FI63743B - FRAMEWORK FOR THE PROCESSING OF THERAPEUTIC THERAPY OF ISCYCLOSPORIN - Google Patents

Description

63743 .Method for the preparation of isocyclosporin G for therapeutic use

This invention relates to a process for the preparation of isocyclosporin G of formula I.

10

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k "VvCKj CHSN. s · *" 'I f «3 CM O .Cm 1 i, cc; -n / CH' /? h» i i i / | | *? CM3-fv · CM- CO-N-Cm — c UN —CM-CO-N — CM-C-tl-CH, 1B L «* li I« - i t u i 15 l0 o Ch3 h o

Cm »· ^ J co CH-CHj-CM l 11

CM3 / I I

CHj- u is I * i o h 20 I, ° i I i II v I.

oc-cm n co-ch-n · —co — CM-s * c — CM — W-CO — CM

III II i i CMa · M CH3 CH, CHJ CM CH,

l CHj NC «j I

CHS CM3 CHj CM3 \ 25 According to the invention, 'isocyclosporin G is prepared by isomerizing cyclosporin G of formula II.

2 63743 c «3 c

II

hTCvch, C "J \ / Ch3 I CM3 CH H ° v / CH« I CHK / CH,,> h '«» \ CH3? "*

CH, CH 2 Cm CH] I CH, CH 2

lii I I I I.

CHj-H-CH-CO-N-CH-C -.- N-CH- CO-U-CH-C-U-CMJ

I «· L II l I L II I

I O HO

co I

CH] I CO

\ I I II

CH-CH 2 -CH 2. I

CH 2 (N-CH)

CH] -N. I

I H OH

I O I 1 t) l «. I I I

OC-CH-N-CO "CM-N-CO-CH-hi-C-c H - N-CO-CH

CHj h CHj CHj CM-i CH CHj

CH C "> ^ CMl CH

c-r, ^ CH3

The isomerization is carried out in a manner known per se, preferably by acid treatment, e.g. using strong organic acids, such as trifluoroacetic acid, preferably methanesulfonic acid or p-to-5-toluenesulfonic acid.

The amount of strong acid used in the isomerization is preferably 1 to 4 moles per mole of cyclosporin G.

Suitable solvents are lower alcohols, e.g. methanol, halogenated hydrocarbons, e.g. chloroform, or ethers, e.g. dioxane.

The reaction temperature may be between 20 and 65 ° C, preferably between 45 and 55 ° C.

The cyclosporin G used as a starting material is prepared by culturing a cyclosporin G-producing strain, for example a Tolypocladium inflatum Gams strain belonging to the Tolypocladium fungus, in a nutrient solution and isolating the cyclosporin G.

The cultivation is carried out by methods known per se, used for culturing the corresponding strains, 20 ° as described, for example, in the example.

Il 63743 3

The preferred cyclosporin G-producing strain is NRRL 8044 belonging to the Toly-pocladium inflatum Gams zion species.

A sample of this strain was submitted to the collections of the United States Department of Agriculture (Northern Research and Development 5 Division), Peoria, 111, U.S.A., under the number NRRL 8044, from which it is generally available. Another sample of this strain was donated to the Fermentation Research Institute, Inage, Chiba City, Japan, where sil-10 le was given the number FRI FERM-P No. 2796. This strain was previously read into the fungus Trichoderma polysporum (Link ex. Pers.) And is described, for example, in English Patent 1,491,509.

Cyclosporin G can be isolated by known methods, as described, for example, in the example. In this case, cyclosporin G is obtained separately from co-occurring natural products, such as less polar cyclosporin D, slightly more polar cyclosporin A (also known as S 7481/20 F1), more polar cyclosporin B (also known as S 7481 / F-). 2) and an even more polar cyclosporin C.

