CA1106303A

CA1106303A - Cyclosporin c from tolypocladium or cylindrocarbon

Info

Publication number: CA1106303A
Application number: CA353,123A
Authority: CA
Inventors: Maz Kuhn; Artur Ruegger
Original assignee: Sandoz AG
Current assignee: Sandoz AG
Priority date: 1975-11-04
Filing date: 1980-05-30
Publication date: 1981-08-04

Abstract

Abstract of the disclosure Cyclosporin C of formula I

I

useful as an intermediate in the production of isocyclo-sporin C.

Description

- ~ \

The present application, which is a divisional of our co-pending application No. 264,703, relates to the compound cyclosporin C of formula I

C~a~C~H
: ' 11 3~ ~CH3 . ~C~
7 C~ ,C,,~~~ ''~` T~3 I H2 C:H~ (.HCH~ ~¦ CH-OH C~l3 C O 1 H ~;--N--C I ~--C O--N--C H--C--~J ~ I I I ?

/C~I_CI~2~
CH3_N , . . N--Cll3 - 01 C 11 N--C O_ ~ H--r~ ---C O--C H - -- - N--C--C I l--N--C O--1 H
Ct-13 H I~HJ C ~2 C1~3 Cll CH;!
~CI~ CH3 CH3 ~1~
C~3 CHJ CH3 CH3 ~ .

.
: Cyclosporin C is a valuable intermediate useful for the production of the pharmacologically active compound dihy-drocyclosporin C. Dihydrocyclosporin C, its production from Cyclosporin C, as well as the pharmacological properties of dihydrocyclosporin C, are described in our above mentioned co-pending application No. 264, 703.
' : ' .- - : -' ' ' ' ' `

3lD3 In acldition to the foregoing the present invention also provides a process for the production of Cyclosporin C
which comprises cultivating a Cyclosporin C producing strain of the fungus species Tolypocla'dium inflatum Gams or Cylin-5. drocarpon lucidum Booth in contact witn a nutrient mediumand isolating Cyclosporin C from the resultant culture broth.

The cultivation may be effectecl in known manner, for exam-ple as described in DOS 2,455, 859 and t,he corresponding Canadian Patent No. 1,030,469 to ~arri et al. granted May 2, 1978. Preferred strains of the fungus species Toly-, pocladium inflatum Gams and Cylindrocarpon Lucidum Booth are the strains NRR~ 8044 and 5670, cultures of which have been deposited at the ~nited States Department of ~griculture (Northern Research and Development Division) Peoria, Ill., U.S.A. and are freely available. The morphological charac-teristics of both strains are fully described in the affore-mentioned Canadian Patent No. 1,030,469.

Cyclosporin C may be isolated from the obtained fermenta-tion broth by chromatography as a compound active against Aspergilius niger and having greater polarity than Cyclo-sporins A.and B (designated as S7481/F-1 and S7481/F-2 in the'above mentioned DOS and Canadian Patent).

In the following example all temperatures are in degrees : 25 centigrade.

~ ' . .

EXAMPLE

400 litres of a culture broth obtained by aerobic sub-mersion cultivation of the strain NRRL 8044 of Tolypocla-dium inflatum Gams [see Example 3 of Canadian Patent No. 1,030,469~ is extracted by stirring with 400 litres of n-butyl acetate. After separation in a "Westfalia-Separator" (trademark), the organic phase is concentrated in a vacuum and the crude extract is defatted by a three stage extraction between petroleum ether and methanol/
water (9:1). The resultant material is dissolved in chloro-form and chromatographed on 4.5 kg silicagel 60 "Merck"(diameter 0.2 to 0.5 mm), using chloroform with increasing amounts of methanol as eluant. Chloroform + 1.5% methanol elutes Cyclosporins A and B, and chloroform ~ 3~ methanol elutes Cyclosporin C. ~The Cyclosporin C may be detected using t.l.c. on silica gel foils "Polygram" using chloro-form/methanol 95:5 as eluant. Rf values - Cyclosporin A
0.44; B 0.37; C 0.26].

After combination and evaporation of the eluant fractions containing Cyclosporin C, the chromatographic purification is repeated. The Cyclosporin C containing fractions are collected and evaporated at 20 - 40~ The residue is treated with 10 times its weight of a mixture of alcohol containing 5% per weight of active charcoal.

., *

Trademark ;

' 6~3 After filtration the mixture is evaporated at 20 to 40 in a vacuum and then dried in a hiqh vacuum at 55C.

Final purification comprises dissolving the resultant resi-du~ in a 5 times amount of ether and by the slow addition of a 30 times amount of hexane. On shaking, a solid sub-stance precipitates and is co:Llected after cooling of the mixture at 0 to 5. This is washed with cold hexane and dr.ied at 55 in a high vacuum. From a 2.5 times amount of acetone the residue affords at -15 colourless, pris-matic needles of crystalline Cyclosporin C.
M.Pt. = 152 - 155 [a]D= -255 (c = 0.5; CHC13) [~]20= -182 (c = 0.5; CH30H)o

Claims

The embodiments of the invention in which an exclusive property or privelege is claimed are defined as follows:

1. A process for the production of Cyclosporin C of formula I
which process comprises cultivating a Cyclosporin C
producing strain of the fungus species Tolypocladium inflatum Gams or Cylindrocarpon lucidum Booth in con-tact with a nutrient medium and isolating Cyclosporin C
from the resultant culture broth.

2. Process according to claim 1 wherein the Cyclosporin C
producing strain is the strain NRRL 8044 of the fungus species Tolypocladium inflatum Gams.

3, Process according to claim 1 wherein the Cyclosporin C
producing strain is the strain NRRL 5760 of the fungus species Cylindrocarpon lucidum Booth.

4. Process according to claim 1 wherein the Cyclosporin C
is isolated in a form substantially free from Cyclo-sporin A and Cyclosporin B.

5. Process according to claim 4 wherein the Cyclosporin C
is isolated in crystalline form.

6. Cyclosporin C whenever prepared by a process as claimed in any one of claims 1 to 3.

7. Cyclosporin C whenever prepared by a process as claimed in claim 4 or 5.