KR19990028724A - Mark Rowledge - Google Patents

Abstract

Immunosuppressive and anti-inflammatory properties

i) a piperidinylcarboxylic acid residue is provided at positions 2 to 6 of the macrocyclic ring, and /

ii) an aromatic? -amino acid residue is provided at positions 7 to 9 of the macrocyclic ring, and /

iii) a novel type of macrolide, wherein aliphatic a-amino acid residues are provided at positions 10 to 12 of the macromocyclic ring, preferably two or all of the structural features i), ii) and iii) More particularly a compound of formula < RTI ID = 0.0 > IX < / RTI >

<Formula IX>

Description

Mark Rowledge

The present invention relates to a novel type of markrolide having useful pharmaceutical and related activities. For convenience, these novel compounds of Markrolide type are collectively referred to herein as " Sanglifehrins ".

The first triglycerin was isolated from Actinomycete fermentation broth. These are Sanglyperin A to D of the formula:

<Sanglipherin A>

<Sanglifeprin B>

<Sanglipherin C>

<Sanglipherin D>

As shown, the macrocyclic ring of Sanglifiperin A to D comprises i) a 3-carboxypiperidinylcarboxylic acid residue at positions 1 to 6, ii) at positions 7 to 9, an aromatic alpha -amino acid Residue, and iii) an aliphatic a-amino acid residue at positions 10-12. The remainder of the macrocyclic ring contains the hydroxycarboxylic acid moiety, and in the case of the sanglyperin A to D, it contains 11 additional carbon atoms in the primary macrocyclic ring.

According to the prior art of markrolide chemistry, the atoms of the triglycerin primary macrocyclic ring start at the carbon atom of the carbonyl group of the macrocyclic lactone linkage at position 1, as in the case of the indicated triglycerin A, It is counted.

Sanglyperin A to D are also characterized by the presence of a novel bicyclic spiro system coupled at the 23 position of the macrocyclic ring through the hydrocarbyl linker group.

Many chemical manipulations can be performed to obtain another markrolide of the &lt; RTI ID = 0.0 &gt; triglycerin &lt; / RTI &gt; Such manipulations include manipulations such as cleavage at the macrolide ring, in particular in the lactoneoxy group, cleavage of the anchor group between the macrolide and the spirocyclic system, and chemical modification of, for example, protection, derivatization or other substituents And is described, for example, in the following. Other modifying means are evident to those skilled in the art.

According to the present invention, in the case of the presence of a spiroglycine system, as in the case of sanglyferin, in particular in the case of sanglyferin A to D, the sanglyferin has a novel and overall novel profile in terms of its biological activity. In particular, they have found that they represent a combination of the following activities.

- cyclophilin binding activity;

- immunosuppressive activity;

- inhibition of B- and T-cell proliferation;

However, they do not have FK binding protein binding activity or calcineurin inhibitory activity.

Thus, it can be understood that phaglycerin provides an interesting new type of immunosuppressive and anti-inflammatory compounds. In particular, Sanglifepin has an activity profile different from the previously known immunosuppressive and anti-inflammatory compounds such as cyclosporin and markrolide, such as rapamycin and the FK506 type, Acting mode. Thus, phaglycerin may be involved in materially expanding the boundaries of immunosuppressive and / or anti-inflammatory therapy; For example, in order to avoid or reduce the undesirable side effects of conventional immunosuppressive agents and antiinflammatory therapies, and / or in terms of both structure and activity for improving or expanding such therapy to new disease areas or new patient ranges Thereby providing a new category of medicinal substance.

For example, when the macrolide ring is in the open ring form, the 26 and 27 positions in the hydrocarbyl linker between the macrolide and the spiro ring system are both hydroxy substituted, or when macrocyclic When the spiro-residue bonded to the ring is cleaved or shortened, the sum glypherin is generally deficient in some or all of the combination of characteristic activities of the triglycerin. For example, sanglyferin in which the spiro residue is truncated usually has cyclophilin binding activity, but does not possess significant immunosuppressive activity. However, as will be apparent to those skilled in the art, these compounds provide effective components, intermediates or key building blocks for the production of novel &lt; RTI ID = 0.0 &gt; topglycerin &lt; / RTI &gt;

In this regard, additional pharmaceutical agents for further derivatization or modification associated with additional &lt; RTI ID = 0.0 &gt; phaglycerin &lt; / RTI &gt; For example, to provide structural components with key biological significance that are useful as structural components for use in derivatization or modification to modify the activity of other immunosuppressive drug substances of the macrolide type, An example of this is the binary spinro system.

As indicated, &lt; RTI ID = 0.0 &gt; triglycerin &lt; / RTI &gt; is a novel whole and represents a novel type of macrolide compound with an overall characteristic structure.

Therefore, as a first aspect of the present invention,

i) a piperidinylcarboxylic acid residue is provided at positions 2 to 6 of the macrocyclic ring, and /

ii) an aromatic? -amino acid residue is provided at positions 7 to 9 of the macrocyclic ring, and /

iii) an aliphatic alpha -amino acid residue is provided at positions 10 to 12 of the macrocyclic ring

There is provided a macrolide of glass or protected form, or a salt thereof.

Suitably, the macrolides of the present invention include two, especially three, of the structural features i), ii) and iii).

The piperidinylcarboxylic acid residue is suitably a 1,2-piperidazine residue, for example a residue of formula I, wherein the carboxy moiety occupies the 1-position and the 1-nitrogen atom is present at the 6 position of the macrocyclic ring Carboxy-1-yl moiety.

(Wherein the numbers given represent the atomic positions of the residues in the macrocyclic ring)

These residues may be substituted or unsubstituted rings. Suitably it is not available.

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

The alpha -amino portion of the aliphatic alpha -amino acid residue occupies the 12-position of the macrocyclic ring. Suitably the aliphatic alpha -amino acid residue is a valine residue in free or protected form.

The remainder of the macrocyclic ring is suitably a hydroxycarboxylic acid residue, an oxy moiety that completes the macrocyclic lactone linkage, and a carbonyl moiety that forms an amide linkage with the [alpha] -amino group at the 12 position of the macrocyclic ring . The hydroxycarboxylic acid residue suitably has a chain length of 6 to 20, more preferably 11. They may be substituted or unsubstituted and may contain one or more unsaturated linkages, especially cumulative double bonds along their length. More preferably, the remainder of the macrocyclic ring is optionally substituted with 11-oxy-endecanoyl-11-yl, especially 11-oxy-6, which is a moiety that may be substituted at the 2, 3, 8-endocadienoyl-11-yl. More preferably, the hydroxyl acid residue is a residue of the formula (II) or a salt thereof in free or protected form.

Wherein R ₁ and R ₂ are both hydrogen or represent an extra bond;

R ₃ is hydrogen;

R ₄ is -CO-CH ₃ or -CH (OH) -CH ₃ or

R ₃ and R ₄ together represent the structure of formula (III)

Thus, preferred macrolides according to the present invention comprise macrolides of formula IV in free or protected form, or salts thereof, or macrolides of formula V in free or protected form Or a salt thereof.

(Wherein X, Y and Z are the residues i), ii) and iii) defined above,

A is a hydroxycarboxylic acid residue as defined above)

Generally, as in Sanglyferin A to D, the macrocyclic ring in the sanglycerin is substituted at the carbon atom adjacent to the oxy moiety of the lactone bridge. Typically, such substituents are selected from the group consisting of 2-oxy (VI) of the formula VI in free or protected form connected to the macrolide ring via a linker comprising a linear array of 6 to 11, typically 9, carbon atoms between the spiro residue and the macrolide ring -2'-aza-3'-oxo-spirobicyclohexan-3-yl residue and salts thereof.

(Me) C = CH- or - (Me) CH-CH (OH) -,

R &lt; ₅ &gt; is H or Me, where Me and Et represent methyl and ethyl, respectively)

The linker group may be substituted or unsubstituted, and / or may contain one or more unsaturated linkages of cumulative double bonds, especially along its length. Suitably, the linker group may be methyl substituted, for example, by two methyl groups. Suitably, the linker group may be further substituted by hydroxy, for example, three hydroxy substituents, and / or may contain ethylenic unsaturation, for example, two carbon-carbon double bonds. More suitably, the linker group is optionally substituted 1-methyl-7-methyl-nonanoyl-9-yl, especially 1-methyl-7-methyl-1-nonenoyl- 9-yl or 1-methyl-7-methyl-1,3-nonadienoyl-9-yl residue. Preferably the linker group is a group of formula VII in free or protected form.

(Wherein c represents the connection to the spiro residue,

d represents the linkage to the macrocyclic ring,

R ₆ and R ₇ each represent OH or an additional bond together)

The linker group may be attached to the carbon atom adjacent to the lactone oxy group of the macrocyclic ring at its 11 position, generally when the macrocyclic group comprises an 11-oxy-endocanoyl-11-yl moiety.

Accordingly, the present invention provides a compound of formula (VIII), or a salt thereof, in free or protected form.

S-L-M

(Wherein S represents a spirobicyclo residue as defined above,

L represents a linker group as defined above,

M represents a macrolide ring as defined above)

Particular compounds of the invention are the compounds of formula (IX) or salts thereof in free or protected form.

(Wherein a-b- is as defined above,

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

-g-h- is as defined in a-b-,

R ₃ , R ₄ and R ₅ are as defined above)

The compounds of formulas I-IX include asymmetric carbon atoms and may therefore exist in various epimer forms. All diastereomeric mixtures as well as all possible epimeric forms are included in the present invention. However, when the macrolide ring is in the ring-closed form and the compounds of formula (VIII) and (IX), which are suitable stereochemically, have activity which is usually characteristic of the triglycerin, Epimers having a triglycerin-specific activity are preferred. Generally, in the case of pharmaceutical applications, for example, in accordance with the present invention, an active epimer containing at least 90%, such as at least 95% of active epimers (i.e. less than 10%, for example less than 5% (Without or substantially free of epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimeric epimorphs).

