DD241908A5 - PROCESS FOR PREPARING A CYCLOSPORIN - Google Patents

Abstract

The invention relates to a process for the preparation of a cyclosporin for use as a medicament. The aim of the invention is the provision of novel cyclosporins with strong immunosuppressive, antiperspirant and antiparasitic effect. According to the invention, novel cyclosporins in which the amino acid residue at position 8 is a (D) -acyloxy-α-amino acid residue are prepared by reacting a cyclosporin in which the amino acid residue at position 8 is a (D) -hydroxy-α-amino acid residue acylated, or that a cyclosporin wherein the amino acid residue is in position 1 -MeBmt- and the residue in position 8 is a (D) -acyloxy-a-amino acid residue is reduced to obtain the corresponding cyclosporin, wherein the radical is in Position 1-dihydro-MeBmt- is.

Description

Field of application of the invention

The invention relates to a process for the preparation of a cyclosporin with valuable pharmacological properties, in particular with immunosuppressive, anti-inflammatory and antiparasitic action. The compounds prepared according to the invention are used as medicaments.

Characteristic of the known technical solutions

Cyclosporins are a series of structurally peculiar, cyclic, poly-N-methylated undecaptides which usually have pharmacological effects, in particular immunosuppressive, antiinflammatory and antiparasitic effects. The first isolated cyclosporin and also the parent compound of this series is the natural fungal metabolite cyclosporin A of formula A.

fMeBmt-oU-bu-Sar-MeLeu-Yal-MeLeu-Ala-CD) AIa-Me Leu-Meleu-1 2 3 4 5 6 7 8 9 10 11

wherein -MeBmt- the radical N-methyl- (4R) -4-but-2E-en-1-yl-4-methyl- (L) threonyl of the formula B,

Γ ²

HO (R) 'CH _(B)

^ CH "(R) ^X

-H CH-CO-

(S) CH ₃

wherein -x-y-represents-CH = CH- (trans) means.

Since the original discovery of cyclosproin A, numerous natural cyclosporins have been isolated and identified, and several other non-naturally occurring cyclosporins have been prepared by total synthesis or semi-synthetic, or by modified culture methods. Because of this, the cyclosporin series has gained importance and now it includes e.g. See, for example, the natural cyclosporins A to Z (see Kobel et al., European Journal of Applied Microbiology and Biotechnology 14, 237-240 [1982] and Traber et al., 24th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washingon, [ 8.-10.10.1984], poster presented), furthermore various cyclosporins of non-natural origin, including dihydro-cyclosporines (in which the -xy-group in the -MeBmt radical - see formula B above - is saturated, eg as in the US Pat. PS 4108985, 4210581 and 4220641), cyclosporins in which the -MeBmt residue is present in isomerized or N-demethylated form (see EP-PS 0034567 and "Cyclosporin A", Proc. Internat. Conference on Cyclosporin A, Cambridge (UK) Semptember 1981, Ed. DJG White, Elsevier Press (1982) - both containing the totally synthetic method of production of cyclosporins developed by R.Wenger) and cyclosporins in which various amino acids have been incorporated at certain positions of the peptide sequence (s before the EP-PS 0056782). As examples of such described in the literature mentioned above, cyclosporins may be mentioned: [Thr] ² -, [Val] ² -, [Nva] ² -and [Nva] ² - [Nva] ^{5-Cyclosporine} (also known as cyclosporin C , D, G or M), [dihydro-MeBmtr- [Val] ² -cyclosporin (also known as dihydro-cyclosporin D) and [(D) Ser] ⁸ - and [dihydro-MeBmtJ ^ HDJSerf-cyclosporin.

In accordance with the current nomenclature for cyclosporins, they are referred to in the present specification and in the claims with reference to cyclosporin A. This is done by first indicating the parts of the molecule which differ from those in cyclosporin A, and then characterizing the remaining parts corresponding to those in cyclosporin A by giving the word "cyclosporin." To this end, the term dihydro-MeBmt used to designate the above-mentioned radical of the formula B in which -XY-TOr-CH ₂ -CH ₂ is ## STR9 ## Thus, [dihydro-MeBmt] ¹ - [Val] ² -cyclosporin refers to that cyclosporin which has the sequence according to formula A, wherein but-MeBmt- [Formula B, -xy- = -CH = CH- (trans)] in position 1 by dihydro-MeBmt- [Formula B, -xy- = -CH ₂ -CH ₂ ] and Similarly, with [(D) Ser] ⁸ cyclosporin, the cyclosporin having the sequence according to Formula A, wherein but- (D) Ala is in position B, is replaced by - ( D) Ser- is replaced, designated.

Further, and in accordance with conventional practice, the amino acids defined by abbreviations, e.g. For example, -Ala-, -MeVaI-etc., The (L) configuration, unless otherwise specified. Residues with the prefix "Me", for example in -MeLeu- are N-methylated radicals The various radicals of the cyclosporin molecule are numbered as in the literature, ie clockwise and starting from the -MeBmt or -dihydro- MeBmt residue in position 1. In the present description and in the claims, the same number sequence is always used.

Object of the invention

The aim of the invention is the provision of new cyclosporins with valuable pharmacological properties, in particular with strong immunosuppressive action.

Explanation of the essence of the invention

The invention has for its object to find new cyclosporins with the desired properties and processes for their preparation.

According to the invention, it has now been found that novel cyclosporins with pharmaceutical applicability can be obtained in which the residue in position 8 consists of an acyloxy-α-amino acid with the (D) -configuration.

Accordingly, the present invention broadly relates to a process for producing a cyclosporin wherein the amino acid residue at position 8 is a (D) -acyloxy-a-amino acid residue, i. H. the residue of an amino acid of the (D) series, wherein the side chain on the a-carbon atom is substituted by acyloxy.

Preferably, the 8 amino acid residue is a (D) -β-acyloxy-α-amino acid residue, i. a residue of an amino acid of the (D) series with an acyloxy group attached to the amino acid via the β-carbon atom. Preferred (D) -β-acyloxyamino acid radicals correspond to the formula II,

R2

Rl-CO-O-CH (ß) ι

• - (II)

-NH-CH-CO-!

Ri is hydrogen, alkyl of 1 to 4 carbon atoms or phenyl and R _{2 is} hydrogen or methyl.

