CH634293A5 - Process for preparing the novel antibiotic derivative dihydroisocyclosporin D - Google Patents

Abstract

The preparation is described of the novel antibiotic derivative dihydroisocyclosporin D (formula I) <IMAGE> which has an antiarthritic effect and is intended to be used for treating immunologically conditioned reactions. The antibiotic dihydrocyclosporin D (formula II) <IMAGE> is subjected to treatment with an acid.

Description

The present invention relates to a process for the preparation of the new antibiotic derivative dihydro-iso-cyclo-sporin D (formula I).

ch3>

CH CK s. f ch3 ch ch3

CH3

ch3

co-

-n ch-

i l h

-c n-

II

ch -.- n

■ chj co i d

OC-CH-

1

t

Cm,

h h

l I

-co-ch n-

i ch3

-co-

l

-ch-

ch2

i ch

-n

I.

ch3

0 II

-c-

H

; h — n - co-

CH

ch3 ch3

n-ch3 -CH

I.

ch2

I.

ch ch3 ch3 ch3 ch3

According to the invention, dihydro-isocyclosporin D is obtained by using dihydro-cyclosporin D (formula II)

ch3n ch.

ch3n ^ ^ CH3

ch

I.

ch2

CH3v ch3

ch3-n-ch-co-n-

co

"iCN: h2

I CK

CH, HO \ / CH \

ch ch3

"ch ch3 i

-L- '

\ / CH3

CH

-c-

1! O

-N-

-ch-L

-co-

-ch-

l ch3

\

CH3

/

ch-ch2-ch l ch3-N

ch3

I.

-c n ch2

II

co

I D

oc-ch-

ch3

-N-

I.

h t

-co-ch -

I.

ch3

H

I.

-n-

-co-

l

-ch—-

I.

ch2

-N

I.

ch3

-ch-

I.

ch h

I.

-n - co-

ch3 ch3

N-CH3 -ch

I.

ch2

I.

ch / \ ch3 CH3

ch

/

ch3 ch3

subject to acid treatment under suitable conditions.

60

Dihydro-iso-cyclosporin D can be obtained, and in-products can then be obtained by chromatography on which dihydro-cyclosporin D (obtained from Hy-silica gel with chloroform-methanol as eluent).

Drying of cyclosporin D) z. B. be cleaned in a suitable Lö. The chromatography fractions can be treated with strong organic acids. For thin layer chromatography checked for uniformity

Example can be dihydro-cyclosporin D in methanol or di- 65, for which z. B. Polygram SIL G-foils with the oxane with methanesulfonic acid for 6 to 20 hours with flow agent chloroform-methanol-glacial acetic acid (90: 6: 4) are suitable 50 ° C. After buffering the acid, e.g. B. (running distance 10 cm). The obtained from the chromatography

with sodium acetate, the resulting mixture of the reaction pure or almost pure fractions become

634 293

4th

Finally absorbed in ether and the product then precipitated with petroleum ether.

Properties of Dihydro-iso-cyclosporin D

Colorless amorphous powder, mp. 145-147 ° [a] D20 = -205 ° (c = 0.51 in CHC13) = - 146.6e (c = 0.60 in CH3OH)

Elemental analysis

C63H115Nai012

Calc .: C 62.1 H 9.5 N 12.6 O 15.8%

Found: C 61.9 H 9.7 N 12.8 O 16.1%

The compound shows end absorption in the UV spectrum. In the IR spectrum (in CH2C12, Fig. 1) an ester band can be seen at 1735 cm-1.

The 'H-NMR spectrum in CDC13 at 90 MHz with tetramethylsilane as the internal standard is shown in Fig. 2.

Dihydro-iso-cyclosporin D is readily soluble in methanol, acetone, ethyl acetate, chloroform; moderately soluble in ether;

very poorly soluble in water and in saturated hydrocarbons.

An Rf value of 0.31 is observed in the thin layer chromatogram on polygrams of SIL G foils with chloroform-methanol-glacial acetic acid (90: 6: 4), running distance 10 cm. The detection is done with iodine vapor.

Dihydro-iso-cyclosporin D is characterized by interesting chemotherapeutic and pharmacological properties and can therefore be used as a remedy. It inhibits the growth of Aspergillus niger and Curvularia lunata.

In particular, the substance is characterized by an immunosuppressive and anti-inflammatory effect.

The immunosuppressive effect of dihydro-iso-cyclosporin D can be shown as follows:

a) In the lymphocyte stimulation test according to Jânossy, a strong inhibition of H3-thymidine incorporation, the proliferation rate and the blastogenesis of lymphocytes stimulated with concanavalin A from mouse spleen was found in vitro in concentrations of 0.01 to 10.0 ng / ml.

b) Oxazolone test on the mouse: The decrease in ear swelling is expressed as a suppressive index (SI); SI = 0.57 after 5 x 70 mg / kg p.o.

Due to its immunosuppressive effect, di-hydro-iso-cyclosporin D can be used for the prophylaxis and treatment of diseases that are related to the negative effects of the defense reaction.

Dihydro-iso-cyclosporin D also has an anti-arthritic effect. So it works e.g. in Freund's ad juvans arthritis latency test on rats in doses of approx. 50 mg / kg body weight / day, strongly anti-swelling.

