RU2264404C2 - Alkylamino-substituted bicyclic nitrogen-containing heterocycles and pharmaceutical composition based on thereof - Google Patents

Abstract

FIELD: organic chemistry of heterocyclic compounds, biochemistry, pharmacy.

SUBSTANCE: invention describes alkylamino-substituted bicyclic nitrogen-containing heterocycles of the general formula (I):

wherein n = 1; R¹ means (C₁-C₆)-alkyl; R² means halogen atom; R³ means (C₁-C₆)-alkoxy-(C₁-C₆)-alkyl, (C₁-C₆)-alkylsulfonyl-(C₁-C₆)-alkyl, hydroxy-(C₁-C₆)-alkyl, dihydroxy-(C₁-C₆)-alkyl, N-heterocyclyl-(C₁-C₆)-alkyl or (C₁-C₆-alkylene)-C(O)R³¹ wherein R³¹ means hydroxy- or (C₁-C₆)-alkoxy-group, and its pharmaceutically acceptable salts. New compounds are inhibitors of protein kinase p38 and can be used in medicine.

EFFECT: valuable medicinal properties of compounds.

8 cl, 13 ex

Description

The present invention relates to bicyclic nitrogen-containing heterocycles. The object of the invention are alkylamino substituted dihydropyrimido [4,5-d] pyrimidinone derivatives, a method for their preparation, their use, pharmaceutical preparations containing them and a method for producing pharmaceutical preparations.

Mitogen-activated protein kinases (MAP kinases) belong to the family of proline directed serine / threonine kinases, which activate their substrates by double phosphorylation. Kinases are activated by various signals, including food and osmotic stress, UV radiation, growth factors, endotoxin and inflammatory cytokines. One group of MAP kinases is a group of p38 kinases, which includes various isoforms (e.g., p38α, p39β and p38γ). Like other kinases, p38α kinases are responsible for phosphorylation and activation of transcription factors, and they themselves are activated by physical and chemical stresses, pro-inflammatory cytokines and bacterial lipopolysaccharides.

Even more importantly, p38 phosphorylation products have been shown to mediate the production of inflammatory cytokines, including TNF (tumor necrosis factor), IL-1 (interleukip-1), and cyclooxygenase-2. Each of these cytokines is involved in various painful conditions and conditions. So, for example, TNF-α is a cytokine produced mainly by activated monocytes and macrophages. Its excessive and unregulated formation plays an important role in the pathogenesis of rheumatoid arthritis. More recently, it has been shown that inhibition of TNF production can be widely used to treat inflammation, inflammatory bowel disease, multiple sclerosis, and asthma.

TNF is also involved in the effects of viral infections such as HIV, influenza virus, herpes virus, including but not limited to herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), chickenpox smallpox (BBO), Epstein-Barr virus, human herpes simplex virus type 6 (HHF-6), human herpes simplex virus type 7 (HHF-7), human herpes simplex virus type 8 (HHF-8), infectious bulbar paralysis and rhinotracheitis.

Similarly, IL-1 is produced by activated monocytes and macrophages and plays a role in many pathophysiological reactions, including rheumatoid arthritis, fever, and decreased bone resorption. Inhibition of these cytokines by p38 kinase inhibitors may be useful in suppressing, attenuating and mitigating many of these painful conditions.

The compounds of formula I and their aforementioned salts are protein kinase inhibitors and in vivo exhibit unexpectedly high activity against p38. The compounds of formula I do not show activity against tyrosine kinases of p56 ^lck T cells at a level below about 10 μM. Such compounds may be effective in the treatment of diseases mediated by pro-inflammatory cytokines, such as TNF and IL-1.

As used herein, “alkyl” means a linear saturated monovalent hydrocarbon radical containing from one to six carbon atoms, or a branched saturated monovalent hydrocarbon radical containing from three to six carbon atoms, for example methyl, ethyl, n-propyl, 2- propyl, tert-butyl, pentyl and the like;

"alkylene" means a linear saturated divalent hydrocarbon radical containing from one to six carbon atoms, or a branched saturated divalent hydrocarbon radical containing from three to six carbon atoms, for example methylene, ethylene, propylene, 2-methylpropylene, pentylene and the like. ;

"alkenyl" means a linear monovalent hydrocarbon radical containing from two to six carbon atoms, or a branched monovalent hydrocarbon radical containing from three to six carbon atoms, including at least one double bond, for example, ethenyl, propenyl, etc .;

"alkynyl" means a linear monovalent hydrocarbon radical containing from two to six carbon atoms, or a branched monovalent hydrocarbon radical containing from three to six carbon atoms, including at least one triple bond, for example ethynyl, propynyl and the like;

“cycloalkyl” refers to a saturated monovalent cyclic hydrocarbon radical containing from three to seven ring carbon atoms; cycloalkyl may be optionally independently substituted with one, two or three substituents selected from alkyl, optionally substituted phenyl or —C (O) R (where R is a hydrogen atom, alkyl, haloalkyl, amino, monosubstituted amino, disubstituted amino, hydroxy -, alkoxy group or optionally substituted phenyl); more specifically, the term “cycloalkyl” encompasses, for example, cyclopropyl, cyclohexyl, phenylcyclohexyl, 4-carboxycyclohexyl, 2-carboxamidocyclohexyl, 2-dimethylaminocarbonylcyclohexyl and the like;

“cycloalkenyl” means an unsaturated non-aromatic monovalent cyclic hydrocarbon radical containing from three to seven ring carbon atoms; typical examples include cyclohexenyl and cyclopentenyl;

“cycloalkylalkyl” means the radical —R ^a R ^b where R ^a is an alkylene group, and R ^b is a cycloalkyl group as described herein, for example cyclopropylmethyl, cyclohexylpropyl, 3-cyclohexyl-2-methylpropyl and the like;

“acyl” means a group —C (O) R ′, where R ′ is alkyl, haloalkyl, heteroalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl;

“alkoxy”, “aryloxy”, “aralkyloxy” or “heteroaralkyloxy” means the radical —OR, where R is respectively alkyl, aryl, aralkyl or heteroaralkyl as described herein, for example methoxy, phenoxy, pyridin-2-ylmethyloxy, benzyloxy and the like;

“halo” or “halogen atom” means a fluorine, chlorine, bromine or iodine atom, preferably fluorine or chlorine; “haloalkyl” means alkyl substituted with one or more identical or different halogen atoms, for example —CH ₂ Cl, —CF ₃ , —CH ₂ CF ₃ , —CH ₃ CCl ₃ and the like, and further encompasses those alkyl groups, such as perfluoroalkyl, in which all hydrogen atoms are replaced by fluorine atoms;

“hydroxyalkyl” means an alkyl radical, as defined herein, substituted with one or more, preferably one, two or three, hydroxyl groups, provided that the same carbon atom does not carry more than one hydroxyl group; typical examples include, although not limited to, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 1- (hydroxymethyl) -2-methylpropyl, 2-hydroxybutple, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl , 1- (hydroxymethyl) -2-hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2- (hydroxymethyl) -3-hydroxypropyl, preferably 2-hydroxyethyl, 2,3-dihydroxypropyl and 1- (hydroxymethyl) - 2-hydroxyethyl, whereby the term “hydroxyalkyl” as used herein, refers to a subgroup of heteroalkyl groups;

“monosubstituted amino group” means the radical —NHR, where R is alkyl, heteroalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heterocyclyl or heterocyclylmethyl, for example methyl etc .;

“disubstituted amino group” means the radical —NRR ′, where each of R and R ′ is independently alkyl, heteroalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, aralkenyl, heteroaryl, heteroalkyl, heteroalkenyl, heterocyclyl or heterocyclyl and R 'together with the nitrogen atom to which they are bonded form a heterocyclic ring; typical examples include, although not limited to, dimethylamino, methylethylamino, di (1-methylethyl) amino groups, piperazin-1-yl, and the like; "aryl" means a monovalent monocyclic or bicyclic aromatic hydrocarbon radical containing from 6 to 10 ring atoms, each of which is independently substituted by one or more substituents, preferably one, two or three substituents selected from alkyl, haloalkyl, heteroalkyl, halo, nitro , cyano groups, methylenedioxy, ethylenedioxy, cycloalkyl, optionally substituted phenyl, heteroaryl, haloalkoxy, optionally substituted phenoxy, heteroaryloxy, -COR (where R is alkyl or optionally ameschenny phenyl), - (CR'R '') n -COOR ( where n is an integer from 0 to 5, each of R 'and R''are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl or cycloalkylalkyl) and - (CR′R ″) _n —CONR ^a R ^b (where N is an integer from 0 to 5, each of R ′ and R ″ is independently a hydrogen atom or alkyl, and each of R ^a and R ^b independently represents a hydrogen atom, alkyl, cycloalkyl or cycloalkylalkyl or R ^a and R ^b together with the nitrogen atom to which they are attached form a heterocyclic ring); more specifically, the term "aryl" covers, although not limited to, phenyl, 1-naphthyl, 2-naphthyl and their derivatives;

“aralkyl” means the radical —R ^a R ^b where R ^a is an alkylene group and R ^b is an aryl group as described herein, for example benzyl, phenylethyl, 3- (3-chlorophenyl) -2-methylpentyl, and t .P.;

“aralkenyl” means the radical —R ^a R ^b where R ^a is an alkenylene group and R ^b is an aryl group as described herein, for example 3-phenyl-2-propenyl and the like;

“arylheteroalkyl” means a radical —R ^a R ^b where R ^a is a heteroalkylene group and R ^b is an aryl group as described herein, for example 2-hydroxy-2-phenylethyl, 2-hydroxy-1-hydroxymethyl-2 phenylethyl and the like;