Preferably, the 3-carboxypiperidinylcarboxylic acid residue i) at positions 1 to 6 of the macrocyclic ring has the following form.

Preferably, the aromatic amino acid ii) at positions 7 to 9 of the macrocyclic ring has an L-form, for example, the following form.

Preferably, the aliphatic amino acid iii) at the 10 to 12 position of the macrocyclic ring has the L-form, for example the following form.

When the remainder of the macrocyclic ring comprises the moiety of formula II, it preferably has the following form.

Wherein R ₃ and R ₄ are together , It preferably has the following form.

Preferably the 2-oxy-2'-aza-3'-oxy-spirobicyclohexan-3-yl moiety has the following form.

(Wherein, when -a-b- is - (Me) CH-CH (OH) -, it preferably has the following form)

When the linker is of formula (VII), it preferably has the following form.

When R ₆ and R ₇ are each an OH group, the form at positions 26 and 27 is preferably 26 (S), 27 (S) or 26 (R), 27 (R). When R ₆ and R ₇ together represent an additional bond, the form at positions 26 and 27 is preferably:

The compounds of the invention of formula (IX) preferably have the following forms.

Here, when -a-b- is - (Me) CH-CH (OH) -, it preferably has the following form:

When the -e-f- is -CH (OH) -CH (OH) -, it preferably has the (S) or (S) form or the (R) or (R) form.

When -g-h- is - (Me) CH-CH (OH) -, it preferably has the following form.

When -g-h- is - (Me) C = CH-, it preferably has the following form.

When R ₃ and R ₄ are fused together, they preferably have the following form.

Preferably, the triglycerin A to C has the following form.

<Sanglipherin A>

<Sanglifeprin B>

<Sanglipherin C>

<Sanglipherin D>

Compounds of the present invention may be in free or protected form, for example, protected forms are described in " Protective Groups in Organic Chemistry Synthesis " by TW Greene and PGM Wuts, 2nd Edition, 1991, John Wiley & New York. In particular, the OH groups may be present, for example, in the form of a silyl ether (as described on pages 68 to 86 of the above document), esters (as described on pages 87 to 103 of the above document) and carbonates May be in protected form. Such protected forms are also protected internally form, e.g. -gh- is _{-CH (CH 3) -CH (OH} ) - of 14 to 17 positions of the macrolide ring in the case of the roll ride mark of formula IX Lt; RTI ID = 0.0 &gt; X &lt; / RTI &gt;

For example &Lt; / RTI &gt;

In addition, for example, the 1,3 diols present in the sanglyperin can be protected with suitable ring structures, for example the appropriate structures described on pages 118 to 142 of the document.

The compounds of the present invention may also be present in the form of a salt. Pharmaceutically acceptable salts suitable for use in accordance with the present invention include suitable acid and base addition salts in connection with the particular substituents present in the present compounds.

As noted above, the macrocyclic ring of the compounds of the present invention may provide compounds, particularly those in which the macrocyclic ring is cleaved at the lactone oxy group. Generally, cleavage of the lactone oxy group proceeds by, for example, hydrolysis (resolved resolution) to provide a compound of formula XI, for example a compound of formula XII, for example a compound of formula IXa.

R ₆ OXYZA-OH

Wherein X, Y, Z, A, R ₃ , R ₄ and R ₅ are as defined above and R ₆ is hydrogen or C _1-4 alkyl, for example methyl.

This open form provides an intermediate for the modification of the basic phase-glycerin macrocyclic ring system, which is also a part of the present invention.

Therefore, in another aspect of the present invention

In the ring-opened form defined above, the ring-opened macrocycle is a macrolide in free or protected form, and its salts,

- a compound R ₆ OXYZA-OH or a salt thereof as defined above in free or protected form,

- a free or protected form of the compound R ₆ OXYZ-A'-CH (OH) -LS wherein -A'-CH (OH) - represents a hydroxycarboxylic acid residue such as a residue of the above- And the other symbols are as defined above) or a salt thereof,

- a compound of formula (XIIa), or a salt thereof, in free or protected form,

A compound of the formula (IXb), or a salt thereof, in free or protected form is provided.

The present invention also relates to compounds of the free or protected form, wherein the 2-oxy-2'-aza-3'-oxo-3'-yl-spirobicyclohexane ring system is a ring- .

(XII)

Wherein a, b, L and M are as defined above.

The ring-opened compounds of the present invention are preferably in the same form as the preferred form classified in the case of the ring-closed compound. The ring spirobicyclo ring system of the compound of formula (XII) preferably has the following form.

In the formula, when a-b- is - (Me) CH-CH (OH) -, it preferably has the following form.

The macrolides according to the invention having spirobicyclo moieties bonded to the macrocyclic ring can also carry out cleavage of the linkage group interposed between residues 26 and 27, for example in the case of formula IX, Bicyclo compounds and further markrolides can be obtained. In addition, as noted above, these compounds are useful as intermediates, particularly as a group, as spirobicycline moieties of the triglycerin, which have an integrative role in the biological activity of the triglycerin.

Therefore,

2-oxy-2'-aza-3'-yl-spirobicyclohexane or its salts, in free or protected form, in free or protected form.

Wherein R ₇ is hydrogen, an optionally protected OH group, a reactive functional group, or a -CH ₂ -CH (OH) -CH (CH ₃ ) -CH ₂ -CH ₂ -CHO group or a delta lactol equivalent thereof or a salt thereof.

Preferably, the compound of formula (VIa) has the following form.

In the formula, when -a-b- is - (Me) CH-CH (OH) -, it preferably has the following form.

The present invention also provides a compound of formula XIIa, especially in the free or protected form, ring opening 2-oxy-2'-aza-3'-oxo-3-yl-spirobicyclohexane or a salt thereof.

<Formula XIIa>

Wherein a, b and R ₇ are as defined above or a salt thereof in free or protected form. A ring opening spiro bicyclo ring system of a compound of formula (XIIa) is a preferred form.

<Formula XIIa>

In the formula, when -a-b- is - (Me) CH-CH (OH) -, it preferably has the following form.

The present invention also provides a macrolide of formula (XIII) in free or protected or ring opened form.

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

And preferably has the following form.

Here, when -g-h- is - (Me) CH-CH (OH) -, it preferably has the following form;

When -g-h- is - (Me) C = CH-, it preferably has the form:

When R ₃ and R ₄ are fused together, they preferably have the following form.

In another aspect, the present invention includes a compound of the present invention in a substantially pure form, for example, at least 90%, preferably at least 95%, particularly at least 98% pure form of the macrolide.

In addition to the above, the present invention also provides a method for producing any of the compounds of the present invention as defined above including the following method.

i) For producing the sanglyferin A, B, C or D, the strain Sanglififerin A, B, C or D-producing actinomycetes is cultivated in a culture medium, and in the obtained culture broth, B, C or D,

ii) To prepare the triglycerin C and D, the triglycerin A and B were cyclized at positions 15 and 16,

iii) To prepare the triglycerin A and B, the triglycerin C and D were opened at the 15 and 16 positions to form the lactol ring,

iv) -gh- is C (CH ₃₎ = CH- the -gh- is _{CH (CH 3) -CH (OH} ) to produce a macrolide of formula IX or IXa - or a protected form of formula (IX) Or &lt; RTI ID = 0.0 &gt; IXa &lt; / RTI &

v) R ₄ is CH (OH) -CH ₃ in order to produce a macrolide of formula IX or IXa, R ₄ is hydrogenated and the C (O) -CH _3, a compound of Formula IX or IXa;

vi) performing the internal protection at the 15 and 17 positions of the compound of formula IX or IXa to produce the markrolide of formula IX or IXa wherein positions 14 to 16 of the macrolide ring comprise a moiety of formula X,

(X)

vii) For the preparation of the macrolides of formula IX or IXa, the compounds of formula IX or IXa in which positions 14 to 16 of the macrolide ring comprise a moiety of formula X are subjected to reversal of internal protection at positions 15 and 17 and,

(X)

viii) to R ₅ is the production of fluoride-methyl of the formula IX or IXa of roll marks, and to perform the R ₅ is H methylating the fluoride of the formula IX or IXa mark roll,

ix) R ₄ O- is to prepare the protected forms of the formula IX or IXa of the macrolide, R ₄ O- is the non-protected form of formula IX or IXa of macrolide O- protection and,

x) R ₄ is performing O- deprotection of O- for the production of a non-protected form of formula IX or IXa of the macrolide, R ₄ is a protected form of O- formula IX or IXa macrolide and

xi) To prepare the macrolylide of formula IX or IXa comprising an O-protected hydroxyphenylalanine residue at positions 7 to 10 of the macromocyclic ring, an O-unprotected Carrying out the protection of the macrolide of formula (IX) or (IXa) comprising a hydroxyphenylalanine residue,

xii) To produce a macrolylide of formula IX or IXa comprising an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, the O-protected Deprotection of the macrolide of formula (IX) or (IXa), which comprises a hydroxyphenylalanine residue,

xiii) For the preparation of the macrolides of formula IX or IXa in which -ef- is CH (OH) -CH (OH) -, oxidation of the macrolides of formula IX or IXa in which -ef- is -CH = CH- &Lt; / RTI &gt;

xiv) In order to prepare a compound of formula (Va) or a compound of formula (XII), the compound of formula (IX) or (IXa) is cleaved with a linker group between the spirobicyclo group and the macrocyclic ring,

xv) reacting the macrocyclic of formula IV or the macrocyclic ring of the compound of formula VIII with a compound of formula VIII in its lactone bridging to produce a compound of formula R ₆ OXYZA-OH or formula R ₆ OXYZ-A'-CH (OH) And,

xvi) Closing the macrocyclic ring of R ₆ OXYZA-OH or the formula R ₆ OXYZ-A'-CH (OH) -LS to prepare the macrolides of formula IX or XII,

xvii) carrying out ring opening in the spirobiocyclic ring system of a compound of formula (IX) or (VIa) to produce a compound of formula (XII) or (XIIa)

xix) In order to prepare compounds of formula IX or VIa, the compounds of formula XII or XIIa are subjected to ring closure in the spirobiocyclic ring system.

