SI9600273A - A PROCESS FOR THE PRODUCTION OF A /3'-DESOXY-3'-OXO-MeBmt/1 CYCLOSPORIN - Google Patents

Abstract

A process for the production of a cyclosporin of formula II wherein A is a 3'-desoxy-3'-oxo-MeBmt residue; B is -alpha Abu-, -Thr-, -Val- or -Nva-; X is -Sar- or the residue of an optically active, alpha-N-methylated alpha-amino acid residue having the (D)-configuration and Y is - Val- or additionally, when B is Nva, Nva, comprises cultivating an organism to produce an intermediate cyclosporin of formula II wherein A is MeBmt, recovering the intermediate cyclosporin and selectively oxidising the intermediate cyclosporin to produce a cyclosporin of formula II wherein A is a 3'-desoxy-3'-oxo-MeBmt residue. This compound is useful in increasing sensitivity to, or in increasing the efficacy of, chemotherapeutic drug therapy, including anti-parasitic and, in particular, antineoplastic or cytostatic chemotherapy.

Description

SANDOZA.G.

A process for the preparation of ^ -deoxy-S ^ oxo-MeBmt] ¹ cyclosporine

The present invention relates to a process for the manufacture of [S ^ deoxy ^ -oxo-MeBmt] ¹ cyclosporin, in particular to a process for the production of (T-deoxy ^ -oxo-MeBnit] ¹ [Nva] ^2- cyclosporine or [T-deoxy-S N-oxo-MeBmtf-fValp-cyclosporine, with MeBmt being N-methyl- (4R) -4-but-2E-en-1-yl-4-methyl- (L) threonyl, and Nva is an abbreviation for the norvaline residue.

The present invention therefore provides a process for the production of [3′-deoxy-3′-oxoMeBmtp-cyclosporine of Formula II

II »-A-B-X-MeLeu-Y-MeLeu-Ala- (D) Ala-MeLeu-MeLeu-MeVal123456 7 8 9 10 11 in which

But the residue is 3′-deoxy-3′-oxo-MeBmt;

B is -aAbu-, -Thr-, -Val-, or -Nva-;

X is the -Sar- (= sarcosine residue) or the residue of an optically active α-Nmethylated α-amino acid residue with (D) -configuration and

Y is -Val- or additionally, if B is Nva, Nva, comprising culturing the organism to form an intermediate cyclosporin of the formula

II in which A is MeBmt, recovering intermediate ciclosporin and selectively oxidizing intermediate ciclosporin to form cyclosporin of formula II, in which A is the residue 3′-deoxy-3′-oxo-MeBmt.

Compounds of formula II in which A is a residue of 3'-deoxy-3'-oxo-MeBmt are useful for increasing the sensitivity for chemotherapeutic drug therapy or for enhancing the effectiveness of this therapy, and are particularly useful for reversing the resistance of various types (e.g. (acquired or congenital) for chemotherapeutic drugs, or for increasing and enhancing sensitivity to therapy with the medicinal products administered. Formulations of chemotherapeutic drug therapies in which these compounds can be used include e.g. antiparasitic, e.g. antiviral, antibacterial or antiprotozoal chemotherapy, and in particular antineoplastic or cytostatic chemotherapy.

Particularly preferred cyclosporins that can be manufactured using the process of the invention are [3'-deoxy-3'-oxo-MeBmt] ^1- [Nva] ^2- cyclosporine or especially [3'-deoxy ^ -oxo-MeBmtp-fVal ^ -cyclosporine. These compounds are prepared by oxidizing [Nva] ² -cyclosporine and [Val] ² -cyclosporine, and these oxidation processes per se are within the scope of the present invention.

Accordingly, a further aspect of the present invention is a process for the production of [3'deoxy ^ -oxo-MeBmtmB ^ -cyclosporin, where B 'is Nva or Val comprising oxidizing [B'] ^2- cyclosporine. [Val] ^2- Cyclosporin is also known as cyclosporin D.

[B '] ² ciclosporin for use in an oxidation process can be obtained by culturing an organism that forms [B'] ^2- cyclosporin, or by partial or complete synthesis involving derivatization of a different ciclosporin or related fermentation product.

Any suitable oxidizing agent, solvent, catalyst or reaction conditions or schemes can be used in the oxidation step of the process of the present invention or process from a further perspective. Suitable reagents for the oxidation reaction include dimethyl sulfide in combination with N-chloro-succinimide or preferably dimethyl sulfoxide in combination with Ν, Ν′-dicyclohexylcarbodiimide, preferably in a non-polar organic solvent such as toluene. More preferably, dodecyl methyl sulfoxide in combination with Ν, Ν′-dicyclohexylcarbodiimide is used as an oxidizing agent in a solvent such as toluene. Dodecyl methyl sulfoxide is preferred over both dimethyl sulfide and dimethyl sulfoxide, since dimethyl sulfide is undesirable for safety and environmental reasons, and dimethyl sulfoxide causes the formation of dimethyl sulfide as a by-product of the reaction. However, there are also problems associated with the odors caused by the use of dodecyl methyl sulfoxide. The preferred oxidation reaction is carried out at low temperature, e.g. -15 to +20 ° C.