Isocyclosporin G did not prove to have an excellent effect on Freund's adjuvant-induced arthritis. In this case, its ED50 value is 18 mg / kg p.o., whereas in the test corresponding to the cyclosporin A of the structure known from e.g. FI patent publication 54-606, for example, 25 mg / kg p.o. In addition, isocyclosporin G has been found to have an immunosuppressive effect. In the lymphocyte stimulation assay (Janossy & Greaves, Clin. Exp. Immunol. 9 (1971) 483 30 and 10 (1972) 525), the degree of inhibition of proliferation in vitro was 63% with concanavalin A-stimulated mouse spleen isolated lymphocytes at a concentration of test compound. 0.1 ug / ml.

Isocyclosporin G can be used as a medicament or as a preparation in admixture with pharmaceutically acceptable inert excipients.

In the following example, all temperatures are in degrees Celsius.

i 4

Example: Isocyclosporin G 63 7 4 3

To a solution of 6.08 cyclosporin G in 40 ml of absolute dioxane is stirred a solution of 1.20 g of methanesulfonic acid in 20 ml of dioxane and the mixture is kept at 50 ° C protected from moisture. The progress of the reaction is monitored by thin layer chromatography (Polygram SIL G foils, chloroform-methanol glacial acetic acid (90: 6: 4), visualized on iodine vapor). After 16 hours, the mixture is cooled to room temperature. To neutralize the acid, 1.13 g of anhydrous sodium acetate are added, the mixture is stirred for 45 minutes, the separated salt is filtered off and the filtrate is evaporated in vacuo at 45 ° C.

8.1 g of the residue are chromatographed on 500 g of silica gel (grain size 0.063-0.2 mm (Merck)) using a chloroform-methanol mixture (98: 2) as eluent. The fractions of substantially pure isocyclosporin G are combined, evaporated in vacuo at 50 ° C and the residue crystallized from ether at + 7 ° C to give isocyclosporin G, m.p. is 143-146 °. Gross formula: cg3Hn3N11012 {XJ 20 = -196 ° (c = 0.72 in chloroform) D -128 ° (c = 0.73 in methanol) 20 UV spectrum in methanol: terminal absorption IR spectrum in methylene chloride: see Figure 3 1 H-NMR spectrum (CDCl 3), 90 MHz, internal standard tetramethylsilane: see Figure 4.

The cyclosporin G used as starting material can be prepared as follows: to 500 liters of nutrient solution containing 40 g of glucose per liter, 2.0 g of sodium caseinate, 2.5 g of ammonium phosphate, 5 g of MgSO4-7H2O, 2 g of KH2PC> 4.3 g of NaNO 0.5 g of KCl, 0.01 g of 30 FeSO 4 and mineral-free water are inoculated with 50 liters of preculture of strain NRRL 8044 and the resulting mixture is incubated in a steel fermentor for 13 days at 27 ° C using agitation (170 l / min) and aeration (1 liters of air / min / liter of nutrient solution) (see DE-A-2 455 859).

35 N-butyl acetate is added to the culture medium, the organic phase is separated and concentrated in vacuo and the concentrate is defatted in three steps by means of a methanol-water mixture (9: 1) and petroleum ether.

