CH637123A5 - Monocyclic peptide, its preparation and use - Google Patents

Abstract

The monocyclic peptide of the formula I is prepared by cultivating, in the presence of a nutrient medium, a strain of the fungal species Tolypocladium inflatum Gams which produces this peptide, and isolating the peptide from the fermentation broth by extractive and/or adsorptive procedures, and then purifying it by chromatography or by means of countercurrent distribution. The peptide of the formula I exhibits immunosuppressive properties. <IMAGE>

Description

       

  
 

** WARNING ** beginning of DESC field could overlap end of CLMS **.

 



   PATENT CLAIMS 1. The monocyclic peptide of formula I.
EMI1.1




  2. A process for the preparation of the monocyclic peptide of the formula I according to claim 1 by culturing a strain of the fungal species Tolypocladium infiatum Gams producing this peptide in the presence of a nutrient medium, characterized in that the peptide is isolated from the fermentation broth by extractive and / or adsorptive working methods and then cleaned chromatographically or by means of countercurrent distribution.



   3. Remedy, characterized in that it contains the monocyclic peptide of formula I according to claim 1.



   The present invention relates to the monocyclic peptide of the formula I,
EMI1.2
 hereinafter also referred to as the new antibiotic cyclosporin F.



   The monocyclic peptide of the formula I is obtained by growing a strain of the fungal species Tolypocladium infiatum Gams producing this peptide in the presence of a nutrient medium and the peptide from the fermenter



  tion broth isolated by extractive and / or adsorptive working methods and then cleaned chromatographically or by means of countercurrent distribution.



   A preferred strain producing cyclosporin F is the freely accessible strain NRRL 8044 of the fungal species Tolypocladium inflatum Gams. A culture of this was deposited in the United States Department of Agriculture (Northern Research and Development Division), Peoria, I11., USA.



  This strain was previously assigned to the fungus species Trichoderma polysporum (Link ex. Pers.) And is e.g. described in DOS 2455859.



   Strains of the fungal species Tolypocladium infiatum Gams can also be used for the production of cyclosporin F, such as those e.g. by selection or mutation of the NRRL 8044 fungal strain under the influence of ultraviolet or X-rays or by application of other measures, e.g. by treating laboratory cultures with suitable chemicals.



   Cyclosporin F can be isolated in a manner known per se, e.g. as described in Example 1. Cyclosporin F can be separated from natural products that are present at the same time, e.g. the cyclosporin D, the more polar cyclosporin A (also known as S 7481 / F-1), the more polar cyclosporin B (also known as S 7481 / F-2) and the even more polar cyclosporin C.



   Characterization of cyclosporin F
Colorless polyhedron (crystallized from ether / petroleum ether 1: 1) mp 183-184 "[oilD = -290" (c = 1.15 in CHCI3) -218 (c = 1.00 in CHOH) UV spectrum in CHOH: final absorption IR spectrum in CH2CI2: see FIG. 1 'H-NMR spectrum in CDCl3, 90 MHz, tetramethylsilane as internal standard: see FIG. 2.



  '3C NMR spectrum in CDCl3, 22.63 MHz, tetramethylsilane as internal standard. Instrument: Bruker HX-90 E. Recording at 22.63 MHz. Spectra width (sweep width): 6000 Hz: see table 1 below.



   Table IO (ppm) 8 (ppm) 8 (ppm) 8 (ppm) 173.15 55.4 36.0 23.8 172.89 55.1 31.7 23.8 172.62 54.9 31.6 23.8 172.62 54.3 31.2 23.5 171.72 49.9 31.2 21.9 170.87 48.7 29.9 21.9 170.58 48.3 29.9 21. 3 170.26 47.9 29.9 20.9 170.14 44.7 29.6 20.4 169.74 40.5 29.6 20.1 169.53 40.5 24.9 19.7 129 , 4 39.2 24.8 18.5 126.6 39.2 24.8 18.3
58.4 37.3 24.5 17.8
57.3 36.7 24.3 17.8
15.1
9.9 mass spectrum: peak at m / e 1187 (molecular peak of C62HXlNxlOt) elemental analysis (dried under high vacuum for 3 hours at 80 ") gross formula: C62HIllNllO analysis: calc .: C 62.8; H 9.4; N 13.0; 0 14.8% Found: C 62.8; H 9.5; N 13.0; 0 14.8%
Behavior in the thin layer chromatogram
Ready-made silica gel panels Merck, layer thickness 0.25 mm, amount of substance applied: 40 y.