Particularly preferred cyclosporins according to the present invention are those wherein the amino acid residue at position 8 is an O-acyl (D) -seryl or O-acyl- (D) -threonyl radical, especially an O-acyl- (D) -seryl or O-acyl- (D) -threonyl radical of the above formula II.

In a group of cyclosporins of the invention, the amino acid residue at position 8 is an O-acyl (D) -seryl radical, especially an O-acyl- (D) -seryl radical, wherein the acyl group corresponds to the formula Ri-CO-, where R _{1 is the} above Has meaning.

In another group of cyclosporins of the invention, the amino acid residue at position 8 is a (D) -β-acyloxy-amino acid residue, especially an O-acyl (D) -seryl residue, most especially a D-acyl (D) -seryl residue, wherein the acyl group corresponds to the formula R ₁ -CO-, where Ri is hydrogen or alkyl of 1 to 4 carbon atoms, and the residue in position 5 is an (L) -norvalyl radical.

Very particularly preferred cyclosporins are those of the formula I,

P-X-Y-Sar-MeLeu-Z-MeLeu-Ala-Q-MeLeu-MeLeu-Val, 123 45 6 789 10 11

(D

X -MeBmt- or dihydro-MeBmt- means

Y - $ Abu -, - AIa -, - Thr -, - VaI-or-Nva-means, Z -VaI-or-Nva-means and

Y represents a radical of the formula II defined above.

In formula I, Q is preferably an O-acyl- (D) -seryl- or O-acyl- (D) -threonyl radical, wherein the acyl group of the formula. R _{1 is} -CO-, where R _{1 has the} above meaning. Y is preferably -aAbu-, -Thr-, -VaI- or -Nva-.

A group of cyclosporins according to the invention form those of the formula I, wherein Y is -aAbu- or -Nva-, Z -VaI- and R _{2 is} hydrogen.

Another group of cyclosporins according to the invention are those of the formula I in which Y is -AaBu or -Nva-, Z is -Nva-, R _{1 is} hydrogen or alkyl having 1 to 4 carbon atoms and R _{2 is} hydrogen.

The present invention further relates to a process for the preparation of a cyclosporin wherein the amino acid residue at position 8 is a (D) -acyloxy-a-amino acid residue, for example a (D) -β-acyloxy-α-amino acid residue, e.g. For the preparation of a cyclosporin of the formula I, characterized in that

a) that a cyclosporin in which the amino acid residue in position 8 is a (D) -hydroxy-a-amino acid residue, for example a (D) -β-hydroxy-α-amino acid residue, acylated, for. B. that a cyclosporin of the formula III,

-X-Y-Sar-MeLeu-Z-MeLeu-Aia-W-MeLeu-MeLeu-MeVal-, 12 3 4 5 6 7 8 9 10 11

(III)

wherein X, Y and Z have the above meaning and W is a radical of the formula IV, R ₂

= .-. HIGH

(IV)

 NH-CH-CO- (D)

wherein R _{2 has the} above meaning acylated by introducing an R-pCO group, wherein Ri has the above meaning, in the β-position, or

b) that a cyclosporin in which the amino acid residue in position 1 is -MeBmt- and the radical in position 8 is a (D) -acyloxyamino acid residue, for example a (D) -β-acyloxy-α-amino acid radical, is reduced, to obtain the corresponding cyclosporin wherein the residue is in the 1-dihydro-MeBmt- position, e.g. B. that a cyclosporin of the formula I, wherein X is

, -MeBmt- is reduced to give the corresponding cyclosporin, wherein X is dihydro-MeBmt-. Process a) can be carried out according to methods known per se for the acylation of hydroxy groups, for. B. by reaction with preferably two equivalents, or, if Y = -Thr-, one equivalent of a suitable acyl, ζ. Β.

, (Ci- ₅₎ alkanoyl or benzoyl halide, or the corresponding anhydride or, for formylation, for example by reaction with acetic formic anhydride, at a temperature of for example about - The reaction takes 10 ⁰ C to 5O ⁰ C. under anhydrous conditions, conveniently in a reaction inert solvent such as methylene chloride and in the presence of a condensing agent such as 4-dimethyl-amino-pyridine. Under these conditions, acylation on the hydroxy group of the amino acid residue occurs at position 8 rather than at the hydroxy group of the amino acid residue at position 1.

Process b) may be carried out per se for the reduction of natural cyclosporines to the corresponding dihydrocyclosporins, for example by catalytic hydrogenation, e.g. according to the general methods described in GB-PS 1 567201.

The hydrogenation is advantageously carried out in the neutral range, at temperatures between about 20 ⁰ C and 3O ⁰ C and under atmospheric pressure or slightly elevated pressure. As a hydrogenation catalyst z. As platinum oxide or palladium catalysts, for. B. palladium on carbon. The hydrogenation may, for. In a reaction inert solvent such as ethyl acetate or a lower aliphatic alkanol such as methanol or isopropanol.

Cyclosporins having a β-hydroxy-α-amino acid residue in position 8, in particular [(D) Ser] ⁸ cyclosporins and [dihydro-MeBmtr-MDlSer ^ cyclosporins, which are suitable as starting materials for process a) are, for. Example, from the above-mentioned EP-PS 0056782, in which also processes for their preparation are described known. Further cyclosporins having a hydroxy-α-amino acid residue in position 8 which are suitable as starting materials for process a) can be prepared in an analogous manner or according to the general methods of cyclosporin total synthesis described in European Patent No. 0034567 (which is referred to in European Patent No. 0056782) be, or even after the methods described below, in particular in the examples.

The cyclosporins which are suitable as starting compounds for process b) can be prepared by the process indicated under a).

