MXPA98000217A - Macroli - Google Patents

Abstract

A novel class of macrolides is provided wherein: i) the positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinylcarboxylic acid residue, and / or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by a residue of - aromatic amino acid, and / or iii) the positions 10-12 inclusive of the macrocyclic ring are provided by an aliphatic amino acid residue, preferably comprising 2, or especially the three characteristic structural features i), ii), and iii), more especially a compound of the formula IX: (see formula) which have immunosuppressive and anti-inflammatory properties, and protected and open ring forms of the same

Description

MACROLIDOS The present invention relates to a class of macrolides having a valuable pharmaceutical and related activity. For convenience, compounds of the novel class of macrolides are collectively referred to herein as "Sanglifehrins". The first of the Sanglifehrins were isolated from actinomycete fermentation broths. These are the Sanglifehrinas A to D of the formulas: As you can see, the macrocyclic ring of the Sanglifehrins A to D is of an entirely novel structure, characterized in that: i) the β-positions comprise a 3-carboxypiperidazinylcarboxylic acid residue, ii) the 7 to 9 positions, an a-amino acid residue, and iii) the 10-positions. to 12 are an aliphatic α-amino acid residue. The rest of the macrocyclic ring is composed of a hydroxycarboxylic acid residue, on the understanding that, in the case of the Sanglifehrins A to D, there are another 11 carbon atoms in the primary macracyclic ring. According to conventional practice in macrolide chemistry, the primary macrocyclic ring atoms of Sanglifehrin are numbered as indicated above for Sanglifehrin A, starting with the carbon atom of the carbonyl group of the macrocyclic lactone bond as position 1. The Sanglifehrinas A to D are also characterized by the presence of a novel bicyclic spiro system at position 23 of the acrocyclic ring, by means of a hydrocarbyl linker group. Sanglifehrins A through D can undergo extensive chemical manipulation to obtain still other macrolides of the Sanglifehrin class. These manipulations include dissociation of the macrocyclic ring, in particular in the oxy group of lactone, dissociation of the linking group between the macrolide and the spiro-ring systems, and manipulation, for example, protection, derivation, or other chemical modification of the groups. of substituents; for example, as described later herein. Experts in this field may think of other means of modification. According to 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, it has been discovered that they exhibit the following combination of activities: cyclophilin binding activity; immunosuppressive activity; inhibition of proliferation of both B cells and T cells; - however, they do not have FK binding protein binding activity or calcineurin inhibiting activity.

Accordingly, it can be seen that Sanglifehrins provide an exciting and novel class of immunosuppressive and apti-inflammatory compounds. In particular, Sanglifehrins have an activity profile that differs from that of previously known immunosuppressive and anti-inflammatory compounds, such as cyclosporins and macrolides, for example, of the class of rapamycin and FK 506, indicating that Sanglifehrins have a mode of action different from those previous compounds. Accordingly, Sanglifehrins provide a novel category of drug substance, both in terms of structure and activity, which can be anticipated to materially extend the limits of immunosuppressive and / or anti-inflammatory therapy; for example, to avoid or reduce the undesirable side effects of previous immunosuppressive and anti-inflammatory therapies, and / or to improve or extend this therapy to new areas of disease or new categories of patients. Sanglifehrins, for example, in which the acrylic ring is in the open ring form, where positions 26 and 27 of the hydrocarbyl linker between the spiro and macrolide ring systems are both substituted by hydroxy, or where the spiro residue bound to the macrocyclic ring has dissociated or truncated, generally lacks some or all of the combination of activities characteristic of Sanglifehrina. For example, Sanglifehrins, in which the spiro residue is dissociated, normally possess cyclophilin binding activity, but do not possess significant immunosuppressive activity. As will be appreciated by those skilled in the art, however, these compounds provide valuable components, intermediates, or key building blocks for the preparation of other novel Sanglifehrins, and therefore, further extend the therapeutic potential of the Sanglifehrin class. In this, its presence seems to be substantial for biological activity, for example, in Sanglifehrinas A to 0, and it can also be seen that the bicyclic spiro system provides a structural component with a key biological significance, useful as a structural component for the derivation or additional modification both in relation to the production of other Sanglifehrins, and for the application in the derivation or modification of other drug substances; for example, to modify the activity of other immunosuppressant drug substances of the macrolide class. As indicated, the Sanglifehrinas represent a novel key of macrolide compounds, of an entirely novel and absolutely characteristic structure. According to the above, in a first aspect, the invention provides: a macrolide wherein: i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a residue of piperidazinylcarboxylic acid; and / or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic α-amino acid residue; and / or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic α-amino acid residue, in free or protected form, or a salt thereof.

Suitably, the macrolides of the invention comprise two, especially the three characteristic structural features i), ii), and iii). The piperidazinylcarboxylic acid residue is suitably a residue of 1,2-piperidazin-3-carboxy-1-yl, of which the carboxy moiety occupies the 1-position, and the 1-nitrogen atom, the 6-position of the macrocyclic ring, by example, a residue of formula I: 4 where the assigned numbers represent the position of the atoms of the residue ep the macrocyclic ring. This residue can be substituted on the ring or unsubstituted. Properly it is unsubstituted. The a-amino moiety of the aromatic α-amino acid residue suitably occupies the 9-position of the macrocyclic ring. Suitably, the aromatic α-amino acid is a phenylalanine residue, especially 3-OH-phenylalanine, either free or protected. The α-amino moiety of the aliphatic α-amino acid residue suitably occupies position 12 of the macrocyclic ring. Suitably, the aliphatic α-amino acid residue is a valine residue in free or protected form. The remainder of the macrocyclic ring suitably comprises a hydroxycarboxylic acid residue, whose frarcínp oxy completes the macrocyclic lactone bond, and whose carbonyl fraction forms an amide bond with the ar-amino group at the 12-position of the macrocyclic ring. This hydroxycarboxylic acid residue suitably has a chain length of 6 to 20, more suitably 11 carbon atoms. It may be substituted or unsubstituted, and / or may contain one or more unsaturated bonds, in particular cumulative double bonds along its length. More suitably the remainder of the macrocyclic ring comprises a residue of 1-oxy-enancanoyl-1-yl, especially 11-oxy-6,8-endecadienoyl-1-yl, optionally substituted, for example in the 2, 3, 4 position, and / or 5. More suitably, the hydrocarboxylic acid residue is a residue of formula II: where: Rx and R2 are both H, or represent an extra link; 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 III: OCH, in free or protected form, or a salt thereof.

Preferred macrolides according to the invention, ep base to the foregoing, are those comprising a macrocyclic ring of formula IV: wherein X, Y, and Z are residues i), ii), and iii) as defined above, and A is a hydrocarboxyl pidon residue as defined above, in free or protected form, or a salt thereof: in particular a macrocyclic ring of the formula V: in free or protected form, or a salt thereof In general, the Sanglifehrinas, co o in the Sanglifehrins A to D. The macrocyclic ring is substituted in the macrocyclic ring adjacent to the oxy portion of the lactone bridge. Typically, this substituent comprises a residue of 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl, for example of the formula VI: ep where: a-b- is - (Me) C = CH- or - (Me) CH-CH (OH) -. And p; is H or Me (where Me and Et represent methyl and ethyl, respectively), in free or protected form, or a salt thereof linked to the macrocide ring by means of a linker comprising a linear sequence of 6 to 11, normally of 9 carbon atoms between the spiro residue and the macrolide ring. The linker group may be substituted or unsubstituted, and / or may contain one or more bonds, particular ep double cumulative bonds along its length. Suitably, the linker g-upo can be substituted methyl, for example by methyl groups. Suitably, the linker group can be further substituted by hydroxy, for example by three hydroxy substituents, and / or can be ethylenically unsaturated, for example, it can contain two carbon-carbon double bonds. More suitably, the linker group comprises a residue of 1-methyl-7-methyl-nonanoyl-9-yl, especially 1-methyl-7-methyl-1-nonenoyl-9-yl, or l-methyl-7- methyl-l, 3-nonadienoyl-9-yl, optionally substituted, for example in the 3, 4, and / or 8 position. Preferably, the linking group is a group of the formula VII: VII | 3 c-CH¿-CH (OH) -CHÍCHj) "(CHj). CH-C rH- • CH = c-d wherein: c represents a bond with the spiro residue, -d represents a bond with the macrocyclic ring, and R6 and R7 are each OH, or together represent an additional bond, in free or protected form.

The linker group will generally bind to the macrocyclic ring at the carbon atom immediately adjacent to the lactone oxy group, that is, when the macrocyclic ring comprises a residue of 11-oxy-enancanoyl-1-yl, at the 11-position thereof.

According to the above, the invention provides compounds of the formula VIII: S- h- M vm wherein: S represents a spirobicyclo residue as defined above; L represents a linker group as defined above, and M represents a macrolide ring as defined above, in free or protected form, or a salt thereof.

The particular compounds of the invention are those of the formula IX: 47 4ß 49 where: -a-b- is as defined above; _e-f- is -CH (OH) -CH (OH) - or -CH = CH-; -g- - is as defined above for -a-b-, and R3 »Rt. and R. are as defined above, in free or protected form, or a salt thereof.

The compounds of formulas I to IX contain asymmetric carbon atoms, and therefore, may exist in a number of epimeric forms. All possible epimers, as well as the diastereoisomeric mixtures thereof, are encompassed in the invention. However, the compounds of the formulas VIII and IX, wherein the macrolide ring is in the form of a closed ring, and which are of an appropriate stereochemistry, normally possess activities that are characteristic of the Sanglifehrinas, as previously referred to herein. The epimers that have characteristic activities of Sanglifehrina, are preferred. In general, for example, for a pharmaceutical use according to the invention, the epimers possessing characteristic activities of Sanglifehrin in a pure or substantially pure form (ie, exempt or substantially free of epimers lacking characteristic activities of Sanglifehrina), for example, which comprise at least 90 percent, for example at least 95 percent active epimer (ie, that comprise less than 10 percent, for example less than 5 percent inactive epimer) will be the preferred ones Preferably, the 3-carboxypiperidazinylcarboxylic acid residue i) in the l to β positions of the macrocyclic ring has the following configuration: Preferably, the amino acid, aromatic ii) in positions 7 to 9 of the macrocyclic ring, has the configuration L, for example it is of the configuration: Preferably, the aliphatic amino acid iii) in positions 10 to 12 of the macrocyclic ring, has the configuration L, for example is of the configuration: When the rest of the macrocyclic ring comprises a residue of formula II, it preferably has the configuration: O.

