AU596071B2 - Novel cyclosporins - Google Patents

Description

C O M MO N W E A L II OF A U T R A rL I A PATENTS ACT 1952 COtl r JVST S PECIFICATION (Original)' FOR OFFICE USE Int. Class Application Number: Lodged: Complete Specification. Lodged: Accepted: Published: Priority: 4 4 i, ,Related Art: fit# 4 t 4 5960'1 Tis document contains the 1ameltlnments made un-kr Section 49 and is correct K)I printing.

Name of Applicant: SANDOZ LTD.

,4 Address of Applicant:

I

Actual Inventor(s): t I Address for Service: 35 Lichtstrasse.

CH-4002 Basle, Switzerland ROLAND WENGER RENE P. TRABER HANS KOBEL HANS HOFMANN DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete specification for the invention entitled: "TMPROVBMBMNa IN OR RfAlbTIJq TO OROANIO COHPOrUHO" The following statement is a full description of this invention, including the best method of performing it known to us -T lll^ ^^4 -la- Case 100-6283 NOVEL CYCLOSPORINS The present invention relates to novel cyclosporins, processes for their production, their use as pharmaceuticals and pharmaceutical compositions comprising them.

0 The cyclosporins comprise a class of structurally distinctive, cyclic, poly-N-methylated undecapeptides commonly possessing pharmacological, in particular immunosuppressive, anti-inflammatory and anti-parasitic activity. The first of the cyclosporins to be isolated and the "parent" compound of the class, was the naturally occurring fungal metabolite Cyclosporine, also known as cyclosporin A, of formula A eBmt-aAbu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala-MeLeu-MeLeu-MeVai 1 2 3 4 5 6 7 8 9 10 11 (A) p wherein -MeBmt- represents the N-methyl-(4R)-4-but-2E-en-1-yl-4methyl-(L)threonyl residue of formula B -L i ~~L 100-6283 H2

CH

CH3 #4 14* 4 4* .4 *r 4 4.4 44 4 44 .4 #444

I

t 414 tf#1 ir HO (R)

XCH

-N-CH-CO-

S(S)

CH

3 in which is -CH=CH- (trans).

I 144 4 4 1 41 4r 4 It 4 4 r Since the original discovery of Cyclosporine a wide variety of naturally occurring cyclosporins have been isolated and identified and many further non-natural cyclosporins have been prepared by total- or semi-synthetic means or by the application of modified culture techniques. The class comprised by the cyclosporins is thus now substantial and includes for example the naturally occurring cyclosporins A through Z Kobel et al. European Journal of applied Microbiology and Biotechnology 14, 237 240 (1982) and poster presented by Traber et al., 24th. Interscience Conference on Antimicrobial Agents and Chemotherpy, Washington, October 8 10, (1984)]; as well as various non-natural or artificial cyclosporins, including dihydro-cyclosporins (in which the group of the -MeBmt- residue see formula B above is saturated, e.g. as disclosed in US Patents Nos. 4,108,985; 4,210,581 and 4,220,641), cyclosporins in which the -MeBmtresidue is present in isomeric or N-desmethyl form European

I

100-6283 patent no. 0 034 567 and "Cyclosporin Proc. Internat.

Conference on Cyclosporin A, Cambridge September 1981, Ed. D.J.G. White, Elsevier Press (1982) both describing the total-synthetic method for the production of cyclosporins developed by R. Wenger] and cyclosporins in which incorporation of variant amino acids at specific positions within the peptide sequence is effected European Patent no. 0 056 782).

o Examples of such cyclosporins as disclosed in the above art references include e.g. [Thr] 2 [Val] 2 [Nva] 2 and [Nva] 2 -[Nva] 5 Cyclosporine (also known as cyclosporins C, D, G and M respectively), [Dihydro-MeBmt]l-[Val] 2 -Cyclosporine (also known as 4 dihydrocyclosporin D) and [(D)Ser] 8 and [Dihydro-MeBmt]l-[(D)- Ser] 8 -Cyclosporine.

[In accordance with now conventional nomenclature for the cyclosporins, these are defined throughout the present specification t and claims by reference to the structure of Cyclosporine (i.e.

cyclosporin This is done by first indicating those residues in the molecule which differ from those present in Cyclosporine Sand then applying the term "Cyclosporine" to characterise the remaining residues which are identical to those present in Cyclosporine. At the same time the term -dihydro-MeBmt- is employed to .t designate the residue of formula B above in which is Si d -CH 2

-CH

2 Thus [Dihydro-MeBmt] 1 -Val] 2 -Cyclosporine is the cyclosporin having the sequence shown in formula A, but in which -MeBmt- [formula B, -CH=CH- (trans)] at the 1-position is replaced by -dihydro-MeBmt- [formula B, -CH2-CH2-] and -aAbu- at the 2-position is replaced by -Val-. Similarly [(D)Ser] 8 -Cyclosporine is the cyclosporin having the sequence shown in formula A, but in which -(D)Ala- at the 8-position is replaced by -(D)Ser-.

