SK498A3 - Macrolides, a method for their preparation and a pharmaceutical composition containing the same - Google Patents

Abstract

A novel class of macrolides 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 alpha -amino acid residue; and/or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic alpha -amino acid residue, preferably comprising two, or especially all three of the characteristic structural features i), ii) and iii), more especially a compound of formula (IX) is provided having immunosuppressant and anti-inflammatory properties and protected and ring-open forms thereof.

Description

Macrolides, process for their preparation and pharmaceutical composition containing them

Area, techniques

The present invention relates to a new class of macrolides having valuable pharmaceutical and related activity. For the preferred compounds of this new class of macrolides, the common name Sanglifehrines is used in the present invention.

BACKGROUND OF THE INVENTION

The first of the Sanglifehrins was mycetic fermentation. These A to D isolated from are the general nutrient formulas of the actinanglifehrin broth

As can be seen, the macrocyclic ring of Sanglifehrin A to D has a completely new structure, characterized in that

(i) at positions 1 to 6 there is a 3-carboxypiperidazynylcarboxylic acid residue; (ii) at positions 7 to 9 is an aromatic α-amino acid residue; and (iii) at positions 10 to 12 is an aliphatic α-amino acid residue. The remainder of the macrocyclic ring consists of a hydroxycarboxylic acid residue, provided that, in the case of Sanglifehrins A to D, there are an additional 11 carbon atoms in the base macrocyclic ring.

In accordance with conventional techniques in macrolide chemistry, the basic macrocyclic ring atoms of the Sanglifehrin number as indicated above for Sanglifehrin A, starting from the carbon atom of the carbonyl group of the macrocyclic lactone linker at position 1.

Sanglifehrins A through D are also characterized by the presence of a novel bicyclic spirosystem attached at the 23-position of the macrocyclic ring via a hydrocarbon linker group.

The sanglifehrins A to D can be subjected to considerable chemical modification to form other sanglifehrin-class macrolides. These modifications include cleavage of the macrocyclic ring, particularly in the lactone oxy group, cleavage of the linker between the macrolide and the spirocyclic system, and variations, for example, protection, derivatization or other chemical modifications of the substituent groups; for example, as described below. Other processing methods will be apparent to those skilled in the art.

In accordance with the present invention, it has been found that Sanglifehrins, particularly those containing a spirocyclic system, such as the case of Sanglifehrins A to D, have characteristic and brand new properties in terms of their biological activity. In particular, they were found to exhibit the following combination of effects:

- cyclophilin binding activity;

- immunosuppressive activity;

inhibition of both B and T cell proliferation;

however, they do not have an FK binding protein binding activity or calcineurin inhibitory effect.

Sanglifehrins are also an interesting and new class of immunosuppressive and anti-inflammatory compounds. In particular, Sanglifehrin have active properties that differ from previously known immunosuppressive and anti-inflammatory compounds, such as cyclosporins and macrolides, for example rapamycin and the FK 506 class, suggesting that Sanglif ehrins act by a different mechanism than the previous compounds. Thus, Sanglifehrins are a new category of therapeutic agents in terms of both structure and activity, which may be. a prerequisite for anticipating immunosuppressive and / or anti-inflammatory therapies; for example, preventing or reducing unwanted side effects from previous immunosuppressive and anti-inflammatory treatments and / or ameliorating or extending this therapy to new groups of diseases or to new categories of patients.

Sanglif ehrins, for example, in which the macrolide ring is in the open form, in which both positions 26 and 27 in the hydrocarbon linking group between the macrolide and the spirocyclic system are hydroxylated, or in which the spiro residue attached to the macrocyclic ring is cleaved or separated or all activities characteristic of Sanglifehrin. For example, Sanglifehrins in which the spiral residue is cleaved typically have cyclophilin binding activity but do not have significant immunosuppressive activity. However, as will be appreciated by those skilled in the art, these compounds provide valuable components, intermediates, or key building blocks for the preparation of other new Sanglifehrin and therefore further enhance the therapeutic potential of the Sanglifehrin class.

Since the bicyclic spiro system has also been shown to be biologically active, for example, with Sanglifehrin A to D, it can be considered as providing a structural component with a key biological significance that is useful as a structural component for further derivatization or modification, as in preparing other Sanglifehrin , as well as for use in variations or modifications of other drugs; for example, to modify the activity of other immunosuppressive drugs of the macrolide class.

As indicated, Sanglifehrins represent a new class of macrolide compounds that are brand new and have a completely characteristic structure.

Therefore, the first aspect of the present invention provides:

macrolide in which

i) a piperidazinylcarboxylic acid residue is attached at positions 2 and 6 of the macrocyclic ring; and / or ii) an aromatic α-amino acid residue is attached at positions 7 to 9 of the macrocyclic ring; and / or iii) at positions 10 to 12 of the macrocyclic ring the aliphatic α-amino acid residue is attached in free or protected form, or in the form of a salt thereof.

Suitable macrolides of the present invention comprise two, preferably all three, of structural features i), ii) and iii).

As the piperidazinylcarboxylic acid residue, a 1,2-piperidazine-3-carboxy-1-yl radical which contains a carboxyl group in the 1-position and a nitrogen atom 1 in the 6-position of the macrocyclic ring, for example a radical of formula I, is suitable:

where the numbers represent the positions of the atoms of the residues on the macrocyclic ring. This residue may have a ring substituted or unsubstituted. Suitable is unsubstituted.

The α-amino group of the aromatic α-amino acid residue preferably occupies position 9 of the macrocyclic ring. A suitable aromatic α-amino acid is a phenylalanine, especially a 3-hydroxy-phenylalanine residue in free or protected form.

The α-amino group of the aliphatic α-amino acid residue preferably occupies position 12 of the macrocyclic ring. A suitable aliphatic α-amino acid is a valine residue in free or protected form.

Suitably, the macrocyclic ring moiety contains hydroxycarboxylic acid residues, the oxy groups that make up the macrocyclic lactone linking groups, and the carbonyl groups that form the amide linking group with the α-amino group at the 12-position of the macrocyclic ring. Said hydroxycarboxylic acid residues have a chain length of 6 to 20 carbon atoms, more preferably 11 carbon atoms. The chain may be substituted or unsubstituted and / or contain one or more unsaturated linking groups, in particular cumulative double bonds along the chain. More preferably, the remaining macrocyclic ring contains an 11-oxyendecanoyl-11-yl radical, preferably 11-oxy-6,8-endecadienoyl-11-yl radically substituted, for example at positions more preferably said hydroxycarbonyl radical is 2, 3, 4 and / or or 5 xylic acid of formula II:

R ³ R ⁴

R ¹ R ² O (CH ₂ ) ₂ IIII CH-ch ₂ -ch = ch-ch = ch-ch-c-ch-ch-codi:

CHa where 1?

R and R are hydrogen or are a special bond;

R is hydrogen;

R ⁴ is -CO-CH ₃ or -CH (OH) -CH ₃ or

R ³ and R ⁴ together represent the structure of formula III:

ch ₃

I

C- (III) and ₃ in free or protected form or in the form of their salts.

Preferred macrolides in accordance with the present invention comprise a macrocyclic ring of formula IV:

(IV)

Xy- _Z wherein X, Y and Z are moieties i), ii) and iii) as defined above and group A is a hydroxycarboxylic acid moiety as defined above in free or protected form or in the form of a salt thereof; a macrocyclic ring of formula V is particularly suitable:

in free or salt form.

Generally, in the Sanglifehrins, such as Sanglifehrins A through D, the macrocyclic ring is substituted on a carbon atom adjacent to the oxy group of the lactone bridge. Typically this substituent comprises a ^{2-oxy-2'-aza-3-one} -oxospirobicyklohexán 3-yl moiety, e.g., of formula VI:

Et kde

-a-b- is - (Me) C = CH- or - (Me) CH-CH (OH) - and f "

R ³ is H or Me (wherein Me and Et is methyl and ethyl) in free or protected form, or in the form of a salt thereof, attached to the macrolide ring via a linker containing a linear sequence of 6 to 11, typically 9, carbon atoms between spiro - residue and macrolium ring.

The linker group may be substituted or unsubstituted and / or may contain one or more unsaturated bonds, in particular cumulative full length double bonds. Suitably, the linker group may be substituted with a methyl group, for example two methyl groups. Further, the linker group may be suitably substituted with a hydroxyl group, for example three hydroxyl substituents and / or may be ethylenically unsaturated, for example it may contain two carbon-carbon double bonds. More preferably, the linker group contains 1-methyl-7-methylnonanoyl-9-yl residues, in particular 1-methyl-7-methylnonanoyl-9-yl or 1-methyl-7-methyl-1,3-nonadienoyl-9-yl, which are optionally substituted, for example in the 3, 4 and / or 8-positions. Preferably, the linker group has the formula VII:

R ⁶ R ⁷ CH ₃

III (VII) c-CH ₂ -CH (OH) -CH (CH ₃ ) - (CH ₂ ) ₂ —CH-CH-CH = Cd wherein c is a bond to a spiro residue;

d represents a bond to the macrocyclic ring and R and R are hydroxyl groups or together represent another bond in free or protected form.

The linking group will generally be attached to a macrocyclic ring on a carbon adjacent to the lactone oxy group, i. j. wherein the macrocyclic ring contains an 11-oxyendecanoyl-11-yl residue at the 11-position.

The present invention therefore provides compounds of formula VIII:

S-L-M (VIII) wherein

S represents a spirobicyclo-residue as defined above,

L represents a linking group as defined above; and

M represents a macrocyclic ring, as defined above, in free or protected form or in salt form.

Particular compounds of the present invention have the general Formula IX:

xs 49

where

-a-b- is as defined above;

-ef- is -CH (OH) -CH (OH) - or -CH = CH-; -gh- has the same meaning as previously defined for -ab-, and, R ⁴ and r ⁵ are as defined above, in free or protected form or in salt form.

The compounds of formulas I-IX contain asymmetric carbon atoms and can therefore exist in a number of epimeric forms. All possible epimers as well as diastereomeric mixtures thereof are included in the present invention. However, compounds of formulas VIII and IX in which the macrolide ring is in the closed form and which have the appropriate stereochemistry have a typical activity that is characteristic of Sanglifehrin as mentioned above. Preferred are epimers having activity characteristic of Saglifehrin. Usually, for example, for pharmaceutical use in accordance with the present invention, epimers having activities characteristic of Sanglifehrins in pure or relatively pure form (i.e., without or almost free of epimers lacking activity characteristic of Sanglifehrins), i.e., will be preferred. j. containing at least 90% by weight, for example 95% by weight, of the active epimer (i.e. containing less than 10% by weight, for example 5% by weight of the inactive epimer).

