RU2413511C2 - Proteinkinase c inhibitors used for treating autoimmune diseases and graft rejection - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmacology, and concerns application of 3-(1-methyl-1H-indole-3-yl)-4-[1-{(1-pyridine-2-ylmethyl)piperidine-4-yl}-1H-indole-3-yl]pyrrole-2,5-dione or 3-(1-methyl-1H-indole-3-yl)-4-[1-( piperidine-4-yl)-1H-indole-3-yl]pyrrole-2,5-dione.

EFFECT: offered is a pharmaceutical composition for high-efficient treatment and prevention of organ or tissue graft rejection.

4 cl, 1 ex

Description

The present invention relates to new uses of protein kinase C inhibitors.

In particular, the invention relates to new uses of protein kinase C inhibitors of the formula I, II, III and IV and their pharmaceutically acceptable salts, hydrates or solvates thereof.

The following are protein kinase C inhibitors of formula I:

wherein

R ₁ and R ′ _{1 are} each independently hydrogen, alkyl, haloalkyl, alkenyl, arylalkyl, alkoxyalkyl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, acylaminoalkyl, acyloxyalkyl, cyanoalkyl, amidinoalkyl, carboxyalkylalkyl, alkyl ), (b) or (c):

in which Het represents a heterocyclyl group; W is NH, S or a bond; T is NH or S; V is O, S, NH or NCN; A represents an alkylthio group, an amino group, a monoalkylamino group or a dialkylamino group; Ar is aryl;

R ₂ and R ′ ₂ each independently of one another are hydrogen, alkyl, alkoxyalkyl, hydroxyalkyl, C ₁ -C ₃ alkylthio, S (O) C ₁ -C ₃ alkyl, CF ₃ ;

or R ₁ and R ₂ together form - (CH ₂ ) _r —X — CH ₂ -, where r is 1, 2 or 3 and X is CHR ₈ or NR ₈ , where R ₈ is (CH ₂ ) _s R ₉ , where R ₉ is hydrogen, hydroxy, alkoxy, amino, monoalkylamino, dialkylamino, trialkylamino, azido, acylamino, alkoxycarbonyl, cyano, amidino or aminocarbonyl and s is 0, 1,2 or 3;

R ₃ is hydrogen or CH ₃ CO;

R ₄ , R ' ₄ , R ₅ , R' ₅ , R ₆ , R ' ₆ , R ₇ and R' _{7 are} each independently hydrogen, halogen, alkyl, hydroxy, alkoxy, -COO (C ₁ -C ₃ alkyl), CF ₃ , nitro group, amino group, acetylamino group, monoalkylamino group, dialkylamino group, alkylthio group, C ₁ -C ₃ alkylthio group or S (O) C ₁ -C ₃ alkyl; and

n is 1, 2, 3, 4, 5, or 6.

The following are protein kinase C inhibitors of formula II:

wherein

R ₁ denotes a group of formula (d), (e) or (f):

in which p and q are each independently of one another 1, 2, 3 or 4;

s is 0, 1, 2 or 3;

t is 1 or 2;

u is 0 or 1; and

R ₁₂ is hydrogen, alkyl, haloalkyl, cycloalkyl, acetyl, aryl, —CH (aryl) ₂ , an amino group, a monomonoalkylamino group, a dialkylamino group, a guanidino group, —C (= N (alkoxycarbonyl)) NH (alkoxycarbonyl), amidino group, hydroxy alkoxycarbonyl or heterocyclyl;

R ′ ₁ is hydrogen, C ₁ -C ₄ alkyl, aminoalkyl, monoalkylaminoalkyl or dialkylaminoalkyl,

R ₂ and R ′ ₂ each independently of one another are hydrogen, alkyl, alkoxyalkyl, hydroxyalkyl, C ₁ -C ₃ alkylthio, S (O) C ₁ -C ₃ alkyl, CF ₃ ;

R ₃ is hydrogen or CH ₃ CO—; and

R ₄ , R ' ₄ , R ₅ , R' ₅ , R ₆ , R ' ₆ , R ₇ and R' _{7 are} each independently hydrogen, halogen, alkyl, hydroxy, alkoxy, -COO (C ₁ -C ₃ alkyl), CF ₃ , nitro group, amino group, acetylamino group, monoalkylamino group, dialkylamino group, alkylthio group, C ₁ -C ₃ alkylthio group or S (O) C ₁ -C ₃ alkyl.

