ATCC 202191 (SC 16250), for example by the prior art. Where microorganisms were employed, the cells may be used in the form of intact moist cells or dry cells such as heat-dried, or spray-dried, lyophilized cells, or in the form of a treated cellular material such as broken cells. or altered or cellular extracts. The use of genetically designed organisms is also contemplated. The host cell can be any cell, for example from 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 deacetylation process of the present invention can be carried out subsequent to the fermentation of the microorganism (two-step fermentation and hydrolysis), or concurrently with it, that is, in the latter case, by fermentation in situ and hydrolysis (single stage fermentation and hydrolysis). The growth of the microorganisms can be achieved by a person with ordinary skill in the art by the use of an appropriate medium. Suitable means for growing microorganisms include those which provide the nutrients necessary for the growth of microbial cells. A typical medium for growth includes carbon sources, nitrogen sources, and elements (for example in trace amounts). You can also add inductors. The term "inducer", when used herein, includes any compound that enhances the formation of the desired enzymatic activity within the microbial cell. The sources of the carbon may include sugars such as maltose, lactose, glucose, fructose, glycerol, sorbitol, sucrose, starch, mannitol, propylene glycol, and the like; organic acids such as sodium acetate, sodium citrate, and the like; and alcohols such as ethanol, propanol and the like. Nitrogen sources may include NZ amine A, corn infusion liquor, soybean meal, beef extracts, yeast extracts, molasses, baker's yeast, tryptone, nutrisoya , peptone, yeast amine, amino acids such as sodium glutamate and the like, sodium nitrate, ammonium sulfate and the like. Trace elements may include the magnesium, manganese, calcium, cobalt, nickel, iron, sodium and potassium salts. The phosphates can also be added in trace amounts or, preferably, in larger amounts than trace amounts. The general biotransformation method described herein that utilizes the microorganism mentioned above, can be illustrated according to the following scheme of the reaction: The specific examples that follow illustrate the synthesis of the representative compounds of the present invention and will not be proposed as limiting the invention in its sphere or scope. The methods can be adapted to the variations to produce intermediates and the compounds encompassed by this invention but not specifically described. In addition, variations of the methods for producing the same compounds in a somewhat different way may also be apparent to one skilled in the art.

Example 1 Deacetylation of 10-deßacetilbaccatine III Medium: 0.5% toasted nutrisoya, 2% glucose, 0. 5% yeast extract, 0.5% K2HP04. 0.5% NaCl, adjusted to pH 7 with HCl (R.V. Smith and J.P. Rosazza, Arch. Biochem. Biophys., 161, 551-558 (1974)). Strain SC16249 (ATCC 202192) isolated from a soil sample collected in Parsippany, N.J. was maintained on a plate containing the above medium plus 1.5% agar. 10 ml of the medium in a 50 ml container was inoculated with a quantity of liquid that can be loaded on a platinum loop of those used in bacteriology, of the culture. After 24 h of incubation at 28 ° C, 200 rpm, the culture was centrifuged at 11951x gravities for 10 minutes. The cell microsphere was resuspended in a 50 ml vessel with 10 ml of 50 mM potassium phosphate buffer solution, pH 7. 2 mg of the 10 desacetylbaccatin III dissolved in 0.2 ml of methanol are added and the vessel is stirred at 28 ° C, 200 rpm for 16 hours. A 0.7 ml sample is diluted with 0.7 ml of methanol and analyzed by the HPLC method given below. The mole yield of 4.10 didesacetylbaccatin III was 86%. The retention time of the HPLC was the same as a chemically prepared standard. The LC / MS analysis showed (M + CH3COO ~) ~ = 561 indicating a molecular weight of 502.