"optionally substituted phenyl" means a phenyl ring which is optionally independently substituted with one or more substituents, preferably one or two substituents selected from alkyl, haloalkyl, heteroalkyl, halo, nitro, cyano groups, methylenedioxy, ethylenedioxy, cycloalkyl, cycloalkylalkyl, -COR ( where R is alkyl or optionally substituted phenyl), - (CR'R "') _n -COOR (where n is an integer from 0 to 5, each of R' and R" is independently a hydrogen atom or alkyl, and R is hydrogen atom, alkyl, cycloalkyl or cycloalkyl) and _{- (CR'R '') n -CONR} a R b ( where n is an integer from 0 to 5, each of R 'and R''are independently hydrogen or alkyl, and each of R ^a and R ^b independently represents a hydrogen atom, alkyl, cycloalkyl or cycloalkylalkyl or R ^a and R ^b together with the nitrogen atom to which they are attached form a heterocyclic ring);

“heteroaryl” means a monovalent monocyclic or bicyclic radical containing from 5 to 12 ring atoms, which has at least one aromatic ring including one, two or three ring heteroatoms selected from N, O and S, with the remaining ring atoms being on C and it must be borne in mind that the site of attachment of the heteroaryl radical is usually located in the aromatic ring; this hsteroaryl ring is optionally independently substituted with one or more substituents, preferably one or two substituents selected from alkyl, haloalkyl, heteroalkyl, halo, nitro, cyano groups, cycloalkyl, cycloalkylalkyl, -COR (where R is alkyl or optionally substituted phenyl), - (CR'R "') _n -COOR (where n is an integer from 0 to 5, each of R' and R" is independently a hydrogen atom or alkyl, and R is a hydrogen atom, alkyl, cycloalkyl or cycloalkylalkyl) and - (CR'R '') _n -CONR ^a R ^b (where n is an integer from 0 d about 5, each of R 'and R "independently represents a hydrogen atom or alkyl, each of R ^a and R ^b independently of each other represents a hydrogen atom, alkyl,

cycloalkyl or cycloalkylalkyl or R ^a and R ^b together with the nitrogen atom to which they are attached form a heterocyclic ring); more specifically, the term “heteroaryl” encompasses, although not limited to, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyrimidinyl, benzofuranyl, tetrahydrobenzofuranyl, isobenzofuazolazole, benzobenzylazolazole, benzoburanothiolazole, benzoburanazole, , isoindolyl, benzoxazolyl, quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl, benzisoxazolyl, benzothienyl and their derivatives;

“heteroaralkyl” means the radical —R ^a R ^b where R ^a is an alkylene group and R ^b is a heteroaryl group as described herein, for example pyridin-3-ylmethyl, 3- (benzofuran-2-yl) propyl, and etc .; “heteroaralkenyl” means the radical —R ^a R ^b where R ^a is an alkenylene group and R ^b is a heteroaryl group as described herein, for example 3- (pyridin-3-yl) propen-2-yl, etc. P.;

“heterocyclyl” means a saturated or unsaturated non-aromatic cyclic radical containing from 3 to 8 ring atoms, in which one or two ring atoms are heteroatoms selected from NR (where R independently represents a hydrogen atom or alkyl), O or S (O) _n (where n denotes an integer from 0 to 2), the remaining ring atoms being in C, where one or two C atoms may, but need not be substituted by a carbonyl group; this heterocyclic ring may optionally be independently substituted with one, two or three substituents selected from alkyl, haloalkyl, heteroalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, monosubstituted amino, disubstituted amino groups, -COR (where R denotes alkyl or optionally substituted phenyl), - (CR′R ″ ″) _n -COOR (where n denotes an integer from 0 to 5, R ″ and R ″ each independently represents a hydrogen atom or alkyl, and R denotes a hydrogen atom, alkyl, cycloalkyl or cycloalkylalkyl) and - (CR′R ″) _n CONR ^a R ^b (where n is an integer from 0 to 5, R ′ and R ″ each independently represents a hydrogen atom or alkyl, and each of R ^a and R ^b independently represents a hydrogen atom, alkyl, cycloalkyl or cycloalkylalkyl or R ^a and R ^b together with a nitrogen atom, with which they are connected form a heterocyclic ring); more specifically, the term "heterocyclyl" covers, although not limited to, tetrahydropyranyl, piperidino, N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl, 3-pyrrolidino, morpholino, thiomorpholino, thiomorpholino-1-oxide , thiomorpholino-1,1-dioxide, pyrrolinyl, imidazolinyl and their derivatives;

“heterocyclylalkyl” means the radical —R ^a R ^b where R ^a is an alkylene group and R ^b is a heterocyclic group as described herein, for example tetrahydropyran-2-ylmethyl, 4-methylpiperazin-1-yl-ethyl, 3-piperidinylmethyl etc.;

“heteroalkyl” means an alkyl radical as defined herein, with one, two or three substituents independently selected from —ORa′NRbR ^c and —S (O) _n R ^d (where n is an integer from 0 to 2) wherein R ^a is a hydrogen atom, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido or mono- or dialkylcarbamo; R ^b represents a hydrogen atom, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl or heteroalkyl; R ^c represents a hydrogen atom, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, alkylsulfonyl, alkoxycarbonyl, aryloxycarbonyl, carboxamide group or mono- or dialkylcarbamoyl; R ^d is a hydrogen atom (provided that n is 0), alkyl. haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, amino, monosubstituted amino, disubstituted amino or hydroxyalkyl; typical examples include, in particular, 2-methoxyethyl, benzyloxymethyl, thiophen-2-ylthiomethyl, 2-hydroxyethyl and 2,3-dihydroxypropyl;

"heteroalkylene" means a linear saturated divalent hydrocarbon radical containing from one to six carbon atoms, or a branched saturated hydrocarbon radical containing from three to six carbon atoms, with one, two or three substituents independently selected from -OR ^a , -NR ^b R ^c and —S (O) _n R ^d (where n is an integer from 0 to 2), where R ^a , R ^b , R ^c and R ^d have the same meanings as indicated in the present description for a heteroalkyl radical; examples include 2-hydroxyethane-1,1-diyl, 2-hydroxypropane-1,1-diyl and the like;

“heterosubstituted cycloalkyl” means a cycloalkyl group in which one, two or three hydrogen atoms are substituted with substituents independently selected from the group consisting of hydroxy, alkoxy, amino, monosubstituted amino, disubstituted amino or —SO _n R (where n is an integer from 0 to 2, and R is a hydrogen atom (provided that n is 0), alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, aralkyl, heteroaryl, heteroalkyl, amino, mono-substituted amino, disubstituted amino or hydro xyalkyl); examples include 4-hydroxycyclohexyl, 2-aminocyclohexyl;

“heteroalkyl-substituted cycloalkyl” means a cycloalkyl group in which one, two or three hydrogen atoms are independently substituted with heteroalkyl groups; examples include 1-hydroxymethylcyclopent-1-yl, 2-hydroxymethylcyclohex-2-yl and the like;

the term "leaving group" is used in the meaning that is usually associated with it in the chemistry of organic synthesis, i.e. meaning an atom or group capable of being replaced by a nucleophile and encompassing a halo group (such as a chlorine, bromine, iodine atom), alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g. acetoxy), arylcarbonyloxy, mesyloxy, tosyloxy, trifluoromethanesulfonyloxy, aryloxy (e.g. 4-dinitrophenoxy), methoxy, N, O-dimethylhydroxylamino group and the like;

“pharmaceutically acceptable excipient” means an excipient that can be used in the preparation of a pharmaceutical composition that is generally harmless, non-toxic and neither biologically nor in any other way is undesirable, and encompasses excipients that are suitable for veterinary use , as well as for human use for pharmaceutical purposes; the term "pharmaceutically acceptable excipient", as used in the present description and claims, covers both one or more than one such excipient;

"pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and which possesses the desired pharmaceutical activity of the parent compound; such salts include:

(1) acid addition salts obtained using mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or obtained using organic acids such as acetic acid, propionic acid , caproic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, 4-toluenesulfonic acid, 4-methylbicyclo [2.2.2] oct-2-en-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis (3-hydroxy-2-en-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid , tert-butylacetic acid, lauryl sulfonic acid, gluconic acid, glutamic acid, hydroxy naphtha acid, salicylic acid, stearic acid, muconic acid, etc., and

(2) salts formed when the acidic proton contained in the parent compound is either replaced by a metal ion, for example an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or forms a coordination bond with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like;

"prodrugs" means any compounds which, when such prodrugs are administered to the body of a mammalian subject, in vivo release the active parent drug substance corresponding to formula (I); prodrugs of the compounds of formula (I) are prepared by modifying the functional groups present in the compounds of formula (I) so that the modifying groups are capable of being cleaved in vivo to isolate the parent compound; prodrugs include compounds of formula (I) in which a hydroxyl, amino, or sulfhydryl group [in the compounds of formula (I)] is bonded to any group that is capable of being cleaved in vivo to reduce a correspondingly free hydroxyl, amino, or sulfhydryl group; examples of prodrugs include, although not limited to, esters (in particular acetate, formate and benzoate derivatives), carbamates (in particular N, N-dimethylaminocarbonyl) hydroxyl functional groups in the compounds of formula (I) and the like. ;

“protecting group” refers to a grouping of atoms which, when it is attached to a reactive group in a molecule, masks, decreases or inhibits this reactivity; examples of protecting groups can be found in T.W. Greene and P.G. Futs, Protective Groups in Organic Chemistry (Wiley, 2nd edition, 1991) and Harrison, Harrison et al., Compendium of Synthetic Organic Methods, Volumes 1-8 (John Wiley and Sons, 1971-1996); typical amino-protecting groups include formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl (BPC), tert-butoxycarbonyl (BOC), trimethylsilyl (TMS), 2-trimethylsilylethanesulfonyl (SES), trityl substituted substituted trityloxycarbonylmethylbenzylcarbonylmethyl, 9-methyl, allyl nitroratryloxycarbonyl (IOC) and the like; typical hydroxyl protecting groups include those in which the hydroxyl group is either acylated or alkylated, such as residues of benzyl and trityl ethers, as well as residues of aliphatic ethers, tetrahydropyranyl ethers, trialkylsilyl ethers and allyl ethers;

A “cure” or “cure” of a disease includes:

(1) the prevention of the disease, that is, the prevention of the development of clinical symptoms of the disease in mammals that may be exposed to or susceptible to the disease, but are not yet susceptible to or exhibit disease symptoms;

(2) inhibition of the disease, i.e. delaying or weakening the development of the disease or its clinical symptoms, or

(3) attenuation of the disease, i.e. an event causing a regression of the disease or its clinical symptoms;

“therapeutically effective amount” means that amount of a compound which, when administered to a mammal to treat a disease, is sufficient for the effect of such a treatment of the disease;

a "therapeutically effective amount" usually varies depending on the compound, the particular disease, its severity, age, weight, etc. mammal to be treated.