Although the starting compounds of the above formula III, which are specifically described in the examples below, fall within the broad scope of the above-mentioned EP-0056782, certain of them are formally new, i. H. that they have never been described specifically so far. According to the invention, it has now also been found that these cyclosporins have a particularly interesting or advantageous spectrum of activity, in particular with regard to their immunosuppressive activity, and more particularly with regard to the prevention of transplantations, e.g. Organ transplants, associated adverse effects (defense), e.g. B. in comparison to the known cyclosporins of the formula Hl, d. H. with those cyclosporins of the formula IM which are specifically described in EP-PS 0056782. Accordingly, in a further aspect, the present invention relates to a cyclosporin of the formula IIIa,

2 3 4 5. 6 7 8 9 10 11 | (purple)

Y 'is -Abu-, -Thr-, -VaI- or -Nva-,

Z 'is -Val.sup.- means ^ ifY'-aAbu or -Nva- means ^ is -Nva-, W' - (D) Ser- or, if Y '-Abu- and Z' -VaI mean, for - (D) Thr is and X 'is -MeBmt- or, if Y' is -Thr, -VaI or -Nva-, Z '-VaI- and W' - (D) Ser-, for -dihydro- MeBmt- stands.

Specific cyclosporins of formula IIIa are

a) [(D) Thr] ⁸ cyclosporin

b) [Thr] ² - [(D) Ser] ⁸ cyclosporin

c) [Diriydro-MeBrntlMThrf-KDiSerf Cyclosporin

d) [Val] ² - [(D) Ser] ⁸ cyclosporin

e) [Dihydro-MeBmtlMValP-KDiSerf cyclosporin

f) [Nva] ² - [(D) Ser] ⁸ cyclosporin

g) [Dihydro-MeBmt] ¹ - [Nva] ² - [(D) Ser] ^8- Cyclosporin h) [Nva] ⁵ - [(D) Ser] ^8- Cyclosporin and

i) [Nva] ² - [Nva] ⁵ - [(D) Ser] ⁸ cyclosporin.

Of the above cyclosporins, the compounds a), b), e), f) and i), in particular a), f) and i), with regard to their effect / spectrum of action, for. B. their immunosuppressive effect, of particular interest, for. As compared with the cyclosporins specifically described in EP-PS 0056782.

Furthermore, the present invention relates to a process for the preparation of a cyclosporin of the formula III a defined above, characterized in that

c) that in a present in O-protected form, defined above cyclosporin of the formula III cleaves the protecting group, or

d) that a straight-chain undecaptaptide containing the sequence

-W-MeLeu-MeLeu-MeVal-X'-Y'-Sar-MeLeu-Z'-MeLeu-AIa-8 9 10 11 123 45 6 7

in which Y ', Z', W 'and X' have the above meaning cyclized, wherein the undecapeptide can be present in unprotected or O-protected form, and if necessary, cleaves off the protective group, or

e) that for the preparation of a cyclosporin of the formula IIIa, in which Y 'is -Thr -, -VaI- or Nva-,

Z 'is -VaI- or, if Y' is -Nva- stands for -Nva-,

W '- (D) Ser- means and

X '-MeBmt- means

a [Thr] ² -cyclosporin, [Val] ² -cyclosporin, [Nva] ² -cyclosporin or [Nva] ² -cyclosporin-producing fungal strain in the presence of a (D) -serine containing nutrient medium and the cyclosporin of the formula III a of the isolated culture broth, or

f) that for the preparation of a cyclosporin of the formula III a, wherein X 'is dihydro-MeBmt-, the corresponding cyclosporin of the formula HIa, wherein X'-MeBmt- means reduced.

Undecapaptides for use in process d) can be prepared analogously to the methods generally used in the abovementioned EP-PS 0056782, for example with reference to the scheme of Example 1a of this patent, by using the peptide sequence consisting of residues 8 to 11 of cyclosporin Molecule having the sequence consisting of residues 1 to 7 combined with the necessary substition of the residues in position 2 and / or 5 and / or 8. Der- (D) Ser or (D) Thr residue in position 8 is suitably in O-protected form, for. B. in the form of the O-t-butyl derivative. The cyclization is carried out using the special methods mentioned in said EP-PS, with subsequent cleavage of the O-protecting groups, if necessary [method c)] by the methods known per se in peptide chemistry.

The preferred strain for use in process e) is strain NRRL 8044 of the fungus species Tolypocladium inflatum (Garns). A culture thereof has been deposited with the United States Department of Agriculture, Peoria, Ill., 111, and is available to the public. Another culture thereof was deposited at the Fermentation Research Institute, Inage, Chiba City, Japan under the culture number FRI FERM-P No. 2796. The morphological features of this strain, formerly associated with the fungal species Trichoderma polysporum (Link ex Pers.), As well as methods for producing preservation of pre- and postcultures, are described extensively in the literature, e.g. In GB-PS 1,491,509.

According to method e), the selected strain, e.g. B. the species Tolypocladium inflatum (yarns), expediently for about 2 weeks in a nutrient medium as described in the following examples and in the presence of added (D) - or (D, L) -serine at 27 ⁰ C grown. The amino acid precursor is conveniently added in an amount of about 1 to about 15 g, preferably about 4 to about 10 g / liter of culture medium. Conveniently, the culture medium also contains added amino acid precursor for the remainder in position 2 of the desired cyclosporin, for example in the amount of about 6.0 to about 10.0, preferably about 8.0 g / liter of culture medium. After the incubation period, the cyclosporin of the formula III a obtained is obtained from the culture broth by methods known per se, e.g. B. by separation of the broth in mycelium and culture filtrate, extraction of the mycelium under homogenization, and centrifuging off the cell material.

The crude cyclosporin obtained can, for. B. purified by chromatography and / or by recrystallization. This removes especially the other cyclosporins, in particular the natural cyclosporins. Method f) can be carried out, for example, by applying the methods described under b).

embodiment

The following examples illustrate the execution of the inventive method.

example 1 Synthesis of [(O-acetyl) - (D) Ser] ⁸ Cyclosporin [Formula I:

X = -MeBmt-, Y = -aAbu-, Z = -VaI-, Q = -O-acetyMDJSer-]:

Manf20204-dimethylaminopyridine to 47mg [(DJSer ^ cyclosporin (prepared in accordance with the method described in Example 1 or 3 of the aforementioned European Patent No. 0056782) dissolved in 3 ml of methylene chloride are then added to 6.1 mg of freshly distilled acetyl chloride in 1 ml of methylene chloride The reaction mixture is stirred for 1 hour at room temperature, diluted with 50 ml of methylene chloride and shaken with 30 ml of water, the organic phase is separated, dried over sodium sulfate, filtered off and evaporated , 20mm) using methylene chloride / 5% methanol as eluent and collecting in 25ml fractions The title compound is recovered from fractions 4-8 by thin layer chromatography using CHCl3 / 5% methanol as the carrier phase

[α] ^{2 0 0} = -202 C (c = 0.92 in CHCI _3).

