2,4-pyrimidinediamine compounds for treating or preventing autoimmune diseases

2,4-Pyrimidinediamine compounds for the treatment or prevention of autoimmune diseases

5 Field of the Invention

The present invention generally relates to 3-hydroxyphenyl-2,4-pyrimidinediamine compounds, pharmaceutical compositions comprising the compounds, intermediates and synthetic methods of preparing the compounds and methods of using the compounds and compositions in a variety of contexts, such as in the treatment or

10 Prevention of various diseases.

Background of the invention

Crosslinking of Fc receptors, such as the high affinity receptor for IgE (FcεRI) and / or the receptor of

fifteen High affinity for IgG (FcγRI) activates a signaling cascade in mast cells, basophils and other immune cells that results in the release of chemical mediators responsible for numerous adverse events. For example, such crosslinking leads to the release of preformed mediators of anaphylactic hypersensitivity reactions of type I (immediate), such as histamine, from granule storage sites by degranulation. It also leads to the synthesis and release of other mediators, including

twenty leukotrienes, prostaglandins and platelet activation factors (PAF), which play important roles in inflammatory reactions. Additional mediators that are synthesized and released after cross-linking of Fc receptors include cytokines and nitric oxide.

The signaling cascade (s) activated by cross-linking of Fc receptors such as FcεRI and / or

25 FcγRI comprises (n) an arrangement of cellular proteins. Among the most important intracellular signal propagators are tyrosine kinases. An important tyrosine kinase involved in the signal transduction mechanisms associated with the cross-linking of the FcεRI and / or FcγRI receptors, as well as other signal transduction cascades, is Syk kinase (see Valent et al., 2002, Intl. J. Hematol 75 (4): 257-362 for review).

30 Recently, several classes of 2,4-pyrimidinediamine compounds have been discovered that inhibit FcεRI and / or FcγRI signaling cascades and have a multitude of therapeutic uses. See, for example, the US application. Serial No. 10 / 355,543 filed on January 31, 2003 (US 2004 / 0029902A1), International Application for Serial No. PCT / US03 / 03022 filed on January 31, 2003 (WO 03/063794 ), the US request Serial No. 10 / 631,029 filed on July 29, 2003, the international application for No. of

35 PCT / US03 / 24087 series (WO 2004/014382), US application. Serial No. 10 / 903,263 filed on July 30, 2004 (US 2005/0234049), the international application for Serial No. PCT / US2004 / 24716, the US application. Serial No. 10 / 903,870 filed July 30, 2004.

Because mediators released as a result of cross-linking of the FcεRI or FcγRI receptor are

40 responsible for, or play important roles in, the manifestation of numerous adverse events, the availability of compounds capable of inhibiting the signaling cascade (s) responsible for their release would be highly desirable. In addition, due to the critical role that Syk kinase plays in this (s) and other receptor signaling cascade (s), the availability of compounds capable of inhibiting Syk kinase would be highly desirable.

Four. Five WO 2005/012294 describes 2,4-pyrimidine diamine compounds for use in the treatment or prevention of autoimmune diseases.

WO 2005/026158 describes 2,4-di (hetero) arylamino-pyrimidine derivatives as inhibitors of ZAP70 and / or Syk.

fifty WO 2005/013996 describes 2,4-pyrimidinediamine compounds and uses as antiproliferative agents.

Summary of the invention

55 The present invention relates to a compound or one of its stereoisomers, salts, hydrates, solvates or N-oxides according to formula I:

in which:

Y is selected from the group consisting of S, O, SO, SO2 and C (R7) 2; 5 each R35 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl and halo,

or both R35 together with the carbon to which they are attached form a carbonyl group;

W is selected from the group consisting of C = O, C = S, C = NH, C (R7) 2 and NR37;

Z is C = O or NR37, with the proviso that Z and W are not both NR37 and with the proviso that when Z is C = O, then W is (CR7) 2 or NR37;

X is CH or N;

fifteen each R31 is independently (C1-C4) alkyl or both R31 together form an (C1-C2) alkylene group optionally substituted with one to two (C1-C4) alkyl groups or substituted with a spirocycloalkyl (C3-C7) group;

each R7 is independently hydrogen or (C1-C4) alkyl; and

R37

it is hydrogen or methyl optionally substituted with phenyl or pyridyl, wherein said phenyl or pyridyl is optionally substituted with (C1-C4) alkoxy.

Various related features, such as methods, are also described herein.

25 compositions and uses that refer to compounds of formula I and II, which will be readily apparent from the following detailed description.

It will be appreciated by the person skilled in the art that the implementations summarized above may be used in conjunction with any appropriate combination to generate implementations not expressly cited above and that said implementations are considered part of the present invention.

Detailed description

Throughout the present application, the text refers to various embodiments of the present compounds and

35 compositions, as well as the methods described herein. It is understood that the various embodiments described provide a variety of illustrative examples and should not be understood as descriptions of alternative species. Instead, it should be appreciated that the descriptions of various embodiments provided herein may be of an overlapping scope. The embodiments discussed herein are illustrative only and do not mean that they limit the scope of the present invention.

Definitions

As used herein, the following definitions will apply unless otherwise indicated.

Four. Five "Alkyl" or "alkanyl" by itself or as part of another substituent refers to monovalent saturated aliphatic hydrocarbyl groups having the claimed number of carbon atoms (ie, C1-C4 means one to four carbon atoms). This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3-CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3) 2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3) 2CHCH2-), sec-butyl ((CH3) (CH3CH2) CH-), t-butyl ((CH3) C-), n-pentyl (CH3CH2CH2CH2CH2-) and neopentyl (( CH3) 3CCH2-).

"Benzyl" by itself or as part of another substituent refers to the group (C6H5) CH2-.

55 "Carbonyl" refers to the group> C = O. "Thiocarbonyl" refers to the group> C = S.

"Cyano" by itself or as part of another substituent refers to the group -CN.

"Halogen" or "halo" by themselves or as part of another substituent, unless otherwise indicated, refer to fluoro, chloro, bromo and iodo.

"Haloalkyl" by itself or as part of another substituent refers to an alkyl group in which one or more of the hydrogen atoms are substituted by a halogen. Thus, it is understood that the term "haloalkyl" includes monohaloalkyl, dihaloalkyl, trihaloalkyl, etc., to perhaloalkyl. For example, the expression

65 "haloalkyl (C1-C2)" includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

"Nitro" by itself or as part of another substituent refers to -NO2.

The aforementioned groups may include prefixes and / or suffixes that are commonly used in the art.

5 to create well recognized substituent groups. By way of examples, "alkyloxy" or "alkoxy" refers to a group of the formula -OR ", wherein R" is alkyl and includes alkoxy groups such as methoxy and ethoxy. As another example, "haloalkoxy" or "haloalkyloxy" refers to a group of formula OR ", wherein R" is haloalkyl. In other examples, "4-methoxybenzyl" refers to substitution of benzyl in the 4-para position with methoxy and "2-pyridylmethyl" refers to the substitution of methyl with a 2-pyridyl group.

"Alkenyl" by itself or as part of another substituent refers to an unsaturated branched, straight chain or cyclic alkyl having at least one carbon-carbon double bond from the removal of a hydrogen atom from an individual carbon atom. of a parental alkene. The group may be either in cis or trans conformation around the double bond (s). Typical alkenyl groups include, but are not limited to,

fifteen ethenyl; propenyl 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 -ilo; cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2en -2-yl, buta-1,3-dien-1-yl, butan-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 As used herein, "lower alkenyl" means (C2-C8) alkenyl.

"Alkynyl" by itself or as part of another substituent refers to an unsaturated branched, straight chain or cyclic alkyl having at least one carbon-carbon triple bond from the removal of a hydrogen atom from an individual carbon atom of a parental alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-in-1-yl, prop-2-in-1-yl, etc .; butyls such as but-1-in-1-yl, but-1in-3-yl, but-3-in-1-yl, etc .; and the like As used herein, "lower alkynyl" means alkynyl.

25 (C2-C8).

"Alkyldiyl" by itself or as part of another substituent refers to a divalent cyclic or straight chain, branched, saturated or unsaturated hydrocarbon group having the claimed number of carbon atoms (ie, C1-C6 means from one to six carbon atoms) from the removal of a hydrogen atom from each of the two different carbon atoms of a parental alkane, alkene or alkyne, or by the removal of two hydrogen atoms from an individual carbon atom from an alkane, alkene or parental alkyne. The two monovalent radical centers or each valence of the divalent radical center can form bonds with the same atoms

or with different atoms. Typical alkyldiyl groups include, but are not limited to, methanediyl; ethyldiyl such as ethan-1,1-diyl, ethan-1,2-diyl, ethe-1,1-diyl, ethe-1,2-diyl; propyldiyl such as propan-1,1-diyl, propan-1,235 diyl, propan-2,2-diyl, propan-1,3-diyl, cyclopropan, 1,1-diyl, cyclopropan-1,2-diyl, prop- 1-en-1,1-diyl, prop-1-en-1,2diyl, 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 .; butyldiyl such as butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl, butan2,2-diyl, 2-methyl-propan-1,1 -diyl, 2-methyl-propan-1,2-diyl, cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3diyl, 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-propan-1,1-diyl, buta-1,3-diene-1,1- diyl, buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4diyl, cyclobut-1-in-1, 2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl, cyclobuta-1,3-diene-1,3-diyl, cyclobuta-1,3dien-1, 3-diyl, but-1-in-1,3-diyl, but-1-in-1,4-diyl, buta-1,3-diin-1,4-diyl, etc .; and the like When specific levels of saturation are intended, the nomenclature of alkyldiyl, alkenyldiyl and / or alkyldiyl is used. When it is specifically intended that the two valences be on the same carbon atom, the nomenclature is used

Four. Five "alkylidene". In some embodiments, the alkyldiyl group is (C 1 -C 8) alkyldiyl. Specific embodiments include saturated acyclic alkyldiyl groups in which the radical centers are in the terminal carbons, for example, methanediyl (methane); ethan-1,2-diyl (ethane); propan-1,3-diyl (propane); butan-1,4-diyl (butane); and the like (also called alkylenes, defined below).

"Alkylene" by itself or as part of another substituent refers to a saturated or unsaturated linear chain alkyldiyl group having two terminal monovalent radical centers from the removal of a hydrogen atom from each of the two terminal carbon atoms. of the alkane, alkene or linear chain parental alkyne. The location of a double bond or triple bond, if present, in a particular alkylene is indicated by square brackets. Typically, alkylene groups include, but are not limited to, methane;

55 ethylenes such as ethane, ethene, ethyne; propylene such as propane, pro [1] eno, propa [1,2] diene, prop [1] ino, etc .; butylenes such as butane, but [1] eno, but [2] eno, buta [1,3] diene, but [1] ino, but [2] ino, buta [1,3] diino, etc .; and the like When specific saturation levels are intended, the nomenclature alkane, alkene and / or alkyne is used. In some embodiments, the alkylene group is (C1-C8) alkylene or (C1-C3). Specific embodiments include saturated straight chain alkane groups, for example, methane, ethane, propane, butane and the like.

"Heteroalkyl", "heteroalkanyl", "heteroalkenyl", "heteroalkynyl", "heteroalkyldiyl" and "heteroalkylene" by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkylene groups, respectively, in which one more carbon atoms are independently substituted by the same group of heteroatoms by different heteroatomic groups. Heteroatoms and / or typical heteroatomic groups 65 that can substitute carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR´-, -PH-, -S (O) -, -S (O) 2-, -S (O) NR´-, -S (O) 2NR´- and the like, including their combinations, in which each R´ is so

hydrogen independent or (C1-C8) alkyl.

"Cycloalkyl" and "heterocycloalkyl" by themselves or as part of another substituent refer to cyclic versions of "alkyl" and "heteroalkyl" groups, respectively. For heteroalkyl groups, a heteroatom can occupy

5 the position that is attached to the rest of the molecule. Typical cycloalkyl groups include, but are not limited to, cyclopropyl; cyclobutyl such as cyclobutanyl and cyclobutenyl; cyclopentyl such as cyclopentyl and cyclopentenyl; cyclohexyl such as cyclohexanyl and cyclohexenyl; and the like Typical heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl (for example, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, etc.), piperidinyl (for example, piperidin-1-yl, piperidin-2-yl, etc.), morpholinyl (for example, morpholin-3-yl, morpholin-4-yl, etc.), piperazinyl (for example, piperazin-1-yl, piperazin-2-yl, etc.) and the like.

"Acyclic Heteroatomic Bridge" refers to a divalent bridge in which the atoms of the main chain are exclusively heteroatoms and / or heteroatomic groups. Typical acyclic heteroatomic bridges include, but are not limited to, -O-, -S-, -SO-, -NR´-, -PH-, -S (O) -, -S (O) 2-, -S (O) NR´-, -S (O) 2NR´ and similar, including their

fifteen combinations, in which each R 'is independently hydrogen or (C1-C8) alkyl.

"Parental Aromatic Ring System" refers to an unsaturated polycyclic or cyclic ring system that has a π electron system. Specifically included in the definition of "parental aromatic ring system" are condensed ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane , indene, phenalene, tetrahydronaphthalene, etc. Typical parental aromatic ring systems include, but are not limited to, aceantrylene, acenaphthylene, acefenantrylene, anthracene, azulene, benzene, chromene, coronenne, fluorantene, fluorene, hexacene, hexaphene, hexalene, indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenanthene, phenanthrene, piceno, pleiadene, pyrene, pyrenean, ruby,

25 tetrahydronaphthalene, triphenylene, trinaphthalene and the like.

"Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon group having the claimed number of carbon atoms (ie, C6-C15 means 6 to 15 carbon atoms) from withdrawal of a hydrogen atom of an individual carbon atom of a parental aromatic ring system. Typical aryl groups include, but are not limited to, groups from aceantrylene, acenaphthylene, acefenantrylene, anthracene, azulene, benzene, chromene, coronen, fluorantene, fluorene, hexacene, hexaphene, hexalene, indacene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, pentacene, pentalene, pentaphene, perylene, phenanene, phenanthrene, piceno, pleiadene, pyrene, pyrenean, rubycene, triphenylene, trinaphthalene and the like, as well as several of its hydroisomers. In preferred embodiments, the

35 aryl group is (C6-C15) aryl, (C6-C10) being more typical. Specific exemplary aryls include phenyl and naphthyl.

"Arylaryl" by itself or as part of another substituent refers to a monovalent hydrocarbon group from the removal of a hydrogen atom from an individual carbon atom from a ring system in which two or more systems of bonding are joined. identical or non-identical parental aromatic rings directly by means of a single bond, in which the number of said direct ring joints is one less than the number of parental aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. When the number of carbon atoms of an arylaryl group is specified, the number refers to the carbon atoms that comprise each parental aromatic ring. For example, an arylaryl (C6-C15) is an arylaryl group in which each aromatic ring

Four. Five it comprises 6 to 5 atoms, for example, biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc. In some embodiments, each parental aromatic ring system of an arylaryl group is independently an aromatic system (C6-C15), more preferably an aromatic system (C6-C10). Specific exemplary arylaryl groups include those in which the parental aromatic ring systems are identical, for example, biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

"Biaryl" by itself or as part of another substituent refers to an arylaryl group having two identical parental aromatic systems linked directly together by means of an individual bond. Typical biaryl groups include, but are not limited to, biphenyl, binaphthyl, biantracenyl and the like. In some embodiments, the aromatic ring systems are aromatic rings (C6-C15), more typically aromatic rings (C6-C10). A

55 Particular exemplary biaryl group is biphenyl.

"Arylalkyl" by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, usually a sp3 carbon atom, is substituted by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenyletan-1-yl, 2-phenyleten-1-yl, naphthylmethyl, 2-naphthyletan-1-yl, 2-naphthylene-1-yl, naphthobenzyl, 2 -naphtophenylethan-1-yl and the like. When specific alkyl moieties are intended, the nomenclature arylalkyl, arylalkenyl and / or arylalkyl. In some embodiments, the arylalkyl group is arylalkyl (C7-C21), for example, an alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C6) and the aryl moiety is (C6-C15). In some specific embodiments the arylalkyl group is (C7-C13), for example, the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) and the aryl moiety is (C6-C10).

65 "Parental Heteroaromatic Ring System" refers to a parental aromatic ring system in which one or

more carbon atoms are independently substituted by the same heteroatom or by different heteroatoms or by different heteroatomic groups. Typical heteroatoms or heteroatomic groups to replace carbon atoms include, but are not limited to, N, NH, P, O, S, S (O), S (O) 2, Si, etc. Specifically within the definition of "parental heteroaromatic ring systems" are condensed ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, benzodioxane , benzofuran, chromane, chromene, indole, indoline, xanthene, etc. Also recognized in the definition of "parental heteroaromatic ring system" are recognized rings that include common substituents, such as, for example, benzopirone and 1-methyl-1,2,3,4-tetrazole. Specifically excluded from the definition of "parental heteroaromatic ring system" are benzene rings fused to cyclic polyalkylene glycols such as cyclic polyethylene glycols. The heteroaromatic ring systems parental typical include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxaxina, benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene , cinoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromen, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole,

fifteen oxazole, pyrimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazol, thiazozole, thiadiazole, thiadiazole, thiadiazole, thiadiazole, thiadiazole xanthene and the like.

"Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaromatic group having the claimed number of ring atoms (eg, "5-14 members" means 5 to 14 ring atoms) from the removal of a hydrogen atom from an individual atom from a parental heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups from acridine, benzimidazole, bencisoxazole, benzodioxane, benzodiaxol, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxaine, carboline, β-chromazole, carzoazoline, β-chromazole, carzoazoline, β-carbonazole, chromazoenozoline, carbolinene, beta-chromazole, carzoazoline, β-carbonazole, chromazoenozine, carboline-cyanozole, carzoazoline, beta-carbonazole, carzoazoline, beta-carbonazole, chromazoenozine, carbolinene, beta-choline, carbonate Cinolina Furan

25 imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazol, thiadiazole, thiazole, thiophene, triazole, xanthene and the like, as well as several of its hydroisomers. In preferred embodiments, the heteroaryl group is a 5-14 membered heteroaryl, with a 5-10 membered heteroaryl being particularly preferred.

"Heteroaryl-Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaromatic group from the removal of a hydrogen atom from an individual atom from a ring system in which two or more parental heteroaromatic ring systems identical or non-identical are joined directly by

35 means of a single bond, in which the number of said ring junctions is one less than the number of parental heteroatomic ring systems involved. Typical heteroaryl-heteroaryl groups include, but are not limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc. When the number of atoms is specified, this refers to the number of atoms comprising each of the parental heteroaromatic ring systems. For example, 5-15-membered heteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which each parental heteroaromatic ring system comprises 5 to 15 atoms, for example, bipyridyl, tripuridyl, etc. In some embodiments, each parental heteroaromatic ring system is, independently, a 5-15 member heteroaromatic system, more usually a 5-10 member heteroaromatic system. Specific exemplary heteroaryl-heteroaryl groups include those in which the parental heteroaromatic ring systems are identical.

Four. Five "Biheteroaryl" by itself or as part of another substituent refers to a heteroaryl-heteroaryl group having two identical parental heteroaromatic ring systems linked directly via a single bond. Typical biheteroaryl groups include, but are not limited to, bipyridyl, bipurinyl, biquinolinyl and the like. In some embodiments, the heteroaromatic ring systems are 5-15 member heteroaromatic rings, more usually they are 5-10 member heteroaromatic rings.

"Heteroarylalkyl" by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms attached to a carbon atom, usually a terminal or sp3 carbon atom, is substituted by a heteroaryl group. . When specific alkyl moieties are intended, the nomenclature is used

55 heteroarylalkyl, heteroarylalkenyl and / or heteroarylalkyl. In some embodiments, the heteroarylalkyl group is a 6-21-membered heteroarylalkyl, for example, the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and the heteroaryl moiety is a 5-15 membered heteroaryl. In some specific exemplary embodiments, the heteroarylalkyl is 6-13 membered heteroarylalkyl, for example, the alkanyl, alkenyl or alkynyl moiety is (C1-C3) alkyl and the heteroaryl moiety is a 5-15 membered heteroaryl.

"Halogen" or "halo" by themselves or as part of another substituent, unless otherwise indicated, refer to fluoro, chloro, bromo and iodo.

"Spirocycloalkyl (C3-C7)" refers to divalent cyclic groups of 3 to 7 carbon atoms that have a ring

65 3 to 7-membered cycloalkyl with a spiro bond (the bond formed by means of an individual atom that is the only common member of the rings) as exemplified by the following structure C3:

"Substituted" when used to modify a specified group or radical, means that one or more atoms of

5 hydrogen of the specified group or radical has each been substituted, independently of each other, by the same substituent or different substituents. Substituent groups useful for the replacement of hydrogens in saturated carbon atoms in the specified or radical group include, but are not limited to, -R60, halo, -O -M +, = O, -OR70, -SR70, -S- M +, = S, -NR80R80, = NR70, = N-OR70, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, = N2, -N3, -S (O) 2R70, - S (O) 2O-M +, -S (O) 2OR70, -OS (O) 2R70, -OS (O) 2O-M +, -OS (O) 2OR70, -P (O) (O-) 2 (M + ) 2, -P (O) (OR70) O-M +,

10 P (O) (OR70) (OR70), -C (O) R70, -C (S) R70, -C (NR70) R70, -C (O) O -M +, -C (O) OR70, -C (S) OR70, -C (O) NR80R80, C (NR70) NR80R80, -OC (O) R70, -OC (S) R70, -OC (O) O -M +, -OC (O) OR70, -OC (S) OR70, -NR70C (O) R70, -NR70C (S) R70, -NR70C (O) O-M +, -NR70C (O) OR70, -NR70C (S) OR70, -NR70C (O) NR80R80, - NR70C (NR70) R70 and -NR70C (NR70) NR80R80, wherein R60 is selected from the group consisting of alkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl; each R70 is independently hydrogen or R60; each R80 is of

fifteen independently R70 or alternatively, the two R80 together with the nitrogen atom to which they are attached,

they form a 5, 6 or 7 membered cycloheteroalkyl which, optionally, may include 1 to 4 of the same or different heteroatoms selected from the group consisting of O, N and S; and each M + is a counterion with positive charge, for example, a positive charge that is independently selected from K +, Na +, + N (R60) 4 and Li +

or two of M + combine to form a divalent counterion, for example a divalent counterion selected from

twenty Ca2 +, Mg2 + and Ba2 +. As specific examples, it is understood that -NR80R80 includes -NH2, -NH-alkyl, N-pyrrolidinyl and N-morpholinyl.

Similarly, substituent groups useful for substituting hydrogen atoms in unsaturated carbon atoms in the specific group or radical include, but are not limited to, -R60, halo, -O -M +, -OR70, -SR70, -S -M +, - NR80R80,

25 trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -S (O) 2R70, -S (O) 2O-M +, -S (O) 2OR70, -OS (O ) 2R70, -OS (O) 2O-M +, -OS (O) 2OR70, -P (O) (O-) 2 (M +) 2, -P (O) (OR70) O-M +, -P (O ) (OR70) (OR70), -C (O) R70, -C (S) R70, -C (NR70) R70, -C (O) O-M +, C (O) OR70, -C (S) OR70 , -C (O) NR80R80, -C (NR70) NR80R80, -OC (O) R70, -OC (S) R70, -OC (O) O -M +, -OC (O) OR70, OC (S) OR70 , -NR70C (O) R70, -NR70C (S) R70, -NR70C (O) O-M +, -NR70C (O) OR70, -NR70C (S) OR70, -NR70C (O) NR80R80, NR70C (NR70) R70 and -NR70C (NR70) NR80R80, in which R60, R70, R80 and M + are as previously defined.

30 Substituent groups, other than Rp, useful for substituting hydrogens on nitrogen atoms in heteroalkyl and cycloheteroalkyl groups include, but are not limited to, -R60, -O-M +, -OR70, -SR70, -S-M +, - NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S (O) 2R70, -S (O) 2O-M +, -S (O) 2OR70, -OS (O) 2R70, -OS (O ) 2O-M +, -OS (O) 2OR70, P (O) (O-) 2 (M +) 2, -P (O) (OR70) O-M +, -P (O) (OR70) (OR70), -C (O) R70, -C (S) R70, -C (NR70) R70, -C (O) OR70, -C (S) OR70,

35 C (O) NR80R80, -C (NR70) NR80R80, -OC (O) R70, -OC (S) R70, -OC (O) OR70, -OC (S) OR70, -NR70C (O) R70, -NR70C (S) R70, NR70C (O) OR70, -NR70C (S) OR70, -NR70C (O) NR80R80, -NR70C (NR70) R70 and -NR70C (NR70) NR80R80, where R60, R70, R80 and M + are as previously defined.

Substituent groups from the above list useful for substituting other groups or atoms specified as "substituted" will be apparent to those skilled in the art.

"Protective group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group. Normally, a protecting group can be selectively removed as desired during the course of the synthesis. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc") , trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, alkoxycarbonyl, 9-fluorenylmethoxycarbonyl ("FMOC"), nitro-veratriloxycarbonyl

fifty ("NVOC") and the like. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is either acylated or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers. (for example, TMS or TIPPS groups) and allyl ethers.

55 "Fc receptor" refers to a member of the family of cell surface molecules that binds to the Fc part (which contains the specific constant region) of an immunoglobulin. Each Fc receptor binds to immunoglobulins of a specific type. For example, the Fc receptor ("FcαR") binds IgA, FcεR binds IgE and FcγR binds IgG.

60 The FcαR family includes the polymeric Ig receptor involved in the epithelial transport of IgA / IgM, the myeloid specific RcαRI receptor (also called CD89), the Fcα / μR receptor and at least two alternative IgA receptors (for a recent review see Monteiro & van de Winkel, 2003, Annu. Rev. Immunol., advanced electronic publication). The FcαRI receptor is expressed on neutrophil, eosinophilic, monocyte / macrophage, dendritic and Kupffer cells. The FcαRI receptor includes an alpha chain and the gamma FcR homodimer carrying an activation motif (ITAM) in the cytoplasmic domain and Syk kinase phosphorylates.

5 The FcεR family includes two types, designated as FcεRI and FcεRII (also known as CD23). FcεRI is a high affinity receptor (it binds to IgE with an affinity of approximately 1010 M-1) found in mast cells, basophilic and eosinophilic cells that bind monomeric IgE to the cell surface. The FcεRI has an alpha chain, a beta chain and a gamma chain homodimer discussed above. FcεRII is a low affinity receptor expressed on mononuclear phagocytes, B lymphocytes, eosinophils and platelets. The FcεRII

10 It comprises an individual polypeptide chain and does not include the gamma chain homodimer.

The FcγR family includes three types, designated as FcγRI (also known as CD64), FcγRII (also known as CD32) and FcγRII (also known as CD16). FcγRI is a high affinity receptor (binds to IgG1 with an affinity of 108M-1) and is found in mast cells, basophilic, mononuclear, neutrophilic, eosinophilic cells,

fifteen dendritic and phagocytes, binding monomeric IgG to the cell surface. The FcγRI includes an alpha chain and the gamma chain dimer shared by FcαRI and FcεRI.

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

twenty FcγRIII is a low affinity receptor (binds to IgG1 with an affinity of 5 x 105M-1) expressed on NK, eosinophils, macrophages, neutrophils and mast cells. It comprises an alpha chain and the gamma homodimer shared by FcαRI, FcεRI and FcγRI.

25 Skilled workers will recognize that the subunit structure and binding properties of these different Fc receptors, as well as the cell types that express them, are not fully characterized. The above discussion simply reflects the current state of the art with respect to these receptors (see, for example, Immunobiology: The Immune System in Health & Disease, 5th edition, Janeway et al., Eds., 2001, ISBM 0-8153- 3642-X, Figure 9.30, on page 371) and are not intended to be limiting with respect to the multitude of

30 receptor signaling cascades that can be regulated with the prodrugs described herein.

"Degranulation with Fc Receiver Mediation" or "Degranulation Induced by the Fc Receiver" refers to degranulation that takes place through a signal transduction cascade of the Fc receiver initiated by

35 crosslinking medium of an Fc receptor.

"IgE induced degranulation" or "FcεRI Mediated Degranulation" refers to degranulation that takes place by means of an IgE receptor signal transduction cascade initiated by means of IgE linked FcεRI crosslinking. Crosslinking can be induced by means of a specific IgE allergen or other multivalent binding agent, such as an anti-IgE antibody. In mast cells and / or basophilic cells, the FcεRI signaling cascade that leads to degranulation can be separated into two stages: upstream and downstream. The upstream stage includes all the processes that take place before the mobilization of the calcium ion. The downstream stage includes the mobilization of calcium ion and all its downstream processes. Compounds that inhibit degranulation with FcεRI mediation can act at any point along the signal transduction cascade with FcεRI mediation. Compounds that selectively inhibit degranulation with upstream FcεRI mediation act to inhibit that part of the FcεRI signaling cascade upstream from the point where calcium ion mobilization is induced. In cell-based assays, compounds that selectively inhibit degranulation with upstream FcεRI mediation inhibit degranulation of cells such as mast cells or basophilic cells that are activated or stimulated with a specific IgE allergen or binding agent (such as

fifty an anti-IgE antibody) but do not appreciably inhibit the degranulation of cells that are activated or stimulated with degranulate agents that cause the deviation of the FcεRI signaling mechanism, such as, for example, calcium ionophore ionomycin and A23187.

"IgG Induced Degranulation" or "FcγRI Mediated Degranulation" refers to degranulate that

55 it passes through the FcγRI signal transduction cascade initiated by crosslinking FcγRI bound to IgG. Crosslinking can be induced by means of a specific IgG allergen or other multivalent binding agent, such as an anti-IgG or antibody fragment. Like the FcεRI signaling cascade, in mast cells and basophilic cells, the FcγRI signaling cascade also leads to degranulation, which can be separated into two stages; upstream and downstream. Similar to degranulation with FcεRI mediation,

60 Compounds that selectively inhibit degranulation with FcγRI mediation upstream act upstream from the point where calcium ion mobilization is induced. In cell-based assays, Compounds that selectively inhibit degranulation with upstream FcγRI mediation inhibit degranulation of cells such as mast cells or basophilic cells that are activated or stimulated with a specific IgG allergen or binding agent (such as an anti-IgG antibody or fragment ) but do not appreciably inhibit the degranulation of cells that are activated or stimulated with degranulate agents that deviate the FcγRI signaling mechanism, such as, for example ionomycin of calcium ionophores and A23187.

"Ionophore-induced degranulation" or "Ionophore-mediated degranulation" refers to degranulation of a cell, such as a mast cell or basophilic cell, which occurs after exposure to a calcium ionophore, such as, for example, ionomycin or A23187 .

"Syk kinase" refers to the well-known 72 kDA splenic protein tyrosine kinase that is not well-known (cytoplasmic) receptor, which is expressed in B cells and other hematopoietic cells. Syk kinase includes two tandem consensus 2 (SH2) Src homology domains that bind to activation motifs based on phosphorylated immunoreceptor tyrosine ("ITAM"), a "binding" domain and a catalytic domain (for a review of the structure and function of Syk kinase, see Sada et al., 2001, J. Biochem (Tokyo) 130: 177-186); see also Turner et al., Immunology Today 21: 148-154). Syk kinase has been extensively studied as an effector of B cell receptor signaling (BCR) (Turner et al., 2000, supra). Syk kinase is also critical for the

fifteen Tyrosine phosphorylation of multiple proteins that regulate important mechanisms that start with imunoreceptors, such as mobilization of Ca2 + and degranulation and cascades of mitogen-activated protein kinase (MAPK). Syk kinase also plays an important role in integrin signaling in neutrophils (see, for example, Mocsai et al., 2002, Immunity 16: 547-558).

As used herein, Syk kinase includes kinases from any species of animal, including but not limited to, Homo sapiens, apes, cattle, pigs, rodents, etc., recognized as belonging to the Syk family. Specifically, isoforms, cutting variants, allelic variants, mutants, both natural and man-made, are included. The amino acid sequences of said Syks kinases are well known and available from GENBANK. Specific examples of mRNA encoding

25 different human Syk kinase isoforms can be found in GENBANK Accession No. gi⏐21361552⏐refNM_003177.2⏐, gi⏐496899⏐emb⏐Z29630.1⏐HSSYKPTK [496899] and gi⏐15030258⏐gb⏐BC011399.1⏐ BC011399 [15030258].

Skilled workers will appreciate that tyrosine kinases that belong to other families may have active sites or hollow junctions that are similar in three-dimensional structure to that of Syk. As a consequence of this structural similarity, said kinases, referred to herein as "Syk mimetics", are expected to catalyze phosphorylation of phosphorylated substrates by means of Syk. Thus, it will be appreciated that said Syk mimetic signal transduction cascades, in which said Syk mimetics play a role, the biological responses carried out by said Syk mimetics and the signaling cascades that depend

35 Syk mimics can be regulated and in particular can be inhibited, with many of the drugs described herein.

"Signaling Cascade that Depends on Syk" refers to a signal transduction cascade in which Syk kinase plays a role. Non-limiting examples of such Syk-dependent signaling cascades include FcαRI, FcεEI, FcγRI, FcγRIII and BCR and integrin signaling cascades.

"Autoimmune disease" refers to those diseases that are commonly associated with non-anaphylactic hypersensitivity reactions (type II, type III and / or type IV hypersensitivity reactions) that generally result in a consequence of the cell-mediated immune response and / or own humoral

Four. Five subject, against one or more immunogenic substances of endogenous and / or exogenous origin. Such autoimmune diseases are distinguished from diseases associated with anaphylactic hypersensitivity reactions (type I or with IgE mediation).

"Prodrug" refers to a derivative of an active compound of 2,4-pyrimidinediamine (drug) that requires a transformation under conditions of use, such as within the body, to release the active drug 2,4-pyrimidinediamine

or one of its active metabolites. Frequently, but not necessarily, prodrugs are pharmacologically inactive until they become the active drug. Normally, prodrugs are obtained by masking one or more functional groups in the 2,4-pyrimidinediamine drug which are thought to be partly necessary for activity with a progroup (defined below) to form

55 a prorate that undergoes a transformation, such as cleavage, under specific conditions of use in order to release the functional group and also the active drug 2,4-pyrimidinediamine. The cleavage of the prorate may occur spontaneously, such as by means of a hydrolysis reaction, or it may be catalyzed or induced by another agent, such as by means of an enzyme, by light, by an acid or a base, or by means of a change or exposure to a physical or environmental parameter, such as a change in temperature. The agent may be endogenous to the conditions of use, such as an enzyme present in the cells in which the prodrug or stomach acid conditions are administered, or it may be delivered exogenously.