The method of the present invention can be carried out, for example, as described in the Examples. It will be appreciated that the above-defined methods for obtaining other mark rolls of the above-described free, protected, ring-opened and ring-closed forms can be applied suitably sequentially or in combination.

Macrolide, for example, the glycidyl Perrin A to D of the present invention can be normal or Streptomyces setae years old child with natural compounds obtained from the group of the (Streptomycetaceae) or derived therefrom.

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

Thus, in another aspect,

i) a piperidonyl carboxylic acid residue is provided at positions 2 to 6 of the macrocyclic ring, and /

ii) an aromatic? -amino acid residue is provided at positions 7 to 9 of the macrocyclic ring, and /

iii) actinomycetes strains which produce markrolides provided with aliphatic a-amino acid residues at positions 10 to 12 of the macrocyclic ring, in particular

-Sanglifiperine A, B, C or D-producing actinomycetes strains are provided.

Suitable Streptomyces strains are Streptomyces setae years old child and, further preferably Streptomyces (Streptomyces), A particularly illustrating Streptomyces species that the or A92-308110 or a mutant, variant, fusion, or a recombinant in which It is derived from the same form as the modified form.

Suitably the strain of the invention is in the form of a biologically pure isomer.

For example, Streptomyces sp. A92-308110 can be mutated or modified in a different form by conventional methods, for example by treatment with ultraviolet radiation or chemical mutagens such as N-methyl-N'-nitro-nitrosoguanidine . Recombinant clones can be obtained by protoplast fusion. All of the mutants or recombinant or modified forms capable of producing the sanglyferin, including mutants and recombinants, which are capable of producing elevated yield of the triglycerin are included within the scope of the present invention.

In certain embodiments of the invention, among others, Sanglyferin A, B, C, and D are isolated from the novel Streptomyces sp . A92-308110. A sample of Streptomyces sp. A92-308110 was prepared according to the Budapest Treaty on May 3, 1995 under the trade name of Doi Pancrasun, Microorganism Mentzelkul Turkel, Mabeo, Germany, And received the deposit number DSM 9954. Samples of Streptomyces sp. A92-308110 are also available from Sandoz, Basel, Switzerland.

It should be noted that access to the samples of DSM 9985 is restricted in accordance with the provisions of Rule 28 (4) and (5) EPC.

Isolation of sanglyperin A, B, C and D from the culture medium of Streptomyces sp. A92-308110 is described in Example 2.

Streptomyces species A92-308110 belongs to the genus Streptomyces according to the description of Bergey's Manual (Volume 4, 1989, Williams and Wilkins, Baltimore) and The Prokaryotes (1992 Springer Verlag, New York). The cell wall contains LL-diaminopimelic acid. Fatty acids are straight and unsaturated in iso-and anteiso-branched. The sugar spectrum is not characteristic. The mycelium of the vegetative growth period is not cut into fragments. The parasitic mycelium forms long chain spores.

According to the cited reference, the strain designated A92-308110 is a novel strain of streptomyces . A92-308110 grows in a variety of organic and inorganic media and in most cases forms parasitic mycelia. The basic substrate mycelium grows as hyphae, and is generally beige to grayish brown. The color of the parasitic mycelium corresponds to No. 4 of the gray series, and this mycelium forms long chain spores belonging to spore type b.

Streptomyces sp. A92-308110 This growth in general biological media, carbon utilization and the ability of its physiological properties are shown in the table below.

Growth in multiple biological media Culture medium Culture characteristics Yeast extract / Malt agar Growth: Excellent Substrate mycelium: brown Parasitic mycelium: gray brown Soluble coloring: None Oatmeal agar Growth: Excellent Substrate mycelium: dark brown Parasitic mycelium: gray brown Soluble coloring: brown Glucose-asparagine Proliferation: Normal Substrate mycelium: brown Parasitic mycelium: gray brown Soluble coloring: None Inorganic salt / starch agar Proliferation: Normal Substrate mycelium: gray Parasitic mycelium: gray brown Soluble coloring: None Sucrose / Nitrate Agar Proliferation: very poor Substrate mycelium: white Parasitic mycelium: poor, gray brown Soluble coloring: None Glycerol / asparagine agar Proliferation: Normal Substrate mycelium: brown Parasitic mycelium: gray brown Soluble coloring: None Nutritional agar Proliferation: Normal Substrate mycelium: beige Parasitic mycelium: none Soluble coloring: None

Carbon use Normal or Good Glucose, fructose, arabinose, xylose, mannose Bad Rhamnose, sucrose, raffinose, cellulose, salicin voice m-inositol

Physiological characteristic Nitrate reduction positivity Starch hydrolysis Inorganic salts - Starch Usually on agar, Oatmeal Agar Negative Tyrosine degradation voice Milk peptone positivity Melamine formation positivity Propagation temperature 18 to 37 DEG C Very poor proliferation at 13 ° C No growth at 45 ° C pH range rich proliferation at pH 5 and 7, excellent proliferation at pH 9 NaCl tolerance Less than 6%, but at 2% concentration, proliferation decreased

The macrolides according to the present invention, which include sanglyferin A, B, C or D, can be prepared by culturing Streptomyces sp. A92-308110 or mutants, recombinant or modified forms thereof in a suitable culture medium . For the purpose of merely describing the present invention in Example 1, a method of culturing Streptomyces sp. A92-308110 was described.

Thus, in another aspect,

a) a biologically pure isomer of strain Streptomyces sp. A92-308110, or a mutant, recombinant or variant thereof capable of producing the inventive markrolide, and

b) culturing the strain Streptomyces sp. A92-308110 (DSM 9954), or a mutant, recombinant or variant thereof, in a suitable culture medium, and optionally recovering the sanglyferin. Manufacturing method

.

The markrolides according to the invention, for example the compounds of the formula IX, Sanglyferin A, B, C and D and their pharmaceutically acceptable salts are generally referred to hereinafter as the preparations of the present invention, Characteristic activity, i. E. A combination of the following activities.

- cyclophilin binding activity,

- immunosuppressive activity,

- inhibition of B- and T-cell proliferation,

- FK binding protein binding activity, and

- Does not inhibit calcinurin activity.

These activities and analyzes for measuring these activities are described in more detail below. The biological activities of the macrolides of the present invention, for example the compounds of formula IX, for example, the activity of Sanglyferin A to D can be expressed in vitro and in vivo by standard methods as follows.

1. Major human immune response to both red blood cells (MD, Michel-Douton)

Rat splenocytes (OF 1, female, 8-10 weeks, 1 × 10 ⁷ ) were cultured in 24-well plates with 1 ml of final volume for 3 days with positive red blood cells (SBRC, 3 × 10 ⁷ ). The lymphocytes were harvested, washed, and placed at a density of 1 × 10 ⁶ cells on a soft agar with new antigen (SBRC). After a reaction period of 60 to 90 minutes, a complement (guinea pig serum) was added, and the reaction was continued for 60 minutes, and the number of plaques was counted (microscope) to evaluate the experiment. During three days of culture, lymphocytes were sensitized to antigen (SBRC). When reacted again with the antigen, B-lymphocytes secrete a specific antibody that binds to the antigen near the secretory lymphocyte. The addition of complement causes haemolysis of antibody coated red blood cells, creating plaque. Each plaque represents a single antibody-producing cell.

Inhibition of flak formation is an indicator of pharmaceutical availability. The compounds of the present invention, for example the compounds of Sanglyferin A to D, are active at constant concentrations for this assay.

<References>

Micell and Dutton (1966) "Immunization in normal mouse spleen cell suspension in vitro" Science 153: 1004-1006.

Micell and Dutton (1967) "Immunization of spleen cell cultures isolated from normal mice" J. Exp. Med. 126: 423-442.

2. Lymphocyte proliferative response to dysmorphic stimulation

Mixed Lymphocyte Reaction (MLR) in rodents:

Splenocytes (2 × 10 ⁵ ) of Balb / c mice (female, 8-10 weeks) were co-cultured with 2 × 10 ⁵ spleen cells of CBA mice (female, 8-10 weeks) for 4 hours. Dendritic cells induce proliferative responses in response spleen cell populations when measured with labeled precursors that are introduced into DNA. Compared to having an IC ₅₀ of about 20 nM in the case of cyclosporin A in this assay, the macrolides of the invention, such as the compounds of formula IX and their pharmaceutically acceptable salts, for example, Sanglyferin A To D have an IC ₅₀ ranging from about 30 to about 200 nM.