When R3 and R4 represent together: -? - OCH3 preferably has the configuration: CH,, OCH3 Preferably, the residue of 2-oxy-2'-aza-3 '-oxo-spirobicyclohexan-3-yl has the configuration: where when -a-b- is - (Me) CH-CH (OH) -, preferably has the configuration: When the linker is of formula VII, it is preferably of the configuration: When R6 and R7 are each OH, the configuration at positions 26 and 27 preferably is either 26 (S), 27 (S) or 26 (R), 27 (R). When R6 and R7 together represent an additional link, the configuration at positions 26 and 27 is preferably: The compounds of the invention of the formula IX, preferably have the following conformation: where when -a-b- is - (Me) CH-CH (OH) -, preferably has the configuration: when -e-f- is -CH (OH) -CH (OH) -, preferably has the configuration (S), (S) or the configuration (R), (R); when -g-h- is - (Me) CH-CH (OH) -, preferably has the configuration: CH r, when -g-h- is - (Me) C = CH-, erably has the configuration: CH X, "and when R3 and R4 condense with each other, they are erably of the configuration: erably, the Sanglifehrins A to C have the following configurations: The compounds of the invention may be in free or protected form, for example 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, the OH groups may be in protected form, for example in the form of silyl ethers (for example, as described on pages 68-86 of Greene and Wuts ibid.), Esters (see, for example, the pages 87-103 by Greene and Wuts ibid), and carbonates (see, for example, pages 104-111 of Greene and Wuts, ibid). These protected forms also include internally protected forms; for example, in the case of macrolides of formula IX wherein -gh- is -CH (CHj) -CH (OH) -, the protected form wherein positions 14 to 17 of the macrocyclic ring comprise a residue of the formula X: -CH-CH-CH-CH- 17 15 14 CH, for example, of the configuration: Also, for example, the 1,3-diols ent in the Sanglifehrins, can be protected as appropriate ring structures, for example, as described in the? 118-142 by Greene Wuts, ibid.

The compounds of the invention also exist in salt form. Examples of pharmaceutically acceptable salts suitable for use in accordance with the invention include acid and base addition salts, as appropriate, considering the particular substituents ent in the compound. As indicated above, the macrocyclic ring of the compounds of the invention can be dissociated, in particular in the lactone oxy group, to provide compounds wherein the macrocyclic ring is in the open ring form. In general, the dissociation of the lactone oxy group proceeds by hydrolysis (solvolysis), for example, to provide compounds of the formula XI: R «O-X-- Y- Zr-A- H XI for example, of formula XII: for example, a compound of the formula IX 1 47 4ß wherein X, Y, Z, A, R :, R (.and R; are as defined above, and R i is H or alkyl of 1 to 4 carbon atoms, for example methyl. intermediary elements for the modification of the basic macroclical ring system of Sanglifehrina, and the ent invention are also part of the ent invention In accordance with the foregoing, in a further aspect, the ent invention provides: a macrolide as defined hereinabove, in the open ring form, the open ring macrocycle being free or protected, or a salt thereof, a compound R.0-XYZA-0H, as iously mentioned, in book or protected form, or a salt of the ism, a compound D.0-yY-2-? '-CH (OH) -LS-, in donop -A'-CH (OH) - is a hydroxycarboxylic acid residue, for example a residue of the formula II, as defined above, and the other symbols are as defined above, in free or protected form ida, or a salt thereof; a compound of formula XII1: in free or protected form, or a salt thereof; a compound of formula IX ": in free or protected form, or a salt thereof.

The invention also includes compounds wherein the ring system of 2-oxy-2'-aza-3 '-oxo-31-yl-spirobi-cyclohexane is in the form of an open ring, for example a compound of the formula XII: wherein a, b, L, and M are as defined above, in free or protected form, or a salt thereof. The open-ring compounds of the invention are erably of one conformation as the erred conformations identified above for the closed-ring compounds. The open ring spirobicyclo ring system of the compounds of formula XII is erably of the conformation: where, when -a-b- is - (Me) CH-CH (OH) -, preferably has the configuration: The macrolides according to the invention, which have a spirobicyclo residue bound to the macrocyclic ring, can also be subjected to dissociation of the intervening linker group, for example, in relation to formula IX, ep epicular the bond between residues 26 and 27, to give novel novel spirobicycle compounds, and other macrolides. Also, as indicated above, these compounds are also useful as intermediates, having the spirobicyclic fraction of Sanglifehrins in particular, an integral functional role in the biological activity of the Sapglifehripas as a class.

In accordance with the foregoing, the present invention provides: un2-oxy-2"-aza-3'-o / o-3'-yl-spirobicyclohe-aro, in free or protected form, or a salt thereof, in particular a compound of the formula VI ': wherein R7 is H, an optionally protected OH group, a reactive functional group, or a group -CH2-CH (OH) -CH (CH3) -CH2-CH2-CHO, or the delta-lacto equivalent thereof, in free or protected form, or a salt thereof. Preferably, the compound of formula VI "has the following configuration: wherein, when -a-b- is - (Me) CH-CH (OH) preferably has the configuration: The invention also includes an open ring 2-oxy-2 • -aza-3'-oxo-3'-yl-spirobicyclohexane, in free or protected form, or a salt thereof, in particular compound of formula XII1: wherein a, b, and R7 are as defined above, in free or protected form, or a salt thereof. The open ring spirobicyclo ring system of the compounds of the formula XII ', preferably is of the conformation: where, when -a-b- is - (Me) CH-CH (OH) -, it preferably has the configuration The invention also provides a macrolide of formula XIII: wherein M is a macrolide ring as defined above, in particular a maleate of the formula XIV preferably of the conformation: wherein, when -g-h- is - (Me) CH-CH (OH) -, preferably has the configuration: and when -g-h- is - (Me) C = CH-, preferably it has the configuration: CH X, and when R; and R. condense each other, preferably they are of the configuration: CH, OCH, in free or protected form, or open ring, or a salt thereof. In a further aspect, the invention includes the macrolides and compounds of the invention, in particular that which are natural products in a substantially purified form, for example in a form at least 90 percent, preferably at least 95 percent by weight. hundred, and especially at least 98 percent pure. In addition to the above, the present invention also provides a process for the production of any compound of the invention as defined hereinabove, said process comprising: i) for the production of any of the Sanglifehrinas A, B, C, or D, cultivate a strain of actinomycetes producing Sapglifehripa A, B, C, or D in a culture medium, and isolate Sanglifehrin A, B, C , or D desired from the obtained culture broth; ii) for the production of Sanglifehrina C and D, subjecting Sanglifehrina A and B to cyclization in positions 15 and 16; iii) for the production of Sanglifehrinas A and B, to subject Sanglifehrinas C and D to ring opening, of the lactol ring, in positions 15 and 16; iv) for the production of a macrolide of the formula IX or IX ', wherein -gh- is -C (CH:) = CH, dehydrating a compound of the formula IX or IX' wherein -gh- is -CH (CH;) - CH (0H) -, or a protected form thereof; v) for the production of a macrolide of the formula IX or IX ', wherein Rf is -CH (0H) -CH3, hydrogenating a compound of the formula IX or IX', wherein 4 is -C (0) -CH3; vi) for the production of a macrolide of the formula IX or IX '. wherein the positions 14 to 16 of the macrolide ring comprise a residue of the formula X: CH, to cause a compound of formula IX or IX 'to undergo internal protection, at positions 15 and 17; vii) for the production of a macrolide of the formula IX or IX ', to make a compound of the formula IX or IX', wherein the positions 14 to 16 of the macrolide ring comprise a residue of the formula X: CH 3 suffer internal protection investment in positions 15 and 17; viii) for the production of a macrolide of the formula IX or IX ', wherein R < is methyl, subjecting to a macrolide of formula IX or IX ', wherein R = is H, a met i 1 ation: ix) for the production of a macrolide of formula IX or IX', wherein R (be in O-protected form, subject to a macrolide of the formula IX or IX ', where Rt is in a 0-unprotected form, to O-proteccióp; x) for the production of a macrolide of the formula IX or IX ', wherein R < is in a 0-unprotected form, subjecting a macrolide of formula IX or IX ', ep where R "is ep the O-protected form, to O-deprotection; xi) for the production of a macrolide of formula IX or IX ', comprising a 0-protected hydroxyphenylalanine residue in positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX', comprising a 0-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, at 0-protection; xii) for the production of a macrolide of the formula IX or IX ', comprising a 0-nn-protected hydroxyphenylalanine residue in the 7 to 10 positions of the macrocyclic ring, subjecting a macrolide of the formula IX or IX', comprising a 0-protected hydroxyphenylalanine residue in the 7-position. to 10 of the macrocyclic ring, to O-deprotection; xiii) for the production of a macrolide of formula IX or IX ', wherein -ef- is -CH (0H) -CH (0H) -, subject to a macrolide of formula IX or IX', wherein -ef - is -CH = CH- to oxidative hydrolysis; xiv) for the production of a compound V, or a compound of the formula XII, subjecting a compound of the formula IX or IX 'to the dissociation of the linker group between the spirobicyclo group and the macrocyclic ring; xv) for the production of a compound of the formula R60-XYZA-0H or of the formula RfcO-XYZ-A'-CH (H) -LS, subjecting a macrolide of the formula IV, or the macrocyclic ring of a compound of formula VIII, to ring opening in the lactone bridge thereof; xvi) for the production of a macrolide of the formula IX or XII ep closed ring form, subjecting a compound of the formula Rt0-XYZA-0H or of the formula R? 0-XYZ-A'-CH (0H) - LS 3 macrocyclic ring closure; xvii) for the production of a compound of the formula XII or XII ', subjecting a compound of the formula IX or VI' to ring opening in the spirobicyclic ring system, and xix) for the production of a compound of the formula IX or VI ', subjecting a compound of the formula XII or XTI' to ring closure inside the spirobicyclic ring system. The processes of the invention can be carried out, for example, as described in the examples. As will be appreciated, the processes defined above can be applied in any appropriate sequence or combination to obtain other macrolides in free, protected, open ring, and closed ring form, as described hereinabove.

