RU2376992C2 - Methods of treatment or prevention of autoimmune diseases by means of 2,4-pyrimidindiamin compounds - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to 2,4-pyrimidindiamins, such as N4-(4-Chlorine-3-methoxyphenyl)-5-fluorine-N2-[3-(N-ethylamino)carbonylmethylenoxyphenyl]-2,4-pyrimidindiamin, N4-(3-Chlorine-4-methjopxycarbonylmethylenoxyphenyl)-5-fluorine- N2-[3-(N-methylamino)carbonylmethylenoxyphenyl]-2,4-pyrimidindiamin, N4-[3-Chlorine-4-(N-methylamino)carbonylmethylenoxyphenyl]-5-fluorine-N2-[3-(]N methylamino)carbonylmethylenoxyphenyl]- 2,4-pyrimidindiamin, N4-[3-Chlorine-4-(2-hydroxyethylenoxy)phenyl]-5-fluorine-N2-[3-(N- methylamino)carbonylmethylenoxyphenyl]- 2,4-pyrimidindiamin and other compounds given in item 1 of claimed invention as Syk-kinase inhibitors, as well as to based on them pharmaceutical composition and their application.

EFFECT: claimed compounds can be applied for treatment of autoimmune diseases, systemic http://lingvo.yandex.ru/?text=lupus%20erythematosus

, rheumatoid arthritis, etc.

12 cl, 27 dwg, 11 tbl, 1797 ex

Description

1. Cross-references to “related” applications

Pursuant to § 119 (e) part 35 of the United States Code (U.S.C.) this application is prevailing with respect to the following applications: registration number 60/399 673, filing date July 29, 2002; registration number 60/443 949, filing date January 31, 2003 and registration number 60/452 339, filing date March 6, 2003.

2. Field of invention

The present invention relates generally to compounds of 2,4-pyrimidinamines, pharmaceutical compositions containing these compounds, intermediates and synthetic methods for the manufacture of these compounds and methods of using these compounds and compositions in various fields, for example in the treatment or prevention of autoimmune diseases and / or symptoms associated with these diseases.

3. Background to the invention

Cross-linking of Fc receptors, such as, for example, a receptor with high affinity for IgE (FcεRI) and / or a receptor with high affinity for IgG (FcγRI), activates the signaling cascade in mast cells, basophils and other immune cells, resulting in the isolation chemical mediators responsible for various adverse events. For example, such cross-linking leads to the release of preformed mediators of type I anaphylactic hypersensitivity reactions (immediate reaction), such as histamine, from the accumulation zones in granules through degranulation. It also leads to the synthesis and secretion of other mediators, including leukotrienes, prostaglandins, and platelet activation factors (PAFs), which play an important role in inflammatory responses. Additional mediators that are synthesized and secreted by cross-linking Fc receptors include cytokines and nitric oxide.

The signal cascade or cascades activated by cross-linking of Fc receptors, such as, for example, FcεRI and / or FcγRI, includes a set of cellular proteins. Tyrosine kinases are one of the most important distributors of intercellular signals. An important tyrosine kinase involved in the establishment of transduction signaling pathways associated with cross-linking of the FcεRI and / or FcγRI receptors, as well as other signaling pathways, is the Syk kinase (see Valent et al. , 2002, Intl. J. Hematol. 75 (4): 257-362).

Since mediators isolated by cross-linking of the FcεRI and FcγRI receptors are responsible for the manifestation of numerous adverse events or play an important role in this, it is highly desirable to have compounds capable of inhibiting the signaling cascade or cascades responsible for their isolation. Moreover, in view of the critical role of Syk kinase in the signaling cascade (s) of these and other receptors, the presence of compounds capable of inhibiting Syk kinase is also highly desirable.

4. Summary of invention

One aspect of the present invention is the provision of new compounds of 2,4-pyrimidinamines, which, as will be described in detail below, exhibit biological activity in a number of ways. These compounds generally consist of a 2,4-pyrimidinamine "core", which has the following structure and numbering system:

These compounds according to the present invention are replaced at the point of attachment of nitrogen (N2) to the C2 atom to form a secondary amine and can be further substituted at one or more positions: at the point of attachment of nitrogen (N4) to the C4, C5 and / or C6 atom. When substituted at the N4 site, the substituent forms a secondary amine. The substituent at the N2 site, as well as possible substituents in other positions, can differ greatly in nature and physicochemical properties. For example, the substituent (s) may be branched, unbranched or cyclic alkyl, branched, unbranched or cyclic heteroalkyl, or a combination of these groups. These substitution groups may be further substituted, as described in detail below.

Substituents N2 and / or N4 may be attached directly to the corresponding nitrogen atoms or may be separated from the corresponding nitrogen atoms by linkers, which may be the same or different. The nature of the linkers can vary widely and can include virtually any combination of atoms or groups suitable for separating one part of a molecule from another. For example, the linker may be a bridge from an acyclic hydrocarbon (e.g., saturated or unsaturated alkene, such as methane, ethane, ethene, propane, 1-propene, butane, 1-butene, 2-butene , 1,3-butadiene and the like), a bridge of monocyclic or polycyclic hydrocarbon (e.g. 1,2-benzene, 2,3-naphthalene and the like), a simple acyclic heteroatom bridge or heteroalkyldiyl bridge (for example, - O -, -S-, -SO-, -NH-, -PH-, -C (O) -, -C (O) NH-, -S (O) -, -S (O) ₂ -, - S (O) NH-, -S (O) ₂ NH-, -O-CH ₂ -, -CH ₂ -O-CH ₂ -, -O-CH = CH-CH ₂ - and the like), monocyclic or polycyclic heteroaryl the second bridge (for example, 3,4-furan-, pyridine-, thiophene, piperidine-, piperazine-, pyrazidine-, pyrrolidine- and the like) or combinations of these bridges.

Substituents at positions N2, N4, C5 and / or C6, as well as potential linkers, may subsequently be substituted with one or more identical or different substitution groups. The nature of these substitution groups can vary widely. An unlimited number of examples of suitable substitution groups include branched, unbranched and cyclic alkyls, mono- and polycyclic aryls, branched, unbranched and cyclic heteroalkyls, mono- and polycyclic heteroaryls, halogenohydrocarbons, branched, unbranched and cyclic hydroxy, trihydroxy, branched, unbranched and cyclic alkoxy groups, branched, unbranched and cyclic halo-alkoxy groups, trifluoromethoxy groups, mono- and olicyclic aryloxy groups, mono- and polycyclic heteroaryloxy groups, esters, alcohols, sulfides, thioethers, sulfanyls (thiols), imines, azo groups, azides, amines (primary, secondary and tertiary), nitriles (any isomers), cyanates (any isomers), thiocyanates (any isomers), nitroso groups, nitro groups, diazes, sulfoxides, sulfonyls, sulfonic acids, sulfamides, sulfonamides, sulfamic acid esters, aldehydes, ketones, carboxylic acids, esters, amides, amidines, formadines, amino acids, acetylenes, carbamates, lactones, lactams, g yukozidy, glyukonuridy, sulfones, ketals, acetals, thioketals, oximes, oxamic acids, oxamic esters, acids, etc., and combinations of these groups. Substitutional groups having functional reactivity can be protected or unprotected, as is well known from previous work.

In one illustrative example, the compounds of 2,4-pyrimidinamines according to the present invention are compounds with the following structural formula (I):

including their salts, hydrates, solvates and N-oxides, in which the structural units L ¹ and L ² , each individually independently from each other, are selected from groups containing a direct bond and a linker;

R ^{2 is} selected from the group consisting of (C1-C6) alkyl, which may be substituted with one or more identical or different R ⁸ groups; (C3-C8) cycloalkyl which may be substituted by one or more identical or different R ⁸ groups; cyclohexyl, which may be substituted by one or more identical or different R ⁸ groups; cycloheteroalkyl with 3-8 members, which may be substituted by one or more identical or different R ⁸ groups; (C5-C15) aryl, which may be substituted by one or more identical or different R ⁸ groups; phenyl, which may be substituted by one or more identical or different R ⁸ groups and heteroaryl with 5-15 members, which may be substituted by one or more identical or different R ⁸ groups;

R ^{4 is} selected from the group consisting of hydrogen, (C1-C6) alkyl, which may be substituted with one or more identical or different R ⁸ groups; (C3-C8) cycloalkyl which may be substituted by one or more identical or different R ⁸ groups; cyclohexyl, which may be substituted by one or more identical or different R ⁸ groups; cycloheteroalkyl with 3-8 members, which may be substituted by one or more identical or different R ⁸ groups; (C5-C15) aryl, which may be substituted by one or more identical or different R ⁸ groups; phenyl, which may be substituted by one or more identical or different R ⁸ groups; and heteroaryl with 5-15 members, which may be substituted by one or more identical or different R ⁸ groups;

R ^{5 is} selected from the group consisting of R ⁶ , (C1-C6) alkyl, which may be substituted with one or more identical or different R ⁸ groups; (C1-C4) alkanyl which may be substituted by one or more identical or different R ⁸ groups; (C2-C4) alkenyl, which may be substituted by one or more identical or different groups of R ⁸ and (C2-C4) alkynyl, which may be substituted by one or more identical or different groups of R ⁸ ;

Each of the R ⁶ groups is independently selected from the group containing hydrogen, an electronegative group, -OR ^d , -SR ^d , (C1-C3) haloalkylalkyloxy group, (C1-C3) perhalo-alkyloxy group, -NR ^c R ^c , halogen, (C1-C3 ) haloalkyl, (C1-C3) perhaloalkyl, —CF ₃ , —CH ₂ CF ₃ , —CF ₂ CF ₃ , —CN, —NC, —OCN, —SCN, —NO, —NO ₂ , —N ₃ , - S (O) R ^d , -S (O) ₂ R ^d , -S (O) ₂ OR ^d , -S (O) NR ^c R ^c ; -S (O) ₂ NR ^c R ^c , -OS (O) R ^d , -OS (O) ₂ R ^d , -OS (O) ₂ OR ^d , -OS (O) NR ^c R ^c , -OS ( O) ₂ NR ^c R ^c , -C (O) R ^d , -C (O) OR ^d , -C (O) NR ^c R ^c , -C (NH) NR ^c R ^c , -OC (O) R ^d , -SC (O) R ^d , -OC (O) OR ^d , -SC (O) OR ^d , -OC (O) NR ^c R ^c , -SC (O) NR ^c R ^c , -OC (NH ) NR ^c R ^c , -SC (NH) NR ^c R ^c , - [NHC (O)] _n R ^d , - [NHC (O)] _n OR ^d , - [NHC (O)] _n NR ^c R ^c and - [NHC (NH)] _n NR ^c R ^c , (C5-C10) aryl optionally substituted with one or more identical or different R ⁸ groups, phenyl optionally substituted with one or more identical or different R ⁸ groups, (C6- C16) arylalkyl optionally substituted with one or more identical or different R ⁸ groups, 5-10 membered heteroaryl optionally substituted with one or bore nly identical or different R ⁸ groups and 6-16 membered heteroarylalkyl optionally substituted with one or more identical or different R ⁸ groups;

R ^{8 is} selected from the group consisting of R ^a , R ^b , in which the group R ^a can be substituted with one or more identical or different groups R ^a or R ^b , the group —OR ^a can be substituted with one or more identical or different groups R ^a or R ^b , -B (OR ^a ) ₂ , -B (NR ^c R ^c ) ₂ , - (CH ₂ ) _m -R ^b , - (CHR ^a ) _m -R ^b , -O- (CH ₂ ) _m -R ^b , -S- (CH ₂ ) _m -R ^b , -O-CHR ^a R ^b , -O-CR ^a (R ^b ) ₂ , -O- (CHR ^a ) _m -R ^b ,

-O- (CH ₂ ) _m -CH [(CH ₂ ) _m R ^b ] R ^b , -S- (CHR ^a ) _m -R ^b , -C (O) NH- (CH ₂ ) _m -R ^b ,

-C (O) NH- (CHR ^a ) _m -R ^b , -O- (CH ₂ ) _m -C (O) NH- (CH ₂ ) _m -R ^b ,

-S- (CH ₂ ) _m -C (O) NH- (CH ₂ ) _m -R ^b , -O- (CHR ^a ) _m -C (O) NH- (CHR ^a ) _m -R ^b ,

-S- (CHR ^a ) _m -C (O) NH- (CHR ^a ) _m -R ^b , -NH- (CH ₂ ) _m -R ^b , -NH- (CHR ^a ) _m -R ^b ,

-NH [(CH ₂ ) _m R ^b ], -N [(CH ₂ ) _m R ^b ] ₂ , -NH-C (O) -NH- (CH ₂ ) _m -R ^b ,

-NH-C (O) - (CH ₂ ) _m -CHR ^b R ^b and -NH- (CH ₂ ) _m -C (O) -NH- (CH ₂ ) _m -R ^b ;

each R ^a group is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl;

each of the R ^b groups is a suitable group selected from the group consisting of = O, -OR ^d , (C1-C3) haloalkylalkyloxy group, -OCF ₃ , = S, -SR ^d , = NR ^d , = NOR ^d , - NR ^c R ^c , halogen, —CF ₃ , —CN, —NC, —OCN, —SCN, —NO, —NO ₂ , = N ₂ , —N ₃ , —S (O) R ^d , —S (O ) ₂ R ^d , -S (O) ₂ OR ^d , -S (O) NR ^c R ^c , -S (O) ₂ NR ^c R ^c , -OS (O) R ^d , -OS (O) ₂ R ^d , -OS (O) ₂ OR ^d , -OS (O) ₂ NR ^c R ^c , -C (O) R ^d , -C (O) OR ^d , -C (O) NR ^c R ^c , -C (NH) NR ^c R ^c , -C (NR ^a ) NR ^c R ^c , -C (NOH) R ^a , -C (NOH) NR ^c R ^c , -OC (O) R ^d , -OC (O) OR ^d , -OC (O) NR ^c R ^c , -OC (NH) NR ^c R ^c , -OC (NR ^a ) NR ^c R ^c , - [NHC (O)] _n R ^d , - [NR ^a C (O)] _n R ^d , - [NHC (O)] _n OR ^d , - [NR ^a C (O)] _n OR ^d , - [NHC (O)] _n NR ^c R ^c , - [NR ^a C (O)] _n NR ^c R ^c , - [NHC (NH)] _n NR ^c R ^c and - [NR ^a C (NR ^a )] _n NR ^c R ^c ;

each of the R ^c groups separately represents a protective group or R ^a , or, conversely, each of the R ^c groups is taken together with the nitrogen atom to which it is attached in order to form a 5-8 membered cycloheteroalkyl or heteroaryl, which may include one or more identical or different additional heteroatoms and which may be substituted by one or more identical or different R ^a groups or suitable R ^b groups;

each R ^d group separately represents a protective group or R ^a ;

each parameter m separately represents an integer from 1 to 3; and

each parameter n separately represents an integer from 0 to 3.

Another aspect of the present invention is the provision of prodrugs of 2,4-pyrimidinamine compounds. Such prodrugs can be active in the form of their prodrugs or inactive until converted to the active dosage form under certain physiological or other conditions of use. In the prodrugs of the present invention, one or more functional groups of 2,4-pyrimidinamine compounds are included in the promoieties, which are cleaved from the molecule under conditions of use, usually by hydrolysis, enzymatic cleavage, or other cleavage mechanism to form functional groups. For example, primary or secondary amino groups may be included in an amide procomponent that cleaves under conditions of use to form a primary or secondary amino group. Thus, the prodrugs of the present invention include special types of protective groups called “progroups” and mask one or more functional groups of 2,4-pyrimidinamine compounds that cleave under conditions of use to form active drug compounds 2 , 4-pyrimidinamines. Functional groups that are part of 2,4-pyrimidinamine compounds that can be masked by progroups for inclusion in a procomponent include, but are not limited to, amines (primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, carbonyls, phenols, catechols, diols, alkynes, phosphates, etc. Currently, there are a huge number of progroups suitable for masking these functional groups in order to form procomponents capable of cleaving under conditions of use. All of these progroups, alone or in combination, may be included in the prodrugs of the present invention. Specific examples of procomponents forming primary or secondary amino groups that may be included in the prodrugs of the present invention include, but are not limited to, the following groups: amides, carbamates, imines, ureas, phosphenyls, phosphorylates, and sulfenyls. Specific examples of sulfonyl group-forming procomponents that may be included in prodrugs of the present invention include, but are not limited to, the following groups: thioethers, for example, S-methyl derivatives (monothio-, dithio-, oxythio-, aminothioacetals, silyl thioethers , thioesters, thiocarbonates, thiocarbamates, asymmetric disulfides, etc. Specific examples of procomponents forming, by cleavage, hydroxyl groups that can be incorporated into prodrugs according to the present and the acquisition include, but are not limited to, the following groups: sulfonates, esters and carbonates Specific examples of procomponents forming carboxyl groups that may be included in prodrugs of the present invention include, but are not limited to, the following groups: complex esters (including silyl esters, oxamic acid esters and thioesters), amides and hydrazides.

In one illustrative example, prodrugs of the present invention are compounds of structural formula (I) in which the protective group R ^c and R ^d is a progroup.

Substitution of hydrogen atoms attached to N2 and N4 in 2,4-pyrimidinamines having structural formula (I) with substituents has an adverse effect on the activity of the compounds. However, this will be appreciated by specialists - these nitrogen atoms can be included in procomponents, which, under the conditions of use, are split and form 2,4-pyrimidinamines according to structural formula (I). Thus, as shown in another illustrative example, prodrugs according to the present invention are compounds with the following structural formula (II):

including their salts, hydrates, solvates and N-oxides in which

R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ and L ² are the groups indicated above in the description of formula (I); and

each of the groups R ^2b and R ^4b individually and independently of one another is a progroup.

A further aspect of the present invention is to provide compositions comprising one or more compounds and / or prodrugs of the present invention, as well as a suitable carrier, excipient or diluent. The exact nature of the carrier, excipient or diluent will depend on the desired use of the composition — from substances used in veterinary practice to substances used to treat a person.

Another aspect of the present invention is to provide intermediates useful in the synthesis of 2,4-pyrimidinamine compounds and prodrugs of the present invention. In one example, the intermediates are 4-pyrimidinamines with the structural formula (III):

including their salts, hydrates, solvates and N-oxides in which R ⁴ , R ⁵ , R ⁶ and L ² are the groups previously described in the definition of structural formula (I); LG is a substituted group, for example, —S (O) ₂ Me, —SMe or halogen groups ( i.e. , F, Cl, Br, I); and R ^4c represents hydrogen or a progroup.

In another example, the intermediates are 2-pyrimidinamines according to structural formula (IV):

including their salts, hydrates, solvates and N-oxides in which R ² , R ⁵ , R ⁶ and L ¹ are the groups previously described in the definition of structural formula (I); LG is a substituted group, for example, —S (O) ₂ Me, —SMe or halogen groups ( i.e. , F, Cl, Br, I); and R ^2c represents hydrogen or a progroup.

In yet another example, the intermediates are 4-amino or 4-hydroxy-2-pyrimidinamines in accordance with structural formula (V):

including their salts, hydrates, solvates and N-oxides in which R ² , R ⁵ , R ⁶ and L ¹ are the groups previously described in the definition of structural formula (I); R ⁷ represents an amino or hydroxyl group and R ^2c represents hydrogen or a progroup.

In the following example, the intermediates are represented by N4-substituted cytosines in accordance with structural formula (VI):

including their salts, hydrates, solvates and N-oxides in which R ⁴ , R ⁵ , R ⁶ and L ² are the groups previously described in the definition of structural formula (I); and R ^4c represents hydrogen or a progroup.

Another aspect of the present invention is to provide methods for the synthesis of compounds of 2,4-pyridinamines and prodrugs according to the present invention. In one example, this method involves the reaction of 4-pyrimidinamines with structural formula (III) with an amine having the formula HR ^2c NL ¹ -R ² , where L ¹ , R ² and R ^2c are the groups previously indicated in the description of structural formula ( IV), with the aim of forming 2,4-pyrimidinamine having structural formula (I), or a prodrug in accordance with structural formula (II).

In another example, this method involves the reaction of 2-pyrimidinamines with structural formula (IV) with an amine having the formula R ⁴ -L ² -NHR ^4c , in which L ⁴ , R ⁴ and R ^4c are the groups indicated previously in the description of the structural formula (III), with the aim of forming 2,4-pyrimidinamines having the structural formula (I), or prodrugs in accordance with the structural formula (II).

In another example, the method involves the reaction of 4-amino-2-pyrimidinamines with the structural formula (V) (in which R ⁷ is an amino group) with an amine having the formula R ⁴ -L ² -NHR ^4c , in which L ⁴ , R ⁴ and R ^4c are the groups indicated previously in the description of structural formula (III), with the aim of forming 2,4-pyrimidinamine having structural formula (I), or prodrugs in accordance with structural formula (II). Alternatively, 4-amino-2-pyrimidinamine may be reacted with a compound having the formula R ⁴ -L ² -LG, in which R ⁴ and L ² are the groups previously described in the description of structural formula (I), and LG is a substituted group.

In another example, said method involves a halogenation reaction of 4-hydroxy-2-pyrimidinamine with the structural formula (V) (R ⁷ is a hydroxyl group) to form 2-pyrimidinamine with the structural formula (IV), followed by the reaction of said pyrimidinamine with the corresponding amine , as mentioned above.

In yet another example, said method involves a halogenation reaction of an N4-substituted cytosine with structural formula (VI) to form 4-pyrimidinamine with structural formula (III) followed by reaction of said pyrimidinamine with the corresponding amine as described above.

The 2,4-pyrimidinamine compounds of the present invention are potent inhibitors of the degranulation of immune cells such as mast cells, basophils, neutrophils and / or eosinophils. Thus, another aspect of the present invention is to provide methods for regulating and, in particular, inhibiting the degranulation of such cells. The proposed method generally consists in bringing the degranulated cell into contact with a compound of 2,4-pyrimidinamines or a prodrug according to the present invention or a corresponding salt, hydrate, MES, N-oxide of these compounds and / or combinations thereof in an amount sufficient to effectively regulate or inhibit cell degranulation. The specified method can be used in vitro or in vivo as a tactic for the treatment or prophylaxis of diseases characterized by cell degranulation caused by or associated with it.

Without claiming to be connected with any theory explaining the principle of action, the available biochemical data confirm that the compounds of 2,4-pyrimidinamines have an inhibitory effect on degranulation, at least in part, by blocking and inhibiting the signal transduction cascade (cascades) initiated by cross-linking of Fc receptors with a high degree of affinity for IgE (“FcεRI”) receptors and / or IgG (“FcγRI”) receptors. In fact, 2,4-pyrimidinamine compounds are potent inhibitors of degranulation mediated by both FcεRI and FcγRI receptors. As a consequence of this, 2,4-pyrimidine compounds can be used to inhibit signaling cascades of these Fc receptors in any type of cell expressing such FcεRI and / or FcγRI receptors, including, but not limited to, the following cells: macrophages, mast cells, basophils neutrophils and / or eosinophils.

These methods also allow you to regulate and, in particular, inhibit the processes occurring in the forward direction, which are the result of the activation of such signaling cascades of Fc receptors. Such direct processes include, but are not limited to, the following; FcεRI- and / or FcγRI- mediated degranulation, cytokine production and / or production and / or isolation of lipid mediators, such as leukotrienes and prostaglandins. Said method generally consists in bringing a cell from the category of the above expressing an Fc receptor into contact with a 2,4-pyrimidinamine compound or prodrug of the present invention, or a corresponding salt, hydrate, solvate, N-oxide of said compounds and / or their combinations in an amount sufficient to efficiently regulate or inhibit the Fc receptor signaling cascade and / or the direct process affected by the activation of this signaling cascade. The specified method can be used in vitro or in vivo as a tactic for the treatment or prophylaxis of diseases characterized by, caused by or associated with the signaling cascade of Fc receptors, such as, for example, diseases caused by the release of chemical mediators of granules during the process of jerking, isolation and / or synthesis of cytokines and / or the isolation and / or synthesis of lipid mediators, such as, for example, leukotrienes and prostaglandins.

Another aspect of the present invention is to provide methods for treating and / or preventing diseases characterized by, caused by or associated with the release of chemical mediators by activating Fc receptor signaling cascades, such as, for example, FcεRI and / or FcγRI receptor signaling cascades. These methods can be applied both to animals and to humans. These methods generally consist in administering to an animal or human a compound of 2,4-pyrimidinamines or a prodrug of the present invention or a corresponding salt, hydrate, solvate, N-oxide of said compounds and / or a combination thereof in an amount sufficient to effectively treat or prevent the disease. As discussed above, activation of the FcεRI or FcγRI receptor signaling cascade in specific immune cells leads to the isolation and / or synthesis of various chemicals that are pharmacological mediators of a wide range of diseases. Any of these diseases can be treated or prevented in accordance with the methods of the present invention.

For example, in mast cells and basophils, activation of the FcεRI or FcγRI receptor signaling cascade leads to the immediate ( i.e. , within 1-3 min from the moment of receptor activation) release of preformed mediators of atopic reactions and / or type I hypersensitivity reactions (e.g. histamine , proteases, such as tryptases, etc.) through a degranulation process. Such atopic and / or type I hypersensitivity reactions include, but are not limited to anaphylactic reactions to the environment and other allergens (e.g., pollen, insect and / or animal venom, food, drugs, contrasting paint, etc.), anaphylactoid reaction, hay fever, allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, eczema, urticaria, dysfunction of the mucous membrane, tissues, as well as a number of gastrointestinal disorders.

Following the immediate release of preformed mediators through degranulation, a number of other chemical mediators are isolated and / or synthesized, including, among others, platelet activating factor (PAF), prostaglandins and leukotrienes (e.g. LTC4), as well as de novo synthesis and cytokine isolation e.g. TNFα, IL-4, IL-5, IL-6, IL-13, etc. The first of these two processes occurs approximately 3-30 minutes after activation of the receptors; the next - approximately 30 minutes - 7 hours after activation of the receptors. These late stage mediators are considered to be partially responsible for the chronic symptoms of the atopic and hypersensitivity reactions of type I listed above and in addition are chemical mediators of inflammation and inflammatory diseases (ie osteoarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, idiopathic inflammatory disease intestines, syndrome of increased gastric irritability, spastic colitis, etc.), weak scarring (i.e., scleroderma, severe fibrosis, keloid formation, last operational scars, pulmonary fibrosis, vascular spasms, migraine, reperfusion injury and post myocardial infarction), and Sjogren's syndrome. All of these diseases can be treated or prevented in accordance with the methods of the present invention.

Additional diseases that can be treated or prevented by the methods of the present invention include diseases associated with the pathology of basophilic cells and / or mast cells. Examples of such diseases include, but are not limited to, skin diseases, such as scleroderma, heart diseases, such as myocardial infarction, lung diseases, such as changes or transformations of lung muscles, or chronic obstructive pulmonary disease (COPD) and bowel disease, such as bowel inflammation (spastic colitis).

The 2,4-pyrimidinamine compounds of the present invention are also potent inhibitors of tyrosine kinase and, in particular, Syk kinase. Thus, another aspect of the present invention is to provide methods for regulating and, in particular, inhibiting the activity of Syk kinase. The method as a whole consists in bringing the Syk kinase or cell containing the Syk kinase into contact with the 2,4-pyrimidinamine compound or prodrug of the present invention or with the corresponding salt, hydrate, MES, N-oxide of these compounds and / or their composition in an amount sufficient to effectively regulate or inhibit the activity of Syk kinase. In one example, a Syk kinase is presented as an isolated or recombinant Syk kinase. In another example, the Syk kinase is presented as an endogenous or recombinant Syk kinase expressed by a cell, as, for example, in the case of a mast cell or basophil. The method can be used in vitro or in vivo as a tactic for the treatment or prophylaxis of diseases characterized by, associated with or associated with Syk kinase activity.

Not limited to any one theory explaining the principle of action, it is believed that the compounds of 2,4-pyrimidinamines according to the present invention have an inhibitory effect on cell degranulation and / or secretion of other chemical mediators mainly due to the inhibition of Syk kinase, which is activated by gamma homodimer FcεRI receptor chains (see, for example, FIG. 2). This gamma chain homodimer is common to other Fc receptors, including FcγRI, FcγRIII and FcαRI. For all of these receptors, the intracellular signaling process is mediated by a common gamma chain homodimer. The binding and aggregation of these receptors leads to the recruitment and activation of tyrosine kinases, such as, for example, Syk kinases. As a result of these common signaling actions, these 2,4-pyrimidinamine compounds can be used to regulate and, in particular, inhibit signaling cascades of Fc receptors with gamma chain homodimers, such as, for example, FcεRI, FcγRI, FcγRIII and FcαRI, as well as cell reactions obtained through these receptors.

Syk kinase is known to play a critical role in other signaling cascades. For example, Syk kinase is a B-cell receptor (BCR) signal effector (Turner et al., 2000, Immunology Today 21: 148-154) and is an essential component of the beta (1), beta (2) and beta (3) signaling pathways ) -integrin in neutrophils (Mocsai et al., 2002, Immunity 16: 547-558). Since these compounds of 2,4-pyrimidinamines are potent inhibitors of Syk kinase, they can be used to regulate, in particular, inhibition, any signal cascades in which Syk kinase plays a role, for example, in signal cascades of Fc receptors, BCR and integrin, as well as cell responses obtained through these receptors. The regulation or inhibition of a particular cell response is partly dependent on the particular cell type and receptor signaling cascade and is well known in the literature. Some of the examples of cell responses that can be regulated or inhibited by 2,4-pyrimidinamine compounds include respiratory burst, cell adhesion, cell degranulation, cell proliferation, cell migration, phagocytosis (e.g. , in macrophages), calcium ion flow (e.g. , in mast cells, basophils, neutrophils, eosinophils and B-cells), platelet aggregation and cell maturation (for example, in B-cells).

Thus, another aspect of the present invention is to provide methods for regulating, in particular inhibiting, signaling cascades in which Syk kinase is involved. The method generally consists in bringing the Syk-dependent receptor or cell expressing the Syk-dependent receptor into contact with the 2,4-pyrimidinamine compound or prodrug of the present invention or the corresponding salt, hydrate, MES, N-oxide of these compounds and / or their combinations in an amount sufficient to effectively regulate or inhibit the signaling cascade. These methods can also be used to regulate and, in particular, inhibit direct processes or cell responses resulting from the activation of a particular Syk-dependent signaling cascade. The methods can be used to regulate any signaling cascades in which the role of the Syk kinase is unknown or is established later. The methods can be used in vitro or in vivo as a tactic for the treatment and prophylaxis of diseases characterized by, associated with or associated with the activation of a Syk-dependent signaling cascade. Some examples of such diseases include the diseases described previously.

The data obtained from cell analysis, together with data obtained from animals, also confirm that the 2,4-pyrimidinamine compounds of the present invention can be used to treat or prevent autoimmune diseases and / or symptoms of these diseases. Appropriate methods generally include administering to a patient suffering from an autoimmune disease or at risk of developing an autoimmune disease, 2,4-pyrimidinediamine or a prodrug of the present invention or an acceptable salt, N-oxide, hydrate, solvate or combination thereof in an amount effective for the treatment or prevention of an autoimmune disease and / or related symptoms. Autoimmune diseases treatable or preventable with 2,4-pyrimidinamine compounds include diseases often associated with non-anaphylactic hypersensitivity reactions (type II, III and / or IV) and / or diseases mediated at least in part, activation of the FcγR signaling cascade in monocytes. Such autoimmune diseases include, but are not limited to, diseases often characterized as autoimmune disorders of a single organ or single cell type, as well as diseases often characterized as including systemic autoimmune disorders. Some examples of diseases, often characterized as autoimmune disorders of a single organ or a single type of cell, include Hashimoto thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyocytoma, paratyphoid arrhythmias, and paratyphoid arthritis, hyperimmunity, parathyroid gland syndrome, autoimmune parasitism, and parasitism syndrome , bazedovy disease, biliary primary cirrhosis of the liver, chronic aggressive hepatitis, ulcerative colitis and membrane glomerulo Atia. Some examples of diseases often characterized as including systemic autoimmune disorders include systemic lupus erythematosus, rheumatoid arthritis, Sjögren’s syndrome, Reiter’s syndrome, polymyositis-dermatomyositis, systemic sclerosis, nodosa polyarteritis, multiple sclerosis, and bullous pemphigoid.

5. Brief description of figures

Figure 1 shows a graphic illustration of the production of IgE under the action of allergens and the subsequent isolation of preformed and other chemical mediators of mast cells;

Figure 2 shows a visual illustration of the cascade of signal transduction of receptors FcεR1, which leads to degranulation of mast cells and / or basophils;

Figure 3 shows a graphic illustration of the proposed sites of action of compounds that selectively inhibit degranulation in the opposite direction, mediated by FcεRI receptors, and compounds that inhibit degranulation, mediated by FcεRI receptors and caused by ionomycin;

4 is a graphical illustration of the effects of certain compounds of 2,4-pyrimidinamines, DMSO (control) and ionomycin on Ca ²⁺ flow in cultured human mast cells (CHMC);

Figure 5 shows a graphical illustration of the speed of the inhibitory effect of compounds R921218 and R926495;

6 is a graphical illustration of the effect of leaching on the inhibitory effect of compounds R921218 and R921302;

Fig. 7 shows data showing that the variable concentration of compounds R921218 (A) and R921219 (B) inhibits the phosphorylation of various proteins in the forward direction from Syk kinase in the cascade of IgE receptor signaling in activated bone marrow mast cells (BMMC cells);

Fig. 8 shows data demonstrating inhibition of phosphorylation of an endogenous substrate (LAT) and a peptide substrate depending on the dose of Syk kinase at increasing concentrations of compounds R921218 (X), R921219 (Y) and R921304 (Z);

Figure 9 shows data demonstrating the competitive in relation to ATP nature of the inhibition of Syk kinase compound R921219;

Figure 10 shows data showing that a variable concentration of compounds R921219 (A) and R218218 (B) inhibits protein phosphorylation in the forward direction from the Syk kinase, but not the LYN kinase, in the cascade of FcεRI receptor signaling in activated CHMC cells ; the figure also shows the inhibition of protein phosphorylation in the forward direction from the LYN kinase, but not the Syk kinase, in the presence of a known LYN kinase inhibitor (PP2);

11A-D are data showing inhibition of protein phosphorylation in the forward direction from Syk kinase in the cascade of FcεRI receptor signaling in BMMC cells;

12 is a graph illustrating the efficacy of compound R921302 in the treatment of arthritis induced by anti-collagen antibody (CAIA) when tested in mice;

13 is a graph illustrating the efficacy of compound R921302 in the treatment of CAIA in mice compared to other agents and control agents;

14 is a graph illustrating the efficacy of compound R921302 in the treatment of collagen-induced arthritis (CIA) when tested in rats;

Fig. 15 is a graph illustrating the efficacy of compound R921302 in inhibiting experimental autoimmune encephalomyelitis (EAE) in mice, a clinical model of multiple sclerosis; and

16 is a graph illustrating the efficacy of compound R921302 when tested on SJL mice that were given 150 μg of PLP 139-151 and 200 μg of MTB (CFA) on the first day of immunization.

6. Detailed Description of Preferred Embodiments

6.1 Definitions

The following terms used in this text have the following meanings:

“ Alkyl ”, alone or as part of another compound, refers to a saturated or unsaturated monovalent hydrocarbon radical with a branched, unbranched or cyclic structure and a fixed number of carbon atoms ( for example , C1-C6 means the number of carbon atoms from one to six) obtained by separation one hydrogen atom from a single carbon atom of the starting alkane, alkene or alkyn. Typical alkyl groups include, but are not limited to, methyl; ethyls, such as ethanyl, ethenyl, ethynyl; propyl, such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en- 1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc .; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-1- silt, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1, 3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobut-1,3-dien-1- silt, but-1-en-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc .; and the like. If desired saturation levels are intended, the term “alkanyl,” “alkenyl,” and / or “alkynyl,” as used below, is used. In preferred embodiments, the alkyl groups are alkyl (C1-C6).

“ Alkanyl ”, alone or as part of another compound, refers to a saturated alkyl with a branched, unbranched or cyclic structure obtained by the separation of one hydrogen atom from a single carbon atom of the starting alkane. Typical alkyl groups include at least methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc .; butanyls such as butan-1-yl, butan-2-yl ( sec- butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl ( tert -butyl), cyclobutane -1-yl, etc .; and the like. In preferred embodiments, the alkyl groups are alkanyl (C1-C6).

