WO2011106168A1 - Purine compounds for treating autoimmune and demyelinating diseases - Google Patents

Purine compounds for treating autoimmune and demyelinating diseases Download PDF

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Publication number
WO2011106168A1
WO2011106168A1 PCT/US2011/024404 US2011024404W WO2011106168A1 WO 2011106168 A1 WO2011106168 A1 WO 2011106168A1 US 2011024404 W US2011024404 W US 2011024404W WO 2011106168 A1 WO2011106168 A1 WO 2011106168A1
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Prior art keywords
purin
indazol
amine
methyl
purine
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PCT/US2011/024404
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French (fr)
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Alfred M. Ajami
Kenneth W. Duncan
Xinqin Fang
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Dcam Pharma Inc
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Publication of WO2011106168A1 publication Critical patent/WO2011106168A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D473/00Heterocyclic compounds containing purine ring systems
    • C07D473/26Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/28Oxygen atom
    • C07D473/30Oxygen atom attached in position 6, e.g. hypoxanthine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders
    • A61P37/02Immunomodulators
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D473/00Heterocyclic compounds containing purine ring systems
    • C07D473/26Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/28Oxygen atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D473/00Heterocyclic compounds containing purine ring systems
    • C07D473/26Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32Nitrogen atom
    • C07D473/34Nitrogen atom attached in position 6, e.g. adenine
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D473/00Heterocyclic compounds containing purine ring systems
    • C07D473/26Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/36Sulfur atom

Definitions

  • DC dendritic cells
  • macrophages and microglia are the homeostatic regulators of the immune system (Liu et al, Science 324:392-397, 2009; Merad and Manz, Blood 113:3418-3427, 2009). Their functions straddle both the initial priming of the immune response, with its attendant burst of inflammatory signaling, and the chronic effector stages of tissue destruction. They also have the capacity to counteract disease by inducing tolerance and permitting tissue repair (Shklovskaya and de St. Groth, Methods Mol. Biol. 380:25-46, 2007). In effect, DCs and their descendant lineages become therapeutic targets because they are hubs in a dysregulated immune system. DCs have the potential to activate autoreactive lymphocytes and secrete cytokines that further influence the status of those lymphocytes.
  • dendritic cells as the cellular target, i.e. mode of action
  • RTKs as the molecular target, i.e. mechanism of action
  • RTK inhibition have been documented in multiple studies, and can be viewed as setting the paradigms for understanding all such immune system diseases sharing similar cellular and molecular etiologies.
  • Multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease are particularly illustrative in this respect, but the same phenomenology applies to psoriasis, graft- versus-host-disease, and other autoimmune diseases.
  • the present invention is directed to a compound of Structural Formula (I):
  • R and R each independently are hydrogen or -L'-R 0 ;
  • R 4 and R 5 taken together with the nitrogen atom to which they are attached, form a 5-membered heteroaryl Ar 2 , optionally including one or two additional heteroatoms selected from N, S or O;
  • Figure 2C is a plot of dendritic cells proliferation as a function of time in the presence of a representative compound of the present invention as well as controls, where the cells counted are limited to the CD 1 lc+CDl lb+ phenotype.
  • Figure 10 is a plot showing time course of body weight change of DBA/1 mice with in the Collagen -Induced Arthritis (CIA) animal model in the presence of representative compounds of the present invention as well as controls.
  • CIA Collagen -Induced Arthritis
  • Figure 12B is a plot showing Mean Clinical Arthritis Score as a function of time for "Non-enrollment Paws" in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as control and comparator methotrexate.
  • Figure 15 shows a histogram of a quantitative measurement of pannus in a joint of animal in the Collagen-Induced Arthritis (CIA) model in the presence of representative compounds of the present invention as well as controls.
  • CIA Collagen-Induced Arthritis
  • Figure 23 is a histogram showing inflammatory score in C57BL/6 mice in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of a representative compound of the present invention as well as controls.
  • DSS dextran sodium sulfate
  • Figure 24 is a histogram showing percent weight loss in C57BL/6 mice in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of representative compounds of the present invention as well as control.
  • DSS dextran sodium sulfate
  • Figure 28 shows two histograms, each histogram representing the amount of a representative biomarker (IL-10, IL-27) determined by qPCR of cells from mesenteric lymph nodes (MLN) collected from a mouse in the dextran sodium sulfate (DSS)- induced murine colitis model, at Day 12 in the presence of representative compounds of the present invention as well as control.
  • IL-10 mesenteric lymph nodes
  • DSS dextran sodium sulfate
  • Embodiments of the present invention provide compounds that can modulate kinase activity, modulate dendritic cell maturation and activation, and can treat or alleviate the symptoms of various autoimmune diseases.
  • a 1 is selected from H, hydroxyl, -C(0)NH 2 , or -C(0)NH(C1-C3)alkyl; and A 2 is selected from H, hydroxyl, or a C1-C6 alkyl, provided that A 1 and A 2 are not simultaneously hydrogens.
  • R and R each independently are hydrogen or -L -R .
  • R and R 3 each independently are hydrogen, phenyl, pyridyl, or benzyl, the phenyl, pyridyl or benzyl being optionally substituted with one to three substituents independently selected from a halogen, C1-C3 alkyl, -CF 3 , hydroxyl, and a (Cl-C3)alkoxy.
  • R 6 is hydrogen, C1-C4 alkyl, or Ar 1 .
  • R 6 is hydrogen, methyl, ethyl, pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, or furyl, the pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, or furyl group, optionally substituted with one to three substituents independently selected from a halogen, C1-C3 alkyl, -CF 3 , hydroxyl, and a (Cl-C3)alkoxy.
  • a C1-C6 alkyl a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl,
  • Q 2 is O, CH 2 , NH, or NR 100
  • R 100 is -C(0)OEt or a C1-C6 alkyl optionally substituted with -CN, -OH, phenyl, or cyclopropyl.
  • the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof.
  • Ar is substituted with one to three substituents R selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two groups R taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
  • the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof.
  • a and A each independently are a C1-C6 alkoxy, optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
  • the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof.
  • R 4 is H or C 1-6 alkyl
  • R 5 is -L 2 -R 7
  • R 7 is a 5- to 6-membered aryl or heteroaryl Ar 3 ;
  • Ar 3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl
  • y is 1 ; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
  • the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof.
  • R 4 is H, y is 2, Ar 3 is phenyl and R K is H or methyl; and the optional substituent R° is, for each occurrence independently, selected from a halogen and a C1-C6 alkoxy group, or, alternatively, two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
  • the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof.
  • R 5 is L 2 -R 7 ; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
  • the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof.
  • R 5 is L 2 -R 7 ;
  • R 7 is -NR M R N ;
  • R M and R N taken together with the nitrogen to which they are attached, form a 5- to 7-membered heterocyclyl C , and further wherein C 3 isselected from the group consisting of
  • the compound of the present invention is represented by Structural For pharmaceutically acceptable
  • the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof.
  • R 1 is NR 4 R 5 ;
  • a 1 is hydroxyl, a C1-C6 alkoxy or H; and
  • a 2 is selected from hydroxyl, a C1-C6 alkoxy, C 1-6 alkyl, C 1-6 haloalkyl, and H;
  • R 4 and R 5 taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclyl C , selected form the roup consisting of
  • the compound of the present invention is represented by Structural Formula (IC), or a pharmaceutically acceptable salt thereof:
  • alkyl groups examples include methyl (Me), ethyl (Et), propyl (e.g., n-propyl, and isopropyl,), butyl (e.g., n- butyl, isobutyl, s-butyl, t-butyl), pentyl groups (e.g., n-pentyl, isopentyl, neopentyl), and the like.
  • a lower alkyl group typically has up to 6 carbon atoms.
  • an alkyl group has 1 to 6 carbon atoms, and is referred to as a "Ci -6 alkyl group", “C1-C6 alkyl group”, or (Cl-C6)alkyl group.
  • C 1-6 alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl and isopropyl), and butyl groups (e.g., n-butyl, isobutyl, s-butyl, t-butyl).
  • a branched alkyl group has at least 3 carbon atoms (e.g., an isopropyl group) and up to 6 carbon atoms, e.g.
  • Substituents on alkyl groups include hydroxyl, oxo, amino, cyano, (Cl-C3)alkylamino, di(Cl-C3)alkylamino, halogen (typically, F, CI, and Br), (Cl-C3)alkoxy, (C3-C6)cycloalkyl, a 5- to 6- membered aryl or heteroaryl, -C(0)OR G2 or -C(0)NR H1 R J1 , wherein R G2 is hydrogen or (Cl-C4)alkyl, and R H1 and R J1 each independently are hydrogen or a (Cl-C3)alkyl.
  • alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like.
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene).
  • a branched alkenyl group has at least 3 carbon atoms, and in various embodiments, has up to 6 carbon atoms, e.g. it is a C 3- 6 alkenyl or (C3-C6)alkenyl group.
  • heteroatom refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, sulfur, phosphorus, and selenium.
  • non-aromatic heterocyclic group or “heterocyclyl” refers to a non-aromatic cycle having 5-7 ring atoms (unless the number of “ring atoms” or “members” is specifically provided), among which 1 to 3 ring atoms (unless specifically provided) are heteroatoms independently selected from oxygen (O), nitrogen (N) and sulfur (S) (unless specifically provided), and that optionally contains one or more, e.g., two, double or triple bonds.
  • R F is hydroxyl, cyano, a C1-C3 alkyl, a C3-C6 cycloalkyl, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, a C1-C3 alkoxy, a 5- to 6- membered heteroaryl or phenyl, -C(0)OR G2 or -C(0)NR H1 R J1 ;
  • R G , R G1 , and R G2 are each independently hydrogen or a C1-C4 alkyl; and
  • R H , R J , R HI , R J1 each independently hydrogen or a C1-C4 alkyl; and R H , R J , R HI , R J1 each
  • Heterocyclyl groups (and, particularly, C 3 ) can further be substituted with a group R 100 selected from a CI- C6 alkyl, a C3-C6 cycloalkyl, phenyl, cyano, hydroxyl and -C(0)OR lul , wherein R 1U1 is methyl or ethyl.
  • heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain 0-0, S-S, or S-0 bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S, S -dioxide).
  • heteroaryl rings include, but are not limited to, pyrrole, furan, thiophene, pyridine, pyrimidine, pyridazine, pyrazine, triazole, tetrazole, pyrazole, imidazole, isothiazole, thiazole, thiadiazole, isoxazole, oxazole, and oxadiazole.
  • the heteroaryl ring may be attached at C-2, C-3, or C-4; that is, be pyridin-2-yl, pyridine-3-yl, or pyridine-4-yl.
  • heteroaryl groups may be C-attached or N-attached (where such is possible).
  • a group derived from pyrrole may be pyrrol-l-yl (N-attached) or pyrrol-3-yl (C-attached).
  • Heteroaryl and aryl groups can further be substituted with one to three substituents R°, wherein each R° is independently selected from:
  • said C1-C6 alkoxy group is optionally substituted with a 5- or 6- membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy, or, alternatively,
  • a “protecting group” refers to modification of a functional group that reduces the reactivity of the functional group in an unwanted reaction.
  • protecting groups for amines include, but are not limited to, tert-butyloxycarbonyl (t-BOC), benzyl (Bn), and carbobenzyloxy (Cbz) groups.
  • C 1-6 alkyl is specifically intended to individually disclose Q, C 2 , C 3 , C 4 , C 5 , C 6 , Ci-C6, Q-C5, Cj-C 4 , C1-C3, C C2, C 2 -C 6 , C 2 -Cs, C 2 -C 4 , C 2 -C 3 , C 3 - C 6 , C3-C5, C 3 -C , C 4 -C 6 , C -C 5 , and C 5 -C 6 alkyl.
  • the term "5-9 membered heteroaryl group” is specifically intended to individually disclose a heteroaryl group having 5, 6, 7, 8, 9, 5-9, 5-8, 5-7, 5-6, 6-9, 6- 8, 6-7, 7-9, 7-8, and 8- 9 ring atoms.
