Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

• the present invention relates to a novel class of kinase inhibitors, including pharmaceutically acceptable salts, prodrugs and metabolites thereof, which are useful for modulating protein kinase activity for modulating cellular activities such as signal transduction, proliferation, and cytokine secretion. More specifically the invention provides compounds which inhibit, regulate and/or modulate kinase activity, in particular JAK3 activity, and signal transduction pathways relating to cellular activities as mentioned above. Furthermore, the present invention relates to pharmaceutical compositions comprising said compounds, for example for the treatment or prevention of an immunological, inflammatory, autoimmune, or allergic disorder or disease or a transplant rejection or a Graft-versus host disease and processes for preparing said compounds.
• Protein kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides and other cellular metabolites and play key roles in all aspects of eukaryotic cell physiology. Especially, protein kinases and lipid kinases participate in the signaling events which control the activation, growth, differentiation and survival of cells in response to extracellular mediators or stimuli such as growth factors, cytokines or chemokines. In general, protein kinases are classified in two groups, those that preferentially phosphorylate tyrosine residues and those that preferentially phosphorylate serine and/or threonine residues. The tyrosine kinases include membrane-spanning growth factor receptors such as the epidermal growth factor receptor (EGFR) and cytosolic non-receptor kinases such as Janus kinases (JAK).
• EGFR epidermal growth factor receptor
• JAK Janus kinases
• JAK Janus kinase
• JAK2 JAK2
• JAK3 Tyrosine kinase 2
• JAK proteins bind to cytokine receptors through their amino-terminal FERM (Band-4.1, ezrin, radixin, moesin) domains. After the binding of cytokines to their receptors, JAKs are activated and phosphorylate the receptors, thereby creating docking sites for signalling molecules, especially for members of the signal transducer and activator of transcription (Stat) family (Yamaoka et al, 2004. The Janus kinases (Jaks). Genome Biology 5(12): 253).
• JAKl, JAK2 and TYK2 are ubiquitously expressed.
• JAK3 is predominantly in hematopoietic cells and it is highly regulated with cell development and activation (Musso et al., 1995. 181(4): 1425-31).
• JAK-deficient cell lines and gene-targeted mice have revealed the essential, nonredundant functions of JAKs in cytokine signalling. JAKl knockout mice display a perinatal lethal phenotype, probably related to the neurological effects that prevent them from sucking (Rodig et al, 1998. Cell 93(3):373-83).
• JAK3 deficiency was first identified in humans with autosomal recessive severe combined immunodeficiency (SCID) (Macchi et al, 1995. Nature 377(6544):65-68).
• SCID autosomal recessive severe combined immunodeficiency
• JAK3 knockout mice too exhibit SCID but do not display non-immune defects, suggesting that an inhibitor of JAK3 as an immunosuppressant would have restricted effects in vivo and therefore presents a promising drug for immunosuppression (Papageorgiou and Wikman 2004, Trends in Pharmacological Sciences 25(11):558-62).
• JAK3 Activating mutations for JAK3 have been observed in acute megakaryoblastic leukemia (AMKL) patients (Walters et al, 2006. Cancer Cell 10(l):65-75). These mutated forms of JAK3 can transform Ba/F3 cells to factor-independent growth and induce features of megakaryoblastic leukemia in a mouse model.
• JAK3 inhibitors have been reported in the literature which may be useful in the medical field (O'Shea et al, 2004. Nat. Rev. Drug Discov. 3(7):555-64).
• a potent JAK3 inhibitor (CP-690,550) was reported to show efficacy in an animal model of organ transplantation (Changelian et al, 2003, Science 302(5646):875-888) and clinical trials (reviewed in: Pesu et al, 2008. Immunol. Rev. 223, 132-142).
• the CP-690,550 inhibitor is not selective for the JAK3 kinase and inhibits JAK2 kinase with almost equipotency (Jiang et al, 2008, J. Med. Chem. 51(24):8012-8018). It is expected that a selective JAK3 inhibitor that inhibits JAK3 with greater potency than JAK2 may have advantageous therapeutic properties, because inhibition of JAK2 can cause anemia (Ghoreschi et al, 2009. Nature Immunol. 4, 356-360).
• a selective JAK3 inhibitor that inhibits JAK3 with greater potency than other kinases may have advantageous therapeutic properties because the inhibition of other kinases may cause unwanted side effects.
• selectivity versus members of the Aurora kinase family may be important.
• Aurora kinases are only expressed and active as kinases during mitosis and inhibitors may therefore have an effect on proliferating normal cells such as epithelial cells of the oral and gut mucosa (Keen and Taylor, 2004. Nat. Rev. Cancer. 4(12):927-36).
• Pyrimidine derivatives exhibiting JAK3 and JAK2 kinase inhibiting activities are described in WO-A 2008/009458.
• WO-A 2008/094602 relates to pyrazolopyrimidine as modulator of mitotic kinases.
• WO-A 2006/074985 relates to 5-membered, annelated hetorocyclic pyrimidines as kinase inhibitors.
• US-A 2009/0203688 relates to pyrolopyrimidine compounds useful in one or more Protein tyrosine kinase mediated diseases.
• JAK inhibitors are known in the art there is a need for providing additional JAK inhibitors having at least partially more effective pharmaceutically relevant properties, like activity, selectivity especially over JAK2 kinase, and ADME properties.
• an object of the present invention is to provide a new class of compounds as JAK inhibitors which preferably show selectivity over JAK2 and may be effective in the treatment or prophylaxis of disorders associated with JAK.
• R is H or F
• Ring A is a 5 membered aromatic heterocycle in which Z 1 , Z 2 and Z 3 are independently selected from the group consisting of C(R 1 ), N, ⁇ R 1 ), O and S, provided that at least one of Z 1 , Z 2 , Z 3 is N;
• Each R 1 is independently H, halogen; CN; C(0)OR 2 ; OR 2 ; C(0)R 2 ; C(0)N(R 2 R 2a );
• T 1 is C3-7 cycloalkyl; or saturated 4 to 7 membered heterocyclyl, wherein T 1 is optionally substituted with one or more R 10 , which are the same or different;
• Y is (C(R 5 R 5a )) n ; n is O, 1, 2, 3 or 4;
• R 5 , R 5a are joined to form an unsubstituted C3-7 cycloalkyl
• X 1 is C(R 6 ) or N
• X 2 is C(R 6a ) or N
• X 3 is C(R 6b ) or N
• X 4 is C(R 6c ) or N
• X 5 is C(R 6d ) or N, provided that at most two of X 1 , X 2 , X 3 , X 4 , X 5 are N;
• R 6 , R 6a , R 6b , R 6c , R 6d are independently selected from the group consisting of H; halogen; CN; C(0)OR 7 ; OR 7 ; C(0)R 7 ; C(0)N(R 7 R 7a ); S(0) 2 N(R 7 R 7a ); S(0)N(R 7 R 7a ); S(0) 2 R 7 ; S(0)R 7 ; N(R 7 )S(0) 2 N(R 7a R 7b ); N(R 7 )S(0)N(R 7a R 7b ); SR 7 ; N(R 7 R 7a ); N0 2 ; OC(0)R 7 ; N(R 7 )C(0)R 7a ; N(R 7 )S(0) 2 R 7a ; N(R 7 )S(0)R 7a ; N(R 7 )C(0)N(R 7a R 7b ); N(R 7 )C(0)OR 7a ; OC(0)N(R 7 R 7a ); T 2 ; Ci
• R 7 , R 7a , R 7b are independently selected from the group consisting of H; T 2 ; Ci_6 alkyl; C 2 -6 alkenyl; and C 2 -6 alkynyl, wherein Ci-6 alkyl; C 2 -6 alkenyl; and C 2 -6 alkynyl are optionally substituted with one or more R 8 , which are the same or different;
• R 8 is halogen; CN; C(0)OR 9 ; OR 9 ; C(0)R 9 ; C(0)N(R 9 R 9a ); S(0) 2 N(R 9 R 9a ); S(0)N(R 9 R 9a ); S(0) 2 R 9 ; S(0)R 9 ; N(R 9 )S(0) 2 N(R 9a R 9b ); N(R 9 )S(0)N(R 9a R 9b ); SR 9 ; N(R 9 R 9a ); N0 2 ; OC(0)R 9 ; N(R 9 )C(0)R 9a ; N(R 9 )S(0) 2 R 9a ; N(R 9 )S(0)R 9a ; N(R 9 )C(0)N(R 9a R 9b ); N(R 9 )C(0)OR 9a ; OC(0)N(R 9 R 9a ); or T 2 ;
• R 9 , R 9a , R 9b are independently selected from the group consisting of H; T 2 ; Ci-6 alkyl; C 2 -6 alkenyl; and C 2 -6 alkynyl, wherein Ci-6 alkyl; C 2 -6 alkenyl; and C 2 -6 alkynyl are optionally substituted with one or more R 12 , which are the same or different;
• T 2 is phenyl; naphthyl; indenyl; indanyl; C3-7 cycloalkyl; 4 to 7 membered heterocyclyl; or 7 to 1 1 membered heterobicyclyl, wherein T 2 is optionally substituted with one or more R 17 , which are the same or different;
• T 3 is phenyl; C3-7 cycloalkyl; or 4 to 7 membered heterocyclyl, wherein T 3 is optionally substituted with one or more R 18 , which are the same or different;
• R 21 , R 21a , R 21b are independently selected from the group consisting of H; Ci_6 alkyl; C 2 - 6 alkenyl; and C 2 - 6 alkynyl, wherein Ci_6 alkyl; C 2 - 6 alkenyl; and C 2 - 6 alkynyl are optionally substituted with one or more halogen, which are the same or different, provided that at least one of R 6 , R 6a , R 6b , R 6c , R 6d is R 6e , wherein R 6e is T 2 ; C(0)T 2 ; N(R 7 )T 2 ; or C 1 -6 alkyl substituted with one or more T 2 .