A wide variety of progroups, as well as the resulting pro-forms, suitable for masking functional groups in active 2,4-pyrimidinediamine compounds to give rise to prodrugs, is well known in the art. For example, a hydroxy functional group can be masked in the form of sulfonate, ester ester

or carbonate, which can be hydrolyzed in vivo to provide the hydroxyl group. An amino functional group can be masked in the form of a prorated amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenyl, which can be hydrolyzed in vivo to provide the amino group. A carboxyl group can be masked in the form of ester pro-salt (including silyl esters and thioesters), amide or hydrazide, which can be hydrolyzed in vivo to provide the carboxyl group. The nitrogen protecting groups and the nitrogen pro-drugs of the invention

5 they can include lower alkyl groups as well as amides, carbamates, etc. Other specific examples of appropriate progroups and their respective pro-forms will be apparent to those skilled in the art.

"Progroup" refers to a type of protective group that, when used to mask a functional group within an active 2,4-pyrimidinediamine drug to form a prorated, convert the drug into a prodrug. Normally, the progroups are linked to the functional group of the drug through links that can be cleaved under the specific conditions of use. Thus, a progroup is that part of a proortion that is cleaved to release the functional group under specific conditions of use. As a specific example, an amide proportion of formula NH-C (O) CH3 comprises the progroup -C (O) CH3.

fifteen Accordingly, the present invention relates to a compound according to formula I:

<dl><dt /><dd>or one of its stereoisomers, salts, hydrates, solvates or N-oxides, in which: Y is selected from the group consisting of S, O, SO, SO2 and C (R7) 2; each R35 is independently selected from the group consisting of hydrogen, (C1-C4) alkyl and halo, </dd></dl>

<dl><dt /><dd>or both R35 together with the carbon to which they are attached form a carbonyl group; </dd></dl>

25 W is selected from the group consisting of C = O, C = S, C = NH, C (R7) 2 and NR37;

Z is C = O or NR37, with the proviso that Z and W are not both NR37 and with the proviso that when Z is C = O, W is C (R7) 2;

X is CH or N;

each R31 is independently (C1-C4) alkyl or both R31 together form an (C1-C2) alkylene group optionally substituted with one to two (C1-C4) alkyl groups or substituted with a spirocycloalkyl (C3-C7) group; Each R7 is independently hydrogen or (C1-C4) alkyl; and

R37

it is hydrogen or methyl optionally substituted with phenyl or pyridyl, wherein said phenyl or pyridyl is optionally substituted with (C1-C4) alkoxy. In one embodiment, Y is O or S. In some aspects, Y is O. In another embodiment, Y is S or an oxidized form of S, such as a sulfoxide SO or a sulfone SO2.

Four. Five In another embodiment, Y is C (R7) 2. In some aspects, Y is C (Me) 2. In one embodiment, both R35 are the same. In some aspects, both R35 are methyl. In other aspects both R35 are hydrogen or both are fluoro. In another aspect, both R35 together with the carbon to which they are attached form a carbonyl group.

In one embodiment, W is C = O or C = S. In some aspects, W is C = O. In another embodiment, W is C (R7) 2. In some aspects, W is CH2. 55 In another embodiment, W is NR37. In some aspects, W is NH.

In one embodiment, Z is NR37. In some aspects, R37 is hydrogen so that Z is NH. In other aspects, R37 it is methyl, 2-pyridylmethyl or 4-methoxybenzyl. In another embodiment, Z is C = O.

In one embodiment, X is N.

In one embodiment each R31 is independently (C1-C2) alkyl. In some aspects, both R31 are 5 methyl.

In another embodiment, both R31 together form an (C1-C2) alkene group optionally substituted with one to two (C1-C4) alkyl groups or substituted with a spirocycloalkyl (C3-C7) group. In some embodiments, both R31 combine to form an acetal or a ketal carbon. When both R31 together form an alkylene group (C1-C2),

10 a bicyclic fused ring system is formed with the phenyl group bearing the OR31 groups. In some embodiments, the bicyclic fused ring system includes the following structures in which each R is (C1-C4) alkyl or two R combine to form a spirocycloalkyl (C3-C7):

In yet another embodiment, the present invention relates to a compound or one of its stereoisomers, salts, hydrates, solvates or N-oxides selected from the group consisting of 5-fluoro-N2- (3-hydroxy-4,5 -dimethoxyphenyl) -N4- [3-oxo-benzo [1,4] thiazin-6-yl] -2,4-pyrimidinediamine;

N4- [2,2-dimethyl-3-oxo-benzo [1,4] thiazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2,4-pyrimidinediamine;

25 N4- [2,2-dimethyl-3-oxo-benz [1,4] oxazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2,4-pyrimidinediamine;

N4- [2,2-dimethyl-3-oxo-pyrid [1,4] oxazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2,4-pyrimidinediamine;

5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [3-oxy-benz [1,4] oxazin-6-yl] -2,4-pyrimidinediamine; N4- [2,2-Difluoro-3-oxo-benz [1,4] oxazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2,4-pyrimidinediamine ;

5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [3-oxo-4- (2-pyridylmethyl) -benz [1,4] oxazin-6-yl] -2,4- pyrimidinediamine;

35 N4- (3,4-dihydro-2H-2,2-dimethyl-5-pyrido [1,4] oxazin-6-yl) -N2- [3,4-dimethoxy-5-hydroxyphenyl] -5-fluoro- 2,4-pyrimidinediamine

5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- (1,3- (2H) -4,4-dimethylisoquinolindione-7-yl) -2,4-pyrimidinediamine;

(R / S) -5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [2-methyl-3-oxo-4- (4-methoxybenzyl) -benz [1,4] oxazin -6-yl] -2,440 pyrimidinediamine;

(R / S) -5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [2-methyl-3-oxo-4- (4-methoxybenzyl) -benzo [1,4] thiazin -6-yl] -2,4 pyrimidinediamine;

Four. Five 5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- (2,2,4-trimethyl-1,1,3-trioxy-benzo [1,4] thiazin-6-yl) - 2,4-pyrimidinediamine; and

5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- (4-methyl-3-oxo-benzo [1,4] thiazin-6-yl) -2,4-pyrimidinediamine.

Those skilled in the art will appreciate that the 3-hydroxyphenyl-2,4-pyrimidinediamine compounds described in the

fifty This report may include functional groups that may be masked with progroups in order to create prodrugs. Normally, but not necessarily, said prodrugs are pharmacologically inactive until they become their active drug form. For example, ester groups commonly undergo acid-catalyzed hydrolysis to give rise to parental carboxylic acid when exposed to acidic stomach conditions, or base-catalyzed hydrolysis when exposed to the basic conditions of the intestine or

55 the blood. Thus, when administered to a subject orally, 3-hydroxyphenyl-2,4-pyrimidinediamines that include ester moieties can be considered prodrugs of their corresponding carboxylic acid, regardless of whether the ester form is pharmacologically active.

In prodrugs related to the compounds of the invention, any functional moiety available with a progroup can be masked to give rise to a prodrug. In the art a multitude of

5 appropriate groups for masking said functional groups in order to give rise to pro-formations that can be cleaved under the desired conditions of use. All these progroups alone or in combinations can be included in prodrugs related to the compounds of the invention.

As described herein, a prodrug related to the compounds of the invention may be a prodrug having the formula II

in which:

fifteen W is selected from the group consisting of C = O, C = S, C = NH, (CR7) 2 and NP1;

Z is selected from the group consisting of C = O, NR37 and NP1; with the proviso that when Z is C = O, then W is C (R7) 2 or NP1 and with the proviso that W and Z are not both NR37 or NP1;

twenty Y, R35, X and R31 are as previously defined for formula I; and

P1, P2, P3 and P4 are independently hydrogen or an Rp progroup, provided that at least one of P1, P2, P3 and P4 is a progroup.

25 In some prodrugs of formula II, P3 is an Rp progroup, in which P3 and the oxygen to which it is attached form a pro-ester ester, thioester, carbonate or carbamate. In some respects, the pro-output -OP3 is an ester.

In some prodrugs of formula II, the 2-amino, 4-amino or Z = N group is linked to an Rp progroup. In some 30 aspects, Z is N-Rp.

In some of the aforementioned prodrugs, the Rp progroup is a phosphorus-containing progroup.

In some of the aforementioned prodrugs, the Rp progroup includes a group or moiety that

35 It metabolizes under the conditions of use to give rise to an unstable intermediate of α-hydroxymethyl, α-aminomethyl or αthiomethyl, which is subsequently further metabolized in vivo to give rise to the active drug of 3-hydroxyphenyl2,4-pyrimidinediamine. In some prodrugs, the progroup includes an α-hydroxyalkyl, α-aminoalkyl or αthioalkyl moiety, for example, an α-hydroxymethyl, α-aminomethyl or α-thiomethyl moiety, which is metabolized under the conditions of use to give rise to the 3-hydroxyphenyl-2,4-pyrimidinediamine active drug. For example, in some

40 prodrugs the progroup Rp is of the formula -CRdRd-AR3, in which each Rd is independently selected from another, from hydrogen, cyano, optionally substituted (C1-C20) alkyl, perfluoroalkyl (C1-C20), arylalkyl (C7 -C30) optionally substituted and optionally substituted 6-30 membered heteroaryl alkyl, wherein each optional substituent, independently of the others, is selected from hydrogen, alkyl, aryl, arylalkyl, heteroaryl and heteroalkyl, or, alternatively, the two Rd are taken together with the carbon atom to which

Four. Five they are joined to form a cycloalkyl containing from 3 to 8 carbon atoms; A is selected from O, S and NR50, in which R50 is selected from hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl and cycloheteroalkyl, or alternatively it is combined with R3 and together with the nitrogen to which they are attached, they form a ring three to seven members and R3 represents a group that can be metabolized in vivo to give rise to a group of formula -CRdRd-AH, in which Rd and A are as defined above.

fifty The identity of R3 is not critical, with the proviso that it can be metabolized under the desired conditions of use, for example in the acidic conditions found in the stomach and / or by means of enzymes found in vivo, to give rise to a group of formula -CRdRd-AH, in which A and Rd are as defined above. In this way, skilled workers will appreciate that R3 can comprise virtually any

55 thiol, amine or hydroxyl protecting group known or to be discovered. Non-limiting examples of appropriate protecting groups can be found, for example, in Protective Groups in Organic Synthesis, Greene &amp; Wuts, 2nd ed., John Wiley &amp; Sons, New York, 1991 (especially pages 10-42 (alcohols, 277- 308 (thiols) and 309-405 (amines)).

In the specific prodrugs, R3 includes, together with A, an ether bond, a thioether bond, a silyl ether bond, a silyl thioether bond, an ester bond, a thioester bond, an amide bond, a carbonate bond, a bond thiocarbonate, a carbamate bond, a thiocarbamate bond or a urea bond, -OCH2SO3R, wherein R is hydrogen, alkyl, aryl, arylalkyl or a metal salt (eg, sodium, lithium, potassium); -GCH2 + N (R51) 3M-, in which

5 G is absent or is -OPO3-, OSO3- or -CO2-, R51 is hydrogen, alkyl, aryl, arylalkyl, cycloheteroalkyl or cycloheteroalkylalkyl and M - is a counterion, usually a halide or the like (acetate, sulfate, phosphate, etc. ). As described herein, specific examples include, but are not limited to, Rp progroups in which R3 is selected from Rf, -C (O) Rf, -C (O) ORf, -C (O) NRfRf and -SiRfRfRf in which each Rf, independently of each other, is selected from hydrogen, optionally substituted lower alkyl, optionally substituted lower heteroalkyl, optionally substituted lower cycloalkyl, optionally substituted lower heterocycloalkyl, optionally substituted aryl (C6-C10), optionally substituted 5-10 membered heteroaryl, optionally substituted aryl (C7-C18) aryl, and optionally substituted 6-18 membered heteroarylalkyl. In a specific embodiment, each Rf is the same.

fifteen The identity of the Rp progroup (s) can be selected to adapt the water solubility and other properties of the active underlying compound of 3-hydroxyphenyl-2,4-pyrimidinediamine, to achieve optimization for a particular mode of administration. It can also be selected to be provided with a view to removal in specific organs and / or tissues inside the body, such as, for example, in the digestive tract, blood and / or serum, or through enzymes residing in specific organs. , such as the liver.

In some prodrugs, Rp progroups that are phosphorus-containing progroups include phosphate residues that can be cleaved in vitro by enzymes such as esterases, lipases and / or phosphatases. These enzymes are dominant throughout the body, residing, for example, in the stomach and digestive tract, blood and / or serum and in almost all tissues and organs. Such Rp progroups containing phosphate generally increase solubility.

25 in water of the underlying active 3-hydroxyphenyl-2,4-pyrimidinediamine compound, making said phosphate-containing prodrugs ideally suited to modes of administration in which water solubility is desirable, such as, for example, modes of oral, oral, intravenous, intramuscular and ocular administration.

In some prodrugs, each Rp progroup containing prodrug phosphate is of the formula - (CRdRd) and OP (O) (OH) (OH), or one of its salts, in which Rd is as previously defined e and is n number integer that varies from 1 to 3, usually 1 or 2. In a specific example described herein, each Rd, independently of the others, is selected from hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted methyl, and substituted or unsubstituted benzyl. In another specific example described herein, each Rd, independently of the others, is selected from

35 hydrogen and unsubstituted lower alkyl. Rp groups containing specific exemplary phosphate include -CH2-OP (O) (OH (OH) and -CH2CH2-OP (O) (OH) (OH) and / or the corresponding salts.

Without claiming to adhere to any theory of operation, when and is 1 in the Rp progroups containing exemplary phosphate, it is believed that prodrugs containing phosphate are converted in vivo by enzymes such as phosphatases, lipases and / or esterases in the corresponding ones. hydroxymethylamines, which are subsequently metabolized in vivo by means of formaldehyde removal to give rise to the active drug compound of 2,4-pyrimidinediamine. The metabolic by-products of phosphate and formaldehyde are harmless.

When y is 2 in prodrugs containing exemplary phosphate, it is believed that prodrugs are metabolized

Four. Five to the active drug compound of 3-hydroxyphenyl-2,4-pyrimidinediamine in vivo by elimination of enol phosphate, which is subsequently metabolized to acetaldehyde and phosphate. The metabolic by-products of phosphate and acetaldehyde are harmless.

Skilled workers will appreciate that certain types of precursors can be converted in vivo into phosphate groups. Such precursors include, by way of example and not limitation, phosphate esters, phosphites and phosphite esters. For example, phosphites can be oxidized in vivo to phosphates. Phosphate esters can be hydrolyzed in vivo to phosphates. Phosphite esters can be oxidized in vivo to phosphate esters, which in turn can be hydrolyzed in vivo to phosphates. As a consequence of the ability of these phosphate precursor groups to convert into phosphates in vivo, prodrugs may also include progroups comprising said phosphates.

55 phosphate precursors. In some prodrugs, phosphate precursor groups can be metabolized directly to the active 2,4-pyrimidinediamine drug, without first becoming a phosphate prodrug. In other prodrugs, prodrugs comprising progroups that include said phosphate precursors are first metabolized to give rise to the corresponding phosphate prodrug, which is subsequently metabolized to the active 3-hydroxyphenyl-2,4-pyrimidinediamine drug by means of a hydroxymethylamine, as discussed above.

In some prodrugs, said phosphate precursor groups are phosphate esters. Phosphate esters can be acyclic or cyclic and can be phosphate tri esters or phosphate diesters. Generally, said esters are less soluble in water than the corresponding acid prodrugs of phosphate and the corresponding active compounds of 3-hydroxyphenyl-2,4-pyrimidinediamine and therefore are normally appropriate for the modes of prodrug administration of the active compounds of 3-hydroxyphenyl-2,4-pyrimidinediamine in which

Desires low water solubility, including, by way of example and without limitation, administration by inhalation. The solubility of the prodrug can be specifically adapted to specific modes of administration by appropriate selection of the number and identity (s) of the esterification groups of the phosphate ester.

5 The mechanism can be controlled by means of which the phosphate ester group is metabolized to the corresponding phosphate group, by appropriate selection of the esterification moieties. For example, it is well known that certain esters are labile against acids (or bases), generating the corresponding phosphate in the acidic conditions found in the stomach and digestive tract. In cases where

10 it is desirable that the phosphate ester prodrug be metabolized to the corresponding phosphate prodrug in the di tract digestive (such as, for example, when the prodrugs are administered orally), it is possible to select the phosphate ester progroups that are labile against acids. Other types of phosphate esters are stable against acids and bases, becoming the corresponding phosphates by means of enzymes found in certain tissues and organs of the body (see, for example, the different phosphate esters

fifteen cyclics described in Erion et al., 2004, J. Am. Chem. Soc. 126: 5154-5163). In cases where it is desirable to convert a phosphate ester prodrug into the corresponding phosphate prodrug into a desired target tissue or site within the body, phosphate esters having desired metabolic properties can be selected.

twenty In some prodrugs, each Rp group containing phosphate ester of the prodrug is an acyclic phosphate ester of the formula - (CRdRd) andOP- (O) (OH) (ORe) or - (CRdRd) and OP) O) (ORe) ( ORe), or one of its salts, in which each Re, independently of others, is selected from substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl (C6-C14) (eg, phenyl, naphthyl, 4 -alkoxyphenyl lower, 4-methoxyphenyl), substituted or unsubstituted (C7-C20) arialkyl (eg, benzyl, 1-phenylethane-1-yl, 2-phenylethane-1-yl), - (CRdRd) and-ORf- (CRdRd) andOC (O) Rf

25 - (CRdRd) and CO (O) ORf, - (CRdRd) and SC (O) Rf; - (CRdRd) and SC (O) ORf, - (CRdRd) and-NH-C (O) Rf, - (CRdRd) and- (NH-C (O) ORf and Si (Rd) 3, where Rd, Rf and y are as defined above In a specific example described herein, each R is selected from hydrogen and unsubstituted lower alkyl and / or each Re is an unsubstituted lower alkanyl or benzyl. As discussed above, specific exemplary phosphate ester groups include, but are not limited to, -CH2-OP (O) (OH) (ORe) -CH2CH2-OP (O) (OH) (ORe), -CH2 -OP (O) (ORe) and

30 CH2CH2-OP (O) (ORe) (ORe) n, in which Re is selected from lower alkanyl, i-propyl and t-butyl.

In other prodrugs, each Rp progroup containing phosphate ester is a cyclic phosphate ester of the formula

35 wherein each Rg, independently of the others, is selected from hydrogen and lower alkyl; each Rh, independently of the others, is selected from hydrogen, substituted or unsubstituted lower alkyl, substituted or unsubstituted lower cycloheteroalkyl, substituted or unsubstituted aryl (C6-C14), substituted or unsubstituted arylalkyl (C7-C20) and 5-14 membered substituted or unsubstituted heteroaryl; z is an integer

40 which varies from 0 to 2; and Rd ey are as defined above. In a specific example described herein, each Rp group containing phosphate ester is a cyclic phosphate ester of the formula

Four. Five in which Rd, Rh and y are as defined above.