The following are protein kinase C inhibitors of formula III:

wherein

R ′ ₁ is hydrogen, C ₁ -C ₄ alkyl, aminoalkyl, monoalkylaminoalkyl or dialkylaminoalkyl;

R ′ ₂ is hydrogen, alkyl, alkoxyalkyl, hydroxyalkyl, C ₁ -C ₃ alkylthio, S (O) C ₁ -C ₃ alkyl, CF ₃ ;

R ₃ is hydrogen or CH ₃ CO—;

R ₄ , R ' ₄ , R ₅ , R' ₅ , R ₆ , R ' ₆ , R ₇ and R' _{7 are} each independently hydrogen, halogen, alkyl, hydroxy, alkoxy, -COO (C ₁ -C ₃ alkyl), CF ₃ , nitro group, amino group, acetylamino group, monoalkylamino group, dialkylamino group, alkylthio group, C ₁ -C ₃ alkylthio group or S (O) C ₁ -C ₃ alkyl;

X is CR ₈ R ₉ , where R ₈ is (CH ₂ ) _s R ₁₀ , where R ₉ is (CH ₂ ) _s R ₁₁ , R ₁₀ and R _{11 are} each independently a hydroxy group, an alkoxy group, a carboxy group, an acyloxy group, an amino group, a monoalkylamino group, a dialkylamino group, a trialkylamino group, an azido group, an acylamino group, an alkoxycarbonyl, a cyano group, an amidino group or an aminocarbonyl, and s is 0, 1, 2 or 3; and

r is 1, 2 or 3.

The following are protein kinase C inhibitors of formula IV:

wherein

R ₁ is an alkyl glycosyl residue or a group of formula (g) or (h):

in which n is 1, 2, 3, 4, 5, or 6;

R ′ ₁ is hydrogen, C ₁ -C ₄ alkyl, cyclopropylmethyl, aminoalkyl, monoalkylaminoalkyl or dialkylaminoalkyl;

R ₂ and R ′ ₂ each independently of one another are hydrogen, alkyl, alkoxyalkyl, hydroxyalkyl, C ₁ -C ₃ alkylthio, S (O) C ₁ -C ₃ alkyl, CF ₃ ;

R ₃ is hydrogen or CH ₃ CO—; and

R ₄ , R ' ₄ , R ₅ , R' ₅ , R ₆ , R ' ₆ , R ₇ and R' _{7 are} each independently hydrogen, halogen, alkyl, hydroxy, alkoxy, -COO (C ₁ -C ₃ alkyl), CF ₃ , nitro group, amino group, acetylamino group, monoalkylamino group, dialkylamino group, alkylthio group, C ₁ -C ₃ alkylthio group or S (O) C ₁ -C ₃ alkyl.

Alkyl, individually or in combination, may be a straight or branched chain alkyl group containing from 1 to 7, preferably from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert- butyl and pentyl. “C ₁ -C ₃ alkyl” means an alkyl that carries from one to three carbon atoms. Alkenyl may be a straight or branched chain hydrocarbon bearing from 2 to 7 carbon atoms, which contains one or more double bonds, preferably one or two double bonds. Examples of alkenyl are ethynylene, propenylene, 1,3-butadienyl and 1,3,5-hexatrienyl.

Cycloalkyl, individually or in combination, may be 3 to 7 carbon atoms cycloalkyl, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The alkoxy group, individually or in combination, may be alkyl covalently linked through an —O— bond. Examples of alkoxy groups are methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group and tert-butoxy group. Alkoxyalkyl may be, for example, CH ₃ (CH ₂ ) -O- (CH ₂ ) _m , such as, for example, tert-butoxycarbonyl or BOC.

Haloalkyl may be alkyl bearing one or more, preferably 1-3, halogen atoms, for example CH ₂ Cl, CF ₃ , CH ₂ CF ₃ , CH ₂ (CF ₂ ) ₂ CF ₃ and the like.

