CH630061A5 - Process for the preparation of an antibiotic derivative - Google Patents

Abstract

The preparation of the antibiotic derivative dihydroisocyclosporin D (formula I) <IMAGE> which acts as antiarthritic and is used for the treatment of immunological reactions is described. The antibiotic isocyclosporin D (formula II) <IMAGE> is hydrogenated.

Description

       

  
 



  The present invention relates to a process for the preparation of the new antibiotic derivative dihydroisocyclosporin D (formula I).
EMI2.1




  According to the invention, dihydroisocyclosporin D is obtained by using isocyclosporin D (formula II)
EMI2.2

The hydrogenation of isocyclosporin D is advantageously carried out by hydrogenating isocyclosporin D in a suitable solvent in the presence of catalytically activated hydrogen. Lower aliphatic alcohols, such as e.g. Methanol, ethanol, isopropanol, in question. The hydrogenation takes place at temperatures between 20 and 30 C and at atmospheric pressure or very slightly elevated pressure. Palladium on carbon is preferably used as the catalyst. The hydrogenation product is then purified in a manner known per se, e.g. by chromatography on silica gel.



  Properties of Dihydroisocyclosporin D Colorless amorphous powder mp 145-147 [X] 20D = -205 (c = 0.51 in CHCl3) = -146.6 (c = 0.60 in CH3OH)
Elemental analysis for C63H115N11O12:
Calc .: C62, l H 9.5 N 12.6 015.8% Ge ±: C 61.9 H 9.7 N 12.8 0 16.1%
The compound shows end absorption in the UV spectrum. In the IR spectrum (in CH2Cl2, Fig. 1) an ester band can be seen at 1735 cm-1.



   The 1H-NMR spectrum in CDCI3 at 90 MHz with tetramethylsilane as internal standard is shown in Fig. 2.



   Dihydroisocyclosporin D is readily soluble in methanol, acetone, ethyl acetate, chloroform; moderately soluble in ether; very sparingly soluble in water and in saturated hydrocarbons.



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



   Dihydroisocyclosporin 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 dihydroisocyclosporin D can be shown as follows: a) In the lymphocyte stimulation test according to Janossy, in vitro in
Concentrations of 0.01 to 10.0 g / ml strongly inhibit the H3-thymidine incorporation, the proliferation rate and the Bla stogenesis of lymphocytes stimulated with Concanavalin A.
Mouse spleen detected.



  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, dihydroisocyclosporin D can be used for the prophylaxis and treatment of diseases which are related to influencing the immune response in a negative sense.



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



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



   Due to its anti-arthritic effect, dihydroisocyclosporin 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 dihydroisocyclosporin D in addition to solid and liquid carriers.



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



   In the following example, which explains the invention in more detail but is not intended to restrict its scope in any way, all the temperatures are given in degrees Celsius.



  Example:
400 mg palladium on carbon (10% Pd) are pre-hydrogenated in 15 ml ethanol for 30 min. The solution of 3.65 g of isocyclosporin D in 30 ml of ethanol is added to this suspension of the palladium catalyst and hydrogenated at 22 ° C. and a pressure of 738 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 ° C. This gives the dihydroisocyclosporin D, which is uniform by thin layer chromatography, as a colorless, amorphous powder which is dried in a high vacuum at 80 ° C. for 4 h.



   The isocyclosporin D used as the starting material is produced as follows:
5001 of a nutrient solution containing 140 g of glucose, 5 g of casein peptone, 5 g of MgSO4 7H, O, g of KH2PO4, 3 g of NaNO3, 0.5 g of KCI, 0.01 g of FeSO4 and demineralized water are mixed with 501 of a preculture of the Inoculated strain NRRL 8044 and incubated in a steel fermenter with stirring (170 rpm) and aeration (1 l air / min / l nutrient solution) for 13 days at 27 "C (see DOS 2455859).



   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 pointed 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 fractions containing cyclosporin A and D are crystallized from 2 to 2.5 times the amount of acetone at up to 15 ° C. and then further separated by double chromatography on silica gel 60, particle size 0.063 to 0.20 mm (Merck) , the fractions first eluted with hexane-acetone (2: 1) containing cyclosporin D in a highly enriched form, which are dissolved in twice the amount of acetone and allowed to crystallize at up to 15 C.

  The crude crystallizate of cyclosporin D thus obtained is dissolved in 1-fold amount of acetone for further purification, 2% by weight of activated carbon is added and the mixture is heated to 60 ° C. for 5 min. The clear and almost colorless filtrate obtained after talc filtration is reduced to a third of the Concentrated volume and let cool to room temperature, during which cyclosporin D crystallizes spontaneously. The crystallization is completed by standing at up to 17 ° C.



  The crystals obtained by filtering are washed with a little ice-cold acetone and then dried in a high vacuum at 80 ° C. for 2 hours.



   To the solution of 18.25 g of antibiotic cyclosporin D in 120 real absolute dioxane, the solution of 3.60 g of methanesulfonic acid in 60 ml of dioxane is added and the mixture is kept at 50 ° C. with exclusion of moisture. The progress of the reaction is monitored in a thin layer chromatogram [Polygram SIL G foils, chloroform-methanol-glacial acetic acid (90: 6: 4), iodine vapor for visualization. After 17 h, the mixture is cooled to room temperature. The acid is blunted by adding 3.38 g of anhydrous sodium acetate, after stirring for 15 min the salt which has separated out is filtered off with suction and the filtrate is evaporated in vacuo at 45 ° C. The 21 g residue is chromatographed on 1.5 kg silica gel, Merck, particle size 0.063 to 0.2 mm, chloroform-methanol (98: 2) being used for the elution.

  The fractions consisting practically of pure isocyclosporin D are combined, evaporated in vacuo at 50 ° C. and the residue is crystallized two to three times from ether, with isocyclosporin D of mp 142 to 144 ° C.


    

Claims (1)

 PATENT CLAIM Process for the production of the new antibiotic derivative dihydroisocyclosporin D (formula I), EMI1.1  characterized in that isocyclosporin D (formula II) EMI1.2  hydrated.