DK173335B1 - Cyclic peptolides, a process for preparing them, pharmaceutical preparations comprising the cyclic peptolides, a corresponding fermentation liquid and the fungal strain NRRL 18230 - Google Patents

Description

DK 173335 B1

The present invention relates to novel cyclic peptolides, a process for their preparation, pharmaceutical compositions containing these cyclic peptolides, a fermentation fluid, and the furigus strain NRRL 18230.

The term "peptolide" here refers to a natural or synthetic compound consisting of o-hyditoxy and α-amino acids which are bound to the gas by both anide and ester bonds. Thus, the structure obtained by substituting an anide linker down for ester linkage in a peptide is a peptide.

10

An important class of peptides are the cyclosporins characterized by a cyclic structure, usually containing 11 amino acid residues, one of which is N-methyl- (4R) -4-bi-t-2E * en-1-yl-4-methyl - (L) -threonyl residue, designated MeBmt, or one derivative thereof. Many cyclosporins have pharmacological properties, especially immunosuppressive and anti-inflammatory properties. The first cyclosporin isolated was the naturally occurring fungal metabolite cyclosporin A (Ciclosporin), which is commercially marketed under the registered trademark Sandimmune ·. This compound has the structure set forth in Formula I.

20

MeBnt-Abu-Sar-HeLeu-Val-MeLeu-Ala- (D) Ala-MeLeu-MeLeu-MeVal -. I I 1 2 3 4 5 6 7 6 9 10 11 | 25 (A complete list of abbreviations is given in Table II at the end of the narrower description.)

Conventionally, the amino acid residues of cyclosporins are numbered clockwise 2Q starting with the MeBmt residue. All α-amino acids have (L) configuration unless otherwise indicated: thus, alanine 1 has position 8 1 of formula I (D) configuration. The symbol Me in front of the abbreviation for an amino acid indicates that the amino acid residue is N-methylated on the nitrogen atom in the amide bond.

35 The cyclic peptolide of the invention is characterized in that it has the structure of a cyclosporin in which an amide bond is replaced by an ester bond.

2 DK 173335 B1

The cyclic peptolide is preferably composed of a MeBmt residue or a derivative thereof, 9 other α-alanoic acid residues and an α-hydroxy acid residue, which is preferably in position 8.

Preferred derivatives of MeBat are the 8'hydroxy derivative (8'OHHeBmt) and the nocturned dihydroderlvate MeBntH2 down the structures shown below / H HOCK, H% il II f2

H - C - CH - c 'I

I 2 ^ CH- I CH- HO fo \ CH, B, | "CH-

Η0 ^ (ΚΚ \ HO ^ CK

ch3 Γ * Γ -C "CHN" h

i 3 1 - N- tH - CO - .3 1 J

- N - CH - CO-- I 1, ς. (S) - N - CH - CO - (S) (S)

HeBmt S'OtMeBMt NeØrntH ^

The preferred cyclic peptides according to the invention have the structure shown in Formula II

: V - Thr - X - T - Z - HeLeu - Ala - A - NeLeu - Leu - MeVal 12 3 4 5 6 7 8 9 10 11 / II

where V represents MeBmt, S'-OHMeBmt or MeBmtH2 X represents Sar or Cly Y represents HeLeu or Leu Z represents Leu, Ile or Val IS and A represents the residue of a ck-hydroxycarboxylic acid,

preferably down to formula III

R

- 0 - ia - CO - III

wherein R is C ^. elk alkyl.