Example 2

In analogy to Example 1, the following compounds are prepared starting from the corresponding non-acylated cyclosporin:

2.1. [(0-benzoyl- (D) Ser] ^8- Cyclosporin [Formula I:

X = -MeBmt-, Y = -OAbu-, Ζ = -Val-, Q = -O-benzoyl- (D) Ser-]: [α] g ° = -20 ° C (c = 1.0 in CHCl ₃ ); 2.2 [O-acetyl- (D) Thr] ^{s-Cyclosporine} [Formally:

X = -MeBmt-, Y = -aAbu-, Z = -VaI-, Q = -O-acetyl-fDi-Ser-]: [a] § ° = -219 ⁰ C (c = 1.0 in CHCl ₃ );

2.3. [NvalMO acetyHDiserf Cyclosporin [Formula I:

X = -MeBmt-, Y = -Nva-, Z = -VaI-, Q = -O-acetyHDJSer-]: [a] g ° = -240 ⁰ C (c = 1.0 in CHCl ₃ ) / - 233 ° C (c = 0.8 in CHCl ₃₎ / ⁰ -177 C (c = 0.76 in CH ₃ OH): mp = 143-147 ⁰ C..

2.4. [Val] ² - [O-acetyl- (D) Ser] ^8- cyclosporin [Formula I:

X = -MeBmt-, Y = -VaI-, Z = -VaI-, Q = -O-acetyl-fDJSer-]: [ag ⁰ = -219 ⁰ C (c = 0.9 in CHCl ₃ );

2.5. [Nvaf-tO-acetyl-IDJSerf-cyclosporin [Formula I:

X = -MeBmt-, Y = - [aAbu-, Z = -Nva-, Q = -O-acetyl-tDJSer-]: [a] g ° = -215 ° C (c = 1.0 in CHCl ₃ ) ;

2.6. [Nvaf-INvaf-IO-acetyl-FDlSerf Cyclosporin [Formula I:

X = -MeBmt-, Y = -Nva-, Z = -Nva-, Q = -O-acetyl- (D) Ser-]: [a] g ° = -169 ° C (c = 1.0 in CHCl ₃ ); and 2.7 [Thr] ² - [O-acetyl- (D) Ser] ^8- cyclosporin [Formula I:

X = -MeBmt-, Y = -Thr-, Z = -VaI-, Q = -O-acetyl-fDJSer-]: [α] ² , ⁰ = -251 ⁰ C (c = 0.86 in CHCl ₃ ) / -174 ° C (c = 0.81 in CH ₃ OH): mp = 143-146 ° C.

Example 3

Synthesis of [dihydro-MeBmt] ¹ - [O-acetyl- (D) Ser] ^8- cyclosporin [Formula I: X = -dihydro-MeBmt, Y = -aAbu, Z = -VaI-, Q = -O- acetyl- (D) Ser]

54 mg of [(O-acetyl) MDI-cyclosporin in 10 ml of ethanol are hydrogenated using 10 mg of palladium / animal charcoal (10%) at room temperature and normal pressure After 20 hours, the resulting reaction solution is filtered through a thin layer of talc and the ethanol is evaporated in vacuo further drying in a high vacuum, the title compound is obtained.

[a] g ° = -205.8 ° C (c = 1.02 in CHCl ₃ ).

Example 4

The following compounds are prepared, either analogously to Example 1, starting from the corresponding non-acylated cyclosporin or analogously to Example 3, by hydrogenating the corresponding cyclosporin described in Example 2:

4.1. [Dihydro-MeßmtlMNva ^ -tO-acetyl-fDJSerf-Cyclosporin [Formula I:

X = -dihydro-Meßmt-, Υ = -Nva-, Z = -VaI-, Q = -O-acetyMDJSer-]: mp = 139-141 ° C; [α] ² , ⁰ = -225 ° (c = 0.88 in CHCl ₃ ) / -163 ° (C = 0.76 in CH ₃ OH);

4.2. [Dihydro-Meßmtr-IVallMO-acetyMDJSerf-Cyclosporin [Formula I:

X = -dihydro-Meßmt-, Y = -VaI-, Z = -Vat-, Q = -O-acetyl-fDJSer-]: [a] g> = -210 ° (c = 0.85 in CHCl ₃ ) ; and

4.3. [Dihydro-Measurin] tThrjlO-acetyl-lDiSerl ^ Cyclosporin [Formula I:

X = -dihydro-Meßmt, Y = -Thr, Z = -VaI, Q = -O-acetyl- (D) Ser-]: [a] g ° = -241 ° (c = 1.0 in CHCl ₃ ) / - 162 ° (c = 1.0 in CH ₃ OH): mp = 148-150 ° C

 Preparation of the starting material:

Example 5

The following starting compounds required for the preparation of the compounds of Examples 2.2 to 2.7 are prepared analogously to the known compound [(D) Ser] ⁸ cyclosporin. The preparation of the latter is described in Example 1 of European Patent No. 0056782, with substitution of the corresponding radicals in the positions 2 and / or 5 and / or 8 in the process sequence, as shown in the reaction scheme to Example 1 a of said patent ,

5.1. [(D) Thr] ^8- Cyclosporin [Formula IIIa: X '= -Meßmt-, Υ' = -aAbu-, Z '= -VaI-, W = - (D) Thr-]: [α] ² , ⁰ = -248.7 ° (c = 1.0 in CHCl ₃ );

5.2. [Nva] ² - [{D) Ser] ⁸ cyclosporin [Formula IHa: X '= -Meßmt-, Y' = -Nva-, Z '= -VaI-, W = - (D) Ser-]: mp = 150-153 ° C; [a] g ° = -262 ° (c = 0.71 in CHCl ₃ ) / - 191 ° (c = 0.73 in CH ₃ OH);

5.3. [Val] ² - [(D) Ser] ⁸ cyclosporin [Formula IIIa: X '= Meßmt, Y' = -VaI, Z '= -VaI, W = - (D) Ser-]: [a ] g ° = -257 ° (c = 1.0 in CHCl ₃ ) / - 255 ° (c = 0.45 in CHCl ₃ ) / - 189 ° (c = 0.42 in CH ₃ OH): mp = 136-14O ⁰ C.