In one aspect of the present invention, there are provided compounds of the formula

wherein

the subscript n denotes an integer from 0 to 3;

R ¹ represents a hydrogen atom, alkyl, alkenyl, alkynyl, alkylcarbonyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl or aralkyl; the values of each R ^{2 are} independently selected from the group consisting of alkyl, halo, heteroalkyl and vinyl;

R ³ is heteroalkyl, heteroalkenyl, heteroalkynyl, geteroalkilkarbonil, heterosubstituted cycloalkyl, heterosubstituted cycloalkylalkyl, heterosubstituted cycloalkylalkenyl, heterosubstituted tsikloalkilalkinil, geteroalkilzameschenny cycloalkyl, heterocyclyl, heterocyclylalkyl, arilgsteroalkil, geteroarilgeteroalkil, - (alkylene) -C (O) R ³¹ or - (heteroalkylene) -C (O) R ³¹ ;

Where

R ³¹ is alkyl, haloalkyl, hydroxy, alkoxy, amino, monosubstituted amino, disubstituted amino, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heteroaryl or heteroalkyl;

and their pharmaceutically acceptable salts.

In formula (I), the symbol R ¹ represents a hydrogen atom, an alkyl, alkenyl, alkynyl, alkple carbonyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl or an aralkyl group.

More preferred R ¹ is a hydrogen atom, alkyl, alkenyl, alkynyl, alkylcarbonyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl or an aralkyl group.

In another preferred embodiment, R ¹ is a hydrogen atom, alkyl, cycloalkyl or aralkyl. More preferred R ¹ is alkyl or cycloalkyl. In preferred embodiments, where R ¹ is alkyl, R ¹ is a branched alkyl group in which the carbon atom bonded to the nitrogen atom is a tetrahedral carbon atom, preferably having 0 or 1 hydrogen atom. More preferred R ^{1 is} usually 2-propyl, cyclohexyl or 1-methylcyclohexyl, most preferably 2-methyl-2-propyl.

In formula I, the symbol R ² denotes alkyl, halo, heteroalkyl or vinyl. R ² may be bound to the phenyl ring of any of the remaining five free bonds, otherwise saturated with hydrogen atoms. The subscript n denotes an integer from 0 to 3, indicating that the phenyl ring is substituted, including from zero to three R ² groups, preferably 1 or 2 R ² groups. In those embodiments in which there are two or three groups of R ² , each may be independent of the other (others). In a preferred embodiment, for R ² in the compounds of formula (I), n is 1 or 2, and each R ² is halo or alkyl, more preferably R ² is halo. Other preferred are those in which - (R ² ) _n is 2-halo or 2,6-dihalo, more preferably 2-chloro or 2,6-dichloro.

As mentioned above, the symbol R ³ represents heteroalkyl, heteroalkenyl, heteroalkynyl, geteroalkilkarbonil, heterosubstituted cycloalkyl, heterosubstituted cycloalkylalkyl, heterosubstituted cycloalkylalkenyl, heterosubstituted tsikloalkilalkinil, geteroalkilzameschenny cycloalkyl, heterocyclyl, heterocyclylalkyl, arilgeteroalkil, geteroarilgeteroalkil, - (alkylene) -C (O) R ^31, or - (heteroalkylene) -C (O) R ³¹ , where R ³¹ is alkyl, haloalkyl, hydroxy, alkoxy, amino, monosubstituted amino, disubstituted amino, cycloalkyl, cycloalkylalkyl, aryl , aralkyl, heteroaryl or heteroaralkyl.

In a preferred embodiment, R ³ in the compounds of formula (I), the values of R ³ are selected from heteroalkyl, heterocyclyl and heterosubstituted cycloalkyl. In one row to which R ³ values are particularly preferred, R ³ is heteroalkyl, more preferably hydroxyalkyl or alkoxyalkyl. Particular hydroxyalkyl and alkoxyalkyl groups are 2-methoxyethyl, 2-hydroxyethyl, 1-hydroxy-2-propyl, 2-hydroxy-1-propyl, 1-hydroxy-2- (hydroxymethyl) -3-propyl, 1,3-dihydroxy- 2-propyl, 1,3-dimethoxy-2-propyl, 1-methoxy-2- (methoxymethyl) -3-propyl, 3,4-dihydroxy-1-cyclopentyl, more specifically 2,3-dihydroxy-1-propyl and 2-methoxyethyl.

In another series in which R ³ values are particularly preferred, R ³ is heterocyclylalkyl. Particular heterocyclylalkyl groups include 2- (N-piperidinyl) ethyl and 2- (N- (2-pyrrolidinonyl)) ethyl.

In yet another particularly preferred embodiment, R ³ is - (alkylene) -C (O) R ³¹ , where R ³¹ is hydroxy, amino, methylamino, dimethylamino, methyl or ethyl. A more preferred alkylene moiety is methylene, ethylene or propylene.

In addition to the compounds described above, the present invention includes all pharmaceutically acceptable salts of these compounds together with prodrug forms of these compounds and all isomers, whether they are in pure chiral form, or in the form of a racemic mixture, or a mixture of another form.

Moreover, combinations of the preferred groups described above for compounds of formula (I) form other preferred variants. In one set of particularly preferred embodiments, R ¹ is alkyl or cycloalkyl, R ² is halo, R ³ is heteroalkyl or - (alkylene) -C (O) R ³¹ , an is 1 or 2. In other embodiments, R ¹ is alkyl or cycloalkyl , R ² is halo, R ³ is heteroalkyl, and n is 1 or 2; or R ¹ is alkyl or cycloalkyl, R ² is halo, R ³ is heterocyclyl, and n is 1 or 2; or R ¹ is alkyl or cycloalkyl,

R ² is halo, R ³ is heterosubstituted cycloalkyl, and n is 1 or 2; or R ¹ is isopropyl, R ² is halo, and n is 1 or 2.

In particular, the compound of formula (I) is selected from the group consisting of

3- (2-chlorophenyl) -1-ethoxycarbonylmethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one,

3- (2-chlorophenyl) -1-carboxymethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one,

3- (2-chlorophenyl) -1- (2-methoxyethyl) -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one,

3- (2-chlorophenyl) -7-isopropylamino-1- (2-methylsulfonylethyl) -3,4-dihydropyrmnido [4,5-d] pyrimidin-2 (1H) -one,

3- (2-chlorophenyl) -1- (2-hydroxyethyl) -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one,

3- (2-chlorophenyl) -1 - [(2S) -2,3-dihydroxyethyl] -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one,

3- (2-chlorophenyl) -1 - [(2R) -2,3-dihydroxyethyl] -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one and 7-isopropylamino -3- (2-chlorophenyl) -1- (2-piperidinylethyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one;

and their pharmaceutically acceptable salts.

In another aspect, the present invention provides methods for preparing the compounds of formula (I) described above. Briefly, these methods include either

(a) treating a compound of formula II

in which n, R ² and R ³ have the meanings given with reference to the above formula I, provided that any available inhibiting reactive group is in an optionally protected form, and L is a leaving group, an amine of formula III

in which R ¹ has the meanings presented with reference to the above formula I,

provided that any existing inhibiting reactive group is in an optionally protected form, and, when necessary, the release of all protected reactive groups, or

(b) treating a compound of formula IV

in which R ¹ , n and R ² have the meanings given for formula I, provided that any available obstructive reactive group is in an optionally protected form,

alkylating agent of formula V

in which R has the meanings given with reference to formula I, a X denotes a leaving group or a hydroxyl group that is activated during the reaction, provided that any available inhibiting reactive group is in an optionally protected form, and, when necessary, release all protected reactive groups; and optionally converting a compound of formula I to a pharmaceutically acceptable salt.

The compounds of the present invention can be prepared by a wide variety of methods using methods that are well known to those skilled in the art. Thus, for example, in one embodiment, compounds are prepared using methods similar to those outlined in Scheme 1.

Scheme 1

Treatment of a compound of formula Ia with a primary amine (R ³ —NH ₂ ) yields compounds of formula Ib. This reaction is conveniently carried out in a solvent which is inert under the reaction conditions, preferably in an acyclic or cyclic ether (such as tetrahydrofuran), a halogenated aliphatic hydrocarbon, in particular dichloromethane, an optionally halogenated aromatic hydrocarbon, formamide or lower alkanol. The reaction is expediently carried out at a temperature of from about -20 to about 120 ° C.