• variable or substituent can be selected from a group of different variants and such variable or substituent occurs more than once the respective variants can be the same or different.
• Alkyl means a straight-chain or branched hydrocarbon chain. Each hydrogen of an alkyl carbon may be replaced by a substituent as further specified.
• Alkenyl means a straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon double bond. Each hydrogen of an alkenyl carbon may be replaced by a substituent as further specified.
• Alkynyl means a straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon triple bond. Each hydrogen of an alkynyl carbon may be replaced by a substituent as further specified.
• Ci-4 alkyl means an alkyl chain having 1 - 4 carbon atoms, e.g. if present at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or e.g.
• C 1 -4 alkyl carbon when two moieties of a molecule are linked by the alkyl group.
• Each hydrogen of a C 1 -4 alkyl carbon may be replaced by a substituent as further specified.
• Ci-6 alkyl means an alkyl chain having 1 - 6 carbon atoms, e.g.
• C 1-4 alkyl methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl; tert-butyl, n-pentyl, n-hexyl, or e.g. -CH 2 -, -CH 2 -CH 2 -, -CH(CH 3 )-, -CH 2 -CH 2 -CH 2 -, -CH(C 2 H 5 )-, - C(CH 3 ) 2 -, when two moieties of a molecule are linked by the alkyl group.
• Each hydrogen of a Ci-6 alkyl carbon may be replaced by a substituent as further specified.
• Each hydrogen of a C 2 - 6 alkenyl carbon may be replaced by a substituent as further specified.
• C 2- 6 alkynyl means an alkynyl chain having 2 to 6 carbon atoms, e.g. if present at the end of a molecule: -C ⁇ CH, -CH 2 -C ⁇ CH, CH 2 -CH 2 -C ⁇ CH, CH 2 -C ⁇ C-CH 3 , or e.g. -C ⁇ C- when two moieties of a molecule are linked by the alkynyl group.
• Each hydrogen of a C 2 - 6 alkynyl carbon may be replaced by a substituent as further specified.
• C 3 - 7 cycloalkyl or "C 3-7 cycloalkyl ring” means a cyclic alkyl chain having 3 - 7 carbon atoms, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl.
• cyloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.
• Each hydrogen of a cycloalkyl carbon may be replaced by a substituent as further specified.
• the term "C 3-5 cycloalkyl” or "C 3 - 5 cycloalkyl ring” is defined accordingly.
• Halogen means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.
• Examples for a 4 to 7 membered heterocycles are azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyr
• “Saturated 4 to 7 membered heterocyclyl” or “saturated 4 to 7 membered heterocycle” means fully saturated “4 to 7 membered heterocyclyl” or "4 to 7 membered heterocycle”.
• heterocycles are furan, thiophene, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, thiadiazole, triazole, tetrazole.
• Examples for a 7 to 1 1 membered heterobicycle are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine or pteridine.
• the term 7 to 11 membered heterobicycle also includes spiro structures of two rings like 2-oxa-6-azaspiro[3.3]heptane or
• Preferred compounds of formula (I) are those compounds in which one or more of the residues contained therein have the meanings given below, with all combinations of preferred substituent definitions being a subject of the present invention. With respect to all preferred compounds of the formula (I) the present invention also includes all tautomeric and stereoisomeric forms and mixtures thereof in all ratios, and their pharmaceutically acceptable salts.
• substituents mentioned below independently have the following meaning. Hence, one or more of these substituents can have the preferred or more preferred meanings given below.
• R 6 , R 6a , R 6b , R 6c , R 6d is R 6e .
• R 6e is T 2 .
• R 6e is C(0)T 2 .
• R 6e is Ci -6 alkyl substituted with one or more T 2 ; preferably, R 6e is CH 2 T 2 .
• R 6e is N(R 7 )T 2 ; preferably R 6e is HT 2 .
• R is H.
• ring A is a pyrazole, an oxazole or an isoxazole. More preferably, ring A is a pyrazole.
• 0, 1, or 2 R 1 which are the same or different, are other than H.
• R 1 is OR 2 or C 1 -4 alkyl, which is optionally substituted with 1 or 2 R 3 , which are the same or different.
• R 1 is unsubstituted C 1 -4 alkyl. More preferably, R 1 is methyl.
• R 1 is C 1 -4 alkyl substituted with 1 or 2 R 3 .
• R 3 is halogen; CN; OR 4 ; C(0)N(R 4 R 4a ); or C(O)! 1 , wherein T 1 is an unsubstituted 4 to 7 membered heterocycle containing at least one ring nitrogen atom which is attached to C(O).
• R 3 is N(R 4 R 4a ); S(0) 2 N(R 4 R 4a ); or T 1 . More preferably, R 3 is T 1 .
• R 4 , R 4a are independently selected from the group consisting of H; methyl; and cyclopropyl.
• n 0, 1 or 2. More preferably, n is 1.
• R 5 , R 5a are H.
• none or one of X 1 , X 2 , X 3 , X 4 , X 5 is N. More preferably, none of X 1 , X 2 , X 3 , X 4 , X 5 is N.
• R 6 , R 6a , R 6b , R 6c , R 6d is R 6e and the others are independently selected from the group consisting of H; unsubstituted Ci- 6 alkyl; halogen. More preferably, one of R 6 , R 6a , R 6b , R 6c , R 6d is R 6e and 0, 1, 2, or 3 of R 6 , R 6a , R 6b , R 6c , R 6d are independently selected from the group consisting of CH 3 ; CI and F, preferably F.
• T 2 is a 4 to 7 membered heterocyclyl.
• T 2 is morpholinyl, piperazinyl, piped dinyl, thiomorpholinyl- 1, 1 -dioxide, tetrahydro-2H-pyranyl, azetidinyl, oxetane, or pyrrolidine, wherein T 2 is optionally substituted with one or more R 17 , which are the same or different. More preferably, T 2 is morpholinyl and wherein T 2 is optionally substituted with one or more R 17 , which are the same or different. Preferably, T 2 is 7 to 11 membered heterobicyclyl.
• T 2 is 8-oxa-3- azabicyclo[3.2.1]octane, 2-oxa-6-azaspiro[3.3]heptane, 2-oxa-6-azaspiro[3.4]octane, or 2,6- diazaspiro[3.3]heptane, wherein T 2 is optionally substituted with one or more R 17 , which are the same or different.
• T 2 is unsubstituted.
• T 2 is substituted with one or more R 17 , which are the same or different.