“ Alkenyl ”, alone or as part of another compound, refers to unsaturated alkyl with a branched, unbranched or cyclic structure having at least one carbon-carbon double bond obtained by separating one hydrogen atom from a single carbon atom of the starting alkene. A group may have a cis or trans configuration in the vicinity of the double bond (double bonds). Typical alkyl groups include, but are not limited to, ethenyl; propenyls, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1- en-1-yl; cycloprop-2-en-1-yl; butenyls, such as, for example, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en- 3-yl, cyclobuta-1,3-dien-1-yl, etc .; and the like. In preferred embodiments, the alkyl groups are alkenyl (C2-C6).

“ Alkynyl, ” alone or as part of another compound, refers to an unsaturated alkyl with a branched, unbranched or cyclic structure having at least one carbon-carbon triple bond obtained by separating one hydrogen atom from a single carbon atom of the starting alkine. Typical alkyl groups include, but are not limited to, ethynyl; propynyls, such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc .; butynyls, such as, for example, but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc .; and the like. In preferred embodiments, the alkynyl group is alkynyl (C2-C6).

“ Alkyldiyl ”, alone or as part of another compound, refers to a saturated or unsaturated divalent hydrocarbon group with a branched, unbranched or cyclic structure and a fixed number of carbon atoms ( i.e., C1-C6 means the number of carbon atoms from one to six) obtained by separating one hydrogen atom from each of two separate carbon atoms of the starting alkane, alkene or alkyn, or by separating two hydrogen atoms from the single carbon atom of the starting alkane, alkene or a Lkina. Two centers of monovalent radicals or each valency of the center of a divalent radical can form bonds with the same or with different atoms. Typical alkyldiyl groups include at least methanediyl; ethyldiyls such as ethane-1,1-diyl, ethane-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl, cyclopropane-1,1-diyl, cyclopropane-1,2- diyl, prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl, prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl, cycloprop-2-en-1,1-diyl, prop-1-in-1,3-diyl, etc. d .; butyl diyls such as butane-1,1-diyl, butane-1,2-diyl, butane-1,3-diyl, butane-1,4-diyl, butane-2,2-diyl, 2-methyl-propan- 1,1-diyl, 2-methyl-propan-1,2-diyl, cyclobutane-1,1-diyl; cyclobutane-1,2-diyl, cyclobutane-1,3-diyl, but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3- diyl, but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl, 2-methanylidene-propane-1,1-diyl, buta-1,3-diene 1,1-diyl, buta-1,3-diene-1,2-diyl, buta-1,3-diene-1,3-diyl, buta-1,3-diene-1,4-diyl, cyclobut- 1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl, cyclobut-1,3-diene-1,2-diyl, cyclobuta- 1,3-diene-1,3-diyl, but-1-en-1,3-diyl, but-1-en-1,4-diyl, buta-1,3-diin-1,4-diyl and etc .; and the like. If predetermined saturation levels are intended, the terms “alkanyldiyl”, “alkenyldiyl” and / or “alkynyldiyl” are used. If it is assumed that two valencies of the same carbon atom are involved, the term “alkylidene” is used. In preferred embodiments, the alkyldiyl group is alkyldiyl (C1-C6). Also preferred are saturated non-cyclic alkanyldiyl groups in which the centers of the radicals are located at the extreme carbon atoms, for example methanediyl (methane-); ethane-1,2-diyl (ethane-); propane-1,3-diyl (propane-); butane-1,4-diyl (butane-); and the like (also referred to as alkylene, as defined below ).

“ Alkylene ”, alone or as part of another compound, refers to an unbranched saturated or unsaturated alkyldiyl group with two extreme centers of monovalent radicals obtained by separating one hydrogen atom from each of their two extreme carbon atoms of an unbranched starting alkane, alkene or alkyn. The designation of a double or triple bond, if it exists, is indicated in square brackets in a separate alkylene group. Typical alkylene groups include, but are not limited to, methane-; ethylene groups, such as, for example, ethane, ethene, ethin; propylene groups, such as propane, prop [1] en-, propa [1,2] diene, prop [1] in-, etc .; butylene groups, such as, for example, butane-, but [1] en-, but [2] en-, buta [1,3] diene, but [1] in-, but [2] in-, buta [1, 3] diene, etc .; and the like. If preset levels of saturation are assumed, the term “alkane-,” “alkene-” and / or “alkine-” is used. In preferred embodiments, the alkylene group is alkylene- (C1-C6) or (C1-C3). Unbranched saturated alkane groups, for example methane, ethane, propane, butane and the like, are also preferred.

“Heteroalkyl” , “Heteroalkanyl” , “Heteroalkenyl” , “ Heteroalkynyl ”, “ Heteroalkyldiyl ” and “ Heteroalkylene ”, as such or as part of other compounds, refer to the groups of alkyls, alkanyls, alkenyls, alkynyls, alkyldiyls or alkylenes, respectively, in which one or more carbon atoms are independently replaced by identical or different heteroatoms or heteroatom groups. Typical heteroatoms and / or heteroatomic groups that can replace carbon atoms include, but are not limited to, —O—, —S—, —SO—, —NR′—, —PH—, —S (O) -, -S (O) ₂ -, -S (O) NR'-, -S (O) ₂ NR'- and the like, including combinations thereof, in which each R 'radical is individually a hydrogen atom or an alkyl group (C1- C6).

“ Cycloalkyl ” and “ Heterocycloalkyl ”, alone or as part of another compound, refer to cyclic versions of “alkyl” and “heteroalkyl” groups, respectively. In the case of a heteroalkyl group, the heteroatom may occupy a position in which it is adjacent to the rest of the molecule. Typical cycloalkyl groups include, but are not limited to cyclopropyl; cyclobutyls, such as cyclobutanyl or cyclobutenyl; cyclopentyls, such as cyclopentanil and cyclopentenyl; cyclohexyls, such as cyclohexanyl and cyclohexenyl; and the like. Typical heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl ( e.g. tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, etc.), piperidinyl ( e.g. piperidin-1-yl, piperidin-2-yl, and t etc.), morpholinyl ( e.g. , morpholin-3-yl, morpholin-4-yl, etc.), piperazinyl ( e.g. , piperazin-1-yl, piperazin-2-yl, etc.), and like that.

“ Acyclic heteroatom bridge ” refers to a divalent bridge in which the atoms of the main target are exclusively heteroatoms and / or heteroatomic groups. Typical non-cyclic heteroatom bridges include, but are not limited to, -O-, -S-, -SO-, -NR'-, -PH-, -S (O) -, -S (O) ₂ -, -S (O) NR'-, -S (O) ₂ NR'- and the like, including combinations thereof, in which each R'-radical is individually a hydrogen atom or an alkyl group (C1-C6).

“ Original aromatic ring system ” refers to an unsaturated cyclic or polycyclic ring system having a conjugated π-electronic system. The definition of “parent aromatic ring system” specifically includes fused ring systems in which one or more rings are aromatic and one or more rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenal, tetrahydronaphthalene, etc. . Typical starting aromatic ring systems include, but are not limited to, aceanthrene, acenaphthylene, acefenanthylene, anthracene, azulene, benzene, chrysene, coronene, fluorantene, fluorene, hexacene, hexafen, hexalene, indacene, s- indacene, indane, indane, indenine, indenine, indenine , octacene, octaphene, octalene, oval, penta-2,4-diene, pentacene, pentalene, pentaphene, phenalen, phenanthrene, pycene, pleiaden, pyrene, pyrantren, rubicene, tetrahydronaphthalene, triphenylene and the like, as well as various hydroisomers of these compounds .

“ Aryl ”, alone or as part of another compound, refers to a monovalent aromatic hydrocarbon group with a fixed number of carbon atoms ( for example , C5-C15 means the number of carbon atoms from 5 to 15) obtained by separating one hydrogen atom from a separate carbon atom of the original aromatic ring systems. Typical aryl groups include, but are not limited to, groups derived from aceantrylene, acenaphtylene, acefenanthrene, anthracene, azulene, benzene, chrysene, coronene, fluorantene, fluorene, hexacene, hexaphene, hexalene, a-indacene, c-indacene, indane, indane, , indene, naphthalene, octene, octaphene, octalene, ovalen, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, pizza, pleiaden, pyrene, pyrantrene, rubicene, triphenylene, trinaphthalene and the like, as well as various hydroisomers of these compounds. In preferred embodiments, the aryl group is an aryl (C5-C15) with the most preferred (C5-C10) structure. The most preferred aryls are cyclopentadienyl, phenyl and naphthyl.

“ Arylaryl, ” alone or as part of another compound, refers to a monovalent hydrocarbon group obtained by separating one hydrogen atom from a separate carbon atom of a ring system in which two or more identical or non-identical starting aromatic ring systems are combined directly with each other using a single a bond in which the number of said straight ring compounds is one less than the number of the starting aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. If the arylaryl group has a given number of carbon atoms, then this number refers to the carbon atoms that make up each of the original aromatic ring. For example, arylaryl (C5-C15) is an arylaryl group in which each aromatic ring consists of 5-15 carbon atoms, for example biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc. It is desirable that each initial aromatic ring system of the arylaryl group be an independent aromatic system (C5-C15), and better, an aromatic system (C5-C10). Arylaryl groups in which all of the starting aromatic ring systems are identical, for example biphenyl, triphenyl, binaphthyl, trinaphthyl, etc., are also desirable.

“ Biaryl ”, alone or as part of another compound, refers to an arylaryl group having two identical starting aromatic ring systems that are connected directly to each other via a single bond. Typical biaryl groups include, but are not limited to, biphenyl, binaphthyl, banthracil, and the like. It is desirable that the aromatic systems of the rings be aromatic rings (C5-C15), and preferably aromatic rings (C5-C10). It is especially desirable that the biaryl group is biphenyl.

“ Arylalkyl ”, alone or as part of another compound, refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, usually a terminal or sp ³ carbon atom, is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylene-1-yl, naphthobenzyl, 2 naphthophenylethan-1-yl and the like. For alkyl compounds, the following terms are used: arylalkanyl, arylalkenyl and / or arylalkynyl. In preferred embodiments, the arylalkyl group is arylalkyl (C6-C21), for example , the alkanyl, alkenyl or alkynyl fragment of the arylalkyl group is a (C1-C6) structure, and the aryl fragment is a (C5-C15) structure. In most preferred embodiments, the arylalkyl group has the structure (C6-C13), for example , the alkanyl, alkenyl or alkynyl fragment of the arylalkyl group has the structure (C1-C3) and the aryl fragment has the structure (C5-C10).

An “ original heteroaromatic ring system ” refers to an initial aromatic ring system in which one or more carbon atoms are independently replaced by the same or different heteroatoms or heteroatom groups. Typical heteroatoms or heteroatom groups replacing carbon atoms include, but are not limited to, N, NH, P, O, S, S (O), S (O) ₂ , Si, etc. The definition of “starting heteroaromatic ring systems” specifically includes fused ring systems in which one or more rings are aromatic and one or more rings are saturated or unsaturated, such as, for example, benzodioxane, benzofuran, chroman, chromene, indole, indoline, xanthan etc. The definition of “starting heteroaromatic ring systems” also includes known rings containing common substitution groups, such as, for example, benzopyrone and 1-methyl-1,2,3,4-tetrazole. Benzene rings condensed to form cyclic polyalkylene glycols such as cyclic polyethylene glycol are specifically excluded from the definition of “starting heteroaromatic ring systems”. Typical starting heteroaromatic ring systems include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxane, benzodioxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline-chromazole, carbazene, carbazene, carbazole , furan, imidazole, indazole, indole, indoline, indolysin, isobenzofuran, isochromen, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline phenaz Ridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolysin, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthan and the like.

“ Heteroaryl ”, alone or as part of another compound, refers to a monovalent heteroaromatic group with a fixed number of ring atoms ( for example , “5-14 membered” means the number of ring atoms from 5 to 14), resulting from the separation of one hydrogen atom from a separate atom of the original heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, benzimidazole, benzisoxazole, benzodioxane, benzodiaxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole, carbazole, β chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthrene ina, phenanthroline, phenanzine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolisin, quinazoline, quinoline, quinolizine, hinexaline, tetrazole, thiadiazole, thiaziazene, thiazole, thiazole, thiazole, thiazole, thiazole, thiazole, thiazole as well as various hydroisomers of these compounds. In preferred embodiments, the heteroaryl group is 5-14 membered heteroaryl, while 5-10 membered heteroaryl is most preferred.

“ Heteroaryl-heteroaryl ”, alone or as part of another compound, refers to a monovalent heteroaromatic group resulting from the separation of one hydrogen atom from a separate atom of a ring system in which two or more identical or non-identical parent heteroaromatic systems are directly connected to each other by a single bond , in which the number of such direct ring compounds is one less than the number of initial heteroaromatic ring systems involved. Typical heteroaryl heteroaryl groups include, but are not limited to, bipyridyl, tripyridyl, pyridylpyrinyl, bipirinyl, etc. For a given number of atoms, these numbers refer to the number of atoms that make up each of the original heteroaromatic ring systems. For example, a 5-15 membered heteroaryl heteroaryl is a heteroaryl heteroaryl group in which each of the starting heteroaromatic ring systems consists of 5-15 atoms, for example bipyridyl, tripyridyl, etc. It is desirable that each initial heteroaromatic ring system be an independent 5-15 membered heteroaromatic system, and better, a 5-10 membered heteroaromatic system. Also preferred are heteroaryl heteroaryl groups in which all of the parent heteroaromatic ring systems are identical.

“ Bigheroaryl ”, alone or as part of another compound, refers to a heteroaryl-heteroaryl group comprising two identical parent heteroaromatic ring systems directly connected to each other by a single bond. Typical bigheroaryl groups include, but are not limited to, bipyridyl, bipirinyl, biquinolinyl, and the like. Preferably, the heteroaromatic ring systems are 5-15 membered heteroaromatic rings, and preferably 5-10 membered heteroaromatic rings.

“ Heteroarylalkyl, ” alone or as part of another compound, refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, usually a terminal or sp ³ carbon atom, is replaced by a heteroaryl group. In the case of alkyl compounds, the terms heteroarylalkanyl, heteroarylalkenyl and / or heteroarylalkynyl are used. In preferred embodiments, the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, for example , the alkanyl, alkenyl or alkynyl fragment of a heteroarylalkyl is alkyl with a structure (C1-C6) and the heteroaryl component is a 5-15 membered heteroaryl. In the most preferred embodiments of the invention, heteroarylalkyl is 6-13 membered heteroarylalkyl, for example , the alkanyl, alkenyl or alkynyl fragment of heteroarylalkyl is alkyl with a structure (C1-C3) and the heteroaryl component is 5-10 membered heteroaryl.

“ Halogen ” or “ Halo ”, alone or as part of another compound, generally refers to fluoro, chloro, bromo and iodo groups.

" Haloalkyl ", alone or as part of another compound, refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen. Thus, the term “haloalkyl” means monohaloalkyls, dihaloalkyls, trihaloalkyls, etc., including perhaloalkyls. For example, the term “(C1-C2) haloalkyl” includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above groups may include prefixes and / or suffixes, which are widely used to create additional well-known substituent groups. For example, “alkyloxy” or “alkoxy” refers to a group of the formula —OR ”,“ alkylamine ”refers to a group of the formula —NHR”, and “dialkylamine” refers to a group of the formula —NR ”R”, where each R ”is an independent alkyl. As another example, “haloalkoxy” or “haloalkyloxy” refers to a group of the formula —OR ″ ″, where R ″ ″ is haloalkyl.

A “ protecting group ” refers to a group of atoms which, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, the protecting group may optionally be selectively excluded during the synthesis process. Examples of protective groups can be found in the book. Greene and Watsa "Protective Groups in Organic Chemistry", 3rd edition, 1999, at publ J. Wiley & Sons, New York (Greene and Wuts, Protective Groups in Organic Chemistry, 3 ^rd Ed., 1999, John Wiley & Sons, NY), as well as in the book. Harrison et al. " A Brief Overview of Synthetic Organic Methods ," Vol. 1-8, 1971-1996, J. Wiley & Sans, New York (Harrison et al. , Compendium of Synthetic Organic Methods , Vols. 1- 8, 1971-1996, John Wiley & Sons, NY). Typical amino-containing protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert -butoxycarbonyl ("BOC"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl (" TEC), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitroveratryloxycarbonyl ("NVOC") and the like. Representative hydroxyl protecting groups include, but are not limited to, where the hydroxyl group is acylated or alkyl, such as, for example, benzyl or trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers ( e.g. , TMC or TIPPS groups) and allyl ethers.

" Prodrug " refers to a derivative of the active compound 2,4-pyrimidinediamine (drug), which requires conversion under conditions of use, for example inside the body, in order to isolate the active 2,4-pyrimidinediamine. Prodrugs are often, but not necessarily, pharmacologically inactive until they are converted into active drugs. Prodrugs are usually obtained by masking the functional group that is part of the 2,4-pyrimidinediamine drug to activate the progroup (defined below) to form the procomponent, which then undergoes transformation, for example, cleavage, under given conditions of use to isolate the functional group and, therefore , the active drug 2,4-pyrimidinediamine. The decomposition of the procomponent can occur spontaneously, as, for example, as a result of a hydrolysis reaction, under the influence of a catalyst or other agent, such as an enzyme, light, acid or base, or as a result of a change or exposure to any physical parameter or environment, for example temperature. The agent may be endogenous (internal), from the point of view of the conditions of use, such as, for example, an enzyme present in the cells for which the prodrug is intended, or acidic conditions in the stomach, or it can be introduced exogenously (externally).

A wide variety of progroups and procomponents suitable for masking functional groups in active components containing 2,4-pyrimidinediamine compounds to produce prodrugs are well known in the art. For example, a hydroxyl functional group may be masked as a sulfonate, ester, or pro-component of carbon dioxide, which can be hydrolyzed in vivo to produce a hydroxyl group. An amino-containing functional group can be masked as an amide, carbamate, imine, urea, phenyl, phosphoryl or pro-component sulfenyl, which can be hydrolyzed in vivo to obtain an amino-containing group. The carboxyl group can be masked as an ester (including silyl esters and thioesters), a pro-component of an amide or hydrazide, which can be hydrolyzed in vivo to produce a carboxyl group. Other representative examples of suitable progroups and their corresponding procomponents are obvious to experts in this field.

“ Progroup ” refers to a type of protecting group which, when used to mask a functional group in an active drug containing 2,4-pyrimidinediamine, converts the drug into a prodrug to form a procomponent. Progroups usually join the functional group of the drug through bonds that can be cleaved under given conditions of use. Thus, the progroup is that part of the procomponent that is cleaved to release a functional group under given conditions of use. As a specific example, an amide-containing procomponent having the formula —NH — C (O) CH ₃ , which contains the progroup —C (O) CH _3, can be cited.

An “ Fc receptor ” refers to a member of a family of cell surface molecule molecules that binds the Fc portion of an immunoglobulin (containing a specific constant region). Each Fc receptor binds a specific type of immunoglobulin. For example, the Fcα receptor (“FcαR”) binds IgA, the FcεR receptor binds IgE, and the FcγR receptor binds IgG.

The FcαR family includes an Ig polymer receptor involved in IgA / IgM epithelial transfer, a mycloid specific RcαRI receptor (also referred to as CD89), an Fcα / μR receptor, and at least two alternative IgA receptors (for a recent review see Monteiro and van de Winkel, 2003. Annual Review of Immunology, Modern Electronic Publications (Monteiro & van de Winkel, 2003, Annu. Rev. Immunol, advanced e-publication)). The FcαRI receptor appears on neutrophils, eosinophils, monocytes / macrophages, dendritic cells and kupferovy cells. FcαRI includes one alpha chain and an FcR gamma homodimer that carries an activation motif (ITAM) in the cytoplasmic domain and phosphorylates the Syk kinase.

The FcεR family includes two types of receptors: named FcεRI and FcεRII (also known as CD23). FcεRI is a high affinity receptor (binds IgE with an affinity of the order of 10 ¹⁰ M ^-1 ), which is found on mast cells, basophilic and eosinophilic cells, and attaches monomeric IgE to the cell surface. FcεRI has one alpha chain, one beta chain and the gamma chain homodimer discussed above. FcεRII is a low affinity receptor expressed on mononuclear phagocytes, lymphocytes, eosinophils and platelets. FcεRII is a single polypeptide chain and does not contain a gamma chain homodimer.

The FcγR family includes three types: named FcγRI (also known as CD64), FcγRII (also known as CD32) and FcγRIII (also known as CD16). FcγRI is a high affinity receptor (binds IgG1 with an affinity of the order of 10 ⁸ M ^-1 ), which is found on mast cells, basophilic, mononuclear, neutrophilic, eosinophilic, dendritic and phagocytic cells, which attaches monometric IgG to the cell surface. FcγRI includes one alpha chain and a gamma chain dimer, which is included in FcαRI and FcεRI.

FcγRII is a low affinity receptor expressed on neutrophils, monocytes, eosinophils, platelets, and B lymphocytes. FcγRII includes one alpha chain and does not contain the gamma chain homodimer discussed above.

The FcγRIII is a low affinity receptor (binds IgG1 with an affinity of the order of 5x10 ⁵ M ^-1 ), expressed on NK, eosinophilic, macrophage, neutrophilic cells and mast cells. It forms one alpha chain and a gamma homodimer, which is part of FcαRI, FcεRI and FcγRI.

Experts in this field will agree that the proposed subunit structure and binding properties of these diverse Fc receptors, as well as the cell types expressed by them, are not fully characterized. The discussion above mainly reflects the current state of the question regarding these receptors ( see, for example, Immunobiology: The immune system in a healthy state and in diseases, 5th edition, Janeway et al., 2001, Fig. 9.30 on page 371 (Immunobiology :. The Immun System in Health & Disease, 5 ^{th Edition, Janeway et al, Eds} , 2001, ISBN 0-8153-3642-x, Figure 9.30 at pp 371)) and is not intended to restrict the set of signaling cascades receptors that. can be regulated by the components described in this application.

“ Fc receptor degranulation” or “ Fc receptor degranulation” refers to degranulation that is performed by means of an Fc receptor signaling cascade initiated by cross-linking of the Fc receptor.

“ IgE- related degranulation” or “ FcεRI degranulation ” refers to degranulation which is carried out by means of an IgE receptor signaling cascade initiated by cross-linking IgE bound by FcεR1. Cross-linking may be caused by an allergen-specific IgE or a multivalent binding agent, such as an anti-IgE antibody. Figure 2 shows that in mast cells and / or basophilic cells, the FcεRI signaling cascade leading to degranulation can be divided into two stages: reverse and direct. The reverse stage includes all the processes that occur before the mobilization of calcium ions (shown as “Ca ²⁺ ” in FIG. 2; see also FIG. 3). The direct stage includes the mobilization of calcium ions and all its subsequent direct processes. Compounds that inhibit degranulation by FcεRI can act at any point along the signaling pathway mediated by FcεRI. Compounds that selectively inhibit degranulation by FcεRI in the opposite direction act to inhibit this portion of the FcεRI signaling cascade in the opposite direction from the point at which calcium ion mobilization is excited. In cell samples, compounds that selectively inhibit degranulation by FcεRI in the opposite direction interfere with the degranulation of cells, such as mast cells or basophil cells, that are activated or stimulated by an allergen specific for IgE or a binding agent (such as an anti-IgE antibody), but they do not significantly inhibit cell degranulation, which is activated or stimulated by degranulating agents that bypass the FcεRI signaling pathway, such as, for example, calcium ionophore ionomycin and A23187.

“ IgG- induced degranulation” or “ FcγRI degranulation ” refers to degranulation that is performed by a signaling cascade initiated by cross-linking IgG bound by FcγRI. Cross-linking can be caused by an allergen-specific IgG or a multivalent binding agent, such as an anti-IgG antibody or a partial antibody. Like the FcεRI signaling cascade, the FcγRI signaling cascade in mast cells and blasophilic cells also leads to degranulation, which can be divided into the same two stages: reverse and direct. Like degranulation by FcεRI, compounds that selectively inhibit degranulation by FcγRI in the opposite direction act in the opposite direction from the point at which calcium ion mobilization is excited. In cell samples, compounds that selectively inhibit degranulation by FcγRI in the opposite direction inhibit the degranulation of cells, such as mast cells or basophilic cells, that are activated or stimulated by an allergen-specific IgG or binding agent (such as an anti-IgG antibody or part thereof), but noticeably they do not inhibit degranulation of cells that are activated or stimulated by degranulating agents that bypass the FcγRI signaling pathway, such as, for example, calcium ionophores iomycin and A23187.

"Degranulation due ionophore," or "ionophore by Degranulation" refers to degranulation of cells such as mastotsit or basophilic cell which appears upon exposure to a calcium ionophore such as ionomycin or A23187.

" Syk kinase " refers to the well-known tyrosine kinase of the 72 kDa non-receptor spleen protein (cytoplasmic) expressed in a B cell and other hematopoietic cells. Syk kinase consists of two consecutive consensus Src homology domains 2 (SH2) that bind to phosphorylated tyrosine-based immunoreceptor motifs, linker domains, and catalytic domains (a review of the structure and function of Syk kinase is given in Sada et al. , 2001, J. Biochem. (Tokyo) 130: 177-186); as well as Turner et al. , 2000, Immunology Today 21: 148-154). Syk kinase has been extensively studied as an effector of B-cell receptor (BCR) signals (see Turner et al. , 2000 above). Syk kinase is also critical for tyrosine phosphorylation of complex proteins that regulate important pathways leading from immunoreceptors, such as Ca ²⁺ mobilization cascades and mitogen activated protein kinases (MAPKs) (see, for example, FIG. 2) and degranulation. Syk kinase also plays a critical role in neutrophil integrin signaling ( see, for example, Mochai et al., 2002, Immunity, vol. 16, pp. 547-558 (Mocsai et al. 2002, Immunity 16: 547- 558)).

As is customary in this work, by Syk kinase is meant the kinase of any animal species, including, but not limited to, humans, monkeys, cows, pigs, rodents, etc., belonging to the Syk family. This group specifically includes isoforms, splice variants, allelic variants, mutants of both natural and artificial origin. The amino acid sequences of such Syk kinases are well known and are available at GENBANK (Genetic Material Bank). Representative examples of mRNA coding of various isoforms of human Syk kinase can be found in GENBANK under the number gi | 21361552 | ref | NM__003177.2 |, gi | 496899 | emb | Z29630.1 | HSSYKPTK [496899] and gi | 15030258 | gb | BC011399 .1 | BC011399 [15030258], which are incorporated herein by reference.

Experienced practitioners will appreciate that tyrosine kinases belonging to other families may have active sites or binding pockets with a three-dimensional structure similar to that of Syk. As a result of this structural similarity, it is believed that such kinases mimicked in this application by " Syk simulators " can serve as catalysts for the phosphorylation of substrates that are phosphorylated by Syk kinase. Thus, it becomes clear that such Syk simulators, signaling cascades influenced by such Syk, and biological reactions from the effects of such Syk simulators and signaling cascades dependent on Syk simulators can be controlled and even inhibited by compounds 2,4-pyrimidinediamine described in this application.

“ Syk-dependent signaling cascade ” refers to a signaling cascade influenced by a Syk kinase. Some examples of such Syk-dependent signaling cascades include FcαRI, FcεRI, FcγRI, FcγRIII, BCR, and integrin signaling cascades.

The term “autoimmune disease” refers to diseases often associated with non-anaphylactic hypersensitivity reactions (type II, III and / or IV hypersensitivity reactions), which are mainly the result of the patient’s own humoral and / or cell-mediated immune response to one or more immunogenic endogenous substances and / or exogenous in nature. Such autoimmune diseases differ from diseases associated with anaphylactic hypersensitivity reactions (type I or mediated IgE).

6.2 Compounds of 2,4-pyrimidinediamines

The structures representing the essence of the invention are basically compounds of 2,4-pyrimidinediamines, which correspond to structural formula (I):

including their salts, hydrates, solvates and N-oxides, in which

L ¹ and L ² , each independently from each other, are selected from the group consisting of one direct link and one linker;

R ^{2 is} selected from the group consisting of (C1-C6) alkyl, which may be substituted with one or more identical or different R ⁸ groups, (C3-C8) cycloalkyl, which may be substituted with one or more identical or different R ⁸ groups cyclohexyl, which may be substituted by one or more identical or different R ⁸ groups, a 3-8 membered cycloheteroalkyl, which may be substituted by one or more identical or different R ⁸ groups, (C5-C15) aryl, which may be substituted by one or more identical or different R ⁸ groups, phenyl, which may be substituted by one or more identical or different R ⁸ groups, and a 5-15 membered heteroaryl, which may be substituted by one or more identical or different R ⁸ groups;

R ^{4 is} selected from the group consisting of hydrogen, (C1-C6) alkyl, which may be substituted with one or more identical or different R ⁸ groups, (C3-C8) cycloalkyl, which may be substituted with one or more identical or different R ⁸ groups, cyclohexyl which may be substituted by one or more identical or different R ⁸ groups, a 3-8 membered cycloheteroalkyl which may be substituted by one or more identical or different R ⁸ groups, (C5-C15) aryl, which may be substituted by one or more identical or different R ⁸ -groups , Phenyl which may be substituted by one or more identical or different R ⁸ -groups, and 5-15 membered heteroaryl, which may be substituted by one or more identical or different R ⁸ -groups;

R ^{5 is} selected from the group consisting of R ⁶ , (C1-C6) alkyl, which may be substituted with one or more identical or different R ⁸ groups, (C1-C4) alkanyl, which may be substituted with one or more identical or different R ⁸ groups, (C2-C4) alkenyl, which may be substituted by one or more identical or different R ⁸ groups, and (C2-C4) alkynyl, which may be substituted by one or more identical or different R ⁸ groups;

each R ^{6 is} independently selected from the group consisting of hydrogen, an electronegative group, -OR ^d , -SR ^d , (C1-C3) haloalkyloxy, (C1-C3) perhaloalkyloxy, -NR ^c R ^c , halogen, (C1-C3) haloalkyl, (C1-C3) perhaloalkyl, —CF ₃ , —CH ₂ CF ₃ , —CF ₂ CF ₃ , —CN, —NC, —OCN, —SCN, —NO, —NO ₂ , —N ₃ , —S (O) R ^d , -S (O) ₂ R ^d , -S (O) ₂ OR ^d , -S (O) NR ^c R ^c , -S (O) ₂ NR ^c R ^c , -OS (O) R ^d , -OS (O) ₂ R ^d , -OS (O) ₂ OR ^d , -OS (O) NR ^c R ^c , -OS (O) ₂ NR ^c R ^c , -C (O) R ^d , -C (O) OR ^d , -C (O) NR ^c R ^c , -C (NH) NR ^c R ^c , -OC (O) R ^d , -SC (O) R ^d , -OC (O) OR ^d , -SC (O) OR ^d , -OC (O) NR ^c R ^c , -SC (O) NR ^c R ^c , -OC (NH) NR ^c R ^c , -SC (NH) NR ^c R ^c , - [NHC (O)] _n R ^d , - [NHC (O)] _n OR ^d , - [NHC (O)] _n NR ^c R ^c and - [NHC (NH)] _n NR ^c R ^c , (C5 -C10) aryl, which may be substituted by one or more identical sludges and various R ⁸ groups, phenyl which may be substituted with one or more identical or different R ⁸ groups, (C6-C16) arylalkyl which may be substituted with one or more identical or different R ⁸ groups, 5-10- member heteroaryl, which may be substituted with one or more identical or different R ⁸ groups, and 6-16 membered heteroaryl, which may be substituted with one or more identical or different R ⁸ groups;

R ^{8 is} selected from the group consisting of R ^a , R ^b , R ^a substituted with one or more identical or different R ^a or R ^b , -OR ^a substituted with one or more identical or different R ^a or R ^b , -B ( OR ^a ) ₂ , -B (NR ^c R ^c ) ₂ , - (CH ₂ ) _m -R ^b , - (CHR ^a ) _m -R ^b , -O- (CH ₂ ) _m -R ^b , -S- (CH ₂ ) _m -R ^b , -O-CHR ^a R ^b , -O-CR ^a (R ^b ) ₂ , -O- (CHR ^a ) _m -R ^b , -O- (CH ₂ ) _m -CH [(CH ₂ ) _m R ^b ] R ^b , -S- (CHR ^a ) _m -R ^b , -C (O) NH- (CH ₂ ) _m -R ^b , -C (O) NH- (CHR ^a ) _m -R ^b , -O- (CH ₂ ) _m -C (O) NH- (CH ₂ ) _m -R ^b , -S- (CH ₂ ) _m -C (O) NH- (CH ₂ ) _m -R ^b , -O- (CHR ^a ) _m -C (O) NH- (CHR ^a ) _m -R ^b , -S- (CHR ^a ) _m -C (O) NH- (CHR ^a ) _m -R ^b , -NH- (CH ₂ ) _m -R ^b , -NH- (CHR ^a ) _m -R ^b , -NH [(CH ₂ ) _m R ^b ], -N [(CH ₂ ) _m R ^b ] ₂ , -NH-C (O) -NH- (CH ₂ ) _m -R ^b , -NH-C (O) - (CH ₂ ) _m -CHR ^b R ^b and -NH- (CH ₂ ) _m -C ( O) -NH- (CH ₂ ) _m -R ^b ;

each R ^{a is} independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) arylalkyl benzyl, 2-6 membered heteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, pipeazinyl, homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 membered heteroaryl and 6-16 membered heteroarylalkyl;

each R ^b represents a suitable group independently selected from the group consisting of = O, -OR ^d , (C1-C3) haloalkyloxy, -OCF ₃ , = S, -SR ^d , = NR ^d , = NOR ^d , -NR ^c R ^c , halogen, —CF ₃ , —CN, —NC, —OCN, —SCN, —NO, —NO ₂ , = N ₂ , —N ₃ , —S (O) R ^d , —S (O) ₂ R ^d , -S (O) ₂ OR ^d , -S (O) NR ^c R ^c , -S (O) ₂ NR ^c R ^c , -OS (O) R ^d , -OS (O) ₂ R ^d , -OS (O) ₂ OR ^d , -OS (O) ₂ NR ^c R ^c , -C (O) R ^d , -C (O) OR ^d , -C (O) NR ^c R ^c , -C ( NH) NR ^c R ^c , -C (NR ^a ) NR ^c R ^c , -C (NOH) R ^a , -C (NOH) NR ^c R ^c , -OC (O) R ^d , -OC (O) OR ^d , -OC (O) NR ^c R ^c , -OC (NH) NR ^c R ^c , -OC (NR ^a ) NR ^c R ^c , - [NHC (O)] _n R ^d , - [NR ^a C ( O)] _n R ^d , - [NHC (O)] _n OR ^d , - [NR ^a C (O)] _n OR ^d , - [NHC (O)] _n NR ^c R ^c , - [NR ^a C ( O)] _n NR ^c R ^c , - [NHC (NH)] _n NR ^c R ^c and - [NR ^a C (NR ^a )] _n NR ^c R ^c ;

each R ^c independently represents R ^a or, alternatively, each R ^c is taken together with the nitrogen atom to which it is bonded to form a 5-8 membered cycloheteroalkyl or heteroaryl, which may include one or more of the same or other additional heteroatoms and which may be substituted by one or more identical or different R ^a or suitable R ^b groups;

each R ^d independently represents R ^a ;

each m independently represents an integer from 1 to 3; and

each n independently represents an integer 0 to 3.

In compounds according to structural formula (I), L ¹ and L ² , independently of one another, represent one direct bond and one linker. Thus, as will be appreciated by qualified practitioners, the substituents R ² and / or R ⁴ can be bonded either directly to their respective nitrogen atoms, or alternatively, spaced from their respective nitrogen atoms via a linker. The identity of the linker is uncritical, and typical suitable linkers include (but are not limited to) (C1-C6) alkyldiyls, (C1-C6) alkanes and (C1-C6) heteroalkyl diyls, each of which may be substituted by one or more identical or different R ⁸ groups, where R ⁸ corresponds, as previously defined, to structural formula (I). In specific embodiments, L ¹ and L ² , each independently from each other, are selected from the group consisting of one direct bond, (C1-C3) alkyl diyl, which may be substituted with one or more identical or different R ^a , suitable R ^b or R ⁹ groups and a 1-3-membered heteroalkyl diyl, which may be substituted by one or more identical or different R ^a , suitable R ^b or R ⁹ groups, where R ^{9 is} selected from the group consisting of (C1-C3) alkyl, ^{oR a, -C (O) oR} a, (C5-C10) aryl, which may be substituted by one or more identical or different halogens, f nile, which may be substituted by one or more identical or different halogens, 5-10 membered heteroaryl, which may be substituted by one or more identical or different halogens, and 6-membered heteroaryl, which may be substituted by one or more identical or different halogens ; and R ^a and R ^b correspond, as previously defined, to structural formula (I). Specific R ⁹ groups that can be used to replace L ¹ and L ² include —OR ^a , —C (O) OR ^a , phenyl, halo phenyl, and 4-halo phenyl, where R ^a corresponds as previously defined structural formula (I).