By reducing the compound of formula Ib, an alcohol of formula Ic is obtained. This reduction is usually carried out using lithium aluminum hydride according to a method that is well known to those skilled in the art (for example, in a solvent that is inert under reduction conditions, preferably in an acyclic or cyclic ether, in particular tetrahydrofuran, at a temperature of from about - 20 to about 70 ° C, preferably at a temperature of from about 0 ° C to about room temperature).

The oxidation of an alcohol of formula Ic in the next step provides the possibility of producing a carboxaldehyde of formula Id. Oxidation is usually carried out using manganese dioxide, although many other methods can also be used [see, for example, Advanced Organic Chemistry, ed. 4th, March, John Wiley & Sons, New York (1992)]. Depending on the oxidizing agent used, the reaction is conveniently carried out in a solvent which is inert under the given specific oxidation conditions, preferably in a halogenated aliphatic hydrocarbon, in particular dichloromethane, or in an optionally halogenated aromatic hydrocarbon. The oxidation is expediently carried out at a temperature of from about 0 to about 60 ° C.

The reaction of a carboxaldehyde of formula Id with substituted aniline makes it possible to obtain a compound of formula Ie. This reaction can be carried out in the presence of an acid, for example aromatic sulfonic acid, preferably 4-toluenesulfonic acid, with azeotropic removal of the water formed during the reaction. Such a reaction is conveniently carried out in a solvent which is inert under the reaction conditions, preferably in an aromatic hydrocarbon, in particular in toluene, or in an optionally halogenated aromatic hydrocarbon and at a temperature of from about 70 to about 150 ° C., preferably at the boiling point of the solvent, to facilitate the aforementioned azeotropic removal of water.

The reduction of the compound of formula Ie to obtain the compound of formula If can be carried out using, for example, sodium borohydride, lithium aluminum hydride or sodium triacetoxyborohydride under conditions that are well known to those skilled in the art. In a preferred embodiment, the compound of formula Ie is not purified, and the reaction mixture in which it is obtained is mainly concentrated and the resulting concentrate is dissolved in a solvent which is inert under the reduction conditions, preferably in an acyclic or cyclic ether, in particular tetrahydrofuran, or optionally halogenated aromatic hydrocarbon, or lower alkanol, and then treated with the aforementioned reducing agents. Recovery is expediently carried out at a temperature of from about 0 to about 100 ° C, preferably at from about 0 to 25 ° C.

By cyclizing a compound of formula If, a bicyclic nitrogen-containing heterocycle of formula Ig is obtained. The cyclization can be carried out by reacting the compound If with phosgene or trichloromethylchloroformate (or phosgene equivalent), suitably in the presence of a tertiary organic base, preferably three (lower) alkyl amines, in particular triethylamine. More specifically, the cyclization is carried out in a solvent which is inert under the reaction conditions, preferably in an acyclic or cyclic ether, in particular tetrahydrofuran, optionally halogenated aromatic hydrocarbon or halogenated aliphatic hydrocarbon. This reaction is conveniently carried out at a temperature of from about −20 to about 50 ° C., preferably at a temperature from about 0 ° C. to about room temperature.

Oxidation of the Ig compound with 3-chloroperbenzoic acid provides the possibility of producing sulfone (Ih), which can be converted into a wide variety of target compounds. Typically, the oxidation of compound Ig is carried out in a solvent which is inert under oxidation conditions, preferably in a halogenated aliphatic hydrocarbon, in particular in chloroform or dichloromethane, and at a temperature of from about −20 to about 50 ° C., preferably from about 0 ° C. to about room temperature temperature.

Finally, treating compound Ih with an amine (R ¹ —NH ₂ ) allows the desired compound of formula I to be prepared. The reaction can be carried out in the presence or absence of a solvent. This reaction is conveniently carried out at temperatures from about 0 to about 200 ° C, more preferably from about room temperature to about 150 ° C.

Thus, the present invention provides a method for preparing a compound of formula I by treating a compound of general formula Ii with an amine (R ¹ —NH ₂ ).

For compound Ii, the symbols R ² and R ³ and the subscript symbol n have the meanings given above with reference to formula I. The letter L denotes a leaving group, which may be a halogen atom, a lower alkensulfonyl group (for example, methanesulfonyl or trifluoromethanesulfonyl) or an aromatic sulfonyl group (e.g. benzenesulfonyl or 4-toluenesulfonyl). Other suitable leaving groups to those skilled in the art are known and can be found, for example, in Advanced Organic Chemistry, ed. 4th, March, John Wiley & Sons, New York (1992). Suitable amines (R ¹ —NH ₂ ) are those in which R represents any of the groups given for R in the description of formula I.

In a preferred embodiment, a bicyclic nitrogen-containing heterocycle can be obtained, and R ³ can be introduced at a later stage of the synthesis, as shown in Scheme 2.

Scheme 2

Compound IIa, the starting material in Scheme 2, can be prepared from commercially available ethyl 4-amino-2-mercaptopyrimidine-5-carboxylate. Briefly, treating the mercapto compound with an acceptable alkylating agent (RX) allows the preparation of a compound of formula Ib (R ³ is H). The steps in Scheme 1 may be followed by the conversion of compound Ib (R ³ is H) to compound IIa.

The cyclization of compound IIa gives a bicyclic nitrogen-containing heterocycle of formula IIb. This cyclization can be carried out by reacting compound IIa with phosgene or trichloromethylchloroformate (or phosgene equivalent), usually in the presence of a tertiary organic base, preferably three (lower) alkyl amines, in particular triethylamine. More specifically, the cyclization is carried out in a solvent which is inert under the reaction conditions, preferably in an acyclic or cyclic ether, in particular tetrahydrofuran, optionally halogenated aromatic hydrocarbon or halogenated aliphatic hydrocarbon. Typically, the reaction is carried out at a temperature of from about −20 to about 50 ° C., preferably at a temperature from about 0 ° C. to about room temperature.

The introduction of the group R ³ in order to obtain a compound of formula IIc can be carried out under a wide variety of conditions. Thus, for example, compound IIb can be treated with an alkali metal hydride, in particular sodium hydride, followed by reaction with a compound of the general formula R ³ -L, in which R ³ has any of the meanings described above for R ³ with the exception of a hydrogen atom, aryl or heteroaryl, and L is a leaving group (e.g., halo, methanesulfonate, toluenesulfonate, trifluoromethanesulfonate, etc.). N-substitution is usually carried out in a solvent which is inert under the reaction conditions, preferably in formamide, in particular in N-methylpyrrolidinone or dimethylformamide, in an acyclic or cyclic ether or optionally halogenated aromatic hydrocarbon. This reaction is expediently carried out at a temperature of from about 50 to about 200 ° C., Preferably at a temperature of from about 50 to about 150 ° C. Alternatively, the alkylation can be carried out with an inorganic base, such as potassium carbonate, in a formamide solvent, such as N-methylpyrrolidinone, at temperatures from about 0 to about 25 ° C.

An alternative and preferred method of introducing R ³ involves alkylation at the pyrimidinone nitrogen atom under the reaction conditions of Mitsonobu. In this method, an alcohol of general formula R ³ —OH is combined with a compound of general formula IIb in the presence of, for example, triphenylphosphine and diethyl azodicarboxylate or diphenylpyridylphosphine and tert-butyl azodicarboxylate (see Tetrahedron Lett., 40: 4497-4500 (1999)).

Alkylation is usually carried out in a solvent which is inert under the reaction conditions, preferably in an acyclic or cyclic ether, at temperatures from about -20 to about 100 ° C, preferably at temperatures from about 0 to about 30 ° C (or at room temperature). As for other alkylation methods, primary and secondary alcohols are most suitable for the reaction under these conditions.

After the introduction of R ³ in order to obtain the target compounds of formula I, the oxidation and substitution steps can be carried out (for the introduction of R ¹ —NH—), as outlined above.

In alternative embodiments, compound IIb can be converted to compound IId first by alkylation of compound IIb under Mitsonobu reaction conditions to introduce R ³ followed by oxidation of the sulfide to the corresponding sulfone IId.

However, in other embodiments, these compounds can be obtained by reversing the order of the alkylation and substitution steps, thereby reversing the order of introduction of —R ³ and —NH — R ¹ shown in Scheme 3.

Scheme 3

Therefore, the compound of formula IIa can be cyclized to give compound IIb (as originally shown in Scheme 2). The oxidation of compound IIb to compound IIIa provides a template for the subsequent substitution and alkylation steps. Thus, treating compound IIIa using R ¹ -NH ₂ under the conditions described above makes it possible to obtain compound IIIb which can be alkylated using compound R ³ -L (in which L has the meanings mentioned above) or R ³ -OH under the reaction conditions of Mitsunobu, obtaining the target compounds of formula I.

One skilled in the art will appreciate that it is possible to make some modifications to the above schemes that are included within the scope of the present invention. For example, some steps include protecting and releasing reactive functional groups that are incompatible with these specific reaction conditions.

In another aspect, the present invention provides compositions comprising a pharmaceutically acceptable excipient and a compound of formula I, as described above.

The compounds of formula I and pharmaceutically acceptable salts of the basic compounds of formula I with acids can be used as pharmaceuticals, for example, in the form of pharmaceutical preparations. Pharmaceutical preparations can be administered enteral, for example, orally in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions, intranasally, for example in the form of an intranasal spray, or rectally, for example in the form of suppositories. However, they can also be administered parenterally, for example, in the form of injection solutions.

