CN1193979A

CN1193979A - Macrolide compounds

Info

Publication number: CN1193979A
Application number: CN96196487A
Authority: CN
Inventors: T·费尔; L·奥贝雷尔; V·奎斯尼奥克斯－赖费尔; J·J·桑利尔; W·舒勒尔; R·瑟拉尼
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 1995-07-04
Filing date: 1996-07-04
Publication date: 1998-09-23
Also published as: AU708004B2; JPH11509092A; TR199800001T1; BR9609324A; AR006514A1; AU6519396A; CZ424297A3; KR19990028724A; IL122844A0; PL324346A1; SK498A3; NO980016L; MX9800217A; NO980016D0; HUP9802320A2; HUP9802320A3; EP0836616A1; CA2224715A1; CO4750713A1; WO1997002285A1

Abstract

Novel macrolides containing 2 of i), ii) and iii) especially all 3 of the characteristic structures, more preferably a compound of formula IX, which have immunosuppressive and anti-infective effects and which may be in protected and ring-opened form, are provided, wherein i) the 2-6 positions of the macrocycle are piperazinylcarboxylic acid residues, and/or ii) the 7-9 positions of the macrocycle are aromatic alpha-amino acid residues, and/or iii) the 10-12 positions of the macrocycle are aliphatic alpha-amino acid residues.

Description

Macrolides compound

The present invention relates to the new macrolides compound of a class, they have important pharmacy and relevant activity.For convenience's sake, such new macrolides compound is referred to as " Sanglifehrins " in this application

From the actinomycete fermentation broth culture, isolate Sanglifehrins first.They are Sanglifehrins A-D of following formula:

As can be seen, big ring Sanglifehrins A-D has new structure fully, it is characterized in that i) 1 to 6 contain 3-carboxyl piperidazinyl carboxylic acid residues (3-carboxypiperidazinylcarboxylic acid residue), ii) 7 to 9 contain aromatic alpha-amino-acid residue, and iii) 10 to 12 contain aliphatic alpha-amino-acid residue.Should contain the hydroxycarboxylic acid residue by big all the other those branches that encircle, 11 other carbon atoms were provided in this main ring under the situation of Sanglifehrins A-D.

According to the practice easily, the atom of the basic big ring of Sanglifehrin is numbered shown in above Sanglifehrin A in the macrolide chemistry, and the carbon atom that is about to macrolide key carbonyl is as 1 open numbering.

Sanglifehrins A-D feature also is, is connected with new dicyclo spiral system by the alkyl linking group on 23 of big ring.

Sanglifehrins A-D can experience chemical reaction widely, obtains the big ring of another kind of Sanglifehrin.Described reaction comprises the cracking of big ring (especially on this lactone oxygen groups), the cracking of linking group between macrolide and spiral ring system, and protective reaction, derivatization reaction or other chemically modifieds of substituted radical as described below.The professional who is familiar with the present technique field understands further chemical modification method.

According to the present invention, have been found that with regard to its biological activity Sanglifehrins (Sanglifehrins that spiral ring is especially wherein arranged, the situation of vk Sanglifehrins A-D) has distinctive and complete new effect.Especially found that they have following common activity:

-cyclophilin is in conjunction with activity;

-immunosuppressive activity;

-inhibition B-cell and T-hyperplasia;

-they do not have, and FK is conjugated protein to suppress active in conjunction with activity or calcineurin.

Therefore, Sanglifehrins can be seen as immunosuppressor and the anti-infective compounds that provides a class challenging and new.Especially Sanglifehrins has the previously known of being different from immunosuppressor and anti-infective compounds (as cyclosporine and Macrolide, for example Wyeth-Ayerst Laboratories and FK-506) active characteristics, show that simultaneously Sanglifehrins has the binding mode that is different from preceding compound.Therefore with regard to structure and activity, Sanglifehrins provides a class new medicine, can expect that they have enlarged the scope of immunosuppression and/or anti-infective therapy in fact, for example they are avoided or have reduced previous immunosuppression and anti-infective therapy's undesirable side effects, and/or improve or enlarged treatment to new diseases range or new patient's type.

Sanglifehrins, for example wherein the macrolide ring is the open loop form, 26 and 27 that wherein are connected with alkyl between the spiral ring system at macrolide all can have hydroxyl to replace, perhaps the volution residue that wherein is connected with big ring is cleaved or block, they usually partially or completely lose Sanglifehrins common feature activity.For example, wherein the cleaved Sanglifehrins of volution residue has the protein binding activity of parent's ring usually, but does not have tangible immunosuppressive activity.Yet, the professional who is familiar with the present technique field understands, such compound provides preparation new in addition Sanglifehrins valuable ingredients, intermediate or crucial structural framework, thereby has further enlarged the possible result of treatment of Sanglifehrin compounds.

For example the dicyclo spiral system can be seen as the constituent with crucial biological effect is provided among Sanglifehrins A～D, because its existence is important to biological activity obviously, therefore it can be used as the constituent that the other Sanglifehrins of preparation further derives or modifies, perhaps be used as the constituent of deriving or modifying other drug, for example can be used to change the activity of other immunosuppressor of Macrolide.

As shown above, the Sanglifehrins new macrolides compound of a class having represented complete novelty and had feature structure fully.

Therefore, first aspect the invention provides a macrolides compound, and they are free or shielded form, or is its salt, wherein

I) 2～6 of big ring is piperidazinyl carboxylic acid residues (piperidazinyl carboxylicacid residue); And/or

Ii) 7～9 of big ring is aromatic alpha-amino-acid residue; And/or

Iii) 10～12 of big ring is aliphatic alpha-amino-acid residue.

Macrolides compound of the present invention comprises feature structure i suitably), ii) and iii) 2, especially comprise whole 3 feature structure i), ii) and iii).

Piperidazinyl (piperidazinyl) carboxylic acid residues is 1 suitably, 2-piperazine pyridazine-3-carboxyl-1-base residue, and wherein carboxyl is positioned at 1 of big ring, and the 1-nitrogen-atoms is positioned at 6 of big ring, formula I residue for example, Wherein numbering is represented the position of atom in big ring of residue.This residue can be that ring is substituted or unsubstituted.It is suitable not being substituted.

The alpha-amino group part of aromatic alpha-amino-acid residue is positioned at 9 of big ring suitably.Suitable aromatic series a-amino acid is a phenylalanine, 3-OH-phenylalanine especially, and residue can be free or shielded form.

The alpha-amino group part of aliphatic alpha-amino-acid residue is positioned at 12 of big ring suitably.Suitable aliphatic alpha-amino-acid residue is the Xie Ansuan of free or protected form.

The rest part of big ring contains the monohydroxy-monobasic acid residue suitably, and its oxygen partly connects the macrolide key, and its carbonyl moiety forms an amido bond with the alpha-amino group on 12 on the big ring.It is suitable that described hydroxycarboxylic acid residue has 6～20 carbon atom chain lengths, has 11 carbon atom chain lengths more suitable.It can be substituted or unsubstituted, and/or it contains one or more undersaturated keys, especially along its chain length a plurality of pairs of keys is arranged.More suitably be, the rest part of big ring contains 11-oxygen-undecylene acyl (endecanoyl)-11-base, especially 11-oxygen-6,8-11 carbon diene acyl (endecadienoyl)-11-bases, this residue can at random be substituted, for example 2,3,4 and/or 5 replacements.Described hydroxycarboxylic acid residue more suitably is a formula II residue, and it can be free or shielded form, or is its salt, R wherein ₁And R ₂Be hydrogen, or represent additional keys;

R ₃Be hydrogen;

R ₄For-CO-CH ₃Or-CH (OH)-CH ₃, or

R ₃With R ₄Represent the formula III structure together,

According to the present invention, preferred macrolides compound contains the big ring of formula IV,

Wherein X, Y and Z are the residue i of above definition), ii) and iii), A is the hydroxycarboxylic acid residue of above definition, it can be free or shielded form, or its salt; Especially the big ring that contains formula V, it can be free or shielded form, or is its salt,

In general, (at Sanglifehrins A～D), big ring is substituted on the carbon atom of contiguous lactone bridging oxygen part at Sanglifehrins.Described substituting group generally include 2-oxa--2 '-azepine-3 '-oxo-spiral shell bis cyclohexane-3-base residue; formula VI for example; it can be free or shielded form; or its salt; it links to each other with the macrolide ring by the linking group that contains 6-11 carbon atom (being generally 9 carbon atoms) chain length between volution residue and the macrolide ring Wherein-a-b-is-(Me) C=CH-or-(Me) CH-CH (OH)-and

R ₅Be H or Me

(wherein Me and Et difference represent methylidene and ethyl)

This linking group can be substituted or unsubstituted, and/or contains one or more unsaturated link(age)s, especially along a plurality of pairs of keys of its chain length.This linking group can be suitably by methyl substituted, for example by 2 methyl substituted.This linking group can suitably further be replaced by hydroxyl, for example replaces by 3 hydroxyls, and/or can alkylene unsaturated, for example can contain 2 carbon-to-carbon double bonds.More suitably be; this linking group contains 1-methyl-7-methyl-nonanoyl-9-base, especially 1-methyl-7-methyl isophthalic acid-nonanoyl-9-base or 1-methyl-7-methyl isophthalic acid, 3-nonadiene acyl group-9-base; residue can at random be substituted, for example 3,4 and/or 8 replacements.Preferred linking group is a formula VII group, and it can be free or shielded form, Wherein the c representative links to each other with the volution residue,

The d representative links to each other with big ring;

R ₆And R ₇The OH that respectively does for oneself, perhaps they represent other key together.

Described linking group links to each other with big ring at the carbon atom place of contiguous lactone oxygen groups usually, and is promptly when big ring contains the basic residue of 11-oxygen-undecylene acyl group-11-, continuous at its 11.

Therefore the invention provides formula VIII compound, can be free or shielded form, or be its salt,

S-L-M VIII is the spiral dicyclo residue of S representative as above definition wherein;

The linking group of L representative as above definition;

The macrolide ring of M representative as above definition.

The concrete compound of the present invention is a compound shown in the formula IX, can be free or shielded form, or is its salt,

Wherein-definition of a-b-is the same;

-e-f-is-CH (OH)-CH (OH)-or-CH=CH-;

-g-h-with the definition of above-mentioned-a-b-and

R ₃, R ₄And R ₅Definition the same.

