AU708004B2 - Macrolides - Google Patents

Description

WO 97/02285 PCT/EP96/02952 The present invention relates to a novel class of macrolides having valuable pharmaceutical and related activity. For convenience compounds of this novel macrolide class are referred to herein collectively as "Sanglifehrins".

The first of the Sanglifehrins were isolated from actinomycete fermentation broths. These are the Sanglifehrins A through D of formulae 54 47 48 49 OH OH Me Me Me Me 21 51 32 28 22 18 1 4" 34' 33 3 o o 9 M; o 0 33 29 27 23 5 5oMe Me 3 0 OH 0 0 *12 0 HN42 38 4 5 o W 6N- 7 ^S-NM5 41 2 1 5 O3 9 6 0 Me 4 59 63 57 Me 60 44 Sanglifehrin A 61 62

OH

OH OH Me Me Me Me Me 0 OH 0 H 0 1 M 0 Me Me 0 NN N Me O 0 Me Me Sanglifehrin

B

WO 97/02285 PCT/EP96/02952 Meo .Me y me H Me 0 MeOH 0 0 V N 0 HNe N. N y Me O0 Me Me Sanglifehrin

C

OH

Me'0 Me OH 0 Me Me Me H T i 1 e a Me 0 OH 0 N 0 H Me O

N

N Me O O Me Me Sanglifehrin

D

OH

As can be seen the macrocyclic ring of Sanglifehrins A to D is of entirely novel structure characterised in that i) positions 1 to 6 comprise a 3-carboxypiperidazinyl carboxylic acid residue, ii) positions 7 to 9 an aromatic a-amino acid residue, and iii) positions 10 to 12 an aliphatic a-amino acid residue. The remainder of the macrocyclic ring is comprised by an hydroxy carboxylic acid residue, providing, in the case of Sanglifehrins A to D a further 11 carbon atoms in the primary macrocyclic ring.

In accordance with conventional practice in macrolide chemistry the atoms of the Sanglifehrin primary macrocyclic ring are numbered as indicated above for Sanglifehrin A, starting with the carbon atom of the carbonyl group of the macrocylic lactone linkage as position 1.

WO 97/02285 PCT/EP96I02952 Sanglifehrins A to D are also characterised by the presence of a novel bicyclic spiro system attached at the 23 position of the macrocyclic ring via a hydocarbyl linker group.

Sanglifehrins A to D may be subjected to extensive chemical manipulation to obtain yet further macrolides of the Sanglifehrin class. Such manipulations include cleavage of the macocyclic ring, in particular at the lactone oxy-group, cleavage of the linker group between the macrolide and spiro ring systems, and manipulation, e.g.

protection, derivatisation or other chemical modification of substituent groupings; for example, as hereinafter described. Further means of modification will be apparent to those skilled in the art.

In accordance with the invention it has been found that Sanglifehrins, in particular those in which the spiro ring system is present, as in the case of Sanglifehrins A to D, have a characteristic and entirely novel profile in terms of their biological activity. In particular they have been found to exhibit the following combination of activities: cyclophilin binding activity; immunosuppressive activity; inhibition of both B-cell and T-cell proliferation they do not, however, have FK binding protein binding activity or calcineurin inhibiting activity.

The Sanglifehrins can accordingly be seen as providing an exciting and novel class of immunosuppressant and antiinflammatory compounds. In particular the Sanglifehrins have an activity profile that differs from that of previously known immunosuppressant and antiinflammatory compounds such as cyclosporins and macrolides, e.g. of the rapamycin and FK 506 class, indicating that the Sanglifehrins have a different mode of action than such previous compounds. Thus the Sanglifehrins provide a novel category of drug substance both in terms of structure and activity which may be anticipated to materially extend the bounds of immunosuppressive and/or antiinflammatory therapy; for example, to avoid or reduce undesirable side effects of WO 97/02285 PCT/EP96/02952 previous immunosuppressive and antiinflammatory therapies and/or to improve or extend such therapy to new disease areas or new patient categories.

Sanglifehrins, e.g. in which the macrolide ring is in ring-opened form, in which the 26 and 27 positions in the hydrocarbyl linker between the macrolide and spiro ring systems are both hydroxy substituted, or in which the spiro residue attached to the macrocyclic ring has been cleaved or truncated, generally lack some or all of the combination of Sanglifehrin characteristic activities. For example, Sanglifehrins in which the spiro residue is cleaved typically possess cyclophilin binding activity but do not possess significant immunosuppressive activity. As will be apparent to those skilled in the art, however, such compounds provide valuable components, intermediates or key building blocks for the preparation of further novel Sanglifehrins, and hence further extend the therapeutic potential of the Sanglifehrin class.

In that its presence appears material to the biological activity, e.g. of the Sanglifehrins A to D, the bicyclic spiro system too may be viewed as providing a structural component with key biological significance, useful as a structural component for further derivatisation or modification both in relation to the production of further Sanglifehrins or for application in the derivatisation or modification of other drug substances; for example, to modify the activity of other immunosuppressive drug substances of the macrolide class.

As indicated, the Sanglifehrins represent a novel class of macrolide compounds of entirely novel and wholly characteristic structure.

Accordingly in a first aspect the invention provides: a macrolide in which i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinyl carboxylic acid residue; and/or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic a-amino acid residue; and/or WO 97/02285 PCT/EP96/02952 iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic a-amino acid residue, in free or protected form, or a salt thereof.

Suitably the macrolides of the invention comprise two, especially all three of the characteristic structural features ii) and iii).

The piperidazinyl carboxylic acid residue is suitably a 1,2 piperidazin-3carboxy-1-yl residue of which the carboxy moiety occupies the 1-position, and the 1nitrogen atom the 6-position of the macrocyclic ring, e.g. a residue of formula I 0

H

-C

3 4 wherein the assigned numbers represent the position of the atoms of the residue in the macrocyclic ring. This residue may be ring substituted or unsubstituted. Suitably it is unsubstituted.

The a-amino moiety of the aromatic a-amino cid residue suitably occupies the 9-position of the macrocyclic ring. Suitably the aromatic a-amino acid is a phenylalanine, especially 3-OH-phenylalanine, residue in free or protected form.

The a-amino moiety of the aliphatic a-amino cid residue suitably occupies the 12-position of the macrocyclic ring. Suitably the aliphatic a-amino cid residue is a valine residue in free or protected form.

The remainder of the macrocyclic ring suitably comprises a hydroxy carboxylic acid residue, the oxy-moiety of which completes the macrocyclic lactone linkage and the carbonyl moiety of which forms an amide linkage with the a-amino group at position 12 of of the macrocyclic ring. The said hydroxy carboxylic acid residue suitably has a chain length of from 6 to 20, more suitably 11 carbon atoms. It WO 97/02285 PCT/EP96/02952 may be substituted or unsubstituted and/or contain one or more unsaturated linkages in particular cumulative double bonds along its length. More suitably the remainder of the macrocyclic ring comprises a 1 l-oxy-endecanoyl-ll-yl, especially 1 l-oxy-6,8endecadienoyl-11-yl, residue optionally substituted, e.g. in the 2, 3, 4 and/or position. More suitably the said hydoxy carboxylic acid residue is a residue of formula

II.

R

RI R2 0 (CH 2 2 CH-CH2-CH=

CH-CH=CH-CH-C-CH-CH-CO-

o

I

Ii CH 3 wherein RI and R 2 are both H or represent an extra bond; R3 is H;

R

4 is -CO-CH 3 or -CH(OH)-CH 3 or

R

3 and R 4 together represent a structure of formula m

CH

-C-

I III

OCH

3 in free or protected form, or salt thereof.

Preferred macrolides in accordance with the invention are accordingly those comprising a macrocylic ring of formula IV IV -A7

Z

wherein X, Y and Z are residues ii) and iii) as defined above and A is a hydroxy carboxylic acid residue as defined above in free or protected form, or salt thereof; in particular a macrocyclic ring of formula V WO 97/02285 PCT1IEP96/02952 CO-

(A)

12 HO CH2 2 V CH 3

CH

3 in free or protected form, or salt thereof Generally in the Sanglifehrins, as in Sanglifehrins A to D, the macrocyclic ring is substituted at the carbon atom adjacent the oxy moiety of the lactone bridge.

Typically this substituent comprises a 2 -oxy- 2 '-aza-3'-oxo-spirobicyclohexan-3-yl residue, e.g. of the formula VI Me sbv 6 a 02

VI

Me 2N-R S I 3' 0 4 Et wherein is -(Me)C=CH- or -(Me)CH-CH(OH)- and

R

5 is H or Me (wherein Me and Et represent methyl and ethyl respectively) in free or protected form, or salt thereof linked to the macrolide ring via a linker comprising a linear sequence of from 6 to 11, typically 9, carbon atoms between the spiro residue and the macrolide ring.

The linker group may be substituted or unsubstituted and/or contain one or more unsaturated linkages in particular cumulative double bonds along its length.

Suitably the linker group may be methyl substituted, e.g. by two methyl groups.

Suitably the linker group may be further substituted by hydroxy, e.g. by 3 hydroxy substituents, and/or may be ethylenically unsaturated, e.g. contain two carbon-carbon double bonds. More suitably the linker group comprises a 1-methyl-7-methylnonanoyl-9-yl, especially a 1-methyl-7-methyl-l-nonenoyl-9-yl or a 1-methyl-7methyl-1,3-nonadienoyl-9-yl, residue optionally substituted, e.g. in the 3, 4, and/or 8 position. Preferably the linker group is a group of formula VII

VII

R

6 7

CH

3 c-CH--CH (OH) -CH (CH 3

(CH

2 CH-CH-CH=C-d wherein c represents linkage to the spiro residue; d represents linkage to the macrocyclic ring and

R

6 and R 7 are each OH or together represent an additional bond, in free or protected form.

The linker group will generally be attached to the macrocyclic ring at the carbon atom immediately adjacent to the lactone oxy group, i.e. when the the macrocyclic ring comprises an 11-oxy-endecanoyl-l1-yl residue, at the 11 position of thereof.