In the preparation of pharmaceutical preparations, the compounds of formula I and their pharmaceutically acceptable salts can be combined with pharmaceutically inert organic or inorganic carriers. As such carriers in the manufacture of tablets, coated tablets, dragees and hard gelatine capsules, for example, lactose, corn starch and its derivatives, talc, stearic acid and its salts and the like can be used. Acceptable carriers for the manufacture of soft gelatin capsules are, for example, vegetable oils, waxes, hard fats, semi-solid and liquid polyols and the like, however, in the case of soft gelatin capsules, depending on the nature of the active substance, usually no carriers are required. In the preparation of solutions and syrups, acceptable carriers are, for example, water, polyols, sucrose, invert sugar, glucose and the like. Suitable carriers for the manufacture of suppositories are, for example, natural or hardened oils, waxes, solid fats, semi-liquid and liquid polyols, and the like.

In addition, pharmaceutical preparations may include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for varying the osmotic pressure, buffers, masking agents and antioxidants. They may also include other therapeutically valuable substances and their aforementioned pharmaceutically acceptable salts.

Medicines that include a compound of formula I or a pharmaceutically acceptable salt of a basic compound of formula I and an acid in combination with a compatible pharmaceutically acceptable carrier material are also an object of the present invention. However, another object of the present invention are methods of preparing drugs that can be used to treat diseases and conditions mediated by p38. Such a method involves giving one or more of these compounds or salts and, if necessary, one or more other therapeutically valuable substances of the dosage form of a galenic preparation together with a compatible pharmaceutically acceptable carrier.

As mentioned above, the compounds of formula I and their aforementioned pharmaceutically acceptable salts can be used in accordance with the invention as therapeutically active substances, in particular as anti-inflammatory agents or for the prevention of implant rejection after transplant surgery. The dose can be varied and wide intervals, and in each case, it, which, obviously, must meet individual needs. Typically, the daily dose for an adult in the case of oral administration should be from about 0.1 to about 100 mg / kg, preferably from about 0.5 to about 5 mg / kg. The daily dose can be administered as a single dose or in divided doses. In addition, when indications exist for this, the above upper limit may be exceeded.

Finally, an object of the invention is the use of compounds of formula I and their aforementioned pharmaceutically acceptable salts for the preparation of drugs, primarily for the treatment and prevention of inflammation, immunological, oncological, bronchopulmonary, dermatological and cardiovascular diseases, for the treatment of asthma, disorders of the central nervous system or diabetic complications, or to prevent transplant rejection after transplant surgery.

The compounds of formula I can be used, although not limited to, for the treatment in humans or other mammals of any disorders or disease states that in such mammals are exacerbated or caused by excessive or unregulated TNF and / or IL-1 or p38 kinase production. Therefore, the present invention provides a method for treating cytokine-mediated diseases, which comprises administering to the body an effective amount of a compound of formula I or a pharmaceutically acceptable salt, or a tautomer thereof, inhibiting cytokine activity.

The compounds of formula I can be used, although not limited to, for treating inflammation in a subject or as an antipyretic in the treatment of fever. The compounds of the present invention can be used to treat arthritis, including, although not limited to, rheumatoid arthritis, spondyloarthropathy, gouty arthritis, osteoarthritis, systemic lupus erythematosus and juvenile arthritis, osteoarthritis, gouty arthritis and other arthritis conditions. Such compounds can be used to treat pulmonary diseases or pneumonia, including adult respiratory distress syndrome, pulmonary sarcoidosis, asthma, silicosis, and chronic pneumonia. These compounds can also be used to treat viral and bacterial infections, including sepsis, septic shock, gram-negative sepsis, malaria, meningitis, secondary cachexia as a result of infection or malignant disease, secondary cachexia to acquired immunodeficiency syndrome (AIDS), AIDS, pre-AIDS prior to Acquired Immunodeficiency Syndrome, ARC (AIDS-related complex), pneumonia and herpes virus. The compounds can also be used to treat diseases associated with resorption bone tissue such as osteoporosis, as well as endotoxic shock, toxic shock syndrome, damage from repeated transfusions, autoimmune diseases, including graft versus host transplant rejection and allograft rejection, cardiovascular diseases including atherosclerosis, thrombosis, congestive heart failure and cardiac damage from repeated transfusions, renal damage from repeated transfusions, liver disease and nephritis, and myalgia as a result of infection.

The compounds can also be used to treat influenza, multiple sclerosis, cancer, diabetes, systemic lupus erythematosus (SLE), conditions associated with skin diseases such as psoriasis, eczema, burns, dermatitis, keloid formations and scar tissue formations. The compounds of the invention can also be used to treat gastrointestinal diseases such as inflammation of the intestine, Crohn's disease, gastritis, irritable bowel syndrome, and ulcerative colitis. These compounds can be used, apparently, in the treatment of ophthalmic diseases such as retinitis, retinopathy, uveitis, photophobia of the eyes and acute damage to the eye tissue. The compounds of the invention can also be used to treat angiogenesis, including neoplasia; metastasis; ophthalmic diseases such as corneal implant rejection, ocular revascularization, renal revascularization, including trauma or infection revascularization, diabetic retinopathy, retrolental fibroplasia and revascular glaucoma; peptic ulcers, such as a stomach ulcer; pathological but not malignant diseases such as hemagiomas, including invantile hemagiomas, nasopharyngeal angiofibroma and avascular necrosis of the bone, diabetic nephropathy and cardiomyopathy; and diseases of the female reproductive system, such as endometriosis. The compounds of the invention can also be used to prevent the production of cyclooxygenase-2, and it is believed that the compounds of the present invention can also be used to prevent and treat cancer, especially colon cancer. It is contemplated that the compounds of the present invention may be effective in the prevention and treatment of Alzheimer's disease.

In addition to the use of these compounds in the treatment of humans, they are also effective in veterinary medicine in the treatment of domestic animals, exotic animals, farm animals, including mammals, rodents, and the like. More preferably for the treatment of animals such as horses, dogs and cats.

The proposed compounds can also be used in joint therapy (partially or completely instead of other traditional anti-inflammatory drugs), in particular together with steroids, cyclooxygenase-2 inhibitors, NSAIDs (non-steroidal anti-inflammatory drugs), DMARDS, immunosuppressive drugs, 5-lipoxygenase inhibitors, leukotriene antagonists B ₄ and leukotriene A ₄ hydrolase inhibitors.

The term “TNF mediated disorder” as used in this work refers to any or all disorders or disease states in which TNF is involved, either TNF itself controls the process or other monokines are released via TNF, such as, for example, IL -1, IL-6 or IL-8, although their list is not limited to them. Therefore, a disease state in which, for example, IL-1 is a major component and whose production or activity is enhanced and secreted in response to TNF, should be considered as a disorder mediated by TNF.

The term “p38 mediated disorder” as used herein refers to any or all of the disorders or disease states in which p38 is involved when either p38 itself controls the process or other factors are released using p38, such as, for example, IL -1, IL-6 and IL-8, although their list is not limited to them. Therefore, a disease state in which, for example, IL-1 is the main component and the production of which or activity is dramatically enhanced and secreted in response to p38, should be considered as a disorder mediated by p38.

Since TNF-β is a close structural homologue of TNF-α (also known as cachectin) and since each of them elicits similar biological responses and binds to the same cell receptors, the compounds of the present invention inhibit the synthesis of both TNF-α and TNF-β, which, therefore, in all cases, unless otherwise indicated, in the present description are referred to collectively as "TNF".

EXAMPLES

In all cases, unless otherwise indicated, in the examples below, the temperature is given in degrees Celsius (° C); the processes were carried out at room temperature or ambient temperature (usually in the range of about 18 to 25 ° C); the evaporation of the solvent was carried out using a rotary evaporator under reduced pressure (usually from 4.5 to 30 mm Hg) at a bath temperature up to 60 ° C; the progress of reactions was usually monitored by TLC, and the duration of the reaction is indicated for illustrative purposes only; melting points were not corrected; products showed satisfactory ¹ H-NMR and / or microanalysis data; product yields are for illustrative purposes only; The following generally accepted abbreviations are also used: t _pl (melting point): l (liter, liters), ml (milliliters), mmol (millimole), g (grams), mg (milligrams), min (minutes) and h (hours).

DEAD stands for diethyl azodicarboxylate.

DIAD stands for diisopropylazodicarboxylate.

Example 1

This example illustrates the preparation of 3- (2-chlorophenyl) -1-ethoxycarbonylmethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one and 3- (2-chlorophenyl) -1 -carboxymethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, using 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrimido as the starting compound [4,5-d] pyrimidin-2 (1H) -one.

1.1 Alkylation of pyrimidinone

1.5 g of sulfide 1a, 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, (3.92 mmol, obtained analogously to example 6 ), dissolved in 15 ml of dimethylformamide and sodium hydride (60%, 0.172 g, 4.31 mmol) was added. The resulting mixture was stirred for twenty minutes, then 0.87 ml of ethyl bromoacetate (7.84 mmol) was added. After 3 hours, the reaction was quenched with water and the mixture was extracted three times with ethyl acetate. The combined extracts were washed five times with water, dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel column chromatography using acetone / hexane in a 30:70 ratio to obtain 1.283 g of ester 1b.

1-2 Oxidation of benzyl sulfide

0.600 g of ester 1b (1.28 mmol) was dissolved in 15 ml of chloroform and 0.883 g of 3-chloroperoxybenzoic acid (50%, 2.56 mmol) was added at room temperature. The mixture was stirred for 2 hours, then washed three times with aqueous sodium sulfite at a concentration of 10 wt.%, Once with saturated aqueous sodium bicarbonate and once with water. The organic layer was dried over MgSO ₄ and concentrated in vacuo to give 0.670 g of benzyl sulfone 1c.