3 DK 173335 B1

Here preferably, R in the formula III is Isopropyl, so that A represents

CH

- o - Ih- co—.

the remainder of an α-hydroxyisovalerianic acid, abbreviated Hiv. The most preferred compound of the invention is the compound of formula II wherein W is KeBmt, X is Sar, Y is MeLeu, Z is Leu, and A is 5 (D) HIV. This compound can be shown by the full structural formula shown in Formula IV

e *

C

CM3 \ / ch> Cl I

CH «0. ^ CM · CH, I Vscm3 I t

cm, CM, Cm Ch, |, β> CH — OH CM j 1V

H-M-CH-CO-M-in-C-M. CH-CO-M-Ih-C-I CH,

| * '4 1 i i 4; IN

CO 10 H 1 3 3 I

e- \ i r

Ch-Cm-Cm in 9/1 1 CH 2 I N-CH,

CH..N 9 7 6 5 * I

'I Q 1 H O H

(o I I i i i i i i i

OC-CH O —CO-Ch — n —— CO — Ch - N—— C — Ch —— H-CO — CH

^ CM CH, CH, CH, Ih, Cm, cm, VcM * / «k ^ CH, Ch, CH, CH, Ch, Ch, 10 or using the now conventional nomenclature for cyclo-sporins based on The structure of Ciclosporin (cyclosporin A) shown in Formula I. This is done by listing in the order of each residue in the molecule that differs from that found in Ciclosporin and adding the term "Ciclosporin *. is thus designated as (Thr) 2 (Leu) 5 (D-Hiv) · (Leu) * ° - Ciclosporin, * · 4 DK 173335 B1, i.e., Ciclosporin, where Thr replaces Abu at position 2, Leu replaces Val at position 5 , (D) HIV replaces D (Ala) at position 8, and Leu replaces MeLeu at position 10, the other residues being identical down to the residues in Ciclosporin.

5

According to the invention, the cyclic peptides can be prepared by growing a producing microorganism strain in the presence of a nutrient medium. Preferred microorganisms are fungal strains of the species Cylindrotrichum Bonorden, especially the strain NRRL 18230, which produces cyclic peptolides of the formula

The stem is isolated from a leaf specimen from tfallenburg in swiss

Law, and a viable culture of the tribe, were deposited on June 17, 1987, with the "U.S. Department of Agriculture" (North Central Region,

Northern Regional Research Center), Peoria, 111. and has been numbered NRRL 18230. The culture can also be obtained from Sandoz Ltd., Basel, Switzerland.

15

The fungal fungus NRRL 18230 is a hyphomycet, and when incubated at 21-24 ° C on 21 oil extract / agar (-MA; 2X malt extract, 0.4X fermentation extract, 21 agar 1 demineralized water), it produces aseptate p. or often 1-septate bacilliform hyaline conidia of size 6-20 15 μη x 1.5-2.7 μη (mostly 9.5-13.5 μη).

The conidiogenic cells are generally cylindrical and have a pronounced colarette; some cells appear sympodial-polyphallic disks. According to the identification key of M.B. Elles (Denatiaceous Hyphomycetes; 25 Commonwealth Mycological Institute, Kew, Surrey, England, 1971) can best classify the strain as belonging to the species Cylindrotrichupi Bonorden.

The fungal strain NRRL 18230 grows relatively slowly, and after 10 days of 2Q incubation at a temperature of 21 ° C, it forms colonies with a diameter of 4-7 mm with a wing gray air mycelium. The optimal

growth temperature is between 18 ° C and 27 ° C, and at temperatures above 33 ° C

there is no growth. The branched and septate aerial mycelium of colonies grown on MA at 21 ° C is usually 1.5-3.5 μια (usually 2-3 μηι) wide; 35 mycelium hyphae with a width of up to 5 can be observed in the substrate. 5 μια.

5 DK 173335 B1

The invention also relates to fermentation whey obtained by growing a cyclic peptide-producing fungus strain of the species Cvlindrotrichum Bonorden. The novel strain NRRL 18230 can be grown by an aerobic surface or Immersion process at suitable temperatures in a wide variety of nutritional media containing the nutrients and minerals in exploitable form.

Thus, the medium should contain an assimilable source of carbon and possibly mineral salts and growth factors. All of these components can be added in the form of well-defined simple compounds or in the form of complex 10 mixtures prepared from biological sources. Cultivation is carried out in accordance with conventional methods and the cyclic peptides formed during fermentation can finally be isolated from the culture medium using well-known chromatographic methods. The cyclic peptolides of the invention may also be prepared by culturing variant * or mutant strains prepared by selection or by the action of mutation inducing agents, e.g., UV light, X-rays or chemical mutant genes on a NRRL 18230 or other strains of the Cylindrotrichum Bohord.