Accordingly the invention provides compounds of formula VIII

VIII

wherein a macrolide of formula VIII

VIII

wherein S represents a 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl residue; L represents a linker comprising a linear sequence of from 6 to 11, carbon atoms, and M represents a macrolide ring of formula V P' (PEISRPB 65193 96. 61 94 6.

CC)o

(A)

2 18 9 10 1 4 N-CO-CH-NHi-CO-CH-NH 7 11 VHO~ CH 3 2CH 3 CN

H

wherein A is a residue comprising a hydroxy carboxylic acid residue which has a chain length of from 6 to 20 carbon atoms, preferably a hydroxy carboxylic acid residue of formula 11.

R4 i3 R1 R~

(CH

2 CH-Cf--WC-CH=CH-CH-C-CH-CI--COwherein R, and R 2 are both H or represent an extra bond; R3is H;

R

4 is -CO-CH 3 or -CH(OH)-CH 3 or

R

3 and R4 together represent a structure of formula MU Y H 3 OCH 3 25 Particular compounds of the invention are those of formula iX WO 97/02285 PCT/EP96/02952 47 48 49 Me Me 26 Me a3 0 OH 2O 21 19 eg O-R Me--38 42

N-R

5 15 30 O 2 2 43 Me 3X o 5 Me 9 HO 0 H NH _1 sMe is -CH(OH)-CH(OH)- or -CH=CH-; is as defined above for and

R

3

R

4 and R 5 are as defined above, in free or protected form or salt thereof The compounds of formulae I to IX contain asymmetric carbon atoms and thus may exist in a number of epimeric forms. All of the possible epimers as well as diastereoisomeric mixtures thereof are encompassed in the invention. However, compounds of formulae VIII and IX in which the macrolide ring is in ring-closed form and which are of appropriate stereochemistry typically possess activities which are characterisic of Sanglifehrins, as hereinbefore referred to. Epimers which possess sanglifehrin characteristic activities are preferred. In general, e.g. for pharmaceutical use in accordance with the invention, epimers which possess sanglifehrin characteristic activities in pure or substantially pure form free or substantially free of epimers which lack sanglifehrin characteristic activities), e.g. comprising at least 90%, e.g. at least 95% of active epimer comprising less than 10%, e.g. less than 5% of inactive epimer) will be preferred.

Preferably the 3 -carboxypiperidazinyl carboxylic acid residue i) at positions 1 to 6 of the macrocyclic ring has the following conformation: WO 97/02285 PCT/EP96/02952

H

II 2 -Vc 4 Preferably the aromatic amino acid ii) at positions 7 to 9 of the macrocyclic ring has the L configuration, e.g. is of configuration 0 8

NH

Preferably the aliphatic amino acid iii) at positions 10 to 12 of the macrocyclic ring has the L configuration, e.g. is of configuration 0 11 NH 12 When the remainder of the macrocyclic ring comprises a residue of formula HI, it preferably has the configuration

R

0 OH 0 0

R

3 or

R

CH

3

CH

2 2

R

3 When R 3 and R 4 together represent WO 97/02285 PCT/EP96/02952

CH

-C-

OCH

3 it preferably has the configuration CH3.

3 OCH 3

C

Preferably the 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl residue has the configuration Me a Me N-R 0 Et wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration

HO

CH

3 When the linker is of formula VII, it is preferably of configuration

CH

3

R

7

CH

3 OH R 6 When R 6 and R 7 are each OH, the configuration at positions 26 and 27 is preferably either 26(S), 27(S) or 26(R), 27(R). When R 6 and R 7 together represent an additional bond, the configuration at positions 26 and 27 is preferably WO 97/02285 PCT/EP96/02952

H

27

H

Compounds of the invention of formula IX preferably have the following conformation 22 20 18 17 Me" 44Me 5-Me

S

HO,

61 wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration:

HO

CH

3 when is -CH(OH)-CH(OH)-, it preferably has the configuration or the configuration; when is -(Me)CH-CH(OH)-, it preferably has the configuration:

>OH

CH

3 when is it preferably has the configuration:

CH

3 and when R 3 and R 4 are fused together they are preferably of configuration Iwdm~ nql~n- of0

WY

PCT/EP96/02952 CH 3 OCH 3 1-C1 c Preferably Sanglifehrins A to C have the following configurations

HOOH

0 N 0 Sanglifehrin

B

Sanglifebrin C WO 97/02285 PCT/EP96/02952 t OH 0 Me Me Me Mee Sanglifehrin D

OH

The compounds of the invention may be in free or protected form, e.g. in protected forms as described in "Protective Groups in Organic Synthesis" by T. W.

Greene and P. G. M. Wuts, 2nd Edition, 1991, John Wiley Sons Inc., New York. In particular OH groups may be in protected form, e.g. in the form of silyl ethers (for instance as described in pages 68-86 of Greene and Wuts ibid.), esters (see e.g. pages 87-103 of Greene and Wuts ibid) and carbonates (see e.g. pages 104-111 of Greene and Wuts ibid). Such protected forms also include internally protected forms; for example, in the case of macrolides of formula IX, wherein is -CH(CH 3 the protected form wherein the 14 to 17 positions of the macrocyclic ring comprise a residue of formula X: WO 97/02285 PCT/EP96/02952

CH

3 0

(CH

2 2 0 16 I x

-CH-CH-C-CH-

17 15 14

CH

3 e.g. of configuration

CH

3 0 0 17

CH

3 14 Also for example, 1,3 diols present in Sanglifehrins may be protected as appropriate ring structures, e.g. as described on pages 118-142 of Greene and Wuts ibid.

Compounds of the invention also exist in salt form. Examples of suitable pharmaceutically acceptable salts for use in accordance with the invention include acid and base addition salts as appropriate having regard to the particular substituents present in the compound.

As previously indicated the macrocyclic ring of the compounds of the invention can be cleaved, in particular at the lactone oxy group, to provide compounds wherein the macrocyclic ring is in ring-open form. Generally cleavage of the lactone oxy group proceeds by hydrolysis (solvolysis), e.g. to provide compounds of formula

XI:

XI

e.g. of formula XII WO 97/02285 PCT/EP96/02952 ROq CO XII

NH

SN CO-CH-NH-CO-CH-NH-A...OH

CE

3 CH 3 e.g. a compound of formula IX' Me' 44 Me S64 63 wherein X, Y, Z, A, R 3

R

4 and R 5 are as defined above and R 6 is H or C 1 4 alkyl, e.g. methyl.

Such ring-opened forms provide intermediate means for modification of the basic Sanglifehrin macrocyclic ring system and are also part of the present invention.

Accordingly in a futher aspect the present invention provides: a macrolide as hereinbefore defined in ring-opened form, said ring-opened macrocycle being in free or protected form, or salt thereof; a compound

R

6 0O X Y Z A OH as defined above, in free or protected form, or salt thereof; WO 97/0l2295 PCT/EP96/02952 acompound R- X Y Z A CH(HL)-Swherin-A'-CH(OH is a hydroxy carboxylic acid residue, e.g. a residue of formnula HI, as defined above, and the other symbols are as defined above, in free or protected form, or a salt thereof; a compound of formula XII'

R

6 0 CO

XII,

NOH

K)CH 3 CH 3 in free or protected form, or salt thereof;, a compound of formula IX" 47 48 Me,

(CH

2

R

4 44Me in free or protected form or a salt thereof.

The invention also includes compounds in which the 2 -oxy-2'-aza-3'-oxo- 3 'yl-spirobicyclo hexane ring system is in ring-opened form, e.g. a compound of formula XII WO 97/02285 PCT/IEP96/02952 Me b L-M a O xlI Me OH o

NH

2 wherein a, b, L and M are as defined above, in free or protected form, or a salt thereof.

The ring-opened compounds of the invention are preferably of conformation as the preferred conformations identified above for ring-closed compounds. The ringopened spiro bicyclo ring system of compounds of formula XII is preferably of conformation: Me b

L-M

a "X Me OH 0

NH

2 wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration:

HO

CH

3 Macrolides in accordance with the invention having a spiro bicyclo residue attached to the macrocyclic ring may also be subjected to cleavage of the intervening 18 WO 97/02285 PCT/EP96/02952 linker group, e.g. in relation to formula IX, in particular at the linkage between residues 26 and 27 to yield separate novel spiro bicyclo compounds and further macrolides. As also previously indicated these compounds too are useful as intermediates the spiro bicyclic moiety of the Sanglifehrins in particular having an integral functional role in the biological activity of the Sanglifehrins as a class.

Accordingly the present invention provides: A 2 -oxy- 2 '-aza-3'-oxo-3'yl-spirobicyclohexane, in free or protected form, or a salt thereof, in particular a compound of formula VI' Me b R7 a 0

VI'

Me

N-R

0 Et wherein R 7 is H, an optionally protected OH group, a reactive functional group, or a -CH2-CH(OH)-CH(CH 3

)-CH

2

-CH

2 -CHO group or the delta lactol equivalent thereof, in free or protected form or salt thereof.

Preferably the compound of formula VI' has the following configuration Me b a 0 Me"' N-R Et wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration: WO 97/02285 PCT/EP96/02952

HO

CH

3 The invention also includes a ring-open 2 -oxy-2'-aza-3'-oxo-3'ylspirobicyclohexane, in free or protected form, or a salt thereof, in particular a compound of formula XII' Me bY R7 a 0

XII'

Me OH o

NH

2 wherein a, b and R 7 are as previously defined, in free or protected form, or a salt thereof. The ring-opened spiro bicyclo ring system of compounds of formula XII' is preferably of conformation: Me b

R

7 a O Me'" OH 0 NH2 wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration:

HO

CH

3 WO 97/02285 PCT/EP96/02952 The invention also provides a niacrolide of formula XII

CH

3 XIII HIO" wherein M is a macolide ring as defined above, in particular a rnacrolide of formnual XIIV 00m xiv NH 0 0

CH

2 i-R 4 N 0 H~ Me Me preferably of conformation Hh ~NHO 0 0 CH 2)i--R4 1 %Me

HO

Me wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration:

CH

3 and when is it preferably has the configuration: WO 97/02285 PCT/EP96/02952

CH

3 and when R 3 and R 4 are fused together they are preferably of configuration

CH

3

OCH

3 in free or protected or ring-opened form, or a salt thereof.