The macrolides of the invention, for example, Sanglifehrins A to D, are, or are derived from, natural compounds normally obtained from members of the Streptococcus family. The microorganisms capable of producing macrolides as defined hereinabove have not been identified previously. Accordingly, in a still further aspect, the present invention provides: a macrolide-producing actinomycete strain, wherein the macrolide is a macrolide wherein: i) positions 2 through 6 inclusive of the macrocyclic ring are provided by a piperidazinylcarboxylic acid residue; and / or ii) positions 7 to 9 inclusive of the macrocyclic ring are provided by an aromatic α-amino acid residue; and / or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic α-amino acid residue, in particular: a strain of actinomycetes producing Sanglifehrin A. B, C, and D. Suitably, the actinomycete strain is from the family of Streptomyceae, more appropriately of the genus Streptomyces, in particular the strain Streptomyces sp. A92-308110 as described hereinafter, or derived therefrom, for example including mutants, variants, condensers, recombinants, or modified forms thereof. Suitably, the strains of the invention are in the form of biologically pure isolates. For example, Streptomyces sp. A92-308110 can be mutated or modified in different ways by conventional techniques, for example by ultraviolet radiation, or by its treatment with a chemical mutagen, such as N-methyl-N '-nitro-nitrosoguanidine. Recombinant clones can be obtained by fusion of the protoplast. All these mutants or recombinants or modified forms, capable of producing Sanglifehrins, including mutants and recombinants capable of producing higher 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 are isolated from Streptomyces sp. A92-308110 novel. Samples of Streptomyces sp. A92-308110 were deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, D-38124 Braunschweig, Germany, on May 3, 1995, in accordance with the terms of the Budapest Treaty, and assigned the number of deposit DSM 9954. Samples of Streptomyces sp. A92-308110 can also be obtained from Sandoz Ltd., CH-4002 Basel, Switzerland.

Hereby, notice is given that access to samples of DSM 9985 is limited in accordance with the provisions of Rule 28 (4) and (5) of EPC. The isolation of Sanglifehrinas A, B, C, and D from cultures of Streptomyces sp. A92-308110 is described in Example 2. Streptomyces sp. strain A92-308110 belongs to the genus Streptomvces according to the description of the Bergey Manual (volume 4, 1989, Williams and Wilkins, Baltimore), and "The Prokaryotes" (1992 Springer Verlag, New York). The cell walls contain L, L-diaminopimelic acid. The fatty acids are also iso- and anteiso-branched, straight, and unsaturated. The sugar spectrum is not distinctive. The vegetative mycelium does not decompose into fragments. The aerial mycelium forms long strings of spores. According to the reference books cited above, the strain designated as A92-308110 is a new Streptomyces. The A92-308110 grows in different organic and inorganic media, and in most cases forms an aerial mycelium. The mycelium of the primary substrate grows like a hyphae, and in general it is beige to grayish chestnut. The color of the aerial mycelium belongs to the series of gray, number 4, and this mycelium forms long strings of spores that belong to the spiral type b. The capacity of Streptomvces sp. A92-308110 to grow in a usual biological medium, its carbon utilization, and its physiological characteristics, are presented in the following tables. Table 1: Growth in Different Biological Media Culture Medium Culture Characteristics yeast extract / growth: good malt agar mycelium from the substrate: chestnut aerial mycelium: grayish chestnut soluble pigment: no oatmeal agar growth: good substrate mycelium: dark brown aerial mycelium: grayish brown soluble pigment: chestnut glucose-asparagine growth: moderate mycelium of substrate: chestnut aerial mycelium: grayish brown soluble pigment: none inorganic salts / agar growth: moderate starch mycelium substrate: gray aerial mycelium: grayish chestnut soluble pigment: none sucrose / agar of nitrate growth: very poor mycelium of the substrate: whitish aerial mycelium: poor, grayish brown soluble pigment: none glycerol / agar of asparagine growth: moderate mycelium of the substrate: chestnut aerial mycelium: grayish brown soluble pigment: no nutrient agar c growth: moderate mycelium of the substrate: beige aerial mycelium: none soluble pigment: chestnut.

Table 2: Use of moderate or good carbon: glucose, fructose, arabinose, xylose, poor mannose: rhamnose, sucrose, raffinose, cellulose, negative salicin: m-inositol.

Table 3: Physiological characteristics reduction of nitrate: positive hydrolysis of starch: moderate in inorganic salts -gar of starch, negative in agar of oatmeal degradation of tyrosine: negative peptonization of milk: positive formation of melanin: positive.

Growth temperatures: 18-37 ° C Very poor growth at 13 ° C. No growth at 45 ° C. pH scale: rich growth at a pH of 5 and 7, good growth at a pH of 9. Resistance to NaCl: up to 6 percent, but growth already reduced at a concentration of 2 percent.

The macrolides of the invention, including Sanglifehrins A, B, C, and D, can be produced by culturing Streptomyces sp. A92-308110 or a mutant, recombinant, or modified form thereof, in an appropriate culture medium. Example 1 describes, by way of illustration only of the invention, a method for the culture of Streptomyces SP. A92-38110. Accordingly, in the additional aspects, the invention includes: a) a biologically pure isolate of the strain Streptomyces SP. 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 the strain Streptomyces s.

A92-308110 (DSM 9954), or a mutant, recombinant, or modified form thereof, in an appropriate culture medium, and optionally recover Sanglifehrin. The macrolides according to the invention, for example the compounds of the formula IX; for example, the Sanglifehrins A, B, C, and D, and their pharmaceutically acceptable salts, generically later in the present "agents of the invention", exhibit the characteristic activities of Sanglifehrina, that is, the following combination of activities: they have a cyclophilin binding activity; they have an immunosuppressive activity; inhibit the proliferation of either B cell or T cell; - but have no FK binding protein binding activity, and do not inhibit calcineurin activity.

These activities and tests to determine these activities are described later in the present in more detail. The biological activity of the macrolides of the invention, for example of the formula IX, for example of the Sapgliffeins A to D, can be demonstrated in standard in vitro and in vivo test methods, for example, as follows. 1. Primary Humoral Immune Response to Bloody Blood Robes of Ovelas (MD, Mishell-Dutton) Mouse spleen cells (OF 1, female, 8 to 10 weeks, 1 x 107) are co-cultured with sheep erythrocytes (SRBC, 3). x 107) for 3 days in a final volume of 1 milliliter in plates of 24 cavities. The lymphocytes are harvested, washed, and coated in a density of 1 x 10 6 cells on soft agar with fresh antigen (SRBC). The complement (guinea pig serum) is added after an incubation period of 60 to 90 minutes, and the incubation is continued for another 60 minutes, after which the test is evaluated by counting (under a microscope) the plates. During the 3-day incubation, the lymphocytes are sensitized to the antigen (SRBC). When they are incubated with antigen again, the B lymphocytes secrete the specific antibody that binds to the antigen in the vicinity of the secretory lymphocyte. The addition of the complement causes the lysis of the erythrocytes coated with antibody, producing a plaque. Each plate represents a single antibody producing cell. The inhibition of plaque formation indicates the pharmaceutical utility. The compounds of the invention, for example, Sanglifehrins A to D, are active in this assay in a concentration of about to nM.

References: R.I. Mishell & R. Dutton (1966) Immunization of normal mouse spleen cell suspensions in vitro, Science 153: 1004-1006. R.I. Mishell & R.W. Dutton (1966) I munizatiop of dissociated spleen cell cultures from normal rnice. J. Exp. Med. 126: 423-442. 2. Proliferative Lymphocyte Response to Allogeneic Stimulation Two-Way MLR (Mixed Murine Lymphocyte Reaction): Balb / c mouse (2 x 105) spleen cells (female, 8 to 10 weeks old) are co-incubated for 4 days with 2 x 10: spleen cells CBA mice (females, 8 to 10 weeks). Allogenic cells induce a proliferative response in the cell population of the responding spleen, which is measured by incorporation of a labeled precursor in the DNA. The macrolides of the invention, for example, the compounds of the formula IX and their pharmaceutically acceptable salts, for example. the? Sanglifehrins A, B, C, and D, have IC50 in the range of approximately 30 to approximately 200 nM, comparing mr, an IC;, of approximately 20 nM for cyclrin A when tested in this assay: Reference: 1. Meo (1979) The MLR ip the mouse. In: "Immunalogical Methods", L. Lefkovits and B. Pernis, Eds., Academic Press, N.Y. pages 227-239. 3. Murine B Cells Stimulated by LPS Spleen cells (2 x 105) of CBA mice are incubated for 48 hours with 50 micrograms / milliliter of LPS plus the test compound. Proliferation is measured by the incorporation of a labeled precursor in the DNA. The macrolides of the invention, for example the compounds of the formula IX and their pharmaceutically acceptable salts, for example, the Sanglifehrins A, B, C, and D, inhibit the proliferation of B cells, and have ICtC on the scale of about 40. to approximately 100 μM.