The mechanism by which cyclic phosphate ester prodrugs that include said cyclic phosphate ester progroups metabolize in vivo to give the active drug compound depends, in part, on the identity of the Rh substituent. For example, the cyclic phosphate ester progroups in which each Rh, so

fifty independent of the others, it is selected from hydrogen and lower alkyl are cleaved in vivo by means of esterases. Thus, in some prodrugs, the cyclic phosphate ester progroups are selected so that they are cleaved in vivo by means of esterases. As described herein, specific examples of said cyclic phosphate ester progroups include, but are not limited to, progroups selected from

55 Alternatively, cyclic phosphate ester prodrugs having progroups in which the substituents

Rh are substituted or unsubstituted aryl, arylalkyl and heteroaryl groups, they are not normally cleaved by means of

esterases, but instead are metabolized to the active prodrug by enzymes, such as 10 cytochrome P450 enzymes, which reside in the liver. For example, a series of ester nucleotide prodrugs

of cyclic phosphate undergoing an oxidative cleavage reaction catalyzed by a cytochrome P450 enzyme

(CYP) expressed predominantly in the liver is described in Erion et al., 2004, J. Am. Chem. Soc. 126:

5154-5163. In some prodrugs, the cyclic phosphate ester groups are selected so that

they can be cleaved by CYP enzymes expressed in the liver. As described herein, embodiments of specific examples of said Rp progroups containing cyclic phosphate ester

include, but not limited to, formula groups

twenty wherein Rh is selected from phenyl, 3-chlorophenyl, 4-pyridyl and 4-methoxyphenyl.

As skilled workers will appreciate, phosphites and phosphite esters can undergo oxidation in vivo to give rise to the corresponding phosphate and phosphate ester analogs. Such reactions can be carried out in vivo, for example, by oxidase enzymes, oxidoreductases enzymes and other oxidative enzymes. 25 Thus, the Rp phosphorus-containing progroups can also include phosphite and phosphite ester analogs of any of the phosphate and phosphate ester progroups described above. In some prodrugs the Rp phosphorus-containing progroups include, but are not limited to, groups of the formula - (CRdRd) and OP- (OH) (OH), - (CRdRd) and P (OH) (ORe) and - (CRdRd) and OP (ORe) (Re) or its salts, in which Rd, Re ey are as defined above. As described herein, specific examples include groups in which each Rd, independently of the others, is selected from hydrogen and unsubstituted lower alkyl and / or each Re, independently of the others, is selected from non-lower alkanyl. substituted and benzyl. As described herein, specific examples of phosphite and phosphite ester progroups include, but are not limited to, -CH2-OO (OH) (OH), -CH2CH2-OP (OH) (OH), - CH2-OP (OH) (ORe) and -CH2CH2-OP (ORe) (ORe), in which each Re is selected from lower alkanyl, i-propyl and t-butyl. As previously mentioned,

35 Specific exemplary cyclic phosphite ester prodrugs include phosphite analogs of the cyclic phosphate ester progroups described above. Conceptually, prodrug compounds that include said phosphite and / or phosphite ester groups can be interpreted as prodrugs of the corresponding phosphate and phosphate ester prodrugs.

40 As mentioned above, it is believed that certain phosphate-containing prodrugs are metabolized in vivo through the corresponding hydroxymethylamine. Although these hydroxymethylamines are metabolized in vivo to the corresponding active compounds of 3-hydroxyphenyl-2,4-pyrimidinediamines, they are stable at pH 7 and can be prepared and administered as prodrugs containing hydroxyalkyl. In some prodrugs, each Rp progroup containing hydroxyalkyl of said prodrugs has the formula -CRdRd-OH, in which Rd is as it is

Four. Five You have previously defined. An Rp progroup containing specific exemplary hydroxyalkyl is -CH2OH.

In an example described herein, Rp has the formula - (CRdRd) and OP (O) (OH) 2, or one of its salts, wherein y is an integer ranging from 1 to 3; each Rd, independently of the others, is selected from hydrogen, optionally substituted lower alkyl, optionally substituted aryl (C6-C14) and optionally substituted aryalkyl (C7C20); wherein the optional substituents, independently of each other, are selected from hydroxyl, lower alkoxy, aryloxy (C6-C14), lower alkoxyalkyl, methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl and halogen, or, alternatively, two Rd are taken attached to the same carbon atom together with the carbon atom to which they are attached to form a cycloalkyl group containing 3 to 8 carbon atoms.

In an example described herein, Rp is selected from -CH2-OP (O) (OH) 2 and -CH2CH2-OP (O) (OH) 2 and its salts.

In an example described herein, Rp comprises a phosphate ester group.

In an example described herein, Rp is selected from - (CRdRd) and OP (O) (ORe) (OH), - (CRdRd) and O-P (O) (ORe) (ORe),

and its salts, wherein each Re, independently of the others, is selected from substituted or unsubstituted lower alkyl, substituted or unsubstituted aryl (C6-C14) (eg, phenyl, naphthyl, 4-lower alkoxyphenyl, 4methoxyphenyl) , substituted or unsubstituted (C7-C20) arylalkyl (for example, benzyl, 1-phenylethane-1-yl, 2-phenylethane-1-yl), (CRdRd) and-ORf, - (CRdRd) andOC (O) Rf , - (CRdRd) and OC (O) ORf, - (CRdRd) and SC (O) Rf, - (CRdRd) and SC (O) ORf, - (CRdRd) and-NHC (O) Rf, - (CRdRd) and-NH-C (O) ORf and -Si (Rd) 3, in which each Rf, independently of the others, is selected from hydrogen, substituted or unsubstituted lower alkyl, aryl (C6-C14) substituted or unsubstituted and aryalkyl (C7-C20) substituted or unsubstituted; each Rg, independently of the others, is selected from hydrogen and lower alkyl; each Rh, independently of the others, is selected from hydrogen, lower alkyl

25 substituted or unsubstituted, substituted or unsubstituted lower cycloheteroalkyl, substituted or unsubstituted aryl (C6-C14), substituted or unsubstituted arial (C7-C20) and substituted or unsubstituted 5-14 membered heteroaryl; z is an integer that varies from 0 to 2; and Rd ey are as defined above.

In an example described herein, Rp is selected from -CH2-OP (O) (OH) 2, -CH2CH2-OP ((OH) 2, -CH2OH and its salts. In some of these aspects Z is N- Rp.

In another embodiment, the present invention provides a composition comprising a compound of formula I and a vehicle, excipient or diluent. A composition comprising a compound of formula II and a vehicle, excipient or diluent is also described herein.

35 Those skilled in the art will appreciate that many of the compounds of the invention and their prodrugs; as well as the various species of compounds specifically described and / or illustrated herein, may exhibit the phenomenon of tautomerism, conformational isomerism, geometric isomerism and / or optical isomerism. For example, the compounds of the invention and their prodrugs may include one or more chiral centers and / or double bonds and as a consequence, they may exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers and diastereoisomers and mixtures thereof, such as racemic mixtures. By way of another example, the compounds of the invention and their prodrugs can exist in various tautomeric forms, including the enol form, the keto form and mixtures thereof. Because the different names of compounds, formulas and drawings of the compounds of the specification and claims

Four. Five they may represent only one of the possible tautomeric, conformational isomeric, optical isomeric or geometric isomeric forms, it should be understood that the invention encompasses any tautomeric, conformational isomeric, optical isomeric and / or geometric isomeric forms of the compounds or prodrugs having one or more of the utilities described herein, as well as the mixtures of these different isomeric forms. In cases of limited rotation around the 2,4-pyrimidinediamine core structure, atropisomers are also possible and are also specifically included in the compounds of the invention.

In addition, skilled workers will appreciate that when the lists of alternative substituents include members in which, due to valence requirements or other reasons, they cannot be used to replace a particular group,

55 the list is intended to be read in context to include those list members that are appropriate to replace the particular group. For example, skilled workers will appreciate that although all the alternatives listed for Rb can be used in order to replace an alkyl group, certain alternatives, such as = 0, cannot be used to replace a phenyl group. It should be understood that only the possible substituent-group combinations are intended.

The compounds of the invention and / or their prodrugs can be identified by any of their chemical structures or their chemical name. When the chemical structure and chemical name conflict, the chemical structure determines the identity of the specific compound.

Depending on the nature of the different substituents, the 2,4-pyrimidinediamine compounds of the invention and their prodrugs may be in the form of salts. Said salts include salts suitable for pharmaceutical uses ("pharmaceutically acceptable salts"), salts suitable for veterinary uses, etc. Such salts may come from acids or bases, as is well known in the art.

In one embodiment, the salt is a pharmaceutically acceptable salt. Generally, pharmaceutically acceptable salts are those salts that substantially retain one or more of the desired pharmacological activities of the parent compound and that are suitable for administration to humans. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids or organic acids. Inorganic acids suitable for the formation of pharmaceutically acceptable acid addition salts include, by way of example and not limitation, hydrohalide acids (for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid , phosphoric acid and the like. Suitable organic acids for the formation of pharmaceutically acceptable acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentapropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (for example, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane disulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (for example, benzenesulfonic acid, 4-chlorobenzenesulfonic acid , 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, cycloalkyl sulfonic acids (for example, camphorsulfonic acid), 4-methylbicyclo [2.2.2] -oct-2en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, acid

25 Muconic and the like.

Pharmaceutically acceptable salts also include salts formed when an acidic proton present in the parent compound is substituted by a metal ion (for example, an alkali metal ion, an alkaline earth metal ion or an aluminum ion), an ammonium ion or coordinates with an organic base (for example,

30 ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine, etc.).

The 2,4-pyrimidinediamine compounds of the invention, as well as their salts, can also be in the form of hydrates, solvates and N-oxides, as is well known in the art. In one implementation, the present invention provides a compound, or stereoisomer, tautomer, solvate or a pharmaceutically salt thereof.

35 acceptable, selected from Table I.

<dl><dt>Table I </dt><dd /></dl>

<dl><dt>Compound No. </dt><dd>AND R35 W Z X R31 </dd></dl>

<dl><dt>R909384 </dt><dd>S H H C = O NH CH Me </dd></dl>

<dl><dt>R909385 </dt><dd>S Me C = O NH CH Me </dd></dl>

<dl><dt>R909390 </dt><dd>OR Me C = O NH CH Me </dd></dl>

<dl><dt>R909391 </dt><dd>OR Me C = O NH N Me </dd></dl>

<dl><dt>R909402 </dt><dd>OR H H C = O NH CH Me </dd></dl>

<dl><dt>R909403 </dt><dd>OR C = O NH CH Me </dd></dl>

<dl><dt>R909404 </dt><dd>OR F, F C = O NH CH Me </dd></dl>

<dl><dt>R909406 </dt><dd>OR H H C = O CH Me </dd></dl>

<dl><dt>R909407 </dt><dd>OR H H, C = O N-CH2C = N CH Me </dd></dl>

<dl><dt>R909408 </dt><dd>S H H, C = O N-CH2C = N CH Me </dd></dl>

<dl><dt>R935879 </dt><dd>OR H H CH2 NH N Me </dd></dl>

<dl><dt>R909414 </dt><dd>C (CH3) 2 = O NH C = O CH Me </dd></dl>

<dl><dt>R909415 </dt><dd>OR Me, h C = O CH Me </dd></dl>

<dl><dt>R909416 </dt><dd>S Me, h C = O CH Me </dd></dl>

<dl><dt>R909417 </dt><dd>SO2 Me C = O N-Me CH Me </dd></dl>

<dl><dt>R909418 </dt><dd>S H H C = O N-Me CH Me </dd></dl>

Synthesis methods

The compounds of the invention and their prodrugs can be synthesized by means of a variety of different

5 synthetic routes, using commercially available starting materials and / or starting materials prepared by means of conventional synthetic methods. Appropriate exemplary methods that can be routinely adapted to synthesize the 2,4-pyrimidinediamine compounds of the invention and their prodrugs are found in US Pat. No. 5,958,935, U.S. Patent Application 10 / 355,543 filed January 31, 2003 (U.S. publication US 20040029902 A1), WO 03/063794, published August 1, 2003,

10 U.S. patent application 10 / 631,029 filed on July 29, 2003 and WO 2004/014382 published on February 19, 2004 and U.S. Patent Application. Serial No. 10 / 903,870 filed July 30, 2004. All compounds of structural formulas (I) and (II) can be prepared by routine adaptation of these methods.

fifteen A variety of exemplary synthetic routes that can be used to synthesize the 3-hydroxy-2,4-pyrimidinediamine compounds of the invention is described in Schemes (I) - (II), below. These methods can be routinely adapted to synthesize prodrugs according to structural formulas (III) and (IV).

In one example, the compounds can be synthesized from substituted or unsubstituted uracils or thiouracils 20 as illustrated in Scheme (I) below:

Scheme (I)

25 In Scheme (I), R35, R31, Y, W, Z and X are as defined above for structural formula (I), X 'is a halogen (for example, F, Cl, Br or I) and each of Q and Q ', independently of each other, is selected from the group consisting of O and S. Referring to Scheme (I), uracil or thiouracil 2 is dihalogenated at positions 2- and 4- using an agent of conventional halogenation PXO3 (or other halogenating agent

30 conventional) under conventional conditions to give rise to 2,4-bishalo pyrimidine 4. Normally, in pyrimidine 4, the halide of the C4 position is more reactive towards nucleophiles than the halide of the C2 position. The differential reactivity to synthesize 2,4-pyridinediamines according to structural formula (I) can be explained by first reacting 2,4-bishalopyrimidine 4 with an equivalent of amine 10, resulting in 2-halo-4-pyrimidinamine 8 with 4N substitution, followed by an amine 6 to give a 2,4-pyridinediamine according to structural formula (I).

Normally, the C4 halide is more reactive towards nucleophiles, as illustrated in the Scheme. However, as the skilled workers will recognize, the regioselectivity of the reaction can be controlled by adjusting the solvent and other synthesis conditions (such as temperature), as is well known in the art.

The reactions shown in Scheme (I) may proceed more quickly when the reaction mixtures are heated by microwave. When heated in this way, the following can be used

10 conditions: heat up to 175-185 ° C in ethanol for 5-60 minutes in a Smith Reactor (Personal Chemistry) in a sealed tube (at 20 bar pressure).

