GB2367553A

GB2367553A - Pharmaceutically Active Macrocycles

Info

Publication number: GB2367553A
Application number: GB0021649A
Authority: GB
Inventors: Klaus Gerth; Gerhard Hoefle; Herbert Irschik; Rolf Jansen; Usama Karama; Brigitte Kunze; Thomas Leibold; Hans Reichenbach; Florenz Sasse; Marc Schinner; Udo Soeker; Heinrich Steinmetz; Larissa Vollbrecht; Peter Washausen; Christoph Heusser; Lukas Oberer
Original assignee: Gesellschaft fur Biotechnologische Forshung Mbh; Novartis AG; Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Current assignee: Gesellschaft fur Biotechnologische Forshung Mbh; Novartis AG; Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 2000-09-04
Filing date: 2000-09-04
Publication date: 2002-04-10
Also published as: GB0021649D0

Abstract

Compounds of the formula: <EMI ID=1.1 HE=68 WI=131 LX=362 LY=562 TI=CF> <PC>wherein R<SP>1</SP>, R<SP>2</SP> and R<SP>3</SP> independently are hydrogen, C<SB>1</SB>-C<SB>4</SB>alkyl which is unsubstituted or substituted by OH, or C<SB>1</SB>-C<SB>4</SB>alkoxy; R<SP>4</SP> is hydrogen, halogen, C<SB>1</SB>-C<SB>4</SB>alkyl which is unsubstituted or substituted by OH, or C<SB>1</SB>-C<SB>4</SB>alkoxy; R<SP>5</SP> is hydrogen or halogen; R<SP>6</SP> is hydrogen or C<SB>1</SB>-C<SB>4</SB>alkyl; and X is C=CH<SB>2</SB> or CHR<SP>6</SP> wherein R<SP>6</SP> is C<SB>1</SB>-C<SB>4</SB>alkyl which is unsubstituted or substituted by -S-C<SB>1</SB>-C<SB>4</SB>alkyl, are useful against autoimmune disorders or diseases.

Description

Organic Compounds The invention is directed to novel macrocycles, their use as pharmaceuticals and to pharmaceutical compositions containing such macrocycles.

More particularly the present invention provides in a first aspect compounds of formula I

wherein R', R2 and R3 independently are hydrogen, C1-C4alkyl which is unsubstituted or substituted by OH, or Ci-C4alkoxy ; R4 is hydrogen, halogen, Cl-C4alkyl which is unsubstituted or substituted by OH, or C1-C4alkoxy ; R5 is hydrogen or halogen ; R6 is hydrogen or Ci-C4alkyl ; and X is C=CH2 or CHR6 wherein R6 is C1-C4alkyl which is unsubstituted or substituted by - S-Ci-C4atky).

It will be understood that the above defined compounds may bear substituents within their structure, e. g. may bear appropriate amino moiety substituents.

Alkyl groups and moieties in the compounds of formula I may be branched or straight chain.

Alkyl groups are suitably straight chain.

In formula I the following significances are preferred independently, collectively or in any combination or sub-combination : (a) R'is hydrogen or unsubstituted CrC4alkyl, e. g. methyl ; (b) R2 is hydrogen or Ci-C4alkyl which is unsubstituted or substituted by OH, e. g. methyl or hydroxymethyl ; (c) R3 is hydrogen or C1-C4alkoxy, e. g. methoxy; (d) R4 is hydrogen or unsubstituted C1-C4alkyl, e. g. methyl ; (e) R5 is hydrogen or bromo; (f) R6 is hydrogen or methyl ; and (g) X is C=CH2 or CHR6 wherein R6 is methyl which is unsubstituted or substituted by -S-ethyl.

