NO310028B1 - Cyclosporins, their use and preparation as well as a biologically pure source - Google Patents

Description

The present invention relates to cyclosporins, their use as pharmaceuticals and pharmaceutical preparations comprising these compounds, as well as methods for their preparation.

The cyclosporins comprise a class of structurally distinctive, cyclic, poly-N-methylated undecapeptides which in common have a pharmacological, especially immunosuppressive, anti-inflammatory and/or antiparasitic activity. The first of the cyclosporins to be isolated was the naturally occurring fungal metabolite "Ciklosporin" or "Cyklosporin-ne", also known as cyclosporin A and which is commercially available under the trade name SANDIMMUN<R> or SAND-IMMUNE<R>. "Ciclosporin" is the cyclosporine of formula A.

wherein MeBmt represents the N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonyl residue of formula B wherein -x-y- is -CH=CH- (trans). Since the original discovery of "Ciklosporin" a wide variety of naturally occurring cyclosporins have been isolated and identified and many additional non-natural cyclosporins have been produced by fully synthetic or semi-synthetic methods or by the use of modified cultivation methods. The class covered by the cyclosporins is thus now large and includes e.g. the naturally occurring cyclosporins A to Z [cf. Traber et al., 1, Heiv. Chim. Acta, 60, 1247-1255 (1977); Traber et al., 2, Heiv. Chim. Acta, 65, 1655-1567 (1982); Kobel et al., Europ. J. Applied Microbiology and Biotechnology, 14, 273-240 (1982); and von Wartburg et al., Progress in Allergy, 38, 28-45, (1986)] as well as various non-natural cyclosporine derivatives and artificial or synthetic cyclosporines including dihydrocyclosporines [wherein the -x-y- portion of the MeBmt residue (formula B in the preceding) is saturated to give -x-y- = CH2-CH2-]; derivatized cyclosporins (in which, for example, the 3'-0-atom MeBm residue is acylated or an additional substituent is introduced at the α-carbon atom of the sarkosyl residue at the 3-position); cyclosporins in which the MeBmt residue is present in isomeric form (eg, in which the configuration across the 6' position and the 7' position of the MeBmt residue is cis rather than trans); and cyclosporins in which varying amino acids are incorporated at specific positions in the peptide sequence, e.g. by using the total synthetic method for the production of cyclosporins developed by R. Wenger - see e.g. Traber et al. , 1, Traber et al., 2 and Koble et al., loe. one.; US Patents 4,108,985, 4,220,461, 4,288,431, 4,554,351, 4,396,542 and 4,798,823, European Patent Publications 34,567A, 56,782A, 300,784A, 300,785A and 414,632A; international patent publication WO 86/02080 and UK patent publications 2,206,119 and 2,207,678; Wenger 1, Transpl. Proe, 15 Suppl. 1:2230 (1983); Wenger 2., Angew. Chem.Int. Oath. 24 77 (1985) and Wenger 3, Progress in the Chemistry of Organic Natural Products, 50, 123 (1986).

The class now covered by the cyclosporins is thus now really large and includes e.g. [Thr]2-, [Val]2-, [Nva]2-and [Nva]2-[Nva] 5-Cyclosporin (also known as cyclosporins, C, D, G and M, respectively) , [3-O -acetyl-MeBmt] ^Cyclosporin (also known as cyclosporine A acetate), [dihydro-MeBmt]<1->

[Val] 2-cyclosporin (also known as dihydro-cyclosporin D), [ (D) Ser] 8-Cyclosporin, [Melle] "-cyclosporin, [ (D) MeVal] 11-Cyclosporin (also known as cyclosporin H), [MeAla]^Cyclosporin, [ (D) Pro] 3-Cyclosporin, etc.

In accordance with the conventional nomenclature for cyclosporins, these are defined throughout the present disclosure and claims with reference to the structure of Cyclosporin (ie cyclosporin-A). This is done by first indicating the residues in the molecule which are different from those present in Ciclosporin and then using the term "Ciclosporin" to characterize the remaining residues which are identical to those present in Ciclosporin. At the same time, the prefix "dihydro" is used to denote cyclosporins in which the MeBmt residue is hydrogenated (dihydro-MeBmt), i.e. where

-x-y- in formula B is -CH2CH2-. Thus, [Thr] is the 2-Cyclosporine with the sequence shown in formula A, but in which aAbu at the 2-position is replaced by Thr, and [dihydro-MeBmt)<1->

[Val]<2->Cyclosporin is the cyclosporine with the sequence shown in formula A, but in which the MeBmt residue at the 1-position is hydrogenated and aAbu at the 2-position is replaced by Val.

In addition, the amino acid residues are named by abbreviations, e.g. Ala, MeVal, aAbu, etc. in accordance with conventional practice, which are understood to have the (L) configuration unless otherwise indicated, e.g. as in the case of "(D)Ala". Abbreviations for residues preceded by "Me" which in the case of "MeLeu" represent α-N-methylated residues. Individual residues of the cyclosporine molecule are numbered as

common in this region in a clockwise direction starting with the residue Me-Bmt or dihydro-MeBmt in the 1-position. It

the same number sequence is used throughout the present presentation and claims.