The cyclic peptolides of the invention can also be prepared by synthetic or semi-synthetic methods, for example, by cyclizing a linear peptide or a linear peptide with a terminal OH group instead of a terminal NH 2 group; or by replacing an amide bond in a natural synthetic or semi-synthetic cyclosporin with an ester bond.

25

The total synthesis of the preferred compounds of formula II may be carried out analogously to the total synthesis of cyclosporin A and analogous to that described, for example, in EP-34 567 and by R.Uenger in "Transplantation Proceedings", vol. XV, pp. 2230-2241 ( 1983). In this method 30, the C-protected heptapeptide of formula V is first prepared

V - Thr - X - Y - 2 - MeLeu Ala - OBr V

Wherein Bz is a benzyl group and W, X, Y and Z have the meanings given above and this compound is then reacted with a tetrapep time corresponding to the sequence 8 * 11.

This tetrapeptide having the formula VI

HO - A - HeLeu - Leu - KeVal VI

5 8 9 10 11 contains 3 normal peptide bonds but has an O-terminal 1 instead of an N-terminal, as the residue at position 6 is derived from an α-hydroxy acid instead of an o-amino acid.

The tetrapeptide can be prepared according to the strategy set out in the following reaction scheme: BOC - HeLeu * Leu - OBz, B BOC - HeLeu - Leu - OBz 9 I 10 Ψ B2 / Pd BOC * HeLeu - Leu - OH 9 10

♦ KeVal - OBz V

BOC - HeLeu - Leu - KeVal - OBz 9 I 10 11 B - HeLeu - Leu - HeVal - OBz 9 10 il

! + R '- A - OH

R '- A - HeLeu - Leu - HeVal - OBz 8 9 i 10 11 ί H: / Pd

R '- A - HeLeu - Leu - HeVal - OH

89 10 11 7 DK 173335 B1

where BOC represents the N-protecting group t-butyloxycarbonyl, and R 'represents a suitable O-protecting group. The reagent shown above sod R '- A - OH is thus an OH-protected α-hydroxycarboxylic acid which, when A has the formula III, forms the parent VII

R

5 I

R '- 0 - CH - COOH VII

where R has the meaning given above.

It is preferred that R 1 is selected from the groups, C 2 H 50 - CH 2, CH 3 CH 2 - and C 1 H 3 tBuSi (CH 3) 2 *. The preferred compounds of formula VII can be prepared by reacting the α-hydroxy acid, in carbonyl protected form, for example as the benzyl ester, with dihydrodofuran, ethoxyethylene, t-butyldimethylchlorosilane or chlorodimethyl ether, respectively.

By OD substitution of the COOH-protected heptapeptide V with the hydroxytetra-peptide VI, in the OH-protected form, a linear hydroxyundeca peptide of formula VIII having the sequence 6-7 is obtained.

R'-A-MeLeu-Leu-HeVal-V-Thr-jK-Y-Z-HeLeu-Ala-OBz VIII

15 89 10 11 1 2 6 5 6 7

Finally, the cyclization of this linear hydroxypeptide can be carried out by removing the protecting groups by acidic or basic hydrolysis and coupling the residue 8 to 7 to form; of an ester bond. The coupling reaction is preferably carried out in methylene chloride using a carbodiimide reagent, for example N-ethyl-N '- (3-dimethylamino) propylcarbodiimide.