In another specific embodiment, L ¹ and L ² , each independently from each other, are selected from the group consisting of methane, ethane and propane, each of which may be monosubstituted by a group R ⁹ , in which R ⁹ corresponds to the above preliminary definition.

In all of the above embodiments, the specific R ^a groups that can be included in the R ⁹ groups are selected from the group consisting of hydrogen, (C1-C6) alkyl, phenyl, and benzyl.

In yet another specific embodiment of the invention, each of L ¹ and L ² represents one direct bond, such that the 2,4-pyrimidiamine compounds of the present invention correspond to structural formula (Ia):

including their salts, hydrates, solvates and N-oxides, in which

R ² , R ⁴ , R ⁵ and R ⁶ correspond, as previously defined, to structural formula (I). Further specific embodiments of the 2,4-pyrimidiamine compounds of the present invention are set forth below.

In the first implementation of the elements of structural formulas (I) and (Ia), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ and L ² are the groups described previously in the definition of structural formulas (I) and (Ia), with the reservation that R ^{2 is} not 3,4,5-trimethoxyphenyl, 3,4,5-tri (C1-C6) alkoxyphenyl or

where R ²¹ , R ²² and R ²³ correspond to the definition for R ¹ , R ² and R ³ according to US patent No. 6 235 746, the disclosure of which is incorporated into this description by reference. In a specific implementation of this first embodiment of the invention, R ²¹ represents hydrogen, a halogen group, a straight or branched (C1-C6) alkyl group which may be substituted by one or more identical or different R ²⁵ groups, a hydroxyl group, (C1-C6) alkoxy group which may be substituted by one or more identical or different phenyl or R ²⁵ groups, thiol (-SH), (C1-C6) alkylthio, which may be substituted by one or more identical or different phenyl or R ²⁵ groups, amine (- NH ₂ ), —NHR ²⁶ or —NR ²⁶ R ²⁶ ; each of R ²² and R ²³ is, independently from each other, unbranched or branched (C1-C6) alkyl, which may be substituted with one or more identical or different R ²⁵ groups; R ^{25 is} selected from the group consisting of halogen, hydroxyl, (C1-C6) alkoxy, thiol, (C1-C6) alkylthio, (C1-C6) alkylamine and (C1-C6) dialkylamine; and each R ²⁶ independently is (C1-C6) alkyl, which may be substituted with one or more identical or different phenyl or R ²⁵ groups or —C (O) R ²⁷ , where R ²⁷ is (C1-C6) alkyl, which may be substituted by one or more identical or different phenyl or R ²⁵ groups.

In another specific embodiment of the first embodiment, R ²¹ is methoxy, which may be substituted with one or more identical or different halo groups and / or each of R ²² and R ²³ , independently of one another, is methyl or ethyl, which may be substituted by one or more identical or different halogen groups.

In a second embodiment of the invention, compounds with structural formulas (I) and (Ia) R ² , R ⁴ , R ⁵ and L ² are the groups previously described in the definition of structural formulas (I) and (Ia), L ¹ represents one a direct bond, and R ⁶ is hydrogen, provided that R ^{2 is} not 3,4,5-trimethoxyphenyl, 3,4,5-tri (C1-C6) alkoxyphenyl or

where R ²¹ , R ²² and R ²³ , as defined above, are associated with the first implementation of the invention.

In a third embodiment of the invention, one or more of the following compounds are excluded from the structural formulas (I) and (Ia) of 2,4-pyrimidiamine compounds:

N2, N4-bis (4-ethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine (R070790);

N2, N4-bis (2-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine (R081166);

N2, N4-bis (4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine (R088814);

N2, N4-bis (2-chlorophenyl) -5-fluoro-2,4-pyrimidinediamine (R088815);

N2, N4-bisphenyl-5-fluoro-2,4-pyrimidinediamine (R091880);

N2, N4-bis (3-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine (R092788);

N2, N4-bis (3-chlorophenyl) -5-fluoro-2,4-pyrimidinediamine (R067962);

N2, N4-bis (2,5-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine (R067963);

N2, N4-bis (3,4-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine (R067964);

N2, N4-bis (4-chlorophenyl) -5-fluoro-2,4-pyrimidinediamine (R0707153);

N2, N4-bis (2,4-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine (R070791);

N2, N4-bis (3-bromophenyl) -5-fluoro-2,4-pyrimidinediamine (R008958);

N2, N4-bis (phenyl) -5-fluoro-2,4-pyrimidinediamine;

N2, N4-bis (morpholino) -5-fluoro-2,4-pyrimidinediamine;

N2, N4-bis [(3-chloro-4-methoxyphenyl)] - 5-fluoro-2,4-pyrimidinediamine.

In a fourth embodiment of the invention, compounds in accordance with the following structural formula (Ib) are excluded from the composition of structural formulas (I) and (Ia):

in which R ²⁴ - (C1-C6) alkyl, and R ²¹ , R ²² and R ²³ , as defined above, are associated with the first implementation of the invention.

In a fifth embodiment of the invention, the compounds described in Examples 1-141 of US Pat. No. 6,235,746, the disclosure of which is incorporated herein by reference, are excluded from the composition of structural formulas (I) and (Ia).

In a sixth embodiment of the invention, compounds which are defined by formula (1) or formula 1 (a) of this US patent No. 6,235,746 are excluded from the composition of structural formulas (I) and (Ia) (see, for example, the description from line 48 of column 1 to rows 49 of column 7 and rows 9-36 of column 8, which is incorporated herein by reference).

In a seventh embodiment of the invention, compounds in which R ⁵ is cyano or —C (O) NHR, where R is hydrogen or (C1-C6) alkyl when R ^{2 is} substituted with phenyl, are excluded from the composition of structural formulas (I) and (Ia) ; R4 is substituted or unsubstituted (C1-C6) alkyl, (C ₃ -C ₈ ) cycloalkyl, a 3-8 membered cycloheteralkyl or 5-15 membered heteroaryl, and R ⁶ is hydrogen.

In an eighth embodiment of the invention, compounds which are defined by formulas (I) and (X) WO 02/04429 or any compound described in WO 02/04429, the disclosure of which is incorporated herein by reference, are excluded from the composition of structural formulas (I) and (Ia) .

In the ninth implementation of the components of structural formulas (I) and (Ia), when R ⁵ is cyano or —C (O) NHR, where R is hydrogen or (C1-C6) alkyl and R ⁶ is hydrogen, then R ² is different from substituted phenyl group.

In a tenth embodiment, those compounds are excluded from structural formulas (I) and (Ia) in which R ² and R ⁴ , each independently of one another, are a substituted or unsubstituted pyrrole or indole ring that is attached to the rest of the molecule through its ring nitrogen atom.

In an eleventh implementation, compounds that are defined by formulas (I) and (IV) shown in US Pat. No. 4,983,608 or any compound described in US Pat. No. 4,983,608, which are excluded from the composition of structural formulas (I) and (Ia), are excluded included in the links of this application.

Experts in this field will be clear that in the composition of formulas (I) and (Ia), R ² and R ⁴ can be the same or different and the degree of difference can vary widely. When R ² and / or R ⁴ are rings that can be substituted, such as optionally substituted cycloalkyls, cycloheteroalkyls, aryls, and heteroaryls, the ring can be attached to the rest of the molecule using any available carbon atom or heteroatom. Additional substituents may be attached to any available carbon atoms and / or heteroatoms.

In a twelfth embodiment, a composition of structural formulas (I) and (Ia) R ² and / or R ⁴ is phenyl or (C5-C15) aryl, which may be substituted, provided that: (1) if R ⁶ is hydrogen, then R ² - is not 3,4,5-trimethoxyphenyl or 3,4,5-tri (C1-C6) alkoxyphenyl; (2) if R ² is 3,4,5-trisubstituted phenyl, then the substituents at the 3- and 4-positions are not simultaneously methoxy or (C1-C6) alkoxy groups; or (3) if R ⁶ is hydrogen and R ⁴ is (C1-C6) alkyl, (C ₃ -C ₈ ) cycloalkyl, 3-8 membered cycloheteroalkyl or 5-15 membered heteroaryl, then R ^{5 is} not cyan. Otherwise, R ² is subject to the reservation given in connection with the first or second implementation examples. Optionally, a substituted aryl or phenyl group may be attached to the rest of the molecule via any available carbon atom. Specific examples of phenyls that can be substituted include phenyls, which may be one-, two- or trisubstituted with the same or different R ⁸ groups, where R ⁸ corresponds to its previously set value for structural formula (I) and the above reservations. If phenyl is monosubstituted, the substituent R ⁸ may be in the ortho , meta, or para position. When in the ortho , meta, or para position, R ^{8 is} preferably selected from the group consisting of (C1-C10) alkyl, (C1-C10) branched alkyl, -OR ^a , which can be substituted with one or more identical or different R ^b groups, -OC (O) OR ^a , -O- (CH ₂ ) _m -C (O) OR ^a , -C (O) OR ^a , -O- (CH ₂ ) _m -NR ^c R ^c , -OC (O) NR ^c R ^c , -O- (CH ₂ ) _m -C (O) NR ^c R ^c , -OC (NH) NR ^c R ^c , -O- (CH ₂ ) _m -C (NH ) NR ^c R ^c and -NH- (CH ₂ ) _m -NR ^c R ^c , where m , R ^a and R ^c correspond to the designations previously established for structural formula (I). In the implementation of these components, —NR ^c R ^c represents a 5-6 membered heteroaryl, which may include one or more identical or different additional heteroatoms. Specific examples of such 5-6 membered heteroaryls include, but are not limited to, oxadiazolyl, triazolyl, thiazolyl, oxazolyl, tetrazolyl and isoxazolyl.

In another example implementation of these components, —NR ^c R ^c is a 5-6 membered saturated cycloheteroalkyl ring that further includes one or more identical or different heteroatoms. Specific examples of such cycloheteroalkyls include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, piperidinyl, piperazinyl and morpholinyl.

In yet another embodiment of these components, each R ^a independently is (C1-C6) alkyl and / or each —NR ^c R ^c is —NHR ^a , where R ^a - is (C1-C6) alkyl. In one specific embodiment, R ⁸ represents an —O — CH ₂ —C (O) NHCH ₃ group. In another specific embodiment, R ⁸ is an —OH group.

If phenyl is bis or trisubstituted, R ⁸ substituents may be in any combination of positions. For example, R ⁸ substituents may be located at 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, 2,3,4-, 2,3, 5-, 2,3,6-, 2,4,5-, 2,4,6-, 2,5,6- or 3,4,5-. In one embodiment of the compound, including disubstituted phenyl, the substituents are located differently than in positions 3.4. In another example implementation, they are located in positions 3.4. In one embodiment of a compound including trisubstituted phenyl, the substituents are located differently from 3,4,5, or, alternatively, none of the two substituents is located at 3,4. In another example implementation, substituents are located at positions 3,4,5.

Specific examples of R ⁸ substituents in such disubstituted and trisubstituted phenyls include various R ⁸ substituents described above in connection with ortho, meta, and para substituted phenyls.

In another specific embodiment example, R ^8- substituents suitable for substituting for such di- and trisubstituted phenyls include (C1-C6) alkyl, (C1-C6) alkoxy, methoxy, halo, chloro, (C1-C6) perhaloalkyl, -CF ₃ , (C1-C6) perhaloalkoxy and -OCF ₃ . In a preferred case, such R ⁸ substituents are located at positions 3.4 or 3.5. Specific examples of preferred disubstituted phenyl rings include 3-chloro-4-methoxyphenyl, 3-methoxy-4-chlorophenyl, 3-chloro-4-trifluoromethoxyphenyl, 3-trifluoromethoxy-4-chlorophenyl, 3,4-dichlorophenyl, 3,4-dimethoxyphenyl and 3,5-dimethoxyphenyl, provided that: (1) if R ⁴ is one of the above phenyls and each of R ⁵ and R ⁶ is hydrogen, then R ^{2 is} not 3,4,5-three (C1- C6) alkoxyphenyl or 3,4,5-trimethoxyphenyl; (2) if R ² is 3,4-dimethoxyphenyl, and each of R ⁵ and R ⁶ is hydrogen, then R ^{4 is} not 3- (C1-C6) alkoxyphenyl, 3-methoxyphenyl, 3,4-di- (C1-C6) alkoxyphenyl or 3,4-dimethoxyphenyl; (3) if R ⁴ represents 3-chloro-4-methoxyphenyl and R ⁵ represents a halo or fluoro group, and further (optionally) R ⁶ represents hydrogen, then R ^{2 is} not 3-chloro-4- (C1 -C6) alkoxyphenyl or 3-chloro-4-methoxyphenyl; (4) if R ⁴ is 3,4-dichlorophenyl, R ⁵ is hydrogen, (C1-C6) alkyl, methyl, halogen or chloro and additionally R ⁶ is hydrogen, then R ^{2 is} not phenyl, monosubstituted in the para- position (C1-C6) alkoxy group, which may be substituted by one or more identical or different groups R ^b , -OH or -NR ^c R ^c , where R ^b and R ^c correspond to their previously established symbols for the structural formula ( I); and / or (5) R ² and / or R ^{4 is} not 3,4,5-tri (C1-C6) alkoxyphenyl or 3,4,5-trimethoxyphenyl, especially if each of R ⁵ and R ⁶ is hydrogen itself.

In another example implementation of the proposed compounds, including trisubstituted phenyl, the latter has the formula:

in which R ³¹ represents methyl or (C1-C6) alkyl; R ³² represents hydrogen, methyl or (C1-C6) alkyl and R ³³ represents a halogen group.

In a thirteenth example of the implementation of the proposed compounds with structural formulas (I) and (Ia), R ² and / or R ⁴ are heteroaryl, which may be substituted. Typical heteroaryl groups corresponding to this thirteenth implementation example contain from 5 to 15, and more typical from 5 to 11 ring atoms and include one, two, three or four identical or different heteroatoms or groups of heteroatoms selected from the group consisting of N , NH, O, S, S (O) and S (O) ₂ . Heteroaryl, which can be substituted, can be attached to its corresponding nitrogen atom, C2 or C4, or to the linker L ¹ or L ² via any available carbon atom or heteroatom, but usually it is attached via a carbon atom. Additional substituents may be the same or different and may be attached to any available carbon atom or heteroatom. In one example of the implementation of these structures, R ^{5 is} not bromo, nitro or trifluoromethyl, as well as cyano or

A —C (O) NHR group, where R is hydrogen or (C1-C6) alkyl. In another example of the implementation of these structures, if each of R ² and R ⁴ is a substituted or unsubstituted pyrrole or indole, then the ring is attached to the rest of the molecule via a ring carbon atom. In another example of the implementation of the proposed structures, including optionally substituted heteroaryl group, heteroaryl is not unsubstituted or substituted by the same or different R ⁸ groups in an amount of one to four, where R ⁸ corresponds to its previously determined value in structural formula (I). Specific examples of such heteroaryls that can be substituted include, but are not limited to, the following groups of heteroaryls:

in which

p is an integer from one to three;

each symbol - - - independently represents a single or double bond;

R ³⁵ is hydrogen or R ⁸ , where R ⁸ corresponds to its value, which was previously determined for structural formula (I);

X is selected from the group consisting of CH, N and N-O;

each Y is independently selected from the group consisting of O, S, and NH;

each Y ^{1 is} independently selected from the group consisting of O, S, SO, SO ₂ , SONR ³⁶ , NH, and NR ³⁷ ;

each Y ^{2 is} independently selected from the group consisting of CH, CH ₂ , O, S, N, NH, and NR ³⁷ ;

R ³⁶ is hydrogen or alkyl;

R ^{37 is} selected from the group consisting of hydrogen and a progroup, preferably hydrogen or a progroup selected from the group consisting of aryl, arylalkyl, heteroaryl, R ^a , R ^b —CR ^a R ^b —OC (O) R ⁸ , —CR ^a R ^b —O — PO (OR ⁸ ) ₂ , —CH ₂ —O — PO (OR ⁸ ) ₂ , —CH ₂ —PO (OR ⁸ ) ₂ , —C (O) —CR ^a R ^b —N ( CH ₃ ) ₂ , —CR ^a R ^b —OC (O) —CR ^a R ^b —N (CH ₃ ) ₂ , —C (O) R ⁸ , —C (O) CF _3, and —C (O) - NR ⁸ -C (O) R ⁸ ;

A is selected from the group consisting of O, NH, and NR ³⁸ ;

R ^{38 is} selected from the group consisting of alkyl and aryl;

R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are each independently selected from the group consisting of alkyl, alkoxy, halogen, haloalkoxy, aminoalkyl and hydroalkyl, or, alternatively, R ⁹ and R ¹⁰ and / or R ¹¹ and R ¹² taken together from ketal;

each Z is selected from the group consisting of hydroxyl, alkoxy, aryloxy, ester, carbamate and sulfonyl;

Q is selected from the group consisting of —OH, OR ⁸ , —NR ^c R ^c , —NHR ³⁹ —C (O) R ⁸ , —NHR ³⁹ —C (O) OR ⁸ , —NR ³⁹ —CHR ⁴⁰ —R ^b , -NR ³⁹ - (CH ₂ ) _m -R ^b and -NR ³⁹ -C (O) -CHR ⁴⁰ -NR ^c R ^c ;

R ³⁹ and R ⁴⁰ , each independently from each other, are selected from the group consisting of hydrogen, alkyl, aryl, alkylaryl; arylalkyl and NHR ⁸ ; and

R ^a , R ^b and R ^c correspond to their designations previously defined for structural formula (I). Preferred R ^b substituents for Q are selected from —C (O) OR ⁸ , —OC (O) R ⁸ , —OP (O) (OR ⁸ ) _2, and —P (O) (OR ⁸ ) ₂ .

In one example of the implementation of the above heteroaryls, as well as other 5-15 membered heteroaryls corresponding to this implementation of the invention, each R ⁸ is independently selected from the group consisting of R ^d , -NR ^c R ^c , - (CH ₂ ) _m - NR ^c R ^c , -C (O) NR ^c R ^c , - (CH ₂ ) _m -C (O) NR ^c R ^c , -C (O) OR ^d , - (CH ₂ ) _m -C (O) OR ^d and - (CH ₂ ) _m —OR ^d , where m , R ^c and R ^d correspond to their designations previously defined for structural formula (I).

In a specific embodiment, R ^d and / or R ^c are selected from the group consisting of R ^a and (C3-C8) cycloalkyl, which may be substituted with one or more of the same or other hydroxyl, amino or carboxyl groups.

, где x=1-8, -CH₂CONHMe, -CH₂CH₂NHMe, In another example implementation of the above heteroaryls, each R ³⁵ represents a hydrogen atom, a carbon chain (C1-C6), which includes methyl, ethyl, isopropyl, cycloalkyl group, which includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or group

where x = 1-8, -CH ₂ CONHMe, -CH ₂ CH ₂ NHMe,

-CH ₂ CH ₂ CONHMe; -CH ₂ CH ₂ CH ₂ NHMe; or -CH ₂ CH ₂ CH ₂ OCH ₃ .

In yet another embodiment of the above heteroaryls, the aromatic ring connectivity is at position 5 or 6. It is important to understand that any of R ² or R ⁴ may use the heteroaryl groups described herein.

In the fourteenth example of the implementation of the composition of structural formulas (I) and (Ia), each R ² and R ⁴ , independently of one another, is phenyl, aryl or heteroaryl, which may be substituted, provided that: (1) if L ¹ represents a direct bond and R ⁶ (and possibly R ⁵ ) represents hydrogen, then R ^{2 is} not 3,4,5-trimethoxyphenyl or 3,4,5-tri (C1-C6) alkoxyphenyl; (2) if both L ¹ and L ² are direct bonds, R ⁶ is hydrogen and R ⁵ is a halogen group, then R ² and R ⁴ are not 3,4,5-trimethoxyphenyl or 3,4,5- tri (C1-C6) alkoxyphenyl; (3) if R ⁴ is 3-methoxyphenyl or 3- (C1-C6) alkoxyphenyl and R ² is 3,4,5-trisubstituted phenyl, then the substituents located at positions 3 and 4 are not simultaneously methoxy or ( C1-C6) alkoxy; (4) if R ² is substituted phenyl and R ⁶ is hydrogen, then R ^{5 is} not a cyano or —C (O) NHR group, where R is hydrogen or (C1-C6) alkyl; and / or (5) if R ² and R ⁴ are each independently or independently substituted or unsubstituted pyrrole or indole, then the latter are attached to the rest of the molecule via a ring carbon atom. Alternatively, R ^{2 is} subject to the comments set forth in connection with the first or second embodiments of the present invention.

In this fourteenth embodiment, the substituents R ² and R ⁴ may be the same or different. Particular phenyl, aryl and / or heteroaryls that may be substituted include those that are illustrated above in the twelfth and thirteenth implementation examples.

In the fifteenth example of the implementation of the composition of structural formulas (I) and (Ia), including the above-described first to fourteenth embodiments, R ⁶ is hydrogen and R ⁵ is an electronegative group. As will be understood by experts in this field, electronegative groups are atoms or groups of atoms with a relatively strong ability to attract electrons to themselves. Specific examples of electronegative groups in accordance with the fourteenth implementation example considered include, but are not limited to, —CN, —NC, —NO ₂ , halogen, bromo, chloro, fluoro groups, (C1-C3) haloalkyl, (C1- C3) perhaloalkyl, (C1-C3) fluoroalkyl, (C1-C3) perfluoroalkyl, -CF ₃ , (C1-C3) haloalkoxy, (C1-C3) perhaloalkoxy, (C1-C3) fluoroalkoxy, (C1-C3) perfluoro —OCF ₃ , —C (O) R ^a , —C (O) OR ^a , —C (O) CF _3, and —C (O) OCF ₃ groups. In this particular case, an electronegative group is an electronegative group containing a halogen, such as, for example, —OCF ₃ , —CF ₃ , bromo, chloro or fluoro groups. In another specific case, R ⁵ represents a fluoro group, provided that in accordance with the third example of implementation, the composition cannot be arbitrary.

In a sixteenth exemplary embodiment, compounds with structural formulas (I) and (Ia) correspond to compounds having structural formula (Ib):

and their salts, hydrates, solvates and N-oxides, where R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , all independently of one another, are selected from the group consisting of hydrogen, hydroxy group, (C1-C6) alkoxy group and -NR ^c R ^c ; and R ⁵ , R ⁶ and R ^c correspond to the previous definition for structural formula (I), provided that if each of R ¹³ , R ⁵ and R ⁶ is hydrogen, then R ¹¹ and R ¹² are not simultaneously a methoxy group, (C1 -C6) alkoxy; or (C1-C6) haloalkoxy.

In a seventeenth exemplary embodiment, compounds with structural formulas (I) and (Ia) are compounds corresponding to structural formula (Ic):

and their salts, hydrates, solvates and N-oxides, where

R ^{4 is} selected from the group consisting of 5-10 membered heteroaryl and 3-hydroxyphenyl;

R ⁵ is F or —CF ₃ ; and

R ⁸ is —O (CH ₂ ) _m —R ^b , where m and R ^{b are} as previously defined for structural formula (I). In a specific implementation, R ⁸ is —O — CH ₂ —C (O) NH — CH ₃ and / or R ⁴ is heteroaryl according to a thirteenth embodiment of the present invention.

In an eighteenth exemplary embodiment, compounds with structural formulas (I) and (Ia) include any compounds selected from Table 1 that suppress the Fc receptor signal transduction cascade, Syk kinase activity, Syk kinase-dependent receptor signal transduction cascade or degranulation of cells, measured in vitro , provided that compounds with the indicated structural formulas are not compounds excluded above in the description of the third and / or other implementation examples. In this particular case, such compounds have an IC _{50 of the} order of 20 μm or less as measured by cell degranulation during an in vitro assay , such as, for example, the degranulation assay described in the Examples section.

In a nineteenth exemplary embodiment, compounds with structural formulas (I) and (Ia) include any compounds selected from Table 1 that suppress the FcγR1 or FcεR1 receptor cascade with an IC _{50 of the} order of 20 μm or less according to the results of an in vitro assay , such as for example, the degranulation analysis described in the Examples section, with the optional condition that compounds with the indicated structural formulas are not compounds excluded above in the description of the third and / or other implementation examples.

In a twentieth example of the implementation of the compounds with structural formula (Ia) include compounds in which R ² selected from the groups consisting of

R ⁴ , R ⁸ , R ^a , R ^b , R ^c , R ^d correspond to those described above, R ⁵ represents a fluorine atom, R ⁶ represents a hydrogen atom and each of R ^{21 is} independently a halogen atom or an alkyl which, at the discretion, may be substituted one or more similar or different halo groups. The groups R ²² and R ²³ , each independently of each other, represent a hydrogen atom, methyl or ethyl, which, at the discretion, can be replaced by one or more similar or different halo groups, each value of m independently represents an integer from 1 to 3 and each n value independently represents an integer from 0 to 3.

, в которой R⁹ и R¹⁰ соответствуют описанным выше и дополнительно каждая в отдельности включают в себя атом водорода, группа R² представляет собой фенильную группу, замещенную одной или более аналогичными группами R⁸, или , где R³⁵ соответствует вышеописанной. В одном конкретном случае, когда R² представляет собой фенильную группу, одна или более R⁸ выбраны из галогена и алкоксигруппы. В одном из примеров фенильная группа является дву- или тризамещенной одной или несколькими идентичными группами R⁸.In a twenty-first example implementation of compounds with structural formula (Ia) include compounds in which R ⁴ represents a group

in which R ⁹ and R ¹⁰ correspond to those described above and additionally each individually includes a hydrogen atom, the group R ² represents a phenyl group substituted by one or more similar R ⁸ groups, or where R ³⁵ corresponds to the above. In one specific case, when R ² represents a phenyl group, one or more R ⁸ selected from halogen and alkoxygroup. In one example, the phenyl group is bis or trisubstituted by one or more identical R ⁸ groups.

и R² представляет фенильную группу, замещенную одной или несколькими идентичными группами R⁸. В одном конкретном случае одна или более R⁸ выбраны из галогена и алкоксигруппы. В одном из примеров фенильная группа является дву- или тризамещенной одной или несколькими идентичными группами R⁸.In a twenty-second example implementation of a compound with structural formula (Ia) include compounds in which R ⁴ represents a group

and R ² represents a phenyl group substituted with one or more identical R ⁸ groups. In one specific case, one or more R ⁸ selected from halogen and alkoxygroup. In one example, the phenyl group is bis or trisubstituted by one or more identical R ⁸ groups.

, в которой R³⁵ соответствует вышеописанной группе. В конкретных примерах фенильная группа R⁴ является дву- или тризамещенной идентичными или различными атомами галогенов. В другом примере R⁴ является монозамещенной группой с атомом галогена. В одном из примеров R³⁵ является гидроксиалкильной группой. В определенных случаях, гидроксиалкильная группа может быть дополнительно превращена в эфирную группу, карбамат, и т.д.In a twenty-third embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ represents a phenyl group substituted by one or more identical R ⁸ groups, where R ² represents

in which R ³⁵ corresponds to the above group. In specific examples, the phenyl group R ⁴ is disubstituted or trisubstituted by identical or different halogen atoms. In another example, R ⁴ is a monosubstituted group with a halogen atom. In one example, R ³⁵ is a hydroxyalkyl group. In certain instances, the hydroxyalkyl group may be further converted to an ester group, carbamate, etc.

, в которой группа R³⁵ соответствует описанной выше и R² представляет фенильную группу, замещенную одной или несколькими идентичными группами R⁸. В одном из примеров R³⁵ является атомом водорода или алкильной группой. В другом примере фенильная группа R² является дву- или тризамещенной одной или несколькими идентичными группами R⁸ и, в частности, атомами галогенов.In a twenty-fourth embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ is

in which the group R ³⁵ corresponds to that described above and R ² represents a phenyl group substituted by one or more identical R ⁸ groups. In one example, R ³⁵ is a hydrogen atom or an alkyl group. In another example, the phenyl group R ² is disubstituted or trisubstituted by one or more identical R ⁸ groups and, in particular, halogen atoms.

, в которой группа R³⁵ соответствует описанной выше и R² представляет собой

, в которой R⁹ и R¹⁰ соответствуют описанным выше и дополнительно каждая в отдельности включают атом водорода. В одном из примеров R³⁵ является атомом водорода или алкильной группой, например метилом, и R⁹ и R¹⁰ представляют алкильные группы, например группы метила.In a twenty-fifth embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ is

in which the group R ³⁵ corresponds to that described above and R ² represents

in which R ⁹ and R ¹⁰ correspond to those described above and additionally each individually includes a hydrogen atom. In one example, R ³⁵ is a hydrogen atom or an alkyl group, for example methyl, and R ⁹ and R ¹⁰ represent alkyl groups, for example methyl groups.

, в которой группа R³⁵ соответствует описанной выше. В конкретных случаях фенильная группа замещена атомом галогена и алкоксигруппой, например, метоксигруппой. В определенных примерах, R³⁵ представляет атом водорода, алкильную группу, например метилгруппу, или гидроксиалкильную группу. В конкретных случаях, гидроксиалкильная группа может быть преобразована в эфирную группу, карбамат и т.д.In a twenty-sixth embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ represents a disubstituted phenyl group substituted by identical or different R ⁸ groups, and R ² represents

in which the group R ³⁵ corresponds to that described above. In specific cases, the phenyl group is substituted by a halogen atom and an alkoxy group, for example, a methoxy group. In certain examples, R ³⁵ represents a hydrogen atom, an alkyl group, for example a methyl group, or a hydroxyalkyl group. In specific cases, the hydroxyalkyl group may be converted to an ester group, carbamate, etc.

, в которых R⁸ и R^с соответствуют описанным выше, и R² представляет фенильную группу, замещенную одной или более идентичными R⁸ группами. В одном конкретном случае R^с является атомом водорода или алкильной группой. В другом случае фенильная группа R² является дву- или тризамещенной одной или несколькими идентичными группами R⁸ и, в частности, атомами галогенов или

.In a twenty-seventh exemplary embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ is

in which R ⁸ and R ^c correspond to those described above, and R ² represents a phenyl group substituted by one or more identical R ⁸ groups. In one specific case, R ^c is a hydrogen atom or an alkyl group. In another case, the phenyl group R ² is disubstituted or trisubstituted by one or more identical R ⁸ groups and, in particular, halogen atoms or

.

, в которой Y¹, Y² и каждая из групп R³⁵ независимо друг от друга соответствуют описанным выше, и R² представляет собой

, в которой R³⁵ соответствует описанной выше. В одном случае двадцать восьмого примера реализации относительно R⁴ Y¹ представляет собой кислород, Y² представляет собой NH и одна или более групп R³⁵ или часть R⁴ являются алкильной группой и, в частности, группой метила. В конкретных случаях двадцать восьмого примера реализации две группы R³⁵, входящие в состав R⁴, образуют гемдиалкилкомпонент и, в частности, гемдиметил компонент, расположенный по соседству с NH группой, представленной в виде

. В конкретных случаях двадцать восьмого примера реализации, касающихся группы R², R³⁵ представляет собой атом водорода или алкильную группу, в частности группу метила.In a twenty-eighth exemplary embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ is

in which Y ¹ , Y ² and each of the groups R ³⁵ independently from each other correspond to those described above, and R ² represents

in which R ³⁵ corresponds to that described above. In one case of a twenty-eighth embodiment, with respect to R ^4, Y ¹ is oxygen, Y ² is NH, and one or more R ³⁵ groups or part of R ⁴ are an alkyl group and, in particular, a methyl group. In specific cases of the twenty-eighth example of implementation, the two groups of R ³⁵ that are part of R ⁴ form a hemodialkyl component and, in particular, a hedimethyl component located adjacent to the NH group represented in the form

. In specific instances of the twenty-eighth exemplary embodiment relating to the R ² group, R ³⁵ represents a hydrogen atom or an alkyl group, in particular a methyl group.

, в которой R⁹ и R¹⁰ соответствуют вышеописанным или представляют замещенную фенильную группу. В одном случае фенильная группа является дву- или тризамещенной одной или несколькими идентичными группами R⁸. В частности, фенильная группа может быть дву- или тризамещенной одной или несколькими атомами галогенов, которые могут быть идентичными или различными. R² в двадцать девятом примере реализации представляет собой

, в которой R³⁵ соответствует вышеописанной. В одном из случаев двадцать девятого примера реализации R³⁵ группы R² не является метилом. В другом случае двадцать девятого примера реализации R² является

.In a twenty-ninth embodiment, compounds of structural formula (Ia) include compounds in which R ⁴ is

in which R ⁹ and R ¹⁰ correspond to the above or represent a substituted phenyl group. In one case, the phenyl group is bis or trisubstituted by one or more identical R ⁸ groups. In particular, the phenyl group may be two- or trisubstituted by one or more halogen atoms, which may be identical or different. R ² in the twenty-ninth example implementation is

in which R ³⁵ corresponds to the above. In one instance of a twenty-ninth embodiment, R ³⁵ of an R ² group is not methyl. In another case of the twenty-ninth example implementation of R ² is

.

In the thirtieth implementation example applicable to the first to twenty-ninth implementation examples, R ⁵ represents a halogen atom, for example fluorine, and R ⁶ represents a hydrogen atom.

Also specifically described are combinations of the above examples of specific implementations of the present invention from the first to the thirtieth.

Those skilled in the art will appreciate that the 2,4-pyrimidinediamine compounds described herein may include functional groups that can be masked by progroups to create prodrugs. Such prodrugs are usually, but not necessarily, pharmacologically inactive until they transition to their active dosage form. Indeed, many of the compounds of active 2,4-pyrimidinediamine described in Table. 1 include compounds that are hydrolyzable or otherwise cleaved under conditions of use. For example, ester groups typically undergo acid-catalyzed hydrolysis to produce the starting carboxylic acid if they are under the conditions of the acidic environment of the stomach, or undergo base-catalyzed hydrolysis if they are in the basic environment of the intestine or blood. Thus, when administered orally, 2,4-pyrimidinediamines, which include esters, can be considered prodrugs of the corresponding carboxylic acid, regardless of whether the ester has a pharmacologically active form. Based on the data in Table 1, the numerous 2,4-pyrimidinediamines of the present invention containing an ester are active in the form of their ester, in the form of a “prodrug”.

In the prodrugs of the subject invention, any functional component present can be masked by the progroup to obtain the prodrug. Functional groups in 2,4-pyrimidinediamine compounds that can be masked by progroups for inclusion in the procomponent include, but are not limited to, amines (primary and secondary), hydroxyls, sulfonyls (thiols), carboxyls, etc. Countless progroups are known in practice that are suitable for masking such functional groups to produce procomponents that cleave under preferred conditions of use. All of these progroups, alone or in combination, may be included in the prodrugs of the present invention.

In one illustrative embodiment, the prodrugs of the present invention are compounds according to structural formula (I) in which R ^c and R ^d may be a progroup in addition to the alternative options previously described.

Substitution of hydrogen atoms attached to N2 and N4 in 2,4-pyrimidinediamines with the structural formula (I) with substituents has the opposite effect on the activity of the components. However, as those skilled in the art will appreciate, these nitrogen atoms can be included in the compounds, which, under the conditions of use, cleave to form 2,4-pyrimidinediamines in accordance with structural formula (I). Thus, in another example implementation of the prodrugs according to the present invention are compounds corresponding to structural formula (II):

including salts, hydrates, solvates and N-oxides, in which

R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ and L ² correspond to the previous definition of structural formula (I); and

R ^2b and R ^4b , each independently from each other, are a progroup. Specific examples of progroups in accordance with this embodiment include, but are not limited to, (C1-C6) alkyl, —C (O) CH ₃ , —C (O) NHR ^36, and -S (O) ₂ R ³⁶ , where R ³⁶ is (C1-C6) alkyl, (C5-C15) aryl and (C3-C8) cycloalkyl.

In prodrugs with structural formula (II), substituents can be selected in accordance with the foregoing when considering various first through twentieth embodiments for structural formulas (I) and (Ia) or combinations of such examples.

