Substituted 3-pyridyl-4-arylpyrazole and associated therapeutic and prophylactic methods

 

(57) Abstract:

This invention relates to new substituted 3-pyridyl-4-allpyral formula I

where R1- H, C1-5of alkyl, C1-5alkylamino, amino groups which are substituted by dimetilan, N-contains1-5Alkylglucoside selected from piperidine, substituted stands, research, pyrrolidine, thiazine, pyrrole, substituted stands, phenyl, phenyl substituted by one or more1-5the alkyl, amino, NHAc, nitro, nitrile and sulfona, and pyridine; R2- H, (CH2)3HE unsubstituted WITH1-5alkylphenyl, and research; R3represents one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, trifloromethyl, hydroxy, dimethylamino; and X is either C or N, or its pharmaceutically acceptable salt. Compound I inhibited the production of TNF-, which allows their use in pharmaceutical compositions for the treatment of inflammatory disorders mediated by inhibition of production of TNF - and/or activity R. 4 N. and 15 C.p. f-crystals, 5 PL.

The scope of the invention

This invention relates to new substituted 3-pyridyl-4-allpyral and altounian data connections, include inflammatory and AIDS-related disorders.

The background to the invention

TNF - and R-related disorder

Inflammatory cytokines, such as TNF-a, produced in the result of the activity of kinases. Such kinases include cytokine suppressive anti-inflammatory drug binding drug protein (CSBP)/p38 kinase, mitogen-activated protein (MAP) kinase family of serine-threonine protein kinases. Inflammatory cytokines play an important role in several inflammatory disorders [1], neurodegenerative disorders [10] and AIDS-related disorders [11-14]. Although the exact mechanism of kinases, such as R, not known, R involved in the production of TNF-, and signal response associated with the receptor of TNF-.

Arthritis is a basic example of inflammatory disorders and is, therefore, an inflammatory disorder, which mainly focus in this section. Millions of people suffer from arthritis and it can affect any joint in the body. Its symptoms vary from mild pain and inflammation in the affected joints to severe and debilitating pain and inflammation. Although this disorder is related mainly to the definition of arthritis involves the use of nonsteroidal anti-inflammatory drugs (NSPA (NSAID's) to relieve symptoms.

However, despite the widespread use of NSPA, many individuals cannot tolerate the dose needed for treatment of this disorder for a long period of time. In addition, NSPA treat only the symptoms of the disorders, not affecting the underlying cause.

When patients show no response to NSPA often use other drugs, such as methotrexate, gold salts, D-penicillamine and prednisone. These medicines also have significant toxicity and the mechanism of their action remains outstanding. It was shown that monoclonal antibodies to TNF, and antagonists of the receptor for interleukin 1 (IL-1) reduce the symptoms of rheumatoid arthritis in clinical trials on humans in small scale [2].

In addition based on protein therapy, there are small molecular agents that inhibit the production of these cytokines, which showed activity in models of rheumatoid arthritis in animals [3]. From these small molecular agents SB 203580 was effective in reducing the production of TNF - and IL-1 in lipopolysaccharide (LPS)-stimulated macrophage cell lines human values IC50then the Finance inflammatory cytokines in rats and mice when values of the IC5015-25 mg/kg [5]. SB 203580 reduces the production of inflammatory cytokines by inhibiting the activity of CSBP/p38 kinase with IC50200 nm [6]. Due to oral activity and efficiency of SB 203580 on animal models, the researchers suggested that any connection with the same activity profile has potential as a viable therapeutic tool against rheumatoid arthritis [5].

Pericerebral and their analogues were also obtained as inhibitors of cytokines and antagonists of glucagon [7] and, in particular, as inhibitors of IL-1, TNF and other cytokines. As inhibitors of cytokines were also obtained arylpyrazole [8] and triallers [9].

The role of CSBP/p38 was observed recently in various neurodegenerative and AIDS-related disorders. As for neurodegenerative disorders, it has been shown that the role R consists in determining survival of whether the cell is, or is exposed to neuronal programmed cell death, or apoptosis [10, 11].

It was shown that also related to SPEED the associated sarcoma herpes virus HHV 8 sarcoma (Kaposi) encodes G-beaxbatons receptor, activating R. It has been suggested that this activation contributes ecograma infection CCR5* T cell lines of human lymphocytes HIV assuming that R may play a role in early viral infection [13]. In addition, it was shown that inhibitors R block HIV replication in vitro in a manner that may be independent of TNF- [14].

The lack of clinically effective agents

In General, arthritis (particularly rheumatoid arthritis and most other inflammatory and AIDS-related disorders takes many lives affected by those people. There is a huge need for small molecular agents for treatment of these disorders. However, at present not identified none of these agents has not shown that he was clinically effective in humans.