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 phytomy- togens, Clin. Exp. Immunol. 9: 483-498. 4. Cytotoxic and Cytostatic Activity in vitro Using the THP1 Cell Line Cytotoxicity is determined using the THP1 human monocytic cell line (5 x 104 cells / well), which is incubated in the presence of IFN ^ (100 Units / milliliter) and LPS (5). micrograms / milliliter) plus the test compound (up to 10 μM) for 24 to 72 hours at 37 ° C. Live cells are quantified using the color-read MTT, which measures the epitoxic activity of mitochondrial dehydrogenase in living cells ( Mossman 1983). The macrolides of the invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example, Sanglifehripas A, B, C, and D, have IC 50 of about 1,000-5,000 nM after a 24 hour incubation in this essay.

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

. Liberation of Tumor Necrosis Factor by Human Peripheral Blood Mononuclear Cells Peripheral blood mononuclear cells are prepared from healthy volunteers using Fipoll-Hypaque density separation according to the method of Hansell et al. (1991). Cells are incubated (iO5 cells / well in 200 microliters of RPMI, 10% FCS by volume), with serial dilutions of the test compounds for 30 minutes at 37 ° C before the addition of the stimulus. Is interferon used? (100 units / milliliter) and LPS (5 micrograms / milliliter) as stimuli, to induce the release of the Tumor Necrosis Factor (TNF), by the mononuclear cells of the peripheral blood. After 3 hours of incubation, the cells are centrifuged (1200 rpm for 10 minutes), and the supernatants are harvested. The amount of tumor necrosis factor present in the cellular supernatants is determined using a commercially available enzyme linked immunosorbent assay kit. The macrolides of the invention, for example the compounds of the formula IX and their pharmaceutically acceptable salts, for example, the Sanglifehrins A, B, C, and D, have ICSO on the scale from about 200 nanometers to about 1,000 nanometers when tested. in this essay. 6. Cyclophilin Linkage Assay A suitable cyclophilin linkage assay is the competitive ELISA assay 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 (recombinant human cyclophilin A) with bovine serum albumin-coated cyclosporin A, and then the concentration required to give a 50 percent inhibition is calculated. of the control reaction without competitor (IC.Q). An alternative assay is the competitive binding test described by Scheinder et al., Ep Biochemistry (1994), 33, 8218-8224, which involves the addition of the test compound during the incubation of biotinylated cyclophilin (recombinant human cyclophilin A) with albumin of bovine serum-cyclosporin A coated. The amount of bound biotinylated cyclophilin in the presence and absence of a test compound is determined by incubation with alkaline phosphatase coupled with Streptavidin. The macrolidaes of the invention, for example the compounds of the formula IX, for example Sanglifehrins A, B, C, and D, have IC =. on the scale of approximately 10 to approximately 100 nM, compared to an IC = E of approximately 80 nM for cyclosporin A, when tested in these assays. Other in vi tro assays that can be used to demonstrate the biological activity of Sanglifehrins are the IL-2 reporter gene assays and the ConA-stimulated spleen cell assays (indicating the effect of cell activation). T). The macrolides of the invention, for example the compounds of the formula TX, for example the Sanglifehrins A, B, C, and D, have no FK binding protein binding activity, and do not inhibit the activity of calcineurin when they tested. in conventional tests for these activities. 7. Localized Response of the Graft versus the Host (GvH) in the Rat [Ford et al., TRANSPL. PROC. 10 (1979) 258]. Spleen cells (1 x 107) of Wistar / Furth rats (WF) females 6 weeks old, are injected subcutaneously on day 0 in the internal left leg of female rats (F344 x WF) F? weighing approximately 100 grams. The 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 to evaluate the reaction. The inhibition of the graft versus host reaction in the previous test indicates a pharmaceutical utility. The acrylics of the invention, for example the compounds of the formula IX and their pharmaceutically acceptable salts, for example, the Sanglifehrins A, B, C, and D, can inhibit the reaction of the graft against the host for up to about 30 patient, when they are administered in a dose of approximately 1 milligram / kilogram subcutaneously. 8. DTH Induced by SRBC-T Cells »Fifty microliters of a 1: 1 mixture (volume / volume) of a clone of T-cells. (red blood cells of sheep primed (2 x 101), and a suspension of red blood cells of 10 percent sheep (SRBC), are injected subcutaneously into the base of the right paw of female C57 BL / 6 mice (6 to 12 weeks of age) 50 microliters of the SRBC cell suspension (diluted 1: 1 by volume / volume with phosphate buffer) are injected subcutaneously at the base of the left leg (to measure the nonspecific increase in swelling of the base of the leg due to the injection procedure.) 24 hours later, the thickness of the base of the left and right leg is measured.The percentage increase in thickness is calculated from the base of the right leg on the base of the left leg (Z), thickness of the base of the right leg = x, thickness of the base of the left leg = y, percentage of specific increase = z.? = ( (xy) / y) .100 The macrolides of the invention, for example the compounds of the formula IX and their pharmaceutically acceptable salts, for example the Sanglifehrins A, B, C, and D, reduce swelling of the DTH mouse by up to about e] 50 percent in doses of order of 5 milligrams / kilogram subcutaneously.

References: A.T.J. Bi anchi, H. Hooij aas, R. Brenner, R. Tees, A.

Nordin & M.H. Schreier (1981) Clones of helper T-cells mediated antigen specific, H-2 restricted DTH. Nature 290: 62-63. P. Herrmann, M.H. Schreier, J.-F. Borsl & 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 Al lergy appl. Immun. 86: 102-105. 9. Altar transplantation of Rat / Mouse The in vivo efficacy of the macrolides of the invention is evaluated in alotransplantation of rat and mouse heart, using Alzet osmotic minipumps for subcutaneous administration. In the mouse heart allotransplant (BALB / ca C3H), the acollides of the invention, for example the compounds of the formula IX and their pharmaceutically acceptable salts, for example the Sanglifehrins A, B, C, and D, prolong the survival of the graft in doses of the order of 30 milligrams / kilogram / day. In the treatment of rat heart halotransplantation (DA to Lewis) with suboptimal doses of cyclosporin A in combination with the macrolides of the invention, for example the compounds of the formula IX and their pharmaceutically salts a to < -, Sanglifehripas A, B, C, and D, prolonged graft survival, as exemplified in the following table.

Ciclosporipa A Sanglifehrina A Survival of (mg / kg) (mg / kg) Graft (days) 1 - 12,12,12,13,13,14 1 10 29,30, 5, 48, > 51, > 46 Control (Placebo) Control (Placebo) Control (Placebo) The agents of the invention are useful as pharmaceuticals, for example as immunosuppressants, as well as anti-inflammatory agents. In particular, they are useful for the prevention of rejection of allo- and geno-transplantation of acute or chronic organ or tissue, for example for the treatment of recipients of heart, lung, heart-lung, liver and kidney transplants. , pancreas, skin, or cornea. They are also indicated for the prevention of graft-versus-host disease, such as following bone marrow transplants. The agents of the invention are also useful for the treatment of autoimmune disease, and inflammatory conditions, in particular inflammatory conditions with an etiology that includes an autoimmune component, such as arthritis (e.g., rheumatoid arthritis, chronic arthritis progrediente, and arthritis deformans). ), and pneumatic diseases. Specific autoimmune diseases for which the agents of the invention may be employed include autoimmune hematological disorders (including, for example, hemolytic anemia, aplastic anemia, pure red cell anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, sclerodoma, Wegener's granulamatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnson syndrome, idiopathic stomatitis, autoimmune inflammatory bowel disease (including, for example, 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 heratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis, glomerulonephritis (with and without nephrotic, for example including or idiopathic nephrotic syndrome or minimal change nephropathy), and asthma. For these and other uses, the agents of the invention may be administered on their own, or together with other immunosuppressive or anti-inflammatory agents, including cyclosporins, rapamycins, FK 506, and steroids. For the above indications, the appropriate dosage and agent of the invention selected, of course, will vary, depending, for example, on the subject to be treated, the mode of administration, and the nature and severity of the condition being treated. I'm trying. However, in general, satisfactory results are obtained in animals at daily dosages of approximately 0.01 to 10 milligrams / kilogram / day orally. In larger mammals, for example humans, an indicated daily dosage is on the scale of about 0.5 to about 500 milligrams of Sanglifehrin administered orally once, or more adequately, in dosages divided two to four times a day. In organ transplantation in humans, oral doses are from 0.1 to 100, preferably from 0.3 to 30, and more preferably from 0.5 to 10 milligrams / kilogram of a compound of the agent of the invention. When an agent of the invention is given together with other immunosuppressants (for example, with corticosteroids or with compounds of the class of ciclosporin or rapamycin as part of a double, triple, or quadruple drug therapy), lower doses may be employed ( for example, 0.1 milligrams / kilogram / day intravenously; 3 milligrams / kilogram / day orally initially). In particular, the agents of the invention can be given with other non-steroidal immunosuppressants, for example with cyclosporin A, rapamycin, or FK 506, with a view to a partial or complete replacement of the steroids. The agents of the invention can be administered by any conventional route, in particular enterally, for example orally, for example in the form of solutions for drinking, tablets or capsules, or parenterally, for example in the form of injectable solutions or suspensions. Normally, for systemic administration, oral dosage forms are preferred, although for some conditions, for example for the prevention of rejection of liver transplants, an intravenously injectable form is desirable. The compounds may also be administered locally or dermally, for example in the form of a dermal cream or gel, or as a preparation, or for the purposes of being applied to the eyes, in the form of an eye cream, gel or preparation thereof. eye drops. Unit dosage forms suitable for oral administration comprise, for example, 0.5 to 100 milligrams of the compound per dosage. In accordance with the foregoing, the present invention also provides, in a further series of embodiments: A. A method for effecting a suppression in a subject in need of such treatment, said method comprising administering to that subject an effective amount of an agent of the invention. invention. B. A method: 1) for the prevention of halo rejection or geno-acute and / or chronic transplantation, for example for the treatment d = recipients of organ transplants of any of the particular types mentioned 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 disease or condition mentioned above; or 4) for the treatment of asthma, in a subject in need of such treatment, said 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 product, for example to be used as a suppressant, or to be used in the treatment of any disease or condition as mentioned in B above. D. A pharmaceutical composition comprising an agent of the invention in association with a pharmaceutically acceptable diluent or carrier. 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 mentioned in B above. In addition, the macrolides of the invention possessing cyclophilin binding activity, may be useful as reagents in the displacement immunoassays for cyclosporins and other cyclophilin binding compounds, for example in the assay procedure described in our patent application. pending Number WO 95/07468. This patent application relates to a test procedure for determining the concentration of an immunophilin binding pharmaceutical product, for example cyclosporin, in the blood; the method comprises adding a binding competitor which displaces the pharmaceutical product from the immunosuppressant-immunophilin complexes in the blood; add a receptor that is linked to the pharmaceutical product, but not in a meaningful way with the link competitor; separating the receptor-pharmaceutical product complex from the sample; and determine the amount of the pharmaceutical product. Sanglifehrins can be used as the binding competitor in these assays; for example, to displace cyclosporins from cyclophilins, thereby releasing cyclosporin for quantitation, for example by a monoclonal antibody that is specific for cyclosporin. The invention is further described by way of illustration only in the following example, which refers to the accompanying figures .- wherein Figure 1 shows the mass spectrum of the compound of Sanglifehrin B; Figure 2 shows the mass spectrum of the Sanglifehrin A compound; Figure 3 shows the mass spectrum of the Sanglifehrin D compound; Figure 4 shows the mass spectrum of the Sanglifehrin C compound; Figure 5 shows the infrared spectrum of the compound of Sanglifehrin B; Figure 6 shows the infrared spectrum of the Sanglifehrin A compound; Figure 7 shows the infrared spectrum of the Sanglifehrin D compound; Figure 8 shows the infrared spectrum of the Sanglifehrin C compound; Figure 9 shows the nuclear magnetic resonance spectrum of the Sanglifehrin A compound; Figure 10 shows the nuclear magnetic resonance spectrum of the compound of Sanglifehrin D; Figure 11 shows the nuclear magnetic resonance spectrum of the compound of Sanglifehrin B, and Figure 12 shows the nuclear magnetic resonance spectrum of the compound of Sanglifehrin C.

