WO2003026666A1 - 2-phenylamino-4- (5-pyrazolylamino)-pyrimidine derivatives as kinase inhibitors, in particular, as src kinase inhibitors - Google Patents

2-phenylamino-4- (5-pyrazolylamino)-pyrimidine derivatives as kinase inhibitors, in particular, as src kinase inhibitors Download PDF

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WO2003026666A1
WO2003026666A1 PCT/US2002/030984 US0230984W WO03026666A1 WO 2003026666 A1 WO2003026666 A1 WO 2003026666A1 US 0230984 W US0230984 W US 0230984W WO 03026666 A1 WO03026666 A1 WO 03026666A1
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alkyl
alkoxy
optionally substituted
halogen
phenyl
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Julie Dixon
Jacques Dumas
Catherine Brennan
Holia Hatoum-Mokdad
Wendy Lee
Robert Sibley
Brian Bear
Brent Chandler
Karl Miranda
Gang Chen
Zhi Chen
David Brittelli
Roger B. Clark
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Bayer Pharmaceuticals Corporation
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    • C07D231/00Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D233/00Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/56Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, attached to ring carbon atoms
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D249/00Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/081,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
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    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D403/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D403/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
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    • C07DHETEROCYCLIC COMPOUNDS
    • C07D405/00Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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    • C07DHETEROCYCLIC COMPOUNDS
    • C07D409/00Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
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    • C07D413/00Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
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    • C07D417/00Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings

Definitions

  • the present invention relates to substituted pyrimidine compounds, and in particular, 2,4-diamine-substituted pyrimidine compounds, and pharmaceutical compositions thereof, and the use of such substituted pyrimidine compounds as inhibitors of src kinase enzymes.
  • Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance, e.g., by increasing the rate of cell proliferation, modulating the rate of cell differentiation or decreasing the rate of cell death, can result in the abnormal growth of cells and is thought to be a major event in the development of cancer, as well as other cell proliferative disorders such as restenosis.
  • NSCLC non-small-cell lung cancer
  • Protein phosphorylation is the result of the transfer of a terminal phosphate of adenosine triphosphate to a particular amino acid of a protein. This transfer is catalyzed by enzymes termed kinases.
  • Protein kinases comprise a large superfamily of homologous proteins. They are related by their kinase or catalytic domains, which consists of approximately 250-300 amino acid residues. There are two main categories within the superfamily of protein kinases: the protein-serine/threonine kinses and the protein-tyrosine kinases (Hanks et al, (1995) FASEB J. 9:576)
  • kinases having an abnormal activity e.g., mutated kinases, or abnormal levels of kinases
  • oncogenes which are capable of transforming cells, are mutated forms of normal genes encoding kinases.
  • oncogenes include the pp60-v-src gene from the Rous avian sarcoma virus, which corresponds to the normal (i.e., proto-oncogene) gene pp60-c-src, containing a deletion that removes the C-terminal 18 amino acids of c-src.
  • Pp60-c-src is also referred to as "src kinase” or "src tyrosine kinase.” Phosphorylation ofa tyrosine residue at position 527 of c-src protein causes a great reduction in its kinase activity, and this site is often altered in oncogenic derivatives of c-src (see, e.g., Brown et al, (1996) Biochem. Biophys. Acta 1287:121).
  • proto-oncogenes encoding tyrosine kinases which when mutated or over-expressed, cause cells to become transformed, include c-yes; c-fps (c-fes); c-abl and c-met. c-abl and c-met are associated with chronic myelogenous leukemia and osteosarcoma, respectively.
  • Proto-oncogenes encoding serine/threonine kinases include c-mos and c-raf (c-mil). Whereas the above-cited proto- oncogenes are intracellular transducers, other proto-oncogenes encode kinases which are cell-surface receptors.
  • proto-oncogenes encoding cell surface receptors with tyrosine kinase activity include c-fins (or Colony Stimulating Factor -1 (CSF-1) receptor); c- erbB, which is an epidermal growth factor receptor; c-neu (or erbB-2), erbB-3 or erbB-4 which are related to epidermal growth factor receptor; and c-ros, which is related to the insulin receptor.
  • CSF-1 Colony Stimulating Factor -1
  • kinases in particular tyrosine kinases.
  • Such inhibitors have been shown to be useful for the treatment of disease states characterized by uncontrolled cell proliferation, e.g., cancer, inflammation, psoriasis, pulmonary fibrosis, glomerulonephritis, atherosclerosis, osteoporosis and restenosis following angioplasty.
  • tyrosine kinase inhibitors with selectivity for the EGF receptor family have been shown to block tumor formation in animals, thus demonstrating their potential usefulness for directly suppressing tumor cell growth in the treatment of human cancer, especially breast carcinoma.
  • tumor metastasis and its associated angiogenesis has been shown to be inhibited by preventing the activation of the vascular endothelial growth factor receptor tyrosine kinase which indicates a utility for tyrosine kinase inhibitors in blocking separate events that occur during carcinogenesis.
  • protein phosphorylation e.g., tyrosine phosphorylation
  • the pp60c-src protein has significant structural homology to about ten proteins (collectively referred to as Src Family kinases or SFKs) which include: Lck, Fyn, Yes, Yrk, Blk, Fgr, Hck, Lyn, and Frk subfamily members Frk/Rak and Iyk/Bsk (Sawyer et al, (2001) Expert Opin. Investig. Drugs 10(7): 1327).
  • Src Family kinases has three major domains: src homology SHI, SH2, and SH3 domains. The SHI domain is most commonly called the catalytic domain or tyrosine kinase domain.
  • the SH3 domain is a binding region for proteins having proline-rich sequences. Both the SH2 and SH3 domains are noncatalytic, but are important in protein-protein recognition. SH2 domains are homologous motifs of approximately 100 amino acids, which recognize and bind to the phosphorylated sequences present on regulatory proteins and growth factor receptors (Anderson et al, Science, 1990, 250, 979).
  • One of the primary purposes of the src family phosphoprotein/SH2 domain interaction is to initiate the association of proteins into an activation complex, often around the intracellular domain of the receptor itself. This role of the src family SH2 domain mediates and organizes the ordered, physical assembly of the various proteins in the activation complex.
  • the Src family of protein kinases which all contain an SH2 domain, are involved in a number of cellular signalling pathways. For example, Src is involved in growth factor receptor signaling; integrin-mediated signaling; T- and B-cell activation; osteoclast activation; cell adhesion; cell motility and cell survival. It is known that the Src SH2 domain binds to several key receptor and nonreceptor tyrosine kinases such as tyrosine kinases containing receptors for PDGF, EGF, HER2 Neu (an oncogene form of EGF), Fibroblast Growth Factor (FGF), focal adhesion kinase, pi 30 protein, and p68 protein. In addition, src has been shown to be involved in the regulation of DNA synthesis, mitosis, and other cellular activities (see, e.g., Susa et al. (2000) Trends Pharm. Sciences 21 :489).
  • kinase inhibitors such as tyrosine kinase inhibitors
  • This invention provides compounds for regulating cellular processes involving a kinase such as a tyrosine kinase, in particular, a src kinase.
  • a kinase such as a tyrosine kinase, in particular, a src kinase.
  • the invention relates to a compound ofthe formula (I)
  • Y 1 represents H, C alkyl, or phenyl optionally substituted up to three times by halogen, C H alkyl, or C alkoxy.
  • Y 2 and Y 3 are independently selected from H; C ⁇ -6 alkyl; C -6 cycloalkyl optionally substituted by C 1-4 alkyl; phenyl optionally substituted up to three times by halogen, C alkyl, or C M alkoxy; adamantyl; CF 3 ; a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C )-6 alkyl; C(O)N(C 1- alkyl) 2 ; C(O)O(C 1- alkyl) ;
  • Y 2 and Y 3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C alkyl, or C alkoxy.
  • X represents halogen or C M alkyl.
  • the subscript n represents 0, 1, or 2.
  • R 1 represents H or C 1-4 alkyl.
  • R 2 represents H or C M alkyl.
  • R 3 represents: C ⁇ -6 alkyl; halogen;
  • CM alkoxy O-phenyl optionally substituted up to two times by halogen, C 1-4 alkyl, C 1-4 alkoxy, or di-(CM alkyl)amino; CN; or N(R') 2 wherein the R 1 moieties are independent or the R 1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle.
  • the subscript p represents 0, 1 , or
  • R 4 represents C ]- alkyl or halogen, and q represents 0, 1, or 2.
  • R 3 and R 4 may be joined and taken together with the carbon atoms to which they are attached, form a 5-6 membered heteroaromatic ring containing up to two heteroatoms selected from N, O, and S, and which is optionally substituted up to two times by halogen, C 1-4 alkyl, or C 1-4 alkoxy.
  • L is a linker selected from: -O-(CH 2 ) 1-4 - ; -S(O) 0-2 -(CH 2 ) 1-4 - ; -N(R')-(CH 2 ) M - ;
  • R 5 represents H, C ⁇ -6 alkyl, or C 1-4 alkoxy-C ⁇ -4 alkyl.
  • R 6 represents H; C 1-6 alkyl; C M alkoxy-substituted C alkyl; C 5-6 cycloalkyl optionally substituted up to 2 times by halogen, C M alkyl, or C M alkoxy; C 3-6 cycloalkyl-substituted C M alkyl; benzyl; phenyl optionally substituted by halogen, C ⁇ -4 alkyl, CM alkoxy,
  • A is N(R 1 ), S, S(O), S(O) 2 , or O , and the bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5 -membered
  • R may also be , in which A represents N(R ), S,
  • G may also be:
  • ⁇ — which may optionally be substituted up to 2 times by C 1-3 alkyl, (C 1-3 alkoxy)(C 1-4 alkyl), C(O)OR 1 , C(O)N(R [ ) 2 , phenyl, or benzyl, and in which A 3 represents N(R'), S, S(O), S(O) 2 ;
  • *Q x 'o-i which may optionally be substituted up to 2 times by oxo, (C 1-3 alkoxy)-(C 1-4 alkyl), C(O)OR', C(O)N(R ! ) 2 , phenyl, or benzyl, or up to 4 times by C ⁇ - alkyl; or
  • L may represent 2-3 or 2 , and in this event,
  • compositions comprising a compound of formula (I) and a pharmaceutically acceptable carrier.
  • the invention provides methods for regulating cellular processes involving a kinase, such as a tyrosine kinase.
  • a kinase such as a tyrosine kinase.
  • the cellular process involves a src kinase.
  • the cellular process can be, e.g., cell proliferation or cell differentiation.
  • the invention provides methods for treating diseases associated with a kinase, e.g., diseases associated with an abnormal kinase activity or level, such as cancers, osteoporosis, and inflammatory disorders.
  • the invention also provides methods for treating diseases associated with abnormal cell proliferation and/or differentiation.
  • the method comprises administering to a subject in need thereof, a pharmaceutically efficient amount of a compound of the invention, such that the subject is treated.
  • kits comprising one or more compounds ofthe invention, optionally in a pharmaceutical composition.
  • the invention is based at least in part on the observation that 2,4-diamino substituted pyrimidine compounds inhibit the activity of src kinases. Exemplary compounds are described herein.
  • Y 1 is preferably H or C )-4 alkyl, and more preferably H.
  • Y 2 is selected from C ⁇ -6 alkyl; C 3-6 cycloalkyl optionally substituted by
  • C M alkyl phenyl optionally substituted up to three times by halogen, C M alkyl, or C M alkoxy; adamantyl; CF 3 ; a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C ⁇ -6 alkyl; and ; and Y 3 is H.
  • Y 2 and Y 3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C 1-4 alkyl, or CM alkoxy.
  • Y 2 is selected from C ⁇ -6 alkyl; C -6 cycloalkyl optionally substituted by C M alkyl; phenyl optionally substituted up to three times by halogen, C 1-4 alkyl, or C alkoxy; adamantyl; and a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C 1-6 alkyl; and Y 3 is H.
  • Y is selected from C 1-6 alkyl, and C 3-6 cycloalkyl optionally substituted by C M alkyl; and Y 3 is H.
  • X preferably represents Cl, F, or C M alkyl, and n is 0, 1, or 2. More preferably, X represents F, and n is 0 or 1.
  • the groups R 1 and R 2 are each preferably H.
  • R is preferably C ⁇ -6 alkyl; halogen; C M alkoxy; CN; or N(R ) 2 in which the R moieties are independent, or the R 1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R 2 ), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. More preferably,
  • R 3 is Ci-6 alkyl; C alkoxy; CN; or N(R*) 2 in which the R 1 moieties are independent, or the
  • R 1 moieties optionally are joined by a linker selected from the group consisting of CH(R 1 ), N(R'), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle.
  • R 3 is C 1-6 alkyl; C 1-4 alkoxy; or
  • the subscript p which represents the number of R 3 groups, is preferably 0, 1 , or 2, more preferably 0 or 1.
  • the phenyl ring of Fig. (I) bears no R 4 groups.
  • L is preferably -O-(CH 2 )M- ; -S(O) 0-2 -(CH 2 ) - ; ; -(CH 2 ) - ;
  • G is preferably: 1) NR 5 R 6 , in which R 5 represents H or C 1-6 alkyl; and R 6 represents: C ⁇ -6 alkyl; C 1-4 alkoxy-substituted C 1-4 alkyl; C 5-6 cycloalkyl optionally substituted up to 2 times by halogen, C M alkyl, or C 1-4 alkoxy; C 3-6 cycloalkyl-substituted C M alkyl; benzyl; phenyl optionally substituted by halogen, C 1-4 alkyl, C 1- alkoxy, -CO 2 R',
  • a 2 represents N(R'), S, S(O), S(O) 2 , or O;
  • L is more preferably -O-(CH 2 )M- ; -S(O) 0-2 -(CH 2 )M- ; -N(R')-(CH 2 )M- ; -(CH 2 ) 1-4 - O-(CH 2 ) 1-4 - ; or -N(R 1 )-C(O)-(CH 2 ) M - •
  • G is more preferably
  • R 5 represents H or C 1-6 alkyl; and R 6 represents C ⁇ -6 alkyl; C 5-6 cycloalkyl optionally substituted up to 2 times by halogen, C M alkyl, or C M alkoxy; benzyl; phenyl optionally substituted by halogen, C M alkyl, C 1-4 alkoxy, -CO 2 R', -C(0)N(R') 2 , -N(R ! ) 2 , y- ⁇
  • A is N(R ), S, S(O), S(O) 2 , or O , and this bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-
  • a 2 represents N(R'), S, S(O), S(O) 2 , or O;
  • a preferred combination of L and G groups is when
  • L is -O-(CH 2 ) - ; -N(R')-(CH 2 ) - ; or -(CH 2 ) 1-4 -O-(CH 2 ) 1-4
  • G is:
  • NR 5 R 6 in which R 5 represents H or C ⁇ -6 alkyl; and R 6 represents C 1-6 alkyl;
  • compounds ofthe invention have the formula (I)
  • Y 1 represents H or C M alkyl.
  • Y 2 is selected from C ]-6 alkyl; C 3-6 cycloalkyl optionally substituted by C M alkyl; phenyl optionally substituted up to three times by halogen, C M alkyl, or C M alkoxy; adamantyl; CF 3 ; a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by
  • halogen or C ⁇ -6 alkyl; and ; andY 3 is H.
  • Y 2 and Y 3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C alkyl, or CM alkoxy.
  • X represents Cl, F, or C M alkyl; and n represents 0, 1, or 2.
  • R 1 and R 2 each represents H.
  • R 3 represents C 1-6 alkyl; halogen; C M alkoxy; CN, or N(R') 2 in which the R 1 moieties are independent, or the R 1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle.
  • the subscript p represents 0, 1, or 2.
  • the subscript q, representing the number of R 4 groups, is 0.
  • L is a linker selected from -O-(CH 2 ) 1-4 - ; -S(O) 0-2 -(CH 2 ) M - ; -N(R 1 )-(CH 2 ) - ;
  • G represents:
  • R 5 represents H or C ⁇ -6 alkyl; and R 6 represents C 1-6 alkyl; C 1-4 alkoxy-substituted C 1-4 alkyl; C 5-6 cycloalkyl optionally substituted up to 2 times by halogen, C M alkyl, or C 1-4 alkoxy; C 3-6 cycloalkyl-substituted C ⁇ -4 alkyl; benzyl; phenyl optionally substituted by halogen, C M alkyl, C M alkoxy,
  • a 2 represents N(R'), S, S(O), S(O) 2 , or O;
  • Y 1 represents H.
  • Y 2 is selected from C 1-6 alkyl; C -6 cycloalkyl optionally substituted by C alkyl; phenyl optionally substituted up to three times by halogen, C alkyl, or C M alkoxy; adamantyl, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C 1-6 alkyl.
  • Y 3 is H.
  • X represents F; and n represents 0 or 1.
  • R and R each represents H.
  • R represents C ⁇ -6 alkyl; C alkoxy; CN; or N(R ) 2 wherein the R 1 moieties are independent, or the R 1 moieties optionally are joined by a linker selected from the group consisting of CH(R 1 ), N(R'), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; and p represents 0 or 1.
  • the subscript q, representing the number of R 4 groups, is O.
  • L is a linker selected from -O-(CH 2 ) - ; -S(O) 0-2 -(CH 2 ) 1-4 - ; -N(R')-(CH 2 ) 1-4 - ; -(CH 2 )M-O-(CH 2 ) 1-4 - ; and .
  • G represents:
  • R 5 represents H or C ⁇ -6 alkyl; and R 6 represents C ⁇ -6 alkyl; C 5-6 cycloalkyl optionally substituted up to 2 times by halogen, C 1-4 alkyl, or C 1-4 alkoxy; benzyl; phenyl optionally substituted by halogen, C 1-4 alkyl, C M alkoxy, -CO 2 R 1 ,
  • a 2 represents N(R ] ), S, S(O), S(O) 2 , or O;
  • Y 1 represents H.
  • Y 2 is C 1-6 alkyl, or C 3-6 cycloalkyl optionally substituted by C ]-4 alkyl.
  • Y 3 is H.
  • X represents F; and n represents 0 or 1.
  • R 1 and R 2 each represents H.
  • R 3 represents C ⁇ -6 alkyl; C 1-4 alkoxy, or N(R*) 2 in which the R 1 moieties are independent, or the R 1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle.
  • the subscript p, representing the number of R 3 groups, is 0 or 1.
  • the subscript q, representing the number of R 4 groups, is 0.
  • L is a linker selected from -O-(CH 2 ) 1- - ; -N(R')-(CH 2 ) 1-4 - ; and
  • G represents: 1) NR 5 R 6 , in which R 5 is H or C )-6 alkyl; and R 6 represents C ⁇ -6 alkyl, or C 5-6 cycloalkyl optionally substituted up to 2 times by halogen, C M alkyl, or C M alkoxy;
  • the group X in the middle ring is preferably on the C-5 position of the pyrimide. Also, in the lower ring, the L-G group is preferably meta to the amino N.
  • the compounds of formula (I) are generally made by coupling a compound of formula (II)
  • Examples of 5-6 membered heteroaromatics containing up to two heteroatoms selected from N, O, and S and employed for groups Y 2 or Y 3 in formula (I) include, but are not limited to, pyridinyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, furanyl, pyrrolyl, thiazolyl, or thienyl.
  • R 1 moieties of a group N(R') are referred to as "independent” it is meant that they are separate groups, each being joined to the N but not joined to each other.
  • R 1 moieties of a group N(R') 2 are referred to as being joined by a linker selected from the group consisting of CH(R'), N(R ! ), S, S(O), S(O) 2 , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle, cyclic moieties such as morpholine, thiomorpholine, pyrrolidine, piperidine, or piperazine are contemplated.
  • R and R 4 may be joined and taken together with the carbon atoms to which they are attached, form a 5-6 membered heteroaromatic ring containing up to two heteroatoms selected from N, O, and S, moieties such as pyridine, pyrimidine, thiozole, imidizole, pyrole, furan, or thiophene are contemplated.
  • analog of a compound refers to a compound having a substantial structural similarity to a particular compound and having essentially the same type of biological activity as the compound.
  • antiproliferative therapeutic or compound refers to a compound or therapeutic which inhibits cell proliferation to at least some extent.
  • cytostatic when referring to the activity of a compound means that the compound causes the cell to cell cycle arrest, but it does not kill the cell. Thus, removal of the drug from the environment ofthe cell results in the resumption of cell proliferation.
  • derivative of a compound or of a small molecule refers to a compound which can be derived, e.g., by chemical synthesis, from the original compound. Thus a derivative ofa compound has certain structural similarities with the compound.
  • Disease associated with an abnormal activity or level of a kinase refers to a disease in which an abnormal activity or protein level of a kinase is present in certain cells, and in which the abnormal activity or protein level of the kinase is at least partly responsible for the disease.
  • a “disease associated with a kinase” refers to a disease that can be treated with a kinase inhibitor.
  • Diseases associated with src kinase-mediated signaling refers to diseases which can be treated with an inhibitor of src kinase-mediated signaling. Such disease can, e.g., be associated with an abnormal src kinase activity or level.
  • Excessive cell proliferation used interchangeably herein with “hyperproliferation” of cells refers to cells which divide more often than their normal or wild-type counterpart. Thus, cells are excessively proliferating when they double in less than 24 hours if their normal counte ⁇ arts double in 24 hours. Excessive proliferation can be detected by simple counting of the cells, with or without specific dyes, or by detecting DNA replication or transcription, such as by measuring incorporation of a labeled molecule or atom into DNA or RNA.
  • Inhibiting cell proliferation refers to decreasing the rate of cell division, by interrupting or slowing down the cell cycle.
  • the term refers to complete blockage of cell proliferation, i.e., cell cycle arrest, as well as to a lengthening of the cell cycle.
  • the period of a cell cycle can be increased by about 10%, about 20%, about 30, 40, 50, or 100%.
  • the duration of the cell cycle can also be augmented by a factor of two, three, 4, 5, 10 or more.
  • Modulating cell differentiation refers to the stimulation or inhibition of cell differentiation.
  • Normalizing cell proliferation refers to reducing the rate of cell proliferation of a cell that proliferates excessively relative to that of its normal or wild-type counte ⁇ art, or increasing the rate of cell proliferation of a cell that proliferates poorly relative to its normal or wild-type counte ⁇ art.
  • a "patient” or “subject” to be treated by the subject method can mean either a human or non-human animal.
  • proliferative disorder refers to any disease/disorder of a tissue marked by unwanted or aberrant proliferation of at least some cells in the tissue. Such diseases include cancer, as well as benign diseases or disorders, such as warts or other benign tumors.
  • a "src inhibitor” is a compound which inhibits at least part of the activity of a src kinase in a cell. The inhibition can be, at least about 20%, preferably at least about 40%, even more preferably at least about 50%, 70%, 80%, 90%, 95%, and most preferably at least about 98% ofthe activity ofthe src kinase.
  • Treating refers to preventing, curing or improving at least one symptom ofa disease.
  • Alkyl means a hydrocarbon radical having up to a maximum of 12 carbon atoms, which may be linear or branched with single or multiple branching. Alkyl is especially lower alkyl.
  • alkyl groups examples include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, and isohexyl.
  • "Halogen” means fluorine, chlorine, bromine, or iodine but is especially fluorine, chlorine, or bromine.
  • Cycloalkyl is a saturated carbocycle that contains between 3 and 12 carbons but preferably 3 to 8 carbons. Examples include the cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl groups.
  • alkoxy means a group in which the alkyl portion is straight or branched and has the designated number of carbon atoms. Examples of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, and isohexoxy.
  • the present invention provides substituted pyrimidine compounds, e.g., 2,4-diamino substituted pyrimidine compounds, which are capable of inhibiting src kinase activity.
  • substituted pyrimidine compounds e.g., 2,4-diamino substituted pyrimidine compounds, which are capable of inhibiting src kinase activity.
  • Exemplary compounds ofthe invention have the IUPAC name set forth below:
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and tr ⁇ ws-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, or by derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group such as amino, or an acidic functional group such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.
  • Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof
  • the compounds of the present invention may be prepared by the methods known to those skilled in the art as illustrated in the general reaction schemes described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
  • the compounds of the invention can be used for treating a disease or condition (generally referred to herein as " disease") associated with a kinase, such as a disease associated with an abnormal activity or level of a kinase.
  • a disease or condition generally referred to herein as " disease”
  • the kinase is a tyrosine kinase, such as a src tyrosine kinase.
  • the compounds of the invention can be used for treating diseases that are associated with a component of the signal transduction pathway in which a kinase is involved.
  • a cell proliferative disease resulting from over-expression of a signal transduction molecule or cell surface receptor that is in the same signal transduction pathway as that in which a kinase which can be inhibited by a compound of the invention is present can also be treated with the compounds of the invention.
  • the compounds of the invention are expected to be effective against a broad range of target cells, and not only target cells having an abnormal activity or level of a kinase.
  • target cell refers to a cell towards which a compound is targeted.
  • at least some of the compounds of the invention may also be effective against cells which proliferate and/or differentiate normally, i.e., wild-type cells. For example, certain compounds could be used to arrest cell proliferation, even if the cell proliferation is not abnormal.
  • the compounds of the invention are useful for treating a disease associated with a src kinase.
  • Src kinases are involved in various cellular functions, including cell proliferation and transformation; cell adhesion, migration and chemotaxis; intracellular trafficking; and cell survival.
  • diseases that can be treated according to the invention include those which are dysfunctional in any of these cellular functions. Exemplary diseases are provided below.