The present invention is further illustrated by the following example, which describes the preparation of [T-deoxy ^ -oxo-MeBmtf-fVal ^ -cyclosporine.

EXAMPLE

a. Preparation of cyclosporine D

Cyclosporin D (also known as [Val] ^2- cyclosporine) is prepared by fermentation of a suitable organism such as Tolypoclad, ium inflatum GAMS (deposited with US Northern Regional Research Laboratories Culture Collection as deposit No. NRRL 8044), recovered from fermentation broth and then cleansing as described below.

500 1 nutrient solution containing per liter: 40 g glucose, 2 g sodium caseinate, 2.5 g ammonium phosphate, 5 g MgSO ₄ .7H ₂ O, 2 g KE1 ₂ PO ₄ , 3 g NaNO ₃ , 0,5 g KC1,0,01 g FeSO ₄ and demineralized water 11 each, inoculated with 501 pre-cultures of NRRL 8044 strain and incubated in stainless steel fermenter with stirring (170 rpm) and aeration (1 1 air / minute / 1 nutrient solution) ) For 13 days at 27 ° C (see published German patent application 2 455 859).

The culture liquor was mixed with the same amount of n-butyl acetate, concentrated by evaporation in vacuo after separation of the organic phase, and the crude extract removed the fats with a three-step distribution between methanol / water (9: 1) and petroleum ether. The methanol phase was separated, concentrated by evaporation in vacuo, and the crude product was precipitated by the addition of water. The material obtained after filtration was chromatographed on silica gel with hexane / acetone (2: 1) as eluant. Initial eluted fractions contain predominantly cyclosporin A and cyclosporin D, and later eluted fractions contain predominantly cyclosporin C. Fractions containing cyclosporin A and D are crystallized from 2- to 2.5-fold acetone at -15 ° C for further purification. The crystallized substance is further chromatographed twice on silica gel, the fractions initially eluting with water-saturated ethyl acetate containing cyclosporin D in a highly enriched form. They were dissolved in 2 times acetone and allowed to crystallize at -15 ° C. The resulting crude crystalline product of cyclosporin D was dissolved for further purification in 10 times the amount of acetone, 2% by weight of activated carbon was added and heated at 60 ° C for 5 minutes. The clear and almost colorless filtrate, obtained after filtration through talc, was concentrated by evaporation to one third of its volume and allowed to cool to room temperature and then cyclosporin D crystallized spontaneously. The crystallization is complete after the mixture is allowed to stand at -17 ° C. The crystals obtained by filtration were washed with a small amount of ice-cold acetone and then dried under high vacuum at 80 ° C for 2 hours.

Labeling of cyclosporine D

Colorless prismatic crystals; mp: 148 ° -151 ° C.

[a] ²⁰ D = -245 ° (c = 0.52 in chloroform) [a] ²⁰ D = -211 ° (c = 0.51 in methanol)

b. Preparation of dodecyl methyl sulfoxide

478 g (2.2 mol) of dodecyl methyl sulfide, 52.5 g of glacial octane and 11 anhydrous ethanol were combined and heated to 70 ° C with stirring. 215 g (2.2 mol) of hydrogen peroxide in 540 ml of water were added with stirring for 90 minutes, then stirring was continued for about 1 hour until the reaction was at least 90% complete, determined by gas chromatographic analysis. The mixture was then cooled to 20-25 ° C and a solution of 27 g of sodium metabisulphite in 41 ml of water was added, and then the resulting mixture was stirred for 20 minutes while maintaining the temperature at 20-25 ° C. Then 3 l of toluene and a solution of 93 g of sodium bicarbonate in 1.07 l of water are added, the mixture is stirred and the phases are separated. The aqueous phase is then discarded, the organic phase is washed with 1 L of water, the rinsing water is discarded and the organic phase is concentrated under reduced pressure to a volume of about 1.25 1. The concentrate is heated to 50-55 ° C and 1.51 hexanes are added. The resulting solution was then grafted with dodecyl methyl sulfoxide crystals and cooled to 2025 ° C for about 1 hour and then stirred at this temperature. The solution was then cooled to 0-5 ° C for about 30 minutes, stirred at this temperature for about 3 hours, and the solid precipitate was collected by centrifugation. The solid was then washed with 400 ml of cold (0 ° C) hexane and dried at a temperature higher than 60 ° C under reduced pressure to give 462 g of dodecyl methyl sulfoxide (approximately 90% of theory).

c. Oxidation of cyclosporin D into [3 ^, -deoxy-3 ^, -oxo-MeBmtU- [Val] -cyclosporine