It is now well established that Ciclosporin works by interfering with the process of T-cell activation by blocking transcriptional initiation by IL-2, although the exact mechanism has not yet been elucidated. Cyclosporine has been shown to form a complex with a 17kD cytosolic protein (cyclophilin) that occurs in many cell types and has been shown to be identical to peptidyl-prolyl-cis-trans isomerase, an enzyme involved in protein folding. Until now, however, it has not been clear whether binding to cyclophilin is directly correlated with immunosuppressive activity in cyclosporins, or indeed whether cyclophilin binding in itself is a sufficient criterion for immunosuppressive activity.

It has now been found that there are cyclosporins that bind strongly to cyclophilin, but above all are not immunosuppressive. It therefore follows that binding to cyclophilin is a necessity but not a sufficient criterion for immunosuppressive activity.

The present invention provides cyclosporins which are active against HIV-1 replication.

Human immunodeficiency virus (HIV) preferentially infects T-helper (T4) lymphocytes, although it also replicates in various other cell types, particularly those of monocytic lineages. It causes a slowly progressing disease in the immune system characterized by gradual T4 cell destruction, known as AIDS. Other immunological abnormalities of AIDS are an increase in cytotoxic/suppressor (T8) lymphocytes, a defect in the antigen presentation/recognition process, and polyclonal activation of B cells. The mechanism of T4 cell destruction is still not clear. Relatively few T4 cells appear to be infected and a direct cytopathic effect caused by the virus need not therefore be the only reason for T4 cell depletion. It has been hypothesized that T4 cell destruction could be enhanced by an autoimmune process triggered by HIV-producing or HIV protein-coated T4 cells. By this continuous antigenic stimulation can lead to a state of permanent activation of T4 cells which would increase HIV replication in these cells and expand T-cytotoxic clones. Uninfected T4 cells can be made antigenic by binding exogenous viral gp120 to their CD4 molecules and would thus be a target for a T-cytotoxic response.

US patent 4,814,323 shows that Ciclosporin has an activity against AIDS and that in general "cyclosporins known as immunosuppressants" can be useful in this indication. There is no suggestion that non-immunosuppressive cyclosporins could be expected to have this property.

Surprisingly, it has now been found that cyclosporins which bind to cyclophilin, but which are not immunosuppressive, exhibit an inhibitory effect on HIV-1 replication.

A ciclosporin is considered to bind to cyclophilin if it binds to human recombinant cyclophilin at least one-fifth as much as Ciclosporin by the competitive ELISA test described by Quesniaux in Eur. J. Immunol. 1987 17 1359-1365. In this test, the cyclosporine being tested is added during the incubation of cyclophilin with coated BSA-Cyclosporin and the concentration required to give a 50% inhibition of the control reaction without a competitor is calculated (IC50). The results are expressed as binding ratio (BR), which is the 10-based log of the ratio between the IC50 of the test compound and the IC50 of a simultaneous test with Ciclosporin itself. Thus, a BR of 1.0 indicates that the test compound binds cyclophilin a factor of ten less well than Ciclosporin, and a negative value indicates a stronger binding than the binding of Ciclosporin.

The cyclosporins active against HIV have a BR below 0.7 (since log10 5 = approximately 0.7), preferably equal to or less than 0.

A ciclosporin is considered to be non-immunosuppressive when it has an activity in the mixed lymphocyte reaction (MLR) of no more than 5%, preferably no more than 2%, of the activity of Ciclosporin. The mixed lymphocyte reaction is described by T. Meo in "Immunological methods", L. Lefko-vits and B. Peris, editors, Academic Press, N.Y. 227-239

(1979). Spleen cells (0.5 x IO<6>) from Balb/c mice (female mice 8-10 weeks) are coincubated for 5 days with 0.5 x IO<6> irradiated (2000 rad) or mitomycin C treated spleen cells from CBA mice (female mice 8-10 weeks). The irradiated allogeneic cells induce a proliferative response in Balb c spleen cells which can be measured by an incorporation of the labeled precursor into DNA. As the stimulator cells are irradiated (or treated with mitomycin), they do not respond to Balb/c cells with proliferation, but retain their antigenicity.

The IC50 found for the test compound by MLR is compared with the IC50 found for Ciclosporin in a parallel experiment.

The activity of cyclosporins as inhibitors of HIV-1 replication can be demonstrated in the following test systems: 1. Inhibition of HIV-1-induced cytopathic effect in MT4 cells

The assay procedure described by Pauwels et al., Virol. meth. 20/309 (1988) is used with slight modifications. The HTLV-I-transformed T4 cell line, MT4, which was previously shown to be very beneficial for HIV infection, is used as the target cell. Inhibition of HIV-1, strain HTLV-IIIB-induced cytopathic effect is determined by measuring the viability of both HIV-infected and mock-infected cells. Viability is assessed spectrophotometrically via in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium boramide (MTT). Virus-infected and uninfected cultures without the compound are included as controls as are uninfected cells treated with the compound. The cell concentration is chosen so that the number of cells per ml increases by a factor of 10 during the 4-day incubation in the mock-infected cultures. The virus inoculum is adjusted so that it causes cell death in 90% of the target cells after 4 days' incubation. The virus is adsorbed onto a 10-fold concentrated cell suspension at 37°C for 1 hour. The infected cells are then diluted 1:10 and microtiter plates containing the test compound are added.