It is understood that many of the compounds and prodrugs of the present invention, as well as various samples of the compounds specifically described and / or illustrated herein, can exhibit tautomerism, conformational isomerism, geometric isomerism and / or optical isomerism. For example, the compounds and prodrugs of the present invention may include one or more chiral centers and / or double bonds and, as a consequence, can exist as stereoisomers, such as, for example, double bond isomers (i.e. geometric isomers), enantiomers and diastereoisomers and mixtures thereof, such as racemic mixtures. As another example, the compounds and prodrugs of the present invention can be considered, which may exist in several tautomeric forms, including the enol form, the ketol form, and a mixture thereof. As various names, the formulas and structures of the compounds in the specification and applications can represent only one of the possible tautomeric forms, forms of conformational isomerism, optical isomerism or geometric isomerism. It should be understood that the invention includes any tautomeric forms, any forms of conformational isomerism, optical isomerism and / or geometric isomerism of compounds and prodrugs having one or more of the advantages described herein, as well as mixtures of these various different isomeric forms. In cases of limited rotation around the structure of the 2,4-pyrimidinediamine core, the formation of atropic isomers becomes possible, which are also specifically included in the compounds of the present invention.

In addition, specialists in this field should understand that when alternative members are included in lists that, due to valency or for other reasons, cannot be used to replace a specific group, only those members of the list that are suitable to replace a specific group should be used. For example, it is obvious that while all of the listed alternative substituents for R ^b can be used to replace the alkyl group, some of the alternative substituents, such as = O, cannot be used to replace the phenyl group. It should be noted that this refers only to possible combinations of pairs of substituent groups.

The compounds and / or prodrugs of the present invention can be identified according to their chemical structure or chemical name. If the chemical structure and chemical name contradict each other, the chemical structure is crucial in identifying a particular compound.

Depending on the nature of the various substituents of the compound and prodrug, the 2,4-pyrimidinediamine constituting the invention may take the form of salts. Such salts include salts suitable for pharmaceutical use (“pharmaceutically acceptable salts”), salts suitable for veterinary use, etc. Such salts can be obtained from acids or bases, which is well known in practice.

In one embodiment, the salt is pharmaceutically acceptable. Typically, pharmaceutically acceptable salts are those salts that retain a substantial portion of one or more of the desired pharmaceutical functions of the parent component and which are suitable for human administration. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids or organic acids. Inorganic salts suitable for forming pharmaceutically acceptable salts with an acid supplement include, but are not limited to, for example, the following: hydrohalide acids (e.g. hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid acid, phosphoric acid and the like. Organic acids suitable for the formation of pharmaceutically acceptable salts with an acid additive include, for example, but are not limited to: acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentane propionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic (hexadecanoic) acid, benzoic acid that, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic (B-phenylacrylic) acid, mandelic acid, alkyl sulfonic acids ( e.g. methanesulfonic acid, ethanesulfonic acid, 1,2-ethane disulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acid ( e.g. , benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenedisulfonic acid, 4-toluenesulfonic acid, cycloalkyl sulfonic acids (e.g. camphorsulfonic acid), 4-methylbicyclo [2.2.2] oct-2-ene-1-carboxylic carboxylic acid 3-phenylpropionic acid acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxy naphtha acid, salicylic acid, stearic (octadecanoic) acid, muconic acid and the like.

Pharmaceutically acceptable salts also include salts formed by replacing the proton of an acid present in the parent compound with a metal ion (e.g., an alkali, alkaline earth metal or aluminum ion), an ammonium ion, or by coordination with an organic base ( e.g. , ethanolamine, diethanolamine , triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine, etc.).

Compounds of 2,4-pyrimidinediamine according to the present invention, as well as their salts, can be in the form of hydrates, solvates and N-oxides, well known in practice.

6.3. Synthesis Methods

The compounds and prodrugs of the invention can be synthesized in various ways using commercially available starting materials and / or starting materials obtained by conventional synthetic methods. Various methods that can be used to synthesize the components of 2,4-pyrimidinediamine and prodrugs of the present invention are described in US Pat. No. 5,958,935, referred to in this application. Specific examples describing the synthesis of numerous components and prodrugs according to the present invention, as well as their intermediates, are presented in the Examples section. All compounds having structural formulas (I), (Ia) and (II) can be obtained by a simple modification of these methods.

A variety of possible synthetic routes that can be used to synthesize the 2,4-pyrimidinediamine compounds of the present invention are described below in Schemes (I) to (XI). In schemes (I) to (XI), compounds with the same numbers have similar structures. These methods can traditionally be used to synthesize prodrugs corresponding to structural formula (II).

In one embodiment, the compounds may be synthesized from substituted or unsubstituted uracils or thiouracils, as shown below in Scheme (I):

Scheme (I)

In Scheme (I), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ and L ² correspond to the previous definition of structural formula (I), X is halogen ( e.g. , F, Cl, Br or I), and Y and Y ', each independently from each other, selected from the group consisting of O and S. Starting from scheme (I), uracil or thiouracil 2 is dihalogenated at the 2- and 4-positions using the standard halogenating agent POX ₃ (or another standard halogenating agent) under standard conditions to produce 2,4-bisgalpirimidina 4. depending upon the R ⁵ substituent in pyrimidine 4, the halide at C4 position is more than D su- relative to the electron-donor substances than halide in C2 position. This difference in reactivity can be used to synthesize 2,4-pyrimidinediamines corresponding to structural formula (I) by reacting 2,4-bishalo-pyrimidine 4 with one equivalent of amine 10 producing 4N-substituted-2-halo-4-pyrimidinamine 8, followed by amine 6 to obtain 2,4-pyrimidinediamine corresponding to structural formula (I). 2N, 4N-bis (substituted) -2,4-pyrimidinediamines 12 and 14 can be obtained by reaction of 2,4-bishalogenopyrimidine 4 with an excess of components 6 or 10, respectively.

In most cases, as shown in the diagram, C4 halide is more reactive with respect to electron-donor substances. However, as should be understood by those skilled in the art, the properties of the substituent R ⁵ may alter this reactivity. For example, if R ⁵ is trifluoromethyl, then a 50:50 mixture of 4N-substituted-4-pyrimidinamine 8 and the corresponding 2N-substituted-2-pyrimidinamine is formed as a result of the reaction. Regardless of what R ⁵ represents, the regioselectivity of the reaction can be controlled by selection of a solvent and other synthesis conditions (such as temperature), which is well known from practice.

The reactions illustrated in Scheme (I) can proceed faster if the reaction mixture is microwave heated. In this case, the following conditions can be used: heating to 175 ° C in ethanol for 5-20 minutes in a Smith reactor (manufactured by Personal Chemistry) in a sealed tube (at a pressure of 20 bar).

The starting materials uracil or thiouracil 2 can be purchased commercially or obtained using a standard technique used in organic chemistry. As an example of commercial uracils and thiouracils that can be used as starting materials for reactions according to Scheme (I), you can use uracil supplied by Aldrich (catalog number 13.078-8; CAS Registry 66-22-8); 2-thiouracil (Aldrich, No. 11,558-4; CAS Registry 141-90-2); 2,4-dithiouracil (Aldrich, No. 15.846-1; CAS Registry 2001-93-6); 5-acetouracil (Chem. Sources Int'l 2000, Registry CAS 6214-65-9); 5-azidouracil; 5-aminouracil (Aldrich No. 85.528-6; Registry CAS 932-52-5); 5-bromouracil (Aldrich No. 85.247-3; Registry CAS 51-20-7); 5- (trans-2-bromovinyl) -uracil (Aldrich No. 45.744-2; Registry CAS 69304-49-0); 5- (trans-2-chlorovinyl) -uracil (Registry CAS 81751-48-2); 5- (trans-2-carboxyvinyl) uracil; uracil-5-carboxylic acid (2,4-dihydroxypyrimidine-5-carboxylic acid hydrate; Aldrich No. 27,770-3; Registry CAS 23945-44-0); 5-chlororacil (Aldrich No. 22,458-8; Registry CAS 1820-81-1); 5-cyanouracil (Chem. Sources Int'l 2000; Registry CAS 4425-56-3); 5-ethyluracil (Aldrich No. 23,044-8; Registry CAS 4212-49-1); 5-ethenyluracil (Registry CAS 37107-81-6); 5-fluorouracil (Aldrich No. 85.847-1; Registry CAS 51-21-8); 5-iodouracil (Aldrich No. 85,785-8; Registry CAS 696-07-1); 5-methyluracil (thymine; Aldrich No. 13,199-7; Registry CAS 65-71-4); 5-nitrouracil (Aldrich No. 85,276-7; Registry CAS 611-08-5); uracil-5-sulfamic acid (Chem. Sources Int'l 2000; Registry CAS 5435-16-5); 5-trifluoromethyluracil (Aldrich No. 22,327-1; Registry CAS 54-20-6); 5- (2,2,2-trifluoroethyl) -uracil (Registry CAS 155143-31-6); 5- (pentafluoroethyl) -uracil (CAS Registry 60007-38-3); 6-aminouracil (Aldrich No. A5060-6; Registry CAS 873-83-6) uracil-6-carboxylic acid (orotic acid; Aldrich No. 0-840-2; Registry CAS 50887-69-9); 6-methyluracil (Aldrich No. D11,520-7; Registry CAS 626-48-2); uracil-5-amino-6-carboxylic acid (5-amino-orotic acid; Aldrich No. 19,121-3; Registry CAS # 7164-43-4); 6-amino-5-nitrosouracil (6-amino-2,4-dihydroxy-5-nitrosopyrimidine; Aldrich No. 27.689-8; Registry CAS 5442-24-0); uracil-5-fluoro-6-carboxylic acid (5-fluorotoric acid; Aldrich No. 42.513-3; Registry CAS 00000-00-0); and uracil-5-nitro-6-carboxylic acid (5-nitroorotic acid; Aldrich No. 18.528-0; Registry CAS 600779-49-9). Additional 5-, 6-, and 5,6-substituted uracils and / or thiouracils are available from General Intermediates of Canada, Inc. (Edmonton, Alberta, Canada) (http://www.generalintermediates.com) and / or Interchim (France) (http://www.interchim.com) that produce chemicals or can be obtained using standard methodology. Below are a large number of links to textbooks that describe suitable synthetic methods .

Amines 6 and 10 can be purchased from commercial sources or, alternatively, can be synthesized using standard techniques. For example, the necessary amines can be synthesized from nitro-precursors using standard chemical methods. Examples of specific reactions are given in the Examples section. See also Vogel, 1989, Practical Organic Chemistry , Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.

It is known from practice that in some cases, amines 6 and 10 and / or substituents R ⁵ and / or R ⁶ on uracil or thiouracil 2 may include functional groups that require protection during synthesis. The specific features of any protective group (s) used will depend on the features of the functional group that needs to be protected, and this will be taken into account by specialists in this field. Guidance on the selection of suitable protecting groups, as well as synthetic strategies for their attachment and removal, can be found, for example, in Green and Watts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sans, New York, 1999 (Greene & Wuts, Protective Groups in Organic Synthesis , 3d Edition, John Wiley & Sons, Inc., New York (1999)) and in the references cited later in the text (hereinafter “Green and Watts” )

A specific example of the implementation of the reaction according to Scheme (I), in which 5-fluorouracil (Aldrich, No. 32,937-1) is used as starting material, is presented below in Scheme (Ia):

Scheme (Ia)

In Scheme (Ia), R ² , R ⁴ , L ¹ and L ² correspond to their previous definition for scheme (I). According to Scheme (Ia), 5-fluorouracil 3 is halogenated with POCl ₃ to give 2,4-dichloro-5-fluoropyridimine 5, which is then reacted with excess amine 6 or 10 to produce N2, N4-bis substituted 5-fluoro-2, 4-pyrimidinediamine 11 or 13, respectively. Alternatively, asymmetric 2N, 4N-disubstituted-5-fluoro-2,4-pyrimidinediamine 9 can be obtained by the reaction of 2,4-dichloro-5-fluoropyridimine 5 with one equivalent of amine 10 (to form 2-chloro-N4-substituted -5-fluoro-4-pyrimidinamine 7), and then with one or more equivalents of amine 6.

In another example implementation of compounds of 2,4-pyrimidinediamine according to the present invention can be synthesized from substituted or unsubstituted cytosines shown below in schemes (IIa) and (IIb):

Scheme (IIa)

Scheme (IIb)

In schemes (IIa) and (IIb), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ , L ² and X correspond to their previous definition for scheme (I), and PG represents a protective group. Based on Scheme (IIa), the exocyclic amine C4 of cytosine 20 is first protected by a suitable PG protecting group to form an N4-protected cytosine 22. For specific guidance on the protective groups used in this context, see Vorbruggen and Ruh-Pohlenz, 2001, Handbook of Nucleoside Synthesis , John Wiley & Sons, NY, pp. 1-631). Protected cytosine 22 halogenates at position C2 under standard conditions using a standard halogenation reagent to form 2-chloro-4N-protected-4-pyrimidinamine 24. The reaction with amine 6, followed by deprotection of exocyclic amine C4 and reaction with amine 10, leads to the formation of 2,4-pyrimidinediamine corresponding to structural formula (I).

Alternatively, based on Scheme (IIb), cytosine 20 may be reacted with amine 10 or protected amine 21 to give N4-substituted cytosine 23 or 27, respectively. These substituted cytosines can then be halogenated, deprotected (in the case of N4-substituted cytosine 27) as described above, and can be reacted with amine 6 to give 2,4-pyrimidinamine corresponding to structural formula (I).

Commercially available cytosines that can be used as starting materials in Schemes (IIa) and (IIb) include, but are not limited to, cytosine (Aldrich No. 14,201-8; Registry CAS 71-30-7); N ⁴ -acetylcytosine (Aldrich No. 37.791-0; Registry CAS 14631-20-0); 5-fluorocytosine (Aldrich No. 27,159-4; Registry CAS 2022-85-7); and 5- (trifluoromethyl) -cytosine. Other suitable cytosines useful as starting materials in Schemes (IIa) are available from General Intermediates of Canada, Inc., (Edmonton, Alberta, Canada) (http://www.generalintermediates.com) and / or Interchim (France) (http://www.interchim.com), or obtained using standard techniques. Below are a large number of links to guides on synthetic methods for their preparation .

In another embodiment, the 2,4-pyrimidinediamine compounds of the subject invention can be synthesized from substituted or unsubstituted 2-amino-4-pyrimidinols, as shown in Scheme (III) below:

Scheme (III)

In Scheme (III), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ , L ^2, and X correspond to their description in Scheme (I), and Z is a substituted group, which is discussed in more detail below in connection with Scheme IV. Based on scheme (III), 2-amino-4-pyrimidinol 30 is reacted with amine 6 (or optionally with protected amine 21) to produce N2-substituted-4-pyrimidinol 32, which is then halogenated as described above, to obtain N2-substituted-4-halogen-2-pyrimidinamine 34. Additional deprotection (for example, if protected amine 21 was used in the first step), accompanied by a reaction with amine 10, leads to the formation of 2,4-pyrimidinamine, corresponding to the structural formula (I). Alternatively, pyrimidinol 30 may react with acylating agent 31.

Suitable and commercially available 2-amino-4-pyrimidinols 30 that can be used as starting materials in Scheme (III) include, but are not limited to, 2-amino-6-chloro-4-pyrimidinol hydrate (Aldrich, No. A4702 -8; Registry CAS 00000-00-0) and 2-amino-6-hydroxy-4-pyrimidinol (Aldrich, No. A5040-1; Registry CAS 56-09-7). Other 2-amino-4-pyrimidinols 30 useful as starting materials in Scheme (III) are available from General Intermediates of Canada, Inc. (Edmonton, Alberta, Canada) (http://www.generalintermediates. com) and / or Interchim (France) (http://www.interchim.com) or obtained using standard techniques. Below are a large number of links to guides on synthetic methods for their preparation .

Alternatively, the 2,4-pyrimidinediamine compounds of the present invention can be synthesized from substituted or unsubstituted 4-amino-2-pyrimidinols, as shown below in Scheme (IV):

Scheme (IV)

In Scheme (IV), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ and L ² correspond to their designations in Scheme (I), and Z represents a substituted group. Starting from Scheme (IV), C2-hydroxyl 4-amino-2-pyrimidinol 40 is more reactive to nucleophiles than C4-amine, so much so that reaction with amine 6 leads to the formation of N2-substituted-2,4-pyrimidinediamine 42. Subsequent reaction with component 44, which includes a good substituted group Z or amine 10, leads to the formation of 2,4-pyrimidinamine corresponding to structural formula (I). Component 44 may include virtually any substitutable group that can be replaced by the C4-amine of an N2-substituted-2,4-pyrimidinediamine 42. Suitable substituted Z groups include, but are not limited to, halogens, methanesulfonyloxy group (mesyloxy; "OM" ), trifluoromethanesulfonyloxy group (“OTf”) and p-toluene sulfonyloxy group (tosyloxy; “OT”), benzenesulfonyloxy group (“besylate”) and methanitrobenzenesulfonyloxy group (“nosylate”). Other suitable substituted groups are well known to those skilled in the art.

The substituted 4-amino-2-pyrimidirol used as starting material can be obtained commercially or synthesized using standard procedures. Below are a large number of links to guides on synthetic methods for their preparation .

In yet another embodiment, the 2,4-pyrimidinediamine compounds of the present invention can be prepared from 2-chloro-4-aminopyrimidine or 2-amino-4-chloropyrimidines, as shown in scheme (V) below:

Scheme (V)

In scheme (V), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ , L ² and X correspond to those previously described in scheme (I), and Z corresponds to its designation in scheme (IV). Starting from Scheme (V), 2-amino-4-chloropyrimidine 50 reacts with amine 10 to form 4N-substituted-2-pyrimidinediamine 52, which, following reaction with component 31 or amine 6, forms 2,4-pyrimidinamine corresponding to structural formula (I). Alternatively, 2-chloro-4-aminopyrimidine 54 may be reacted with component 44 and then with an amine to produce a compound corresponding to structural formula (I).

Examples of commercially available starting materials for use in Scheme (V) include, but are not limited to, pyrimidines 50 and 54, including 2-amino-4,6-dichloropyrimidine (Aldrich No. A4860-1; Registry CAS 56-05-3) ; 2-amino-4-chloro-6-methoxypyrimidine (Aldrich No. 51,864-6; Registry CAS 5734-64-5); 2-amino-4-chloro-6-methylpyrimidine (Aldrich No. 12,288-2; Registry CAS 5600-21-5) and 2-amino-4-chloro-6-methylthiopyrimidine (Aldrich No. A4600-5; Registry CAS 1005-38 -5). Additional pyrimidine-based starting material can be obtained from General Intermediates of Canada, Inc., (Edmonton, Alberta, Canada) (http://www.generalintermediates.com) and / or Interchim (France) (http: // www.interchim.com), or obtain using standard methods. Below are a large number of links to guides on synthetic methods for their preparation .

Alternatively, 4-chloro-2-pyrimidinamines 50 may be prepared as shown in scheme (Va) below:

Scheme (Va)

In Scheme (Va), R ⁵ and R ⁶ correspond to their notation in structural formula (I). In Scheme (Va), dicarbonyl 53 reacts with guanidine to produce 2-pyrimidinamine 51. The reaction with peracids such as m-chloroperbenzoic acid, trifluorooracetic acid or a complex of hydrogen peroxide with urea leads to the formation of N-oxide 55, which then halogenates to give 4-chloro-2-pyrimidinamines 50. The corresponding 4-halo-2-pyrimidinamines can be prepared using suitable halogenating agents.

In another embodiment, the 2,4-pyrimidinediamine compounds of the present invention can be prepared from substituted or unsubstituted uridines, as shown below in Scheme (VI):

Scheme (VI)

In Scheme (VI), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ , L ^2, and X correspond to their designations in Scheme (I), and the superscript PG represents a protecting group, considered in connection with Scheme (IIb) . According to Scheme (VI), uridine 60 has a C4 reactive center such that reaction with amine 10 or protected amine 21 leads to the formation of an N4-substituted cytidine 62 or 64, respectively. Deprotection of N4-substituted 62 or 64 with an acid catalyst (when “PG” is an acid-unstable protecting group) gives N4-substituted cytosine 28, which can then be halogenated at the C2 position and can react with amine 6 to obtain 2,4-pyrimidinadiamine corresponding to structural formula (I).

Similarly, cytidines can also be used as starting material, as shown in scheme (VII) below:

Scheme (VII)

In Scheme (VII), R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ , L ^2, and X correspond to their designations in Scheme (I), and the superscript PG represents a protecting group, as indicated above. Starting from scheme (VII), like iridine 60, cytidine 70 has a C4 reactive center such that reaction with amine 10 or protected amine 21 leads to the formation of an N4-substituted cytidine 62 or 64, respectively. These cytidines 62 and 64 are then processed as described above for scheme (VI) to obtain 2,4-pyrimidinediamine corresponding to structural formula (I).

Despite the fact that schemes (VI) and (VII) are given as an example with ribosyl nucleosides, it is clear to a person skilled in the art that the corresponding 2'-deoxyrib and 2 ', 3'-dideoxyribonucleosides, as well as nucleosides including sugars or sugar analogues other than ribose.

Numerous uridines and cytidines suitable for use as starting material in Schemes (VI) and (VII) are known to those skilled in the art and include, for example, 5-trifluoromethyl-2'-deoxycytidine (Chem. Sources, No. ABCR F07669; Registry CAS 66.384-66-5); 5-bromuridine (Chem. Sources Int'l 2000, Registry CAS 957-75-5); 5-iodo-2'-deoxyuridine (Aldrich, No. 1-775-6; Registry CAS 54-42-2); 5-fluoruridine (Aldrich, No. 32.937-1; Registry CAS 316-46-1); 5-ioduridine (Aldrich, No. 85,259-7; Registry CAS 1024-99-3); 5- (trifluoromethyl) uridine (Chem. Sources Int'l 2000; Registry CAS 70-00-8); 5-trifluoromethyl-2'-deoxyuridine (Chem. Sources Int'l 2000; Registry CAS 70-00-8). Additional uridines and cytidines that can be used as starting material in Schemes (VI) and (VII) are available from General Intermediates of Canada, Inc. (Edmonton, Alberta, Canada) (http: // www .generalintermediates.com) and / or Interchim (France) (http://www.interchim.com) or obtained using standard methods. Below are a large number of links to guides on how to artificially produce them .

Compounds of 2,4-pyrimidinediamine according to the present invention can also be synthesized from substituted pyrimidines, such as chlorosubstituted pyrimidines, as shown below in schemes (VIII) and (IX):

Scheme (VIII)

Scheme (IX)

In schemes (VIII) and (IX), R ² , R ⁴ , L ¹ , L ² and R ^a correspond to their designations in structural formula (I), and “Ar” represents a group of aryls. Based on scheme (VIII), the reaction of 2,4,6-trichloropyrimidine 80 (Aldrich No. T5,620-0; CAS No. 3764-01-0) with amine 6 leads to the formation of three compounds: substituted pyrimidine mono-, di- and triamines 81, 82 and 83, which can be separated and isolated by high pressure liquid chromatography (HPLC) or other conventional technique. Mono and diamines 81 and 82 may subsequently react with amines 6 and / or 10 to form N2, N4, N6-trisubstituted-2,4,6-pyrimidine triamines 84 and 85, respectively.

N2, N4-bis-substituted-2,4-pyrimidinediamines can be obtained by a method similar to that shown in scheme (VIII) using 2,4-dichloro-5-methylpyrimidine or 2,4-dichloropyrimidine as starting materials. In this case, the monosubstituted pyrimidinamine corresponding to compound 81 is not formed. Instead, the direct formation of N2, N4-bis-substituted-2,4-pyrimidinediamine takes place.

Based on scheme (IX), 2,4,5,6-tetrachloropyrimidine 90 (Aldrich No. 24.671-9; CAS No. 1780-40-1) interacts with excess amine 6 to give a mixture of three components: 91, 92, and 93, which can be separated and isolated using HPLC or standard methods. As shown, N2, N4-bis-substituted -5,6, -dichloro-2,4-pyrimidinediamine 92 then, in the presence of halide C6, can react, for example, with nucleophilic substance 94 to give compound 95. Alternatively, compound 92 can be converted to N2, N4-bis-substituted -5-chloro-6-aryl-2,4-pyrimidinediamine 97 using the Suzuki reaction. 2,4-pyrimidinediamine 95 can be converted to 2,4-pyrimidinediamine 99 by reaction with Bn ₃ SnH.

It will be understood by those skilled in the art that the 2,4-pyrimidinediamines of the present invention synthesized using the methods described above or other well known methods can also be used as starting materials and / or intermediates for synthesizing additional components 2, 4-pyrimidinediamine according to the invention. A specific example is shown below in diagram (X):

Scheme (X)

In Scheme (X), R ⁴ , R ⁵ , R ⁶ , L ^2, and R ^a correspond to their notation in structural formula (I). Each R ^{a ′} independently represents R ^a and may be the same or different from that represented by R ^a . Based on scheme (X), the carboxylic acid or ester 100 can be converted to amide 104 by reaction with amine 102. In amine 102, R ^{a ′} may be the same or different from R ^a acid or ester 100. Similarly, carbonate ester 106 can be converted to carbamate 108.

A second specific example is shown below in diagram (XI):

Scheme (XI)

In Scheme (XI), R ⁴ , R ⁵ , R ⁶ , L ^2, and R ^c correspond to their notation in structural formula (I). Based on Scheme (XI), amide 110 or 116 can be converted to amine 114 or 118, respectively, due to a decrease in boron content using boron methyl sulfide complex 112. Other suitable reactions for synthesizing 2,4-pyrimidinediamine compounds from 2,4-pyrimidinediamine starting material understandable to experts in this field.

Although many of the synthetic schemes discussed above do not reflect the use of protective groups, it is clear that in some cases, the substituents R ² , R ⁴ , R ⁵ , R ⁶ , L ¹ and / or L ² may include functional groups requiring protection. The exact properties of the protective group used will depend, inter alia, on the properties of the functional group that needs to be protected and on the conditions of the reaction used in a particular synthetic scheme, which is obvious to those skilled in the art. Guidance on the selection of protecting groups and attachment mechanisms suitable for a particular application can be found, for example, in Greene & Wuts.

Prodrugs corresponding to structural formula (II) can be obtained using the usual modification of the above methods. Otherwise, such prodrugs can be obtained by the reaction of a suitably protected 2,4-pyrimidinediamine corresponding to structural formula (I) with a suitable progroup. The conditions for carrying out such reactions and for deprotecting the reaction product to obtain a prodrug corresponding to formula (II) are well known.

A large number of training methods are known in the literature, mainly applicable for the synthesis of both pyrimidines and the starting materials described in schemes (I) - (IX). For specific guidance, the reader may consult the following sources: Brown, DJ, "The Pyrimidines", in The Chemistry of Heterocyclic Compounds, Volume 16 (Weissberger, A., Ed.), 1962, Interscience Publishers, (A Division of John Wiley & Sons), New York (“Brown I”); Brown, DJ, "The Pyrimidines", in The Chemistry of Heterocyclic Compounds, Volume 16, Supplement I (Weissberger, A. and Taylor, EC, Ed.), 1970, Wiley-Interscience, (A Division of John Wiley & Sons) , New York (Brown II "); Brown, DJ, "The Pyrimidines", in The Chemistry of Heterocyclic Compounds, Volume 16, Supplement II (Weissberger, A. and Taylor, EC, Ed.), 1985, An Interscience Publication (John Wiley & Sons), New York ( “Brown III”); Brown, DJ, "The Pyrimidines" in The Chemistry of Heterocyclic Compounds, Volume 52 (Weissberger, A. and Taylor, EC, Ed.), 1994, John Wiley & Sons, Inc., New York, pp. 1-1509 (Brown IV "); Kenner, GW and Todd, A., in Heterocyclic Compounds , Volume 6, (Elderfield, RC, Ed.), 1957, John Wiley, New York, Chapter 7 (Pyrimidines); Paquette, LA, Principles of Modern Heterocyclic Chemistry , 1968, WA Benjamin, Inc., New York, pp. 1-401 (synthesis of uracil - p. 313, 315; synthesis of pyrimidine - p. 313-316; synthesis of pyrimidinamine - p. 315); Joule, JA, Mills, K. and Smith, GF, Heterocyclic Chemistry, 3 ^rd Edition, 1995, Chapman and Hall, London, UK , pp. 1-516; Vorbruggen, H. and Ruh-Pohlenz, C., Handbook of Nucleoside Synthesis , John Wiley & Sons, New York, 2001, pp. 1-631 (protection of pyrimidines by acylation - p. 90-91; silylation of pyrimidines - p. 91-93); Joule, JA, Mills, K. and Smith, GF, Heterocyclic Chemistry , 4 ^th Edition, 2000, Blackwell Science, Ltd, Oxford, UK, pp. 1-589; and Comprehensive Organic Synthesis , Volumes 1-9 (Trost, BM and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, UK.

6.4. Inhibition of Fc receptor signaling cascades

The active 2,4-pyrimidinediamine compounds of the present invention suppress Fc receptor signaling cascades, which leads, inter alia, to cell degranulation. As a specific example, suppression of FcεRI and / or FcγRI signals by these compounds can result in degranulation of immune cells, such as neutrophilic, eosinophilic cells, mast cells and / or basophilic cells. Both mast cells and basophilic cells play a central role in disorders caused by allergens, including, for example, allergic rhinitis and asthma. Based on figure 1, under the influence of allergens, which, among other things, may be pollen or parasites, allergen-specific IgE antibodies are synthesized by B cells that are activated using IL-4 (or IL-13) and other messengers in order to go on to the synthesis of a specific IgE class antibody. These allergen-specific IgE antibodies bind to FcεRI having a high affinity. After antigen binding, FcεRI-associated IgE antibodies cross-link and the IgE receptor signal pathway is activated, which leads to cell degranulation and subsequent isolation and / or synthesis of a variety of chemical mediators, including histamine, proteases (e.g. tryptase and chymase), lipid intermediates, such as leukotrienes (e.g. LTC4), platelet activation factors (PAF) and prostaglandins (e.g. PGD2), as well as a series of cytokines, including TNF-α, IL-4, IL-13, IL-5, IL-6, IL -8, GMCSF, VEGF and TGF-β. Isolation and / or synthesis of these mediators from mast cells and / or basophilic cells is the cause of early or late responses caused by allergens and is directly related to processes in the forward direction that lead to a prolonged inflammatory state.

Molecular processes in the FcεRI receptor signaling pathway that cause the release of preformed mediators through degranulation and the isolation and / or synthesis of other chemical mediators are well known and illustrated in FIG. 2. Based on figure 2, FcεRI is a heterotetrameric receptor composed of IgE-binding alpha subunits, beta subunits and two gamma subunits (gamma homodimer). Cross-linking of FcεRI-bound IgE with multivalent binders (including, for example, IgE-specific allergens, or anti-IgE antibodies, or fragments) causes rapid association and activation of the Src-dependent Lyn kinase. Lyn phosphorylates tyrosine-induced immunoreceptor activation motifs (“ITAM”) on the intercellular beta and gamma subunits, leading to the recruitment of an additional Lyn kinase to the beta subunit and Syk kinase to the gamma homodimer. These receptor-related kinases, which are activated by intra- and intermolecular phosphorylation, phosphorylate other pathway components, such as Btk kinase, LAT, and C-gamma (PLC-gamma) phospholipase. Activated PLC-gamma initiates a pathway that leads to the activation of protein C-kinase and the mobilization of Ca ²⁺ , which is necessary for degranulation. Cross-linking FcεRI also activates the three main classes of mitogen activated protein kinases (MAP), i.e., ERK1 / 2, JNK1 / 2, and p38. Activation of these pathways is important in the transcriptional regulation of pro-inflammatory mediators, such as TNF-α and IL-6, as well as leukotriene CA (LTC4) of the lipid mediator.

Although not shown in the figure, it is believed that the FcγRI receptor signaling cascade has several common elements with the FcεRI receptor signaling cascade. It is significant that, like FcεRI, the FcγRI receptor includes a gamma homodimer that phosphorylates and recruits the Syk kinase, as in the case of FcεRI, and activation of the FcγRI receptor signaling cascade leads, among other things, to degranulation. Other Fc receptors that are involved in the gamma homodimer and can be regulated by the active compounds of 2,4-pyrimidinediamine include, but are not limited to, FcαRI and FcγRIII.

The ability of the 2,4-pyrimidinediamine compounds of the present invention to inhibit Fc receptor signaling cascades can be easily determined or confirmed by in vitro assay. Samples convenient for confirming the inhibition of FcεRI-mediated degranulation are described in the Examples section. In one typical sample, cells capable of undergoing FcεRI mediated degranulation, such as mast cells or blazophiles, are first grown in the presence of IL-4, stem cell factor (SCF), IL-6 and IgE to increase the expression of FcεRI exposed to the test compound 2,4-pyrimidinediamine according to the present invention and stimulated with anti-IgE antibodies (or, alternatively, an IgE-dependent allergen). Following the incubation, the amount of chemical mediator or other chemical reagent isolated and / or synthesized by activation of the FcεRI signaling cascade can be determined using standard methods and can be equated to the amount of mediator or reagent isolated from control cells (i.e. cells that stimulated but not exposed to the test compound). The concentration of the test compound, which leads to a 50% reduction in the amount of mediator or reagent measured relative to control cells, is equal to the IC _{50 of the} test compound. The origin of the mast cells or basophilic cells used in the sample will depend, in particular, on the preferred use of the compounds, which is obvious to specialists in this field. For example, if the compounds will be used to treat or prevent a specific disease characteristic of a person, a convenient source of mast cells or basophilic cells is a person or other animal, which is considered to be an acceptable or well-known clinical model for a specific disease. Thus, depending on the particular application, mast cells or basophilic cells can be obtained from various animal sources, from lower mammals such as mice and rats to dogs, sheep and other mammals commonly used in clinical trials, and higher mammals such as monkeys, chimpanzees, anthropoids and even humans. Specific examples of cells suitable for in vitro sampling include, but are not limited to, rodent or human basophilic cells, rat leukemia basophilic cell lines, basic mouse mast cells (such as mouse bone marrow mast cells (“BMMCs)), and basic cultured from human cord blood mast cells (“CHMCs”) or other tissues, such as lung tissue. Methods for isolating and culturing these types of cells are well known or are presented in the Examples section ( see, for example , Demo et al. , 1999, Cytometry 36 (4): 340-348 and one of several patent applications pending simultaneously). , registration number 10 / 053,355, filing date - November 8, 2001, the exposition of which is included in the references to this work). Of course, other types of immune cells that degranulate after activation of the FcεRI signaling cascade can also be used, including, for example, eosinophils.

Those skilled in the art will agree that a quantified neurotransmitter or reagent is not required. The only requirement is that the mediator or reagent is isolated and / or synthesized as a result of the initiation or activation of the Fc receptor signaling cascade. For example, based on Fig. 1, activation of the FcεRI signaling cascade in mast cells and / or basophilic cells leads to numerous phenomena that occur in the forward direction. For example, as a result of the activation of the FcεRI signaling cascade, various preformed chemical mediators and reagents are released instantly (that is, within 1-3 minutes after activation of the receptor) using degranulation. Thus, in one embodiment of the invention, a quantified neurotransmitter or reagent may be characteristic of the granules (that is, usually present in the granules, and not in the cytoplasm of the cell). Examples of granule-specific mediators or reagents that can be quantified to determine and / or confirm the activity of the 2,4-pyrimidinediamine compound of the present invention include, but not limited to, granule-specific enzymes, such as hexosaminidase and tryptase, and granule-specific components, such like histamine and serotonin. Samples for quantifying such factors are well known and, in many cases, commercially available. For example, the isolation of tryptase and / or hexosaminidase can be quantified by growing cells with cleavable substrates that fluoresce after cleavage, by determining the amount of fluorescence obtained by conventional methods. Such cleavable fluorogenic substrates are commercially available. For example, fluorogenic substrates Z-Gly-Pro-Arg-AMC (where Z = benzyloxycarbonyl and AMC = 7-amino-4-methylcoumarin, manufactured by BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA 19462, Catalog No. P-142 ) and Z-Ala-Lys-Arg-AMC (manufactured by Enzyme Systems Products, a Division of ICN Biomedicals, Inc., Livermore, CA 94550, Catalog No. AMC-246) can be used to estimate the amount of tryptase secreted. The fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (supplier: Sigma, St. Louis, MO, Catalog No. 69585) can be used to determine the amount of hexosaminidase released. Histamine release can be quantified using a commercially available enzyme-linked immunosorbent assay ELISA, such as, for example, the Immunotech histamine ELISA assay No. IM2015 (Beckman-Coulter, Inc.). Specific methods for quantifying the release of tryptase, hexosaminidase and histamine are presented in the Examples section. Each of these samples can be used to determine or confirm the activity of the 2,4-pyrimidinediamine compounds of the present invention.