Preferably, it has a 3-carboxypiperidazinylcarboxylic acid residue

(i) at positions 1 to 6 of the macrocyclic ring, the following conformation:

Preferably, the aromatic amino acid (ii) has a configuration at positions 7 to 9 of the macrocyclic ring L, for example having the configuration:

Preferably, the aliphatic amino acid iii) has a configuration at positions 10-12 of the macrocyclic ring L, for example having the configuration:

A. i 'NH kΎ 12'

If the macrocyclic ring moiety contains a moiety of formula II, it preferably has the configuration:

or

If R ³ and R ⁴ together represent ch ₃

I

-οΙ och ₃ preferably have the configuration:

CH _n

Rest of 2-oxy-2 ':

ase-3 ' spirobicyclohexan-3-yl

Me has configuMe b

T

ABOUT

Et where, when -a-b- is a group of the general formula - (Me) CH-CH (OH) -, preferably has the configuration:

HO ^ ch ₃

If the linker group is of the general formula VII, it is preferably of the configuration:

c n

When R ° and R are hydroxyl groups, the configuration at positions 26 and 27 is preferably either 26 (S), 27 (S) or 26 (R), 27 (R). If R 1 * 7 and R 2 together represent another bond, the configuration at positions 26 and 27 is preferably:

H

H

The compounds of the present invention of formula IX preferably have the following conformation:

Wherein, when -a-b- is a group of the general formula - (Me) CH-CH (OH) -, the group preferably has the configuration:

HO.

CH.

if

-e-f of the general formula is a group

-CH (OH) -CH (OH) -, this group preferably has the (S), (S) or (R), (R) configuration;

when -g-h- is a group of the general formula (Me) CH-CH (OH) -, this group preferably has the configuration:

, 0H

ch ₃ when -gh- is a group of the general formula - (Me) C = CH-, this group preferably has the configuration:

.H

CH.

and when R ³ and R ⁴ are joined, they are preferably of the configuration:

Sanglifehrin A

The compounds of the present invention may be in free or protected form, for example in protected forms as described in Protective Groups in Organic Synthesis, TW Greene and PGM Wuts, second edition, eds. John Wiley & Sons, New York. The hydroxyl groups may in particular be in protected form, for example in the form of silyl ethers (as described, for example, on pages 68-86, Greene and Wuts, also there), esters (see for example pages 87-103, Greene and Wuts, also there) and carbonates (see for example pages 104-111, Greene and Wuts, also there). Such protected forms also include internally protected forms; for example, in the case of a macrolide of formula IX, wherein -gh- is a group of formula -CH (CH ₃ ) -CH (OH) -, in the protected form of position 14 to 7 of the macrocyclic ring, contain a radical of formula X:

CH.

CK ₂ ) ₂

1S (X)

CH — CH — CH — CH '17 Jan 15 14

CH.

for example, with a configuration

Also, for example, 1,3-diols present in Sanglifehrins can be protected as suitable cyclic structures, for example as described on pages 118-142, Greene and Wuts as mentioned above.

The compounds of the present invention also exist in the form of salts. Examples of suitable pharmaceutically acceptable salts for use in accordance with the present invention include acidic and basic complementary salts which are suitably related to the individual substituents present in the compound.

As mentioned above, the macrocyclic ring of the compounds of the present invention may be cleaved, especially in the lactone oxy group, to form compounds wherein the macrocyclic ring is in the open form. Usually, the cleavage of the lactone oxy group is carried out by hydrolysis (solvolysis), for example to give compounds of formula XI:

R ⁶ -XYZA-OH (XI) for example with the general formula XII:

R ⁶ 0 0

NH

N-CO-CH-NH-CO "CH-NH-A OH

HO

CK.

(XII)

CH,

For example, compounds of formula (IX '):

wherein X, Y, Z, A, R ³ , R ⁴ and R ³ are as defined above and R ³ is hydrogen or C 1 -C 4 alkyl, for example methyl.

These open ring forms provide intermediates for modifying the basic macrocyclic ring of Sanglifehrin and are also part of the present invention.

Therefore, another aspect of the present invention provides:

an macrolide as defined above in the form of an open ring; said open ring macrocycle is in free or protected form or in salt form;

a compound of the formula R 10 -O-X-Y-Z-A-OH as defined above in free or protected form or in salt form;

a compound of formula R 6 OXYZ-A'-CH (OH) -LS, wherein the group of formula -A'-CH (OH) - is a hydroxycarboxylic acid residue, for example a residue of formula II as defined above, and the other symbols are as defined above, in free or protected form or in salt form;

R ⁶ 0 compound of formula XII '

WHAT

NH i

N CO-CH-NH-CO

HO

CH

A '—CH-Li

OH (xii ') in free or protected form or in salt form;

- a compound of formula IX ''

in free or protected form or in salt form.

The invention also includes compounds in which the 2-oxy-2'-aza-3'-oxo-3 ¹ -ylspirobicyklohexánový ring system is in ring-opened form, e.g. a compound of formula XII:

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

The compounds of the present invention with an open ring preferably have the same conformation as that of the preferred ring-closed compounds. The open ring spirobicyclic ring system of the compounds of formula (XII) preferably has the conformation:

Me

Me '

o nh ₂ wherein, when -ab- is a group of the formula - (Me) CH-CH (OH) -, the group is preferably of the configuration:

Macrolides in accordance with the present invention having a spirobicyclo- residue attached to a macrocyclic ring may also react at cleavage of the linking group, for example, as a compound of formula IX, especially at the bond between residues 26 and 27 to form separate new spirobicyclic compounds. and other macrolides. As indicated above, these compounds are also useful as intermediates; In particular, the spirobicyclic moieties of Sanglifehrin have an integral functional role in the biological activity of Sanglifehrin as a class.

Therefore, the present invention provides:

2-Oxy-2'-ase-3'-oxo-3'-ylspirobicyclohexane, in free or protected form or in salt form, in particular a compound of formula VI ¹ :

Me

Me (vi -)

Et -7 wherein R is an optionally protected hydroxyl, reactive functional group or a group of the formula -CH ₂ -CH (OH) -CH (CH ₃ ) -CH ₂ -CH ₂ -CHO or its delta lactone equivalent, in in free or protected form or in salt form.

Preferably, the compound of formula VI 'has the following configuration:

wherein, when -a-b- is a group of the general formula - (Me) CH-CH (OH) -, the group preferably has the configuration:

OS

The present invention further includes an open ring of 2-oxy-2'-base-3'-oxo-3'-ylspirobicyclohexane, in free or protected form or in salt form, in particular a compound of formula XII ':

(XII ¹ ) wherein a, b and R ⁷ are as defined above in free or protected form or in salt form. The open ring spirobicyclic ring system of the compounds of formula XII 'preferably has the conformation:

wherein, when -a-b- is a group of the general formula - (Me) CH-CH (OH) -, the group preferably has the configuration:

X ^CH 3

The present invention further provides a macrolide of formula XIII:

CH.

CO (XIII) wherein M is a macrolide ring as defined above, in particular with formula XIV:

(XIV) which preferably has the conformation:

wherein, when -g-h- is - (Me) CH-CH (OH) -, the group of the general formula preferably has a configuration of:

and when -g-this group is a group of the general formula preferably the configuration:

(Me) C = CH-, has

CH, and when R ³ and R ⁴ are joined, they preferably have the configuration:

CH ₃ and O ₃ in free or protected open ring form or in salt form.

A further aspect of the present invention comprises the macrolides and compounds of the present invention, in particular compounds which are natural products in substance-pure form, for example containing at least 90% by weight, preferably at least 95% by weight, in particular at least 98% by weight of pure form.

In addition, the present invention also provides a process for the preparation of all compounds of the present invention as defined above, comprising:

i) for the preparation of Sanglifehrin A, B, C and D, culturing a strain of actinomycetes producing Sanglifehrin A, B, C or D in a culture medium and isolating the desired Sanglifehrin A, B, C, or D from the culture solution obtained;

ii) for the preparation of Sanglifehrin C and D cyclization

Sanglifehrin A and B at positions 15 and 16;

iii) for the preparation of Sanglifehrins A and B, opening the lactone ring of Sanglifehrins C and D at positions 5 and 16;

iv) for the preparation of macrolides of the general formula IX or IX ', wherein -gh- is C (CH 2) 2 CH-, dehydrating a compound of the general formula IX or IX ¹ , wherein -g -h is C (CH 3) -CH (OH) - or a protected form thereof;

v) for the preparation 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 preparation of a macrolide of formula IX or IX ', wherein the positions 14 and 16 of the macrolide ring contain a residue of formula X:

CH

CH,),

I —CH — CH — CH — CHI 15 14

CH (X) reacting a compound of formula IX or IX 'for internal protection;

vii) for the preparation of a macrolide of formula IX or IX ¹ , reacting a compound of formula IX or IX ', wherein the positions 14 to 16 of the macrolium ring contain a residue of formula X

CH.