Formula I～IX compound contains unsymmetrical carbon, therefore many epimer forms can be arranged.Epimer that all these are possible and their non-enantiomer mixture include in the present invention.But wherein the macrolide ring is that closed loop and the formula VIII that the suitable stereo chemistry is arranged and IX compound generally have the activity of Sanglifehrins feature as previously discussed.Preferably has the active epimer of Sanglifehrin feature.In general, for pharmaceutical use of the present invention, preferred pure or pure in fact (be free or the active epimer that lacks of free Sanglifehrin feature) in fact has the active epimer of Sanglifehrin feature, for example contain at least 90%, for example contain at least 95% active epimer (promptly contain and be less than 10%, for example contain the epimer that is less than 5% non-activity).

3-carboxyl piperidazinyl carboxylic acid residues on 1～6 on big ring preferably has the following formula configuration:

Die aromatischen Aminosaeuren on 7～9 on big ring ii) is preferably the L configuration, for example is preferably the following formula configuration,

Aliphatic amino acid on 10～12 on big ring iii) is preferably the L configuration, for example is preferably the following formula configuration,

When the rest part of big ring contained formula II residue, it preferably had the following formula configuration,

Or Work as R ₃And R ₄When representing following formula together,

It preferably has the following formula configuration,

2-oxa--2 '-azepine-3 '-oxo-spiral shell bis cyclohexane-3-base residue preferably has the following formula configuration, Wherein when-a-b-be-(Me) CH-CH (OH)-time, it is preferably the following formula configuration,

When linking group was formula VII, it was preferably the following formula configuration,

Work as R ₆And R ₇When respectively doing for oneself OH, the configuration on 26 and 27 is preferably 26 (S), 27 (S) or 26 (R), 27 (R).Work as R ₆And R ₇When representing other key together, the configuration on 26 and 27 preferably as shown in the formula,

Formula IX compound of the present invention preferably has the following formula configuration, Wherein when-a-b-be-(Me) CH-CH (OH)-time, it is preferably the following formula configuration,

When-e-f-be-CH (OH)-CH (OH)-time, it preferably has (S), (S) configuration or (R), (R) configuration,

When-g-h-be-(Me) CH-CH (OH)-time, it preferably has the following formula configuration,

When-g-h-be-(Me) during C=CH-, it preferably has the following formula configuration,

Work as R ₃And R ₄When condensing together, they preferably have the following formula configuration,

Sanglifehrins A～C preferably has the following formula configuration,

The compounds of this invention can be free or shielded form, and for example " ProtectiveGroups in Organic Synthesis " (write second edition, 1991, John Wiley by T.W.Greene and P.G.M.Wuts; Sons Inc., New York) protected form described in.Especially hydroxyl can be shielded form, for example is the form of silyl ether (for example an above-mentioned book of writing at Green and Wuts is p.68-86 described), ester (see an above-mentioned book that Green and Wuts write p.87-103) and carbonic ether (see an above-mentioned book that Green and Wuts write p.104-111).Described shielded form also comprises inner shielded form; For example the situation of formula IX macrolide (wherein-g-h-is-CH (CH ₃)-CH (OH)-), 14～17 shielded forms of wherein big ring comprise formula X residue,

For example it is configured as

Again for example, in Sanglifehrins 1, the 3-glycol can be protected with suitable ring structure, for example an above-mentioned book of writing referring to Greene and Wuts is p.118-142 described.

The compounds of this invention can also exist with the form of salt.Be used for the pharmaceutically example of suitable salt of the present invention, comprise acid salt and base addition salt according to being present in substituting group specific in the compound.As previously discussed, the big ring of The compounds of this invention can cleaved (especially on lactone oxygen base), the compound that to obtain wherein big ring be the open loop form.The cracking of lactone oxygen base generally can be undertaken by hydrolysis (solvolysis), obtains formula XI compound,

R ₆O-X-Y-Z-A-OH XI is formula XII compound for example,

Formula IX ' compound for example, Wherein X, Y, Z, A, R ₃, R ₄And R ₅Definition the same, R ₆Be hydrogen or C _1-4Alkyl, for example methyl.

Described open loop form provides the method that changes basic Sanglifehrin macrocycle systems intermediate, and they also belong to part of the present invention.

Therefore, on the other hand, the present invention also provides

The macrolide of-above-mentioned open loop form, the big ring of described open loop is free or shielded form, or is its salt;

-above-claimed cpd R ₆O-X-Y-Z-A-OH, it can be free or shielded form, or is its salt;

-compound R ₆O-X-Y-Z-A '-CH (OH)-L-S is free or shielded form, or is its salt, wherein-A '-CH (OH)-be the hydroxycarboxylic acid residue, formula II residue for example defined above, the definition of other symbols is the same;

Formula XII ' compound is free or shielded form, or is its salt,

-Shi IX " compound is free or shielded form, or is its salt,

The present invention also comprise wherein 2-oxa--2 '-azepine-3 '-oxo-3 '-Ji-spiral shell bis cyclohexane system is the compound of open loop form, formula XII compound for example, it can be free or shielded form, or is its salt, Wherein the definition of a, b, L and M is the same.

The configuration of opened loop compound of the present invention is preferably the preferred configuration that above closed loop compound is determined.Open loop spiral shell bicyclic system compound formula XII preferably has the following formula configuration,

Wherein when-a-b-be-(Me) CH-CH (OH)-time, it preferably has the following formula configuration,

Macrolide of the present invention with the spiral shell dicyclo residue that is connected with big ring can also experience relate to linking group cracking (for example about formula IX, especially the key between residue 26 and 27), the result obtains independently new spiral shell dicyclic compound and another macrolides compound.As previously discussed, above-claimed cpd also can be used as intermediate, especially has the spiral shell dicyclo part of the Sanglifehrins of main functionallization in the biological activity of Sanglifehrins.

Therefore, the invention provides:

-2-oxa--2 '-azepine-3 '-oxo-3 '-Ji-spiral shell bis cyclohexane, it can be free or shielded form, or is its salt, formula VI ' compound especially, it can be free or shielded form, or is its salt, R wherein ₇Be H, any shielded OH group, active function groups or-CH ₂-CH (OH)-CH (CH ₃)-CH ₂-CH ₂-CHO group, or its equivalent δ lactol

Preferred formula VI ' compound has following configuration,

The present invention also comprise the 2-oxa--2 of open loop '-azepine-3 '-oxo-3 '-Ji-spiral shell bis cyclohexane, it is free or shielded form, or is its salt, formula XII ' compound especially, it is free or shielded form, or is its salt,

Wherein a, b and R ₇Definition the same.The spiral shell bicyclic system formula XII ' compound of open loop is preferably the following formula configuration,

The present invention also provides formula XIII macrolide, and it is free or shielded form, or is its salt,

Wherein M is macrolide ring, especially formula XIV macrolide ring as defined above,

Especially the macrolide ring that has the following formula configuration,

Wherein when-g-h-be-(Me) CH-CH (OH)-time, it preferably has the following formula configuration,

On the other hand, the present invention includes Macrolide of the present invention and related compound, pure especially in fact (for example at least 90%, at least 95% is better, and especially at least 98% is pure) natural product.

Except the above, the present invention also provides the method for preparing arbitrary the invention described above compound, and this method comprises:

I) in order to prepare any one product among Sanglifehrins A, B, C, the D, the actinomycetes strain that can produce Sanglifehrin A, B, C, D is cultivated in substratum, and separates needed Sanglifehrin A, B, C or D from the broth culture that obtains;

Ii), make Sanglifehrins A and B carry out cyclization at 15 and 16 in order to prepare Sanglifehrins C and D;

Iii) in order to prepare Sanglifehrins A and B, make Sanglifehrins C and D carry out open loop at 15 and 16 lactol ring;

Iv) for prepare wherein-g-h-is-C (CH ₃The formula IX of)=CH-or IX ' macrolide, will be wherein-g-h-is-CH (CH ₃)-CH (OH)-formula IX or IX ' compound or its protected form dewater;

V) in order to prepare wherein R ₄For-CH (OH)-CH ₃Formula IX or IX ' macrolide, will be wherein R ₄For-C (O)-CH ₃Formula IX or IX ' compound carry out hydrogenation;

Vi), make formula IX or IX ' compound carry out internal protection at 15 and 17 in order to prepare wherein 14～16 formula IX or IX ' macrolides that contain formula X residue of macrolide ring,

Vii) for preparation formula IX or IX ' macrolide, make 14～16 of macrolide ring wherein contain the formula IX of formula X residue or IX ' compound at 15 and 17 deprotections that carry out internal protection,

Viii) in order to prepare wherein R ₅Be the formula IX or the IX ' macrolide of methyl, wherein R ₅For the formula IX or the IX ' macrolide of hydrogen methylates;

Ix) in order to prepare wherein R ₄Formula IX or IX ' macrolide for oxygen-protected form make wherein R ₄For the formula IX or the IX ' macrolide of oxygen-not protected form changes oxygen-protected form into;

X) in order to prepare wherein R ₄Formula IX or IX ' macrolide for oxygen-not protected form make wherein R ₄For the formula IX or the IX ' macrolide of oxygen-protected form carries out the oxygen deprotection;

Xi), make wherein the formula IX or the IX ' macrolide of the hydroxy phenyl alanine residue of 7～10 aerobics at big ring-not protected carry out oxygen-protection in order to prepare the formula IX or the IX ' macrolide of aerobic-hydroxyl and protected phenylalanine residue on 7～10 on big ring;

Xii) for the formula IX or the IX ' macrolide of the hydroxy phenyl alanine residue for preparing aerobic on 7～10 on big ring-not protected, make formula IX or IX ' macrolide carry out oxygen-deprotection at 7～10 aerobic-hydroxyl and protected phenylalanine residues of big ring;

Xiii) for prepare wherein-e-f-for-CH (OH)-CH (OH)-formula IX or IX ' macrolide, make wherein-e-f-carries out oxydrolysis for formula IX or the IX ' macrolide of-CH=CH-;

Xiv), make the spiral shell dicyclo of formula IX or IX ' and the linking group between the big ring carry out cracking for preparation formula V ' compound or formula XII compound;

Xv) for preparation formula R ₆O-X-Y-Z-A-OH or formula R ₆O-X-Y-Z-A '-CH (OH)-L-S compound makes big ring of formula IV or the big ring of formula VIII carry out open loop in ester bridge place within it;

Xvi) formula IX or the XII macrolide in order to prepare closed loop makes formula R ₆O-X-Y-Z-A-OH compound or formula R ₆The closure that O-X-Y-Z-A '-CH (OH)-the L-S compound encircles greatly;

Xvii), make formula IX or VI ' compound in the spiral shell bicyclic system, carry out open loop for preparation formula XII or XII ' compound;

Xix), make formula XII or XII ' compound in the spiral shell bicyclic system, carry out closure for preparation formula IX or VI ' compound.