  • Representative compounds of Formula (I) in accordance with embodiments of the present invention include, but are not limited to, the compounds presented in Table 1 below. Table 1
  • step d acetic anhydride and triethylorthoformate
  • Invitrogen, Inc. (Carlsbad, CA, USA). Animal models for various diseases, including autoimmune diseases, are known in the art, and include, for example, Collagen- Induced Arthritis (CIA) and Collagen Antibody-Induced Arthritis (CAIA) for rheumatoid arthritis (RA), Experimental Autoimmune Encephalomyelitis (EAE) for multiple sclerosis (MS), dextran sodium sulfate (DSS)-induced murine colitis for inflammatory bowel disease (IBD) and 2,4,6-trinitrobenzene sulfonic acid (TNBS)- induced murine colitis for Crohn's Disease, Ova-Induced- Asthma for chronic obstructive pulmonary disease (COPD) or asthma, and granuloma and air pouch models for inflammation, among others.
  • CIA Collagen- Induced Arthritis
  • CAIA Collagen Antibody-Induced Arthritis
  • EAE Experimental Autoi
  • RTKs such as Flt3, CSF-1R, ACVR1 (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRK1 (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), and RIPK2, and ROS1.
  • RTKs such as Flt3, CSF-1R, ACVR1 (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRK1 (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), and RIPK2, and ROS1.
  • Flt3 has been linked to the negative regulation of IL-10, a principal regulator of the immunogenic-tolerogenic balance toward tolerogenic Th2 and T-reg biology, wherein silencing Flt3 downregulates T-cell reactivity and T-reg signaling by upregulation of IL-10 (see, Astier et al, J.Immunology, 184:685 -693, 2010).
  • IL-10 has a role in many diseases such as IBD, MS, and asthma, among others.
  • IL-10-deficiency in mice leads to the development of colitis while IL- 10 treatments have been shown effective in inhibiting the development of type I diabetes and EAE.
  • IL-10 may reduce Thl7 and macrophages that cause joint and bone erosion, while having only a limited impact on reducing joint inflammation.
  • Compounds of the present invention can be used to modulate production of IL- 10.
  • compounds of Formula (I) can modulate the activity of the following kinases: Flt3, CSF-IR, ACVRl (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRKl (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), ROS1, and RIPK2.
  • a variety of pathological conditions, states, disorders or diseases, including autoimmune diseases, can be treated by modulating the activity of one or more of these kinases.
  • Compounds of Formula (I) can be used in the treatment of disorders mediated by one or more kinase selected from Flt3, CSF-IR, ACVRl (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRKl (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), ROS1, and RIPK2.
  • kinase selected from Flt3, CSF-IR, ACVRl (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRKl (TRKA), NTR
  • ROS1 has been shown to be activated in gliobastomas and astrocytomas.
  • disorders mediated by one or more of these kinases are disorders in which one or more symptoms can be inhibited, alleviated, reduced or whose onset can be delayed by completely or partially inhibiting the protein kinase.
  • the RTK is Flt3 or CSF-IR
  • compounds of the present invention are useful in the treatment of a Flt3-mediated disorder or a CSF-1R- mediated disorder.
  • Flt3 -mediated disorder and “CSF-lR-mediated disorder” refer to disorders in which one or more symptoms can be inhibited, alleviated, reduced or whose onset can be delayed by completely or partially inhibiting the protein kinase Flt3 or the protein kinase CSF-IR, respectively.
  • Flt3-mediated disorders and conditions include autoimmune disorders such as ankylosing spondylitis, arthritis, aplastic anemia, Behcet's disease, type 1 diabetes mellitus, graft-versus-host disease, Graves' disease, autoimmune hemolytic anemia, Wegener's granulomatosis, hyper IgE syndrome, idiopathic thrombocytopenia purpura, rheumatoid arthritis, Crohn's disease, multiple sclerosis, Myasthenia gravis, psoriasis, and lupus, transverse myelitis, and amyotrophic lateral sclerosis; neurodegenerative diseases such as infantile spinal muscular atrophy and juvenile spinal muscular atrophy, Creutzfeldt- Jakob disease; and subacute sclerosing panencephalitis; and cancers such as leukemia including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocy
  • CSF-lR-mediated disorders and conditions include autoimmune disorders such as sarcoidosis, asthma, psoriasis, diabetes, Sjogren's syndrom, and uveitis;
  • cardiovascular disease involving chronic inflammation (e.g. atherosclerosis ); cancers such as osteolytic sarcoma, myeloma, breast cancer, tumor metastasis to bone, uterine cancer, stomach cancer, and hairy cell leukemia; diseases with an inflammatory component including glomerulonephritis, prosthesis failure, congestive obstructive pulmonary disease, pancreatitis, HIV infection, tumor related angiogenesis, age- related macular degeneration, diabetic retinopathy, restenosis, schizophrenia, skeletal pain caused by tumor metastasis or osteoarthritis, or visceral, inflammatory, and neurogenic pain, osteoporosis, Paget's disease, and prosthesis failure.
  • diseases with an inflammatory component including glomerulonephritis, prosthesis failure, congestive obstructive pulmonary disease, pancreatitis, HIV infection, tumor related angiogenesis, age- related macular degeneration, diabetic retinopathy, restenosis, schizophrenia, skeletal pain caused by tumor met
  • the present invention is a method of treating a patient suffering from an autoimmune disease by administering to a patient suffering from such disorders a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
  • IBD inflammatory bowel disease
  • IBD Crohn's disease and ulcerative colitis
  • UC primarily affects the mucosal lining of the large intestine and rectum, while Crohn's disease may involve any part of the GI tract.
  • Extraintestinal manifestations are often associated with IBD, and include inflammatory conditions such as peripheral arthritis, ankylosing spondylitis, sacroiliitis, uveitis, and primary sclerosing cholangitis, among others.
  • treatment of IBD and symptoms thereof includes treatment of both intestinal and extraintestinal aspects.
  • preferred compounds useful for the treatment of a patient with IBD, UC, or Crohn's disease, or symptoms thereof are represented by Structural Formula(IAl), (IA2), (IB1) or (IB2).
  • compounds represented by Structural Formula (IA1) and (IB2) useful for the treatment of a patient with IBD, UC, or Crohn's disease , or symptoms thereof are Compounds 12, 36, 47, 129.
  • compounds of Formula (I) useful for the treatment of a patient with RA or psoriatic arthritis , or symptoms thereof have structure (IA1), (IA2), (IB2) or (IB1).
  • compounds of Formula (I) useful for the treatment of a patient with RA or psoriatic arthritis , or symptoms thereof are Compounds 5, 36, 12, 47, 50, 54, 100, and 129.
  • MS Multiple sclerosis
  • MS is characterized by patches of demyelination in the brain and spinal cord, and is an example of a demyelinating condition.
  • a demyelinating condition is a condition that destroys, breaks the integrity of or damages a myelin sheath, the insulating layer surrounding vertebrate peripheral neurons that increases the speed of conduction and formed by Schwann cells in the peripheral or by oligodendrocytes in the central nervous system.
  • demyelinating autoimmune conditions include Chronic Immune Demyelinating Polyneuropathy (CIDP); multifocal CIDP; inflammatory demylinating
  • compounds useful for the treatment of a patient with MS or demyelinating autoimmune conditions have structure (IA1) or (IB1).
  • compounds of Formula (IA1) and (IB1) useful for the treatment of a patient with MS or demyelinating autoimmune conditions are
  • compounds of Formula (I) can be used in the treatment of cancer.
  • cancer refers to the uncontrolled growth of abnormal cells that have mutated from normal tissues.
  • a cancerous tumor (malignancy) is of potentially unlimited growth and expands locally by invasion and systemically by metastasis.
  • cancers examples include: breast cancer, colorectal cancer, non-small cell lung cancer, ovarian, renal, sarcoma, melanoma, head & neck, hepatocellular, thyroid, leukemia, lymphoma, multiple myeloma, esophageal, large bowel, pancreatic, mesothelioma, carcinoma (e.g. adenocarcinoma, including esophageal adenocarcinoma), sarcoma (e.g.
  • the patient can be treated for certain leukemias, including Flt3 -mediated leukemias such as acute myeloid leukemia characterized by one or more Flt3 mutations.
  • Flt3 -mediated leukemias such as acute myeloid leukemia characterized by one or more Flt3 mutations.
  • Treating a subject suffering from cancer includes achieving, partially or substantially, one or more of the following: arresting the growth or spread of a cancer, reducing the extent of a cancer (e.g., reducing size of a tumor or reducing the number of affected sites), inhibiting the growth rate of a cancer, and ameliorating or improving a clinical symptom or indicator associated with a cancer (such as tissue or serum components).
  • the patient can be treated for bone metastases.
  • Bone is one of the most common metastatic sites for cancers, such as breast, lung and prostate cancers.
  • metastatic tumour cells in bone enhance bone resorption (osteolysis) by inducing and activating osteoclasts.
  • This in turn releases growth factors, such as TGF- ⁇ and IGF-1 from the bone matrix that promote tumour growth.
  • Osteoclasts are multinucleated cells formed by fusion of Flt3 -positive monocyte-macrophage cells, which can also differentiate to dendritic cells and macrophages. Therefore suppression of hyperactive osteoclasts (OCL), and thus osteolysis, may be effective against bone metastasis.
  • OCL hyperactive osteoclasts
  • Treating bone metastases refers to reducing (partially or completely) the size of the bone metastases, slowing the growth of the metastases relative to the absence of treatment and reducing the extent of further spread of the cancer, and also includes pain reduction, decreased incidents of fractures, relief of spinal cord compression, control of hypercalcaemia, and/or restoration of normal blood cell counts.
  • the method includes administering to a mammal a pharmaceutical composition that comprises a compound disclosed herein in combination or association with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition that comprises a compound disclosed herein in combination or association with a pharmaceutically acceptable carrier.
  • mammal refers to any warm blooded species, such as a human.
  • the compound of the present teachings can be administered alone or in combination with other therapeutically effective compounds or therapies for the treatment of such
  • an effective dosage can vary depending upon the particular compound utilized, the mode of administration, and severity of the condition being treated, as well as the various physical factors related to the individual being treated.
  • a compound of the present teachings can be provided to a patient already suffering from a disease in an amount sufficient to treat the symptoms of the disease and its complications.
  • the dosage to be used in the treatment of a specific individual typically must be subjectively determined by the attending physician.
  • the variables involved include the specific condition and its state as well as the size, age and response pattern of the patient.
  • pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable.
  • Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
  • Compounds of the present teachings can be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers.
  • Applicable solid carriers can include one or more substances which can also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents, or encapsulating materials.
  • the compounds can be formulated in conventional manner, for example, in a manner similar to that used for known anti-inflammatory agents.
  • Oral formulations containing an active compound disclosed herein can comprise any conventionally used oral form, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions.
  • the carrier can be a finely divided solid, which is an admixture with a finely divided active compound.
  • an active compound can be mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets can contain up to about 99% or greater of the active compound.
  • Surface modifying agents can include nonionic and anionic surface modifying agents.
  • Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine.
  • Oral formulations herein can utilize standard delay or time-release formulations to alter the absorption of the active compound(s).
  • the oral formulation can also consist of administering an active compound in water or fruit juice, containing appropriate solubilizers or emulisifiers as needed.
  • Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups, and elixirs.
  • An active compound described herein can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a mixture of both, or pharmaceutically acceptable oils or fats.
  • the liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, and osmo-regulators.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intrathecal, intramuscular,
  • compositions for oral administration can be in either liquid or solid form.
  • the pharmaceutical composition is in unit dosage form, for example, as tablets, capsules, powders, solutions, suspensions, emulsions, granules, or suppositories.
  • the pharmaceutical composition can be sub-divided in unit dose(s) containing appropriate quantities of the active compound.
  • the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids.