CH ₂ (X) 1a

-CH 2 CH-CH-CH 17 I 15 µ

CH.

to remove the internal protecting group;

viii) for the preparation of a macrolide of formula IX or IX ', wherein R ⁵ is methyl, methylation of the macrolide with

C is of formula IX or IX ', wherein R is hydrogen;

ix) for the preparation of a macrolide of formula IX or IX 'in which R ^{4 is} in the O-protected form, reacting a macrolide of formula IX or IX' in which r ⁵ is in the O-unprotected form to introduce an O-protecting group ;

x) for the preparation of a macrolide of formula IX or IX in which R ^{4 is} in the O-unprotected form, reacting a macrolide of formula IX or IX ¹ in which R ^{5 is} in the O-protected form to remove the O-protecting groups;

(xi) for the preparation of a macrolide of formula (IX) or (IX ') containing an O-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, reacting a macrolide of formula (IX) or (IX') containing an O-unprotected at position 7 to 10 a hydroxyphenylalanine residue to introduce an O-protecting group;

(xii) for the preparation of a macrolide of formula IX or IX 'containing an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, reacting a macrolide of formula IX or IX' containing at the positions 7 to 10 of the macrocyclic ring O- a protected hydroxyphenylalanine residue to remove the O-protecting group;

xiii) for the preparation of a macrolide of formula IX or IX 'in which -ef- is a CH (OH) -CH (OH) - group, oxidative hydrolysis of a macrolide of formula IX or IX' in which -ef- is a group - CH = CH;

xiv) for preparing a compound of formula V or a compound of formula XII, reacting a compound of formula IX or IX 'to cleave a linking group between a spirobicyclo group and a macrocyclic ring;

xv) for the preparation of a compound of the general formula

R 6 -O-X-Y-Z-A-OH or of the formula R 6 -O-X-Y-Z-A'-CH (OH) -L-S, opening the ring of the lactone bridge of the macrocycle of formula IV or the macrocyclic ring of a compound of formula VIII;

xvi) for the preparation of a closed ring macrolide of formula IX or XII, reacting the compound with

C with the formula R ⁶ OXYZA-OH or with the formula R ⁶ OXYZ-A'-CH (OH) -LS to close the macrocyclic ring;

xvii) for preparing a compound of formula XII or XII ', reacting a compound of formula IX or VI ¹ to open a ring within a spirobicyclic ring system; and xviii) for preparing a compound of formula IX or VI ¹ , reacting a compound of formula XII or XII 'to ring-closure within a spirocyclic ring system

The processes of the present invention can be performed, for example, as described in the examples. As will be pointed out below, the above defined can be applied in any suitable order or combination to give other macrolides in free form, protected form, open ring form and closed ring form as described above.

The macrolides of the present invention, for example Sanglifehrins A to D, are natural compounds or are derived therefrom, which are typically obtained from various members of the Streptomycetaceae family.

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

Therefore, yet another aspect of the present invention provides:

a macrolide producing strain of actinomycetes, wherein the macrolide is a compound in which

i) a piperidazinylcarboxylic acid residue is attached at positions 2 to 6 of the macrocyclic ring; and / or ii) an aromatic α-amino acid residue is attached at positions 7 to 9 of the macrocyclic ring; and / or iii) an aliphatic α-amino acid residue is attached at positions 10-12 of the macrocyclic ring

- A strain of Aktinomycetes producing Sanglifehrin A, B, C or D.

Suitably, the actinomycetes strain is a representative of the group Streptomycetaceae, more preferably belonging to the genus Streptomyces, in particular the strain Streptomyces sp. A92308110 as described below or derived therefrom, for example including mutants, variants, fusants, combinations, or modified forms thereof.

Suitably, the strains of the present invention are biologically pure separated.

For example, Streptomyces sp. A92308110 can be mutated or modified to other forms by conventional techniques, for example by UV radiation or by treatment with a chemical mutagen such as N-methyl-N ¹ -nitronitroso-guanidine. Recombined clones can be obtained by protoplast fusion. All such mutants and recombinants or modified forms which they are capable of producing. Sanglifehrins, including mutants and recombinants capable of producing increased amounts of Sanglifehrins, are part of the present invention.

In a particular embodiment of the present invention, Sanglif ehrins A, B, C and D are isolated from the novel Streptomyces sp. A92308110. Samples of Streptomyces sp. A92308110 were deposited with the Deutche Sammlung von Microorganisms and Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany, on May 3, 1995 under the Budapest Treaty and were assigned the deposit number DSM 9954. Samples of Streptomyces sp. A92308110 can also be obtained from Sandoz Ltd. CH-4002 Basel, Switzerland.

As a note, access to the DSM 9985 samples is restricted in accordance with the provisions of Article 28 (4) and (5) of the EPC.

Isolation of Sanglif ehrins A, B, C and D from Streptomyces sp. A92308110 is described in Example 2.

The strain Streptomyces sp. A92308110 belongs to the genus Streptomyces as described in Bergey's Manual (Volume 4, Williams and Wilkins, Baltimore) and The Prokaryotes (1992 Springer Verlag, New York). Cell walls contain acid

LL-diaminopimelic. Fatty acids are iso- and anteis-branched, straight and unsaturated. The spectrum of sugar is not clear.

The growth mycelium does not shred into pieces. The air spawn is formed by long chains of spores.

In accordance with the references to the books cited above, the strain designated in various oroads

A92308110 is organic and forms the aerial new Streptomyces. A92308110 grows in inorganic environment and in most mycelium. The primary substrate of the mycelium grows as a dung and is usually grayish brown. The color of the air mushroom belongs to the gray series, number 4, and this mushroom is formed by long chains of spores that belong to the spira b type.

The ability of Streptomyces sp. A92308110 to grow in usable biological environments, utilizing their carbon-degrading capacity and their physiological characteristics are listed in the following tables.

Table 1. Growth in different biological environments

Culture environment__ Characteristics of culture

Yeast extract / malt agar

Oat agar growth: good substrate mycelium: dark brown airborne: dark brown soluble dye: no growth: good substrate mycelium: dark brown airborne: gray brown soluble dye: brownish

Glucose-asparagine

Inorganic salts / starch agar

Sucrose / nitrate agar growth: average substrate spawn: gray air spawn: gray brown soluble dye: no growth: average substrate spawn: gray air spawn: gray brown soluble dye: no growth: very poor substrate spawn: whitish air spawn: whitish air spawn: : none

Glycerol / asparagine agar growth: average substrate mycelium: gray

Nutrient agar air spawn: gray-brown soluble dye: no growth: average substrate spawn: beige air spawn: no soluble dye: brown

Table 2: Carbon utilization

Moderate or good: glucose, fructose, arabinose, xylose, mannose

Bad: rhamnose, sucrose, raffinose, cellulose, salicin

Negative: m-inositol

Table 3: Physiological characteristics

Reduction of nitrate: Hydrolysis of starch:

Degradation of tyrosine Degradation of milk proteins:

Melanin formation: Growth temperature:

positive medium on agar from inorganic salts - starch negative on oat agar negative positive positive

18-36 ° C

Very poor growth at 13 ° C. No growth at 45 ° C.

PH range: rich growth at pH 5 and 7, good growth at pH 9 NaCl: resistance up to 6% by weight, but reduced growth already at 2% by weight concentration

The macrolides of the present invention, including Sanglifehrins A, B, C and D, can be prepared by culturing Streptomyces sp. A92308110 or a mutant, recombinant or modified forms thereof in a suitable culture medium. Example 1 describes a procedure for culturing Streptomyces sp. A92308110, which illustrates the present invention.

Therefore, further aspects of the present invention include:

(a) isolating a biologically pure strain of Streptomyces sp. A92308110 (DSM 9954) or a mutant, recombinant, or modified form thereof capable of producing the macrolide of the present invention, and

b) a process for preparing a macrolide according to the present invention, which comprises culturing Streptomyces sp. A92308110 (DSM 9954) or a mutant, recombinant, or modified form thereof in a suitable culture medium, optionally obtaining Sanglifehrin.

Macrolides in accordance with the present invention, for example compounds of Formula IX; for example, Sanglifehrins A, B, C and D, and their pharmaceutically acceptable salts, hereinafter generally referred to as agents of the present invention, exhibit activities characteristic of Sanglifehrins, i. the following combination of activities:

- have cyclophilin binding activity

have immunosuppressive activity to inhibit the proliferation of both B cells and T cells;

lack binding activity to the FK binding protein, and

- do not inhibit calcineurin activity

These activities and assays for detecting these activities are described in more detail below, of the present invention, by Formula IX, e.g.

The biological activity of the macrolides according to, for example, the compound with the general Sanglifehrin A to D may be:

demonstrated by standard in vitro and in vivo test methods, for example, as described below.

Primary humoral immune responses to sheep red blood cells

Mouse spleen cells (OF1, female, 8-10 weeks, 1x10 ⁷ ) are cultured together with sheep red blood cells (SRBC, 3 x 10 ⁷ ) for three days in 1 ml total volume on a 24-well plate. Lymphocytes are separated, washed and plated on fresh antigen (SRBC) soft agar at a density of 1 x 10 6 cells. After 60 to 90 minutes, supplement (guinea pig serum) is added and incubation is continued for a further 60 minutes, after which the test is evaluated by spot counting (microscope). Within three days of incubation, lymphocytes are desensitized to antigen (SRBC). When incubated with the antigen again, B cells secrete a specific antibody that binds to the antigen in the vicinity of the secreting lymphocyte. Addition of the supplement will rupture the cell membrane of the red blood cells coated with the antibody, creating spots. Each spot represents one antibody-producing cell.

Inhibition of staining is one of the pharmaceutical uses. The compounds of the present invention, for example Sanglifehrins A to D, are active in this assay.

references:

R. I. Mishell & R. V. Dutton (1996): Immunization of normal mouse spleen cell suspension in vitro. Science 153: 1004-1006.

R. I. Mishell & R. V. Dutton (1996): Immunization of dissociated mouse spleen cell culture from normal mice. J. Exp. Med. 126: 423-442.

. Lymphocyte proliferative response to allogeneic irritation

Two methods of MLR (mixed mouse lymphocyte reaction):

Spleen cells (2 x 10 5) Balb / c mice (female, 8-10 weeks) are incubated with spleen cells of CBA mice (2 x 10 ⁵ ; female, 8-10 weeks) for four days. Allogeneic cells cause an antibody response to a population of spleen cells, which is measured by incorporating the labeled precursor into DNA. The macrolides of the present invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example

Sanglifehrins A, B, C and D have an IC ₅₀ in the range of 30 to 200 nm compared to an IC _{50 of} about 20 nm for cyclosporin A when tested with this assay.

references:

T. Meo (1979): The MLR in the Mouse. In Immunological Methods, L. Lefkovits and B. Pernis, eds. Academic Press, N.Y., p. 227-239.

LPS-stimulated mouse B cells

The spleen cells (2 x 10 $) of CBA mice are incubated for 48 hours with 50 gg / ml LPS with the test compound. Growth is measured by incorporating the labeled precursor into DNA. The macrolides of the present invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example Sanglifehrins A, B, C and D, inhibit B cell proliferation and have ICgQs in the range of 40 to 100 μΜ.

references:

M. F. Greaves, J. Janossy (1972): Elicitation of selective T and B lymphocyte response by cell surface binding ligands. Transplant Rev.