The heptapeptide of formula V and the tetrapeptide down formula VI can also be prepared by controlled hydrolysis of cyclic peptolides of formula II prepared by fermentation. This low temperature trifluoroacetic acid treatment cleaves the bond between residues 11 and 1 and yields a linear undecapeptolide containing residues 1 (N-terminal) through 11 (C-terminal) with an ester linkage at 7-8. Alkaline hydrolysis yields the 1-7 heptapeptide and 8 * 11 hydroxytetrapeptide. Then, semi-synthetic cyclic peptolides can be prepared, for example, by reacting the hydroxytetrapeptide thus produced with a synthetic heptapeptide, or vice versa, and cyclizing the linear product.

For the cyclization reaction, the peptide may, if desired, be in O-protected form, i.e., the hydroxy groups in 1-MeBmt or derivatives thereof and / or in the 2-threonine residue, only protecting groups, as described in EP-34 567, can be removed. then by conventional methods after the ring closure. The removal of, for example, a benzyl group by hydrogenation will also lead to hydrogenation of MeBmt to MeBmtH2 · and in any case, the first prepared cyclic peptolides containing a MeBmt residue at position 1 can be converted to the corresponding MeBmtH2 compound by hydrogenation.

The invention thus relates to a process for preparing a cyclic peptide of the above Formula II which comprises a) removing the O-protecting groups from a cyclic peptide of Formula II in O-protected form; b) cyclizing a straight-chain hydroxy-endecapeptide of Formula VIII, 1 unprotected form or O-protected at one or both residues 1 and 2, and, if necessary, performing step (a); and, if desired, c) hydrogenating a cyclic peptide of formula II thus obtained, wherein W is HeBmt, to form the corresponding cyclic peptide, wherein W is HeBatl

The cyclic peptolides of the invention exhibit pharmacological action and can therefore be used as pharmaceuticals. Compounds 9 DK 173335 B1 exhibit immunosuppressive, anti-inflammatory, and antiparasitic activity by the following tests: IN VITRO MODELS (1-3) 1. Interleukin 2 (IL-2> Inhibition 5 Interleukin 2 is induced by mitogen stimulation of mouse spleen cells) 48 Hours of growth of assayants are assayed and assayed to determine their content of IL-2 using an IL-2-dependent cell line (CTTL) .The growth of these cells is determined after 48 hours using an enzymatic assay measuring 10 ml. -activity.

[T. Mosmann J. Immunol. Methods 65: 55-63 (1983)]

The compounds of the invention have an inhibitory effect at concentrations of IC 0.1 µg / ml.

2. Lymphocyte proliferative response to allogeneic stimulation

Murine "mixed lymphocyte" reaction (MLR)

Spleen cells (0.5 x 10 6) from Balb / c mice (female, 8-10 weeks) are co-incubated for 5 days with 0.5 x 10 6 irradiated (2000 rad) or mitomycin C treated spleen cells from CBA mice (female , 8-10 20 weeks). The irradiated allogeneic cells induce a proliferative response in the Balb c spleen cells, which response can be measured by market precursor incorporation into DNA. As the stator cells are irradiated (or treated with mitomycin C), they do not respond to the Balb / c cells with proliferation but retain their antigenicity.

] T. Meo "Immunological Methods", L. Lefkovits and B. Pemis, Eds., Academic Press, N.Y., pp. 227-239 (1979)]

The compounds of the invention have an inhibitory effect at concentrations of from IC5Q - 0.0001 to approx. 0.001 µg / ml.

10 DK 173335 B1 3. Prinuer humoral immune response in vitro to red blood cells from fAr (Mishell-Dutton Assav)

Mouse spleen cells (OFI, female, 8-10 weeks, 1 x 107) are cultured with sheep erythrocyte (SRBC, 3 x 107) for 3 days in a final grape capture 5 of 1 ml in 24 well plates. Lymphocytes were harvested, washed and seeded with a cached of 1 x 10 6 cells on soft agar with fresh antigen (SBRC). Complement (guinea pig serum) was added after an incubation period of 60-90 minutes, and the incubation was continued for an additional 60 minutes, after which the test result is assessed by counting 10 numerous plaques under a microscope. During the 3 days of incubation, the lymphocytes have been made sensitive to the antigen (SRBC). When incubated with the antigen again, the B lymphocytes specifically secrete antibody which binds to the antigen close to the secretory lymphocyte. Upon addition of 15 complement, the antibody-coated erythrocytes are lysed, resulting in a plaque. Each plaque represents a single antibody producing cell.