I

-4- In addition, amino acid residues referred to by abbreviation, e.g. -Ala-, -MeVal- etc... are, in accordance with conventional practice, to be understood as having the (L)-configuration unless otherwise indicated. Residue abbreviations preceded by as in the case of -MeLeu- represent N-methylated residues, The individual residues of the cyclosporin molecule are numbered, as in the art, clockwise and starting with the residue -MeBmt- or I, dihydro-MeBmt- in position 1. The same numerical sequence is employed throughout the present specification and claims.] 'S 15 In accordance with the present invention it has now been found that novel cyclosporins may be obtained having pharmaceutical r tc utility, in which the residue at the 8-position comprises an acyloxy a-amino acid residue having the (0)-configuration.

Accordingly, in its broadest aspect, the present invention pro- G vides: a cyclosporin wherein the amino acid residue at the 6* 8-position is a (D)-3-acyloxy-a-amino acid residue of formula II

A

I

Rl-CO-O-CH(3) -NH-CH,-COv-

(D)

(11) 4* I I I II .4 I I I III 1f I 14 It 1444 I 44 4~ wherein R1 is hydrogen, Cl.4alkyl or phenyl and R2 is hydrogen or me thyl.

Especially preferred cyclosporins in accordance with the present 15 invention are those of formula I 114* 1

III

44 *0 U II I 9 II

II

I.

S S

I

)I0t ,014.db40272.res.5 4 A ,I -6- .X-Y-Sar-MeLeu-Z-MeLeu-Al a-Q-MeLeu-Meleu-Val- 12 3 4 5 6 7 8 9 10 11] (1) 9m 4 49 4 *4 *4 9 a 4*4 0a 4 .4 44 4.4.

4 *44, 4449 9 944* *999 9 9 444* *94 4 #4 94 9 9* 9 9 9 *9 94 9 4 999 9 wherein X is -Me~mt- or -dihydro-MeBmt-, Y is -cObu-, -Ala-, -Thr-, -Val-. or -Nva- Z is -Val- or -Nva-, and Q is a residue of formula 11 as defined above.

A group of cyclosporins in accordance with the present invention are those of formula I as defined above, wherein Y is -aAbu- or 20 Z is -Val- and R2 is hydrogen.

A further group of cyclosporins in accordance with the present invention are those of formula I as defined above, wherein Y is -axAbu- or -Nva-, Z is -Nva-, R1 is hydrogen or C1i4alkyl and R2 is hydrogen.

A cyclosporin wherein the amino acid residue at the 8position is a (D-1-acyloxy-a-amino acid residue of formula 11, a cyclosporin of formula I as defined above, may be produced by a process which comprises: a) Acylating a cyclosporin wherein the amino acid residue at the 8-position, is

LU

(aL 1 4

K

1, r 100-6283 s>F G:(rmu\c,. II a (D)-B-hydroxy-a-amlno acid residue e.g. acylating a cyclosporin of formula III X-Y-Sar-MeLeu-Z-MeLeu-Ala-W-MeLeu-MeLeu-MeVal 1 2 3 4 5 6 7 8 9 10 11 (III) I Ilrllul~ee-l,"elullulll 9 I t I I I wherein X, and W is a Y and Z have the meanings given above for formula I residue of formula IV

R

2

HO-CH

-NH-CH-CO-

(IV)

4$ 'I 49* 4 4~ 4 4* 4 *r 49. 4

I

II

4% 4 414C I

(D)

wherein R 2 has the meaning given above for formula II, to introduce a group R 1 wherein R 1 has the meaning given above for formula II, at the 1-position of said residue IV; or b) Reducing a cyclosporin wherein the amino acid residue at the 1-position is -MeBmt- and the residue at the 8-position is a a6,ylxy a amino cid rciduo, for OAmp8 a acyloxy-a-amino acid residue, to produce the corresponding cyclosporin wherein the residue at the 1-position is -dihydro-MeBmt-, e.g. reducing a cyclosporin of formula I as hereinbefore defined, wherein X is -MeBmt-, to produce the corresponding cyclosporin wherein X is -dihydro-MeBmt-.

Process step a) above may be carried out in accordance with standard procedures for the acylation of hydroxy groups, for 100-6283 #4 .,r 4 *4i #9 41 4*r Irr It I( ii i *4 4* P *i P .1 4 4.

4e I 14 1: I I I example by reaction with (preferably 2 equivalents or, when Y -Thr-, 1 equivalent) of an appropriate acyl-, e.g. Cl-ialkanoylor benzoyl-halide, or corresponding -anhydride or, for formylation, by reaction with e.g. acetic-formic anhydride, at a temperature of e.g. from about -10 to 50'C. The reaction is carried out under anhydrous conditions, suitably in the presence of an inert solvent or diluent such as methylene chloride, and in the presence of a condensation agent such as 4-dimethyl-amino-pyridine. In this connection it is to be noted that the reaction proceeds with acylation occurring at the OH group of the amino acid residue at the 8-position, in preference to the hydroxy group of the amino acid residue at the 1-position.

Process step b) may be carried out analogously to known methods for reducing naturally-occurring cyclosporins to the corresponding dihydrocyclosporins, for example by catalytic hydrogenation, e.g. in accordance with the general methods disclosed in U.K.

Patent Specification No. 1,567,201.

Hydrogenation is suitably effected under neutral pH conditions at temperatures of from about 20' to about 30'C and at atmospheric or slightly elevated pressure, in the presence of a catalyst such as platinum or, preferably, palladium palladium on charcoal) in the presence of an inert solvent or diluent such as ethyl acetate or lower aliphatic alkanols such as methanol or iso-propanol.