• T 2 is substituted with one R 17 .
• R 17 is unsubstituted
• T 2 is pyrazolyl (more preferably pyrazol-4-yl), wherein T 2 is unsubstituted or substituted with one or more R 17 , which are the same or different. More preferably, T 2 is unsubstituted Ci -4 alkyl; or Ci -4 alkyl, substituted with OR 21 or C(0)N(R 21 R 21a ).
• a compound has formula (lb)
• Prodrugs of the compounds of the present invention are also within the scope of the present invention.
• Prodrug means a derivative that is converted into a compound according to the present invention by a reaction with an enzyme, gastric acid or the like under a physiological condition in the living body, e.g. by oxidation, reduction, hydrolysis or the like, each of which is carried out enzymatically.
• Examples of a prodrug are compounds, wherein the amino group in a compound of the present invention is acylated, alkylated or phosphorylated to form, e.g., eicosanoylamino, alanylamino, pivaloyloxymethylamino or wherein the hydroxyl group is acylated, alkylated, phosphorylated or converted into the borate, e.g.
• metabolites refers to all molecules derived from any of the compounds according to the present invention in a cell or organism, preferably mammal. Preferably the term relates to molecules which differ from any molecule which is present in any such cell or organism under physiological conditions.
• tautomerism e.g. keto-enol tautomerism
• the individual forms e.g. the keto and enol form
• stereoisomers e.g. enantiomers, cis/trans isomers, conformers and the like.
• isomers can be separated by methods well known in the art, e.g. by liquid chromatography. The same applies for enantiomers by using e.g. chiral stationary phases. Additionally, enantiomers may be isolated by converting them into diastereomers, i.e. coupling with an enantiomerically pure auxiliary compound, subsequent separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of a compound of formula (I) may be obtained from stereoselective synthesis using optically pure starting materials.
• the compounds of formula (I) may exist in crystalline or amorphous form. Furthermore, some of the crystalline forms of the compounds of formula (I) may exist as polymorphs, which are included within the scope of the present invention. Polymorphic forms of compounds of formula (I) may be characterized and differentiated using a number of conventional analytical techniques, including, but not limited to, X-ray powder diffraction (XRPD) patterns, infrared (IR) spectra, Raman spectra, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and solid state nuclear magnetic resonance (ss MR).
• XRPD X-ray powder diffraction
• IR infrared
• Raman spectra Raman spectra
• DSC differential scanning calorimetry
• TGA thermogravimetric analysis
• ss MR solid state nuclear magnetic resonance
• the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts.
• the compounds of the formula (I) which contain acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids.
• Compounds of the formula (I) which contain one or more basic groups i.e.
• acids which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids.
• suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art.
• the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions).
• the respective salts according to the formula (I) can be obtained by customary methods which are known to the person skilled in the art like, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.
• the present invention also includes all salts of the compounds of the formula (I) which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.
• the term "pharmaceutically acceptable” means that the corresponding compound, carrier or molecule is suitable for administration to humans.
• this term means approved by a regulatory agency such as the EMEA (Europe) and/or the FDA (US) and/or any other national regulatory agency for use in animals, preferably in humans.
• the present invention furthermore includes all solvates of the compounds according to the invention.
• JAK comprises all members of the JAK family (e.g. JAK1, JAK2, JAK3, and TYK2).
• JAK1 or "JAK1 kinase” means "Janus kinase 1".
• the human gene encoding JAK1 is located on chromosome lp31.3.
• the expression "JAK2" or “JAK2 kinase” means “Janus kinase 2".
• the human gene encoding JAK2 is located on chromosome 9p24.
• the expression “JAK3” or “JAK3 kinase” means “Janus kinase 3”.
• the gene encoding JAK3 is located on human chromosome 19pl3.1 and it is predominantly in hematopoietic cells.
• JAK3 is a cytoplasmic protein tyrosine kinase that associates with the gamma-chain of the interleukin 2 (IL-2) receptor.
• IL-2 interleukin 2
• This chain also serves as a component for the receptors of several lymphotropic cytokines, including interleukins IL-4, IL-7, IL-9, IL-15 and IL-21 (Schindler et al, 2007. J. Biol. Chem. 282(28):20059-63). JAK3 plays a key role in the response of immune cells to cytokines, especially in mast cells, lymphocytes and macrophages. Inhibition of JAK3 has shown beneficial effects in the prevention of transplant rejection (Changelian et al., 2003, Science 302(5646):875-888).
• the expression "JAK3" or “JAK3 kinase” includes mutant forms of JAK3, preferably JAK3 mutants found in acute megakaryoblastic leukemia (AMKL) patients. More preferred, these mutants are single amino acid mutations. Activating JAK3 mutations were observed in acute megakaryoblastic leukemia (AMKL) patients (Walters et al, 2006. Cancer Cell 10(l):65-75). Therefore, in a preferred embodiment, the expression "JAK” also includes a JAK3 protein having a V7221 or P132T mutation.
• TYK2 or "TYK2 kinase” means "Protein-Tyrosine kinase 2".
• the JAK3 and TYK2 genes are clustered on chromosome 19pl3.1 and 19pl3.2, respectively.
• compounds of the invention were tested for their selectivity for JAK3 over JAK2 kinases.
• all tested compounds bind JAK3 more selectively than, JAK2 (see table 6 below).
• the compounds of the present invention are considered to be useful for the prevention or treatment of diseases and disorders associated with JAK, for example immunological, inflammatory, autoimmune, or allergic disorders, transplant rejection, Graft- versus-Host-Disease or proliferative diseases such as cancer.
• diseases and disorders associated with JAK for example immunological, inflammatory, autoimmune, or allergic disorders, transplant rejection, Graft- versus-Host-Disease or proliferative diseases such as cancer.
• the compounds of the present invention are selective JAK3 inhibitors.
• the compounds of the present invention may be further characterized by determining whether they have an effect on JAK3, for example on its kinase activity (Changelian et al, 2003, Science 302(5646):875-888 and online supplement; Yang et al, 2007. Bioorg. Med. Chem. Letters 17(2): 326-331).
• JAK3 kinase activity can be measured using a recombinant GST-JAK3 fusion protein comprising the catalytic domain (JH1 catalytic domain).
• JAK3 kinase activity is measured by ELISA as follows: Plates are coated overnight with a random L-glutamic acid and tyrosine co-polymer (4: 1; 100 ⁇ g/ml) as a substrate. The plates are washed and recombinant JAK3 JH1 :GST protein (100 ng/well) with or without inhibitors is incubated at room temperature for 30 minutes.
• HPR-conjugated PY20 anti-phosphotyrosine antibody (ICN) is added and developed by TMB (3,3',5,5'-tetramethylbenzidine) (Changelian et al, 2003, Science 302(5646):875-888 and online supplement).
• TMB 3,3',5,5'-tetramethylbenzidine
• a cell-based assays (TF-1 cell proliferation) was described to assess the inhibitory activity of small molecule drugs toward JAK2 or JAK3 -dependent signal transduction (Chen et al., 2006. Bioorg. Med. Chem. Letters 16(21): 5633-5638).
• the present invention provides pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as active ingredient together with a pharmaceutically acceptable carrier, optionally in combination with one or more other pharmaceutical compositions.
• “Pharmaceutical composition” means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.
• the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally.
• Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained- release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
• a pharmaceutical composition of the present invention may comprise one or more additional compounds as active ingredients like one or more compounds of formula (I) not being the first compound in the composition or other JAK inhibitors.
• Further bioactive compounds may be steroids, leukotriene antagonists, cyclosporine or rapamycin.
• the compounds of the present invention or pharmaceutically acceptable salt(s) thereof and the other pharmaceutically active agent(s) may be administered together or separately and, when administered separately, this may occur separately or sequentially in any order.
• the two compounds When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation.
• they When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.
• the compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I) is administered in combination with another drug or pharmaceutically active agent and/or that the pharmaceutical composition of the invention further comprises such a drug or pharmaceutically active agent.
• drug or pharmaceutically active agent includes a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.
• Combined or “in combination” or “combination” should be understood as a functional coadministration, wherein some or all compounds may be administered separately, in different formulations, different modes of administration (for example subcutaneous, intravenous or oral) and different times of administration.