A similar effect is observed in Freund's adjuvant arthritis therapy trial in rats at doses of 10 mg / kg / day.

Due to its arthritis-inhibiting effect, dihydro-iso-cyclosporin D can be used for the prophylaxis and treatment of arthritis and rheumatic diseases.

The doses to be used naturally vary depending on the type of administration and the condition to be treated. In general, however, satisfactory results are obtained with test animals with a dose of 10 to 200 mg / kg body weight. If necessary, this dose can be administered in 2 to 3 portions or as a slow-release form.

For larger mammals, the daily dose is around 50 to 900 mg. For oral applications, the partial doses can contain, for example, about 25 to 300 mg of the dihydro-isocyclosporin D in addition to solid and liquid carrier substances.

As a remedy, dihydro-isocyclosporin D can be administered alone or in a suitable pharmaceutical form with pharmacologically indifferent auxiliaries.

The dihydro-cyclosporin D used as the starting material is produced as follows:

400 mg palladium-carbon (10% palladium) are pre-hydrogenated in 15 ml ethanol for 20 minutes. The solution of 3.66 g of cyclosporin D in 30 ml of ethanol is added to this suspension of the palladium catalyst and then hydrogenated at 24 ° and a pressure of 736 mm of mercury until the hydrogen uptake has ended. The catalyst is then filtered off and the filtrate is evaporated to dryness in vacuo at 20 to 40 °. The thin-layer chromatography-uniform dihydro-cyclosporin D is obtained as a colorless amorphous powder, which is dried under high vacuum at 70 ° for 4 hours.

The cyclosporin D used as the starting material is produced as follows:

5001 of a nutrient solution containing 40 g of glucose, 5 g of casein peptone, 5 g of MgS04-7H2Ò, 2 g of KH2P04, 3 g of NaN03, 0.5 g of KCl, 0.01 g of FeS04 and demineralized water are mixed with 501 of a preculture of the NRRL 8044 strain and incubated in a steel fermenter with stirring (170 rpm) and aeration (1 liter air / min. / liter nutrient solution) at 27 ° for 13 days (see DOS 2 455 859).

The culture broth is stirred with the same amount of n-butyl acetate, after the organic phase has been separated off, it is concentrated in vacuo and the crude extract is degreased by 3-stage distribution between methanol-water (9: 1) and petroleum ether. The methanolic phase is separated off, concentrated in vacuo and the crude product is precipitated by adding water. The material obtained after the filtration is chromatographed on the 5- to 7-fold amount of Sephadex LH-20 with methanol as the eluent. The peak fractions are then chromatographed on silica gel 60, particle size 0.063-0.20 mm (Merck) with hexane-acetone (2: 1), the fractions eluted first predominantly containing cyclosporin A and cyclosporin D, the later eluted portions predominantly cyclosporin C. For further purification, the cyclosporin A and D-containing fractions are crystallized from the 2 to 2.5 times the amount of acetone at -15 ° and then further separated by double chromatography on silica gel 60, particle size 0.063-0.20 mm (Merck), the fractions which were eluted first with hexane-acetone (2: 1) contain cyclosporin D in a highly enriched form. These are dissolved in twice the amount of acetone and left to crystallize at -15 °. The crude crystals of cyclosporin D thus obtained are dissolved in 10 times the amount of acetone for further purification, 2% by weight of activated carbon are added and the mixture is heated to 60 ° for 5 minutes. The clear and almost colorless filtrate obtained after filtration over talc is concentrated to a third of the volume and allowed to cool to room temperature, cyclosporin D spontaneously crystallizing out. The crystallization is completed by standing at -17 °. The crystals obtained by filtering are washed with a little ice-cold acetone and then dried in a high vacuum at 80 ° for 2 hours.

Mp 148-151 °

[a] D20 = -245 ° (c = 0.52 in CHC13)

[a] D20 = -211 ° (c = 0.51 in CH3OH).

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

S

2 sheets of drawings

Claims (4)

634 293 PATENT CLAIM Process for the preparation of the new antibiotic derivative dihydro-isocyclosporin D (formula I), ch3. CH " CH3> ^ ^ CH3 ch CH3 ^ ^ CH] ch2 ch3 ch x: H2 ch ch3 CH ch3 CH3-n-ch-co-n ch c hn ch co n ch c n 'ch? CO I L CH, O CH '. \ ch-ch, -ch l ch3 / CHj-N CO n-ch3 H oc-ch n co-ch n co ch n III ■ J I I ch3 h ch3 ch2 ch3 ^ ch h I. ch / \ CH3 ch3 characterized in that dihydrocyclosporin D (formula II) h-n-co-ch I. ch2 ch3 ch3 CH chi ch3 CKi ch3 ^ / ch3 ch T1 HZC ^ i ch3 ^ ch-j ch2 ch3 ch HO. H? ! ch. CH ch ch3 \ ch3 CH 3. / CH3 CH3-N-CH-CO-N- -CH C N- L ü o ch3 ch- \ ch CO • chj-ch L ch3 ch-j— n -ch co n ch c n ch2 l 1 L II h o CO oc-ch- I I ch3 h ■ N CO-CH N CO CH for \! c CH N-CO CH ch- 1 I ch2 ch3 I. ch ch3 ch3 ch ch3 ch3 ch 2 I. CH Ch3 ch3 subjected to an acid treatment. 3rd 634 293