Cyclosporins having a B-hydroxy-c-amino acid residue at the 8-position, in particular [(D)Ser) 8 -Cyclosporine and [Dihydro- MeBmt]l-[(D)Ser] 8 -Cyclosporine, suitable for use as starting materials in process step a) above are known and have been described together with processes for their production, e.g. in the aforementioned European patent no. 0 056 782. Other cyclosporins having a hydroxy-a-amino acid residue at the 8-position f 9 and required as starting materials for process step a), may be prepared analogously or in accordance with the general procedures of the cyclosporin total-synthetic method described in European patent no. 0 034 567 to which publication 0 056 782 cross-refers, or in accordance with the procedures hereinunder described in particular in the accompanying examples.

The cyclosporins starting materials for use in process step b) above are obtainable in accordance with the method of process step a).

The following examples are illustrative of the processes of the present invention.

*0 0 a S *o 0* 0 0 S10,db40272.res,9 i 0 0 \0 -44- 100-6283 EXAMPLE 1 Synthesis of [(O-acetyl)-(D)Ser] 8 -Cyclosporine [Formula I: X -MeBmt-, Y -aAbu-, Z -Val-, Q -0-acetyl-(D)Ser-].

mg 4-dimethylaminopyridine are added to 47 mg [(D)Ser8]-Cyclosporine (prepared in accordance with the method described in Example 1 or 3 of the above mentioned European Patent No. 0 056 782) dissolved in 3 ml methylene chloride. 6.1 mg of freshly distilled acetylchloride in 1 ml methylene chloride are then added and the obtained reaction mixture is stirred for 1 hour at room temperature. The reaction mixture is diluted with ml methylene chloride and shaken with 30 ml H20. The organic phase is separated, dried over Na 2 SO4, filtered off and evaporated. The residue is filtered on 60 g silica gel (0.062 0.20 mm) using methylene chloride/5 methanol as eluant and collected in 25 ml fractions. The title compound is recovered from fractions 4 to 8 by thin layer chromatography using CHC1 3 methanol as carrier phase: [a]20 -202° (c 0.92 in CHC13).

EXAMPLE 2 The following compounds may be prepared analogously to example 1 starting from the corresponding non-acylated cyclosporin: 2.1 [(O-benzoyl-(D)Ser]8-Cyclosporine [Formula I: X -MeBmt-, Y -aAbu-, Z -Val-, Q -0-benzoyl- [a] 20 -220° (c 1.0 in CHC13); j

-T-

100- 6283 2.2 L0-acetyl-(D)Thr]B-Cyclosporine [Formula I: X =-MeBmt-, Y= -aAbu-, Z Q -0-acetyl-(D)-Ser-1: (c =1.0 in CHC13) 2.3 ENva) 2 -[0-acetyl-(D)Ser) 8 -Cyclosporine [Formula I: X =-MeBmt-, Y -Nva-, Z Q -0-acetyl-(D)Ser-J: [a]2 -400(c 1.0 in CHC1 3 233* (c 0.8 in CHCl 3 )/-177 =0.76 in CH 3 0H): m.p. 143 147 *C.

2.4 [Val] 2 -[0-acetyl-(D)Ser) 8 -Cyclosporine [Formula 1: X =-MeBmt-, Y Z Q -0-acetyl-(D)Ser-J: -219* (c =0.9 in GHCl 3 [Nva]5-[0-acetyl-(D)Ser) 8 -Cyclosporine [Formula I: X =-MeBmt-, Y -czAbu-, Z Q -0-acetyl-(D)Ser-]: 4 -215' (c =1.0 in CHC1 3 A 4.

[Nva] 2 -[Nva) 5 -[0-acetyl-(D)Ser)S-Cyclosporine [Formula I: X -MeBmt-, Y Z -Nva-, Q -0-acety-(O)Ser-): [a] 20 -196.9 (c =1.0 in CHCl 3 and

D

tot,2.7 [Thr) 2 -[0-acetyl-(D)Ser] 8 -Gyclosporine [Formula 1: X -MeBmt-, Y -Thr-, Z =-Val- Q =-0-acetyl-(D)Ser-]: -251 0.86 in CHCl 3 174 (c 0.81 in CH 3

OH):

m.p. =143 -146 C.

l i 100-6283 EXAMPLE 3 Synthesis of [Dihydro-MeBmt] l 0-acetyl-(D)Ser]8-Cyclosporine [Formula I: X -dihydro-MeBmt-, Y -aAbu-, Z -Val-, Q -0-acetyl-(D)Ser-]: 9* S 4' I I 99r 4 54 mg of [(0-acetyl)-(D)Ser 8 ]-Cyclosporine in 10 ml ethanol are hydrogenated using 10 mg palladium/charcoal (10 at room temperature and under normal pressure. After 20 hours the obtained reaction solution is filtered through a thin layer of talc and the ethanol is evaporated off under vacuum. After further drying under high vacuum, the title compound is obtained: [a] 2 0 -205.8' (c 1.02 in CHC13).