Based on figure 1, degranulation is only one of several reactions initiated by the FcεRI signaling cascade. In addition, activation of this signaling pathway leads to de novo synthesis and release of cytokines and chemokines, such as IL-4, IL-5, IL-6, TNF-α, IL-13 and MIP1-α, and the release of lipid mediators such such as leukotrienes (e.g., LTC4), platelet activation factors (FAT), and prostaglandins. Therefore, the 2,4-pyrimidinediamine compounds of the present invention can also be evaluated for their activity by assessing the amount of one or more of these isolated mediators and / or synthesized using activated cells.

In contrast to the granule-specific components considered above, these late-phase mediators are not released immediately after the activation of the FcεRI signaling cascade. Thus, when assessing the number of these late stage mediators, measures should be taken to ensure that the culture of the activated cell is grown for a time sufficient to allow synthesis (if necessary) and isolation of the estimated mediator. Typically, FAT and lipid mediators, such as leukotriene C4, are released 3-30 minutes after activation of FcεRI. Cytokines and other late phase mediators are secreted approximately 4-8 hours after FcεRI activation. Growth times corresponding to specific mediators are known to those skilled in the art. Specific guidance and samples are provided in the Examples section.

The amount of specific late-phase mediator isolated can be determined using any standard method. In one embodiment of the present invention, this amount (s) can be estimated using an ELISA assay. An ELISA kit suitable for the quantification of isolated TNFα, IL-4, IL-5, IL-6 and / or IL-13 can be obtained, for example, from Biosource International, Inc., Camarillo, CA 93012 (see, for example, catalog numbers No. KHC3011, KHC0042, KHC0052, KHC0061 and KHC0132). An ELISA kit suitable for the quantification of C4 leukotriene (LTC4) isolated from cells can be obtained from Cayman Chemical Co., Ann Arbor, MI 48108 (see, for example, catalog number 520211).

Typically, the active 2,4-pyrimidinediamine compounds of the present invention will have an IC ₅₀ concentration with respect to FcεRI receptor mediated degranulation and / or a mediator isolation or synthesis level of about 20 μm or less in accordance with in vitro measurements such as one of the in vitro samples discussed above or in the Examples section. Of course, it will be understood by those skilled in the art that compounds that exhibit low IC ₅₀ values, for example of the order of 10 μm, 1 μm, 100 nM, 10 nM, 1 nM or even lower, are particularly useful.

Those skilled in the art will also agree with the fact that the various mediators discussed above can cause different negative effects or different levels of the same negative effect. For example, the lipid mediator LTC4 is a powerful vasoconstrictor: its potency in terms of narrowing blood vessels is approximately 1000 times greater than the potency of histamine. As another example, in addition to mediating atopic or type I hypersensitivity reactions, cytokines can also be cited, which can also cause tissue transformation and cell proliferation. Thus, despite the possibility of using compounds that inhibit the excretion and / or synthesis of one of the previously considered chemical mediators, specialists in this field will agree that compounds that inhibit the excretion and / or synthesis of most or even all of the mediators discussed above will undoubtedly find wider application due to their ability to improve or completely suppress most or even all of the adverse effects caused by individual mediators. For example, compounds that inhibit the release of all three types of mediators — granulospecific mediators, lipids, and cyclotins — are effective in treating or preventing the immediate type I hypersensitivity reaction, as well as associated chronic symptoms.

Compounds of the present invention capable of inhibiting the release of more than one type of mediator (e.g., a granule-specific mediator or a late phase mediator) can be identified by determining the IC ₅₀ relative to the model mediator of each class using the various in vitro samples discussed above (or other equivalent in vitro samples). Compounds of the present invention that can suppress mediators of more than one type will typically exhibit IC ₅₀ values of less than 20 μm for mediators of each type tested. For example, a compound with an IC ₅₀ value of 1 μm relative to histamine release (IC ₅₀ ^histamine ) and with an IC ₅₀ value of 1 nM relative to LTC4 leukotriene synthesis and / or isolation (IC ₅₀ ^LTC4 ) is characterized by both immediate release of mediators (inherent in granules) and release late phase mediators. As another specific example, a compound with IC ₅₀ ^tryptase 10 μm, IC ₅₀ ^LTC4 - 1 μm and IC ₅₀ ^IL-4 - 1 μm, which inhibits the instant release of mediators (characteristic of granules), as well as the release of lipid and cytokine mediators . Despite the fact that the above specific examples use the IC _{50 of} one mediator - a representative of each class, experts in this field will agree that the IC ₅₀ values of most or even all mediators that make up one or more classes can also be obtained. For specialists in this field it will be obvious the number (s) and variety (s) of mediators for which data on the IC ₅₀ should be established in the case of a particular connection and application.

Such samples can be used to test the inhibition of signaling cascades initiated by other Fc signaling receptors, such as FcαRI, FcγRI and / or FcγRIII with the usual modification. For example, the ability of compounds to inhibit FcγRI signaling can be tested using samples similar to those discussed above, except that the FcγRI signaling cascade is activated, for example, by growing cells with IgG and allergen-specific IgG or an antibody instead of IgE and an allergen-specific IgE or antibody. Suitable cell types, activating reagents, and reagents are known to those skilled in the art for the quantification needed to test the inhibition of other Fc receptors, such as, for example, the Fc receptors that make up the gamma homodimer.

But the most useful class of compounds includes compounds of 2,4-pyrimidinediamines, which inhibit the release of immediate-type granulator mediators and late-phase mediators with approximately equivalent IC ₅₀ values. “Approximately equivalent” means that the IC ₅₀ values for the mediator of each type differ from each other by no more than 10 times. Another particularly useful class of compounds includes 2,4-pyrimidinediamine compounds that inhibit the release of immediate type granule mediators and cytokine mediators with approximately equivalent IC ₅₀ values. In specific embodiments of the present invention, such compounds inhibit the secretion of the following mediators with approximately equivalent IC ₅₀ values: histamine, tryptase, hexosaminidase, IL-4, IL-5, IL-6, IL-13, TNFα and LTC4. Such compounds are especially effective, inter alia, for improving or completely eliminating both early and late responses associated with atopic or immediate type I hypersensitivity reactions.

Ideally, the ability to inhibit the release of mediators of all desirable types will be inherent in one compound. However, mixtures of compounds can be formulated to provide the same result. For example, a first compound that inhibits the release of granule-specific mediators can be used in combination with a second compound that inhibits the release and / or synthesis of cytokine mediators.

In addition to the FcεRI or FcγRI degranulation pathways discussed above, degranulation of mast cells and / or blasophilic cells can be caused by other reagents. For example, ionomycin, which is a calcium ionophore that bypasses the early signal transmission mechanism of FcεRI or FcγRI cells, directly causes a flow of calcium, which initiates degranulation. As follows from figure 2, activated PLCγ initiates paths leading, inter alia, to the mobilization of calcium ions and subsequent degranulation. As shown in this figure, such Ca ²⁺ mobilization is triggered at a later stage in the FcεRI signal transmission path. As noted above and shown in figure 3, ionomycin directly causes the mobilization of Ca ²⁺ and the flow of Ca ²⁺ , which leads to degranulation. Other ionophores that cause degranulation in this manner include A23187. The ability of ionophores, such as ionomycin, to cause degranulation to bypass the early stages of the FcεRI and / or FcγRI signaling cascades allows them to be used as a counter screen to identify active compounds of the present invention that especially affect their activity in degranulation and inhibition by blocking or suppressing the early signaling cascades of FcεRI or FcγRI, as discussed above. Compounds that specifically inhibit such early degranulation mediated by FcεRI or FcγRI receptors inhibit not only degranulation and subsequent rapid release of histamine, tryptase and other components containing granules, but also proinflammatory activation pathways causing TNFα, IL-4, IL -13 and lipid mediators, such as LTC4. Thus, compounds that specifically inhibit such early degranulation mediated by FcεRI or FcγRI receptors block or inhibit not only acute atopic or type I hypersensitivity reactions, but also late responses involving numerous inflammatory mediators.

Compounds of the present invention that suppress the early degranulation mediated by FcεRI and / or FcγRI receptors to the greatest extent possible are degranulation mediated inhibitors mediated by FcεRI and / or FcγRI receptors (having, for example , IC ₅₀ values less than 20 μm relative to excretion granule-specific mediator or component according to in vitro measurements in a sample with cells that are stimulated by the binding reagent IgE or IgG) but slightly inhibit ionophore degranulation. In one embodiment of the present invention, the compounds are considered to be slightly inhibiting ionophore-induced degranulation if the in vitro value of the IC _{50 of} ionophore-induced degranulation does not exceed 20 μm. Of course, active compounds that exhibit even higher IC ₅₀ values due to ionophore degranulation or do not suppress ionophore degranulation at all are particularly effective. In another embodiment of the present invention, the compounds are considered to be slightly inhibiting ionophore-induced degranulation if they exhibit more than a 10-fold difference in the IC ₅₀ values of the degranulation mediated by FcεRI and / or FcγRI and due to the ionophore degranulation measured in vitro in the sample. Samples suitable for determining the IC ₅₀ value of ionophore-induced degranulation include any of the previously considered degranulation samples, provided that the cells are stimulated or activated by a calcium ionophore, such as ionomycin or A23187 (AG Scientific, San Diego, CA), causing degranulation, instead of anti-IgE antibodies or allergen-specific IgE. Specific tests for evaluating the ability of a particular 2,4-pyrimidinediamine compound of the present invention to inhibit ionophore degranulation are presented in the Examples section.

Those skilled in the art will understand that compounds that exhibit a high degree of selectivity for FcεRI-mediated degranulation find particular use, since such compounds selectively act on the FcεRI cascade and do not affect other degranulation mechanisms. Similarly, compounds that exhibit a high degree of selectivity for FcγRI receptor mediated degranulation find particular use, since such compounds selectively act on the FcγRI cascade and do not affect other degranulation mechanisms. Compounds that exhibit a high degree of selectivity are generally 10 or more times more selective with respect to the mediated FcεRI or FcγRI receptors than with ionophore degranulation, such as, for example, ionomycin degranulation.

Biochemical and other data confirm that the 2,4-pyrimidinediamine compounds described in this work are effective inhibitors of Syk kinase activity. For example, in experiments with isolated Syk kinase from twenty-four tested 2,4-pyrimidinediamine compounds, all but two inhibited the phosphorylation of the peptide substrate catalyzed by Syk kinase in the submicromolar range of IC ₅₀ values. The remaining compounds inhibited micromolar phosphorylation. In addition, all of the sixteen in vitro mast cell assays tested inhibited phosphorylation of Syk kinase substrates (e.g. PLC-gamma1, LAT) and forward Syk kinase proteins (e.g. JNK, p38, Erk1 / 2 and PKB after testing ), but not proteins in the reverse direction of the cascade Syk kinase flow ( e.g., Lyn). Phosphorylation of Lyn substrates was not inhibited by the tested 2,4-pyrimidinediamine compounds. However, for the following compounds, a high correlation was observed between their ability to inhibit Syk kinase activity in biochemical samples (IC ₅₀ ranged from 3 to 1850 nM) and the ability to inhibit FcεRI-mediated mast cell degranulation (IC ₅₀ ranged from 30 to 1650 nM): R950373, R950368, R921302, R945371, R945370, R945369, R945365, R921304, R945144, R945140, R945071, R940358, R940353, R940352, R940351, R94040, R940940921, R940340, R940921, R940340, R940921, R940340, R940340, R940921 R940275, R940269, R940255, R935393, R935372, R935366, R935310, R935309, R935307, R935304, R935302, R935293, R935237, R935199, R935196, R93519926, R93519995935939, R926931, R926891, R926839, R926834, R926816, R926813, R926791, R926782, R926780, R926757, R926753, R926745.

Accordingly, the activity of the 2,4-pyrimidinediamine compounds of the present invention can also be confirmed by biochemical or cellular activity tests of Syk kinase. Based on FIG. 2, in the FcεRI signaling cascade in mast cells and / or basophilic cells, Syk kinase phosphorylates LAT and PLC-gamma1, which leads, among other things, to degranulation. Any of these activities may be used to confirm the activity of the 2,4-pyrimidinediamine compounds of the present invention. In one embodiment of the present invention, this activity is confirmed by combining an isolated Syk kinase or its active fragment with a 2,4-pyrimidinediamine compound in the presence of a Syk kinase substrate (for example, a synthetic peptide or protein, if it is known to be phosphorylated with using Syk in the signaling cascade) as a result of the assessment of substrate phosphorylation by Syk kinase. Alternatively, a sample can be obtained using cells that express Syk kinase. Cells can express Syk kinase endogenously, or they can be designed to express recombinant Syk kinase. Cells can also additionally express Syk kinase substrate. Cells suitable for obtaining such control samples, as well as methods for creating suitable cells, are obvious to specialists in this field. Specific examples of biochemical or cell samples convenient for confirming the activity of 2,4-pyrimidinediamine compounds are presented in the Examples section.

In general, Syk kinase inhibitors will have IC ₅₀ values relative to Syk kinase activity, such as, for example, the ability of Syk kinase to phosphorylate a synthetic or endogenous support in vitro or in a cell sample of the order of 20 μM or less. Those skilled in the art will agree that compounds with lower IC _50s , for example of the order of 10 μM, 1 μm M, 100 nM, 10 nM, 1 nM or even less, are particularly effective.

6.5 Application and composition

As noted previously, the active compounds of the present invention inhibit Fc receptor signaling cascades and, in particular, Fc receptors, including a gamma homodimer, such as FcεRI and / or FcγRI signaling cascades, which lead, inter alia, to isolation and / or synthesis chemical mediators from cells through degranulation or other processes. It has also been discussed that active compounds are also effective inhibitors of Syk kinase. Therefore, the active compounds of the present invention can be used under various in vitro, in vivo and ex vivo conditions for the regulation or inhibition of Syk kinase, signaling cascades in which Syk kinase plays the role of Fc receptor signaling cascades, and biological reactions caused by such signaling cascades . For example, in one embodiment of the present invention, the compounds can be used to inhibit Syk kinase in vitro or in vivo in virtually any type of cell expressing the Syk kinase. They can also be used to regulate signaling cascades in which the Syk kinase plays a role. Such Syk-dependent signaling cascades include, but not limited to, FcεRI, FcγRI, FcγRIII, BCR, and integrin signaling cascades. The compounds can also be used in vitro or in vivo to regulate and, in particular, suppress cellular or biological responses caused by such Syk-dependent signaling cascades. Such cellular or biological reactions include but are not limited to respiratory bursts, cell adhesion, cell degranulation, cell spreading, cell migration, cell aggregation, phagocytosis, cytokine synthesis and secretion, cell maturation, and Ca ²⁺ ion flux. It is significant that these compounds can be used to inhibit Syk kinase in vivo as a therapeutic approach to the treatment or prevention of diseases mediated in whole or in part by Syk kinase activity. Some examples of Syk kinase-mediated diseases that can be treated or prevented with such compounds are discussed in more detail below.

In another embodiment, the active compounds can be used to regulate or inhibit Fc and / or FcεRI and / or FcγRI-mediated degranulation signaling cascades as a therapeutic approach to the treatment or prevention of diseases characterized by, caused by and / or associated with isolation or synthesis of chemical mediators of such signaling cascades or degranulation of Fc receptors. Such treatment can be prescribed to animals in veterinary conditions or to people. As an example, diseases that are characterized, caused by, or associated with it by such isolation, synthesis or degranulation of a mediator and, therefore, treatable or preventable by active compounds, include, but are not limited to, a hereditary predisposition to allergic diseases, anaphylactic hypersensitivity or allergic reactions, allergies (e.g., allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis and food allergies), mild Goes scarring (e.g., scleroderma, increased fibrosis, the formation of keloids, post-operative scarring, lung fibrosis, vascular spasms, migraine, reperfusion injury and post-infarction period), diseases associated with tissue destruction (e.g., chronic obstructive pulmonary disease (COPD), kardiobronhit and post-infarction period), diseases associated with tissue inflammation ( for example , irritable bowel syndrome, spastic colon and inflammatory bowel disease, inflammation and scarring.

In addition to the many diseases mentioned above, the results obtained from cell analysis and animal testing confirm that the 2,4-pyrimidinamine compounds described in the present invention are also useful in the treatment and prevention of autoimmune diseases, as well as various symptoms associated with such diseases. Types of autoimmune diseases that can be treated or prevented with 2,4-pyrimidinamine compounds in total include tissue damage disorders resulting from a humoral and / or cell-mediated reaction to endogenous and / or immunogens or antigens exogenous nature. Such diseases are often characterized as diseases associated with non-anaphylactic hypersensitivity reactions (for example, type II, III and / or IV).

In accordance with the discussion above, type I hypersensitivity reactions mainly arise from the release of pharmacologically active substances, for example, histamine, mast cells and / or basophils as a result of contact with a specific exogenous antigen. As noted above, such type I reactions play a role in a large number of diseases, which include allergic asthma, allergic rhinitis, etc.

Type II hypersensitivity reactions (also known as cytotoxic, cytolytic, complement-induced or cell-stimulated hypersensitivity reactions) occur when immunoglobulins react with antigenic components of cells or tissues or with antigen or haptens that are inextricably linked to cells or tissues. Diseases that are typically associated with type II hypersensitivity reactions include, but are not limited to, autoimmune hemolytic anemia, congenital neonatal anemia, and Goodpasture syndrome.

Type III hypersensitivity reactions (also known as hypersensitivity reactions caused by toxic, soluble or immune complexes) result from the deposition of antigen-immunoglobulin in the vessels or tissues of soluble circulating complexes, which is accompanied by acute inflammatory reactions at the sites of deposition of the immune complexes. Unlimited examples of prototypes of diseases resulting from type III reactions include the Arthus reaction, rheumatoid arthritis, serum sickness, systemic lupus erythematosus, certain types of glomerulonephritis, multiple sclerosis, and bullous pemphigoid.

Type IV hypersensitivity reactions (often referred to as cellular, cell-mediated, delayed or tuberculin hypersensitivity reactions) are caused by sensitized T-lymphocytes, which are formed as a result of contact with a specific antigen. Unlimited examples of diseases in which type IV reactions are involved include contact dermatitis and allograft rejection.

Autoimmune diseases associated with any of the above non-anaphylactic hypersensitivity reactions can be treated or prevented with the 2,4-pyrimidinamine compounds of the present invention. In particular, these methods can be used to treat or prevent autoimmune diseases, often characterized as autoimmune disorders of a single organ or a single cell type, which include, but are not limited to, Hashimoto thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis with pernicious anemia, encephalomyelitis, autoimmune orchitis, Goodpasture syndrome, autoimmune thrombocytopenia, metastatic ophthalmia, bulbospinal paralysis, bazedova bo disease, biliary primary cirrhosis of the liver, chronic aggressive hepatitis, ulcerative colitis and membrane glomerulopathy, as well as autoimmune diseases, often characterized by the presence of systemic autoimmune disorders, which include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome , polymyositis-dermatomyositis, systemic sclerosis, nodose polyarteritis, multiple sclerosis and bullous pemphigoid.

Specialists in this field will agree that many of the above autoimmune diseases are associated with serious symptoms, the weakening of which represents significant progress in treatment even in cases where it is not possible to improve the condition of the underlying disease. Many of these symptoms, as well as the stages of the underlying disease, are the result of activation of the FcγR signaling cascade in monocytes. Since the 2,4-pyrimidinamine compounds of the present invention are potential inhibitors of such FcγR signaling cascades in monocytes and other cells, the described methods can be used in the treatment and / or prevention of a variety of undesirable symptoms associated with the above autoimmune diseases.

As an example, rheumatoid arthritis (RA) usually causes swelling, pain, loss of mobility and increased sensitivity of damaged joints of the whole body. RA is characterized by the presence of a chronically inflamed synovial membrane densely populated by lymphocytes. The synovial membrane, which usually has a thickness of one cell layer, becomes saturated with cells and takes on a shape similar to lymphoid tissues, including dendritic cells, T, B, and NK cells, macrophages, and plasmocyte clusters. This process, combined with an abundance of immunopathological mechanisms, which include the formation of antigen-immunoglobulin complexes, ultimately leads to a violation of the integrity of the joint, which leads to deformation, the final loss of functioning and / or erosion of the joint bone or in the immediate vicinity of the joint. The described methods can be used to treat or alleviate the condition of any particular, several or all of the symptoms of RA. Thus, within the context of RA, treatment modalities (described more generally below ) are considered to help alleviate the onset or improve the symptoms often associated with RA, regardless of the concomitant success in treating the underlying disease (RA) and / or reducing the amount of circulating rheumatoid factor (RF).

Another specific example is systemic lupus erythematosus (SLE), which is often associated with symptoms such as fever, joint pain (arthralgia), arthritis, and serositis (pleurisy or pericarditis). Within the context of the SLE, treatment methods are considered that contribute to alleviating the manifestation or improving the condition of symptoms often associated with SLE, regardless of the concomitant success in the treatment of underlying disease (SLE).

As another specific example, multiple sclerosis (MS) can be cited, the damage from which includes impaired visual acuity of the patient; the development of double vision; impaired mobility, which affects the ability to walk and act with hands; incontinence of the stomach and bladder; spasticity; as well as limited sensitivity (to touch, pain and temperature). Within the context of MS, treatment methods that improve or slow down the development of any specific or several damaging factors, often associated with MS, are considered, regardless of the concomitant success in the treatment of the underlying disease (MS).

When used to treat or prevent such diseases, the active compounds may be administered separately as mixtures of one or more active compounds or as a mixture or combination with other substances effective in the treatment of such disorders or diseases. These active compounds are also prescribed in a mixture or combination with substances that are used to treat other disorders or diseases, such as steroids, membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptor inhibitors, IgE isotype switching inhibitors or IgE synthesis, inclusion of IgG isotype or synthesis IgG, β-agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, rituxan, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, antihistamines, etc. These active compounds can be prescribed per se (ca by itself) in the form of prodrugs or as medicaments forming the active compound or prodrug.

Medicines containing the active compounds of the present invention (or prodrugs thereof) can be prepared by conventional mixing, dissolving, granulating, dragee-eluting, emulsifying, encapsulating, entrainment or lyophilization processes. These compositions can be obtained in the usual way using one or more physiologically acceptable carriers, diluents, excipients or excipients that facilitate the processing of the active compounds into drugs that can be used for pharmaceutical purposes.

The active compound or prodrug can be obtained in the form of pharmaceutical compositions per se or in the form of a hydrate, solvate, N-oxide or a pharmaceutically acceptable salt, as described above. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, while salts that are less soluble than the corresponding free acids and bases can be formed.

The medicaments according to the present invention can take a form convenient for virtually any type of application, including, for example, external, ophthalmic, internal, buccal, systemic, nasal, injectable, transdermal, rectal, vaginal, etc., or a form convenient for appointments in the form of inhalation or insufflation.

For external use, the active compound (s) or prodrug (prodrugs) can be prepared in the form of solutions, gels, ointments, creams, suspensions, etc., which is well known in the literature.

Systemic administration of such compounds includes those which are intended for use in the form of injections, for example, subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injections, as well as those intended for transdermal administration, administration through the mucous membrane, for internal or pulmonary use.

Effective injectable forms of drugs include sterile suspensions, solutions or emulsions of the active compound (s) in aqueous or oily solvents. The compositions may also contain formulating agents, such as a suspending agent, a stabilizing agent and / or a dispersant. The preparation of injectable dosage forms can be presented in unit dose form, for example, in ampoules or in multi-dose containers, and may contain added preservatives.

In an alternative embodiment, the injectable drug can be prescribed in the form of a powder, in which, to restore the initial consistency, a convenient solvent is added before use, including sterile, pyrogen-free water, buffer, dextrose solution, etc. To this end, the active compound (s) can be dried using any known technology, such as lyophilization, and returned to its previous form before use.

For use through the mucous membrane in the drug, wetting agents corresponding to the existing biological barrier are used. Such substances are known in the literature.

For internal use, pharmaceutical compositions may take the form of, for example, lozenges, lozenges, tablets or capsules prepared by conventional methods using pharmaceutically acceptable excipients, such as binders (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or quartz); disintegrants (e.g. potato starch or sodium starch glycolate) or wetting agents (e.g. sodium lauryl sulfate). Tablets can be coated using well-known methods with substances such as, for example, sugars, films or enteric coatings. Compounds that are particularly suitable for internal use include compounds R940350, R935372, R935193, R927050 and R935391.

Liquid medicinal products for internal use may take the form of, for example, elixirs, solutions, syrups or suspensions, or may be supplied as a dry product which, before use, dissolves in water or in another convenient solvent. Such liquid drugs can be prepared by conventional methods using pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); anhydrous solvents (e.g., almond oil, oily esters, ethyl alcohol, cremophor ^TM or fractionated vegetable oils) and preservatives (e.g. methyl or propyl p-hydroxybenzoates or sorbic acid). If necessary, medicines may also contain buffering salts, preservatives, flavoring, coloring and sweetening agents.

As is well known, drugs for internal use can be conveniently prepared to obtain a controlled release of the active compound or prodrug.

For buccal administration, the compositions may be shaped into tablets or lozenges prepared in a conventional manner.

For rectal or vaginal administration, the active compound (s) can be prepared in the form of solutions (for holding enemas), suppositories (suppositories), or ointments containing a suppository base such as cocoa butter or other glycerides.

For intranasal administration or administration by inhalation or injection, the active compound (s) or prodrug (s) may conveniently be supplied as aerosols for spraying from pressurized cans or a nebulizer using a convenient compressed liquid, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons carbon dioxide or other suitable gas. If aerosols are used under pressure, the dosage can be determined using a valve to dispense a measured amount of the drug. Capsules and sprays used in an inhaler or blower (for example, capsules and sprays consisting of gelatin) can be formulated to contain a powder mixture of a drug compound and a convenient powder base such as lactose or starch.

A specific example of a medicament in the form of an aqueous suspension suitable for intranasal administration using commercially available aerosol intranasal devices includes the following ingredients: active compound or prodrug (0.5-20 mg / ml); benzalkonium chloride (0.1-0.2 mg / ml); polyoxyethylene fatty acid ester 80 (TWEEN® 80; 0.5-5 mg / ml); sodium carboxymethyl cellulose or microcrystalline cellulose (1-15 mg / ml); phenylethanol (1-4 mg / ml) and dextrose (20-50 mg / ml). The pH of the final suspension can be adjusted in the range of about pH5 to pH7, usually at pH 5.5.

Another specific example of an aqueous suspension suitable for administration of the compounds by inhalation, and in particular for such administration of the compound, R921218 contains 1-20 mg / ml of the compound or prodrug, 0.1-1% (volume) of polyoxyethylene fatty acid ester 80 (TWEEN ®80), 50 mmol is a salt of citric acid and / or 0.9% sodium chloride.

For ophthalmic use, the active compound (s) or prodrug (s) can be prepared in the form of a solution, emulsion, suspension, etc., suitable for ophthalmic use. Many solvents are known in the literature that are convenient for use with compounds used as ophthalmic agents. Some specific examples are described in US patent No. 6,261,547; 6,197,934; 6,056,950; 5,800,807; 5,776,445; 5,698,219; 5,521,222; 5,403,841; 5,077,033; 4,882,150 and 4,738,851.

For sustained delivery, the active compound (s) or prodrug (s) can be prepared as a sustained absorption drug taken by implantation or intramuscular injection. The active ingredient may be prepared using suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or sparingly soluble derivatives, for example sparingly soluble salts. Alternatively, transdermal delivery systems, such as an adhesive disk or sticker, that slowly release the active compound (s) for subcutaneous absorption may be used. To this end, penetration stimulants can be used to improve subcutaneous absorption of the active compound (s). Convenient transdermal areas are described, for example, in US patent No. 5 407 713; 5,352,456; 5 332 213; 5,336,168; 5,290,561; 5,254,346; 5,164,189; 5,163,899; 5,088,977; 5,087,240; 5,008,110 and 4,921,475.

Alternatively, other drug delivery systems may be used. Liposomes and emulsions are well known examples of carrier solvents that can be used to deliver the active compound (s) or prodrug (s). Certain organic solvents such as dimethyl sulfoxide (DMSO) can also be used, although this usually results in higher toxicity.

Medicines, if necessary, can be presented in the form of a package or a dispensing device, which may contain one or more dosage forms with the active compound (s). This package may contain, for example, metal or plastic foil as a blister pack. The package or dispenser may contain instructions for use with this medication.

6.6 Effective Dosages

The active compound (s) or prodrug (s) of the present invention or mixtures thereof will generally be used in amounts effective to achieve the intended results, for example, in an amount sufficient to treat or prevent a particular disease. The compound (s) may be administered therapeutically to achieve a therapeutic result or prophylactically to achieve a prophylactic result. The therapeutic result means the elimination or reduction of the underlying disorder being treated and / or the elimination or alleviation of one or more symptoms associated with the underlying disorder to such an extent that the patient feels an improvement in well-being or condition, even if he continues to suffer from this disorder . For example, administering the active compound to a patient suffering from an allergy provides a therapeutic result not only when the main allergic reaction is eliminated or weakened, but also when the patient experiences a weakening severity or duration of allergy symptoms as a result of exposure to the allergen. As another example, a therapeutic outcome in the case of asthma includes improving breathing after an asthmatic attack or reducing the frequency or severity of asthmatic episodes. The therapeutic result also includes the suspension or slowdown of the development of the disease, regardless of whether an improvement has occurred.

For prophylactic administration, the compound may be prescribed to a patient at risk of developing one of the previously described diseases. For example, if it is not known whether the patient is prone to an allergic reaction to a particular drug, the compound may be administered prior to prescribing the drug to avoid or reduce the allergic reaction to that drug. Alternatively, prophylactic administration may be used to avoid the patient having symptoms associated with the underlying disorder. For example, a compound may be administered to an allergy sufferer prior to anticipated exposure to an allergen. The compounds can also be prophylactically prescribed to healthy people who are repeatedly exposed to agents associated with one of the above diseases in order to avoid the onset of the disorder. For example, a compound can be administered to a healthy person who is repeatedly exposed to an allergen known for its ability to cause allergies, such as latex, with the intention of preventing the development of an allergy in that person. Alternatively, the compound may be administered to a patient suffering from asthma prior to initiating activities that cause asthmatic attacks in order to reduce their severity or to completely avoid the manifestation of asthmatic episodes.

The amount of compound administered depends on many factors, including, for example, specific indicators, method of use, whether the treatment is prophylactic or therapeutic, the severity of the disease, the age and weight of the patient, bioaccumulation of the specific active compound, etc. The accuracy of determining the effective dose is easily determined by a specialist.

Effective doses can initially be estimated using in vitro samples. For example, the initial dose for animals can be formulated so that the concentration of the active compound circulating in the blood or serum is the same or exceeds the IC ₅₀ value for a particular compound in accordance with the results of measurements in an in vitro sample, such as, for example, in vitro CHMC or BMMC, as well as in other in vitro samples described in the Examples section. The accuracy of the dosage calculation to achieve such circulating concentrations in the blood or serum, taking into account the bioaccumulation of a particular compound, is easily determined by an experienced physician. For further information, the reader may refer to the publication: Fingl & Woodbury, “General Principles,” In: Goodman and Gilman's The Pharmaceutical Basis of Therapeutics , Section 1, pp. 1-46, latest edition, Pagamonon Press, as well as the links provided in the specified book.

Initial doses can also be estimated from in vivo analysis using, for example, experimental animal models. Experimental animal models used to test the effects of compounds for treating or preventing the various diseases described above are well known in the literature. Useful animal models for investigating hypersensitivity or allergic reactions are described in Foster, 1995, Allergy 50 (21 Suppl): 6-9, discussion 34-38, and Tumas et al. , 2001, J. Allergy Clin. Immunol. 107 (6): 1025-1033. Useful animal models for the study of allergic rhinitis are described in the following sources: Szelenyi et al. 2000, Arzneimittelforschung 50 (11): 1037-42); Kawaguchi et al. 1994, Clin. Exp. Allergy 24 (3): 238-244) and Sugimoto et al. , 2000, Immunopharmacology 48 (1): 1-7. Useful animal models for the study of allergic conjunctivitis are described in the following sources: Carreras et al. 1993, Br. J. Ophthalmol. 77 (8): 509-514; Saiga et al. 1992, Ophthalmic Res. 24 (1): 45-50; and Kunert et al. , 2001, Invest. Ophthalmol. Vis Sci. 42 (11): 2483-2489. Useful animal experimental models for studying systemic mastocytosis are described by O'Keefe et al. , 1987, J. Vet. Intern. Med. 1 (2): 75-80 and Bean-Knudsen et al. 1989, Vet. Pathol. 26 (1): 90-92. Useful animal experimental models for investigating hyper-IgE syndrome are described by Claman et al. 1990, Clin. Immunol. Immunopathol. 56 (1): 46-53. Useful animal experimental models for investigating B-cell lymphoma are described by Hough et al. 1998, Proc. Natl. Acad. Sci. USA 95: 13853-13858 and Hakim et al. , 1996, J. Immunol. 157 (12): 5503-5511. Useful animal experimental models for investigating atopic disorders such as atopic dermatitis, atopic eczema, and atopic asthma are described by Chan et al. , 2001, J. Invest. Dermatol. 117 (4): 977-983 and Suto et al. , 1999, Int. Arch. Allergy Immunol. 120 (Suppl 1): 70-75. Those of ordinary skill in the art can easily use this information to determine the dosage suitable for human administration. Additional useful experimental animal models are described in the Examples section.

Dose sizes can usually range from about 0.0001 or 0.001 or 0.01 mg / kg / day to 100 mg / kg / day, but they can be more or less depending, among other factors, on the activity of the compound, its bioaccumulation, method of administration and various factors described above. Dose size and administration intervals can be individually adjusted in order to provide plasma levels of the compound (s) sufficient to maintain a therapeutic or prophylactic effect. For example, the compounds may be prescribed once a week, several times a week (for example, every other day), once a day, or several times a day, depending, inter alia, on the method of application, the specific indications of treatment and the decision of the patient a doctor. In cases of local administration or selective administration, such as topical administration, the effective local concentration of the active compound (s) may not be related to the plasma concentration. Specialists in this field will be able to optimize the effective local dose, without resorting to experimental verification.

Preferably, the compound (s) has a therapeutic or prophylactic effect without causing significant toxicity. The toxicity of the compound (s) can be determined using standard pharmaceutical procedures. The relationship between toxic and therapeutic (or prophylactic) dose effects is a therapeutic indicator of a drug. Preference should be given to the compound (s) with high therapeutic performance.

In addition to the description of the invention, the following examples are provided illustrating its effect, but not limiting its use.

7. EXAMPLES

7.1. Synthesis of starting materials and intermediates useful for synthesizing 2,4-pyrimidinediamine compounds in accordance with schemes (I) to (V)

A large number of starting materials and N4-monosubstituted-2-pyrimidinamines and N2-monosubstituted-4-pyrimidinediamines [aromatic nucleophilic substitution reaction products (RANZ)] useful for the synthesis of 2,4-pyrimidinediamine compounds according to the present invention in accordance with schemes (I) - (V) was obtained as described below (see graphic part of description). Conditions suitable for synthesizing mono-SNAR products are demonstrated using 2-chloro-N4- (3,4-ethylenedioxyphenyl) -5-fluoro-4-pyrimidinamine (R926087).

7.5 The 2,4-pyrimidinediamine compounds of the invention are inhibitors of FcεRI receptor mediated degranulation.

The ability of the 2,4-pyrimidinediamine compounds of the present invention to inhibit IgE-induced degranulation has been demonstrated by various cell assays on cultured human mast cells (CHMC) and / or cells derived from mouse bone marrow (BMMC). Inhibition of degranulation was measured both at low and at high cell population densities using quantitative analysis of the isolation of granule-specific factors: tryptase, histamine, and hexosaminidase. Inhibition of the release and / or synthesis of lipid mediators was evaluated by measuring the release of leukotriene LTC4, and inhibition of the release and / or synthesis of cytokines was observed by quantitative analysis of TNF-α, IL-6 and IL-13; quantitative analysis of tryptase and hexosaminidase was performed using fluorogenic substrates in accordance with the description given in specific examples. Quantification of histamine, TNFα, IL-6, IL-13, and LTC4 was performed using the following commercial kits for detecting specific antibodies or antigens using an antigen or antibody immobilized enzyme (ELISA): histamine (supplier company: Immunotech, catalog No. 2015, Beckman Coulter), TNFα (supplier company: Biosource, catalog number KHC3011), IL-6 (supplier company: Biosource, catalog number KMC0061), IL-13 (supplier company: Biosource, catalog number KHC0132) and LTC4 (supplier: Cayman Chemical, catalog number 520211). The protocols for the various analyzes are given below.