<References>

Meo (1979), " The MLR in the mouse. In: Immunological Methods ". Lefkovitz and Pernis, Academic Press, N.Y. pp.227-239 "

3. LPS-stimulated rodent B-cells

Splenocytes (2 × 10 ⁵ ) of CBA mice were incubated with 50 μg / ml LPS-treated experimental compound for 48 hours. Proliferation was measured with the precursor introduced into the DNA. The macrolides of the invention, for example the compounds of formula IX and their pharmaceutically acceptable salts, for example, Sanglyferin A, B, C and D, inhibit B-cell proliferation and inhibit B- It has a range of IC ₅₀ μM of.

<References>

Graves and Zanoshi (1972), "Induction of selective T and B lymphocytes by cell surface binding ligands", Transplant Rev., 11:87

Janoshi and Grebis (1971), "Lymphocyte Activity, I, Reaction of T and B Lymphocytes to Phytomaitogen", Clin., Exp. Immunol. 9: 483-498.

4. In vitro cytotoxicity and cytostatic activity using THPl cell line

Cytotoxicity was assessed using the human mononuclear cell line THP1 (5 x 10 &lt; ⁴ &gt; cells / ml) cultured at 37 &lt; 0 &gt; C for 24-72 hours in the presence of IFN gamma (100 U / ml) and LPS Cell / well). Live cells were quantified using MTT assay (Morman, 1983). Colorimetric assay to measure mitochondrial dehydrogenase enzyme activity in living cells. The markrolide of the present invention, for example a compound of formula IX and a pharmaceutically acceptable salt thereof, such as, for example, sanglifiperin A, B, C, and D, after incubation for 24 hours in this assay, It has an IC _50.

<References>

Morman (1983), "Rapid Colorimetric Assay for Intracellular Growth and Survival: Application to Proliferation and Cytotoxicity Analysis", J. Imm. Methods, 65, 55-63.

5. TNF release by human peripheral blood mononuclear cells

Mononuclear cells were prepared from peripheral blood vessels of healthy volunteers using Ficoll-Hypaque density separation according to the method of Hansel et al. (1991). Cells (10 ⁵ cells per well in RPMI 200 ml of 10 vol% FCS) were reacted with serial dilutions of the test compound for 30 min at 37 [deg.] C prior to the addition of stimulation. Interferon γ (100 U / ml) and LPS (5 μg / ml) were used as stimuli to induce tumor necrosis factor (TNF) α release by peripheral blood mononuclear cells. After 3 hours of reaction, the cells were centrifuged (1200 rpm for 10 minutes) and the supernatant was collected. The amount of TNF present in the cell supernatant was measured using an enzyme-linked immuno-solvent assay kit, which was colorimetrically available. The macrolides of the present invention, for example the compounds of formula IX and the pharmaceutically acceptable salts thereof, such as, for example, Sanglifiperin A, B, C and D, a has an IC _50.

6. Analysis of cyclophilin binding

Suitable cyclophilin binding assays are competitive ELISA assays as described in Quasniow (Eur. J. Immunol. 1987 17 1359-1365). In this experiment, the compound to be tested was added during the reaction of cyclophilin (human recombinant cyclophilin A) coated with BSA-Cyclosporin A to calculate the concentration (IC ₅₀ ) required to inhibit 50% of the competitor-free control reaction. Other assays were performed during the incubation of biotinylated cyclophilin (human recombinant cyclophilin A) coated with BSA-Cyclosporin A in a competitive binding experiment described in Schneider et al. (Biochemistry, 1994, 33, 8218-8224) Addition of an experimental compound. The amount of biotinylated cyclophilin bound in the presence or absence of the test compound was determined by reacting with streptavidin-coupled alkaline phosphatase. The macrolides of the present invention, for example the compounds of formula IX, and the pharmaceutically acceptable salts thereof, such as, for example, Sanglyferin A, B, C and D, when tested in this assay, It has an IC ₅₀ of about 10 to about 100 nM compared to an IC ₅₀ range.

Additional in vitro assays that can be used to demonstrate the biological activity of the triglycerin are the IL-2 reporter gene assay and the ConA-stimulated splenocyte assay (an indicator of the effect on T-cell activity).

The macrolides of the present invention, for example the compounds of formula IX and the pharmaceutically acceptable salts thereof, for example, Sanglyferin A, B, C, and D, when tested in standard experiments on this activity, Does not have binding activity, and does not inhibit calcineurin activity.

7. Ubiquitous graft-versus-host response (GvH) in rats (Ford et al., Transpl. Proc. 10 (1979) 258)

Splenocytes from six-week-old female Wistar / Furth (WF) mice were injected subcutaneously into the left hind paw of a female F1 mouse (F344 × WF) weighing approximately 100 g on day 0. After 4 days of continuous treatment, the popliteal implants were removed and weighed on the 7th day. The weight difference between the two impose clauses is regarded as a measure of the response evaluation.

Inhibition of the GvH response in these experiments is an indicator of pharmaceutical use. The macrolides of the invention, for example the compounds of formula IX and the pharmaceutically acceptable salts thereof, for example sanglyferin A, B, C and D, are also administered at doses of about 1 mg / kg subcutaneously The GvH response can be inhibited to about 30% or less.

8. SRBC-T _H Cell-induced DTH

50 μl of a 1: 1 mixture (v: v) of T _H cells (2 × 10 ⁶ ) and 10% red blood cell (SBRC) suspension was mixed with female C57 BL / 6 mice ) On the right hind paw. 50 μl of SRBC cell suspension (diluted 1: 1 v / v with PBS) was injected subcutaneously in the left hind paw (to measure the nonspecific increase in swelling according to the injection method). The thickness of the right and left hind feet was measured after 24 hours. The% increase in the right foot thickness (z) for the left foot was calculated. The thickness of the right foot is x; When the thickness of the left foot is y and the% specific increase is z, z = ((xy) / y) x 100.

For example, Sanglyferin A, B, C, and D are administered at subcutaneous doses of about 5 mg / kg to about 50 or less subcutaneous doses of about 5 mg / kg of a compound of formula IX and a pharmaceutically acceptable salt thereof, To reduce the swelling of DTH mice.

<References>

(1981) " Clones of helper T-cells mediated antigen specific, H-2 restricted DTH " Nature 290: 62-63

Herman, Schleier, Borrell, and Poer (1988), "Mast Cell Degranulation as a Major Event in the Effector Phase of Delayed-Type Hypersensitivity Induced by Cloned Helper T Cells" Int. Archs. Allergy appl. Immun. 86: 102-105

9. Rat / mouse heart allograft

The in vivo efficacy of the macrolides of the present invention was evaluated by the rat and mouse cardiac transplantation method using an Aljette osmotic small pump for subcutaneous administration. The macrolides of the present invention, e.g., compounds of formula IX, and pharmaceutically acceptable salts thereof, such as, for example, Sangliffein A, B, C, and D, The survival rate was prolonged at a dose of 30 mg / kg / day. A rat heart homologue using an optimal level of cyclosporin A and the macrolides of the invention, such as the compound of formula IX, and a pharmaceutically acceptable salt thereof, such as, for example, Sanglyferin A, B, C and D The transplantation treatments prolonged the survival rate as illustrated in the table below.

Cyclosporin A (mg / kg) Sanglipherin A (mg / kg) Change survival rate (days) One - 12 12 12 13 13 14 One 10 29 30 45 48 > 51 > 46 Control (placebo) Control (placebo) Control (placebo)

The preparation of the present invention is useful as a medicament, for example, an anti-inflammatory agent as well as an immunosuppressive agent.

In particular, they can be used for the treatment of the prophylaxis of acute and / or chronic organ or tissue allograft or xenotransplant rejection, such as heart, lung, combined heart-lung, liver, kidney, pancreas, It is useful for. They are also prescribed for the prevention of graft-versus-host disease, such as bone marrow transplantation.

The agents of the present invention may also be used in the treatment of inflammatory diseases of the etiology including autoimmune diseases and inflammatory diseases, especially autoimmune parts such as arthritis (for example, rheumatoid arthritis, chronic prodrugal arthritis and arthritis) and rheumatic diseases It is useful for treatment. Certain autoimmune diseases in which the agents of the present invention may be used include autoimmune hematologic diseases (e.g., hemolytic anemia, anemic anemia, simple erythropoiesis and spontaneous thrombocytopenia), systemic lupus erythematosus, Psoriasis, Steven-Johnson syndrome, spontaneous spontaneous, autoimmune inflammatory bowel disease (eg, ulcerative colitis and Crohn's disease), endocrine disorders such as Crohn's disease, Parkinson's disease, (Diabetic type I), uveitis (anterior and posterior), dry keratoconjunctivitis and spring and / or allergic keratoconjunctivitis, interstitial pulmonary fibrosis (Kidney Disease), seborrheic dermatitis, Graves Disease, Graves Disease, sarcopenia, multiple sclerosis, primary biliary cirrhosis, , Psoriatic arthritis, glomerulonephritis (with or without nephrotic syndrome, e.g., spontaneous nephrotic syndrome or minimal mutant nephropathy) and asthma.

For these and other uses, the agents of the invention may be administered alone or in combination with other immunosuppressants or anti-inflammatory agents, including cyclosporine, rapamycin, FK506, and steroids.

In the case of such a regimen, the appropriate dosage and the formulations of the present invention will, of course, vary depending on, for example, the subject to be treated, the mode of administration and the nature and severity of the disease to be treated. In general, however, when administered orally at a daily dose of about 0.01 to 10 mg / kg / day, satisfactory results are obtained in animals. For larger mammals, for example, daily doses prescribed to humans are administered orally once per day, and more suitably two to four divided doses per day, ranging from about 0.5 to about 500 mg.