  • a therapeutic method comprises administering to a subject having a disease associated with a kinase, a pharmaceutically effective amount of a compound of the invention, such that the disease is treated.
  • the subject is preferably a mammal, e.g., a human, non-human primate, bovine, ovine, porcine, feline, canine, mouse or rat.
  • the compounds can be administered via various routes depending on the disease to be treated. Methods of administration are further described herein.
  • Non-mammalian cells which share essentially the same signal transduction pathways as those in mammalian cells, e.g., yeast cells, can also be target cells ofthe invention.
  • Compounds of the invention may specifically inhibit the activity of a single kinase, e.g., src kinase, or they may inhibit the activity of more than one kinase or more than one type of kinase. Accordingly, a compound of the invention could be used for treating one or more diseases associated with one or more kinases.
  • the efficacy of the compounds of the invention against a broad range of target cells allows for broad applications for these compounds.
  • Src tyrosine kinase has specifically been implicated in the development, growth, progression, and metastasis of a number of human cancers such as colon, breast, pancreas and brain (see, e.g., Irby and Yeatman (2000) Oncogene 19:5636), and these cancers are expected to be treatable with the compounds of the invention.
  • a src kinase activity from 4-20 fold higher than normal has been found in mammary carcinomas (Irby and Yeatman, supra; Egan et al. (1999) Oncogene 18: 1227 and Nerbeek et al. (1996) J. Pathol. 180:383).
  • c-src has also frequently been implicated in the initiation and progression of human colon cancer and in resultant metastases (see, e.g., Cartwright et al. (1994) J. Clin. Invest. 93:509; Talamonti et al. (1991) J. Clin. Invest. 91 :53; and Termuhlen et al. (1993) J. Surg. Res. 54).
  • Src is increased 5-8 fold in the majority of colon tumors. Elevated src activity is also present in pre-cancerous colon lesions, e.g., adenomatous polyps (Pena et al. (1995) Gastroenterol. 108: 1 17).
  • pancreatic cancer Flossmann-Kast et al. (1998) Cancer Res. 8:3551); and Visser et al. (1996) Lab. Invest. 74:2)
  • lung cancer Mazurenko et al. (1992) Eur. J. Cancer 28:372)
  • neural cancer Bjelfinan et al. (1990) Cancer Res. 50:6908
  • ovarian cancer Wiener et al. (1999) Clin. Cancer Res. 5:2164)
  • esophageal adenocarcinomas and Barrett's Kumble et al. (1997) Gastroeneterology 112:348)
  • gastic cancers Takeshima et al.
  • the compounds of the invention can also be used to treat diseases associated with defects in cell adhesion and motility, such as angiogenesis, inflammation and bone reso ⁇ tion.
  • Src has been shown to play a role in signal transduction via cell-adhesion receptors (integrins). Src dependent cell migration is important for the function of many cell types, e.g., the motility of osteoclasts and metastasizing cells (Chellaiah et al. (2000) J. Biol. Chem. 275 : 11993 and Susa and Teri (2000) Drug News Pe ⁇ ect. 13:169).
  • Src dependent cell migration may also be important for the recruitment of vascular smooth muscle cell precursors in response to PDGF produced by endothelial cells during blood vessel formation (Hirschi et al. (1998) J. Cell. Biol. 141 :805).
  • Src kinase is also involved in endocytosis, e.g., transcytosis, such as that which occurs in osteoclasts (Nesbitt and Horton (1997) Science 276:266). Src assists endocytosis of certain growth factor receptors, e.g., EGF receptors (Wilde et al. (1999) Cell 96:677). Blood vessel hype ⁇ ermeability induced by vascular endothelial growth factor (VEGF) is also dependent on src (Eliceiri et al. (1999) Mol. Cell 4:915). Src has been shown to also be involved in cell survival (reviewed in Susa et al. (2000) Trends in Pharmacol. Sci. 21 :489). Accordingly, diseases related to any of these exemplary src biological activities can be treated with the compounds ofthe invention.
  • endocytosis e.g., transcytosis, such as that which occurs in osteoclasts (Ne
  • a preferred use for the compounds of the invention is for the treatment of osteoporosis, which involves bone reso ⁇ tion.
  • Osteoporosis is a widespread disease of low bone mass that particularly affects post-menopausal women (see, e.g., Gowen et al. (2000) Emerging Drugs 5: 1).
  • the role of src in bone metabolism was first demonstrated in src- deficient mice and has been confirmed using small molecular weight inhibitors in animal models of osteoporosis.
  • Src-deficient mice have defective bone reso ⁇ tion, resulting in excessive bone mass and osteopetrosis (see, e.g., Thomas and Brugge (1997) Annu. Rev. Cell. Dev. Biol., 13: 513).
  • src The role of src in bone reso ⁇ tion is well recognized.
  • a src inhibitor has been shown to reduce bone reso ⁇ tion in an animal model of osteoporosis (Missbach et al. (1992) Bone 24:437).
  • the disorder is believed to be caused by dysfunctions in osteoclasts and osteoblasts, as well as in osteoclast survival and osteoclast formation (reviewed in Susa et al, supra).
  • cancers of the brain include histiocytic lymphoma, lung adenocarcinoma, pancreatic carcinoma, colo-rectal carcinoma, bladder cancers, head and neck cancers, acute and chronic leukemias, melanomas, neurological tumor, myeloid leukemias (for example, acute myelogenousleukemia), sarcomas, thyroid follicular cancer, and myelodysplastic syndrome.
  • malignancies e.g., cancers of the brain, genitourinary tract, prostate, skin, lymphatic system, rectum, stomach, larynx, ovary, bladder, and liver.
  • cancers include histiocytic lymphoma, lung adenocarcinoma, pancreatic carcinoma, colo-rectal carcinoma, bladder cancers, head and neck cancers, acute and chronic leukemias, melanomas, neurological tumor, myeloid leukemias (for example, acute myelogenousleukemia), sarcoma
  • the compounds ofthe invention can also be used for treating disease associated with abnormal activity and/or expression of members of a growth factor family or receptors thereof.
  • compounds of the invention are expected to be effective against diseases associated with a defect in a growth factor or receptor of the EGF receptor family, such as Neu-erb2-related genes.
  • the compounds ofthe invention are believed to be effective against the following diseases.
  • amplification and/or over-expression of human erbB2 gene has been shown to correlate with a poor prognosis in breast and ovarian cancers, in particular, carcinomas (see, e.g., Slamon et al, Science 235:177-82 (1987); Slamon et al, Science 244:707-12 (1989)).
  • ErbB2 Overexpression of erbB2 has also been correlated with other carcinomas including carcinomas ofthe stomach, endometrium, salivary gland, lung, kidney, colon and bladder. ErbBl has been causally implicated in human malignancy, e.g., aggressive carcinomas ofthe breast, bladder, lung, and stomach. ErbB gene amplification or overexpression, or a combination of both, has also been demonstrated in squamous cell carcinomas and glioblastomas (Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M.D., Ullrich, A.
  • ErbB3 has been found to be overexpressed in breast (Lemoine et al, Br. J. Cancer 66:1116-21 (1992)), gastrointestinal (Poller et al, J. Pathol. 168:275-80 (1992); Rajkumer et al, J. Pathol. 170:271-78 (1993); Sanidas et al, Int. J. Cancer 54:935- 40 (1993)), and pancreatic cancers (Lemoine et al, J. Pathol.
  • the hyper-proliferative disorders that can be treated by the disclosed substituted pyrimidine compounds, salts, prodrugs and compositions thereof include, but are not limited to solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include, but are not limited to lymphomas, sarcomas, and leukemias.
  • breast cancer examples include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
  • cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.
  • brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor.
  • Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer.
  • Tumors ofthe female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma ofthe uterus.
  • Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallblader, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
  • Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, and urethral cancers.
  • Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
  • liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
  • Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
  • Head-and-neck cancers include, but are not limited to laryngeal / hypopharyngeal / nasopharyngeal / oropharyngeal cancer, and lip and oral cavity cancer.
  • Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma ofthe central nervous system.
  • Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.
  • Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. These disorders have been well characterized in man, but also exist with a similar etiology in other mammals, and can be treated by pharmaceutical compositions ofthe present invention.
  • proliferative disorders that can be treated according to the invention include non malignant cell proliferative disorders, such as those associated with an abnormal production of, or response to a growth factor, e.g., platelet derived growth factor (PDGF), fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF) and vascular endothelial growth factor (VEGF).
  • a growth factor e.g., platelet derived growth factor (PDGF), fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF) and vascular endothelial growth factor (VEGF).
  • exemplary diseases include restinosis, glomerulonephritis, neurofibromatosis, glaucoma, psoriasis, rheumatoid arthritis, inflammatory bowel disease, and chemotherapy-induced alopecia and mucositis. Restenosis following coronary angioplasty is one major unsolved problem of interventional cardiology.
  • the compounds of the invention are used for treating inflammatory diseases, e.g., rheumatoid arthritis (R.A.).
  • Synovial tissues of RA patients express high levels of FGF and PDGF compared with synovial tissues of osteoarthritis patients, a non invasive joint disease (Sano et al, J. Cell. Biol. 110:1417-1426, 1990). These data are consistent with the theory that PDGF and FGF play a role in generating an invasive tumor-like behavior in arthritic joints of RA synovial connective tissues (Sano et al, J. Clin. Invest. 91 :553-565 1993).
  • PDGF is considered to be a principal growth-regulatory molecule responsible for smooth muscle cell proliferation.
  • One smooth muscle disorder is atherosclerosis, which is a disease characterized by focal thickening of the inner portion of the artery wall, predisposing an individual to myocardial infarction (heart attack), cerebral infarction (stroke), hypertension (high blood pressure) and gangrene ofthe extremities.
  • atherosclerosis is a disease characterized by focal thickening of the inner portion of the artery wall, predisposing an individual to myocardial infarction (heart attack), cerebral infarction (stroke), hypertension (high blood pressure) and gangrene ofthe extremities.
  • lesions of atherosclerosis are surrounded by large amounts of lipid-laden macrophages, varying numbers of lymphocytes and large amounts of connective tissue.
  • PDGF has been found in numerous cells in such lesions, and it is believed that PDGF plays a critical role in the atherosclerosis disease process.
  • FGF and VEGF are potent angiogenic factors that induce formation of new capillary blood vessels. Accordingly, the compounds of the invention may be useful in inhibiting vascularization, e.g., in tumors.
  • the instant compounds may also be useful in the treatment of certain viral infections, in particular in the treatment of hepatitis C or delta and related viruses (Glenn et al. Science, 256:1331-1333 (1992)). Numerous viruses also induce non cancerous cell proliferation. Examples include papilloma viruses (HPV), which create skin lesions. Such viral infections may also be treatable with the compositions ofthe invention.
  • the compounds of the invention can also be used for treatment of hype ⁇ roliferative cutaneous diseases, e.g., keratosis and psoriasis.
  • the compounds of the invention can be used as anti-fungal agents to treat fungal infections on animals, e.g., humans.
  • the compounds can also be used for stopping fungus growth on objects, e.g., mold or mildew growth on shower curtains.
  • Salts especially pharmaceutically acceptable salts, ofthe compounds ofthe invention such as, for example, organic or inorganic acid addition salts, are also provided by the invention.
  • Suitable inorganic acids include but are not limited to halogen acids (such as hydrochloric acid), sulfuric acid, or phosphoric acid.
  • Suitable organic acids include but are not limited to carboxylic, phosphonic, sulfonic, or sulfamic acids, with examples including acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, 2- or 3 -hydroxy butyric acid, ⁇ -aminobutyric acid (GAB A), gluconic acid, glucosemonocarboxylic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azeiaic acid, malic acid, tartaric acid, citric acid, glucaric acid, galactaric acid, amino acids (such as glutamic acid, aspartic acid, N-methylglycine, acetytaminoacetic acid, N- acetylasparagine or N-acetylcysteine), pyruvic acid, acetoacetic acid, phosphoserine, and 2-
  • prodrugs Formation of prodrugs is well known in the art in order to enhance the properties of the parent compound; such properties include solubility, abso ⁇ tion, biostability and release time (see “Pharmaceutical Dosage Form and Drug Delivery Systems” (Sixth Edition), edited by Ansel et al, publ. by Williams & Wilkins, pgs. 27-29, (1995)).
  • prodrugs ofthe disclosed 2,4-diamino-pyrimidine compounds can be designed to take advantage ofthe major drug biotransformation reactions and are also to be considered within the scope of the invention.
  • Major drug biotransformation reactions include N-dealkylation, O-dealkylation, aliphatic hydroxylation, aromatic hydroxylation, N-oxidation, S-oxidation, deamination, hydrolysis reactions, glucuronidation, sulfation and acetylation (see Goodman and Gilman's The Pharmacological Basis of Therapeutics (Ninth Edition), editor Molinoff et al, publ. by McGraw-Hill, pages 11-13, (1996)).
  • the invention also includes pharmaceutical compositions comprising one or more of the compounds of the invention, or their salts or prodrugs forms thereof, with a pharmaceutically acceptable ingredient.
  • compositions can be prepared so that they may be administered orally, dermally, parenterally, nasally, ophthalmically, otically, sublingually, rectally or vaginally.
  • Dermal administration includes topical application or transdermal administration.
  • Parenteral administration includes intravenous, intraarticular, intramuscular, and subcutaneous injections, as well as use of infusion techniques.
  • One or more compounds of the invention may be present in association with one or more non-toxic pharmaceutically acceptable ingredients and optionally, other active anti-proliferative agents, to form the pharmaceutical composition.
  • These compositions can be prepared by applying known techniques in the art such as those taught in Remington's Pharmaceutical Sciences (Fourteenth Edition), Managing Editor, John E. Hoover, Mack Publishing Co., (1970) or Pharmaceutical Dosage Form and Drug Delivery Systems (Sixth Edition), edited by Ansel et al, publ. by Williams & Wilkins, (1995).
  • Commonly used pharmaceutical ingredients which can be used as appropriate to formulate the composition for its intended route of administration include: acidifying agents, examples include but are not limited to acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid; alkalinizing agents, examples include but are not limited to ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine; adsorbents, examples include but are not limited to powdered cellulose and activated charcoal; aerosol propellants, examples include but are not limited to carbon dioxide, CC1 2 F 2 , F 2 C1C- CC1F 2 and CC1F 3 ; air displacement agents, examples include but are not limited to nitrogen and argon; antifungal preservatives, examples include but are not limited to benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium be
  • caramel and ferric oxide red examples include but are not limited to bentonite; emulsifying agents, examples include but are not limited to acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyethylene 50 stearate; encapsulating agents, examples include but are not limited to gelatin and cellulose acetate phthalate; flavorants, examples include but are not limited to anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin; humectants, examples include but are not limited to glycerin, propylene glycol and sorbitol; levigating agents, examples include but are not limited to mineral oil and glycerin; oils, examples include but are not limited to arachis oil, mineral oil, olive oil, peanut oil, sesame oil and vegetable oil; ointment bases, examples include but are not limited to lanolin, hydrophil
  • compositions can take the form of aerosols, capsules, creams, elixirs, emulsions, foams, gels, granules, inhalants, lotions, magmas, ointments, peroral solids, powders, sprays, syrups, suppositories, suspensions, tablets and tinctures.
  • the therapeutic methods of the invention generally comprise administering to a subject in need thereof, a pharmaceutically effective amount of a compound.
  • the compounds of the invention can be administered in a amount effective to inhibit the activity of a kinase, e.g., a tyrosine kinase, such as src kinase.
  • the compounds of the invention can also be administered in a "growth inhibitory amount," i.e., an amount of the compound which is pharmaceutically effective to inhibit or decrease proliferation of target cells.
  • the compounds can also be administered in a "differentiation modulating amount", e.g., “differentiation-inducing amount” or “differentiation-inhibiting amount,” which is an amount of the compound which is pharmaceutically effective to modulate differentiation of target cells.
  • the compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice.
  • the compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
  • Toxicity and therapeutic efficacy of the compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such reagents to the site of affected tissue in order to minimize potential damage to normal cells and, thereby, reduce side effects.
  • the dosage of such reagents lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
  • compositions containing a compound of the invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate
  • granulating and disintegrating agents for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid
  • binding agents for example starch, gelatin, polyvinyl-pyrrolidon
  • the tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and abso ⁇ tion in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
  • Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
  • preservatives for example ethyl, or n-propyl p- hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p- hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p- hydroxybenzoate
  • sweetening agents such as sucrose, saccharin or aspartame.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation.
  • These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol .
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound of the invention in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent, suspending agent and one or more preservatives are exemplified by those already mentioned above.
  • Additional excipients for example sweetening, flavoring and coloring agents, may also be present.
  • These compositions may be preserved by the addition of an anti- oxidant such as ascorbic acid.
  • compositions of the invention may also be in the form of an oil-in- water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • sweetening agents for example glycerol, propylene glycol, sorbitol or sucrose.
  • Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
  • compositions may be in the form of sterile injectable aqueous solutions.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • Sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the compound of the invention is dissolved in the oily phase.
  • the active ingredient may be first dissolved in a mixture of soybean oil and lecithin.
  • the oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
  • the injectable solutions or microemulsions may be introduced into a patient's bloodstream by local bolus injection.
  • a continuous intravenous delivery device may be utilized.
  • An example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous pump.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this pu ⁇ ose any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions of the invention may also be administered in the form of a suppositories for rectal administration ofthe drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • creams, ointments, jellies, solutions or suspensions, etc., containing the compound of the invention can be employed.
  • topical application shall include mouth washes and gargles.
  • the compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art.
  • the dosage administration will preferably be continuous rather than intermittent throughout the dosage regimen.
  • the compounds ofthe invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated.
  • the compounds may be administered simultaneously or sequentially.
  • the instant compounds may be useful in combination with known anti-cancer and cytotoxic agents.
  • the instant compounds may be useful in combination with agents that are effective in the treatment and prevention of osteoporosis, inflammation, neurofibromatosis, restinosis, and viral infections.
  • the instant compounds may also be useful in combination with inhibitors of other components of signaling pathways of cell surface growth factor receptors.
  • Drugs can be co-administered to a subject being treated with a compound of the invention include antineoplastic agents selected from vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine, cytochalasin B, emetine, maytansine, or amsacrine.
  • antineoplastic agents selected from vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine, cytochalasin B, emetine, maytansine, or amsacrine.
  • chemotherapeutic agents are described in the "Physicians' Desk Reference” (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA).
  • Optional anti-proliferative agents that can be added to the composition include but are not limited to compounds listed on the cancer chemotherapy drug regimens in the 11 th Edition of the Merck Index, (1996), which is hereby inco ⁇ orated by reference, such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinote
  • anti-proliferative agents suitable for use with the composition of the invention include but are not limited to those compounds acknowldeged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Ninth Edition), editor Molinoff et al, publ.
  • anti-proliferative agents suitable for use with the composition of the invention include but are not limited to other anti-cancer agents such as epothilone, irinotecan, raloxifen and topotecan.
  • the daily oral dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the daily dosage for administration by injection including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight.
  • the daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily.
  • the transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg.
  • the daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight.
  • the particular method of administration will depend on a variety of factors, all of which are considered routinely when administering therapeutics. It will also be understood, however, that the specific dose level for any given patient will depend upon a variety of factors, including, but not limited to the activity of the specific compound employed, the age of the patient, the body weight of the patient, the general health ofthe patient, the gender ofthe patient, the diet ofthe patient, time of administration, route of administration, rate of excretion, drug combinations, and the severity of the condition undergoing therapy.
  • the optimal course of treatment i.e., the mode of treatment and the daily number of doses of a compound of formulae (I) or (II) or a pharmaceutically acceptable salt thereof given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests.
  • Radiation therapy including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with a compound ofthe invention to treat a disease, e.g., cancer.
  • a disease e.g., cancer.
  • the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response ofthe individual patient, as well as the severity ofthe patient's symptoms.
  • compounds of the invention and/or materials and reagents required for administering the compounds of the invention may be assembled together in a kit.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly prefe ⁇ ed.
  • the kit may further comprise one or more other drugs, e.g., chemo- or radiotherapeutic agent.
  • drugs e.g., chemo- or radiotherapeutic agent.
  • These normally will be a separate formulation, but may be formulated into a single pharmaceutically acceptable composition.
  • the container means may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, or injected into an animal, or even applied to and mixed with the other components ofthe kit.
  • compositions of these kits also may be provided in dried or lyophilized forms.
  • reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
  • the kits of the invention may also include an instruction sheet defining administration of the agent and, e.g., explaining how the agent will decrease proliferation of cells.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention also may comprise, or be packaged with a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • Other instrumentation includes devices that permit the reading or monitoring of reactions.
  • the resulting crude solid is purified either by silica gel column chromatography or washing with other solvents to afford the N-(3-substituted-lH-pyrazol-5-yl)-2-chloro-5- substituted-4-pyrimidinamine intermediate whose structure is confirmed by LC/MS and ⁇ ⁇ MR.
  • Preparative thin-layer silica gel chromatography, silica gel column chromatography, and or preparative HPLC are used to purify final products.
  • LC/MS and ⁇ NMR spectroscopy are used to confirm the structures of the final 2,4-substituted pyrimidinediamines.
  • a solution of the substituted nitrobenzene (1 equiv) in ethanol (0.2 M) is added via syringe to a flask containing palladium on carbon (10 mol%).
  • the reaction vessel is fitted with a balloon adapter and charged with hydrogen and evacuated three times until the reaction is under a H 2 atmosphere.
  • the reaction is allowed to stir overnight and then purged with As and evacuated three times until an Ar atmosphere has been achieved.
  • the reaction solution is filtered through a pad of Celite and washed with copious amounts of ethanol. The filtrate is concentrated in vacuo to afford the desired aniline whose structure is confirmed by LC/MS and ⁇ NMR.
  • Nitrophenylbromide (1 equiv), phenol (2 equiv), CsCO 3 (2 equiv), Cu(OTf) 2 ThH (0.03 equiv), 1-naphthoic acid (2 equiv), molecular sieves (5 A, 2 equiv), EtOAc (0.05 equiv), and toluene (1.6 M) are combined and heated at 100 °C overnight.
  • the mixture is cooled to ambient temperature before being taken up in CH C1 2 .
  • the mixture is washed with IN NaOH (lx), H O (lx), and brine (lx).
  • the organics are dried over Na 2 SO 4 and concentrated. Purification by flash silica gel chromatography provids the desired biaryl ether intermediate whose structure is confirmed by LC/MS and ⁇ NMR.
  • This compound was prepared by the general method described in D using 2-(2-chloroethyl)- 1 -methylpyrrolidine hydrochloride as the starting aminoalkyl halide to give 1 -methyl -4-(3- nitrophenoxy)azepane.
  • Examples 2-220 were prepared by general method C where a heterocyclic substituted pyrimidine (prepared by general methods A and B) is reacted with an aniline sidechain (prepared by general methods D-X):
  • Analytical HPLC were obtained using a Gilson HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (50 x 4.6 mm, 12 ⁇ m).
  • the eluents were A: acetonitrile w/0.1% TFA and B: H 2 O w/0.1% TFA.
  • Gradient elution from 10% B to 90% over 4 min at a flowrate of 4.0 mL/min was used with an initial hold of 0.5 min and a final hold at 90% B of 0.5 minutes. Total run time was 5 min.
  • HPLC - electrospray mass spectra were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonirile with 0.018% TFA. Gradient elution from 10% B to 95% over 3.5 minutes at a flowrate of 1.0 mL/min was used with an initial hold of 0.5 minutes and a final hold at 95% B of 0.5 minutes. Total run time was 6.5 minutes.
  • HPLC - electrospray mass spectra were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonirile with 0.018% TFA. Gradient elution from 10% B to 95% over 3.5 minutes at a flowrate of 1.0 mL/min was used with an initial hold of 0.5 minutes and a final hold at 95% B of 0.5 minutes. Total run time was 6.5 minutes.
  • the silica-coated crude was then purified on a plug of silica gel (1 L), eluting with a gradient of 100% CH 3 CN (2 L) -» 5% MeOH/CH 3 CN (1 L) -» 10% MeOH/CH 3 CN (1 L) -» 20% MeOH/CH 3 CN (6 L) -> 50% MeOH/CH 3 CN (1 L) - 50% MeOH/CH 3 CN (2 L).
  • the fractions containing product were pooled and concentrated in vacuo to give a brown solid. This was triturated in MeOH (700 ml) with water (300 ml) and the insoluble salts filtered off and washed with MeOH.
  • the filtrate was again coated on silica gel (300 ml) and the column purification repeated as above to give an amber oil/solid. This was again suspended in MeOH/water (2:1) and concentrated until a precipitate formed. The salts were again filtered off and washed with CH 3 CN and acetone. The filtrate was concentrated in vacuo to a brown gum which solidified after drying in vacuo under P 2 ⁇ 5 . The solid was pulverized with a mortar and pestle then suspended in acetone (1 L), sonicated, and filtered washing with acetone. The desired product was obtained as a light tan solid in 65% yield (44.04 g, 210.1 mmol) after drying in vacuo under P 2 O 5 for 24 h.
  • the internal temp was kept at ⁇ 5 °C during the quench. This was stirred from 0 °C -> rt over 20 h with vigorous stirring.
  • the acidic solution was then cooled to 0 °C with an ice bath, and quenched by portionwise addition of powdered K 2 CO 3 (380 g) over 1.5 h with vigorous stirring to a final pH of 1 1.5 (internal temp, always kept at ⁇ 8 °C during the quench; vigorous bubbling observed).