Method of the present invention can be undertaken by for example embodiment is described.Can understand that the above method can be used for the arbitrary suitable order or the combination of proper order, the result obtains another macrolides compound, and it can be the above free, shielded, open loop and form closed loop.

Macrolide of the present invention, for example Sanglifehrins A～D is natural compound, or by the natural compounds deutero-, they can obtain among the member by Streptomycetaceae usually.

As previously discussed, bacterium can produce macrolide, this former not being determined.

Therefore, on the one hand, the invention provides again:

-produce the actinomycetes strain of macrolide, wherein macrolide is following macrolide, wherein

I) 2～6 of big ring is the piperidazinyl carboxylic acid residues; And/or

Ii) 7～9 of big ring is aromatic alpha-amino-acid residue; And/or

Iii) 10～12 of big ring is aliphatic alpha-amino-acid residue, especially the invention provides

The ray bacteria strain of-generation Sanglifehrin A, B, C or D.

Suitable actinomyces strain is a Streptomycetaceae, more suitably is streptomyces, the especially described streptomycete sp.A in back 92-308110 strain, or, for example comprise the form of its mutantion line, variant, fusion, recombinant strain or modification by its deutero-bacterial strain.

The suitable bacterial strain that the present invention uses is biologically pure isolated strains.

By method commonly used, for example, can make streptomycete bacterial strain A92-308110 variation or be varied to different forms by uviolizing or by with chemical mutagen (as N-methyl-N '-nitro-nitroso-guanidine).Merge the clone that can obtain recombinating by protoplasma.Can produce all variants of Sanglifehrins or the form of recombinant strain or variation, comprise that the variant and the recombinant strain that can increase yield generation Sanglifehrins include within the scope of the invention.

In certain embodiments of the present invention, wherein Sanglifehrins A, B, C and D separate to obtain from new streptomycete bacterial strain A92-308110.Streptomyces A92-308110 bacterial classification with Budapest Treaty name storage at Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany's (May 3 nineteen ninety-five), its storage is numbered DSM 9954.Streptomyces A92-308110 bacterial classification also can obtain from Sandoz Ltd.CH4002 Basel Switzerland.

Must be noted that according to supplying with rule 28 (4) and (5) EPC, it is conditional utilizing DSM 9985 bacterial classifications.

Narration separates SanglifehrinsA, B, C and D from streptomyces A92-308110 in embodiment 2.

According to Bergey ' s Manual (the 4th volume, 1989, Williams and Wilkins, Baltimore) and the narration of The Prokaryotes (1992 Springer Verlag, New York), streptomyces A92-308110 strain belongs to Streptomycetaceae.The cell walls of this bacterium contains the LL-diaminopimelic acid.This lipid acid is different-and anteiso--side chain, straight chain and undersaturated.Sugar spectrum is not special.Vegetative mycelium does not resolve into fragment.Aerial mycelium forms the long-chain gemma.

In above reference of drawing, the bacterial strain that is designated as A92-308110 is new streptomycete.A92-308110 is grown in the various organic and inorganic mediums, and in most of the cases forms aerial mycelium.The primary substrate mycelium is as mycelial growth, and is generally cream-coloured to light taupe brown.The color of aerial mycelium belongs to grey colour pattern, ordinal number 4, and this mycelium forms gemma long-chain, that belong to spiral b type.

In following table, listed the energy for growth of streptomyces A92-308110 in Biomedia commonly used, its carbon is used and its physiological characteristic.

The growth of table 1. in various Biomedias

Culture medium culturing thing feature

Yeast extract/growth: good

Malt agar substrate mycelium: light brown

Aerial mycelium: light ash-brown

Soluble pigment: do not have

Oat agar growth: good

Substrate mycelium: burgundy

Aerial mycelium: light ash-brown

Soluble pigment: light brown

Glucose-l-asparagine growth: appropriateness

Substrate mycelium: light brown

Aerial mycelium: light ash-brown

Soluble pigment: do not have

Inorganic salt/Starch Agar growth: appropriateness

Substrate mycelium: grey

Aerial mycelium: light ash-brown

Soluble pigment: do not have

Sucrose/nitrate agar growth: very bad

Substrate mycelium: canescence

Aerial mycelium: bad, light taupe brown

Soluble pigment: no glycerine/l-asparagine agar growth: appropriateness

Substrate mycelium: light brown

Aerial mycelium: light ash-brown

Soluble pigment: no nutrient agar medium growth: appropriateness

Substrate mycelium: cream-coloured

Aerial mycelium: do not have

Soluble pigment: brown

Table 2: carbon is used appropriateness or good: glucose, fructose, pectinose, wood sugar, seminose are bad: rhamnosyl, sucrose, raffinose, Mierocrystalline cellulose, salicin feminine gender: m-inositol

Table 3: the positive starch hydrolysis of physiological characteristic nitrate reductase is an appropriateness to inorganic salt-Starch Agar

To the negative tyrosine of oat agar degrade negative milk peptonize positive melanocyte generate 18～37 ℃ of positive growth temperatures 13 ℃ of growths very bad 45 ℃ of pH scopes of not growing in pH5 and 7, rich growth

At pH9, well-grown NaCl resistance is up to 6%, but is that growth in 2% o'clock is slowed down in concentration

Macrolides compound of the present invention comprises that Sanglifehrins A, B, C and D can obtain by cultivation streptomyces A92-308110 or its variant, recombinant strain or modified form on suitable medium.The method of cultivation streptomyces A92-308110 strain that embodiment 1 has narrated (only being for narration of the present invention).

Therefore, on the one hand, the present invention includes again:

A) can produce biologically pure streptomyces A92-308110 (DSM 9954) strain of macrolide of the present invention or the form of its variant, recombinant strain or modification,

B) prepare the method for macrolide of the present invention, this method is included in the form of cultivating streptomyces A92-308110 (DSM 9954) bacterial strain or its variant, recombinant strain or modification in the suitable medium, and reclaims Sanglifehrin selectively.

Macrolides compound of the present invention, formula IX compound for example, for example the salt that pharmaceutically is suitable for of SanglifehrinsA, B, C and D and they (back on kind back be called " medicine of the present invention ") has Sanglifehrin feature activity, promptly has following common activity:

-cyclophilin is arranged in conjunction with activity;

-immunosuppressive activity arranged;

-inhibition B-cell and T-hyperplasia effect;

-there is not FK conjugated protein in conjunction with active;

-do not suppress the calcineurin activity.

In order to measure their activity, narrate test and their activity below in more detail.Macrolides compound of the present invention, for example the biological activity of formula IX compound and Sanglifehrins A, B, C and D can use-case such as following common external and In vivo assay Cells show.To the elementary humoral immune reaction of sheeps blood erythrocyte (MD, Mishell-Dutton)

In 24 well culture plates, (OF 1, and is female, 8-10 age in week, 1 * 10 with mouse boosting cell with the last volume of 1ml ⁷) and sheep red blood cell (SRBC, 3 * 10 ⁷) cultivated jointly 3 days.Collect lymphocyte, washing, and with 1 * 10 ⁶The density of individual cell is made plate and is cultivated on the soft agar that has fresh antigen (SRBC).After cultivating 60-90 minute, add complement (guinea pig serum), continue again to cultivate 60 minutes, then by plaque counting (microscopically) evaluation test result.In 3 days cultivation, lymphocyte is by antigen (SRBC) sensitization.When bringing up jointly with antigen once more, B-lymphocytic emiocytosis specific antibody, and combine with near this secretion lymphocyte antigen.Adding complement causes the erythrocytic dissolving of antibody sandwich and produces plaque.Each plaque is then represented a cell that produces antibody.

The restraining effect that plaque is formed is the pointer of medicament validity.Compound of the present invention is as Sanglifehrins A to D, approximately ... extremely ... the concentration of nM has activity in this test.

Reference:

R.I.Mishell ﹠amp; F.W.Dutton (1966), the normal mouse splenocyte suspension is in external immunization.Scierce?153：1004-1006。

R.I.Mishell ﹠amp; F.W.Dutton (1967) is from the immunization of the isolating spleen cell cultures thing of normal mouse.J.Exp.Med.126：423-442。2. lymphocyte is to the proliferative response of heterology stimulation

Two-way MLR (mouse mixed lymphocyte reacion)

To derive from the splenocyte (2 * 10 of Balb/c mouse (female, 8-10 week age) ⁵) with derive from 2 * 10 of CBA mouse (female, 8-10 week age) ⁵Splenocyte is common to be cultivated 4 days.The allos cell brings out proliferative response to reactive splenocyte group, can mix DNA by the labelled precursor thing and measure.Macrolide of the present invention, compound and the acceptable salt of pharmaceutics thereof as structural formula IX as Sanglifehrins A, B, C and D, have from the IC of about 30up to 200nM scope ₅₀, by comparison, Sandimmune records IC in this test ₅₀Be about 20nM.

T.Meo (1979) mouse MLR reaction.In: " immunological method ", L.Lefkovitsand B.Pernis, Eds, Academic Press, N.Y.pp.227-239.3.LPS activated mouse B cell

The splenocyte (2 * 10 of CBA mouse will be derived from ⁵) mix with 50 μ g/ml LPS and testing compound and to cultivate 48 hours.Precursor by mark mixes DNA mensuration cell proliferative response.Macrolide of the present invention as structural formula IX compound and the acceptable salt of pharmaceutics thereof, as Sanglifehrins A, B, C and D, suppresses B cell proliferation, has from the IC of about 40up to 100 μ M scope ₅₀

Reference:

Greaves, M.and J.Janossy, 1972, by selectivity T and the bone-marrow-derived lymphocyte reaction that the cell surface binding partner causes, Transplant Rev.11:87.