5.4. [Nva] ⁵ - [(D) Ser] ^8- Cyclosporin [Formula lilac: X '= -Meßmt-, Y' = -aAbu-, Z '= -Nva-, W = - (D) Ser-]: [ a] g ° = -212 ° (c = 1.0 in CHCl ₃ );

5.5. [Nva] ² - [Nva] ⁵ - [(D) Ser] ⁸ cyclosporin [Formula lilac: X '= -Meßmt-, Y' = -Nva-, Z '= -Nva-, W = - | D) Ser-]: [a] g> = -217 ° (C = 1.0 in CHCl ₃ ); and

5.6. [lhr] ² - [(D) Ser] ^8- Cyclosporin [Formula IHa: X '= -Meßmt-, Y' = -Thr-, Z = -VaI-, W = - (D) Ser-]: [α ] ² , ⁰ = 258 ° (c = 0.39 in

Example 6

The compound of example 5.2. may be otherwise prepared microbiologically as follows:

a) 10 liters of a nutrient medium containing 50 g maltose, 5 g (DL) -norvaline, 8 g (D) -serine, 0.75 g KH ₂ PO ₄ , 0.5 g MgSO ₄ -7 H ₂ O; 0.1 CaCl ₂ 6 H ₂ O and 8 g per liter of casein peptone is inoculated with 1 liter of a conidia and mycelia suspension of the fungal strain NRRL 8044, which was taken from a three-day-old pre-culture. The inoculated production medium is filled into 100 ml portions in 100 Erlenmeyer flasks, which are then incubated for 14 days at 27 ° C on a rotary shaker with 180 rpm. The mycelium is separated from the culture medium and extracted in a Turrax by crushing and stirring with 3 x 3 liters of 90% methanol. The minced mycelium is separated from the solvent by suction filtration and the combined filtrates are concentrated by evaporation in vacuo at a temperature of 40 ^° C. until the vapor consists mainly of water. The resulting mixture is extracted 4x using 0.5 liter of 1,2-dichloroethane in each extraction. The combined 1,2-dichloroethane solutions are concentrated by evaporation under vacuum at a temperature of 4O ⁰ C.

The residue obtained is subjected to gel filtration on Sephadex LH-20 (1.4 kg, Pharmacia) with methanol and then collected in 280 ml fractions. Fractions 9-11 containing a cyclosporin mixture are combined and then separated by silica gel column chromatography (1 kg of silica gel, grain size 0.063-0.2 mm) using ethyl acetate saturated with water as eluent (fractions of 500 ml). In accordance with their polarity, [Nva ² ] cyclosporin (fractions 7-9) is first eluted, followed by a mixture containing [Nva ² ] - [(D) Ser ⁸ ] cyclosporin and cyclosporin. The separation of [Nva ² ] - [(D) Ser ⁸ ] cyclosporin and cyclosporin is carried out by silica gel chromatography (280 g, 0.063-0.2 mm) using chloroform / methanol (98: 2) as eluant (fractions of 100 ml). Fractions 20-30 containing crude [Nva ² H (D) Ser ⁸ ] cyclosporin are further purified by pressure chromatography on a phase-reversed silica gel column LiChropep RP 18.260g, grain size 0.04-0.063 mm) using methanol / water ( 85:15) as eluans and collected in 25 ml fractions. The pooled fractions 45-55 give pure [Nva ² ] - [(D) Ser ⁸ ] cyclosporin as an amorphous white powder

 The preculture required for the above process can be obtained as follows:

b) The spore and mycelial suspension used for inoculation, prepared from a culture of the originally isolated strain NRRL 8044, is grown for 21 days at 21 ° C on the following agar medium: 20 g malt extract, 20 g agar, 4 g yeast extract per liter of demineralized water. The spores of this culture are taken up in a physiological saline solution to give a final concentration of 5 x 10 ⁶ spores / ml. 10 ml of this suspension are used for the inoculation of 1 liter of a nutrient solution with the same composition as the culture medium of Example 6a, with the exception of the (D) -serine and the (DL) -norvaline component. The incubation takes place for 3 days at 27 ° C on a rotary shaker (200 rpm). This culture is used to inoculate the production culture. [Nva ² ] - [(DJSer1-cyclosporin can be produced in the fermenter as follows:

c) Approx. 10 ⁹ spores of a slant culture of strain NRRL 8044 are placed in a stainless steel fermenter containing 20 liters of a preculture medium of the following composition:

fructose 75 g Amber EHC 25g KH ₂ OP ₄ 5g KCI 2.5 g Dest. Water ad 1 liter (pH = 5.5

Previously, sterilization is carried out at 120 ° C for 20 minutes. Favorable incubation conditions are a temperature of 27 ° C, an air rate of 16 liters per minute at a pressure of 0.5 bar and a speed of 200 rpm. The developing preculture is incubated for 6 days and then 15 liters are placed in a stainless steel fermenter containing 300 liters of a production medium of the following composition:

maltose 75 g Amber EHC 25 g KH ₂ PO ₄ 5g KCI 2.5 g (DL) -Norvalin 5g (D) -serine 8g Dest. Water ad 1 liter (pH = 5.5)

Previously, sterilization is carried out at 120 ° C for 20 minutes. The culture is maintained at a temperature of 27 ° C, vented at an air rate of 120 liters of air per minute, at an overpressure of 0.5 bar and stirred at a speed of 70 rpm. The foam control is carried out by adding a silicone emulsion. After incubation for 14 days, the culture is cooled to 10X and the mycelium removed using a Westfalia Separator. The filtrate is extracted by stirring with ethyl acetate for 2m. The extracts are washed with a little water, combined and dried in vacuo. The mycelium is mixed with methanol, homogenized and filtered. This extraction is repeated 2x using 90% methanol. The methanolic extracts are combined and concentrated under vacuum with the addition of water. The remaining aqueous concentrate is extracted 2x with ethyl acetate, the extracts washed with a little water, combined and concentrated in vacuo. The extracted aqueous phase is extracted 2 times more with ethyl acetate / isopropanol (8: 2). These extracts are combined and again evaporated in vacuo.