G. Janossy, M.F. Greaves (1971): Lymphocyte activation I, Response of T and B lymphocytes to phytomitogens. Clin. Exp. Immunol. 9: 483-498.

4. Cytotoxic and cytostatic activity using a series of THP1 cells

Cytotoxicity is determined using a human monocyte series of THP1 cells (5 x 10 ⁴ cells / well) that are incubated in the presence of IFNt (100 U / ml) and LPS (5 µg / ml) and test compound (up to 100 µΜ) 24 to 72 hours at 37 ° C. The amount of viable cells is determined by calorimetric reading of MTT, which measures the enzymatic activity of mitochondrial dehydrogenase in viable cells (Mossman, 1983). The macrolides of the present invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example Sanglifehrins A, B, C and D, have an IC ₅₀ of from 1000 to 5000 nM after 24 hours of incubation in this assay.

references:

Mossman, T. J. (1983): Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J. Imm. Methods 65, 55-63.

5. TNF release by many human peripheral blood nuclear cells

Many nuclear cells are prepared from peripheral blood of healthy volunteers using Ficoll-Hypaque density division according to the procedure of Hansell et al., 1991. Cells (ZLO ⁴ cells / well in 200 μΐ RPMI 10% volume. FCS) are incubated with a series of dilutions of test compounds. 30 minutes at 37 ° C before the stimulant is added. Interferon r (100 U / ml) and LPS (5 gg / ml) are used as stimulants to release tumor necrosis factor (TNF) (oi) by peripheral circulatory mononuclear cells. After three hours of incubation, cells are centrifuged (10 minutes at 1200 rpm) and supernatants are collected. The amount of TNF present in the cell supernatant is determined using a commercially available enzyme immunosorbent assay kit. The macrolides of the present invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example Sanglif ehrins A, B, C and D have an IC ₅₀ ^{in the} range of 200 to 1000 nm when tested in this assay.

. Cyclophilin Binding Assay

A suitable cyclophilin binding assay is the competitive ELISA described by Quesniaux in Eur. J. Immunol. 17, 1987. 1359-1365. In this assay, test compounds are added during the incubation of cyclophilin (recombinant human cyclophilin A) with coated BSA-cyclosporin A, and then the desired concentration is calculated for 50% inhibition of the non-competing control reaction, i.e. IC ₅₀ value. An alternative assay is the competitive binding assay described by Schneider et al. in Biochemistry 33, 1994, 8218-8224, which involves the addition of a test compound during incubation of biotinylated cyclophilin (recombinant human cyclophilin A) coated with BSA-cyclosporin A. The amount of biotinylated cyclophilin bound in the presence and absence of the test compound is determined by incubation with alkaline phosphatase. associated with streptaviain. Macrolides of the invention, e.g. compounds of formula IX and their pharmaceutically acceptable salts, e.g. Sanglif ehríny A, B, C, and D, have to test using the assay IC q in the range of 10 to 100 nm, compared to the _IC50 of cyclosporin A about 80 nm when tested with this assay.

Other in vitro assays that can be used to demonstrate the biological activity of Sanglifehrin are IL-2 reference gene assays and assays of ConA stimulated spleen cells (showing an effect on T cell activation).

Macrolides of the present invention, for example compounds of formula IX, for example Sanglif ehrins A, B, C and D, do not have FK protein binding activity or inhibitory activity and do not inhibit calcineurin activity when tested by standard assays for this activity.

7. Localized implant versus host (GvH) response in rats [Ford et al., TRANSPL. PROC. 10 (1989) 258]

Spleen cells (1 x 10 ⁷ ) of six week old female Wistar / Furth rats (WF) were injected subcutaneously on day 0 into the left hind paw of female rats (F344 x WF) weighing about 100 g. The animals were treated for four consecutive days and the lymph node was removed and weighed on day 7. The difference between the masses of the two lymph nodes was considered as a parameter to evaluate the response.

Inhibition of the GvH reaction in the assay described above is an example of pharmaceutical utility. The macrolides of the present invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example Sanglifehrins A, B, C and D are capable of limiting the GvH response by a maximum of 30% at a dose of about 1 mg / kg.

8. DTH induced by SRBC-T cells _H μΐ of a 1: 1 (v / v) mixture Tjj (red blood cell cells)

Of the sheep, a cell clone (2 x 10) and a 10% by weight suspension of sheep red blood cells (SRBC) were injected subcutaneously into the foot of the right hind limb of female C57 BL / 6 mice (six to twelve weeks of age). The left hind foot (to measure nonspecific growth of the paw swelling due to injection) was injected subcutaneously with 50 μΐ of a sheep red blood cell (SRBC) suspension (diluted 1: 1 v / v with PBS). 24 hours later, the thickness of the right and left hind feet was measured.

The percentage growth of the right foot thickness versus the left foot (z) was calculated. Thickness of right foot = x; left foot thickness = y; percent specific growth = z z = ((x-y) / y) .100

The macrolides of the present invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example Sanglifehrins A, B, C and D, reduce the swelling of DTH mice by a maximum of 50% at a dose of 5 mg / kg subcutaneously.

references:

A.T.J. Bianchi, H. Hooijkaas, R. Brenner, R.Tees, A.A. Nordin and 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, J.-F. Borel and C. Freuer (1988): Mast cell degranulation as a major event in the effector phase of delayed-type hypersensitivity induced by cloner helper T cells. Int. Archs Allergy Appl. Immun. 86: 102-105.

. Allotransplantation of rat / mouse heart

The in vivo effect of the macrolides of the present invention was evaluated in allotransplantation of rat and mouse hearts using an osmotic minipump for subcutaneous administration. In mouse heart allotransplantation (BALB / c to C3H), the macrolides of the present invention, for example, compounds of formula IX, pharmaceutically acceptable salts, for example, Sanglifehrins

A, B, C and D prolong the survival of mice after transplantation at 30 mg / kg / day. In rat heart allotransplantation (from DA for Lewis) treatment with less than an optimal dose of cyclosporin A in combination with the macrolides of the present invention, for example, compounds of formula IX and their pharmaceutically acceptable solar salts, for example Sanglifehrin A,

B, C, and D, prolongs the survival of the transplanted rat as shown in the table below.

Cyclosporin A Sanglifehrin A Survival of transpl. rat (mg / kg) (mg / kg) (days)

- 12, 12, 12, 13, 13, 14

10 29, 30, 45, 48, 51, 46

Control (placebo) Control (placebo) Control (placebo)

The agents of the present invention are useful as pharmaceutical compositions, for example, as immunosuppressants as well as anti-inflammatory agents.

They are particularly useful in preventing acute and / or chronic organ or tissue rejection in allo- or xenotransplantation, for example in the treatment of a recipient following a heart, lung, heart and lung transplant, liver, kidney, pancreas, skin or corneal transplant. They are also useful in preventing transplant versus host disease, such as the consequences of bone marrow transplantation.

arthritis, arthritis) disease, of the present invention

The agents of the present invention are also useful in the treatment of autoimmune disease and inflammatory conditions, in particular inflammatory conditions with an etiology comprising autoimmune components such as arthritis (for example, rheumatic progressive chronic arthritis and deforming and rheumatic diseases. hematologic disorders (including, for example, haemolytic anemia, aplastic anemia, pure red blood cell anemia and idiopathic platelet deficiency of peripheral blood), systemic erythematous ulcers, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, lupus mycosis, chronic activitis, mycosis, chronic activisia Johnson syndrome, idiopathic malabsorption syndrome, autoimmune inflammatory bowel disease (including, for example, ulceration of part of the colon and Crohn's disease), endo chronic ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, diabetes mellitus I, uveitis (anterior and posterior), keratoconjunctivitis (dry and spring) and / or allergic keratoconjunctivitis, interstitial glomerulitis, psoriasis fibrosis, psoriasis, psoriasis, psoriasis, psoriasis nephrotic syndrome, for example including idiopathic nephrotic syndrome or minimal change nephropathy) and asthma.

In these and other uses, the present invention may be administered, in particular, by immunosuppressive or anti-inflammatory being compounds of the invention according to or in admixture with other agents including cyclosporins, rapamycins, FK 506 and steroids.

Of course, for the above indications, the appropriate dosage and selection of the agent of the present invention will vary depending upon, for example, the subject being treated, the route of administration, and the nature and severity of the condition of the subject. Typically, satisfactory results in animals were achieved at daily dosages of about 0.01 to 10 mg / kg / day by oral administration. In larger mammals, for example in humans, the indicated daily dose ranges from 0.5 to 500 mg of Sanglifehrin administered orally one or more times38, in divided doses, two to four times daily.

For human organ transplantation, daily dosages of the compounds of the present invention administered orally are in the range of 0.1 to 100, preferably 0.3 to 30, more preferably 0.5 to 10 mg / kg. When the agent of the present invention is co-administered with another immunosuppressant (e.g., corticosteroids or cycliosporine or rapamycin class compounds as part of a double, triple or quadruple therapy), lower doses (e.g., 0.1 mg / kg / day) may be used. injection, mg / kg / day orally). The agents of the present invention may be. administered in particular with other non-steroidal immunosuppressants, such as cyclosporin A, rapamycin or FK 506, with respect to partial or complete replacement of steroids.

The agents of the present invention may be administered by any conventional means, particularly internally, for example orally, for example in the form of drinking solutions, tablets or capsules, or intravenously, for example in the form of injectable solutions or suspensions. Oral administration is normally preferred for systemic administration, although in some cases, for example, to prevent rejection of a transplanted liver, an intravenous injection form is suitable. The compounds may also be administered topically or via the skin, for example in the form of a cream or gel or the like, for the purpose of ophthalmic application in the form of an eye cream, gel or eye drops.

A suitable unit dosage form for oral administration contains 0.5 to 10 mg of the compound per dose.

In accordance with the foregoing, the compounds of the present invention provide another series of embodiments:

A. Method of effecting immunosuppression in patients in need of such treatment comprising administering to the patient an effective amount of an agent of the present invention.

B. Method:

1) preventing acute and / or chronic organ rejection after allo- or xenotransplantation, for example, to treat a transplant recipient of all the specific types described above; or

2) prevention of transplanted tissue versus host disease, for example, in bone marrow transplant recipients; or

3) for treating diseases of the autoimmune reaction or for treating any such diseases or conditions listed above; or

4) for treating asthma in a patient in need of such treatment comprising administering to the patient an effective amount of an agent of the present invention.