The acyl radical of an acylamino group or acylaminoalkyl group is derived from an alkanoic acid containing at most 7, preferably at most 4 carbon atoms, for example acetyl, propionyl or butyryl, or from aromatic carboxylic acid, for example benzoyl. An acyloxy group is a group, such as an acyl linked via an —O— bond, for example an acetyloxy group, CH ₃ C (= O) O—. The acylamino group is, for example, CH ₃ (C = O) NH- (acetylamino group). Similarly, acylaminoalkyl denotes CH ₃ (C = O) NH (CH ₂ ) _m -.

Aryl, individually or in combination, may be an unsubstituted phenyl group or a phenyl group bearing one or more, preferably from 1 to 3 substituents, independently selected from the group consisting of halogen, alkyl, hydroxy group, benzyloxy group, alkoxy group, haloalkyl, nitro group, amino group, acylamino group, monoalkylamino group, dialkylamino group, alkylthio group, alkylsulfinyl, alkylsulfonyl and cyano groups. Arylalkyl is preferably benzyl.

Halogen may be fluoro, chloro, bromo or iodo.

A heterocyclic group designated as “Het” or “heterocyclyl” may be a stable, saturated, partially unsaturated or aromatic 5- or 6-membered heterocyclic group. The heterocyclic ring consists of carbon atoms and 1-3 heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur. The heterocyclic group may be substituted by 1 to 3 substituents independently selected from the group consisting of halogen, alkyl, hydroxy group, alkoxy group, haloalkyl, nitro group, amino group, acylamino group, monoalkylamino group, dialkylamino group, alkylthio group, alkyl sulfonyl group, and alkyl sulfonyl group represents an aromatic nitrogen-containing heterocyclic group, a nitrogen atom can carry an oxide group. Examples of such heterocyclyl groups are imidazolyl, imidazolinyl, thiazolinyl, pyridyl, indolyl, furyl and pyrimidinyl.

An “alkyl glycosyl residue” may be a glycosyl radical attached at the C-1 position to an indolyl via C ₂ -C ₄ alkyl. The glycoses included in the alkyl glycosyl residue may be either 5 or 6 sugar carbon atoms, either naturally occurring or not naturally occurring, preferably selected from the group consisting of allosyl, altrosyl, glucosyl, mannosyl, gulosyl, idosyl, galactosyl, talosyl, arabinosyl, xylosyl , lyxosyl, rhamnosyl, ribosyl, deoxyfuranosyl, deoxypyranosyl and deoxyribosyl. Glycose may be substituted by an azido group, O-acetylated, O-methylated, substituted by an amino group, a mono- and dialkylamino group, or substituted by an acylamino group. For example, an alkyl glycosyl residue includes

The compounds of formulas I-IV may be in free or salt form, for example an acid addition salt, for example a salt of an organic or inorganic acid, for example, hydrochloride, phosphate, acetate, mesylate, citrate or tartrate. According to the invention, the compounds of formulas I-IV in free or salt form can be used in the form of a hydrate or solvate, in amorphous or crystalline form.

Particularly preferred protein kinase C inhibitors are compounds of formulas Ia, Ib, IIa and IIIa or their salts.

The following are compounds of formula Ia:

wherein

R ₁ is hydrogen, aminoalkyl, monoalkylaminoalkyl or dialkylaminoalkyl;

R ′ ₁ is hydrogen, C ₁ -C ₄ alkyl, aminoalkyl, monoalkylaminoalkyl or dialkylaminoalkyl; and

R ₂ is hydrogen or methyl.

The following are compounds of formula Ib:

wherein

R ′ ₁ is hydrogen or C ₁ -C ₄ alkyl;

X is CR ₈ R ₉ or NR ₈ , where R ₈ is (CH ₂ ) _s R ₁₀ , where R ₉ is (CH ₂ ) _s R ₁₁ , R ₁₀ and R _{11 are} each independently hydrogen, hydroxy, amino a monoalkylamino group or a dialkylamino group, and s is 1; and

r is 1 or 2.

The following are compounds of formula IIa:

wherein

R ₁ is

where either s 'is 0 and R' ₁₂ is hydrogen or C ₁ -C ₄ alkyl; or s ′ is 1 and R ′ ₁₂ is pyridyl, preferably 2-pyridyl, and

R ′ ₁ is hydrogen, C ₁ -C ₄ alkyl.