  • the unit dosage form can be a capsule or tablet itself, or it can comprise the appropriate number of any such compositions in package form.
  • Such unit dosage form may contain from about 1 mg/kg of active compound to about 500 mg/kg of active compound, and can be given in a single dose or in two or more doses.
  • Such doses can be administered in any manner useful in directing the active compound(s) to the recipient's bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, vaginally, and transdermally.
  • Such administrations can be carried out using the compounds of the present teachings including pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (e.g., rectal and vaginal).
  • the compounds of the present teachings can be formulated, for example, into an aqueous or partially aqueous solution.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations typically contain a preservative to inhibit the growth of microorganisms.
  • Compounds described herein can be administered transdermally, i.e., administered across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administration can be carried out using the compounds of the present teachings including pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (e.g., rectal and vaginal). Topical formulations that deliver active compound(s) through the epidermis can be useful for localized treatment of inflammation and arthritis.
  • Transdermal administration can be accomplished through the use of a transdermal patch containing an active compound and a carrier that can be inert to the active compound, can be non-toxic to the skin, and can allow delivery of the active compound for systemic absorption into the blood stream via the skin.
  • the carrier can take any number of forms such as creams and ointments, pastes, gels, and occlusive devices.
  • the creams and ointments can be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active compound can also be suitable.
  • occlusive devices can be used to release the active compound into the blood stream, such as a semi-permeable membrane covering a reservoir containing the active compound with or without a carrier, or a matrix containing the active compound.
  • Other occlusive devices are known in the literature.
  • Compounds described herein can be administered into a body cavity, (e.g., rectally or vaginally) in the form of a conventional suppository.
  • Suppository formulations can be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin.
  • Water-soluble suppository bases such as polyethylene glycols of various molecular weights, can also be used.
  • a compound can be desirable to combine a compound with other agents effective in the treatment of the target disease.
  • other active compounds i.e., other active ingredients or agents
  • active compounds of the present teachings can be administered with active compounds of the present teachings.
  • the other agents can be administered at the same time or at different times than the compounds disclosed herein.
  • Drugs useful in the treatment of IBD include 5-ASA compounds,
  • 5-ASA compounds include sulfasalazine and mesalamine.
  • Antibiotics include metronidazole and ciprofloxacin.
  • compounds of Formula (I) are used in combination with anti-inflammatory agents for the treatment of an autoimmune disease such as IBD, Crohn's disease, UC, or RA.
  • compositions of the present teachings also can consist essentially of, or consist of, the recited components, and that the processes of the present teachings also consist essentially of, or consist of, the recited processing steps.
  • an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
  • Compounds 1-142 can be prepared using the appropriate starting materials in accordance with the following examples. Selected compounds are shown in Table 3 below. It is understood by those skilled in the art of organic synthesis that the substitution patterns of the starting materials determines the substitution patterns of the products, and the skilled practitioner will be able to exercise routine judgment for the selection of suitable starting materials in order to prepare specific products, the order of synthetic steps, and the need for protecting groups for remote functionalities.
  • reaction mixture was filtered through celite and the filtrate concentrated under vacuum. The residue was treated with a small amount of water and the solid filtered off. Whilst moist, the solid was re-crystallized from water, the solid filtered off and dried under vacuum overnight to yield the desired product
  • the title compound was prepared according to the methods in Tanji, K.I. et al., Chem. Pharm. Bull. 35:4972-4976, 1987: 5-Amino-2,4-dichloropyrimidine (9.3 g, 57.1 mmol) and 5-aminoindazole (7.5 g, 57.1 mmol) were combined and dissolved in a mixture of ethanol (30 mL), water (200 mL) and concentrated HC1 (3.0 mL). The reaction mixture was then heated to 90 °C for 20 hours at which point no starting materials remained by t.l.c.
  • the reaction mixture was cooled and the precipitate filtered off washed with water (50 mL), saturated sodium bicarbonate solution (100 mL) and again with water (2 x 100 mL). The solid was dried under vacuum for 20 hours (10. 6g, 40.7 mmol, 71%).
  • the resulting solid was filtered off and washed with water (100 mL), saturated sodium bicarbonate solution (60 mL) and water (100 mL) before drying in a vacuum oven at 40 °C overnight.
  • the solid was shown by HPLC to be at least 95% pure and not purified further (10.6 g, 39.2 mmol, 94% yield).
  • Representative compounds of Formula (I) are screened for activity in several standard pharmacological test procedures. Based on the activity shown in the standard pharmacological test procedures, the compounds of the present teachings can be useful for treating chronic inflammatory and autoimmune diseases.
  • Symadex is used for comparison.
  • "Symadex” and “C-1311” are both names for 5-(2-(diethylamino)ethylamino)-8- hydroxy-6H-imidazo[4,5,l-de]acridin-6-one.
  • the use of Symadex as a Flt3 inhibitor and immune system modulator has been described previously by Ajami, A.M., Boss, M.A. and Paterson, J. in US Patent Appl. 2006/0189546A1.
  • EXAMPLE 1 DETERMINATION OF PROTEIN TYROSINE KINASE ACTIVITY TARGETING IN VITRO.
  • Compounds that are active in inhibition or modulation of Flt3 activity can be used to treat inflammatory and autoimmune diseases.
  • IC 50 the 50% inhibitory concentration
  • DR dose response
  • the most common approach exemplified by the Z-Lyte® assay is based on treating each specific kinase with a unique substrate and optical reporter system in the presence of ATP at apparent Km for each kinase, typically ranging from 5 to 500 ⁇ .
  • the biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non- phosphorylated peptides to proteolytic cleavage.
  • the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide.
  • a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides.
  • Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET- peptides maintain FRET.
  • a ratiometric method which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress versus a baseline response obtained in controls.

Abstract

A compound of Formula (I), or a pharmaceutically acceptable salt thereof. Values and preferred values of variables A1, A2 and R1 are provided herein.

Description

PURINE COMPOUNDS FOR TREATING AUTOIMMUNE AND
DEMYELINATING DISEASES
RELATED APPLICATION
This application claims the benefit of Provisional Application No. 61/307,515, filed February 24, 2010. The entire teachings of the above application are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
Autoimmune diseases are a significantly prevalent but poorly understood group of diseases in which an individual's immune system either 1) begins recognizing self antigens as foreign and starts destroying tissues expressing such antigens thereby causing a disease, or 2) forms immune complexes with these antigens which then deposit in tissues and cause inflammatory pathology. As such, autoimmune diseases represent an imbalance in one or more of the homeostatic mechanisms that regulate activity of the innate or acquired immune systems, tolerance to self-antigens vs. immunogenicity, and tissue repair vs. tissue destruction. Such autoimmune diseases include, for example, multiple sclerosis, wherein the target antigen is the myelin sheath protecting neurons leading to destruction of function of motor neurons; inflammatory bowel disease, presenting as Crohn's disease or ulcerative colitis, where the target is the endothelial lining of the gut mucosa;
rheumatoid arthritis, where the target organ is cartilage; psoriasis, where the target of the immune system is skin; graft-versus-host disease, where the target is the transplanted tissue or organ that becomes rejected; systemic lupus erythematosus, sarcoidosis, granulomatosis and vasculitis conditions which present as targeting a variety of tissues with no apparent specificity or selectivity, although the target antigens themselves are extremely consistent and characteristic; and diabetes wherein the immune system turns against and destroys insulin producing pancreatic islet cells.
Because the mechanisms leading to the development of autoimmune diseases in general are mostly unknown, their treatment is often directed to generally suppressing the immune system. Such general immunosuppressive therapies often cause a variety of undesirable side effects including cancer, infertility, and increased susceptibility to infections by viruses, fungi, yeast, and bacteria. Therefore, it would be desirable to harness the mechanisms that cause the immune system to turn against itself to enable development of more specific therapies (see, St. Clair, Curr. Opinion Immunol. 21 : 1-10, 2009).
One preferred mechanism involves modulation of dendritic cells (DC), and closely related cell types, including macrophages and microglia (Hackstein and Thomson, Nature Revs. Immunol. 4:24-34, 2004). These cells are the homeostatic regulators of the immune system (Liu et al, Science 324:392-397, 2009; Merad and Manz, Blood 113:3418-3427, 2009). Their functions straddle both the initial priming of the immune response, with its attendant burst of inflammatory signaling, and the chronic effector stages of tissue destruction. They also have the capacity to counteract disease by inducing tolerance and permitting tissue repair (Shklovskaya and de St. Groth, Methods Mol. Biol. 380:25-46, 2007). In effect, DCs and their descendant lineages become therapeutic targets because they are hubs in a dysregulated immune system. DCs have the potential to activate autoreactive lymphocytes and secrete cytokines that further influence the status of those lymphocytes.
DCs are the most potent antigen presenting cells (APC) endowed with the ability to stimulate and polarize naive T cells to either Thl or Th2 phenotypes. DCs also play a critical role in the maintenance of self tolerance by curtailing T cell responses directly or indirectly through the generation of T-regulatory cells (T-reg). The difference between DC subsets that stimulate and those that suppress immune responses seems to reside in the expression of co-stimulatory molecules and cytokines. The subset of DCs called tolerogenic DCs (Tol-DCs) have a distinct phenotype, suppress activation of conventional T cells and activate T-regulatory cells in an antigen-specific manner. Tol-DCs possess reduced expression of the co- stimulatory molecules, such as CD40, CD80 and CD86, and reduced ability to secrete T cell activating cytokines, such as interleukin-12 (IL-12), and chemotactic factors. Generally, IL-12 seems to stimulate Thl activation, whereas production of interleukin-10 (IL-10) by DCs stimulates Th2 activation and in some cases T-reg generation. Understanding this duality in function has already led to DC based immunotherapies, which have been used to potentiate T cell responses (in the case of cancer vaccines) or diminish them (in autoimmune disorders and transplantation). More relevant to this invention, the ability to affect the proportions of DCs at different stages of maturation or differentiation, and therefore to exert downstream regulatory effects upon the course of various immune diseases, provides a focal point for pharmacological intervention.
Coincident with this increased understanding about the role of DCs as immune modulators, it is now widely understood that DC development itself is susceptible to regulation by several classes of small molecule inhibitors of protein kinases ("PKs'Or "PK") (Gaestel et al., Nature Rev.. Drug Discov. 8:480-499, 2009). Flt3 inhibitors drugs in clinical development such as PKC-412, a Flt3 inhibitor, and SU11657, an oxindole Flt3 inhibitor, have been used to demonstrate the modulation of,
respectively, human CD34+ derived DCs (Weisel et al. Ann. Hematol. 88:203-21 1, 2009) and mouse DCs (Tussiwand et al. J. Immunol. 175:3674-3680, 2005).
PKs are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. An important subset of these kinases, the protein receptor tyrosine kinases ("RTKs" or "RTK"), act primarily as growth factor receptors and play a central role in the signal transduction pathways integrating a number of cellular functions, such as cell cycle, cell growth, cell differentiation and cell death. In a more specific context relevant to diseases of the immune system, RTKs provide a switching mechanism for the expansion,
proliferation, differentiation, activation and trafficking of both myeloid and lymphoid DCs. The Flt3 and CSF-1R receptors and their ligand growth factors, Flt3L and GM- CSF, are especially noteworthy in this context (Kingston et al., Blood 114:835-843, 2009). In turn, macrophages, microglia, mast cells, eosinophils, monocytes, T-reg and T-helper cells each with differing phenotypes, including the now widely recognized autoimmune-mediating Thl7 cell subsets, are the kinds of processes that are governed by RTK signaling, either directly or via the cascading effects of altered costimulation by affected DCs. The connection between dendritic cells as the cellular target, i.e. mode of action, and RTKs as the molecular target, i.e. mechanism of action, for RTK inhibition have been documented in multiple studies, and can be viewed as setting the paradigms for understanding all such immune system diseases sharing similar cellular and molecular etiologies. Multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease are particularly illustrative in this respect, but the same phenomenology applies to psoriasis, graft- versus-host-disease, and other autoimmune diseases.
Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease that affects the central nervous system. It is a clinically and pathologically heterogeneous disease most often characterized as a consequence of CD4+ Thl -mediated
autoimmune dysfunction. Several biologic immunomodulatory agents have been approved as treatments for relapsing-remitting MS, but inhibiting disease progression remains the central challenge. From the perspective of modern drug development for a chronic disease, this challenge is even more daunting when considering therapeutic modalities based on small molecules suitable for oral administration.
As reported by Lopez-Diego and Weiner (Nature Rev. Drug Discov. 7:909- 925, 2008) in their recent review on novel therapeutic strategies for MS within the broader constant of all autoimmune disease, new targets have emerged that challenge the traditional views of MS simply as a T-cell mediated disease. Instead, a broader stage has been set for understanding the molecular and cellular factors affecting the subtle balance between autoimmunity, with its corollary tissue destruction, and tolerance, with its accompanying repair processes, and the coordination of multiple immune components responsible for tipping that balance towards the latter outcome.
At the center of this new scenario is the interplay among T-helper effector subsets, Thl, Th2 and Thl 7, and T-reg with antigen presenting DCs as the common denominator in their cross-signaling cascade. DCs have been shown to have an important role across the spectrum of autoimmune diseases. Notably in the case of MS, dendritic cells have been identified as specific targets for therapeutic intervention (Whartenby et al., Expert Opin. Investig. Drugs 17: 1685-1692, 2008) based on the pioneering work of Whartenby and Small (Whartenby et al., Proc. Nat. Acad. Sci. 102: 16741-16746, 2005; Skarika et al., J. Immunol. 182:4192-4199, 2009) on the application of Flt3 inhibitors.
These authors have shown that small molecule inhibition of Flt3 with a small molecule compound blocks Flt3-mediated DC maturation (Waskow et al., Nature Immunol. 9:676-683, 2008) and ultimately shifts the activation and differentiation of T-cells from autoreactive, immunogenic phenotypes towards tolerogenic phenotypes thereby effecting reversal of disease in a mouse model of MS. In another recent study with the same compound, Weisel et al. (Ann. Hematol. 88:203-211, 2009) demonstrated that Flt3 inhibition during the stimulated expansion and differentiation of CD34+ precursors impaired cell growth, increased apoptosis and downregulated the proportion of lineage-committed, maturing cells versus primitive progenitor counts.
The beneficial effect of Flt3 inhibition in the mouse model of disease has been confirmed with another small molecule in the oxindole class (Tussiwand et al., J. Immunol. 175:3674-3680, 2005). In more general terms, inhibition of DCs development, maturation or antigen presenting capacity are becoming the ascribed mechanisms for a spectrum of experimental therapies proposed for MS (Zinser et al., Immunobiol. 209:89-97, 2004; Horstmann et al, Immunobiol. 212:839-853, 2008) as well as for the established MS therapy, the tolerogenic amino acid tetramer, glatiramer (Weber et al., Nature Med. 13:935-943, 2007). In this latter case, it is becoming increasingly accepted that localized immunomodulation with glatiramer extends beyond multiple sclerosis into inflammatory bowel disease (Aharoni et al. J. Pharmacol. Exp. Ther. 318:68-78, 2006), an autoimmune condition in which DCs play an important pathogenic role (Denning et al., Nature Immunol. 8: 1086-1094, 2007).
Completing the major trilogy of autoimmune diseases in which there exists an emerging central role for Flt3 and DCs, Dehlin et al. (PLos ONE 3:e3633,l-5, 2008) have demonstrated for the first time that Flt3 ligand, and by implication its receptor mediated signaling, is a driving force in rheumatoid arthritis induction and progression. A similar mode and mechanism of action has been described for DCs as regulatory hubs in the presentation of psoriasis and the regulation of the Thl7 inflammatory phenotypes. Fully mature DCs, exhibiting the full complement of surface co-stimulatory molecules, drive pathogenesis, while immature DCs promote counter-regulatory tolerance (Zaba et al., Investig. Dermatol. 129:79-88, 2009). In an earlier observation, the small molecule fumarate drug BG-12, had been shown to be a potent inhibitor of DC maturation, resulting in the downregulation of co-stimulatory antigens, e.g. CD40, CD80, CD86, and consequently of activated Thl cells via the concomitant lowering of cytokine, e.g. IL-6 and IL-12, secretion (Litjens et al., Cutan. Biol. 154:211-217, 2006). In graft-vs-host-disease, suppression of these same co- stimulatory antigens and cytokines cause the arrest of DCs maturation and change phenotypic progression towards the macrophage lineage and is also considered a desirable therapeutic effect (Vicenti and Kirk, Am. J. Transplant. 8: 1972-1981, 2008). From these findings one can reasonably expect that Flt3 signaling blockade and its effect on DC development, should have a beneficial therapeutic result in rheumatoid arthritis, psoriasis (and psoriatic arthritis), and graft rejection disorders similar to that observed in the multiple sclerosis and gastrointestinal autoimmune disease settings.
Dendritic cells share the CD11 lineage with macrophages and their
proliferation, maturation and activation is co-stimulated by colony-stimulating factor 1 (CSF-1). The receptor CSF-1R is a close homolog of Flt3, and this RTK also plays an important regulatory role in the survival, proliferation, and differentiation of mononuclear phagocyte lineages. It shares a signaling cascade similar to, and intertwined with that of Flt3 and its expression is upregulated in a number of human pathologies that involve chronic activation of tissue macrophage populations, such as that which exists in inflamed CNS, mucosal, and synovial tissues.
In view of the large multitude of RTK-mediated proliferative, inflammatory, and immune function diseases that are modulated by DCs, there is an ever-existing need to provide novel classes of compounds that are useful as RTK inhibitors and thus in the treatment of RTK related and DC modulated diseases, as discussed herein. There is a need for new pharmaceutically acceptable therapies for immune system disorders, including autoimmune and chronic inflammatory disorders with etiologies in DCs dysfunction. SUMMARY OF THE INVENTION
The present invention is directed to a compound of Structural Formula (I):
Figure imgf000008_0001
(i); nd pharmaceutically acceptable salt thereof.
A1 is selected from H, hydroxyl, a C1-C6 alkoxy, or -C(0)NRARB, wherein RA and RB each independently are hydrogen or a C1-C3 alkyl,
A2 is selected from H, hydroxyl, a C1-C6 alkoxy or a C1-C6 alkyl, optionally substituted with a halogen;
or
A1 and A2 taken together with the intervening atoms form a lH-pyrazole ring or a pyrrol-2-one ring;
provided that A1 and A2 are not simultaneously hydrogens;
R1 is a halogen, -OR2, -SR3, or -NR4R5;
R and R each independently are hydrogen or -L'-R0;
L1 is -(CH2)X-, wherein x is 0, 1, 2, 3, or 4;
6 is independently for each occurrence selected from hydrogen, C1-C4
Figure imgf000008_0002
wherein R is hydrogen or a C1-C3 alkyl, optionally substituted with amino, a (Cl- C3)alkylamino, or a di(Cl-C3)alkylamino, and further wherein, optionally, the alkyl portions of the di(Cl-C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl;
or, alternatively,
R4 and R5, taken together with the nitrogen atom to which they are attached, form a 5-membered heteroaryl Ar2, optionally including one or two additional heteroatoms selected from N, S or O;
or, alternatively,
R4 and R5, taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclyl C2, optionally including an additional heteroatom selected from N, O or S, wherein S is optionally oxidized into S(02);
L2 is -(CHRK)y-, wherein y is 0, 1, 2, 3 or 4, and each RK is independently hydrogen or a C1-C4 alkyl;
R7 is:
- hydrogen,
- a CI -C4 alkyl,
- -C(0)ORL, wherein RL is hydrogen or a C 1 -C4 alkyl,
- a Cl-C4 alkoxy,
- -NRMRN,
- a 5- to 6-membered heterocyclyl C1 including one or two heteroatoms independently selected from N, O or S, or
a 5- to 6-membered aryl or heteroaryl Ar ,
wherein
RM and RN each independently are hydrogen or a C1-C4 alkyl, or, alternatively,
RM and RN, taken together with the nitrogen to which they are attached, form a 5- to 7-membered heterocyclyl C3, optionally including one additional heteroatom selected from N, O or S. The present teachings also provide methods of making the compounds of Formula (I), as well as methods of using the compounds for the treatment of autoimmune diseases and for modulating the activity of Flt3 or CSF-1R. The methods of using the compounds generally include administering a therapeutically effective amount of a compound of Formula (I) to a mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
Figure 1 A is a plot of dendritic cells proliferation as a function of time in the presence of representative compounds of the present invention as well as controls.
Figure IB is a histogram of the dendritic cell counts at day 15 in the presence of representative compounds of the present invention as well as controls.
Figure 2 A presents the result shown in Figure 1 A, where the cells counted are limited to the CD1 lc+CDl lb+ phenotype.
Figure 2B presents the result shown in Figure IB, where the cells counted are limited to the CD 1 lc+CDl lb+ phenotype.
Figure 2C is a plot of dendritic cells proliferation as a function of time in the presence of a representative compound of the present invention as well as controls, where the cells counted are limited to the CD 1 lc+CDl lb+ phenotype.
Figure 2D a histogram of the dendritic cell counts at day 15 in the presence of a representative compound of the present invention as well as controls, where the cells counted are limited to the CD 11 c+CD 1 lb+ phenotype. Figure 3 A shows the time course for the expression on the dendritic cells of the CD83 maturation marker coexpressed with the CD1 lc dendritic cell hallmark in the presence of representative compounds of the present invention as well as controls.
Figure 3B shows the histogram of the CD1 lc/CD83 dendritic cell counts at day 15 in the presence of representative compounds of the present invention as well as controls.
Figure 4A shows the time course for the dendritic cell viability in the presence of representative compounds of the present invention as well as controls.
Figure 4B shows the histogram of the dendritic cell viability counts at day 15 in the presence of representative compounds of the present invention as well as controls.
Figure 5 is a plot of a Total Clinical Score, in the Collagen Antibody-Induced Arthritis (CAIA) animal model, as a function of time in the presence of a
representative compound of the present invention as well as controls.
Figure 6 is a histogram of Clinical Scores of mouse ankle arthritis for the indicated categories, in the Collagen Antibody-Induced Arthritis (CAIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 7 is a histogram of macrophage infiltration in the mouse joint tissue, in the Collagen Antibody- Induced Arthritis (CAIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 8A shows four histograms, each histogram representing the amount of a representative biomarker (IL-4, IL-6) in a tissue sample (plasma or joint extract) collected from a mouse in the Collagen Antibody-Induced Arthritis (CAIA) animal model, in the presence of a representative compound of the present invention as well as controls. Figure 8B shows four histograms, each histogram representing the amount of a representative biomarker (IL-10, IL-12p70) in a tissue sample (plasma or joint extract) collected from a mouse in the Collagen Antibody-Induced Arthritis (CAIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 9 shows four histograms, each histogram representing the amount of a representative biomarker (Monocyte Chemoattractant Protein-3 and Macrophage Inflammatory Protein- 1 beta) in a tissue sample (plasma or joint extract) collected from a mouse in the Collagen Antibody-Induced Arthritis (CAIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 10 is a plot showing time course of body weight change of DBA/1 mice with in the Collagen -Induced Arthritis (CIA) animal model in the presence of representative compounds of the present invention as well as controls.
Figure 11 A is a plot showing Mean Clinical Arthritis Score as a function of time for "All Paws" in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as control and imatinib.
Figure 1 IB is a plot showing Mean Clinical Arthritis Score as a function of time for "Non-enrollment Paws" in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as control and comparator imatinib.
Figure 12A is a plot showing Mean Clinical Arthritis Score as a function of time for "All Paws" in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as control and comparator methotrexate.
Figure 12B is a plot showing Mean Clinical Arthritis Score as a function of time for "Non-enrollment Paws" in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as control and comparator methotrexate.