Brief description of the invention

This invention provides compounds having the structure

or their pharmaceutically acceptable salts, with the formula:

(a) R1selected from the group consisting of (i) hydrogen, (ii) C1-5of alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl, independently substituted with one or more of the RAS group, consisting of (i) hydrogen, (ii) and (CH2)3HE, (iii) substituted or unsubstituted C1-5alkylphenyl, and (iv) N-containing C1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole;

(c) R3represents one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, nitro, trifloromethyl, hydroxy, dimethylamino and methylsulfoxide; and

(d) X is either C or n

This invention also provides another group of compounds having the structure:

or their pharmaceutically acceptable salts, in which:

(a) R1selected from the group consisting of (i) hydrogen, (ii) C1-5of alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl, independently substituted by one or more C1-5the alkyl, amino, substituted amino, nitro, nitrile and sulfona, and (vii) pyridine;

(b) R2selected from the group consisting of (i) hydrogen, (ii) and (CH2(c) R4represents a substituted or unsubstituted a heterocycle selected from pyridine, pyrimidine, furan, or thiophene; and

(d) X is either C or n

In addition, this invention provides a pharmaceutical composition containing one of these compounds and a pharmaceutically acceptable carrier.

In addition, this invention also provides a method of treatment of a subject having the disorder, improving by reducing the production of TNF - and/or activity R in corresponding cells, which includes the introduction of this subject a therapeutically effective dose of the present pharmaceutical compositions.

Finally, this invention provides a method of preventing an inflammatory response in the subject, comprising the introduction of this subject prophylactically effective amount of this pharmaceutical composition, either before or after the event, which is expected to cause the subject's inflammatory response.

Detailed description of the invention

This invention provides n is emich by reducing the production of TNF - and/or activity R and therefore, useful for the treatment of inflammatory disorders such as rheumatoid arthritis, and AIDS-related disorders.

Specifically, this invention provides a first group of compounds having the structure

or their pharmaceutically acceptable salts, in which:

(a) R1selected from the group consisting of (i) hydrogen, (ii) C1-5of alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi) phenyl, independently substituted by one or more C1-5the alkyl, amino, substituted amino, nitro, nitrile and sulfona, and (vii) pyridine;

(b) R2selected from the group consisting of (i) hydrogen, (ii) and (CH2)3HE, (iii) substituted or unsubstituted C1-5alkylphenyl, and (iv) N-containing C1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole;

(c) R3represents one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, bat is 2">In one variant embodiment of the first group of ports:

(a) R1selected from the group consisting of (i) hydrogen, (ii) C1-5of alkyl, (iii) substituted or unsubstituted C1-5alkylamino, (iv) N-containing C1-5Alkylglucoside selected from piperidine, research and pyrrolidine, and (v) phenyl, substituted Deputy selected from the group consisting of amino, substituted amino, nitro and nitrile.

(b) r2selected from the group consisting of hydrogen and (CH2)3phenyl;

(c) R3selected from the group consisting of halogen, nitro and trifloromethyl; and

(d) X is S.

In the preferred embodiment, the first compound is selected from the group of compounds represented in Table 1.

This invention also provides a second group of compounds having the structure

or farmatsevticheskii acceptable salts, in which:

(a) R1selected from the group consisting of (i) hydrogen, (ii)1-5of alkyl, (iii) substituted or unsubstituted WITH1-5alkylamino, (iv) N-containing1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole, (v) phenyl, (vi, and (vii) pyridine;

(b) R2selected from the group consisting of (i) hydrogen, (ii) and (CH2)3HE, (iii) substituted or unsubstituted C1-5alkylphenyl, and (iv) N-containing C1-5Alkylglucoside selected from thiazolidine, piperidine, research, piperazine, thiomorpholine, pyrrolidine, thiazine, pyrrole and imidazole;

(c) R4represents a substituted or unsubstituted a heterocycle selected from pyridine, pyrimidine, furan, or thiophene; and

(d) X is either C or n

In one of the embodiments of the second group of ports:

(a) R1selected from the group consisting of (i) C1-5of alkyl, (ii) substituted or unsubstituted C1-5alkylamino, (iii) substituted or unsubstituted C1-5alkylchlorosilanes amino, (iv) phenyl, and (v) phenyl, independently substituted by one or more amino, substituted amino, nitro, or nitrile.

(b) R2selected from the group consisting of hydrogen and (CH2)3phenyl; and

(c) X is S.

These compounds can be extracted and used in the form of free bases. They can also be extracted and used in the form of pharmaceutically acceptable salts. Examples of such salts include salts of Hydrobromic, what antinoi, almond, methansulfonate, hydrometallurgically, benzosulfimide, oxalic, pambou, 2-naphthalenesulfonate, p-toluenesulfonic acid, cyclohexanesulfamic and sugar acids.