2. Cytotoxicity

To assess the anti-HIV potential in further cell lines and in particular in a monocytic cell line (U937), the cellular toxicity of the test compound against these cell lines is first assessed. Jurkat and U937 cell suspensions are adjusted to 1 x 10<5> cells/ml and incubated in the presence of various concentrations of test compound. After 48 hours, the amount of cells per ml is compared by staining with MTT. Cytotoxicity in MT4 cells can be measured in the same way.

3. Inhibition of HIV-1 replication in Jurkat and U937 cells The T4 cell line Jurkat and the monocytic cell line U937 are infected by suspending the cells 10 times concentrated in virus solution. Adsorption is allowed for 2 hours at 37°C. The cells are then centrifuged down, the inoculum is removed and the cells are resuspended at their original concentration in fresh culture medium containing the test compound. Thus, the substance is added after adsorption. At days 2, 5, 8, 12, 15 and 19 after infection, sub-samples of the infected cultures are removed. The cells are centrifuged down and the supernatants are collected. The concentration of viral p24 antigen is determined in the supernatants using a commercial ELISA kit and serves as a parameter for virus production. After each removal of aliquots, the cells are counted and adjusted to 2 x 10<5> cells/ml by adding fresh medium containing the test compound at the particular concentration.

Of the compounds in accordance with the invention, i.e. non-immunosuppressive, cyclophilin-binding cyclosporins which were active against HIV-1 (active compound), some are new and others are known. However, the anti-HIV activity of the known active compounds has not previously been revealed and in many cases the known active compounds have not been shown to have any pharmaceutical activity whatsoever.

It has been found that many of the active compounds have structures that differ from the structure of Ciclosporin specifically at the 4- and/or 5-positions.

A group of active compounds are cyclosporins in which the MeLeu group at the 4-position is replaced by another N-methylated amino acid, e.g. γ-hydroxy-MeLeu, Melle, MeVal, MeThr or MeAla. In addition to Melle and meThr, the alloforms Mealle and MeaThr can also be used. In the allo-form, the stereochemistry at the S-position is opposite to the configuration of the natural amino acid, so that the normal form and the allo-form form a pair of diastereoisomers.

A further group of active compounds is that in which Val at the 5-position is replaced by an N-alkyl, preferably N-methyl, amino acid. Preferred is the amino acid which is N-alkylated Val or Leu. Preferably, the hydrogen of the imino group in [Bal]<5> is replaced with an unbranched C-L-galkyl group, preferably methyl, ethyl or n-propyl, especially methyl. The latter preferred group of active compounds are all new.

Additionally or alternatively, certain active compounds may differ from Ciclosporin at the 1-, 2-, 3- and/or 6-positions. The present invention comprises a compound characterized by formula I:

in which

W is MeBmt, dihydro-MeBmt or 8'-hydroxy-MeBmt

X is aAbu, Val, Thr, Nva or O-methylthreonine (MeOThr)

R is Sar or (D)-MeAla

Y is MeLeu, γ-hydroxy-MeLeu, Melle, MeVal, MeThr, MeAla,

Mealle or MeaThr

Z is Val, Leu, MeVal or MeLeu, and

Q is MeLeu, γ-hydroxy-MeLeu or Meala

with the proviso that when Y is MeLeu, Z is either MeVal or MeLeu, or W is 8'-hydroxy-MeBmt, for use as a pharmaceutical agent.

The groups W, X, Y, Z, Q and R independently have the following preferred meanings: W is preferably W where W is MeBmt or dihydro-MeBmt

X is preferably X' when X' is aAbu or Nva, more

preferably X" where X" is aAbu

R is preferably R' where R<1> is Sar

Y is preferably Y<1> where Y<1> is y-hydroxy-MeLeu, MeVal, MeThr or Melle,

Z is preferably Z' wherein Z' is Val or MeVal, and

Q is preferably Q<1> where Q' is MeLeu.

A particularly preferred group of active compounds are the compounds of formula I in which W is W, X is X', Y is Y', Z is Z', Q is Q' and R is R'.

The invention comprises a compound characterized in that it is selected from the group comprising:

a) [dihydro-MeBmt]<1-> [γ-hydroxy-MeLeu] 4-cyclosporine

b) [MeVal]<4->cyclosporine

c) [Melle]" cyclosporine

d) [MeThr]"-cyclosporine

e) [γ-hydroxy-MeLeu] "-cyclosporine

f) [Nva]<2-> [γ-hydroxy-MeLeu] "-Cyclosporin

g) [γ-hydroxy-MeLeu]<4-> [γ-hydroxy-MeLeu] <6->cyclosporine

h) [MeVal]<5->cyclosporine

i) [MeOThr]<2>- [ (D) MeAla]3- [MeVal] <5>-cyclosporine

j ) [8'-hydroxy-MeBmt] ^cyclosporin

k) [γ-hydroxy-Me-Leu] 9-cyclosporine,

for use as a pharmaceutical agent.