Janossy, G.and M.F.Greaves, 1971, lymphocyte activator effect.I.T and bone-marrow-derived lymphocyte are to the reaction of phytomitogen.4. do vitro cytotoxicity and cell inhibitory activity test with the THP1 cell strain

With person monocytic cell's strain THP1 (5 * 10 ⁴Individual cells/well) makes cytotoxic assay, exist IFN γ (100U/ml) and LPS (5 μ g/ml) to add under the situation of compound to be determined (to 10 μ M), it was cultivated 24-72 hour at 37 ℃.With colorimetric MTT reading method, survivaling cell is carried out quantitatively (Mossman1983) by the activity of measuring mitochondrial dehydrogenase in the viable cell.After this test was cultivated through 24 hours, macrolide of the present invention as structural formula IX compound and the acceptable salt of pharmaceutics thereof, as Sanglifehrins A, B, C and D, had the IC of about 1000-5000nM ₅₀

Mossman T.J. (1983), the rapid colorimetric determination method of cell growth and survival: be used for cell proliferation and toxicity test, J.Imm.Methods, 65, the 55-635. human peripheral blood mononuclear cell discharges TNF

According to the method for Hansell et al. (1991),, prepare monocyte from healthy volunteer's peripheral blood by Ficoll-Hypaque density separation technology.Before adding stimulant, with cell (10 ⁵Individual cells/well is in containing the 200 μ l RPMI of 10% volume FCS) cultivated 30 minutes jointly at 37 ℃ with the testing compound of serial dilution.Make stimulant with IFN-(100U/ml) and LPS (5 μ g/ml), bring out peripheral blood lymphocytes and discharge cachectin (TNF).After cultivating 3 hours,, collect supernatant liquor with cell centrifugation (1200rpm, 10 minutes).With the enzyme-linked immunosorbent assay (ELISA) reagent box that can buy, measure the content of TNF in the cell conditioned medium liquid.When measuring with this method, macrolide of the present invention as structural formula IX compound and the acceptable salt of pharmaceutics thereof, as Sanglifehrins A, B, C and D, has from the IC of about 200nM to 1000nM scope ₅₀6. cyclophilin binding assay

The cyclophilin binding assay that is fit to is to be tested at the competitive ELISA described in Eur.J.Immunol.1987 17 1359-1365 by Quesniaux.In this test, altogether add testing compound in the process of incubation, calculate then and reach the control reaction 50% that does not have competitor and suppress needed concentration (IC at the BSA-Sandimmune of cyclophilin (the human cyclophilin A of reorganization) and bag quilt ₅₀).Another kind of assay method be Schneider et al in Biochemistry (1994), 33, the competition described in the 8218-8224 is in conjunction with test.It is included in the BSA-Sandimmune of biotinylated cyclophilin (the people A of reorganization) and bag quilt is altogether added testing compound in the process of incubation.Can be by being total to incubation with streptavidin link coupled alkaline phosphatase, the amount of combined biotinylation cyclophilin when measuring existence and not having testing compound.Macrolide of the present invention as structural formula IX compound, as Sanglifehrin A, B, C and D, has from the IC of about 10 to 100nM scopes ₅₀, by comparison.When Sandimmune is measured with these detection methods, has the IC of about 80nM ₅₀

Can be used for proving that bioactive other in vitro testss of Sanglifehrins are, IL-2 indicator detection method and ConA stimulate splenocyte detection method (indication is to the effect of T-cell-stimulating).

When measuring with the test of standard, macrolide of the present invention as structural formula IX compound such as Sanglifehrin A, B, C and D, does not have the protein bound activity in conjunction with FK, can not suppress the activity of neurocalcin.7. part transplanting-antagonism-host (GvH) of rat reaction

〔Ford?et?al.，TRANSP?L.PROC.10(1979)258〕

From initial day (0 day), will be from the splenocyte (1 * 10 of 6 all female Wistar/Furth in age (WF) rats ⁷), to the female (F of F344 * WF) of the about 100g of body weight ₁The left back claw of rat is done subcutaneous injection.Animal was handled 4 days more continuously, took out Shuan Ce lymphonodi poplitei at the 7th day and weighed.With the parameter of the weight differential between two lymphoglandula as evaluation response.

In above-mentioned test, the restraining effect that GvH is reacted is the pointer of pharmacy effect.Macrolide of the present invention as structural formula IX compound and the acceptable salt of pharmaceutics thereof, as Sanglifehrins A, B, C and D, when with the dosage subcutaneous administration of about 1mg/kg, can suppress the GvH reaction and reach about 30%.8.SRBC-T _HThe DTH that cell brings out

To the female C57 BL/6 mouse right back palmula subcutaneous injection T in (6-12 age in week) _H(standby sheep red blood cell) cell clone (2 * 10 ⁶) and 1: 1 (v/v) mixture 50 μ l of 10% sheep red blood cell (SRBC) suspension.And to the left back pawl subcutaneous injection 50 μ l SRBC cell suspensions of mouse (with 1: 1 v/v of PBS dilution) (be used to measure because the non-specific palmula swelling that injection operation causes increases).After 24 hours, measure the thickness of left and right back palmula.

Calculate the percentage ratio (z) that right palmula increases than left palmula thickness.Right palmula thickness=x, left palmula thickness=y, % is than increase=z

z＝((x-y)/y)·100

Macrolide of the present invention, as structural formula IX compound and the acceptable salt of pharmaceutics thereof, as Sanglifehrins A, B, C and D, when the about 5mg/kg dosage of subcutaneous injection, the swelling of DTH mouse reduces and reaches about 50%.

Reference

A.T.J.Bianchi, H.Hooijkaas, R.Brenner, R.Tees, AA.Nordin ﹠amp; M.H.Schreier (1981) helper cell clone intermediary antigen is specific, H-2 limitation DTH.Nature?290：62-63。

P.Herrmann, M.H.Schreier, J.-F.Borel ﹠amp; C.Feurer (1988) mast cell degranulation is the main incident in mutually during as the delayed hypersensitivity effect of being brought out by the pure lines helper cell.Int.Archs?Allergy?appl.Immun.86：102-105。

The heterotransplantation of rat/mouse heart

The in vivo test effect of macrolide of the present invention, available Alzet infiltration minipump subcutaneous administration is measured by rat and mouse heart heterotransplantation.In the mouse heart heterotransplantation, (C3H is transplanted) with BALB/c, macrolide of the present invention, as structural formula IX compound and the acceptable salt of pharmaceutics thereof, Sanglifehrins A, B, C and D can prolong the transplanting survival time under approximately 30mg/kg/ days dosage.In the rat heart heterotransplantation, (Lewis is transplanted) with DA, Sandimmune and macrolide of the present invention with suboptimal dosage, as structural formula IX compound and the acceptable salt of pharmaceutics thereof, as Sanglifehrins A, B, C and D, the compatibility administration can prolong the transplanting survival time, and is as shown in the table.

Sandimmune (mg/kg) 11 contrasts (placebo)

Sanglifehrin A (mg/kg)-10 contrasts (placebo)

The transplanting survival time (my god) 12,12,12,13,13,14 29,30,45,48,＞51,＞46 contrasts (placebo)

The compounds of this invention can be used as medicine, for example as immunosuppressor and as anti-inflammatory agent.

The compounds of this invention especially can be used for preventing and treating the allotransplantation and the Xenogeneic rejection of acute and/or chronic organ or tissue, for example is used for the transplanting of cardiopulmonary, liver, kidney, pancreas, skin or the cornea of the heart, liver, merging.Show that also The compounds of this invention also can be used for preventing and treating the graft versus host disease as following bone marrow transplantation.

Medicine of the present invention also can be used for treating autoimmune disease and inflammation, especially with the relevant inflammation of etiology (comprising the autoimmunization composition), as sacroiliitis (for example rheumatoid arthritis, chronic arthritis and joint deformity) and rheumatism.The adaptable concrete autoimmune disease of medicine of the present invention comprises that the autoimmunization hematologic disease (comprises for example hemolytic anemia, aplastic anemia, pure red-cell anemia and spontaneous thrombocytopenia), the whole body lupus erythematosus, polychondritis, the sclerderm disease, wegner's granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Stevens-Johnson syndrome, spontaneous sprue, autoimmunization inflammatory bowel disease (comprising for example ulcerative colitis and Crohn disease), endocrine illness in eye, Graves disease, sarcoidosis, the multiple sclerosis disease, primary biliary cirrhosis, juvenile diabetes (diabetes I type), ommochrome urine scorching (preceding and back), keratoconjunctivitis keratoconjunctivitis sicca and spring and/or hypersensitive, the pulmonary fibrosis in gap, psoriatic arthritis, glomerulonephritis (have or do not have nephrotic syndrome, for example comprise the spontaneous nephrotic syndrome or the ephrosis of minimum change) and asthma.

For such use and other purposes, medicine of the present invention can be used separately or use with other immunosuppressor or anti-inflammatory agent (comprising S-Neoral, Wyeth-Ayerst Laboratories, FK 506 and steroide).

For above-mentioned indication, proper dosage can change according to the situation of selected medicine of the present invention, the object of receiving treatment, the mode of administration, the characteristics of need treatment disease and serious degree.But in animal experiment obtained satisfied result by the administration of 0.01～10mg/kg dosage oral administration every day in general.In bigger Mammals (for example people), every day, the dosage range of 1 oral Sanglifehrin was 0.5～500mg, every day with equal divided dose divide take for 2～4 times more suitable.

Can be in people's organ transplantation by 0.1～100mg/kg oral dose medicine of the present invention, it is better to press 0.3～30mg/kg oral dose, is more preferably the oral dose by 0.5～10mg/kg.Reinstate two, three or tetrad medicine when treating when medicine of the present invention and other immunosuppressor (as corticosteroid compound or cyclosporine or Wyeth-Ayerst Laboratories compounds), can use low dosage (for example 0.1mg/kg/ days, intravenous injection; 3mg/kg/ days begin oral) medicine of the present invention especially can with the immunosuppressor of other on-steroidals, for example use with cyclosporin A, Wyeth-Ayerst Laboratories or FK506, purpose is replacement steroide partially or completely.

Medicine of the present invention can be by common administration, especially through gastrointestinal administration, for example with oral solution form, tablet or Capsule form administration, perhaps with non-solution or the suspendible liquor form administration of for example injecting through the stomach and intestine form.Though for some situation (for example preventing and treating the rejection of liver transplantation) intravenous injection form is suitable, for the whole body drug administration oral administration normally preferably.All right topical of The compounds of this invention or percutaneous dosing for example with creme or gelifying agent or similarly dosage form application through percutaneous drug delivery, for ophthalmic applications, can be used eye creme, gelifying agent or eye drip preparation.