[R.I. Mishell & R.V. Dutton J. Exp. With. 126: 243-442 (1967))

The suppression of plaque-forming cells is observed at concentrations of the compound of the invention of from 0.1 µg / ml.

IN VIVO MODELS (4-9) 4. Generation of plaque-forming cells (humoral immune response)

Female rats (OFA) are immunized by intravenous injection of (1 x 25 10®) sheep erythrocytes (SRBC) and treated for 3 consecutive days with the drugs being investigated. Spleen cell suspensions are prepared 6 days after immunization, and 1 x 10 6 lymphocytes are seeded on soft agar in the decay of indicator cells (SRBC) and complement. Lysis of the indicator cells due to the secretion of specific antibody and the presence of complement 11 gives plaques. The number of plaques per plate is counted and corrected for the number of plaques per spleen.

[N.K. Iron and A.A. Hordih Science 140: 405 (1969); N. K. Jerne, A.A. Nordin & C. Henry (1) 563) In: "Cell Bound Antibodies", B.

5 Amos & H. Koprowski, ed., Ulster Inst. Press, Philadelphia, p.

109-125 (19963)).

The compounds of the invention produce this effect in rats when administered orally at an ED 6.0-8.0 mg / kg.

5. Localized graft-versus-host reaction 10 Spleen cells (1 x 10 6) from 6-week-old female Wistar / Furth (UF) rats are injected subcutaneously on day 0 1 the left hind paw of female (F344 x WF) FI. rot'ts weighing approx. The animals are treated for 4 consecutive days, and the poplitea lymph nodes are removed and weighed on day 7. The difference in weight between 15 and 2 lymph nodes applies the seam parameter for evaluation of the reaction.

[U.L. Ford, U. Burr & M. Simonsen Transplantation 10: 258-266 (1970)]

Compounds of the invention have an oral ED 20-30 mg / kg.

6. Freund's adiuvans arthritip O FA and Uist rats (male and female, weighing 150 g) are injected intracutaneously at the tail of the tail or into the hind paw with 0.1 ml of mineral oil containing 0.6 mg lyophilized, heat-killed 25 Mycobacterium smegmatls. treatment is started on day 14 when the secondary inflammation is well developed (days 14-20). At the end of the experiment, the joint swelling must be assisted by a micro-caliper. ED5Q is the oral dose in mg / kg orally, which reduces the port (primer or secondary) to half of the port for the control rats. For compounds of the invention, the oral ED50 is up to 30 mg / kg.

[C.A. Winter & G.W. Nuss Arthritis and Rheumatism 9: 394-404 (1966)] S 7. Kidney allograft reaction in 1 rat

A kidney from a female Fischer 344 rat is transplanted into the renal vessel of an unlateral (left side) nephrectarized Wlstar / Furth recurrence rat using end-to-end anastomosis. Ureter anastomosis is also end-to-end. The treatment begins on the transplant day and continues for 14 days. A contralateral nephrectomy is performed 7 days after the transplant, leaving the recipient to function in the donomyr. Recipient survival is taken as a parameter for a functional graft.

[P.C. Hiestand, All Immunology £ 249-255 (1985)]

The compounds of the invention are effective in rats at an oral ED 9 mg / kg.

8. UV Ervthem test

The test is performed on female albino guinea pigs with a guard p in approx. 150 20 g. The animals are shaved on both sides using a Braun micro-razor and without causing skin irritation.

For each test substance, 5 test animals are subjected to a defined intensity of UV irradiation for 10 seconds on each of four skin areas (diameter 10 mm). Immediately after, 50 mlcroliter of a solution of the test substance in ethanol / propylene glycol / dimethylacetamide (19: 19: 2 v / v) is applied to two of the irradiated areas of each animal and 50 ml of the solvent mixture is applied to the other two as a control. . Four hours after application, the degree of erythema is assessed visually. 1 [Raake, W. Arzneim.-Forsch. 34 (1) No. 4 (1984)] 13 DK 173335 B1

The compounds of the invention are effective at a concentration of from 1 to 10X.