• the individual compounds of such combinations may be administered either sequentially in separate pharmaceutical compositions as well as simultaneously in combined pharmaceutical compositions.
• combination with other chemotherapeutic or antibody agents is envisaged.
• Suitable examples of pharmaceutically active agents which may be employed in combination with the compounds of the present invention and their salts for rheumatoid arthritis therapy include: immunosuppresants such as amtolmetin guacil, mizoribine and rimexolone; anti-T Fa agents such as etanercept, infliximab, Adalimumab, Anakinra, Abatacept, Rituximab; tyrosine kinase inhibitors such as leflunomide; kallikrein antagonists such as subreum; interleukin 11 agonists such as oprelvekin; interferon beta 1 agonists; hyaluronic acid agonists such as RD-101 (Aventis); interleukin 1 receptor antagonists such as anakinra; CD8 antagonists such as amiprilose hydrochloride; beta amyloid precursor protein antagonists such as reumacon; matrix metalloprotease inhibitors such as cipemastat and other disease
• the treatment defined herein may be applied as a sole therapy or may involve, in addition to the compounds of the invention, conventional surgery or radiotherapy or chemotherapy.
• the compounds of the invention can also be used in combination with existing therapeutic agents for the treatment proliferative diseases such as cancer. Suitable agents to be used in combination include:
• antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea and gemcitabine); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like
• cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5a-reductase such as finasteride;
• antioestrogens for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene
• anti-invasion agents for example c-Src kinase family inhibitors like 4-(6-chloro- 2,3 - methylenedioxyanilino)-7- [2-(4-methylpiperazin- 1 -yl)ethoxy] -5 -tetrahydropyran- 4-yloxy- quinazoline (AZD0530) and N-(2-chloro-6-methylphenyl)-2- ⁇ 6- [4-(2- hydroxyethyl)piperazin-l-yl]-2-methylpyrimidin- 4-ylamino ⁇ thiazole-5-carboxamide
• c-Src kinase family inhibitors like 4-(6-chloro- 2,3 - methylenedioxyanilino)-7- [2-(4-methylpiperazin- 1 -yl)ethoxy] -5 -tetrahydropyran- 4-yloxy- quinazoline (AZD0530) and N-(2-chloro-6-
• dasatinib (dasatinib, BMS-354825), and metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function);
• inhibitors of growth factor function include growth factor antibodies and growth factor receptor antibodies (for example the anti-erbB2 antibody trastuzumab [HerceptinTM] and the anti-erbBl antibody cetuximab [C225]); such inhibitors also include, for example, tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as N-(3- chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD 1839), A/-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-aciylamido-A/-(3-chloro-4-fluorophenyl)-7-(3-
• antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, for example the anti-vascular endothelial cell growth factor antibody bevacizumab (AvastinTM) and VEGF receptor tyrosine kinase inhibitors such as 4-(4-bromo- 2-fiuoroanilino)-6-methoxy-7-( 1 -methylpiperidin-4-ylmethoxy)quinazoline (ZD6474; Example 2 within WO 01/32651), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3- pyrrolidin-l-ylpropoxy)quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO 98/35985) and SUl 1248 (sunitinib; WO 01/60814), and compounds that work by other mechanisms (for example linomide, inhibitor
• vascular damaging agents such as combretastatin A4 and compounds disclosed in International Patent Application WO 99/02166;
• antisense therapies for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense agent;
• gene therapy approaches including approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and(ix) immunotherapeutic approaches, including ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.
• GDEPT gene-directed enzyme pro-drug therapy
• immunotherapeutic approaches including ex-vivo and in
• the compounds of formula (I) can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
• the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
• any of the usual pharmaceutical media may be employed, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.
• oral liquid preparations such as, for example, suspensions, elixirs and solutions
• carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.
• tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions and preparations should contain at least 0.1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be between about 2 percent to about 60 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that an effective dosage will be obtained.
• the active compounds can also be administered intranasally, for example, as liquid drops or spray.
• the tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin.
• a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as fatty oil.
• tablets may be coated with shellac, sugar or both.
• a syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor.
• Compounds of formula (I) may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropyl-cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
• Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dose of a compound of the present invention.
• oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed.
• Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
• compounds of formula (I) are administered orally.
• the effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.
• a therapeutically effective amount of a compound of the present invention will normally depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration.
• an effective amount of a compound of formula (I) for the treatment of an inflammatory disease for example rheumatoid arthritis (RA) will generally be in the range of 0.1 to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 1 to 10 mg/kg body weight per day.
• the actual amount per day would usually be from 70 to 700 mg and this amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same.
• An effective amount of a pharmaceutically acceptable salt, prodrug or metabolite thereof may be determined as a proportion of the effective amount of the compound of formula (I) per se. It is envisaged that similar dosages would be appropriate for treatment of the other conditions referred to above.
• the term "effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.
• therapeutically effective amount means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder.
• the term also includes within its scope amounts effective to enhance normal physiological function.
• Another aspect of the present invention is a compound of the present invention or a pharmaceutically acceptable salt thereof for use as a medicament.
• Another aspect of the present invention is a compound of the present invention or a pharmaceutically acceptable salt thereof for use in a method of treating or preventing a disease or disorder associated with JAK.
• a disease or disorder associated with JAK is defined as a disease or disorder where JAK is involved.
• the diseases or disorder is associated with JAK is an immunological, inflammatory, autoimmune, or allergic disorder or disease of a transplant rejection or a Graft-versus host disease.
• another aspect of the present invention is a compound or a pharmaceutically acceptable salt thereof of the present invention for use in a method of treating or preventing an immunological, inflammatory, autoimmune, or allergic disorder or disease of a transplant rejection or a Graft-versus host disease.
• Inflammation of tissues and organs occurs in a wide range of disorders and diseases and in certain variations, results from activation of the cytokine family of receptors.
• Exemplary inflammatory disorders associated with activation of JAK include, in a non-limiting manner, skin inflammation due radiation exposure, asthma, allergic inflammation and chronic inflammation.
• an autoimmune disease is a disease which is at least partially provoked by an immune reaction of the body against own components, for example proteins, lipids or DNA.
• organ-specific autoimmune disorders are insulin- dependent diabetes (Type I) which affects the pancreas, Hashimoto's thyroiditis and Graves' disease which affect the thyroid gland, pernicious anemia which affects the stomach, Cushing's disease and Addison's disease which affect the adrenal glands, chronic active hepatitis which affects the liver; polycystic ovary syndrome (PCOS), celiac disease, psoriasis, inflammatory bowel disease (IBD) and ankylosing spondylitis.
• non-organ- specific autoimmune disorders are rheumatoid arthritis, multiple sclerosis, systemic lupus and myasthenia gravis.
• Type I diabetes ensues from the selective aggression of autoreactive T-cells against insulin secreting beta-cells of the islets of Langerhans.
• Targeting JAK3 in this disease is based on the observation that multiple cytokines that signal through the JAK pathway are known to participate in the T-cell mediated autoimmune destruction of beta-cells.
• a JAK3 inhibitor, JA EX-1 was shown to prevent spontaneous autoimmune diabetes development in the NOD mouse model of type I diabetes.
• the autoimmune disease is selected from the group consisting of rheumatoid arthritis (RA), inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis, systemic lupus erythematosus (SLE), and multiple sclerosis (MS).
• RA rheumatoid arthritis
• IBD inflammatory bowel disease
• SLE systemic lupus erythematosus
• MS multiple sclerosis
• IBD Inflammatory bowel disease
• Crohn's disease involves most frequently the terminal ileum and colon, is transmural and discontinuous.
• ulcerative colitis the inflammation is continuous and limited to rectal and colonic mucosal layers.
• definitive classification of Crohn's disease or ulcerative colitis cannot be made and are designated 'indeterminate colitis.
• Both diseases include extraintestinal inflammation of the skin, eyes, or joints. Neutrophil-induced injuries may be prevented by the use of neutrophils migration inhibitors (Asakura et al., 2007, World J Gastroenterol. 13(15):2145-9).