The unit dosage form that oral preparations is suitable contains for example 0.5～100mg The compounds of this invention for each dosage.

According to the above, the present invention also provides a series of concrete methods:

A. give the effective immunosuppressant method of object that needs above-mentioned treatment, this method comprises the medicine of the present invention of using effective dose to described object.

B.1) method of control acute and/or chronic organ allotransplantation or Xenogeneic rejection, above for example treatment is accepted shown in arbitrary type organ transplantation recipient's the method for rejection;

2) method of control graft versus host disease, the method for for example preventing and treating the graft versus host disease among the bone marrow transplantation recipient;

3) treatment autoimmune disease or treat the method for above-mentioned arbitrary disease or sufferer;

4) method of treatment asthma in the object of the described treatment of needs, this method comprises the medicine of the present invention of using effective dose to described object.

C. be used as the The compounds of this invention of medicine, for example be used as the The compounds of this invention of immunosuppressor, or be used for the treatment of the The compounds of this invention of described disease of above B or sufferer.

The medicinal compositions of the present invention of the diluent or carrier that D. contains The compounds of this invention and pharmaceutically be suitable for.

E. the application of The compounds of this invention in the preparation medicine, described medicine is used as immunosuppressor or is used for the treatment of described disease of above-mentioned B or sufferer.

In addition, have cyclophilin and can also be used as the reagent of the displacement immunity test of S-Neoral and other cyclophilin binding compounds, for example in test method described in our the common pending application application WO 95/07468, use as reagent in conjunction with active macrolides compound of the present invention.This patent application relates to the test method of measuring immunophilins bound drug (as ciclosporin) concentration in the blood, and this method comprises and adds one in conjunction with competitor so that from the mid-thing of changing dressings of immunosuppressor one immunophilins mixture of blood; Adding one can combine with medicine but not obvious and competitor bonded acceptor; From sample, separate acceptor-medicinal composition; Measure the amount of medicine.Sanglifehrins can be used as in conjunction with competitor in this test, and for example D-loop spore rhzomorph from cyclophilin, so the S-Neoral that discharges can be used for quantitative assay, for example uses the single-minded monoclonal antibody of S-Neoral is carried out quantitative assay.

Further narrate the present invention with the following examples, following embodiment only is in order to narrate, also to relate to accompanying drawing among the embodiment, wherein

Fig. 1 shows the mass spectrum of compound S anglifehrin B;

Fig. 2 shows the mass spectrum of compound S anglifehrin A;

Fig. 3 shows the mass spectrum of compound S anglifehrin D;

Fig. 4 shows the mass spectrum of compound S anglifehrin C;

Fig. 5 shows the infrared spectra of compound S anglifehrin B;

Fig. 6 shows the infrared spectra of compound S anglifehrin A;

Fig. 7 shows the infrared spectra of compound S anglifehrin D;

Fig. 8 shows the infrared spectra of compound S anglifehrin C;

Fig. 9 shows the nuclear magnetic resonance spectrum of compound S anglifehrin A;

Figure 10 shows the nuclear magnetic resonance spectrum of compound S anglifehrin D;

Figure 11 shows the nuclear magnetic resonance spectrum of compound S anglifehrin B;

Figure 12 shows the nuclear magnetic resonance spectrum of compound S anglifehrin C.

Embodiment

Culture condition

Place the various substratum of suitable nutrition and inorganics under suitable temperature, to cultivate streptomyces A92-308110 bacterial strain with aerobism or dipping culture method.Fermention medium contains available carbon source, nitrogenous source usually and comprises the inorganic salt of various trace elements, and the product that all these all can be definite or the form of compounding mixture add, and for example can find them in various original organism products.

Embodiment 1 narration obtains the inherent condition of formula I compound.The best by culture condition is selected (quantity of the quality of ventilation, temperature, pH, carbon source and nitrogenous source and quantity, inorganic salt and trace element) and by the fermentation condition in the control biological reactor, can be improved output.The cultivation of embodiment 1 A92-308110 bacterial strain

A. initial agar culture

Grew 10～14 days in the underlaid nutrient agar of slant agar culture with A92-308110:

Glucose 10.0g

Zulkovsky starch 20.0g

Yeast extract (Gistex, Gist Brocades) 5.0g

NZ-amine, A type (Sheffield) 5.0g

Lime carbonate 1.0g

Agar (Bacto) 15.0g

Demineralized water adds to 1000ml

Use NaOH/H ₂SO ₄Above-mentioned substratum is transferred to pH6.6～6.8, then in 121 ℃ of sterilizations 20 minutes.

Culture is stored in-25 ℃～-70 ℃, and the suspended matter in glycerine one peptone is stored under the liquid nitrogen condition.

B. cultivate in advance

Gemma and mycelial 10 parts of starting cultures are suspended in 100ml 0.9% salts solution.Contain the above-mentioned suspension inoculation of each personal 50ml of two 2 liter Erlenmeyer flasks of 1 liter pre-culture medium separately.The composition of pre-culture medium is as follows:

Glucose (industry) 7.5g

Glycerine 7.5g

Yeast extract (BBL) 1.35g

Fructus Hordei Germinatus extraction liquid (Wander) 7.50g

Zulkovsky starch 7.50g

NZ-amine, A type (Sheffield) 2.50g

Soy-protein 2.50g

L (-) l-asparagine 1.00g

CaCO ₃ 0.050g

NaCl 0.050g

KH ₂PO ₄ 0.250g

K ₂HPO ₄ 0.500g

MgSO ₄·7H ₂O 0.100g

Trace element solution A 1ml

Agar (Bacto) 1g

Demineralization water adds to 1000ml

Use NaOH/H ₂SO ₄Above-mentioned substratum is transferred to pH6.8～7.2, and in 121 ℃ of sterilizations 20 minutes.

The composition of trace element solution A is as follows:

FeSO ₄·7H ₂O 5.0g

ZnSO ₄·7H ₂O 4.0g

MnCl ₂·4H ₂O 2.0g

CuSO ₄·5H ₂O 0.2g

CoCl ₂·6H ₂O 2.0g

H ₃BO ₃ 0.1g

KI 0.05g

H ₂SO ₄(95％) 1ml

Demineralization water adds to 1000ml

Is that 200rpm, excentricity are in 27 ℃ of fermentations 24 hours on the gyrate shaker of 50mm with above-mentioned pre-culture at rotating speed.

C. the first intermediate culture

To contain two 75 liter bio-reactors of 50 liter pre-culture mediums separately respectively with the pre-culture inoculation of 1 liter and in 27 ℃ of fermentations 96 hours.Fermented product rotates with the 150rpm rotating speed.Speed introducing air with every liter substratum per minute 0.5 liter.

D. the second intermediate culture

Two 750 liter fermenting containers that contain 500 liter pre-culture mediums are separately inoculated with 50 liters, the first intermediate culture respectively.The second intermediate culture was incubated 70 hours in 27 ℃ of temperature.Make fermented product rotation and with the speed introducing air of every liter substratum per minute 0.8 liter with the 100rpm rotating speed.

E. main culture

To contain two 5000 liter bio-reactors of 3000 liter main mediums separately respectively with 250 liters and the inoculation of 300 liters, the second intermediate culture.Main culture was incubated 96 hours in 24 ℃ of temperature.Bio-reactor is with 45rpm rotating speed rotation, and with the speed introducing air of every liter substratum per minute 0.5 liter.

The composition of main medium is as follows:

Glucose (industry) 20g

Fructus Hordei Germinatus extraction liquid (Wander) 2g

Yeast extract (Bacto) 2g

Soybean (Bacto) 2g

KH ₂PO ₄ 0.2g

K ₂HPO ₄ 0.4g

MgSO ₄·7H ₂O 0.2g

NaCl 0.05g

CaCl ₂·6H ₂O 0.05g

Trace element solution B 1ml

Agar (Bacto) 1g

Demineralization water adds to 1000ml

With KOH/HCl pH is transferred to 6.3.This is cultivated based on 121 ℃ of sterilizations 20 minutes.

The composition of trace element solution B is as follows:

FeSO ₄·7H ₂O 5.0g

ZnSO ₄·7H ₂O 4.0g

MnCl ₂·4H ₂O 2.0g

CuSO ₄·5H ₂O 0.2g

(NH ₄) ₆Mo ₇O ₂₄ 0.2g

CoCl ₂·6H ₂O 1.0g

H ₃BO ₃ 0.1g

KI 0.05g

H ₂SO ₄(95％) 1ml

Demineralization water adds to 1000ml

The optimal medium of main culture is as follows:

Soyabeen grists 20.0g

Glycerine 40.0g

MES 0.1M

Demineralization water adds to 1000ml, pH6.8.Embodiment 2 separates Sanglifehrins_A, B, C and D from streptomyces A92-308110 bacterial strain

With the directed classification of activity, HPLC and thin-layer chromatographic analysis, from two 3000l fermentor tanks, finish the initial gross separation and the CHARACTERISTICS IDENTIFICATION of 4 new CBA active metabolites.Above-mentioned CBA (parent's ring factor is in conjunction with test) can be used as bioactive test.

With two parts of 3000l tunnings separately.From every part of tunning, get 1500l, and in the 2000l ethyl acetate places the 4000l stainless steel vessel, stirred 20 hours.Separate organic phase with SA-20 type Westfalia-Saparator.With acetic acid ethyl acetate extract washing 2 times, and vapourisation under reduced pressure is to doing with 80l water, obtains 1.64 and the 2kg extract.With three step extraction processs two parts of thick extracts are carried out the degreasing fat with 40l methanol (9: 1) and 40l hexane.Vapourisation under reduced pressure obtains the 1.34kg extract to doing.