The following are compounds of formula IIIa:

wherein

R ′ ₁ is hydrogen, alkyl, aminoalkyl, monoalkylaminoalkyl or dialkylaminoalkyl;

X is CR ₈ R ₉ or NR ₈ , where R ₈ is (CH ₂ ) _s R ₁₀ , where R ₉ is (CH ₂ ) _s R ₁₁ , R ₁₀ and R _{11 are} each independently hydroxy, carboxy, alkoxycarbonyl , an amino group, a monoalkylamino group or a dialkylamino group, and s is 0 or 1; and r is 1 or 2.

Even more preferred compounds are 3- (1-methyl-1H-indol-3-yl) -4- [1 - {(1-pyridin-2-ylmethyl) piperidin-4-yl} -1H-indol-3-yl ] pyrrole-2,5-dione, also designated LY 317615 (hereinafter referred to as compound A) and 3- (1-methyl-1H-indol-3-yl) -4- [1- (piperidin-4-yl) - 1H-indol-3-yl] pyrrole-2,5-dione (hereinafter referred to as compound B), in free or salt form, for example, hydrochloride.

Compounds of formula I, II, III, and IV can be synthesized using methods known in the art, for example, those described in US 5,545,636.

It has been found that protein kinase C inhibitors of formula I, II, III or IV and their pharmaceutically acceptable salts, hydrates or solvates due to their detected activity, for example, the ability to inhibit protein kinase C isoenzymes, such as protein kinase C β-1 and β-2, which are described , for example, in US 5545636, can be used to treat conditions associated with diabetes mellitus and its complications, as well as other diseases associated with increased levels of β-1 and β-2 isoenzymes, such as ischemia, inflammation, diseases of the central nervous system, heart but vascular disease, dermatological disease, Alzheimer's disease and cancer.

When the invention was created, it was found that protein kinase C inhibitors of formula I, II, III or IV, for example, formulas Ia, Ib, IIa and IIIa, and their pharmaceutically acceptable salts, hydrates or solvates can be used to treat and prevent organ, tissue or cells, for example, for treating recipients of dense organs or tissues, for example, heart, lung, heart, lung, kidney, pancreas, skin, or cornea grafts, or cells, for example, stem cells or insulin producers ruyuschih cells, e.g. pancreatic islet cells. They may also be indicated to prevent a graft versus host reaction, for example, after bone marrow transplantation.

Specific embodiments of the present invention are:

1.1. A method for treating organ, tissue or cell transplant rejection, for example, transplantation of a heart, lung, transplant of a heart, lung, liver, kidney, pancreas, skin or cornea transplant, and for preventing a transplant versus host transplant reaction in an individual in need thereof consisting in the fact that a protein kinase inhibitor C of the formula I, II, III or IV, for example of the formula Ia, Ib, IIa and IIIa, preferably a compound A or B or its pharmaceutically, is administered to the individual in a therapeutically effective amount acceptable salt, hydrate or solvate.