More particularly the present invention provides in a further aspect a compound of formula la

wherein R', R2, R3, R4, R5, R6 and X have the above meanings including the preferences. Examples for compounds of formula la include

compound R1 R2 R3 R4 R5 R6 X A1 H CH3 OCH3 H H CH3 C=CH2 A2 CH3 CH3 OCH3 H H CH3 C=CH2 A3 H CH3 H H H CH3 C=CH2 A4 OCH3 H H CH3 C=CH2 AS CH3 CH20H OCH3 H H CH3 C=CH2 A6 H H OCH3 H H CH3 C=CH2

A7 C=CH2 A8 CH3 CH3 OCH3 CH3 H CH3 C=CH2 A9 H CH3 OCH3 H H CH3 CHCH3 A10 H CH3 OCH3 H H CH3 CHCH2SCH2CH3 A11 H CH3 OCH3 H Br CH3 C=CH2 A12 H CH3 OCH3 Br H CH3 C=CH2 A13 CH3 CH3 H H H CH3 C=CH2 A14 CH3 CH3 OCH3 H H H C=CH2 Compounds of formula I may be prepared synthetically by methods known to the skilled person in the art of peptide chemistry, e. g. by a process comprising ring formation of a suitable oligopeptid, or, alternatively, may be isolated from the culture broth of a suitable microorganism. Preparation may include an additional modification step, e. g. hydration of an exocyclic double bond, an addition reaction step or a halogenation step. A suitable microorganism may be identified by culturing a variety of different microorganisms, e. g. selected from the group of myxobacteria and screening the resulting culture broths for the presence of a compound of formula 1, in particular for a compound of formula I wherein R', R2, R3 and R4 independently are hydrogen, Cl-C4alkyl which is unsubstituted or substituted by OH, or d4alkoxy ; R5 is hydrogen; and X is C=CH2. Isolation may follow methods commonly known to the skilled person. A suitable approach may, e. g. , comprise culturing the microorganisms in the presence of a solid phase, e. g. a resin, e. g. amberlite, adsorbing the compound of formula 1, and, having stopped culturing, eluting the compound of formula I from the solid phase.

A very suitable microorganism for the purposes of the present invention is a strain of the species Cystobacter minor (in the present literature, the organism is normally labelled Cystobacter minus, which is, however, grammatically not correct) of the order Myxococcales, family Cystobacteriaceae. This species may be found in soil and rotting plant material worldwide and may be isolated by standard methods.

Cystobacter minor strain Cbm 20, was deposited according to the Budapest Treaty with the DSM (Deutsche Sammlung von Mikroorganismen) in Braunschweig, Germany, on September 4,2000 by the Gesellschaft für Biotechnologische Forschung under the identification number Cb m20.

The characteristics of the strain correspond to those of the species description as outlined, e. g. in Bergey's Manual of Systematic Bacteriology, volume 3, p. 2150, Baltimore : Williams & Wilkins (1989). Vegetative cells are long, slender, needle-shaped, 0.6 to 0.8/5 to 10 Am.

Myxospores are rod-shaped, short to very short, well rounded, in phase contrast optically refractile, 1.0 to 1. 3/1. 2 to 1. 5 Am in size. Fruiting bodies consist of yellow-brown to red brown spherical or somewhat elongated sporangioles, 40 to 60 Am in size, which are arranged in short chains or dense clusters up to 0.5 mm2 in size. In culture, the fruiting bodies tend to degenerate into undifferentiated knots and narrow ridges, 20 to 30/40 to 80 Am in size and often densely packed and fused. The colonies are spreading swarms with long, delicated, radiating veins, often intensely yellow-brown. Cystobacter minor, strain Cbm 20 does not degrade chitin. The strain is resistant to ampicillin, sodium salt (50 mg/1) and kanamycin sulfate (250 mg/1).

Suitable culture media include solid and liquid media, e. g. yeast agar, e. g. yeast agar with cells of baker's yeast, which are vigorously decomposed, media containing an enzymatically

digested peptone, e. g. Difco Casitone (0. 3 %), VY/2-agar (yeast agar, DSM medium 9), CYagar (DSM medium 67). Cultures may be incubated for, e. g. 2 to 6 days at a temperature of, e. g. 30 C or room temperature, under aerobic conditions. They may be stored frozen, e. g. in a solution of casitone (e. g. 1 %) at-80 C or in liquid nitrogen. If fruiting bodies or at least myxospores are present cultures may be Iyophilysed.

Cystobacter minor, strain Cbm 20, and any strain derived therefrom, in particular any strain derived therefrom according to Rule 28 (3) second paragraph European Patent Convention, is also an object of the present invention.