On the post of the methanol solution of 10kg Sephadex H filling, divide two parts (every part of 670g) that the extract of above-mentioned degreasing fat is carried out column chromatography.When being added to every part of degreasing fat extract in the post, make every part to be dissolved in the 3.3l methyl alcohol.At the 15l elutriant (as fraction 1) of collecting beginning afterwards, during collecting 2l elutriant part, proceed chromatography.The active fraction of tool is 2,3 and 4, therefore with its merging, obtains 146g.(Merck, an enterprising step of 0.04～0.063mm) post is carried out chromatography, uses methyl tertiary butyl ether (MTBE), MTBE/5g methyl alcohol and MTBE/10% methyl alcohol as eluent at 1kg silica gel again with sample.Collect the 2l fraction.Fraction 5～9th, tool is active, with its merging, obtains the 43.8g sample.Further (Merck separates on 0.04～0.063mm) post, carries out gradient elution with hexane/acetone (7: 3)～acetone at 1kg silica gel with this sample.With the part 6 (7.0g) of this chromatography at 3kg Lihroprep R P 18 (Merck, 40～63 μ m) further separate on the post, carry out wash-out with methanol (94: 6), get fraction 4～7 2.16g altogether, and then on 100g silica gel H post chromatographic separation, with methylene dichloride and 3% methanol-eluted fractions, obtain 733mg, chromatographic separation on 3kg Lichroprep R P 18 posts again, obtain 621mg, chromatographic separation on 100g Lichroprep R P 18 posts is carried out wash-out with acetonitrile/water (1: 1) then, obtain the pure Sanglifehrin A of 324mg (m.p.142～145 ℃ (unbodied), (α) _D ²⁵=-67.30 (c=0.988, methyl alcohol).

Merge (7.1g) from chromatography column with the fraction 5 and 7 that the hexane/acetone wash-out obtains, and on 3kg Lichroprep R P18 (40-63 μ m) post, carry out purifying, with methanol (9: 1) wash-out, obtain 769mg, chromatography on the post of 100g silica gel H, use the MTBE/3% methanol-eluted fractions, obtain 309mg, at last chromatography on 100g silica gel H post, with methylene dichloride and 3% methanol-eluted fractions, obtain 90mg purifying Sanglifehrin B, m.p.117～121 ℃ (unbodied), (α) _D ²⁵=-52.8 (c=1.128, methyl alcohol)

Chromatography on 3kg Lichroprep R P 18 posts, the fraction 9 and 10 (2.147g) that obtains with methanol (94: 6) wash-out is purifying on 100g silica gel H post further, with methylene dichloride/5% methanol-eluted fractions, obtain 800mg, chromatography on 3kg Lihroprep R P 18 posts with methanol (9: 1) wash-out, obtains 480mg Sanglifehrin C at last, m.p.165～170 ℃, (α) _D ²⁵=-35.60 (c=0.736, methyl alcohol).

Chromatography on 3kg Lichroprep R P 18 posts, the fraction 11 and 12 (835mg) that obtains with methanol (94: 6) wash-out is purifying on the 100g silica gel H further, uses the MTBE/5% methanol-eluted fractions, obtains 140mg Sanglifehrin D, mp.137～142 ℃, unbodied.

The feature of representing Spnglifehrin A, B, C and D then with ultraviolet, infrared, mass spectrum and nuclear sulphur resonance spectrum.What obtain the results are shown in following table 4 and accompanying drawing.

Table 4Sanglifehrin A molecular formula: C ₆₀H ₉₁N ₅O ₁₃(1090.4) UV (MeOH): 275 (1962), 242 (54500), 197 (75755)

H ⁺：275(1635)，242(51884)，

OH ^-: 292 (1973), 242 (60495) IR-spectrum: Fig. 6 mass spectrum: FAB 1096[MH+Li] ⁺Fig. 2 NMR spectrum: Fig. 9 Sanglifehrin B molecular formula: C ₆₀H ₈₉N ₅O ₁₂(1072.4) UV (MeOH): 273 (4395), 242 (50600), 197 (78577) IR-spectrum: Fig. 5 mass spectrum: FAB 1098[MH+Li] ⁺Fig. 1 NMR spectrum: Figure 11 Sanglifehrin C molecular formula: C ₆₁H ₉₃N ₅O ₁₃(1104.4) UV (MeOH): 275 (1876)), 242 (51557), 197 (72643)

H ⁺：275(1391)，242(50120)

OH ^-: 292 (1832), 242 (57960) IR-spectrum: Fig. 8 mass spectrum: FAB 1110[MH+Li] ⁺Fig. 4 NMR spectrum: Figure 12 Sanglifehrin D molecular formula: C ₆₁H ₉₁N ₅O ₁₂(1086.4) UV (MeOH): 273 (3 194), 242 (47584), 197 (73766)

H ⁺：273(3237)，242(46389)

OH ^-: 285 (2600), 242 (52907) IR-spectrum: Fig. 7 mass spectrum: FAB 1092[MH+Li] ⁺Fig. 3 NMR spectrum: Figure 10

Embodiment 3 is transformed into Sanglifehrin C with Sanglifehrin A

To what stir, be chilled in 0 ℃ the 0.5ml methanol solution of 20mg (18.3 μ mol) Sanglifehrin A and add the tosic acid hydrate crystallization.The yellow solution that obtains was stirred 1 hour, use the saturated sodium bicarbonate aqueous solution termination reaction.The mixture that obtains ethyl acetate extraction secondary.Organic solution is washed with saturated brine, toward anhydrous sodium sulfate drying, filters, and concentrates down in decompression.Resistates is used methyl tertiary butyl ether through purification by silica gel column chromatography: methyl alcohol (95: 5) wash-out, obtain Sanglifehrin C, and be white amorphous powder.It is Sanglifehrin C and C thereof ₅₃4: 1 mixtures of epimer show (S) configuration (R=Me) under Sanglifehrin C has.

4: 1 mixtures of diastereomer

In addition, above-mentioned transformation can with other protonic acid (as tosic acid pyridine, hydrochloric acid or sulfuric acid) or Lewis acid (as zinc chloride, magnesium bromide or magnesium chloride.Titanium tetraisopropylate or boron trifluoride) in methyl alcohol, carry out.Use other alcoholic solvent or cosolvent such as ethanol.Virahol, butanols, vinyl carbinol, propargyl alcohol, benzylalcohol, by obtain in the following formula result, wherein reaching the homologue of ethyl, sec.-propyl, butyl, allyl group, propargyl, benzyl respectively with sample loading mode.

By above-mentioned same mode, Sanglifehrin B can be transformed into Sanglifehrin D embodiment 4 Sanglifehrin C is transformed into Sanglifehrin A

The solution of 550mg (0.5mmol) Sanglifehrin C in 4: 1 THF-water of 5ml is handled and stirred 1.5 hours with 0.5ml 2N dilute sulphuric acid.Use the saturated sodium bicarbonate aqueous solution termination reaction, the mixture that obtains ethyl acetate extraction secondary.Organic layer with the saturated sodium bicarbonate washing once, washs secondary with saturated brine earlier then, through anhydrous sodium sulfate drying, filters, and concentrates down in decompression.Resistates is used methyl tertiary butyl ether through purification by silica gel column chromatography: methyl alcohol (90: 10) wash-out, obtain Sanglifehrin A, and be white amorphous powder.

Other inorganic or organic acid can be used to contain the medium of water and any organic cosolvent.Suitable acid comprises hydrochloric acid, tosic acid or other sulfonic acid, tosic acid pyridine, acetate, trifluoroacetic acid, formic acid.Suitable organic cosolvent has acetonitrile, trimethylammonium methane amide, dimethyl sulfoxide (DMSO), diox.

The formula XV compound of formation without quantity followed in described reaction, especially depends on the reaction times (production XV compound is method preferably, and face embodiment 5 as follows).

Equally, Sanglifehrin D can be transformed into Sanglifehrin B.Embodiment 5 changes accepted way of doing sth XV compound with Sanglifehrin A

In the solution of 1.9ml acetonitrile, add the 0.1ml hydrogen fluoride-pyridine to 50mg (46 μ mmol) the Sanglifehrin A that stirs, be chilled to 0 ℃.The yellow solution that obtains stirred 1 hour, and used the saturated sodium bicarbonate aqueous solution termination reaction.The mixture that obtains ethyl acetate extraction secondary.Organic layer washs secondary with saturated brine, through anhydrous sodium sulfate drying, filters and concentrating under reduced pressure.Resistates is through purification by silica gel column chromatography, with 95: 5 methyl tertiary butyl ethers: methanol-eluted fractions, obtain formula XV compound, be white amorphous powder.

By similar approach, Sanglifehrin B can be changed accepted way of doing sth XVI compound.The C of described material ₅₃The single epimer form in position exists, but absolute configuration is when clear and definite the determining in end. Formula XV:MS m/z 1078[M+Li] ⁺(relative intensity 100); ¹H NMR (DMSO) (just list and provide characteristic signal) δ 0.40 (3H, d, H-50), 1.20 (3H, s, H-54), 1.69 (3H, s, H-49), 4.20 (1H, t, H-15), 4.58 (1H, dd, H-17), 5.19 (1H, dd, H-18), 5.28 (1H, dd, H-23), 5.62 (1H, m, H-21), 5.67 (1H, m, H-27), 5.99 (1H, d, H-25), 6.03 (1H, dd, H-19), 6.14 (1H, dd, H-20), 6.22 (1H, dd, H-26). embodiment 6 is transformed into Sanglifehrin A with formula XV compound

In the solution of 4: 1 THF-water of 0.5ml, add 50 μ l 2N dilute sulphuric acids to 54mg (μ mol) the formula XV compound that stirs.The solution that obtains stirred under room temperature 12 hours, used the saturated sodium bicarbonate aqueous solution termination reaction.Mixture ethyl acetate extraction 2 times.The organic layer that merges is used saturated sodium bicarbonate aqueous solution and salt water washing successively, through anhydrous sodium sulfate drying, filters and concentrates down in decompression.Resistates is through silica gel column chromatography, with 90: 10 methyl tertiary butyl ethers: methanol-eluted fractions, obtain Sanglifehrin A, be white amorphous solid.

Equally, formula XVI compound can be transformed into Sanglifehrin B.