9. Antal malarate test

House (OFI, male) is infected intraperitoneally on day 0 with 0.2 5 ml of a suspension of erythocytes containing 10 ^ cells parasitized by Plasmodiup Berghei (strain NK 65). The test substance is administered subcutaneously at varying doses, using 5-10 mice per dose. dosage. The survival time is recorded and the minimum effective dose (MED) is calculated by comparing the survival time with the survival time of untreated control animals. For the control animals, the survival time is approx. 7 days.

MED is the dose by which the survival time is doubled.

[L. Rane in "Chemotherapy and'Drug Resistance in Malaria", ed.

W. Feters, Academic Press, Nejw York, (1979)] 15 Because of their immunosuppressive effect, the compounds are indicated for use in the prophylaxis and treatment of conditions and diseases requiring a suppression of the immune response, eg in the treatment of autoimmune diseases, the prevention of tissue rejection during transplants, eg bone marrow and kidney transplants.

Specific autoimmune diseases to which the compounds of the invention may be used are, for example, aastatic anemia, pure red blood cell anemia, idiopathic thrombocytopenia, systemic lupus erythematosus, polychondritis, scleroderma, Wegner's granulomatosis, dermatomyositis, chronic active hepatitis gravis, Steven-Johnson 25 'syndrome, psoriasis, ideopathic sphincter, Crohn's disease, Graves' opthalmopathy, sarcoidosis, multiple sclerosis, primer biliary cirrhosis, primer Juvenile diabetes, posterior uveitis, interstitial pulmonary fibrosis and psoriasis arthritis.

Because of their anti-inflammatory effects, compounds 30 of the invention are indicated for use in the treatment of inflammatory conditions, especially inflammatory conditions with an aetiology comprising an autoimmune component, e.g., the treatment of arthritis and rheumatic diseases such as chronic progressive arthritis.

Because of their antiparasitic effect, the compounds are also indicated for use in the treatment of parasitic diseases, e.g.

The compounds of the invention are also indicated for use in the treatment of certain skin diseases and conditions which, in addition to the above, include alopecia areata, urticaria, vasculitis, erythema, atopic dermatitis, eczema, cutaneous eosinophilia and angioderma.

For these conditions, an indicated daily dose of 1 ranges from approx. 70 to approx. 3000 mg of a compound of the invention which is conveniently administered in divided doses up to 4 times daily.

The compounds of the invention may be administered by any conventional route, e.g., orally, e.g., in the form of tablets or capsules, parenterally in the form of injectable solutions or suspensions, or topically 1 in the form of lotion, cream or gel.

The invention also relates to pharmaceutical compositions containing a compound of the invention together with at least one pharmaceutical bar or at least one pharmaceutical diluent. Such compositions may be prepared in a conventional manner. Unit dosage forms contain e.g. 20 mg to approx. 1500 mg of a compound of the invention.

The invention is further illustrated by the following Examples: EXAMPLE 1

Cultivation of strain NBHL l5_230_i_ Erlenmeverkolbe

Starting cultures of strain NRRL 18230 are incubated at 21 ° C for 14 days on AM in oblique culture. A praculture is then prepared by homogenizing the total contents of an initial culture under sterile conditions, transferring it to a 50Q ml Erlenmeyer flask containing *. in 200 ml nutrient solution op (2X malt extract, 0.4X g * extract in demineralized water) and incubate on a rotary shaker at 5,200 rpm for 10 days at 21 ° C. 1

Intermediate cultures are then obtained by transferring 20 ml of the pre-culture to a 500 ml Erlenmeyer flask containing 200 ml of nutrient solution H and incubating on a rotary shaker at 200 rpm for 7 days at 21 ° C.