1.3 Sulfone Substitution

0.520 g of benzylsulfone 1c (1.04 mmol) and 0.18 ml of isopropylamine (2.08 mmol) were combined and kept at 90-100 ° C for one hour. The reaction mixture was cooled to room temperature and the mixture was purified by silica gel column chromatography using CH ₂ Cl ₂ / MeOH in an 18: 1 ratio. The product-containing fractions were combined and concentratedron to give 0.366 g of compound 1d, 3- (2-chlorophenyl) -1-ethoxycarbonylmethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidium-2 (1H) - she (mass spectrogram: MH ⁺ = 404. t _pl .: 188.8-191.4 ° C).

1.4 Saponification of ethyl ether

To a solution of 0.266 g of compound 1d (0.66 mmol) in 10 ml of MeOH was added 0.026 g of sodium hydroxide (0.66 mmol) and 3 ml of water. The reaction mixture was stirred at room temperature for 12 hours. The mixture was concentrated in vacuo and triturated with ethyl acetate, then redissolved in MeOH and concentrated in vacuo. The residue was triturated with diethyl ether, filtered and dried to give 0.225 g of compound 1e, 3- (2-chlorophenyl) -1-carboxymethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 ( 1H) -one (mass spectrogram: MN = 376, _mp : 172.0-188.0 ° C).

Example 2

This example illustrates a process for the preparation of 3- (2-chlorophenyl) -1- (2-methoxyethyl) -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, in which alkylation is carried out using the Mitsunobu reaction conditions.

To a solution of 0.500 g of pyrimidinone 2a (1.57 mmol obtained by treating 7-benzylsulfonyl-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one with isopropylamine under conditions as in section 1.3 above), 0.413 g of triphenylphosphine (1.57 mol) and 0.12 ml of 2-methoxyethanol (1.57 mmol) were added to 20 ml of THF. The mixture was cooled to 0 ° C. and 0.31 ml of DIAD (1.57 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred for 12 hours. 0.12 ml of 2-methoxyethanol (1.57 mmol), 0.413 g of triphenylphosphine (1.57 mmol) and 0.31 ml of DIAD (1) were added as additional portions. , 57 mmol) and the mixture was stirred for another 12 h at room temperature, then kept at 55-60 ° C. After 2 hours, the mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography using hexane / ethyl acetate in a 1: 1 ratio as eluent. The product fractions were combined and concentrated to a residue as an oil, which was redissolved in ethyl acetate. Hydrochloric acid (1.0 M / Et ₂ O, 0.6 ml) was added to give a salt. The mixture was stirred for 12 hours, then concentrated in vacuo. The residue was triturated in diethyl ether over dry ice and allowed to slowly warm to room temperature while the mixture was stirred for 12 hours. The solid was filtered to give 0.139 g of the hydrochloride salt of the title compound, 2b (mass spectrogram: MH ⁺ = 376, t _pl .: 126.0-131.6 ° C).

Example 3

This example illustrates the preparation of 3- (2-chlorophenyl) -7-isopropylamino-1- (2-methylsulfonylethyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one.

3.1 Mapping of Pyrimidinone by Mitsunobu

500 mg of benzyl sulfide 1a (1.31 mmol) was dissolved in 2 ml of THF with 114 μl of 2- (methylthio) ethanol (1.31 mmol), 343 mg of triphenylphosphine (1.31 mmol) and 0.21 ml of DEAD (1, 31 mmol). The mixture was stirred at RT for 4 days, evaporated in vacuo and purified by silica gel chromatography using 5-20% acetone / hexanes as eluent to give 529 mg of compound Za (1.16 mmol, 88%).

3.2 Oxidation of benzyl sulfide

Oxidized 3a was carried out using 1.19 g of m-CPBA (meta-chloroperbenzoic acid) (5.8 mmol) in CH ₂ Cl ₂ with stirring at RT for 5 h. The reaction was quenched with 10% Na ₂ SO ₃ (aq. , 50 ml) and was extracted three times with CH ₂ Cl ₂ . The combined extracts were washed with saturated NaHCO ₃ and concentrated in vacuo to give compound 3b, which was used without purification.

3.3 Sulfone Substitution

Crude product 3b was dissolved in 5 ml of isopropylamine (58 mmol) and stirred at almost RT for 2 days. The mixture was concentrated in vacuo and purified by silica gel chromatography using 10-50% acetone / hexanes as the eluent, to give 287 mg of compound 3c (0.732 mmol, yield: 63% in terms of compound 3a, 56% in terms of compound 1a). The purified product was dissolved in ethyl acetate and processed using 1 equiv. HCl / Et ₂ O precipitating the HCl salt of compound 3c (mass spectrogram: MH = 424).

Example 4

This example illustrates the preparation of 3- (2-chlorophenyl) -1- (2-hydroxyethyl) -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one.

4.1 Alkylation of pyrimidone according to Mitsunobu

500 mg of benzyl sulfide 1a (1.31 mmol) was dissolved in 7 ml of DMF and treated with 555 mg of triisopropylsilyl protected iodoethanol (1.57 mmol obtained in accordance with the methods described in J.Am. Chem. Soc., 112 ( 10), 4078-9 (1990) and J. Chem. Soc. Perkin Trans., 1 (6), 1417-23, (1998)), and cooled to 0 ° C. Sodium hydride (60%, in oil, 63 mg, 1.57 mmol) was added and the reaction mixture was heated from 0 to 30 ° C, then stirred overnight at 30 ° C. An additional 63 mg of 60% sodium hydride was added. After another 6 hours, the reaction was quenched by the addition of 10 ml of water. The resulting mixture was extracted with EtOAc and CH ₂ Cl ₂ and the combined extracts were dried over MgSO ₄ , concentrated in vacuo and purified by silica gel chromatography using 10-20% acetone / hexanes as eluent, to give 430 mg 4a (0.738 mmol, 56%).

4.2 Oxidation of benzyl sulfide

The oxidation of compound 4a was carried out with 500 mg m-CPBA (2.46 mmol) in CH ₂ Cl ₂ . After quenching the reaction, Na ₂ SO ₃ (aq), crude product 4b was extracted with CH ₂ Cl ₂ , washed with saturated NaHCO ₃ , concentrated in vacuo and used without purification.

4.3 Sulfone Substitution

Crude benzylsulfone 4b was dissolved in 3 ml of CH ₂ Cl ₂ containing 1 ml of isopropylamine (11.7 mmol), stirred at 80 ° C overnight, concentrated in vacuo and purified by silica gel chromatography using 5-20% acetone / hexanes in as eluent, obtaining 281 mg of compound 4c (0.542 mmol, yield: 73%, calculated as compound 4a).

4.4 Release of the hydroxyl group

Compound 4c was released by 0.54 ml of tetrabutylammonium fluoride (1 M / THF, 0.542 mmol) in THF with stirring at RT for 4 hours. The mixture was evaporated in vacuo and purified by chromatography on silica gel using 10-40% acetone / hexanes in as eluent, obtaining 179 mg of compound 4d (0.495 mmol). The purified product was dissolved in ethyl acetate and processed using 1 equiv. HCl / Et ₂ O, precipitating the HCl salt of compound 4d (mass spectrogram: MH ⁺ = 362).

Example 5

4-amino-2-benzylthiopyrimidine-5-carboxaldehyde

a) 272 g (4.0 mol) of sodium ethoxide (Lancaster company) was mixed in 1 l of ethanol and treated with 304 g (4.0 mol) of thiourea (Avocado company). 676 g (4.0 mol) of ethyl ethoxymethylene cyanoacetate (Avocado) were added and the mixture was refluxed for 8 hours. After cooling to room temperature and holding overnight, the reaction mixture was sequentially treated with 2 L of water and 400 ml of acetic acid. The reaction mixture was refluxed for 30 minutes, cooled to room temperature and the suspension was filtered. The solid was washed with three 500 ml portions of water, two 500 ml portions of acetone and 500 ml of diethyl ether. The product was dried to give 473.3 g (yield: 60%) of 4-amino-5-carbethoxypyrimidin-2-thiol as a cream solid with a melting point> 250 ° C.

b) A mixed suspension of 473 g (2,377 mol) of 4-amino-5-carbethoxypyrimidin-2-thiol in 3.5 L of ethanol was treated with 180.4 g (1.307 mol) of potassium carbonate and 447.1 g (2.615 mol) of benzyl bromide. The mixture was refluxed for 2 hours, then allowed to cool to room temperature and allowed to stand overnight. The suspension was filtered and the solid was washed with two 500 ml portions of ethanol, 2 L of water and two 500 ml portions of water. The product was dried under vacuum over phosphorus pentoxide at 50 ° C to obtain 416 g (yield: 61%) of ethyl 4-amino-2-benzylthiopyrimidine-5-carboxylate as a cream solid with a melting point of 117-118 ° C.

c) A solution of 462.4 g (1.6 mol) of ethyl 4-amino-2-benzylthiopyrimidine-5-carboxylate in 2.3 L of tetrahydrofuran dried over a molecular sieve in a nitrogen atmosphere with ice cooling was slowly introduced into 1.6 with stirring l (1.6 mol) of a 1 M solution of lithium aluminum hydride in tetrahydrofuran. The solution was added at such a rate as to maintain a temperature of 18 to 20 ° C. Upon completion of the addition, the mixture was heated to 60 ° C and carefully treated for 1.5 hours with 60.8 ml of water. 60.8 ml of 15% aqueous sodium hydroxide was added over 30 minutes, and then 182.5 ml of water was added over 30 minutes. The suspension was stirred at 60 ° C. overnight, then, while still hot, was filtered through a HyfLo filter aid and the solid was washed with two 1 liter portions of tetrahydrofuran. Evaporation of the filtrate to dryness gave 392.5 g (yield: 99%) of 4-amino-2-benzylthiopyrimidine-5-methanol as an off-white solid, which was used in the next step without further purification.

d) A suspension of 392.5 g (1.59 mol) of 4-amino-2-benzylthiopyrimidine-5-methanol in 7.75 L of dichloromethane was treated with 1.382 kg (15.9 mol) of activated manganese dioxide (Acros) in a nitrogen atmosphere. The reaction mixture was stirred at room temperature overnight, then filtered through a HyfLo filter aid. The solid was washed with three 1-liter portions of dichloromethane and the combined filtrates were evaporated to give 340.5 g (yield: 88%) of 4-amino-2-benzylthiopyrimidine-5-carboxaldehyde as a pale yellow solid with a melting point of 136-139 ° FROM.