Dissolve 269 g (0.22 mol) of pure cyclosporin D in 2.95 l of toluene, add 283 g (1.22 mol) of dodecyl methyl sulfoxide prepared as described above, cool the mixture to 0-5 ° C and add 78 g of dichloroacetic acid together with a further 50 ml of toluene. The temperature of the reaction mixture was maintained at 0-5 ° C for about 45 minutes and then a solution of 205 g (0.99 mol) of Ν'-dicyclohexylcarbodiimide in 290 ml of toluene was added. The reaction mixture was stirred for about 30 minutes until the reaction was at least 95% perfected as determined by HPLC. A solution of 81 g (0.64 mol) of oxalic acid in 630 ml of water was added and the resulting mixture was stirred at 0-5 ° C for about 3 hours. The solids formed were separated by centrifugation and washed with 440 ml of cold (5 ° C) toluene and the solids were then discarded. The liquid fraction of the reaction mixture and the leaching fluid were then combined, the phases were separated, the aqueous phase discarded, and an aqueous solution of sodium bicarbonate (80 g in 1.25 l) added to the organic phase to pH 7-8. After stirring, the phases were allowed to separate, the aqueous phase was discarded and the organic phase was washed with 650 ml of water. The wash water is discarded and the organic phase is evaporated to dryness under reduced pressure at a temperature not higher than 60 ° C. The residue was dissolved in a mixture of 560 ml of isopropyl acetate and 10 ml of water at 55-60 ° C. The solution was cooled to about 40 [deg.] C. in about 30 minutes and cleaved with dodecyl methyl sulfoxide crystals. The mixture was then cooled to -10 to -15 ° C in about 20 minutes and stirred at this temperature for about 4 hours. The solids were removed by centrifugation, washed with 390 ml of isopropyl acetate and then discarded. The liquid fraction of the reaction mixture and the washing liquid were then combined to give 1.28 kg of solution containing about 256 g of [3′deoxy-S ^ oxo-MeBmtp-fVal ^ -cyclosporine (approximately 95% theory).

The solution obtained as described above is enriched by silica gel chromatography. Prepare two columns each containing 1 kg of silica gel suspended in 2.5 l of isopropyl acetate and each filled with 162 g of solution. The columns were eluted at a flow rate of 500 ml / hour with isopropyl acetate saturated with water and fractions were collected in the range of the first 4 l (first eluate), the next 500 ml (second eluate), the next 4.5 l (third eluate) and the next 11 ( the fourth eluate). HPLC determines that the desired product is in the third eluate. Thus, the first eluates are discarded and the second and fourth ones are retained for reprocessing. The third eluates from the two columns were combined, concentrated under reduced pressure to a volume of about 320 ml and then evaporated to dryness under reduced pressure at a temperature not higher than 60 ° C to give about 40 g of product.

This product was further purified by crystallization from tert-butyl methyl ether. The resulting purified [3'-deoxy-3'-oxo-MeBmt] '- [Val] ² -cyclosporine is determined to have a melting point in the range of 195-198 ° C, an optical rotation of [a] ²⁰ D = -255.1 ⁰ (c = 0.5 in CHC1 ₃ ) and a molecular weight of 1214.65.

[3'-deoxy-3'-oxo-MeBmt] '- [Nva] ^2- cyclosporine can be prepared analogously to the oxidation of [Nva] ² -cyclosporin essentially as described above for the oxidation of [Val] ^2- cyclosporine.

Claims (6)

A process for the manufacture of cyclosporine of formula II II i-A-B-X-MeLeu-Y-MeLeu-Ala- (D) Ala-MeLeu-MeLeu-MeVal-t 123 4 5 6 7 8 9 10 11 in which But the residue is 3′-deoxy-3′-oxo-MeBmt; B is -aAbu-, -Thr-, -Val-, or -Nva-; X is a -Sar- or a residue of an optically active α-N-methylated α-amino acid residue with (D) -configuration and Y is -Val- or additionally, if B is Nva, Nva, characterized in that it comprises culturing the organism to form an intermediate cyclosporin of formula II in which A is MeBmt, recovering the intermediate cyclosporin, and selectively oxidizing the intermediate cyclosporin to give A cyclosporin of formula II is formed in which A is a residue of 3'-deoxy-3'-oxoMeBmt. Process according to claim 1, characterized in that it is useful for the manufacture of [3'-deoxy3'-oxo-MeBmt] ^1- [Nva] ^{2 3 4 5} -cyclosporine or ( ⁴ -deoxy ^ -oxo-MeBmtp-JVal ] ² cyclosporine. A process for the manufacture of p ^ deoxy ^ -oxo-MeBintp-fB ^ -cyclosporine, wherein B 'is Nva or Val, characterized in that it comprises oxidizing [B'] ^2- cyclosporine. A process according to claim 1, characterized in that dimethylsulfide in combination with N-chlorosuccinimide is used as an oxidizing agent. Process according to any one of claims 1 to 3, characterized in that dimethyl sulfoxide in combination with Ν, Ν′-dicyclohexylcarbodimide or dodecyl methyl sulfoxide in combination with Ν, Ν′-dicyclohexylcarbodimide is used as an oxidizing agent. A process according to claim 4 or 5, characterized in that it is carried out at a temperature between -15 ° C and +20 ° C in a non-polar organic solvent such as toluene.