Figure 13 is a photomicrograph comparing the toluidine-blue stained tibio- tarsal joint slices of a animals in the Collagen-Induced Arthritis (CIA) model in the presence of Compound 12 as well as vehicle control.
Figure 14 shows a histogram of a quantitative measurement of inflammation in tissues of a joint of animal in the Collagen- Induced Arthritis (CIA) model in the presence ofrepresentative compounds of the present invention as well as controls.
Figure 15 shows a histogram of a quantitative measurement of pannus in a joint of animal in the Collagen-Induced Arthritis (CIA) model in the presence of representative compounds of the present invention as well as controls.
Figure 16 shows a histogram of a quantitative measurement of cartilage erosion in a joint of animal in the Collagen-Induced Arthritis (CIA) model in the presence of representative compounds of the present invention as well as controls.
Figure 17 shows a histogram of a quantitative measurement of exostotic bone growth in a joint of animal in the Collagen- Induced Arthritis (CIA) model in the presence of representative compounds of the present invention as well as controls.
Figure 18 shows a histogram representing the amount of IL-6 in a tissue sample (plasma or joint extract) collected from a mouse in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 19 shows a histogram representing the amount of IL-10 in a tissue sample (plasma or joint extract) collected from a mouse in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 20 shows a histogram representing the amount of MIP-2 in a tissue sample (plasma or joint extract) collected from a mouse in the Collagen-Induced Arthritis (CIA) animal model, in the presence of a representative compound of the present invention as well as controls.
Figure 21 is a plot showing weight of C57BL/6 mice in in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of representative compounds of the present invention as well as controls.
Figure 22 is a histogram showing percent weight loss in C57BL/6 mice in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of selected representative compounds of the present invention as well as controls.
Figure 23 is a histogram showing inflammatory score in C57BL/6 mice in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of a representative compound of the present invention as well as controls.
Figure 24 is a histogram showing percent weight loss in C57BL/6 mice in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of representative compounds of the present invention as well as control.
Figure 25A is a photomicrograph (20x) of a colon from a mouse in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of Compound 47.
Figure 25B is a photomicrograph (20x) of a colon from a mouse in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of Compound 129.
Figure 25C is a photomicrograph (20x) of a colon from a mouse in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of vehicle control.
Figure 26 is a histogram showing histological score for severity of
inflammation, depth of injury/inflammation, and crypt damage in C57BL/6 mice in the dextran sodium sulfate (DSS)-induced murine colitis model, in the presence of representative compounds of the present invention as well as control. Figure 27 shows two histograms, each histogram representing the amount of a representative biomarker (IL-Ιβ and TNF-D) determined by qPCR of cells from mesenteric lymph nodes (MLN) collected from a mouse in the dextran sodium sulfate (DSS)-induced murine colitis model, at Day 5 in the presence of representative compounds of the present invention as well as control.
Figure 28 shows two histograms, each histogram representing the amount of a representative biomarker (IL-10, IL-27) determined by qPCR of cells from mesenteric lymph nodes (MLN) collected from a mouse in the dextran sodium sulfate (DSS)- induced murine colitis model, at Day 12 in the presence of representative compounds of the present invention as well as control.
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention provide compounds that can modulate kinase activity, modulate dendritic cell maturation and activation, and can treat or alleviate the symptoms of various autoimmune diseases.
As used herein, the terms "autoimmune disease" and "autoimmune disorder" comprise conditions in which the immune system of a patient becomes dysregulated or compromised within one or more of its innate or adaptive components.
Autoimmune disease may have systemic or highly localized presentations and may variously derive its etiology from the involvement of one or more cell types from among dendritic cells, macrophages, T- or B-lymphocytes, mast cells, neutrophils and related effector cells of lymphoid or myeloid origin in the presence of self or pathogenic antigens. Various disorders are recognized as autoimmune diseases by the American Autoimmune Related Diseases Association.
The present invention is directed to a compound represented by the Structural Formula (I), or a pharmaceutically acceptable salt thereof. Values and alternative values for the variables in Structural Formula I, or a pharmaceutically acceptable salt thereof are provided in the following paragraphs. It is understood that the invention encompasses all combinations of the substituent variables defined herein. A1 is selected from H, hydroxyl, a C1-C6 alkoxy, or -C(0)NRARB, wherein RA and RB each independently are hydrogen or a C1-C3 alkyl; and A2 is selected from H, hydroxyl, a C1-C6 alkoxy or a C1-C6 alkyl, optionally substituted with a halogen,
1 9 1 provided that A and A are not simultaneously hydrogens. Alternatively, A and A each independently are a C1-C6 alkoxy, optionally substituted with a halogen or a 5-
1
or 6-membered aryl or heteroaryl. In yet another alternative, A and A each independently are selected from -O-benzyl,- O-tButyl, -G-Methyl, -O-Ethyl,
1 9
-O-iPropyl and -0-CF3. In yet another alternative, A and A taken together with the intervening atoms form a lH-pyrazole ring or a pyrrol-2-one ring. In yet another alternative, A1 is hydroxyl, a C1-C6 alkoxy or H; and A2 is selected from hydroxyl, a C1-C6 alkoxy, C1-C6 alkyl, C1-C6 haloalkyl, and H, provided that A1 and A2 are not simultaneously hydrogens. In yet another alternative, A1 is selected from H, hydroxyl, -C(0)NH2, or -C(0)NH(C1-C3)alkyl; and A2 is selected from H, hydroxyl, or a C1-C6 alkyl, provided that A1 and A2 are not simultaneously hydrogens.
R1 is a halogen, -OR2, -SR3, or -NR4R5. Alternatively, R1 is -OR2 or -SR3. In another alternative, R1 is halogen, C1, -NH(CH2)0-2Ar3, -NHCH(CH3) Ar3.
1 1 1 1
-0(CH2)o-4Ar , or -S(CH2)o-4Ar . In yet another alternative, R is halogen, C , -NH(CH2)0-4Ar3, -NHCH(CH3) Ar3. -OCCH^Ar1, or -S(CH2)0-4Ar1. In yet another alternative, R1 is C1, -NH(CH2)0-2Ar3, -NHCH(CH3) Ar3.
9 "3 1 9
R and R each independently are hydrogen or -L -R . Alternatively, R and R3 each independently are hydrogen, phenyl, pyridyl, or benzyl, the phenyl, pyridyl or benzyl being optionally substituted with one to three substituents independently selected from a halogen, C1-C3 alkyl, -CF3, hydroxyl, and a (Cl-C3)alkoxy.
L1 is -(CH2)X-, wherein x is 0, 1, 2, 3, or 4. Alternatively, x is 0 or 1.
R6 is hydrogen, C1-C4 alkyl, or Ar1. Alternatively, R6 is hydrogen, methyl, ethyl, pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, or furyl, the pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, or furyl group, optionally substituted with one to three substituents independently selected from a halogen, C1-C3 alkyl, -CF3, hydroxyl, and a (Cl-C3)alkoxy.
Alternatively, R6 is hydrogen, C1-C4 alkyl, phenyl, or pyridyl, optionally substituted with one to three substituents independently selected from a halogen, C1-C3 alkyl, - CF3, hydroxyl, and a (Cl-C3)alkoxy. R is hydrogen or a C1-C4 alkyl.
R5 is selected from hydrogen, -L2-R7, and -C(0)RD. Alternatively, R5 is hydrogen, -C(0)H, -C(0)(Cl-C3)alkyl (optionally substituted with amino), - C(0)(ClC3)alkylamino, (Cl-C3)alkylpiperidine, a 5- to 6-membered heterocyclyl C1 including one or two heteroatoms independently selected from N, O or S, -(CHRK)o_ 4H, -(CHRK)0-4C(O)OH, -(CHRK)0-4C(O)(Cl-C4)alkyl, -(CHRK)0-4(C1-C4)alkoxy, - K)o-4NH2, -(CHRK)o-4NH Cl-C4)alkyl, -(CHRK)0-4C3, -(CHRK)0-4Ar3,
Figure imgf000017_0001
wherein R is hydrogen or a (Cl-C4)alkyl, Ar is a 5- to 6-membered aryl or heteroaryl, and C3 is a 5- to 7-membered heterocyclyl, including one nitrogen atom bonded to -(CHRK)o-4 and optionally one additional heteroatom selected from N, O, or S. In another alternative, R5 is hydrogen, -C(0)H, -C(0)(Cl-C3)alkyl (optionally substituted with amino), -C(0)(ClC3)alkylamino, (Cl-C3)alkylpiperidine, piperidine, tetrahydro-2H-pyran (wherein the piperidine and tetrahydro-2H-pyran are
independently optionally substituted with a substituent selected from a C1-C3 alkyl, phenyl, benzyl or -C(0)ORE, wherein RE is hydrogen or a C1-C4 alkyl), -(CHRK)0-4H, -(CHRK)0-4C(O)OH, -(CHRK)0-4C(O)(Cl-C4)alkyl, -(CHRK)0-4(C1-C4)alkoxy, - K)0-4NH2, -(CHR )0-4NH(C1-C4 alkyl, -CHRK)0-4C3, -(CHRK)0-4Ar3,
Figure imgf000017_0002
wherein RK is hydrogen or a (Cl-C4)alkyl, Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl, wherein the pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl groups are optionally substituted with one to three substituents independently selected from
a halogen, hydroxyl,
cyano,
- -S(02)NH2 or -S(02)N(CH3)2;
amino,
a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl, a 5-6-membered aryl or heteroaryl;
a 5- to 7-membered non-aromatic heterocyclic ring including one or two heteroatoms selected form O, N and S;
- -C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl,
- (Cl-C3)alkylamino,
di(Cl-C3)alkylamino, wherein, optionally, the alkyl portions of the di(Cl- C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl, optionally including one additional heteroatom selected from N, O or S; and
a CI -C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a 5- or 6- membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy; and C3 is selected from the group consisting of
Figure imgf000018_0001
wherein Q2 is O, CH2, NH, or NR100, and R100 is -C(0)OEt or a C 1 -C6 alkyl optionally substituted with -CN, -OH, phenyl, or cyclopropyl.
Alternatively, R4 and R5, taken together with the nitrogen atom to which they are attached, form a 5-membered heteroaryl Ar2, optionally including one or two additional heteroatoms selected from N, S or O. In another alternative, R4 and R5, taken together with the nitrogen atom to which they are attached form a 5- to 7- membered heterocyclyl C2, optionally including an additional heteroatom selected from N, O or S, wherein S is optionally oxidized into S(02). In yet another alternative, R4 and R5, taken together with the nitrogen atom to which they are attached, form a piperidine, piperazine, thiomorpholine, thiomorpholine- 1 ,1 -dioxide, morpholine, pyrrolidine, or imidazole ring, wherein the piperidine, piperazine, thiomorpholine, thiomorpholine- 1,1 -dioxide, morpholine, pyrrolidine, or imidazole ring are each optionally independently substituted with one to three substituents selected from a halogen, C1-C3 alkyl (optionally substituted with hydroxyl, halogen, cyano or cyclopropyl), hydroxyl, phenyl (optionally substituted with halogen, (Cl-C3)alkyl, or (Cl-C3)alkoxy), benzyl, -C(0)0(Cl-C3)alkyl, -C(0)OH, and (Cl-C3)alkoxy. In yet another alternative, R4 and R5, taken together with the nitrogen to which they are attached, form N-morpholinyl, N-thiomorpholinyl, N-piperidinyl, N-piperazinyl, or N-pyrrolidinyl, each independently optionally substituted with one to three substituents selected from a halogen, C1-C3 alkyl (optionally substituted with hydroxyl, halogen, cyano or cyclopropyl), hydroxyl, phenyl (optionally substituted with halogen, (Cl-C3)alkyl, or (Cl-C3)alkoxy), benzyl, -C(0)0(Cl-C3)alkyl, - C(0)OH, and (Cl-C3)alkoxy.