In the transplantation of a human organ, 0.1 to 100, preferably 0.3 to 30, more preferably 0.5 to 10 mg / kg of the compound of the present invention is administered orally. When the agents of the present invention are administered in combination with other immunosuppressive agents (e. G., With a corticosteroid or cyclosporine or rapamycin type of compound as part of a dual, triple, or quadrupial medicament therapy) For example, 0.1 mg / kg / day intravenously; 3 mg / kg / day initial oral dose) may be used. In particular, the agents of the present invention may be used in combination with other non-steroidal immunosuppressants such as cyclosporine, rapamycin or FK506 in terms of partially or completely replacing the steroid.

The formulations of the present invention may also be administered parenterally, either parenterally, e. G. In the form of a solution, tablet or capsule, or in the form of, for example, an injection solution or suspension, . In the case of systemic administration, in the case of some diseases, for example, prevention of liver transplant rejection, intravenous injection forms are preferable, but oral dosage forms are usually preferred. The compounds may also be administered topically or in the form of, for example, creams, gels or similar formulations for the skin, or in the form of creams, gels or eyedrops, for the purposes of the eyes.

Suitable unit dosage forms for oral administration comprise from 0.5 to 100 mg of compound per administration.

In accordance with the above description, the present invention further provides a series of implementations.

A. A method of performing immunosuppression comprising administering an effective amount of an agent of the invention to a subject in need thereof.

B. administering an effective amount of an agent of the invention to a subject in need of treatment

1) methods for the treatment of recipients of acute and / or chronic bronchial allografts or xenotransplantation rejection, for example, any of the above-described specific types of organ transplants; or

2) methods of preventing graft-versus-host disease in recipients of bone marrow transplantation, for example; or

3) a method of treating an autoimmune disease or a disease described above; or

4) Treatment of asthma.

C. An agent of the present invention used as a medicament, for example, as an immunosuppressive agent or for the treatment of any disease or disease described in B) above.

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

E. Use of the present invention for the manufacture of a medicament for use as an immunosuppressive agent, or for use in the treatment of any disease or disorder described in B above.

In addition, the macrolides of the present invention having cyclophilin binding activity can be useful in immunoassay of cyclosporin and other cyclophilin binding compounds, for example, in any of the assays described in co-pending patent application WO95 / 07468 . The present patent application relates to an analytical procedure for determining the concentration of an immunopilline binding restriction in blood, for example a cyclosporin; The procedure comprises adding a binding competitor to displace the drug in the immunosuppressant-immunofillin complex in the blood; Isolating the receptor-drug complex from the sample by adding a receptor that binds to the drug without significantly binding to the binding competitor; And measuring the amount of the drug. Cangliopherin can be used as a binding competitor in this assay: for example, the cyclosporin is displaced from the cyclophilin to release the cyclosporin, and its amount is quantified as a monoclonal antibody specific for the cyclosporin.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is further illustrated by the following examples, which are for illustrative purposes only, in accordance with the accompanying drawings.

Brief Description of the Drawings Figure 1 is a mass spectrum of a compound of Sangliferin B;

2 is a mass spectrum of a compound of Sanglyferin A;

Figure 3 is a mass spectrum of a compound of Sanglifiperin D;

4 is a mass spectrum of a compound of Sanglifiperin C. Fig.

5 shows the IR spectrum of the compound of Sanglyferin B;

Figure 6 shows IR spectra of compounds of Sanglyferin A;

Figure 7 shows IR spectra of the compounds of Sanglyferin D;

8 is IR spectrum of the compound of Sanglifiperin C. Fig.

9 is an NMR spectrum of a compound of Sanglyferin A;

10 shows the NMR spectrum of a compound of Sanglyferin D. Fig.

11 is an NMR spectrum of a compound of Sanglyferin B;

FIG. 12 is an NMR spectrum of a compound of Sanglifiperin C. FIG.

Culture conditions

Streptomyces sp. A92-308110 can be cultured at a suitable temperature using aerobic conditions and liquid culture methods in various culture media using suitable nutrients and inorganic materials. The fermentation medium contains inorganic salts, including carbon sources, nitrogen sources and trace elements, which are commonly available, all of which may be added, for example, as well-defined products or complex mixtures found in biological products of various origins.

Example 1 illustrates the basic conditions under which the compounds of formula (I) are obtained. Improved yields can be obtained by optimizing the culture conditions (aeration, temperature, pH, quality and quantity of carbon and nitrogen sources, amounts of inorganic salts and trace elements), and controlling fermentation in biological reactors.

&Lt; Example 1 >

Strain A 92-308110

a. Agar-starting culture

The strain A 92-308110 was subjected to agar slant culture at 27 ° C in the following agar medium.

Glucose 10.0 g Soluble starch 20.0 g Yeast extract (Gistex, Gist Brocades) 5.0 g NZ-amine, Type A (Sheffield) 5.0 g Calcium carbonate 1.0 g Agar (bamboo) 15.0 g Desalted water Remaining amount 1000 ml

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

The culture can be stored at -25 to -70 &lt; 0 &gt; C. The suspension in glycerol-peptone can be stored in liquid nitrogen.

b. Preliminary culture

Spores and mycelium of 10 starting culture solutions were suspended in 100 ml of 0.9% salt solution. Two 2 liter Ellenmeyer flasks each containing 1 liter of pre-culture medium were each inoculated with 50 ml of the suspension. The composition of the pre-culture medium is as follows.

Glucose 7.50 g glycerin 7.50 g Yeast extract (BBL) 1.35 g Malt extract (Wander) 7.50 g Soluble starch 7.50 g NZ-amine, Type A (Sheffield) 2.50 g Soy protein 2.50 g L (-) asparagine 1.00 g CaCO ₃ 0.050 g NaCl 0.050 g KH ₂ PO ₄ 0.250 g K ₂ HPO ₄ 0.500 g MgSO ₄ .7H ₂ O 0.100 g Trace element solution A 1 ml Agar (bamboo) 1 g Desalted water Remaining amount 1000 ml

The pH of the medium was adjusted to 6.8 to 7.2 with NaOH / H ₂ SO ₄ and sterilized at 121 ° C for 20 minutes.

The composition of the trace element solution A is as follows:

FeSO ₄ .7H ₂ O 5.0 g ZnSO ₄ .7H ₂ O 4.0 g MnCl ₂ .4H ₂ O 2.0 g CuSO ₄ .5H ₂ O 0.2 g CoCl ₂ .6H ₂ O 2.0 g H ₃ BO ₃ 0.1 g KI 0.05 g H ₂ SO ₄ (95%) 1 ml Desalted water Remaining amount 1000 ml

The preliminary culture was fermented at 27 DEG C for 24 hours at 200 rpm on a rotary shaker having an eccentricity of 50 mm.

c. First intermediate culture

Two 75 liter bioreactors, each containing 50 liters of pre-culture medium, were each inoculated with 1 liter of pre-culture and fermented at 27 ° C for 96 hours. The fermenter was rotated at 150 rpm. Air was introduced in an amount of 0.5 liters per liter of the medium.

d. Second intermediate culture

Two 750 liter fermentation vessels each containing 500 liters of pre-culture medium were each inoculated with 50 liters of the first intermediate culture. The second intermediate culture was incubated at 27 DEG C for 70 hours. The fermenter was rotated at 100 rpm, and air was introduced in an amount of 0.8 liters per liter of the medium.

e. Main culture

Two 5000 liter bioreactors each containing 3000 liters of the main medium were inoculated with 250 and 300 liters of the second intermediate medium, respectively. The main culture was incubated at 24 DEG C for 96 hours. The bioreactor was rotated at 45 rpm and air was introduced in an amount of 0.5 liters per liter of the medium.

The composition of the main culture medium is as follows.

Glucose 20 g Malt extract (Wander) 2 g Yeast extract (bark) 2 g Soiton (basto) 2 g KH ₂ PO ₄ 0.2 g K ₂ HPO ₄ 0.4 g MgSO ₄ .7H ₂ O 0.2 g NaCl 0.05 g CaCl ₂ .6H ₂ O 0.05 g Trace element solution B 1 ml Agar (bamboo) 1 g Desalted water Remaining amount 1000 ml

The pH of the medium was adjusted to 6.3 with KOH / HCl. Lt; RTI ID = 0.0 &gt; 121 C &lt; / RTI &gt;

The composition of the trace element solution B is as follows:

FeSO ₄ .7H ₂ O 5.0 g ZnSO ₄ .7H ₂ O 4.0 g MnCl ₂ .4H ₂ O 2.0 g CuSO ₄ .5H ₂ O 0.2 g (NH ₄ ) ₆ Mo ₇ O ₂₄ 0.2 g CoCl ₂ .6H ₂ O 1.0 g H ₃ BO ₃ 0.1 g KI 0.05 g H ₂ SO ₄ (95%) 1 ml Desalted water Remaining amount 1000 ml

The optimized culture medium of the main culture is as follows.

Soybean wheat 20.0 g Glycerol 40.0 g MES 0.1 M Desalted water (pH 6.8) Remaining amount 1000 ml

&Lt; Example 2 >

Isolation of Sanglyperin A, B, C and D from Streptomyces sp. A92-308110

The first separation and characterization of the four new CBA active metabolites was carried out from the two 3000 liter fermentation tanks by the indicated active fractions and HPLC and thin layer chromatography analysis. The CBA (cyclophilin binding assay) as described above was used as an experiment for biological activity.