In addition, when the invention was created, it was found that protein kinase inhibitors of formula I, II, III or IV, for example, formulas Ia, Ib, IIa and IIIa, and their pharmaceutically acceptable salts or solvates can be used to treat and prevent autoimmune diseases, such as such as sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, obstructive airway disease, including conditions such as asthma, hereditary asthma, acquired asthma, dust-related asthma, especially chronic or difficult to cure May asthma (e.g., delayed-type asthma and hypersensitivity of the respiratory tract), bronchitis, including bronchial asthma, juvenile asthma, allergic rheumatoid arthritis, systemic lupus erythematosus, nephrotic lupus erythematosus syndrome, Hashimoto thyroiditis, multiple sclerosis, severe pseudoparalytic nephrotic lameness, uveitis steroid-dependent and steroid-resistant nephrosis, pustular acrodermatitis, allergic encephalomyelitis, glomerulonephritis, psoriasis, psoriatic arthritis, atopic eczema (atopic th dermatitis), contact dermatitis and other eczematous dermatitis, seborrheic eczema, lichen planus, pemphigus, bullous pemphigoid, congenital bullous epidermolysis, urticaria, angioneurotic edema, vasculitis, erythema, cutaneous eosinophilia, atrophyotia, nemorrhagia, nemorrhoidis, nemorrhoidis, nemorrhoidis, nemorrhoidis, nemorrhoidis, nemorrhosis, nemorrhoidis, nemorrhoidis, nasal echinosciosis, erytherosis, nemorrhoidis, nodosa, nodosa, nodosa, naprilosis, nemorrhoidis, nodosa, nodosa, nodosa, nodosa, naprilosis, nemorrhoidis, acne, erythematosus, acne , keratoconjunctivitis, keratitis, spring conjunctivitis, uveitis associated with Behcet’s disease, herpetic keratitis, conical cornea, epithelial corneal dystrophy, white corneal opacity, ocular pu A stigma that corrodes a corneal ulcer, scleritis, severe intraocular inflammation, inflammation of the mucous membrane or blood vessels, such as leukotriene-mediated B4 diseases, stomach ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel disease, inflammatory disease, e.g. bowel disease and ulcerative colitis), necrotizing enterocolitis, kidney disease, including interstitial nephritis, Goodpasture syndrome, hemolytic uremic syndrome m and diabetic nephropathy, nervous diseases selected from the group including dermatomyositis, Guillain-Barré disease, Meniere's disease and radiculopathy, diseases associated with collagen disorders, including scleroderma, Wegener's granulomatosis and Sjogren's syndrome, chronic autoimmune liver diseases, including autoimmune hepatitis, hepatitis primary biliary cirrhosis and sclerosing cholangitis, partial liver resection, acute liver necrosis (e.g., necrosis caused by toxins, viral hepatitis, shock or hypoxia), type B viral hepatitis, he non-A, non-B, and cirrhosis, rapidly developing hepatitis, pustular psoriasis, Behcet’s disease, active chronic hepatitis, Evans syndrome, hay fever, idiopathic hypoparathyroidism, Addison’s disease, autoimmune atrophic gastritis, lupus hepatitis, tubulo-nephritis, nephritis amyotrophic lateral sclerosis or rheumatic attack, especially inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis; amyotrophic lateral sclerosis (AMK); multiple sclerosis; rheumatoid arthritis or hepatitis C.

Thus, the present invention provides:

1.2. A method of treating or preventing autoimmune diseases, for example, as described above, in an individual who needs it, comprising administering to said individual a protein kinase C inhibitor of formula I, II, III or IV, for example of formula Ia, Ib, IIa and IIIa, or a pharmaceutically acceptable salt, hydrate or solvate thereof in a therapeutically effective amount.

In the context of the present description, the terms “treatment” or “treatment” both refer to prophylactic or preventive treatment, as well as therapeutic or disease-modifying treatment, including the treatment of patients who are at risk of being in contact with the disease or who are expected to be able to have contact with the disease, as well as patients who are already sick or who have been diagnosed with a disease or medical condition.

Specific embodiments of the present invention are also:

2. A protein kinase C inhibitor of formula I, II, III or IV, preferably Compound A or B, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use as an immunosuppressant or immunomodulator, for example, in the above methods.

3. A protein kinase C inhibitor of formula I, II, III or IV, preferably compound A or B, or a pharmaceutically acceptable salt, hydrate or solvate thereof, for the manufacture of a pharmaceutical composition that is used as an immunosuppressant or immunomodulator, for example, in the above methods .

4. A pharmaceutical composition intended for use as an immunosuppressant or immunomodulator, for example, in the above methods, which contains a protein kinase inhibitor C of the formula I, II, III or IV, preferably compound A or B, or a pharmaceutically acceptable salt, hydrate or solvate thereof in combination with one or more pharmaceutically acceptable diluents or carriers.

The possibility of using the compounds of the invention for the treatment and / or prevention of the above diseases and conditions can be demonstrated using standard animal experiments or clinical trials, which are described below.

In vitro: MLR (mixed cell lymphocyte response)

To demonstrate the immunosuppressive effect of the compounds that are intended for use in the method of the invention, a murine MLR model was used, for example, described by T. Meo in “Immunological Methods”, ed. L. Lefkovits and B. Peris, Academic Press, NY, 1979, pp. 227-239. Spleen cells (0.5 × 10 ⁶ ) from Balb / c mice (females, 8-10 weeks old) were incubated together for 5 days with 0.5 × 10 ⁶ irradiated (2000 rad) or mitomycin C treated splenic cells of mice line CBA (females, 8-10 weeks old). Irradiated allogeneic cells induce a proliferative response in the spleen cells of the Balb / c line, which can be assessed by the inclusion of a labeled precursor in the DNA. Since stimulant cells are irradiated (or treated with mitomycin C), they do not respond to Balb / c cells with a proliferative response, but retain their antigenicity. The antiproliferative effect of the compounds on Balb / c cells was evaluated using various dilutions and the concentration resulting in 50% inhibition of cell proliferation was calculated (IC ₅₀ ). The IC ₅₀ value for compound B was found to be 195 nm.