B TP QE Culture conditions Streptomyces s. A92-308110 can be grown at appropriate temperatures in different culture media, using appropriate mineral nutrients, using aerobic or immersion culture procedures. The fermentation medium usually contains a usable source of carbon, nitrogen sources and mineral salts, including trace elements, all of which can be added in the form of well-defined products, or as complex mixtures, for example, as found in biological products of different origins. Example 1 describes the original conditions under which the compounds of the formula I were obtained. Better yields can be obtained by optimizing the culture conditions (aeration, temperature, pH, quality and quantity of the carbon and nitrogen sources, amount of mineral salts and trace elements), and by controlling fermentation conditions in bioreactors.

Example 1. Cultivation of Strain A 92-308110 to. Initial agar culture The agar cultures of strain A 92-308110 are grown for 10 to 14 days at 27 ° C in the following agar medium: Glucose 10.0 grams soluble starch 20.0 grams yeast extract 5.0 grams (Gist x , Gist Brocadas) NZ-amine, Type A (Sheffield) 5.0 grams Calcium carbonate 1.0 grams Agar (Bacto) 15.0 grams Demineralized water up to 1,000 milliliters The medium is adjusted to a pH of 6.6 to 6.8 with NaOH / H2SO4, and then sterilized for 20 minutes at 121 ° C. The cultures can be stored at -25 to -70 ° C. A suspension in glycerol-peptone can be stored under liquid nitrogen. b. Preculture The spores and mycelium of 10 starting cultures are suspended in 100 milliliters of a 0.9 percent salt solution. Two 2 liter Erlenmeyer flasks, each containing 1 liter of preculture medium, are each inoculated with 50 milliliters of this suspension. The composition of the preculture medium is as follows: technical glucose 7.50 grams glycerin 7.50 grams yeast extract (BBL) 1.35 grams liquid malt extract (Wander) 7.50 grams soluble starch 7.50 grams NZ-amine, type A (Sheffield) 2.50 grams protein of soybeans 2.50 grams L (-) asparagine 1.00 grams CaC03 0.050 grams NaCl 0.050 grams KH2P04 0.250 grams K2HP04 0.500 grams MgS04 «7H20 0.100 grams trace element solution A 1 milliliter Agar (Bacto) 1 gram Demineralized water up to 1,000 milliliters The medium is adjusted to a pH of 6.8 to 7.2 with NaOH / H2SO4, and sterilized for 20 minutes at 121 ° C. The composition of trace element solution A is as follows: FeS04 »7H20 5.0 grams ZnS04e7H20 4.0 grams MnCl2-» 4H20 2.0 grams CuS04e5H20 0.2 grams CoCl2e6H20 2.0 grams H3BO3 0. l grams Kl 0.05 grams H2S04 (95%) 1 milliliter water demineralized to 1,000 milliliters The precultures are fermented for 24 hours at 27 ° C on a rotary shaker at 200 rpm with an eccentricity of 50 millimeters. c. First intermediate culture Two 75-liter bioreactors, each containing 50 liters of preculture medium, are each inoculated with 1 liter of preculture, and fermented for 96 hours at 27 ° C. The fermenter is centrifuged at 150 rpm. Air is introduced at a rate of 0.5 liters per minute per liter of the medium. d. Second intermediate culture Two fermentation vessels of 750 liters, each containing 500 liters of preculture medium, are each inoculated with 50 liters of the first intermediate culture. The second intermediate cultures are incubated for 70 hours at 27 ° C. The fermenters are centrifuged at 100 rpm, and air is introduced at a rate of 0.8 liters per minute per liter of the medium. and. 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 for 96 hours at 24 ° C. The bioreactors are centrifuged at 45 rpm, and air is introduced at a rate of 0.5 liters per minute per liter of the medium. The composition of the main culture medium is as follows: technical glucose 20 grams liquid malt extract (Wander) 2 grams yeast extract (Bacto) 2 grams Soytone (Bacto) 2 grams KH2P04 0.2 grams K2HP04 0.4 grams MgS04 »7H20 0.2 grams NaCl 0.05 grams CaCl2 # 6H20 0.05 grams trace element solution B 1 milliliter Agar (Bacto) 1 gram Demineralized water up to 1,000 milliliters The pH is adjusted to 6.3 with KOH / HCl. The medium is sterilized for 20 minutes at 121 ° C. The composition of trace element solution B is as follows: FeS04 «7H20 5.0 grams ZnS04e7H20 4.0 grams MnCl2e4H20 2.0 grams CuS04» 5H20 0.2 grams (NH4) 6 o7024 0.2 grams CoCl2 «6H20 1.0 grams H3BO3 0.1 grams KJ 0.05 grams H2S0t ( 95%) 1 milliliter demineralized water up to 1,000 milliliters An optimized culture medium for the main crop is as follows: soybean meal 20.0 grams glycerol 40.0 grams MES 0.1 M demineralized water up to 1,000 milliliters at a pH of 6.8. Example 2 - Isolation of Sanglifehrins A, B, C, and D, from Streptomyces sp. A92-308110 The first isolation and characterization of the 4 new active metabolites of CBA was made from two tank fermentations of 3,000 liters by activity-guided fractionation and analysis of high performance liquid chromatography and thin layer chromatography. The CBA (cyclophilin binding assay), as described above, was used to test the biological activity. The two 3,000 liter fermentations were processed separately. 1,500 liters of each fermentation are stirred with 2,000 liters of ethyl acetate in a 4,000 liter stainless steel vessel for 20 hours. The separation of the organic phase is done with a type-Separator typ SA-20. 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 kilograms of extracts. The two crude extracts are degreased by a three step extraction with 40 liters of methanol / water, 9: 1, and 40 liters of hexane. Evaporation to dryness under reduced pressure gives 1.34 kilograms of extract. The defatted extract is passed through chromatography in two portions (670 grams) on a 10 kilogram column of Sephadex H in a methanol solution. Each portion dissolves in 3.3 liters of methanol when added to the column. After collecting the first 15 liters of the eluate as fraction 1, the chromatography is continued, collecting fractions of 2 liters. The most active fractions were 2, 3, and 4, and therefore, combine to give 146 grams. This sample is chromatographed further on 1 kilogram of Merck silica gel 0.04-0.063 millimeters, with methyl tertiary butyl ether (MTBE), MTBE / 5% methanol, and MTBE / 10 percent ethanol. Fractions of 2 liters are collected. The? fractions 5 to 9 are the most active, and combine to give a sample of 43.8 grams. This sample is further separated on a column of 1 kilogram of silica gel (Merck) of 0.04 to 0.063 millimeters, with a hexane / acetone gradient of 7: 1 to acetone. From this chromatographyS separates the fraction 6 (7.0 grams) onto a column of ~ kilograms of Lichroprep RP 18 (Merck) from 40 to 63 microns with methanol / water, 94: 6 (fractions 4-7, 2.16 grams). , and then on a column of 100 grams of silica gel H, with methylene chloride, and 3 percent methanol (773 milligrams), a 3 kilogram column of Lichroprep RP18 with metapol / water, 9: 1 (621 milligrams ), and then over 100 grams of Lichroprep RP 18 with acetonitrile / water, 1: 1, to give 324 milligrams of pure Sanglifehrin A (mp 142-145 ° C (amorphous), () D25 = 67.30 (c = 0- 988, methanol)). Fractions 5 and 7 of the hexane / acetone column are combined (7.1 grams), and further purified on a 3 kilogram column of Lichroprep RP 18, 40 to 63 microns, with methanol / water, 9: 1 ( 769 milligrams), on a column of 100 grams of silica gel H with MTBE / 3 percent methanol (309 milligrams), and finally on 100 grams of silica gel H with methylene chloride and 3 percent methanol, give 90 milligrams of pure Sanglifehrina B (mp 117-121 ° C (amorphous), (a) D25 = 52.80 (c = l-128 in methanol)). Fractions 9 and 10 (2.147 grams) of the chromatogram with methanol / water, 94: 6, on 3 kilograms of Lichroprep RP 18, are further purified from 100 grams of ICICP gel with methylene chloride / 5% methanol. (800 milligrams), and finally about 3 kilograms of Lichroprep RP 18 with methanol / water, 9: 1, to give 480 milligrams of the Sanglifehripa C (mp 165 -70 ° C, (a) D25 = 35.60 (c-0- 736 in methanol)). Fractions 11 and 12 (835 milligrams) of the methanol / water chromatogram. 94: 6 on 3 kilograms of Lichroprep RP18, are purified on 100 grams of silica gel H with MTBE / 5% methanol, to give 140 milligrams of Sanglifehrin D (p.f. 137-142 ° C, amorphous). Sanglifehrins A, B, C, and D were then characterized by ultraviolet, infrared, mass spectroscopy, and nuclear magnetic resonance. The results obtained are given in the following table 4, and in the accompanying figures. Table 4 Sanglifehrin A Molecular Formula: C60H91N5O13 (1090.4) UV (MeOH): 275 (1962), 242 (54500), 197 (75755) H +: 275 (1635), 242 (51884), OH: 292 (1973), 242 (60495) Infrared spectrum: Figure 6 Mass spectrum: FAB 1096 [MH + Li] +: Figure 2 Nuclear magnetic resonance spectrum: Figure 9 Sanglifehrin B Molecular formula: C60H89N5O12 (1072.4) UV (MeOH): 273 (4395), 242 (50600), 197 (78577) Infrared spectrum: Figure 5 Mass spectrum: FAB 1098 [MH + Li] +: Figure 1 Nuclear magnetic resonance spectrum: Figure 11 Sanalifehrin C Molecular formula: C61Hg3N5013 (1104.4) UV (MeOH): 275 (1876), 242 (51557), 197 (72643) H +: 275 (1391), 242 (50120), OH: 292 (1832), 242 (57960) Infrared spectrum: Figure 8 Mass spectrum: FAB 1110 [MH + Li] +: Figure 4 Nuclear magnetic resonance spectrum: Figure 12 Sanglifehrin D Molecular formula: C6lH91N5012 (1086.4) UV (MeOH): 273 (3194), 242 (47584), 197 (73766) H +: 273 (3237), 242 (46389), OH: 285 (2600), 242 (52907) ) Infrared spectrum: Figure 7 Mass spectrum: FAB 1092 [MH + Li] +: Figure 3 Nuclear magnetic resonance spectrum: Figure 10 Example 3 - Transformation of Sanalifehrin A into Sanglifehrin C To a cooled (0 ° C) stirred solution of 20 milligrams (18.3 micromoles) of Sanglifehrin A in 0.5 milliliters of methanol, a crystal of paratoluenesulfonic acid monohydrate is added. The resulting yellow solution is stirred for 1 hour, and the reaction is quenched with a saturated aqueous solution of 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 tertiary butyl ether: methanol) to give Sanglifehrin C as an amorphous white powder. The latter consisted of a 4: 1 mixture of Sanglifehrin C and its C53 epimer, Sanglifehrin C having the (S) configuration as illustrated below (R = Me). 4: 1 mixture of diastereomers Alternatively, this transformation can be carried out by the use of other protic acids (such as pyridinium paratoluensulfonate, hydrochloric acid, or sulfuric acid), or Lewis acids (such as zinc chloride, magnesium bromide or chloride, tetraisopropoxide of titanium, or boron trifluoride) in methanol. The use of other alcohol solvents or cosolvents such as ethanol, isopropanol, butanol, allyl alcohol, propargyl alcohol, benzyl alcohol, leads in the same way to analogues wherein R in the above structure is ethyl, isopropyl, butyl, allyl, propargyl, benzyl In the same manner as described above, Sanglifehrina B can be transformed into Sanglifehrina D.