RD is hydrogen or a C1-C3 alkyl, optionally substituted with amino, a (Cl- C3)alkylamino, or a di(Cl-C3)alkylamino, and further wherein, optionally, the alkyl portions of the di(Cl-C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl. Alternatively, RD is hydrogen, a C1-C3 alkyl ( optionally substituted with amino, a (Cl-C3)alkylamino, or a di(Cl-C3)alkylamino), or piperidinyl(Cl-C3)alkyl. In another alternative, RD is hydrogen, unsubstituted C1-C3 alkyl, or piperidinyl(Cl-C3)alkyl.
L2 is -(CHRK)y-, wherein y is 0, 1, 2, 3 or 4, and each RK is independently hydrogen or a C1-C4 alkyl. Alternatively, L is -(CH2)y-, wherein y is 0, 1 , 2, 3 or 4.
R7 is hydrogen, a C1-C4 alkyl, -C(0)ORL (wherein RL is hydrogen or a C1-C4 alkyl), a C1-C4 alkoxy, -NRMRN, a 5- to 6-membered heterocyclyl C1 including one or two heteroatoms independently selected from N, O or S, a 5- to 6-membered aryl or heteroaryl Ar3, wherein RM and RN each independently are hydrogen or a C1-C4 alkyl, or, alternatively, RM and RN, taken together with the nitrogen to which they are attached, form a 5- to 7-membered heterocyclyl C3, optionally including one additional heteroatom selected from N, O or S. Alternatively, R7 is hydrogen, a Cl- C4 alkyl, -C(0)OH, -C(0)0(C 1 -C4)alkyl, a C1-C4 alkoxy, -NH2, -NH(C1-C4)alky, piperidine, tetrahydro-2H-pyran (wherein the piperidine and tetrahydro-2H-pyran are independently optionally substituted with a substituent selected from a C1-C3 alkyl, phenyl, benzyl or -C(0)ORE, wherein RE is hydrogen or a C1-C4 alkyl), pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl (wherein the pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl groups are optionally substituted with one to three substituents independently selected from
a halogen,
hydroxyl,
cyano,
-S(02)NH2 or -S(02)N(CH3)2;
amino,
a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl,
a 5-6-membered aryl or heteroaryl;
a 5- to 7-membered non-aromatic heterocyclic ring including one or two heteroatoms selected form O, N and S;
-C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl, (Cl-C3)alkylamino,
di(Cl-C3)alkylamino, wherein, optionally, the alkyl portions of the di(Cl-C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl, optionally including one additional heteroatom selected from N, O or S; and
a C 1 -C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a 5- or 6-membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy),
Figure imgf000020_0001
Figure imgf000021_0001
wherein Q2 is O, CH2, NH, or NR100, and R100 is -C(0)OEt or a C1-C6 alkyl optionally substituted with -CN, -OH, phenyl, or cyclopropyl.
In a 1st specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar is substituted with one to three substituents R selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two groups R taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 2nd specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar is phenyl, or pyridyl, optionally substituted with one to three substituents R selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two groups R taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro- 1 ,4-dioxine ring, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 3rd specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. C1 is substituted with a substituent selected from a C1-C3 alkyl, phenyl, benzyl or -C(0)ORE, wherein RE is hydrogen or a C1-C4 alkyl, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 4th specific embodiment, the compound of the present invention is represented b Structural Formula (I), or a pharmaceutically acceptable salt thereof.
C1 is
Figure imgf000021_0002
, wherein R is benzyl, methyl, or -C(0)OEt, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 5th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar2 is substituted with one to three substituents RC1 selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two groups R taken together with the intervening atoms form a 1,3 -dioxole ring or a 2,3-dihydro-l,4-dioxine ring, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 6th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar2 is phenyl, pyridyl or imidazolyl, each optionally substituted with one to three substituents RCI selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl- C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two groups RC1 taken together with the intervening atoms form a 1,3- dioxole ring or a 2,3-dihydro-l,4-dioxine ring, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 7th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. C2 is substituted with one to three substituents R8, wherein R8, for each occurrence independently, is selected from hydroxyl, cyano, a halogen, a C1-C6 alkyl,
-C(0)ORG1, or a 5- to 6-membered aryl or heteroaryl, wherein said C1-C6 alkyl is optionally substituted with RF and said 5- to 6-membered aryl or heteroaryl is optionally substituted with a hydroxyl, a C1-C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, -C(0)ORG, or -C(0)NRHRJ, wherein RF is hydroxyl, cyano, a C1-C3 alkyl, a C3-C6 cycloalkyl, amino, a (Cl-C3)alkylamino, a di(Cl- C3)alkylamino, a C1-C3 alkoxy, a 5- to 6-membered heteroaryl or phenyl,
-C(0)ORG2 or -C(0)NRH1RJ1; RG, RG1, and RG2 are each independently hydrogen or a C1-C4 alkyl; and RH, RJ RH1, RJ1 each independently are selected from hydrogen or a C1-C3 alkyl, and values and alternative values for the remainder of the variables are as described for Structural Formula (I). In a 8th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. C is a piperidine, piperazine, thiomorpholine, thiomorpholine 1,1 -dioxide, morpholine, pyrrolidine, or imidazole ring, wherein the piperidine, piperazine, thiomorpholine, thiomorpholine 1,1 -dioxide, morpholine, pyrrolidine, or imidazole ring is substituted with one to three substituents R , wherein R , for each occurrence independently, is selected from hydroxyl, cyano, a halogen, a C1-C6 alkyl,
-C(0)ORG1, or a 5- to 6-membered aryl or heteroaryl, wherein said C1-C6 alkyl is optionally substituted with RF and said 5- to 6-membered aryl or heteroaryl is optionally substituted with a hydroxyl, a C1-C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, -C(0)ORG, or -C(0)NRHRJ, wherein RF is hydroxyl, cyano, a C1-C3 alkyl, a C3-C6 cycloalkyl, amino, a (Cl-C3)alkylamino, a di(Cl- C3)alkylamino, a C1-C3 alkoxy, a 5- to 6-membered heteroaryl or phenyl,
-C(0)ORG2 or -C(0)NRH1RJ1; RG, RG1, and RG2 are each independently hydrogen or a C1-C4 alkyl; and RH, RJ RH1, RJ1 each independently are selected from hydrogen or a C1-C3 alkyl, and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 9th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar3 is substituted with one to three substituents R°, wherein each R° is independently selected from:
a halogen,
hydroxyl,
- cyano,
- -S(02)NH2 or -S(02)N(CH3)2;
amino,
- a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally
substituted with a halogen or a 5- or 6-membered aryl or heteroaryl, a 5-6-membered aryl or heteroaryl;
a 5- to 7-membered non-aromatic heterocyclic ring including one or two heteroatoms selected form O, N and S;
- -C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl, - (Cl-C3)alkylamino,
di(C 1 -C3)alkylamino, wherein, optionally, the alkyl portions of the di(C 1 - C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl, optionally including one additional heteroatom selected from N, O or S; and
a C1-C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a 5- or 6- membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy, or, alternatively,
two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 10th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl, each optionally substituted with one to three substituents R°, wherein each R° is independently selected from:
a halogen,
hydroxyl,
cyano,
- -S(02)NH2 or -S(02)N(CH3)2;
amino,
- a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally
substituted with a halogen or a 5- or 6-membered aryl or heteroaryl, a 5-6-membered aryl or heteroaryl;
a 5- to 7-membered non-aromatic heterocyclic ring including one or two heteroatoms selected form O, N and S;
- -C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl,
- (Cl-C3)alkylamino,
- di(Cl-C3)alkylamino, wherein, optionally, the alkyl portions of the di(Cl- C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl, optionally including one additional heteroatom selected from N, O or S; and
a C1-C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a 5- or 6- membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy, or, alternatively,
two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In an 11th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl, each optionally substituted with one to three substituents R°, wherein each R° is independently selected from:
a halogen;
hydroxyl;
cyano;
- -S(02)NH2 or -S(02)N(CH3)2;
amino;
- a C 1 -C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl;
phenyl, pyridyl;
piperazinyl;
- -C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl;
- (Cl-C3)alkylamino;
piperidinylethyl; and
a CI -C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a phenyl or pyridyl, said phenyl or pyridyl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy, or, alternatively, two groups R taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 12th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof.
C includes an additional heteroatom selected from O or N, and wherein if said additional nitrogen heteroatom is nitrogen, then said additional heteroatom is optionally substituted with a group R100 selected from a C1-C6 alkyl, a C3-C6 cycloalkyl, a phenyl, cyano, hydroxyl and -C(0)OR101, wherein R101 is methyl or ethyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 13th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof.
C is selected from the group consisting of
Figure imgf000026_0001
wherein
Q2 is O, CH2, NH, or NR100; R100 is a C1-C6 alkyl, a C3-C6 cycloalkyl, a phenyl, cyano, hydroxyl or -C(0)OR101, wherein R101 is methyl or ethyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 14th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. A and A each independently are a C1-C6 alkoxy, optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 14th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. A1 and A2 each independently are selected from -O-benzyl,- O-tButyl, -O-Methyl, -O-Ethyl, -O-iPropyl and -0-CF3; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 15th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. R1 is -NR4R5; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 16th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA) or (IB), or a pharmaceutically acceptable salt thereof:
Figure imgf000027_0001
(IA) (IB);
and values and alternative values for the variables are as described for Structural Formula (I).