Example 6

7-benzylthio-3- (2-chlorophenyl) -3.4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one

6.1. Preparation of 5- (2-chlorophenyl) aminomethyl-4-amino-2-benzylthiopyrimidine

A mixture of 5 g (20.4 mmol) of 4-amino-2-benzylthiopyrimidine-5-carboxaldehyde, 2.25 ml (21.4 mmol) of 2-chloroaniline and 0.1 g (0.5 mmol) of 4-toluenesulfonic acid monohydrate 60 ml of toluene was boiled with azeotropic removal of water for 3 hours. The mixture was cooled to 0 ° C and the precipitate was collected by filtration in vacuo, washed with hexanes and dried in air. Then this solid was dissolved in 100 ml of THF and the reaction mixture was cooled to 0 ° C. 0.735 g of lithium aluminum hydride (18.8 mmol) was added in small portions over 45 minutes. After the addition was complete, the mixture was stirred for another 15 minutes and carefully cautiously treated with 0.8 ml of H ₂ O, 0.8 ml of 15% aqueous NaOH and then 2.4 ml of H ₂ O. The mixture was stirred for 30 minutes, was filtered through celite and the filtrate was concentrated in vacuo. The solid was stirred with diethyl ether, filtered and dried in air to give 6.1 g of 5- (2-chlorophenyl) aminomethyl-4-amino-2-benzylthiopyrimidine as a white solid.

6.2 Preparation of 3- (2-chlorophenyl) -7-benzylthio-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one

To a stirred solution cooled to -10 ° C, 4.3 g (12.1 mmol) of 5- (2-chlorophenyl) aminomethyl-4-amino-2-benzylthiopyrimidine in 100 ml of tetrahydrofuran was added 3.1 ml (22.2 mmol) of triethylamine. Then this solution was treated by adding dropwise a solution of 6.15 ml of phosgene (20% solution in toluene; 11.8 mmol). After stirring for 30 minutes, an additional 1.0 ml of triethylamine (7.1 mmol) was added, followed by 2.0 ml of phosgene (20% solution in toluene, 3.8 mmol). The reaction mixture was warmed to room temperature, treated with 0.5 ml of H ₂ O and stirred for 30 minutes. Next, the reaction mixture was filtered and the mother liquor was concentrated and stirred with dichloromethane. The product was then collected by suction filtration and dried in vacuo to give 3.83 g of 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) as a white solid )-she.

Example 7

7-benzylsulfonyl-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one

A suspension of 1 g (2.61 mmol) of 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrmpido [4,5-d] pyrimidin-2 (1H) -one in 10 ml of dichloromethane was cooled on ice and treated 1.29 g (5.23 mmol) of 70% 3-chloroperbenzoic acid. The mixture was stirred at room temperature for 2 hours, then treated with 25 ml of 10% aqueous Na ₂ S ₂ O ₃ and allowed to mix for 30 minutes. The reaction mixture was diluted with 100 ml of dichloromethane and the phases were separated. The organic phase was washed with 10% aqueous K ₂ CO ₃ , brine, and then dried over magnesium sulfate and filtered. By concentration of the filtrate under reduced pressure, 0.73 g of benzylsulfonyl-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one was obtained as a white solid.

Example 8

3- (2-chlorophenyl) -1 - [(2S) -2,3-dihydroxyethyl] -7-isopropylamino-3.4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one

Stage 1

A mixture of 1.0 g (2.6 mmol) of 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 1.5 g (5 , 2 mmol) of L-α, β-isopropylindenglycerol-γ-tosylate and 1.44 g (10.4 mmol) of potassium carbonate were mixed in 20 ml of DMF and kept at 80 ° in a nitrogen atmosphere. After 16 hours, the reaction mixture was cooled to room temperature, poured into a solution of sodium chloride, extracted with ethyl acetate and dried over sodium sulfate. The solution was concentrated in vacuo and purified by flash chromatography (eluting with 40% EtOAc / Hexane) to give 1.1 g of isopropylinene ketal adduct as an oil (mass spectrogram: MH ⁺ = 496).

Stage 2

To a solution of 1.2 g (2.4 mmol) of the isopropylinene ketal adduct obtained as described above in 25 ml of methylene chloride, cooled in a wet ice bath under a nitrogen atmosphere, 1.8 g (9.6 mmol) of m-chloroperbenzoic acid were added in individual portions. . The resulting suspension was stirred and allowed to warm to room temperature. After 16 hours, the reaction mixture was cooled in a wet ice bath and 50 ml of a 10% aqueous solution of sodium bisulfite was added dropwise. The mixture was stirred for 30 minutes and the layers were separated. The aqueous layer was extracted with methylene chloride and the combined organic fractions were washed with brine, dried over sodium sulfate and concentrated to dryness. The residue was purified by flash chromatography (gradient elution: 60-100% ethyl acetate / hexane). 0.73 g of sulfonic product was isolated as a foam (mass spectrogram: MH ⁺ = 529).

Stage 3

A solution of 0.7 g (1.3 mmol) of sulfone, prepared as described above, in 10 ml of isopropylamine was kept at 40 ° in a nitrogen atmosphere. After 3 hours, the reaction mixture was cooled to room temperature, concentrated to dryness and purified by flash chromatography (eluting with 60% ethyl acetate / hexane) to give 0.42 g of isopropylamine adduct as a foam.

Stage 4

To a solution of 0.4 g (0.93 mmol) of the isopropylamine adduct obtained as described above, 0.05 g of p-toluenesulfonic acid was added in 15 ml of methanol and 7 ml of water, and the mixture was kept at 50 °. After 16 hours, methanol was removed under reduced pressure and the resulting aqueous solution was extracted with ethyl acetate. The organic fractions were washed with 5% aqueous sodium bicarbonate solution and brine, then dried over sodium sulfate, concentrated and purified by flash chromatography (gradient elution: 40-100% ethyl acetate / hexane) to give 0.2 g of product, 3- (2 -chlorophenyl) -1 - [(2S) -2,3-dihydroxyethyl] -7-isopronylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one (mass spectrogram: M ⁺ = 391, _mp : 130.8-134.7).

Example 9

3- (2-chlorophenyl) -1 - [(2R) -2,3-dihydroxyethyl] -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one

Stage 1

Mixture of 1.0 g (2.6 mmol) of 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrmpido [4,5-d] pyrimidin-2 (1H) -one, 1.5 g (5 , 2 mmol) of D-α, β-isopropylindenglycerol-γ-tosylate and 1.44 g (10.4 mmol) of potassium carbonate were mixed in 25 ml of DMF and kept at 80 ° in a nitrogen atmosphere. After 16 hours, the reaction mixture was cooled to room temperature, poured into a sodium chloride solution, extracted with ethyl acetate, and dried over sodium sulfate. The solution was concentrated in vacuo and purified by flash chromatography (eluting with 40% EtOAc / Hexane) to give 1.2 g of isopropylinene ketal adduct as an oil.

Stage 2

To a solution of 1.2 g (2.4 mmol) of the isopropylinene ketal adduct obtained as above, in 30 ml of methylene chloride, cooled in a wet ice bath, 1.1 g (5.6 mmol) of m-chloroperbenzoic acid were added in separate portions in a nitrogen atmosphere. . The resulting suspension was stirred and allowed to warm to room temperature. After 6 hours, the reaction mixture was cooled in a wet ice bath and 50 ml of a 10% aqueous solution of sodium bisulfite was added dropwise. The mixture was stirred for 30 minutes and the layers were separated. The aqueous layer was extracted with methylene chloride and the combined organic fractions were washed with brine, dried over sodium sulfate and concentrated to dryness. The residue was purified by flash chromatography (gradient elution: ethyl acetate-10% methanol / ethyl acetate). 1.1 g of sulfone product was isolated as a foam (mass spectrogram: MH ⁺ = 529).

Stage 3

A solution of 0.22 g (0.42 mmol) of sulfone, prepared as described above, in 5 ml of isopropylamine was kept at 40 ° in a nitrogen atmosphere. After 3 hours, the reaction mixture was cooled to room temperature, concentrated to dryness and purified by flash chromatography (elution with ethyl acetate) to obtain 0.088 g of product, 3- (2-chlorophenyl) -1 - [(2R) -2.3-, as a foam. dihydroxyethyl] -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one (mass spectrogram: MH ⁺ = 392).

Example 10

7-isopropylamino-3- (2-chlorophenyl) -1- (2-piperidinylethyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one

500 mg of 7-benzylthio-3- (2-chlorophenyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one (1.31 mmol) was dissolved in 2 ml of THF with 0.173 ml of 1- piperidinethanol (1.31 mmol), 343 mg triphenylphosphine (1.31 mmol) and 0.21 ml of DEAD (1.31 mmol) and stirred at RT for 4 days, then purified by silica gel chromatography using 2-5% methanol / dichloromethane as an eluent, obtaining 350 mg of N- (2-piperidinylethyl) adduct.

Sulfide oxidation was carried out using 0.02 g m-CPBA (0.708 mmol) in dichloromethane with stirring at RT for 1 h. The reaction was quenched with 0.5 ml of 25% aqueous sodium sulfite and the mixture was extracted with dichloromethane. The extract was dried with magnesium sulfate and concentrated in vacuo to give sulfoxide, which was used without purification.