In a further aspect the invention includes the macrolides and compounds of the invention, in particular those which are natural products in substantially purified form, e.g. at least 90%, preferably at least 95%, especially at least 98% pure form.

In addition to the foregoing the present invention also provides a process for the production of any compound of the invention as hereinbefore defined, which process comprises: for the production of any one of Sanglifehrins A, B, C or D, cultivating a Sanglifehrin A, B, C or D producing actinomycete strain in a culture medium and isolating the desired Sanglifehrin A, B, C or D from the obtained culture broth; ii). for the production of Sanglifehrins C and D subjecting Sanglifehrins A and B to cyclisation at positions 15 and 16; iii). for the production of Sanglifehrins A and B subjecting Sanglifehrins

C

and D to ring opening of the lactol ring at positions 15 and 16; iv). for the production of a macrolide of formula IX or IX', wherein is

C(CH

3 dehydrating a compound of formula IX or IX' wherein is

CH(CH

3 or a protected form thereof; WO 97/02285 PCT/EP96/02952 for the production of a macrolide of formula IX or IX' wherein R 4 is

CH(OH)-CH

3 hydrogenating a compound of formula IX or IX' wherein R 4 is

C(O)-CH

3 vi). for the production of a macrolide of formula IX or IX' wherein the 14 to 16 positions of the macrolide ring comprise a residue of formula X 1 O 16 I

-CH-CH-CH-CH-

17 15 14

CH

3 causing a compound of formula IX or IX' to undergo internal protection at positions 15 and 17; vii). for the production of a macrolide of formula IX or IX', causing a compound of formula IX or IX' wherein the 14 to 16 positions of the macrolide ring comprise a residue of formula X

CH

3 O

CH

2 2 0 I 16 1

-CH-CH-CH-CH--

17 15 14

CH

3 to undergo reversal of internal protection at positions 15 and 17; viii). for the production of a macrolide of formula IX or IX', in which Rs is methyl, subjecting a macrolide of formula IX or IX', in which R 5 is H to methylation; WO 97/02285 PCT/EP96/02952 ix). for the production of a macrolide of formula IX or IX', in which R 4 is in 0-protected form, subjecting a macrolide of formula IX or IX', in which R 4 is in 0-unprotected form to O-protection; for the production of a macrolide of formula IX or IX', in which R 4 is in 0unprotected form, subjecting a macrolide of formula IX or IX', in which R 4 is in 0-protected form to O-deprotection; xi). for the production of a macrolide of formula IX or IX', which comprises an 0-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX', in which comprises an O-unprotected hydroxyphenylalanine residue at positions 7 to of the macrocyclic ring to O-protection; xii). for the production of a macrolide of formula IX or IX', which comprises an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX' which comprises an 0-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring to O-deprotection; xiii). for the production of a macrolide of formula IX or IX', in which is CH(OH)-CH(OH)-, subjecting a macrolide of formula IX or IX' in which -e-fis -CH=CH- to oxidative hydrolysis; xiv). for the production of a compound of formula V' or a compound of formula XII, subjecting a compound of formula IX or IX' to cleavage of the linker group between the spiro bicyclo group and the macrocyclic ring.

xv). for the production of a compound of formula R 6 0 X Y Z A OH or of formula R 6 0 X Y Z A' CH(OH) L S, subjecting a macrocycle of formula IV or the macrocyclic ring of a compound of formula VIII to ringopening at the lactone bridge thereof; WO 97/02285 PCT/EP96/02952 xvi). for the production of a macrolide of formula IX or XII in ring-closed form, subjecting a compound of formula R 6 0O X Y Z A OH or of formula R 6 0 X Y Z A' CH(OH) L S to closure of the macrocyclic ring.

xvii). for the production of a compound of formula XII or XII', subjecting a compound of formula IX or VI' to ring-opening within the spiro bicyclic ring system, and xix). for the production of a compound of formula IX or VI', subjecting a compound of formula XII or XII' to ring-closure within the spiro bicyclic ring system.

Processes of the invention may be performed, e.g. as described in the examples. As will be appreciated the processes defined above may be applied in any appropriate sequence or combination to obtain other macrolides in free, protected, rigopen and ring-closed form as hereinbefore described.

The macrolides of the invention, e.g. Sanglifehrins A to D, are, or are derived from, natural compounds typically obtained from members of the family Streptomycetaceae.

Microorganisms capable of producing macrolides as hereinbefore defined not previously been identified.

Accordingly in a yet further aspect the present invention provides: a macrolide producing actinomycete strain wherein the macrolide is a macrolide in which i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinyl carboxylic acid residue; and/or WO 97/02285 PCT/EP96/02952 ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic a-amino acid residue; and/or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic a-amino acid residue, in particular a Sanglifehrin A, B, C or D producing actinomycete strain.

Suitably the actinomycete strain is of the family Streptomycetaceae, more suitably of the genus Streptomyces, in particular the strain Streptomxces sp. A92- 308110 as hereinafter described, or is derived therefrom, e.g. including mutants, variants, fusants, recombinants or modified forms thereof.

Suitably the strains of the invention are in the form of biologically pure isolates.

For example Streptomyces s. A92-308110 may be mutated or modified into different forms by conventional techniques, e.g. by UV radiation or by treatment with a chemical mutagen such as N-methyl-N'-nitro-nitrosoguanidine. Recombinant clones may be obtained by protoplast fusion. All such mutants or recombinants or modified forms, capable of producing sanglifehrins, including mutants and recombinants capable of producing increased yields of, sanglifehrins are included within the scope of the present invention.

In a particular embodiment of the invention Sanglifehrins A, B, C, and D, amongst others, are isolated from the novel Streptomvces s. A92-308110. Samples of Streptomyces sp. A92-308110 were deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany on the 3 May 1995 under the terms of the Budapest Treaty and have been assigned deposition number DSM 9954. Samples of Streptomyces sp. A92- 308110 may also be obtained from Sandoz Ltd. CH-4002 Basel, Switzerland.

WO 97/02285 PCT/EP96/02952 Notice is hereby given that access to samples of DSM 9985 is limited in accordance with the provisions of Rule 28 and EPC.

The isolation of Sanglifehrins A, B, C, and D from cultures of Streptomyces A2.

A92-308110 is described in Example 2.

Streptomyces strain A92-308110 belongs to the genus Streptomyces according to the description in Bergey's Manual (Volume 4, 1989, Williams and Wilkins, Baltimore) and The Prokaryotes (1992 Springer Verlag, New York). The cell walls contain LL-diaminopimelic acid. The fatty acids are iso- and anteiso-branched, straight and unsaturated. The sugar spectrum is non distinctive. The vegetative mycelium does not break down into fragments. The aerial mycelium forms long chains of spores.

According to the reference books cited above, the strain designated A92-3081 is a new Streptomyces. A92-3081 10 grows on various organic and inorganic media and in most cases forms aerial mycelium. The primary substrate mycelium grows as hyphae and is generally beige to greyish-brown. The color of the aerial mycelium belongs to the grey series, number 4, and this mycelium forms long chains of spores which belong to the type spira b.

The ability of Streptomyces s. A92-3081 10 to grow on usual biological media, its carbon utilization, and its physiological characteristics are presented in the following tables.

Table 1. Growth on various biological media Culture medium Culture charcteristics yeast extract/ growth: good malt agar substrate mycelium: brownish aerial mycelium: grayish-brown soluble pigment: none WO 97/02285 PCTIEP96/02952 oatmeal agar glucose-asparagine Inorganic salts/ starch agar Sucrose/ nitrate agar growth: good substrate mycelium: dark brown aerial mycelium: greyish-brown soluble pigment: brownish growth: moderate substrate mycelium: brownish aerial mycelium: greyish-brown soluble pigment: none growth: moderate substrate mycelium: grey aerial mycelium: greyish-brown soluble pigment:none growth: very poor substrate mycelium: whitish aerial mycelium: poor, greyish brown soluble pigment: none growth: moderate substrate mycelium: brownish aerial mycelium: greish-brown soluble pigment: none growth: moderate substrate mycelium: beige aerial mycelium: none soluble pigment: brown Table 2: carbon utilization Glycerol/ asparagine agar Nutrient agar WO 97/02285 PCT/EP96/02952 moderate or good: poor: negative: glucose, fructose, arabinose, xylose, mannose, rhammnose, sucrose, raffinose, cellulose, salicin m-inositol Table 3: physioloeical characteristics nitrate reduction: starch hydrolysis: tyrosine degradation: milk peptonisation: melanin formation: positive moderate on inorganic salts-starch agar, negative on oatmeal agar.

negative positive positive growth temperatures: 18-37°C.

Very poor growth at 13 0

C.

No growth at pH-range: rich growth at pH 5 and 7, good growth at pH 9 up to but reduced growth already at a 2% concentration.

NaCI resistance: Macrolides of the invention, including Sanglifehrins A, B, C and D may be produced by cultivating Streptomyces s. A92-308110 or a mutant, recombinant or modified form thereof on an appropriate culture medium. Example 1 describes, by way of illustration only of the invention, a procedure for the cultivation of Streptomyces sp.

A92-308110.

Thus in further aspects the invention includes: WO 97/02285 PCT/EP96/02952 a) a biologically pure isolate of strain Streptomvces s A92-308110

(DSM

9954) or a mutant, recombinant or modified form thereof which is capable of producing a macrolide of the invention, and b) a process for the production of a macrolide of the invention, which comprises cultivating strain Streptomyces sp. A92-3081 10 (DSM 9954) or a mutant, recombinant or modified form thereof in an appropriate culture medium and optionally recovering the sanglifehrin.

Macrolides in accordance with the invention, e.g. compounds of formula IX; for example, Sanglifehrins A, B, C and D, and their pharmaceutically acceptable salts, hereinafter generically "agents of the invention", exhibit sanglifehrin characteristic activities, i.e. the following combination of activities: have cyclophilin binding activity; have immunosuppressive activity; inhibit proliferation of both B-cells and T-cells; but do not have FK binding protein binding activity, and do not inhibit calcineurin activity.