Example 1: Preparation of compounds of formula la wherein X is C=CH2 and R5 is hydrogen (a) A 350 I bioreactor is charged with 300 I of culture medium containing 0.5 % Probion (single cell protein, Hoechst), 0.1 % calcium chloride, 0.1 % magnesium chloride, 0.1 % yeast extract, 0.5 % starch, 0.2 % glucose, 30 mg of vitamin B12, 31 of amberlite XAD 16 (Rohm + Haas), and 10 g of 1-docosanol (n-C22H460H) as antifoam agent. After sterilization the pH is adjusted to pH 7.2 with potassium hydroxide, and after inoculation with 2 I of shaking culture of Cystobacter minor, strain Cbm 20 kept at pH 7.6 by titration with acetic acid. As antifoam a mixture of 1-dodecanol and methanol 8: 2 is added throughout cultivation at a rate of 0.6 ml/h. The temperature is adjusted to 30 C, aeration is 30 I/min, the impeller is set initially to 150 rpm later adjusted to give an oxygen saturation of 30 %. After 5 days the adsorber resin is harvested by filtration through a 0.5 m2 process filter (210 urn).

(b) The resin is washed with water to remove adherent cells and is then extracted with 20 lof methanol. The methanol is evaporated and the residual water phase extracted three times with each of 3 1 of ethyl acetate. The organic layer is evaporated in vacuo to give a crude extract of 70 g.

This is dissolved in 1 1 of methanol and extracted twice with each 0. 8 1 of n-heptane. The methanol phase is evaporated in vacuo to give a refined extract of 55 g.

Further enrichment is achieved by chromatography on Sephadex LH 20 (column 80 x 20 cm, eluent methanol, 35 ml/min, detection at 254 nm). Based on UV adsorption and tic monitoring two fractions are taken, a first fraction at 450-555 min and a second fraction at 555-600 min.

The first fraction is separated on a Merck Präpbar 100 chromatograph (reversed phase column YMC ODS AQ, 120 A, 15 urn, column dimensions 40 x 10 cm, solvent system methanol/0. 05 M Ammonium-acetate buffer 65: 35, flow 200 ml/min, detection 227 nm). The first fraction collected (75-90 min) yields compound A1 (R1 and R4 are hydrogen, R2 is methyl and R3 is methoxy), the second fraction (95-120 min) yields compound A2 (R1 and R2 are methyl, R3 is methoxy and R4 is hydrogen).

Purification and partial separation of the individual components of the second fraction from the Sephadex column is achieved by RP-18 chromatography (ODS AQ 120 A column from Kronlab, 16 um, column dimensions 48 x 3 cm, solvent system methanol/0. 05 M ammonium acetate buffer 6: 4, flow 18 ml/min, detection 227 nm). A first and a second fraction are taken after 80-110 min and 110-160 min.

Preparative HPLC (Nucleosil C18, Machery Nagel, 7 um, column 250 x 20 mm, solvent system acetonitrile/water 35: 65, flow 8 ml/min, detection 227) nm of the first fraction gives compound A3 (Rl, R3 and R4 are hydrogen, R2 is methyl). Similarly the second fraction gives compounds A4 (Rl and R 4 are hydrogen, R2 is hydroxymethyl and R3 is methoxy), A5 (R'is methyl, R2 is hydroxymethyl, R3 is methoxy and R4 is hydrogen) and A6 (Rl, R2 and R4 are hydrogen, R3 is methoxy).

In analogy to the above procedure compounds A7 (Rl is hydrogen, R2 and R4 are methyl and

R3 is methoxy) and A8 (R', R2 and R4 are methyl and R3 is methoxy) may be prepared.

The above compounds are found to exhibit the following HPLC retention data [min] [methanol/water 65: 35 + 0.5 % acetic acid/Nucleosil (Macherey Nagel) 125/2 120-5-C18/0. 3 ml/min/detection : diode array] :

compound [min] A1 5.0 A4 3.4 A7 6.3 A2 5.9 A5 3.8 A8 8.2 A3 3.0 A6 3.9 Example 2: Preparation of compound A9, a compound of formula la wherein R\ R4 and R5 are hydrogen, R2 is methyl, R3 is methoxy, and X is CHR6 wherein R6 is methyl 30 mg (36 pmol) of compound A1 are dissolved in 4 ml of methanol and palladiumoxid is added (1 spatula tip). The mixture is stirred for six days under an atmosphere of hydrogen, adding fresh catalyst (1 spatula tip) each day. After six days the palladiumoxid is filtered off and the methanol is removed by using a rotary evaporator. The residue is suspended in 500 pu methanol and subjected to preparative HPLC (RP-18, methanol/water = 70/30).