In addition, certain compounds that are not within the scope of formula I are active compounds, e.g. 1) [MeAla] ^cyclosporine and m) [γ-hydroxy-MeLeu] 9-cyclosporine.

The present invention also includes a compound of formula II:

wherein W is MeBmt or dihydro-MeBmt,

X is aAbu, Val, Thr, Nva or MeOThr

R is Sar or (D)-MeAla

Y is MeLeu, γ-hydroxy-MeLeu, Melle, MeVal, MeThr, MeAla,

Mealle or MeaThr and

Z is Val, Leu, MeVal or MeLeu,

with the condition that

1) when Y is MeLeu or MeAla, Z is MeVal or MeLeu, and 2) when W is MeBmt, R is Sar and Y is y-hydroxy-MeLeu, then Z has a meaning other than Val.

A preferred group of new active compounds consists of compounds of formula II in which Y is Y' and Z is Z', with the proviso that when W is MeBmt Y' has a meaning other than γ-hydroxy-MeLeu.

Particularly preferred new active compounds are the compounds a), b), c), d), f) and h) in the foregoing. Certain of these compounds, e.g. compounds b) and c) are found to block the immunosuppressive effect of Ciclosporin by blocking its binding to cyclophilin and thus act as Ciclosporin antagonists.

The present invention comprises a compound characterized in that it is selected from the following group: [dihydro-MeBmt]1- [γ-hydroxy-MeLeu]"-cyclosporine [MeVal]"-cyclosporine

[Melle]"-Cyclosporin

[MeThr]”-cyclosporine

[Nva]<2-> [γ-hydroxy-MeLeu] "-cyclosporine

[γ-hydroxy-MeLeu]"- [γ-hydroxy-MeLeu] 6-cyclosporine [MeVal]<5->cyclosporine, and

[MeOThr]<2-> [ (D) MeAla]3- [MeVal] 5-Cyclosporin.

The invention also includes a method for the production of [Melle] "-cyclosporin, characterized by growing the fungal strain DSM 6627 in a nutrient medium and isolating the product from the fermentation liquid.

Furthermore, the invention includes a biologically pure culture of a Tolyplocadium inflatum Cy E 4556, characterized by the fact that the strain is Tolyplocadium inflatum, DSM 662 7.

The invention also includes a method for the production of cyclosporins with one or more γ-hydroxy MeLeu residues, characterized by growing the fungal strain DSM 6182 in a nutrient medium, adding a cyclosporin that has one or more MeLeu residues and isolating the product from the fermentation liquid.

The active compounds can be obtained in a number of different ways which can be classified as:

1) fermentation

2) biotransformation

3) derivatization

4) partial synthesis

5) total synthesis

1) Certain of the active compounds are produced as by-products by fermentation of original or modified strains of cyclosporin-producing organisms such as Tolypocladium inflatum Gams, as exemplified by the production of compound c) described in the following example. 1. 2) Other active compounds, including the known compounds j) and 1) are metabolites of cyclosporine and can be isolated using chromatographic methods from urine of humans or animals that have been given cyclosporine. Furthermore, these and other metabolic transformations are possible using microorganisms, e.g. production of compound e) and g) by biotransformation of cyclosporine A, as described in examples 2 and 3, or production of compound f) by biotransformation of cyclosporine G (ex. 4). These examples demonstrate the novel method for preparing cyclosporins with one or more γ-hydroxy MeLeu residues, comprising the steps of growing a new modified strain of Sebekia benihana, adding cyclosporine with one or more MeLeu residues, and isolating the product from the fermentation liquid. 3) By derivatization is meant that natural or synthetic cyclosporins can be converted into active compounds by means of one or more chemical reactions in which one or more of the amino acids are modified without the peptide bonds being opened and converted. E.g. the class of active compounds in which Val at the 5-position is N-alkylated can be obtained by reacting the corresponding cyclosporine with Val at the 5-position with butyllithium, followed by reaction with an alkylating agent, as exemplified for compound h) in ex.

5 and for the compound i) in ex. 6.

As further examples, the compound a) can be prepared by hydrogenating the compound e) (ex. 7) and the compound j) which is also a main metabolite of Ciclosporin, can be prepared from the known compound Ciclosporin acetate as described in ex. 8. 4) The term partial synthesis is used to denote a series of chemical reactions in which the ring of natural cyclosporin is opened, one or more amino acids are removed and different amino acids are added, and the ring is closed again. 5) The total synthesis of cyclosporins can be carried out by building up a linear undecapeptide and ring closure as described by Wenger (loe. eit.), see also US patent documents 4.3 96,542 and 4,798,823. In principle, any cyclosporine can be prepared by the total synthesis method, although where one of the other methods is available, this may be more advantageous than total synthesis. Total synthesis can be used for the preparation of compound d) (ex. 9) and for the known metabolite compound 1).

The compound k) is a known substance with properties described by Quesniaux et al., (Mol. Immuno. 24 1159 1987) and can also be produced by total synthesis. E.g. is a total synthesis of this compound described in USP 4,914,188.