In addition, the present invention provides a pharmaceutical composition comprising any of these compounds and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to specialists in this field and include, but are not limited to, from about 0.01 to about 0.1 M, and preferably 0.05 M phosphate buffer or 0.8% saline. Such pharmaceutically acceptable carriers may submit an aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. Aqueous carriers include water, ethanol, spirit aqueous solutions, glycerin, emulsions or suspensions, including saline or buffer medium. Oral media can imagine elixirs, syrups, capsules, tablets and the like. Typical solid support is an inert substance, such as lanie carriers include sodium chloride solution, dextrose of Injera (Ringer''s dextrose, dextrose and sodium chloride, electrovanne Riegerovy and thick oil. Intravenous carriers include fillers liquid and nutrients, DOPOLNITEL electrolyte, such as on the basis of dextrose Ringer and similar. May also contain preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases and similar. All media can be mixed if necessary with leavening agents, diluents, granulating agents, lubricating agents, binders and the like using conventional techniques known in this field.

In addition, the present invention provides a method of treatment of a subject having the disorder, improving by reducing the production of TNF - and/or activity R in appropriate cells, which comprises the administration to the subject a therapeutically effective dose of the present pharmaceutical compositions.

In one variation of the embodiment of the disorder is an inflammatory disorder. In yet another variant embodiment of the disorder is associated with AIDS disorder. Examples of disorders that can be treated this is ergicheskoe inflammation, periodontal disorder, inflammatory intestinal disorders, septic shock, insulin-dependent diabetes mellitus, insulin-independent diabetes, cachexia, pulmonary fibrosis, myasthenia gravis, Crohn's disease, hepatitis, primary biliary cirrhosis, acute pancreatitis, allograft rejection, glioblastoma, alopecia alopecia, psoriasis, ischemia, congestive heart failure, restenosis, atherosclerosis, systemic erythematous lupus, nephritis syndrome of Gillen-Barr (Guillain-Barre), viral myocarditis, HIV replication, depletion of T-lymphocytes during HIV infection, impaired cognitive ability, called neural inflammation, multiple sclerosis, stroke, or shock, neuropathic pain, HIV dementia and Alzheimer's disease. In the preferred embodiment the disorder is rheumatoid arthritis.

Used herein, the term “subject” includes, without limitation, any animal or artificially modified animal with the disorder, improving by reducing the production of TNF - and/or activity R in the respective cells. In the preferred embodiment of the subject is the man.

Used herein, the term “appropriate cells include, for example, cells that kind of what to include, without limitation, monocytes, macrophages, T lymphocytes, fibroblasts, dendritic cells, Langerhans ' cells (Langerhans), Kupffer cells (Kuppfer) and astroglial cells.

Introduction the present pharmaceutical compositions can be executed or implemented using any of various methods known to experts in this field. These compounds can be entered, for example, intravenously, intramuscularly, orally and subcutaneously. In a preferred method embodiment of the present pharmaceutical composition is administered orally. In addition, the introduction may include providing the subject for taking multiple doses over a suitable period of time. These modes of introduction can be defined in the usual way.

Used herein, “therapeutically effective dose of the pharmaceutical composition is an amount sufficient to halt, to normalize or reduce the development of the disorder. “Prophylactically effective dose of the pharmaceutical composition is an amount sufficient to prevent disorder, that is, termination, improvements and/or delay the onset of the disorder. In this area there are ways to determine those is to introduce the pharmaceutical compositions of the person, for example, you can define mathematically the results of animal studies.

In one of the embodiments of therapeutically and/or prophylactically effective dose is a dose sufficient to deliver the present pharmaceutical composition in an amount of from about 0.05 mg/kg body weight to about 200 mg/kg of body weight. In another embodiment therapeutically and/or prophylactically effective dose is a dose sufficient to deliver from about 0.5 mg/kg body weight to about 50 mg/kg of body weight. More specifically, in one of the embodiments of the oral dose is changed in the range from about 0.05 mg/kg to about 100 mg/kg / day. In yet another embodiment of an oral dose is in the range of from about 0.05 mg/kg to about 50 mg/kg / day and in the following embodiment from about 0.05 mg/kg to about 20 mg/kg / day. In another embodiment the dose for infusion are in the range of from about 1.0 μg/kg/min to about 1,0104mcg/kg/min of inhibitor in a mixture with a pharmaceutical carrier over a period of time in the range from several minutes to about several days. According to another embodiment for the local introduction of the present compound can be combined with a pharmaceutical carrier in the ratio of drug medium spans the treatment of inflammatory response in the subject, providing an introduction to the subject prophylactically effective amount of this pharmaceutical composition, either before or after the event, which is expected to cause the subject's inflammatory response. This event may be an insect bite or animal bite.

Used here the following chemical terms have the meanings set forth in this paragraph, “independent”, in relation to chemical substituents, means that if there are more than one substituent, these substituents may be the same or different; “alkyl” means alkyl with linear, cyclic or branched chain; “alkoxy” means-O-alkyl; “halogen” represents fluorine, chlorine, bromine or iodine; “Ph” means phenyl; “TCA” (TCA) means trichloroacetic acid; “TCF” (FCS) mean fetal calf serum; and “RPMI” means the environment of the Roswell Park Memorial Institute (Sigma cat # R0833).

The invention will be better understood upon reference to the following experimental details, but experts in this field obviously easy to understand that they are only illustrative of the invention, described more fully in the claims which follows. In addition, in this application mean what about the description of the state of technology, to which the invention relates.