7.5.1 Cultivation of human mastocytes and basophils

Human mast cells and basophils were cultured from CD34-negative progenitor cells in accordance with the description below (see also the methods set forth in U.S. Patent Application Serial No. 10 / 053,355, filed November 8, 2001, the description of which is included to the text of this document by reference to the source).

7.5.1.1 Preparation of the complete nutrient medium STEMPRO-34

To prepare a complete culture medium (CM), STEMPRO-34, 250 ml of STEMPRO-34 ^TM serum-free medium (SFM) (supplier company: GibcoBRL, catalog No. 10640) was placed in a filter flask. To the same flask was added 13 ml of the STEMPRO-34 Nutrient Supplement (NS) (supplier company: GibcoBRL, catalog No. 10641) (the preparation process is described in more detail below). The nutritional supplement jar was rinsed with approximately 10 ml of serum-free medium (SFM) and added to the flask for filtration. After adding 5 ml of L-glutamine (200 mmol; supplier company: Mediatech, catalog number MT 25-005-CI) and 5 ml of 100X penicillin / streptomycin (“pen-strep”; supplier company: HyClone, catalog No. SV30010), the volume was adjusted to 500 ml by the addition of serum-free medium (SFM) and the solution was filtered.

The largest number of variables in the preparation of a complete nutrient medium (CM) contains the method by which the nutritional supplement (NS) is thawed and mixed before being introduced into serum-free medium (SFM). Defrosting a nutritional supplement (NS) must be done in a water bath at 37 ° C with circular stirring without swirling or shaking until completely dissolved. When mixing, it is necessary to monitor whether insoluble lipids remain. If there are any and the solution is heterogeneous, it is necessary to place the additive in the water bath again and continue the mixing process until a homogeneous solution is obtained. Sometimes this component dissolves immediately, sometimes after one or two cycles of mixing, and sometimes it does not dissolve at all. If after one or two hours the nutritional supplement does not change to a uniform solution, it should be discarded and a new one thawed. Do not use a nutritional supplement (NS) that is not uniform after defrosting.

7.5.1.2 Increasing CD34 + cell population

The initial and rather small population of CD34-positive (or CD34 +) cells (1-5 x l0 ⁶ cells) was increased to a relatively high number of CD34-negative progenitor cells (about 2-4 x l0 ⁹ cells) using a nutrient medium and methodology, described below. CD34 + cells (from one donor) were supplied by Allcells (Berkeley, CA). Due to a certain degree of variation in terms of quality and number of cells commonly supplied by this company, the resulting cell cultures were placed in a 15 ml conical tube and brought to a volume of 10 ml in a complete culture medium before use.

On day 0, viable (refractile) cells were counted, which were then placed in a centrifuge and rotated at a speed of 1200 rpm to pelletize. After this, the cells were re-suspended in a suspension with a density of 275,000 cells / ml in complete culture medium (CM) containing 200 ng / ml recombinant human stem cell factor (SCF) (company-supplier: Peprotech, catalog No. 300-07) and 20 ng / ml human flt-3 ligand (company-supplier: Peprotech, catalog No. 300-19) (“CM / SCF / flt-3 medium”). On approximately the fourth or fifth day, the culture density was checked by cell counting and the culture was diluted to a density of 275,000 cells / ml with fresh CM / SCF / flt-3 medium. On approximately the seventh day, the culture was transferred into a sterile tube and cell counts were performed. Cells were centrifuged at 1200 rpm and re-suspended in a density of 275,000 cells / ml in fresh CM / SCF / flt-3 medium.

This cycle was repeated, starting from day No. 0, a total of 3 to 5 times during the period of increasing population.

If it is necessary to re-suspend a large number of cultures contained in several flasks, the contents of all flasks were combined in one vessel before cell counting. Thus, the accuracy of cell counting was ensured, as well as a unified approach to the entire population. Before combining the contents, the flask was examined under a microscope for contamination in order to prevent possible contamination of the entire population.

 Between the 17th and 24th day, the deterioration of the state of the culture is possible (i.e., 5 to 10% of the total number of cells dies) and population growth slows down. During this period, cells are monitored daily, because in just a day the culture can completely die. After the onset of its deterioration, the cells are counted, centrifuged at 850 rpm for 15 minutes and re-suspended in a density of 350,000 cells / ml in a complete nutrient medium CM / SCF / flt-3 medium in order to obtain one or two divisions of culture. The cells are monitored daily in order to prevent the death of the culture.

When the percentage of cell death in the culture of the progenitor cells exceeds 15% and a certain amount of decomposition products is observed in the culture, CD34-negative progenitor cells are ready for differentiation.

7.5.1.3 Differentiation of CD34-negative progenitor cells into mucosal mast cells

The second phase is performed to convert the grown CD34-negative progenitor cells into differentiated mucosal mast cells. These mucous cultured human mast cells are obtained from CD34 + cells isolated from cord blood and processed to produce a large population of CD34-negative progenitor cells, as described above. In order to obtain CD43-negative progenitor cells, the culture suspension re-cycle was identical to the above except that the culture was seeded at a density of 425,000 cells / ml and about 15% additional culture medium was added on the fourth or fifth day without cell counting. In addition, the cytokine composition of the medium was changed so that it contained stem cell factor (200 ng / ml) and human recombinant IL-6 (200 ng / ml; company-supplier: Peprotech, catalog No. 200-06), restored up to 100 μm / ml in sterile acetic acid (10 mmol) (“CM / SCF / IL-6 medium”).

 Phases I and II take about 5 weeks in duration. Cell death and the presence of decomposition products in the culture are visible during the first to third week; in addition, from the second to the fifth week there is a period during which some part of the culture is no longer in suspension, but attached to the surface of the vessel in which the culture is grown.

As during phase I, if necessary, re-suspension of the culture, on the seventh day of each cycle, the contents of all flasks were combined in one vessel before cell counting to ensure uniformity of the entire population. Each flask was pre-checked under a microscope in order to identify contaminants and prevent contamination of the entire population.

 When combining the contents of the flasks, approximately 75% of the volume of the flask was poured into a common vessel, leaving about 10 ml in the flask. Then, the flask with the remaining liquid was tapped abruptly in the transverse direction in order to release the cells adhering to the surface. Repeated tapping was carried out at right angles to the first in order to finally move the cells from the place.

Before overflowing the remaining liquid into the vessel for counting, the flask was tilted at an angle of 45 degrees for several minutes. Before plating in a volume of 35-50 ml per flask (at a density of 425,000 cells / ml), the cells were centrifuged at 950 rpm for 15 minutes.

7.5.1.4 Differentiation of CD34-negative progenitor cells into connective tissue mast cells

An increased population of CD34-negative progenitor cells was prepared according to the above procedure and processed to form a tryptase / chemo-positive phenotype (connective tissue). In this case, methods similar to those described above for mast cells of the mucous membranes were used, with the exception of adding IL-4 instead of IL-6 to the culture medium. The resulting cells are typical for connective tissue mast cells.

7.5.1.5 Differentiation of CD34-negative progenitor cells into basophils

A multiplied population of CD34-negative progenitor cells was prepared according to the method described in section 7.5.1.3 above and used to form an increased basophil population. CD34-negative cells were treated in a manner similar to that described above for mucosal mast cells, except for the addition of IL-3 (20-50 ng / ml) instead of IL-6 in the culture medium.

7.5.2 Activation of CHMC (cultured human mast cells) using IgE at low cell density: tryptase and LTC4 analysis

65 μl of solutions of the prepared compounds or control samples prepared in MT [137 mmol NaCl, 2.7 mmol KCl, 1.8 mmol CaCl _2, 1.0 mmol MgCl ₂ should be added to two 96-well round-bottom plates (Costar 3799 model) , 5.6 mmol of glucose, 20 mmol of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (pH 7.4), 0.1% bovine serum albumin, (Sigma A4503)] with 2% MeOH and 1% DMSO . This is followed by the process of pelletizing CHMC cells in a centrifuge (980 rpm, 10 min) and re-suspension in a pre-heated MT medium. Next, 65 μl of cells should be poured into each 96-well round-bottom plate. Depending on the degranulation activity of each individual CHMC donor, place 1,000-1,500 cells in each well. Stir four times, followed by incubation for 1 hour at 37ºC. Prepare a 6X anti-IgE solution [rabbit anti-human IgE rabbit immunoglobulin (1 mg / ml, supply company: Bethyl Laboratories, catalog number A80-109A), diluted 1: 167 in MT buffer solution, in the hour required for incubation. ]. Stimulate cells by adding 25 μl of a 6X anti-IgE solution to the appropriate plates. Add 25 µl MT to control tubes not subject to stimulation. After adding anti-immunoglobulin E, mix twice. Incubate at 37ºC for 30 minutes. During a 30-minute incubation, dilute a solution of 20 mmol broth with tryptase substrate [(Z-Ala-Lys-Arg-AMC ^. 2TFA; Enzyme Systems Products, No. AMC-246)] 1: 2000 in a buffer solution for quantitative tryptase analysis [0 , 1 M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (pH 7.5), 10% m / v. glycerol, 10 μmol of heparin (Sigma H-4898) 0.01% NaN ₃ ]. Treat the plates in a centrifuge at 1,000 rpm for 10 minutes to pelletize the cells. Transfer 25 μl of the supernatant to a 96-well black bottom plate and add 100 μl of freshly diluted tryptase substrate solution to each well. Incubate for 30 minutes at room temperature. Using a spectrophotometer to read the tablets, determine the optical density of the tablets at 355nm / 460nm.

Quantitative analysis of leukotriene C4 (LTC4) is also carried out using ELISA kits on properly diluted samples of the supernatant (determined empirically for the cell population of each donor so that the sample measurements are within the standard curve) in accordance with the manufacturer's instructions.

7.5.3 Activation of CHMC with IgE at high cell density: degranulation assays (tryptase, histamine), leukotriene (LTC4) and cytokine (TNFalpha, IL-13)

Cultured human mast cells (CHMCs) are sensitized for 5 days using IL-4 (20 ng / ml), stem cell factor (200 ng / ml), IL-6 (200 ng / ml) and human IgE CP 1035K (company- supplier company: Cortx Biochem, 100-500 ng / ml, depending on generation) in an air-conditioned environment. After sensitization, the cells are counted, centrifuged (1000 rpm, 5-10 minutes) and reconverted into a suspension with a density of 1-2 x10 ⁶ cells / ml in MT buffer solution. Add 100 μl of cell suspension to each well and 100 μl of solutions of the prepared compositions. The final concentration of the medium is 0.5% DMSO. Incubate at 37ºC (5% CO ₂ ) for 1 hour. After an hour of reagent treatment, stimulate 6X anti-IgE cells. Mix the tubes with the cells and leave the plates for incubation at 37ºC (5% CO ₂ ) for one hour. After incubation for an hour, treat the cells in a centrifuge (10 minutes, 1000 rpm) and take 200 μl of the supernatant from each tube, while being careful not to touch the pellet. Place the supernatant plate on ice. When performing an item requiring 7 hours (see below), determine the tryptase activity of the supernatant diluted 1: 500, resuspend the cells in 240 μl of conditioned medium containing 0.5% DMSO and the corresponding concentration of the compound. Incubate CHMC cells for 7 hours at 37 ° C (5% CO ₂ ). After incubation, centrifuge the cells at 1,000 rpm, 10 minutes), withdraw 225 μl from each tube and store at -80ºC until ready for ELISA assays. ELISA is carried out on properly diluted samples (determined empirically for the population of cells of each donor so that the measurements of the samples are within the standard curve) in accordance with the manufacturer's instructions.

7.5.4 Activation of BMMC (mouse bone marrow cells) with IgE at high cell density: degranulation assays (hexosiminidase, histamine), leukotriene (LTC4) and cytokine (TNFalpha, IL-6)

7.5.4.1 Preparation of WEHI-conditioned media

WEHI-conditioned medium was obtained by growing WEHI-3B murine myelomonocytic cells (American Type Culture Collection, Rockville, Md.) In the Isagov-modified Eagles medium (supplier company: Mediatech, Gernandon, pieces of Virginia) with the addition of 10% bovine fetal serum, inactivated by heat treatment (manufactured by JRH Biosciences, Kansas City, Missouri), 50 μmol of 2-mercaptoethanol (company supplier: Sigma, St. Lewis, Mo.) and 100 IU / ml penicillin-streptomycetes for (company supplier company: Mediatech) in an incubator at 37ºC with a humidified gas mixture of 5% CO _2/95% air. An initial cell suspension was plated at approximately 200,000 cells / ml and then divided 1: 4 every 3-4 days for two weeks. Cell-free supernatants were collected, aliquoted and stored at -80 ° C until needed.

7.5.4.2 Preparation of BMMC medium

BMMC media consists of 20% WEHI conditioned medium, 10% bovine fetal serum inactivated by heat treatment (supplier: JHR Biosciences), 25 mmol N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4 (supplying company: Sigma), 2 mmol of L-glutamine (supplying company: Mediatech), 0.1 mmol of non-essential amino acids (supplying company: Mediatech), 1 mmol of sodium pyruvate (supplying company: Mediatech) , 50 μmol of 2-mercaptoethanol (company-supplier company: Sigma) and 100 IU / ml penicillin-streptomycin (company-company-pos vendor: Mediatech) in RPMI 1640 environment (company-supplier: Mediatech). To prepare the BMMC medium, all components are combined in a sterile IL filtration vessel and passed through a 0.2 μm filter before use.

7.5.4.3 Protocol

Bone marrow derived mast cells (BMMCs) are sensitized for about half a day with mouse stem cell factor (20 ng / ml) and a monoclonal anti-DNA protein complex (10 ng / ml, Clone SPE-7, company-company supplier: Sigma, catalog No. D-8406) in BMMC medium at a cell density of 666 × 10 ³ cells / ml. After sensitization, the cells are counted, centrifuged (1,000 rpm, 5-10 minutes) and re-turned into a suspension with a density of 1-3 × 10 ⁶ cells / ml in MT-buffer. Add 100 μl of cell suspension to each well and 100 μl of compound solutions. The final concentration of filler is 0.5% DMSO. Incubate at 37ºC (5% CO ₂ ) for 1 hour. After 1 hour of treatment with the composition, stimulate cells with 6X stimulus (60 ng / ml bovine serum DNA-protein complex-albumin). Mix the wells with the cells and incubate the plates at 37ºC (5% CO ₂ ) for one hour. After one hour of incubation, centrifuge the cells (10 minutes, 1,000 rpm), take 200 μl of the supernatant from each tube; while doing this, be careful not to touch the pellet and transfer to a clean tube of a 96-well plate. Place the tablet with the supernatant on ice. During the 4-5 hour (see below) procedure, perform an analysis of hexosimidase. Re-prepare the cell suspension in 240 μl of WEI conditioned medium containing 0.5% DMSO and the appropriate concentration of the compound. Incubate BMMC cells for 4-5 hours at 37 ° C (5% CO ₂ ). After incubation, treat the cells in a centrifuge (1,000 rpm, 10 minutes), withdraw 225 μl from each tube and store at - 80ºC until ready for ELISA. ELISA is carried out on properly diluted samples (determined empirically for the population of cells of each donor so that the measurements of the samples are within the standard curve) in accordance with the manufacturer's instructions.

Hexosaminidase analysis: Pipette 50 μl of a hexosaminidase substrate (4-methylumbelliferyl-N-acetyl-β-D-glucosaminide; 2 mmol) into each well of a black 96-well plate. Add 50 μl of the BMMC cell supernatant (see above) to the hexosaminidase substrate, incubate at 37ºC for 30 minutes and analyze the plate at 5, 10, 15, and 30 minutes on a spectrophotometer.

7.5.5 Activating Basophils with IgE or a House Tick Allergen: Histamine Analysis

Basophil activation analysis was performed using whole human peripheral blood obtained from donors who are allergic to house mites; most red blood cells were removed by dextran precipitation. Human peripheral blood was mixed in a 1: 1 ratio with 3% T500 dextran and erythrocyte sedimentation was performed for 20-25 minutes. The upper fraction was diluted with three volumes of physiological saline buffered with Dulbecco phosphate, after which the cells were centrifuged for 10 min at 1500 rpm at room temperature. The supernatant was removed with an aspirator and the cells were washed with an equal volume of MT buffer. Finally, the cells were resuspended in MT buffer containing 0.5% DMSO in the original blood volume. 80 μl of cells were mixed with 20 μl of the compound in the presence of 0.5% DMSO in triplicate in a deep-bottom 96-well plate for culturing tissue cultures. A number of dosages of concentrates of 8 compounds were tested, as a result of which a 10-point response curve was obtained for the dosage, including the maximum (stimulated) and minimum (unstimulated) reaction. Cell incubation was carried out after adding compounds for 1 hour at 37 ° C with 5% CO ₂ , after which 20 μl of 6x stimulator [1 μg / ml anti-IgE (supplier company: Bethyl Laboratories) 667 au / ml house tick was added (company-supplier company: Antigen Laboratories)]. Cells were stimulated for 30 minutes at 37 ° C, 5% CO ₂ , the plate was centrifuged for 10 minutes at 1,500 rpm at room temperature, and 80 μl of supernatant was collected for histamine analysis, for which histamine ELISA kit was used (company- supplier company: Immunotech). ELISA was performed in accordance with the instructions of the supplier company.

7.5.6 Results

The results of the analysis of low-density CHMC populations (Section 7.5.2), the high-density CHMC populations (Section 7.5.4) and the analysis of basophils (Section 7.5.5) are shown in Table 1, the results of the analysis of high-density CHMC populations (Section 7.5.3) are given in Table 2. All values in tables 1 and 2 are expressed in IC ₅₀ (in µmol). The indicator “9999” corresponds to an IC ₅₀ > 10 μmol in the absence of measurable activity at a concentration of 10 μmol. Most of the analyzed compounds had IC ₅₀ values of less than 10 μmol, and in many IC ₅₀ values were observed in the submicromolar range.

7.6 2,4-Pyrimidinediamine compounds inhibit FcγRI receptor mediated degranulation

The ability of the 2,4-pyrimidinediamine compounds described in the present invention to inhibit FcγRI-mediated degranulation was demonstrated using the compositions R921218, R921302, R921303, R940347, R920410, R927050, R940350, R935372, R920323, R9262, R9261, R9262, R926 Section 7.5, except that the cells were not pretreated with IgE immunoglobulin and were activated with the Fab fragment of the rabbit anti-human immunoglobulin IgG (supplier company: Bethyl Laboratories, catalog number A80-105).

All analyzed compounds had IC ₅₀ values in the submicromolar range.

7.7 Selective inhibition of the 2,4-pyrimidinediamine compounds of the invention by an inverse cascade of IgE receptor reactions

To confirm that many of the 2,4-pyrimidinediamine compounds of the invention exhibit their inhibitory ability by blocking or inhibiting the transmission cascade of the initial IgE receptor signals, some of the compounds were tested by ionomycin-induced degranulation cell analysis as described below .

7.7.1 Activation of low density CHMC by ionomycin, tryptase assay

The analysis of mast cell degranulation induced by ionomycin was carried out similarly to the analysis of low density IgE CHMC activation (Section 7.5.2 above ) except that a solution of 6X ionomycin [5 mmol of ionomycin (Signma I-0634) in MeOH (stock solution) diluted 1: 416.7 in MT buffer (final concentration 2 μmol)] and cells were stimulated by adding 25 μl of 6X ionomycin solution to the appropriate plates.

7.7.2 Ionomycin Basophil Activation: Histamine Release Analysis

Assays for ionomycin-induced basophil degranulation were performed similarly to the method described for analyzing basophil activation with IgE immunoglobulin or a house tick allergen (Section 7.5.5 above ), except that after incubation with the compound, the cells were stimulated with 20 μl of 2 μmol ionomycin solution .

7.7.3 Results

The results of ionomycin-induced degranulation tests, presented as IC ₅₀ values (in μmol), are shown in Table 1. The vast majority of active compounds analyzed (ie, inhibiting immunoglobulin E-induced degranulation) do not inhibit ionomycin-induced degranulation, which confirms the selective inhibition by these active compounds of the cascade of transmission of early (or reverse) IgE receptor signals.

These results were confirmed for certain compounds by measuring the fluxes of calcium ions in CHMC caused by anti-immunoglobulin IgE and ionomycin. When testing the Ca ²⁺ fluxes, it was found that the compounds R921218 (10 μmol) and R902420 (10 μmol) inhibit the Ca ²⁺ flux induced by anti-immunoglobulin IgE, but do not affect the Ca ²⁺ flux induced by ionomycin (see Fig. .four).

7.8 Immediate manifestation of the inhibition effect produced by the 2,4-pyrimidinediamine compounds of the present invention

To test the immediate effect of the inhibition, some of the 2,4-pyrimidinediamines of the invention were added to the above formulations to analyze cell activity simultaneously with an anti-IgE antibody activator. All analyzed compounds blocked the induced IgE degranulation of CHMC to the same extent that was observed upon preincubation of the CHMC compounds for 10 or 30 min before cross-linking of the receptors.

7.9 Kinetics of pharmacological activity in vitro

Compounds R921218, R921302, R921219, R926240, R940277, R926742, R926495, R909243 and R926782 were analyzed in washing experiments. During the experiments, CHMC cells were either immediately activated with an anti-IgE antibody in the presence of 1.25 μmol of the compound (time zero) or the compound was washed out, followed by activation with an anti-IgE antibody after 30, 60 or 120 minutes. The inhibition activity of the aforementioned compounds fell sharply 30 minutes after removal of the compound, which indicates the need for continuous exposure of these compounds to mast cells to maximize the suppression of degranulation. Tests of other formulations yielded similar results.

7.10 Toxicity: T and B cells

The ability of the compounds of the invention to exhibit inhibitory activity in the absence of toxicity to cells of the immune system was demonstrated in the course of cell analyzes with B and T cells. Test reports are provided below.

7.10.1 Toxicity to Jurkat cells (T-cell)

Jurkat cells were diluted to a concentration of 2x10- ⁵ cells / ml in complete medium RPMI (10% fetal calf serum inactivated by heat treatment) and incubated at 37 ° C in the presence of 5% CO ₂ for 18 hours. Then, 65 μl of cells with a density of 7.7 x 10 ⁵ cells / ml was added to a 96-well plate with deep bottom wells (treated with TC (tissue culture), vendor: Costar), which contained 65 μl of compound 2X (final carrier concentration : 0.5% DMSO, 1.5% MeOH). The mixture was stirred and incubated for 18-24 hours at 37 ° C in an atmosphere of 5% CO ₂ . Toxicity was evaluated by flow cytometric analysis of cell light scattering.

7.10.2 Toxicity to the "BJAB" cell (B-cell)

The BJAB B cell line was cultured in log phase in RPMI1640 + 10% fetal bovine serum, heat-inactivated, 1x L-glutamine, 1x penicillin, 1x streptavidin and 1x beta-mercaptoethanol at 37 ° C, 5% CO ₂ . First, BJAB cells were harvested, centrifuged, and resuspended in culture medium at a concentration of 7.7 × 10 ⁵ cells / ml. 65 μl of cells were mixed with 65 μl of the compound in duplicate in the presence of 0.1% DMSO in a 96-well plate with deep bottom wells. Cells were incubated with the compound at various concentrations at 37 ° C in an atmosphere of 5% CO _2. Toxicity was evaluated by flow cytometric analysis of cell light scattering.

7.10.3 Toxicity: Glo cell titer analysis

50 μl of cells (1 × 10 ⁶ / ml) was placed in each well containing 50 μl of compound. The final concentration of the carrier is 0.5% DMSO, 1.5% MeOH. The plates were shaken for one minute to mix the cells and mix. The plates were then incubated at 37 ° C (5% CO ₂ ) for 18 hours. The next day, 50 μl of cells were collected from each well and poured into 50 μl of Glo cell titer reagent (supplier: Invitrogen). The tablets were shaken for one minute, after which they were read with a luminometer.

7.10.4 Results

The results of toxicity tests for T and B cells, presented as indicators of IC ₅₀ (in μmol), are shown in Table 2. With a few exceptions (see Table 1), all analyzed compounds did not show toxicity to T - and B -cells at an effective concentration for inhibiting. Assays performed on embryonic B cells gave similar results.

7.11 Tolerance of 2,4-pyrimidine compounds in animals

The ability of the compounds of the invention to exhibit inhibitory activity at doses below toxic to animals was demonstrated by the example of compounds R921218, R921219 and R921302.

7.11.1 Connection R921218

Compound R921218 was investigated as part of a large-scale program of non-clinical safety studies that showed that this compound is well tolerated by rodents and other animals. Summary of Toxicological / Nonclinical Studies on the Safety of Compound R921218: This chemical does not cause dose-limiting toxicity, whether intranasally administered to non-rodent animals (rabbits and primates), or oral administration to rodents (mice and rats) during 14-day toxicological studies with repeated administration of doses many times higher than the expected effective doses sufficient for humans. A negative effect on the main indicators of the functions of the cardiovascular, respiratory and (or) central nervous system, studied to determine pharmacological safety, was not found. Genetic toxicological analyzes showed no evidence of either mutagenic or clastogenic potential; no adverse effects were found after exposure to the eyes and skin. A brief description of the main toxicological studies is provided.

A 14-day toxicology study with multiple dosing intranasally was performed on Cynomolgus monkeys at dosages of 2.1, 4.5, or 6.3 mg / kg / day. The parameters included clinical observations, body weight, food intake, ophthalmological parameters, blood pressure, electrocardiography, hematological parameters, clinical biochemical parameters, urinalysis, immunotoxicological assessment, general autopsy, organ mass, toxicokinetic assessments and histopathology (including nasal cavity). None of the study parameters revealed negative effects associated with the use of R921218, and the dose at which there were no observed negative manifestations was set at 6.3 mg / kg / day.

A 14-day toxicological study with multiple dosing intranasally was carried out on rabbits of the breed "White New Zealand" with dosages of 1.7, 3.4 or 5.0 mg / kg / day. Elements of observation included clinical observations, body weight, food consumed, ophthalmological parameters, hematological parameters, clinical biochemical parameters, general autopsy, organ mass, toxicokinetic assessments and histopathology (including nasal cavity). None of the study parameters revealed negative effects associated with the use of R921218, and the dose at which there were no observed negative manifestations was set at 5.0 mg / kg / day.

7.11.2 Connection R921219

In experimental dosage studies, a single oral dose of 600 mg / kg was taken as the dose at which there were no observed effects, while multiple (over 7 days) doses of 200 mg / kg / day and above were not tolerated.

An in vitro analysis of Salmonella-Escherichia coli / mammalian microsome reverse mutation (Ames test) showed a positive result for R921219 in the TA1537 line analyzer with and without metabolic activation, which confirms the results of a previous study. No adverse effects of R921219 on any of the other four analyzer lines were detected. It was found that R921219 lacks clastogenic potential in an in vitro study of chromosomal aberrations.

7.11.3 Connection R921302

Several experimental toxicological studies have been carried out on rodents in an unproven laboratory method. For mice, an oral dose tolerance of up to 1,000 mg / kg for up to 7 days was detected. A 14-day toxicological study was carried out in mice with oral doses of 100, 300 and 1,000 mg / kg. A dose of 1,000 mg / kg was not tolerated, and a dose of 300 mg / kg led to the appearance of histopathological changes in the vulva. A dose of 100 mg / kg was considered the dose of the absence of observed adverse events in the study. A 28-day toxicological study was conducted using oral doses of 100 mg / kg once a day, 100 mg / kg twice a day, 300 mg / kg once a day, and 300 mg / kg twice a day. Intolerance to R921302 appeared at dosages of 300 mg / kg once a day and twice a day. Lower doses (100 mg / kg per day or twice daily) were well tolerated (the results of clinical and histopathological analyzes are not yet known). A rat study using oral doses of 50, 150 and 300 mg / kg for 32 days showed good tolerance (the results of clinical and histopathological analyzes are not yet known).

An in vitro analysis of Salmonella-Escherichia coli / mammalian microsome reverse mutation (Ames test) showed a positive result of R921302 in the TA98 line analyzer with and without S9 and TA1537 metabolic activation. No negative effects of R921302 on any of the other three analyzer lines were detected. An in vitro study of chromosomal aberrations found that R921302 lacks clastogenic potential.

7.12 Oral bioavailability of 2,4-pyrimidinediamine compounds

More than 50 compounds of 2,4-pyrimidinediamine provided in the present invention, were analyzed for oral bioavailability. During the study, the preparations were dissolved in various solvents (for example, a PEG 400 solution and a suspension of carboxymethyl cellulose) for intravenous and oral administration to rats. After drug administration, plasma samples were taken and examined. The concentration of compounds in the plasma was determined by high performance liquid chromatography / tandem mass spectrometry (LC / MS / MS). Pharmacokinetic analyzes were performed based on plasma concentration data. Pharmacokinetic parameters of interest included the following: clearance (CL), homeostasis distribution volume (Vss), terminal elimination half-life (t _½ ), and oral bioavailability (% F).

Pharmacokinetic studies indicate that many 2,4-pyrimidinediamine compounds are available orally, with% F reaching about 50% (in the range of 0-50%). The elimination half-life ranged from 30 minutes to 3 hours. In particular, compounds R940350, R935372, R935193, R927050 and R935391 showed good oral bioavailability and elimination half-life results in rat studies. Thus, studies confirm: these 2,4-pyrimidinediamine preparations are suitable for oral use.

7.13 The effectiveness of compounds in the treatment of allergies

The in vivo efficacy of compounds R926109, R921218, R921219, R921302, R926495, R926508, R926742, R926745, and R945150 in the treatment of allergic phenomena was evaluated in a mouse model of passive skin anaphylaxis (PCA). This model provides a direct measurement of tissue mast cell degranulation caused by IgE. In this model, animals sensitized to IgE are exposed to an allergen, and the change in the permeability of the skin vasculature as a result of the release of histamine by mast cells is measured by the change in the amount of dye penetration into the surrounding tissue. Inhibition of neurotransmitter ejection by compounds modulating mast cell degranulation is readily measured by extracting dye from the tissue.

7.13.1 Protocol and study results

In the analysis of passive cutaneous anaphylaxis, mice are passively sensitized with an intradermal injection of antidinitrophenol (DNP) IgE antibodies (day minus 1). At a predetermined point in time, the animals receive the test substance (Day 0). The modulating effect of the drug on the degranulation of skin mast cells is measured after intravenous administration of DNP coupled with human serum albumin (HSA - DNP), together with Evans blue dye. The resulting cross-linking of the IgE receptor and a subsequent increase in vascular permeability caused by mast cell degranulation is determined by measuring the volume of dye transudation into adjacent tissues. The dye is removed from the fabric with formamide and the absorption coefficient of the extract at 620 nm is read. The inhibitory effect of drug treatment is expressed as the percentage of inhibition compared to treatment with a neutral agent, i.e. as a percentage reduction A _620.

Two drugs were examined as positive controls: the histamine antagonist diphenhydramine and the serotonin antagonist cyproheptadine. Both mediators (histamine and serotonin) are released from mouse mast cells after IgE-mediated degranulation. Both controls inhibit passive cutaneous anaphylaxis; cyproheptadine was commonly used in subsequent experiments. Cyproheptadine with a reproducible result inhibited the reaction of passive cutaneous anaphylaxis by 61% +/- 4% (8 mg / kg, intraperitoneal, 30 minutes — premedication time, n = 23 experiments).

7.13.1.1 Results

Dose-dependent inhibition of FcεR-mediated vascular permeability was observed with increasing doses of R921218, R926109, R921219 and RR921302. These preparations were used either as a solution (67% PEG / 33% citrate buffer) or as an aqueous suspension (1.5% Avicel). The results show a pronounced relationship between plasma drug levels, in vivo efficacy, and in vitro activity. The most potent drug R921219 showed activity at a level of exposure to the circulatory system of about 10 μg / ml (68% inhibition when applying a dose of 100 mg / kg) compared with R921302, a relatively less active molecule that reduced plasma extravasation by 42% when applying a dose of 100 mg / kg Moreover, the duration of exposure to the drug affected the duration of inhibitory activity. R921302, which was considered the most metabolically stable drug in the course of pharmacokinetic studies, inhibited vascular permeability for 1-2 hours prior to the occurrence of receptor signals caused by antigen, after which the efficiency began to decrease. All these data are shown in table 3 and table 4.

TABLE 3
The effectiveness of R921218, R926109, R921219 and R921302 in the analysis of passive cutaneous anaphylaxis A drug Mode of application Filler Sedation time (min) Dose (mg / kg) % Inhibition Contents in plasma (μg / ml) R921218 Peror. 67% PEG / 33% citrate buffer 10 fifty 7 3 one hundred eleven four 200 fifty eighteen R926109 Peror. 67% PEG / 33% citrate buffer fifteen fifty 22 BUT one hundred 32 200 48 R921219 Peror. 1.5% Avicel / water fifteen thirty 25 0.4 one hundred 68 four 300 92 eleven R921302 Peror. 1.5% Avicel / water 60 fifty 35 25 one hundred 42 38 150 56 64 200 93 105 TABLE 4
Duration of action of R921219 and R921302 in analysis of passive cutaneous anaphylaxis A drug Mode of application Filler Dose (mg / kg) Sedation time (min) % Inhibition Contents in plasma (μg / ml) RR921302 Peror. 1.5% Avicel / water 200 thirty 89 88 60 83 53 120 82 61 240 37 8

Similar in vivo activity was observed in preparations R926495, R926508, R926742, R926745 and R926150, which were able to inhibit the passive skin anaphylaxis reaction when administered orally in the PEG-based formula (data not shown).

7.14 Effectiveness of Compounds for the Treatment of Asthma

The efficacy of compounds R921218, R921302, R926495, R926508, R926742 and R921219 for the treatment of asthma was demonstrated in a sheep allergic asthma model. Within a few minutes after exposure to the inhaled antigen ( Ascaris suum ), sheep develop bronchostenosis, with maximum airway obstruction observed during the initial allergic reaction. Most likely, this initial phase of airway obstruction is caused by the release of preformed mast cell mediators. In addition to the initial allergic reaction, the sheep model allowed us to evaluate the effect of the effects of our preparations on the late asthmatic reaction and nonspecific hypersensitivity of the respiratory tract, which develops as a result of local or local exposure of the allergen to the respiratory tract. In sheep, nonspecific airway hypersensitivity develops within a few hours after exposure to antigen and can last up to two weeks. The results presented below demonstrate the potential of the analyzed compounds to inhibit a cascade of events that can develop as a result of the release of cytokines from mast cells.

7.14.1 Research Protocol

In a model of allergic asthma, sheep are exposed to animals tested in an aerosol form through an endotracheal tube, followed by exposure to an aerosol antigen obtained from Ascaris suum roundworm, for which sheep have a natural allergy. Allergen stimulation leads to direct bronchostenosis (both an initial allergic reaction and a late asthmatic reaction) and sustained nonspecific airway hypersensitivity. These three characteristics are similar to symptoms in people with allergic asthma. The activity of the test drug is determined by the change in lung resistance (R _L ), which is calculated by the measured pulmonary pressure, air flow and tidal volume. Control data for previous periods obtained from the same sheep after treatment with saline, compared with exposure to the allergen, show a sharp increase in R _L during the initial allergic reaction, which lasts about 2-3 hours after irritation with the allergen. Late asthmatic reaction gives a less significant increase in R _L , occurring about 5-6 hours after exposure to the allergen and disappearing within 8 hours after exposure. Twenty-four hours after exposure, a response to a carbachol dose is measured to measure nonspecific airway hypersensitivity. This is expressed as the dose of carbachol needed to increase R _L by 400% compared to the baseline. (This measurement is called the provocative carbachol concentration necessary to increase R _L by 400% compared to the baseline value (PC ₄₀₀ ). The data are compared with control data from previous periods for the same subject with the introduction of a control aerosol and exposure to Ascaris suum .

7.14.2 Result

All analyzed drugs showed the effect of inhibiting late asthmatic reactions and persistent nonspecific hypersensitivity, and some of these drugs also inhibited the initial allergic reaction. The optimal results for each of the preparations obtained as a result of a number of studies to evaluate activity in the case of different durations of premedication and different compositions of solutions and suspensions are shown in Table 5 (see the graphic part of the description). The effectiveness of the effect of R921218 on the initial allergic reaction depends on the composition, with the greatest effect observed at a dosage of 30 mg / sheep, the introduction of an aerosol solution in 10% ethanol. R926495, R926742, R926508, and R921219, applied to four different sheep at a dosage of 45 mg / sheep in an aqueous suspension 60 minutes before exposure to the allergen, showed blocking of late asthmatic reaction and nonspecific airway hypersensitivity. In addition to these late manifestations, the initial allergic reaction was also significantly reduced by treatment with R921219, R926508 or R926495. The effectiveness of the drug RR921302 was investigated using a filler of 45% PEG400 / 55% citrate buffer. Under such conditions, R921302, administered at a dose of 30 mg / sheep 60 minutes before exposure, blocked the asthmatic reaction and nonspecific airway hypersensitivity, but the initial allergic reaction remained unchanged.