  • the still-cold light brown solution is extracted with EtOAc (2L), and this was dried (Na 2 SO 4 ), filtered, and concentrated in vacuo to give an amber oil.
  • the combined organics were dried ( ⁇ a 2 SO 4 ) and the solvent removed in vacuo.
  • the crude product was purified by silica gel chromatography (100% acetonitrile - 5% water/methanol gradient). The fractions containing product were concentrated in vacuo, and the residue dissolved in dichloromethane and filtered to remove silica gel. Hexane was added, and the solvent removed in vacuo to give a solid. The product was isolated as a light pink solid from hexane in 68% yield (88 mg, 0.195 mmol).
  • Examples 220-258 are prepared by general method AC or AD where a 4-chloropyrimidine (prepared by methods Y-AB) is reacted with an amino pyrazole (prepared by general method A): Table 3. Compounds Prepared by general methods AC or AD
  • d Pyrazole is commerically available.
  • d Pyrazole was prepared as follows: To a solution of 5-nitro-lH-pyrazole-3-carboxylic acid (1 equiv) in T ⁇ F (0.32 M) was added CDI (1 equiv). The reaction was allowed to stir for 5 min and then diethylamine (1.5 equiv) was added via syringe. The reaction mixture was stirred overnight at room temperature and concentrated. The residue was purified by preparative ⁇ PLC using 10-90%ACN/ ⁇ 2 O to afford the intermediate nitro pyrazole whose structure was confirmed by LC/MS and ⁇ NMR.
  • Example 25 Preparation of N -(3-tert-butyl-l-methyl-iH-pyrazol-5-yl)-N 2 - ⁇ 3-[2- (diethyl-amino)ethoxy] phenyl ⁇ -N 2 ⁇ V 4 -dimethyl-2,4-py rimidinediamine
  • Example 260 N 4 -(3-tert-butyl-l-methyl-lH-pyrazol-5-yl)-N 2 - ⁇ 3-[2- (diethylamino)ethoxy]-phenyl ⁇ -N -methyl-2,4-pyrimidine-diamine
  • the organic layer was extracted with water (2 x 25 mL) and brine (1 x 25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure.
  • the crude organic concentrate was purified using MPLC (Biotage) eluted with 10% methanol - dichloromethane with 1 % ammonium hydroxide followed by 20% methanol - dichloromethane with 1% ammonium hydroxide.
  • src kinase activity assays e.g., assays for testing the activity of compounds on kinases other than src; and assays for testing the activity of compounds on cell proliferation and differentiation.
  • a preferred method for measuring src kinase activity uses ATP (5 ⁇ M/well) mixed with biotinylated poly-GAT substrate (10 nM/well), Streptavidin- APC (15 nM/well) and European-labeled anti-phosphotyrosine antibody (2.5 nM/well).
  • 10 ⁇ l of a mixture of these components is added to each well of a black 96-well plate, with or without test compound (5 ⁇ l desired concentration of compound in DMSO).
  • 75 ⁇ l of assay buffer 50 mM HEPES pH 7.5, 0.1 mM EDTA, 0.015% BRIJ 35 solution, 0.1 mg/mL BSA, 0.1% beta-mercaptoethanol, 10 mM magnesium chloride
  • assay buffer 50 mM HEPES pH 7.5, 0.1 mM EDTA, 0.015% BRIJ 35 solution, 0.1 mg/mL BSA, 0.1% beta-mercaptoethanol, 10 mM magnesium chloride
  • the src kinase (0.1 units/well) (Upstate Biotech, Lake Placid, NY) is added (10 ⁇ l) to a final volume of 100 ⁇ l.
  • a specific signal is the ratio ofthe value of the signal at 665 and the value of the signal at 615 multiplied by 10,000 (i.e., (signal at 665/signal at 615) x 10,000).
  • Compounds that cause the specific signal to decrease inhibit the kinase activity of src. Percent inhibitions and/or IC 50 values can then calculated based on specific signals from wells that have no compound added, i.e., zero percent inhibition.
  • a specific signal is the ratio of the value of the signal at 665 and the value of the signal at 615 multiplied by 10,000 (i.e., (signal at 665/signal at 615) x 10,000).
  • Compounds that cause the specific signal to decrease inhibit the kinase activity of src. Percent inhibitions and/or IC 50 values can then calculated based on specific signals from wells that have no compound added, i.e., zero percent inhibition.
  • the assay can also be adapted to determine the inhibitory activity of compounds towards kinases other than src kinases.
  • the src kinase enzyme in the above assay can be replaced with another kinase.
  • the antibody in the assay may also have to be replaced with an antibody that is specific for the phosphorylated residue, which has been phosphorylated by the kinase.
  • the effect of compounds on cell proliferation can be determined, e.g., by incubating cells with varying amounts of the compounds and counting the cells over time. Viable cells can be counted by staining the cells with a specific dye, e.g., Trypan Blue, according to methods well known in the art. Other methods include measuring the incorporation of a labeled molecule into DNA or RNA or protein of cells. For example, cell proliferation is often measured by H thymidine or 5-bromodeoxyuridine incorporation assays, also well known in the art.
  • the effect of a compound on cell differentiation can be determined by visualization of the cells after having been contacted with the compound, preferably by comparison with cells which have not been contacted with the compound.
  • the differentiation of certain cells is visible by the naked eye (e.g., that of 3T3L1 cells), whereas that of other cells may require the use of a microscope.
  • Specific dyes can also be used to evaluate the state of differentiation of cells.
  • Cell differentiation can also be monitored by measuring the expression level of certain genes, whose expression is known to vary during differentiation ofthe cells.
  • the effect of a compound on a cell can be determined in a cell that contains an abnormal kinase, e.g., a mutated kinase gene, or a cell which over-expresses a kinase.
  • the cell can be a cell expressing a mutated form ofa tyrosine kinase, e.g., src kinase, thereby transforming the cell.
  • the cell can also be a cell that has an abnormal proliferation which is not caused by an abnormal activity or level of a kinase.
  • Cells that can be used for testing compounds of the invention include cell lines and primary cell cultures.
  • a proto-oncogene which encodes, e.g., a kinase
  • ATCC American Type Culture Collection
  • Cell lines over-expressing a gene e.g., a kinase
  • Nucleic acids for use in transforming cells e.g., nucleic acids encoding kinases, are also publicly available or can readily be obtained.
  • Cell lines can also be obtained from transgenic animals, e.g., animals overexpressing a kinase or expressing a mutated kinase.
  • MG 1361 is a breast carcinoma cell line obtained from the MMTV-neu transgenic mouse (Sacco et al, Breast Cancer Res. Treat., 47:171-180 (1998)).
  • Primary cell cultures can be established from biopsies obtained from patients, e.g., patients having cancer.
  • the present invention also provides methods of testing a compound (e.g., the candidate drug) for its inhibition of src, its antiproliferative effect, its effect on cell differentiation and/or its toxicity on normal or wild-type cells in animals, e.g., transgenic animals, e.g., mice.
  • a compound e.g., the candidate drug
  • transgenic animals e.g., mice.
  • Transgenic mice are produced that express a transforming agent (e.g., a growth factor receptor) under the control of a promoter, e.g., a tissue specific promoter.
  • a promoter e.g., a tissue specific promoter.
  • Such mice develop carcinomas that have genetic and pathological features that closely resemble human cancers.
  • mice expressing viral polyoma middle T antigen under the control of the MMTV promoter produces highly metastatic mammary tumors with elevated c-src kinase activity (Guy et al. (1994) Genes and Dev. 8:23).
  • Nude mice in which tumor cell lines have been administered can also be used.
  • breast cancer cell lines over-expressing c-src can be administered to nude mice (see, e.g., Biscardi et al. (1998) Mol. Carcinog. 21 : 261).
  • the ability of the compound to inhibit tumor formation or growth is then ascertained.
  • the size of the tumor is monitored by determining the tumor size and/or weight.
  • the compounds can be administered by a variety of ways including orally, subcutaneously, or intraperitoneally. Generally, at least two groups of animals are used in the assay, with at least one group being a control group which is administered the administration vehicle without the compound.

Abstract

The invention provides novel substituted 2,4-diaminopyrimidine compounds (I), in which L is a linker selected from: -O-(CH2)1-4- ; s(O)0-2-(CH2)1-4- ; -N(R1)-(CH2)1-4-(CH2)1-4-O-(CH2)1-4- ; -N(R1)-C(O)-(CH¿2?)1-4- ; (I) and (I) G represents: Alternatively, L may represent (I) or (I), and in this event, G represents (I) and pharmaceutical compositions thereof. The invention also provides methods of use of the novel substituted 2,4-diaminopyrimidine compounds and pharmaceutical compositions thereof as inhibitors of src kinase enzymes. Exemplary diseases that can be treated by the compounds of the invention include cell proliferative diseases, such as cancer and non-malignant cell proliferative diseases, osteoporosis and inflammatory diseases. Also provided are methods for preparing the compounds of the present invention.

Description

APPLICATION FOR PATENT
2-PHENYLAMINO-4-(5-PYRAZOLYLAMINO) -PYRIMIDINE DERIVATIVES AS KINASE INHIBITORS, IN PARTICULAR, AS SRC KINASE INHIBITORS
Field ofthe Invention
The present invention relates to substituted pyrimidine compounds, and in particular, 2,4-diamine-substituted pyrimidine compounds, and pharmaceutical compositions thereof, and the use of such substituted pyrimidine compounds as inhibitors of src kinase enzymes.
Background ofthe Invention
Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance, e.g., by increasing the rate of cell proliferation, modulating the rate of cell differentiation or decreasing the rate of cell death, can result in the abnormal growth of cells and is thought to be a major event in the development of cancer, as well as other cell proliferative disorders such as restenosis.
Proliferative disorders, e.g., cancer, cause significant numbers of deaths. For example, cancer causes over half a million deaths per year in the United States alone. Conventional strategies for the treatment of cancer include chemotherapy, radiotherapy, surgery or combinations thereof, however further advances in these strategies are limited by lack of specificity and excessive toxicity to normal tissues. In addition, certain cancers are refractory to treatments such as chemotherapy, and some of these strategies such as surgery are not always viable alternatives. For example, non-small-cell lung cancer (NSCLC), which includes squamous cell carcinoma, adenocarcinoma and large-cell carcinoma, accounts for 75-80% of all lung cancers (American Cancer Society, 1993). Current multimodality therapeutic strategies applied to regionally advanced NSCLC are minimally effective with the overall cure rate being only about 10% (Belani (1993) Semin Oncol. 20:302 and Roth et al. (1994) Lung Cancer 11 Suppl 3:S25).
Cell growth, differentiation and other cell processes are regulated by signal transduction pathways involving protein phosphorylation. Protein phosphorylation is the result of the transfer of a terminal phosphate of adenosine triphosphate to a particular amino acid of a protein. This transfer is catalyzed by enzymes termed kinases. Protein kinases comprise a large superfamily of homologous proteins. They are related by their kinase or catalytic domains, which consists of approximately 250-300 amino acid residues. There are two main categories within the superfamily of protein kinases: the protein-serine/threonine kinses and the protein-tyrosine kinases (Hanks et al, (1995) FASEB J. 9:576)
Kinases having an abnormal activity, e.g., mutated kinases, or abnormal levels of kinases, have been associated with abnormal cellular processes, which result in specific diseases. For example, several oncogenes, which are capable of transforming cells, are mutated forms of normal genes encoding kinases. Examples of such oncogenes include the pp60-v-src gene from the Rous avian sarcoma virus, which corresponds to the normal (i.e., proto-oncogene) gene pp60-c-src, containing a deletion that removes the C-terminal 18 amino acids of c-src. Pp60-c-src is also referred to as "src kinase" or "src tyrosine kinase." Phosphorylation ofa tyrosine residue at position 527 of c-src protein causes a great reduction in its kinase activity, and this site is often altered in oncogenic derivatives of c-src (see, e.g., Brown et al, (1996) Biochem. Biophys. Acta 1287:121). Other proto-oncogenes encoding tyrosine kinases, which when mutated or over-expressed, cause cells to become transformed, include c-yes; c-fps (c-fes); c-abl and c-met. c-abl and c-met are associated with chronic myelogenous leukemia and osteosarcoma, respectively. Proto-oncogenes encoding serine/threonine kinases include c-mos and c-raf (c-mil). Whereas the above-cited proto- oncogenes are intracellular transducers, other proto-oncogenes encode kinases which are cell-surface receptors. Examples of proto-oncogenes encoding cell surface receptors with tyrosine kinase activity include c-fins (or Colony Stimulating Factor -1 (CSF-1) receptor); c- erbB, which is an epidermal growth factor receptor; c-neu (or erbB-2), erbB-3 or erbB-4 which are related to epidermal growth factor receptor; and c-ros, which is related to the insulin receptor.
The role of abnormal kinase activity or protein levels in diseases has been abundantly documented. This has been demonstrated, e.g., by using inhibitors of kinases, in particular tyrosine kinases. Such inhibitors have been shown to be useful for the treatment of disease states characterized by uncontrolled cell proliferation, e.g., cancer, inflammation, psoriasis, pulmonary fibrosis, glomerulonephritis, atherosclerosis, osteoporosis and restenosis following angioplasty. For example, tyrosine kinase inhibitors with selectivity for the EGF receptor family have been shown to block tumor formation in animals, thus demonstrating their potential usefulness for directly suppressing tumor cell growth in the treatment of human cancer, especially breast carcinoma. Also, tumor metastasis and its associated angiogenesis has been shown to be inhibited by preventing the activation of the vascular endothelial growth factor receptor tyrosine kinase which indicates a utility for tyrosine kinase inhibitors in blocking separate events that occur during carcinogenesis. Thus, protein phosphorylation, e.g., tyrosine phosphorylation, plays an important role in cell regulatory processes, e.g., cell proliferation, and in diseases.
The pp60c-src protein has significant structural homology to about ten proteins (collectively referred to as Src Family kinases or SFKs) which include: Lck, Fyn, Yes, Yrk, Blk, Fgr, Hck, Lyn, and Frk subfamily members Frk/Rak and Iyk/Bsk (Sawyer et al, (2001) Expert Opin. Investig. Drugs 10(7): 1327). The Src family of tyrosine kinases, has three major domains: src homology SHI, SH2, and SH3 domains. The SHI domain is most commonly called the catalytic domain or tyrosine kinase domain. The SH3 domain is a binding region for proteins having proline-rich sequences. Both the SH2 and SH3 domains are noncatalytic, but are important in protein-protein recognition. SH2 domains are homologous motifs of approximately 100 amino acids, which recognize and bind to the phosphorylated sequences present on regulatory proteins and growth factor receptors (Anderson et al, Science, 1990, 250, 979). One of the primary purposes of the src family phosphoprotein/SH2 domain interaction is to initiate the association of proteins into an activation complex, often around the intracellular domain of the receptor itself. This role of the src family SH2 domain mediates and organizes the ordered, physical assembly of the various proteins in the activation complex. The activity of a number of immunologically important src family SH2 domain-containing proteins, including, Fyn, Fgr, Yes, Lyn, Hck and Lck, is mediated in this way. P561ck is of particular interest because it has been associated with the signal transduction cascade needed for T-cell activation mediated by the T-cell receptor (TCR) (Straus et al. (1992) Cell, 70, 585).
The Src family of protein kinases, which all contain an SH2 domain, are involved in a number of cellular signalling pathways. For example, Src is involved in growth factor receptor signaling; integrin-mediated signaling; T- and B-cell activation; osteoclast activation; cell adhesion; cell motility and cell survival. It is known that the Src SH2 domain binds to several key receptor and nonreceptor tyrosine kinases such as tyrosine kinases containing receptors for PDGF, EGF, HER2 Neu (an oncogene form of EGF), Fibroblast Growth Factor (FGF), focal adhesion kinase, pi 30 protein, and p68 protein. In addition, src has been shown to be involved in the regulation of DNA synthesis, mitosis, and other cellular activities (see, e.g., Susa et al. (2000) Trends Pharm. Sciences 21 :489).
Current cancer therapies utilize a battery of cytotoxic agents and radiation regimens to both decrease and eradicate tumors. The therapeutic index associated with these therapies is narrow and patients suffer from toxic side effects such as hair loss, bone marrow toxicity, loss of intestinal epithelium and mucositis. Many patients derive a therapeutic benefit from such treatment with an initial reduction in tumor mass and stabilization of the disease. However, recurrence is common and many times the tumors acquire a drug resistant phenotype and are refractory to future treatment with chemotherapeutic agents.
The need exists for kinase inhibitors, such as tyrosine kinase inhibitors, that overcome the above-mentioned deficiencies.
Summary ofthe Invention
This invention provides compounds for regulating cellular processes involving a kinase such as a tyrosine kinase, in particular, a src kinase. In its broad aspect, the invention relates to a compound ofthe formula (I)
Figure imgf000006_0001
in which Y1 represents H, C alkyl, or phenyl optionally substituted up to three times by halogen, CH alkyl, or C alkoxy. Y2 and Y3 are independently selected from H; Cι-6 alkyl; C -6 cycloalkyl optionally substituted by C1-4 alkyl; phenyl optionally substituted up to three times by halogen, C alkyl, or CM alkoxy; adamantyl; CF3 ; a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C)-6 alkyl; C(O)N(C1- alkyl)2 ; C(O)O(C1- alkyl) ;
CN; halogen; and
Figure imgf000007_0001
.
Alternatively, Y2 and Y3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C alkyl, or C alkoxy. X represents halogen or CM alkyl. The subscript n represents 0, 1, or 2. R1 represents H or C1-4 alkyl. R2 represents H or CM alkyl. R3 represents: Cι-6 alkyl; halogen;
CM alkoxy; O-phenyl optionally substituted up to two times by halogen, C1-4 alkyl, C1-4 alkoxy, or di-(CM alkyl)amino; CN; or N(R')2 wherein the R1 moieties are independent or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. The subscript p represents 0, 1 , or
2. R4 represents C]- alkyl or halogen, and q represents 0, 1, or 2.
Alternatively, R3 and R4 may be joined and taken together with the carbon atoms to which they are attached, form a 5-6 membered heteroaromatic ring containing up to two heteroatoms selected from N, O, and S, and which is optionally substituted up to two times by halogen, C1-4 alkyl, or C1-4 alkoxy.
L is a linker selected from: -O-(CH2)1-4- ; -S(O)0-2-(CH2)1-4- ; -N(R')-(CH2)M- ;
-(CH2)M-O-(CH2)1-4- ; -(CH2) - ; -NCR'KXOMCH^M- ;
~C=C-(CH2)ι_4- .
Figure imgf000007_0002
. a Ό-I G represents:
1) NR5R6 , and a number of additional groups to be discussed below. In the substituent group NR5R6 , R5 represents H, Cι-6 alkyl, or C1-4 alkoxy-Cι-4 alkyl. R6 represents H; C1-6 alkyl; CM alkoxy-substituted C alkyl; C5-6 cycloalkyl optionally substituted up to 2 times by halogen, CM alkyl, or CM alkoxy; C3-6 cycloalkyl-substituted CM alkyl; benzyl; phenyl optionally substituted by halogen, Cι-4 alkyl, CM alkoxy,
-CO2R', -C(O)N(R')2 , -N(Rl)2 , or by a bivalent group
Figure imgf000007_0003
. In the foregoing bivalent groups, A is N(R1), S, S(O), S(O)2, or O , and the bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5 -membered
heterocycle. R may also be
Figure imgf000008_0001
, in which A represents N(R ), S,
S(O), S(O)2 , or O; or
Figure imgf000008_0002
? ιn whjch A2 represents N(R'), S, S(O), S(O)2 , or O. G may also be:
2)
Figure imgf000008_0003
9 which may optionally be substituted up to 2 times by C1-3 alkyl, (Cι-3 alkoxy)(C1-4 alkyl), CCOJOR1, C(O)N(R')2 , phenyl, or benzyl;
|- A3
3) ^ — , which may optionally be substituted up to 2 times by C1-3 alkyl, (C1-3 alkoxy)(C1-4 alkyl), C(O)OR1, C(O)N(R[)2 , phenyl, or benzyl, and in which A3 represents N(R'), S, S(O), S(O)2 ;
4) *Q x 'o-i, which may optionally be substituted up to 2 times by oxo, (C1-3 alkoxy)-(C1-4 alkyl), C(O)OR', C(O)N(R!)2 , phenyl, or benzyl, or up to 4 times by Cι- alkyl; or
5)
Figure imgf000008_0004
s which may optionally be substituted up to 2 times by halogen, C1-4 alkyl, or CM alkoxy, and in which v is 0 or 1 ; or
|-N 6) ^^ , which may optionally be substituted up to 2 times by CM alkyl.
, /(CH2)- . ,(CV12)-
-O- X-3 _N 2 2
(CH,)- (CH2)-
Alternatively, L may represent 2-3 or 2 , and in this event,
G represents
N-R1
Pharmaceutically acceptable salts are also within the scope ofthe invention. In another aspect, the invention relates to a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier.
In yet another embodiment, the invention provides methods for regulating cellular processes involving a kinase, such as a tyrosine kinase. In a preferred embodiment, the cellular process involves a src kinase. The cellular process can be, e.g., cell proliferation or cell differentiation.
The invention provides methods for treating diseases associated with a kinase, e.g., diseases associated with an abnormal kinase activity or level, such as cancers, osteoporosis, and inflammatory disorders. The invention also provides methods for treating diseases associated with abnormal cell proliferation and/or differentiation. In a preferred embodiment, the method comprises administering to a subject in need thereof, a pharmaceutically efficient amount of a compound of the invention, such that the subject is treated.
The invention also provides methods for preparing the compounds of the present invention. Also within the scope of the invention are kits comprising one or more compounds ofthe invention, optionally in a pharmaceutical composition.
Detailed Description ofthe Invention
The invention is based at least in part on the observation that 2,4-diamino substituted pyrimidine compounds inhibit the activity of src kinases. Exemplary compounds are described herein.
In formula (I), Y1 is preferably H or C)-4 alkyl, and more preferably H.
Preferably, Y2 is selected from C ι-6 alkyl; C3-6 cycloalkyl optionally substituted by
CM alkyl; phenyl optionally substituted up to three times by halogen, CM alkyl, or CM alkoxy; adamantyl; CF3; a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or Cι-6 alkyl; and
Figure imgf000010_0001
; and Y3 is H. As a preferred alternative, Y2 and Y3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C1-4 alkyl, or CM alkoxy.
More preferably, Y2 is selected from C ι-6 alkyl; C -6 cycloalkyl optionally substituted by CM alkyl; phenyl optionally substituted up to three times by halogen, C1-4 alkyl, or C alkoxy; adamantyl; and a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C1-6 alkyl; and Y3 is H.
Most preferably, Y is selected from C 1-6 alkyl, and C3-6 cycloalkyl optionally substituted by CM alkyl; and Y3 is H.
X preferably represents Cl, F, or CM alkyl, and n is 0, 1, or 2. More preferably, X represents F, and n is 0 or 1.
The groups R1 and R2 are each preferably H.
R is preferably Cι-6 alkyl; halogen; C M alkoxy; CN; or N(R )2 in which the R moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R2), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. More preferably,
R3 is Ci-6 alkyl; C alkoxy; CN; or N(R*)2 in which the R1 moieties are independent, or the
R1 moieties optionally are joined by a linker selected from the group consisting of CH(R1), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. Most preferably, R3 is C1-6 alkyl; C 1-4 alkoxy; or
N(R*)2 in which the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O) , and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. The subscript p, which represents the number of R3 groups, is preferably 0, 1 , or 2, more preferably 0 or 1. Preferably, the phenyl ring of Fig. (I) bears no R4 groups.
L is preferably -O-(CH2)M- ; -S(O)0-2-(CH2) - ;
Figure imgf000010_0002
; -(CH2) - ;
-(CH2)M-O-(CH2)M- ; v '°-' ; or
Figure imgf000010_0003
.
G is preferably: 1) NR5R6 , in which R5 represents H or C1-6 alkyl; and R6 represents: Cι-6 alkyl; C1-4 alkoxy-substituted C1-4 alkyl; C5-6 cycloalkyl optionally substituted up to 2 times by halogen, CM alkyl, or C1-4 alkoxy; C3-6 cycloalkyl-substituted CM alkyl; benzyl; phenyl optionally substituted by halogen, C1-4 alkyl, C1- alkoxy, -CO2R',
-C(O)N(R , 1K)2 , -N vτ(/Rn K')2 , o _r b!.y.. a_ b !.i:v..a.l,ent. g _ro.u.-p_
Figure imgf000011_0001
i -n- w~.hi-i.c-hi. A Λ is_ Nτ(R'),
S, S(O), S(O) , or O , and this bivalent group is connected to the phenyl ring at
adjacent carbon atoms to form a fused 5-membered heterocycle; or
Figure imgf000011_0002
; in which A2 represents N(R'), S, S(O), S(O)2 , or O;
2)
Figure imgf000011_0003
5 optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(C1-4 alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl;
|-N A3
3) \/ , optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(CM alkyl), C(O)OR1, C(O)N(R')2 , phenyl, or benzyl, and in which A3 represents N(R]), S, S(O), S(O)2 , or O;
4) yo-i, optionally substituted up to 2 times by oxo, (C1-3 alkoxy)-(C1-4 alkyl), C(O)OR', C(O)N(R')2 , phenyl, or benzyl, or up to 4 times by C1-3 alkyl; or
5)
Figure imgf000011_0004
, optionally substituted up to 2 times by halogen, CM alkyl, or CM alkoxy, and in which v is 0 or 1.