Experimental details

I. General scheme of synthesis

Typical representatives of compounds of the present invention can be obtained in accordance with the General methods of synthesis described below and illustrated by the General schemes 1-6, listed at the end of the description. Some products of schemes can be used as intermediates for more than one of these compounds. In these cases, the choice of intermediate compounds used to obtain further compounds of the present invention, it is at the discretion and competence of specialists in this field.

Scheme 1 can be used to obtain the compounds of the invention in which R1represents methyl. As a starting material in scheme 1 can be used with compound 1a, such as 1,2-disubstituted alkyne. 1,2-Disubstituted alkynes can be obtained using known procedures. The substituents X and R3compounds of the invention are defined by substituents of the compounds 1A. Compound 1A together with the N-oxide, trimethylamine and dissolved in a dry solvent such as THF, and cooled to 0C. Add a base, such as d is oduct.

Scheme 2 may be used to obtain compounds of the invention in which R1represents hydrogen. As in the original scheme 2, you can use the connection type 2A 1-[(1-isocyano-2-(3-methoxyphenyl)ethynyl)sulfonyl]-4-methylbenzol. Compound 2A can be obtained by known methods. Deputy R3compounds of the invention typically is determined by the substituents at the phenyl atenolol group of compounds 2A; atom X is determined heteroaromatic Deputy acetate groups in the compound 2b. Compound 2A was dissolved in a dry solvent such as dimethyl ether of ethylene glycol, and added dropwise to a mixture of compound 2b, such as ethyl 4-pyridylacetate, and base, such as tert-butyl potassium, in a dry solvent such as dimethyl ether of ethylene glycol, at 0C. Upon completion of addition the reaction mixture was warmed up to 25 ° C and stirred for 3 hours, obtaining an intermediate compound 2C. When R3represents methoxy, intermediate compound 2C can be handled demetrious agent such as VVG3in an inert solvent such as CH3CL2when-S, giving compound 2d.

Scheme 3 can be used to obtain the compounds of the invention, in substance, in figure 3, you can use the connection 3A, diarylethylenes ,-unsaturated ester. The compounds of this type can be obtained by known methods. Compound 3b can be another source material, substituted toiletrieschoice derived (tosMIC), which can be obtained using known procedures.

For example, compound 3A can be ethyl ester 4-fluoro--[(4-pyridyl)methylene ]benzooxazol acid, and compound 3b can be 1-(4-tamilselvan)-1-(3-phenylpropyl)methylisocyanate. The compound 3A and compound 3b was dissolved in a dry solvent such as dimethyl ether of ethylene glycol, and added dropwise to-40C-Noah mixture of reasons, such as trebuchet potassium, in a dry solvent such as dimethyl ether of ethylene glycol. The stirring at-40C continue for 1 hour before give the temperature to rise to-20C. Accordingly, the resulting compound 3C is 4-(4-forfinal)-2-(3-phenylpropyl)-3-(4-pyridyl)-pyrrole (compound 27).

As shown in scheme 4, compound 2 and compound 3 can be used as intermediates for other compounds of the present invention.

Scheme 4 can be used to obtain the compounds of the invention in which R1substituted. Prom the body, such as dimethylformamide, at 0C. Upon completion of addition the reaction mixture is stirred at 0C for 15 minutes, then add portions of an alkylating agent, such as hydrochloride of 4-(2-chloroethyl)of the research. The reaction mixture is heated to 60C for 16 hours before give the temperature to drop to 25C with obtaining compounds 4A.

Scheme 5 can be used to obtain the compounds of the invention in which R1substituted. When R1is 4-nitrophenyl, intermediate compound 4A can be restored with the use of a catalyst, such as palladium on charcoal, in a solvent such as ethyl acetate, giving the compound 5A. Amine compound 5A can be handled allermuir agent such as acetic anhydride, in a solvent such as water, giving the product connection 5b.

Scheme 6 can be used to obtain the compounds of the invention in which R2represents an alkyl chain containing heteroatoms. Intermediate compound 3C expose restoration conditions using a catalyst such as palladium on charcoal, in a solvent such as ethanol, to which is added a catalytic amount of acid, such as concentraion agent, such as methylchloride, and a base, such as triethylamine, in a solvent such as CH2Cl2to obtain compounds 6b. Compound 6b can then be heated with a nucleophile, such as morpholine, in a solvent such as CH2Cl2to obtain compounds 6C.

II. The synthesis of specific compounds

Specific compounds, which are typical representatives of this invention can be obtained, as described in the following examples. No attempt to optimize the yields obtained in these reactions, was not done. However, based on the following, the expert knows how to increase the outputs using the normal variation in the duration of the reaction, temperatures, solvents and/or reagents.

Some products of the synthesis methods can be used as intermediates for more than one of these compounds. In these cases, the choice of intermediate compounds used to obtain further compounds of the present invention, is provided at the discretion of the specialists in this area and is within their competence.