The product containing fractions are combined, concentrated and extracted four times with ethyl acetate. The ethyl acetate containing phase is dried over sodium sulfate and the solvent is removed using a rotary evaporator. The residue is dried in high vacuum yielding compound A9.

'H-NMR (300 MHz, d6-Aceton) : Ala2 : sH= 1.55 (d, J3. 2 = 7.0 Hz, 3-H3), 4.63 (m, 2-H) Example 3: Preparation of compound A10, a compound of formula la wherein R\ R4 and R5 are hydrogen, R2 is methyl, R3 is methoxy, and X is CHR wherein R6 is-CH2SCH2CH3

15 mg (18 mmol) of compound A1 are dissolved in 100 ui of methanol and 300 ut (41 mmol) of ethanethiol are added. Potassium hydride (1 spatula tip) is added and the solution is stirred at 60oC. After 2 h a further 100 pl ethanethiol and a spatula tip potassium hydride are added followed by stirring for another 2 h.

The mixture is dried in vacuo, the residue is dissolved in dichloromethan and washed with brine. The organic phase is dried with sodium sulfate and the solvent is evaporated.

According to HPLC/MS analysis (RP-18, methanol/water = 70/30) two isomers are obtained (relative ratios): Rt = 2.68 Isomer 1 (46%); Rt = 3.37 Isomer 2 (38%) The residue is dissolved in 200 pi methanol and separated by preparative HPLC (RP-18, methanol/water = 70/30).

Isomer 1 : 1H-NMR (300 MHz, de-Aceton) : #H = 1.29 (m, 5-H3), 2.72 (q, 4-H2), 4.73 (m, 2-H) Isomer 2 : 1H-NMR (300 MHz, de-Aceton) : 8H = 1.18 (m, 5-H3), 2.56 (q, 4-H2), 4.86 (m, 2-H) Example 4 : Preparation of compound A11, a compound of formula la wherein R1 and R4 are hydrogen, R2 is methyl, R3 is methoxy, R5 is bromine and X is C=CH2 15 mg (18 mol) of compound A1 are dissolved in 300 RI dichloromethan and 64 fl. 1 (40

Jlmol) of a solution of bromine in dichloromethan are added. After 10 min of stirring dichloromethan is added and the mixture is washed twice with an aqueous solution of sodium thiosulfate, twice with brine and once with water. The organic phase is dried with sodium sulfate and the solvent is evaporated in vacuo. The residue is dissolved in 200 Al of methanol and separated by preparative HPLC (RP-18 ; methanol/water = 70/30).

Product containing fractions are united, concentrated to the water phase and extracted four times with ethyl acetate. The ethyl acetate phase is dried over sodium sulfate and the solvent is removed in vacuo.

'H-NMR (300 MHz, de-Aceton) : 8H = 6. 83 (d, Jg. s = 8. 6 Hz, 9-H), 7. 40 (d, Ja, g = 8. 1 Hz, 8-H) Example 5 : Preparation of compound A12, a compound of formula la wherein R1 and R5 are hydrogen, R2 is methyl, R3 is methoxy, R4 is bromine and X is C=CH2 38, 6 mg (46 mmol) of compound A1 are dissolved in 200 pl of dichloromethane. 9, 2 mg (52 mol) N-bromosuccinimide (NBS) are dissolved in 420 u) dichloromethane and added at 0 C while stirring.

HPLC (methanol/water = 70/30): Rt = 3. 49.

The solvent is evaporated in vacuo and the residue is dissolved in 300 pi of methanol. The products are separated by preparative HPLC (RP-18, methanol / water = 70/30).

1H-NMR (de-Aceton, 300 MHz): 8H = 6.89 (dd, Je. 7/9 = 4.7, 3.9 Hz, 8-H) 7.23 (m, 7-H, 9-H) Starting from compounds All or A12 compounds A7 and A8 may be prepared by alkylation following procedures known to the skilled artisan.