  Rf values: see table 2 below. (Detection is carried out using Munier iodine vapor or Dragendorff spray reagent.)
Table 2 Metabolite chloroform-meth-acetone-hexane 1: 1 acetic ester anol 96: 4 a) a) b) Cyclosporin FC) 0.51 0.45 0.50 Cyclosporin D 0.47 0.43 0.58 Cyclosporin A 0.40 0.35 0.39 a) Running distance: 1 x 10 cm b) Running distance: 3 X 10 cm c) Staining in iodine weaker than cyclosporin A.



   Amino acid analysis: In the hydrolyzate, which is obtained by heating cyclosporin F with 6N hydrochloric acid for 16 hours at 115, the amino acids alanine, or-aminobutyric acid, valine and sarcosine can be detected using an amino acid analyzer due to the retention times.



   Solubility: Cyclosporin F is easily soluble in methanol, ethanol, acetone and chlorinated hydrocarbons; moderately soluble in ether; practically insoluble in water and saturated hydrocarbons.



   The new antibiotic Cyclosporin F 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 effect.



   The immunosuppressive effect can be shown as follows: a) In the lymphocyte stimulation test according to Janossy, in vitro concentrations of 0.01 to 10.0 Fg / ml show a strong inhibition of H3-thymidine incorporation, the proliferation rate and the blastogenesis of with concanavalin A. stimulated lymphocytes from mouse spleen were found.



   b) Oxazolone test on the mouse:
The decrease in ear swelling is expressed as a suppressive index (SI); SI = 0.62 after 5 x70 mg / kg p.o.



   Due to its immunosuppressive effect, the substance can be used for the prophylaxis and treatment of diseases that are related to the negative effects of the defense reaction.



   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 60 to 300 mg / kg of 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 300 to 900 mg. For oral applications, the partial doses can contain, for example, about 150 to 300 mg of the new antibiotic cyclosporin F in addition to solid and liquid carriers.



   As a remedy, the new antibiotic cyclosporin F 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
Cyclosporin F
500 liters of a nutrient solution containing 40 g glucose, 2.0 g sodium caseinate, 2.5 g ammonium phosphate, 5 g MgSO4-7H20, 2 g KH2PO4, 3 g NaNO3, 0.5 g KCI, 0.01 g FeSO4 and contains demineralized water, are inoculated with 50 liters of a pre-culture of strain NRRL 8044 and incubated in a steel fermenter with stirring (170 rpm) and aeration (1 liter air / min. / liter nutrient solution) for 13 days at 27 (see DOS 2455 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 top fractions are then chromatographed on silica gel 60, particle size 0.063-0.2 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-fold amount of acetone at -15 and then further separated by double chromatography on silica gel 60, particle size 0.063-0.2 mm (Merck), the Fractions cyclosporin D first eluted with hexane-acetone (2: 1) contained in a highly enriched form. These are dissolved in twice the amount of acetone and left to crystallize at - 15. The crude crystallizate obtained in this way consists of very strongly enriched cyclosporin D, the mother liquor contains, in addition to cyclosporin D, further components, so cyclosporin F.

  To obtain cyclosporin F, the mother liquor evaporated to dryness is chromatographed on silica gel 60, particle size 0.063-0.2 mm (Merck), with water-saturated ethyl acetate, the fractions which eluted first contain cyclosporin D and the fractions which later eluted cyclosporin F in a mixture, among others Components. For further enrichment, the mixture is first chromatographed on silica gel 60, particle size 0.063-0.2 mm (Merck), with chloroform-methanol (98: 2) and then for separation with hexane-acetone (2: 1); the fractions eluted first contain cyclosporin F in a very enriched form.

  For purification, the cyclosporin F fractions are crystallized twice from ether petroleum ether (1: 4) at room temperature, pure cyclosporin F in the form of colorless polyhedra of mp.


    

Claims (3)

 PATENT CLAIMS 1. The monocyclic peptide of formula I. EMI1.1 2. A process for the preparation of the monocyclic peptide of the formula I according to claim 1 by culturing a strain of the fungal species Tolypocladium infiatum Gams producing this peptide in the presence of a nutrient medium, characterized in that the peptide is isolated from the fermentation broth by extractive and / or adsorptive working methods and then cleaned chromatographically or by means of countercurrent distribution.    3. Remedy, characterized in that it contains the monocyclic peptide of formula I according to claim 1.  The present invention relates to the monocyclic peptide of the formula I, EMI1.2  hereinafter also referred to as the new antibiotic cyclosporin F.  The monocyclic peptide of the formula I is obtained by growing a strain of the fungal species Tolypocladium infiatum Gams producing this peptide in the presence of a nutrient medium and the peptide from the fermenter ** WARNING ** End of CLMS field could overlap beginning of DESC **.