Example 1 [Medium]<4->Cyclosporin (compound c)

Producing strain

Compound c) is obtained by fermentation of the fungal strain Tolypocladium inflatum Cy E 4556, deposited at the Deutsche Sammlung fur Mikroorganismen under the conditions of the Budapest Agreement on 24 July 1991 under accession number DSM 6627. This strain is a mutant of the strain NRRL 8044 of the species Tolypocladium inflatum Chamois, and is taxonomically identical to the parental strain that has been fully described, e.g. in British patent 1,491,509.

Culture 1. Agar release culture:

Agar slant cultures of the strain DSM 6627 are grown for 14 days at 27°C on the following agar medium:

The medium has a pH of 5.4-5.6 and is sterilized for 20 minutes. at 120°C.

2. Preculture:

Spores from the mycelium of 4 starting cultures are suspended in 40 ml of a 0.9% saline solution. A series of 500 ml Erlenmeyer flasks each containing 100 ml of pre-culture medium are each inoculated with 20 ml of this suspension. The composition of the pre-culture medium is as follows:

demineralized water to 1000 ml

The medium is adjusted to pH 5.2-5.5 with HC1 and then sterilized for 20 min. at 12 0°C. The pre-cultures are fermented for 24 hours at 27°C on a rotary shaker at 200 rpm. with an eccentricity of 50 mm.

3. Intermediate culture:

A 25 liter steel fermenter containing 20 1 of pre-culture medium is inoculated with 200 ml of pre-culture. The intermediate culture is fermented for 5 days at 27°C with a stirring rate of 150 revolutions per minute. and an air flow of 0.5 l/min. per 1 medium at 0.5 bar pressure.

4. Main culture:

10 0 1 pre-culture medium is inoculated with 10 1 intermediate culture and fermented in a 120 1 steel fermenter. 4 g/l D-Threonine, sterilized by filtration, is added to this medium.

Fermentation is carried out at 27°C for 14 days with a stirring rate of 70 revolutions per minute. and an air flow of 0.4 l/min. per 1 medium at 0.5 bar pressure used for the first 5 days, with the stirring rate then being increased to 100 revolutions per minute. and the air flow to 0.5 l/min. for the rest of the fermentation period.

Insulation:

The mycelium that is separated from the culture medium and extracted in a Turrax apparatus by crushing and stirring 3 times with 10 1 methanol/water (9:1 volume basis). The crushed mycelium is separated from the solvent by suction filtration and the combined filtrates are concentrated by evaporation under water at a temperature of 40°C until the vapor consists mainly of water alone. The resulting mixture is extracted 4 times using 2 L of 1,2-dichloroethane in each extraction and the combined 1,2-dichloromethane solutions are concentrated by evaporation under vacuum at a temperature of 40°C.

The residue is subjected to silica gel column chromatography (10 kg silica gel, granule size 0.02 - 0.045 mm, "Grace") using ethyl acetate/water as eluent (fractions of 2.5 1). Fractions 20 - 23 containing

[Melle] "-Cyclosporin is collected and then further separated by means of silica gel column chromatography (600 g silica gel, granule size 0.04 - 0.063 mm, "Merck") using chloroform/methanol (98:2 volume basis) as eluent (fraction size 3 00 (ml). pure [Melle]"-Cyclosporin as an amorphous white powder with m.p. 155-188°C [<x]20/D = -235° (c = 0.68 in CHCl3) and -193°

(c 0 0.74 in CH 2 OH).

IR spectrum in CH 2 Cl 2 as shown in fig. 1 and proton NMR spectrum in CDCl3 is as shown in fig. 2.

Example 2 [γ-Hydroxy-MeLeu]"-Cyclosporin (compound e)

Compound e) is obtained by biotransformation of Ciclosporin using the microorganism Sebikia benihana. The original strain used is named NRLL 11111 and belongs to the genus Sebikia benihana (Dietz and Li: Sebekia, a new genus of the family Actinoplanaceae. Abstrs. 82nd Ann.Meet:-Amer.Soc. Microbiol., 163, Atlanta, 1982) . This strain is able to hydroxylate novobiocin. The subcultured strain used for the production of compound e) and related compounds has been deposited at the German Collection of Microorganism (D-3300 Braunschweig) under number DSM 6182.

1. Aqar output culture:

Agar slant cultures of the strain DSM 6182 are grown for 10 days at

2 7°C on the following agar medium:

Demineralized water up to 1 1.

pH: neutralized at 7 with NaOH/H2SO4

sterilization 120°C/20 min.

Tracer solution no. 235:

Demineralized water up to 999 ml

H2S04 1 ml

The pre-cultures are fermented for 3 days at 27°C on a rotary shaker at 200 rpm. with an eccentricity of 5 0 mm.

3. Intermediate culture:

A series of 200 ml Erlenmeyer flasks each containing 50 ml of the pre-culture medium are each inoculated with 5 ml of the pre-culture. The intermediate culture is fermented for 3 days at 27°C on a rotary shaker at 200 rpm. with an eccentricity of 50 mm.

4. Main culture:

A series of 500 ml Erlenmeyer flasks each containing 50 ml of the main medium (12 L in total) are inophorcultured each with 5 ml of intermediate culture. These cultures are fermented for 3 days at 27°C on a rotary shaker at 200 rpm. with an eccentricity of 50 mm. After 24 hours, cyclosporin A (7.5 mg) dissolved in methanol is added to each main culture (= 150 mg/l).