EXAMPLE 4 Ir 4* 9 9 9 i t i 4C The following compounds may be prepared either analogously to example 1, starting from the corresponding non-acylated cyclosporin or analogously to example 3, by hydrogenation of the corresponding cyclosporin described in example 2: 4.1 [Dihydro-MeBmt]1-[Nva] 2 -[O-acetyl-(D)Ser] 8 -Cyclosporine [Formula I: X -dihydro-MeBmt-, Y -Nva-, Z -Val-, Q -0-acetyl-(D)Ser-]: m.p. 139 141'C; [ca]0 -225' (c 0.88 in CHC13)/-163' (c 0.76 in CH 3 0H); 4.2 [Dihydro-MeBmt]1-[Val] 2 -[O-acetyl-(D)Ser] 8 -Cyclosporine [Formula I: X -dihydro-MeBmt-, Y -Val-, Z -Val-, Q -0-acetyl-(D)Ser-]: [al]0 -210' (c 0.85 in CHC13); and ~AL;q 4 yz2 100- 6283 4.3 [Dihydro-MeBmt~l-EThr] 2 -L0-acetyl-(D)Ser 8 -Cyclosporile [Formula 1: X =-dihydro-MeBmt-, Y -Thr-, Z Q= 0-acetyl-(0)Ser-]: [a]2 0 -241 (c 1.0 in CHC13)/ 162*(c =1.0in CH 3 OH): m.p. 148-150 *C.

Preparation of starting materials: EXAMPLE $4 The following compounds required as starting materials for the production of the compounds of examples 2.2 through 2.7 may be t~vtrprepared analogously to the known compound [(D)Ser] 8 -Cyclosporine, the preparation of which is described in Example 1 of European Patent No. 0 056 782, with substitution of the appropriate residues at positions 2 and/or 5 and/or 8 In the process sequence set forth in the flow chart to Example la of said patent: 41, 4. 5.1 [(D)Thr] 8 -Cyclosporine [Formula Mia: X' -MeBmt-, Y! -czAbu-0 Z' -Val-, W' [a]2 0 -248.7*(c in CHCl3); 5.2 (Nva] 2 _[(D)Ser] 8 -Cyclosporine [Formula 111a: X' -MeBmt-, Y' -Nva-, Z' W1 m.p. =150 153*C; -262* (c 0.71 in CHCl3)/-191 0 (c 0.73 in

CH

3

OH);

5.3 [Val] 2 -E(0)Ser) 8 -Cyclosporine [Formula I11a: X' -MeBmt-, Y' -Val-, Z' -Val-, W' [a] 20 =-257* (c

D

in CHCl 3 2550 (c =0.45 in CHCl 3 189 (c =0.42 in CH 3 OH): m.p. =136 140 *C.

-w 100-6283 5.4 [Nva]5-[(0)Ser] 8 -Cyclosporine [Formula liIa: X' -MeBmt-, Y' -aAbu-, Z' -Nva-, W' [a]20 -212° (c in CHC13); [Nva]2Nva 2 -va]5-[(D)Ser] 8 -Cyclosporine [Formula liIa: X' -MeBmt-, Y' -Nva-, Z' -Nva-, W' [a]60 -217' (c 1.0 in CHC1 3 and 5.6 [Thr] 2 -[(D)Ser] 8 -Cyclosporine [Formula liIa: X' -MeBmt-, Y' -Thr-, Z' -Val-, W' [a]0 258 (c 0.39 in CHC13)/- 178 (c 0.40 in CH3OH): m.p. 147 152 C.

EXAMPLE 6 The compound of example 5.2 may alternatively be produced microbiologically as follows: a) 10 litres of a nutrient medium containing 50 g maltose; 5 g (DL)-norvaline; 8 g (D)-serine; 0.75 g KH2P04; 0.5 g i, MgS04.7H20; 0.1 CaC12.6H20 and 8 g caseinpeptone per litre are t* inoculated with 1 litre of a suspension of conidia and mycelia of the fungus strain NRRL 8044 taken from a 3 day old pre-culture. The incoculated production-medium is filled in 100 ml portions into 100 Erlenmeyer flasks which are then incubated c, t for 14 days at 27° on an agitator rotating at 180 r.p.m. The Sr mycelium is separated from the culture medium and extracted in a Turrax apparatus by crushing and stirring with 3 x 3 litres of 90 methanol. The crushed mycelium is separated from the solvent by suction-filtration and the combined filtrates are concentrated by evaporation under vacuum at a temperature of 1.

1 0 i ^t I^ i. i i i 9. 100-6283 °C until the vapour consists mainly of water alone. The obtained mixture is extracted 4 x using 0.5 litre 1,2-dichloroethane at each extraction and the combined 1,2-dichloroethane solutions are concentrated by evaporation under vacuum at a temperature of The obtained residue is subjected to gel filtration on Sephadex LH-20 (1.4 kg; Pharmacia) with methanol, and collected in 280 ml fractions. Fractions 9 11, containing a cyclosporin mixture are pooled and then separated by silica gel column chromatography (1 kg of silica gel, granulate size 0.063 0.2 mm, "Merck") using water saturated ethyl acetate as eluent (fractions of 500 ml). In accordance with their polarity, [Nva 2 ]-Cyclosporine elutes first (fractions 7 followed by a mixture comprising [Nva2]-[(D)Ser8]-Cyclosporine and Cyclosporine. Separation of [Nva 2 ]-[(D)Ser 8 ]-Cyclosporine and Cyclosporine is achieved by silica gel chromatography (280 g, S"Merck", 0.63 0.2 mm) using chloroform/methanol (98 2) as eluent (fractions of 100 ml). Fractions 20 30, containing 'crude [Nva 2 ]-[(D)Ser8]-Cyclosporine, are further purified by medium-pressure chromatography on a reversed-phased silica gel column ("Merck" LiChropep RP 18, 260 g, granulate size 0.04 0.063 mm) with methanol/water (85 15) as eluent, with collection in 25 ml fractions. The combined fractions 45 c yield pure [Nva 2 ]-[(D)Ser 8 ]-Cyclosporine as an amorphous white t powder.