A separate process was performed with two 3000 liter fermentation tanks. 1500 liters of each fermentation broth was stirred in a 4000 liter stainless steel vessel with 2000 liters of ethyl acetate for 20 hours. Organic phase isolation was performed with a BestPalia-separator type SA-20. The ethyl acetate extract was washed twice with 80 liters of water and evaporated to dryness under reduced pressure to give 1.64 and 2 kg of an extract. The two extracts were defatted in three stages of extraction using 40 liters of methanol / water 9: 1 and 40 liters of hexane. Evaporated under reduced pressure and dried to give 1.34 kg of an extract.

The deacidified extract was chromatographed with two portions (670 g) of a methanol solution on a 10 kg Sephadex H column. Each portion was dissolved in 3.3 liters of methanol and added to the column. After collecting the first 15 liters of eluant into fractions 1, chromatography was continued to collect 2 liters of fractions. The most active fractions were 2, 3 and 4, collecting 146 g. This sample was further chromatographed on 1 kg of silica gel Merck 0.04-0.063 mm using methyl-t-butyl ether (MTBE), MTBE / 5% methanol and MTBE / 10% methanol. Two liters of fractions were collected. Fractions 5 to 9 were the most active, and they were collected to collect 43.8 g of the sample. This sample was further separated on a 1 kg silica gel Merck 0.04-0.063 mm column using a gradient of hexane / acetone 7: 3 to acetone. This chromatographic fraction 6 (7.0 g) was further separated on a 3 kg Lichroprep RP 18 (Merck) 40-63 占 퐉 column using methanol: water 94: 6 (fractions 4 to 7, 2.16 g) A 100 g silica gel H column using methylene and 3% methanol (733 mg), 3 kg of Lichroprep Rp18 column (621 mg) using methanol / water 9: 1 and 100 &lt; RTI ID = 0.0 &gt; g of Lichroprep RP18 to give 324 mg of pure Sanglycerine A (melting point 142-145 C, amorphous, (?) D25 = 67.30 (c = 0-988, methanol)).

Fractions 5 and 7 from the hexane / acetone column were pooled (7.1 g), 3 kg of Lichroprep RP18 40-63 占 퐉 column (769 mg) using methanol: water 9: 1, MTBE 3% methanol Further purified on a column of 100 g of a silica gel H column (309 mg) and finally 100 g of silica gel H using methylene chloride and 3% methanol to obtain 90 mg of pure Shangrelperin B (melting point 117-121 [deg.] C, Amorphous, (?) D25 = 52.80 (c = 1-128, methanol).

Fractions 9 and 10 (2.147 g) of the chromatography using methanol / water on a column of 3 kg of Lichroprep RP18 were combined with 100 g of silica gel H phase (800 mg) using methylene chloride / 5% methanol and finally methanol / Further purification on a 3 kg Lichroprep RP18 column using water 9: 1 yielded 480 mg of pure Sanglyferin C (melting point 165-170 ° C, (?) D25 = 35.60 (c = 0-736, methanol) Respectively.

Fractions 11 and 12 (835 mg), chromatographed on a 3 kg Lichroprep RP18 column with methanol / water 94: 6, were purified on 100 g of silica gel H using MTBE / 5% methanol to give 140 mg of Sanglyferin D ( Melting point 137, amorphous).

Subsequently, the triglycerin A, B, C and D were characterized by ultraviolet, infrared mass and NMR spectroscopy. The results obtained are shown in Table 4 and the accompanying drawings.

Sanglyperin A Molecular formula C ₆₀ H ₉₁ N ₅ O ₁₃ (1090.4) UV (MeOH) H ⁺ OH ^- 275 (1962), 242 (54500), 197 (75755) 275 (1635), 242 (51884) 292 (1973), 242 (60495) IR-spectrum 6 Mass-spectrum: FAB 1096 [MH + Li] ⁺ 2 NMR spectrum 9 Sanglyferin B Molecular formula C ₆₀ H ₈₉ N ₅ O ₁₂ (1072.4) UV (MeOH) 273 (4395), 242 (50600), 197 (78577) IR-spectrum 5 Mass-spectrum: FAB 1098 [MH + Li] ⁺ 1 NMR spectrum 11 Sanglyferin C Molecular formula C ₆₁ H ₉₃ N ₅ O ₁₃ (1104.4) UV (MeOH) H ⁺ OH ^- 275 (1876), 242 (51577), 197 (72643) 275 (1391), 242 (50120) 292 (1832), 242 (57960) IR-spectrum 8 Mass-spectrum: FAB 1110 [MH + Li] ⁺ 4 NMR spectrum 12 Sanglyferin D Molecular formula C ₆₁ H ₉₁ N ₅ O ₁₂ (1086.4) UV (MeOH) H ⁺ OH ^- 273 (3194), 242 (47584), 197 (73766) 273 (3237), 242 (46389) 285 (2600), 242 (52907) IR-spectrum 7 Mass spectrum: FAB 1092 [MH + Li] ⁺ 3 NMR spectrum 10

<Sanglipherin A>

&Lt; Example 3 >

Conversion of Sanglyferin A to Sanglyferin C

To a stirred cold (0 ° C) solution of 20 mg (18.3 μM) of Sanglycerin A in 0.5 ml of methanol was added one crystal of paratoluenesulfonic acid monohydrate. The resulting yellow solution was stirred for 1 hour and the reaction quenched with saturated aqueous sodium bicarbonate. The resulting mixture was extracted twice with ethyl acetate. The organic solution was washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (95: 5 methyl-t-butyl ether: methanol) to give Sanglyferin C as a white amorphous powder. It consists of a 4: 1 mixture of the triglycerin C and its C53 epimer, and the sanglyperin C has the (S) form (R = Me) as shown below.

In addition, such conversion may be accomplished using other protonic acids in methanol (e.g. pyridinium para-toluenesulfonate, hydrochloric acid or sulfuric acid) or Lewis acids (such as zinc chloride, magnesium bromide, magnesium chloride, titanium tetraisopropoxide, Or boron trifluoride). The use of other alcoholic solvents or cosolvents such as ethanol, isopropanol, butanol, allyl alcohol, propargyl alcohol, benzyl alcohol and the like is likewise preferred in which the radicals R in the structure are each ethyl, isopropyl, butyl, allyl, Resulting in homologues.

In the same way as described above, Sanglyferin B can be converted to Sanglyferin D.

<Example 4>

Conversion to phosglycerin C to phosglycerin A

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

Other inorganic or organic acids may be used as the medium containing water and any organic co-solvent. Suitable acids include hydrochloric acid, para-toluenesulfonic acid or other sulfonic acids, pyridinium para-toluenesulfonate, acetic acid, trifluoroacetic acid, formic acid. Suitable organic co-solvents include acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane.

With this reaction, compounds of formula (XV) can be formed in varying amounts according to the reaction time (see Example 5 below for better methods of producing compounds of formula (XV)). Similarly, Sanglyferin D can be converted to Sanglyferin B.

&Lt; Example 5 >

Conversion of Sanglyferin A to the compound of formula XV

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

In a similar manner, the sanglycerin B can be converted to a compound of formula XVI. These materials are present as one epimer in C53, but the absolute morphology is not clearly determined.

XV: MS m / z 1078 [M + Li] ⁺ (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signals listed a) δ0.40 (3H, d, H -50), 1.20 (3H, s, H-54), 1.69 (3H, s, H-49), M), 5.62 (1H, d, H-15), 4.58 (1H, dd, H-21), 5.67 (1H, m, H-27), 5.99 (1H, d, H-25), 6.03 (1H, dd, H-20).

&Lt; Example 6 >

Conversion of a compound of formula XV to &lt; RTI ID = 0.0 &gt;

To 50 mL of a 2 N aqueous sulfuric acid solution was added to a stirred solution of 54 mg (50 [mu] M) of the compound of formula (XV) in 0.5 ml of 4: 1 THF-water. The resulting solution was stirred at ambient temperature for 12 hours and the reaction quenched with saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted twice with ethyl acetate. The organic solution was collected, washed with a saturated aqueous sodium bicarbonate solution, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (90:10 methyl-t-butyl ether: methanol) to give Sanglyperin A as a white amorphous solid.

Similarly, the compound of formula (XVI) was converted to &lt; RTI ID = 0.0 &gt;

As the selective intramolecular protection-deprotection proceeding, the methods described in Examples 3-6 were used. Thus, in the reaction described in Example 5, the hydroxyl at the 15-position can be selectively protected and the residual free hydroxyl can be selectively manipulated. The procedure of Example 5 can selectively protect both hydroxyls at positions 15 and 17. All of these procedures can also be used as intermolecular protection of C53 ketones. The hydroxyl and ketone may be regenerated by the reactions described in Examples 4 and 6. Sanglyferrin C and D as well as compounds of formulas XV and XVI are important intermediates for the production of another top glyphelin.

&Lt; Example 7 >

Preparation of 16-dehydro-17-dehydroxy-phosglycerine A (Formula XVII)

54 mg (50 [mu] M) of the compound of formula XV and 1 ml of 4: 1 acetonitrile-water and a crystal solution of paratoluenesulfonic acid monohydrate were heated to 80 [deg.] C for 1.5 hours. The reaction was quenched with a saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted twice with ethyl acetate. The organic layer was washed with a saturated aqueous sodium bicarbonate solution, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (90:10 methyl-t-butyl ether: methanol) and reverse-phase chromatography (RP18, 50:50 acetonitrile-water to acetonitrile, 45 min) To give the pure title compound as a white amorphous solid.

MS m / z 1078 [M + Li] &lt; ⁺ &gt; (relative intensity 100); (3H, s, H-54), 4.03 (2H, s). &Lt; ¹ &gt; H NMR (DMSO) OH, d, H-15 and C31-OH), 5.57 (2H, m, H-21 and C35-OH), 5.72 (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).