• Psoriasis is a chronic inflammatory dermatosis that affects approximately 2% of the population. It is characterized by red, scaly skin patches that are usually found on the scalp, elbows, and knees, and may be associated with severe arthritis. The lesions are caused by abnormal keratinocyte proliferation and infiltration of inflammatory cells into the dermis and epidermis (Schon et al., 2005, New Engl. J. Med. 352: 1899-1912).
• SLE Systemic lupus erythematosus
• T cell- mediated B-cell activation results in glomerulonephritis and renal failure.
• Human SLE is characterized at early stages by the expansion of long-lasting autoreactive CD4+ memory cells (D'Cruz et al., 2007, Lancet 369(9561):587-596).
• MS Multiple sclerosis
• JAK3 was shown to be a valid target in the treatment of mast cell mediated allergic reaction.
• Allergic disorders associated with mast cell activation include Type I immediate hypersensitivity reactions such as allergic rhinitis (hay fever), allergic urticaria (hives), angioedema, allergic asthma and anaphylaxis, for example anaphylatic shock. These disorders may be treated or prevented by inhibition of JAK3 activity, for example, by administration of a JAK3 inhibitor according to the present invention.
• Transplant rejection includes, without limitation, acute and chronic allograft rejection following for example transplantation of kidney, heart, liver, lung, bone marrow, skin and cornea. It is known that T cells play a central role in the specific immune response of allograft rejection. Hyperacute, acute and chronic organ transplant rejection may be treated. Hyperacute rejection occurs within minutes of transplantation. Acute rejection generally occurs within six to twelve months of the transplant. Hyperacute and acute rejections are typically reversible where treated with immunosuppressant agents. Chronic rejection, characterized by gradual loss of organ function, is an ongoing concern for transplant recipients because it can occur anytime after transplantation.
• GVDH graft-versus-host disease
• BMT bone marrow transplantation
• JAK3 plays a key role in the induction of GVHD and treatment with a JAK3 inhibitor, JA EX-1, was shown to attenuate the severity of GVHD (reviewed in Cetkovic- Cvrlje and Ucken, 2004).
• the inflammatory disease is an eye disease.
• Dry eye syndrome (DES, also known as keratoconjunctivitis sicca) is one of the most common problems treated by eye physicians. Sometimes DES is referred to as dysfunctional tear syndrome (Jackson, 2009. Canadian Journal Ophthalmology 44(4), 385-394). DES affects up to 10% of the population between the ages of 20 to 45 years, with this percentage increasing with age. Although a wide variety of artificial tear products are available, these products provide only transitory relief of symptoms. As such, there is a need for agents, compositions and therapeutic methods to treat dry eye.
• dry eye disorder is intended to encompass the disease states summarized in a recent official report of the Dry Eye Workshop (DEWS), which defined dry eye as "a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. It is accompanied by increased osmolality of the tear film and inflammation of the ocular surface.” (Lemp, 2007. "The Definition and Classification of Dry Eye Disease: Report of the Definition and Classification Subcommittee of the International Dry Eye Workshop", The Ocular Surface, 5(2), 75-92). Dry eye is also sometimes referred to as keratoconjunctivitis sicca.
• the treatment of the dry eye disorder involves ameliorating a particular symptom of dry eye disorder, such as eye discomfort, visual disturbance, tear film instability, tear hyperosmolarity, and inflammation of the ocular surface.
• Uveitis is the most common form of intraocular inflammation and remains a significant cause of visual loss.
• Current treatments for uveitis employs systemic medications that have severe side effects and are globally immunosuppressive.
• Clinically chronic progressive or relapsing forms of non-infectious uveitis are treated with topical and/or systemic corticosteroids.
• macrolides such as cyclosporine and rapamycin are used, and in some cases cytotoxic agents such as cyclophosphamide and chlorambucil, and antimetabolites such as azathioprine, methotrexate, and leflunomide (Srivastava et al, 2010.
• Uveitis Mechanisms and recent advances in therapy. Clinica Chimica Acta, doi: 10.1016/j .cca.2010.04.017).
• the disease or disorder associated with JAK is a proliferative disease, especially cancer.
• JAK Diseases and disorders associated especially with JAK are proliferative disorders or diseases, especially cancer.
• cancer comprises a group of diseases characterized by uncontrolled growth and spread of abnormal cells. All types of cancers generally involve some abnormality in the control of cell growth, division and survival, resulting in the malignant growth of cells. Key factors contributing to said malignant growth of cells are independence from growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis, and genome instability (Hanahan and Weinberg, 2000. The Hallmarks of Cancer. Cell 100, 57-70).
• cancers are classified as hematological cancers (for example leukemias and lymphomas) and solid cancers such as sarcomas and carcinomas (for example cancers of the brain, breast, lung, colon, stomach, liver, pancreas, prostate, ovary).
• hematological cancers for example leukemias and lymphomas
• solid cancers such as sarcomas and carcinomas (for example cancers of the brain, breast, lung, colon, stomach, liver, pancreas, prostate, ovary).
• the JAK inhibitors of the present invention may also useful in treating certain malignancies, including skin cancer and hematological malignancy such as lymphomas and leukemias.
• cancers in which the JAK-STAT signal transduction pathway is activated are expected to respond to treatment with JAK3 inhibitors.
• JAK3 inhibitors include acute megakaryoblastic leukemia (AMKL) (Walters et al, 2006. Cancer Cell 10(l):65-75) and breast cancer (Jeong et al, 2008. Clin. Cancer Res. 14, 3716-3721).
• Proliferative diseases or disorders comprise a group of diseases characterized by increased cell multiplication as observed in myeloprolifetative disorders (MPD) such as polycythemia vera (PV).
• MPD myeloprolifetative disorders
• PV polycythemia vera
• diseases and disorders associated with JAK are as defined above.
• Yet another aspect of the present invention is a method for treating, controlling, delaying or preventing in a mammalian patient in need thereof one or more conditions selected from the group consisting of diseases and disorders associated with JAK, wherein the method comprises the administration to said patient a therapeutically effective amount of a compound according to present invention or a pharmaceutically acceptable salt thereof.
• Yet another aspect of the present invention is a method for treating, controlling, delaying or preventing in a mammalian patient in need thereof one or more conditions selected from the group consisting of an immunological, inflammatory, autoimmune, or allergic disorder or disease or a transplant rejection or a Graft-versus host disease, wherein the method comprises the administration to said patient a therapeutically effective amount of a compound according to present invention or a pharmaceutically acceptable salt thereof.
• Yet another aspect of the present invention is a method for treating, controlling, delaying or preventing in a mammalian patient in need thereof a proliferative disease, especially cancer, wherein the method comprises the administration to said patient a therapeutically effective amount of a compound according to present invention or a pharmaceutically acceptable salt thereof.
• diseases and disorders associated with JAK are as defined above.
• treating or “treatment” is intended to refer to all processes, wherein there may be a slowing, interrupting, arresting, or stopping of the progression of a disease, but does not necessarily indicate a total elimination of all symptoms. All embodiments discussed above with respect to the pharmaceutical composition of the invention also apply to the above mentioned first or second medical uses or methods of the invention.
• compounds of the present invention may be prepared according to a method comprising the steps of
• B' is a suitable leaving group, like chloro, and R has the meaning as indicated above with a compound of formula
• NMR spectra were obtained on a Brucker dpx400.
• LCMS was carried out on an Agilent 1 100 using a Gemini C18, 3 x 30 mm, 3micron. Column flow was 1.2mL/min and solvents used were water and acetonitrile (0.1% formic acid- high pH, 0.1% ammonia- low pH) with an injection volume ⁇ 3 ⁇ ⁇ . Wavelengths were 254 and 210nm.
• l-(3-(dimethylamino)propyl)-lH-pyrazol-4-amine was prepared by Procedure A using 3- chloro-N,N-dimethylpropan-l -amine, HC1 as alkylating agent.
• 3-morpholinobenzyl methanesulfonate was prepared according to Procedure E (Step ii) using (3-morpholinophenyl)methanol.
• 2-(4-amino-lH-pyrazol-l-yl)ethanol was prepared according to Procedure A using 2- bromoethanol as alkylating agent.