These data clearly demonstrated that the mentioned drugs have the ability to block the asthmatic reaction in sheep with allergies. All drugs showed significant inhibition of nonspecific airway hypersensitivity and late asthmatic reactions compared with control data for previous periods. The initial allergic reaction was significantly inhibited by R921219, R926508 and R926495 (54%, 21% and 33%, respectively). In contrast, R921218, R921302 and R926742 did not inhibit the initial allergic reaction when using preparations in the form of an aqueous suspension.

7.15 The effectiveness of drugs in the treatment of asthma

The efficacy of R921304 and R921219 in the treatment of asthma was also demonstrated in a mouse allergic asthma model.

7.15.1 Research Protocol

Mice are sensitized to ovalbumin (chicken protein) in the presence of an adjuvant (alum) intraperitoneally on day 0 and day 7. After a week, ovalbumin is administered to the mice intranasally - on days 14, 15 and 16 (stricter model) or on day 14 (less strict model ) Such sensitization and exposure regimen lead to airway hypersensitivity and inflammation in the lungs, and these are two dominant characteristics of human allergic asthma. In a mouse model, the in vivo airway response is measured by a common plethysmograph that determines PENH (enhanced Pause, Buxco Electronics). PENH is a dimensionless indicator consisting of the maximum inspiratory speed, maximum expiratory flow, inspiratory time, expiratory time, relaxation time, and is considered a confirmed parameter of the sensitivity of the respiratory tract. Allergen response (OVA) is compared with animals exposed to pure saline. Twenty-four hours after exposure, mice are given increasing doses of methacholine (a muscarinic receptor agonist), which causes a reduction in smooth muscle tissue. In mice exposed to ovalbumin, there is a significant hypersensitivity of the respiratory tract to methacholine compared to mice treated with saline. In addition, cell infiltrate was detected in the airways of mice exposed to ovalbumin, unlike mice injected with saline. Infiltrate consists mainly of eosinophils, but there are also some neutrophils and mononuclear cells.

The use of this model for the assessment of small-molecule inhibitors of mast cell degranulation was justified in several ways. First, in mice with mast cell deficiency (W / W ^v ), ovalbumin-induced reactions have been shown to depend on the presence of mast cells. In mice with mast cell deficiency, sensitization and exposure to ovalbumin did not lead to airway hypersensitivity and eosinophil secretion. Secondly, it was possible to block hypersensitivity and inflammation of the airways caused by ovalbumin, using a stabilizer of mast cells, cromolin, (data not shown). The use of this model for the evaluation of drugs for the treatment of asthmatic reactions that can be mediated by mechanisms other than stabilization of mast cells is also justified by the inhibitory effect of the use of steroids, dexamethasone and budesonide in case of methacholine-induced bronchostenosis.

7.15.2 Results

The efficacy of R921304 was evaluated with intrananasal administration for 10 consecutive days, from day 7 to day 16, at a dosage of 20 mg / kg, with the last three doses being administered 30 minutes before exposure to either saline or ovalbumin. R921304 successfully inhibited ovalbumin-induced airway hypersensitivity to methacholine compared to excipient-treated mice.

Under a less stringent protocol, in which ovalbumin was administered to mice once, on day 14, R921219, administered subcutaneously at a dose of 70 mg / kg in 67% PEG400 / 33% citrate buffer 30 minutes before exposure to ovalbumin or saline, proved that R921219 was completely blocks airway hypersensitivity and cell infiltration caused by ovalbumin.

These results clearly demonstrate the efficacy of R921219 and R921304 in inhibiting the respiratory tract response in a mouse model of allergic asthma.

7.16 Inhibitory effect of 2,4-pyrimidinediamine compounds on protein phosphorylation in the forward direction from Syk kinase in activated mast cells

The inhibitory effect of 2,4-pyrimidinediamine compounds on protein phosphorylation in the forward direction from Syk kinase was studied using R921218, R218219 and R921304 preparations in BMMC activated by IgE receptors.

For analysis, BMMC cells were incubated in the presence of various concentrations of the test drug (0.08 μmol, 0.4 μmol, 2 μmol and 10 μmol) for 1 hour at 37 ° C. The cells were then stimulated with an anti-IgE antibody as described above. After 10 minutes, the cells were lysed and cell proteins were isolated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS PAGE). After electrophoresis, phosphorylation of the proteins shown in FIG. 7, 10 and 11A-D were determined by immunoblot. Antibodies were purchased from Cell Signaling Technology, Beverly, pc. Massachusetts.

The studied preparations indicated in FIGS. 7, 10 and 11A-D inhibited protein phosphorylation in the direct but not in the opposite direction from the Syk kinase in the signaling cascade of IgE receptors, which confirms both the inhibition of IgE-induced reverse degranulation by drugs and the implementation inhibitory activity of drugs by inhibiting Syk kinase.

7.17 Inhibition of Syk kinase by 2,4-pyrimidinediamine compounds during biochemical analyzes

Several 2,4-pyrimidinediamine compounds were analyzed to determine the ability to inhibit the phosphorylation of a peptide substrate catalyzed by a Syk kinase by the method of biochemical analysis of fluorescence polarization with an isolated Syk kinase. In this experiment, the compounds were diluted to 1% DMSO in kinase buffer (20 mmol N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4, 5 mmol MgCl ₂ , 2 mmol MnCl ₂ , 1 mmol dithiothreitol, 0.1 mg / ml acetylated bovine gamma globulin). The preparation in 1% DMSO (0.2% DMSO - finally) was mixed with adenosine triphosphate / substrate solution at room temperature. Syk kinase (Upstate, Lake Placid NY) was added to the final reaction volume, 20 μl, and the reaction was incubated for 30 minutes at room temperature. Final enzyme reaction conditions: 20 mmol N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.4, 5 mmol MgCl ₂ , 2 mmol MnCl ₂ , 1 mmol dithiothreitol, 0.1 mg / ml acetylated bovine gamma globulin 0.125 ng Syk kinase, 4 μmol of adenosine triphosphate, 2.5 μmol of peptide substrate (Biotin-EQEDEPEGDYEEVLE-CONH ₂ , supplier: SynPep Corporation). EDTA (10 mmol - final concentration) / antiphosphothyrosine antibody (1X - terminal) / fluorescent phosphopeptide label (0.5X - final) was added to the phosphopeptide buffer solution to terminate the reaction and obtain a total volume of 40 μl according to the manufacturer's instructions (PanVera Corporation ) The plate was incubated for 30 minutes in the dark at room temperature. The tablets were read on a fluorescence polarization apparatus for Tecan tablets Polarion. The data were recalculated according to the amount of available phosphopeptide according to the calibration curve obtained by competition with the competitor phosphopeptide provided in the tyrosine kinase assay kit (green) (supplier company: PanVera Corporation),

The results of the analysis are shown in Table 6 (see the graphic part of the description).

These data show that all studied drugs with the exception of R945142 and R909236 inhibit the phosphorylation of Syk kinase IC ₅₀ s in the submicromolar range. All studied drugs inhibit the phosphorylation of Syk kinase IC ₅₀ s in the micromolar range

7.18 The effectiveness of compounds in the treatment of autoimmunity

The in vivo efficacy of individual 2,4-pyridinediamine compounds with respect to autoimmune diseases was obtained using the Arthus reverse passive reaction as a model of severe tissue damage mediated by an antigen-antibody pair, as well as several models of diseases associated with autoimmunity and inflammation. These models are similar in that an antibody to a specific antigen mediates an inflammatory disease caused by the immune complex (IR) and associated tissue destruction. Deposition of IR in certain anatomical areas (central nervous system (CNS) in the case of experimental autoimmune encephalomyelitis (EAE) and the synovial membrane in the case of collagen-induced arthritis (CIA)) leads to the activation of cells expressing surface receptors FcγR and FcεR, mainly mast cells, macrophages and neutrophils, which leads to the release of the cytokine and chemotaxis of neutrophils. The activation of the inflammatory response is responsible for the forward response of nerve endings, including edema, hemorrhage, neutrophil infiltration, and the release of pro-inflammatory mediators. In the case of autoimmune disorders, the consequences of these events provoked by IC are difficult to establish; however, many researchers have shown that inhibition of FcγR receptor signaling pathways in these animal models led to a significant weakening of the development of the disease and its severity.

7.18.1 Efficacy of compounds in the Arthus reaction in mice

In vivo, the efficacy of compounds R921302, R926891, R940323, R940347, and R921303, in terms of inhibiting the inflammatory cascades provoked by IC, was demonstrated using the Arthus reverse passive response in mice (RPA).

7.18.1.1 Model

Acute inflammatory tissue injury mediated by the immune complex (IR) is part of many different human autoimmune diseases, including vasculitis syndrome, serum sickness, systemic lupus erythematosus (SLE), rheumatoid arthritis, Goodpasture syndrome and glomerulonephritis. The classic experimental model of IC-mediated tissue damage is Arthus's reverse passive response. The model of this reaction is a convenient in vivo method for studying localized inflammation provoked by immune complexes in the absence of systemic effects. Subcutaneous injection of antibodies (AT) specific for chicken albumin (anti-rabbit IgG immunoglobulin IgG), followed by intravenous injection of (IV) antigens (AG), namely chicken albumin (egg albumin, OVA), causes circulatory deposition of immune complexes and An immediate inflammatory reaction characterized by edema, neutrophil infiltration, and hemorrhage at the injection site. Some aspects of the mouse RPA model are similar to the inflammatory response of patients with rheumatoid arthritis, SLE, and glomerulonephritis.

7.18.1.2 Study Protocol

In this model, the test compounds are administered at specific points in time before the introduction of antibodies and antigens. The rabbit anti-IgA immunoglobulin IgG antiOVA solution (50 μg in 25 μl / mouse) was subcutaneously followed by an intravenous injection of albumin obtained from chicken egg (20 mg / kg body weight) in a solution containing 1% Evans blue dye. The degree of swelling and hemorrhage is measured in the skin of the back of C57BL / 6 mice using Evans blue dye as an indicator of local tissue damage. Purified polyclonal rabbit IgG immunoglobulin was used as a control.

The time interval between the administration of the test compounds and the antibody / antigen complex depends on the pharmacokinetic (FC) properties of each individual compound. Four hours after the induction of the Arthus reaction, the mice were euthanized and a tissue sample was taken to evaluate edema. This model allows you to quickly track in vivo activity of most inhibitors.

7.18.1.3 Results

All tested compounds were administered orally.

The drug R921302 when administered in an amount of 50 mg / kg, 100 mg / kg and 200 mg / kg 60 minutes before administration of the antibody / antigen when tested in C57Bl6 mice, showed the dependence of inhibition of edema formation on the administered dose (49.9%, 93, 2% and 99.1% respectively). Moreover, the drug R921302 showed not only prophylactic inhibition of edema, but also therapeutic efficacy consisting in 77.5% inhibition of edema with the administration of the compound 30 minutes after immunization at a dose of 100 mg / kg.

Compounds R940323 and R926891 showed effective inhibition of edema development by 32.4% and 54.9%, respectively, when administered in the amount of 200 mg / kg 60 minutes before exposure to the antibody / antigen. These compounds are significantly less bioavailable when administered orally, and systemic exposure levels are about 50 times lower compared to compound R921302 (data not shown). Compound R940347 inhibited edema by 89% when administered in an amount of 100 mg / kg 2 hours before exposure to antibody / antigen.

Compound R921303 showed 100%, 100% and 3.6% inhibition of edema formation at a dose of 200 mg / kg when taken for 30, 60 and 120 minutes, respectively. This compound also demonstrated the dependence of the percent inhibition (65.4%, 81.2% and 100%) on the dose of administration, which were 50 mg / kg, 100 mg / kg and 200 mg / kg, respectively. The test results of various compounds are presented in Table 7 (see the graphic part of the description).

7.18.2 Efficacy of compounds in mouse modeling of arthritis induced by antibodies to collagen

In vivo, the efficacy of compound R921302 against autoimmune diseases was demonstrated in a model of arthritis induced by anti-collagen antibodies (CAIA) in mice.

7.18.2.1 Model

Collagen-induced arthritis (CIA) in rodents is often used as one of the experimental models in the case of tissue damage mediated by the immune complex (IR). The administration of type II collagen to mice or rats leads to an immune response, which is characterized by the presence of inflammatory damage to the cartilage and bones of the peripheral joints, which is accompanied by a tumor of the surrounding tissues. CIA is often used to evaluate the effectiveness of compounds that can potentially be used as medications for the treatment of rheumatoid arthritis and other chronic inflammatory conditions.

Over the past years, a new CIA modeling technology has emerged, based on the use of antibodies to collagen antotype II to induce antibody-mediated CIA. The advantages of this method are as follows: a short period of time required to induce the disease (the disease develops within 24-48 hours after intravenous (IV) injection of antibodies); arthritis can be induced in both CIA-susceptible and CIA-resistant mouse breeds; and in addition, the procedure is ideal for quickly checking the performance of anti-inflammatory therapeutic drugs.

The Arthrogen-CIA® monoclonal antibody mixture (supplier: Chemicon International Inc.), which induces arthritis, is administered intravenously to Balb / c mice (2 mg / mouse) on day 0. Forty-eight hours later, an intraperitoneal injection of 100 μl LPS ^* ( 25 mcg). On the fourth day, claw swelling may occur. On the fifth day, one or both legs (especially the hind legs) begin to redden and swell. On the sixth day and for the next 1 to 2 weeks, the redness and swelling of the legs will persist. In the course of the study, the clinical characteristics of inflammation are recorded in order to assess the intensity of paw edema. The severity of the occurrence of arthritis is recorded as the sum of the points characterizing the condition of both hind legs of each animal (the maximum possible number of points is 8). The degree of inflammation of the damaged legs is assessed by measuring the diameter of the legs. Also keep track of changes in body weight.

Treatment of animals begins with the induction of arthritis, starting from day zero. Experimental and control compounds are administered once or twice a day orally (PO), depending on previously established pharmacokinetic properties.

At the end of the study (1-2 weeks after inducing arthritis), the mice were euthanized, paws were cut off at the site of the distal tibia using guillotine scissors and weighed. The average ± standard error (SD) of the averaged characteristics for each group is determined every day based on the clinical indicators of each individual animal, and at the end of the study, the weight of the hind legs for each experimental group is calculated and recorded. A histopathological assessment of the legs is also obtained.

7.18.2.2 Results

The administration of compound R921302 significantly suppressed the development of arthritis and the severity of the disease (p <0.005), as evidenced by changes in the average daily values of the clinical parameters of arthritis (Fig. 12). The average daily values of the clinical parameters of arthritis in the treated group for the period from day 4 to day 14 were 71-92% lower compared to the control group. Based on the weight of the legs, the degree of their inflammation in animals that were injected with the drug R921302 turned out to be less in comparison with animals of the control group (Fig.13). At the final stage of the study, the degree of inflammation was estimated based on the weight of the paws and a 99.9% decrease was found in the group in which the animals were injected with R921302 compared to the average weight of the paws of the animals of the control group (p <0.002).

As a result of a histopathological examination of the cut off legs, the fact of pronounced synovitis characteristic of CIA was established. While in animals that were injected with saline or excipient, there was a pronounced lesion, in the group of animals that received R921302 injections, the severity of the lesion was less. Thickening of the joints with marked proliferation of the synovial membrane was observed. An increase in fibroblasts with dense infiltration of neutrophils, lymphocytes, monocytes, macrophages and plasma cells was also observed. Vascular proliferation with blockage, hemorrhage and edema was observed. The formation of pannus in the joint space was noted, as well as the destruction of cartilage. In the group of animals treated, the condition of the joints was close to normal or with limited inflammation, but without spreading to the cartilage.

Table 8
The average histopathological indicators in the group (0-15 points) Method of treatment Averaged overall metrics ± standard deviation Saline solution 9.8 ± 2.1 Filler 9.3 ± 4.5 R921302 (100 mg / kg), twice daily 5.1 ± 1.9 No drugs 0,0 ± 0,0

Clinical indicators of arthritis and swelling of the legs were on average 20% less in the group of animals that were injected with R050 twice a day in the amount of 100 mg / kg, compared with animals in the control group who did not receive treatment (excipient was used, p = 0.1) . Swelling of the legs was inhibited by about 26% compared with animals in the control group (where the vehicle was used), which was established by measuring the thickness of the hind legs (p = 0.1). R050 had no effect on arthritis at a dose level of 30 mg / kg.

The drug R070, which is the salt of R050, which was administered in an amount of 50 or 100 mg / kg twice a day, inhibited clinical disease by an average of 39.75% (p <0.0002) or 35.28% (p <0.0004 ), respectively, compared with the used control substance (filler). The thickness of the legs was reduced by about 50%.

The drug R429, which is the salt of R363, which was administered twice daily in the amount of 50 or 100 mg / kg, showed an average of 23.81% (p <0.05) or 20.82% (p = 0.05) inhibition of clinical indicators of arthritis, respectively, compared with animals of the control group, which were administered a control substance (excipient). As in the previous case, a decrease in the thickness of the legs was observed.

R347 did not significantly affect arthritis at the dose levels used in the trials (30 and 100 mg / kg twice daily).

7.18.3 The efficacy of compounds in the treatment of collagen-induced arthritis in rats

In vivo, the efficacy of compound R921302 against autoimmune diseases was demonstrated in a model of collagen-induced arthritis (CIA) in rats.

7.18.3.1 Model Description

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints, which ultimately leads to irreversible destruction of the cartilage. The synovial tissues of patients suffering from RA contain a large amount of IR with immunoglobulin IgG. Despite ongoing debate about the role of these complexes in the etiology and pathology of this disease, the communication of IR with hematopoietic cells is carried out using FcγR receptors.

CIA is a widely recognized animal model of RA, the result of which is chronic inflammatory synovitis, characterized by pannus formation and joint degradation. In this model, subcutaneous administration of native type II collagen emulsified with incomplete Freund's adjuvant leads to the development of inflammatory arthritis within 10 or 11 days and subsequent destruction of the joints in the period from 3 to 4 weeks.

7.18.3.2 Study Protocol

On day 0, a group of isogenic rats of the LOU line were immunized with native type II collagen and the efficacy of R921302 was evaluated in the prophylactic and treatment regimen. According to the prophylaxis protocol, each excipient or different doses of R921302 was administered orally, starting from the day of immunization (day 0). According to the treatment protocol, treatment with R921302 (300 mg / kg orally) was started after the development of clinical signs of arthritis on the tenth day and continued until the end of the study (day 28), when the animal was euthanized. According to each of the protocols, clinical indicators were recorded on a daily basis and body weight measurements were performed twice a week. On the twenty-eighth day, X-ray data were obtained and serum levels of type II collagen antibodies in the serum were measured using an ELISA ^* assay.

7.18.3.3 Results

After 10 days after immunization in rats, clinical CIA developed, which can be judged by the increase in arthritic indices (Fig. 14). In rats that were administered only vehicle, the average value of arthritic indices gradually increased after the tenth day and reached 6.75 ± 0.57 by the twenty-eighth day. In animals that were injected with a high dose of R921302 (300 mg / kg / day) from the day of immunization (day 0), during the period from the 10th to the 28th day, these indicators were significantly lower (p <0.01) by compared with the performance of the control group. In rats that were injected with 300 mg / kg of R921302 from the onset of the development of the disease, a significantly lower level of arthritis was observed starting from day 16, and this difference remained until the end of the experiment on day 28. The X-ray analysis indices (0-6 scale) obtained on the 28th day of the CIA were 4.8 ± 0.056 in the control group compared with 2.5 ± 0.016, 2.4 ± 0.006, and 0.13 ± 0.000001 in animals to which the drug was administered in the amount of 75, 150 and 300 mg / kg / day once a day, respectively, in the prophylactic regimen, and 0.45 ± 0.031 in animals to which the drug was administered 300 mg / kg / day once a day with the moment the disease develops. Treatment with a dose of R921302 in the amount of 300 mg / kg / day for the purpose of prevention (with immunization) or after the onset of the development of the disease prevented erosion and reduced soft tissue swelling. In a similar manner, treatment with R921302 resulted in a significant decrease in serum anti-collagen II antibodies (data not shown).

7.18.4 Efficacy of compounds in the treatment of experimental autoimmune encephalomyelitis in mice

In vivo, the efficacy of R921302 in relation to autoimmune disease was demonstrated in a mouse model of experimental autoimmune encephalomyelitis (EAE).

7.18.4.1 Model Description

EAE is a useful model for the study of multiple sclerosis (MS), which is an autoimmune disease of the central nervous system caused by the infiltration of the white matter of the central nervous system by immune cells. Inflammation and subsequent destruction of myelin leads to progressive paralysis. Similar to human disease, EAE is associated with peripheral activation of T cells that are self-reactive with myelin proteins, such as myelin base protein (MBP), proteolipid protein (PLP) or myelin oligodendrocyte protein (MOG). Activated neuroantigen-specific T cells cross the blood-brain barrier and lead to focal mononucleic cell infiltration and demyelination. EAE can be induced in predisposed breeds of mice by immunization with myelinspecific proteins in combination with an adjuvant. In the SJL mouse model used in the present studies, hind limb and tail paralysis occurs on the 10th day after immunization, while the peak of the disease occurs on the 10th to 14th day, and the cycles of partial spontaneous remission with subsequent relapses can be observed up to the 35th day. The results below demonstrate the potential ability of the study drug (R921302) to suppress the severity of the disease and prevent the recurrence of disease symptoms, which may be the result of FcγR-mediated release of cytokine by immune cells.

7.18.4.2 Study Protocol

According to the EAE model, using SJL mice, each mouse was sensitized with PLP / CFA (to induce EAE in four places on the posterior lateral part of the mouse, 0.2 ml of an emulsion consisting of 150 μg of compound PLP139-151 in combination with 200 μg of CFA at 0 was injected. 05 ml homogenate). According to the suppression protocol, control excipient or various doses of R921302 was administered orally from the day of immunization (day 0). In accordance with the treatment protocol, at the beginning of the development of the disease, animals were divided into groups having similar averaged clinical indicators at the beginning of the development of the disease, and a control excipient or various doses of the studied drugs were orally administered at different frequencies. With both protocols, clinical indicators were checked on a daily basis and body weight was measured twice a week.

7.18.4.3 Results

After 10 days from the time of PLP immunization, SJL mice developed clinical EAE, as evidenced by an increase in their average clinical indicators (Fig. 15). Paralytic parameters in animals that were injected with only the control excipient from the day of immunization (day 0) gradually increased and reached a peak by day 14, which amounted to 5.1 + 0.3. At the peak of the disease (day 14), the average clinical indicators in animals that were administered 100 mg / kg daily or 100 mg / kg twice a day were significantly less (p <0.05, 4.3 + 1.3, and 4 , 3 + 1.4, respectively). After the 16th day, all animals showed partial remission of moderate severity, which is typical for the SJL model. Significantly reduced clinical indices in animals to which R921302 was administered twice daily at 100 mg / kg were maintained (p <0.05) throughout the entire experiment until the animals were euthanized on day 30. These lower rates throughout the treatment period were reflected in a significant decrease in the cumulative disease index (CIB) and an increase in the cumulative weight index (CIV), as shown in Table 9 (see the graphic part of the description). In the group of animals that were administered only the control vehicle, 2/5 of the mice experienced a relapse. In the group receiving 100 mg / kg / day, 3/8 of the mice experienced a relapse. In the group receiving 100 mg / kg / day twice a day, no relapse was observed.

SJL mice treated with R921302 at the beginning of the disease (day 11) at a dose of 200 mg / kg twice a day showed a significant decrease (p = 0.003) in the CIB level (53.5 ± 16.9 in animals treated with R921302 , compared with 72.9 ± 8.9 in animals receiving only vehicle). Moreover, there was a sharp decrease in the number of relapses in animals treated with R921302 (2/12), compared with the number of relapses in animals treated with filler (7/11). The generalized results are shown in Table 10 (see the graphic part of the description) and in Fig.16.

7.18.5. The ability of the 2,4-pyridinediamine compounds of the present invention to inhibit T-cell activation

7.18.5.1 Description

The ability of the 2,4-pyridinediamine compound of the present invention to inhibit T cell activation has been demonstrated by various assays using Jurkat T cells and primary T cell cultures. Inhibition of Jurkat T cell activation in response to stimulation of T cell receptors (TCR) was measured by quantitatively analyzing the increasing regulation of the CD69 cell surface marker. Primary T cell activation inhibition was measured by quantitative analysis of cytokine excretion, including tumor necrosis factor alpha (TNF), interleukin 2 (IL-2), interleukin 4 (IL-4), interferon gamma (IFNg) and granulocyte macrophage colony stimulating factor ( GMSCF), in response to TCR / CD28 co-stimulation.

7.18.5.2 Testing the inhibition of activation of Jurkat T cells

Human Jurkat T cells (clone N) were routinely cultured on RPMI 1640 medium (supplier company: Mediatech), to which 10% fetal calf serum (FBS) (supplier company: Hyclone), penicillin and streptamycin were added. The verification process took three days.

On the first day of testing, cultured cells were centrifuged (1000 rpm, 5 minutes) and re-placed in suspension at a concentration of 3.0 × 10 ⁵ cells / ml in RPMI + 5% FBS. On the second day of the test, the cells were centrifuged at 1000 rpm for 5 minutes and re-suspended in RPMI + 5% FBS at a concentration of 1.3 × 10 ⁵ cells / ml. 85 μl of the cell suspension was added to the wells of a 96-well plate (vendor: Corning). 85 μl of the compound or diluted RPMI + 5% FBS (as a control) was added to each well and incubated at 37 ° C for 1 hour. The cells were then stimulated with antiTCR (C305) at 500 ng / ml by adding 8X solution in 25 μl to the cells of the tablet. Cells were then incubated at 37 ° C for 20 hours.

On the third day of testing, the plates were rotated in a centrifuge (Beckman GS-6R model) at 2500 rpm for 1 minute, after which the medium was removed. Next, 50 μl of a staining solution (diluted 1: 100 solution of anti-CD69-APC antibody (supplier: Becton Dickenson) in phosphate buffered saline (PBS + 2% FBS) was added to each well, after which the plates were incubated at 4 ° C for 20 minutes in the dark, after which 150 μl of buffer solution (PBS + 2% FBS) was added to each well and the plate was rotated at 3000 rpm for 1 minute, then the supernatant was again removed and the compacted material was transferred into suspension by gentle mixing. 75 μl of PBS + 2% FBS + Cyto solution was added X (diluted in the ratio 1: 4) was stirred gently and the contents of the plates were wrapped in aluminum foil cell assay was performed with a flow cytometer, connected to an automatic liquid supply system..

Different concentrations of the compound were compared with the solvent itself to determine the IC ₅₀ inhibition of T-cell activation by various compounds. Exemplary IC ₅₀ values for the 2,4-pyridinediamine compounds of the present invention are shown in Table 11.

7.18.5.3 Isolation of primary T cells

2E8-4E8 PBMCs or proliferating T cells grown in rIL-2 from healthy human donors were suspended in PBS, rotated at 1500 rpm for 8-10 minutes and resuspended in 100 ml of complete RPMI medium (1 % Pen-Strep, 1% L-Glutamine, 10 mmol HEPES). After this, the cells were placed in T175 flasks (37 ° C, 5% CO ₂ ) and allowed the monocytes to contact for 2-3 hours. After monocyte attachment, unbound cells were collected, counted using a hemocytomer, washed several times with PBS and resuspended in complete Yssel medium (Iskova medium modified with Dulbeko (modified IMDM) with 1% human AB serum, 1% Pen-Strep, 1% L- glutamine, 10 mmol HEPES) at a concentration of 1.5 4 x 10 ⁶ cells / ml. 90 μl of the diluted cells was then added to the compounds diluted to 2X in Yssel medium and incubated for 30 minutes at 37 ° C (5% CO ₂ ). After this pre-incubation step, the compound / cell mixture was transferred to stimulation plates as described below.

7.18.5.4 Testing for inhibition of cytokine production in stimulated primary T cells

Cell stimulation plates were prepared by coating 96-well plates with 5 μg / ml αCD3 solution (supplier: BD PharMingen, catalog No. 555336) + 10 μg / ml αCD28 solution (supplier: Beckman Coulter, catalog No. IM1376) in PBS (in the absence of Ca ²⁺ / Mg ^{2+ ions} ) at 37 ° C (5% CO ₂ ) for 3-5 hours. Upon completion of incubation in the presence of stimulating antibodies, the mixture was removed and the plates were washed 3 times with PBS before adding a mixture of primary T cells with the drug.

A mixture of primary T cells with the preparation was placed on stimulation plates and incubated for 18 hours at 37 ° C (5% CO ₂ ). Upon completion of cell stimulation, ~ 150 μl of the supernatant from each well was transferred to a 96-well filter plate (supplier: Corning PVDF Filter Plates), centrifuged (2000 rpm, 2-3 minutes) and immediately used for analysis ELISA or LUMINEX assays were either frozen at -80 ° C for future use.

ELISA analyzes of IL-2 were performed using the Quantikine Human IL-2 ELISA kit (supplier: R&D Systems, catalog number D2050) according to the manufacturer's instructions and absorbance was measured using a spectrophotometer at a wavelength of 450 nm. After subtracting the noise, the absorbance was converted to pg / ml using a calibration curve.

Luminex immunoassay multiplexing for TNF, IL-2, GMSCF, IL-4 and IFNg was carried out in accordance with the manufacturer's instructions (Upstate Biotechnology). In particular, 50 μl of the sample was diluted with 50 μl of experimental diluent and 50 μl of incubation buffer, after which they were incubated in the presence of 100 μl of a diluted antibody-detector solution for 1 hour at room temperature in the dark. The filter plate was washed 2 times with wash buffer and incubated in the presence of 100 μl of diluted secondary reagent (SAV-RPE ^* ) for 30 min at room temperature in the dark. Finally, the plates were washed 3 times and measurements were performed using labeled microspheres and a fluorescent compound RPE using a Luminex instrument.

Different concentrations of the compound were compared solely with the solution in order to determine the IC ₅₀ values of various compounds characterizing the inhibition of T-cell activation. Representative IC ₅₀ values for the 2,4-pyridinediamine compounds of the present invention are shown in Table 11.

7.18.6 The ability of compounds of 2,4-pyridinediamine according to the present invention to inhibit the activation of B cells

7.18.6.1 Description

The ability of the 2,4-pyridinediamine compounds of the present invention to inhibit B-cell activation has been demonstrated by primary B-cells by analyzing cell surface markers using a fluorescent activation cell sorter (FACS). Inhibition of activation of primary B cells in response to stimulation of B cell receptors (BCR) was measured by quantifying the CD69 cell surface marker.

7.18.6.2 Isolation of primary B cells

Primary human B cells were isolated from a light layer of a blood clot, which is a layer of white blood cells that forms between red blood cells and platelets as a result of rotation of non-coagulating blood in a centrifuge, or from fresh blood using CD19-Dynal® microspheres and FACS sorter. A light layer of a blood clot was purchased from the Blood Bank of the Stanford University Medical Center, where it was prepared on the same day, stored and taken out in a refrigerated state (with ice). A light clot layer (approximately 35 ml) was placed in a 500 ml conical sterile centrifuge container and ice-cooled, after which the contents were diluted with cold PBS containing 0.2% BSA ^* (supplier: Sigma, catalog No. A7638) and sodium citrate (0.1 %, supplier company: Sigma, catalog number S-5570) (PBC), to a total volume of 200 ml and gently mixed. Fresh blood came from donors in 10 ml vacuum containers containing heparin (1 vacuum container contains approximately 8.5 ml of blood). Blood was ice-cooled, transferred to 50 ml tubes (20 ml / tube) or a 500 ml conical sterile centrifuge container, and then diluted with an equal volume of PBC.

25 ml of diluted blood or a light layer of a blood clot was applied in layers to 15 ml of chilled ficoll and again placed on ice. Layer Ficoll blood was centrifuged (Beckman GS-6R model) for 45 minutes at 2000 rpm, 4 ° C to separate peripheral blood mononuclear cells (PBMCs) from red blood cells (red blood cells - RBCs) and granulocytes. The upper aqueous layer was then aspirated to a level of 1 inch (25.4 mm) above the PBMC layer. Thereafter, PBMC cells were transferred from every 2 Ficoll tubes into one clean 50 ml Falcon tube (approximately 10 ml / tube). The transferred PBMC cells were then diluted in a 5-fold proportion with ice-cold PBS with 0.2% BSA (P-B) and centrifuged for 20 min at 1400 rpm and 4 ° C. The supernatant (possibly clouding) was then aspirated, PBMC cells were suspended in 25 ml of P-B solution, counted (by dilution in a ratio of 1: 5) and stored with ice.

Further, the cells were positively selected using anti-D19 antibodies applied to magnetic microspheres (Dynal®) in accordance with the manufacturer's instructions. The approximate required number of CD19-Dynal® microspheres (dynna beads M-450 microspheres (pabB) manufactured by Dynal CD19 coated) were calculated assuming that the number of B cells was 5% of the calculated PBMC number, and about 10 microspheres were added per cell from a microsphere container containing 4 × 10 ⁸ microspheres / ml. The CD19-Dynal® microspheres were washed 2 times with PB solution in a 5 ml tube using a Dynal magnet, and then added to the PBMC cell suspension. This mixture was then passed through a Dynal magnet and washed several times to separate cells adhering to microspheres.

7.18.6.3 Testing Compounds for Inhibition of B-Cell Activation

After separation, the microspheres and antibodies were removed using Dynal's CD19-DETACHaBEAD® compound for 45 minutes at 30 ° C. The result was approximately 2 × 10 ⁷ B-cells per light layer of a blood clot. B cells were washed and transferred to a suspension with a concentration of 1 × 10 ⁶ cells / ml in RPMI1640 + 10% FBS + penicillin / streptavidin + 1 ng / ml IFNα8. The cells were left overnight at 37 ° C and 5% CO _2,

The next day, the cells were washed and suspended in RPMI1640 + 2.5% FBS medium to a concentration of 1 × 10 ⁶ cells / ml. Next, the cells were transferred to a multiple of the wedge-shaped wells of a 96-well plate (supplier: Corning) at 65 μl of cells per well. Using an automatic setup, 65 μl of 2x compounds was added to the cells to a DMSO concentration of 0.2% and incubated for 1 hour at 37 ° C. After that, the cells were stimulated in the presence of 20 μl of 7.5x α-IgM immunoglobulin from Jackson laboratories (final concentration of 5 μg / ml) for 24 hours. On day 3, the cells were centrifuged, tinted for CD69 and analyzed by FACS by gating on living cells (by light scattering).

Different concentrations of the compound were compared solely with the solution in order to determine the IC ₅₀ values of various compounds characterizing inhibition of B-cell activation. Representative IC ₅₀ values for the 2,4-pyridinediamine compounds of the present invention are shown in Table 11.

7.18.7 The ability of the 2,4-pyridinediamine compounds of the present invention to inhibit macrophage activation

7.18.7.1 Description

The ability of the 2,4-pyridinediamine compounds of the present invention to inhibit the activation of various macrophages has been demonstrated by measuring the release of cytokines by stimulated macrophages. The isolation of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) was quantified in response to IgG or LPS stimulation.

7.18.7.2 Purification and culture of human macrophages

CD14 + monocytes were purified from PBMC (supplier company: Allcells, catalog number PB002) using a monocyte isolation kit (supplier company: Miltenyi biotec, catalog number 130-045-501) in accordance with the manufacturer's instructions. The degree of purity was assessed by measuring the percentage of CD14 + cells using flow cytometry. Typically, the degree of purity was> 90%. The purified CD14 + cells were then plated (6 × 10 ⁶ cells per 150 cm diameter Petri dish in the presence of 15 ml of medium) in macrophage serum-free medium (M-SFM) (supplier company: Gibco, catalog No. 12065-074) at a concentration of M-CSF ^* 100 ng / ml (supplier: Pepro Tech, catalog number 300-25) and differentiated for five days. After this period, cell morphology and cell surface markers (CD14, HLA-DR, B7.1, B7.2, CD64, CD32, and CD16) reflected the presence of matured differentiated macrophages.