L is more preferably -O-(CH2)M- ; -S(O)0-2-(CH2)M- ; -N(R')-(CH2)M- ; -(CH2)1-4- O-(CH2)1-4- ; or -N(R1)-C(O)-(CH2)M- •
G is more preferably
1) NR5R6 , in which R5 represents H or C1-6 alkyl; and R6 represents Cι-6 alkyl; C5-6 cycloalkyl optionally substituted up to 2 times by halogen, CM alkyl, or CM alkoxy; benzyl; phenyl optionally substituted by halogen, CM alkyl, C1-4 alkoxy, -CO2R', -C(0)N(R')2 , -N(R!)2 ,
Figure imgf000012_0001
y-Λ
^ N x in which A is N(R ), S, S(O), S(O)2, or O , and this bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-
membered heterocycle; or
Figure imgf000012_0002
5 in which A2 represents N(R'), S, S(O), S(O)2 , or O;
2)
Figure imgf000012_0003
, optionally substituted up to 2 times by Cι-3 alkyl, (C1-3 alkoxy)(CM alkyl),
Figure imgf000012_0004
, phenyl, or benzyl;
Hsr A3
3) ^ — f , optionally substituted up to 2 times by C)- alkyl, (C1-3 alkoxy)(CM alkyl), C(O)OR!, C(0)N(R')2 , phenyl, or benzyl, and in which A3 represents N(R'), S, S(O), S(O)2 , or O;
4)
Figure imgf000012_0005
, optionally substituted up to 2 times by oxo, (C1.3 alkoxy)-(C1-4 alkyl), C(O)OR*, C(O)N(R')2 , phenyl, or benzyl, or up to 4 times by Cι-3 alkyl;
5)
Figure imgf000012_0006
, optionally substituted up to 2 times by halogen, CM alkyl, or C1-4 alkoxy, and in which v is 0 or 1.
A preferred combination of L and G groups is when
t oXCHX ^
L represents 1 XCH2) 2-~3 an Λd G represents X ^ R
Most preferably, L is -O-(CH2) - ; -N(R')-(CH2) - ; or -(CH2)1-4-O-(CH2)1-4 Most preferably, G is:
1) NR5R6 , in which R5 represents H or Cι-6 alkyl; and R6 represents C1-6 alkyl;
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C1-4 alkyl, or
CM alkoxy;
2)
Figure imgf000013_0001
, optionally substituted up to 2 times by C1-3 alkyl, (C1-3 alkoxy)(C alkyl), C(O)OR', O R1^ , phenyl, or benzyl;
Hsr A3
3) ^ — / , optionally substituted up to 2 times by C1-3 alkyl, (C1-3 alkoxy)(C alkyl), C(O)OR', C^N R1^ , phenyl, or benzyl, and in which A3 represents N(R!), S, S(O), S(O)2 , or O;
Figure imgf000013_0002
alkyl), C(O)OR', C(0)N(R')2 , phenyl, or benzyl, or up to 4 times by C1-3 alkyl;
5)
Figure imgf000013_0003
, optionally substituted up to 2 times by halogen, C1-4 alkyl, or
CM alkoxy, and in which v is 0 or 1.
In a preferred embodiment, compounds ofthe invention have the formula (I)
Figure imgf000013_0004
in which
Y1 represents H or CM alkyl. Y2 is selected from C ]-6 alkyl; C3-6 cycloalkyl optionally substituted by CM alkyl; phenyl optionally substituted up to three times by halogen, CM alkyl, or CM alkoxy; adamantyl; CF3 ; a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by
halogen or Cι-6 alkyl; and
Figure imgf000014_0001
; andY3 is H. Alternatively, Y2 and Y3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C alkyl, or CM alkoxy.
X represents Cl, F, or CM alkyl; and n represents 0, 1, or 2.
In this embodiment, R1 and R2 each represents H.
R3 represents C1-6 alkyl; halogen; C M alkoxy; CN, or N(R')2 in which the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. The subscript p represents 0, 1, or 2. The subscript q, representing the number of R4 groups, is 0.
L is a linker selected from -O-(CH2)1-4- ; -S(O)0-2-(CH2)M- ; -N(R1)-(CH2) - ;
Figure imgf000014_0002
; -(CH2)M-O-(CH2)1-4- ; -(CH2) - ; and
Figure imgf000014_0003
. G represents:
1) NR5R6 , in which R5 represents H or Cι-6 alkyl; and R6 represents C1-6 alkyl; C1-4 alkoxy-substituted C1-4 alkyl; C5-6 cycloalkyl optionally substituted up to 2 times by halogen, CM alkyl, or C1-4 alkoxy; C3-6 cycloalkyl-substituted Cι-4 alkyl; benzyl; phenyl optionally substituted by halogen, CM alkyl, CM alkoxy,
Figure imgf000014_0004
-C(O)N(R')2 , -N(R')2 , or by a bivalent group
Figure imgf000014_0005
, or in which A is N(R'),
S, S(O), S(O) , or O , and this bivalent group is connected to the phenyl ring at
adjacent carbon atoms to form a fused 5-membered heterocycle; or
Figure imgf000014_0006
j in which A2 represents N(R'), S, S(O), S(O)2 , or O;
2)
Figure imgf000014_0007
, optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(d-4 alkyl),
Figure imgf000014_0008
C(O)N(R')2 , phenyl, or benzyl; f-N A3
3) \ — / , optionally substituted up to 2 times by Cι- alkyl, (C]-3 alkoxy)(C1-4 alkyl),
Figure imgf000015_0001
C(O)N(R*)2 , phenyl, or benzyl, and wherein A3 represents N(R'), S, S(O), S(O)2 , or O;
4) O-I , optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(C)-4 alkyl), C(O)OR', C(O)N(R')2 , phenyl, or benzyl, or up to 4 times by d-3 alkyl;
5)
Figure imgf000015_0002
, optionally substituted up to 2 times by halogen, C1-4 alkyl, or C]-4 alkoxy, and in which v is 0 or 1.
In a more preferred embodiment, compounds ofthe invention have the formula (I)
Figure imgf000015_0003
in which
Y1 represents H. Y2 is selected from C 1-6 alkyl; C -6 cycloalkyl optionally substituted by C alkyl; phenyl optionally substituted up to three times by halogen, C alkyl, or CM alkoxy; adamantyl, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C1-6 alkyl. Y3 is H.
X represents F; and n represents 0 or 1.
R and R each represents H. R represents Cι-6 alkyl; C alkoxy; CN; or N(R )2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R1), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; and p represents 0 or 1. The subscript q, representing the number of R4 groups, is O.
L is a linker selected from -O-(CH2) - ; -S(O)0-2-(CH2)1-4- ; -N(R')-(CH2)1-4- ; -(CH2)M-O-(CH2)1-4- ; and
Figure imgf000016_0001
. G represents:
1) NR5R6 , in which R5 represents H or Cι-6 alkyl; and R6 represents Cι-6 alkyl; C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C1-4 alkyl, or C1-4 alkoxy; benzyl; phenyl optionally substituted by halogen, C1-4 alkyl, CM alkoxy, -CO2R1,
-C(O)N(R')2 , -N(R')2 , or by a bivalent group
Figure imgf000016_0002
) θr X in which A is N(R'), S, S(O), S(O)2, or O , and this bivalent group is connected to the phenyl ring at
adjacent carbon atoms to form a fused 5 -membered heterocycle; or
Figure imgf000016_0003
, ιn which A2 represents N(R]), S, S(O), S(O)2 , or O;
2)
Figure imgf000016_0004
, optionally substituted up to 2 times by Cι- alkyl, (Cι-3 alkoxy)(C alkyl),
Figure imgf000016_0005
C(O)N(R1)2 , phenyl, or benzyl;
|-N A3 3) x — ' , optionally substituted up to 2 times by Cι-3 alkyl, (C1-3 alkoxy)(C1- alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl, and in which A3 represents N(R'),
S, S(O), S(O)2 , or O;
4) , optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(CM alkyl),
Figure imgf000016_0006
O)N(R')2 , phenyl, or benzyl, or up to 4 times by C1-3 alkyl;
5)
Figure imgf000016_0007
, optionally substituted up to 2 times by halogen, C alkyl, or CM alkoxy, and wherein v is 0 or 1. or i oXCHX ^ .
L represents 2-3 and G represents ^
In the most preferred embodiment, compounds ofthe invention have the formula (I)
Figure imgf000017_0001
in which
Y1 represents H. Y2 is C 1-6 alkyl, or C3-6 cycloalkyl optionally substituted by C]-4 alkyl. Y3 is H.
X represents F; and n represents 0 or 1.
R1 and R2 each represents H. R3 represents Cι-6 alkyl; C 1-4 alkoxy, or N(R*)2 in which the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle. The subscript p, representing the number of R3 groups, is 0 or 1. The subscript q, representing the number of R4 groups, is 0. L is a linker selected from -O-(CH2)1- - ; -N(R')-(CH2)1-4- ; and
-(CH2)1-4-O-(CH2)1-4- -
G represents: 1) NR5R6 , in which R5 is H or C)-6 alkyl; and R6 represents Cι-6 alkyl, or C5-6 cycloalkyl optionally substituted up to 2 times by halogen, CM alkyl, or CM alkoxy;
2)
Figure imgf000017_0002
optionally substituted up to 2 times by Cι- alkyl, (C)-3 alkoxyXCM alkyl), C^OR1, C(O)N(R')2 , phenyl, or benzyl; 3)
Figure imgf000018_0001
, optionally substituted up to 2 times by C1-3 alkyl, (d-3 alkoxy)(CM alkyl),
Figure imgf000018_0002
C(O)N(R1)2 , phenyl, or benzyl, and in which A3 represents N(R'), S, S(O), S(O)2 , or O;
4) 'o-i , optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(Cι-4 alkyl), C(O)OR1, C(O)N(R1)2 , phenyl, or benzyl, or up to 4 times by Cι-3 alkyl; or
5)
Figure imgf000018_0003
, optionally substituted up to 2 times by halogen, CM alkyl, or C1- alkoxy, and in which v is 0 or 1.
When present, the group X in the middle ring is preferably on the C-5 position of the pyrimide. Also, in the lower ring, the L-G group is preferably meta to the amino N.
The compounds of formula (I) are generally made by coupling a compound of formula (II)
Figure imgf000018_0004
with a compound of formula (III)
Figure imgf000018_0005
, and, i -n the case where R 2 is an alkyl group, alkylating the resulting secondary amine, to yield a compound of formula (I); or coupling a compound of formula (IV)
Figure imgf000019_0001
with a compound of formula (V)
Figure imgf000019_0002
and, in the case where R is an alkyl group, alkylating the resulting secondary amine, to yield a compound of formula (I). In formulae (II), (III), (IV), and (V), the meanings of the substituent groups are as described above.
Examples of 5-6 membered heteroaromatics containing up to two heteroatoms selected from N, O, and S and employed for groups Y2 or Y3 in formula (I) include, but are not limited to, pyridinyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, furanyl, pyrrolyl, thiazolyl, or thienyl.
When R1 moieties of a group N(R') are referred to as "independent" it is meant that they are separate groups, each being joined to the N but not joined to each other.
When the R1 moieties of a group N(R')2 are referred to as being joined by a linker selected from the group consisting of CH(R'), N(R!), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle, cyclic moieties such as morpholine, thiomorpholine, pyrrolidine, piperidine, or piperazine are contemplated.
When it is stated that R and R4 may be joined and taken together with the carbon atoms to which they are attached, form a 5-6 membered heteroaromatic ring containing up to two heteroatoms selected from N, O, and S, moieties such as pyridine, pyrimidine, thiozole, imidizole, pyrole, furan, or thiophene are contemplated.
Definitions For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
The terms "a" and "an" refer to "one or more" when used in this application, including the claims. "Abnormal growth of cells" means cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition).
The term "analog" of a compound refers to a compound having a substantial structural similarity to a particular compound and having essentially the same type of biological activity as the compound. The term "antiproliferative" therapeutic or compound refers to a compound or therapeutic which inhibits cell proliferation to at least some extent.
The term "cytostatic" when referring to the activity of a compound means that the compound causes the cell to cell cycle arrest, but it does not kill the cell. Thus, removal of the drug from the environment ofthe cell results in the resumption of cell proliferation. The term "derivative" of a compound or of a small molecule refers to a compound which can be derived, e.g., by chemical synthesis, from the original compound. Thus a derivative ofa compound has certain structural similarities with the compound.
"Disease associated with an abnormal activity or level of a kinase" refers to a disease in which an abnormal activity or protein level of a kinase is present in certain cells, and in which the abnormal activity or protein level of the kinase is at least partly responsible for the disease.
A "disease associated with a kinase" refers to a disease that can be treated with a kinase inhibitor.
"Diseases associated with src kinase-mediated signaling" refers to diseases which can be treated with an inhibitor of src kinase-mediated signaling. Such disease can, e.g., be associated with an abnormal src kinase activity or level.
The terms "excessive cell proliferation," used interchangeably herein with "hyperproliferation" of cells refers to cells which divide more often than their normal or wild-type counterpart. Thus, cells are excessively proliferating when they double in less than 24 hours if their normal counteφarts double in 24 hours. Excessive proliferation can be detected by simple counting of the cells, with or without specific dyes, or by detecting DNA replication or transcription, such as by measuring incorporation of a labeled molecule or atom into DNA or RNA.
"Inhibiting cell proliferation" refers to decreasing the rate of cell division, by interrupting or slowing down the cell cycle. The term refers to complete blockage of cell proliferation, i.e., cell cycle arrest, as well as to a lengthening of the cell cycle. For example, the period of a cell cycle can be increased by about 10%, about 20%, about 30, 40, 50, or 100%. The duration of the cell cycle can also be augmented by a factor of two, three, 4, 5, 10 or more.
"Modulating cell differentiation" refers to the stimulation or inhibition of cell differentiation.
"Normalizing cell proliferation" refers to reducing the rate of cell proliferation of a cell that proliferates excessively relative to that of its normal or wild-type counteφart, or increasing the rate of cell proliferation of a cell that proliferates poorly relative to its normal or wild-type counteφart. A "patient" or "subject" to be treated by the subject method can mean either a human or non-human animal.
The term "proliferative disorder" refers to any disease/disorder of a tissue marked by unwanted or aberrant proliferation of at least some cells in the tissue. Such diseases include cancer, as well as benign diseases or disorders, such as warts or other benign tumors. A "src inhibitor" is a compound which inhibits at least part of the activity of a src kinase in a cell. The inhibition can be, at least about 20%, preferably at least about 40%, even more preferably at least about 50%, 70%, 80%, 90%, 95%, and most preferably at least about 98% ofthe activity ofthe src kinase.
"Treating" a disease refers to preventing, curing or improving at least one symptom ofa disease.
The following definitions pertain to the structure ofthe compounds:
The abbreviations Me, Et, Ph, and OMe represent methyl, ethyl, phenyl, and methoxy respectively. A more comprehensive list ofthe abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry (i.e. J. Org. Chem. 1995, 60, 12a.). This list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in this list are hereby incoφorated by reference. "Alkyl" means a hydrocarbon radical having up to a maximum of 12 carbon atoms, which may be linear or branched with single or multiple branching. Alkyl is especially lower alkyl. Examples of such alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, and isohexyl. "Halogen" means fluorine, chlorine, bromine, or iodine but is especially fluorine, chlorine, or bromine.
"Cycloalkyl" is a saturated carbocycle that contains between 3 and 12 carbons but preferably 3 to 8 carbons. Examples include the cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl groups. The term "alkoxy" means a group in which the alkyl portion is straight or branched and has the designated number of carbon atoms. Examples of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy, hexoxy, and isohexoxy.
The term "optionally" means that the subsequent desribed event(s) may or may not occur, and includes both event(s), which occur, and event(s) that do not occur. The term "oxo" refers to the group =O.
Abbreviations and Acronyms
When the following abbreviations are used throughout the disclosure, they have the followiri meaning:
ADDP 1 , 1 ' -(azodicarbonyl)dipiperidine
ATP adenosine triphosphate
Ar argon BRIJ polyoxyethylene(23) lauryl ether
BSA bovine serum albumin
H-BUOH 1-butanol
CBr4 carbon tetrabromide
CD3OD methanol-^ CDC13 chloroform-d
CH2C12 methylene chloride
CH3CN acetonitrile Cs2CO3 cesium carbonate
Cu(OTf)2-Ph copper(I) trifluoromethanesulfonate benzene complex
DEAD diethyl azodicarboxylate
DMF dimethylformamide
DMSO dimethylsulfoxide
EDTA ethylenediaminetetraacetic acid
ESI-MS electrospray-mass spectrometry ionization
EtOAc ethyl acetate
Et2O diethyl ether
Et3N triethylamine
H2 hydrogen gas
HBr hydrobromic acid
HCl hydrochloric acid
HEPES 4-(2 -hydroxyethyl)- 1 -piperazineethane-sulfonic acid
HEX hexanes
1H NMR proton nuclear magnetic resonance
HPLC high pressure liquid chromatography
Hz hertz
K2CO3 potassium carbonate
KOAc potassium acetate
KOH potassium hydroxide
LC/MS liquid chromatography / mass spectrometry
MeOH methanol
MgSO4 anhydrous magnesium sulfate
MMTV murine mammary tumor virus
MPLC medium pressure liquid chromatography
MS ES mass spectroscopy with electrospray
NaH sodium hydride
NaHCO3 sodium bicarbonate
Nal sodium iodide
NaOH sodium hydroxide
P2O5 phosphorous pentoxide POCl3 phosphorous oxychloride
Poly-GAT poly glycine, alanine, tyrosine
PPh3 triphenyl phosphine
RNA ribonucleic acid rt room temperature
SnCl2 tin(II) chloride
Streptavidin-APC steptavadin conjugated allopycocyanin
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin layer chromatography
Compounds ofthe Invention
The present invention provides substituted pyrimidine compounds, e.g., 2,4-diamino substituted pyrimidine compounds, which are capable of inhibiting src kinase activity. Exemplary compounds ofthe invention have the IUPAC name set forth below:
Example IUPAC NAME
N*-(3-tert-butyl-lH-pyrazol-5-yl)-N2-{3-[2- (diethylamino)ethoxy]phenyl}-2,4-pyrimidinediamine
4-( {4-[(3-tert-butyl- lH-pyrazol-5-yl)amino]-2- pyrimidinyl}amino)-2-[2-(diethylamino)ethoxy]benzonitrile
4-( {4-[(3-tert-butyl- lH-pyrazol-5-yl)amino]-5-fiuoro-2- pyrimidinyl}amino)-2-[2-(diethylamino)ethoxy]benzonitrile
N2- {3-[2-(diethylamino)ethoxy]phenyl} - Λ^-p-tert-pentyl- 1H- pyrazol-5-yl)-2,4-pyrimidinediamine
^-^-^-(diethylamino^thoxyJ^methoxyphenylj- Λ^-β-tert- pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
N2- {3-[2-(diethylamino)ethoxy]-4methoxyphenyl}-5-fluoro- N4- (3-tert-pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
2-[2-(diethylamino)ethoxy]-4-( {4-[(3-tert-pentyl- lH-pyrazol-5- yl)amino]-2-pyrimidinyl}amino)benzonitrile
N2-{4-methoxy-3-[2-(l-pyrrolidinyl)ethoxy]phenyl}- N4-(3-tert- pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
5-fluoro- N2- {4-methoxy-3-[2-( 1 -pyrrolidinyl)ethoxy]phenyl} ■ N4-(3-tert-pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine Λ^-β-tert-pentyl- 1 H-pyrazol-5-yl)- N2- {3-[2-( 1 - piperidinyl)ethoxy]phenyl } -2,4-pyrimidinediamine
N2- {4-methoxy-3-[2-( 1 -piperidinyl)ethoxy]phenyl} -N*-(3-tert- pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
5-fluoro- N^^-methoxy-S-^- -piperidiny^ethoxyjphenylJ-N4- (3-tert-pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
N2- {3-[2-(4-methyl- 1 -piperidinyl)ethoxy]phenyl} - N*-(3-tert- pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
N2- {3 - [( 1 -methyl-3 -piperidinyl)methoxy]phenyl }- N4-(3 -tert- pentyl-lH-pyrazol-5-yl)-2,4-pyrimidinediamine
N2- {3-[2-(diethylamino)ethoxy]phenyl} - N*-[3-(l -ethyl-1 - methylpropyl)-lH-pyrazol-5-yl]-2,4-pyrimidinediamine
N2- {3-[2-(diethylamino)ethoxy]-4methoxyphenyl}- N*-[3-(l - ethyl- 1 -methylpropyl)- 1 H-pyrazol-5 -yl] -2 ,4-pyrimidinediamine
N2- {3-[2-(diethylamino)ethoxy]-4methoxyphenyl} - N4-[3-(l - ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl]-5-fluoro-2,4- pyrimidinediamine
2-[2-(diethylamino)ethoxy]-4-[(4- {[3-( 1 -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl]amino}-2-pyrimidinyl)amino]benzonitrile
N4-[3-( 1 -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl ]- N2- {4- methoxy-3-[2-( 1 -pyrrolidinyl)ethoxy]phenyl} -2,4- pyrimidinediamine
N4-[3-( 1 -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl ]-5-fiuoro- N2- {4- methoxy-3-[2-(l-pyrrolidinyl)ethoxy]phenyl}-2,4- pyrimidinediamine
N4-[3-(l-ethyl-l-methylpropyl)-lH-pyrazol-5-yl]- N2-{3-[2-(l- piperidinyl)ethoxy]phenyl}-2,4-pyrimidinediamine
N*-[3-( 1 -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl]- N2- {4- methoxy-3-[2-(l-piperidinyl)ethoxy]phenyl}-2,4- pyrimidinediamine
N4-[3-( 1 -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl]-5-fluoro- N2- {4- methoxy-3-[2-(l-piperidinyl)ethoxy]phenyl}-2,4- pyrimidinediamine
N4-[3-(l -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl]- N2- {3-[2-(4- methyl- 1 -piperidinyl)ethoxy]phenyl} -2,4-pyrimidinediamine
N2-(3- {2-[(3 ?,5S)-3,5-dimethyl- 1 -piperidinyl] ethoxy} phenyl)- N4-[3-( 1 -ethyl- 1 -methylpropyl)- lH-pyrazol-5-yl]-2,4-
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trøws-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group such as amino, or an acidic functional group such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.
Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof
(e.g., functioning as src kinase inhibitors), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in inhibiting src kinases.
In general, the compounds of the present invention may be prepared by the methods known to those skilled in the art as illustrated in the general reaction schemes described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
Methods of use ofthe compounds ofthe invention
The compounds of the invention, including substituted pyrimidine compounds, salts, prodrugs, and compositions thereof, can be used for treating a disease or condition (generally referred to herein as " disease") associated with a kinase, such as a disease associated with an abnormal activity or level of a kinase. In a preferred embodiment, the kinase is a tyrosine kinase, such as a src tyrosine kinase. Generally, the compounds of the invention can be used for treating diseases that are associated with a component of the signal transduction pathway in which a kinase is involved. For example, it is expected that a cell proliferative disease resulting from over-expression of a signal transduction molecule or cell surface receptor that is in the same signal transduction pathway as that in which a kinase which can be inhibited by a compound of the invention is present, can also be treated with the compounds of the invention. At least for this reason, the compounds of the invention are expected to be effective against a broad range of target cells, and not only target cells having an abnormal activity or level of a kinase. The terms "target cell" refers to a cell towards which a compound is targeted. Furthermore, at least some of the compounds of the invention may also be effective against cells which proliferate and/or differentiate normally, i.e., wild-type cells. For example, certain compounds could be used to arrest cell proliferation, even if the cell proliferation is not abnormal.
In a preferred embodiment, the compounds of the invention are useful for treating a disease associated with a src kinase. Src kinases are involved in various cellular functions, including cell proliferation and transformation; cell adhesion, migration and chemotaxis; intracellular trafficking; and cell survival. Accordingly, diseases that can be treated according to the invention include those which are dysfunctional in any of these cellular functions. Exemplary diseases are provided below. In one embodiment, a therapeutic method comprises administering to a subject having a disease associated with a kinase, a pharmaceutically effective amount of a compound of the invention, such that the disease is treated. The subject is preferably a mammal, e.g., a human, non-human primate, bovine, ovine, porcine, feline, canine, mouse or rat. The compounds can be administered via various routes depending on the disease to be treated. Methods of administration are further described herein. Non-mammalian cells, which share essentially the same signal transduction pathways as those in mammalian cells, e.g., yeast cells, can also be target cells ofthe invention.
Compounds of the invention may specifically inhibit the activity of a single kinase, e.g., src kinase, or they may inhibit the activity of more than one kinase or more than one type of kinase. Accordingly, a compound of the invention could be used for treating one or more diseases associated with one or more kinases.
The efficacy of the compounds of the invention against a broad range of target cells allows for broad applications for these compounds. The following are exemplary therapeutic applications for the compounds of the invention. These exemplary therapeutic applications focus first on diseases associated with src tyrosine kinase and then describe other diseases that may also be treated with the compounds of the invention.