The starting materials of uracil or thiouracil 2 can be purchased from commercial sources or can be prepared using conventional organic chemistry techniques. Commercially available uracils and thiouracils 15 that can be used as starting materials in Scheme (I) include, by way of example and not limitation, uracil (Aldrich No.: 13, 078-8; CAS Registry 66-22- 8); 2-thio-uracil (Aldrich No.: 11, 558-4; CAS Registry 141-90-2); 2,4-thiouracil (Aldrich No.: 15, 846-1; CAS Registry 2001-93-6); 5-acetouracil (Chem. Sources Int´l 12000; CAS Registry 6214-65-9); 5-azidouracil; 5-aminouracil (Aldrich No.: 85, 528-6, CAS Registry 932-52-5); 5-bromouracil (Aldrich No.: 85, 247-3; CAS Registry 51-20-7); 5- (trans-2-bromovinyl) -uracil (Aldrich No.: 45, 744-2; CAS Registry 20 69304-49-0); 5- (trans-2-chlorovinyl) -uracil (CAS Registry 81751-48-2): 5- (trans-2-carboxyvinyl) -uracil; uracil-5-carboxylic acid (hydrated 2,4-dihydroxypyrimidin-5-carboxylic acid; Aldrich No.: 27, 770-3; CAS Registry 23945-44-0); 5-chlorouracil (Aldrich No.: 22, 458-8; CAS Registry 1820-81-1); 5-cyanouracil (Chem. Sources Int´l 2000; CAS Registry 4425-56-3); 5-ethyluracil (Aldrich No.: 23, 044-8; CAS Registry 4212-49-1); 5-ethenyluracil (CAS Registry 3710781-6); 5-fluorouracil (Aldrich No.: 85, 847-1; CAS Registry 51-21-8); 5-iodouracil (Aldrich No.: 85, 785-8; Registration 25 CAS 696-07-1); 5-methyluracil (thymine; Aldrich No.: 13, 199-7; CAS Registry 65-71-4); 5-nitrouracil (Aldrich No.: 85, 276-7; CAS Registry 611-08-5); uracil-5-sulfamic acid (Chem. Sources Int´l 2000; CAS Registry 5435-16-5); 5 (trifluoromethyl) -uracil (Aldrich No.: 22, 327-1; CAS Registry 54-20-6); 5- (2,2,2-trifluoroethyl) -uracil (CAS Registry 155143-31-6); 5- (pentafluoroethyl) -uracil (CAS Registry 60007-38-3); 6-aminouracil (Aldrich No.: A5060-6; ​​CAS Registry 873-83-6), uracil-6-carboxylic acid (orotic acid: Aldrich No.: 0-840-2; CAS Registry 50887-69-9 ); 630 methyluracil (Aldrich No.: D11, 520-7; CAS Registry 626-48-2); Uracil-5-amino-6-carboxylic acid (5 aminoorotic acid; Aldrich No.: 18, 121-3; CAS Registry No.: 7164-43-4); 6-amino-5-nitrosouracil (6-amino-2,4-dihydroxy5-nitrosopyrimidine; Aldrich No.: 27, 689-8; CAS Registry 5442-24-0); uracil-5-fluoro-6-carboxylic acid (5fluoroorotic acid; Aldrich No.: 42, 513-3; CAS Registry 00000-00-0); and uracil-5-nitro-6-carboxylic acid (5nitroorotic acid; Aldrich No.: 18, 528-0; CAS Registry 600779-49-9). Additional 5-substituted Uracils and / or Thiouracils, 6

35 substituted and 5,6-substituted are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA (www.generalintermediates.com) and / or Interchim, France (www.interchim.com), or can be prepared using techniques conventional. Multiple textbook references that show appropriate synthetic methods are provided below.

40 Amines 6 and 10 can be purchased from commercial sources or, alternatively, they can be synthesized using conventional techniques. For example, appropriate amines can be synthesized from nitro precursors using conventional chemistry. Specific exemplary reactions are provided in the Examples section. See also Vogel, 1989, Practical Organic Chemistry, Addison Wesley Longman, Ltd. and John Wiley &amp; Sons, Inc.

Four. Five Skilled workers recognize that in some cases, amines 6 (such as the 3-hydroxy moiety) and 10 and / or other substituents of uracil or thiouracil 2, may include functional groups that require protection during synthesis. The exact identity of any / any protective group (s) used will depend on the identity of the functional group that is being protected and will be apparent to the person skilled in the art. Guidance for the choice of appropriate protecting groups, as well as synthetic strategies for their union and withdrawal, can be found,

fifty for example, in Greene &amp; Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley &amp; Sons, Inc., New York (1999) and references cited therein (hereinafter "Greene &amp; Wuts").

A specific example of Scheme (I) using 5-fluorouracil (Aldrich No.: 32, 937-1) as a starting material is illustrated in Scheme (I) below:

55 Scheme (II)

In Scheme (II), Y, Z and X are as previously defined for Scheme (I). According to Scheme (II), 5-fluorouracil 3 is halogenated with POCl 3 to give 2,4-dichloro-5-fluoropyrimidine 5, which is subsequently reacted with an equivalent of 10 'amine (to give rise to 2-Chloro-N4-substituted-5-fluoro-4-pyrimidinediamine 8 ') followed by one or more equivalents of amine 6 to give the compounds of formula (II).

Prodrugs can be prepared according to structural formula (II) by means of routine modification of the methods described above. Alternatively, said prodrugs can be prepared by reacting

10 an appropriately protected 2,4-pyrimidinediamine of structural formula (I) with an appropriate progroup. The conditions for carrying out said reactions and the deprotection of the product to give rise to a prodrug of formulas (III) and (IV) are well known, and include, for example, those shown in the US patent application. UU. No.: 2006-0211657, PCT Publication WO 2006/078846 and the US application. Serial No. 11 / 295,752 filed on December 6, 2005.

fifteen As described herein, a prodrug of formula V is prepared

twenty wherein Y, Z and X are as defined above for formula I and P3 and the oxygen atom to which they are attached form an ester pro-salt, reacting a 3-hydroxy phenyl compound of formula V in the that P3 is hydrogen with an appropriate acid halide or anhydride and optionally in the presence of a base such as an amine. In another example disclosed herein the compound is reacted with an appropriate acid in the presence of an acid catalyst or a coupling reagent. In some examples described in the

25 Here, the acid catalyst is sulfuric acid or HCl. In other examples described herein, the coupling agent is a carbodiimide such as dicyclohexylcarbodiimide or is 1,1'-carbonyldiimidazole.

Many references are known in the art that show useful methods for synthesizing pyrimidines in general, as well as the starting materials described in Schemes (I) - (II). For specific guidance, 30 directs the reader to Brown, DJ, "The Pyrimidines," in The Chemistry of Heterocyclic Compounds, Volume 16 (Weissberger, A., Ed.), 1962, Interscience Publishers, (a division of John Wiley &amp; 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 &amp; Sons), New York ("Brown II"); Brown, D. J., "The Pyrimidines", in The Chemistry of Heterocyclic Compounds, Volume 16, 35 Supplement II (Weissberger, A. and Taylor, EC, Ed.), 1985, An Intersecience Publication (John Wiley &amp; Sons), New York ( "Brown III"); Brown, DJ, "The Pyrimidines" in The Chemistry of Heterocyclic Compounds, Volume 52 (Weissbergar, A. and Taylor, EC Ed.), 1994, John Wiley &amp; Sons, Inc. New York, pp. 1-1509 ("Brown IV"); Kenner, G.

W. 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., 40 New York, pp. 1-401 (synthesis of uracil pp. 313, 315; synthesis of pyrimidine pp. 313-316; synthesis of amino pyrimidine pp. 315); Joule, JA, Mills, K. and Smith, G. F., Heterocyclic Chemistry, 3rd edition, 1995; Chapman and Hall, London, United Kingdom, pp., 1-516; Vobrüggen, H. and Ruh-Pohlenz, C., Handbook of Nucleoside Synthesis, John Wiley &amp; Sons, New York, 2001, pp. 1-631 (protection of pyrimidines by means of acylation pp. 90-91; silylation of pyrimidines pp. 91-93); Joule, JA, Mills, K. and Smith, GF, Heterocyclic Chemistry, 4th edition, 2000, Blackwell

Four. Five Science, Ltd., Oxford, United Kingdom, pp. 1-589; and Comprehensive Organic Synthesis, Volumes 1-9 (Trost, BM and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, United Kingdom.

Inhibition of Fc receptor signal cascades

fifty The active 2,4-pyrimidinediamine compounds of the invention inhibit the Fc receptor signaling cascades that lead to, among other things, cell degranulation. As a specific example, the compounds inhibit the FcεRI and / or FcγRI signal cascades that lead to the degranulation of immune cells such as neutrophil, eosinophilic, mast cells and / or basophilic cells. Both mast cells and basophilic cells play a central role in allergen-induced disorders, including, for example, allergic rhinitis and asthma. After exposure to allergens, which may be, among other things, pollen or parasites, IgE antibodies are synthesized

5 Allergen-specific B-cells activated by IL-4 (or IL-3) and other messengers to activate the synthesis of specific IgE class antibodies. These allergen-specific IgEs bind to high affinity FcεRI. After antigen binding, cross-linking of IgEs with bound FcεRI occurs and the signal transduction mechanism of the IgE receptor is activated, which leads to cell degranulation and subsequent release and / or synthesis of a mediator host chemicals, including histamine, proteases (for example, tryptase and chymase), lipid mediators such as leukotrienes (for example, LTC4), platelet activation factor (PAF) and prostaglandins (for example, PGD2) and a series of cytokines, including TNF-α, IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF-β. The release and / or synthesis of these mediators from mast cells and / or basophilic cells is taken into account for allergen-induced early and late stage responses and is directly related to the downstream episodes that lead to a prolonged inflammatory state.

fifteen Molecular episodes in the FcεRI signal transduction pathway that lead to the release of preformed mediators by means of degranulation and release and / or synthesis of other chemical mediators are well known. FcεRI is a heterotetrameric receptor formed by an IgE binding subunit, a beta subunit and two gamma subunits (gamma homodimer). Crosslinking of IgE bound to FcεRI by means of multivalent binding agents (including, for example, specific IgE allergens or anti-IgE fragments or antibodies) induces the rapid association and rapid activation of Src-related Lyn kinase. Lyn produces the phosphorylation of the tyrosine-based activation motifs of the immunoreceptor (ITAM) on the beta and gamma intracellular subunits, which leads to the recruitment of additional Lyn for the Syk kinase and beta subunit for the gamma homodimer. These receptor-associated kinases, which are activated by intra-e phosphorylation

25 inter-molecular, phosphorylate other components of the route, such as Btk kinase, LAT and C-gamma PLCgamma phospholipase. Activated PLC-gamma initiates mechanisms that lead to the activation of protein kinase C and the mobilization of Ca2 +, both of which are required for degranulation. FcεRI crosslinking also activates the three main classes of mitogen-activated protein kinases (MAP), that is, ERK1 / 2, JNK 1/2 and p38. The activation of these mechanisms is important in the transcriptional regulation of proinflammatory mediators, such as TNF-α and IL-6, as well as the leukotriene CA (LTC4) of lipid mediator.

Although not shown, it is believed that the FcγRI signaling cascade shares certain common elements with the FcεRI signaling cascade. Importantly, FcγRI includes a gamma homodimer that undergoes phosphorylation and recruits Syk, and like FcεRI, activation of the FcγRI cascade leads to, between

35 other things, degranulate. Other Fc receptors that share the same gamma homodimer and that can be regulated by means of the active 2,4-pyrimidinediamine compounds include, but are not limited to, FcαRI and FcγRIII.

The ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit Fc receptor signaling cascades can be determined simply or confirmed by in vitro assays. Appropriate tests are provided to confirm the inhibition of FcεRI-mediated degranulation in the Examples section. In a typical assay, cells capable of experiencing FcεRI-mediated degranulation, such as mast cells or basophilic cells, are first grown in the presence of IL-4, Stem Cell Factor (SCF), IL-6 and IgE to increase FcεRI expression , are exposed to the 2,4-pyrimidinediamine test compound of the invention and stimulated with anti-IgE antibodies (or, alternatively, an IgE-specific allergen). After incubation, the amount of chemical mediator or other chemical agent released and / or synthesized can be quantified as a result of the activation of the FcεRI signaling cascade, using conventional techniques and can be compared with the amount of mediator or agent released to from the control cells (i.e., cells that are stimulated but not exposed to the test compound). The concentration of the test compound that results in a 50% reduction in the amount of mediator or agent measured, compared to the control cells is the IC50 of the test compound. The origin of mast cells or basophilic cells used in the assay depends, in part, on the desired use of the compounds and will be apparent to those skilled in the art. For example, if the compounds are used to treat or prevent a particular disease in humans, an appropriate source of mast cells or basophilic cells is a human or other animal that constitutes an accepted or known clinical model for the particular disease. Thus, depending on the particular application, mast cells or basophilic cells can come from a wide variety of animal sources, ranging from, for example, lower mammals such as mice and rats, to dogs, sheep and other mammals commonly used in the conduct of clinical trials, higher mammals such as monkeys, chimpanzees and apes, to humans. Specific examples of cells suitable for carrying out in vitro assays include, but are not limited to, basophil cells of rodents or humans, cell lines of rat basophilic leukemia, primary mouse mast cells (such as mouse mast cells from bone marrow "BMMC") and primary human mast cells isolated from umbilical cord blood ("CHMC") or other tissues such as lung. Methods for isolating and culturing these cell types are well known or provided in the Examples section (see, for example, Demo et al., 1999, Cytometry 36 (4): 340-348 and the related application for No. of

65 Series 10 / 053.355 filed on November 8, 2001). Of course, other types of immune cells that undergo degranulation upon activation of the FcεRI signaling cascade can also be used including, for example, eosinophils.

As skilled workers will recognize, the mediator or quantified agent is not critical. The only requirement is

5 that is a mediator or agent released and / or synthesized as a result of the initiation or activation of the signaling cascade of the Fc receptor. For example, activation of the FcεRI signaling cascade in mast cells or basophilic cells leads to numerous episodes downstream. For example, activation of the FcεRI signal cascade leads to the immediate release (i.e., in 1-3 minutes after receptor activation) of a variety of preformed chemical mediators and agents by means of degranulation. Of this

10 Thus, in one embodiment, the mediator or quantified agent may be specific for the granules (ie, present in the granules but not in the cellular cytoplasm in general). Examples of specific mediators of granules or agents that can be quantified to determine and / or confirm the activity of the 2,4-pyrimidinediamine compound of the invention include, but are not limited to, specific granule enzymes such as hexosaminidase and tryptase and specific components of granules such as histamine and serotonin. They know each other

fifteen Well, trials to quantify these factors and in many cases are commercially available. For example, the release of tryptase and / or hexosaminidase can be quantified by incubating the cells with substrates suitable for excision that undergo fluorescence after excision and quantifying the amount of fluorescence produced using conventional techniques. Such cleavage suitable fluorogenic substrates are commercially available. For example, Z-Gly-Pro-Arg fluorogenic substrates can be used

twenty AMC (Z = benzyloxycarbonyl; AMC = 7-amino-4-methylcoumarin; BiOMOL Research Laboratories, Inc., Plymouth Meeting, PA 19462, Catalog No. P-142) and Z-Ala-Lys-Arg-AMC (Enzyme Systems Products, a division of ICN Biomedicals, Inc., Livermore, CA 94550, Catalog No. AMC-246) to quantify the amount of tryptase released. Fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma, St. Louis, MO, Catalog No. 69585) to quantify the amount of hexosaminidase released. The release of

25 histamine using a commercially available enzyme-linked immunosorbent assay (ELISA) such as an Immunotech histamine ELISA assay #: M2015 (Beckman-Coluter, Inc.). Specific methods for quantifying the release of tryptase, hexosaminidase and histamine are provided in the Examples section. Any of these assays can be used to determine or confirm the activity of the 2,4-pyrimidinediamine compounds of the invention.

30 Degranulation is only one of the different responses initiated by the FcεRI signaling cascade. In addition, the activation of this signaling mechanism leads to de novo synthesis and the 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 as leukotrienes (eg, LTC4), platelet activation factor (PAF) and prostaglandins. Therefore,

35 The activity of the 2,4-pyrimidinediamine compounds of the invention can also be evaluated by quantifying the amount of one or more of these mediators released and / or synthesized by activated cells.

Unlike the specific granule components discussed above, these "late stage" mediators are not released immediately after activation of the FcεRI signaling cascade. Therefore, when quantification of these late stage mediators occurs, caution should be taken to ensure that the activated cell culture is incubated for sufficient time to result in synthesis (if necessary) and release of the mediator object of quantification. In general, PAF and lipid mediators such as C4 leukotriene are released in 3-30 minutes after activation of FcεRI. Cytokines and other late stage mediators are released approximately 4-8 hours after activation of FcεRI. The

Four. Five appropriate incubation times for an appropriate mediator will be apparent to those skilled in the art. Specific guidance and essays are provided in the Examples section.

The amount released from a particular late stage mediator can be quantified using any conventional technique. In one embodiment, the quantity (s) can be quantified using ELISA assays. The kits of

fifty Appropriate ELISA assay to quantify the amount of TNF-α, IL-4, IL-5, IL-6 and / or IL-13 released are available in, for example, Biosource International, Inc., Camarillo, CA 93012 ( see, for example, Catalog No. KHC3011, KHC0042, KHC0052, KHC0061 and KHC0132). Appropriate ELISA test kits to quantify the amount of leukotriene C4 (LTC4) released by the cells are available from Cayman Chemical Co., Ann Arbor, MI 48108 (see, for example, Catalog No. 520211).

55 Normally, the 2,4-pyrimidinediamine active compounds of the invention will exhibit IC50 with respect to FcεRI-mediated degranulation and / or with respect to the release of mediator or synthesis of approximately 20 μM

or less, as measured in an in vitro assay, such as one of the in vitro assays described above in the Examples section. Of course, skilled workers will appreciate that compounds that exhibit values

60 of lower IC50s, for example of the order of 10 μM, I μM, 100 nM, 10 nM, 1 nM or even less, are particularly useful.

Skilled workers will also appreciate that the different mediators discussed above can induce different adverse effects or exhibit different potencies with respect to the same adverse effect. For example, the LTC4 lipid mediator is a potent vasoconstrictor - it is approximately 1000 times more potent.

to induce vasoconstriction than histamine. As another example, in addition to mediating the atopic reactions

or of type I hypersensitivity, cytokines can also cause tissue remodeling and cell proliferation. Thus, although the compounds that inhibit the release and / or synthesis of any one of the chemical mediators previously discussed are useful, skilled workers will appreciate that the compounds

5 which inhibit the release and / or synthesis of a plurality, or even all, of the previously described mediators find a particular use, since said compounds are useful for completely alleviating or preventing a plurality, or even all, of the adverse effects induced by private mediators For example, compounds that inhibit the release of the three types of mediators - specific for granules, lipids and cytokines are useful for treating or preventing immediate type I hypersensitivity reactions as well as the chronic symptoms associated with them.