In a 17th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA) or (IB), or a pharmaceutically acceptable salt thereof. R1 is -NR4R5; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In an 18th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof:
Figure imgf000027_0002
(IA2) (IB2) wherein:
R4 is H or C1-6 alkyl,
R5 is -L2-R7,
R7 is a 5- to 6-membered aryl or heteroaryl Ar3; and Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 19th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H or C1-6 alkyl, R5 is -L2-R7, R7 is a 5- to 6-membered aryl or heteroaryl Ar3; and Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl; y is 1 ; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 20th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H, y is 1, Ar3 is phenyl; and the optional substituent R° on Ar3 for each occurrence independently, is selected from a halogen, C1-6 alkyl, Ci-6 haloalkyl, amino, (Cl-C3)alkylamino, di(Cl-C3)alkylamino, phenyl, and a C1-C6 alkoxy group, or, alternatively, two groups R° taken together with the intervening atoms form a 1,3- dioxole ring or a 2,3-dihydro-l,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 20th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (ΓΒ2), or a pharmaceutically acceptable salt thereof. R4 is H, y is 1 , Ar3 is pyridyl and RK is H or methyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 21st specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H, y is 1, Ar3 is thiophen-2-yl and RK is H or methyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I). In a 22st specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H, y is 2, Ar3 is pyridyl and R is H or methyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 22nd specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H, y is 2, Ar3 is phenyl and RK is H or methyl; and the optional substituent R° is, for each occurrence independently, selected from a halogen and a C1-C6 alkoxy group, or, alternatively, two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 23rd specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H or C1-6 alkyl; R5 is -L2-R7; y is 0; R7 is a 5- to 6-membered aryl or heteroaryl Ar3; and Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 24th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 is H; R5 is -L2-R7; y is 0; R7 is phenyl optionally substituted with one to three R°, wherein the optional substitutent R°, for each occurrence independently, is selected from halogen, C2-6 alkynyl, a 5 to 7 membered non-aromatic heterocyclic ring including one or two heteroatoms selected from N, O and S, phenyl, benzyl; -SO2NH2 , -S02N(CH3)2 or a C1-C6 alkoxy group, or, alternatively, two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro- 1,4-dioxine ring; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 25th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 and R5, taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclyl C2, selected form the group consisting of
Figure imgf000030_0001
wherein Q1 is O, S, S02, CH2, CHR8A, NH, or NR8B;
R8A is Ci-6 alkyl, phenyl, benzyl, CN, or -C(0)ORG1, wherein said C1-C6 alkyl optionally substituted with a C1-C2 alkoxy; and R8B is Ci-6 alkyl, phenyl, or - C(0)ORG1, wherein said C1-6 alkyl is optionally substituted with -CN, hydroxyl, a Cl- C3 alkoxy, or cyclopropyl, and wherein said phenyl is optionally substituted with Cl- C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 26th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R4 and R5, taken together with the nitrogen to which they are attached, form N-morpholinyl, N-thiomorpholinyl, N-piperidinyl, N-piperazinyl, or
N-pyrrolidinyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 27th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R5 is L2-R7; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 28th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R5 is L2-R7; R7 is -NRMRN; RM and RN, taken together with the nitrogen to which they are attached, form a 5- to 7-membered heterocyclyl C , and further wherein C3 isselected from the group consisting of
Figure imgf000030_0002
wherein Q2 is O, CH2, NH, or NR100;R100 is -C(0)OEt or a Ci-6 alkyl optionally substituted with -CN, -OH, phenyl, or cyclopropyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 29th specific embodiment, the compound of the present invention is is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R5 is L2-R7; R7 is -NRMRN; RM and RN, taken together with the nitrogen to which they are attached, form a 5- to 7-membered heterocyclyl C , and further wherein 3 isselected from the group consisting of
Figure imgf000031_0001
wherein y is 2; Q2 is O, CH2, or NR100;R100 is -C(0)OEt or a C1-6 alkyl optionally substituted with -CN, -OH, phenyl, or cyclopropyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 30th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R5 is L2-R7; R7 is -NRMRN; RM and RN, taken together with the nitrogen to which they are attached, form N-morpholinyl, N-piperidinyl, or N-piperazinyl; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 3 lth specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R5 is a 5- to 6-membered heterocyclyl C1 including one or two heteroatoms independently selected from N, O or S; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 32nd specific embodiment, the compound of the present invention is represented by Structural For pharmaceutically acceptable
salt thereof, y is 0 and R5 is
Figure imgf000031_0002
wherein R50 is benzyl, methyl, or -C(0)OEt; and values and alternative values for the remainder of the variables are as described for Structural Formula (I). In a 33rd specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof. R5 is R5 is represented by the following structural formula:
Figure imgf000032_0001
; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 34th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA2) or (IB2), or a pharmaceutically acceptable salt thereof, y is 0 and C1 is n ; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 35th specific embodiment, the compound of the present invention is represented by Structural Formulas (I), or a pharmaceutically acceptable salt thereof R1 is NR4R5; A1 is hydroxyl, a C1-C6 alkoxy or H; and A2 is selected from hydroxyl, a C1-C6 alkoxy, C1-6 alkyl, C1-6 haloalkyl, and H; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 36th specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. R1 is NR4R5; A1 is hydroxyl, a C1-C6 alkoxy or H; and A2 is selected from hydroxyl, a C1-C6 alkoxy, C1-6 alkyl, C1-6 haloalkyl, and H; R4 and R5, taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclyl C , selected form the roup consisting of
Figure imgf000032_0002
wherein Q1 is O, S, S02, CH2, CHR8A, NH, or NR8B; R8A is C1-6 alkyl, phenyl, benzyl, CN, or -C(0)ORG1, wherein said C1-C6 alkyl optionally substituted with a C1-C2 alkoxy; and R is Ci.6 alkyl, phenyl, or -C(0)OR , wherein said C .e alkyl is optionally substituted with -CN, hydroxyl, a C1-C3 alkoxy, or cyclopropyl, and wherein said phenyl is optionally substituted with C1-C3 alkoxy, amino, a (Cl- C3)alkylamino, a di(Cl-C3)alkylamino; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 37th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA) or (IB), or a pharmaceutically acceptable salt thereof. R1 is -OR2 or -SR3; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 38th specific embodiment, the compound of the present invention is represented by Structural Formulas (IA) or (IB), or a pharmaceutically acceptable salt thereof. R1 is -OR2 or -SR3, and R2 and R3 each independently are hydrogen or -I^-R6; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 39th specific embodiment, the compound of the present invention is represented by Structural Formula (IC), or a pharmaceutically acceptable salt thereof:
Figure imgf000033_0001
(IC).
Values and alternative values for the variables are as described for Structural Formula (I).
In a 40th specific embodiment, the compound of the present invention is represented by the following Structural Formula, or a pharmaceutically acceptable salt thereof:
Figure imgf000034_0001
Values and alternative values for the variables are as described for Structural Formula (I).
In a 41st specific embodiment, the compound of the present invention is represented by Structural Formula (I), or a pharmaceutically acceptable salt thereof. R1 is a halogen; and values and alternative values for the remainder of the variables are as described for Structural Formula (I).
In a 42nd specific embodiment, the compound of the present invention, is selected from the group consisting of:
2-Chloro-9-(lH-indazol-5-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-propane-l,3-diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(3-morpholin-4-yl-propyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-dimethyl-amine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-N'-methyl-ethane-l,2- diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(2-methoxy-ethyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-phenethyl-amine;
Diethyl-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-amine;
9-(lH-Indazol-5-yl)-2-mo holin-4-yl-9H-purine;
9-( 1 H-Indazol-5-yl)-2-(4-methyl-piperazin- 1 -yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-piperidin-l-yl-9H-purine;
9-(lH-Indazol-5-yl)-2-pyrrolidin-l-yl-9H-purine;
9-(lH-Indazol-5-yl)-9H-purin-2-ylamine;
[9-( 1 H-Indazol-5 -yl)-9H-purin-2-yl] - [3 -(4-methyl-piperazin- 1 -yl)-propyl] - amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine; 2-(4-tert-Butyl-piperidin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[2-(l-methyl-piperidin-4-yl)-ethyl]- amine;
2- {4- [9-( 1 H-Indazol-5 -yl)-9H-purin-2-yl] -piperazin- 1 -yl } -ethanol;
2-(4-Cyclopropylmethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
3 - {4- [9-( 1 H-Indazol-5 -yl)-9H-purin-2-yl] -piperazin- 1 -yl} -propionitrile;
9-( 1 H-Indazol-5 -yl)-2- [4-(3 -methoxy-p enyl)-piperazin- 1 -yl] -9H-purine;
9-(lH-Indazol-5-yl)-2-[4-(2-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
9-(lH-Indazol-5-yl)-2-methoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenylsulfanyl-9H-purine;
9-( 1 H-Indazol-5 -yl)-2-piperazin- 1 -yl-9H-purine;
1 -[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid ethyl ester;
9-(lH-Indazol-5-yl)-2-(2-methyl-imidazol-l-yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-(pyridin-4-ylsulfanyl)-9H-purine;
2-Chloro-9-(lH-indazol-6-yl)-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-dimethyl-amine;
2-(4-Ethyl-piperazin- 1 -yl)-9-(lH-indazol-6-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-N'-methyl-ethane-l,2- diamine;
N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-N,N,,N'-trimethyl-propane-l,3- diamine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -pyridin-3 -ylmethyl-amine;
9-(lH-Indazol-6-yl)-2-morpholin-4-yl-9H-purine;
N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-N,N N'-trimethyl-ethane-l,2-diamine; 9-(lH-Indazol-6-yl)-2-piperidin-l-yl-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(4-methoxy-phenyl)-methyl-amine; (3-Chloro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-phenethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-[2-(3-methoxy-phenyl)-ethyl]-amine; [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-2-yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-methyl-amine;
2- { 4- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -piperazin- 1 -yl} -ethanol;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]-(3 -methoxy-benzyl)-amine;
[2-(3-Chloro-phenyl)-ethyl]-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine; (3 ,4-Dichloro-benzyl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
3- {[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-methyl-amino} -benzoic acid;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -(2-pyridin-3 -yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-2-ylmethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-4-yl-ethyl)-amine;
9-( 1 H-Indazol-6-yl)-2- [4-(2-methoxy-phenyl)-piperazin- 1 -yl] -9H-purine;
4- [9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-piperidine-l-carboxylic acid ethyl ester;
(4-Dimethylamino-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl] -amine; 4-(2-Chloro-purin-9-yl)-phenol;
4- { 2- [(2-Diethylamino-ethyl)-methyl-amino] -purin-9-yl } -phenol;
4-(2-Dimethylamino-purin-9-yl)-phenol;
4-(2-Pyrrolidin- 1 -yl-purin-9-yl)-phenol;
4-(2-Piperidin- 1 -yl-purin-9-yl)-phenol;
4-[2-(4-Ethyl-piperazin-l-yl)-purin-9-yl]-phenol;
4-(2-Morpholin-4-yl-purin-9-yl)-phenol;
4- {2- [(3 -Dimethylamino-propyl)-methyl-amino] -purin-9-yl } -phenol;
4-{2-[(2-Dimethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
4-{2-[4-(2-Hydroxy-ethyl)-piperazin-l-yl]-purin-9-yl}-phenol;
4-[2-(4-Methyl-piperazin-l-yl)-purin-9-yl]-phenol;
4-{2-[(Pyridin-3-ylmethyl)-amino]-purin-9-yl}-phenol;
4- [2-(3,4-Dichloro-benzylamino)-purin-9-yl] -phenol;
4-(2-Chloro-purin-9-yl)-2-methyl-phenol;
4-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-2 -methyl-phenol; 4-(2-Dimethylamino-purin-9-yl)-2-methyl-phenol; 2-Methyl-4- [2-(3 -morpholin-4-yl-propylamino)-purin-9-yl] -phenol; 4-[2-(4-Ethyl-piperazin-l-yl)-purin-9-yl]-2-methyl-phenol;
2-Methyl-4- {2- [3 -(4-methyl-piperazin- 1 -yl)-propylamino] -purin-9-yl } - phenol;
2-Methyl-4-(2-pyrrolidin- 1 -yl-purin-9-yl)-phenol;
2- Methyl-4-(2-piperidin- 1 -yl-purin-9-yl)-phenol;
4- [2-(3 -Diethylamino-propylamino)-purin-9-yl] -2-methyl -phenol;
3- (2-Chloro-purin-9-yl)-phenol;
3 - { 2- [(2-Diethylamino-ethyl)-methyl-amino] -purin-9-yl } -phenol;
3-(2-Dimethylamino-purin-9-yl)-phenol;
3-(2-Chloro-purin-9-yl)-benzamide;
3-(2-Dimethylamino-purin-9-yl)-benzamide;
3- {2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-benzamide;
4- (2-Methylamino-purin-9-yl)-benzamide;
4-(2-Piperidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Pyrrolidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Diethylamino-purin-9-yl)-benzamide;
4- [2-(3 -Diethylamino-propylamino)-purin-9-yl] -benzamide;
4-(2-Isobutylamino-purin-9-yl)-benzamide;
4- [2-(3 -Methyl-butylamino)-purin-9-yl] -benzamide;
4- [2-(4-Ethyl-piperazin- 1 -yl)-purin-9-yl] -benzamide;
5- (2-Chloro-purin-9-yl)-2,3-dihydro-isoindol-l-one;
N- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -3 -piperidin- 1 -yl-propionamide;
9-(lH-Indazol-6-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
(l-Benzyl-piperidin-4-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
9-( 1 H-Indazol-6-yl)-2-pyrrolidin- 1 -yl-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3,4,5-trimethoxy-phenyl)-amine;
(4-Chloro-3-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
{[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-methyl-amino} -acetic acid;
(S)-l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyrrolidine-2-carboxylic acid;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(l-methyl-piperidin-4-yl)-amine;
(3-Chloro-4-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine; 4-(2-Dimethylamino-purin-9-yl)-N-methyl-benzamide;
4- [2-(3 -Diethylamino-propylamino)-purin-9-yl] -N-methyl-benzamide;
(3-Ethynyl-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-trifluoromethyl-phenyl)-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl-amine;
{ l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl} -methanol;
9-(lH-Indazol-6-yl)-2-phenylsulfanyl-9H-purine;
l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carbonitrile;
(2,3 -Dihydro-benzo [ 1 ,4]dioxin-6-yl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] - amine;
Benzo[l,3]dioxol-5-yl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl-amine;
1- [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid methyl ester;
4-[9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-benzenesulfonamide;
Biphenyl-4-ylmethyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
2- { 4- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -piperazin- 1 -yl} -acetamide;
{4-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl}-acetic acid methyl ester;
9-(lH-Indazol-6-yl)-2-[4-(2-methoxy-ethyl)-piperazin-l-yl]-9H-purine; 9-(lH-Indazol-6-yl)-2-piperazin-l-yl-9H-purine;
(3-Chloro-4-methoxy-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[3 -Chloro-4-(3 -fluoro-benzyloxy)-phenyl] - [9-( 1 H-indazol-6-yl)-9H-purin-2- yl] -amine;
[4-(3-Fluoro-benzyloxy)-3-methoxy-phenyl]-[9-(lH-indazol-6-yl)-9H-purin- 2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-[4-(4-methyl-piperazin-l-yl)-phenyl]- amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methyl-benzyl)-amine;
(3 -Fluoro-benzyl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-methoxy-benzyl)-amine;
(2,4-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine; (2,6-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(3 , 5 -Difluoro-benzyl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
Benzo[l,3]dioxol-5-ylmethyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methoxy-benzyl)-methyl-amine; 4-(2-Chloro-purin-9-yl)-N-methyl-benzamide ;
N-Methyl-4-(2-thiomorpholin-4-yl-purin-9-yl)-benzamide;
N-Methyl-4-(2-piperidin-l-yl-purin-9-yl)-benzamide;
4-(2-Amino-purin-9-yl)-N-methyl-benzamide;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -((R)- 1 -phenyl-ethyl)-amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -((S)- 1 -phenyl-ethyl)-amine;
N-Methyl-4-(2-morpholin-4-yl-purin-9-yl)-benzamide;
9-(lH-indazol-6-yl)-2-((3-(trifluoromethyl)benzyl)thio)-9H-purine;
4-(4-(9-( 1 H-indazol-6-yl)-9H-purin-2-yl)piperazin- 1 -yl)aniline;
4-((9-(lH-indazol-6-yl)-9H-purin-2-yl)amino)-2-methoxyphenol;
4-(9-(lH-indazol-6-yl)-9H-purin-2-yl)thiomorpholine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-N-methyl-9H-purin-2-amine; 9-(lH-indazol-6-yl)-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine;
9-( 1 H-indazol-5 -yl)-2-(4-(2-methoxyethyl)piperazin- 1 -yl)-9H-purine;
N-((6-chloropyridin-3-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine; 2-(4-benzylpiperidin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-((4-methoxybenzyl)thio)-9H-purine;
2-((3-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
2-((4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-(4-phenylpiperidin-l-yl)-9H-purine;
4-(9-(lH-indazol-6-yl)-9H-purin-2-yl)thiomorpholine 1,1 -dioxide;
N-((2,3-dihydrobenzo[b][l,4]dioxin-5-yl)methyl)-9-(lH-indazol-6-yl)-9H- -2-amine;
N-(5-bromo-2-fluorobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-((2-methoxypyridin-4-yl)methyl)-9H-purin-2-amine; N-((2-fluoropyridin-4-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine; 2-((3-chloro-4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine; 9-(lH-indazol-5-yl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-2-amine;
9-( 1 H-indazol-6-yl)-2-(2-methyl- 1 H-imidazol- 1 -yl)-9H-purine; and
N-methyl-4-(2-(pyrrolidin-l-yl)-9H-purin-9-yl)benzamide, or a
pharmaceutically acceptable salt thereof.