Embodiment 3～6 described methods can be used as selectable intramolecularly protection-deprotection procedure.Therefore, press embodiment 5 described reactions, 15 hydroxyls can be protected selectively, makes all the other free hydroxyls selectively to handle like this.Method among the embodiment 5 is considered the selective protection of 15 and 17 two hydroxyls.Two methods can also be used as C ₅₃The intramolecularly protection of

ketone.Press embodiment

4 and 6 described reactions, hydroxyl and ketone are lived again.Therefore Sanglifehrins C and D and formula XV and formula XVI compound are the important intermediates of other Sanglifehrin compounds of preparation.Embodiment 7 preparation 16-dehydrogenation-17-dehydroxylation-Sanglifehrin A (formula XVII)

54mg (50 μ mol) formula XV compound and the tosic acid monohydrate crystal solution in 4: 1 acetonitrile-waters of 1ml is heated to 80 ℃ and kept 1.5 hours.Add saturated sodium bicarbonate aqueous solution and make reaction terminating.The mixture that obtains ethyl acetate extraction 2 times.Organic layer, filters and concentrates through anhydrous sodium sulfate drying with saturated sodium bicarbonate aqueous solution and salt water washing.Resistates through purification by silica gel column chromatography (with 90: 10 methyl-tertbutyl ethers: methanol-eluted fractions),, obtain pure title compound, be the unbodied solid of white with (RP18, with 50: 50 acetonitrile-waters～acetonitrile wash-out 45 minutes) purifying after reversed phase chromatography.MS m/z 1078[M+Li] ⁺(relative intensity 100); ¹H NMR (DMSO) (only listing characteristic signal) 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, C15-OH), 6.03 (1H, d, H-25), 6.09-6.28 (4H, m, H-18, H-19, H-20 and H-26), 6.37 (1H, d, H-17). embodiment 8 preparation 42-N-methyl-Sanglifehrin A (formula XVIII)

To 109mg (0.1mmol) the Sanglifehrin A and the 67 μ l (0.3mmol) 2 that stir and cool off (15 ℃), the 6-di-tert-butyl pyridine adds 16.5 μ l methyl trifluoro methanesulfonates in the solution of 1ml methylene dichloride.The mixture temperature to room temperature and continuously stirring 6 hours, is added saturated sodium bicarbonate aqueous solution then and makes reaction terminating.The mixture that obtains ethyl acetate extraction 2 times.Organic layer salt water washing through anhydrous sodium sulfate drying, is filtered and is concentrated.Resistates through 2 continuous chromatography purifying of silica gel (with 90: 10 methyl-tertbutyl ethers: methyl alcohol, 95: 5 methyl-tertbutyl ethers then: methanol-eluted fractions), obtain pure title compound, be the unbodied solid of white.MS m/z 1110[M+Li] ⁺(relative intensity 100); ¹H NMR (DMSO) (only listing characteristic signal) 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). embodiment 9 preparations 53 dihydro Sanglifehrin A (formula XIX)

54mg (50 μ mol) Sanglifehrin A to stirring and cooling (0 ℃) adds 2.8mg (75 μ mol) sodium borohydride in the solution of 0.5ml methyl alcohol.Continuously stirring 1 hour, and add saturated sodium bicarbonate aqueous solution.Mixture ethyl acetate extraction 2 times.Organic solution salt water washing through anhydrous sodium sulfate drying, is filtered and is concentrated.Resistates through the silica gel column chromatography purifying (earlier with 95: 5 methyl-tertbutyl ethers: methyl alcohol, use 90: 10 methyl-tertbutyl ethers then: methanol-eluted fractions), obtain pure title compound, be the unbodied solid of white.Separated products is equivalent to the mixture M S m/z 1098[M+Li of about 1: 1 C-53 diastereomer] ⁺(relative intensity 63), 1104[M+2Li-H] ⁺(relative intensity 100); ¹HNMR (DMSO) (only listing characteristic signal) δ 0.62 (3H, d, H-50), 1.02 (3H, d, H-54), 3.55 and 3.59 (1H, 2m, H-53). embodiment 10 preparation 53-tosyl group hydrazone-Sanglifehrin A (formula XX)

55mg (50 μ mol) Sanglifehrin A and 23mg (the 125 μ mol) mixture of tosyl group hydrazine in the 0.5ml methylene dichloride were at room temperature stirred 6 hours.Remove and to desolvate, resistates through the silica gel column chromatography purifying (with 90: 10 methyl-tertbutyl ethers: methanol-eluted fractions), obtain title compound, be the unbodied powder of white.MS m/z 1264[M+Li] ⁺(relative intensity 100); ¹H NMR (DMSO) (only listing characteristic signal) δ 1.70 (3H, s, H-49), 1.77 (3H, s, H-54), 2.37 (3H, s ,-NSO ₂C ₆H ₄CH 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, position protons between tosyl group), 7.73 (2H, d, tosyl group contraposition protons).Embodiment 11 preparation 26S, 27S-dihydroxyl-Sanglifehrin A (formula XXI) and 26R, 27R-dihydroxyl-Sanglifehrin A (formula XXII)

To 495mg (1.5mmol) Tripotassium iron hexacyanide that stirs and cool off (0 ℃), 207mg (1.5mmol) salt of wormwood, 19.5mg (0.025mmol) (DHQ) ₂PHAL, 65 μ l (0.005mmol) 0.08M perosmic anhydrides solution and 95mg (1mmol) the methyl sulphonamide 2.5ml t-butanol solution that in the solution of 2.5ml butanols and 5ml water, adds 545mg (0.5mmol) Sanglifehrin A in the trimethyl carbinol.With the two-phase mixture temperature that obtains to room temperature and stirred 3 hours.Add 1.08g (8.6mmol) S-WAT then, add ethyl acetate and water subsequently, mixture vigorous stirring 15 minutes.Separates two, water layer ethyl acetate extraction 2 times.The organic layer that merges, filters and concentrates through anhydrous sodium sulfate drying with saturated sodium bicarbonate aqueous solution and salt water washing.Resistates obtains 26S through reversed phase chromatography purifying (RP18, with 30: 70 acetonitrile-waters～acetonitrile wash-out 60 minutes), and the 27S-glycol is unbodied powder.

Use aforesaid method, but with (DHQD) ₂PHAL replaces (DHQ) ₂PHAL makes corresponding 26R, the 27R-glycol.

26S, the 27S-glycol; MS m/z 1130[M+Li] ⁺(relative intensity 100); ¹H NMR (DMSO) (only listing characteristic signal) δ 1.64 (3H, s, H-49), 2.06 (3H, s, H-54), 3.20 (1H, wide m, H-27), 3.45 (1H, wide m, H-31), (3.94 3H, m, H-17, H-26 and C31-OH), 4.30 (1H, d, C27-OH), 4.57 (1H, d, C26-OH), 5.20 (1H, t, H-23), 5.33 (1H, d, H-25), (5.57 3H, m, H-18, H-21 and C35-OH), 6.03 (1H, dd, H-19), 6.14 (1H, dd, H-20) .26R, 27R-glycol: MS m/z 1130[M+Li] ⁺(relative intensity 100); ¹H NMR (DMSO) (

Only list characteristic signal) and δ 1.64 (3H, s, H-49), 2.06 (3H, s, H-54), 3.16 (1H, wide m, H-27), 3.48 (1H, wide m, H-31), (3.94 3H, m, H-17, H-26 and C31-OH), 4.30 (1H, d, C27-OH), 4.57 (1H, d, C26-OH), 5.20 (1H, 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 (1H, dd, H-20). embodiment 12 26S, the cracking of glycol among 27S-dihydroxyl-Sanglifehrin A

To 90mg (79 μ mol) 26S, the 27S glycol adds 33.7mg (157 μ mol) sodium periodate in the solution of 2: 1 THF-water of 0.9ml.Continuously stirring 1 hour, and add saturated sodium bicarbonate aqueous solution.Mixture ethyl acetate extraction 2 times.Organic solution salt water washing through anhydrous sodium sulfate drying, is filtered and is concentrated.Resistates is through silica gel purification (95: 5 methyl-tertbutyl ethers: methyl alcohol), obtain formula XXIII compound (spumescence) and formula (XXIV) compound (Powdered).

Formula XXIII:MS m/z 366[M+H-H ₂O] ⁺(relative intensity 100); ¹H NMR (DMSO) is (at different center OH _Ax: Oh _Eq2: 1 mixtures of epimer)

δ 3.54 and 4.08 (1H, 2m, H-31), 3.57 (1H, wide m, H-35), 3.66 (1H, m, H-33), 4.38 (0.67H, ddd, H-27 _Ax), 4.95 (0.33H, wide m, H-27 _Eq), 5.40 (0.33H, d, C27-OH _Eq), 5.59 (0.33H, d, C35-OH), 5.61 (0.67H, d, C35-OH), 5.96 (0.67H, d, C27-OH _Ax), 7.89 (0.67H, s, NH-42), 7.91 (0.33H, s, NH-42). formula XXIV:MS m/z 745[M+Li] ⁺ ¹H NMR (DMSO) (only lists characteristic signal

δ 0.64 (3H, d, H-50), 0.81 (6H; d, H-56 and H-57), 2.06 (3H, s; H-54), 2.17 (4H, s, H-14 and H-49); 3.80 (1H, wide m, H-15), 3.94 (1H; dd, H-17), 5.33 (1H, wide 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). embodiment 13 Sanglifehrin A acetylizes obtain 61-O-ethanoyl-Sanglifehrin A (formula XXV)

54mg (50 μ mol) Sanglifehrin A and 50 μ l pyridines to stirring and cooling (0 ℃) add 5.2 μ l (55 μ mol) acetic anhydride in the solution of 0.5ml methylene dichloride.Reaction solution kept 1 hour in 0 ℃, then with its temperature to room temperature, and continuously stirring 12 hours.Add saturated sodium bicarbonate aqueous solution, the mixture ethyl acetate extraction that obtains.Organic layer filters and concentrates through anhydrous sodium sulfate drying.Resistates obtains title compound through reversed phase chromatography purifying (RP18, with 40: 60 acetonitrile-waters～acetonitrile wash-out 45 minutes), is amorphous powder.MS m/z 1132[M+H] ⁺(relative intensity 100); ¹H NMR (DMSO) (just list and provide characteristic signal) δ 1.68 (3H, s, H-49), 2.06 (3H, s, H-54), 2.25 (3H, s, CH ₃CO 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), 5.57 (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 (1H, dd, H-63), 7.51 (1H, d, H-12), 7.89 (1H, s, H-42), 8.23 (1H, d, H-9).

Claims

1. macrolide, wherein

I) 2～6 of big ring is piperazine pyridazine carboxylic acid residues; And/or

Ii) 7～9 of big ring is aromatic alpha-amino-acid residue; And/or

Iii) 10～12 of big ring is aliphatic alpha-amino-acid residue, and described macrolide is free or shielded form, or is its salt.

2. the described macrolide of claim 1, it contains above-mentioned feature structure i), ii) and iii) 2, or especially whole 3.

3. claim 1 or 2 described macrolides, wherein the rest part of big ring contains 6～20 carbon atoms, especially the hydroxycarboxylic acid residue of 11 carbon atom chain lengths.

