CY1258A

CY1258A - Cyclosporin derivatives

Info

Publication number: CY1258A
Application number: CY125878A
Authority: CY
Original assignee: Sandoz Ltd
Priority date: 1977-05-10
Filing date: 1978-05-08
Publication date: 1984-11-23
Also published as: HK79684A; KE3442A; SG49284G; GB1591933A; MY8500646A

Description

PATENT SPECIFICATION <u) 1 591 933

(21) Application No. 18159/78 (22) Filed 8 May 1978

OS

ITi

(31) Convention Application No's. 5822/77 (32) Filed 10 May 1977 in

5823/77 '

(33) Switzerland (CH)

(44) Complete Specification Published 1 Jul. 1981

(51) INT. CL.3

C07C 103/52 A61K 37/02

(52) Index at Acceptance C3H 311 350 A7

(72) Inventors: RENE P. TRABER MAX KUHN HANS HOFMANN EUGEN HARRI

(54) CYCLOSPORIN DERIVATIVES

(71) We, SANDOZ LTD., of 35 Lichtstrasse, 4002 Basle, Switzerland, a Swiss Body Corporate, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:

The present invention relates to cyclosporin derivatives. More particularly the present invention relates to compounds of formula I

C"*V'XH3 _ CH, CH ;CH CH^ .CH. "3 , i ;L ;L „Lri v J / ;3 X / ;CH. CM Cll CH ;10 1 2 « 3 I I I 3 10 ;Ctl—W-CM-C— N LH A — C N - CH—C N CM ;3 It » L II I L » I 2 ;0-C ° OHO c-0 ;CH3S I I <H ;1, /CH-ayp, L ;0,3 CH -N 0 H 0 0 H 0 I 3 I D g L lit *1. I II U 0-C-CH— N-t —CH— N-C—CH — N — C-CH — H — t CH

III I 1 » I

CH H CH, CH, OL th

3 3 I 2 3 CH. CH

I 2

20 /»s 013 ^ /CH\ 20

CH3 ch3- cfT3 ch3

wherein A is a group of formula II

CH,

CH?

25 »i 25

2\

CH2 (II)

30 \R/30

CH, CH 3

— C N CH —

0 L

or of formula III

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CH, H C

c v \n-. (ill)

X

s\

Cli CH,

I 3

10 —C HN-CH- 10

o I L

3

which compounds arc hereinafter referred to as dihydrocyclosporin D and isocyclosporin D, respectively.

15 The present invention also provides a process for the production of:- 15

a) dihydrocyclosporin D which comprises hydrogenating a compound of formula I above wherein A is a group of formula IV,

CH, H \ /

20 c 20

a f"2 (IV)

I R

5 HO „ CH 25

1 3 I 3

— C N CH—

II <-

30 ° 30

which compound is hereinafter referred to as cyclosporin D, and b) isocyclosporin D which comprises isomerising cyclosporin D by subjection to acid conditions.

(The symbols "D" and "L" in the formulae I to IV above and in the accompanying claims 35 indicate D and L optically active centres in accordance with standard usage). 35

The required starting material for the above process, cyclosporin D, may be produced by cultivating a cyclosporin D producing strain of the species Tolypocladium inflatum Gams in a nutrient medium and isolating cyclosporin D.

The cultivation process may be effected in conventional manner for analogous strains, 40 e.g. as described in Example 1 hereinafter. 40

A preferred cyclosporin D producing strain is the freely available strain NRRL 8044 of the species Tolypocladium inflatum Gams, a culture of which is available from United States Department of Agriculture (Northern Research and Development Division), Peoria, 111., USA. This strain was formerly described as a strain of the species Trichoderma 45 polysporum (Link ex Pcrs.) and is described in the literature, e.g. in U.K. Patent 45 Specification No. 1,491,509.

Alternatively cyclosporin D producing strains obtained by selection or mutation of NRRL 8044, or treatment of this strain by ultraviolet light or X-rays, or treatment of this strain with laboratory chemicals, may be used.

50 Cyclosporin D may be isolated in conventional manner, e.g. as described in Example 1, 50 from other natural products that may be produced in greater amounts, e.g. the somewhat more polar cyclosporin A (also known as S 7481/F-l), the more polar cyclosporin B (also known as S 7481/F-2) and the yet more polar cyclosporin C.