The hydrobromide 3-(3-hydroxyphenyl)-4-(4-pyridyl)pyrrole

Compound 13 (0.15 g, 0,006 mol) ur up to 25S. The reaction mixture was extinguished Meon (20 ml) and evaporated to a solid. The solid is triturated with ether and filtered, obtaining compound 12 (0.15 g, yield 79%).1H NMR (DMSO-d6) 11,91 (1H, s, NH), for 9.47 (1H, Shir.S., IT), 8,66 (2H, d, J=8.6 Hz), 7,86-7,86 (3H, m), 7,25-7,14 (1H, m), 7,06 (1H, s), 6,80 of 6.66 (3H, m).

3-(3-methoxyphenyl)-4-(4-pyridyl)pyrrole

1-[(1-Isocyano-2-(3-methoxyphenyl)ethynyl)sulfonyl]-4-methyl-benzene (9.0 g, 0,0285 mol) was dissolved in dry DME (200 ml) and added dropwise to a cooled (0 C) a mixture of ethyl 4-pyridylacetate (9.0 g, 0,545 mol) and of potassium tert-butylate (7,1 g, 0,0633 mol) in dry DME (100 ml). Upon completion of addition the reaction mixture was warmed up to 25 ° C and was stirred for 3 h Then the reaction mixture was poured into ice water (1200 ml) and was extracted with CH2CL2(3500 ml). The combined organic extracts were dried over Na2SO4, was filtered and was evaporated in vacuum, obtaining a solid substance. This solid is then triturated with ether and filtered, obtaining 3.0 g of pure 3-(3-methoxyphenyl)-4-(4-pyridyl)pyrrole. The filtrate was again evaporated in vacuo and then triturated with a mixture of 50/50 CH2Cl2and ether, receiving additional 1.5 g of pure product. Finally the filtrate was evaporated in vacuum and purified on SiO2The p (DMSO-d6) 11,38 (1H, Shir.S., NH), of 8.37 (2H, d, J=6,1 Hz), 7.24 to 7,17 (4H, m), 7,02-7,00 (1H, m), 6,80-6,77 (3H, m), 3,68 (3H, s).

4-(4-forfinal)-2-(3-phenylpropyl)-3-(4-pyridyl)pyrrole

1-(4-Tamilselvan)-1-(3-phenylpropyl)methylisocyanate (10,9 g, 0,0346 mol) and ethyl 4-fluoro--[(4-pyridyl)methylene]-benzooxazol acid (9.4 g, 0,0346 mol) was dissolved in dry DME (250 ml) and added dropwise to a cooled (-40 ° C) mixture of potassium tert-butylate (9.5 g, 0,0847 mol) in dry DME (50 ml). The mixture was stirred for 1 hour allowing the temperature to rise to-20C. The mixture was poured into H2O (1800 ml) and was extracted with CH2CL2(3500 ml). The combined organic extracts were dried over Na2SO4, was filtered and was evaporated in vacuum, obtaining a solid substance. Rubbing solids with acetonitrile gave pure compound 27 (6.0 g, yield 49%). 1H NMR (DMSO-d6) 11,17 (1H, s, NH), scored 8.38 (2H, d, J=5.8 Hz), 7,27-6,92 (N, m), 2,61 is 2.51 (4H, m), 1.93 and of-1.83 (2H, m).

1-methyl-3-(4-forfinal)-4-(4-pyridyl)pyrrole

4-[(4-Forfinal)ethinyl]pyridine (2.0 g, 0,0101 mol) and N-trimethylamine oxide (1.0 g, 0,0133 mol) was dissolved in dry THF (200 ml) and cooled to 0C. Added diisopropylamide lithium (1.5 M in THF, 14 ml) and stirred the reaction mixture at 0C for 1 h Then the reaction mixture was extinguished H2O (20 ml) and getting extra oil. Purification on SiO2with elution EtOAc gave 0,356 g (yield 14%) of compound 28.1H NMR (Dl3) to 8.41 (2H, d, J=5.4 Hz), 7,19 (2H, DD, J=5,7, 6,0 Hz), 7,11 (2H, d, J=5.4 Hz), of 6.99 (2H, DD, J=8,7, 8.5 Hz), 6.87 in (1H, d, J=2.1 Hz), 6,69 (1H, d, J=2.4 Hz), 3,71 (3H, s).

