RU2170741C2 - Method of preparing cyclosporin - Google Patents

Abstract

FIELD: biochemistry. SUBSTANCE: described is method of preparing cyclosporin of formula II:

Description

The invention relates to a method for producing [3'-deoxy-3'-oxo-MeBmt] ¹ cyclosporine, in particular to a method for producing [3'-deoxy-3'-oxo-MeBmt] ¹ - [Nva] ² -cyclosporine or [3 '-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporin.

где A представляет собой остаток 3'-дезокси-3'-оксо-MeBmt;
B представляет собой -αAbu-, -Thr-, -Val- или -Nva-;
X представляет собой -Sar- или остаток оптически активной α-N-метилированной α-аминокислоты в (D)-конфигурации; и
Y представляет собой -Val- или, когда B является Nva, Nva, при котором культивируют организм с получением промежуточного диклоспорина формулы II, где A является MeBmt, выделяют указанный промежуточный циклоспорин и селективно окисляют указанный промежуточный циклоспорин с получением циклоспорина формулы II, где A является остатком 3'-дезокси-3'-оксо-MeBmt.According to the present invention, a method for producing [3'-deoxy-3'-oxo-MeBmt] ¹ cyclosporin of the formula

where A represents the remainder of 3'-deoxy-3'-oxo-MeBmt;
B is -αAbu-, -Thr-, -Val- or -Nva-;
X represents —Sar— or the remainder of the optically active α-N-methylated α-amino acid in the (D) configuration; and
Y is -Val- or, when B is Nva, Nva, wherein the body is cultured to obtain an intermediate diclosporin of formula II, where A is MeBmt, the indicated intermediate cyclosporin is isolated and selectively oxidized the indicated intermediate cyclosporin to obtain cyclosporin of the formula II, where A is residue 3'-deoxy-3'-oxo-MeBmt.

 The compounds of formula II, where A is a 3'-deoxy-3'-oxo-MeBmt residue, are useful for increasing sensitivity (or increasing effectiveness) to chemotherapeutic drug therapy and, in particular, are useful in controlling various types of chemotherapeutic drug resistance (e.g. acquired or congenital) or to enhance or restore sensitivity to prescribed drug therapy. Forms of chemotherapeutic drug therapy in which these compounds can be used include, for example, antiparasitic, in particular antiviral, antibacterial or antiprotozoal chemotherapy, as well as antineoplasma and cytostatic chemotherapy.

Particular preferred cyclosporins that can be prepared by the process of the invention are [3'-deoxy-3'-oxo-MeBmt] ¹ - [Nva] ² -cyclosporin or especially [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporin. These compounds are prepared by oxidation of [Nva] ² -cyclosporin and [Val] ² -cyclosporin, and the oxidation methods themselves are part of this invention.

Accordingly, a further aspect of the invention provides a process for the preparation of [3'-deoxy-3'-oxo-MeBmt] ¹ - [B '] ² -cyclosporin, where B is Nva or Val, by oxidizing [B'] ² -cyclosporin. [Val] ² -cyclosporin is also known as cyclosporin D.

The [B '] ² -cyclosporin used in the oxidation process can be obtained by culturing organisms producing the [B'] ² -cyclosporin, or by partial or complete synthesis, including derivatives of another cyclosporin or a suitable fermentation product.

Any suitable oxidizing agent, solvent, catalyst, as well as reaction conditions and reaction scheme can be used in the oxidation step of the method according to the invention or the method according to a further aspect. Suitable reagents for the oxidation reaction include dimethyl sulfide in combination with N-chloro-succinimide or preferably dimethyl sulfoxide in combination with N, N'-dicyclohexylcarbodiimide, preferably in a non-polar organic 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 increases the yield of dimethyl sulfide as a reaction by-product. There are also less undesirable odor problems associated with the use of dodecylmethyl sulfoxide. Preferably, the oxidation reaction is carried out at low temperatures, for example from -15 to 20 ^o C.

The invention is further described by way of illustration in the following example, which describes the preparation of [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporin.

 Example.

a) Obtaining cyclosporin D
Cyclosporin D (also known as [Val] ² -cyclosporin) is obtained by fermentation of a suitable organism, such as Tolypocladium inflatum GAMS (deposited at US Northern Regional Research Laboratories Culture Collection under number NRRL 8044), isolated from the fermentation broth and then purified as described below.