E-ißnmlo 4 - Transformation of Sanglifehrina C in Sanslifehrina A A solution of 550 milligrams (0.50 millimoles) of Sanglifehrin C in 5 milliliters of 4: 1 tetrahydrofuran-water, treated with 0.5 milliliters of 2N aqueous sulfuric acid, and stirred for 1.5 hours. 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 a saturated aqueous solution of sodium bicarbonate, 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 tertiary butyl ether: methanol) to give 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 paratoluensulfonate, acetic acid, trifluoroacetic acid, formic acid. Suitable organic cosolvents are acetonitrile, dimethyl formamide, dimethyl sulfoxide, dioxane. These reactions are accompanied by the formation of different amounts of the compound of formula XV, depending, among other things, on the reaction time (for a better procedure leading to the compound of formula XV, see example 5 below). In an analogous way, Sanglifehrina D can be transformed into Sanglifehrina B.

Example 5 - Transformation of Sanslifehrin A into the Compound of Formula XV To a cooled (0 ° C) and stirred solution of 50 milligrams (46 micromoles) of Sanglifehrin A in 1.9 milliliters of acetonitrile, 0.1 milliliters of hydrogen fluoride are added. -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 tertiary butyl ether: methanol), to give the compound of formula XV as a white amorphous powder.

In an analogous manner, Sanglifehrin B can be transformed into the compound of formula XVI. These substances exist as a single epimer in C53, but the absolute configuration has not been determined in an unambiguous way.

Formula XV: MS m / z 1078 [M + Li] * (relative intensity 100); 1 H NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) d 0.40 (3 H, d, H-50), 1.20 (3 H, s, H-54), 1.69 (3 H, s, H-49), 4.20 (ÍH, t, H-15), 4.58 (ÍH, dd, H-17), 5.19 (ÍH, dd, H-18), 5.28 (ÍH, dd, H-23), 5.62 (ÍH, m, H -21), 5.67 (ÍH, m, H-27), 5.99 (ÍH, d, H-25), 6.03 (ÍH, dd, H-19), 6.14 (ÍH, dd, H-20), 6.22 ( ÍH, dd, H-26).

Example 6 - Transformation of the Compound of Formula XV in Sanslifehrina AA a stirred solution of 54 milligrams (50 micromoles) of the compound of formula XV in 0.5 milliliters of 4: 1 tetrahydrofuran-water, is added 50 microliters of aqueous sulfuric acid 2N. The resulting solution is stirred at room temperature for 12 hours, and the reaction is quenched with a saturated aqueous solution of sodium bicarbonate. This mixture is extracted twice with ethyl acetate. The combined organic solution is washed with a saturated aqueous solution of sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Column chromatography of the residue on silica gel (90:10 methyl tertiary butyl ether: methanol) produces Sanglifehrin A as a white amorphous solid. In an analogous manner, the compound of formula XVI can be transformed into Sanglifehrin B.

The procedures described in Examples 3 to 6 can be employed as selective intramolecular protection-deprotection sequences. Accordingly, by the reaction described in Example 5, the hydroxyl can be selectively protected at position 15, which allows selective manipulation of the remaining free hydroxyls. The procedure of Example 5 allows selective protection of both hydroxyls at positions 15 and 17. Both methods can be employed as an intramolecular protection of C53 ketone. The hydroxyl and the ketone can be regenerated by the reactions described in 4 and 6. The Sanglifehrins C and D, as well as the compounds of the formulas XV and XVI, are therefore important intermediaries for the generation of other Sanglifehrinas.

Example 7 - Preparation of 16-dehydro-17-dehydroxy-Sanolifehrin A (Formula XVII) A solution of 54 milligrams (50 micromoles) of the compound of the formula XV, and a crystal of paratoluenesulfonic acid monohydrate ep 1 milliliter of acetonitrile-water 4: 1, is heated at 80 ° C for 1.5 hours. The reaction is quenched by the addition of a saturated aqueous solution of sodium bicarbonate. 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 tertiary butyl ether methanol), followed by reverse phase chromatography (RP18, 50:50 acetonitrile-water to acetonitrile for 45 minutes), give the pure title compound as an amorphous white solid.

MS m / z 1078 [M + Li] * (relative intensity 100); ? NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) d 1.58 (3 H, s, H-50), 1.71 (3 H, s, H-49), 2.08 (3 H, s, H-54), 4.03 ( 2H, d, H-15 and C31-0H), 5.57 (2H, rn, H-21 and C35-0H), 5.72 (ΔH, dt, H-27), 5.96 (ΔH, d, C15-0H), 6.03 (ÍH, d, H-25), 6.09-6.28 (4H, m, H-18, H-19, H-20, and H-26), 6.37 (1H, d, H-17). - • »-j -,« rir- 8 - Preparation of 42-N-methyl-Sanalifehrins? (Formula XVIII) To a cooled (-15 ° C) and stirred solution of 109 milligrams (0.1 millimoles) of Sanglifehrin A and 67 microliters (0.3 millimoles) of tertiary 2,6-dibutyl pyridine in 1 milliliter of ethylene chloride was added 16.5 microliters of methyl triflate. The mixture is allowed to warm to room temperature, and stirring is continued for 6 hours, after which the reaction is quenched by the addition of a saturated aqueous solution of sodium bicarbonate. 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 tertiary butyl ether: methanol, and then 95: 5 methyl tertiary butyl ether: methanol), to give the pure title compound as a solid amorphous white MS m / z 1110 [M + Li] + (relative intensity 100); 1 H NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) 6 1.70 (3 H, s, H-49), 2.06 (3 H, S, H-54), 3.53 (3 H, s, 42 N-Me), 3.98 (HH, d, C31-OH), 4.50 (HH, d, H-65), 4.77 (HH, d, C17-0H), 5.43 (HH, d, C15-0H), 5.49 (HH, d, C35-OH), 7.50 (H, d, H-12), 8.11 (H, d, H-9), 9.22 (H, s, C61-OH).