In vitro: Analysis of protein kinase C (PCS)

The activity of the compounds proposed in the invention, in relation to the PCD is evaluated using the following method. The analysis is carried out in white 384-well microtiter plates with a transparent bottom, which do not have a binding surface. The reaction mixture (25 μl) contains 1.5 μM acceptor-acceptor substrate that mimics the sequence of pseudosubstrate PKSα with Ala → Ser, 10 μM ³³ P-ATP, 10 mM Mg (NO ₃ ) ₂ , 0.2 mM CaCl ₂ , PKC protein, the concentration of which varies from 25 to 400 ng / ml (depending on the isotype used), lipid vesicles (containing 30 mol.% phosphatidylserine, 5 mol.% DAG and 65 mol.% phosphatidylcholine) in a final lipid concentration of 0.5 mM, in 20 mM Tris-HCl buffer, pH 7.4 + 0.1% BSA. Incubation is carried out for 60 minutes at room temperature. The reaction is stopped by adding 50 μl of the stop mixture (100 mM EDTA, 200 μM ATP, 0.1% Triton X-100, 0.375 mg / well streptavidin-coated SPA granules in phosphate buffered saline, in the presence or absence of Ca, Mg (w / o)). After incubation for 10 min at room temperature, the suspension is stirred for 10 min at 300 × g. The incorporated radioactivity was evaluated using a Trilux type counter for 1 minute. IC ₅₀ values are determined by a conventional method by incubation of an inhibitor, the concentration of which is obtained by serial dilution from 1 to 1000 μM. IC ₅₀ values are calculated from a graph obtained by fitting a curve using the XL fit ^® program.

In this analysis, it was found that the inhibitory effect against PKC of the compounds of the invention, for example the compounds of formula IIa, is characterized by an IC ₅₀ value of ≤1 μM, preferably ≤10 nM. For example, the inhibitory effect of compound B against PKSα characterized IC ₅₀ value of 3.0 nM, PKSβ - IC ₅₀ value of 2.0 nM, PKSδ - IC ₅₀ value of 2.0 nM and PKSε - IC ₅₀ value of 2.0 nM.

In vivo: Heart Transplantation for Rabbits

The combination of lines used: males of the Lewis line (RT ¹ - haplotype) and line DA (RT ¹ - haplotype). Animals are anesthetized with isofluorane inhalation. After heparinization of the donor rat through the ventral inferior vena cava with simultaneous bleeding through the aorta, the chest is opened and the heart is quickly cooled. A ligature is applied to the aorta and dissected distally from the first branch, and the brachiocephalic trunk is dissected at the level of the first bifurcation. A ligature is placed on the left pulmonary artery and dissected, and the right side is dissected, but the left side is left open. All other vessels are not dissected, ligated on them and separated and the donor heart is transferred to physiological saline cooled on ice.

The recipient is prepared by dissection and clamping of the abdominal aorta in the area under the kidney and vena cava. The graft is implanted using an end-to-side anastomosis using a 10/0 single-stitch suture between the donor’s brachiocephalic stem and the recipient’s aorta and the donor’s right pulmonary artery and the recipient vena cava. The clamps are removed, the graft is placed retroabdominally, the contents of the abdominal cavity are washed with warm saline and the animal is sutured and allowed to recover under a warming lamp. The graft viability is assessed by daily patching of the pulsating heart of the donor through the abdominal wall. It is believed that rejection is complete when the heart stops beating. In animals that were orally administered a compound of formula I, II, III or IV or a pharmaceutically acceptable salt or solvate thereof in a daily dose of 10 to 30 mg / kg twice daily (bid), an extension of the transplant viability was found. In this model, an extension of the graft viability by 14, 25, 26 days was found when using compound A when it was administered at a dose of 30 mg / kg bid.