500 l of a nutrient solution containing 1 l: 40 g of glucose, 2 g of sodium caseinate, 2.5 g of ammonium phosphate, 5 g of MgSO ₄ • 7H ₂ O, 2 g of KH ₂ PO ₄ , 3 g of NaNO ₃ , 0.5 g of KCl, 0.01 g of FeSO ₄ and demineralized water to 1 l, inoculate 50 l of the preliminary culture of strain NRRL 8044 and incubate in a steel fermenter with stirring (170 min ^-1 ) and aeration (1 l of air / min / 1 l of nutrient solution ) for 13 days at 27 ^o C.

The culture fluid is mixed with the same amount of n-butyl acetate, concentrated by evaporation in vacuo after separation of the organic phase and the crude extract is degreased by 3-stage separation in methanol / water (9: 1) and petroleum ether. The methanol phase is separated, concentrated by evaporation in vacuo and the crude product is precipitated by adding water. The material obtained after filtration was subjected to silica gel chromatography using hexane / acetone (2: 1) as an eluent. The fractions eluted first mainly contain cyclosporin A and cyclosporin D, the fractions eluted later mainly contain cyclosporin C. For further purification, cyclosporin A- and D-containing fractions crystallize from 2-2.5-fold amount of acetone at a temperature of -15 ^o C, The crystallate was subjected to silica gel chromatography twice, the first fractions eluted with water saturated with ethyl acetate were significantly enriched with cyclosporin D. They were dissolved in double amount of acetone and left to crystallize at 15 ^o C. The resulting crude the cyclosporin D product is dissolved for further purification in 10-fold amount of acetone, 2% by weight of active charcoal is added and heated for 5 minutes to 60 ^° C. After filtration through talc, a clear and almost colorless filtrate is obtained, and concentrated by evaporation to one third of its volume and left to cool at room temperature, whereby the spontaneously crystallized cyclosporin D. Crystallization is completed by placing at -17 ^o C. The resulting crystals after filtration washed with a little cooled to du acetone and then dried under high vacuum at 80 ^o C for 2 hours.

Characteristics of cyclosporin D: colorless prismatic crystals, T. pl. 148-151 ^o C; [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ = -245 ^o (c = 0.52 in chloroform); [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ = -211 ^° C (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 acetic acid and 1 L of anhydrous ethanol are mixed and heated to 70 ^° C. with stirring. A solution of 215 g (2.2 mol) of hydrogen peroxide in 540 ml of water is added with stirring over 90 minutes and stirring is continued for about 1 hour until the reaction is completed by at least 90%, as determined by gas chromatography. Then the mixture is cooled to 20-25 ^o C and a solution of 27 r sodium metabisulfite in 41 ml of water is added, the mixture is stirred for 20 minutes, maintaining the temperature at 20-25 ^o C. Then add 3 l of toluene and a solution of 93 g of sodium bicarbonate in 1 , 07 l of water, the mixture is stirred and allowed to separate phases. The aqueous phase is removed, and the organic phase is washed with 1 L of water, the washing water is also removed and the organic phase is concentrated under reduced pressure to a volume of about 1.25 L. The concentrate is heated to 50-55 ^° C. and 1.5 L of hexane is added. Dodecyl methyl sulfoxide crystals are added to the resulting solution, cooled to 20-25 ^° C. for 1 hour and then stirred at this temperature. Then, the resulting solution was cooled to 0-5 ^° C. for about 30 minutes, stirred at this temperature for about 3 hours, and a solid precipitate was collected by centrifugation. The solid was then washed with 400 ml of cold hexane (0 ^° C.) and dried at a temperature not exceeding 60 ^° C. under reduced pressure to obtain 462 g of dodecylmethyl sulfoxide (about 90% of theoretical yield).