C. The agent of the present invention for use as a pharmaceutical composition, for example, for use as an immunosuppressant or for the treatment of any of the diseases or conditions mentioned in point B.

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

E. Use of the agent of the present invention for the preparation of medicaments useful as an immunosuppressant or useful for the treatment of any of the diseases or conditions mentioned in point B.

The macrolides of the present invention having cyclophilin binding activity may be useful as reagents in cyclosporine substitution immune assays and other cyclophilin binding compounds, for example, in carrying out the assay described in patent application WO 95/07468. This patent application. relates to performing an assay to determine the blood concentration of pharmaceutical compositions binding an immunophilin, such as cyclosporin; the process comprises adding a competing binding agent to replace the pharmaceutical composition in the immunosuppressant-immunophilin complex in the blood; adding a receptor that binds to the pharmaceutical composition but not significantly to the binding competitor; separating the receptor-pharmaceutical composition complex from the sample; and determining the amount of the pharmaceutical composition. Sanglifehrins can be used as competitors in these tests; for example, they can replace cyclosporins from cyclophilin to release the cyclosporin and determine its amount, for example, using a monoclonal antibody that is specific for cyclosporin.

The present invention is further described by way of illustration only, with reference to the accompanying drawings, in which:

using the following and related

Oois attached pictures

Figure 1 display e weight spectrum compound sanelifehrin B; Figure 2 shows weight spectrum compound sanelifehrin A; Figure 3 display e weight spectrum compound sanelifehrin D; Figure 4 shows weight spectrum compound sanelifehrin C;

A picture

A picture

A picture

A picture

A picture

A picture

A picture

The figure shows the IR spectrum of Sanglifehrin B;

shows the IR spectrum of Sanglifehrin A;

shows the IR spectrum of Sanglifehrin D;

shows the IR spectrum of Sanglifehrin C;

shows the NMR spectrum of Sanglifehrin A;

shows the NMR spectrum of Sanglifehrin D;

shows the NMR spectrum of Sanglifehrin B; and shows the NMR spectrum of Sanglifehrin C.

DETAILED DESCRIPTION OF THE INVENTION

Characteristics of cultures

Streptomyces sp. A 92308110 is cultured at a suitable temperature in various culture media using appropriate nutrients and minerals and anaerobic and immersion cultivation procedures. The fermentation medium typically comprises a suitable carbon source, nitrogen sources, and mineral salts, including salts containing trace elements, all of which may be added in the form of individual substances or as complex mixtures, for example occurring in biological products from different sources.

Example 1 of the compound to achieve properties describes the original conditions under which formula I was obtained. Improvement of yield can be achieved by optimizing the culture (aeration, temperature, pH, and amount of carbon and nitrogen sources, amount of mineral salts and trace elements).

and changes in fermentation

Example 1

Cultivation of species A 92308110.

(a) initial agar culture

Agar cultures of species A 92308110 are cultured for 10-14 days at 27 ° C on the following agar medium:

glucose ii, o g soluble starch 20,0 g yeast extract 5.0 g (Gistex, Gist Brocades) NZ-amine, type A (Sheffield) 5.0 g calcium carbonate 1.0 g agar (Bacto) 150.0 g demineralized water up to 1000 ml The pH of the medium is adjusted to 6.6-6.8 with NaOH / H ₂ SO ₄ and then with

sterilized for 20 min at 121 ° C.

The cultures can be stored at -25 ° C to -70 ° C. The suspension in the glycerol-peptone mixture can be stored in liquid nitrogen.

(b) preculture

The spores and mycelium of the ten starting cultures are suspended in 100 ml of 0.9% saline. 50 ml of this solution is then inoculated into two 2000 ml Erlenmeyer flasks, each containing 1 liter of preculture medium.

Composition of preculture medium:

technical glucose 7.50 g glycerin 7.50 g yeast extract (BBL) 1.35 g liquid malt extract (Waer) 7.50 g soluble starch 7.50 g NZ-amine, type A (Sheffield) 2.50 g soy protein 2.50 g L (-) asparagine 1.00 g CaCO ₃ 0.05 g NaCl 0.05 g kh ₂ Mon ₄ 0.25 g k ₂ hpo ₄ 0.50 g MgSO ₄ .7H ₂ O 0.10 g trace element solution 1 ml agar (Bacto) 1 g demineralized water to 1000 ml

The pH of the medium is adjusted to 6.6-6.8 with NaOH / H ₂ SO ₄ and then sterilized for 20 min at 121 ° C.

Composition of trace element solution A:

FeSO ₄ .7H ₂ O ZnSO ₄ .7H ₂ O MnCl ₂ .4H ₂ O CuSO ₄ .5H ₂ O CaCl ₂ -6H ₂ O

5.0 g 4.0 g

20.0 g 0.2 g 2.0 g

Η ₃ ΒΟ ₃

ΚΙ

H ₂ SO ₄ (95%) ο, ι g

0.05 g 1 ml demineralized water to 1000 ml

The pre-cultured cells are fermented for 24 h at 27 ° C on a rotary shaker at 200 rpm with an eccentricity of 50 mm.

(c) the first intermediate

Each of the two 75 liter bioreactors containing 50 liters of preculture medium is inoculated with 1 liter of intermediate and fermented at 270 ° C for 96 hours. The machine is rotated at 150 rpm. Air is supplied at a rate of 0.5 l / min per liter of medium.

d) a second intermediate

Each of the two 750-liter fermentation vessels containing 500 liters of preculture medium is inoculated with 50 liters of the first intermediate. The second intermediate was incubated at 27 ° C for 70 hours. The machine is rotated at 100 rpm. Air is fed at a rate of 0.8 l / min per liter of medium.

(e) main cultivation

Each of the two 5000 liter bioreactors containing 3,000 liters of the main medium is inoculated with 250 and 250 ml, respectively. 300 liters of the second intermediate and incubated at 24 ° C for 96 hours. The apparatus was rotated at 45 rpm. Air is supplied at a rate of 0.5 l / min per liter of medium.

The composition of the main culture medium is as follows:

technical glucose 20 g liquid malt extract (Waer) 2 g yeast extract (Bacto) 2 g

Soytone (Bacto) 2 g

KH ₂ PO ₄

0,2 g and _{2 to 4}

_MgSO4 .7Η ₂ Ο

NaCl

CaCl ₂ .6H ₂ O trace element solution B agar (Bacto) demineralized water The pH is adjusted with KOH / HCl for 20 min at 121 ° C.

0.4 g 0.2 g

0.05 g 0.05 g ml i g to 1000 ml at 6.3. The medium is sterilized

Composition of the trace element solution

FeSO ₄ .7H ₂ O

ZnSO ₄ .7H ₂ O

MnCl ₂ .4H ₂ O

CuSO ₄ .4H ₂ O (NH ₄ ) gMO-γ ₂₄

CaCl ₂ · 6H ₂ O h ₃ or ₃

KI

H ₂ SO ₄ (95%) demineralized water

Optimized culture soy flour glycerol

MES demineralized water is as follows:

5.0 g 4.0 g 2.0 g 0.2 g 0.2 g 1/0 g 0.1 g

0.05 g 1 ml to 1000 ml main culture medium

20,0 g

40.0 g 0.1 M to 1000 ml at pH 6.8

Example 2

Isolation of Sanglifehrin A, B, C and D from Streptomyces sp. A 92308110

The first isolation and characterization of the 4 new CBA active metabolites was performed from two 3000 L fermentation tanks by active fractionation and HPLC and thin layer chromatography analysis. The above-described CBA (cyclophilin binding assay) was used to test for biological activity.

In particular, two fermentations of 3000 liters are carried out. 1500 liters of each fermentation were stirred with 2000 liters of ethyl acetate in a 4000 liter stainless steel vessel for 20 hours. The separation of the organic phase is carried out by means of an apparatus

Westfalia-Separator, type SA-20. The ethyl acetate extract was washed twice with 80 liters of water and evaporated to dryness under reduced pressure. 1.64 resp. 2 kg of extract. Both crude extracts are defatted by three-stage extraction with 40 liters of ethanol / water 9: 1 and 40 liters of hexane. Evaporation to dryness under reduced pressure gives 1.34 kg of extract.

The fat-free extract is purified twice by chromatography (670 kg each) on a 10 kg Sefadex H column in methanol. Each portion was dissolved in 3.3 liters of methanol when applied to the column. After separation of the first 15 liters of eluate as fraction 1, chromatography is continued by collecting 2 liter fractions. The most active fractions were 2, 3 and 4 and were therefore combined to give 146 g. This material was further chromatographed on 1 kg of silica gel (Merck 0.04-0.063 mm) with the eluent: methyl t-butyl ether (MTBE), MTBE / 5% methanol and MTBE / 10% methanol. Fractions of two liters are collected. Fractions 5 to 9 are the most active and therefore combined to give 44.8 g. This material was subdivided into a 1 kg silica gel column (Merck) 0.04-0.063 mm with a hexane / acetone gradient of 7: 3 to acetone. Fraction 6 (0.7 g) was further separated from this chromatography on a 3 kg column of Lichroprep RP18 (Merck) 40-63 μπι with a 94: 6 methanol / water eluent (fractions 4-7 → 2.16 g). and then on a 100 g silica gel column

H with eluent methylene chloride and 3% methanol (733 mg), a 3 kg column of Lichoprep RP18 with 9: 1 methanol / water (621 mg) and then per 100 g of Lichoprep RP18 with 1: 1 acetonitrile / water eluent to give 324 mg pure Sanglifehrin A (m.p. 142-145 ° C (amorphous material) (a) D 25 = -67.30 (c = 0-988, methanol)).

Fractions 5 and 7 from the hexane / acetone column were combined (7.1 g) and purified on a 3 kg Lichoprep RP18 40-63 μπι column with a 9: 1 methanol / water eluent (769 mg). column of 100 g silica gel H with eluent MTBE / 3% methanol (309 mg) and finally on 100 g silica gel H with eluent methylene chloride and 3% methanol to give 90 mg of pure Sanglifehrin B (m.p. 117-121 ° C (amorphous)), (o:) D25 = -52.80 (c = 1-128 in methanol)).