The mycelium and filter extracts are filtered using the amount of Sephadex LH-20 with methanol as eluent. The peak fractions are then purified by chromatography, 10Ox the amount of silica gel 60 (particle size = 0.04-0.063 mm) and water saturated ethyl acetate is used as eluent. First, [Nva ² ] cyclosporin elutes, followed by cyclosporin and [Nva ² ] - [(D) Ser ⁸ ] cyclosporin. These later fractions will become another

chromatographic purification using 14Ox the amount of silica gel 60 (particle size 0.063-0.20mm) and chloroform / methanol (98: 2) as eluent. Pure [Nva ² ] - [(D) Ser ⁸ ] cyclosporin is obtained.

Example 7

The compound of example 5.3 can also be produced microbiologically by proceeding analogously to example 6a), but with the following modifications:

a) In the nutrient medium, the (DL) -norvaline is replaced by 10 g (L) -valine. After the separation of the mycelium from the culture medium is extracted as follows:

The crude mycelium is separated from the solvent by suction filtration and the combined filtrates are concentrated with the addition of water by evaporation in vacuo at a temperature of 40 ⁰ C until the steam consists mainly of water. The resulting mixture is extracted 3 times using 5 liters of ethyl acetate on each extraction. The combined ethyl acetate solutions are concentrated by evaporation under vacuum at a temperature of 40 ° C. The resulting residue is subjected to gel filtration on Sephadex LH-20 (1.4 kg, Pharmacia) with methanol The fractions containing a cyclosporin mixture are combined and then separated by silica gel column chromatography (3 kg silica gel, granule size 0.020-0.045 mm, "Grace") using water-saturated ethyl acetate as eluent. In accordance with their polarity, [Val ² ] cyclosporin is first eluted, followed by a mixture containing [Val ² ] - [(D) Ser ⁸ ] cyclosporin as the main component. Further purification of [Val ² ] - [(D) Ser ⁸ ] cyclosporin is carried out by silica gel chromatography (80 g, "Grace", 0.020-0.0045 mm) using acetone / hexane (1: 1) as The fractions containing crude [Val ² ] - [(D) Ser ⁸ ] cyclosporin are further purified by pressure chromatography on a phase-reversed silica gel column ("Merck" LiChropep RP18, 160g, granule size 0.04-0.063 mm) Use of methanol / water (80:20) as eluent to give pure [Val ² ] - [(D) Ser ⁸ ] cyclosporin as an amorphous white powder.

b) The required preculture is obtained as in Example 6 b).

[Val ² ] - [(D) Ser ⁸ ] cyclosporin can be prepared at the fermentor level by following the procedure of Example 6c), but with the following modifications:

c) In the composition of the production medium, the (DL) -norvaline is replaced by 10 g (L) -VaMn. After adding methanol to mycelium, homogenizing and filtering (2x with 90% methanol), proceed as follows:

The methanolic extracts are combined and concentrated under vacuum with the addition of water. The remaining, aqueous concentrate is extracted 3x with ethyl acetate, the extracts washed with a little water, combined and evaporated in vacuo.

The mycelium and filtrate extracts are filtered using 50 times the amount of Sephadex LH-20 with methanol as eluent. The peak fractions are then chromatographed using 40 times the amount of silica gel 60 (particle size = 0.04-0.063 mm) and water saturated ethyl acetate as eluent. First, [VaI ² ] cyclosporin elutes, followed by cyciosporin and [Val ² ] - [(D) Ser ⁸ ] cyclosporin. These later fractions are subjected to further chromatographic purification using 100x the amount of silica gel 60 and acetone / hexane (1: 1) and reverse phase silica gel chromatography ("Merck" LiChroprep RP 18, particle size 0.04 to 0.063 mm using methanol / water (80:20) as eluent to give pure [Val ² ] - [(D) Ser ⁸ ] cyclosporin.

Examples

The compound of Example 5.6 can also be prepared microbiologically by proceeding analogously to Example 6a), but with the following deviations:

a) In the nutrient medium, the (DL) -norvaline is replaced by 5g (L) -thronin. After incubation, extract as follows: The mycelium is separated from the culture medium and extracted in a Turrax by crushing and stirring with 3 x 9 liters of 90% methanol. The minced mycelium is separated from the solvent by suction filtration and concentrated the combined filtrates with the addition of water by evaporation in vacuo at a temperature of 4O ⁰ C, until the steam consists mainly of water. The resulting mixture is extracted 3 times using 5 liters of ethyl acetate on each extraction. The combined ethyl acetate solutions are concentrated by evaporation under vacuum at a temperature of 40 ⁰ C.

The residue obtained is subjected to gel filtration on Sephadex LH-20 (2 kg, Pharmacia) with methanol. Extraction containing a cyclosporin mixture is combined and then separated by silica gel column chromatography (2 kg silica gel, 0.02-0.045 mm, Grace grain size) using water saturated ethyl acetate as eluent then [(E) Ser ⁸ ] cyclosporin followed by [Thr ² ] cyclosporin and finally [Thr ² ] - [(D) Ser ⁸ ] cyclosporin in crude form. Further purification of [Thr ² ] - [ (D) Ser ⁸ ] cyclosporin is prepared by silica gel chromatography (50g, "Grace", 0.02-0.45 mm) using acetone / hexane (2: 1) as eluant, with pure [Thr ² ] - [(D) Ser ⁸ ] cyclosporin is obtained as an amorphous white powder.

b) The required preculture is obtained as in Example 6 b). [Thr ² ] - [(D) Ser ⁸ ] cyclosporin can be prepared at the fermenter level by following the procedure of Example 6c), but with the following deviations:

c) In the composition of the production medium, the (DL-Novalin is replaced by 5 g of (L) -Threonin After incubation and removal of the mycelium using a Westfalia Separator, the procedure is as follows:

The mycelium is mixed with methanol, homogenized and filtered. This extraction is repeated 2x using 90% methanol. The methanolic extracts are combined and concentrated under vacuum with the addition of water. The remaining aqueous concentrate is extracted 2x with ethyl acetate, the extracts washed with a little water, combined and concentrated in vacuo.