Finally, to obtain a production culture, inoculate 100 Erlenmeyer flasks (each 500 ml), each containing 200 ml of nutrient solution M with 20 ml of the interstellar cultures. The flasks are incubated at 21 ° C in a rotary shaker at 200 rpm. After 10 days, the 20 liters of fenugreek washes are combined for 15 extraction of the product.

EXAMPLE 2

Isolation of (Thr112 (Leu) 5 (D-HIV) 8 (Leul2-Ciclosporin

The 20 liters of culture medium prepared in Example 1 is subjected to mixing under high shear conditions in a 20 rod mixer (Lutz, Verthelm, Germany) to break up the cells and extracted three times with 20 liters of ethyl acetate. The 60 liters organic phase is combined and dried over anhydrous sodium sulfate and evaporated to dryness in vacuo to give a residue of 17.4 g. 1 2 3 4 5 6

The residue is dissolved in 80 ml of methanol and chromatographed on a 90 mm diameter column 2 containing 1 $ 00 g of Sephadex LH-20 (Pharmacia Fine 3

Chemicals AB, Uppsala, Sweden). $ are extracted fractions of 100 ml, 4 whereby (Thr) ^ (Leu) ^ (D-Hiv) 8 (Léu) ^ - Ciclosporin appears in the fractions 22-30, These fractions are combined and Evaporated under 6 vacuum to give 8400 mg of a slightly colored solid foam. This residue is dissolved in wet ethyl acetate and chromatographed on a column down to a diameter of 55 mm containing 500 g of silica gel (Merck) down to a particle size of 0.04-0.063 mm. The desired product is detected in fractions 9-14 (fraction size 100 ml). Upon evaporation a pale yellow residue (4200 mg) is obtained. By active charcoal (Merck) treatment in diethyl ether and filtration through a thin layer of talc, (Thr) 2 (Leu) ^ (D-Hiv) ® (Leu) ^ ®-Ciclosporin is obtained as a white powder which is recrystallized from ether.

mp. 163-164 ° C (transmitter division); [.alpha.] @ 20 to -186 \ (c - 1 in MeOH); [a] 20 - 223 'in CHCl

EXAMPLES 3-7

By modifying the above-described chromatographic methods, the following compounds can be isolated in minor quantities from the production fermentation scrubber:

Eks.nr. Compound 3 (Thr) J (D-Hiv) · (Leu) ie-Ciclosporin 4 (Thr) J (Ile) 5 (D-Biv) · (Leu) le-Ciclosporin 5 (Thr) 2 (Leu) 4 (Leu) (5-D-Biv) · (Leu) 10-Cyclosporin 6 (Thr) 2 (Gly) J (Leu) 5 (D-Biv) · (L «u) ie-Cyclosporin 7 (8'-0HHeBmt) 1 ( Thr) 2 (Leu) 5 (D-Hiv) · (Leu) 10-Ciclosporin 1

The compounds have the properties shown in Table I: DK 173335 B1

Table I

17

Compound calculated found melt- [a) 0

according to ex.no. formula molecular point * C (c-1 in MeO

guard guard * 3 C «sBti, N | 0Ol4 1233, $ 1233.9 164 ** -184» 4 C „Hll4KxoOx« 1247.7 1247.6 151.3 ** -180 »5 Ce3Bli2N10Ol4 1233.6 1233.6 166.8 ** -174 · 6 CoHujNioOu 1233.6 1233.6 140.43 ** '-154 · 7 C ^ HIuNmOxs 1263.7 1263 158 *** -174 · 195 ** 5 * by FAfi (Fast Atomic Bombardment) mass spectrometry ** - transmitter division *** - sinks EXAMPLE 8

Synthesis of (Thr) 2 (Leu) 5 (D-Hlv) 8 (Leu) 10-Clclosporin by cyclization.