4. the described macrolide of claim 3, wherein said hydroxycarboxylic acid residue is a formula II residue,

R wherein ₁And R ₂Be hydrogen, or represent an extra key;

R ₃Be hydrogen;

R ₄For-CO-CH ₃Or-CH (OH)-CH ₃, or

R ₃And R ₄Represent the formula III structure together, Described macrolide is free or shielded form, or is its salt.

5. any one described macrolide in the claim 1～5, it comprises the big ring of formula IV,

Wherein X, Y and Z are the residue i described in the claim 1), ii) and iii),

A is the hydroxycarboxylic acid residue described in claim 3 or 4,

Described macrolide is free or shielded form, or is its salt.

6. the described macrolide of claim 5, it comprises the big ring of formula V Wherein A is described with claim 5,

Described macrolide is free or shielded form, or is its salt.

7. any one described macrolide during aforesaid right requires, the carbon atom of contiguous lactone bridging oxygen part by 2-oxa--2 '-azepine-3 '-oxo-spiral shell bis cyclohexane-3-base residue replaces.

8. the described macrolide of claim 7, wherein spiral shell bis cyclohexane base residue is a formula VI group, Wherein-a-b-is-(Me) C=CH-or-(Me) CH-CH (OH)-, R ₅Be hydrogen or methyl,

Described macrolide is free or shielded form, or is its salt, and it is by containing 6～11 between volution residue and the macrolide, and the linking group that is generally 9 carbon atom chain lengths links to each other with macrolide.

9. the described macrolide of claim 8, wherein the linking group between volution residue and macrolide is a formula VII group, Wherein the c representative is connected with the volution residue,

The d representative is connected with big ring,

R ₆And R ₇Hydroxyl, perhaps R respectively do for oneself ₆And R ₇Represent other key together;

Described macrolide is free or shielded form.

10. formula VIII compound,

S-L-M VIII wherein S represent 2-oxa--2 '-azepine-3 '-oxo-spiral shell bis cyclohexane-3-base residue,

The L representative contains 6～11, is generally the linking group of 9 carbon atom chain lengths,

M represents any one described macrolide in the claim 1～6,

Described compound is free or shielded form, or is its salt.

11. the described compound of claim 10, it has formula IX structure,

Wherein-definition of a-b-is the same;

-e-f-is-CH (OH)-CH (OH)-or-CH=CH-;

The definition of-g-h-is with-a-b-; With

R ₃, R ₄And R ₅Definition the same,

Described compound is free or shielded form, or is its salt.

12. the described compound of claim 11, it shows configuration under having,

Wherein when-a-b-be-(Me) CH-CH (OH)-time, it preferably has following configuration,

When-e-f-be-CH (OH)-CH (OH)-time, it preferably has (S), (S) configuration or (R), (R) configuration;

When-g-h-be-(Me) CH-CH (OH)-time, it preferably has following configuration,

When-g-h-be-(Me) during C=CH-, it preferably has following configuration, Work as R ₃And R ₄When condensing together, they preferably have following configuration,

13. be selected from the Sanglifehrin of Sanglifehrin A, B, C and D.

14. any one described compound in any one described macrolide or the claim 10～12 in the claim 4～9, wherein 14～17 of big ring contain formula X residue,

For example, it is configured as

15. formula XI compound,

R ₆O-X-Y-Z-A-OH XI is formula XII for example

Formula IX ' compound for example

Wherein X, Y, Z, A, R ₃, R ₄And R ₅Define the same,

R ₆Be hydrogen or C _1-4Alkyl, methyl for example,

Described compound is free or shielded form, or is its salt.

16. any one described macrolide among the claim 1-9, it is the open loop form, and the big ring of described open loop is free or shielded form, or is its salt.

17. compound R ₆O-X-Y-Z-A-OH, wherein X, Y, Z and A define with claim 5,

R ₆Be hydrogen or C _1-4Alkyl,

Described compound is free or shielded form, or is its salt.

18. compound R ₆O-X-Y-Z-A '-CH (OH)-L-S, wherein-A '-CH (OH)-be the hydroxycarboxylic acid residue of claim 3 definition, other symbol is with claim 17,

Described compound is free or shielded form, or is its salt.

19. formula XII ' compound,

Described compound is free or shielded form, or is its salt.

20. formula IX " compound,

Described compound is free or shielded form, or is its salt.

21. wherein 2-oxa--2 '-azepine-3 '-oxo-3 '-Ji-spiral shell bis cyclohexane ring system is the compound of open loop form, formula XII compound for example,

Wherein the definition of a, b, L and M is the same,

Described compound is free or shielded form, or is its salt.

22.2-oxa--2 '-azepine-3 '-oxo-3 ' base-spiral shell bis cyclohexane compound, it is free or shielded form, or is its salt; Especially formula VI ' compound, R wherein ₇Be hydrogen or hydroxyl and protected arbitrarily, activity functional groups or-CH ₂-CH (OH)-CH (CH ₃)-CH ₂-CH ₂-CHO group or corresponding δ-lactol,

Described compound is free or shielded form, or is its salt.

23. the 2-oxa--2 of open loop '-azepine-3 '-oxo-3 ' base-spiral shell bis cyclohexane, it can be free or shielded form, or is its salt, formula XII ' compound especially,

Wherein a, b and R ₇Define the same,

Described compound is free or shielded form, or is its salt.

24. formula XIII macrolide, Wherein M is the above macrolide, formula XIV macrolide especially,

Described macrolide is free or shielded form, or is its salt,

25. prepare the method for above-mentioned arbitrary The compounds of this invention, this method comprises:

I) in order to prepare among Sanglifehrins A, B, C or the D any one, in substratum, cultivate the actinomycetes strain that can produce Sanglifehrin A, B, C or D, from the broth culture that obtains, separate needed Sanglifehrin A, B, C or D;

Ii), make Sanglifehrins A and B 15 and 16 cyclizations in order to prepare Sanglifehrins C and D;

Iii) in order to prepare Sanglifehrins A and B, make Sanglifehrins C and D 15 and 16 lactol ring open loop;

Iv) for prepare wherein-g-h-is-C (CH ₃The formula IX of)=CH-or IX ' macrolide, make wherein-g-h-is-CH (CH ₃)-CH (OH)-or the formula IX of its shielded form or the dehydration of IX ' compound;

Vi), make formula IX or IX ' compound carry out internal protection in order to prepare wherein 14～16 formula IX or IX ' macrolides that contain formula X residue of macrolide ring,

Vii) for preparation formula IX or IX ' macrolide, make wherein 14～16 formula IX or IX ' compounds that contain formula X residue of macrolide ring slough internal protection,

Viii) in order to prepare wherein R ₅Formula IX or IX ' macrolide for methyl make wherein R ₅For the formula IX or the IX ' macrolide of hydrogen methylates;

Ix) in order to prepare wherein R ₄Formula IX or IX ' macrolide for the O-protected form make wherein R ₅Formula IX or the IX ' macrolide not protected for O-carry out the O-protection;

X) in order to prepare wherein R ₄Formula IX or IX ' macrolide for the not protected form of O-make wherein R ₅For the formula IX or the IX ' macrolide of O-protected form carries out the O-deprotection;

Xi), make wherein and carry out the O-protection at 7～10 that encircle greatly formula IX or the IX ' macrolides that contain the not protected hydroxy phenyl alanine residue of O-in order to prepare formula IX or the IX ' macrolide that wherein on 7～10 of big ring, contains O-hydroxyl and protected phenylalanine residue;

Xii), make wherein 7～10 formula IX or IX ' macrolides that contain O-hydroxyl and protected phenylalanine residue carry out the O-deprotection at big ring in order to prepare wherein 7～10 formula IX or IX ' macrolides that contain the not protected hydroxy phenyl alanine residue of O-at big ring;

Xiv), make the spiral shell bicyclic radicals of formula IX or IX ' compound and the linking group between the big ring carry out cracking for preparation formula V ' compound or formula XII compound;

Xv) for preparation formula R ₆O-X-Y-Z-A-OH or formula R ₆O-X-Y-Z-A '-CH (OH)-L-S compound makes big ring of formula IV or the big ring of formula VIII carry out open loop;

Xvi) formula IX or the XII macrolide in order to prepare not busy loop type makes formula R ₆O-X-Y-Z-A-OH or formula R ₆The closure that O-X-Y-Z-A '-CH (OH)-the L-S compound encircles greatly;

Xvii) for preparation formula? does compound make formula? compound carries out open loop in the spiral shell bicyclic system;

Xix) for preparation formula? does compound make formula? compound carries out closed loop in the spiral shell bicyclic system.

26. produce the actinomycetes strain of macrolide, this macrolide is wherein here

I) 2～6 of this big ring is the piperidazinyl carboxylic acid residues; And/or

Should big 7～9 of encircling be aromatic alpha-amino-acid residue ii); And/or

Should big 10～12 of encircling be aliphatic alpha-amino-acid residue iii),

27. can produce the biology of macrolide of the present invention pure isolated strains streptomycete bacterial strain A92-308110 (DSM 9954) or its mutant strain, its reorganization or modified form.

28. prepare the method for macrolide of the present invention, this method is included in the form of cultivating streptomycete bacterial strain A92-308110 (DSM 9954) or its mutant strain, its reorganization or modification in the suitable medium, and randomly reclaims Sanglifehrin.

Need the object of described treatment to carry out immunosuppressant method 29. give, this method comprises the The compounds of this invention of using effective dose to this object.

30. in the object of the described treatment of needs:

I) prevent acute and/or chronic organ allotransplantation or Xenogeneic rejection, for example treat above-mentioned arbitrary particular type organ transplantation recipient;

Ii) prevent graft versus host disease, for example this disease in the bone marrow transplantation recipient;

Iii) treat autoimmune disease or arbitrary above-mentioned disease or illness;

Iv) treat the method for asthma, this method comprises the The compounds of this invention of using effective dose to described object.

31. be used as the The compounds of this invention of medicine, for example be used as immunosuppressor or be used for the treatment of described arbitrary disease of top B or illness.

32. contain the The compounds of this invention and the medicinal compositions of suitable diluents or carrier pharmaceutically.

33. the application of The compounds of this invention in the medicine of the preparation described arbitrary disease of B or illness or above being used for the treatment of as immunosuppressor.

34. any one described compound is used as reagent in S-Neoral or other displacement immunity tests in conjunction with the compound of cyclophilin in the claim 1～6,8～10,15 or 16.