These activities and assays to determine these activities are described hereinafter in greater detail. Biological activity of macrolides of the invention, e.g. of formula IX, e.g of Sanglifehrins A to D, may be demonstrated in standard in vitro and in vivo test methods, e.g. as follows.

1. Primary Humoral Immune Response to Sheep Red Blood Cells (MD, Mishell-Dutton) Mouse spleen cells (OF 1, female, 8-10 weeks, 1 x 107) are co-cultured with sheep erythrocytes (SRBC, 3 x 10 7 for 3 days in 1 ml final volume in 24 well plates.

Lymphocytes are harvested, washed and plated at a density of 1 x 10 6 cells onto soft-agar with fresh antigen (SRBC). Complement (guinea pig serum) is added after a 60-90 minute incubation period and incubation is continued for a further 60 minutes after which the test is evaluated by counting (microscope) the plaques. During the 3 day incubation, the lymphocytes are sensitized to the antigen (SRBC). When incubated with antigen again, B-lymphocytes secrete specific antibody which binds to the antigen in the vicinity of the secretory lymphocyte. Addition of complement causes lysis of the antibody-coated erythrocytes yielding a plaque. Each plaque represents a single antibody-producing cell.

References: R.I. Mishell R.W. Dutton (1966) Immunization of normal mouse spleen cell suspensions in vitro. Science 153: 1004-1006 R.I. Mishell R.W. Dutton (1967) Immunization of dissociated spleen cell cultures from normal mice. J.Exp.Med. 126:423-442 2. Proliferative Response Of Lymphocytes to Allogenic Stimulation Two-way MLR (Murine Mixed Lymphocyte Reaction): Spleen cells (2 x 105) from Balb/c mice (female, 8-10 weeks) are co-incubated for 4 days with 2 x 105 spleen cells from CBA mice (female, 8-10 weeks). The allogenic cells induce a proliferative response in the responder spleen cell population which is measured by labelled precursor incorporation into the DNA. Macrolides of the invention, e.g. compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, have IC 5 os in the range from about 30 up to about 200 nM as compared with an ICso of about 20 nM for cyclosporin A when tested in this assay.

Reference: WO 97/02285 PCT/EP96/02952 T. Meo (1979) The MLR in the mouse. In: "Immunological Methods", L. Lcfkovits and B. Pernis, Eds., Academic Press, N.Y. pp. 227- 239 3. LPS- stimulated murine B-cells Spleen cells (2 x 10 5 from CBA mice are incubated for 48 hours with 50 gg/ml LPS plus test compound. Proliferation is measured by labelled precursor incorporation into DNA. Macrolides of the invention, e.g. compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, inhibit B-cell proliferation and have IC 50 s in the range from about40 up to about 100 iM.

References: Greaves, M. and J. Janossy, 1972, Elicitation of selective T and B lymphocyte response by cell surface binding ligands, Transplant Rev., 11: 87 Janossy, G. and M. F. Greaves, 1971, Lymphocyte activation, I, Response of T and B lymphocytes to phytomitogens, Clin. Exp. Immunol. 9: 483-498 4. Cytotoxic and cvtostatic activity in vitro using the THP1 cell line Cytotoxicity is determined using the human monocytic cell line THP1 (5 x 104 cells/well) which are incubated in the presence of IFNy (100 U/ml)and LPS (5 gg/ml) plus test compound (up to 10 pM) for 24 to 72 h at 37 0 C. Living cells are quantified using the colourimetric read-out MTT which measures mitochondrial dehydrogenase enzymatic activity in living cells (Mossman 1983). Macrolides of the invention, e.g.

compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, have IC 50 s of about 1000-5000 nM after 24 h incubation in this assay.

Reference: WO 97/02285 PCT/EP96/02952 Mossman T. J. (1983), Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxic assays, J. Imm. Methods, 65, 55-63.

TNF release by human peripheral blood mononuclear cells Mononuclear cells are prepared from the peripheral blood of healthy volunteers using Ficoll-Hypaque density separation according to the method of Hansell et al.

(1991). Cells (105 cells/well in 200 pl RPMI 10% FCS by volume) are incubated with serial dilutions of the test compounds for 30 min at 37°C prior to the addition of stimulus. Interferon y (100 U/ml) and LPS (5tg/ml) are used as stimuli to induce Tumour Necrosis Factor (TNF) a release by peripheral blood mononuclear cells. After 3 h incubation, the cells are centrifuged (1200 rpm for 10 min) and the supematants are collected. The amount of TNF present in the cell supernatants is determined using a commercially available enzyme-linked immunosorbent assay kit. Macrolides of the invention, e.g. compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, have ICsos in the range from about 200 nm to about 1000 nm when tested in this assay.

6. Cyclophilin Binding Assay A suitable cyclophilin binding assay is the competitive ELISA test described by Quesniaux in Eur. J. Immunol. 1987 17 1359-1365. In this test, the compound to be tested is added during the incubation of cyclophilin (human recombinant cyclophilin A) with coated BSA-cyclosporin A and the concentration required to give a 50% inhibition of the control reaction without competitor is then calculated (ICso). An alternative assay is the competitive binding test described by Schneider et al. in Biochemistry (1994), 33, 8218-8224, which involves addition of test compound during the incubation of biotinylated cyclophilin (human recombinant cyclophilin A) with coated BSAcyclosporin A. The amount of biotinylated cyclophilin bound in the presence and absence of a test compound is determined by incubation with streptavidin-coupled alkaline phosphatase. Macrolides of the invention, e.g. compounds of formula IX, e.g.

WO 97/02285 PCT/EP96/02952 Sanglifehrin A, B, C and B, have IC5os in the range from about 10 to about 100 nM, compared with an IC5o of about 80 nM for cyclosporin A when tested in these assays.

Further in vitro assays which may be used to demonstrate the biological activity of Sanglifehrins are IL-2 reporter gene assays and ConA-stimulated spleen cell assays (indicative of effect on T-cell activation).

Macrolides of the invention, e.g. compounds of formula IX, e.g. Sanglifehrin A, B, C and B, do not have FK binding protein binding activity and do not inhibit calcineurin activity when tested in standard tests for these activities.

7. Localised Graft-versus-Host (GvH) Reaction in the rat [Ford et al., TRANSPL. PROC. 10 (1979) 258].

Spleen cells (1 x 107) from 6 week old female Wistar/Furth (WF) rats are injected subcutaneously on day 0 into the left hind-paw of female (F344 x WF)F rats weighing about 100g. Animals are treated for 4 consecutive days and the popliteal lymph nodes are removed and weighed on day 7. The difference in weight between the two lymph nodes is taken as the parameter for evaluating the reaction.

Inhibition of GvH reaction in the above test is indicative of pharmaceutical utility.

Macrolides of the invention, e.g. compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, are able to inhibit the GvH reaction by up to about 30% when administered at a dose of about 1 mg/kg s.c..

8. DTH induced by SRBC-TH cells Fifty microliters of a 1:1 mixture of a TH (sheep red blood cell primed) cell clone (2 x 106) and a 10 sheep red blood cell (SRBC) suspension are injected s.c. into the right hind footpad of female C57 BLJ6 mice (6-12 weeks old). 50 pl of the SRBC cell suspension (diluted 1:1 v/v with PBS) is injected s.c. into the left hind footpad (to 34 WO 97/02285 PCT/EP96/02952 measure non specific increase in footpad swelling due to the injection procedure). Right and left hind footpad thickness is measured 24 hours later.

The percent increase in thickness of the right footpad over the left footpad is calculated. Thickness of right footpad x; thickness of left footpad y; specific increase z 100 Macrolides of the invention, e.g. compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, reduce swelling of the DTH mouse by up to about 50% at doses of the order of 5 mg/kg s.c..

References: A.T.J. Bianchi, H. Hooijkaas, R. Brenner, R. Tees, AA. Nordin M.H. Schreier (1981) Clones of helper T-cells mediate antigen specific, H-2 restricted DTH. Nature 290:62-63 P. Herrmann, M.H. Schreier, Borel C. Feurer (1988) Mast cell degranulation as a major event in the effector phase of delayed-type hypersensitivity induced by cloned helper T cells. Int. Archs Allergy appl. Immun. 86: 102- 105 9. Rat/Mouse Heart Allotransplantation The in vivo efficacy of macrolides of the invention is assessed in rat and mouse heart allotransplantation using Alzet osmotic minipumps for s.c. administration. In mouse heart allotransplantation (BALB/c to C3H), macrolides of the invention, e.g.

compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, prolong graft survival at doses of the order of 30 mg/kg/day. In rat heart allotransplantation (DA to Lewis) treatment with suboptimal doses of cyclosporin A in combination with Macrolides of the invention, e.g. compounds of formula IX and their WO 97/02285 PCT/EP96/02952 pharmaceutically acceptable salts, e.g. Sanglifehrins A, B, C and D, prolonged graft survival as exemplified in the table below.

Cyclosporin A Sanglifehrin A Graft survival (mg/kg) (mg/kg) (days) 1 12, 12, 12, 13, 13, 14 1 10 29, 30, 45, 48, >51, >46 Control (Placebo) Control (Placebo) Control (Placebo) Agents of the invention are useful as pharmaceuticals, e.g. as immunosuppressive as well as an anti-inflammatory agents.

They are, in particular, useful for the prevention of acute and/or chronic organ or tissue allo- and xenotransplant rejection, e.g. for the treatment of recipients of heart, lung, combined heart-lung, liver, kidney, pancreatic, skin or corneal transplants. They are also indicated for the prevention of graft-versus-host disease, such as following bone marrow transplants.

Agents of the invention are also useful for the treatment of autoimmune disease and of inflammatory conditions, in particular inflammatory conditions with an aetiology including an autoimmune component such as arthritis (for example rheumatoid arthritis, arthritis chronica progrediente and arthritis deformans) and rheumatic diseases. Specific auto-immune diseases for which the agents of the invention may be employed include autoimmune haematological disorders (including e.g. haemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, sclerodoma, Wegener granulamatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (including e.g. ulcerative colitis and Crohn's disease) endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal and/or allergic keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis, glomerulonephritis (with WO 97/02285 PCT/EP96/02952 and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy) and asthma.