The composition of the main culture medium is as follows:

Demineralized water up to 1 1.

pH: adjusted to 7.2-7.5 (KOH/H2SO4)

sterilization: 120°C/20 min.

Tracer solution of AC-1: This solution has the same composition as solution no. 235 with the addition of (NH4)6Mo7024 0 , 2 g.

5. Insulation:

The mycelium is separated from the culture medium and the resulting culture filtrate (13 L) is extracted three times with 1,2-dichloroethane using 1.5 L in each extraction. The combined 1,2-dichloroethane solutions are evaporated under vacuum at a temperature of 40°C. The crude residue is subjected to Sephadex LH-20 gel filtration using methanol as eluent. The fractions containing cyclosporin compounds (525 mg) are collected and chromatographed on silica gel (50 g, granule size 0.04-0.063 mm, "Merck") using chloroform/methanol as eluent. Repeated chromatography using the same system affords pure [γ-hydroxy-MeLeu]"-Cyclosporin as an amorphous white powder, mp 150-153°, [a] D20 = -2 2 5° (c = 0.53 in CHCl3 ), -171° (c = 0.44 in CH 3 OH) .

Example 3 [γ-Hydroxy-MeLeu]4- [γ-Hydroxy-Leu] 6-Cyclosporin

(compound g)

The more polar side fractions resulting from the purification from [γ-hydroxy-MeLeu]"-Cyclosporin are further separated by repeated silica gel column chromatography (granule size 0.04 - 0.0063 mm) using acetone/hexane 2:1 ' as eluent followed by chromatography on silica gel using methyl t-butyl ether/methanol/water 90:9:1 as eluent. The first fractions contain [γ-hydroxyLeu] "-cyclosporin which is further purified by decolorization with charcoal to give the pure compound as an amorphous white powder, m.p. 162-164°C, [α] 20D = -211° (c = 0.50 in CHCl 3 ), -157° (c = 0.52 in CH 3 OH).

The later fractions from the above silica gel column chromatography contain [γ-hydroxy-MeLeu]"-[γ-hydroxy-MeLeu] 6-Cyclosporin and are further purified by decolorization with charcoal to give the pure title compound as an amorphous white powder with m.p. 157-160°C, [α] <20>D -217° (c = 0.54 in CHCl 3 ), - 176° (c = 0.42 in CH 3 OH) .

Example 4 [Nva]2-[γ-hydroxy-MeLeu]"-Cyclosporin

(connection f)

This compound is prepared using a procedure analogous to the preparation of compound e ( [γ-hydroxy-MeLeu] "-Cyclosporin) , but using cyclosporin G

([Nva] 2-Cyclosporin) as starting material. After purification by means of repeated silica gel column chromatography using ethyl acetate saturated with water or acetone/hexane 2:1 as eluent, the compound mentioned in the title is obtained as an amorphous white powder with m.p. 138-141°C, [a]<20>D -213° (c = 0.69 in CHCl3), -168° (c = 0.70

in CH3OH).

Example 5 [MeVal] 5-Cyclosporin (compound h)

Cyclosporin A (0.60 g = 0.5 mmol) dissolved in tetrahydrofuran (20 ml) is incubated with 0.63 ml of a 1.6 M solution of butyllithium (1.0 mmol) in hexane. The resulting solution is reacted with dimethyl sulfate (0.1 mL, 1.5 mmol) at -78°C. The reaction mixture is slowly warmed to room temperature and stirred overnight.

Isolation by flash chromatography (SiO 2 , 5% methanol/ether), followed by HPLC (reverse phase) gave the title compound. The compound is characterized by a 300 MHz <*>H NMR spectrum in CDCl3 as shown in fig. 3.

Example 6 [MeOThr]2- [ (D) MeAla]3- [MeVal] 5-Cyclosporin

(connection in)

A mixture of 480 ml of THF (absolute) and 6.96 g (49.2 mmol) of diisopropylamine is cooled to -80°C and 33.5 ml of a 1.33 M solution of butyllithium in hexane (= 44.5 mmol) is added slowly through a syringe needle. The mixture is stirred for 30 min. at -80°C and then a solution of 8 g is added

(6.6 mmol) cyclosporin C ([Thr] 2-Cyclosporin) in 120 ml of absolute THF through a syringe needle during 2 - 3 min. The clear solution is stirred for a further hour at -80°C and then slowly added 2 .06 ml of methyl iodide.

The mixture is warmed to room temperature within 2 hours and then 40 ml of water is added and the solvents are evaporated on a rotary evaporator at 30°C/15 mm hg. The residue is distributed between water and ether and the ether layer is washed four times with half-saturated salt solution, dried over MgSO 4 and evaporated to give a residue of 8.1 g.

The residue is chromatographed on 1200 g of silica gel with water-saturated ethyl acetate to give a crude product which, after further chromatography on 200 g of silica gel with 5% MeOH/CH2Cl2, gives the pure product mentioned in the title, [a] <20>D = -195°C ( c = 1.0 in CHCl3).