The compounds of the invention and their pharmaceutically acceptable acid addition salts (hereinafter: Pharmaceutical Compounds) have pharmacological activity and are useful as pharmaceutical as may be demonstrated in animal test methods, e. g. in accordance with the following test methods: I. Xenotransplantation of syrian hamster heart into rats The hamster-to-rat heart xenotransplantation model is a so-called difficult concordant model, in which antibody-mediated rejection (3 to 4 days) is the first event. The antibody synthesis occurs in a T-cell independent fashion; congenitally athymic ru/mu rats reject a hamster heart in a similar way as euthymic rats.

A compound of formula I is dissolved in a 1+1 mixture of dimethylsulfoxide and a Concentrate For Infusion [650 mg/ml rhicinus oil, polyethoxylated (= Cremophor EL) plus 94 % ethanol ad 1 ml] (COMPOSITION).

Male Syrian hamsters are anaesthetised and the heart is removed into cold physiological saline (4OC).

Male congenitally athymic (rnulrnu) rats are anaesthetised. A mid-line incision is made from xiphisternum to pubis. The abdominal contents are retracted to the right, to expose the infrarenal abdominal aorta and inferior vena cava. The vessels are dissected free from the fascia for a length of 0.5 to 1 cm, ligating and dividing any small branches (retroaortic spinal branches are preserved). The vessels are clamped individually, first the abdominal aorta.

Arteriotomy and venotomy are performed, and lumens are flushed with heparinised physiological saline. Then, the graft is placed in position and correctly oriented. End-to-side aortic anastomosis is made. Then, an end-to-side anastomosis of donor right pulmonary to recipient inferior vena cava is made. The venous clamps are removed, and then distal and proximal aortic clips. Physiological saline is infused intravenously. The abdomen is flooded with warm saline (37OC) and the wound is closed.

Subsequently, COMPOSITION is administered, e. g. by continuous administration for 14 days, e. g. by osmotic minipump.

The graft is monitored daily by palpation of the abdomen. Rejection is concluded in case of cessation of heart beat.

At weekly intervals markers for the presence of antibody-mediated rejection, including destruction of blood vessels and myocyte parenchyma, extravasation of erythrocytes and infiltration by polymorphonuclear granulocytes, are monitored. Compounds of formula I are effective in this assay when used at a daily dose of in the range of from 0.1 to 200 mg/kg, e. g. 5 to 50 mg/kg, e. g. 10 mg/kg.

II. Allotransplantation of arteries in mice A compound of formula I is dissolved in a 1 +1 mixture of dimethylsulfoxide and Concentrate For Infusion (COMPOSITION).

A mouse donor carotid artery segment is attached in a loop onto the carotid artery of a histoincompatible recipient mouse.

Grafts are harvested 30 days after transplantation and markers of vasculopathies including area of the neointima and smooth muscle cell number as described, e. g. in Shi et al., Circ.

Res. 75: 199-207 (1994) are monitored. Compounds of formula I are effective in this assay when used at a daily dose of in the range of from 0.1 to 200 mg/kg, e. g. 5 to 50 mg/kg, e. g.

10 mg/kg. The inhibition of lymphocyte antigen responses is in the range of about 0.1 to 200 mg/kg, e. g. 5 to 50 mg/kg. The IC50 data are in a dosage range of about 0.1 to about 1 J. lmol.

For compound A2 an IC50 of 0.003 J. lmol has been determined.

Compounds of formula I are, therefore, useful, e. g. as a lymphocyte inhibitor, e. g. a B cell inhibitor.

Compounds of formula I are useful for preventing development of, delaying onset of, delaying progression of, attenuating severity of, suppressing, mitigating or treating graft rejection, e. g. antibody-mediated graft rejection (acute rejection), for preventing graft-versus host diseases, or for treating or preventing transplant vasculopathies (late allograft rejection) in a recipient of e. g. an unmodified or modified organ, tissue or cell allo-or xenograft, e. g. heart, lung, combined heart-lung, trachea, liver, kidney, pancreas, Islet cell, bowel, e. g. small bowel, skin, muscles or limb, bone marrow, oesophagus, cornea or nervous tissue allo-or xenograft.