The compound cyclosporin D is production and compositions containing the same and 55 the subject of our co-pending application No. 8006316 (Serial No. 1591934). 55

The hydrogenation process (a) may be effected in conventional manner, e.g. by catalytic hydrogenation. Preferred solvents include methanol, ethanol, isopropanol or ethyl acetate. The proccss is preferably effected in a neutral medium at a temperature of from 20 to 30°C and at atmospheric or slightly elevated pressure. A preferred catalyst is palladium on 60 charcoal. 60

The acid treatment of process (b) may be effected in conventional manner, e.g. with trifluoroacctic acid or preferably methanesulphonic acid or p-toluenesulphonic acid. The mole ratio of acid to cyclosporin D is preferably from 1:1 to 4:1. Preferred solvents include methanol, chloroform, and dioxane. Preferred temperatures are from 20 to 65°C, more 65 preferably from 40 to 55°C. 65

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1 591 933

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In the following examples all temperatures are in degrees Centigrade. All ratios are by volume unless otherwise stated.

EXAMPLE 1 5 Preparation of starting material - cyclosporin D

500 litres of a nutrient solution containing per litre, 40 g of glucose, 2 g of sodium caseinate, 2.5 g of ammonium phosphate, 5 g of MgS04.7H20, 2 g of KH2P04, 3 g of NaN03, 0.5 g of KC1, 0.01 g of FeS04 and demineralized water to 1 litre, are inoculated with 50 litres of a pre-culture of the strain NRRL 8044 and are incubated in a steel 10 fermenter under stirring (170 rpm) and aeration (1 litre air/minute/litre nutrient solution) for 13 days at 27° (see U.K. Patent Specification No. .1,491,509).

The culture liquor is stirred with the same amount of n-butyl acetate, concentrated by evaporation in a vacuum after separation of the organic phase and the crude extract is de-fatted by a 3-stage partition between methanol/water (9:1) and petroleum ether. The 15 methanolic phase is separated, concentrated by evaporation in a vacuum and the crude product is precipitated by the addition of water. The material obtained upon filtration is chromatographed on silica gel with n-hexane/acetone (2:1) as eluant. The initially eluted fractions contain predominantly cyclosporin A and cyclosporin D and the later eluted fractions contain predominantly cyclosporin C. For further purification the cyclosporin 20 A-and D-containing fractions are crystallized from a 2- to 2.5-fold amount of acetone at —15°. The crystallate is further chromatographed twice on silica gel, the fractions initially eluted with water-saturated ethyl acetate, containing cyclosporin D in greatly enriched form. These are dissolved in twice the amount of acetone ana are allowed to crystallize at —15°. The resulting crude crystalline product of cyclosporin D is dissolved for further 25 purification in a 10-fold amount of acetone, 2% by weight of active charcoal are added and heating is effected for 5 minutes to 60°C. The clear and almost colourless filtrate obtained upon filtration over talc is concentrated by evaporation to a third of its volume and is allowed to cool down at room temperature, whereupon cyclosporin D crystallizes spontaneously. The crystallization is completed by allowing to stand at -17°. The crystals 30 obtained by filtration are washed with a small amount of ice-cold acetone and are subsequently dried in a high vacuum at 80° for 2 hours.

CHARACTERIZATION OF cyclosporin D:

Colourless, prismatic crystals. M.P. 148-151°

35 [a]2£ = -245° (c = 0.52 in chloroform)

[a]2g = —211° (c = 0.51 in methanol)

EXAMPLE 2

40 Preparation of end-product: dihydrocyclosporin D

400 mg of palladium on charcoal [10% (w/w) palladium] are pre-hydrogenated in 15 ml of ethanol during the course of 20 minutes. To this suspension of the palladium catalyst, there is added the solution of 3.66 g of cyclosporin D in 30 ml of ethanol. The mixture is hydrogenated at 24° and at a pressure of 736 mm of Hg until the hydrogen take-up is 45 complete. Subsequently the catalyst is filtered off and the filtrate is evaporated to dryness in a vacuum at 20 to 40°C. The dihydrocyclosporin D, which is pure by thin layer chromatography, precipitates as colourless amorphous powder, which is dried in a high vacuum for 4 hours at 70°.

CHARACTERIZATION OF dihydrocyclosporin D: M.P. 153-156°

50

[a]2o = -237° (c = 0.56 in chloroform)

[a]2o = —196° (c = 0.58 in methanol)

55 EXAMPLE 3

Preparation of end-product: isocyclosporin D

A solution of 3.6 g of methanesulphonic acid in 60 ml of dioxane is added to the solution of 18.25 g of antibiotic cyclosporin D in 120 ml of absolute dioxane and the mixture is kept at 50° in the absence of moisture. The reaction course is followed by thin layer 60 chromatography [polygram SIL G-foils; chloroform/methanol/glacial acetic acid (90:6:4); iodine vapour for detection]. After 17 hours, the mixture is cooled to room temperature. After 3.38 g of anhydrous sodium acetate is added the precipitated salt is filtered with suction after stirring for 15 minutes and the filtrate is concentrated by evaporation in a vacuum at 45°. The 21 g of residue are chromatographed on 1.5 kg of silica gel, using 65 chloroform/methanol (98:2) for elution. The fractions consisting of practically pure

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isocyclosporin D are combined, concentrated by evaporation in a vacuum at 50° and the residue is crystallized twicc or thrice from diethyl ether, whereupon the isocyclosporin D precipitates out.