HPLC method column: Hewlett Packard Hypersil 5 (ODS C 18 200x4.6 mm) mobile phase: 45% methanol; 55% water flow rate: 1 ml / min detection: 235 nm temperature: 40 ° C TABLE 1 Compound retention time 10 minutes-desacetylbaccatin III 10.221 4, 10-didesacetylbaccatine III 5.131 Example 2 Deacetylation of 10-desacetylbaccatin III Strains SC16249 (ATCC 202192) and SC 16250 (ATCC 202191) (both isolated from a soil sample collected in Parsippany, NJ) were grown in 500 ml containers containing 100 ml of the medium given in Example 1. The containers were inoculated with a quantity of liquid that can be loaded on a platinum loop of those used in bacteriology, from the culture, and stirred for 64 h at 28 ° C, 200 rpm. The cells were collected by centrifugation, washed with 50 mM of the potassium phosphate buffer, pH 7, and centrifuged again. The cellular microspheres were resuspended at a concentration of 10% w / v in 50 mM potassium phosphate buffer solution, pH 7. Samples of the cell suspension containing 2 mg of 10-deacetylbaccatin III and 2%, 5%, 10 % or 20% methanol in a total volume of 10 ml were stirred in 50 ml containers at 28 ° C, 200 rpm. The 0.5 ml samples were diluted with 0.5 ml of methanol and analyzed by the HPLC method given in Example 1. The molar yield of 4,10-didesacetylbaccatin III was 96% after 2 h for strain SC16249 ( ATCC 202192) with 10% methanol. The molar yield of 4,10-didesacetylbaccatine III was 89% after 18 h for strain SC 16250 (ATCC 202191) with 5% methanol.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (11)

1. A method for the preparation of 4-desacetyltaxanes, characterized in that it comprises the steps of contacting a taxane with a microorganism or an enzyme derived therefrom which is capable of deacetylating the taxane in position 4 thereon to obtain the 4-desacetyltaxane.

2. The method according to claim 1, characterized in that the taxane is 10-deacetylbaccatin III, paclitaxel, cephalomannin, taxol c, 10-desacetylpaclitaxel, 10-desacetyl-cephalomannine, 7-ß-xylosylpaclitaxel, baccatin-III, 7-ß-xylosyl-10-desacetyl-cephalomannine, 7-ß-xylosyl-10-deacetylbaccatin 111 / and 10-desacetyltaxol c.

3. The method according to claim 1, characterized in that the 4-deacetyltaxane is 4, 10-didesacetylbaccatine III, 4-desacetylpaclitaxel, 4-desacetylcefalomananine, 4-deacetyltaxol c, 4,10-didesacetylpaclitaxel, 4,10-didesacetylcefalomannin, 4 -desacetyl-7, ß-xylosylpaclitaxel, 4-desacetylbaccatine III, 4-desacetyl-7-ß-xylosyl-10-desacetylcephalomannine, 4-desacetyl-7-ß-xylosyl-10-deacetylbaccatin III, 4-desacetyl-7-β-xylosylbaccatine III. and 4,10-disesacetyltaxol c.

4. The method according to claim 1, characterized in that the taxane is obtained by the cell culture of, and / or the extraction of the tissue from the plant, wherein the plant is a member of the genus Taxus.

5. The method according to claim 1, characterized in that the microorganism is within the genus Rhodococcus.

6. The method according to claim 5, characterized in that the microorganism is Rhodococcus sp. ATCC 202192 (SC16249) or Rhodococcus sp. ATCC 202191 (SC 16250).

7. The method according to claim 1, characterized in that the enzyme is derived from a microorganism which is within the genus Rhodococcus.

8. The method according to claim 7, characterized in that the enzyme is derived from the microorganism Rhodococcus sp. ATCC 202192 (SC16249) or Rhodococcus sp. ATCC 202191 (SC 16250).

9. An enzyme isolated from a microorganism which is capable of catalyzing the deacetylation of C-4 of a taxane.

10. The enzyme according to claim 9, characterized in that the microorganism is within the genus Rhodococcus.

11. The enzyme according to claim 9, characterized in that the microorganism is Rhodococcus sp. ATCC 202192 (SC16249) or Rhodococcus sp. ATCC 202191 (SC 16250).