Example 7 [dihydro-MeBmt]x-[γ-hydroxy-MeLeu]"-Cyclosporin

(compound a)

To a suspension of 200 mg of pre-hydrogenated 10% palladium/charcoal in 4 ml of ethanol is added 1.2 g of [γ-hydroxy-MeLeu]"-Cyclosporin (compound e) in 10 ml of ethanol and hydrogenation is carried out at room temperature until hydrogen no longer After removal of the catalyst by filtration, the solution is evaporated to give the title compound as an amorphous white powder with mp 154-156°C, [a]<20>D -225° (c = 0.87 in CHCl3) , -169° (c = 0.70 in CH 3 OH) .

Example 8 [8 1 -hydroxy-MeBmt] ^Cyclosporin (compound j)

1. [ Q- acetvl- cobromo- MeBmt] ^ Cyclosporine

A mixture of 25.0 g (20 mMol) [O-acetyl-MeBt] ^Cyclosporin (Traber et al, Heiv. Chim. Acta 1982, 65, 1653), 4.4 g (25 mMol) N-bromosuccinimide and 400 mg azobisisobutyronitrile

1,250 ml of carbon tetrachloride are heated under reflux for 2.5 hours. The solvent is evaporated and replaced with ether, the solid is filtered off, washed with water, dried over magnesium sulphate and evaporated to dryness. The remainder is chromatographed

over silica gel with ethyl ether/ethyl acetate (4:1) to give 10.7 g (40%) of amorphous product which is crystallized from ether/hexane to give 8.4 g pure substance with mp 207-209°C. Later fractions of the chromatogram contained a further 11.2 g of product of somewhat lower quality.

2. [ O- acetvloo- acetoksv- MeBmtl ^ Cyclosporine:

A mixture of 4.31 g (3.3 mmol) of the product from step 1 (contaminated with an estimated 15-20% starting material) and 2.1 g (8 mmol) of tetraethylammonium acetate tetrahydrate in 30 mL of methyl ethyl ketone, containing a catalytic amount of sodium iodide, heated in an oil bath at 105°C for 3 hours and kept at room temperature over the weekend. The solvent is diluted with methyl-t-butyl ether and washed with water and saline. The organic layer is dried over MgSO 4 and evaporated leaving 4.0 g of crude product which is purified on an RP-18 reverse phase column (240 g) to give 3.07 g of the title product mp 191-192°C.

3. [ co- hydroxy- MeBmt 1 ^ Cyclosporine:

A solution of 1.72 g (1.3 mmol) of the product from step 2 in 75 ml of methanol and a solution of 1.2 g of sodium in 50 ml of methanol are mixed and kept at room temperature for 2.5 hours. The solution is then acidified with acetic acid. The solvent is evaporated under reduced pressure and the residue is dissolved in methyl-t-butyl ether, washed in sequence with water, brine and sodium bicarbonate solution, dried and MgSO 4 and evaporated. The crude product (1.6 g) is eluted from an RP-18 column (240 g) with methanol/water 75:15 to give 1.5 g of crude product. A sample is crystallized from ether/hexane to give a crystalline product with mp 181-183°C. Example 9 [MeThr] "-Cyclosporin (compound d) The total synthesis of Ciclosporin as described in USP 4.3 96,542 and 4,798,823 is carried out using MeThr in the 4-position instead of MeLeu. The product has [a] 20D = -249.6 ° (c = 1.0 in CHCl 3 ).

Example 10 [MeVal] "-Cyclosporin (compound b)

The total synthesis of Ciclosporin as described in USP 4.3 96,542 and 4,798,823 is carried out using MeVal in the 4-position instead of MeLeu. The product has [a] 20D = -226°

(c = 0.358 in CHCl3.

Example 11 immunosuppression and cyclophilin binding of active compounds in relation to Ciclosporin

Table I provides examples of (1) cyclophilin binding ratios (BR) of active compounds as measured by an ELISA assay and (2) the immunosuppressive activity of active compounds relative to Ciclosporin as measured by an MLR assay and expressed as a percentage activity relative to Ciclosporin (Immunosuppressive ratio or IR).

Further explanation of the meaning of these values and the procedures for conducting these tests are set forth above.

Compound 12 Anti-HIV activity and cytotoxicity of active compounds

Examples of the activity of active compounds and Ciclosporin as inhibitors of HIV-1 replication in MT4 cells and the cytotoxicity of active compounds and Ciclosporin in MT4 cells are given in Table II. The meaning of these values and the relevant methods have been discussed above.

The active compounds are indicated both for the prevention of AIDS in asymptomatic HIV-positive patients and in the treatment of patients suffering from AIDS. In the patients in whom AIDS has developed, administration of the active compound should reverse the T4 cell depletion associated with AIDS, induce regression of AIDS-related disorders such as Kaposi's sarcoma, and reduce the likelihood of new occasional infections.

Method for the treatment and prevention of acquired immunodeficiency syndrome and other disorders induced by HIV-1 virus in a patient who is infected by said virus can be used, comprising administering to the patient an effective amount of an active compound in accordance with the invention.