At room temperature, 2.4 g (2.20 mmol) of the unprotected hydroxy undecapeptide HO- (D) Blv-MeLeu-L «uM * Val-MeBmt-iThr-Sar-MeLeu-Leu-MeLeu-Ala-OH are dissolved. 0.84 g (6.88 mmol) of 4-dimethylalhopyridine and 0.66 g (1.5 aq valents, 3.44 amol) of N-ethyl-N- (3-4imethylamino) propylcarbodiimide are dissolved in 145 ml of methylene chloride. and reacted for 1 3 days with stirring and moisture exclusion. The resulting solution is diluted with 300 ml of methylene chloride, shaken with 1.00 µl of water acidified to pH 2 with 1N HCl, filtered through talc and evaporated. The residue is chromatographed on 100 g of sieve gel using 10X Me-20 OH / CH 2 Cl 2. to give the title compound (2.55 g, 89%). Identity of the compound of Example 2 is demonstrated by HHR spectroscopy and [α] 20 (-220 °, c-1 in CHCl 3).

18 DK 173335 B1

Table II Abbreviations

Abu a-amino butyric acid

Ala alanine BOC t-butyloxycarbonyl

Bz benzyl

Gly glycine

HIV α-hydroxyisovaleric acid

Ile isoleucine

Leu leucine

MeBmt N-methyl- (4R) -4-but-2H-en-1-yl-4-Bethyl- (L) -threonine

HeBmtHj N -ethyl- (4R) -4-but-1-yl-4-Bethyl- (L) -threenin 8'0HMeBBt N-Bethyl- (4R) -4- (4'-hydroxybut-2E-en-1 yl) -4-

Bethyl- (L) -threonine Sar sarcosin

Thr threonine

Selections selected

Claims (10)

1. Cyclic peptolide, characterized in that it has the structure of a cyclosporin, in which an amide bond is replaced by an ester bond. 2. Cyclic peptide according to claim 1, characterized in that it consists of a MeBmt residue, or a derivative thereof, 9 other o-amino acid residues and an α-hydroxy acid residue. A cyclic peptide according to claim 2, characterized in that it has the formula II - V-Thr-X-Y-Z - MeLeu - Ala - A - HeLeu - Leu - MeVal -. 1 2 3 4 5 6 7 8 9_10 11 | II 10 where V represents MeBmt, 8'-0HMeBn | t or MeBmtH2 X represents Sar or Gly Y represents HeLeu or Leu Z represents Leu, Ile or Vel and A represents the residue of an o-hydroxycarboxylic acid. Cyclic peptolide according to claim 3, characterized in that A is (D) Hlv. 5. (Thr) 2 (Leu) 5 (D-Hlv) 1 2 3 <Leu) 44-Ciclosporin. A process for producing a cyclic peptide according to any one of the preceding claims, characterized in that one produces a producing microorganism in the manufacture of a nutrient medium. Process for producing a cyclic peptide According to any one of claims 3-5, 3, characterized in that njan cultivates a producing fungus of about -25 mm of the species Cyllndrotrlchum Bonorden in the presence of a nutritional medium. DK 173335 B1 8. Fermentation bag obtained by growing a cyclic peptide-producing fungus strain of the species Cvlindrotrlchum Bonorden. A pharmaceutical composition containing a cyclic peptide according to any one of claims 1 * 5 together with a pharmaceutically acceptable diluent or pharmaceutically acceptable bars. 10. Biologically pure culture of the fungal strain NRRL 18230. A process for producing a cyclic peptide according to any one of claims 1-5, characterized in that a linear peptide 10 or a peptide with a hydroxy terminal group is cyclized instead of an amino terminal group. A process for preparing a cyclic peptide of the formula II as claimed in claim 3, characterized in that it comprises a) removing the O-protecting groups from a cyclic peptide of the formula II-O-protected form; b) cyclizing a low chain hydroxy endecapeptide of formula VIII in unprotected form or O-protected on one or both residues 1 and 2, and, if necessary, performing step (2); 20 and, if desired, c) hydrogenating a peptide thus obtained of formula II, where V is MeBmt, to form the corresponding cyclic peptide, where W is MeBmt1 t