Src tyrosine kinase has specifically been implicated in the development, growth, progression, and metastasis of a number of human cancers such as colon, breast, pancreas and brain (see, e.g., Irby and Yeatman (2000) Oncogene 19:5636), and these cancers are expected to be treatable with the compounds of the invention. For example, a src kinase activity from 4-20 fold higher than normal has been found in mammary carcinomas (Irby and Yeatman, supra; Egan et al. (1999) Oncogene 18: 1227 and Nerbeek et al. (1996) J. Pathol. 180:383). c-src has also frequently been implicated in the initiation and progression of human colon cancer and in resultant metastases (see, e.g., Cartwright et al. (1994) J. Clin. Invest. 93:509; Talamonti et al. (1991) J. Clin. Invest. 91 :53; and Termuhlen et al. (1993) J. Surg. Res. 54). Src is increased 5-8 fold in the majority of colon tumors. Elevated src activity is also present in pre-cancerous colon lesions, e.g., adenomatous polyps (Pena et al. (1995) Gastroenterol. 108: 1 17).
Other cancers that can be treated include pancreatic cancer (Flossmann-Kast et al. (1998) Cancer Res. 8:3551); and Visser et al. (1996) Lab. Invest. 74:2), lung cancer (Mazurenko et al. (1992) Eur. J. Cancer 28:372), neural cancer (Bjelfinan et al. (1990) Cancer Res. 50:6908); ovarian cancer (Wiener et al. (1999) Clin. Cancer Res. 5:2164); esophageal adenocarcinomas and Barrett's (Kumble et al. (1997) Gastroeneterology 112:348); gastic cancers (Takeshima et al. (1991) Jpn. J. Cancer Res. 82:1428); melanomas (Bjorge et al. (1996) Biochem. Cell Biol. 74:477) and Kaposi's sarcoma (Munshi et al. (2000) J. Immunol. 164:1169). Src probably also contributes to tumor growth in synergy with receptor tyrosine kinases, such as c-met and those of the ErbB family (Biscardi et al. (1999) Adv. Cancer Res. 76: 6). Accordingly, all of the above are exemplary cancers that can be treated with the compounds ofthe invention. The compounds of the invention can also be used to treat diseases associated with defects in cell adhesion and motility, such as angiogenesis, inflammation and bone resoφtion. Src has been shown to play a role in signal transduction via cell-adhesion receptors (integrins). Src dependent cell migration is important for the function of many cell types, e.g., the motility of osteoclasts and metastasizing cells (Chellaiah et al. (2000) J. Biol. Chem. 275 : 11993 and Susa and Teri (2000) Drug News Peφect. 13:169). Src dependent cell migration may also be important for the recruitment of vascular smooth muscle cell precursors in response to PDGF produced by endothelial cells during blood vessel formation (Hirschi et al. (1998) J. Cell. Biol. 141 :805).
Src kinase is also involved in endocytosis, e.g., transcytosis, such as that which occurs in osteoclasts (Nesbitt and Horton (1997) Science 276:266). Src assists endocytosis of certain growth factor receptors, e.g., EGF receptors (Wilde et al. (1999) Cell 96:677). Blood vessel hypeφermeability induced by vascular endothelial growth factor (VEGF) is also dependent on src (Eliceiri et al. (1999) Mol. Cell 4:915). Src has been shown to also be involved in cell survival (reviewed in Susa et al. (2000) Trends in Pharmacol. Sci. 21 :489). Accordingly, diseases related to any of these exemplary src biological activities can be treated with the compounds ofthe invention.
A preferred use for the compounds of the invention is for the treatment of osteoporosis, which involves bone resoφtion. Osteoporosis is a widespread disease of low bone mass that particularly affects post-menopausal women (see, e.g., Gowen et al. (2000) Emerging Drugs 5: 1). The role of src in bone metabolism was first demonstrated in src- deficient mice and has been confirmed using small molecular weight inhibitors in animal models of osteoporosis. Src-deficient mice have defective bone resoφtion, resulting in excessive bone mass and osteopetrosis (see, e.g., Thomas and Brugge (1997) Annu. Rev. Cell. Dev. Biol., 13: 513). The role of src in bone resoφtion is well recognized. A src inhibitor has been shown to reduce bone resoφtion in an animal model of osteoporosis (Missbach et al. (1992) Bone 24:437). The disorder is believed to be caused by dysfunctions in osteoclasts and osteoblasts, as well as in osteoclast survival and osteoclast formation (reviewed in Susa et al, supra).
Other diseases that may also be treated according to the invention include other types of malignancies, e.g., cancers of the brain, genitourinary tract, prostate, skin, lymphatic system, rectum, stomach, larynx, ovary, bladder, and liver. More particularly, such cancers include histiocytic lymphoma, lung adenocarcinoma, pancreatic carcinoma, colo-rectal carcinoma, bladder cancers, head and neck cancers, acute and chronic leukemias, melanomas, neurological tumor, myeloid leukemias (for example, acute myelogenousleukemia), sarcomas, thyroid follicular cancer, and myelodysplastic syndrome.
The compounds ofthe invention can also be used for treating disease associated with abnormal activity and/or expression of members of a growth factor family or receptors thereof. For example, compounds of the invention are expected to be effective against diseases associated with a defect in a growth factor or receptor of the EGF receptor family, such as Neu-erb2-related genes. The compounds ofthe invention are believed to be effective against the following diseases. For example, amplification and/or over-expression of human erbB2 gene, has been shown to correlate with a poor prognosis in breast and ovarian cancers, in particular, carcinomas (see, e.g., Slamon et al, Science 235:177-82 (1987); Slamon et al, Science 244:707-12 (1989)). Overexpression of erbB2 has also been correlated with other carcinomas including carcinomas ofthe stomach, endometrium, salivary gland, lung, kidney, colon and bladder. ErbBl has been causally implicated in human malignancy, e.g., aggressive carcinomas ofthe breast, bladder, lung, and stomach. ErbB gene amplification or overexpression, or a combination of both, has also been demonstrated in squamous cell carcinomas and glioblastomas (Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M.D., Ullrich, A. & Schlessinger, J., 1985, Nature 313:144-147). Accordingly, the compounds of the invention are believed to be useful for treating these malignancies. ErbB3 has been found to be overexpressed in breast (Lemoine et al, Br. J. Cancer 66:1116-21 (1992)), gastrointestinal (Poller et al, J. Pathol. 168:275-80 (1992); Rajkumer et al, J. Pathol. 170:271-78 (1993); Sanidas et al, Int. J. Cancer 54:935- 40 (1993)), and pancreatic cancers (Lemoine et al, J. Pathol. 168:269-73 (1992), and Friess et al, Clinical Cancer Research 1:1413-20 (1995)). Plowman et al. found that Increased erbB4 expression have been found to closely correlate with certain carcinomas of epithelial origin, including breast adenocarcinomas (Plowman et al, PNAS 90:1746-50 (1993) and Plowman et al, Nature 366:473-75 (1993)).
The hyper-proliferative disorders that can be treated by the disclosed substituted pyrimidine compounds, salts, prodrugs and compositions thereof include, but are not limited to solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. Those disorders also include, but are not limited to lymphomas, sarcomas, and leukemias.
Examples of breast cancer include, but are not limited to invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ.
Examples of cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma.
Examples of brain cancers include, but are not limited to brain stem and hypophtalmic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor. Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer.
Tumors ofthe female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma ofthe uterus.
Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallblader, gastric, pancreatic, rectal, small-intestine, and salivary gland cancers.
Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, and urethral cancers.
Eye cancers include, but are not limited to intraocular melanoma and retinoblastoma.
Examples of liver cancers include, but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma.
Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer.
Head-and-neck cancers include, but are not limited to laryngeal / hypopharyngeal / nasopharyngeal / oropharyngeal cancer, and lip and oral cavity cancer. Lymphomas include, but are not limited to AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma ofthe central nervous system.
Sarcomas include, but are not limited to sarcoma of the soft tissue, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia. These disorders have been well characterized in man, but also exist with a similar etiology in other mammals, and can be treated by pharmaceutical compositions ofthe present invention.
Other types of proliferative disorders that can be treated according to the invention include non malignant cell proliferative disorders, such as those associated with an abnormal production of, or response to a growth factor, e.g., platelet derived growth factor (PDGF), fibroblast derived growth factor (FGF), epidermal derived growth factor (EGF) and vascular endothelial growth factor (VEGF). Exemplary diseases include restinosis, glomerulonephritis, neurofibromatosis, glaucoma, psoriasis, rheumatoid arthritis, inflammatory bowel disease, and chemotherapy-induced alopecia and mucositis. Restenosis following coronary angioplasty is one major unsolved problem of interventional cardiology. Of the nearly 400,000 angioplasties currently performed in the United States each year, 25-34% fail within the first five years, of which most occur during the first year, due to restenosis (Geschwind H.J. (1995) Interv. Cardiol. 8:756 and The Merck Manual of Diagnosis and Therapy, 16th Ed. (1992) Merck Res. Lab., p. 406. The process of restenosis involves the reocclusion of an atherosclerotic artery which in many cases is due to the proliferation of smooth muscle cells which is mediated by growth factors such as PDGF and FGF. In animal models of restenosis, antibodies which block the activation of PDGF or FGF receptor tyrosine kinase activity prevent smooth muscle cell proliferation and the formation of neointima. These studies indicate that tyrosine kinase inhibitors that block PDGF or FGF receptor function could have utility in treating human restenosis.
In experimental models of glomerulonephritis, a 20-fold increase in PDGFR expression is associated with mesangial cell proliferation. Neutralization of PDGF which prevents the activation of its tyrosine kinase receptor limits the amount of renal degeneration which normally occurs. These studies demonstrate that a tyrosine kinase inhibitor which blocks PDGFR could have potential for the treatment of human glomerulonephritis. Johnson et al. (1992) J. Exp. Med. 175:1413. In another embodiment, the compounds of the invention are used for treating inflammatory diseases, e.g., rheumatoid arthritis (R.A.). Synovial tissues of RA patients express high levels of FGF and PDGF compared with synovial tissues of osteoarthritis patients, a non invasive joint disease (Sano et al, J. Cell. Biol. 110:1417-1426, 1990). These data are consistent with the theory that PDGF and FGF play a role in generating an invasive tumor-like behavior in arthritic joints of RA synovial connective tissues (Sano et al, J. Clin. Invest. 91 :553-565 1993).
It is further expected that the compounds of the invention are useful for treating smooth muscle cell hyper-proliferation, at least in part since PDGF is considered to be a principal growth-regulatory molecule responsible for smooth muscle cell proliferation. One smooth muscle disorder is atherosclerosis, which is a disease characterized by focal thickening of the inner portion of the artery wall, predisposing an individual to myocardial infarction (heart attack), cerebral infarction (stroke), hypertension (high blood pressure) and gangrene ofthe extremities. In addition to consisting primarily of proliferated smooth muscle cells, lesions of atherosclerosis are surrounded by large amounts of lipid-laden macrophages, varying numbers of lymphocytes and large amounts of connective tissue. PDGF has been found in numerous cells in such lesions, and it is believed that PDGF plays a critical role in the atherosclerosis disease process. Other smooth muscle diseases include diabetic vascular pathologies.
Both FGF and VEGF are potent angiogenic factors that induce formation of new capillary blood vessels. Accordingly, the compounds of the invention may be useful in inhibiting vascularization, e.g., in tumors.
In addition, the instant compounds may also be useful in the treatment of certain viral infections, in particular in the treatment of hepatitis C or delta and related viruses (Glenn et al. Science, 256:1331-1333 (1992)). Numerous viruses also induce non cancerous cell proliferation. Examples include papilloma viruses (HPV), which create skin lesions. Such viral infections may also be treatable with the compositions ofthe invention. The compounds of the invention can also be used for treatment of hypeφroliferative cutaneous diseases, e.g., keratosis and psoriasis.
Also within the scope of the invention are methods for inhibiting growth of non- mammalian cells, which have similar signal transduction pathways as those in mammalian cells. Exemplary cells include yeast cells. Accordingly, the compounds of the invention can be used as anti-fungal agents to treat fungal infections on animals, e.g., humans. The compounds can also be used for stopping fungus growth on objects, e.g., mold or mildew growth on shower curtains.
A person of skill in the art would understand, based on the instant description, that other diseases can also be treated according to the invention.
Description of the Pharmaceutical Compositions and Methods of Administration of the Compounds ofthe Invention
Pharmaceutically acceptable salts of these compounds as well as commonly used prodrugs of these compounds are also within the scope ofthe invention.
Salts, especially pharmaceutically acceptable salts, ofthe compounds ofthe invention such as, for example, organic or inorganic acid addition salts, are also provided by the invention. Suitable inorganic acids include but are not limited to halogen acids (such as hydrochloric acid), sulfuric acid, or phosphoric acid. Suitable organic acids include but are not limited to carboxylic, phosphonic, sulfonic, or sulfamic acids, with examples including acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, 2- or 3 -hydroxy butyric acid, γ-aminobutyric acid (GAB A), gluconic acid, glucosemonocarboxylic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azeiaic acid, malic acid, tartaric acid, citric acid, glucaric acid, galactaric acid, amino acids (such as glutamic acid, aspartic acid, N-methylglycine, acetytaminoacetic acid, N- acetylasparagine or N-acetylcysteine), pyruvic acid, acetoacetic acid, phosphoserine, and 2- or 3-glycerophosphoric acid.
Formation of prodrugs is well known in the art in order to enhance the properties of the parent compound; such properties include solubility, absoφtion, biostability and release time (see "Pharmaceutical Dosage Form and Drug Delivery Systems" (Sixth Edition), edited by Ansel et al, publ. by Williams & Wilkins, pgs. 27-29, (1995)). Commonly used prodrugs ofthe disclosed 2,4-diamino-pyrimidine compounds can be designed to take advantage ofthe major drug biotransformation reactions and are also to be considered within the scope of the invention. Major drug biotransformation reactions include N-dealkylation, O-dealkylation, aliphatic hydroxylation, aromatic hydroxylation, N-oxidation, S-oxidation, deamination, hydrolysis reactions, glucuronidation, sulfation and acetylation (see Goodman and Gilman's The Pharmacological Basis of Therapeutics (Ninth Edition), editor Molinoff et al, publ. by McGraw-Hill, pages 11-13, (1996)).
The invention also includes pharmaceutical compositions comprising one or more of the compounds of the invention, or their salts or prodrugs forms thereof, with a pharmaceutically acceptable ingredient.
The pharmaceutical compositions can be prepared so that they may be administered orally, dermally, parenterally, nasally, ophthalmically, otically, sublingually, rectally or vaginally. Dermal administration includes topical application or transdermal administration. Parenteral administration includes intravenous, intraarticular, intramuscular, and subcutaneous injections, as well as use of infusion techniques. One or more compounds of the invention may be present in association with one or more non-toxic pharmaceutically acceptable ingredients and optionally, other active anti-proliferative agents, to form the pharmaceutical composition. These compositions can be prepared by applying known techniques in the art such as those taught in Remington's Pharmaceutical Sciences (Fourteenth Edition), Managing Editor, John E. Hoover, Mack Publishing Co., (1970) or Pharmaceutical Dosage Form and Drug Delivery Systems (Sixth Edition), edited by Ansel et al, publ. by Williams & Wilkins, (1995).
Commonly used pharmaceutical ingredients which can be used as appropriate to formulate the composition for its intended route of administration include: acidifying agents, examples include but are not limited to acetic acid, citric acid, fumaric acid, hydrochloric acid, nitric acid; alkalinizing agents, examples include but are not limited to ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium hydroxide, triethanolamine, trolamine; adsorbents, examples include but are not limited to powdered cellulose and activated charcoal; aerosol propellants, examples include but are not limited to carbon dioxide, CC12F2, F2C1C- CC1F2 and CC1F3; air displacement agents, examples include but are not limited to nitrogen and argon; antifungal preservatives, examples include but are not limited to benzoic acid, butylparaben, ethylparaben, methylparaben, propylparaben, sodium benzoate; antimicrobial preservatives, examples include but are not limited to benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate and thimerosal; antioxidants, examples include but are not limited to ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorus acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite; binding materials, examples include but are not limited to block polymers, natural and synthetic rubber, polyacrylates, polyurethanes, silicones and styrene-butadiene copolymers; buffering agents, examples include but are not limited to potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate anhydrous and sodium citrate dihydrate; carrying agents, examples include but are not limited to acacia syrup, aromatic syrup, aromatic elixir, cherry syrup, cocoa syrup, orange syrup, syrup, corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride injection and bacteriostatic water for injection; chelating agents, examples include but are not limited to edetate disodium and edetic acid; colorants, examples include but are not limited to FD&C Red No. 3, FD&C Red No. 20,
FD&C Yellow No. 6, FD&C Blue No. 2, D&C Green No. 5, D&C Orange No. 5, D&C Red
No. 8, caramel and ferric oxide red; clarifying agents, examples include but are not limited to bentonite; emulsifying agents, examples include but are not limited to acacia, cetomacrogol, cetyl alcohol, glyceryl monostearate, lecithin, sorbitan monooleate, polyethylene 50 stearate; encapsulating agents, examples include but are not limited to gelatin and cellulose acetate phthalate; flavorants, examples include but are not limited to anise oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil and vanillin; humectants, examples include but are not limited to glycerin, propylene glycol and sorbitol; levigating agents, examples include but are not limited to mineral oil and glycerin; oils, examples include but are not limited to arachis oil, mineral oil, olive oil, peanut oil, sesame oil and vegetable oil; ointment bases, examples include but are not limited to lanolin, hydrophilic ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white ointment, yellow ointment, and rose water ointment; penetration enhancers (transdermal delivery), examples include but are not limited to monohydroxy or polyhydroxy alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalin, teφenes, amides, ethers, ketones and ureas; plasticizers, examples include but are not limited to diethyl phthalate and glycerin; solvents, examples include but are not limited to alcohol, corn oil, cottonseed oil, glycerin, isopropyl alcohol, mineral oil, oleic acid, peanut oil, purified water, water for injection, sterile water for injection and sterile water for irrigation; stiffening agents, examples include but are not limited to cetyl alcohol, cetyl esters wax, microcrystalline wax, paraffin, stearyl alcohol, white wax and yellow wax; suppository bases, examples include but are not limited to cocoa butter and polyethylene glycols (mixtures); surfactants, examples include but are not limited to benzalkonium chloride, nonoxynol 10, oxtoxynol 9, polysorbate 80, sodium lauryl sulfate and sorbitan monopalmitate; suspending agents, examples include but are not limited to agar, bentonite, carbomers, carboxymethylcellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, kaolin, methylcellulose, tragacanth and veegum; sweetening agents, examples include but are not limited to aspartame, dextrose, glycerin, mannitol, propylene glycol, saccharin sodium, sorbitol and sucrose; tablet anti-adherents, examples include but are not limited to magnesium stearate and talc; tablet binders, examples include but are not limited to acacia, alginic acid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatin, liquid glucose, methylcellulose, povidone and pregelatinized starch; tablet and capsule diluents, examples include but are not limited to dibasic calcium phosphate, kaolin, lactose, mannitol, microcrystalline cellulose, powedered cellulose, precipitated calcium carbonate, sodium carbonate, sodium phosphate, sorbitol and starch; tablet coating agents, examples include but are not limited to liquid glucose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose, ethylcellulose, cellulose acetate phthalate and shellac; tablet direct compression excipients, examples include but are not limited to dibasic calcium phosphate; tablet disintegrants, examples include but are not limited to alginic acid, carboxymethylcellulose calcium, microcrystalline cellulose, polacrillin potassium, sodium alginate, sodium starch glycollate and starch; tablet glidants, examples include but are not limited to colloidal silica, corn starch and talc); tablet lubricants (examples include but are not limited to calcium stearate, magnesium stearate, mineral oil, stearic acid and zinc stearate; tablet/capsule opaquants, examples include but are not limited to titanium dioxide; tablet polishing agents, examples include but are not limited to carnuba wax and white wax; thickening agents, examples include but are not limited to beewax, cetyl alcohol and paraffin; tonicity agents, examples include but are not limited to dextrose and sodium chloride; viscosity increasing agents, examples include but are not limited to alginic acid, bentonite, carbomers, carboxymethylcellulose sodium, methylcellulose, povidone, sodium alginate and tragacanth; and wetting agents, examples include but are not limited to heptadecaethylene oxycetanol, lecithins, polyethylene sorbitol monooleate, polyoxyethylene sorbitol monooleate, and polyoxyethylene stearate.
Depending on the route of administration, the compositions can take the form of aerosols, capsules, creams, elixirs, emulsions, foams, gels, granules, inhalants, lotions, magmas, ointments, peroral solids, powders, sprays, syrups, suppositories, suspensions, tablets and tinctures.
The therapeutic methods of the invention generally comprise administering to a subject in need thereof, a pharmaceutically effective amount of a compound. The compounds of the invention can be administered in a amount effective to inhibit the activity of a kinase, e.g., a tyrosine kinase, such as src kinase. The compounds of the invention can also be administered in a "growth inhibitory amount," i.e., an amount of the compound which is pharmaceutically effective to inhibit or decrease proliferation of target cells. The compounds can also be administered in a "differentiation modulating amount", e.g., "differentiation-inducing amount" or "differentiation-inhibiting amount," which is an amount of the compound which is pharmaceutically effective to modulate differentiation of target cells. The compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration. Toxicity and therapeutic efficacy of the compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5o/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such reagents to the site of affected tissue in order to minimize potential damage to normal cells and, thereby, reduce side effects.
Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such reagents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any reagent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture. Based on these assays, it is possible to derive an appropriate dosage for administration to subjects by combining IC50 data with appropriate pharmacokinetic evaluation. Pharmaceutical compositions containing a compound of the invention may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absoφtion in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol .
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the compound of the invention in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti- oxidant such as ascorbic acid.
Pharmaceutical compositions of the invention may also be in the form of an oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
Pharmaceutical compositions may be in the form of sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the compound of the invention is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin.
The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
The injectable solutions or microemulsions may be introduced into a patient's bloodstream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this puφose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Compounds of the invention may also be administered in the form of a suppositories for rectal administration ofthe drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of the invention can be employed. For puφoses of this application, topical application shall include mouth washes and gargles. The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will preferably be continuous rather than intermittent throughout the dosage regimen.
The compounds ofthe invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. The compounds may be administered simultaneously or sequentially. For example, the instant compounds may be useful in combination with known anti-cancer and cytotoxic agents. Similarly, the instant compounds may be useful in combination with agents that are effective in the treatment and prevention of osteoporosis, inflammation, neurofibromatosis, restinosis, and viral infections. The instant compounds may also be useful in combination with inhibitors of other components of signaling pathways of cell surface growth factor receptors.
Drugs can be co-administered to a subject being treated with a compound of the invention include antineoplastic agents selected from vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine, cytochalasin B, emetine, maytansine, or amsacrine. Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, N.J. 07645-1742, USA). Optional anti-proliferative agents that can be added to the composition include but are not limited to compounds listed on the cancer chemotherapy drug regimens in the 11th Edition of the Merck Index, (1996), which is hereby incoφorated by reference, such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarbazine, raloxifen, streptozocin, tamoxifen, thioguanine, topotecan, vinblastine, vincristine, and vindesine.
Other anti-proliferative agents suitable for use with the composition of the invention include but are not limited to those compounds acknowldeged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Ninth Edition), editor Molinoff et al, publ. by McGraw-Hill, pages 1225-1287, (1996), such as aminoglutethimide, L-asparaginase, azathioprine, 5-azacytidine cladribine, busulfan, diethylstilbestrol, 2', 2'-difluorodeoxycytidine, docetaxel, erythrohydroxynonyladenine, ethinyl estradiol, 5-fluorodeoxyuridine, 5-fluorodeoxyuridine monophosphate, fludarabine phosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate, idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate, melphalan, mitotane, paclitaxel, pentostatin, N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, teniposide, testosterone propionate, thiotepa, trimethylmelamine, uridine, and vinorelbine.
Other anti-proliferative agents suitable for use with the composition of the invention include but are not limited to other anti-cancer agents such as epothilone, irinotecan, raloxifen and topotecan.
For all regimens of use disclosed herein for the invention, the daily oral dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous and parenteral injections, and use of infusion techniques will preferably be from 0.01 to 200 mg/kg of total body weight. The daily rectal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The daily vaginal dosage regimen will preferably be from 0.01 to 200 mg/kg of total body weight. The daily topical dosage regimen will preferably be from 0.1 to 200 mg administered between one to four times daily. The transdermal concentration will preferably be that required to maintain a daily dose of from 0.01 to 200 mg/kg. The daily inhalation dosage regimen will preferably be from 0.01 to 100 mg/kg of total body weight.
It will be appreciated by those skilled in the art that the particular method of administration will depend on a variety of factors, all of which are considered routinely when administering therapeutics. It will also be understood, however, that the specific dose level for any given patient will depend upon a variety of factors, including, but not limited to the activity of the specific compound employed, the age of the patient, the body weight of the patient, the general health ofthe patient, the gender ofthe patient, the diet ofthe patient, time of administration, route of administration, rate of excretion, drug combinations, and the severity of the condition undergoing therapy. It will be further appreciated by one skilled in the art that the optimal course of treatment, i.e., the mode of treatment and the daily number of doses of a compound of formulae (I) or (II) or a pharmaceutically acceptable salt thereof given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests.
Radiation therapy, including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with a compound ofthe invention to treat a disease, e.g., cancer.
When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response ofthe individual patient, as well as the severity ofthe patient's symptoms.