In vivo: transplant versus host reaction model

Spleen cells (2 × 10 ⁷ ) from Wistar / F rats are injected subcutaneously into the pad of the right hind paw of hybrid rats of the Wistar / F × Fischer 344 generation F ₁ . The pad of the left paw is not treated. Animals are treated with test compounds for 4 consecutive days (0-3). The popliteal lymph nodes are removed on day 7 and the difference in mass of the two corresponding lymph nodes is determined. The results are expressed as inhibition of lymph node enlargement (in percent), comparing the change in the mass of the lymph node in the experimental groups and the change in the mass of the corresponding lymph nodes in the groups of animals that were not treated with the test compound. In this analysis, it was found that compound A, when administered at a dose of 30 mg / kg bid, inhibits node enlargement by 60-80%, preferably 70-80%.

In vivo: Treatment of Multiple Sclerosis: a model obtained using SJL / J mice, a chronic progressive experimental autoimmune encephalomyelitis (EAE)

Immunization: on day 0, female SJL / J mice were immunized (subcutaneous injection in the flank) with 200 μl of an inoculum containing 500 μg of bovine myelin basal protein (MBP) emulsified in Freund's complete adjuvant (CFA). On day 9, mice were subjected to a second MBP booster injection and an adjuvant containing 200 ng of B. ertussis toxin was additionally administered intravenously. The final injection of pertussis toxin is carried out on day 11.

In most mice immunized with MBP, a serious EAE attack occurs by day 21. This is followed by a recovery phase that starts at about day 25, during which the mice have no symptoms for about 20 days. Then after 45-47 days, approximately 50% of the animals begin a progressive phase of the disease. Therefore, therapeutic treatment with the test compounds begins on day 21, when the presence of the disease is fully proven, and continues until day 70, unless otherwise indicated. Recombinant murine interferon beta (INFβ, Calbiochem / Biosciences) was dissolved in physiological saline and administered by intraperitoneal injection 3 × per week. Compounds of the invention, for example Compound A, are administered p.o. 5 × per week by feeding through a gastric tube. Mice in the vehicle-treated control group were immunized with MBP and treated with water.

Each experimental group consists of mice in which the clinical symptoms of EAE are evaluated daily. The incidence rate and day of occurrence of EAE are also recorded. The clinical manifestation of EAE is assessed using a scale from 0 to 3. All cases of illness-related death that occurred after the start of drug therapy were recorded, giving them a maximum score of 3.

The daily doses required to put into practice the method of the present invention should depend, for example, on the compound used, the host, the route of administration and the severity of the condition to be treated. A preferred daily dose is from about 1 to about 1000 mg of active ingredient in a single dose or in divided doses.

Compounds of the formula I, II, III or IV, for example, the formulas Ia, Ib, IIa or IIIa, can be administered in free form or in the form of a pharmaceutically acceptable salt, for example the above salt. Such salts can be obtained by the conventional method, and they have the same level of activity as the free-form compounds.

Compounds of formula I, II, III or IV, for example, formulas Ia, Ib, IIa or IIIa, preferably compound A or compound B, or their pharmaceutically acceptable salts or solvates, may be administered as a single active ingredient or in combination with other drugs for the purpose immunomodulations, for example, for treating or preventing acute or chronic rejection of an allo or xenograft or autoimmune disease. For example, they can be used in combination with a calcineurin inhibitor, for example, cyclosporin A, ISA Tx247, FK506, ABT-281, ASM 981; mTOR inhibitor, for example rapamycin, 40-O- (2-hydroxyethyl) rapamycin, CCI779, АВТ578 or rapalogu, for example AP23573, AP23464, AP23675, AP23841, TAPA-93, biolimus 7 or biolimus 9, etc .; corticosteroids; cyclophosphamide; azathioprene; methotrexate; an S1P receptor agonist, for example FTY 720 or an analogue thereof; leflunomide or its analogues; misoribine; mycophenolic acid or its salt; for example, sodium salt, mycophenolate mofetil; 15-deoxyspergulin or its analogues; immunosuppressive monoclonal antibodies, for example, monoclonal antibodies to leukocyte receptors, for example, MHCs CD2, CD3, CD4, CD 11a / CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB, or their ligands, for example CD 154; or other immunomodulators, for example, recombinant binding molecules that carry at least a portion of the extracellular domain of CTLA4 or its mutant, for example at least an extracellular fragment of CTLA4 or its mutant, linked to a non-CTLA4 protein sequence, for example CTLA4Ig (e.g., deposited under number ATCC 68629) or a mutant thereof, for example LEA29Y, or with other adhesion molecule inhibitors, for example, with Mabs or low molecular weight inhibitors, including LFA-1 antagonists, selectin antagonists and VLA-4 antagonists.