For these and other uses agents of the invention may be administered on their own or together with other immunosuppressant or antiinflammatory agents, including cyclosporins, rapamycins, FK 506, and steroids.

For the above indications the appropriate dosage will, of course, vary depending, and the agent of the invention chosen, for example, on the subject to be treated, the mode of administration and the nature and severity of the condition being treated. However, in general, satisfactory results in animals are obtained at daily dosages of from about 0.01 to 10 mg/kg/day In larger mammals, for example humans, an indicated daily dosage is in the range of from about 0.5 to about 500 mg of sanglifehrin administered orally once or, more suitably, in divided dosages two to four times/day.

In organ transplantation in humans, oral doses of 0.1 to 100, preferably 0.3 to 30, more preferably 0.5 to 10, mg/kg of a compound of agent of the invention. When an agent of the invention is given along with other immunosuppressants with corticosteroids or with compounds of the cyclosporin or rapamycin class as part of a double, triple or quadruple drug therapy) lower doses 0.1 mg/kg/day 3 mg/kg/day oral initially) may be used. In particular agents of the invention may be given with other non-steroidal immunosuppressants, e.g. with cyclosporin A, rapamycin or FK 506, with a view to the partial or complete replacement of steroids.

Agents of the invention may be administered by any conventional route, in particular enterally, e.g. orally, for example in the form of solutions for drinking, tablets or capsules or parenterally, for example in the form of injectible solutions or suspensions.

Normally for systemic administration oral dosage forms are preferred, although for some conditions, for example for prevention of rejection of liver transplants, an intravenously injectable form is desirable. Compounds may also be administered topically or dermally, e.g. in the form of a dermal cream or gel or like preparation or, for the purposes of application to the eye, in the form of an occular cream, gel or eye-drop preparation.

WO 97/02285 PCT/EP96/02952 Suitable unit dosage forms for oral administration comprise e.g. from 0.5 to 100 mg of the compound per dosage.

In accordance with the foregoing the present invention also provides in a further series of embodiments: A. A method of effecting immunosuppression in a subject in need of such treatment which method comprises administering to said subject an effective amount of an agent of the invention.

B. A method: 1) for the prevention of acute and/or chronic organ allo- or xenotransplant rejection, for example for the treatment of recipients of organ transplants of any of the particular types listed above; or 2) for the prevention of graft-versus-host disease, for example in recipients of bone marrow transplants; or 3) for the treatment of autoimmune disease or for the treatment of any such disease or condition listed above; or 4) for the treatment of asthma in a subject in need of such treatment, which method comprises administering to said subject an effective amount of an agent of the invention.

C. An agent of the invention for use as a pharmaceutical, e.g. for use as an immunosuppressant or for use in the treatment of any disease or condition as set forth under B above.

D. A pharmaceutical composition comprising an agent of the invention in association with a pharmaceutically acceptable diluent or carrier.

WO 97/02285 PCT/EP96/02952 E. The use of an agent of the invention.for the preparation of a medicament for use as an immunosuppressant or for use in the treatment of any disease or condition as set forth under B above.

In addition macrolides of the invention which possess cyclophilin binding activity, may be useful as reagents in displacement immunoassays for cyclosporins and other cyclophilin binding compounds, for example in the assay procedure described in our copending patent application WO 95/07468. This patent application relates to an assay procedure for determining the concentration of an immunophilin-binding pharmaceutical, e.g. Ciclosporin, in blood; the procedure comprising adding a binding competitor that displaces the pharmaceutical from immunosuppressant-immunophilin complexes in the blood; adding a receptor that binds to the pharmaceutical but not significantly to the binding competitor; separating the receptor-pharmaceutical complex from the sample; and determining the amount of the pharmaceutical. Sanglifehrins may be used as the binding competitor in such assays; for instance, to displace cyclosporins from cyclophilins, thereby releasing the cyclosporin for quantitation, e.g. by a monoclonal antibody which is specific for the cyclosporin.

The invention is further described by way of illustration only in the following Example which refers to the accompanying Figures: in which Figure 1 shows the Mass spectrum of the compound of Sanglifehrin

B;

Figure 2 shows the Mass spectrum of the compound ofSanglifehrin

A;

Figure 3 shows the Mass spectrum of the compound ofSanglifehrin

D;

Figure 4 shows the Mass spectrum of the compound of Sanglifehrin

C;

Figure 5 shows the IR spectrum of the compound of Sanglifehrin

B;

Figure 6 shows the IR spectrum of the compound of Sanglifehrin

A;

Figure 7 shows the IR spectrum of the compound of Sanglifehrin

D;

Figure 8 shows the IR spectrum of the compound of Sanglifehrin

C;

Figure 9 shows the NMR spectrum of the compound of formula Sanglifehrin

A;

Figure 10 shows the NMR spectrum of the compound of Sanglifehrin

D.

Figure 11 shows the NMR spectrum of the compound of SanglifehrinB, and Figure 12 shows the NMR spectrum of the compound of Sanglifehrin C.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

S

ft 9* a a a a a

V

*V

a..

S

Va oo *o «oe *o WO 97/02285 PCT/EP96/02952

EXAMPLES

Culture conditions Streptomyces sp. A 92-308110 may be cultured at suitable temperatures on various culture media using appropriate nutrients and mineral substances, using aerobic or immersion culture procedures. The fermentation media typically contains a utilisable source of carbon, sources of nitrogen and mineral salts including trace elements, all of which can be added in the form of well defined products or as complex mixtures, for instance as are found in biological products of various origins.

Example 1 describes the original conditions under which compounds of formula

I

were obtained. Improved yields may be obtained by optimisation of the culture conditions (aeration, temperature, pH, quality and quantity of the carbon and nitrogen sources, quantity of the mineral salts and of the trace elements) and by controlling the fermentation conditions in bioreactors.

Example ICulture of strain A 92-308110.

a. Agar starting culture Agar slant cultures of the strain A 92-308110 are grown for 10 to 14 days at 27 0 C on the following agar medium: Glucose 10.0g Soluble starch 2 0.Og Yeast extract (Gistex, Gist Brocades) NZ-Amine,Type A (Sheffield) 5 .0g Calcium carbonate Agar (Bacto) 15.0g Demineralised water to 1000ml WO 97/02285 PCTI.EP96/02952 The medium is adjusted to pH 6.6-6.8 with NaOH/Hi 2 50 4 then sterilised for 20 min. at 121 0

C.

The cultures can be stored at -25--70*C. A suspension in glycerol-peptone can be stored under liquid nitrogen.

b Preulture Spores and mycelium of 10 starting cultures are suspended in 100 ml of a 0.9% salt solution. Two 2 Liter-Erleneyer flasks each containing I liter of preculture medium are inoculated each with 50 ml of this suspension. The composition of the preculture medium is as follows: Glucose techn 7 Glycerin 7 Yeast extract (BBL) 1.35g Malt extract liquid (Wander) 7 Starch soluble 7 NZ-Amnine,Type A (Sheffield) 2.50g Soya protein 2 Asparagine l.OOg CaCO 3 0.050g NaCI 0- 05 0g

KH

2

PO

4 0.

2

K

2

HPO

4 0.500g MgSO 4 .7H 2 o 0. l OOg Trace element solution A imI Agar (Bacto) Ig Demnineralised water to lOO0ml The medium is adjusted to pH 6.8-7.2 with NaOHIH 2

SO

4 and sterilised for 20m at 121 0

C.

WO 97/02285 PCT/EP96/02952 The composition of the trace element solution A is as follows: FeSO 4 .7H 2 0 ZnSO 4 .7H 2 0 MnC1 2 .4H 2 0 CuSO 4 .5H 2 0 0.2g CoC1 2 .6H 2 0

H

3

BO

3 O.1g KI 0.05g

H

2 SO4 iml Demineralised water to 1000 ml The precultures are fermented for 24 hr. at 27 0 C on a rotary shaker at 200 rpm with an eccentricity of c First intermediate culture Two 75 Liter bioreactors containing each 50 liters of preculture medium are inoculated each with I liter of the preculture and fermented for 96 hr. at 27 0 C. The fermenter is rotated at 150 rpm. Air is introduced at a rate of 0.5 liter per minute per liter medium.

d Second intermediate culture Two 750 liter fermentation vessels each containing 500 liters of the preculture medium are each inoculated with 50 liter of the first intermediate culture. The second intermediate cultures are incubated for 70 hr at 27°C. The fermenters are rotated at 100 rpm and air intoduced at a rate of 0.8 liter per minute per liter medium.

WO 97/02285 PCT/EP96/02952 e. Main culture Two 5'000 Liter bioreactors each containing 3'000 liters of the main medium are inoculated respectively with 250 and 300 liters of the second intermediate cultures. The main cultures are incubated during 96 hr at 24 0 C. The bioreactors are rotated at 45 rpm and air introduced at a rate of 0.5 liter per minute per liter medium.

The composition of the main culture medium is as follows: Glucose techn Malt extract liquid (Wander) 2g Yeast extract (Bacto) 2g Soytone (Bacto) 2g

KH

2

PO

4 0.2g

K

2

HPO

4 0.4g MgSO 4 .7H 2 0 0.2g NaCl 0.05g CaCI 2 .6H 2 0 0.05g Trace element solution B iml Agar (Bacto) lg demineralised water to 1000ml.

The pH is adjusted to 6.3 with KOH/HCI. The medium is sterilised for 20 min at 121°C.

The composition of the trace element solution B is the following: FeSO 4 .7H 2 0 ZnSO 4 .7H 2 0 MnC1 2 .4H 2 0 CuSO 4 .5H 2 0 0.2g

(NH

4 6 M070 24 0.2g WO 97/02285 PCTEP96O2!j52 CoC1 2 .6H 2 0 1.Og

H

3 B0 3 0.lg KJ 0.05g

H

2 S0 4 imi Dernineralised water to 1000 ml An optimised culture medium for the main culture is as follows: Soybean meal Glycerol MIES 0. IM Demineralised water to 1000 mlI at pH 6.8 WO 97/02285 PCT/EP96/02952 Example 2 Isolation of Sanelifehrins A. B, C and D from Streptomyces sp. A92- 308110 The first isolation and characterization of the 4 new CBA active metabolites was done from two 3000 1 tank fermentations by activity guided fractionation and HPLC and thinlayer chromatographic analysis. CBA (cyclophilin binding assay) as described above was used to test for biological activity.