The crude sulfoxide was dissolved in 5 ml of isopropylamine and stirred at 40 ° C. overnight, then at 80 ° C. for 4 days. The mixture was purified by silica gel chromatography using 1-10% methanol / dichloromethane as the eluent to give 48 mg of amine (0.112 mmol). The purified product was dissolved in ethyl acetate and treated with 1 equiv. HCl / Et ₂ O, precipitating the HCl salt of 7-isopropylamino-3- (2-chlorophenyl) -1- (2-piperidinylethyl) -3,4-dihydropyrmpido [4,5-d] pyrimidin-2 (1H) -one.

Example 11

In vitro p38 MAP kinase inhibition test

This example illustrates an in vitro method for determining the p38 MAP kinase activity used to evaluate the compounds of the present invention.

In order to evaluate the inhibitory activity of the compounds of the present invention with respect to p38 kinase MAP, the in vitro transfer of γ-phosphate from γ- ³³ P-ATP to the myelin basic protein (MBP) catalyzed by p38 kinase was determined using the method described by Ahn with some modifications et al., J. Biol. Chem. 266: 4220-4227 (1991).

The phosphorylated form of recombinant p38 MAP kinase was coexpressed with SEK-1 and MEKK in E. Coli (see Khokhlatchev et al., J. Biol. Chem. 272: 11057-11062 (1997)) and then purified by affinity chromatography using nickel columns .

The phosphorylated MAP kinase p38 was diluted with kinase buffer [20 mM 3- (N-morpholino) propanesulfonic acid, pH 7.2, 25 mM β-glycerol phosphate, 5 mM ethylene glycol bis-beta-aminoethyl ether N, N, N ', N' -tetraacetic acid, 1 mM sodium orthovanadate, 1 mM dithiothreit, 40 mM magnesium chloride]. The test compound dissolved in DMSO or only DMSO (reference) was added and the samples were incubated for 10 min at 30 ° C.

The kinase reaction was initiated by the addition of a substrate cocktail containing MBP and γ- ³³ P-ATP. After incubation for another 20 min at 30 ° C, the reaction was stopped by the addition of 0.75% phosphoric acid. Then, the phosphorylated MBP was separated from the residual γ- ³³ P-ATP using a phosphocellulose membrane (Millipore, Bedford, Minnesota) and quantified using a scintillation counter (Packard, Meridian, Connecticut).

The inhibitory activity of the compounds of the present invention to p38 (expressed as IC ₅₀ , which is the concentration of the compound causing 50% inhibition of the p38 enzyme used in the test) presented in Examples 1-4 was less than 10 μM.

Example 12

In vitro TNF Inhibition Test

This example illustrates an in vitro method for assessing the inhibition of the production of TNF-α induced by LPS in THP1 cells.

The ability of the compounds of the present invention to inhibit the release of TNF-α was determined using, with minor modification, the method described by Blifeld et al. Transplantation, 51: 498-503 (1991).

a) Induction of TNF biosynthesis

THP1 cells were suspended in culture medium [RPMI (Gibco-BRL, Gailthersburg, MD), contained 15% fetal bovine serum, 0.02 mm 2-mercaptoethanol] at a concentration of 2.5 × 10 ⁶ cells / ml and then placed in 96-well plate (0.2 ml aliquots in each well). The test compound was dissolved in DMSO and then diluted with culture medium so that the final concentration of DMSO reached 5%. Aliquots of twenty-five microliters of the test solution or only DMSO medium (standard) were added to each well. Cells were incubated at 37 ° C for 37 minutes. LPS (Sigma, St. Louis, Missouri) was added to the wells to a final concentration of 0.5 μg / ml and the cells were incubated for another 2 hours. At the end of the incubation period, the culture supernatant was collected and the amount of TNF-α present was determined using the procedure described below is the ELISA method.

b) ELISA method

The amount of human TNF-α present was determined by specific binding according to the ELISA method using two anti-TNF-α antibodies (2TNF-H12 and 2TNF-H34) described by Reimund, J.M. et al., GUT, Volume 39 (5), 684-689 (1996).

50 μl of 2TNF-H12 antibody in RBF (solution containing phosphate buffer) (10 μg / ml) was sorbed into the wells of a polystyrene 96-well plate and incubated at 4 ° C overnight in a humidified chamber. The tablet was washed with RBF and then blocked with 5% skimmed milk powder in RBF for 1 h at room temperature and washed with 0.1% BSA (bovine serum albumin) in RBF.

TNF standards were prepared from a stock solution of recombinant human TNF-α (R&D Systems, Minneapolis, Minnesota). The initial concentration of standards in the tests was 10 ng / ml, and then 6 semi-logarithmic serial dilutions followed.

Aliquots of 25 μl of the above supernatant, or TNF standards, or medium alone (standard) were mixed with aliquots of 25 μl of 2TNH-H34 biotinylated monoclonal antibody (2 μg / ml in RBF containing 0.1% BSA) and then added to each well. Samples were incubated with gentle shaking at room temperature for 2 h and then washed 3 times with 0.1% BSA in RBF. 50 μl of a streptavidin peroxidase solution (Zymed, S., San Francisco, Calif.) Containing 0.416 μg / ml streptovidin peroxidase and 0.1% BSA in RBF was injected into each well. Samples were additionally incubated for 1 h at room temperature and then washed 4 times with 0.1% BSA in RBF. Fifty microliters of a solution of o-phenylenediamine (1 μg / ml o-phenylenediamine and 0.03% hydrogen peroxide in 0.2 M citrate buffer, pH 4.5) were injected into each and the samples were incubated for 30 min at room temperature in the dark . The optical densities of the sample and the comparative cell were determined at 450 nm and 650 nm, respectively. TNF-α levels were determined from the curve of optical density at 450 nm versus the concentration used.

The IC ₅₀ value was determined as the concentration of the test compound, corresponding to a half reduction in maximum adsorption at 450 nm.

Example 13

This example illustrates an in vivo method used to evaluate the inhibition of LPS induced TNF-α production in mice (or rats).

The ability of the compounds of the present invention to inhibit the in vivo release of TNF-α was determined using, with minor modification, the method described by Zanetti et al., J. Immunol., 148: 1890 (1992) and Sekut et al., J. Lab.Clin Med. 124: 813 (1994).

Female BALB / c mice weighing 18-21 g (Charles River, Hollister, Calif.) Acclimatized for one week. Animals of groups of 8 mice were orally administered either a test compound in suspension or dissolved in an aqueous vehicle containing 0.9% sodium chloride, 0.5% sodium carboxymethyl cellulose, 0.4% polysorb 80, 0 , 9% benzyl alcohol (carrier for CMC), or only the carrier (control group). After 30 minutes, 20 μg LPS (Sigma, St. Louis, Missouri) was injected intraperitoneally into mice. After 1.5 hours, the mice were sacrificed by inhalation of CO ₂ and blood was collected by puncture of the heart. Blood was clarified by centrifuging at 15600 g for 5 min, the blood serum was transferred to clean tubes, frozen at -20 ° C and stored until analysis for TNF-α was performed. ELISA method (Biosource International, Camarillo, Calif.) in accordance with the manufacturer's instructions.

It must be borne in mind that the examples and embodiments of the invention given in the present description serve only illustrative purposes, therefore, taking them into account, at the suggestions of specialists in the given field of technology, a wide variety of modifications or changes can be made that do not go beyond and the scope of this application and the scope of the attached claims.

Claims (8)

1. Alkylamino-substituted bicyclic nitrogen-containing heterocycles of the general formula I

in which the subscript n denotes the integer 1; R ¹ is C ₁ -C ₆ alkyl; R ² is halo; R ³ is C ₁ -C ₆ alkoxy-C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkylsulfonyl-C ₁ -C ₆ -alkyl, hydroxy-C ₁ -C ₆ -alkyl, dihydroxy-C ₁ - C ₆ -alkyl, N-heterocyclyl-C ₁ -C ₆ -alkyl or (C ₁ -C ₆ -alkylene) -C (O) R ³¹ , where R ³¹ is hydroxy or C ₁ -C ₆ -alkoxy; and its pharmaceutically acceptable salts. 2. The compound according to claim 1, in which - (R ² ) _n is a 2-halo. 3. The compound according to claim 1, in which R ³ denotes C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl. 4. The compound according to claim 1, in which R ³ denotes N-heterocyclyl-C ₁ -C ₆ -alkyl. 5. The compound according to claim 1, in which R ¹ denotes isopropyl. 6. The compound according to claim 1, selected from the group including 3- (2-chlorophenyl) -1-ethoxycarbonylmethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 3- (2-chlorophenyl) -1-carboxymethyl-7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 3- (2-chlorophenyl) -1- (2-methoxyethyl) -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 3- (2-chlorophenyl) -7-isopropylamino-1- (2-methylsulfonylethyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 3- (2-chlorophenyl) -1- (2-hydroxyethyl) -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, and its pharmaceutically acceptable salts. 7. The compound according to claim 1, selected from the group including 3- (2-chlorophenyl) -1 - [(2S) -2,3-dihydroxyethyl] -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 3- (2-chlorophenyl) -1 - [(2R) -2,3-dihydroxyethyl] -7-isopropylamino-3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, 7-isopropylamino-3- (2-chlorophenyl) -1- (2-piperidinylethyl) -3,4-dihydropyrimido [4,5-d] pyrimidin-2 (1H) -one, and its pharmaceutically acceptable salts. 8. A pharmaceutical composition having p38 protein kinase inhibitory activity, comprising a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, and a compatible pharmaceutically acceptable carrier.