The mycelial extracts are filtered using the amount of Sephadex LH-20 with methanol as eluent. The peak fractions are then chromatographed using 3Ox the amount of silica gel 60 (particle size = 0.04-0.063 mm) and water saturated ethyl acetate as eluent. First, cyciosporin elutes, followed by [(D) Ser ⁸ ] cyclosporin followed by [Thr ² ] cyclosporin and ultimately [Thr ² ] - [(D) Ser ⁸ ] cyclosporin. These later fractions are subjected to further chromatographic purification, 25% being the amount

Silica gel 60 (grain size 0.02-0.045mm) and acetone / hexane (2: 1) is used as eluent. Pure [Thr ² ] - [(D) Ser ⁸ ] cyclosporin is obtained.

Example 9

The following compounds, which can be used as starting material for the preparation of the compounds listed in Examples 4.1 to 4.3, can be prepared from the cyclosporines given in Examples 5 to 7 by proceeding analogously to Example 3.

9.1. [Dihydro-Meßmt] ¹ - [Nva] ² - [(D) Ser] ^8- Cyclosporin [Formula IHa: X '= -dihydro-Meßmt-, Y' = Nva-, Z '= -VaI-, W = - (D) Ser-] prepared from the product of Example 5.2 or 6: [α] ² ) ⁰ = -251 ° (c = 1.23 in CHCl ₃ , / -! 79 ° (c = 1.16 in CH ₃ OH):

Mp = 155-157 ° C.

9.2. [Dihydro-Meßmt] ¹ - [Val] ² - [(D) Ser] ^8- Cyclosporin [Formula lilac: X '= -dihydro-Meßmt-, Y' = -VaI-, Z '= -VaI-, W = (D) Ser-] prepared from the product of Example 5.3 or 7: [α] * ⁰ = -224 ° (c = 1.0 in CHCl ₃ ).

9.3. [Dihydro-Meßmt] ¹ - [Thr] ² - [(D) Ser] ^8- Cyclosporin [Formula lilac: X '= -dihydro-Meßmt-, Y' = -Thr-, Z '= -VaI-, W = - (D) Ser-] prepared from the product of Example 5.6 or 8: [a] g ° = -262 ° (c = 0.73 in CHCl ₃ ) / - 173 ° (c = 0.79 in CH ₃ OH): mp = 156-158 ° C.

The cyclosporins wherein the amino acid residue at position 8 is a (D) -acyloxy-a-amino acid residue, as previously defined and described, hereinafter referred to as cyclosporins of the present invention, have pharmacological activity as shown in the following test methods.

1. Immunosuppressive effects:

1.1 Local Hemolysis in vitro in Gel [R. I. Mishell and R.W. Dutton, J. Exp. Medicine, 126, 423-442 (1976)]. The cyclosporins of the invention inhibit haemolysis zones compared to untreated controls in concentrations of 0.01 to 10.0 g / ml.

1.2 Lymphocyte Stimulation Test According to Janossy and Greaves [Clin. Exp. Immunol., 9, 483 (1971) and 10,552 (1072)]: The cyclosporins of the present invention inhibit Concanaval-A-stimulated DNA synthesis (inhibition of H ³ - thymidine incorporation), cell proliferation and blastogenesis in mouse spleen cells Comparison with untreated controls in concentrations of 0.001 to 10.0pg / ml.

1.3 Mixed lymphocyte reaction [Bach et al., J. Exp. Med. 136, 1430 (1972)]:

The reaction (ie proliferation and differentiation) of lymphocytes [mouse (Balb / c) spleen cells] when co-incubated for 5 days with allogeneic spleen cells from irradiated mice (CBA t) is measured in the presence and absence of the test substance. The reaction in the presence is expressed in the percentage change of the reaction compared with 100% of the control reaction. When using cyclosporins according to the invention an inhibition of the reaction at a concentration of 0.001 to 10.0μg / mΓ ^{1 is} observed.

1.4 Suppression of Organ Repulsion:

Kidney and donor rats (F 344, Figure 9) are transplanted into recipient rats (Wistar-Furth, SJ) The test substance is administered to the recipient rats for 14 days, after which the treatment is discontinued and 7 days after transplantation Since the life of the test animals depends on the acceptability and functioning of the transferred organ, the increase in survival time is used as a parameter for the efficacy of the test substance as compared to that of control animals receiving placebo only, animals, the cyclosporins of the invention at doses of 2.5 to 10 mg / kg po have a survival span of 60 to 250 days compared to untreated controls, all of which die as a result of organ rejection within about 9 to 10 days.

2. Anti-inflammatory effect

The anti-inflammatory effect can be demonstrated with the rat adjuvant arthritis test. In this test, adjuvant arthritis is determined by the method of Pearson and Wood, "Arthr. Rheum. ", 2,440 (1959) The cyclosporins of the present invention are shown to be effective in developing and in established arthritis at doses of 10 to 30 mg / kg / day p.o.

3. Antiparasitic action

Anti-Malaria Test near L. Rane, "Chemotherapy and Drug Resistance in Malaria" ed. W Peters, Academic Press, New York,

1970. Mice (OF1: male) are infected on day 0 with 0.2 ml of a suspension containing 10 ~ ⁷ parasitic cells of the species Plasmodium berghei (strain NK 65). Administration is ip On day 3, the test substance is administered at variable doses using 5 to 10 mice per dose sc. Survival time is recorded and the minimum effective dose (MED) calculated by comparing survival time to untreated control animals. In the control animals, the survival time is about 7 days. The MED is the dosage at which the survival time is doubled. The cyclosporins of the present invention are shown to be effective at doses of 25 to 100 mg / kg / day in this test; s; c.

Because of their immunosuppressive activity, the cyclosporins of the invention can be used for the prophylaxis and treatment of conditions and diseases which require a reduction of the immune response. Thus, the cyclosporins of the present invention find application in suppressing the proliferation of lymphocytes and immunocytes, such. In the treatment of autoimmune diseases, to prevent tissue rejection in transplants, e.g. Skin, lung, heart, heart, bone marrow, kidneys, spleen and corneal transplants. Specific autoimmune diseases in which cyclosporins of the invention can be used are those for which the treatment with cyclosporin A has been proposed or has already been used, e.g. Eg, aplastic anemia, pure red cell anemia, idiopathic trombocytopathy, systemic lupus erythematosus, polychrondritis, scleroderma, Wegener granulomatosis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue, Crohn's disease, Graves opthalmopathy, sacoidosis Multiple sclerosis, primary billiare cirrhosis, primary juvenile diabetes, posterior uveitis, interstitial pulmonary fibrosis and psoriatic arthritis.