The pre-culture required for the above process may be obtained as follows: i "c'i i; 4e9- 100-6283 b) The spore and mycelium suspension used for inoculation is produced from a culture of the originally isolated strain NRRL 8044, cultivated for 21 days at 27'C on an agar medium containing 20 g of malt extract, 20 g of agar, 4 g of yeast extract per litre of demineralised water. The spores of this culture are taken up in a physiological NaCl solution to give a final concentration of 5 x 106 spores/ml. 10 ml of this suspension are used for inoculation of 1 litre of a nutrient solution having the same composition as the culture medium of Examlple 6a, with the exception of the (D)-serine and (DL)-norvaline components, and incubation is effected at 27*C for 3 days on a rotary shaker (200 This culture is used as inoculum for the producing culture, [Nva 2 ]-[(D)Ser 8 t Cyclosporine may be produced on fermenter scale as follows: c) Ca. 109 spores from an agar slant of the strain NRRL 8044 are transferred into a stainless-steel-fermenter containing litres of a pre-culture-medium comprising: Fructose 75 g Amber EHC 25 g

KH

2 0P 4 5 g KC1 2.5 g r Dist. Water to 1 litre S6 (pH previously sterilized for 20 minutes at 120'C. Favourable incubation conditions are a temperature of 27'C, airflow of 16 litres per minute at an overpressure of 0.5 bar and stir rotation of 200 r.p.m. The developing pre-culture is incubated for 6 days and 15 litres are then transferred to a stainless- I I1 i; i i~

I

-4- 100-6283 steel-fermenter prising: Maltose Amber EHC

KH

2

PO

4 KC1 (DL)-norvaline (D)-serine Dist. Water to (pH holding 300 litres of production medium com- 75 g 25 g 5 g 2,5 g 5 g 8 g 1 litre 0* 0 *0 09 00 4r t 4 I te.

r

I

Icc I OI II *I 9 It I Ir I I previously sterilized for 20 minutes at 120°C.

The culture is held at a temperature of 27°C, aerated with 120 litres air per minute at an overpressure of 0.5 bar and stirred at 70 r.p.m. Foam control is performed by addition of a silicone emulsion.

After incubation for 14 days the culture, which has a total volume of 275 litres is cooled to 10"C and the mycelia removed using a Westfalia separator. The filtrate is extracted by stirring 2 x with ethyl acetate, the extracts are washed with a little water, combined and dried under vacuum. The mycelium is combined with methanol, homogenised and filtered. This extraction is repeated 2 x using 90 methanol. The methanolic extracts are combined and, with addition of water, concentrated under vacuum. The remaining, aqueous concentrate is extracted 2 x with ethyl acetate, the extracts washed with a little water, combined and concentrated under vacuum. The extracted aqueous phase is re-extracted 2 x with ethyl

U~

~i 1% 100-6283 acetate/isopropanol (8 These extracts are combined and again evaporated under vacuum.

The mycelial and filtrate extracts are filtered using 50 x the amount of Sephadex LH-20 with methanol as eluant. The peakfractions are then purified chromatographically using 100 x the amount of silica gel 60 (particle size 0.04 0.063 mm) $o using water-saturated ethyl acetate as eluant. [Nva2]-Cyclosporine elutes first followed by Cyclosporine and [Nva 2 Ser 8 ]-Cyclosporine. These later fractions are subjected to S',i further chromatographic purification using 140 x the amount of '"er silica gel 60 (particle size 0.063 0.20 mm) and chloroform/ methanol (98 2) as eluant, to yield pure [Nva 2 3-[(D)Ser 8 Cyclosporine.

EXAMPLE 7 The compound of example 5.3 may also be produced microbiologically proceeding analogously to example 6a) but with the following modifications: a) In the nutrient medium replacement of (DL)-norvaline with I 10 g (L)-valine. Following separation of the mycelium from the e culture medium extraction as follows: The crushed mycelium is separated from the solvent by suction-filtration and the combined filtrates are concentrated (under addition of water) by evaporation under vacuum at a temperature of 40 C until the vapour consists mainly of water alone. The obtained mixture is extracted 3 x using litres ethyl acetate at each extraction and the combined

I

Npt 1 It s 100-6283 ethyl acetate solutions are concentrated by evaporation under vacuum at a temperature of 40 C.