Compounds of the invention capable of inhibiting the release of more than one type of mediator (e.g., granule-specific or late-stage) can be identified by determining IC50 with respect to a representative mediator of each class, using the various in vitro assays described above (or another 15 equivalent in vitro assays). Normally, the compounds of the invention that are capable of inhibiting the release of more than one type of mediator will exhibit an IC50 for each type of mediator tested less than about 20 µM. For example, a compound that exhibits an IC50 of 1 μM with respect to histamine release (IC50histamine) and an IC50 of 1 nM with respect to the synthesis and / or release of LTC4 from leukotriene (IC50 LCT4) inhibits mediator release both immediate (granule specific) and late stage. By way of another specific example, a compound exhibiting a 10 µM IC50triptase, a 0.1 µM IC50LTC4 and a 1 µM IC50IL-4 inhibits the immediate release of cytokine and lipid mediator. Although the specific examples above use the IC50 values ​​of a representative mediator of each class, skilled workers will appreciate that the IC50 values ​​of a plurality, or even all, of mediators comprising one or more classes can be obtained. The quantity (s) and identity (s) of the mediators for whom the data should be determined

25 IC50 for a particular application and compound will be apparent to those skilled in the art.

Similar assays can be used to confirm the inhibition of signal transduction cascades initiated by other Fc receptors, such as FcαRI, FcγRI and / or FcγRIII signaling, with routine modification. For example, the ability of the compounds to inhibit FcγRI signal transduction can be confirmed in assays similar to those described above, with the exception that the FcγRI signaling cascade is activated, for example by incubating the cells with IgG and an IgG-specific antibody or allergen, instead of IgE and an IgE-specific antibody or allergen. Appropriate cell types, activating agents and quantifying agents in order to confirm the inhibition of other Fc receptors, such as Fc receptors comprising a gamma homodimer, will be apparent to those skilled in the art.

35 technique.

A particularly useful class of compounds includes 2,4-pyrimidinediamine compounds that inhibit the release of specific immediate granule mediators and late stage mediators with approximately equivalent IC50 values. By approximately equivalent it is understood that the IC50 values ​​for each type of mediator are within a range of about 10 times with respect to each other. Another particularly useful class of compounds includes 2,4-pyrimidinediamine compounds that inhibit the release of specific immediate granule mediators, lipid mediators and cytokine mediators with approximately equivalent IC50. In a specific embodiment, said compounds inhibit the release of the following mediators with approximately equivalent IC50: histamine, tryptase, hexosaminidase, IL-4, IL-5, IL-6,

Four. Five IL-13, TNFα and LTC4. Such compounds are particularly useful for, among other things, alleviating or completely preventing both early and late stage responses associated with atopic or immediate type I hypersensitivity reactions.

Ideally, the ability to inhibit the release of all desired types of mediators resides in an individual compound. However, mixtures of compounds that achieve the same result can also be identified. For example, a first compound that inhibits the release of specific granule mediators can be used in combination with a second compound that inhibits the release and / or synthesis of cytokine mediators.

55 In addition to the FcεRI or FcγRI degranulation mechanisms discussed above, degranulation of mast cells and / or basophil cells can be induced by other agents. For example, ionomycin, a calcium ionophore that diverts the FcεRI or FcγRI signal early transduction cell machinery, directly induces a calcium flux that activates degranulation. Activated PLCγ initiates the mechanisms that lead to, among others, calcium ion mobilization and subsequent degranulation. As illustrated, this mobilization of Ca2 + is activated late in the FcεRI signal transduction mechanism. As mentioned above, ionomycin directly induces the mobilization of Ca2 + and the flow of Ca2 + that leads to degranulation. Other ionophores that induce degranulation in this manner include A23187. The ability of granulated inducing ionophores such as ionomycin can be used to divert early stages of FcεRI and / or FcγRI signaling cascades, as a counting screen to identify active compounds

65 of the invention that specifically exert their degreasing inhibitory activity by blocking or inhibiting the early signaling cascades of FcεRI or FcγRI, as discussed above. Compounds that specifically inhibit said early degranulation mediated by FcεRI or FcγRI inhibit not only the degranulation and subsequent rapid release of histamine, tryptase and other granule contents, but also inhibit the pro-inflammatory activation pathways that cause TNFα release , IL-4, IL-13 and

5 lipid mediators such as LTC4. Thus, compounds that specifically inhibit early degranulation mediated by FcεRI and / or FcγRI block or inhibit not only acute atopic and hypersensitivity type I reactions, but also late responses involving multiple inflammatory mediators.

10 Compounds of the invention that specifically inhibit early degranulation mediated by FcεRI and / or FcγRI are the compounds that inhibit degranulation mediated by FcεRI and / or FcγRI (for example, having an IC50 of less than about 20 μM with respect to release of the specific granule mediator or component, measured in an in vitro assay with cells stimulated with an IgE or IgG binding agent) but which do not appreciably inhibit ionophore-induced degranulation. In one embodiment, the

fifteen Compounds do not appreciably inhibit ionophore-induced degranulation if they exhibit an IC50 of ionophore-induced degranulation greater than about 20 μM, as measured in an in vitro assay. Of course, active compounds that exhibit even higher IC50 values ​​of ionophores induced degranulation, or that do not inhibit ionophores induced degranulation at all, are particularly useful. In another embodiment, the compounds are considered to not appreciably inhibit ionophore induced degranulation.

twenty if they exhibit a difference of more than 10 times in their IC50 values ​​of FcεRI and / or FcγRI mediated degranulation and ionophore induced degranulation, as measured in an in vivo assay. Appropriate assays to determine the IC50 of the ionophore-induced degranulate include any of the degranulate assays previously described, with the modification of the cells being stimulated or activated with a calcium ionophore that induces degranulation such as ionomycin or A23187 (AG Scientific, San Diego, CA) instead of anti-IgE antibodies or with

25 a specific IgE allergen. Specific tests to evaluate the ability of a particular 2,4-pyrimidinediamine compound of the invention to inhibit ionophore-induced degranulation are provided in the Examples section.

As will be recognized by skilled workers, compounds that exhibit a high degree of selectivity

30 of FcεRI-mediated degranulation find a particular use, since said compounds selectively direct the FcεRI cascade and do not interfere with other degranulation mechanisms. Similarly, compounds that exhibit a high degree of FcγRI-mediated degranulation selectivity find particular use, since said compounds selectively direct the FcγRI cascade and do not interfere with other degranulation mechanisms. Compounds that exhibit a high degree of selectivity are generally 10 times or more.

35 selective for FcεRI and / or FcγRI mediated degranulation with respect to ionophore induced degranulation, such as ionomycin induced degranulation.

Accordingly, the activity of the 2,4-pyrimidinediamine compounds of the invention can also be confirmed in biochemical or cellular assays of Syk kinase activity. In the FcεRI signaling cascade in 40 mast cells and / or basophilic cells, Syk kinase produces phosphorylation of LAT and PLC-gamma 1, which leads, among other things, to degranulation. In one embodiment, the activity is confirmed by contacting an isolated Syk kinase, or one of its active fragments with a 2,4-pyrimidinediamine compound in the presence of a Syk kinase substrate (e.g., a synthetic peptide or a protein that it is known that it undergoes phosphorylation by Syk in a signaling cascade) and assessing whether Syk kinase produces phosphorylation of the substrate. Alternatively, the assay can be carried out with cells expressing a Syk kinase. Cells can express Syk kinase endogenously or can be subjected to technical study to express a recombinant Syk kinase. Optionally, the cells also express the Syk kinase substrate. Appropriate cells for carrying out such confirmation assays, as well as the methods for technical examination, appropriate cells will be apparent to those skilled in the art. Specific examples of biochemical and cellular assays

fifty Appropriate for confirming the activity of 2,4-pyrimidinediamine compounds are provided in the Examples section.

In general, compounds that are Syk kinase inhibitors exhibit an IC50 with respect to Syk kinase activity, such as the ability of Syk kinase to phosphorylate a synthetic endogenous substrate, in an in-house assay.

55 vitro or cell assay within the range of approximately 20 μM or less. Skilled workers will appreciate that compounds that exhibit lower IC50 values, such as within the range of 10 μM, 1 μM, 100 nM, 10 nM, 1 nM or even less, are particularly useful.

Uses and compositions

60 As previously discussed, the active compounds of the invention inhibit Fc receptor signaling cascades, especially those Fc receptors that include gamma homodimer, such as FcεRI and / or FcγRI signaling cascades, which lead to, between other things, the release and / or synthesis of chemical mediators from cells, either by means of degranulation or by other methods. As also

65 It has been commented, the active compounds are also potent Syk kinase inhibitors. As a consequence of

These activities, the active compounds of the invention can be used in a variety of contexts in vitro, in vivo and ex vivo to regulate or inhibit Syk kinase, signaling cascades in which Syk kinase plays an important role, receptor signaling cascades of Fc and the biological responses made by said signaling cascades. For example, in one embodiment, the compounds may be for use in order to inhibit Syk kinase 5, either in vitro or in vivo, in virtually any cell type that expresses Syk kinase. They can also be used to regulate signal transduction cascades in which Syk kinase plays a role. Such signal transduction cascades that depend on Syk include, but are not limited to, FcεRI, FcγRI, FcγRIII, BCR and integrin signal transduction cascades. The compounds may also be useful for use in vitro or in vivo to regulate and in particular inhibit cell or biological responses carried out by signal transduction cascades that depend on Syk. Such cellular or biological responses include, but are not limited to, respiratory impulse, cell adhesion, cell degranulation, cell dispersion, cell migration, cell aggregation, phagocytosis, cytokine synthesis and release, cell maturation and Ca2 + flow. Importantly, the compounds can be used to inhibit Syk kinase in vivo as a therapeutic approach to the treatment or prevention of diseases with total or partial mediation of Syk kinase activity. Examples no

fifteen Limitations of Syk kinase-mediated diseases that can be treated or prevented with the compounds are discussed in more detail below.

In another embodiment, the active compounds can be used to regulate or inhibit Fc and / or FcγRI-mediated receptor signaling cascades as a therapeutic approach to the treatment or prevention of diseases characterized by, caused by and / or associated with the release or synthesis of chemical mediators of said Fc receptor signaling cascades or degranulation. Such treatments can be administered to animals in veterinary contexts or to humans. The diseases that are characterized by, are caused by or associated with said mediator release, synthesis or degranulation and that, therefore, can be treated or prevented with the active compounds include, by way of example and without limitation,

25 atopic or anaphylactic or allergic hypersensitivity reactions, allergies (for example, allergic conjunctivitis, allergic rhinitis, atopic asthma, atopic dermatitis and food allergies), low-grade pathological scarring (for example, scleroderma, increased fibrosis, keloids, post-scars surgical, pulmonary fibrosis, vascular spasms, migraines, reperfusion injury and post-myocardial infarction), diseases associated with tissue destruction (e.g. COPD, cardiobronchitis and post-myocardial infarction), diseases associated with inflammation of tissues (for example, irritable bowel syndrome, irritable bowel disease and inflammatory bowel disease), inflammation and scarring.

In addition to the multitude of diseases discussed above, cellular and animal empirical data confirm that the 2,4-pyrimidinediamine compounds described herein are also useful for the

35 treatment or prevention of autoimmune diseases, as well as the different symptoms associated with said diseases. The types of autoimmune diseases that can be treated or prevented with 2,4-pyrimidinediamine compounds generally include those disorders that involve tissue injury that occurs as a result of a humoral response and / or with cellular mediation against immunogens or antigens of origin. endogenous and / or exogenous. Often, such diseases are called diseases that involve non-anaphylactic hypersensitivity reactions (ie, type II, type III and / or type IV).

As previously mentioned, type I hypersensitivity reactions generally result in the release of pharmacologically active substances, such as histamine, from mast cells and / or basophilic cells after contact with a specific exogenous antigen. As previously mentioned, said

Four. Five Type I reactions play a role in numerous diseases, including allergic asthma, allergic rhinitis, etc.

Type II hypersensitivity reactions (also called cell stimulation, cytotoxic or cytolytic complement hypersensitivity reactions) occur when immunoglobulins react with antigenic components of cells or tissues, or with an antigen or hapten that has been coupled in a way narrows to cells or tissue. Diseases that are commonly associated with type II hypersensitivity reactions include, but are not limited to, autoimmune hemolytic anemia, fetal erythroblastosis and Goodpasture's disease.

Type III hypersensitivity reactions (also called complex hypersensitivity reactions

55 immune, toxic complex or soluble complex) take place from the deposition of circulating soluble antigen-immunoglobulin complexes in blood vessels or tissues, with associated inflammatory water reactions at the site of immune complex deposition. Non-limiting examples of prototype type III reaction diseases include Arthus reaction, rheumatoid arthritis, serum sickness, systemic lupus erythematosus, certain types of glomerulonephritis, multiple sclerosis and vesicular pemphigoid.

Type IV hypersensitivity reactions (often referred to as tuberculin-type hypersensitivity reactions, cellular, with cellular mediation or delayed) are caused by sensitized T-lymphocytes, which are the result of contact with a specific antigen. Non-limiting examples of the diseases cited that involve type IV reactions are contact dermatitis and allograft rejection.

65 Autoimmune diseases associated with any of the non-anaphylactic hypersensitivity reactions

The above can be treated or prevented with the 2,4-pyrimidinediamine compounds of the invention. In particular, the compounds can be used to treat or prevent those autoimmune diseases frequently characterized as autoimmune disorders of the individual cell type or individual organ that include, but are not limited to: Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia 5, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Grave disease, primary biliary cirrhosis, chronic aggressive colitis, chronic aggressive ulcer hepatitis membranous glomerulopathy, as well as autoimmune diseases frequently characterized by involving a disorder of the immune system, including but not limited to: systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis

10 dermatomyositis, systemic sclerosis, nodular polyarteritis, multiple sclerosis or vesicular pemphigoid.

Skilled workers will appreciate that many of the aforementioned autoimmune diseases are associated with severe symptoms, the relief of which provides significant therapeutic benefit even in cases where it is not possible to relieve the underlying autoimmune disease. Many of these

fifteen Symptoms, as well as their underlying disease states, are the result of activation of FcγRI signaling cascades in monocytic cells. Because the 2,4-pyrimidinediamine compounds described herein are potent inhibitors of said FcγRI signaling cascades in monocytes and other cells, the compounds can be used in the treatment and / or prevention of a multitude of associated adverse symptoms. to the aforementioned autoimmune diseases.

twenty As a specific example, rheumatoid arthritis (RA) usually results in inflammation, pain and loss of mobility and pain with palpation in the target joints of the body. RA is characterized in that the chronic inflamed synovium is densely populated with lymphocytes. The synovial membrane, which is normally of a thick cell layer, becomes intensely cellular and assumes a shape similar to that of a lymphoid tissue,

25 including dendritic cell, T-, B- and NK cells, macrophages and plasma cell associates. This process, as well as a plethora of immunopathological mechanisms that include the formation of antigen-immunoglobulin complexes, ultimately result in the destruction of joint integrity, resulting in permanent loss of function with deformity and / or bone erosion in the joint or in its vicinity. The compounds can be used to treat or alleviate any one, several or all of these.

30 RA symptoms. Thus, in the context of RA, the compounds are considered to provide a therapeutic benefit (discussed more generally below) when a reduction or relief of the symptoms commonly associated with RA is achieved, regardless of whether the treatment has as resulted in a concomitant treatment of underlying RA and / or a reduction in the amount of circulating rheumatoid factor ("RF").

35 By way of another specific example, systemic lupus erythematosus ("SLE") is usually associated with symptoms such as fever, joint pain (arthralgia), arthritis and serositis (pleurisy or pericarditis). In the context of SLE, the compounds are considered to provide a therapeutic benefit when a reduction or relief of any of the symptoms commonly associated with SLE is achieved, regardless of whether the treatment results in a concomitant treatment of underlying SLE.

40 By way of another specific example, multiple sclerosis ("MS") negatively affects the patient by altering visual acuity; stimulating double vision; altering the motor functions that affect the actions of walking and hand use; producing intestinal and bladder incontinence; spasms and sensory deficiencies (sensitivity to touch, pain and temperature). In the context of MS, the compounds are considered to provide an effect

Four. Five therapeutic when an improvement or reduction in the progression of any one or more than one of the limiting effects commonly associated with MS is achieved, regardless of whether the treatment results in a concomitant treatment of the underlying MS.

When used to treat or prevent such diseases, the active compounds can be administered in

fifty individually, in the form of mixtures of one or more active compounds or in mixture or combination with other agents useful for the treatment of said diseases and / or the symptoms associated with said diseases. The active compounds can also be administered in a mixture or in combination with agents useful for treating other disorders or diseases, such as steroids, membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype activation. or synthesis of IgE, activation of

55 IgE isotype or IgE synthesis, β-agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, Rituximab, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors and antihistamines, to name a few. The active compounds can be administered by themselves in the form of prodrugs or as pharmaceutical compositions, which comprise an active compound or prodrug.

60 Pharmaceutical compositions comprising the active compounds of the invention (or their prodrugs) can be manufactured by means of conventional mixing, solution, granulation, levigation methods for the manufacture of dragees, emulsified, encapsulated, imprisoned or lyophilized. The compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliary substances that facilitate the processing of the active compounds to give rise to preparations that can be used.

65 from the pharmaceutical point of view.

The active compound or prodrug can be formed in the pharmaceutical compositions by itself, or in the form of hydrate, solvate, N-oxide or pharmaceutically acceptable salt as described above. Normally, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts can also be formed having a lower solubility than the corresponding bases and acids.

5 free.