Because of their anti-inflammatory effect cyclosporins of the invention can find application in the treatment of inflammatory conditions, in particular inflammatory conditions with an ethology that a

Autoimmune component includes, ζ. As the treatment of arthritis and rheumatic diseases such as arthritis chronica progrediens.

Due to their anti-parasitic effect cyclosporins of the invention can be used as antiparasitic remedies, eg. B. for the treatment of parasitic infections of various types, especially in protozoa as well as trematode and Neumatodeninfektionen. Specific types of parasitic infections of various types, especially in protozoa as well as trematode and nematode infections. Specific types of parasitic infections in which the cyclosporins of the invention can be used are those for which treatment with cyclosporins has already been suggested in the literature. Schistomosomiasis, filariasis, leishmania, coccidioidomycosis, and especially malaria.

For the above indications, the daily dose is about 75 to about 5000, preferably about 2000, most preferably about 1500 mg. When using a unit dose, eg. For oral administration, a dosage of from about 25 to about 2500, preferably about 1 to 20, more preferably about 800 mg of a cyclosporin of the present invention is suitable, admixed with a pharmaceutically acceptable diluent or carrier.

The cyclosporins of the present invention can be administered by any conventional route, particularly in accordance with current methods of administering cyclosporin A, especially by intravenous infusion,

z. In the case of organ transplantation, before and immediately after transplantation, as well as in the presence of a gastrointestinal disorder, which would otherwise worsen the absorption or orally, ζ. B. in the form of an oral solution.

As described above, the cyclosporins of the formula IIIa are also new. In addition to their use as intermediates, they exhibit a pharmacological effect and / or profile, especially in relation to immunosuppressive activity, and particularly in relation to their use in preventing graft rejection. This makes them particularly interesting in relation to other cyclosporins as specifically disclosed in European Patent No. 0056782. The pharmacological action of cyclosporins of the formula IIIa can be demonstrated, for example, in the above-described test methods 1.1.1.2.1.3.2 or 3. The cyclosporins of the formula IIIa are active in accordance with the present invention:

In Test 1.1 at concentrations of 0.01 to 10 ^ g / ml;

in test 1.2 at concentrations of 0.001 to 10 / ng / ml;

in test 1.3 at concentrations of 0.001 to 10 μg / ml

in Test 2 at doses of 10 to 30 mg / kg / day p.o .; and

in Test 3 at doses of 50 to 100 mg / kg / day.

In view of their immunosuppressive activity, cyclosporins of the formula are useful for the prophylaxis and treatment of diseases and conditions which require a reduction of the immune response, e.g. To suppress the proliferation of lymphocytes and immunocytes, e.g. In the treatment of autoimmune diseases, e.g. In the treatment of specific autoimmune diseases as listed above in connection with the use of the cyclosporins of the invention or in the prevention of transplant rejection, e.g. In the various specific species cited above in connection with the use of the cyclosporins of the invention.

With regard to their anti-inflammatory activity, cyclosporins of the formula IIIa are also useful for the treatment of inflammatory conditions, in particular of inflammatory conditions with an etiology comprising or including an auto-immune component, e.g. B. for the treatment of arthritis and rheumatic diseases ₍ such as polyarthritis chronica

Progrediens.

In view of their antiparasitic action, cyclosporins of the formula IIIa are also useful as antiparasitic agents, e.g. For the treatment of parasitic infections of various types, in particular as described above in connection with the use of the cyclosporins according to the invention.

For the indications mentioned above, the daily dose ranges from about 75 to about 5000 mg and when using a unit dose, e.g. For oral administration, a dose of about 25 to about 2500 mg cyclosporin of formula IIIa is suitable, mixed with a pharmaceutically acceptable diluent or carrier.

The cyclosporins of Formula IIIa can be administered by any conventional route, particularly in accordance with current methods of administering cyclosporin, particularly by intravenous infusion,

z. In the case of organ transplantation, before and immediately after transplantation, as well as in the presence of a gastrointestinal disorder, which would otherwise worsen the absorption or orally, ζ. B. in the form of an oral solution.

Claims (2)

Invention claim: A process for the preparation of a cyclosporin, wherein the amino acid residue in position 8 is a (D) -acyloxy-a-amino-acid radical, characterized by a) acylating a cyclosporin in which the amino acid residue at position 8 is a (D) -hydroxy-a-amino acid residue, or b) reducing a cyclosporin wherein the amino acid residue in position 1 is -MeBmt- and the residue in position 8 is a (D) -acyloxyamino acid residue, to obtain the corresponding cyclosporin wherein the residue in position 1 is dihydro -MeBmt-is. 2. Process for the preparation of a cyclosporin of the formula I, "X" Y-Sar-MeLeu-Z-Meleu-Ala-Q-MeLeu-MeLeu-VaI-123 456789 10 11 (D X -MeBmt- or dihydro-MeBmt- means Y -aAbu -, - AIa -, - Thr -, - VaI-or-Nva-means, Z is -VaI- or -NVa- means and Q is a radical of the formula II. R ₁ -CO-O-CH (S) -HH-CH-CO- (D) R _{1 is} hydrogen, alkyl having 1 to 4 carbon atoms or phenyl and R _{2 is} hydrogen or methyl, characterized by a) that a cyclosporin of the formula III, IX-Y-Sai ^ MeLeu-Z-MeLeu-Ala-W-Meleu-MeLeu-MeYal · 12 3 4 5 6 7 8 9 10 11 (ID (III) X -MeBmt- or -dihydro-MeBmt- means Y is -AAbu, -AIa, -Thr, VaI or -Nva-, Z is -VaI- or -NVa- means and W is a radical of formula IV. HIGH -HH-CH-CO- (D) wherein R _{2 is} hydrogen or methyl, acylated by introducing a R _r CO group, wherein Ri is hydrogen, alkyl having 1 to 4 carbon atoms or phenyl, in the β-position, or b) that a cyclosporin of the formula I, wherein X is -MeBmt- reduced to give the corresponding cyclosporin, wherein X is dihydro-MeBmt.