Kits ofthe invention
In one embodiment, compounds of the invention and/or materials and reagents required for administering the compounds of the invention may be assembled together in a kit. When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly prefeπed.
The kit may further comprise one or more other drugs, e.g., chemo- or radiotherapeutic agent. These normally will be a separate formulation, but may be formulated into a single pharmaceutically acceptable composition. The container means may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, or injected into an animal, or even applied to and mixed with the other components ofthe kit.
The compositions of these kits also may be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration of the agent and, e.g., explaining how the agent will decrease proliferation of cells.
The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective ofthe number or type of containers, the kits of the invention also may comprise, or be packaged with a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. Other instrumentation includes devices that permit the reading or monitoring of reactions.
The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application) are hereby expressly incoφorated by reference.
Examples 1-260
General Method A. Preparation of 5-amino-3-substituted pyrazoles
Figure imgf000060_0001
To a mixture of NaH (2.1 equiv) and THF (0.15 M) is added CH3CN (2.1 equiv) and the required ester (1 equiv). The suspension is stirred at 65 °C for 16 h. The reaction is then quenched with an alcohol such as EtOH at 0 °C. Volatiles are evaporated and water added to the residue. This solution is cooled to 0 °C and the pH adjusted to ~3 with cone. HCl. The solution is extracted with Et2O (3x) to give the crude β-ketonitrile intermediate. The crude β-ketonitrile (1 equiv) is treated with EtOH (0.3 M) and hydrazine hydrate (1.3 equiv) and stirred at 70 °C for 15 h. Volatiles are evaporated and the crude residue is purified by flash column chromatography (1/9 MeOH/CH2Cl2) to give the required pyrazole whose structure is confirmed by LC/MS and Η NMR.
General Method B. Coupling of 5-amino-3-substituted pyrazoles with 5-substituted-2,4- dichloropyrimides
Figure imgf000061_0001
A solution of 5-substituted-2,4-dichloφyrimidine (1 equiv), KOAc (1.3 equiv) and 5-amino- 3-substituted pyrazole (1.1 equiv) in THF/H2O (2/1, 0.15 M) is heated at 40 °C for 24 h. The reaction mixture is allowed to cool to rt, dissolved in EtOAc and washed with aqueous NaHCO3. The combined organic layers are dried (MgSO4) and concentrated under reduced pressure. The resulting crude solid is purified either by silica gel column chromatography or washing with other solvents to afford the N-(3-substituted-lH-pyrazol-5-yl)-2-chloro-5- substituted-4-pyrimidinamine intermediate whose structure is confirmed by LC/MS and Η ΝMR.
General Method C. Coupling of substituted anilines with N-(3-substituted-lH-pyrazol- 5-yl)-2-chloro-5-substituted-4-pyrimidinamines
Figure imgf000061_0002
A solution of N-(3-substituted-lH-pyrazol-5-yl)-2-chloro-5-substituted-4-pyrimidinamine (1 equiv) and a substituted aniline (1 equiv) in an alcohol such as n-BuOΗ (0.08 M) with a catalytic amount of cone. ΗC1 is heated at 100 °C for 24 h. The reaction is cooled to rt then concentrated under reduced pressure. The crude residue is dissolved in CΗ2C1 and washed with aqueous ΝaHCO3. The combined organic layers are dried (MgSO4) and concentrated under reduced pressure. Preparative thin-layer silica gel chromatography, silica gel column chromatography, and or preparative HPLC are used to purify final products. LC/MS and Η NMR spectroscopy are used to confirm the structures of the final 2,4-substituted pyrimidinediamines.
General Method D. Preparation of substituted-nitrophenoxyamines from substituted- nitrophenols
Figure imgf000062_0001
A slurry of 3-nitrophenol (1 equiv) and NaOH pellets (1 equiv) in H2O (7 M) is stirred for ten min after which time p-xylem (1.4 M), K2CO3 (1.5 equiv) and aminoethylchloride HCl (1 equiv) are added and the reaction heated to 100 °C for 4 h. The reaction was cooled to rt then concentrated under reduced pressure. The crude residue is dissolved in p-xylene and washed with IN NaOH (2x) and H2O (lx). The organic layer is dried (MgSO ) and concentrated in vacuo to furnish the resulting crude material whose structure is confirmed by LC/MS and Η NMR.
General Method E. Hydrogenation of substituted nitrobenzenes with palladium to substituted anilines
Figure imgf000062_0002
A solution of the substituted nitrobenzene (1 equiv) in ethanol (0.2 M) is added via syringe to a flask containing palladium on carbon (10 mol%). The reaction vessel is fitted with a balloon adapter and charged with hydrogen and evacuated three times until the reaction is under a H2 atmosphere. The reaction is allowed to stir overnight and then purged with As and evacuated three times until an Ar atmosphere has been achieved. The reaction solution is filtered through a pad of Celite and washed with copious amounts of ethanol. The filtrate is concentrated in vacuo to afford the desired aniline whose structure is confirmed by LC/MS and Η NMR.
General Method F. Hydrogenation of substituted nitrobenzenes with tin chloride to substituted anilines
Figure imgf000063_0001
A solution of intermediate nitro derivative (1 equiv) and SnCl2 (6 equiv) in ethanol (0.18 M) is heated to reflux over 4h. The reaction mixture is allowed to cool to rt, concentrated and dissolved in EtOAc. Satd NaHCO3 is then added to precipitate the tin salts. The liquid is decanted and poured into a separatory funnel, diluted with EtOAc and washed with H2O. The combined organic layers are dried (MgSO4) and filtered. The filtrate is concentrated in vacuo to afford the intermediate aniline whose structure is confirmed by LC/MS and Η NMR.
General Method G. Deprotection of substituted nitroanisole with borontribromide
Figure imgf000063_0002
To a solution of 2-substituted-5-nitroanisole (1 equiv) in CH2C12 (0.12 M) is added borontribromide (1.0 M in CH Cl2, 1 equiv) at -65 °C. The reaction is slowly allowed to reach room temperature and stir for 16 h. The reaction mixture is quenched slowly with H2O, poured into a separatory funnel and extracted with CH2C12. The combined organic layers are washed with satd NaHCO3, dried (MgSO4), filtered and concentrated in vacuo to give crude organic residue. The residue is purified by flash silica column chromatography using 10%EtOAc/90%Hex as eluent to afford the desired phenol whose structure is confirmed by LC/MS and Η NMR.
General Method H. Deprotection of substituted nitroanisole with hydrogen bromide
Figure imgf000064_0001
To a solution of 48% HBr (0.5 M) is added 2-substituted-5-nitroanisole (1 equiv). The solution is heated at reflux for 3 d. The reaction mixture is allowed to cool to rt, diluted with H2O, and extracted with EtOAc. The organic layer is dried (MgSO4), filtered and concentrated in vacuo to give crude organic residue. Purification by silica gel column chromatography using 20%EtOAc/80%Hex as eluent affords the desired phenol whose structure is confirmed by LC/MS and 1H NMR. General Method I. Preparation of substituted 2-bromoethoxy-nitrobenzenes
Figure imgf000064_0002
To a solution of substituted 3-nitrophenol (1 equiv) in acetonitrile (0.72 M) is added 1,2- dibromoethane (1 equiv) and Cs2CO3 (3 equiv). The mixture is refluxed overnight. After cooling to rt, the reaction mixture is diluted by EtOAc and washed with IN NaOH (3x), water (lx) and brine (2x). The organic layer is dried (MgSO4) and concentrated in vacuo to afford the crude product whose structure is confirmed by 1H NMR and which is used without further purification.
General Method J. Preparation of substituted nitrophenoxyethanamines without sodium iodide
Figure imgf000065_0001
A solution of bromoether intermediate (1 equiv), amine (2 equiv) and K2CO3 (8 equiv) in acetone (0.10 M) is heated to 65 °C for 24 h. The reaction mixture is allowed to cool to rt, diluted with EtOAc and washed with H2O. The organic layer is concentrated in vacuo and the resulting crude solid or oil is purified by silica gel column chromatography to furnish the nitro derivative whose structure is confirmed by LC/MS and 1H NMR.
General Method K. Preparation of substituted nitrophenoxyethanamines using sodium iodide
Figure imgf000065_0002
A solution of bromoether intermediate (1 equiv), amine (5 equiv), Nal (1 equiv), and Na2CO3 (5 equiv) in acetone (0.10 M) is heated to 65 °C for 24 h. The reaction mixture is allowed to cool to rt, evaporated to dryness, diluted with EtOAc, and washed with H O. The organic layer is concentrated in vacuo and the resulting crude solid or oil is purified by silica gel column chromatography to furnish the nitro derivative whose structure is confirmed by
LC/MS and 1H NMR.
General Method L. Preparation of substituted nitrophenyl-l,2-ethanediamines
Figure imgf000065_0003
To a vigorously stirring mixture of the hydrochloride salt of the chloroamine (1.5 equiv ) in Et O (1.0 M) is added Et3N (1 equiv). A white precipitate forms and is filtered off. The filtrate is concentrated under reduced pressure to obtain an oil. To the resulting free base is added 3-nitroaniline (1 equiv) and xylenes (0.5 M). The reaction mixture is heated to 130 °C for 24 h. After reaction cooling to rt, the mixture is concentrated under reduced pressure. The crude residue is dissolved in 30% EtOAc/Hex and passed through a pad of silica gel to afford the nitroamine derivative whose structure is confirmed by LC/MS and Η NMR.
General Method M. Preparatio of amino substituted nitrophenyl-l,2-ethanediamines
Figure imgf000066_0001
A flask charged with the nitro compound (1 equiv), Pd2(dba) (0.01 equiv), r c-BINAP (0.02 equiv) and Cs CO3 (1.4 equiv) is flushed with Ar before toluene (degassed with Ar, 0.3 M) and the amine (1.2 equiv) are added via syringes to form a red suspension (Wolfe, J.P.; Buchwald, S.L. J. Org, Chem. 2000, 65, 1144). The reaction is heated at 80 °C overnight. The reaction solution is filtered through a pad of celite and washed with EtOAc. The filtrate is concentrated in vacuo. The residue is purified by flash silica column chromatography (EtOAc/MeOH 9:1) to afford the pure product whose structure is confirmed by LC/MS and 1H NMR.
General Method N. Preparation of substituted nitrophenoxyethanamines from substituted nitrophenols.
Figure imgf000066_0002
To a solution of the nitrophenol (1 equiv) and aminoalcohol (1 equiv) in THF (0.20 M) is added PPh3 (1.5 equiv) and ADDP (1.5 equiv) at ambient temperature. The reaction mixture is allowed to stir overnight under Ar. The mixture is placed in an ice bath and a volume of hexanes is added to double the reaction volume. The white precipitate is filtered off and the filtrate is concentrated in vacuo. The residue is purified by silica gel column chromatography to furnish the desired nitro derivative whose structure is confirmed by LC/MS and 1H NMR.
General Method O. Preparation of substituted nitrophenyl-2-propyn-l-amine
Figure imgf000067_0001
A mixture of aminopropyne (1 equiv), bromonitrobenzene (1.1 equiv), copper(I) iodide (0.25 equiv), triphenylphosphine (0.25 equiv), and tra«.s-dichlorobis(triphenylphosphine)- ρalladium(II) (0.25 equiv) in DMF (0.5 M) and Et3N (0.8 M) is stirred at 80 °C under argon overnight. The mixture is cooled to rt and diluted with EtOAc and water. The layers are separated and the organic phase is washed with brine, dried (MgSO ), and concentrated in vacuo. Purification by column chromatography using 30-70% EtOAc in hexanes as eluent affords the alkyne intermediate whose structure is confirmed by LC/MS and Η NMR.
General Method P. Preparation of substituted nitrophenoxymethyl piperidines
Figure imgf000067_0002
A solution of the nitrophenol (1 equiv), l-methylpiperidine-2-methanol (1.25 equiv) and PPh3 (1 equiv) is dissolved in THF (0.13 M) and cooled to 0 °C. After 10 min DEAD (1.25 equiv) is added slowly via syringe. The cold bath is removed and the reaction mixture is allowed to stir overnight under Ar at room temperature. The mixture is concentrated and purified by flash silica gel chromatography (5/95 MeOH/CH2Cl ) to furnish the intermediate nitro derivative whose structure is confirmed by LC/MS and Η NMR. General Method Q. Preparation of substituted 7-nitro-2,3-dihydro-4H-l,4-benzoxazin- 4-yl ethanamines
Figure imgf000068_0001
A solution of 7-nitro-2H-l,4-benzoxazin-3(4H)-one (1 equiv) in TΗF (1.3 M) is cooled to 0
°C and borane-TΗF complex (1.3 equiv) is added slowly via syringe. The reaction mixture is allowed to warm slowly to rt and then fitted with a reflux condensor and heated to reflux for
2 h. The reaction is cooled, diluted with Et2O and washed with satd. NaΗCO3. The combined organics are dried (MgSO4), filtered and concentrated to yield the desired intermediate as a bright orange solid whose structure is confirmed by LC/MS and 1H NMR. The nitro intermediate (1 equiv) is dissolved in THF (0.1 M), cooled to 0 °C and NaH (2 equiv, 60%wt in mineral oil) is added. The reaction solution is allowed to stir for 5 min and then the substituted aminoethylchloride is added. The cold bath is removed and the reaction is allowed to reach rt. The flask is fitted with a reflux condenser and heated to reflux overnight. The reaction mixture is cooled to rt and quenched with a slow addition of water. The mixture is extracted with EtOAc (3x) and the combined organic layers are dried (MgSO4), filtered and concentrated. Purifcation by flash silica chromatography (5/95 MeOH/CH2Cl2) furnishes the desired product as a yellow oil whose structure is confirmed by LC/MS and Η NMR.
General Method R. Preparation of substituted phenoxy-5-nitrophenoxyethanamines
Figure imgf000068_0002
Nitrophenylbromide (1 equiv), phenol (2 equiv), CsCO3 (2 equiv), Cu(OTf)2ThH (0.03 equiv), 1-naphthoic acid (2 equiv), molecular sieves (5 A, 2 equiv), EtOAc (0.05 equiv), and toluene (1.6 M) are combined and heated at 100 °C overnight. The mixture is cooled to ambient temperature before being taken up in CH C12. The mixture is washed with IN NaOH (lx), H O (lx), and brine (lx). The organics are dried over Na2SO4 and concentrated. Purification by flash silica gel chromatography provids the desired biaryl ether intermediate whose structure is confirmed by LC/MS and Η NMR.
General Method S. Preparation of substituted {[2-(amino)ethyl]sulfanyl}aniline
Figure imgf000069_0001
A slurry of aminothiophenol (1 equiv) and NaOH pellets (1 equiv) in H2O (7 M) is stirred for ten min, after which time p-xy ene (1.4 M), K CO (1.5 equiv) and aminoethylchloride HCl (1 equiv) is added and the reaction is heated to 100 °C for 2 h. After cooling to rt, the reaction is concentrated under reduced pressure. The crude residue is dissolved in p-xylene and washed with IN NaOH (2 x) and H2O (1 x). The organic layer is dried (MgSO4) and concentrated in vacuo to furnish the resulting crude material whose structure is confirmed by LC/MS and 1H NMR.
General Method T. Preparation of substituted (2-bromoethyl)sulfonyl-nitrobenzene
Figure imgf000069_0002
A solution ofthe nitrothiophenol (1 equiv), 2-bromoethanol (1 equiv) and K2CO3 (1.5 equiv) in toluene (0.10 M) is heated to 100 °C for 12 h. The reaction mixture is allowed to cool to rt, diluted with EtOAc and washed with H2O. The organic layer is dried (MgSO4) and concentrated in vacuo to furnish the intermediate alcohol product whose structure is confirmed by LC/MS and Η NMR. The crude alcohol (1 equiv) is then dissolved in THF (0.03 M) and treated with CBr4 (1.25 equiv) and triphenyl phosphine (1.25 equiv). The reaction is left to stir for 1.5 h then another batch of CBr4 (1.25 equiv) and triphenyl phosphine (1.25 equiv) is added and the mixture is allowed to stir for lh. The reaction is diluted with H2O and extracted with EtOAc. The organic layer is dried (MgSO4), filtered and concentrated in vacuo to give the crude organic residue. Purification by silica gel column chromatography using 20%EtOAc/80%Hex as eluent affords the bromoethyl sulfide intermediate which is confirmed by LC/MS and Η NMR. To a rt solution of the sulfide (1 equiv) in acetone (1.1 M) is added Oxone™ (3 equiv). After stirring for 3 days, the reaction is diluted with H2O and extracted with EtOAc. The organic layer is dried (MgSO4), filtered and concentrated in vacuo to give the crude bromo phenylsulfone whose structure is confirmed by LC/MS and Η NMR. Product is used in subsequent reactions without purification.
General Method U. Preparation of substituted (nitrobenzyl)oxyethanamines
Figure imgf000070_0001
To a solution of the aminoalcohol (1 equiv) in anhydrous DMF (0.2 M) is added NaH (1.5 equiv) in one portion, and the reaction mixture is stirred at rt under argon. After lh, nitrobenzyl bromide (1 equiv) is added and the reaction is stirred for 4h at rt. The reaction is partitioned with 0.2 M HCl and dichloromethane. The organic layer is washed with brine and concentrated under reduced pressure to obtain an oil. The crude residue is purified by MPLC (Biotage) to afford the corresponding intermediate whose structure is confirmed by LC/MS and Η NMR.
General Method V. Preparation of substituted nitrophenylmethanamines
Figure imgf000071_0001
A solution of the secondary amine (2 equiv) and nitrobenzyl bromide (1 equiv) in anhydrous THF (0.20 M) is stirred under argon for 15 h. The resultant white precipitate is filtered off and the filtrate is concentrated in vacuo. The reaction is partitioned between water and dichloromethane. The organic layer is washed with brine and concentrated under reduced pressure to obtain an oil. The crude residue is purified by MPLC (Biotage) to afford the corresponding intermediate whose structure is confirmed by LC/MS and !H NMR.
General Method W. Preparation of substituted bromo-N-(nitrophenyl)acetamides
Figure imgf000071_0002
To a 0 °C suspension of the nitroaniline (1 equiv) and sodium bicarbonate (4 equiv) in chloroform (1 M) is added bromoacetyl bromide (1.1 equiv) dropwise. The thick suspension is stirred at 0 °C for 30 min. The reaction is then diluted with dichloromethane and water. The layers are separated and the organic phase is washed with brine, dried (MgSO4), and concentrated in vacuo to furnish the intermediate nitro-α-bromoketone derivative whose structure is confirmed by Η NMR.
General Method X. Preparation of substituted Nl-[2-(amino)ethyl]-l,3-benzenediamine
Figure imgf000071_0003
A solution of the amide (1 equiv) in THF (0.1 M) is treated with borane dimethylsulfide (5 equiv). The reaction is refluxed under argon overnight at which time it becomes a suspension. The reaction mixture is cooled to rt and quenched with EtOH (15 equiv) and 2 M HCl (6 equiv). The resulting solution is refluxed for 1 h, cooled to rt, and basified with 1 N KOH solution. The product is then extracted with CH2C1 , dried (MgSO4), and concentrated in vacuo to give crude product. Purification by column chromatography using 2-8% MeOH in CH C12 as eluent affords the aniline intermediate whose structure is confirmed by Η NMR.
Preparation of l-methyl-4-(3-nitrophenoxy)azepane
Figure imgf000072_0001
This compound was prepared by the general method described in D using 2-(2-chloroethyl)- 1 -methylpyrrolidine hydrochloride as the starting aminoalkyl halide to give 1 -methyl -4-(3- nitrophenoxy)azepane. Η NMR (CDC13) δ (7.78, d, IH), (7.68, s, IH), (7.37, dd, IH), (7.18, d, IH), (4.63, m, IH), (2.72, m, IH), (2.63, m, 2H), (2.55, m, IH), (2.33, s, 3H), (2.16, m, 2H), (1.96, m, IH), (1.87, m, 2H), (1.36, m, IH); MS (ESI-MS) 251 (M+H)+.
Example 1: Preparation of N4-(3-tert-butyl-lH-pyrazol-5-yl)-N2-{3-f2-('diethylamino)- ethoxy1phenyl}-2, 4-pyrimidinediamine
Figure imgf000072_0002
p-xylene
Figure imgf000073_0001
A slurry of 3-nitrophenol (1.0 g, 7.19 mmol) and NaOH pellets (288 mg, 7.19 mmol) in H2O (1 mL) was stirred for 10 min after which time »-xylene (5 mL), K2CO3 (1.5 g, 10.78 mmol) and 2-diethylaminoethylchloride HCl (1.24 g, 7.19 mmol ) were added and the mixture was heated to 100 °C for 4 h. The reaction was cooled to rt then concentrated under reduced pressure. The crude residue was dissolved in/?-xylene (20 mL) and washed with IN NaOH (2 x 20 mL) and H2O (1 x 20 mL). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure to yield 1.51 g (88%) of N,N-diethyl-N-[2-(3- nitrophenoxy)ethyl]amine as a solid. Η ΝMR (300 MHz, CDC13) δ 7.81-7.76 (m, IH), 7.73- 7.72 (m, IH), 7.40 (t, J= 8.1 Hz, IH), 7.23-7.19 (m, IH), 4.09 (t, J= 6.2 Hz, 2H), 2.88 (t, J = 5.9 Hz, 2H), 2.63 (q, J = 6.92 Hz, 4H), 1.60 (t, J = 6.8 Hz, 6H); MS (ESI-MS) 239 (M+H)+.
A solution of N,N-diethyl-N-[2-(3-nitrophenoxy)ethyl]amine (1.5 g, 6.30 mmol) in ethanol (35 mL) was added via syringe to a flask containing palladium on carbon (150 mg). The reaction vessel was fitted with a balloon adapter and charged with hydrogen and evacuated three times until the reaction was under a H2 atmosphere. The reaction was allowed to stir overnight and then purged with Ar and evacuated three times until an Ar atmosphere had been achieved. The reaction solution was filtered through a pad of Celite and washed with copious amounts of ethanol. The filtrate was concentrated in vacuo to afford 1.27 g (97%) of 3-[2-(diethylamino)ethoxy]aniline as an oil. Η ΝMR (300 MHz, CDC13) δ 7.02 (t, J= 8.2 Hz, IH), 6.32-6.22 (m, 3H), 3.98 (t, J= 6.4 Hz, 2H), 2.83 (t, J= 6.4 Hz, 2H), 2.60 (q, J=6.81 Hz, 4H), 1.04 (t, J= 6.9 Hz, 6H); MS (ESI-MS) 209 (M+H)+.
A solution of 2,4-dichloropyrimidine (11.92 g, 80.0 mmol), KOAc (9.42 g, 96.0 mmol, 1.2 equiv) and 5-amino-3-tert-butylpyrazole (11.14 g, 80.0 mmol) in THF/H2O (225 mL, 2/1) was heated at 45 °C for 24 h. The reaction mixture was allowed to cool to rt, dissolved in EtOAc (200 mL) and washed with aquous ΝaHCO3 (2 x 200 mL). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure. The resulting crude solid was purified by silica gel column chromatography (MeOH/CH2Cl2, 1/19) to give 8.62 (43%) of N-(3-tert-butyl-lH-pyrazol-5-yl)-2-chloro-4-pyrimidinamine. Η ΝMR (300 MHz, DMSO) δ 12.2 (s, IH), 10.3 (s, IH), 8.16 (s, 1 H), 1.26 (s, 9H); Mp>250 °C; MS (ESI-MS) 252 (M+H)+. tR 2.20 min. (10-90% CH3CΝ/H2O). A solution of N-(3-tert-butyl-lH-pyrazol-5-yl)-2-chloro-4-pyrimidinamine (6.0 g,
23.84 mmol) and N-[2-(3-aminophenoxy)ethyl]-NN-diethylamine (5.0 g, 23.84 mmol ) in n- BuOΗ (160 mL) with cone. ΗC1 (2 mL) was heated at 100 °C for 48 h. The reaction was cooled to rt and a precipitate formed. The reaction mixture was filtered and the filtercake washed with n-BuOΗ (100 mL). The resulting white solid was dissolved in CΗ2C12 (150 mL) and washed with aquous ΝaHCO (2 x 150 mL). The combined organic layers were dried (MgSO4) and concentrated under reduced pressure and dried in a vacuum oven overnight to afford 7.2 (70%) of N4-(3-tert-butyl-lH-ρyrazol-5-yl)-N2-{3-[2-(diethylamino)- ethoxy]ρhenyl}-2,4-pyrimidine-diamine as a solid. 1H ΝMR (300 MHz, CD3OD) δ 7.94 (br s, IH), 7.26 (br s, IH), 7.18 (br s, 1 H), 6.59-6.56 (m, IH), 6.35-6.28 (m, 2H), 4.08 (t, J= 5.9 Hz, 2H), 2.90 (t, J= 5.8 Hz, 2H), 2.67 (q, 7= 7.1 Hz, 4H), 1.30 (t, J= 7.4 Hz, 9H), 1.08 (t, 6H); Mp=160 °C MS (ESI-MS) 424 (M+H)+; tR 1.74 min (10-90% CH3CΝ/H2O).
The compounds of examples 2-220 were prepared by general method C where a heterocyclic substituted pyrimidine (prepared by general methods A and B) is reacted with an aniline sidechain (prepared by general methods D-X):
Table 1. Compounds Prepared by general method C.