The two 3000 liter fermentations are processed separately. 1500 liter from each fermentation is stirred with 20001 ethyl acetate in 4000 liter stainless steel vessel for hours. The separation of the organic phase is done with a Westfalia-Separator typ SA- The ethyl acetate extracts are washed twice with 80 liters of water and evaporated to dryness under reduced pressure to give 1.64 and 2 kg extracts. The two crude extracts are defatted by a three step extraction with 40 liter methanol/water 9:1 and 40 liter of hexane. Evaporation to dryness under reduced pressure gives 1.34 kg extract.

The defatted extract is chromatographed in two portions (670 g) on a column of 10 kg Sephadex H in methanol solution. Each portion is dissolved in 3.3 liters of methanol when added to the column. After collection of the first 15 liters eluate as fraction 1 the chromatography is continued by collecting 2 liter fractions.

The most active fractions were 2,3 and 4 and are therefore combined to give 146 g.

This sample is further chromatographed on 1 kg Silicalgel Merck 0.04-0063 mm with methyl-tertiary-butyl-ether (MTBE), MTBE/5 methanol and MTBE 10 methanol. Fractions of 2 liters are collected. Fractions 5 to 9 are the most active ones and are combined to give a sample of 43.8 g. This sample is further separated on a column of 1 kg Silicagel (Merck) 0.04-0.063 mm with a gradient of hexane/acetone 7:3 to acetone. From this chromatography fraction 6 (7.0 g) is further separated on a column of 3 kg Lichroprep RP 18 (Merck) 40-63 um with methanol/water 94:6 (fraction 4-7 2.16 then on a column of 100 g Silicagel H with methylenechloride and 3 methanol (733 mg), a column of 3 kg Lichroprep RP18 with methanol/water 9:1 (621 mg) and then on 100 g Lichroprep RP18 with acetonitrile/water 1:1 to yield 324 mg of 46 WO 97/02285 PCT/EP96I02952 Pure Sanglifehrin A (mnp 142-145- C (amorphous), (a)D25=-67.30 (c=0-988, methanol)).

Fractions 5 and 7 from the hexane/acetone column are combined (7.1 g) and further purified on a column of 3 kg lichroprep RPl 8 40-63 pin with methanol/water 9: 1 (769 mg), on a column of 100 g silicagel H with MTBE 3 methanol (309 mg) and finally on 100 g silicagel H with methylenechionide and 3 methanol to yield 90 mg pure Sanglifehrin B (mp. 11l7-121l* C (amorphous), (a)D25=-52.80 I- 128 in methanol)).

Fractions 9 and 10 (2.147 g) of the chromatogrammj with methanol/water 94:6 on 3 kg Lichroprep RP18 are further purified on 100 g Silicagel H with methylenechloride/5 methanol (800 mg) and finally on 3 kg Lichroprep RP18 with methanol/water 9:1 to give 4 80 mg of Sanglifehrin C (mnp. 165-170* C, (aX)D25=-35.60 (c=0-736 in methanol).

Fractions I11 and 12 (835 mg) of the chromatograrnm with methanol/water 94:6 on 3 kg Lichroprep RP18 are purified on 100 g Silicagel H with MTBE/5 methanol to give 140 mg of Sanglifehrin D (mnp. 137-142' C, amorphous).

Sanglifehrin A, B, C and D were then characterised by LTV, IR, Mass and NMR spectroscopy. The results obtained are given in Table 4 below and in the accompagying figures.

WO 97/02285 PCT/EP96/02952 Table4 Sanglifehrin A Molecular formula: C 60

H

91

N

5 0 13 (1090.4) UV (MeOH): 275 (1962), 242 (54500), 197 (75755) W:275 (1635), 242 (51884), OIT 292 (1973), 242 (60495) JR-spectra: Figure 6 Mass-spectra: FAB 1096[M+Li] 4 Figure 2 NMR spectra: Figure 9 Sanglifehrin B molecular formula: C 6 oHs 9

N.

5 0 12 (1072.4) UJV (MeOH): 273 (4395), 242 (50600), 197 (78577) JR-spectra: Figure Mass-spectra: FAB 1098[MH+Li]+ Figure I NMR spectra: Figure I11 Sanglifehrin C molecular formula: C 61

H

93

N

5 0 13 (1104.4) UV (MeOH): 275 (1876)), 242 (51557), 197 (72643) H+ 275 (1391), 242 (50120) Off: 292 (1832), 242 (57960) JR-spectra: Figure 8 Mass-spectra: FAB 11 I0[MH+Ui]+ Figure 4 NMR spectra: Figure 12 WO 97/02285 PCT/EP96/02952 San lifehrin D Molecular formula: C 61

H

91

N

5 0 12 (1086.4) UV (MeOfi): 273 (3194), 242 (47584), 197 (73766) HW 273 (3237), 242 (46389) 01-: 285 (2600), 242 (52907) JR-SPectra Figure 7 Mass-spectra: FALB 1092[MH+LiI+: Figure 3 NMR spectra: Figure 49 WO 97/02285 PCT/EP96/02952

,OH

.OH

I**

39 44 -"43

HO

61 Sanglifehrin

A

Example 3 Transformation of Sanglifehrin A into Sanglifehrin

C

To a stirred, cooled solution of 20 mg (18.3 mol) of Sanglifehrin A in 0.5 mL of methanol is added one crystal of paratoluenesulfonic acid monohydrate. The resulting yellow solution is stirred for one hour and the reaction is quenched with saturated aqueous sodium bicarbonate solution. The resulting mixture is extracted twice with ethyl acetate. The organic solution is washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by column chromatography on silica gel (95:5 methyl-tert.-butyl ether:methanol) to yield Sanglifehrin C as a white amorphous powder. The latter consisted of a 4:1 mixture of Sanglifehrin C and its C53 epimer, Sanglifehrin C having the configuration as depicted below (R Me).

4:1 mixture of diastereomers Alternatively, this transformation can be carried out by using other protic acids (such as pyridinium paratoluenesulfonate, hydrochloric acid or sulfuric acid) or Lewis acids WO 97/02285 PCT/EP96/02952 (such as zinc chloride, magnesium bromide or chloride, titanium tetraisopropoxide or boron trifluoride) in methanol. Use of other alcoholic solvents or cosolvents like ethanol, isopropanol, butanol, allyl alcohol, propargyl alcohol, benzyl alcohol lead in the same manner to analogues where R in the structure above is respectively ethyl, isopropyl, butyl, allyl, propargyl, benzyl.

In the same manner as described above, Sanglifehrin B can be transformed into Sanglifehrin

D.

Example 4 Transformation of Sanlifehrin C into Sanglifehrin

A

A solution of 550 mg (0.50 mmol) of sanglifehrin C in 5 mL of 4:1 THF-water is treated with 0.5 mL of 2N aqueous sulfuric acid and stirred for 1.5 h. The reaction is quenched with saturated aqueous sodium bicarbonate and the resulting mixture is extracted twice with ethyl acetate. The organic solution is washed with saturated aqueous sodium bicarbonate solution and twice with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by column chromatography on silica gel (90:10 methyl-tert.-butyl ether:methanol) to yield sanglifehrin A as a white amorphous powder.

Other inorganic or organic acids can be used in a medium containing water and optionally an organic cosolvent. Suitable acids include hydrochloric acid, paratoluenesulfonic acid or other sulfonic acids, pyridinium paratoluenesulfonate, acetic acid, trifluoroacetic acid, formic acid. Suitable organic cosolvents are acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane.

These reactions are accompanied by the formation of varying amounts of the compound of formula XV, depending among others on the reaction time (for a better procedure leading to the compound of formula XV see Example 5 below).

Analogously, sanglifehrin D can be transformed into sanglifehrin

B.

Example 5 Transformation of Sanglifehrin A into the compound of formula XV WO 97/02285 PCT/EP96/02952 To a stirred, cooled (0 0 C) solution of 50 mg (46 pmmol) of sanglifehrin A in 1.9 mL of acetonitrile is added 0.1 mL of hydrogen fluoride pyridine. The resulting yellow solution is stirred for 1 hour and the reaction is quenched with saturated aqueous sodium bicarbonate.. The resulting mixture is extracted twice with ethyl acetate. The organic solution is washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by column chromatography on silica gel (95:5 methyl-tert.-butyl ether:methanol) to yield the compound of formula XV as a white amorphous powder.

HOH

0 0 OH 0 0 0 O NH0 xv HO H In an analogous manner sanglifehrin B can be transformed into the compound of formula XVI. These substances exist as a single epimer at C53, but the absolute configuration has not been unambiguously determined.

6 0 53 HO H xvi

I

Formula XV: MS m/z 1078 [M+Li] (rel. intensity 100); 'H NMR (DMSO) (only characteristic signals listed) 8 0.40 (3H, d, H-50), 1.20 (3H, s, H-54), 1.69 (3H, s, H- WO 97/02285 PCT/EP96/02952 49), 4.20 (1H, t, H-15), 4.58 (1H, dd, H-17), 5.19 (1H, dd, H-18), 5.28 (IH, dd, H-23), 5.62 (1H, m, H-21), 5.67 (1H, m, H-27), 5.99 (1H, d, H-25), 6.03 (1H, dd, H-19), 6.14 (1H, dd, H-20), 6.22 (1H, dd, H-26).

Example 6 Transformation of the compound of formula XV into Sanglifehrin

A

To a stirred solution of 54 mg (50 pmol) of the compound of formula XV in 0.5 mL of 4:1 THF-water is added 50 pL of 2N aqueous sulfuric acid. The resulting solution is stirred at ambient temperature for 12 hours and the reaction is quenched with saturated aqueous sodium bicarbonate solution. This mixture is extracted twice with ethyl acetate. The combined organic solution is washed with saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfated, filtered and concentrated under educed pressure. Column chromatography of the residue on silica gel gel (90:10 methyl-tert.-butyl ether:methanol) yields sanglifehrin A as a white amorphous solid.