The obtained residue is subjected to gel filtration on Sephadex LH-20 (1.4 kg; Pharmacia) with methanol. Those fractions which contain a cyclosporin mixture are pooled and then separated by silica gel column chromatography (3 kg of silicagel, granulate size 0.020 0.045 mm, "Grace") using water-satu- S* rated ethyl acetate as eluent. In accordance with their polarity, [Val2-Cyclosporine elutes first followed by a mixture comprising [Val 2 ]-(D)Ser8]-Cyclosporine as the majnr compo- 2 t nent. Further purification of [Val23-[(D)Ser 8 Cyclosporine is achieved by silica gel chromatography (80 g, "Grace", 0.020 0.0045 mm) using acetone/hexane (1 1) as eluent.

Those fractions containing crude [Val 2 ]-[(D)Ser 8 ]-Cyclosporine, ire further purified by medium-pressure chromatography on a reversed-phased silica gel column ("Merck" LiChroprep RP 18, 160 g, granulate size 0.04 0.063 mm) with methanol/water 20) as eluent, yielding pure [Val 2 ]-[(D)Ser 8 ]-Cyclosporine as an amorphous white powder.

b) The pre-culture required is obtained as in examiiple 6 b).

[Val 2 ]-[(0)Ser8]-Cyclosporine may be produced on fermenter scale proceeding analogously to example 6 c) but with the following modifications: c) In the production medium replacement of (DL)-norvaline with g (L)-valine. Following combination of the mycelium with methanol, homogenisation and filtration (repeated 2 x using methanol) further processing as follows: ~lr~~rr. 100-6283 The methanolic extracts are combined and, with addition of water, concentrated under vacuum. The remaining, aqueous concentrate is extracted 3 x with ethyl acetate, the extracts washed with a little water, combined and evaporated under vacuum.

The mycelial and filtrate extracts are filtered using 50 x the amount of Sephadex LH-20 with methanol as eluant. The 'S peak-fractions are then purified chromatographically using 40 x the amount of silica gel 60 (particle size 0.04 0.063 mm) using water-saturated ethyl acetate as eluant.

[Val 2 ]-Cyclosporine elutes first followed by Cyclosporine and t [Val 2 ]-[(D)Ser8]-Cyclosporine. These later fractions are subjected to further chromatographic purification using 100 x the amount of silica gel 60 and acetone/hexane (1 1) as eluant, and medium-pressure chromatography on reversed-phased Ssilica gel ("Merck" LiChroprep RP 18, granulate size 0.04 0.063 mm) with methanol/water (80 20) as eluant, to yield pure [Val 2 ]-[(D)Ser8]-Cyclosporine.

EXAMPLE 8 The compound of example 5.6 may also be produced microbiologically proceeding analogously to example 6 a) but with the following modifications: a) In the nutrient medium replacement of (DL)-norvaline with g (L)-threonine. Following incubation extraction as follows: A f77-77 100-6283 The mycelium is separated from the culture medium and extracted in a Turrax apparatus by crusing and stirring with 3 x 9 litres of 90 methanol. The crushed mycelium is separated from the solvent by suction-filtration and the combined filtrates are concentrated (under addition of water) by evaporation under vacuum at a temperature of 40 C until the vapour consists mainly of water alone. The obtained mixture is extracted 3 x using 5 litres ethyl acetate at each extraction and the combined ethyl acetate solutions are concentrated by evaporation under vacuum at a temperature of 40 C.

k t c The obtained residue is subjected to gel filtration on Sephadex LH-20 (2 kg; Pharmacia) with methanol. Those fractions containing a cyclosporin mixture are pooled and then separated by silica gel column chromatography (2 kg of silica gel, granulate size 0.02 0.045 mm, "Grace") using watersaturated ethyl acetate as eluent. In accordance with their polarity, Cyclosporine elutes first, followed by [(D)Ser 8 Cyclosporine, followed by [Thr 2 ]-Cyclosporine and finally [Thr 2 ]-[(D)Ser 8 ]-Cyclosporine in crude form. Further purification of [Thr 2 ]-[(D)Ser 8 ]-Cyclosporine is achieved by silica gel chromatography (50 g, "Grace", 0.02 0.45 mm) using acetone/hexane (2 1) as eluent yielding pure [Thr 2 [(D)Ser 8 ]-Cyclosporine as an amorphous white powder.

b) The pre-culture required is obtained as in example 6 b).

[Thr 2 ]-[(D)Ser 8 ]-Cyclosporine may be produced on fermenter scale proceeding analogously to example 6 c) but with the following modifications: i

C~~II

100-6283 too r 4 44 4 4, *4 44 4 (r c) In the production medium replacement of (DL)-norvaline with g (L)-threonine. Following incubation and removal of the mycelia using a Westfalia separator further processing as follows: The mycelium is combined with methanol, homogenised and filtered. This extraction is repeated 2 x using 90 methanol. The methanolic extracts are combined and, with addition of water, concentrated under vacuum. The remaining, aqueous concentrate is extracted 3 x with ethyl acetate, the extracts washed with a little water, combined and concentrated under vacuum.