4-(4-forfinal)-1-(4-nitrophenyl)-2-(3-phenylpropyl)-3-(4-pyridyl)pyrrole

Sodium hydride (60% suspension in mineral oil, 0,80 g, 0,0209 mol) was washed 3 times with hexane and then dissolved in DMF (15 ml). Then portions were added compound 27 (6.0 g, 0,01683 mol) at 0 C under stirring. Upon completion of addition the reaction mixture was stirred at 0C for 15 minutes, then was added dropwise 4-ftorirovannom (2.4 g, 0,170 mol). The reaction mixture was stirred at 0C for 1 h before to give the temperature to rise to 25C. The reaction mixture was poured into 1.5 liters of water and was extracted with CH2Cl2(3500 ml). The combined organic extracts were washed with water (4500 ml) and dried over Na2SO4. Evaporation in vacuo gave a yellow solid, which was triturated with acetonitrile, filtered and dried in the air, gaining 6.6 g (yield of 82.5%) of pure compound 30.1H NMR (CDCl3) charged 8.52 (2H, d, J=5.8 Hz), compared to 8.26 (2H, d, J=8,9 Hz), 7,46 (2H, d, J=8,9 Hz), 7,16-7,13 (3H, m), to 7.09-7.03 is (4H, m), 6,95-6,82 (5H, m), 2,73-to 2.67 (2H, m), 2,35 SS="ptx2">The connection 32 of 0.85 g, 0,0019 mol) was stirred for 16 hours in acetic anhydride (20 ml) and water (50 ml). The solution was extracted with ethyl acetate (100 ml), washed with water (350 ml), then washed with saturated sodium bicarbonate (350 ml) and then washed with N2O (250 ml). Organic matter was dried over Na2SO4and was evaporated in vacuum, obtaining a connection 31 (0,89 g, yield 96%) allocated in the form of butter.1H NMR (l3) to 8.41 (2H, d, J=5.6 Hz), with 8.05 (1H, s), 7,73 (2H, d, J=8.6 Hz), 7,28 (2H, d, J=8.7 Hz), 7,17? 7.04 baby mortality (7H, m), 6,91-6,85 (4H, m), 6,79 (1H, s), 2,66-2,61 (2H, m), 2,35-of 2.30 (2H, m), 2,22 (3H, s), 1,59-is 1.51 (2H, m).

1-(4-AMINOPHENYL)-4-(4-forfinal)-2-(3-phenylpropyl)-3-(4-pyridyl)pyrrole

Compound 30 (6.6 g, 0,0138 mol) is suspended in ethanol (200 ml) and ethyl acetate (50 ml) and subjected to the conditions of recovery for 16 hours in hydrogenator Parra at 50 lb./square inch (3,515 kg/cm2). The mixture was filtered through celite and evaporated in vacuum, obtaining oil. Rubbing this oil with acetonitrile and filtering the obtained solid substance gave 3.2 g of compound 32. The filtrate was evaporated in vacuum and purified on SiO2by elution with 50% ethyl acetate in hexane, obtaining additional of 1.75 g of product (total yield 79%).1H NMR (Dl3A solution of 2-(3-benzyloxyphenyl)-4-(4-forfinal)-3-(4-pyridyl) pyrrole (0.95 g, 0,0025 mol) in ethanol (125 ml) containing concentrated Hcl (0.2 ml) was added Pd on coal (0.2 g). This mixture was placed in an atmosphere of hydrogen for 16 hours in hydrogenator Parra at 50 lb./square inch (3,515 kg/cm2). The mixture was filtered through celite and was added to the obtained solution of triethylamine (0.5 ml), then was evaporated in vacuum, obtaining a solid substance. The solid was extracted with ethyl acetate (100 ml) and washed with water (350 ml). The organic fraction was dried over Na2SO4and was evaporated in vacuum, obtaining a light yellow solid (0.7 g, yield 96%).1H NMR (DMSO-d6) 11,10 (1H, s, NH), 8,44 (2H, d), 7,05 (6N, m) 6,91 (1H, d), a 4.53 (1H, Shir.with IT), 3,49 (2H, Shir.C) of 2.64 (2H, t), 1,72 (2H, m).

4-(4-forfinal)-2-(3-morpholinopropan)-3-(4-pyridyl)pyrrole

4-(4-Forfinal)-2-(3-methyloxirane)-3-(4-pyridyl)pyrrole (0.25 g, 0,0007 mol) was heated under reflux for 16 hours in CH2Cl2(50 ml) and morpholine (0.25 ml). The solution was cooled and diluted with CH2Cl2(~100 ml), then washed with H2O (350 ml). Organic matter was dried over Na2SO4and was evaporated in vacuum, obtaining oil. This oil was purified on SiO2, elwira 10% Meon substances (0,088 g, yield 36%).1H NMR (DMSO-d6) 11,12 (1H, s, NH), 8,42 (2H, d), 7,05 (6N, m), to 6.95 (1H, d), 3,55 (4H, t), 2,62 (2H, t), 2,29 (6N, m, 1,71 (2H, m).

4-(4-forfinal)-2-(3-methyloxirane)-3-(4-pyridyl)pyrrole

4-(4-Forfinal)-2-(3-hydroxypropyl)-3-(4-pyridyl)pyrrole (0.55 g, 0,0019 mol) was combined with triethylamine (0,52 ml, 0,0037 mol) in CH2Cl2(50 ml) and was cooled to 10C. Was added dropwise methanesulfonanilide (0.16 ml, 0,0020 mol) and gave received the mixture warmed to room temperature. This mixture was diluted with CH2Cl2(50 ml) and washed with water (30 ml). Organic matter was dried over Na2SO4and was evaporated in vacuum, obtaining oil. This oil was dissolved in ethyl acetate and purified on a layer of SiO2(~ 20 ml), elwira EtOAc. Evaporation of the solvent in vacuo gave a yellow solid (0,63 g, yield 91%).1H NMR (DMSO-d6) of 11.25 (1H, s, NH), to 8.45 (2H, d), 7,09 (6N, m) 6,94 (1H, d), 4,19 (2H, t), 3,17 (3H, s), a 2.71 (2H, t), to 1.98 (2H, m).