Example 9 - Preparation of 53 -dihydro- Sanali ehrina A (Formula XIX) To a cooled (0 ° C) and stirred solution of 54 milligrams (50 micromoles) of Sanglifehrin A, in 0.5 milliliters of methanol, 2.8 milligrams (75 micromoles) of sodium borohydride are added. Stirring is continued for 1 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 residue is purified by chromatography on silica gel (95: 5 methyl tertiary butyl ether: methanol, followed by 90:10 methyl tertiary butyl ether: methanol), to give the pure title compound as a white amorphous solid. . The isolated product corresponds to a mixture of approximately 1: 1 of diastereoisomers at C-53.

MS m / z 1098 [M + Li] + (relative intensity 63), 1104 [M + 2Li-H] + (relative intensity 100); - "" H NMR (dimethyl sulfoxide) (only the characteristic signals are mentioned) d 0.62 (3H, d, H-50), 1.02 (3H, d, H-54), 3.55 and 3.59 (H, 2m, H -53).

E-example 10 - Preparation of 53 -t? 8 -hydrorazone-Sanalifehrin A (Formula XX) A mixture of 55 milligrams (50 micromoles) of Sanglifehrin A, and 23 milligrams (125 micromoles) of tosylhydrazide in 0.5 milliliters of methylene chloride, is stirred at room temperature for 6 hours. The solvent is removed, and the residue is purified by chromatography on silica gel (90:10 methyl tertiary butyl ether: methanol), to give the title compound as a white amorphous powder.

MS m / z 1264 [M + Li] + (relative intensity 100); 1 H NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) d 1.70 (3 H, S, H-49), 1.77 (3 H, S, H-54), 2.37 (3 H, S, -NS02C6H4CH3), 6.51 ( ÍH, S, H-60), 6.59 (2H, 2d, H-62 and H-64), 7.06 (ÍH, dd, H-63), 7.35 (2H, d, tosyl protons meta), 7.73 (2H , d, tosyl protons for).

Example 11 - Preparation of 26S, 27S-dihydroxy-Sanalifehrin A (Formula XXI) v 26R.27R-dihydroxy-Sanslifehrin A (Formula XXII.

To a cooled (0r, C) and stirred solution of 49 milligrams (1.5 millimoles) of potassium ferricyanide, 20 milligrams (1.5 millimoles) of potassium carbonate, 19. milligrams (0.025 ml) of (DMG): PH4L, 65 microliters (0.00 millimoles) of 0.08 M osmium tetroxide in tertiary butanol, 95 milligrams (1 millimole) of methyl sulfonamide in 2. milliliters of tertiary butanol, and 5 milliliters of water, is it added a solution of 545 milligrams (0.5 millimoles) Sangl ifehrlpa? in 2.5 milliliters and tertiary hutanol. The resulting m-pi biphasic is allowed to warm to room temperature, and is stirred for 3 hours. Then add 1.08 grams (8 millimoles) of sodium sulfite, followed by ethyl acetate water, and the mixture is stirred vigorously for 15 minutes. The layers are separated, and the aqueous poop is extracted twice with ethyl acetate. The combined organic layer is washed not 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 for 60 minutes, yielding 26S, 27S-diol as an amorphous powder.) The corresponding 26R, 27R-diol is obtained by the procedure above, but using (DHQD) 2PHAL instead of (DHQ) 2PHAL. 26S, 27S-diol: MS m / z 1130 [M + Li] + (relative intensity 100); 1 H NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) d 1.64 (3 H, s, H-49), 2.06 (3 H, s, H-54), 3.20 (H, broad m, H-27), 3.45 (ÍH, broad m, H-31), 3.94 (3H, m, H-17, H-26, and C31-0H), 4.30 (ÍH, d, C27-OH), 4.57 < 1H, d, C26-OH), 5.20 (H, t, H-23), 5.33 (H, d, H-25), 5.57 (3 H, m, H-18, H-21, and C35-OH) , 6.03 (ÍH, dd, H-19), 6.14 (ÍH, dd, H-20). 26R, 27R-diol: MS m / z 1130 [M + Li] + (relative intensity 100); 1 H NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) d 1.64 (3 H, s, H-49), 2.06 (3 H, s, H-54), 3.16 (H, broad m, H-27), 3.48 (HH, broad m, H-31), 3.94 (3H, m, H-17, H-26, and C31-OH), 4.30 (HH, d, C27-OH), 4.57 (HH, d, C26 -OH), 5.20 (HH, dd, H-23), 5.35 (HH, d, H-25), 5.57 (3H, m, H-18, H-21, and C35-OH), 6.03 (HH, dd, H-19), 6.14 (ÍH, dd, H-20).

Example 12 - Dissociation «l? Ifil-83 2 $ S. SVg-a.Utiflr? Qi-SftaqUf'Pfarina A To a solution of 90 milligrams (79 micromoles) of the 26S, 27S-diol in 0.9 milliliters of 2: 1 tetrahydrofuran-water, 33.7 milligrams (157 micromoles) of sodium periodate are added. Stirring is continued for 1 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 tertiary butyl ether: methanol) yields the compounds of formulas XXIII (foam) and XXIV (powder).

XXIII Formula XXIII: MS m / z 366 [M + H-H20] + (relative intensity 100); XH NMR (dimethyl sulfoxide) (2: 1 mixture of OH- ^ OHgq epimer in the anomeric center) (only the characteristic signals are mentioned) d 3.54 and 4.08 (ÍH, 2m, H-31), 3.57 (ÍH, m broad, H-35), 3.66 (ÍH, m, H-33), 4.38 (0.67H, ddd, H-27a?), 4.95 (0.33H, broad m, H-27eq), 5.40 (0.33H, d, C27-0Heq), 5.59 (0.33H, d, C35-OH), 3.61 (0.67H, d, C35-OH), 5.96 (0.67H, d, C27-0Hax), 7.89 (0.67H, S, NH-42), 7.91 (0.33H, s, NH-42).

Formula XXIV: MS m / z 745 [M + Li] +; 1 H NMR (dimethyl sulfoxide) (only characteristic signals are mentioned) d 0.64 (3 H, d, H-50), 0.81 (6 H, d, H-56 and H-57), 2.06 (3 H, s, H- 54), 2.17 (4H, s, H-14 and H-49), 3.80 (ÍH, broad m, H-15), 3.94 (ÍH, dd, H-17), 5.33 (ÍH, broad d, H- 23), 5.62 (2H, m, H-18 and H-21), 6.89 (ÍH, d, H-25), 6.10 (ÍH, dd, H-19), 6.18 (ÍH, dd, H-20) , 10.0 (ÍH, d, H-26).

Example 13 - Acetylation of Sanslifehrin A for der 61-O-acetyl-Sanalifehrin A (Formula XXV) To a stirred (0 ° C) and stirred solution of 54 milligrams (50 micromoles) of Sanglifehrin A and 50 microliters of pyridine in 0.5 milliliters of methylene chloride are added . 2 microliters (55 micromoles) of acetic anhydride. The reaction is maintained at 0 ° C for 1 hour, then it is allowed to warm to room temperature, and stirring is continued for 12 hours. Saturated aqueous sodium bicarbonate is added, and the resulting mixture is extracted with ethyl acetate. The organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by reverse phase chromatography (RP 18, 40:60 acetonitrile-water, to acetonitrile for 45 minutes), giving the title compound as an amorphous powder.

MS m / z 1132 [M + H] + (relative intensity 100); 1 H NMR (dimethylsulfoxide) (only characteristic signals are mentioned) d 1.68 (3 H, S, H-49), 2.06 (3 H, s, H-54), 2.25 (3 H, S, CH 3 C 0 2), 4.04 (H) , d, C31-0H), 4.67 (HH, d, C2-NH), 4.76 (HH, d, C17-OH), 5.42 (2H, m, H-8 and C15-0H), 5.57 (3H, m , H-18, H-21, and C35-OH), 6.85 (H, s, H-60), 6.98 (H, D, H-62), 7.06 (H, D, H-64), 7.31 ( HH, dd, H-63), 7.51 (HH, d, H-12), 7.89 (HH, S, H-42), 8.23 (HH, d, H-9).

Claims (34)

REI INDICATIONS

1. A macrolide wherein: i) positions 2 to 6 inclusive of the macrocyclic ring are provided by a piperidazinylcarboxylic acid residue; and / or ii) positions 7 through 9 inclusive of the macrocyclic ring are provided by an aromatic α-amino acid residue; and / or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by a residue of aliphatic tx-a-naphtha, in free or protected form, or a salt thereof.

2. A macrolide according to claim 1, which comprises two, or especially the three characteristic structural features i), ii), and iii).

3. A macrolide according to claim 1 or 2, wherein the rest of the macrocyclic ring comprises a hydroxycarboxylic acid residue, having a chain length of dp 6 to 20, preferably of 11 carbon atoms.

4. A macrolide according to claim 3, wherein the hydroxycarboxylic acid residue is a residue of formula II: O CH2) 2 I -CH- -CH-CO- CiH- • CH-CH = CH-CH = CH-CH-C- CH, II where: R; and Ri are both H, or represent an extra link; R, is H; R? is -CO-CH »or -CH (0H) -CH :. or Rj and P (together represent a structure of the formula III: CH, III OCH, in free or protected form, or a salt thereof.

5. A macrolide according to any of claims 1 to 5, which comprises a macrocyclic ring of the formula IV: wherein 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 form or protected, or a salt thereof.

6. A macrolide according to claim 5, which comprises a macrocyclic ring of the formula V: wherein A is cone is defined in claim 5 in free or protected form, or a salt thereof.