c) Oxidation of cyclosporin D to [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporin
269 g (1.22 mol) of pure cyclosporin D are dissolved in 2.95 L of toluene, 283 g (1.22 mol) of dodecyl methyl sulfoxide obtained as described above are added, the mixture is cooled to 0-5 ^° C. and 78 g of dichloroacetic acid are added together. with 50 ml of toluene. The temperature of the reaction mixture was maintained at 0-5 ^° C. for about 45 minutes, after which a solution of 205 g (0.99 mol) of N, N'-dicyclohexylcarbodiimide in 290 ml of toluene was added. The reaction mixture is stirred for about 30 minutes until the reaction is 95% complete, as determined by HPLC chromatography. A solution of 81 g (0.64 mol) of oxalic acid in 630 ml of water is added and the resulting mixture is stirred at 0-5 ^o C for 3 hours. The resulting solid is separated by centrifugation, washed with 440 ml of cold toluene (5 ^o C), after whereby the solid is thrown away. The liquid fraction of the reaction mixture and the washing liquid are combined, the phases are allowed to separate, the aqueous phase is discarded and an aqueous solution of sodium bicarbonate (80 g in 1.25 L) is added to the organic phase until the pH reaches 7-8. After stirring, the phases are allowed to separate, the aqueous phase is discarded, and the organic phase is washed with 650 ml of water. The washing water is discarded and the organic phase is evaporated to dryness under reduced pressure at a temperature not exceeding 60 ^o C. The residue is dissolved in a mixture of 560 ml of isopropyl acetate and 10 ml of water when heated to 55-60 ^o C. For about 30 minutes, the solution is cooled to about 40 ^o C and add crystals of dodecyl methyl sulfoxide to it. After about 90 minutes, the mixture was cooled to a temperature of from -10 to -15 ^° C and stirred at this temperature for about 4 hours. The solid was removed by centrifugation, washed with 390 ml of isopropyl acetate and discarded. The liquid fraction of the reaction mixture and the washing liquid are combined to obtain 1.28 kg of a solution containing about 256 g of [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporine (about 95% of theoretical yield) .

The solution obtained as described above is enriched by chromatography on silica gel. Two columns are prepared, each of which contains 1 kg of silica gel suspended in 2.5 L of isopropyl acetate, and 162 g of the indicated solution are each loaded. The columns are eluted with water-saturated isopropyl acetate at a flow rate of 500 ml / h and the fractions are collected in the following order: first 4 L (first eluate), next 500 ml (second eluate), next 4.5 L (third eluate) and the next 1 L (fourth eluate). As determined by HPLC, the desired product is in the third eluate. Thus, the first eluate is discarded, and the second and fourth are left for reprocessing. The third eluate from both columns was combined, concentrated under reduced pressure to a volume of about 320 ml, and then evaporated to dryness under reduced pressure and at a temperature not exceeding 60 ^° C. to obtain 40 g of product.

The product is further purified by crystallization from tert-butyl methyl ether. It was found that the resulting purified [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporin has a melting point in the range 195-198 ^o C, optical rotation [α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ = -255.1 ^° (c = 0.5 in CHCl ₃ ) and a molecular weight of 1214.65.

[3'-deoxy-3'-oxo-MeBmt] ¹ - [Nva] ² -cyclosporin can be prepared in a similar manner by oxidizing [Nva] ² -cyclosporin essentially the same way as the oxidation of [Val] ² -cyclosporin is described.

Claims (8)

1. The method of obtaining cyclosporin formula II

where A is the remainder of 3'-deoxy-3'-oxo-MeBmt;
B is -αAbu-, -Thr-, -Val- or -Nva-;
X is —Sar— or the remainder of the optically active α-N-methylated α-amino acid in the (D) configuration;
Y is -Val- or, when B is Nva, Nva,
which includes the selective oxidation of intermediate cyclosporin of formula II, where A is MeBmt, dimethyl sulfoxide in combination with N, N'-dicyclohexylcarbodiimide or dodecylmethyl sulfoxide in combination with N, N'-dicyclohexylcarbodiimide.  2. The method according to claim 1, wherein the intermediate cyclosporin of formula II is obtained by culturing an organism producing an intermediate cyclosporin and recovering said intermediate. 3. The method according to claim 1 or 2, characterized in that receive [3'-deoxy-3'-oxo-MeBmt] ¹ - [Nva] ² -cyclosporine or [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporin. 4. The method according to claim 3, characterized in that receive [3'-deoxy-3'-oxo-MeBmt] ¹ - [Val] ² -cyclosporine. 5. The method according to claim 4, characterized in that the oxidation reaction is carried out at 0 ^o C and 5 ^o C. 6. A method of producing [3'-deoxy-3'-oxo-MeBmt] ¹ - [B '] ² -cyclosporin, where B' is Nva or Val, in which [B '] ² -cyclosporin dimethyl sulfoxide is oxidized in combination with N , N'-dicyclohexylcarbodiimide or dodecylmethyl sulfoxide in combination with N, N'-dicyclohexylcarbodiimide.  7. The method according to claims 1 to 6, characterized in that dodecylmethyl sulfoxide is used as an oxidizing agent in combination with N, N'-dicyclohexylcarbodiimide. 8. The method according to claim 7, characterized in that it is carried out at a temperature of from -15 to +20 ^o C in a non-polar organic solvent, such as toluene.