The pharmaceutical compositions of the invention may take an appropriate form for virtually any mode of administration, including, for example, topical, ocular, oral, oral, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or an appropriate form. administration by inhalation or insufflation.

For topical administration, the active component (s) can be formulated in the form of solutions, gels, ointments, creams, suspensions, etc., as is well known in the art.

Systemic formulations include those designed for administration by injection, for example,

fifteen subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound (s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and / or dispersing agent. Formulations for injection may be presented in unit dosage form, for example, in ampoules in multi-dose containers and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder form for reconstitution with a

25 appropriate vehicle, including but not limited to sterile pyrogen-free water, a buffer solution, a dextrose solution, etc., before use. To this end, the active compound (s) can be dried by any known technique, such as lyophilization and reconstituted before use.

For transmucosal administration, penetrating substances suitable for the permeate barrier are used in the formulation. Such penetrating substances are known in the art.

For oral administration, the pharmaceutical compositions may take the form of, for example, pills, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch

or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, enteric sugars, films or coatings.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other appropriate vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (eg, sorbitol syrup, derivatives

Four. Five of cellulose or hydrogenated edible fats); emulsifying agents (for example, lecithin or gum arabic); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol, cremophoretm or separate vegetable oils); and preservatives (for example, methyl or propyl p-hydroxybenzoates or ascorbic acid). The preparations may also contain buffer salts, preservatives, flavorings, coloring agents and sweeteners as appropriate.

Properly, preparations for oral administration can be formulated to provide controlled release of the active compound or prodrug, as is well known.

For oral administration, the compositions may take the form of tablets or tablets formulated in a conventional manner.

For rectal or vaginal administration routes, the active compound (s) can be formulated in the form of solutions (for retention enemas), suppositories or ointments containing conventional suppository bases such as butter of cocoa or other glycerides.

For nasal administration or administration by inhalation or insufflation, the active compound (s) or prodrug (s) may be appropriately administered in the form of an aerosol spray in pressurized containers or a nebulization with the use of an appropriate propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other appropriate gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for administering a measured amount. Capsules and cartridges can be formulated for use in an inhaler or insufflator (for

example capsules and cartridges formed by gelatin) so as to contain a powder mixture of the compound and an appropriate powder base such as lactose or starch.

A specific example of an aqueous suspension formulation suitable for nasal administration using 5 commercially available nasal spray devices includes the following ingredients: active compound

or prodrug (0.5-20 mg / ml); benzalkonium chloride (0.1-0.2 mg / ml); polysorbate 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 to vary within approximately pH 5 to pH 7, with pH 5.5 being a typical value.

Another specific example of aqueous suspension suitable for the administration of the compounds by means of inhalation and in particular for said administration of a compound of the invention, contains 1-20 mg / ml of the compound or prodrug, 0.1-1% (v / v) Polysorbate 80 (TWEEN®80), 50 nM citrate and / or 0.9 5 sodium chloride.

fifteen For ocular administration, the active compound (s) or prodrug (s) can be formulated as a solution, emulsion, suspension, etc., suitable for administration to the eye. A variety of appropriate vehicles are known for administering compounds to the eye. Specific non-limiting examples are described in US Pat. No.: 6,261,547; U.S. Patent No. 6,197,934; U.S. Patent No.: 6,056,950; U.S. Patent N. 5,800,807; U.S. Patent No.: 5,776,445; U.S. Patent No.: 5,698,219; U.S. Patent No.: 5.52 1,222; U.S. Patent No. 5,403,841; U.S. Patent No. 5,077,033; U.S. Patent :

4,882,150 and U.S. Pat. No.: 4,738,851.

For prolonged administration, the active compound (s) or prodrug (s) can be formulated as a prolonged release preparation for administration by means of intramuscular implantation or injection. He

25 it can formulate the active ingredient with appropriate polymeric or hydrophobic materials (for example, in the form of an acceptable oil emulsion) or ion exchange resins, or as moderately soluble derivatives, for example, a moderately soluble salt. Alternatively, transdermal delivery systems manufactured in the form of an adhesive disc or patch that slowly release the active compound (s) for percutaneous absorption can be used. To this end, permeability improvers can be used to facilitate the penetration of the active compound (s). Appropriate transdermal patches are described for example in US Pat. No. 5,407,713; U.S. Patent No. 5,352,456; U.S. Patent No.: 5,332,213; U.S. Patent No. 5,336,168; U.S. Patent No.: 5,290,561; U.S. Patent No.: 5,254,346; U.S. Patent No.: 5,164,189; U.S. Patent No. 5,163,899, U.S. Pat. No. 5,088,977; U.S. Patent No.: 5,087,240; U.S. Patent No.: 5,008,110; and U.S. Pat. No.: 4,921,475.

35 Alternatively, other pharmaceutical administration systems can be used. Liposomes and emulsions are well known examples of delivery vehicles that can be used to administer the active compound (s) or prodrug (s). Certain organic solvents such as dimethylsulfoxide (DMSO) can also be used, although usually at higher toxicity cost.

If desired, the pharmaceutical compositions may be presented in a package or dispensing device that may contain one or more dosage forms containing the active compound (s). For example, the package may comprise metal or metallic foil, such as a blister pack. The container or dispensing device may be accompanied by instructions for administration.

Four. Five Effective dosages

The active compound (s) of the invention or its prodrug (s), or its compositions, are generally used in an amount effective to achieve the intended result, for example, in an amount effective for treat or prevent the particular disease being treated. The compound (s) can be administered therapeutically to achieve the therapeutic benefit or prophylactically to achieve a prophylactic benefit. Therapeutic benefit is the eradication or relief of the underlying disorder being treated and / or the eradication or relief of one or more of the symptoms associated with the underlying disorder so that the patient experiences an improvement in sensation or condition, despite that the patient may still be affected by the underlying disorder. For example, the

55 administration of a compound to a patient suffering from an allergy provides a therapeutic benefit not only when the underlying allergic response is eradicated or relieved, but also when the patient experiences a decrease in the severity or duration of symptoms associated with allergy after exposure to the allergen By way of another example, the therapeutic benefit in the context of asthma includes an improvement in breathing after the onset of an asthmatic attack, or a reduction in the frequency or severity of asthmatic episodes. The therapeutic benefit also includes the detection or slowdown of disease progression, regardless of whether the improvement is carried out.

For prophylactic administration, the compound can be administered to a patient at risk of developing one of the diseases described above. For example, if it is unknown if a patient is allergic to a particular drug 65, the compound can be administered before drug administration to prevent or alleviate an allergic response to the drug. Alternatively, prophylactic administration can be applied to prevent

appearance of symptoms in a patient diagnosed with the underlying disorder. For example, a compound can be administered to a patient suffering from allergy before the expected exposure to the allergen. Compounds can also be administered prophylactically to healthy individuals who are repeatedly exposed to known agents to one of the above-mentioned evils in order to prevent the onset of the disorder. For example, you can

5 administer to a healthy individual who is repeatedly exposed to a known allergen to induce allergies, such as latex, in an effort to prevent the individual from developing an allergy. Alternatively, a compound can be administered to a patient suffering from asthma before taking part in activities that trigger asthma attacks to soften the severity, or to completely prevent, an asthmatic episode.

The amount of compound administered depends on several factors, including, for example, the particular indication being treated, the mode of administration, if the desired benefit is prophylactic or anaphylactic, the severity of the indication being treated and the age and weight. of the patient, the bioavailability of the particular active compound, etc. The determination of an effective dosage is within the capabilities of those skilled in the art.

fifteen Initially, effective dosages can be estimated from in vitro assays. For example, an initial dosage may be formulated for use in animals in order to achieve a concentration of active compound in the bloodstream or in the serum that is equal to or greater than IC 50 of the particular compound, measured in an in vitro assay, such as CHMC or BMMC in vitro and other in vitro assays described in the Examples section. The calculation of dosages to achieve serum or bloodstream concentrations, taking into account the bioavailability of the particular compound, is within the capabilities of skilled workers. By way of guidance, the reader is referred to Fing &amp; Woodbury, "General Principles," In: Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latest edition, Pagarnonon Press and references cited therein.

25 The initial dosages can also be estimated from in vivo data. Animal models useful for testing the efficacy of the compounds for treating or preventing the different diseases described above are well known in the art. Appropriate animal models of hypersensitivity or allergic reactions are described in Foster, 1995, Allergy 50 (Suppl. 21): 6-9, discussion 34-38 and Tumas et al., 2001, J. Allergy Clin. Immunol 107 (6): 1025-1033. Appropriate animal models of allergic rhinitis are described in 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. Appropriate animal models of allergic conjunctivitis are described in Carreras et al., 1993, Br. J. Opthalmol. 77 (8): 509-514; Saiga et al. 1992, Opthalmic Res. 24 (1): 45-50; and Kunert et al., 2001, Invest. Opthalmol Vis. Sci. 42 (1 1): 2483-2489. Appropriate animal models of mastocytosis

35 systemically described in 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. Appropriate animal models of hyper IgE syndrome are described in Claman et al., 1990, Clin. Immunol Immunopathol 56 (1): 46-53. Appropriate animal models of B-cell lymphoma are described in Hough et al., 1998, Proc. Natl Acad. Sci. USA 95: 13853-13858 and Hakin et al., 1996, J. Immunol. 157 (12): 55035511. Appropriate animal models of atopic disorders such as atopic dermatitis, atopic eczema and atopic asthma are described in 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. Skilled workers can routinely adapt such information to determine appropriate dosages for administration to humans. Additional appropriate animal models are described in the Examples section.

Four. Five Normally, the dosage amounts are in the range of about 0.0001 or 0.001 or 0.01 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending, among other factors, on the activity of the compound, its bioavailability, mode of administration and various factors discussed above. The dosage amount and interval can be adjusted individually to provide plasma levels of the compound (s) that are sufficient to maintain the therapeutic or prophylactic effect. For example, the compounds can be administered once a week, several times a week (for example, on alternate days), once a day or several times a day, depending, among others, on the mode of administration, the specific indication object of treatment and the judgment of the doctor responsible for the prescription. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of the active compound (s) may not be referred to the plasma concentration. The

55 Skilled workers will be able to optimize effective local dosages without unnecessary experimentation.

Preferably, the compound (s) provides a therapeutic or prophylactic benefit without causing substantial toxicity. The toxicity of the compound (s) can be determined using conventional pharmaceutical procedures. The dosage ratio between the toxic and therapeutic (or prophylactic) effect is the therapeutic index. The compound (s) exhibiting high therapeutic indices are preferred.

The above aspects and other aspects of the present invention will be better understood in relation to the following representative examples.

65 Examples

Example 1. 5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [3-oxo-benzo [1,4] thiazin-6-yl] -2,4-pyrimidinediamine

5 A mixture of 40 mg of 2-chloro-5-fluoro-N4- [3-oxo-benzo [1,4] thiazin-6-yl] -4-pyrimidinediamine and 48 mg of 3-hydroxy hydrochloride salt was heated 4,5-dimethioxyaniline in 700 µl of EtOH in the microwave at 180 ° C for 1 hour. The precipitate was collected by means of suction filtration, dried, suspended in deionized water and the pH adjusted to 5 with a dilute sodium bicarbonate solution, brine was added and subsequently subjected to

10 Ultrasonic treatment the suspension briefly, the solid was collected by suction filtration and dried resulting in a 43% yield of the desired product 5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [3oxo-bezno [1,4] thiazin-6-yl] -2,4-pyrimidinediamine. 1H NMR (DMSO-d6): 8.06 (d, 1H, J = 2.7 Hz), 7.58 (s, 1H), 7.40 (dd, 1H, J = 8.4 Hz, J = 1.5 Hz), 7.19 (s, 1H), 6.92 (s, 1H), 6.76 (d, 1H, J = 1.5 Hz), 3.60 (s, 3H) , 3.58 (s, 3H), 3.43 (s, 2H); purity 92%; MS (m / e): 444 (MH +).

fifteen Examples 2-13 were prepared according to the procedure of Example 1.

Example 2. N4- [2,2-dimethyl-3-oxo-benzo [1,4] thiazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2.4 -pyrimidine

1H NMR (DMSO-d6): 5 8.07 (d, 1H, J = 3.3 Hz), 7.67 (s, 1H), 7.46 (dd, 1H, J = 9 Hz, J = 2.1 Hz), 7.18 (d, 1H, J = 9 Hz), 6.92 (s, 1H), 6.78 (d, 1H, J = 2.1 Hz), 3.62 (s , 3H), 3.58 (s, 3H), 1.35 (s, 6H); purity 94%; MS (m / e): 472 (MH +).

25 Example 3. N4- [2,2-dimethyl-3-oxo-benz [1,4] oxazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2.4 -pyrimidindiamine

1H NMR (DMSO-d6): 8.3 (d, 1H, J = 3.9 Hz), 7.38 (dd, 1H, J = 9 Hz, J = 2.1 Hz), 7.29 (d, 1H, J = 1.8 Hz), 6.81 (m, 30 2H), 6.77 (d, 1H, J = 2.1 Hz), 3.59 (s, 3H), 3, 58 (s, 3H), 1.38 (s, 6H); 95% purity; MS (m / e): 455 (MH +).

Example 4. N4- [2,2-dimethyl-3-oxo-pyrid [1,4] oxazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2.4 -pyrimidindiamine

35 1H NMR (DMSO-d6): 8.09 (d, 1H, J = 3.6 Hz), 7.72 (d, 1H, J = 8.1 Hz), 7.31 (d, 1H, J = 8.1 Hz), 6.91 (d, 1H, J = 2.7 Hz), 6.75 (d, 1H, J = 2.7Hz), 3.63 (s, 3H), 3, 58 (s, 3H), 1.41 (s, 6H); purity 96%; MS (m / e): 457 (MH +).

Example 5. 5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [3-oxo-benz [1,4] oxazin-6-yl] -2,4-pyrimidinediamine

1H NMR (DMSO-d6): 5 8.02 (d, 1H, J = 3.9 Hz), 7.34 (dd, 1H, J = 9 Hz, J = 2.1 Hz), 7.25 (d, 1H, J = 1.8 Hz), 6.84 (m, 2H), 6.78 (d, 1H, J = 2.1 Hz), 4.52 (s, 2H), 3.57 (s, 3H), 3.56 (s, 3H); purity 97%; MS (m / e): 428 (MH +).

Example 6. N4- [2,2-Difluoro-3-oxo-benz [1,4] oxazin-6-yl] -5-fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -2.4 -pyrimidindiamine

1H NMR (DMSO-d6): 5 8.15 (d, 1H, J = 3.6 Hz), 7.68 (dd, 1H, J = 9.3 Hz, J = 2.1 Hz), 7 , 58 (d, 1H, J = 2.1 Hz), 7.25 5 (d, 1H, J = 9.3 Hz), 6.93 (d, 1H, J = 2.1 Hz), 6, 84 (d, 1H, J = 2.7 Hz), 3.64 (s, 3H), 3.63 (s, 3H); 95% purity; MS (m / e): 464 (MH +).

Example 7. 5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [3-oxo-4- (2-pyridylmethyl) -benz [1,4] oxazin-6-yl] -2 , 4pyrimidinediamine

= 3.9 H ), 8.07 (d, 1H, J = 4.2 Hz), 7.70 (m, 1H), 7.38 (m, 2H), 7.26 (m, 2H) , 6.96 (d, 1H, J = 8.7 Hz), 6.68 (m, 2H), 5.08 (s, 2H), 4.76 (s, 2H), 3.61 (s, 3H), 3.59 (s, 3H); 95% purity; MS (m / e): 15 519 (MH +).

Example 8. 5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- (1,3- (2H) -4,4-dimethylisoquinolindione-7-yl) -2,4-pyrimidinediamine

1H NMR (DMSO-d6): 8.36 (m, 1H), 8.10 (m, 2H), 7.56 (d, 1H, J = 9.0 Hz), 6.86 (d, 1H, J = 2.4 Hz), 6.745 (d, 1H, J = 2.7 Hz), 3.58 (s, 3H), 3.54 (s, 3H), 1.51 (s, 6H) ; purity 93%; MS (m / e): 468 (MH +).

25 Example 9. (R / S) -5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [2-methyl-3-oxo-4- (4-methoxybenzyl) -benz [1, 4] oxazin-6-yl] 2,4-pyrimidinediamine

30 1H NMR (DMSO-d6): 8.02 (d, 1H, J = 3.2 Hz), 7.44 (m, 2H), 7.06 (m, 2H), 6.92 (m, 2H), 6.76 (m, 3H), 4.86 (s, 2H), 4.79 (c, 1H, J = 7.2 Hz), 3.64 (s, 3H), 3.63 ( s, 3H), 3.54 (s, 3H), 1.47 (d, 3H, J = 7.2 Hz); purity 92%; MS (m / e): 562 (MH +).

Example 10. (R / S) -5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- [2-methyl-3-oxo-4- (4-methoxybenzyl) -benzo [1, 4] thiazin-635 il] -2,4-pyrimidinediamine

1H NMR (DMSO-d6): 5 8.06 (d, 1H, J = 3.3 Hz), 7.59 (m, 2H), 7.26 (d, 1H, J = 10.8 Hz) , 6.99 (m, 2H), 6.76 (m, 4H), 4.94 (s, 2H), 3.75 (c, 1H, J = 7.2 Hz), 3.63 (s, 3H), 3.58 (s, 3H), 3.55 (s, 3H), 1.36 (d, 3H, J = 7.2 Hz); purity 90%; MS (m / e): 578 (MH +).