The methanol phase is collected, concentrated in vacuo and the crude product is precipitated by adding water. The product recovered by filtration is chromatographed with a 5-7 yellow amount of "Sephadex LH-20" using methanol as eluent. The tip fractions are then chromatographed on silica gel 60 (grain size 0.063-0.2 mm (Merck)) 10 using acetone-hexane (2: 1), with the first fractions eluted containing mainly cyclosporin A and cyclosporin D and in the subsequently eluted portions mainly cyclosporin C. For further purification, the fractions containing cyclosporin A and 15 D are crystallized from 2-2.5 times the amount of acetone at -15 ° C, the product is chromatographed twice on silica gel 60 (grain size 0.063-0.2 mm (Merck )) using acetone-hexane (2: 1) as eluent, eluting cyclosporin 20 D in high enrichment in the first fractions. These fractions are dissolved in twice the amount of acetone and allowed to crystallize at -15 ° C. The crude crystalline mass consists of very highly enriched cyclosporin D and the mother liquor contains, in addition to cyclosporin D, other components such as cyclosporin G. To separate cyclosporin G, the mother liquor is evaporated to dryness, the residue is chromatographed on silica gel 60 (grain size 0.063-0.2 mm (Merck)) using ethyl acetate saturated with water as eluent, the first eluted fractions containing cyclosporin D and the subsequently eluted fractions cyclosporin G and other components. Enrichment is continued by chromatography on silica gel 60 (grain size 0.063-0.2 mm (Merck)) using first a mixture of chloroform-methanol (98: 2) and then hexane-acetone (63: 2) as eluent. ), in which case the fractions eluted later contain cyclosporin G in a highly enriched form. Purification is continued by crystallizing the cyclosporin G-containing fractions twice at room temperature from a mixture of ether and petroleum ether (1: 1) to give a thin layer chromatographically uniform, pure (> 95% pure) cyclosporin G as colorless polygons, m.p. 193-194 ° (crystals dried under high vacuum at 80 ° C for 2 hours).

10-245 ° (c = 1.00 in chloroform) -191 ° (c = 1.04 in methanol).

UV spectrum in methanol: terminal absorption IR spectrum in methylene chloride: Cf. Figure 1. 1 H-NMR spectrum (CDCl 3), 90 MHz, internal standard 15 tetramethylsilane: see Figure 2.

Gross formula:.

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Claims (4)

A process for the preparation of a therapeutically useful isocyclosporin G of formula I, CH 2 I H n H; 10 CH * \. | CH3 CH „O vCH. * I CH ^ / CH '/ CHi CH3 CM / \ CU, CHj I | I / I I I CMj-W-Crt-CO-W - CH— C HH-CH-CO-N — CM - O —- M-CH · »I t II I L Iti! , _ IO CH HO 15 co u '3 CM \' I? ° CM-CHj-CM t J cn:> I L-CHj IV OH I *> «I t li t I i. On OC-CH - N CO-CH-N CO —CH- »4 c CH N-CÖ CM iii iii ί c ,, 3 H CHj CHj CHj CH CHj I CHj '' '' CH} I CM · * CH / \ / 'vc * <3 chj CHa CHa 25 characterized in that the cyclosporin G of the formula II is 63743 cm3 ^ c li / CH3 I 9h3 CH HO yCH silicon I CH3 \ / CM5 t> H '"' \ h3, 2 CHj Ch3 cm CH, I CM , CH3!. II II t 2 I CHj-N-cM-CO-iy-'CM - cN-CM-CO-N —CM - C - N-CM j I «- L II u lii» IIO MO co I CH3 ^ I · CO CH-CHj-ch l I II CHJ j N-CH, CH «- N · ä IHOM 1 i> i I ini I i OC-CH-N-CO-CM - N-CO-CH- Nc-CM-N - CO-CH III III I Cl <3 H CH3 CHj CH3 cm CH} 1 I CHj I CH J CH / \ / \ CHj CMJ CHj CH] isomerized Patent claim 63743 For the preparation of therapeutically active isocyclosporin G with formula I, 5 cf 10 ch3 CHj.H CH- \ / I · I 3 CM ^ O. yCH 'I _ ^ CH, / CH? CHj · CM SI CH, CHJ I 11/1 \ \ CMj-W- CH'CO-M-'CH-C HN - CH-CC-N-CH-C-lv-CH, II «. L 11 L ° C ,, 3 M ° C "J. I CO 1K \ I 1 i Le h, CHj-ll «I M OH I ° i I i li t I i! OC-CM - N-CO-CH-N - CO-CH --- U - e - CH-N-CO-CH 111 III I CHj H CM- * CMj CHi CM CH, 20. I CHj NCH5 l CM CM / \ / S. CHa CMj CHj CMj 25 kännetecknat därav, att man isomeriserar cyklosporin G med formuleln II