The active compound can be administered by any conventional means, especially enterally, e.g. orally, e.g. in the form of solutions for drinking, tablets or capsules for parenteral administration, e.g. in the form of injectable solutions or suspensions. With the intravenous method, an indicated daily dose can be from 1 to 20 ml/kg, preferably from 3 to 10 mg/kg and with the oral method from 1 to 50 mg/kg, preferably from 7 to 20 mg/kg.

The toxicity of the active compounds is believed to be similar to Cyclosporine. As the active compounds are not immunosuppressive, certain side effects of Ciclosporin in connection with immunosuppression are avoided. However, other side effects associated with Ciclosporin, particularly nephrotoxicity with long-term use, may also be associated with the active compounds.

Galenic preparations for the active compounds which can be used include preparations based on microemulsions as described in British Patent Application 2,222,770A which include topical as well as oral forms. Also oral and injectable forms obtained from solid solutions comprising a fatty acid saccharide monoester, e.g. sucrose monolaurate, as described in British patent application 2,209,671A can be used. Suitable unit dosage forms for oral administration include e.g. from 25 to 200 mg of active compound per dose.

Composition example A:

Composition example B:

Composition example C:

Composition example D:

The individual components of these compositions, as well as the methods of their preparation, are fully described in British Patent Application 2,222,770, the contents of which are incorporated herein by reference.

Claims (10)

1. A compound, characterized in that it has formula I in which W is MeBmt, dihydro-MeBmt or 8<1>hydroxy-MeBmt X is aAbu, Val, Thr, Nva or O-methylthreonine (MeOThr) R is Sar or (D)-MeAla Y is MeLeu, γ-hydroxy-MeLeu, Melle, MeVal, MeThr, MeAla, Mealle or MeaThr Z is Val, Leu, MeVal or MeLeu, and Q is MeLeu, γ-hydroxy-MeLeu or MeAla with the proviso that when Y is MeLeu, Z is either MeVal or MeLeu, or W is 8'-hydroxy-MeBmt, for use as a pharmaceutical agent. 2. A compound as specified in claim 1 is characterized by the fact that it has formula I' in which W is MeBmt or dihydro-MeBmt X<1> is α-Abu or Nva, Y' is γ-hydroxy-MeLeu, MeVal, MeThr or Melle, and Z' is Val or MeVal for use as a pharmaceutical agent. 3. A compound, characterized in that it is selected from the group comprising: [dihydro-MeBmt]<1-> [γ-hydroxy-MeLeu] "-cyclosporine [MeVal] "-cyclosporine [Melle] "-cyclosporine [MeThr] "-cyclosporine γ-hydroxy-MeLeu] "-cyclosporine [Nva]<2-> [γ-hydroxy-MeLeu] "-cyclosporine [γ-hydroxy-MeLeu]"- [γ-hydroxy-MeLeu]<6->cyclosporine [MeVal] <s->cyclosporin [MeOThr]<2-> [ (D) MeAla]3- [MeVal] "-cyclosporin [8 1 -hydroxy-MeBmt]1-cyclosporin, and [γ-hydroxy-MeLeu] 9-cyclosporin for use as a pharmaceutical agent. 4. Use of a compound as stated in claim 1, 2, or 3 in the preparation of a drug for the treatment and prevention of AIDS and AIDS-related disorders. 5 . A compound of formula II, wherein W is MeBmt or dihydro-MeBmt, X is aAbu, Val, Thr, Nva or MeOThr R is Sar or (D)-MeAla Y is MeLeu, γ-hydroxy-MeLeu, Melle, MeVal, MeThr, MeAla, Mealle or MeaThr and Z is Val, Leu, MeVal or MeLeu, with the condition that 1) when Y is MeLeu or MeAla, Z is MeVal or MeLeu, and 2) when W is MeBmt, R is Sar and Y is y-hydroxy-MeLeu, then Z has a different meaning than Val. 6. A compound as stated in claim 5, characterized in that it has formula II 1 in which W MeBmt or dihydro-MeBmt Y' is γ-hydroxy-MeLeu, MeVal, MeThr or Melle and Z' is Val or MeVal, with the proviso that when W is MeBmt, Y' has a meaning other than y-hydroxy-MeLeu. 7 . A compound characterized in that it is selected from the following group: [dihydro-MeBmt]<1-> [γ-hydroxy-MeLeu] "-cyclosporine [MeVal] "-cyclosporine [Melle] "-cyclosporine [MeThr] "-cyclosporine [ Nva]2- [γ-hydroxy-MeLeu] "-cyclosporine [γ-hydroxy-MeLeu]"- [γ-hydroxy-MeLeu] 6-cyclosporine [MeVal] 5-cyclosporine, and [MeOThr]<2-> [ ( D) MeAla]3-[MeVal]5-Cyclosporin. 8 . A process for the production of [Melle] "-cyclosporin, characterized by growing the fungal strain DSM 6627 in a nutrient medium and isolating the product from the fermentation liquid. 9. A biologically pure culture of a Tolyplocadium inflatum Cy E 4556, characterized in that the strain is Tolyplocadium inflatum, DSM 6627. 10 . A method for the production of cyclosporins with one or more γ-hydroxy-MeLeu residues, characterized by growing the fungal strain DSM 6182 in a nutrient medium, adding a cyclosporine having one or more MeLeu residues and isolating the product from the fermentation broth.