Fractions 9 and 10 (2.147 g) from chromatography with methanol / water 94: 6 per 3 kg Lichoprep RP18 are then purified on 100 g silica gel H with methylene chloride / 5% methanol (800 mg) and finally on 3 kg Lichoprep RP18 with the mixture methanol / water 9: 1 to give 480 mg of Sanglifehrin C (mp 165-170 ° C, (a) D 25 = -35.60 (c = 0-736 in methanol)).

Fractions 11 and 12 (835 mg) from chromatography with eluent methanol / water 94: 6 per 3 kg Lichoprep RP18 are purified on 100 g silica gel H with MTBE / 5% methanol to give 140 mg of Sanglifehrin D (m.p. 137-142 ° C). , amorphous substance)

The sanglifehrins A, B, C and D were then characterized by UV, IR, mass and NMR spectroscopy. The results obtained are shown in Table 4 and in the accompanying Figures 4.

Table 4

Sanglifehrin A summary formula: C 8 H ₉₁ N ₅ O ₁₃ (1090.4).

UV (MeOH): 275 (1962), 242 (54500), 197 (75755);

H ⁺ : 275 (1635), 242 (51883)

OH: 292 (1973), 242 (60495)

IR spectrum: Figure 6 Mass spectrum: FAB 1096 [MH + Li] ⁺ : Figure 2

NMR spectrum: Figure 9

Sanglifehrin B Summary formula: ^c 6o ^H 9O ^N 5 ° 12 ( ^1072/4 )

UV (MeOH): 273 (4395), 242 (50600), 197 (78577);

IR spectrum: Figure 5 mass spectrum: FAB 1098 [MH + Li] ⁺ : Figure 1 NMR spectrum: Figure 11

Sanglifehrin C summary formula: ^c gi ^H 93 ^N 5 ° i3 (1104.4)

UV (MeOH): 275 (1876), 242 (51557), 197 (72643) H ^{@ +} : 275 (1391), 242 (50120)

OH: 292 (1832), 242: 57960

IR spectrum: Figure 8 mass spectrum: FAB 1110 [MH + Li] ⁺ : Figure 4 NMR spectrum: Figure 12

Sanglifehrin D summary formula: Cg ₁ Hg] _ Ν ₅ Ο ₁₂ (1086,4)

UV (MeOH): 273 (3194), 242 (47584), 197 (73766) H < ^{+ >} : 273 (3237), 242 (46389)

OH: 285 (2600), 242: 52907

IR spectrum: Figure 7 mass spectrum: FAB 1092 [MH + Li] ⁺ : Figure 3 NMR spectrum: Figure 10

Sanglifehrin A

Example 3 - Transformation of Sanglifehrin A to Sanglifehrin C

To a stirring cold (0 ° C) solution of 20 mg (18.3 μιηοΐ) of Sanglifehrin A in 0.5 mL of methanol was added 1 crystal of paratoluenesulfonic acid monohydrate. The resulting yellow solution was stirred for 1 hour and quenched with saturated aqueous sodium bicarbonate. The resulting mixture was extracted twice with ethyl acetate. The organic solution was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (95: 5 methyl tert-butyl ether: methanol) to give Sanglifehrin C and its C53 epimer, Sanglifehrin C having the (S) configuration as described below (R = Me).

mixture of diastereomers 4: 1

HO

HO

Alternatively, this transformation is carried out using another antidote acid (such as pyridiniumparatoluene sulfonate, hydrochloric acid or sulfuric acid) or Lewis acid (such as zinc chloride, bromide or magnesium chloride, titanium tetraisopropoxide or boron trifluoride) in methanol. The use of other alcoholic solvents or co-solvents such as ethanol, isopropanol, butanol, allyl alcohol, propargyl alcohol, benzyl alcohol leads in the same way to analogues where R in the above formula is 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 Sanglifehrin C to Sanglifehrin A

A solution of 550 mg (0.50 mmol) of Sanglifehrin C in 5 mL of a 4: 1 THF-water solution was treated with 0.5 mL of 2N appropriate sulfuric acid and stirred for 1.5 hours. Quench the reaction with saturated aqueous sodium bicarbonate and extract twice with ethyl acetate. The organic solution was washed with saturated aqueous sodium bicarbonate solution and twice with saturated brine solution, dried over anhydrous magnesium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (90:10 methyl tert-butyl ether: methanol) to obtain Sanglifehrin A as a white amorphous powder.

Suitable paratoluenesulfonic acids

Other inorganic and organic acids, preferably organic additional acids, may be used on the water-containing carrier.

are hydrochloric acid, acid or other sulfonic acids, pyridiniumparatoluene sulfonate, acetic acid, trifluoroacetic acid, formic acid. Suitable organic co-solvents are acetonitrile, dimethylformamide, dimethylsulfoxide, dioxane.

These reactions are accompanied by the formation of varying amounts of a compound of formula XV, inter alia depending on the reaction time (a more suitable procedure leading to a compound of formula XV see Example 5 below).

Analogously, Sanglifehrin D can be transformed into Sanglifehrin B.

Example 5 - Transformation of Sanglifehrin A to Compound of Formula XV

To a stirred cold (0 ° C) solution of 50 mg (46 gmol) of San 50 glifehrin Αν, 1.9 mL of acetonitrile was added 0.1 mL of hydrogen fluoride in pyridine. The resulting yellow solution was stirred for 1 hour and quenched with saturated aqueous sodium bicarbonate. The resulting mixture was extracted twice with ethyl acetate. The organic solution was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue is purified by silica gel column chromatography (95: 5 methyl tert-butyl ether: methanol) to give the compound of formula XV as a white amorphous powder.

(XV)

Convert Sanglifehrin B to XVI. These substances exist as an absolute configuration was not

In an analogous manner, a compound of the general formula can be one epimer per C53, but unambiguously determined.

HO ti (xvi)

Compound of Formula XV: MS m / z 1078 [M + Li] ⁺ (rel.

intensity 100); ^{@ 1} H NMR (DMSO) only characteristic signals) δ 0.40 (3H, d, H-50), 1.20 (3H, with, H-54), 1.69 (3H, s, H-49) 4.20 (1 H, t, H-15), 4.58 (1 H, dd, H-17), 5.19 (1 H, dd, H-18) 5.28 (1 H, dd, H-23), 5.62 (1 H, m. H-21), 5.67 (1 H, m, H-27) dd, H-20) 5, 99 6.22 (1 H, (1 H, d, H-25), 6.03 (1 H, dd, H-26). dd, H-19), 6.14 (1 H,

Example 6 - Transformation of Compound (XV) to Sanglifehrin A

To a stirred solution of 54 mg (50 μιηοΐ) of the compound of formula XV in 0.5 mL of THF-water 4: 1 was added 50 μΐ of 2N aqueous sulfuric acid. The resulting yellow solution was stirred at ambient temperature for 12 hours and quenched with saturated aqueous sodium bicarbonate. The resulting mixture was extracted twice with ethyl acetate. The combined organic solution was washed with saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (90:10 methyl tert-butyl ether: methanol) to afford Sanglifehrin A as a white amorphous solid.

In an analogous manner, a compound of formula XVI can be converted to Sanglifehrin B.

The procedures described in Examples 3 to 6 can be used as a selective sequence of intramolecular protecting reactions and reactions by which protecting groups are removed. Thus, by the reaction described in Example 5, the hydroxyl group at the 15-position can be selectively protected, allowing selective manipulation of the remaining free hydroxyl groups. The procedure described in Example 5 allows selective protection of the hydroxyl groups at both the 15-position and the 17-position. Both procedures can also be used in the intramolecular protection of the C53 ketone. The hydroxyl and ketone groups can be regenerated by the reactions described in Examples 4 and 6. Sanglifehrins C and D, as well as compounds of formulas XV and XVI, are therefore important intermediates for the formation of other Sanglifehrins.

Example 7 - Preparation of 16-dehydro-17-dehydroxySanglifehrin A (Compound of Formula XVII)

A solution of 54 mg (50 µmol) of the compound of formula XV and a crystal of paratoluenesulfonic acid monohydrate in 1 mL of 4: 1 acetonitrile-water was heated at 80 ° C for 1.5 h. The reaction mixture was quenched with saturated aqueous sodium bicarbonate. The resulting mixture was extracted twice with ethyl acetate. The organic solution was washed with saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (90:10 methyl tert-butyl ether: methanol) and reverse phase chromatography (RP18, 50:50 acetonitrile-water to acetonitrile over 45 minutes) to give the pure title compound as a white amorphous solid.

MS m / z 1078 [M + Li] ⁺ (rel intensity 100), 1 H NMR (DMSO) (only the characteristic signals are shown) δ 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 (1H, d, C 15 -OH), 6.03 (1H, d, H-25), 6.09-6.28 (4H, m,

H-18, H-19, H-20 and H-26), 6.37 (1 H, d, H-17).

Example 8 - Preparation of 42-N-methyl-Sanglifehrin A (Compound of Formula XVIII)

To a stirred, cold (-15 ° C) solution of 109 mg (0.1 mmol) of Sanglifehrin A and 67 μΐ (0.3 mmol) of 2,6-di-tert-butylpyridine in 1 mL of CH ₂ Cl ₂ is added 16.5 μΐ methyl triflate. The mixture was allowed to warm to room temperature and stirred for an additional 6 hours and then quenched with saturated aqueous sodium bicarbonate. The resulting mixture was extracted twice with ethyl acetate. The organic solution was washed with brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by two successful silica gel column chromatography (90:10 methyl tert-butyl ether: methanol, then 95: 5 methyl tert-butyl ether: methanol) to give the pure title compound as a white amorphous solid.

MS m / z 1110 [M + Li] ⁺ (rel intensity 100), 1 H NMR (DMSO) (only the characteristic signals are shown) δ 1.70 (3H, s, H-49), 2.06 ( 3H, s, H-54), 3.53 (3H, s, 42 N-Me), 3.98 (1H, d,

C31-OH), 4.50 (1H, d, H-65), 4.77 (1H, d, C17-OH), 5.43 (1H, d, C15-OH), 5.49 (1H, d, C35-OH), 7.50 (1H, d, H-12), 8.11 (1H, d, H-9), 9.22 (1H, s, C61-OH).