CHARACTERIZATION OF isocyclosporin D: M.P. 142-144°

[a]2!! = -205.5° (c = 0.51 in chloroform)

[ci32d = -144.4° (c = 0.64 in methanol)

The compounds dihydrocyclosporin D and isocyclosporin D exhibit pharmacological activity. In particular, they exhibit anti-inflammatory activity and anti-arthritic activity as indicated in standard tests with animals, for example by an inhibition of swellings in the Freunds adjuvant arthritis test in rats on p.o. administration of 3 to 100 mg/kg of the compounds.

The compounds are therefore indicated for the treatment and prophylaxis of chronic inflammations, e.g. arthritis and rhcumatic disorders. For this use an indicated daily dose is from 50 to 900 mg, conveniently administered in divided doses 2 to 4 times a day in unit dosage form containing from 15 to 450 mg, or in sustained release form.

The present invention also provides a pharmaceutical composition comprising dihydrocyclosporin D or isocyclosporin D in association with a pharmaceutical carrier or diluent.

Such compositions may be in the form of, for example, a solution or a tablet.

The compound of Example 3 exhibits especially interesting activity.

WHAT WE CLAIM IS:

1. A proccss for the production of a compound of formula I,

C"^C,^CM3 CH CH3 ch.a

CH CHA/CH3 ^ yr J

CH Cli Cli CH.

("2 T"'S 7" T" 7 "3

CH-N-CM-C—N C.H A c N- t:n C U Cli

3 I L II L |l I L » I 2

CM DI

o.c w 0 C-0

•as I I <*>;/CH-CiyCM L;3 CH -N 0 M 0 0 H 0;» L »jL It »«U;0-C-CH — N-C — CH— N-C —CH —N — C- CM — N— C CK;3 I D;III I " I J;CH H CH. CH Ol, /CHV CH;3 5 ' 2 3 of CH, 1 2;wherein A is a group of formula II,;CHT;tH.,;ch3 ch3- cfi3 ch3;I;H,C;2 \;CHJ (II);or of formula 111,;CH, CH cn3 I 3 i;N CH —;Clt, H ^ /;C;n \H, '*«>

2

CH ^CH.

I

-C UN—CH —

I L

CH3

O

which compounds arc hereinafter referred to as hvdrocyclosporin D or isocyclosporin D

5

1 591 933

5

5

respectively, which process comprises a) for the production of dihydrocyclosporin D, hydrogenating a compound of formula I, above, wherein A is a group of formula IV,

CHj H

5

10

L2

(IV)

10

N CH—

C

15

il

L

15

o which compound is hereinafter referred to as cyclosporin D, or b) for the production of isocyclosporin D, isomerising cyclosporin D by subjection to acid conditions.

20 2. A process as claimed in Claim 1, substantially as hereinbefore described with 20 reference to Example 2 or 3.

3. Dihydrocyclosporin D or isocyclosporin D according to Claim 1 whenever prepared by a process as claimed in Claim 1 or Claim 2.

4. Dihydrocyclosporin D of formula I as illustrated in Claim 1, wherein A is a group of

25 formula II as illustrated in Claim 1. 25

5. Isocyclosporin D of formula I as illustrated in Claim 1, wherein A is a group of formula III as illustrated in Claim 1.

6. A pharmaceutical composition comprising dihydrocyclosporin D as claimed in Claim 4 in association with a pharmaceutical carrier or diluent.

30 7. A pharmaceutical composition comprising isocyclosporin D as claimed in Claim 5 in 30 association with a pharmaceutical carrier or diluent.

Chartered Patent Agents, 98, The Centre, Feltham, Middlesex TW13 4EP Agents for the Applicants.

B.A. YORKE & CO.

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Primed for Her Majesty's Stationery Office, by Croydon Printing Company Limited. Croydon. Surrey, 1981. Published by The Patent Office, 25 Southampton Buildings. London. WC2A 1 AY, from which copies may be obtained.

BNSDOCID: <GB 1591933A__I_>