• Example 7 N-( 1 -Methyl- lH-pyrazol-4-yl)- 1 -(3 -(morpholinomethyl)benzyl)- 1H- pyrazolo[3,4-d]pyrimidin-6-amine
• Procedure E Steps ii-iii) using (3- (mo holinomethyl)phenyl)methanol:
• Example 8 N-( 1 -Methyl- lH-pyrazol-4-yl)- 1 -(3 -(piperidin- 1 -yl)benzyl)- lH-pyrazolo[3 ,4- d]pyrimidin-6-amine
• reaction mixture was diluted with methanol, passed through a thiol column, washed with methanol and evaporated. The residue was dissolved in DCM, the organic phase washed with water, dried over sodium sulfate and concentrated in vacuo to afford l-(3-(3,6- dihydro-2H-pyran-4-yl)benzyl)-N-(l -methyl- lH-pyrazol-4-yl)-lH-pyrazolo[3,4-d]pyrimidin- 6-amine.
• Example 10 N-( 1 -Methyl- 1 H-pyrazol-4-yl)- 1 -(3 -(4-methylpiperazin- 1 -yl)benzyl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• the following compound was made according to the procedure in Example 2 using N- methylpiperazine in Step (ii):
• Step (i) (2-methyl-3-mo holinophenyl)methanol was prepared as in Procedure H using 2-methyl-3- morpholinobenzoic acid.
• Example 12 N-Methyl-2-(4-((l-(3-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- lH-pyrazol- 1 -yl)acetamide
• 3-n ⁇ holinobenzyl methanesulfonate was prepared according to Procedure E (Step ii) using (3-n ⁇ holinophenyl)methanol.
• Step (ii) 2-(4-amino-lH-pyrazol-l-yl)acetamide was prepared according to Procedure A using 2- bromo-N-methylacetamide.
• Example 14 3-(4-((l-(3-Mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-lH- pyrazol- 1 -yl)propanamide
• Example 15 1 -(2-Fluoro-5 -morpholinobenzyl)-N-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• Example 16 N,N-Dimethyl-3-(4-((l-(3-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- lH-pyrazol- 1 -yl)propanamide
• Example 17 N-( 1 -Methyl- 1 H-pyrazol-4-yl)- 1 -(2,3 , 6-trifluoro-5 -morpholinobenzyl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• Example 18 3-(4-((l-(3-Mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-lH- pyrazol- 1 -yl)propan- 1 -ol
• Example 19 1 -(3 -(4,4-Difluoropiperidin- 1 -yl)benzyl)-N-( 1 -methyl- lH-pyrazol-4-yl)- 1H- pyrazolo[3,4-d]pyrimidin-6-amine
• Example 20 3-(4-((l-(3-Mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-yl)amino)-lH- pyrazol- 1 -yl)- 1 -(piperidin- 1 -yl)propan- 1 -one
• Example 21 1 -(2-Fluoro-3 -morpholinobenzyl)-N-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• Example 22 N-( 1 -Methyl- 1 H-pyrazol-4-yl)- 1 -(3 -(piperazin- 1 -yl)benzyl)- 1 H-pyrazolo [3 ,4- d]pyrimidin-6-amine
• tert-Butyl-4-(3-(ethoxycarbonyl)phenyl)piperazine-l-carboxylate (0.20g, 0.60mmol) was dissolved in THF (lmL) and lithium borohydride (0.10g, 7.5eq) was added in three portions. The reaction mixture stirred at rt for 48h then quenched with saturated ammonium chloride under ice-cooling. The aqueous phase was extracted with DCM and the combined organic phases dried over sodium sulfate and evaporated to afford tert-butyl 4-(3- (hydroxymethyl)phenyl)piperazine-l-carboxylate (0.16mg, 0.54mmol, 90%).
• tert-Butyl-4-(3-(((methylsulfonyl)oxy)methyl)phenyl)piperazine-l-carboxylate was made according to Procedure E (Step ii) using tert-butyl 4-(3-(hydroxymethyl)phenyl)piperazine-l- carboxylate.
• Step (iii) tert-Butyl-4-(3 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 - yl)methyl)phenyl)piperazine-l-carboxylate was made according to Procedure D using tert- Butyl 4-(3-(((methylsulfonyl)oxy)methyl)phenyl)piperazine-l-carboxylate.
• Example 24 N-Methyl-3-(4-((l-(3-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- lH-pyrazol- 1 -yl)propanamide
• the following compound was made according to the procedure in Example 5, using 3-chloro- N-methylpropanamide in Step (ii):
• Example 25 4-(3 -((6-(( 1 -Methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 - yl)methyl)phenyl)thiomorpholine 1 , 1 -dioxide
• the title compound was made according to the procedure in Example 2 using thiomorpholine 1, 1 -dioxide in Step (ii):
• Example 27 4-(3-((6-((l -Methyl- lH-pyrazol-4-yl)amino)- 1 H-pyrazolo [3, 4-d]pyrimidin- 1 - yl)methyl)phenyl)morpholin-3 -one
• Step (ii) To a solution of l-(3-iodobenzyl)-N-(l-methyl-lH-pyrazol-4-yl)-lH-pyrazolo[3,4- d]pyrimidin-6-amine (50mg, 0.12mmol) in dioxane (lmL) were added morpholin-3-one (15mg, 1.25eq), copper iodide (4.4mg, 0.2eq), potassium phosphate (49mg, 2eq) and ⁇ , ⁇ '- dimethylethylene diamine (5 ⁇ 1, 0.4eq). After stirring for 16h at 90°C, the reaction mixture was partitioned between 0.5M EDTA and DCM.
• Example 28 4-(3 -((6-(( 1 -(3 -(Dimethylamino)propyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• l-(3-(dimethylamino)propyl)-lH-pyrazol-4-amine was prepared by Procedure A using 3- chloro-N,N-dimethylpropan-l -amine, HC1 as alkylating agent.
• Step (iv) The title compound was made according to the procedure in Procedure D (Step ii), using 4-(3- (bromomethyl)phenyl)morpholin-3-one and 6-chloro-lH-pyrazolo[3,4-d]pyrimidine followed by Procedure D (Step i) using l-(3-(dimethylamino)propyl)-lH-pyrazol-4-amine.
• Example 29 4-(3-((6-((l-(2-Hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d] pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Example 30 1 -(3 -(4-(( 1 -(3 -Morpholinobenzyl)- lH-pyrazolo[3 ,4-d]pyrimidin-6-yl)amino)- lH-pyrazol- 1 -yl)propyl)pyrrolidin-2-one
• Example 31 N-( 1 -((3 -((Dimethylamino)methyl)oxetan-3 -yl)methyl)- 1 H-pyrazol-4-yl)- 1 -(3 - mo holinobenzyl)-lH-pyrazol -d]pyrimidin-6-amine
• Step (i) 4-nitro-lH-pyrazole (0.5g, 4.42mmol) and K 2 C0 3 (1.22g, 8.84mmol) were suspended in acetonitrile (30mL) in a 2-necked flask under nitrogen and (3-(bromomethyl)oxetan-3- yl)methanol (1.36g, 7.52mmol) was added dropwise. The reaction was heated at 60°C for 16h and the solution was concentrated to about 1/3 of the volume under vacuum and then partitioned between DCM (50mL) and water (50mL).
• Example 32 N-( 1 -(3 -(Dimethylamino)-2-methylpropyl)- 1 H-pyrazol-4-yl)- 1 -(3 - mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine
• Example 33 l-(Dimethylamino)-3-(4-((l-(3-mo holinobenzyl)-lH-pyrazolo[3,4- d] pyrimidin-6-yl)amino)- 1 H-pyrazol- 1 -yl)propan-2-ol
• tert-butyl (3-(4-amino-lH-pyrazol-l-yl)propyl)carbamate was prepared as in Procedure A using tert-butyl (3-bromopropyl)carbamate.
• tert-butyl (3-(4-((l-(3 -morpholinobenzyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-yl)amino)- 1 H- pyrazol-l-yl)propyl)carbamate was made according to the procedure in Procedure D using tert-butyl (3-(4-amino-lH-pyrazol-l-yl)propyl)carbamate in Step (i) and 3-morpholinobenzyl methanesulfonate in Step (ii).