III. Biological assays and activity

A. Inhibition R in enzymatic analysis in vitro

Solution (38 ml) purified recombinant R (6xHis-p38, expressed in E. coli), myelin basic protein substrate (determined empirically) and buffer with pH 7.5 (Hepes: 25 mm MgCl2: 10 mm MnCl2: 10 mm) was added to the based on linear interval analysis and acceptable ratio of signal and noise. The remaining wells were used for control (“COUNTER” CTRL) and background (“BACKGROUND” BKG). COUNTER was prepared using the enzyme substrate buffer and 2% DMSO, and the BACKGROUND was prepared using the substrate buffer and 2% DMSO.

Solution (12 μl) of test compound in DMSO was added to the test wells. Compounds were diluted to 125 microns with a mixture of 10% DMSO/N2Oh and analyzed at 25 μm, when the final concentration of DMSO was 2%. All wells were added to the solution ATP/33P ATP (10 μl, containing 50 μm unlabeled ATP and 1 MX33P-ATP) and tablets were mixed and incubated at 30C for 30 minutes In each well was added ice 5% TCA/10 mm sodium phosphate (60 μl) and the tablets were kept on ice for 15 minutes, the Contents of each well was transferred into wells of 96-well filterplate (Millipore, MultiScreen-DP) and filterplate was placed in the vacuum manifold equipped with a tray for collecting waste. The wells are washed five times with a mixture of 10% TCA/10 mm sodium phosphate (200 μl) in a vacuum. Added scintillator MicroScint-20 and the plates were sealed using sheets Topseal-S and was made a count in scintillation counter Packard TopCount using33R liquid program with correction for quenching colors, where the output signal of the th test connection in table 2, was calculated by the following formula: % inhibition = [1-(sample - BACKGROUND)/(COUNTER - BACKGROUND)]100.

Although the compounds were initially tested at 20 μm, for reliability compounds were tested at concentrations of 4 times larger and 4 times smaller than the specified concentration. In addition, some compounds were calculated IC50using a 4-parameter receiving curve on Deltagraph.

Century Complete cell analysis for inhibition of TNF - in vitro

Their venous blood was anticoagulatory heparin, diluted with an equal volume of phosphate-saline buffer solution (“PBS”, PBS) and placed in a sterile test tube or other vessel. Aliquots (30 ml) of this mixture was transferred into centrifuge tubes amended Ficoll-Hypaque (15 ml). Prepared in this way, the tubes were centrifuged at 400g without interruption for 30 min at room temperature. Approximately 1/2 to 2/3 of the platelet layer over a layer of mononuclear cells was removed by pipette. A large part of the layer of mononuclear cells was carefully removed with a pipette, and the data RVMS were diluted with PBS and centrifuged at 600g for 15 min Obtained RVMS washed one more is ulali nutrient medium RPMI/1% FCS (fetal, bovine serum) with low content of endotoxin, and got a cell concentration of 0.5-2,0106RUMS/ml. a Small amount of this suspension was taken for counting on hemocytometer, and the remaining product was centrifuged at 200g for 15 min at room temperature. The obtained precipitation RVMS re-suspended in RPMI/1% FCS to a concentration of 1,67106/ml.

For this analysis, the suspension RVMS (180 ál) was transferred into a double wells of 96-well flat-bottomed microtitration tablet, and incubated for 1 h at S. A solution of the test compound (10 μl: cooked in a 20-fold concentration from the desired final concentration) was added to each well, and incubated tablet for 1 h at S. Solution was added (10 ál) of LPS in RPMI/1% FCS (200 ng/ml), and incubated in the wells overnight at S. Supernatant (100 μl) was removed from each well and diluted with RPMI/1% FCS (400 µl). The samples were analyzed for TNF - using an ELISA kit (solid immunofermentnyi analysis) (Genzyme).

Some compounds according to the invention are listed in table 3. These compounds were tested for their ability to inhibit the production of TNF-. For these compounds the results IC2were bled by cardiac puncture and collected whole blood (0.1 to 0.7 ml). Blood was allowed to clot, and serum was transferred into a centrifuge tube. This sample was centrifuged, and serum was collected, divided into aliquots and frozen at-80C. The samples were tested using industrial set ELISA for TNF- (endogen for TNF in mice and bioresource for TNF - a in rats). The test results in vivo for some compounds according to the invention are given in table 4. These compounds were tested for their ability to inhibit the production of TNF - a in mice, and the data are given as % inhibition at 25 mg/kg

The results of in vivo tests for some of the compounds according to the invention are given in table 5. Compounds were investigated for their ability to inhibit the production of TNF - a in mice, and the data are given as % inhibition at 10 mg/kg

LINKS

1. C. Dinl., Treatment of Rheumatoid Arthritis with Chimeric Monoclonal Antibodies to Tumor Necrosis Factor . Arthritis Rheum. 1993, 36, 1681-90.