7. A macrolide according to any of the preceding claims, which is substituted on the carbon atom adjacent the oxy moiety of the lactone bridge by a residue of 2-i-2'-aza '-c o-espirobiciclohexan-3 -i what.

8. A macrolide according to claim 7, P? Where P1 residue from I R formula 1: wherein: -sb- is - (Me) C = CH- or - (Me) CH-CH (OH) -, and R5 is H or Me, in free or protected form, or a salt of the ism, linked to the macrolide ring by means of a linker comprising a linear sequence of 6 to 11, usually 9 carbon atoms, between the spiro residue and the brownish ring.

9. A macrolide according to claim 8, wherein the linker between the spiro residue and the macrolide ring is a group of the formula VII: c-CH2-CH (OH) where: c represents uo in a e ccn the spiro residue; d represents a bond with the macrocyclic ring, and Rj and R? they are each OH, or they represent an additional pnl c in free form or Droteaida.

10, A compound of formula VIII wherein: S represents a residue of 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl; L represents a linker comprising a linear sequence of 6 to 11, usually of 9 carbon atoms, and M represents a macrolide ring as defined P? any of claims 1 to 6, in free or protected form, or a salt thereof.

11. A compound according to claim m, of formula IX: where: -a-b- is as defined above; -e-f- is -CH (OH) -CH (OH) - or -CH = CH-; -g-h- is as defined above for -a-b-, and 3; R "and R5 are as defined above, in free or protected form, or a salt thereof.

12 A compound according to claim 11, of the following conformation: where, when -a-b- is - (Me) CH-CH (OH) -, preferably has the configuration: when -e-f- is -CH (OH) -CH (OH) -, preferably has the configuration (S), (S) or the configuration (R), (R); when -g-h- is - (Me) CH-CH (OH) -, preferably has the configuration: when -g-h- is (Me) C = CH-, preferably it has the configuration: and when R; and R? they condense with each other. of preference! are from the CH configuration, .... OCH3

13. A Sanglifehrina selected from the group consisting of Sanglifehrina A, B, C, and D ep.

14 A macrolide according to any of claims 4 to 9, or a compound according to any of claims 10 to 12, in where positions 14 to 17 of the macrocyclic ring comprise a residue of formula X: - • CCHH - CCHH --- CCCHHH-CH- CH 17 14 15, for example, of the configuration:

15. A compound of formula XI: R «O-X-Y-Z-A-OH XI for example, of formula XII IX1 where X, Y, Z, A, R :, Rt, and R; they are as defined above, and R; is H or alkyl of 1 to 4 carbon atoms, for example methyl, in free or protected form, or a salt thereof.

16. A macrolide as defined in any one of claims 1 to 9, in the form of an open ring, wherein the ring is open or free, or a salt thereof.

17. A compound R.O-X-Y-Z-A-OH, where X, Y, Z, and A are as defined in claim 5, and R i is H or alkyl of 1 to 4 carbon atoms, in free or protected form, or a salt thereof.

18. A compound R.O-X-Y-Z-A '-CH (OH) -L-S. in where < -? A'-CH (OH) - is a residue of the hydroxy acid > The code is defined in claim 3, and the other symbols are as defined in claim 17, in free or protected form, or a salt thereof.

19. A compound of formula XII1: in free or protected form, or one of the same.

20. A compound of formula IX ": 47 in free or protected form, or a salt thereof.

21. A compound wherein the ring system of 2-oxy-2'-aza-3 '-oxo-3' -yl-spirobicyclohexane is in the open-ring form, for example, a compound of formula XII: where a, b, L and M, are as defined above, in free or protected form, or a salt thereof.

22. Un2-oxy-2 * -aza-3 * -oxo-3 '-yl-spirobicyclohexane, in free or protected form, or a salt thereof, in particular a compound of formula VI': Et where R- is H, an optically protected OH group, a reactive functional group, or a group -CH: -CH (OH) -CH (CH;) -CH ~ CHi-CHO, or the equivalent of delta-lacto of the same, in lihre or protected form, or a salt thereof.

23. An open-ring 2-oxy-2 '-a? A-3' -oxo-3'-yl-spirobicyclohexane, in free or protected form, or a salt thereof, in particular a compound of the formula XII ": wherein a, b, and R7 are as defined above, in free or protected form, or a salt thereof.

24. An acrylic of formula XIII: wherein M is a ring of macrolide as pfinio above, in particle .-- ur - a chloride of the formula v H > : in free or protected form, or a salt thereof

25. A process oara production of any compound cerno ds the invention defined above] p present, said process comprising: i) for the production gives either Sanglifehripas A, B, C, or D. Cultivate a strain of Sangl producing actinomycetes if A, B, C, or D in a culture medium, and isolate the desired Sanglifehrin A, B, C, or D from the breeding broth obtained; ii) for the production of Sanglifhrina C and (1) to subject Sanglifehrina A and B to cyclization in positions 15 and 16, iii) for the production of Sanglifehripas A and B, to submit to Sanglifhrinas C and D a ring opening, dl lactol ring, in positions 15 and 16; i) for the production of a macrolide of the formula IX or IX '. where -g-h- p -c (C;) = CH. dehydrating a compound of formula IX or IX 'in dnnd ° -gh- is -CH (CH -CH (OH) -, n a protected form thereof, v) for the production of a macrolide of formula IX or IX ', where R? is -CH (OH) -CH;, hydrogenate an HP compound of formula IX or IX ', wherein R? is -C (0) -CH5; vi) for the production of a macrolide of the formula IX or IX ', wherein the positions 14 to 16 of the macrolide ring comprise a residue of the formula X: CH, make a compound of the order IX or IX "suffer internal protection; vii) for the production of a macrolide of the formula IX or IX ', make a compound of the formula IX or IX', ep riopHp the positions 14 to 16 of the macrolide ring comprise a residue of formula X: CH_ suffer investment of internal protection. viii) for the production of a macrolide of the formula IX or IX ', wherein R5 is methyl, subjecting a macrolide of the formula IX or IX', wherein R $ is H, to methylation; ix) for the production of a macrolide of the formula IX or IX ', wherein R ^ is in O-protected form, subjecting a macrolide of formula IX or IX', wherein R, is in a 0-unprotected form to O-protection; x) for the production of a macrolide of the formula IX or IX ', wherein R | is in a 0-unprotected form, subjected to a macrolide of formula IX or IX ', wherein R5 is in the O-protected form, to O-deprotection; xi) for the production of a macrolide of formula IX or IX ', comprising a 0-protected hydroxyphenylalanine residue in positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX', comprising a 0-unprotected hydroxyphenylalanine residue in positions 7 to 10 ring macrocyclic ring, to O-proteccióp; xii) for the production of a macrolide of formula IX or IX ', comprising a 0-unprotected hydroxyphenylalanine residue in positions 7 to 10 of the macrocyclic ring, subjecting a macrolide of formula IX or IX', comprising a 0-protected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, at O-deprotection; xiii) for the production of a macrolide of the formula IX or IX ', wherein -ef- is -CH (OH) -CH (OH) -, subject to a macrolide of the formula IX or IX', wherein -ef - is -CH = CH- to oxidative hydrolysis; xiv) for the production of a compound V, or a compound of the formula XII, subjecting a compound of the formula IX or IX 'to the dissociation of the linker group between the spirobicyclo group and the macrocyclic ring; xv) for the production of a compound of the formula RtO-XYZA-OH or of the formula RtO-XYZ-A'-CH (OH) -LS, subjecting a macrolide of the formula IV, or the macrocyclic ring of a compound of formula VIII, to ring opening; xvi) for the production of a macrolide of formula IX or XII in the form of a closed ring, subjecting a compound of the formula Rfc0-XYZA-0H or of the formula Ré0 ~ XYZA '-CH (0H) -LS at closure of the macrocyclic ring; xvii) for the production of a compound of the formula XII or XII ', subjecting a compound of the formula IX or VI' to ring opening in the spirobicyclic ring system, and xix) for the production of a compound of the formula IX or VI ', subjecting a compound of formula XII or XII' to ring closure inside the spirobicyclic ring system.

26. A strain of actinomycetes that produces a macrolide, wherein the macrolide is a macrolide wherein: i) the positions 2 to 6 inclusive of the ring rnacropi cl i co e ^ t-an provided by a residue of piperidazipilcarbsxil; and / or ii) positions 7 through 9 inclusive of the macrocyclic ring are provided by an aromatic α-amino acid residue; and / or iii) positions 10 to 12 inclusive of the macrocyclic ring are provided by an aliphatic α-amino acid residue.

27. A biologically pure isolate of the strain Streptomyces sp. A92-308110 (DSM 9954), or a mutantp, recombinant, or modified form thereof, which is capable of producing a macrolide of the invention.

28. A process for the production of an acrylic acid of the invention, which comprises cultivating the strain Streptomyces sp. A92-308110 (DSM 9954), or a mutant, recombinant, or modified form thereof, ep an appropriate culture medium, and optionally recover Sanglifehrin.

29. A method for effecting a suppression in a subject in need of such treatment, said method comprising administering to that subject an effective amount of an agent of the invention.

30. A method: 1) for the prevention of halo rejection or geno-acute and / or chronic transplantation, for example for the treatment of recipients of organ transplants of any of the particular types mentioned 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 disease or condition mentioned above; or 4) for the treatment of asthma, in a subject in need of such treatment, said method comprises administering to said subject an effective amount of an agent of the invention.

31. An agent of the invention for use as a pharmaceutical product, for example to be used as an immunosuppressant, or to be used in the treatment of any disease or condition as mentioned in B above.

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

33. 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 mentioned in B above.

34. The use of a compound according to any of claims 1-6, 8-10, 15 or 16, as a reagent in a displacement immunoassay for cyclosporins or other oxyphilin binding compounds.