According to the foregoing, the following objects of the present invention are:

5. The above method, which consists in administering, for example, simultaneously or sequentially, in a therapeutically effective amount, a protein kinase inhibitor C of the formula I, II, III or IV, for example of the formula Ia, Ib, IIa or IIIa, for example, compound A or compound B, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a second drug substance, which is an immunosuppressant or immunomodulator, for example, the aforementioned substance.

6. A therapeutic combination, for example, a kit comprising a) a protein kinase C inhibitor of formula I, II, III or IV, for example, formula Ia, Ib, IIa or IIIa, for example compound A or compound B, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and b) at least one second agent selected from a drug having an immunosuppressive or immunomodulatory effect. Component a) and component b) can be used simultaneously or sequentially. The kit may include instructions for its use.

When a protein kinase C inhibitor of formula I, II, III or IV, for example, formula Ia, Ib, IIa or IIIa, or a pharmaceutically acceptable salt or solvate thereof, is administered in combination with another drug having an immunosuppressive or immunomodulatory effect, for example, to prevent or treat acute or chronic transplant rejection or the above autoimmune disease, the dose of the immunosuppressant or immunomodulator used together, of course, should depend on the type used for co-administration Ia drug, for example on whether it is a steroid or a cyclosporin, specificity drug employed, the condition being treated, etc. or

Compound A and compound B are preferred, especially for use to treat or prevent transplant rejection and to prevent the graft versus host reaction.

Claims (4)

1. Use of a compound selected from 3- (1-methyl-1H-indol-3-yl) -4- [1 - {(1-pyridin-2-ylmethyl) piperidin-4-yl} -1H-indol-3 -yl] pyrrol-2,5-dione or 3- (1-methyl-1H-indol-3-yl) -4- [1- (piperidin-4-yl) -1H-indol-3-yl] pyrrole- 2,5-dione or a pharmaceutically acceptable salt, hydrate or solvate thereof for the preparation of a pharmaceutical composition for the treatment and prevention of organ or tissue transplant rejection and for preventing the graft versus host reaction. 2. Use of a compound selected from 3- (1-methyl-1H-indol-3-yl) -4- [1 - {(1-pyridin-2-ylmethyl) piperidin-4-yl} -1H-indol-3 -yl] pyrrol-2,5-dione or 3- (1-methyl-1H-indol-3-yl) -4- [1- (piperidin-4-yl) -1H-indol-3-yl] pyrrole- 2,5-dione or a pharmaceutically acceptable salt, hydrate or solvate thereof for the treatment and prevention of organ or tissue graft rejection and for preventing the graft versus host reaction. 3. A pharmaceutical composition that contains (a) a compound selected from 3- (1-methyl-1H-indol-3-yl) -4- [1 - {(1-pyridin-2-ylmethyl) piperidin-4-yl } -1H-indol-3-yl] pyrrol-2,5-dione or 3- (1-methyl-1H-indol-3-yl) -4- [1- (piperidin-4-yl) -1H-indole -3-yl] pyrrole-2,5-dione or a pharmaceutically acceptable salt, hydrate or solvate thereof, and (b) at least one second agent, which is a drug having an immunosuppressive or immunomodulating effect. 4. A method of treating or preventing organ or tissue transplant rejection or an autoimmune disease, or to prevent a graft versus host reaction in an individual who needs it, comprising administering to the individual a therapeutically effective amount of a compound selected from 3- (1-methyl-1H-indol-3-yl) -4- [1 - {(1-pyridin-2-ylmethyl) piperidin-4-yl} -1H-indol-3-yl] pyrrole-2,5- dione or 3- (1-methyl-1H-indol-3-yl) -4- [1- (piperidin-4-yl) -1H-indol-3-yl] pyrrole-2,5-dione, or their pharmaceutically acceptable salt, hydrate or solvate.