Analogously the compound of formual XVI can be transformed into sanglifehrin

B.

The procedures described in examples 3 to 6 can be used as selective intramolecular protection-deprotection sequences. Thus, by the reaction described in example 5, the hydroxyl at position 15 can be selectively protected, which allows the selective manipulation of the remaining free hydroxyls. The procedure in example 5 allows for the selective protection of both the hydroxyls in the 15 and 17 positions. Both procedures can also be used as an intramolecular protection of the C53 ketone. The hydroxyls and the ketone can be regenerated by the reactions described in 4 and 6.

Sanglifehrins C and D, as well as the compounds of formulae XV and XVI are therefore important intermediates for the generation of further sanglifehrins.

WO 97/02285 PCT/EP96/02952 Example 7 PreDaration of 16-Dehvdro-17-Dehydroxy-sanalifehrin A (Formula

HO

H 0 0 1 OH

NH

A solution of 54 mg (50 pmol) of the compound of formula XV and a crystal of paratoluenesulfonic acid monohydrate in 1 mL of 4:1 acetonitrile-water is heated to for 1.5 hours. The reaction is quenched by the addition of saturated aqueous sodium bicarbonate solution. The resulting mixture is extracted twice with ethyl acetate. The organic layer is washed with saturated aqueous sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by column chromatography on silica gel (90:10 methyl-tert.-butyl ether:methanol), followed by reverse phase chromatography (RP18, 50:50 acetonitrile-water to acetonitrile over 45 minutes) to yield the pure title compound as a white amorphous solid.

MS m/z 1078 [M+Li] (rel. intensity 100); 1 H NMR (DMSO) (only characteristic signals listed) 8 1.58 (3H, s, H-50), 1.71 (3H, s, H-49), 2.08 (3H, s, H-54), 4.03 (2H, d, H-15 and C31-OH), 5.57 (2H, m, H-21 and C35-OH), 5.72 (1H, dt, H-27), 5.96 d, C15-OH), 6.03 (1H, d, H-25), 6.09-6.28 (4H, m, H-18, H-19, H-20 and H-26), 6.37 (1H, d, H-17).

WO 97/02285 PCTI]EP96Io29S2 Exam~le 8 Preparation of 4 2 -N-methvl-san~iehrijn A (Formula

XVHII

N

To a stirred, cooled solution of 109 mg (0.1 nimol) of sanglifehrin A and 67 P1. (0.3 mmol) of 2 6 -di-tert.-butylpyridine in 1 MiL of methylene chloride is added 16.5 p1. of methyl triflate. The mixture is allowed to warm to room temperature and stirring is continued for six hours, after which the reaction is quenched by addition of saturated aqueous sodium bicarbonate solution. The resulting mixture is extracted twice with ethyl acetate. The organic layer is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by two successive chromatographies on silica gel (90:10 methyl-tert.-butyl ether:methanol then 95:5 methyl-tert.-butyl ether:methanol) to yield the pure title compound as a white amorphous solid.

MS m/z 1110 (rel. intensity 100); 'H NMR (DMSO) (only characteristic signals listed) 5 1.70 (3H, s, H-49), 2.06 (3H, s, H-54), 3.53 (3H, s, 42 N-Me), 3.98 (lH, d, C31-OH), 4.50 (1H, d, H-65), 4.77 (lH, d, C17-OH), 5.43 (1H, d, 5.49 (1IH, d, C35-OH), 7.50 (1 H, d, H- 12), 8.11 (1 H, d, 9.22 (1 H, s, C61 -OH).

WO 97/02285 PCT/EP96/02952 Example 9 Preparation of 53 Dihydro sanglifehrin A (Formula XIX) O OH 0 0

OH

N H OH XIX HO To a stirred, cooled solution of 54 mg (50 pmol) of sanglifehrin A in 0.5 mL of methanol is added 2.8 mg (75 pmol) of sodium borohydride. Stirring is continued for one hour and saturated aqueous sodium bicarbonate is added. The mixture is extracted twice with ethyl acetate. The organic solution is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The The residue is purified by chromatography on silica gel (95:5 methyl-tert.-butyl ether:methanol followed by 90:10 methyl-tert.-butyl ether:methanol) to yield the pure title compound as a white amorphous solid. The isolated product corresponds to a ca. 1:1 mixture of diastereoisomers at C-53) MS m/z 1098 [M+Li] (rel. intensity 63), 1104 [M+2Li-H] (rel. intensity 100); 'H NMR (DMSO) (only characteristic signals listed) 8 0.62 (3H, d, H-50), 1.02 (3H, d, H-54), 3.55 and 3.59 (1H, 2m, H-53).

WO 97/02285 PCT1EP96/02952 EXample 10 Pre~aration of 53- Tosv~hdrazone-san~Iifehirin A (formulaX) 0I 0 A mixture of 55 mg (50 pmol) Of sanglifehrin A and 23 mg (125 jimol) of tosyihydrazide in 0.5 ML of methylene chloride is stirred at room temperature for six hours. The solvent is removed and the residue is purified by chromatography on silica gel (90: 10 methyl-tert.-lbutyl ether:methanol) to yield the title compound as a white amorphous powder.

MS m/z 1264 [M+LiI' (rel. intensity 100); 'H NMR (DM50) (only characteristic signals listed) 8 1.70 (3H, s, H-49), 1.77 (3H, s, H-54), 2.37 (3H, s, -NS02C 6 H4CACN 6.51 (1IH, s, H-60), 6.59 (2H, 2d, H-62 and H-64), 7.06 (1 H, dd, H-63), 7.35 (2H, d, tosyl meta protons), 7.73 (2H, d, tosyl para protons).

Example 11 Prearation of 2 6 S 2 7 S-Dihvdroxv.san2ifehrin A (Formula

XXI)

and 2 6 R 2 7 R-Dihdroxy.sanelifehrin A (Formula

XXI)

HO

H

NHI

57 WO 97/02285 PCT/EP96/02952 HO

OOH

0° O H OH 0 0

OH

N H 0" NH 0 ~o 0 0 0 xxII

HO

To a stirred, cooled solution of 495 mg (1.5 mmol) of potassium ferricyanide, 207 mg (1.5 mmol) of potassium carbonate, 19.5 mg (0.025 mmol) of (DHQ) 2

PHAL,

pL (0.005 mmol) of 0.08 M osmium tetroxide in t-butanol and 95 mg (1 mmol) of methyl sulfonamide in 2.5 mL of t-butanol and 5 mL of water is added a solution of 545 mg (0.5 mmol) of sanglifehrin A in 2.5 mL of t-butanol. The resulting biphasic mixture is allowed to warm to room temperature and stirred for three hours. Then 1.08 g (8.6 mmol) of sodium sulfite is added, followed by ethyl acetate and water, and the mixture is vigorously stirred for 15 minutes. The layers are separated and the aqueous layer is extracted twice with ethyl acetate. The combined organic layer is washed with saturated aqueous sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue is purified by reverse phase chromatography (RP18, 30:70 acetonitrile-water to acetonitrile over 60 minutes) yielding the 26S,27Sdiol as an amorphous powder.

The corresponding 26R,27R-diol is obtained by the above procedure, but using

(DHQD)

2 PHAL instead of (DHQ) 2

PHAL.

26S,27S-diol: MS m/z 1130 [M+Li] (rel. intensity 100); 1H NMR (DMSO) (only characteristic signals listed) 8 1.64 (3H, s, H-49), 2.06 (3H, s, H-54), 3.20 (1H, broad m, H-27), 3.45 (1H, broad m, H-31), 3.94 (3H, m, H-17, H-26 and C31-OH), 4.30 (1H, d, C27-OH), 4.57 (1H, d, C26-OH), 5.20 (1H, t, H-23), 5.33 (1H, d, H-25), 5.57 (3H, m, H-18, H-21 and C35-OH), 6.03 (1H, dd, H-19), 6.14 (1H, dd, WO 97/02285 PCT/EP96/02952 26R,27R-diol: MS m/z 1130 (rel. intensity 100); 1 H NMR (DMSO) (only characteristic signals listed) 8 1.64 (3H, s, H-49), 2.06 (3H, s, H-54), 3.16 (111, broad m, H-27), 3.48 (1H, broad mn, H-31), 3.94 (3H, mn, H1-17, H-26 and C31I-OH), 4.30 (111, d, C27-OH), 4.57 (1H1, d, C26-OH), 5.20 (1H1, dd, H-23), 5.35 (1H1, d, 5.57 (3H, m, H-18,1H-21 and C35-OH), 6.03 (lii, dd, H-19), 6.14 (11, dd, Example 12 Cleavaee of the diol in 2 6 S.27S-Dihydrou-sanglifelirn

A

To a solution of 90 mg (79 pmol) of the 26S,27S diol in 0.9 mL of 2:1 THF-water is added 33.7 mg (157 ptmol) of sodium periodate. Stirring is continued for one hour and saturated aqueous sodium bicarbonate is added. The mixture is extracted twice with ethyl acetate. The organic solution is washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. Purification of the residue on silica gel (95:5 methyl-tert.-butylether:methanol) yields the compounds of formula XXII (foam) and XXIV (powder).

XXIII

OH

OH

X IV formula XXII: MS m/z 366 [M+H-H 2 Oj+ (rel. intensity 100); 'H NMR (DM50) (2:1 mixture of OH,,:OHq epimers at the anomeric center) (only characteristic signals WO 97/02285 PCT/EP96/02952 listed) 8 3.54 and 4.08 (111, 2m, H-3 3.57 (1Hi, broad m, H-35), 3.66 (1H, m, H1-33), 4.38 (0.67H, ddd, 4.95 (0.33H1, broad m, 5.40 (0.33H, d, C27-OHq), 5.59 (0.33H, d, C35-OH), 5.61 (0.67H1, d, C35-OH), 5.96 (0.67H, d, C27-OH"), 7.89 (0.67H, s, NH-42), 7.91 (0.33H, s, NH-42).

formula XXJV: MS m/z 745 'H NMR (DMSO) (only characteristic signals listed) 8 0.64 (311, d, H-50), 0.81 (6H, d, H1-56 and H1-57), 2.06 (3H, s, H-54), 2.17 (4H, s, H-14 and H1-49), 3.80 (1H, broad m, H-15), 3.94 (lH, dd, H-17), 5.33 (111, broad d, H-23), 5.62 (2H1, m, H1-18 and 11-21), 6.89 (111, d, H1-25), 6. 10 (114, dd, H- 19), 6.18 (11H, dd, 11-20), 10.0 (111, d, 11-26).