Example 11. 5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- (2,2,4-trimethyl-1,1,3-trioxo-benzo [1,4] thiazin-6- il) -2,4 pyrimidinediamine

10 1H NMR (DMSO-d6): 8.22 (d, 1H, 3.3 Hz), 8.04 (d, 1H, J = 2.1 Hz), 7.92 (dd, 1H, J = 8.4 Hz, J = 2.1 Hz), 7.52 (d, 1H, J = 8.4 Hz), 6.85 (d, 1H, J = 2.1 Hz), 6.71 (d , 1H, J = 2.1 Hz), 3.59 (s, 3H), 3.58 (s, 3H), 3.25 (s, 3H), 1.41 (s, 6H); purity 98%; MS (m / e): 518 (MH +).

fifteen Example 12. 5-Fluoro-N2- (3-hydroxy-4,5-dimethoxyphenyl) -N4- (4-methyl-3-oxo-benzo [1,4] thiazin-6-yl) -2,4-pyrimidinediamine

1H NMR (DMSO-d6): 8.09 (d, 1H, 3.6 Hz), 7.71 (d, 1H, J = 1.8 Hz), 7.67 (dd, 1H, J = 8.1 Hz, J = 2.1 Hz), 7.28

twenty (d, 1H, J = 8.1 Hz), 6.83 (d, 1H, J = 1.8 Hz), 6.75 (d, 1H, J = 2.1 Hz), 3.58 (s , 3H), 3.57 (s, 3H), 3.47 (s, 2H), 3.18 (s, 3H); 95% purity; MS (m / e): 458 (MH +).

Example 13. N4- (3,4-Dihydro-2H-2,2-dimethyl-5-pyrido [1,4] oxazin-6-yl) -N2- [3,4-dimethoxy-5-hydroxyphenyl] -5 -fluoro-2,4-pyrimidinediamine

1H NMR (DMSO-d6): 5 8.92 (s, 1H), 8.89 (s, 1H), 8.64 (s, 1H), 8.02 (d, 1H, J = 3.5 Hz), 7.33 (d, 1H, J = 8.2 Hz), 6.92 (d, 1H, J = 2.3 Hz), 6.88 (d, 1H, J = 8.2 Hz) , 6.78 (d, 1H, J = 8.2 Hz), 6.56 (s, 1H), 3.63 (s, 3H), 3.59 (s, 3H),

30 3.12 (d, 1H, J = 2.3 Hz), 1.24 (s, 6H). LCMS: retention time: 9.36 min .; purity: 97%; MS (m / e): 443 (MH +).

Inhibition of degranulation with FcεRI receptor mediation

The capacity of 2,4-pyrimidinediamine compounds of the invention is demonstrated by inhibiting degranulation

35 IgE-induced in a variety of cell assays with cultured human mast cells (CHMC) and / (or cells from mouse bone marrow (BMMC). Inhibition of degranulation at both high and low cell density is measured by quantification of the release of tryptase from the specific factors of granule, histamine and hexosaminidase. The inhibition of the release and / or synthesis of lipid mediators is evaluated by measuring the release of leukotriene LTC4 and the inhibition of the release and / or synthesis of

40 cytokine through the quantification of TNF-α, IL-6 and IL-13. Tryptase and hexosaminides are quantified using fluorogenic substrates as described in their respective examples. Histamine, TNFa, IL-6, IL-13 and LTC4 are quantified using the following commercial ELISA kits: histamine (Immunotech No.: 2015, Beckman Coulter), TNFα (Biosource No.: KHC 3011), IL-6 (Biosource No.: KMC0061), IL-13 (Biosource No.: KHC0132) and LTC4 (Cayman Chemical No.: 520211). The protocols of various tests are provided below.

Four. Five Cultivation of mast cells and basophilic cells

Mast cells and basophil cells of CD34-negative progenitor cells are subjected to culture as described below (see also the methods described in the related application of US Serial No. 10 / 053,355,

fifty filed on November 8, 2001, the description of which is incorporated by reference herein).

Preparation of STEMPRO-34 complete medium Preparing STEMPRO-34 complete medium ("CM"), 250 ml of STEMPRO-34TM serum free medium ("SFM"; GibcoBRL, Catalog No. 10640) was added to a filter flask . To this, 13 ml of STEMPRO-34 Nutrient Supplement ("NS"; GibcoBRL, Catalog No. 10641) (prepared as described in more detail, below) was added. The NS vessel was washed with approximately 10 ml of SFM and the wash was added to the flask with

5 filter. After the addition of 5 ml of L-glutamine (200 mM; Mediatech, Catalog No. MT 25-005-CI) and 5 ml of penicillin 100X / streptomycin ("pen-strep"; HyClone, Catalog No. SV30010), the volume was brought to 500 ml with SFM and the solution was filtered.

The most variable aspect of the CM preparation is the method by which NS is thawed and mixed before the addition of SFM. You should thaw NS in a 37 ° C water bath and generate eddies, without agitation or vortexing, until the complete solution is achieved. At the time that eddies are generated, it is necessary to appreciate if there are lipids that are not yet in the solution. If lipids are present and NS lacks a uniform appearance, it is necessary to return to the water bath and repeat the swirling process until a uniform appearance is achieved. Sometimes, the component goes into solution immediately, sometimes after a couple of cycles of

fifteen swirling and sometimes nothing at all. If after a couple of hours NS is not yet in the solution, a new unit is discarded and thawed. NS that seems non-uniform after defrosting should not be used.

CD34 + cell expansion

A starting population of CD34-positive (CD34 +) cells from a relatively small number (1-5 x 106 cells) was expanded to a relatively large number of CD34-negative progenitor cells (approximately 2-4 x 109 cells) using the medium of culture and the methods described below. CD34 + cells (from an individual donor) were obtained from Allcells (Berkeley, CA). Because there is a degree of variation in the

25 quality and number of CD34 + cells normally provided by Allcells, the newly supplied cells were transferred to a 15 ml conical tube and carried up to 10 ml in CM before use.

On day 0, a viable cell count (bright-phase) was carried out and the cells were centrifuged at 1200 rpm to settle. The cells were resuspended to a density of 275,000 cells / ml, containing CM 200 ng / ml of Recombinant Human Stem Cell Factor ("SCF"; Peprotech, Catalog No. #: 300-07) and 20 ng / ml of human flt-3 ligand (Peptrotech, Catalog No. 300-19) ("CSM / SCF / flt-3 medium"). Approximately on day 4 or 5, the density of the culture was checked by carrying out a cell count and the culture was diluted to a density of 275,000 cells / ml with fresh CM / SCF / flt-3 medium. Approximately on day 7, the culture was transferred to a sterile tube and a cell count was carried out. The cells were centrifuged at 1200 rpm

35 and resuspended to a density of 275,000 cells / ml with fresh CM / SCF / flt-3 medium.

This cycle was repeated, starting on day 0, a total of 3-5 times during the expansion period.

When the culture is large and held in multiple flasks and has to be resuspended, the contents of all flasks are combined in an individual container before carrying out a cell count. This ensures that a precise cell count is achieved and a degree of uniformity of treatment of the entire population is provided. The contamination of each flask is checked separately under a microscope before combining avoiding contamination of the entire population.

Four. Five Between days 17-24, the culture may begin to decay (that is, approximately 5-10% of the total number of cells dies) and the failure occurs as to the expansion rapidly as previously. Subsequently, the cells are monitored on a daily basis during this time, as the complete failure of the culture takes place in a period as short as 24 hours. Once the decay has begun, the cells are counted, centrifuged at 850 rpm for 15 minutes and resuspended at a density of 350,000 cells / ml in CM / SCF / flt-3 medium inducing one or more divisions of the culture. The cells are controlled daily avoiding the failure of the culture.

When more than 15% cell death is evident in the culture of progenitor cells and some residues are present in the culture, the CD34-negative progenitor cells are ready for

55 differentiate.

Differentiation of CD34-negative progenitor cells in mucosal mast cells

A second phase is carried out by converting the expanded CD34-negative progenitor cells into differentiated mucosal mast cells. These human mucosal cultured mast cells ("CHMC") are derived from CD34 + cells isolated from blood from the umbilical cord and are treated forming a proliferated population of CD34-negative progenitor cells, as described above. Producing negative CD34 progenitor cells, the culture resuspension cycle was the same as described above, except that the culture was sown at a density of 425,000 cells / ml and an additional 15% medium was added approximately at day 65 four or five without carry out a cell count. Likewise, the cytokine composition of the medium was modified to contain SCF (200 ng / ml) and recombinant human IL-6 (200 ng / ml; Peprotech, No. of

Catalog 200-06 reconstituted up to 100 μg / ml in sterile 10 mM acetic acid) (“CM / SCF / IL-6 medium”).

Phases I and II were centrifuged together approximately 5 weeks. During weeks 1-3 some cases of death and crop residues were evident and there is a period during weeks 2-5, over which a small percentage of the crop is no longer in suspension, but rather joins the surface of the culture vessel.

Just as during Phase I, when the culture has to be resuspended on day seven of each cycle, all the contents of the flasks are combined in an individual container before carrying out a cell count in order to ensure uniformity. of the entire population. The contamination of each flask is checked separately under a microscope before the combination in order to avoid contamination of the entire population.

When the flasks are combined, approximately 75% of the volume is transferred to the communal vessel, leaving approximately 10 ml or the like in the flask. The flask containing the volume that was taking off the bound cells was suddenly and laterally shaken. The shake was repeated at the correct angle with respect to the first shake, completely taking off the cells.

The flask was tilted at an angle of 45 degrees for a couple of minutes before transferring the remaining volume to the counting vessel. The cells were centrifuged at 950 rpm for 15 min before seeding at 35-50 ml per 20 flask (at a density of 425,000 cells / ml).

Differentiation of CD34-negative progenitor cells in mast cells of connective tissue type

A proliferated population of CD34-negative progenitor cells is prepared as discussed above.

25 and are treated by forming a positive tryptase / chymase phenotype (connective tissue). The methods are performed as described above for mucosal mast cells, but with the substitution of IL-6 for IL-4 in the culture medium. The cells obtained are typical of connective tissue mast cells.

Differentiation of CD34-negative progenitor cells in basophilic cells

30 A differentiated population of CD34-negative progenitor cells is prepared as described above and used to form a proliferated population of basophilic cells. CD34-negative cells are treated as described for mucosal mast cells, but with the substitution of IL-3 (at 20-50 ng / ml) with IL-6 in the culture medium.

35 CHMC low cell density IgE activation: tryptase and LTC4 assays

Duplicating 96-well U-shaped bottom plates (Costar 3799) add 65 μl of compound dilutions or control samples that have been prepared in MT [137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, MgCl2 1.0 mM, 40.6 mM Glucose, 20 mM Hepes (pH 7.4), 0.1% bovine serum albumin, (Sigma A4503)] containing 2% MeOH and 1% DMSO. CHMC cells were pelleted (980 rpm, 10 min.) And resuspended in preheated MT. Add 65 μl of cells to each 96-well plate. Depending on the degranulation activity for each particular CHMC donor, load 1000-1500 cells / well. Mix four times in a row for incubation for 1 hour at 37 ° C. During the 1 hour incubation, prepare a solution of 6X anti-IgE [rabbit anti-human IgE (1 mg / ml, 45 Bethyl Laboratories A80-109A) diluted 1: 167 in MT buffer]. Stimulate the cells by adding 25 µl of 6X anti-IgE solution to the appropriate plates. Add 25 μl of MT to the non-stimulated control wells. Mix twice after the addition of anti-IgE. Incubate at 37 ° C for 30 minutes. During the 30-minute incubation, dilute the 20 mM tryptase substrate stock solution [(Z-Ala-Lys-Arg-AMC2TFA; Enzyme Systems Products, No.: AMC-246)] 1: 2000 in assay buffer Tryptase [0.1 M Hepes (pH 7.5), 10% glycerol weight / volume, heparin

fifty 10 μM (Sigma H-4898) 0.01% NaN3]. Centrifuge plates at 1000 rpm for 10 minutes until cells settle. Transfer 25 μl of supernatant to a 96-well plate with black bottom and add 100 μl of diluted tryptase substrate solution back to each well. Incubate the plates at room temperature for 30 minutes. Read the optical density of the plates at 355 nm / 460 nm in a spectrometric plate reader.

55 Leukotriene C4 (LTC4) is also quantified using an ELISA kit on appropriately diluted supernatant samples (empirically determined for each donor cell population so that the sample measurement falls within the standard curve) following the manufacturer's instructions.

CHMC high cell density IgE activation: degranulation assays (tryptase, histamine), leukotriene 60 (LTC4) and cytokine (TNFalfa, IL-13)

Cultured human mast cells (CHMC) were sensitized for 5 days with IL-4 (20 ng / ml), SCF (200 ng / ml), IL-6 (200 ng / ml) and human IgE (CP 1035K from Cortx Biochem , 100-500 ng / ml depending on the generation) in CM medium. After sensitization, the cells are counted, sedimented (1000 rpm, 5-10 minutes) and resuspended at 1-2 x 10 6 cells / ml in MT buffer. Add 100 µl of cell suspension to each well and 100 µl of compound dilutions. The final vehicle concentration is 0.5% DMSO. Incubate at 37 ° C (5% CO2) for 1 hour. After 1 hour of treatment of the compound, stimulate the cells with 6X anti-IgE. Mix the wells with the cells and allow the plates to incubate at 37 ° C (5% CO2) for one hour. After 1 hour of incubation, sediment the cells (10 minutes, 1000 rpm) and collect 200 μl per well of the supernatant, being careful not to alter the sediment. Place the supernatant plate on ice. During the 7 hour step (see below) carry out the tryptase test on the supernatant that had been diluted 1: 500. Resuspend the cell pellet in 240 μl of CM medium containing 0.5% DMSO and the corresponding product concentration. Incubate CHMC cells for 7 hours at 37 ° C (5% CO2). After incubation, sediment the cells (1000 rpm, 10 minutes) and collect 225 μl per well and place at -80 until they are ready to

10 carry out ELISA. ELISAs are carried out in appropriately diluted samples (empirically determined for each population of donor cells so that the sample measurement falls within the standard curve) following the supplier's instructions.

Inhibition of the upstream IgE receptor cascade

fifteen Assays for ionomycin-induced mast cell degranulation are performed as described in the CHMC Low Density IgE Activation assays, with the exception that for 1 hour of incubation, a 6X ionomycin solution [ionomycin 5 nM (Sigma 1-0634) in MeOH (stock) diluted 1: 416.7 in TM buffer (2 µM final)] and cells were stimulated by adding 25 µl of 6X ionomycin solution to

twenty the appropriate plates.

Inhibition of Syks kinase in biochemical assays

The capacity of the compounds is tested by inhibiting phosphorylation of a catalyzed peptide substrate

25 by Syk kinase in a biochemical polarization assay subjected to fluorescence with isolated Syk kinase. In this experiment, the compounds are diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg / ml acetylated bovine gamma globulin ). The compound is mixed in 1% DMSO (final 0.2% DMSO) with ATP / substrate solution at room temperature. Syk kinase (Upstate, Lake Placid NY) is added to a final reaction volume of 20 μl and the reaction is incubated for 30 minutes at temperature

30 ambient. The final conditions of the enzyme reaction were 20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg / ml acetylated bovine gamma globulin, 0.125 ng Syk, 4 μM ATP , 2.5 μM peptide substrate (biotin-EQEDEPEGDYEEVLE-CONH2, SynPep Corporation). EDTA (final 10 nM) / anti-phosphotyrosine antibody (final 1X) / fluorescent phosphopeptide tracer (final 0.5X) in FP Dilution Buffer is stopped by stopping the reaction for a total volume of 40 μl according to the manufacturer's instructions (PanVera Corporation). He

35 incubate this plate for 30 minutes in the dark at room temperature. The plates are read in a Polarion fluorescence polarization plate reader (Tecan). The data is converted to the amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Test Kit, Green (PanVera Corporation).

40 When LD Triptase was tested, all 3-hydroxyphenyl-2,4-pyrimidinediamine compounds of Examples 1-13 were found to have an activity less than 5 µM in the assay, as shown in Table 1 below, in the A indicates an activity less than 1 μM and B indicates an activity less than 5 μM. In addition, the 2-hydroxyphenyl compounds that were tested for their 3,4,5-trimethoxyphenyl counterparts showed between about 10% and 500% improved potency.

Four. Five Table 1 12

<dl><dt>Example of Compound No. </dt><dd>LD Triptase, CHMC, IgE, 8 pt </dd></dl>

<dl><dt>1 </dt><dd>TO </dd></dl>

<dl><dt>2 </dt><dd>TO </dd></dl>

<dl><dt>3 </dt><dd>TO </dd></dl>

<dl><dt>4 </dt><dd>TO </dd></dl>

<dl><dt>5 </dt><dd>TO </dd></dl>

<dl><dt>6 </dt><dd>TO </dd></dl>

<dl><dt>7 </dt><dd>B </dd></dl>

<dl><dt>8 </dt><dd>TO </dd></dl>

<dl><dt>9 </dt><dd>B </dd></dl>

<dl><dt>10 </dt><dd>B </dd></dl>

<dl><dt>11 </dt><dd>TO </dd></dl>

TO

TO

Although the above invention has been described in some detail to facilitate compression, it is evident that certain changes and modifications can be made within the scope of the appended claims. Accordingly, the described embodiments are considered to be illustrative and not restrictive and the invention is not limited to the details given herein, but can be modified within the scope of the appended claims.