&Lt; Example 8 >

Preparation of 42-N-methyl-phosglycerin A (Formula XVIII)

16.5 [mu] l of methyl triflate was added to a stirred cooled (-15 [deg.] C) solution of 109 mg (0.1 mmol) of Sanglycerin A in 1 ml methylene chloride and 67 l (0.3 mmol) The mixture was allowed to warm to room temperature and stirred for 6 hours, then quenched by addition of saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted twice with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified twice by column chromatography on silica gel (90:10 methyl-t-butyl ether: methanol, then 95: 5 methyl-t-butyl ether: methanol) to give pure The title compound was obtained.

MS m / z 1110 [M + Li] ⁺ (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signals listed) δ1.70 (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 -OH), 7.50 (1H, d, H-12), 8.11 (1H, d, H-9), 9.22 (1H, s, C61-OH).

&Lt; Example 9 >

53 Preparation of dihydroglycerin A (Formula XIX)

To a stirred cold (0 C) solution of 54 mg (50 [mu] M) of superglycerin A in 0.5 ml methanol was added 2.8 mg (75 [mu] M) sodium borohydride. The mixture was stirred for 1 hour and saturated aqueous sodium bicarbonate solution was added. The resulting mixture was extracted twice with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (95: 5 methyl-t-butyl ether: methanol, then 90:10 methyl-t-butyl ether: methanol) to give the pure title compound as a white amorphous solid &Lt; / RTI &gt; The isolated product corresponded to about a 1: 1 mixture of diastereomers at about C-53.

MS m / z 1098 [M + Li] ⁺ (relative intensity 63), 1104 [M + 2 Li-H] ⁺ (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signal reference)? 0.62 (3H, d, H-50), 1.02 (3H, d, H-54), 3.55 and 3.59 (1H, 2m, H-53).

&Lt; Example 10 >

Preparation of 53-tosylhydrazone-sanglyperin A (XX)

A mixture of 55 mg (50 μM) of Sanglifepin A and 23 mg (125 μmol) of tosyl hydrazide in 0.5 ml of methylene chloride was stirred at room temperature for 6 hours. The solvent was removed and the residue was subjected to column chromatography on silica gel (90:10 methyl-t-butyl ether: methanol) to give the pure title compound as a white amorphous powder.

MS m / z 1264 [M + Li] &lt; ⁺ &gt; (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signals listed) δ1.70 (3H, s, H -49), 1.77 (3H, s, H-54), 2.37 (3H, s, NSO 2 C 6 H 4 CH _{3), 6.51 (1H, s} , H-60), 6.59 (2H, 2d, H-62 and H-64), 7.06 (1H , dd, H-63), 7.35 (2H, d, tosyl meta protons) , 7.73 (2H, d, Tosyl group).

&Lt; Example 11 >

Preparation of 26S, 27S-dihydroxy-topglycerin A (Formula XXI) and 26R, 27R-dihydroxy-topglycerin A (Formula XXII)

(DHQ) ₂ PHAL, 65 [mu] l (0.025 mmol) of potassium ferricyanide, 207 mg (1.5 mmol) potassium carbonate, 19.5 mg (0.025 mmol) of potassium ferricyanide in 2.5 ml of t-butanol and 5 ml of water (0 &lt; 0 &gt; C) solution of 0.08 M osmium tetraoxide in tetrahydrofuran and 95 mg (1 mmol) methylsulfonamide in 2.5 ml of t-butanol was added dropwise a solution of 545 mg (0.5 mmol) Perine A was added. The resulting mixture was allowed to warm to room temperature and stirred for 3 hours. 1.08 g (8.6 mmol) of sodium sulfite was added followed by ethyl acetate and water, and the mixture was stirred vigorously for 15 minutes. This layer was separated and the aqueous layer was extracted twice with ethyl acetate. The organic layers were combined, washed with a saturated aqueous sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by reverse phase column chromatography (RP18, 30:70 acetonitrile-water to acetonitrile, 60 min) to yield 26S, 27S-diol as an amorphous powder.

The corresponding 26R, 27R-diol was obtained according to the above procedure except that (DHQD) ₂ PHAL was used in place of (DHQ) ₂ PHAL.

26S, 27S-diol: MS m / z 1130 [M + Li] ⁺ (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signals listed) δ1.64 (3H, s, H -49), 2.06 (3H, s, H-54), 3.20 (1H, br. M, H-27), (1H, d, C27-OH), 3.45 (1H, d, C27-OH) (1H, d, H-25), 5.57 (3H, m, H-18, H-21 and C35-OH), 6.03 , H-19), 6.14 (1H, dd, H-20).

26R, 27R-Diol: MS m / z 1130 [M + Li] ⁺ (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signals listed) δ1.64 (3H, s, H -49), 2.06 (3H, s, H-54), 3.20 (1H, br. M, H-27), (1H, d, C27-OH), 3.45 (1H, d, C27-OH) H), 6.03 (1H, d, H-25), 5.57 (3H, m, H-18, H-21 and C35- , H-19) and 6.14 (1H, dd, H-20).

&Lt; Example 12 >

Cleavage of the diol in 26S, 27S-dihydroxy-phase glyphelin A

To a solution of 90 mg (79 [mu] M) of 26S, 27S-diol in 0.9 ml of 2: 1 THF-water was added 33.7 mg (157 [mu] M) sodium periodate. After stirring for 1 hour, a saturated aqueous sodium bicarbonate solution was added. The resulting mixture was extracted twice with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified on silica gel (95: 5 methyl-t-butyl ether: methanol) to give the compounds of formulas XXIII (foam) and XXIV (powder).

Formula XXIII: MS m / z 366 [M + H + H ₂ O] ⁺ (relative intensity 100); &Lt; ¹ &gt; H NMR (DMSO) (a 2: 1 mixture of OH _ax : OH _eq at the center of the anomer) m, H-35), 3.66 (1H, m, H-33), 4.38 (0.67H, ddd, H-27 ax), 4.95 (0.33H, br 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 (0.67 H, s, NH-42), 7.91 (0.33H, s, NH-42).

Formula XXIV: MS m / z 745 [M + Li] ⁺ ; ¹ H NMR (DMSO) (only characteristic signals listed) δ0.64 (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, br m, H-15), 3.94 (1H, dd, H-20), 5.62 (2H, m, H-18 and H-21), 6.89 10.0 (1H, d, H-26).

&Lt; Example 13 >

Acetylation of the &lt; RTI ID = 0.0 &gt; 61-O-acetyl-topglycerin A &lt; / RTI &

To a stirred cold (0 ° C) solution of 54 mg (50 μM) of Sanglycerin A in 50 ml of methylene chloride and 50 μl of pyridine in 0.5 ml of methylene chloride was added 5.2 μl of acetic anhydride. The reaction system was maintained at 0 &lt; 0 &gt; C for 1 hour, warmed to room temperature and then stirred for 12 hours. A saturated aqueous sodium bicarbonate solution was added and the resulting mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was subjected to reverse phase chromatography (RP18, 40:60 acetonitrile-water to acetonitrile, 45 min) to give the title compound as an amorphous powder.

MS m / z 1132 [M + Li] ⁺ (relative intensity 100); ¹ H NMR (DMSO) (only characteristic signals listed) δ1.68 (3H, s, H -49), 2.06 (3H, s, H-54), 2.25 (3H, s, CH 3 CO 2), 4.04 (1H, d, C31-OH), 4.67 (1H, d, C2- , m, H-18, H-21 and C35-OH), 6.85 (1H, s, H-60), 6.98 1H, dd, H-63), 7.51 (1H, d, H-12), 7.89 (1H, s, H-42), 8.23 (1H, d, H-9).

Claims (34)