7.18.7.3 Stimulation of IgG immunoglobulin

Immulon 4HBX 96-well plates (supplier company: VWR, catalog number 62402-959) were coated with human IgG (supplier company: Jackson Immunoresearch lab, catalog number 009-000-003) at a concentration of 10 μg / well overnight at 4ºC or for 1 h at 37ºC. The negative control, consisting of the F (ab ') 2 fragment, was also covered in order to evaluate the background stimulation. Unbound antibody was washed twice with 200 μl of PBS. 20 μl of 5X compound was added to each well, after which 15,000 differentiated macrophage cells were added in 80 μl, which were collected from the walls of the plates. Cells were incubated for 16 hours at 37 ° C in an incubator and supernatants were collected for further analysis using Luminex instruments for IL-6 and TNFα, which was carried out similarly as described above in the case of primary T cells.

7.18.7.4 Stimulation of LPS

For stimulation using LPS, 10 μl of a 10X solution was added to the pre-incubated mixture of cells with the preparation until a final concentration of 10 ng / ml was reached. Cells were then incubated for 16 hours at 37 ° C. Supernatants were analyzed in the same manner as described above.

Different concentrations of the compound were compared solely with the solution in order to determine the IC ₅₀ value of each compound for each cytokine. Representative IC ₅₀ values for the 2,4-pyridinediamine compounds of the present invention are shown in Table 11 (see the graphic part of the description).

Despite the detailed description of the above invention in order to facilitate understanding, it is obvious that certain changes and modifications are possible within the scope of the attached claims. Accordingly, the described examples should be considered as illustrative and not limiting, and the invention should not be limited to the details given in these examples, which can be modified within and in accordance with the equivalents of the appended claims.

All literature and patent sources referenced in this application are included in this application by reference for any purpose.

Claims (12)

1. A compound selected from the following group of compounds, or a pharmaceutically acceptable salt thereof:
N4- (4-Chloro-3-methoxyphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxycarbonylmethyleneoxyphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- [3-Chloro-4- (N-methylamino) carbonylmethyleneoxyphenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- [3-Chloro-4- (2-hydroxyethylene ethylene) phenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3,5-Dichloro-4-methoxyphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (2-aminopyrid-6-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- [2- (N-Acetylamino) pyrid-6-yl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- (3,5-dichloro-4-hydroxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-trifluoromethoxyphenyl) -N2- (3,5-dichloro-4-hydroxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dichloro-4-hydroxyphenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
N2- (3,5-Dichloro-4-hydroxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- (3,5-dichloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-trifluoromethoxyphenyl) -N2- (3,5-dichloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- (3,5-dichloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dichloro-4-methoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
N2- (3,5-Dichloro-4-methoxyphenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-3-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-trifluoromethoxyphenyl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- [2-Aminopyrid-6-yl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- (indol-6-yl) -2,4-pyrimidinediamine
N4- [2- (N-Acetylamino) pyrid-6-yl] -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dichlorophenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (3-methoxy-5-trifluoromethylphenyl) -2,4-pyrimidinediamine
N2- (2,6-Dimethoxypyrid-3-yl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
N2- (2,6-Dimethylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (2-aminopyrid-6-yl) -N2- (2,6-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- (2,6-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (2,6-Dimethylphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (2,6-Dimethylphenyl) -5-fluoro-N4- (indol-6-yl) -2,4-pyrimidinediamine
N4- [2- (N-Acetylamino) pyrid-6-yl] -N2- (3,5-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dimethylphenyl) -5-fluoro-N4- [2- (N-methylamino) carbonylaminopyrid-6-yl] -2,4-pyrimidinediamine
N2- (3,5-Dimethylphenyl) -5-fluoro-N4- [1- (N-methylamino) carbonylindol-6-yl] -2,4-pyrimidinediamine
N4- (2-aminopyrid-6-yl) -N2- (3-chloro-4-trifluoromethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-trifluoromethoxyphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (2-aminopyrid-6-yl) -N2- (3-chloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -5-fluoro-N4- (indol-6-yl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -5-fluoro-N4- [1- (N-methylaminocarbonyl) indol-6-yl] -2,4-pyrimidinediamine
N4- (2-aminopyrid-6-yl) -5-fluoro-N2- (3-hydroxyphenyl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2 [1- (N-methylaminocarbonyl) indol-6-yl] -2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N2- (4-Chloro-3,5-dimethylphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (4-Chloro-3,5-dimethylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
(+) - N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [2- (N-methylaminocarbonyl) -2,3-dihydrobenzofuran-5 -yl] -5-fluoro-2,4-pyrimidinediamine
(+) - N4- (3-Chloro-4-methoxyphenyl) -N2- [2- (N, N-dimethylaminocarbonyl) -2,3-dihydrobenzofuran-5-yl] -5-fluoro-2,4-pyrimidinediamine
(+) - N4- (3-Chloro-4-trifluoromethoxyphenyl) -N2- [2- (N, N-dimethylaminocarbonyl) -2,3-dihydrobenzofuran-5-yl] -5-fluoro-2,4-pyrimidinediamine
(+) - N4- (3-Chloro-4-methoxyphenyl) -N2- [2- (N-methylaminomethylene) -2,3-dihydrobenzofuran-5-yl] -5-fluoro-2,4-pyrimidinediamine
5-Fluoro-N2- [2 (R) - {(1R, 2S, 5R) -methyloxycarbonyl} -2,3-dihydrobenzofuran-5-yl] -N4- (4-isopropoxyphenyl) -2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [2 (R) - {(1R, 2S, R) -methyloxycarbonyl} -2, 3-dihydrobenzofuran-5-yl] -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine salt p- toluenesulfonic acid
N2- (3,5-Dimethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine salt of methanesulfonic acid
N2- (3,5-Dimethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine salt of benzenesulfonic acid
Hydrochloride N2- (3,5-dimethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz
[1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine salt DL- camphorsulfonic acid
N2- (3,5-Dimethylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine salt p- toluenesulfonic acid
N2- (3,5-Dimethylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine salt of benzenesulfonic acid
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (indol-6-yl) -2,4-pyrimidinediamine salt of p-toluenesulfonic acid
N4- (3,4-Ethylenedioxyphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine salt of p-toluenesulfonic acid
(+) N2- (2-Carboxyl-2,3-dihydrobenzofuran-5-yl) -N4- (3-chloro-4-trifluoromethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- (3-Chloro-4-trifluoromethoxyphenyl) -5-fluoro-N2- [2- (N-2,3-dihydroxypropylamino) carbonyl-2,3-dihydrobenzofuran-5-yl] -2,4- pyrimidinediamine
(+) N4- (3-Chloro-4-trifluoromethoxyphenyl) -5-fluoro-N2- [2- (N-2-hydroxyethylamino) carbonyl-2,3-dihydrobenzofuran-5-yl] -2,4-pyrimidinediamine
(+) N4- (3-Chloro-4-trifluoromethoxyphenyl) -5-fluoro-N2- [2- (N-2-hydroxyethyl-N-methylamino] carbonyl-2,3-dihydrobenzofuran-5-yl] -2, 4-pyrimidinediamine
N4- (3-Chloro-4-trifluoromethoxyphenyl) -5-fluoro-N2- [2- (N-isopropylamino) carbonyl-2,3-dihydrobenzofuran-5-yl] -2,4-pyrimidinediamine
5-Fluoro-N4- (2-isopropoxypyridin-5-yl) -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- [3- (N-2-hydroxyethylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(+) N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- [3- (N-2,3-dihydroxypropylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N2, N4-Bis (4-benzyloxy-3-chlorophenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (4-Benzyloxy-3-chlorophenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- [3- (N-cyclopropylamino) carbonylmethyleneoxyphenyl] -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -N4- (3-chloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- [3- (N-cyclobutylamino) carbonylmethyleneoxyphenyl] -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2, 4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- (3-chloro-4-methoxy-5-methylphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-isopropoxyphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxy-5-methylphenyl) -5-fluoro-N2- [3 - [(N-methylamino) carbonylmethyleneoxy] phenyl] -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- (indol-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (indol-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (indol-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- [2- (N, N-dimethylaminomethyl) benzofuran-5-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [2- (N, N-dimethylaminomethyl) benzofuran-5-yl] -5-fluoro -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- [2- (N, N-dimethylaminomethyl) benzofuran-5-yl] -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (indol-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [2- (N, N-dimethylaminocarbonyl) -2,3-dihydrobenzofuran-5-yl ] -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Diftor-3-oxo-4H-benz [l, 4] oxazin-6-yl) -5-fluoro-N2- (3-methoxy-5-trifluoromethylphenyl) -2,4-pyrimidinediamine
N2- (3,5-Dichlorophenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- [3-Chloro-4- (N-morpholino) phenyl] -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- [3-Chloro-4- (N-morpholino) phenyl] -5-fluoro-N2- (3-methoxy-5-trifluoromethylphenyl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine salt of p-toluenesulfonic acid
N4- (3-Chloro-4-methoxyphenyl) -N2- (3,5-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- (3,5-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (3-methoxy-5-trifluoromethylphenyl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (3,4,5-trimethylphenyl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (3,4,5-trimethylphenyl) -2,4-pyrimidinediamine
5-Fluoro-N4- [1- (N-methylaminocarbonyl) indol-6-yl] -N2- (3,5-dimethoxyphenyl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2, 4-pyrimidinediamine
N2- (3-Chloro-4-methoxy-5-methylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2, 4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
(+) N4- (2-Ethoxycarbonyl-2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- [3- (methoxycarbonylmethyleneoxy) phenyl] -2, 4-pyrimidinediamine
5-Fluoro-N4- [2-methyl-2- (N-methylaminocarbonyl) -3-oxo-4H-benz [1,4] oxazin-6-yl] -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (N1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- (2,2-dimethyl-3-oxo-4H-benz [1,4] oxazine -6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -N2- (3-chloro-4-methoxyphenyl) -5-fluoro-2,4- pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,4-Dichlorophenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4 - [(2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
N2- (3-tert-Butylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4 - [(2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (3-hydroxyphenyl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -N2- (3-fluoro-4-methoxyphenyl) -5-fluoro-2,4- pyrimidinediamine
N2- (3-Chlorophenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,5-Dichlorophenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4- pyrimidinediamine
N2- (3-Chloro-4-trifluoromethoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
N2- (3-Chloro-4-methoxy-5-methylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro- 2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (3-methoxycarbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro- 2,4-pyrimidinediamine
N2- (3,5-Dimethyl-4-methoxyphenyl) -N4- (2,2-dimethyl-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2, 4-pyrimidinediamine
N2- (3,5-Dimethyl-4-methoxyphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxy-5-methylphenyl) -N4- (3,4-ethylenedioxyphenyl) -5-fluoro-2,4-pyrimidinediamine
5-Fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -N4- [3- (N-morpholino) carbonyl-4-trifluoromethoxyphenyl] -2,4-pyrimidinediamine
N4- [3- (N-2-aminoethylamino) carbonyl-3-trifluoromethoxyphenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
5-Fluoro-N4- [3- (N-methylamino) carbonyl-4-trifluoromethoxyphenyl) -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
5-Fluoro-N4- (3- [N- (2- (N-methylamino) ethyleneamino) carbonyl-4-trifluoromethoxyphenyl] -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
5-Fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -N4- [3- (N-piperidinecarbonyl-4-trifluoromethoxyphenyl] -2,4-pyrimidinediamine
(R) -N4- (3- [N- (1,2-Dihydroxypropylamino) carbonyl-4-trifluoromethoxyphenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(+) N4- [4- (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(+) 5-Fluoro-N4- [4- (N-methyl) amino-1-benzopyran-6-yl] -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(+) N4- [4- (N-tert-butoxycarbonyl-N-methyl) amino-1-benzopyran-6-yl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4 pyrimidinediamine
(S) -N4- [4- (N-Benzyloxycarbonyl) amino-1-benzopyran-6-yl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(R) -N4- [4- (N-Benzyloxycarbonyl) amino-1-benzopyran-6-yl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(S) -N4- (4-amino-1-benzopyran-6-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(R) -N4- (4-amino-1-benzopyran-6-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(+) N4- [4- (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl] -5-fluoro-N2- (indazol-6-yl) -2,4-pyrimidinediamine
(+) N4- (4-amino-1-benzopyran-6-yl) -5-fluoro-N2- (indazol-6-yl) -2,4-pyrimidinediamine
(+) N4- (4-amino-1-benzopyran-6-yl) -N2- (3,5-dichloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- [4- (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl] -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- (4-amino-1-benzopyran-6-yl) -N2- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- [4- (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl] -N2- (3-chloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- (4-amino-1-benzopyran-6-yl) -N2- (3-chloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- [4- (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl] -N2- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N4- (4-amino-1-benzopyran-6-yl) -N2- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N2- [4- (N-tert-butoxycarbonylamino-1-benzopyran-6-yl] -N4- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N2- [4- (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl] -N4- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
N2- [4 (R, S) - (N-tert-butoxycarbonyl) amino-1-benzopyran-6-yl] -5-fluoro-N4- [2- (S) -methyl-3-oxo-4H-benz [1,4] oxazin-6-yl] -2,4-pyrimidinediamine
(+) N2- (4-amino-1-benzopyran-6-yl] -N4- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
(+) N2- (4-amino-1-benzopyran-6-yl) -N4- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
N2- [4 (R, S) -Amino-1-benzopyran-6-yl) -5-fluoro-N4- (2 (S) -methyl-3-oxo-4H-benz [1,4] oxazin-6 -yl] -2,4-pyrimidinediamine
N2 - [(1R, 2R) -2-Aminocyclohex-1-yl) -N4- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2 - [(1R, 2R) -2-Aminocyclohex-1-yl) -N4- (3,5-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
N2 - ((1R, 2R) -2-Aminocyclohex-1-yl) -5-fluoro-N4 - [(2S) -2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl ) -2,4-pyrimidinediamine
(R, R) -N4- (4,4-Dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-N2- (2-aminocyclohexan-1-yl) -2, 4-pyrimidinediamine
N2 - ((1R, 2R) -2-Aminocyclohex-1-yl) -5-fluoro-N4 - [(2R, S) -2- (2-hydroxy) ethyl-3-oxo-4H-benz [1, 4] oxazin-6-yl] -2,4-pyrimidinediamine
N4- (3,5-Dimethoxyphenyl) -N2- [4- (2-N, N-diethylaminoethylenamino) carbonylphenyl] -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- [4 (2-N, N-diethylaminoethylenamino) carbonylphenyl] -5-fluoro-2,4-pyrimidinediamine
(S) -N2- [4- (2-N, N-Diethylaminoethylenamino) carbonylphenyl] -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4- (4,4-Dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-N2- [4- (N, N-diethylaminoethyleneaminocarbonyl) phenyl] -2,4-pyrimidinediamine
(+) - N2- [4- (2-N, N-Diethylaminoethylenamino) carbonylphenyl] -5-fluoro-N4 - [(2-hydroxyethyl) -3-oxo-4H-benz [1,4] oxazin-6- il] -2,4-pyrimidinediamine
N2- [4- (2-N, N-Diethylaminoethylenamino) carbonylphenyl] -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2 4-pyrimidinediamine
N2- (4-Aminocarbonylphenyl) -N4- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (4-Aminocarbonylphenyl) -N4- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
(S) -N2- (4-Aminocarbonylphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4- (4,4-Dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-N2- (4-aminocarbonylphenyl) -2,4-pyrimidinediamine
(+) - N2- (4-Aminocarbonylphenyl) -5-fluoro-N4 - [(2-hydroxyethylene) -3-oxo-4H-benz [1,4] oxazin-6-yl] -2,4-pyrimidinediamine
N2- (4-Aminocarbonylphenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N2- [4- (N-tert-butoxycarbonylamino) methylenephenyl] -N4- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- [4- (N-tert-butoxycarbonylamino) methylenephenyl] -N4- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
(S) -N2- [4- (N-tert-Butoxycarbonylamino) methylene phenyl] -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2 4-pyrimidinediamine
N2- [4- (N-tert-butoxycarbonylamino) methylene phenyl] -N4- (4,4-dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-2,4-pyrimidinediamine
N2- [4- (N-tert-butoxycarbonylaminomethylene) phenyl] -N4- (2,2-difluoro-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4 pyrimidinediamine
N2- (4-Aminomethylene phenyl) -N4- (3,5-dimethoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (4-Aminomethylenephenyl) -N4- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
(S) -N2- (4-aminomethylene phenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (4-Aminomethylene phenyl) -N4- (4,4-dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-2,4-pyrimidinediamine
N2- (4-Aminomethylenephenyl) -N4- (2,2-difluoro-3-oxo-4H-benz [l, 4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,5-Dimethoxyphenyl) -N2- (3-N, N-diethylaminopropyl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- (3-N, N-diethylaminopropyl) -5-fluoro-2,4-pyrimidinediamine
(S) -N2- (3-N, N-Diethylaminopropyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4- pyrimidinediamine
N4- (4,4-Dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -N2- (3-N, N-diethylaminopropyl) -5-fluoro-2,4-pyrimidinediamine
(+) - 5-Fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -N4- [4- (N-p-toluenesulfonyl) amino-1-benzopyran-6-yl) -2,4-pyrimidinediamine
(+) - 5-Fluoro-N4- [4- (N-methanesulfonyl) amino-1-benzopyran-6-yl) -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
(+) - N4- [4-N- (N, N-Dimethylaminomethylene-carbonyl) amino-1-benzopyran-6-yl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2, 4-pyrimidinediamine
(+) - N4- [4-N- (N, N-Dimethylaminomethylene-carbonyl) -N-methylamino-1-benzopyran-6-yl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4-Cyclopropyl-5-fluoro-N2- (4-morpholinophenyl) -2,4-pyrimidinediamine
N2-Cyclopropyl-5-fluoro-N4- (4-morpholinophenyl) -2,4-pyrimidinediamine
N2-Cyclobutyl-5-fluoro-N4- (4-morpholinophenyl) -2,4-pyrimidinediamine
N2- [3- (N-Cyclopropylamino) carbonylmethyleneoxyphenyl] -5-fluoro-N4- (4-morpholinophenyl) -2,4-pyrimidinediamine
5-Fluoro-N2- (3-methoxycarbonylmethyleneoxyphenyl) -N4- (4-morpholinophenyl) -2,4-pyrimidinediamine
N2- [3- (N-Cyclobutylamino) carbonylmethyleneoxyphenyl] -5-fluoro-N4- (4-morpholinophenyl) -2,4-pyrimidinediamine
N4-Cyclopropyl-N2- [3- (N-cyclopropylamino) -carbonylmethyleneoxyphenyl] -5-fluoro-2,4-pyrimidinediamine
N2- [3- (N-Cyclobutylamine) Carbonylmethyleneoxyphenyl] -N4-Cyclopropyl-5-fluoro-2,4-pyrimidinediamine
N4-Cyclopropyl-5-fluoro-N2- [3- (4-morpholinophenyl) aminocarbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-hydroxy-5-methylphenyl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
5-Fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -N4- (4-morpholinophenyl) -2,4-pyrimidinediamine
5-Fluoro-N4- [4- (4-methoxycarbonylpiperazino) phenyl] -N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
N2- [4- (N-Acetyl-N-methylamino) phenyl] -N4-cyclopropyl-5-fluoro-2,4-pyrimidinediamine
N2- [4- (4-Acetylpiperazino) phenyl] -N4-cyclopropyl-5-fluoro-2,4-pyrimidinediamine
N4-Cyclopropyl-5-fluoro-N2- [4- (4-methoxycarbonylpiperazino) phenyl] -2,4-pyrimidinediamine
N4-Cyclopropyl-5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
N2-Cyclopropyl-5-fluoro-N4- [4- (4-methoxycarbonylpiperazino) phenyl] -2,4-pyrimidinediamine
N2-Cyclobutyl-5-fluoro-N4- [4- (4-methoxycarbonylpiperazino) phenyl] -2,4-pyrimidinediamine
N4- [4- (N-Acetyl-N-methylamino) phenyl] -N2-cyclopropyl-5-fluoro-2,4-pyrimidinediamine
N2, N4-Bis (cyclopropyl) -5-fluoro-2,4-pyrimidinediamine
N4- [4- (N-Acetyl-N-methylamino) phenyl] -5-fluoro-N2- [3-N-methylamino) carbonylmethyleneoxyphenyl] -2,4-pyrimidinediamine
N2, N4-Bis (3-methylaminocarbonylmethyleneoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4- [4- (N-Acetyl-N-methylamino) phenyl] -N2-cyclobutyl-5-fluoro-2,4-pyrimidinediamine
N2, N4-Bis (cyclobutyl) -5-fluoro-2,4-pyrimidinediamine
N4- [4- (4-Acetylpiperazino) phenyl] -N2-cyclopropyl-5-fluoro-2,4-pyrimidinediamine
N4- [4- (4-Acetylpiperazino) phenyl] -N2-cyclobutyl-5-fluoro-2,4-pyrimidinediamine
N2- [4- (N-Acetyl-N-methylamino) phenyl] -N4-cyclobutyl-5-fluoro-2,4-pyrimidinediamine
cis / trans-N4- [4- [4- (tert-butoxycarbonylamino) cyclohexyloxy] -3-chlorophenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
N2- [4- (4-Acetylpiperazino) phenyl] -N4-cyclobutyl-5-fluoro-2,4-pyrimidinediamine
N4-Cyclobutyl-5-fluoro-N2- [4- (4-methoxycarbonylpiperazino) phenyl] -2,4-pyrimidinediamine
N4-Cyclobutyl-5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
N2- [3- (N-Cyclobutylamino) carbonylmethyleneoxyphenyl] -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- [3- (N-Cyclopropylamino) carbonylmethyleneoxyphenyl] -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- [4- (4-Acetylpiperazino) phenyl] -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N2- [4- (4-methoxycarbonylpiperazino) phenyl] -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4-Cyclobutyl-N2- (3-cyclopropylaminocarbonylmethyleneoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N4-Cyclobutyl-N2- (3,4-dichlorophenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -N4-cyclobutyl-5-fluoro-2,4-pyrimidinediamine
N4-Cyclobutyl-N2- [3- (N-cyclobutylamino) carbonylmethyleneoxyphenyl] -5-fluoro-2,4-pyrimidinediamine
N4-Cyclobutyl-N2- (3,5-dichloro-4-methoxyphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3,4-Dichlorophenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3-Chlorop-4-methoxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [l, 4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3,5-Dichloro-4-methoxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N2- (3-fluoro-4-methoxyphenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
cis / trans-N4- [4- (4-aminocyclohexyloxy) -3-chlorophenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
5-Fluoro-N2- (4-methoxyphenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3,4-Ethylenedioxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -N2- (4-trifluoromethoxyphenyl) -2,4-pyrimidinediamine
N2- (4-Ethoxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (4-Butoxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N2- (4-phenoxyphenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (4-Benzyloxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
cis / trans-N4- [3-Chloro-4- [4- (N-ethylamino) cyclohexyloxy] phenyl] -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2,4-pyrimidinediamine
5-Fluoro-N2- (4-morpholinophenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N2- (4-isopropoxyphenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Difluoro-2H-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2.4 -pyrimidinediamine salt of p-toluenesulfonic acid
N4- (2,2-Difluoro-2H-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl) -2.4 -pyrimidinediamine salt of benzenesulfonic acid
N4- (2,2-Dimethyl-2H-3-oxo-4H-5-pyrido [1,4] oxazin-6-yl] -5-fluoro-N2- [4- (4-methoxycarbonylpiperazino) phenyl] -2 4-pyrimidinediamine
N2- [4- (N-Acetyl-N-methylamino) phenyl] -N4- (2,2-dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5 -fluoro-2,4-pyrimidinediamine
N2- [4- (N-Acetyl-N-ethylamino) phenyl] -N4- (2,2-dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5 -fluoro-2,4-pyrimidinediamine
N2- [4- (4-Acetylpiperazino) phenyl] -N4- (2,2-dimethyl-2H-3-oxo-4H-5-pyrido [1,4] oxazin-6-yl) -5-fluoro-2 4-pyrimidinediamine
N2- [4- (N-Acetyl-N-methylamino) phenyl] -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2.4 pyrimidinediamine
N2- [4- (N-Acetyl-N-ethylamino) phenyl] -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2.4 pyrimidinediamine
N2- (3,4-Dimethoxyphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (4-morpholinophenyl) -2,4-pyrimidinediamine
N2- [3- (N-Cyclobutylamino) carbonylmethyleneoxyphenyl] -N4- (2,2-dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2 4-pyrimidinediamine
5-Fluoro-N2- [4- (4-methylpiperazino) phenyl] -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- [4- (4-methylpiperazino) phenyl] -2 4-pyrimidinediamine
N2- (3,5-Dimethylphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3,5-Dimethylphenyl) -N4- (2,2-dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-2,4- pyrimidinediamine
5-Fluoro-N2- (3-isopropylphenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-methylphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N2- (3-methoxy-5-trifluoromethylphenyl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N2- (indol-6-yl) -N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -5-fluoro-N2- (indol-6-yl) -2,4- pyrimidinediamine
N2- (3,5-Dichlorophenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3-Bromophenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3-tert-Butylphenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3,4-Difluorophenyl) -5-fluoro-N4- (2H-3-oxo-4H-5-pyrid [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
(S) -5-Fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2.4 pyrimidinediamine
N4- (4,4-Dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-N2- [3- (N-methylamino) carbonylmethylenedioxyphenyl] -2,4-pyrimidinediamine
(R) -5-Fluoro-N2- [3- (N-methylamino) carbonylmethyleneoxyphenyl] -N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2.4 pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -N4- (4,4-dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-2,4- pyrimidinediamine
(S) -N2- (3-Chloro-4-methoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4- pyrimidinediamine
(R) -N2- (3-Chloro-4-methoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4- pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -N4- (4,4-dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-2,4-pyrimidinediamine
(S) -N2- (3,5-Dichloro-4-methoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] -oxazin-6-yl) -2 4-pyrimidinediamine
(R) -N2- (3,5-Dichloro-4-methoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] -oxazin-6-yl) -2 4-pyrimidinediamine
(+) - N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- [2- (2-hydroxyethyl) -3-oxo-4H-benz [1,4] oxazin-6-yl] -2, 4-pyrimidinediamine
(+) - N2- (3-Chloro-4-methoxyphenyl) -5-fluoro-N4- [2- (2-hydroxyethyl) -3-oxo-4H-benz [1,4] oxazin-6-yl] - 2,4-pyrimidinediamine
(S, S) -N2, N4-Bis- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
(S) -N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
(R) -N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4-pyrimidinediamine
N2- (3,5-Dichloro-4-methoxyphenyl) -N4- (4,4-dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (4,4-Dimethyl-1,3-dioxo-2H, 4H-isoquinolin-7-yl) -N2- (indazol-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,3-Dimethyl-4H-benz [1,4] oxazin-6-yl) -N2- (3,5-dimethylphenyl) -5-fluoro-2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxyphenyl) -N4- (3,3-dimethyl-4H-benzo [1,4] oxazin-6-yl) -5-fluoro-2,4-pyrimidinediamine
N4- (3,3-Dimethyl-1,4-benzoxazin-6-yl) -5-fluoro-N2- (indazol-6-yl) -2,4-pyrimidinediamine
N4- (3,3-Dimethyl-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (N1-methylindazol-6-yl) -2,4-pyrimidinediamine
(R) -N2- (3-Chloro-4-methoxyphenyl) -5-fluoro-N4- (2-methyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -2,4- pyrimidinediamine salt of toluenesulfonic acid
N4- (2,6-Dimethoxypyrid-3-yl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-5-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (4-Chlorophenyl) -5-fluoro-N2- {1- [2- (N-methylamino) carbonylethyl] indazolin-5-yl} -2,4-pyrimidinediamine
N4- (4-Chlorophenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-5-yl] -2,4-pyrimidinediamine
N4- (3,4-Differphenyl) -N2- [1- (2-ethoxycarbonylethyl) -indazolin-5-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Differphenyl) -5-fluoro-N2- {1- [2- (N-methylaminocarbonyl) ethyl] indazolin-5-yl} -2,4-pyrimidinediamine
N4- (3,4-Difluorophenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-5-yl] -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-5-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- {1- [2 (N-methylaminocarbonyl) ethyl] indazolin-5-yl} -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-5-yl] -2,4-pyrimidinediamine
N4- (3,4-Ethylenedioxyphenyl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-hydroxy-5-methylphenyl) -5-fluoro-N4- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- [2- (2-methoxy-4-methoxycarbonylbenzyl) indazolin-6-yl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- [2- (2-methoxy-4-methoxycarbonylbenzyl) indazolin-6-yl] -2,4-pyrimidinediamine
N4- (3,4-Ethylenedioxyphenyl) -5-fluoro-N2- {1- [2-methoxy-4- (N-methylaminocarbonyl) benzyl] indazolin-6-yl} -2,4-pyrimidinediamine
N4- (3,4-Difluorophenyl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Difluorophenyl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-6-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- {1- [2 (N-methylamino) carbonylethyl] indazolin-6-yl} -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-6-yl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-6-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-6-yl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- {1- [2- (N-methylaminocarbonyl) ethyl] indazolin-6-yl} -2,4-pyrimidinediamine
5-Fluoro-N4- (4-fluoro-3-methoxyphenyl) -5-fluoro-N2- (1-methylindazolin-5-yl) -2,4-pyrimidinediamine
N4- (4-Chloro-3-fluorophenyl) -5-fluoro-N2- (1-methylindazolin-5-yl) -2,4-pyrimidinediamine
N4- (3,4-Dimethoxyphenyl) -5-fluoro-N2- (1-methylindazolin-5-yl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxy-5-methylphenyl) -5-fluoro-N4- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (4-Chloro-3-fluorophenyl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (4-Chloro-3-fluorophenyl) -5-fluoro-N2- (indazolin-5-yl) -2,4-pyrimidinediamine
5-Fluoro-N4- (4-fluoro-3-methoxyphenyl) -N2- (indazolin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N4- (4-fluoro-3-methoxyphenyl) -N2- (indazolin-5-yl) -2,4-pyrimidinediamine
N2- (3-Chloro-4-methoxy-5-methylphenyl) -5-fluoro-N4- {4H-imidazo [2,1-c] benz [1,4] oxazin-8-yl} -2.4 pyrimidinediamine
N2- (3-Chloro-4-methoxy-5-methylphenyl) -5-fluoro-N4- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N2- (4-Chlor-2,5-dimethoxyphenyl) -5-fluoro-N4- (l-methylindazolin-6-yl) -2,4-pyrimidinediamine
N2- (3,5-Dimethoxyphenyl) -5-fluoro-N4- (indazolin-6-yl) -2,4-pyrimidinediamine
N2- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N4- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-fluorophenyl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-fluorophenyl) -5-fluoro-N2- (1-methylindazolin-5-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-fluorophenyl) -5-fluoro-N2- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-fluorophenyl) -5-fluoro-N2- (indazolin-5-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N4- (indazolin-6-yl) -2,4-pyrimidinediamine salt of benzenesulfonic acid
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N4- (indazolin-6-yl) -2,4-pyrimidinediamine salt p- toluenesulfonic acid
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-5-yl] -5-fluoro-2 4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-5-yl] -2 4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- {1- [2 (N-methylaminocarbonyl) ethyl] indazolin-5- silt} -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-6-yl] -5-fluoro-2 4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- {1- [2- (N-methylaminocarbonyl) ethyl] indazolin-6 -yl} -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [1- (methoxycarbonyl) methylindazolin-5-yl] -5-fluoro-2,4 pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- [1- (N-methylaminocarbonyl) methylindazolin-5-yl} -2 4-pyrimidinediamine
N2- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N4- (indazolin-5-yl) -2,4-pyrimidinediamine
N2- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N4- (1-methylindazolin-5-yl) -2,4-pyrimidinediamine
N2- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N4- (indazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Dimethoxyphenyl) -5-fluoro-N2- (indazolin-5-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- [1- (2-methoxy-4-methoxycarbonylbenzyl) indazolin-6- il] -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine salt of p-toluenesulfonic acid
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- (2-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- (2-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (2-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -N2- [2- (2-ethoxycarbonylethyl) indazolin-5-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -N2- [2- (2-ethoxycarbonylethyl) indazolin-5-yl] -5-fluoro-2 4-pyrimidinediamine
5-Fluoro-N4- (4-fluoro-3-methoxyphenyl) -N2- (1-methylindazolin-6-yl) -2,4-pyrimidinediamine
5-Fluoro-N4- (4-fluoro-3-methoxyphenyl) -N2- (2-methylindazolin-6-yl) -2,4-pyrimidinediamine
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-5-yl] -2,4-pyrimidinediamine salt of bis (p) -toluenesulfonic acid
N4- (3,4-Dichlorophenyl) -5-fluoro-N2- {2- [2- (N-methylaminocarbonyl) ethyl] indazolin-5-yl} -2,4-pyrimidinediamine
N4- (2,2-Dimethyl-3-oxo-4H-benz [1,4] oxazin-6-yl) -5-fluoro-N2- {2- [2- (N-methylaminocarbonyl) ethyl] indazolin-5 -yl} -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (1-methylindazolin-5-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- (indazolin-5-yl) -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -N2- [1- (2-ethoxycarbonylethyl) indazolin-5-yl] -5-fluoro-2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- [1- (3-hydroxypropyl) indazolin-5-yl] -2,4-pyrimidinediamine
N4- (3-Chloro-4-methoxyphenyl) -5-fluoro-N2- {1- [2- (N-methylaminocarbonyl) ethyl] indazolin-5-yl} -2,4-pyrimidinediamine. 2. A pharmaceutical composition for treating an autoimmune disease, comprising in an effective amount a compound according to claim 1 and a pharmaceutically acceptable carrier, excipient and / or diluent. 3. The use of a compound according to claim 1 for the manufacture of a medicament for the treatment of an autoimmune disease. 4. Use of a compound selected from the group consisting of the following compounds:
N2, N4-bis (3-aminophenyl) -5-fluoro-2,4-pyrimidinediamine (R921302);
N4- (3-chloro-4-methoxyphenyl) -5-fluoro-N2- [3 - [(N-methylamino) carbonylmethylene] phenyl] -2,4-pyrimidinediamine (R926891);
N4 - [(2,2-dimethyl-4H-benzo [1,4] oxazin-3-one) -6-yl] -5-fluoro-N2- [3- (methylaminocarbonylmethyleneoxy) phenyl] -2,4-pyrimidinediamine (R940323);
N4 - [(2,2-dimethyl-4H-5-pyrido [1,4] oxazin-3-one) -6-yl] -5-fluoro-N2- [3- (methylaminocarbonylmethyleneoxy) phenyl] -2,4 -pyrimidinediamine (R940347) and
N4 - [(2,2-difluoro-4H-benzo [1,4] oxazin-3-one) -6-yl] -5-fluoro-N2- [3- (methylaminocarbonylmethyleneoxy) phenyl] -2,4-pyrimidinediamine (R921303)
for the manufacture of a medicament for the treatment of an autoimmune disease. 5. The use according to any one of claims 3 and 4, according to which the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitomasenoma, syndrome of autoimmunity, anomitosis, anomitosis, and syndrome of autoimmunity, arrhythmias, and syndrome of autoimmunity, arrhythmias, and syndrome of autoimmunity, arrhythmias, and others. asthenic bulbar paralysis, bazedovy disease, biliary primary cirrhosis of the liver, glomerulonephritis, chronic aggressive hepatitis, membrane glomerulopathy and autooi munnyh diseases involving systemic autoimmune disorder. 6. The use according to any one of claims 3 and 4, according to which the autoimmune disease is selected from the group consisting of autoimmune diseases, often characterized as autoimmune disorders of a single organ or single cell type, as well as diseases, often characterized as including systemic autoimmune disorders . 7. The use according to any one of claims 3 and 4, according to which the autoimmune disease is selected from the group consisting of Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitomasenoma, syndrome of autoimmunity, anomitosis, anomitosis, and syndrome of autoimmunity, anomitosis, and anemia syndrome, anomitosis, anomitosis, and anomitosis, and others. bulbospinal paralysis, bazedovy disease, biliary primary cirrhosis of the liver, chronic aggressive hepatitis and membrane glomerulopathy. 8. The use according to any one of claims 3 and 4, according to which the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, Sjogren’s syndrome, Reiter’s syndrome, polymyositis-dermatomyositis, systemic sclerosis, nodose polyarteritis, multiple sclerosis, and bullous . 9. The use of claim 8, according to which the autoimmune disease is systemic lupus erythematosus. 10. The use of claim 8, according to which the autoimmune disease is rheumatoid arthritis. 11. The use of claim 8, according to which the autoimmune disease is multiple sclerosis. 12. The use according to any one of claims 3 and 4, according to which the autoimmune disease is glomerulonephritis.

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