The mycelial extract is filtered using 50 x the amount of Sephadex LH-20 with methanol as eluant. The peak-fractions are then purified chromatographically using 30 x the amount of silica gel 60 (particle size 0.04 0.063 mm) using watersaturated ethyl acetate as eluant. Cyclosporine elutes first followed by [(D)Ser 8 ]-Cyclosporine, followed by [Thr2]-Cyclosporine and finally [Thr 2 ]-[(D)Ser 8 ]-Cyclosporine. These later fractions are subjected to further chromatographic purification using 250 x the amount of silica gel 60 (particle size 0.02 0.045 mm) and acetone/hexane (2 1) as eluant, to yield pure [Thr2]-[(D)Ser 8 ]-Cyclosporine.

0 yz L -24-100- 6283 EXAMPLE 9 The following compounds, which may be employed as starting materials for production of the compounds of examples 4.1 through 4.3, may be prepared from the indicated cyclosporins of examples through 7, proceeding analogously to example 3.

9 9 9.1 [Dihydro-Me~mt] 1 -[Nvaj 2 -[(D)Ser] 8 -Cyclosporine [Formula lIIa: X= -dihydro-Me~mt-, Y' Z' -Val-, W' -(D)Ser-] prepared from the product of example 5.2 or 6: [a] 20 251 (c =1.23 in CHCl3)/- 179 =1.16 in CH3OH): 444m.p. =155 -157 o C 9.2 [Dihydro-MeBmt] 1 -[Val] 2 -[(D)Ser] 8 -Gyclosporine [Formula 111a: P= -dihydro-MeBmt-, Y' Z' W' -(D)Ser-] prepared from the product of example 5.3 or 7: -224 o (c =1.0 in CHCl 3 4.4 9.3 [Dihydro-MeBmt] 1 -[Thr] 2 -[(D)Ser] 8 -Cyclosporine [Formula Ila: P=-dihydro-MeBmt-, Y' -Thr-, P -Val-, W' prepared from the product of example 5.6 or 8: 20 262 0(c =0.73 in CHCl 3 173 (c =0.79 in CH 3 0H): Sm.p. =156 158e C.

*L ul -I 1 -100-6283 End product cyclosporins, e.g. of formula I, as hereinbefore defined and described exhibit pharmacological activity as may be shown in the following test methods: 1. Immunosuppressive activity: 1.1 Local haemolysis in vitro in gel Mishell and R.W.

Dutton, J. Exp. Medicine, 126, 423 442 (1976)].

Sr Cyclosporins of formula I in accordance with the invention inhibit haemolysis zones compared with untreated controls at concentrations of from 0.01 to 10.0 pg/ml.

1.2 Lymphocyte stimulation test according to Janossy and Greaves [Clin. Exp. Immunol., 9, 483 (1971) and 10, 525 (1972)]:- Cyclosporins of formula I in accordance with the invention inhibit concanavalin A stimulated DNA-synthesis (inhibition .of H 3 -thymidine incorporation), cell-proliferation and blastogenesis in mouse-spleen lymphocytes compared witi untreated controls at concentrations of from 0.001 to 10.0 Pg/ml.

1.3 Mixed lymphocyte reaction [Bach et al., J. Exp. Med. 136, 1430 (1972)]:- The reaction proliferation and differentiation) of lymphocytes [mouse (Balb/c) spleen cells] on co-incubation for 5 days, with allogenic spleen cells from irradiated mice (CBAj) is measured in the presence and absence of testsubstance. Reaction in the absence of test-substance serves as control and is taken as 100 Reaction in the presence of test-substance is expressed as the change compared with the 100 control reaction. Inhibition of reaction is observed 100-6283 using cyclosporins of formula I in accordance with the invention at a concentration of from 0.001 to 10.0 pg/ml- 1 1.4 Suppression of organ-rejection:- Kidneys from donor rats (F 344, are transplanted in recipient (Wistar-Furth,?) rats. Test-substance is administered p.o. to recipient rats for 14 days, after which treatment is discontinued. Test animals are subjected to bilateral nephrectomy seven days after transplant. Since the life of Sotest animals depends on acceptance and functioning of the ,J grafted organ, increase in survival time compared with control animals receiving placebo only serves as a parameter for test-substance efficiency. In the above test r2thod animals receiving cyclosporins of formula I in accordance with the present invention at dosages of from 2.5 to mg/kg p.o. exhibit a survival span from 60 to >250 days as compared with untreated controls all of which die as a o, result of organ rejection within ca. 9 to 10 days.

2. Anti-inflammatory activity Anti-inflammatory activity may be shown in the adjuvant :arthritis test in the rat. For this test adjuvant arthritis 6 is induced according to the method of Pearson and Wood, "Arthr. Rheum" 2, 440 (1959). Cyclosporins of formula I in accordance with the invention are active in this test against developing and established arthritis at doses of from 10 to mg/kg/day p.o.

A LI

_W

I I =31- 100-6283 3. Anti-parasitic activity Anti-malaria test according to 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 107 parasitic cells of the species Plasmodium berghei (strain NK 65) administered i.p.

P Test substance is administered s.c. on day 3, at varying dosages using 5 to 10 mice/dose. The survival time is recorded, and the minimum effective dosage (MED) calculated t by comparison of survival time with that for untreated controls. For controls, survival time ca. 7 days. The MED is the dosage at which survival time is doubled. Cyclosporins of formula I in accordance with the invention are effective in this test at dosages of form 25 to 100 mg/kg/day, s.c.