i) a piperidinylcarboxylic acid residue is provided at positions 2 to 6 of the macrocyclic ring, and / ii) an aromatic? -amino acid residue is provided at positions 7 to 9 of the macrocyclic ring, and / iii) an aliphatic alpha -amino acid residue is provided at positions 10 to 12 of the macrocyclic ring Or a salt thereof. The markrolide of claim 1 comprising two, or especially all three of the structural features i), ii) and iii). The macrolide of claim 1 or 2, wherein the remainder of the macrocyclic ring comprises a hydroxycarboxylic acid residue having a chain length of 6 to 20, preferably 11. 4. The macrolide of claim 3, wherein the hydroxycarboxylic acid residue is a residue of formula (II), or a salt thereof, in free or protected form. &Lt; Wherein R ₁ and R ₂ are both hydrogen or represent an extra bond; R ₃ is hydrogen; R ₄ is -CO-CH ₃ or -CH (OH) -CH ₃ or R ₃ and R ₄ together represent the structure of formula III (III) 6. A macrolide or salt thereof according to any one of claims 1 to 5, which comprises a macrocyclic ring of formula (IV) in free or protected form. (IV) Wherein X, Y and Z are the residues i), ii) and iii) defined in claim 1, A is a hydroxycarboxylic acid residue as defined in the above-mentioned 3 or 4. 6. A macrolide or salt thereof according to claim 5, in free or protected form, comprising a macrocyclic ring of formula (V). (V) Wherein A is as defined in claim 5. 7. A process according to any one of claims 1 to 6, wherein the carbon atom adjacent to the oxy moiety of the lactone bridge is replaced with a 2-oxy-2'-aza-3'-oxo-spirobicyclohexane- Roll ride. 8. A compound according to claim 7, wherein the spirobicyclohexanyl moiety is a residue of formula VI bonded to the macrolide ring through a linker comprising a linear arrangement of 6 to 11, usually 9, carbon atoms between the spiro residue and the macrolide ring The macrolide or its salt in free or protected form. &Lt; Formula (VI) Wherein -a-b- is - (Me) C = CH- or - (Me) CH-CH (OH) -, R &lt; ₅ &gt; is H or Me. 9. The macrolide of claim 8, wherein the linker between the spiro residue and the macrolide ring is a group of formula VII, in glass or protected form. (VII) Wherein c represents a link to the spiro residue, d represents the linkage to the macrocyclic ring, R ₆ and R ₇ are each OH or together represent an additional bond. Compound of formula (VIII), or a salt thereof, in free or protected form. &Lt; Formula (VIII) S-L-M Wherein S represents 2-oxy-2'-aza-3'-oxo-spirobicyclohexan-3-yl residue, L represents a linker comprising an array of straight lines having from 6 to 11, usually 9, carbon atoms, And M represents a macrolide ring as defined in any one of claims 1 to 6 above. 11. Compounds of formula (IX) according to claim 10, in free or protected form, or salts thereof. <Formula IX> Wherein -a-b- is as defined above, -e-f- is -CH (OH) -CH (OH) - or -CH = CH-, -g-h- is defined in the above-a-b-, R ₃ , R ₄ and R ₅ are as defined above 12. A compound according to claim 11 having the form. In the formula, when -ab- is - (Me) CH-CH (OH) -, Shaped, When the -e-f- is -CH (OH) -CH (OH) -, it preferably has the form of (S), (S) or (R) When -gh- is - (Me) CH-CH (OH) -, preferably Shaped, When -gh- is - (Me) C = CH-, preferably Shaped, When R &lt; ₃ &gt; and R &lt; ₄ &gt; are fused together, . &Lt; / RTI &gt; Sanglifeprin selected from the group consisting of A, B, C and D. The 14th to 17th positions of the macrocyclic ring are, for example, Or a compound according to any one of claims 10 to 12 according to any one of claims 4 to 9, wherein R &lt; 1 &gt; &Lt; Formula 10 > A compound of formula (XI) in free or protected form, or a salt thereof, for example a compound of formula (XII), for example a compound of formula (IXa). (XI) R ₆ OXYZA-OH (XII) <Formula IXa> Wherein X, Y, Z, A, R ₃ , R ₄ and R ₅ are as defined above and R ₆ is H or C _1-4 alkyl, for example methyl. 9. The macrolide or its salt according to any one of claims 1 to 9, wherein the ring-opened macrocycle is in free or protected form. Or a salt thereof, in free or protected form R ₆ OXYZA-OH, wherein X, Y, Z and A are as defined in claim 5 and R ₆ is H or C _1-4 alkyl. Glass or of _{R 6 OXYZ-A'-CH (} OH) in protected form -LS (here, -A'-CH (OH) -, and a is as defined in claim 3 wherein the hydroxy acid residue, and other symbols are the 17) or a salt thereof. A compound of formula (XIIa), or a salt thereof, in free or protected form. <Formula XIIa> The compound of formula (IXb), or a salt thereof, in free or protected form. <Formula IXb> A free or protected form of the compound, for example a compound of formula (XII) or a salt thereof, wherein the 2-oxy-2'-aza-3'-oxo-3 'yl-spirobicyclohexane ring system is in the ring-opened form. (XII) Wherein a, b, L and M are as defined above. 2-oxy-2'-aza-3'-oxo-3 'yl-spirobicyclohexane or a salt thereof in free or protected form, especially a compound of formula (VIa) in free or protected form or a salt thereof. &Lt; Formula VIa & Wherein R ₇ is hydrogen, an optionally protected OH group, a reactive functional group, or a -CH ₂ -CH (OH) -CH (CH ₃ ) -CH ₂ -CH ₂ -CHO group or a delta lactol equivalent thereof. Free ring-opening 2-oxy-2'-aza-3'-oxo-3 'yl spirobicyclohexane or a salt thereof, in free or protected form, especially a compound of the formula XIIa or its salt in free or protected form. <Formula XIIa> Wherein a, b and R &lt; ₇ &gt; are as defined above. A macrolide of formula XIII in free or protected form, or a salt thereof, especially a macrolide of formula XIV, or a salt thereof, in free or protected form. &Lt; Formula (XIII) <Formula XIV> Wherein M is a macrolide ring as defined above. i) culturing the strain of actinomycetes A, B, C or D producing actinomycetes in a culture medium in order to produce any one of the triglycerin A, B, C or D, Glycerin A, B, C or D is isolated, ii) To prepare the triglycerin C and D, the triglycerin A and B were cyclized at positions 15 and 16, iii) To prepare the triglycerin A and B, the triglycerin C and D were opened at the 15 and 16 positions to form the lactol ring, iv) -gh- is C (CH ₃₎ CH- to = the production of the formula IX or IXa of the macrolide, -gh- is _{CH (CH 3) -CH (OH} ) - or the formula His-protected form The compound of IX or IXa is dehydrated, v) hydrogenating a compound of formula (IX) or (IXa) wherein R ₄ is C (O) -CH ₃ to produce a markrolide of formula IX or IXa wherein R ₄ is CH (OH) -CH ₃ ; vi) carrying out an internal protection of a compound of formula (IX) or (IXa) in order to prepare a markrolide of formula (IX) or (IXa) wherein positions 14-16 of the macrolide ring comprise a moiety of formula X, (X) vii) In order to prepare the macrolides of formula IX or IXa, the 14 to 16 positions of the macrolide ring carry out the reversal of the internal protection of the compounds of formula IX or IXa comprising the moiety of formula X, (X) viii) to R ₅ is the production of fluoride-methyl of the formula IX or IXa of roll marks, R ₅ is a methylated and a macrolide of formula IX or IXa H, ix) R ₄ O- is to prepare the protected forms of the formula IX or IXa of the macrolide, R ₅ O- is the non-protected form of formula IX or IXa of macrolide O- protection and, x) R ₄ is performing O- deprotection of O- for the production of a non-protected form of formula IX or IXa of the macrolide, R ₅ O- is in the protected form of Formula IX or IXa macrolide and xi) To prepare the macrolylide of formula IX or IXa comprising an O-protected hydroxyphenylalanine residue at positions 7 to 10 of the macromocyclic ring, an O-unprotected Protecting the macrolides of formula (IX) or (IXa) comprising a hydroxyphenylalanine residue, xii) To produce a macrolylide of formula IX or IXa comprising an O-unprotected hydroxyphenylalanine residue at positions 7 to 10 of the macrocyclic ring, the O-protected O-deprotection of the macrolide of formula (IX) or (IXa), which comprises a hydroxyphenylalanine residue, xiii) For the preparation of the macrolides of formula IX or IXa in which -ef- is CH (OH) -CH (OH) -, oxidation of the macrolides of formula IX or IXa in which -ef- is -CH = CH- &Lt; / RTI &gt; xiv) In order to prepare a compound of formula Va or a compound of formula XII, the linker group between the spirobicyclo group and the macrocyclic ring of the compound of formula IX or IXa is cleaved, xv) carrying out the ring opening of the macrocyclic ring of the formula IV or of the compound of the formula VIII in order to prepare a compound of the formula R ₆ OXYZA-OH or the formula R ₆ OXYZ-A'-CH (OH) -LS , xvi) The macrocyclic ring of the R ₆ OXYZA-OH or the formula R ₆ OXYZ-A'-CH (OH) -LS compound is closed to prepare the ring-closed form of the macrolide of formula IX or XII, xvii) In order to prepare a compound of formula XII or XIIa, the compound of formula IX or VIa is subjected to ring opening in a spirobicyclic ring system, xix) A process for the preparation of a compound of the present invention as defined above which comprises carrying out ring closure in a spirobicyclic ring system of a compound of formula (XII) or (XIIa), in order to prepare a compound of formula (IX) or (VIa). i) a piperidinylcarboxylic acid residue is provided at positions 2 to 6 of the macrocyclic ring, and / ii) an aromatic? -amino acid residue is provided at positions 7 to 9 of the macrocyclic ring, and / iii) Actinomycetes strains that produce markrolide in which aliphatic a-amino acid residues are provided at positions 10 to 12 of the macrocyclic ring. The biological parent strain, or a mutant, recombinant or variant thereof, of the strain Streptomyces sp. A92-308110 (DSM 9954) capable of producing the markrolide of the present invention. Culturing the strain Streptomyces sp. A92-308110 (DSM 9954) or a mutant, recombinant or variant thereof in a suitable culture medium, and optionally recovering the sanglyferin. Comprising administering to a subject in need of an immunosuppressive treatment an effective amount of the agent of the invention. Comprising administering to a subject in need thereof an effective amount of the invention agent i) for the treatment of acute and / or chronic bronchitis allografts or xenotransplantation rejection, for example, any of the above-mentioned specific types of organ transplant recipients, or ii) for the prevention of graft-versus-host disease in recipients of bone marrow transplantation, for example, or iii) for the treatment of autoimmune diseases or for the treatment of any of the diseases or disorders described above, or iv) Methods for the treatment of asthma. An agent of the invention used in the treatment of any disease or disorder, such as those used as medicaments, e.g. immunosuppressants, or as described under B above. A pharmaceutical composition comprising an agent of the invention together with a pharmaceutically acceptable diluent or carrier. As an immunosuppressive agent, or for the manufacture of a medicament for use in the treatment of any disease or disorder described under B above. Use of a compound according to any one of claims 1 to 6, 8 to 10, 15 or 16 as a reagent in a substitution immunoassay of a cyclosporin or other cyclophilin binding compound.