• Example 35 N-Cyclopropyl-3-(4-((l-(3-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- lH-pyrazol- 1 -yl)propanamide
• Example 36 1 -(3 -((6-(( 1 -Methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 - yl)methyl)phenyl)piperazin-2-one
• Example 37 1 -(2, 3 -Difluoro-5 -morpholinobenzyl)-N-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H pyrazolo[3,4-d]pyrimidin-6-amine
• Example 38 1 -(2, 6-Difluoro-3 -morpholinobenzyl)-N-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• Example 39 2-(4-((l-(2-Fluoro-3-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• Example 40 4-(2-Fluoro-3 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4- d] pyrimidin- 1 -yl)methyl)phenyl)mo holin-3 -one
• Example 41 N-( 1 -((3-((Methylamino)methyl)oxetan-3 -yl)methyl)- lH-pyrazol-4-yl)- 1 -(3 - mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine
• N-methyl- 1 -(3 -((4-nitro- 1 H-pyrazol- 1 -yl)methyl)oxetan-3 -yl)methanamine was formed following the procedure in Example 31 (Steps i-iii) using methylamine.
• N-((3 -((4-amino- 1 H-pyrazol- 1 -yl)methyl)oxetan-3 -yl)methyl)-2,2,2-trifluoro-N- methylacetamide was synthesised following the procedure in Example 20 (Step iii) using 2,2,2-trifluoro-N-methyl-N-((3-((4-nitro-lH-pyrazol-l-yl)methyl)oxetan-3- yl)methyl)acetamide.
• Example 42 N-( 1 -(3 -(Dimethylamino)propyl)- 1 H-pyrazol-4-yl)- 1 -(2, 3 , 6-trifluoro-5 - mo holinobenzyl)-lH-pyrazol -d]pyrimidin-6-amine
• l-(3-(dimethylamino)propyl)-lH-pyrazol-4-amine was prepared by Procedure A using 3- chloro-N,N-dimethylpropan-l -amine. HC1 as alkylating agent.
• Example 43 N-( 1 -(3 -(Dimethylamino)propyl)- 1 H-pyrazol-4-yl)- 1 -(2-fluoro-3 - mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine
• Example 44 2-(4-((l-(2-Fluoro-5-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• Example 45 2-(4-((l-(2,3,6-Trifluoro-5-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• Example 46 4-(2-Fluoro-3 -((6-(( 1 -(2-hydroxy ethyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Example 48 l-(3-Morpholinobenzyl)-N-(l -(piped din-3-ylmethyl)- lH-pyrazol-4-yl)- 1H- pyrazolo[3,4-d]pyrimidin-6-amine
• 3-morpholinobenzyl methanesulfonate was prepared according to Procedure E (Step ii) using (3-morpholinophenyl)methanol.
• Step (ii) tert-butyl 3-(hydroxymethyl)piperidine-l-carboxylate was prepared following Procedure H using l-(tert-butoxycarbonyl)piperidine-3-carboxylic acid.
• tert-butyl 3-((4-amino-lH-pyrazol-l-yl)methyl)piperidine-l-carboxylate was prepared following Procedure G using tert-butyl 3-(hydroxymethyl)piperidine-l-carboxylate.
• tert-butyl 3-((4-((l-(3 -morpholinobenzyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-yl)amino)- 1 H- pyrazol-l-yl)methyl)piperidine-l-carboxylate was made according to Procedure D (Step ii), using 3-morpholinobenzyl methanesulfonate and 6-chloro-lH-pyrazolo[3,4-d]pyrimidine followed by Procedure D (Step i) using tert-butyl 3-((4-amino-lH-pyrazol-l- yl)methyl)piperidine- 1 -carboxylate.
• Example 49 1 -(3 -Fluoro-5 -morpholinobenzyl)-N-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• Step (ii) The title compound was formed following Procedure D using 4-(3-(bromomethyl)-5- fluorophenyl)morpholine in Step (ii).
• Example 50 4-(3 -((6-(( 1 -(3 -(Dimethylamino)propyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)-2-fluorophenyl)morpholin-3 -one
• l-(3-(dimethylamino)propyl)-lH-pyrazol-4-amine was prepared by Procedure A using 3- chloro-N,N-dimethylpropan-l -amine. HC1 as alkylating agent.
• Example 51 2-(4-((l-(3-Fluoro-5-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• Example 52 4-(3 -Fluoro-5 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4- d] pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Example 54 1 -(2-Fluoro-3 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4- d]pyrimidin-l-yl)methyl)phenyl)piperazin-2-one
• Step (i) 2-(4-amino-lH-pyrazol-l-yl)ethanol was made following Procedure A using 2-bromoethanol as alkylating agent.
• tert-butyl 4-(2-fluoro-3-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)methyl)phenyl)-3-oxopiperazine-l-carboxylate was made following the procedure in Example 27 using tert-butyl 3-oxopiperazine-l-carboxylate and 2-(4-amino-lH- pyrazol- 1 -yl)ethanol.
• Example 56 2-(4-((l-(2,3-Difluoro-5-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• tert-butyl 4-(3-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d]pyrimidin-l-yl)methyl)phenyl)-3-oxopiperazine-l-carboxylate was made according to the procedure in Example 27 using tert-butyl 3-oxopiperazine-l-carboxylate and 2-(4-amino-lH- pyrazol- 1 -yl)ethanol.
• Example 58 4-(3-Fluoro-5-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Example 59 4-(3,4-Difluoro-5-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Example 60 l-(3-Mo holinobenzyl)-N-(l-(pyrrolidin-3-ylmethyl)-lH-pyrazol-4-yl)-lH- pyrazolo[3,4-d]pyrimidin-6-amine
• tert-butyl 3-((4-amino-lH-pyrazol-l-yl)methyl)pyrrolidine-l-carboxylate was prepared as in Procedure G using tert-butyl 3-(hydroxymethyl)pyrrolidine-l-carboxylate
• tert-butyl 3-((4-((l-(3 -morpholinobenzyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-yl)amino)- 1 H- pyrazol-l-yl)methyl)pyrrolidine-l-carboxylate was made according to Procedure D (Step ii), using 3-mo holinobenzylmethanessulfonate followed by Procedure D (Step i) using tert- butyl 3 -((4-amino- lH-pyrazol- 1 -yl)methyl)pyrrolidine- 1 -carboxylate .
• Example 61 4-(2,4,5-Trifluoro-3-((6-((l -methyl- lH-pyrazol-4-yl)amino)-l H-pyrazolo [3, 4- d] pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• n-Butyllithium (2.5M in hexane, 10.5mL, l . leq.) was added dropwise over 15min to a solution of diisopropylamine (4.0mL, 1.2eq.) in THF (25mL) at 0°C. After stirring for 15min, the LDA solution was added dropwise over 40min to a solution of 2,4,5- trifluorobromobenzene (5.0g, 24mmol) in THF (50mL) at -78°C. The solution was stirred for lOmin and then transferred to a slurry of dry ice (50g) in diethylether (65mL). The reaction was allowed to warm to rt and treated with 1M HCl.
• Example 62 4-(2,4,5-Trifluoro-3-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• the title compound was made according to the procedure in Example 61, using 2-(4-amino- lH-pyrazol-l-yl)ethanol (in Step v) which was prepared by Procedure A using 2- bromoethanol as alkylating agent:
• Example 63 1 -(3 -Fluoro-5 -((6-(( 1 -(2 -hydroxy ethyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo[3,4-d]pyrimidin-l-yl)methyl)phenyl)piperazin-2-one
• Step (ii) 2-(4-(( 1 -(3 -bromo-5 -fluorobenzyl)- 1 H-pyrazolo [3 ,4-d]pyrimidin-6-yl)amino)- 1 H-pyrazol- 1 - yl)ethanol was made according to Procedure D, using 2-(4-amino-lH-pyrazol-l-yl)ethanol in Step (i) and l-bromo-3-(bromomethyl)-5-fluorobenzene in Step (ii).
• Example 64 1 -(3 -Fluoro-5 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 , 4- d]pyrimidin-l-yl)methyl)phenyl)piperazin-2-one
• Step (ii) tert-butyl 4-(3-fluoro-5-((6-((l-methyl-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4-d]pyrimidin- l-yl)methyl)phenyl)-3-oxopiperazine-l-carboxylate was made according to the procedure in Example 27 (Step ii) using tert-butyl 3-oxopiperazine-l-carboxylate.