Figure imgf000074_0001
Figure imgf000074_0002
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
u
A (M+H)+ 450
162 H t-butyl H U,E tR 2.00 min.c
(M+H)+ 468
163 t-butyl H U,E tR 2.75 min.c
(M+H)+ 480
164 H t-butyl OMe U,E tR2.13min.c
(M+H)+ 498
165 t-butyl OMe U,E tR2.13min.c
(M+H)+ 468
166 H t-butyl OMe U,E k tR 1.83min.°
(M+H)+ 486
167 t-butyl OMe U,E tR 1.84min.c
A (M+H)+ 496
168 H t-butyl OMe U,E tR 1.92min.c
(M+H)+514
169 t-butyl OMe U,E tR 1.94min.c
(M+H)+ 436
170 H t-butyl Me A M N, E tR 1.79min.c
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
aThe structures of the final compounds were confirmed by 1H NMR spectroscopy and the spectra were consistent with the desired chemical structures.
Analytical HPLC were obtained using a Gilson HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (50 x 4.6 mm, 12μm). The eluents were A: acetonitrile w/0.1% TFA and B: H2O w/0.1% TFA. Gradient elution from 10% B to 90% over 4 min at a flowrate of 4.0 mL/min was used with an initial hold of 0.5 min and a final hold at 90% B of 0.5 minutes. Total run time was 5 min.
CHPLC - electrospray mass spectra (HPLC ES-MS) were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonirile with 0.018% TFA. Gradient elution from 10% B to 95% over 3.5 minutes at a flowrate of 1.0 mL/min was used with an initial hold of 0.5 minutes and a final hold at 95% B of 0.5 minutes. Total run time was 6.5 minutes.
Table 2. Other Compounds Prepared by general method C.
Figure imgf000098_0001
Figure imgf000098_0002
Figure imgf000099_0001
'The structures ofthe final compounds were confirmed by !H NMR spectroscopy and the spectra were consistent with the desired chemical structures. bAnalytical HPLC were obtained using a Gilson HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (50 x 4.6 mm, 12μm). The eluents were A: acetonitrile w/0.1% TFA and B: H2O w/0.1% TFA. Gradient elution from 10% B to 90% over 4 min at a flowrate of 4.0 mL/min was used with an initial hold of 0.5 min and a final hold at 90% B of 0.5 minutes. Total run time was 5 min.\ CHPLC - electrospray mass spectra (HPLC ES-MS) were obtained using a Hewlett-Packard 1100 HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (2 x 23 mm, 120A), and a Finnigan LCQ ion trap mass spectrometer with electrospray ionization. Spectra were scanned from 120-1200 amu using a variable ion time according to the number of ions in the source. The eluents were A: 2% acetonitrile in water with 0.02% TFA and B: 2% water in acetonirile with 0.018% TFA. Gradient elution from 10% B to 95% over 3.5 minutes at a flowrate of 1.0 mL/min was used with an initial hold of 0.5 minutes and a final hold at 95% B of 0.5 minutes. Total run time was 6.5 minutes.
Method Y. Preparation of l-[2-(l-pyridiniumyl)-4-pyrimidinyl]pyridinium dichloride
Figure imgf000100_0001
A solution of 2,4-dichloropyrimidine (84.6 g, 567.9 mmol) in anhydrous pyridine (2.5 L) was heated slowly to reflux over 2 h with mechanical stirring and kept at reflux for 10 min. Over the course of the reaction, a thick precipitate formed which became pink in color. The reaction was cooled to rt over 24 h, then the solid filtered through a coarse sintered glass funnel. The cake washed with copious amounts of ether then dried in vacuo to give the desired product as a light purple / brown fluffy solid in 99% yield (173.3 g, 564.2 mmol). 1H NMR (DMSO) δ 10.33 (2H, d, J = 6.9 Hz), 10.18 (2H, d, J = 7.2 Hz), 9.78 (IH, d, J = 5.1 Hz), 9.07 and 9.02 (2H, t overlapping, J - 6.0 and 7.8 Hz), 8.95 (IH, d, J = 5.4 Hz), 8.49 (4H, m).
Method Z. Preparation of l-(4-hydroxy-2-pyrimidinyl)pyridinium chloride
Figure imgf000100_0002
A solution of l-[2-(l-pyridiniumyl)-4-pyrimidinyl]pyridinium dichloride (100 g, 325.5 mmol) in 0.33 M NaHCO3 solution (109.4 g NaHCO3 in 3.91 L dH2O) was stirred at 23 °C for 24 h. The brown solution slowly evolved CO2 (final reaction pH after this time = 7-7.5). The reaction was concentrated by rotary evaporation at 6 mm vacuum at 30 °C and the crude brown sludge (still containing water) suspended in methanol and coated on silica gel (700 ml) by concentration in vacuo. The silica-coated crude was then purified on a plug of silica gel (1 L), eluting with a gradient of 100% CH3CN (2 L) -» 5% MeOH/CH3CN (1 L) -» 10% MeOH/CH3CN (1 L) -» 20% MeOH/CH3CN (6 L) -> 50% MeOH/CH3CN (1 L) - 50% MeOH/CH3CN (2 L). The fractions containing product were pooled and concentrated in vacuo to give a brown solid. This was triturated in MeOH (700 ml) with water (300 ml) and the insoluble salts filtered off and washed with MeOH. The filtrate was again coated on silica gel (300 ml) and the column purification repeated as above to give an amber oil/solid. This was again suspended in MeOH/water (2:1) and concentrated until a precipitate formed. The salts were again filtered off and washed with CH3CN and acetone. The filtrate was concentrated in vacuo to a brown gum which solidified after drying in vacuo under P2θ5. The solid was pulverized with a mortar and pestle then suspended in acetone (1 L), sonicated, and filtered washing with acetone. The desired product was obtained as a light tan solid in 65% yield (44.04 g, 210.1 mmol) after drying in vacuo under P2O5 for 24 h. TLC: Rf = 0.40 (33% methanol/dichloromethane); Rf = 0.20 (33% methanol/acetonitrile with 1% water); MS (ESI-MS): 174 (M+H)+; tR 0.74 min.; Η NMR (DMSO) δ 9.89 (2H, d, J = 6.9 Hz), 8.84 (IH, t, J = 7.5 Hz), 8.41 (IH, d, J = 5.7 Hz), 8.29 (2H, t, J = 7.2 Hz), 6.79 (IH, d, J = 6.0 Hz).
General Method AA. Preparation of substituted 2-({3-[2-(diethylamino)- ethoxy] phenyl} amino)-4-py rimidinols
Figure imgf000101_0001
2-({3-[2-(diethylamino)ethoxy]phenyl}amino)-4-pyrimidinol (R3 = H)
A suspension of l-(4-hydroxy-2-pyrimidinyl)pyridinium chloride (14.19 g, 67.69 mmol) and 3- [2-(diethylamino)ethoxy] aniline (14.10 g, 67.69 mmol) in anhydrous rc-BuOH (600 ml) was stirred under argon. To this was added cone. HCl (16.92 ml, 203.1 mmol, 3 eq) and the brown suspension stirred at 90 °C (internal temp.) for 1 h (solids dissolve). More l-(4-hydroxy-2- pyrimidinyl)pyridinium chloride (14.19 g, 67.69 mmol) was then added followed by n-BuOH (150 ml). The brown solution was stirred at 90 °C (internal temp.) for 16 h. A white ppt formed in the reaction. The solvent was removed by rotary evaporation at 2 mm vacuum and 30 °C, and the oily residue dried in vacuo. The crude product was quenched with sat. K2CO3 (300 ml) and immediately extracted with EtOAc (2 X IL) (note: a white ppt formed in the biphase - this may be excess starting aniline). The combined extracts were dried (Na2SO4), filtered, and concentrated in vacuo to give a brown gum. This was purified by silica gel chromatography (flush column) (800 ml silica gel, eluting with a gradient of 100% dichloromethane -> 50% methanol / dichloromethane (streaks off). Fractions containing product were pooled and concentrated in vacuo to give the desired product as a solid amber foam (after drying in vacuo) in 79% yield (16.20 g, 53.58 mmol). TLC: Rf = 0.25 (33% methanol / dichloromethane), Rf= 0.05 (25% water / acetonitrile); MS (ESI-MS): 303 (M+H)+; tR 0.71 min.; Η NMR (DMSO) δ 9.49 (IH, vbs), 7.76 (IH, d, J = 6.6 Hz), 7.42 (IH, s), 7.18 (IH, t, J = 8.4 Hz), 7.13 (IH, d, J = 8.4 Hz), 6.58 (IH, d, J = 9.0 Hz), 5.80 (IH, d, J = 6.3 Hz), 4.01 (2H, t. J = 6.3 Hz), 2.84 (2H, t, J = 6.0 Hz), 2.61 (4H, quart, J = 7.2 Hz), 1.00 (6H, t, J = 6.9 Hz).
2-({3-[2-(diethylamino)ethoxy]-4-methoxyphenyl}amino)-4-pyrimidinol (R3 = OCH3)
The general procedure used in the preparation of 2-({3-[2- (diethylamino)ethoxy]phenyl}amino)-4-pyrimidinol (R = H) was also used in the preparation of 2-({3-[2-(diethylamino)ethoxy]-4-methoxyphenyl}amino)-4-pyrimidinol (R = OCH3) using 1- (4-hydroxy-2-pyrimidinyl)pyridinium chloride (32.50 g, 153.46 mmol), 3-[2-
(diethylamino)ethoxy]-4-methoxyaniline (35.00 g, 139.51 mmol), and cone. HCl (34.9 ml, 418.5 mmol) in anhydrous n-BuOH (500 ml). Product was obtained as a solid amber foam in 40% yield (18.99 g, 57.13 mmol). TLC: Rf = 0.15 (50/49/1 methanol/acetonitrile/water); MS (ESI-MS): 333 (M+H)+; tR = 0.75 min.; Η NMR (CD3OD) δ 7.62 (IH, d, J = 6.3 Hz), 7.25 (IH, d, J = 2.4 Hz), 6.95 (2H, m), 5.81 (IH, d, J = 6.6 Hz), 4.15 (2H, t. J = 5.7 Hz), 3.81 (3H, s), 3.02 (2H, t, J = 5.7 Hz), 2.78 (4H, quart, J = 7.5 Hz), 1.13 (6H, t, J = 6.9 Hz).
General Method AB. Preparation of substitutued 4-chloro-N-{3-[2-
(diethylamino)ethoxy]phenyl}-2-pyrimidin-amines
Figure imgf000102_0001
-chloro-N-{3-[2-(diethylamino)ethoxy]phenyl}-2-pyrimidinamine (R3 = H) A suspension of 2-({3-[2-(diethylamino)ethoxy]phenyl}amino)-4-pyrimidinol (5.40 g, 17.86 mmol, R = H) in phosphorous oxychloride (200 ml) was heated to reflux (solid dissolves) and the POCl distilled off over lh to give a concentrated reaction volume of 50 ml POCl3. The dark brown solution was quenched by dropwise addition to ice water (mostly ice) (1.5 L) with vigorous stirring over 15 min. (note: the secondary container also contained ice). The internal temp, was kept at <5 °C during the quench. This was stirred from 0 °C -> rt over 20 h with vigorous stirring. The acidic solution was then cooled to 0 °C with an ice bath, and quenched by portionwise addition of powdered K2CO3 (380 g) over 1.5 h with vigorous stirring to a final pH of 1 1.5 (internal temp, always kept at <8 °C during the quench; vigorous bubbling observed). The still-cold light brown solution is extracted with EtOAc (2L), and this was dried (Na2SO4), filtered, and concentrated in vacuo to give an amber oil. This was purified by a silica gel plug (flush column) (200 ml silica gel, eluting with a gradient of 100% acetonitrile -> 50% water/acetonitrile). Fractions containing product were pooled and concentrated in vacuo to give an amber oil. This crystallized to an amber solid over 48 h of drying in vacuo to give the desired product in 77% yield (4.18 g, 13.03 mmol). TLC: Rf = 0.20 (25% water/acetonitrile); MS (ESI-MS): 321 (M+H)+; tR = 1.97 min.; 1H NMR (DMSO) δ 9.99 (IH, s), 8.42 (IH, d, J = 5.4 Hz), 7.39 (IH, s), 7.16 (IH, t, J = 8.1 Hz), 7.23 (IH, d, J = 8.4 Hz), 6.94 (IH, d, J = 4.8 Hz), 5.56 (IH, d, J = 7.8 Hz), 3.98 (2H, t. J = 5.7 Hz), 2.80 (2H, b s), 2.47 (4H, b s), 0.97 (6H, t, J = 7.2 Hz).
4-chloro-N-{3-[2-(diethylamino)ethoxy]-4-methoxyphenyl}-2-pyrimidinamine (R3 =OCH3) The general procedure used in the preparation of the above 4-chloro-N-{3-[2- (diethylamino)ethoxy]phenyl}-2-pyrimidin-amine was also used in the preparation of compound 4-chloro-N- {3-[2-(diethylamino)ethoxy]-4-methoxyphenyl}-2-pyrimidinamine using 2-({3-[2-(diethylamino)ethoxy]-4-methoxyphenyl}-amino)-4-pyrimidinol (12.27 g, 35.81 mmol) in phosphorous oxychloride (250 ml), except the reaction was stirred at ambient temp for 20 h instead of being refluxed. The product was obtained as a brown oil which crystallized to a brown in 86% yield (11.10 g, 31.64 mmol). TLC: Rf = 0.35 (50/49/1 methanol/acetonitrile/water); MS (ESI-MS): 351/353 (M+H)+; tR - 1.83 min.; Η ΝMR (CD3OD) δ 8.25 (IH, d, J = 5.4 Hz), 7.42 (IH, d, J = 2.7 Hz), 7.13 (IH, dd, J = 2.4, 8.7 Hz), 6.90 (IH, d, J = 8.7 Hz), 6.76 (IH, d, J = 5.4 Hz), 4.14 (2H, t. J = 5.7 Hz), 3.81 (3H, s), 2.97 (2H, t, J = 5.7 Hz), 2.73 (4H, quart, J = 7.5 Hz), 1.11 (6H, t, J = 6.9 Hz). General Method AC. Coupling of 5-amino-3-substituted pyrazoles with substitutued 4- chloro-N-{3-[2-(diethylamino)ethoxy]phenyl}-2-pyrimidin-amines using dilute conditions
Figure imgf000104_0001
4-Chloropyrimidine intermediate (1 equiv), aminopyrazole (1.25 equiv), «-BuOH (0.15 M), and HCl (cone, 1 drop) are combined and heated at 100 °C overnight. The mixture is taken up in CH2CI2 and washed with NaHCO3 before being dried (Na2SO4) and concentrated. Purification of the residue by flash silica gel chromatography provides the desired 2,4- diaminopyrimidienes whose structures are confirmed by LC/MS and lH NMR.
General Method AD. Coupling of 5-amino-3-substituted pyrazoles with substitutued 4- chloro-N-{3-[2-(diethylamino)ethoxy]phenyl}-2-pyrimidin-amines using concentrated conditions
Figure imgf000104_0002
4-Chloropyrimidine intermediate (1 equiv), aminopyrazole (1.25 equiv), and HCl (cone, 1 drop) are combined and melted at 100 °C overnight. The mixture is taken up in CH2CI2 and washed with NaHCO3 before being dried (MgSO4) and concentrated. Purification of the residue by flash silica gel chromatography or preparative HPLC provides the desired 2,4- diaminopyrimidienes whose structures are confirmed by LC/MS and H NMR
7 Λ
Example 221: N -{3-f2-(diethylamino)ethoxyI-4-methoxyphenyl}-N -I3-(l- methylcvclopropyl)-lH-pyrazol-5-yll-2,4-pyrimidinediamine
Figure imgf000105_0001
To a homogenized mixture of 4-chloro-N-{3-[2-(diethylamino)ethoxy]-4-methoxyphenyl}-2- pyrimidinamine (100 mg, 0.280 mmol) and 5-amino-3-(l-methylcyclopropyl)-lH-pyrazole (1 17 mg, 0.830 mmol) was added cone. ΗC1 (0.048 ml, 0.576 mmol) and the vial sealed and melted to an amber oil at 100 °C for 1 h. The reaction was cooled, dissolved in MeOΗ (10 ml), quenched with sat. K2CO3 (50 ml), and extracted with EtOAc (2 X 150 ml). The combined organics were dried (Νa2SO4) and the solvent removed in vacuo. The crude product was purified by silica gel chromatography (100% acetonitrile - 5% water/methanol gradient). The fractions containing product were concentrated in vacuo, and the residue dissolved in dichloromethane and filtered to remove silica gel. Hexane was added, and the solvent removed in vacuo to give a solid. The product was isolated as a light pink solid from hexane in 68% yield (88 mg, 0.195 mmol). TLC: Rf = 0.20 (50/49/1 methanol/acetonitrile/water); MS (ESI- MS): 452 (M+H)+; tR = 1.60 min.; 1H NMR (CD3OD) δ 7.89 (IH, bs), 7.25 (IH, bs), 7.05 (IH, dd, J = 2.4, 8.7 Hz), 6.92 (IH, d, J = 9.0 Hz), 6.22 (2H, bs), 4.09 (2H, t. J = 6.3 Hz), 3.82 (3H, s), 2.94 (2H, t, J = 5.4 Hz), 2.69 (4H, quart, J = 6.9 Hz), 1.38 (3H, s), 1.09 (6H, t, J = 7.5 Hz), 0.87 (2H, bs), 0.74 (2H, bs).
The compounds of examples 220-258 are prepared by general method AC or AD where a 4-chloropyrimidine (prepared by methods Y-AB) is reacted with an amino pyrazole (prepared by general method A): Table 3. Compounds Prepared by general methods AC or AD
Figure imgf000106_0001
Figure imgf000106_0002
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
3The structures of the final compounds were confirmed by Η NMR spectroscopy and the spectra were consistent with the desired chemical structures. b Analytical HPLC were obtained using a Gilson HPLC equipped with a quaternary pump, a variable wavelength detector set at 254 nm, a YMC pro C-18 column (50 x 4.6 mm, 12μm). The eluents were A: acetonitrile w/0.1% TFA and B: H2O w/0.1% TFA. Gradient elution from 10% B to 90% over 4 min at a flowrate of 4.0 mL/min was used with an initial hold of 0.5 min and a final hold at 90% B of 0.5 minutes. Total run time was 5 min. cPyrazole is commerically available. dPyrazole was prepared as follows: To a solution of 5-nitro-lH-pyrazole-3-carboxylic acid (1 equiv) in TΗF (0.32 M) was added CDI (1 equiv). The reaction was allowed to stir for 5 min and then diethylamine (1.5 equiv) was added via syringe. The reaction mixture was stirred overnight at room temperature and concentrated. The residue was purified by preparative ΗPLC using 10-90%ACN/Η2O to afford the intermediate nitro pyrazole whose structure was confirmed by LC/MS and Η NMR.
Example 259. Preparation of N -(3-tert-butyl-l-methyl-iH-pyrazol-5-yl)-N2-{3-[2- (diethyl-amino)ethoxy] phenyl} -N2^V4-dimethyl-2,4-py rimidinediamine Example 260. N4-(3-tert-butyl-l-methyl-lH-pyrazol-5-yl)-N2-{3-[2- (diethylamino)ethoxy]-phenyl}-N -methyl-2,4-pyrimidine-diamine
Figure imgf000111_0001
To N4-(3-tert-butyl-lH-pyrazol-5-yl)-N -{3-[2-(diethylamino)ethoxy]phenyl}-2,4- pyrimidine-diamine (75 mg, 0.17 mmol) in anhydrous NN-dimethylformamide (1.8 mL) was added 60% sodium hydride in mineral oil (25 mg, 0.62 mmol, 3.5 eq). After lh, methyl iodide (16mL, 0.26mmol, 1.5 eq) was added, and the reaction mixture was stirred at room temperature for 16h. The reaction was quenched with saturated ammonium chloride (1 mL) and poured into ethyl acetate. The organic layer was extracted with water (2 x 25 mL) and brine (1 x 25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude organic concentrate was purified using MPLC (Biotage) eluted with 10% methanol - dichloromethane with 1 % ammonium hydroxide followed by 20% methanol - dichloromethane with 1% ammonium hydroxide. The first product to elute was N^-(3-tert- butyl-l-methyl-lH-pyrazol-5-yl)-N2-{3-[2-(diethylamino)ethoxy]phenyl}-N2,N¥-dimethyl- 2,4-pyrimidinediamine (10.0 mg, 12.1% yield) as a white solid; MS (ESI-MS): 466 (M+Η)+; Rf = 0.74 (3/1 CH2Cl2/MeOH) followed by N*-(3-tert-butyl-l -methyl- lH-pyrazol-5-yl)-N2- {3-[2-(diethylamino)ethoxy]-phenyl}-N?-methyl-2,4-pyrimidinediamine (5.0 mg, 6.2%) as a white solid; MS (ESI-MS): 452, (M+Η)+; Rf= 0.59 (3/1 CH2Cl2/MeOH).
Assays for testine the activity ofthe compounds
This section describes assays that can be used to characterize compounds of the invention, e.g., src kinase activity assays; assays for testing the activity of compounds on kinases other than src; and assays for testing the activity of compounds on cell proliferation and differentiation. A preferred method for measuring src kinase activity (a "src biochemical assay") uses ATP (5 μM/well) mixed with biotinylated poly-GAT substrate (10 nM/well), Streptavidin- APC (15 nM/well) and European-labeled anti-phosphotyrosine antibody (2.5 nM/well). 10 μl of a mixture of these components is added to each well of a black 96-well plate, with or without test compound (5 μl desired concentration of compound in DMSO). 75 μl of assay buffer (50 mM HEPES pH 7.5, 0.1 mM EDTA, 0.015% BRIJ 35 solution, 0.1 mg/mL BSA, 0.1% beta-mercaptoethanol, 10 mM magnesium chloride) is then added to each well. Last, the src kinase (0.1 units/well) (Upstate Biotech, Lake Placid, NY) is added (10 μl) to a final volume of 100 μl. After 3-hour incubation at room temperature, plates are read on Wallac 1420 Victor Multilabel Counter (Perkin Elmer™ Life Sciences, Boston, MA) at 665 and 615 nm. A specific signal is the ratio ofthe value of the signal at 665 and the value of the signal at 615 multiplied by 10,000 (i.e., (signal at 665/signal at 615) x 10,000). Compounds that cause the specific signal to decrease inhibit the kinase activity of src. Percent inhibitions and/or IC50 values can then calculated based on specific signals from wells that have no compound added, i.e., zero percent inhibition.
A specific signal is the ratio of the value of the signal at 665 and the value of the signal at 615 multiplied by 10,000 (i.e., (signal at 665/signal at 615) x 10,000). Compounds that cause the specific signal to decrease inhibit the kinase activity of src. Percent inhibitions and/or IC50 values can then calculated based on specific signals from wells that have no compound added, i.e., zero percent inhibition.
Compounds of examples 1, 4, 6, 9-13, 15-21, 23-28, 49-52, 54-56, 58, 59, 61-63, 70, 74, 75, 78, 80-88, 90-94, 96, 98-100, 107, 111, 115, 116, 132, 136-141, 144-147, 150, 151, 153-156, 159-161, 179, 180, 183, 185, 191, 195-197, 199, 201, 203, 205-208, 210, 211, 224, 228, 237, 240, 244, and 257 demonstrate an IC50 less than 150 nM in the src biochemical assay. Compounds of examples 2, 3, 5, 7, 8, 14, 22, 29-32, 34-42, 44-48, 53, 57, 60, 64-69, 71-73, 76, 77, 79, 89, 95, 97, 101-103, 106, 108-110, 112-114, 117-131, 133, 135, 142, 148, 149, 152, 157, 158, 162, 163, 165-169, 171-176, 178, 181, 182, 184, 186-190, 192-194, 198, 200, 202, 204, 209, 215, 216, 219-223, 225-227, 229-232, 234, 236, 238, 239, 241, 243, 245- 248, 250-252, 254-256, and 258 exhibit an IC50 greater than 150 nM but less than 1.0 μM in the src biochemical assay. Compounds of examples 33, 43, 104, 105, 134, 143, 164, 170, 177, 212-214, 217, 218, 233, 235, 242, 249, 253, 259, and 260 show an IC50 greater than 1 μM and/or percent inhibition less than 50 but greater than 30 at 1 μM in the src biochemical assay.
It will be understood by a person of skill in the art that modified versions of the src biochemical assay described above can be conducted. These alternative assays can also be used to test the inhibitory activity of compounds of the invention or analogs or derivatives thereof.
The assay can also be adapted to determine the inhibitory activity of compounds towards kinases other than src kinases. For example, the src kinase enzyme in the above assay can be replaced with another kinase. When testing the inhibitory activity on kinases that are not tyrosine kinases, the antibody in the assay may also have to be replaced with an antibody that is specific for the phosphorylated residue, which has been phosphorylated by the kinase.
The effect of compounds on cell proliferation can be determined, e.g., by incubating cells with varying amounts of the compounds and counting the cells over time. Viable cells can be counted by staining the cells with a specific dye, e.g., Trypan Blue, according to methods well known in the art. Other methods include measuring the incorporation of a labeled molecule into DNA or RNA or protein of cells. For example, cell proliferation is often measured by H thymidine or 5-bromodeoxyuridine incorporation assays, also well known in the art. An increase in 3H thymidine or 5-bromodeoxyuridine incorporation in cells incubated with a test compound that is similar to that in cells non incubated with the test compound indicates that the test compound is essentially not inhibiting the proliferation ofthe cells. On the contrary, a lower 3H thymidine or 5-bromodeoxyuridine incorporation in cells incubated with a test compound relative to cells that were not treated with the test compound indicates that the test compound inhibits cell proliferation.