In view of their immunosuppressive activity, end product cyclosporins e.g. of formula I are indicated for use in the prophylaxis and treatment of diseases and conditions requiring a reduct1 tion of the immune response for example to suppress the proliferation of lymphocytes and immunocytes, e.g. in treatment of autoimmune diseases or in preventing the rejection of transplants, e.g. skin, lung, heart, heart-lung, bone-marrow, kidney, spleen and corneal transplants.

Specific auto-immune diseases for which the cyclosporins of formula I are indicated include all of those for which treatment with Cyclosporine has been proposed or used, for example, aplastic anaemia, pure red cell anaemia, idiopathic thrombocytopaenia, systemic lupus erythematodes, polychondritis, sclerodoma, Wegener granulamatosis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue,

I

100-6283 Crohn's disease, Graves opthalmopathy, sarcoidosis, multiple sclerosis, primary billiary cirrhosis, primary juvenile diabetes, uveitis posterior, interstitial lung fibrosis and psoriatic arthritis.

In view of their anti-inflammatory activity, end product cyclosporins, e.g. of formula I, are also indicated for use in the 2 treatment of inflammatory conditions, in particular inflammatory conditions with an aetiology comprising or including an autoimmue component, e.g. for the treatment of arthritis and rheumatic diseases such as polyarthritis chronica progrediens.

In view of their anti-parasitic activity, end product cyclosporins, e.g. of formula I, are also indicated for use as anti-parasitic agents, i.e. for the treatment of parasitic infection of varying type, in particular for the treatment of protozoan as well as trematodal and nematodal parasitic infection. Specific types of parasitic infection include all of those for which treatment with Cyclosporine has been previously proposed in the literature, including schistomosomiasis, filariasis, leishmania, coccidioidomycosis and in particular malaria.

For the above-mentioned uses an indicated daily dosage is in the range of from about 75 up to about 5,000, preferably up to about 2,000 and most preferably up to about 1,500 mg conveniently administered once or in divided doses 2 to 3x a day. Unit dosage forms, e.g. for oral administration, suitably comprise from about up to about 2,500, preferably up to about 1,000 and most preferably up to about 800 mg cyclosporin of formula I admixed with a pharmaceutically acceptable diluent or carrier therefor.

-28- End product cyclosporins e.g. of formula I may be administered by any conventional route, in particular in accordance with means currently practiced in relation to administration of Cyclosporine, in particular via intravenous infusion e.g. in the case of organ transplant, pre- and immediately post-transplant, as well as during episodes of gastrointestinal disturbance which might otherwise impair absorption, or orally, e.g. in the form of an oral solution.

In accordance with the foregoing the present invention S. also provides a method of inducing immunosuppression, of Streating inflammation or of treating parasitic 15 infection, in a subject in need of such treatment, which 15 method comprises administeringto said subject an Sit effective amount of a cyclosporin of formula I as f hereinbefore defined.

*t S* Ii l i S

Claims (5)

1. A cyclosporin wherein the amnino acid residue at the 8-position is a -1-acyloxy-a-amino acid residue of formula 11 R140040 -NH-CH-CO- *0,40whierein Ri is hydrogen, C1j 4 alkyl or phenyl and 4 f f R 2 is hydrogen or methyl.

2.A cyclosporin according to claim 1 of formula I 00 I Y-Sar-MeLeu-Z-MeLeu-A1 a-Q-MeLeu-MeLeu-MeVal- 1 2 3 4 5 6 7 8 9 10 11j (1) tt t where in X is -MeBmt- or -dihydro-MeBmt-, Y is -cxAbu-, -Ala-, -Thr-, -Val- or -Nva-, Z is -Val- or -Nva-, and Q is a residue of formula Il as defined in claim I. ,17 7-- i 'l 4 V 30

3. A cYclosporin according to claim 1 which is tO- acetyl- (D )-Ser] 8 -Cyclosporine,

4. A cyclosporin according to claim 1 selected from the group consisting of: (Nvaj (-acetyl- (D )SerJ 8 -Cyclosporine; (Dihydro-MeBmtJ 1 -(0-acetyl-(D)Ser] 8 -CylosporfleI (0-benzoyl-(D)Ser) 8 -Cyclosporine; and [Dihydro-MeBmt)1-(Nva) 2 -(O-acety..(D)SerJ 8 Cyclosporine; [Nva) 5 -(0.acetyl-(D)Ser] 8 -Cyclosporine; S (0-acetyl-(D)Thr) 8 -Cyclosporine,* tVal) (-acetyl- D)Ser) 8 -Cyclosporine; [Dihydro..MeBmt)1..(Val]2..(Q.acetyl..(D)Ser) 8 Cyclosporine; moo* [NvaJ2..(Nva)5..O.acetyl.(D)Ser 8 cycloporine; [Thr) 2 -(0-acetyl-(D)Ser] 8 -Cyclosporins; and [Dihydro..Mesmt)1..(ThrJ2..(0.acetyl..(D)SerJS- Cyclosporine. @444

5. A method of inducing immunosuppression, of treating inflammation or of treating parasitic infection in a 25 subject in need of such treatment, which method comprises administering to said subject an effective amount of a cyclosporin of formula as defined in claim 2. o o .090 2 February, 1990 SANDOZ LIMITED By 1"ts Patent Attorneys DAVIES COILLISON 01o, db4 v7ares,30