Compounds of formula I are useful for preventing development of, delaying onset of, delaying progression of, attenuating severity of, suppressing, mitigating or treating autoimmune disorders or diseases. Examples for autoimmune disorders and diseases include antibody or NK cells mediated autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, hashimoto's thyroidis, multiple sclerosis, myasthenia gravis, type I diabetes and uveitis, and allergies, asthma, psoriasis, sarcoidosis, dermatitis of various etiology, crohn's disease, irritable bowel disease and colitis.

Compounds of formula I are useful for inducing immunological tolerance in a subject in need of such a treatment, e. g. in a cell, tissue or organ transplant recipient. The term "immunological tolerance"refers to a state of unresponsiveness by the immune system of a patient subject to challenge with the antigen to which tolerance has been induced. In the transplant setting, in particular, it refers to the inhibition of the graft recipient's ability to mount an immune response which would otherwise occur in response to the introduction of non-self MHC antigen of the graft into the recipient.

Compounds of formula I are also useful for the treatment of infections, e. g. infections with Pseudomonas.

The dosage range of a compound of formula I to be employed for inhibiting lymphocyte antigen responses and the treatment or prevention of graft rejection or treatment of autoimmune disorders or diseases depends upon factors known to the person skilled in the art including host, nature and severity of the condition to be treated and the mode of admini stration. However, in view of the results in the animal test, satisfactory results are anticipated at daily dosages from about 0.1 to 200 mg/kg, e. g. 5 to 50 mg/kg, preferably administered once daily or continuously via, e. g. minipump. In the case of transplantation administration may start at a day within the range of from, e. g. 14 days prior to transplantation to 14 days post transplantation, in particular at the day of transplantation, and last up to, e. g. 4 weeks, in particular 14 days. Unit dosage forms for administration thus suitably comprise, e. g. from about 0.002 to 100 mg of a compound of formula I together with a pharmaceutically acceptable diluent or carrier therefor.

Compounds of formula I may be administered systemically or topically, by any conventional route, in particular enterally, e. g. orally, e. g. in the form of tablets or capsules, parenterally, e. g. in the form of injectable solutions or suspensions, topically, e. g. in the form of lotions, gels, ointments or creams, or in nasal or a suppository form. Pharmaceutical compositions comprising a compound of formula I may be manufactured in conventional manner, e. g. in the form of a lyophilized powder. The solution for parenteral administration may conveniently be prepared shortly before administration.

The present invention thus provides in further aspects (1 a) a method for inhibiting lymphocyte antigen responses in a subject in need of such a treatment, e. g. in a cell, tissue or organ transplant recipient or in a subject with auto-immune disorders or diseases, which method comprises administering to said recipient or subject an effective amount of a compound of formula l ;.

(1 b) a method for preventing development of, delaying onset of, delaying progression of, attenuating severity of, suppressing, mitigating or treating graft rejection, e. g. antibodymediated graft rejection (acute rejection), for preventing graft-versus host diseases, or for treating or preventing transplant vasculopathies (late allograft rejection), in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound of formula 1 ; (1c) a method for inducing immunological tolerance in a subject in need of such a treatment, e. g. in a cell, tissue or organ transplant recipient, which method comprises administering to said subject an effective amount of a compound of formula 1.

In a still further aspect the present invention provides (1d) a method for preventing development of, delaying onset of, delaying progression of, attenuating severity of, suppressing, mitigating or treating autoimmune disorders or diseases in a subject in need of such treatment, which method comprises administering to said subject an effective amount of a compound of formula 1.

In a preferred embodiment the invention provides: (1e) a method of preventing or treating antibody-mediated rejection in an allo-or xeno-cell,

- tissue or-organ recipient which method comprises administering to said recipient an effective amount of a compound of formula 1.

(1f) a method of transplanting an allo-or xeno-cell,-tissue or-organ in a recipient which method comprises the step of administering to said recipient an effective amount of a compound of formula 1.