3. J. C. Boehm, et al., 1-Substituted 4-Aryl-5-pyridinyl-imidazoles: A New Class of Cytokine Suppressive Drugs with Low 5-Lipoxygenase and Cyclooxygenase Inhibitory Potency, J. Med. Chem., 1996, 39, 3929-37.

4. International Publication No. WO 93/14081.

5. A. M. Badger, et al., Pharmacological Profile of SB 203580, A Selective Inhibitor of Cytokine Suppressive Binding Protein p38 Kinase, in Animal Models of Arthritis, Bone Resorption, Endotoxin Shock and Immune Function, The Journal of Pharmacology and Experimental Therapeutics, 1996, 279, 1453-61.

6. D. Griswold, et al., Pharmacology of Cytokine Suppressive Anti-Inflammatory Drug Binding Protein (CSBP), A Novel Stress-Induced Kinase, Pharmacology, Communications, 1996, 7, 323-29.

7. U. S. Patent No.5776954.

8. International Publication No. WO 97/05877.

9. International Publication No. WO 97/05878.

10. Davis, Roger J., et al., Opposing effects of ERK and JNK-p38 MAP Kinases on Apoptosis, Science, 1995, 270 (5240), 1326-31.

11. Heidenreich, Kim A., et al., Inhibition of p38 Mitogen-Activated Protein Kinase by Insulin in Cultured Fetal Neurons. J. Biol. Chem., 1996, 271 (17), 9891-4.

12. Arvanitakis L., et al., G-Protein-Coupled Receptor of Kaposi's Sarcoma-Associated Herpesvirus is a Viral Oncogene and Angiogenesis Activator. Nature, 1998, 391 (6662), 86-89.

13. Pitha, Paula M., et al., Eariy Activation of Mitogen-Activated Protein Kinase Kinase, increasing interest among Signal-Regulated Kinase, p38 Mitogen-Activated Protein Kinase, and c-Jun N-terminal Kinase in Response to Binding of Simian Immunodeficiency Virus to Jurkat T Cells Expressing CCR5 Receptor, Virology, 1998, 252 (1), 210-217.

14. Bukrinsky, M., The Critical Role of p38 MAP Kinase in T Cell HIV-1 Replication, Mol. Med., 1997, 3 (5), 339-346.

in which R1selected from the group consisting of (i) hydrogen, (ii) C1-5of alkyl, (iii) C1-5alkylamino, amino groups which are substituted by dimetilan, (iv) N-containing C1-5Alkylglucoside selected from piperidine, substituted stands, research, pyrrolidine, thiazine, pyrrole, substituted stands, (v) phenyl, (vi) phenyl, substituted with one or more C1-5the alkyl, amino, NHAc, nitro, nitrile and sulfona, and (vii) pyridine;

R2selected from the group consisting of (i) hydrogen, (ii) and (CH2)3HE, (iii) unsubstituted C1-5alkylphenyl, and (iv) research;

R3represents one or more substituents independently selected from the group consisting of hydrogen, halogen, methoxy, trifloromethyl, hydroxy, dimethylamino;

X is either C or N

or its pharmaceutically acceptable salt.

2. Connection on p. 1, in which R1selected from the group consisting of (i) hydrogen, (ii)1-5of alkyl, (iii)1-5alkylamino, amino groups which are substituted by dimetilan, (iv) N-containing1-5Alkylglucoside selected from piperidine, substituted stands, the research and pyrrolidine, and (v) phenyl, substituted amino, NHAc, nitro and nitrile;

R2selected the alogena and trifloromethyl;

X represents S.

3. Connection on p. 1, having the structure

4. Connection on p. 1, having the structure

5. Connection on p. 1, having the structure

6. Connection on p. 1, having the structure

7. Connection on p. 1, having the structure

8. Connection on p. 1, having the structure

9. Connection on p. 1, having the structure

10. Connection on p. 1, having the structure

11. Connection on p. 1, having the structure

12. Connection on p. 1, having the structure

13. Connection on p. 1, having the structure

14. Connection on p. 1, having the structure

15. Connection on p. 1, having the structure

16. Connection on p. 1, having the structure

17. Substituted 3-pyridyl-4-arylpyrol formula

in which R1means1-5alkyl;

R2means hydrogen;

R4represents a heterocycle selected from pyridyl or thiophene;

X means,

or its pharmaceutically acceptable salt.

18. Pharmaceutical composition for inhibiting the production of TNF-including soedinenii disorder, mediated by inhibition of production of TNF - and/or activity R in appropriate cells, which comprises the administration to the subject a therapeutically effective dose of the pharmaceutical composition under item 18.

 

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