The effect of a compound on cell differentiation can be determined by visualization of the cells after having been contacted with the compound, preferably by comparison with cells which have not been contacted with the compound. The differentiation of certain cells is visible by the naked eye (e.g., that of 3T3L1 cells), whereas that of other cells may require the use of a microscope. Specific dyes can also be used to evaluate the state of differentiation of cells. Cell differentiation can also be monitored by measuring the expression level of certain genes, whose expression is known to vary during differentiation ofthe cells.
i l l The effect of a compound on a cell can be determined in a cell that contains an abnormal kinase, e.g., a mutated kinase gene, or a cell which over-expresses a kinase. For example the cell can be a cell expressing a mutated form ofa tyrosine kinase, e.g., src kinase, thereby transforming the cell. The cell can also be a cell that has an abnormal proliferation which is not caused by an abnormal activity or level of a kinase. Cells that can be used for testing compounds of the invention include cell lines and primary cell cultures. Numerous cell lines that are transformed, e.g., by over-expression of a proto-oncogene, which encodes, e.g., a kinase, are available, e.g., from the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Virginia 20110. Cell lines over-expressing a gene, e.g., a kinase, can be prepared by transient, or preferably, stable transfection of cells with an expression plasmid containing the gene, according to methods well known in the art. Nucleic acids for use in transforming cells, e.g., nucleic acids encoding kinases, are also publicly available or can readily be obtained. Cell lines can also be obtained from transgenic animals, e.g., animals overexpressing a kinase or expressing a mutated kinase. For example, MG 1361 is a breast carcinoma cell line obtained from the MMTV-neu transgenic mouse (Sacco et al, Breast Cancer Res. Treat., 47:171-180 (1998)). Primary cell cultures can be established from biopsies obtained from patients, e.g., patients having cancer.
The present invention also provides methods of testing a compound (e.g., the candidate drug) for its inhibition of src, its antiproliferative effect, its effect on cell differentiation and/or its toxicity on normal or wild-type cells in animals, e.g., transgenic animals, e.g., mice. Transgenic mice are produced that express a transforming agent (e.g., a growth factor receptor) under the control of a promoter, e.g., a tissue specific promoter. Such mice develop carcinomas that have genetic and pathological features that closely resemble human cancers. For example, mice expressing viral polyoma middle T antigen under the control of the MMTV promoter produces highly metastatic mammary tumors with elevated c-src kinase activity (Guy et al. (1994) Genes and Dev. 8:23). Nude mice in which tumor cell lines have been administered can also be used. For example, breast cancer cell lines over-expressing c-src can be administered to nude mice (see, e.g., Biscardi et al. (1998) Mol. Carcinog. 21 : 261). The ability of the compound to inhibit tumor formation or growth is then ascertained. In one embodiment the size of the tumor is monitored by determining the tumor size and/or weight. The compounds can be administered by a variety of ways including orally, subcutaneously, or intraperitoneally. Generally, at least two groups of animals are used in the assay, with at least one group being a control group which is administered the administration vehicle without the compound.
An animal model for osteoporosis that can be used for testing the activity of compounds is described, e.g., in Missbach et al. (1999) Bone 24:437 and in Sims et al. (1999) J. Bone Miner. Res. 14: S183.
Other embodiments of the invention will be apparent to the skilled in the art from a consideration of this specification or practice ofthe invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit ofthe invention being indicated by the following claims.

Claims

We claim
1. A compound of formula (I)
Figure imgf000116_0001
wherein
Y represents H, C alkyl, or phenyl optionally substituted up to three times by halogen, C alkyl, or C1-4 alkoxy; Y2 and Y3 are independently selected from H, C ι-6 alkyl,
C3-6 cycloalkyl optionally substituted by C1- alkyl, phenyl optionally substituted up to three times by halogen, CM alkyl, or
Cι-4 alkoxy, adamantyl, CF3, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C1-6 alkyl, C(O)N(C alkyl)2 , C(O)O(C alkyl) ,
CN, halogen, and
Figure imgf000116_0002
or Y2 and Y3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C1-4 alkyl, or C1-4 alkoxy, X represents halogen or CM alkyl; n represents 0, 1, or 2; R1 represents H or CM alkyl;
R2 represents H or CM alkyl; R3 represents
Ci-6 alkyl, halogen, C M alkoxy,
O-phenyl optionally substituted up to two times by halogen, C1- alkyl, C1- alkoxy, or di-(C1- alkyl)amino, CN, or
N(R^2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'),
N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; p represents 0, 1, or 2; R4 represents CM alkyl or halogen; q represents 0, 1, or 2; or
R3 and R4 may be joined and taken together with the carbon atoms to which they are attached, form a 5-6 membered heteroaromatic ring containing up to two heteroatoms selected from N, O, and S, and which is optionally substituted up to two times by halogen, C1- alkyl, or C1-4 alkoxy, L is a linker selected from
-O-(CH2)M- ,
Figure imgf000117_0001
-S(O)0-2-(CH2)M- -N(R')-(CH2)M-
Figure imgf000118_0001
-(CH2) - ,
_C≡C-(CH2)1-4- and
•>
-N(R1)-C(O)-(CH2) - , and G represents
NR5R6 wherein
R5 represents H, Cι-6 alkyl, or C1-4 alkoxy-CM alkyl; and R6 represents
H,
C1-6 alkyl,
C1-4 alkoxy-substituted C1-4 alkyl,
C5- cycloalkyl optionally substituted up to 2 times by halogen, C1-4 alkyl, or CM alkoxy,
C3-6 cycloalkyl-substituted C1-4 alkyl, benzyl, phenyl optionally substituted by halogen, CM alkyl, CM alkoxy, -CO2R', -C(O)N(R1)2 , -N(R')2 , or by a
Figure imgf000118_0002
wherein
A is N(R'), S, S(O), S(O)2, or O , and said bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-membered heterocycle,
-(C.^ alky -N^ A1 x/ , wherein A represents N(R ), S, S(O),
S(O)2 , or O, or
Figure imgf000119_0001
? wherein A2 represents N(R'), S, S(O), S(O)2 , or O,
Figure imgf000119_0002
, optionally substituted up to 2 times by Cι- alkyl, (C1- alkoxy)(C alkyl), C^OR1, C(O)N(R')2 , phenyl, or benzyl,
j-N A3 \/ , optionally substituted up to 2 times by C1-3 alkyl, (C1-3 alkoxy)(C alkyl), C(O)OR1, C(O)N(R')2 , phenyl, or benzyl, and wherein A3 represents NCR1), S, S(O), S(O)2 , or O,
NA ) O-i , optionally substituted up to 2 times by oxo, (Cι- alkoxy)-(CM alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl, or up to 4 times by Cι-3 alkyl,
Figure imgf000119_0003
optionally substituted up to 2 times by halogen, C1- alkyl, or Ci-4 alkoxy, and wherein v is 0 or 1 , and
|-N ' ^ , optionally substituted up to 2 times by C1-4 alkyl,
or
L represents
Figure imgf000119_0004
K(CH (CIH" and G represents
A;
N-R1
X .
or a pharmaceutically acceptable salt thereof.
2. A compound ofclaim 1 wherein Y1 represents H or CM alkyl.
3. A compound ofclaim 1 wherein Y1 represents H.
4. A compound ofclaim 1 wherein Y2 is selected from
C ι-6 alkyl, C3-6 cycloalkyl optionally substituted by C]- alkyl, phenyl optionally substituted up to three times by halogen, G alkyl, or
C1-4 alkoxy, adamantyl, CF3, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C1-6 alkyl, and Me Me
X N
and Y3 is H; or Y2 and Y3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C alkyl, or C1- alkoxy.
5. A compound of claim 1 wherein Y2 is selected from C ι-6 alkyl,
C -6 cycloalkyl optionally substituted by CM alkyl, phenyl optionally substituted up to three times by halogen, C1-4 alkyl, or
C1-4 alkoxy, adamantyl, and a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C1-6 alkyl, and Y3 is H.
6. A compound of claim 1 wherein Y2 is selected from
C ι-6 alkyl, and
C3-6 cycloalkyl optionally substituted by CM alkyl, and Y3 is H.
7. A compound of claim 1 wherein
X represents Cl, F, or C alkyl; and n represents 0, 1, or 2.
8. A compound of claim 1 wherein X represents F; and n represents 0 or 1.
9. A compound ofclaim 1 wherein R1 represents H and R »2 represents H.
10. A compound ofclaim 1 wherein R3 represents C1-6 alkyl, halogen, C M alkoxy, CN, or
N(R')2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'),
N(R*), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; and p represents 0, 1, or 2.
11. A compound of claim 1 wherein R3 represents
-6 alkyl, C M alkoxy, CN, or N(R')2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R!), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; and p represents 0 or 1.
12. A compound ofclaim 1 wherein R3 represents
Ci-6 alkyl, C ι-4 alkoxy, or N(R wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; and p represents 0 or 1.
13. A compound ofclaim 1 wherein L is selected from
-O-(CH2)M- , -S(O)0-2-(CH2)M- , -N(R1)-(CH2)M- ,
Figure imgf000123_0001
-(CH2)M-O-(CH2)M- , -(CH2)1-4- , or
-N(R')-C(O)-(CH2)1-4- , and G represents NR5R6 , wherein
R5 represents H or Cι-6 alkyl; and
R6 represents
-6 alkyl, CM alkoxy-substituted CM alkyl,
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C alkyl, or C1-4 alkoxy, C -6 cycloalkyl-substituted CM alkyl, benzyl, phenyl optionally substituted by halogen, CM alkyl,
CM alkoxy, -CO2R', -C^^R^Z , -N(R*)2 , or by a
bivalent group
Figure imgf000123_0002
, 0r wherein
A is N(R'), S, S(O), S(O)2, or O , and said bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-membered heterocycle, or
Figure imgf000124_0001
, wherein A2 represents N(R!), S, S(O), S(O)2 , or O,
Figure imgf000124_0002
, optionally substituted up to 2 times by Cι-3 alkyl, (C1- alkoxy)(C1-4 alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl,
j-N A3 \/ , optionally substituted up to 2 times by Cι-3 alkyl, (C1-3 alkoxy)(C1-4 alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl, and wherein A3 represents N(R'), S, S(O), S(O)2 , or O,
Figure imgf000124_0003
, optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(C1-4 alkyl), C(O)OR', C(0)N(R')2 , phenyl, or benzyl, or up to 4 times by C,.3 alkyl,
Figure imgf000124_0004
, optionally substituted up to 2 times by halogen, C1-4 alkyl, or C1-4 alkoxy, and wherein v is 0 or 1. 14. A compound ofclaim 1 wherein L is selected from -O-(CH2)M- ,
-S(O)O-2-(CH2)M- , -N(R')-(CH2)M- , -(CH2)M-O-(CH2)I-4- , and -N(R')-C(O)-(CH2)M- , and G represents NR5R6 , wherein
R5 represents H or C1-6 alkyl; and R6 represents Ci-6 alkyl,
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C alkyl, or C alkoxy, benzyl, phenyl optionally substituted by halogen, CM alkyl, C1-4 alkoxy, -CO2R', -C(O)N(R1)2 , -NCR1^ , or by a
bivalent group N , or
Figure imgf000125_0001
wherein
A is NCR1), S, S(O), S(O)2, or O , and said bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-membered heterocycle, or
Figure imgf000125_0002
wherein A2 represents N(R!), S, S(O), S(O)2 , or O,
Figure imgf000125_0003
, optionally substituted up to 2 times by C1-3 alkyl, (C1-3 alkoxy)(C1-4 alkyl),
Figure imgf000125_0004
C(0)N(R')2 , phenyl, or benzyl,
-N \ — / , optionally substituted up to 2 times by Cι-3 alkyl, (C1-3 alkoxy)(CM alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl, and wherein A3 represents NCR1), S, S(O), S(O)2 , or O,
Figure imgf000125_0005
, optionally substituted up to 2 times by oxo, (Cj-3 alkoxy)-(CM alkyl),
Figure imgf000125_0006
C(0)N(R')2 , phenyl, or benzyl, or up to 4 times by Ci-3 alkyl,
Figure imgf000126_0001
optionally substituted up to 2 times by halogen, C alkyl, or C1-4 alkoxy, and wherein v is 0 or 1, or
L represents
Figure imgf000126_0002
G represents
1 N-R1
X
15. A compound of claim 1 wherein L is selected from
-O-(CH2)1-4- , -N(R')-(CH2)M- , -(CH2)M-O-(CH2)M- , and
G represents
NR5R6 , wherein
R5 represents H or C1-6 alkyl; and R6 represents
C1-6 alkyl, or
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C1-4 alkyl, or C1-4 alkoxy,
Figure imgf000126_0003
, optionally substituted up to 2 times by .3 alkyl, (C1-3 alkoxy)(C1-4 alkyl), C(O)OR' , C(O)N(R')2 , phenyl, or benzyl,
Figure imgf000127_0001
, optionally substituted up to 2 times by Cι-3 alkyl, (C1- alkoxy)(C1-4 alkyl), C(O)OR', C(0)N(R')2 , phenyl, or benzyl, and wherein A3 represents N(R!), S, S(O), S(O) , or O,
Figure imgf000127_0002
o-i, optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(C1-4 alkyl), C(O)OR*, C(O)N(R1)2 , phenyl, or benzyl, or up to 4 times by Cι-3 alkyl,
Figure imgf000127_0003
optionally substituted up to 2 times by halogen, C alkyl, or C - alkoxy, and wherein v is 0 or 1. 16. A compound of claim 1 wherein Y1 represents H or C1-4 alkyl; Y2 is selected from C ι.6 alkyl,
C -6 cycloalkyl optionally substituted by CM alkyl, phenyl optionally substituted up to three times by halogen, C alkyl, or
CM alkoxy, adamantyl, CF3, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or Cι-6 alkyl, and
Figure imgf000127_0004
Y3 is H; or Y and Y are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, C1-4 alkyl, or C1- alkoxy, X represents Cl, F, or CM alkyl; n represents 0, 1, or 2;
1
R and R each represents H; R3 represents C1-6 alkyl, halogen, C M alkoxy, CN, or
N(R')2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'),
N(R!), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; p represents 0, 1, or 2; q is O; L is a linker selected from
-O-(CH2)1-4- , -S(O)0-2-(CH2)1-4- , -N(R')-(CH2)M- ,
Figure imgf000128_0001
-(CH2)M- , and -N(R1)-C(O)-(CH2)M- , and G represents
NR5R6 , wherein
R5 represents H or Cι-6 alkyl; and R6 represents
Ci-6 alkyl,
CM alkoxy-substituted CM alkyl, C5-6 cycloalkyl optionally substituted up to 2 times by halogen, Ci-4 alkyl, or G alkoxy,
C3-6 cycloalkyl-substituted CM alkyl, benzyl, phenyl optionally substituted by halogen, CM alkyl,
C1-4 alkoxy,
Figure imgf000129_0001
-C(O)N(R1)2 , -N(R!)2 , or by a
bivalent group N 7> , 0r
Figure imgf000129_0002
wherein
A is NCR1), S, SCO), S(O)2, or O , and said bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-membered heterocycle, or
r l
Figure imgf000129_0003
represent .s x Nτ(/R'>), S, SCO), S(O)2 , or O,
Figure imgf000129_0004
, optionally substituted up to 2 times by C)-3 alkyl, (Cι-3 alkoxy)(CM alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl,
Hsr A3 \ — , optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(C1-4 alkyl), C(O)OR', C(O)N(R1)2 , phenyl, or benzyl, and wherein A3 represents N(R'), S, S(O), S(O)2 , or O,
Figure imgf000129_0005
, optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(CM alkyl), C(O)OR1, C(O)N(R')2 , phenyl, or benzyl, or up to 4 times by
C,.3 alkyl,
Figure imgf000129_0006
, optionally substituted up to 2 times by halogen, C1- alkyl, or C alkoxy, and wherein v is 0 or 1.
7. A compound of claim 1 wherein Y1 represents H; Y is selected from C ι-6 alkyl,
C3-6 cycloalkyl optionally substituted by CM alkyl, phenyl optionally substituted up to three times by halogen, C1-4 alkyl, or
Ci-4 alkoxy, adamantyl, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or Cι-6 alkyl, Y3 is H; X represents F; n represents 0 or 1 ;
R1 and R2 each represents H; R3 represents
Ci-6 alkyl, C alkoxy, CN, or
N(R*)2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'), N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; p represents 0 or 1 ; q is O;
L is a linker selected from
Figure imgf000130_0001
-S(O)0.2-(CH2)M- , -N(R1)-(CH2)1-4- ,
-(CH2)M-O-(CH2)I-4- , and -N(R')-C(O)-(CH2)M- , and G represents
NR5R6 , wherein
R5 represents H or Cι-6 alkyl; and R6 represents
Ci-6 alkyl,
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, Ci-4 alkyl, or CM alkoxy, benzyl, phenyl optionally substituted by halogen, CM alkyl,
CM alkoxy, -CO2R', -C(O)NCR1)2 , -N(R')2 , or by a
bivalent group N , or
Figure imgf000131_0001
wherein
A is NCR1), S, S(O), S(O)2, or O , and said bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-membered heterocycle, or
Figure imgf000131_0002
, wherein A2 represents NCR1), S, S(O), S(O)2 , or O,
Figure imgf000131_0003
, optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(C alkyl), C(O)OR1, C(O)N(R1)2 , phenyl, or benzyl,
|-N A3
^ — , optionally substituted up to 2 times by Cι- alkyl, (Cι-3 alkoxy)(CM alkyl), C(O)OR', C(0)N(R')2 , phenyl, or benzyl, and wherein A3 represents N(R!), S, S(O), S(O)2 , or O,
Figure imgf000131_0004
o-i , optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(CM alkyl), C(O)OR", C(O)N(R1)2 , phenyl, or benzyl, or up to 4 times by
CM alkyl,
Figure imgf000132_0001
, optionally substituted up to 2 times by halogen, C alkyl, or Ci-4 alkoxy, and wherein v is 0 or 1 ,
or
L represents i /(CH2)-
-O-< 1-3
(CH2)- 2 2-3 and
G represents
N-R1
X
18. A compound ofclaim 1 wherein Y1 represents H; Y2 is selected from C ι-6 alkyl, and
C3-6 cycloalkyl optionally substituted by CM alkyl, Y3 is H; X represents F; n represents 0 or 1 ; R1 and R2 each represents H;
R3 represents
Ci-6 alkyl, C alkoxy, or
N(R')2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R'),
N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; p represents 0 or 1 ; q is O;
L is a linker selected from
-O-(CH2-4- ,
-N(R')-(CH2)M- ,
-(CH2)1-4-O-(CH2)1-4- , and
G represents
NR5R6 , wherein
R5 represents H or Cι-6 alkyl; and
R6 represents
Ci-6 alkyl, or
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C1-4 alkyl, or G-4 alkoxy,
Figure imgf000133_0001
optionally substituted up to 2 times by Cι- alkyl, (C]-3 alkoxy)(C alkyl), C(O)OR', C(0)N(R')2 , phenyl, or benzyl,
|-N A3
\ — , optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(C alkyl), C(O)OR1, C(O)N(R1)2 , phenyl, or benzyl, and wherein A3 represents NCR1), S, S(O), S(O)2 , or O,
Figure imgf000133_0002
optionally substituted up to 2 times by oxo, (Cι-3 alkoxy)-(CM alkyl), C(O)OR1, C(O)N(R*)2 , phenyl, or benzyl, or up to 4 times by C1-3 alkyl,
Figure imgf000133_0003
, optionally substituted up to 2 times by halogen, C alkyl, or G-4 alkoxy, and wherein v is O or l.
19. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.
20. A method of inhibiting Src kinase receptors in a subject comprising contacting said receptors with the compound according to claim 1.
21. A method for treating a disease associated with a src kinase in a subject, comprising administering to said subject a therapeutically effective amount of a compound according to claim 1, such that the disease is treated.
22. The method of claim 21 wherein said disease is cancer or osteoporosis.
23. A method for treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of a compound according to claim 1 , such that the cancer is treated.
24. The method of claim 23, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, lung cancer, neural cancer, esophageal cancer, gastric cancer, melanoma and Kaposi's sarcoma.
25. A method for treating a non-malignant proliferative disease in a subject, comprising administering to said subject a therapeutically effective amount of a compound according to claim 1 , such that the non-malignant proliferative disease is treated.
26. A method for treating osteoporosis in a subject, comprising administering to said subject a therapeutically effective amount of a compound according to claim 1, such that the osteoporosis is treated.
27. A method for preparing a compound of formula (I)
Figure imgf000135_0001
wherein
Y r l represents H, Cμ alkyl, or phenyl optionally substituted up to three times by halogen, CM alkyl, or C]- alkoxy; Y and Y are independently selected from
H,
C ι-6 alkyl,
C3-6 cycloalkyl optionally substituted by C alkyl, phenyl optionally substituted up to three times by halogen, C1- alkyl, or C alkoxy, adamantyl,
CF3, a 5-6 membered heteroaromatic containing up to two heteroatoms selected from N, O, and S, and optionally substituted up to two times by halogen or C i -6 alkyl,
C(O)N(C1-4 alkyl)2 ,
C(O)O(C1-4 alkyl) ,
CN, halogen, and
Figure imgf000135_0002
or
Y τ-2 and A v Y3 are joined and together represent a fused aromatic ring optionally substituted up to two times by halogen, CM alkyl, or G-4 alkoxy, X represents halogen or Cι-4 alkyl; n represents 0, 1, or 2; R1 represents H or CM alkyl; R2 represents H or CM alkyl; R3 represents
Ci-6 alkyl, halogen, C M alkoxy,
O-phenyl optionally substituted up to two times by halogen, G-4 alkyl, CM alkoxy, or di-(G- alkyl)amino,
CN,
N(R^2 wherein the R1 moieties are independent, or the R1 moieties optionally are joined by a linker selected from the group consisting of CH(R ),
N(R'), S, S(O), S(O)2, and O, and taken together with the N to which they are attached, form a 5-6 membered nonaromatic heterocycle; p represents 0, 1, or 2; R4 represents CM alkyl or halogen; q represents 0, 1, or 2; or
R3 and R may be joined and taken together with the carbon atoms to which they are attached, form a 5-6 membered heteroaromatic ring containing up to two heteroatoms selected from N, O, and S, and which is optionally substituted up to two times by halogen, G-4 alkyl, or C1-4 alkoxy, L is a linker selected from
Figure imgf000136_0001
-S(O)0.2-(CH2)M- ,
-N(R1)-(CH2)M-
Figure imgf000136_0002
-(CH2)1-4-O-(CH2-4- , -(CH2)ι-4- , -C≡C-(CH2),.4- or
-N(R')-C(O)-(CH2)M- , and G represents NR5R6 , wherein
R5 represents H, Cι_6 alkyl, or C]-4 alkoxy-CM alkyl; and R6 represents H, Cι-6 alkyl,
CM alkoxy-substituted CM alkyl,
C5-6 cycloalkyl optionally substituted up to 2 times by halogen, C alkyl, or G-4 alkoxy, C3-6 cycloalkyl-substituted CM alkyl, benzyl, phenyl optionally substituted by halogen, CM alkyl, CM alkoxy,
Figure imgf000137_0001
-C(O)N(R1)2 , -N(R')2 , or by a
bivalent group N , 0r
Figure imgf000137_0002
wherein
A is N(R'), S, S(O), S(O)2, or O , and said bivalent group is connected to the phenyl ring at adjacent carbon atoms to form a fused 5-membered heterocycle,
-(C^ alkylM^A1 — , wherein A represents N(R ), S, S(O),
S(O)2 , or O, or
Figure imgf000137_0003
> wherein A2 represents N(R'), S, S(O), S(O)2 , or
O,
Figure imgf000138_0001
, optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alkoxy)(C1-4 alkyl), C(O)OR', C(0)N(R')2 , phenyl, or benzyl,
\ 3
-N A3
, optionally substituted up to 2 times by Cι-3 alkyl, (Cι-3 alk ooxxyy))((CC aallkkyyll)),, CC((OO))OORR'',, CC((OO))NN((RR11))22 ,, pphheennyl, or benzyl, and wherein A3 represents N(R'), S, S(O), S(O)2 , or O,
Figure imgf000138_0002
optionally substituted up to 2 times by oxo, (Cι- alkoxy)-(CM alkyl), C(O)OR1, C(O)N(R')2 , phenyl, or benzyl, or up to 4 times by
C,-3 alkyl,
Figure imgf000138_0003
, optionally substituted up to 2 times by halogen, G-4 alkyl, or G-4 alkoxy, and wherein v is 0 or 1 , and
-N. ,. optionally substituted up to 2 times by CM alkyl,
or
L represents i κ(CH2)-
-O-XX ι-3 (CH2)-
2-3 or
Figure imgf000138_0004
G represents
N-R1 compnsmg coupling a compound of formula (II)
Figure imgf000139_0001
with a compound of formula (III)
Figure imgf000139_0002
is an alkyl group, alkylating the resulting secondary amine, to yield a compound of formula (I); or coupling a compound of formula (IV)
Figure imgf000139_0003
with a compound of formula (V)
Figure imgf000139_0004
i P (v) and, in the case where R is an alkyl group, alkylating the resulting secondary amine, to yield a compound of formula (I).
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