Example 13 Acetylation of Sanlifehrin A to Live 6 Acetl-sanlifehrin

A

(formula

XXV)

N HH To sired c old 0 oui no 4 m 5 o)o a giern A a d5 Lo pyrdin in 05 m of m t y e e c l rd is a d d 52 ±L 5 mO o a e an ydie. Th eatonisk pta 0 C o o e orten alo edt w rm t r o te m era ur a n d st rr n is c n i u d f r t e v o r S t r t d a u o s s d u signal sted) co1.68 (311, solto of-9) 2.06 (1 2.2 (311,fi A, andO) .04o WO 97/02285 PCTfIEP96Io2952 (111, d, C31-QH), 4.67 (1H, d, C2-NH), 4.76 (1H, d, C17-OH), 5.42 (2H, m, H1-8 and 5.57 (3H, m, H-18, H1-21 and C35-OH), 6.85 (1H, s, H1-60), 6.98 (111, d, H- 62), 7.06 (111, d, H-64), 7.31 (111, dd, H-63), 7.51 (1H, d, H- 12), 7.89 (1H, s, H1-42), 8.23 (111, d, H-9).

61

Claims (3)

1. 2. A compound according to claim 1, wherein S reprweets a 2-oxy-2'-az-3'- oxo-spirobicyclohexan-3-yl residue of formula VI Me 6 a 02 vi me '2N- '3 0 Et wherein is -(Me)C=CH- or -(Me)CH-CE(OH)-; and Rsis H or Me, in free or protected formn, or salt thereof.
2. 3. A compound according to claim 1 or 2 of fornila IX 47 48 49 *Me Me 26 Me 2 2018 b S30 0 2 T~ h 0a~ OH 27 0g 4s Me
3. 141- i *39 40 3 NH 0 41 0 1 2 (H2i N 0 St4Mje ix HOe a 63 wherein is -(Me)C-CH- or -(Me)CH-CH(OH)-; is -CH(OH)-CH(OH)- or -CH=CH-; is as defined above for R 3 are as defined in claim 1; and is H or Me, in free or protected form or salt theof. 4. A compound according to claim 3 having the following conformation 47 48 49 Me Me a n Me b 30 e P4 3142 i 20 a7 =O OH 2 7 I O-R 4s Me a 42N-R i 40M e5] 6 5 14 3W 4 39a 1 3 NH CO (CH13-R 4 43 61 Mie wherein when is -(Me)CH-CH(OH)-, it preferably has the configuration: HO *C 61 IS9M 3 when is -CH(OH)-CH(OH)-, it preferably has the configuration or the configuration; when is -(Me)CH-CH(OH)-, it preferably has the configuration: 99** OH CH 9 C 3 when is it preferably has the conflguration: :64 N 64 CH 3 and when 3 and R 4 are fused together they are preferably is of configuration, CH 34 4' OCH 3 A compound selected from the group consisting of Sanglifebrin A, B, C and D 54 HOo 3 3 9 2 65 N4 2 19 ~1352 mee 7 0 OH 36 39 0 me SagieSnA6 1 6 'S.4 S H OH OH Me MeMe M S0 h. 00 0~S 0 agieai O M Me Sanglifehrin C Sanglifehrin D 6. A compound of formula XII" R 6 0 I CH 3 CE' 3 wherein Ais asdefinedinclaimlI and R6is Hor C.lkyt in free or protected form or salt thereof, 7. A compound according to claim 6 which is of formula IX' 2 4 39N 40 R 6 3 4Nj> CHJi-R 4 S 41 02 4 N 44Me Ix' M 63 wherein R 3 and R 4 are as defined in claim 3 and Pis H or Q- 4 alkyl, in free or protected form, or a salt thereof. 8. A compound according to claim 6 which is of formula IX" S. *SS S S S.. S 45 Me ~64 wherein R 3 R 4 and are as defined in claim 3 aud RG is H or C 1 4 alkyl, in free or protected form or a sat thereof. 9. A compound of formula XlV o me Hh 0 0 R 3 NH rQ(CH 2 -R 4 HOHO wherein R 3 and R 4 are as defined in claim 3, in free or protected form, or salt thereof. A process for the production of a compound according to any one of claims 1 to 8, which process comprises: for the production of any one of Sanglifebrins A, B, C or D as defined in claim 5, cultivating a Sangifbrin A. B, C or D producing actinomycete strain in a culture mezdium and isolating the desired Sanglifehrin A. B. C or D from the obtained culture broth; for the production of Sanglifebins. C and D as defined in claim 5 subjecting Sanglifebrins A and B as defined in claim 5 to cyclisation at positions 15 and 16; iii). for the production of Sangliferins A and B as defined in claim subjecting Sanglifebrins C and D as defined in claim 5 to ring opening of the lactol ring at positions 15 and 16: aa.iv). for the production of a compound of foml EK or IX' as defined in claim 3 or 7, respectively, wherein is dehydrating a compound 68 of formula IX or IX' wherein is -CH(CH 3 or a protected formn thereof; for the production of a compound of fortmula DC or EK' as defined in claimn 3 or 7, respectively, wherein R4 is -CH(OH)-CH 3 hydrogenating a compound of formula IX or EX' wherein 1.is vi). for the production of acomnpound of ftmuila IX or IX' as defined in claim 3 or 7, respectively, in which Rs is methyl subjecting a compound of formula IX or DC' in which RS is H to methylation; vii). for the production of a compound of formula IX or IX' as defined in claim 3 or 7, respectively, in which RZA is in 0-protected form, subjecting a compound of formula IX or DC, in which R is in 0-unprotected form to 0-protection; viii). for the production of a compound of formula IX or IX' as defined in claim 3 or 7, respectively, in which R 4 is in 0-unprotected form, subjecting a compound of formula. M or IX', in which R 5 is in 0-protected form to 0- deprotection; for the production of a compound of rmla IX or IX' as defined in claim. 3 or 7, respectively, which comprises an 0-protected hydroxyphenylalaziine residue at positions 7 to 10 of the macrocyclic ring, subjecting a compound of formula IX or IX', which comprises an 0-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the niacrocyclic ring to 0-protection; for the production of a compound of formula IX or IX' as defined in claim 3 or 7, respectively, which comprises an 0-unprotected hydroxyphenylaanine residue at positions 7 to 10 of the umacoyclic ring, subjecting a compound of formula IX or IX' which comprises an 0-protected hydroxyphenylalnin residue at positions 7 to 10 of the macrocyduc ring to 0-doprotection; 69 xi). for the production of a compound of formula IX or IX' as defined in claim 3 or 7, respectively, in which is -CH(OH)-CH(OH)-, subjecting a compound of formula IX or IX' in which is -CH=CH- to oxidative hydrolysis; xii). for the production of a compound of fornuila XJJ as defined in claim 6. subjecting a compound of fornukl IX as defined in clam 3 to cleavage of the linker group between the spiro bicyclo group and the inacrocyclic ring. xiii). for the production of a compound of formmla XII as defined in claim 6 subjecting a compound of formla, IX as defined in claim 3 to ring-opening within the spiro bicyclic ring system, and xiv). for the production of a compound of formula IX as defined in claim 3 subjecting a compound of formula XLI as defined in claim 6 to ring-closure within the spiro bicyclic ring system. 11. A biologically pure isolate of strai so A92-3081 10 (DSM 9954) or a mutant, recombinant or miodified form thereof which is capable of producing a ~.compound accrding to claim I. 12. A process for the production of a compound according to claim 1, which comprises cultivating strain Stremycs A92-308 110 (DSM 9954) or a nuitant, rcmirxit or modified form thereof in an appropriate culture irnedium and optionally recovering the desired compound. 13. A method of effccting imsprsinin asubject in need of such treatment which uzthod couprises ad-n-er to said subject an eff~ctive amount of a compound according to ay one of clains I to 8. 14. A nxhdxL i) for the prevention of acute and/or chronic organ allo- or xenotransplarit rejection, for example for the treatn~nt of recipients of organ transplants of any of the particular types listed above; or ii) for the prevention of graft-versus-host disease, for example in recipients of bone marrow transplantsZ or iii) for the treatmrent of autoinunie disease or for the treatnent of any such diseas or condition listed above; or iv) for the treatrvent of asthma. in a subject in need of such treatment, which method comprises administering to said subject an effective amoxunt of a compound according to any one of claims 1 to 8. A pharmaceutical compiosition Comprising a compound according to any one of claims 1 to 8 in association with'a pharmaceutically acceptable diluent or carrier. 16. Use of a compound according to claim 1 as a reagent in a displacement immunoassay for a cyclosporins or other cyclophilin binding compounds 17. Use of a compound according to any one of claims 1 to 8 in the manufacture of a medicament for effecting immunosuppression in a subject. 18. Use of a compound according to any one of claims 1 to 8 in the manufacture of a a..medicament: 71 P:\OPER\PDB\65193-96.099 9/4/99 for the prevention of acute and/or chronic organ allo- or xenotransplant rejection, for example for the treatment of recipients of organ transplants of any of the particular types listed above; or (ii) for the prevention of graft-versus-host disease, for example in recipients of bone marrow transplants; or (iii) for the treatment of autoimmune disease or for the treatment of any such disease or condition listed above; or (iv) for the treatment of asthma. 19. A macrolide according to claim 1 or a process according to claim 10 or claim 12 substantially as hereinbefore described with reference to the Examples. An isolate according to claim 11 or a composition according to claim 21. A method according to claim 13 or 14 or a use according to any one of claims 16 to 20 18 substantially as hereinbefore described with reference to the Examples. DATED this 9th day of April 1999 Novartis AG. °By its Patent Attorneys DAVIES COLLISON CAVE ,iA"Q