In further or alternative embodiments the invention provides (2) a compound of formula I for use as a pharmaceutical ; (2a) a compound of formula I for use as a lymphocyte inhibitor, e. g. a B cell inhibitor, e. g. to prevent or reduce antibody-mediated responses; (2b) a compound of formula I for use in the prevention or treatment of antibody-mediated rejection of an allo-or xenograft, e. g. an unmodified or modified organ, tissue or cell allo-or xenograft, e. g. heart, lung, combined heart-lung, trachea, liver, kidney, pancreas, Islet cell, bowel, e. g. small bowel, skin, muscles or limb, bone marrow, oesophagus, cornea or nervous tissue allo-or xenograft, in a method as described above; (2c) a compound of formula I for use in the induction of immunological tolerance ; (2d) a compound of formula I for use as a lymphocyte inhibitor to prevent development of, delay onset of, delay progression of, attenuate severity of, suppress, mitigate or treat autoimmune disorders or diseases; (2e) a compound of formula I for use in the manufacture of a composition, e. g. for use in a method as described above;

(2f) a compound of formula I for use in the manufacture of a medicament for use as a lymphocyte inhibitor, e. g. in any of the methods as described above; (3) a pharmaceutical composition comprising a compound of formula I together with one or more pharmaceutically acceptable carriers or diluents therefor; (3a) a pharmaceutical composition as above for use in any of the methods as described above.

Compounds of formula I may be administered as the sole active ingredient or in conjunction with, e. g. as an adjuvant to, other drugs in immunomodulating regimens or other antiinflammatory agents for the treatment or prevention of allo-or xenograft acute or chronic rejection or inflammatory or autoimmune disorders. For example, compound of formula I may be used in combination with cyclosporin, rapamycins or ascomycins, or their immunosuppressive derivatives, e. g. cyclosporin A, cyclosporin G, FK-506, ABT-281,

ASM981, rapamycin, 40-0- (2-hydroxy) ethyl-rapamycin etc. ; corticosteroids ; cyclophosphamide ; azathioprene; methotrexate; FTY720 ; leflunomide ; mizoribine; mycophenolic acid; mycophenolate mofetil ; 15-deoxyspergualine ; immunosuppressive monoclonal antibodies, e. g., monoclonal antibodies to leukocyte receptors, e. g., MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD40, CD45 or CD58 or their ligands ; or other immunomodulatory compounds, e. g. CTLA41g, or other adhesion molecule inhibitors, e. g. mAbs or low molecular weight inhibitors including Selectin antagonists and VLA-4 antagonists. A preferred composition is with Cyclosporin A, FK506, rapamycin or 40-0- (2- hydroxy) ethyl-rapamycin. Dosages of the co-administered immunosuppressant or immunomodulatory compound will of course vary depending on the type of co-drug employed, e. g. whether it is a steroid or a cyclosporin. Preferably the co-drug employed is an immunosuppressive drug or regimen as used in transplantation, e. g. di-or tri-therapy.

In accordance with the foregoing the present invention provides in a yet further aspect: (a) a method as defined above comprising co-administration, e. g. concomitantly or in sequence, of a therapeutically effective amount of a compound of formula I and a second drug substance, said second drug substance being an immunosuppressant or immunomodulator drug, e. g. as indicated above; (b) a therapeutic combination, e. g. a kit, for use in any method as defined above, including a pharmaceutical composition comprising a compound of formula I and a pharmaceutical composition comprising an immunosuppressant or immunomodulatory drug. The kit may comprise instructions for its administration.

Investigations so far indicate that compounds of formula I of the invention are well tolerated at dosages required for use in accordance with the present invention.

Claims

Claims : 1. A compound of formula I

wherein R', R2 and 3 independently are hydrogen, Dialkyl which is unsubstituted or substituted by OH, or Ci4alkoxy ; R4 is hydrogen, halogen, Ci-C4alkyl which is unsubstituted or substituted by OH, or Ci4alkoxy ; R5 is hydrogen or halogen ; R6 is hydrogen or Ci-C4alkyl ; and X is C=CH2 or CHR6 wherein R6 is C1-C4alkyl which is unsubstituted or substituted by -S-C1-C4alkyl.
2. A compound of formula la

wherein R', R2, R3, R4, R5, R and X have the meanings as defined in claim 1.
3. Cystobacter minor strain Cbm 20, deposited according to the Budapest Treaty with the DSM (Deutsche Sammlung von Mikroorganismen) in Braunschweig, Germany, on September 4,2000 by the Gesellschaft für Biotechnologische Forschung under the identification number Cb m20, and any strain derived therefrom.
4. Pharmaceutical composition comprising, or use of, a compound according to claim 1 substantially as hereinbefore described.
5. A compound according to claim 1 for any use substantially as hereinbefore described.