Example 9 - Preparation of 53-Dihydro-Sanglifehrin A (Compound of Formula XIX) (XIX)

Η (

To a stirred cold (0 ° C) solution of 54 mg (50 gmol) of Sanglifehrin A in 0.5 mL of methanol was added 2.8 mg (75 µmol) of sodium borohydride. Stirring is continued for 1 hour and saturated aqueous sodium bicarbonate solution is added. The resulting mixture was extracted twice with ethyl acetate. The organic solution was washed twice with brine, dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (95: 5 methyl tert-butyl ether: methanol, then 90:10 methyl tert-butyl ether: methanol) to give the pure title compound as a white amorphous solid. The isolated product corresponds approximately to a 1: 1 mixture of diastereoisomers on C-53.

MS m / z 1098 [M + Li] ⁺ (rel intensity 63), 1104 [M + 2 Li-H] ⁺ (rel intensity 100); ¹ H NMR (DMSO) (only the characteristic signals are given) δ 0.62 (3H, d, H-50), 1.02 (3H, d, H-54), 3.55 and 3.59 (1H 2m, H-53).

Example 10 - Preparation of 53-tosyloiddrazone-Sanglifehrin A (Compound of Formula XX)

HC

(XX)

A mixture of 55 mg (50 µl) of Sanglifehrin A and 23 mg (125 µl) of tosyl hydrazide in 0.5 ml of dichloromethane was stirred at room temperature for 6 hours. The solvent was removed and the residue was purified by silica gel column chromatography (90:10 methyl tert-butyl ether: methanol) to give the title compound as a white amorphous powder.

MS m / z 1264 [M + Li] ⁺ (rel intensity 100), ¹ H NMR (DMSO) (only the characteristic signals are shown) δ 1.70 (3H, s, H-49), 1.77 (3H) , s, 54-H), 2.37 (3H, s, -NSO ₂ C _g H ₄ CH _3), 6.51 (1H, s, H-60), 6.59 (2H, 2d, H 62 and H-64), 7.06 (1H, dd, H-63), 7.35 (2H, d, tosyl meta protons), 7.73 (2H, d, tosyl para protons).

Example 11 - Preparation of 26S, 27S-dihydroxy-Sanglifehrin A (Compound of Formula XXI) and 26R, 27R-dihydroxy-Sanglifehrin A (Compound of Formula XXII)

(XXII)

To a stirring cold (0 ° C) solution of 495 mg (1.5 mmol) of potassium ferrocyanide, 207 mg (1.5 mmol) of potassium carbonate, 19.5 mg (0.025 mmol) (DHQ) ₂ PHAL, 65 μΐ (0.005) mmol)

0.08 M osmium tetroxide in t-butanol and 95 mg (1 mmol) of methylsulfonamide in 2.5 ml of t-butanol and 5 ml of water are added a solution of 545 mg (0.5 mmol) of Sanglifehrin A in 2.5 ml of t-butanol. butanol. The resulting biphasic mixture was allowed to warm to room temperature and stirred for 3 hours. 1.08 g (8.6 mmol) of sodium sulfite was added, followed by ethyl acetate and water, and the mixture was vigorously stirred for 15 minutes. The layers were separated and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by reverse phase chromatography (RP18, 30:70 acetonitrile-water to acetonitrile over 60 min) to give 26S, 27S-diol 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), ¹ H NMR (DMSO) (only the characteristic signals are shown) δ 1.64 (3H, s, H-49) 2.06 (3H, s, H-54), 3.20 (1H, broad, H-27), 3.45 (1H, broad, H-31), 3.94 (3H, m, H- 17, H-26 and C 31 -OH), 4.30 (1H, d, C 27 -OH), 4.57 (1H, d, C 26 -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 (1 H, dd, H-20).

26R, 27R-diol: MS m / z 1130 [M + Li] ⁺ (rel intensity 100), ¹ H NMR (DMSO) (only the characteristic signals are shown) δ 1.64 (3H, s, H-49) 2.06 (3H, s, H-54), 3.16 (1H, broad, H-27), 3.48 (1H, broad, H-31), 3.94 (3H, m, H- 17, H-26 and C 31 -OH), 4.30 (1H, d, C 27 -OH), 4.57 (1H, d, C 26 -OH), 5.20 (1H, dd, H-23),

5.35 (1H, d, H-25), 5.57 (3H, m, H-18, H-21 and C35-OH), 6.03 (1H, dd, H-19), 6.14 (1 H, dd, H-20).

Example 12 - Diol Cleavage in 26S, 27S-Dihydroxy-Sanglifehrin A

To a solution of 90 mg (79 gmol) of 26S, 27S-diol in 0.9 mL of mixture

THF-water 2: 1 was added 33 mg (157 gmol) of sodium periodate stirring was continued for 1 hour and saturated aqueous sodium bicarbonate solution was added. The mixture was extracted twice with ethyl acetate. The organic anhydrous sulfate by purifying the solution was washed with brine, dried over sodium, filtered and evaporated. The residue is chromatographed on silica gel (95: 5 methyl tert-butyl ether: methanol) to give compounds of general formula XXIII (foam) and XXIV (powder).

(XXIII)

(XXIV)

A compound of formula XXIII: MS m / z 366 [M + HH ₂ O] ⁺ (rel. Intensity 100); ¹ H NMR (DMSO) (2: 1 mixture of OH _{and x} OH _eg epimers at the anomeric center) (referred to as characteristic signals only) δ 3.54 and 4.08 (1H, 2m, H-31), 3.57 (1H, broad m,

H-35), 3.66 (1H, m, H-33), 4.38 (0.67H, ddd, H-27 _ax ), 4.95 (0.33H, broad, H-27 _eg ), 5.40 (0.33H, d, C 27 -OH _eg ), 5.59 (0.33H, d, C 35 -OH), 5.61 (0.67H, d, C 35 -OH), 5.96 ( 0.67H, d, C 27 -OH _ax ), 7.89 (0.67H, s, NH-42), 7.91 (0.33H, s, NH-42).

Compound of Formula XXIV: MS m / z 745 [MH-Li] ^{+ 1} H NMR (DMSO) (only the characteristic signals are shown) δ 0.64 (3H, d, H-50), 0.81 (6H, d, H-56 and H-57), 2.06 (3H, s, H-54),

2.17 (4', s, H-14 and H-49), 3.80 (1H, broad m, H-15), 3.94 (1H, dd, H-17), 5.33 (1H, broad d, H-23), 5.62 (2H, m, H-18 and H-21), 6.89 (1H, d, H-25), 6.10 (1H, dd, H-19) 6.18 (1H, dd, H-20); 10.0 (1H, d, H-26).

Example 13 - Acetylation of Sanglifehrin A to form 61-O-acetyl-Sanglifehrin A (Compound of Formula XXV)

To a stirred cold (0 ° C) solution of 54 mg (50 μπιοί) Sanglif ehrin A and 50 μΐ pyridine in 0.5 ml dichloromethane is added 5.2 μΐ (55 μπιοί) acetic anhydride. The reaction mixture is kept at 0 ° C for 1 hour, then allowed to warm to room temperature and stirred for a further 12 hours. Saturated aqueous sodium hydrogen carbonate solution was added and the resulting mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by reverse phase chromatography (RP18, 40:60 acetonitrile / water to acetonitrile over 45 min) to give the title compound as an amorphous powder.

MS m / z 1132 [M + H] ⁺ (rel intensity 100), ¹ H NMR (DMSO) (only the characteristic signals are given) δ 1.68 (3H, s, H-49),

2.06 (3H, s, H-54), 2.25 (3H, s, CH ₃ CO ₂ ), 4.04 (1H, d,

C31-OH), 4.67 (1H, d, C2-NH), 4.76 (1H, d, C17-OH), 5.42 (2H, m, H-8 and C15-OH), 57 (3H, m, H-18, H-21 and C35-OH), 6.85 (1H, s, H-60), 6.98 (1H, d, H-62), 7.06 (1H) d, H-64), 7.31 (1 H, dd, H-63), 7.51 (1 H, d, H-12), 7.89 (1 H, s, H-42), 8.23 (1 H, d, H-9).

Industrial usability

The agents of the present invention are useful as pharmaceutical compositions, for example, as immunosuppressants as well as anti-inflammatory agents.

They are particularly useful in preventing acute and / or chronic organ or tissue rejection in allo- or xenotransplantation, for example in the treatment of a recipient following a heart, lung, heart and lung transplant, liver, kidney, pancreas, skin or corneal transplant. They are also useful in preventing graft-versus-host diseases, such as the consequences of bone marrow transplantation.

Claims (27)