• Example 65 1 -(3 -(3 -Methoxy azetidin- 1 -yl)benzyl)-N-( 1 -methyl- 1 H-pyrazol-4-yl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-ami
• Example 66 N-( 1 -Methyl- 1 H-pyrazol-4-yl)- 1 -(2, 3 , 6-trifluoro-5 -(piperazin- 1 -yl)benzyl)- 1 H- pyrazolo[3,4-d]pyrimidin-6-amine
• tert-butyl 4-(3-(chloromethyl)-2-fluorophenyl)piperazine-l-carboxylate was synthesized following the procedure in Example 11 (Step ii) using tert-butyl 4-(2-fluoro-3- (hydroxymethyl)phenyl)piperazine- 1 -carboxylate.
• tert-butyl 4-(2-fluoro-3 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4-d]pyrimidin- l-yl)methyl)phenyl)piperazine-l -carboxylate was synthesized following Procedure D using tert-butyl 4-(3-(chloromethyl)-2-fluorophenyl)piperazine-l -carboxylate in Step (ii).
• Example 68 2-(4-((l-(3-Fluoro-5-(3-oxomo holino)benzyl)-lH-pyrazolo[3,4-d]pyrimidin- 6-yl)amino)- lH-pyrazol- 1 -yl)-N-methylacetamide
• tert-butyl 4-nitro-lH-pyrazole-l-carboxylate was stirred with palladium on carbon (10%, 170mg) in ethanol (20mL) under an atmosphere of hydrogen at 20°C for 18h. The palladium was removed by filtration and the solvent was removed in vacuo to give tert-butyl 4-amino- lH-pyrazole-l-carboxylate (1.48g).
• 1H MR (d 6 -DMSO) ⁇ 7.35 (d, 1H), 7.33 (d, lH), 4.40 (s, 2H), 1.54 (s, 9H).
• Example 69 4-(2,4-Difluoro-3-((6-((l-methyl-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d] pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Example 70 4-(3 -((6-(( 1 -(3 -(Dimethylamino)propyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo[3,4-d]pyrimidin-l-yl)methyl)-2,4,5-trifluorophenyl)mo holin-3-one
• tert-butyl 4-(3,4-difluoro-5-(hydroxymethyl)phenyl)-3-oxopiperazine-l-carboxylate was prepared following the procedure in Example 27 (Step ii) using (5-bromo-2,3- difluorophenyl)methanol and tert-butyl 3-oxopiperazine-l-carboxylate.
• tert-butyl 4-(3,4-difluoro-5-(((methylsulfonyl)oxy)methyl)phenyl)-3-oxopiperazine-l- carboxylate was prepared following Procedure E, Step (ii) using tert-butyl 4-(3,4-difluoro-5- (hydroxymethyl)phenyl)-3-oxopiperazine-l-carboxylate.
• N-(l-methyl-lH-pyrazol-4-yl)-lH-pyrazolo[3,4-d]pyrimidin-6-amine was prepared as in Procedure D (Step i) followed by Procedure D (Step ii) using tert-butyl 4-(3,4-difluoro-5- (((methylsulfonyl)oxy)methyl)phenyl)-3-oxopiperazine-l-carboxylate to afford tert-butyl 4- (3 ,4-difluoro-5 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4-d]pyrimidin- 1 - yl)methyl)phenyl)-3-oxopiperazine-l-carboxylate.
• Example 72 l-(3,4-Difluoro-5-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)methyl)phenyl)piperazin-2-one
• 2-(4-amino-lH-pyrazol-l-yl)ethanol was prepared according to Procedure A using 2- bromoethanol as alkylating agent.
• Example 73 4-(2,5-Difluoro-3-((6-((l-methyl-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4- d] pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• Step (iii) (3-bromo-2,5-difluorophenyl)methanol was prepared as in Procedure E (Step i) using 3- bromo-2,5-difluorobenzaldehyde.
• Example 74 4-(2, 5 -Difluoro-3 -((6-(( 1 -(2-hydroxy ethyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo [3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)morpholin-3 -one
• 2-(4-amino-lH-pyrazol-l-yl)ethanol was prepared according to Procedure A using 2- bromoethanol as alkylating agent.
• Step (iii) The title compound was made following the procedure in Example 73 using 2-(4-((lH- pyrazolo[3,4-d]pyrimidin-6-yl)amino)-lH-pyrazol-l-yl)ethanol in Step (iii).
• Example 75 4-(3 -((6-(( 1 -(3 -(Dimethylamino)propyl)- 1 H-pyrazol-4-yl)amino)- 1 H- pyrazolo[3,4-d]pyrimidin-l-yl)methyl)-2,4-difluorophenyl)mo holin-3-one
• l-(3-(dimethylamino)propyl)-lH-pyrazol-4-amine was prepared by Procedure A using 3- chloro-N,N-dimethylpropan-l -amine. HCl as alkylating agent.
• Example 76 1 -(2,4-Difluoro-3 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4- d]pyrimidin-l-yl)methyl)phenyl)piperazin-2-one
• 3-bromo-2,6-difluorobenzoic acid was prepared following the procedure in Example 61 (Step i) using l-bromo-2,4-difluorobenzene.
• Example 77 1 -(3 -(4-Methoxypiperidin- 1 -yl)benzyl)-N-( 1 -methyl- lH-pyrazol-4-yl)- 1H- pyrazolo[3,4-d]pyrimidin-6-amine
• Example 78 2-(4-((l-(2,3-Difluoro-5-(piperazin-l-yl)benzyl)-lH-pyrazolo[3,4-d]pyrimidin- 6-yl)amino)- lH-pyrazol- 1 -yl)ethanol
• tert-butyl 4-(3-(bromomethyl)-4,5-difluorophenyl)piperazine-l-carboxylate was formed following procedure F using tert-butyl 4-(3,4-difluoro-5-(hydroxymethyl)phenyl)piperazine- 1-carboxylate.
• 2-(4-amino-lH-pyrazol-l-yl)ethanol was prepared according to Procedure A using 2- bromoethanol as alkylating agent.
• Step (vi) tert-butyl 4-(3,4-difluoro-5-((6-((l-(2-hydroxyethyl)-lH-pyrazol-4-yl)amino)-lH- pyrazolo[3 ,4-d]pyrimidin- 1 -yl)methyl)phenyl)piperazine- 1 -carboxylate was prepared following Procedure D using 2-(4-amino-lH-pyrazol-l-yl)ethanol in Step (i) and tert-butyl 4- (3-(bromomethyl)-4,5-difluorophenyl)piperazine-l-carboxylate in Step (ii).
• Example 79 (3 -Fluoro-5 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4- d]pyrimidin-l-yl)methyl)phenyl)(mo holino)methanone
• Example 80 (3 -Fluoro-5 -((6-(( 1 -methyl- 1 H-pyrazol-4-yl)amino)- 1 H-pyrazolo [3 ,4- d]pyrimidin- 1 -yl)methyl)phenyl)(piperazin- 1 -yl)methanone
• tert-butyl 4-(3-fluoro-5-((6-((l-methyl-lH-pyrazol-4-yl)amino)-lH-pyrazolo[3,4-d]pyrimidin- l-yl)methyl)benzoyl)piperazine-l-carboxylate was made according to Example 6 using 1- bromo-3-(bromomethyl)-5-fluorobenzene in Step (i) and tert-butyl piperazine-l-carboxylate in Step (ii).
• Example 82 2-(4-((l-(2,6-Difluoro-3-mo holinobenzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• Example 83 2-(4-((l-(2-Fluoro-3-(piperazin-l-yl)benzyl)-lH-pyrazolo[3,4-d]pyrimidin-6- yl)amino)- 1 H-pyrazol- 1 -yl)ethanol
• Example 84 4-(3-((6-((lH-Pyrazol-4-yl)amino)-lH-pyrazolo[3,4-d]pyrimidin-l-yl)methyl)- 5-fluorophenyl)morpholin-3-one
• 2-(4-amino-lH-pyrazol-l-yl)ethanol was prepared as in Procedure A using 2-bromoethanol as alkylating agent.

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