PATENT CLAIMS 1. A macrolide in which (i) a piperidazinylcarboxylic acid residue is attached at positions 2a to 6 of the macrocyclic ring; and / or ii) an aromatic α-amino acid residue is attached at positions 7 to 9 of the macrocyclic ring; and / or iii) at positions 10-12 of the macrocyclic ring, an aliphatic α-amino acid residue, in free or protected form, or in the form of a salt thereof, is attached. A macrolide according to claim 1 comprising two or preferably all three structural features i), ii) and iii). A macrolide according to claim 1 or 2, wherein the macrocyclic ring moiety comprises a hydroxycarboxylic acid residue having a chain length of from 6 to 20, preferably 11 carbon atoms. A macrolide according to claim 3, wherein the hydroxycarboxylic acid residue is a radical of formula II: R ³ R ⁴ R ¹ R ² 0 _{(CH2) 2} I CH-CH ₂ -CH = CH-CH = CH-CH-C-CH-CH-CO- (II) II o ch ₃ where R ¹ and R ² are hydrogen or together represent a bond; Q R is hydrogen; R ⁴ is -CO-CH-j -CHĺOíO or a group-CH or R ^S and R ⁴ together represent a radical of the formula III: ch ₃ I C '(III) OCH ₃ in free or protected form or in the form of A macrolide according to any one of claims 1 to 5, a macrocyclic ring of formula IV: salt. containing (IV) wherein the groups X, Y and Z are residues i), ii) and iii) as defined in claim 1 and A is a hydroxycarboxylic acid residue as defined in claim 3 or 4, in free or protected form, or in the form of a salt. A macrolide according to claim 5 comprising a macrocyclic ring of formula V: KO (V) wherein the group A is as defined in claim 5, in free or protected form or in the form of a salt thereof. A macrolide according to any one of the preceding claims which is substituted on a carbon atom adjacent to the oxy group of the lactone bridge by a 2-oxy-2'-base-3'-oxospirobicyclohexan-3-yl radical. The macrolide according to claim 7, wherein the spirobicyclohexyl radical has the general formula VI wherein -ab- is - (Me) C = CH- or - (Me) CH-CH (OH) and R ₅ is hydrogen or methyl, in free or protected form, or a salt thereof, attached to the macrolide ring via a linker a group comprising a linear sequence of 6 to 11, typically 9 carbon atoms between the spiro-residue and the macrolide ring. The macrolide of claim 8, wherein the linking group between the spiro moiety and the macrolide ring is a group of formula VII R ⁶ R ⁷ CH ₃ c-CH ₂ -CH (OH) -CH (CH ₃ ) - (CH ₂ ) ₂ - CH-CH-CH = Cd (VII) wherein c is a bond to a spiro residue; d represents a bond to the macrocyclic ring and R and R are hydroxy or together represent another bond, in free or protected form. A compound of formula VIII S-L-M (VIII) wherein S represents a 2-oxy-2'-base-3'-oxospirobicyclohexan-3-yl radical L represents a linker comprising a linear sequence of 6 to 11, typically 9 carbon atoms, and M represents a macrolide ring as defined in any one of claims 1 to 6, in free or protected form or in salt form. A compound according to claim 10 having the formula IX wherein -a-b- is as defined above; -e-f- is -CH (OH) -CH (OH) - or -CH = CH-; -g-h- has the same meaning as defined above for the group -a-b-, and R ³ , R ⁴ and R ⁵ are as defined above, in free or protected form or in salt form. A compound according to claim 11 having the following conformation: wherein, when -a-b- is - (Me) CH-CH (OH) -, the group preferably has the configuration: X CH 3 when -e-f- is -CH (OH) -CH (OH) -, the group preferably has the (S), (S) or (R), (R) configuration; when -g-h- is - (Me) CH-CH (OH) -, this group preferably has the configuration: CH ₃ when -gh- is - (Me) C = CH-, preferably has the following configuration: and when and R- are connected, they are preferably of the configuration: 13. 14. ,, v 'ock ₃ Sanglifehrin selected from the group consisting of Sanglifehrin A, B, C and D. A macrolide according to any one of claims 4 to 9 or a compound according to any one of claims 10 to 12, wherein the positions 14 to 17 of the macrocyclic ring contain a radical of formula X: CH, CH (X) 16 I - CH — CH — CH — CH17 I 15 1 < CH for example with the configuration: 15 Dec Compound of formula XI: R ⁶ -XYZA-OH (XI) for example a compound of formula XII: (XII) for example a compound of formula IX 'wherein X, Y, Z, A, R ³ , R ⁴ and R ⁵ are as defined above and R ⁶ is a hydrogen atom or a C 1 -C 4 alkyl group, for example a methyl group, in free or protected form or in salt form. A macrolide according to any one of claims 1 to 9 in the form of an open ring, wherein the open macrocyclic ring is in free or protected form or in salt form. A compound of the formula R 6 O-X-Y-Z-A-OH, wherein X, Y, Z and A are as defined in claim 5 and R ⁶ is hydrogen or C 1-4 alkyl, in free or protected form, or in the form of a salt thereof. A compound of the general formula R ⁶ OXYZ-A'-CH (OH) -LS, wherein the group A'-CH (OH) - is a hydroxycarboxylic acid residue as defined in claim 3 and the other symbols are as defined in claim 17, in free or protected or salt form. 19. A compound of formula XII ': R 2 O: or-N-CO-CH-NH-CO-CH-NH-A'-CH-L S. ° i 'n O CH ₂ t ha ck _{3 CH3} (XII') in free or protected form or salt form. 20. Compound of formula (IX ''): in free or protected form or in salt form. A compound in which the 2-oxy-2'-aza-3'-oxo-3 ¹ -ylspirobicyklohexánový ring system in the form of an open ring, 21st for example, a compound of formula XII M (XII) wherein a, b, L and M are as defined above, in free or protected form or in salt form. 2-Oxy-2'-base-3'-oxo-3'-ylspirobicyclohexane in free or protected form or in salt form, in particular a compound of the general formula VI ¹ : (VI -) wherein R ₇ is a hydrogen atom, an optionally protected hydroxyl group, a reactive functional group, or a -CH ₂ -CH (OH) -CH (CH ₃ ) -CH ₂ -CH ₂ -CHO group, or a lactol delta thereof equivalent, in free or protected form or in salt form. 23. 2-oxy-2'-aza-3'-oxo-3 ¹ -ylspirobicyklohexán open ring, in free or protected form, or a salt thereof, especially a compound of formula XII ': wherein a, b and R are as defined above, in free or protected form or in salt form. 24. Macrolide of the general formula XIII: CH _of COr, in which M (XIII) wherein M is a macrolide ring is as defined above, in particular a macrolide of formula XIV: (XIV) in free or protected form or in salt form. 25. A process for the preparation of compounds as defined above, comprising: ΊΟ i) for the preparation of any Sanglifehrin A, B, C and D, culturing the Sanglifehrin A, B, C or D producing actinomycetes strain in the culture medium and isolating the desired Sanglifehrin A, B, C, or D from the culture solution obtained; ii) for the preparation of Sanglifehrins C and D, cyclizing Sanglifehrins A and B at positions 15 and 16; iii) for the preparation of Sanglif ehrins A and B, opening the lactone ring of Sanglif ehrins C and D at positions 5 and 16; iv) for the preparation of a macrolide of formula IX or IX ', wherein -gh- is C (CH 2) = CH-, dehydrating a compound of formula IX or IX', wherein -g -h is C (CH ₃ ) -CH (OH) - or a protected form thereof; v) for the preparation of a macrolide of formula IX or IX 'wherein R * is ΟΗ (ΟΗ) -ΟΗβ, hydrogenating a compound of formula IX or IX' wherein R ₄ is C (O) -CH ₃ ; (vi) for the preparation of a macrolide of formula IX or IX ', wherein the positions 14 and 16 of the macrolide ring contain a residue of formula X: CH CH) ^Λ 2 ¹ 2 (X) CH-CH "CH-CH- 15 14 CHreaction causing internal protection of a compound of formula IX or IX '; vii) for preparing a macrolide of formula IX or IX ¹ , reacting a compound of formula IX or IX IX ', wherein the positions 14 to 16 of the macrolide ring contain a radical of formula X CH. J oo is i - CH - CH - CK 17 | 15 ch ₃ (X) • CH · to remove the internal protecting group; viii) for preparing a macrolide of formula IX or IX ', wherein R ⁵ is methyl, methylating a macrolide of formula IX or IX', wherein R is hydrogen; ix) for the preparation of a macrolide of formula IX or IX 'in which R ^{4 is} in the O-protected form, an O-protection of a macrolide of formula IX or IX' in which R ^{5 is} in the O-protected form; x) for the preparation of a macrolide of formula IX or IX wherein R ^{4 is} in the O-protected form, removing the O-protecting group of the macrolide of formula IX or IX 'wherein R ^{5 is} in the O-protected form; (xi) for the preparation of a macrolide of formula IX or IX 'containing an O-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, O-protection of a macrolide of formula IX or IX 'containing an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring; (xii) for the preparation of a macrolide of formula IX or IX 'containing an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, removal of the O-protecting group of a macrolide of formula IX or IX' containing at the 7 to 10 positions of a macrocyclic ring an O-protected hydroxyphenylalanine residue; xiii) for the preparation of a macrolide of formula IX or IX 'in which -ef- is a CH (OH) -CH (OH) - group, oxidative hydrolysis of a macrolide of formula IX or IX' in which -ef- is a group - CH = CH; xiv) for preparing a compound of formula V or a compound of formula XII, reacting a compound of formula IX or IX 'to cleave a linking group between a spirobicyclo group and a macrocyclic ring; xv) for the preparation of a compound of formula R ⁵ OXYZA-OH or of formula R 6 OXYZA '-CH (OH) -LS, ring opening of a macrocycle of formula IV or a macrocyclic ring of a compound of formula VIII; xvi) for preparing a macrolide of formula IX or XII in the form of a closed ring, reacting a compound of formula R OXYZA-OH or formula R ⁶ OXYZ-A'-CH (OH) -LS to close the macrocyclic ring; xvii) for preparing a compound of formula XII or XII ', reacting a compound of formula IX or VI' to open a ring within a spirobicyclic ring system; and xviii) for preparing a compound of formula IX or VI ', reacting a compound of formula XII or XII' to ring-closure within a spirocyclic ring system 26. The strain of Aktinomycetes, which produces a macrolide in which (i) a piperidazinylcarboxylic acid residue is attached at positions 2a to 6 of the macrocyclic ring; and / or ii) an aromatic α-amino acid residue is attached at positions 7 to 9 of the macrocyclic ring; and / or iii) an aliphatic α-amino acid residue is attached at positions 10-12 of the macrocyclic ring 27. A biologically pure isolate of Streptomyces sp. A92308110 (DSM 9954) or a mutant, recombinant, or modified form thereof capable of producing a macrolide according to the present invention. 28. A process for the preparation of a macrolide according to the present invention comprising culturing a strain of Streptomyces sp. A92308110 (DSM 9954) or a mutant, recombinant, or modified form thereof, in a suitable culture medium and optionally isolating Sanglinfehrin. 29. The method comprising administering to a patient an effective amount of an agent of the present invention. 30. Method: (i) preventing acute and / or chronic organ rejection following allo- or xenotransplantation, for example, to treat a transplant recipient of any particular type described above; or ii) preventing disease transplanted tissue versus recipient, for example, in bone marrow transplant recipients; or iii) treating diseases of the autoimmune response or treating any such diseases or conditions listed above; or 4) treating asthma in patients in need of such treatment comprising administering to the patient an effective amount of a compound of the present invention. A compound of the present invention for use as a medicament, for example, for use as an immunosuppressant or for the treatment of any of the diseases or conditions mentioned in Part B above. 32. A pharmaceutical composition comprising an active ingredient of the present invention in association with a pharmaceutically acceptable diluent or carrier. 33. Use of a compound of the present invention for the preparation of medicaments for use as an immunosuppressant or for use in the treatment of any of the diseases or conditions mentioned in B above. Use of a compound according to any one of claims 1 to 6, 8 to 10, 15 or 16 as substitution immunoassay reagents for compounds that bind cyclosporins or other cyclophilin.