In a 43rd specific embodiment, the compound of the present invention, is selected from the group consisting of: 9-(lH-Indazol-5-yl)-2-pyrrolidin-l-yl-9H- purine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-3-ylmethyl-amine, 9-(lH- Indazol-6-yl)-2-morpholin-4-yl-9H-purine, 9-(lH-Indazol-6-yl)-2-piperidin-l-yl-9H- purine, [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -(3 -methoxy-benzyl)-amine, [9-( 1 H- Indazol-6-yl)-9H-purin-2-yl]-pyridin-2-ylmethyl-amine, [9-(lH-Indazol-6-yl)-9H- purin-2-yl]-(2-pyridin-4-yl-ethyl)-amine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3,4,5- trimethoxy-phenyl)-amine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl- amine, (2,3-Dihydro-benzo[l,4]dioxin-6-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]- amine, 4-[9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-benzenesulfonamide, (3-Fluoro- benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine. (2,6-Difluoro-benzyl)-[9-(lH- indazol-6-yl)-9H-purin-2-yl]-amine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-((R)-l- phenyl-ethyl)-amine, and 4-((9-(lH-indazol-6-yl)-9H-purin-2-yl)amino)-2- methoxyphenol,
or a pharmaceutically acceptable salt thereof.
In a 44th specific embodiment, the compound of the present invention, is selected from the group consisting of: 2-Chloro-9-(lH-indazol-5-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-propane-l,3-diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(3-morpholin-4-yl-propyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-dimethyl-amine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-N'-methyl-ethane-l,2- diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(2-methoxy-ethyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-phenethyl-amine;
Diethyl-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-amine;
9-(lH-Indazol-5-yl)-2-morpholin-4-yl-9H-purine;
9-(lH-Indazol-5-yl)-2-(4-methyl-piperazin-l-yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-piperidin-l-yl-9H-purine; 9-( 1 H-Indazol-5 -yl)-2-pyrrolidin- 1 -yl-9H-purine;
9-(lH-Indazol-5-yl)-9H-purin-2-ylamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[3-(4-methyl-piperazin-l-yl)-propyl]- amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
2-(4-tert-Butyl-piperidin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[2-(l-methyl-piperidin-4-yl)-ethyl]- amine;
2-{4-[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperazin-l-yl}-ethanol;
2-(4-Cyclopropylmethyl-piperazin- 1 -yl)-9-( 1 H-indazol-5-yl)-9H-purine; 3 - {4- [9-( 1 H-Indazol-5 -yl)-9H-purin-2-yl] -piperazin- 1 -yl } -propionitrile;
9-(lH-Indazol-5-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine; 9-(l H-Indazol-5-yl)-2-[4-(2-methoxy-phenyl)-piperazin- 1 -yl]-9H-purine; 9-(lH-Indazol-5-yl)-2-methoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenylsulfanyl-9H-purine;
9-( 1 H-Indazol-5-yl)-2 -piperazin- 1 -yl-9H-purine;
1 -[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid ethyl ester;
9-( 1 H-Indazol-5-yl)-2-(2-methyl-imidazol- 1 -yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-(pyridin-4-ylsulfanyl)-9H-purine;
2-Chloro-9-(lH-indazol-6-yl)-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-dimethyl-amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-N'-methyl-ethane-l52- diamine;
N- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -Ν,Ν',Ν'-trimethyl-propane- 1,3- diamine;
[9-(l H-Indazol-6-yl)-9H-purin-2-yl] -pyridin-3 -ylmethyl-amine;
9-(lH-Indazol-6-yl)-2-morpholin-4-yl-9H-purine;
N-[9-(m-Indazol-6-yl)-9H-purin-2-yl]-N,N',N'-trimethyl-etliane-l,2-diamine; 9-(lH-Indazol-6-yl)-2-piperidin-l-yl-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(4-methoxy-phenyl)-methyl-amine;
(3 -Chloro-benzyl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-phenethyl-amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] - [2-(3 -methoxy-p enyl)-ethyl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-2-yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-methyl-amine;
2- {4- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -piperazin- 1 -yl } -ethanol;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methoxy-benzyl)-amine;
[2-(3-Chloro-phenyl)-ethyl]-[9-(lH-indazol-6-yl)-9H-purin-2-yl] -amine; (3,4-Dichloro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
3- {[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-methyl-amino}-benzoic acid;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-3-yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-2-ylmethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-4-yl-ethyl)-amine;
9-(lH-Indazol-6-yl)-2-[4-(2-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
4- [9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-piperidine-l-carboxylic acid ester;
(4-Dimethylamino-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine; 4-(2-Chloro-purin-9-yl)-phenol;
4-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
4-(2-Dimethylamino-purin-9-yl)-phenol;
4-(2-Pyrrolidin- 1 -yl-purin-9-yl)-phenol;
4-(2-Piperidin- 1 -yl-purin-9-yl)-phenol;
4-[2-(4-Ethyl-piperazin-l-yl)-purin-9-yl]-phenol;
4-(2-Morpholin-4-yl-purin-9-yl)-phenol;
4-{2-[(3-Dimethylamino-propyl)-methyl-amino]-purin-9-yl}-phenol; 4-{2-[(2-Dimethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
4- { 2- [4-(2-Hydroxy-ethyl)-piperazin- 1 -yl] -purin-9-yl } -phenol;
4-[2-(4-Methyl-piperazin-l-yl)-purin-9-yl]-phenol; 4-{2-[(Pyridin-3-ylmethyl)-amino]-purin-9-yl}-phenol;
4- [2-(3,4-Dichloro-benzylamino)-purin-9-yl] -phenol;
4-(2-Chloro-purin-9-yl)-2-methyl-phenol;
4-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-2-methyl-phenol;
4-(2-Dimethylamino-purin-9-yl)-2-methyl-phenol;
2-Methyl-4- [2-(3 -morpholin-4-yl-propylamino)-purin-9-yl] -phenol;
4-[2-(4-Ethyl-piperazin- 1 -yl)-purin-9-yl]-2-methyl-phenol;
2-Methyl-4- { 2- [3 -(4-methyl-piperazin- 1 -yl)-propylamino] -purin-9-yl} - phenol;
2-Methyl-4-(2-pyrrolidin- 1 -yl-purin-9-yl)-phenol;
2- Methyl-4-(2-piperidin- 1 -yl-purin-9-yl)-phenol;
4-[2-(3-Diethylamino-propylamino)-purin-9-yl]-2-methyl-phenol;
3- (2-Chloro-purin-9-yl)-phenol;
3-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
3 -(2-Dimethylamino-purin-9-yl)-phenol;
3-(2-Chloro-purin-9-yl)-benzamide;
3-(2-Dimethylamino-purin-9-yl)-benzamide;
3- {2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-benzamide;
4- (2-Methylamino-purin-9-yl)-benzamide;
4-(2-Piperidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Pyrrolidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Diethylamino-purin-9-yl)-benzamide;
4- [2-(3 -Diethylamino-propylamino)-purin-9-yl] -benzamide;
4-(2-Isobutylamino-purin-9-yl)-benzamide;
4- [2-(3 -Methyl-butylamino)-purin-9-yl] -benzamide;
4- [2-(4-Ethyl-piperazin- 1 -yl)-purin-9-yl]-benzamide;
5- (2-Chloro-purin-9-yl)-2,3-dihydro-isoindol-l-one;
N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-3-piperidin-l-yl-propionamide; 9-(lH-Indazol-6-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine; (l-Benzyl-piperidin-4-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine; 9-(lH-Indazol-6-yl)-2-pyrrolidin-l-yl-9H-purine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]-(3 ,4,5-trimethoxy-phenyl)-amine; (4-Chloro-3-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl] -amine;
{[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-methyl-amino} -acetic acid;
(S)-l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyrrolidine-2-carboxylic acid;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(l-methyl-piperidin-4-yl)-amine;
(3 -Chloro-4-fluoro-phenyl)-[9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
4-(2-Dimethylamino-purin-9-yl)-N-methyl-benzamide;
4- [2-(3 -Diethylamino-propylamino)-purin-9-yl] -N-methyl-benzamide;
(3 -Ethynyl -phenyl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-trifluoromethyl-phenyl)-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl-amine;
{l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl}-methanol;
9-(lH-Indazol-6-yl)-2-phenylsulfanyl-9H-purine;
l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carbonitrile;
(2,3-Dihydro-benzo[l,4]dioxin-6-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]- amine;
Benzo[l,3]dioxol-5-yl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl-amine;
1- [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid methyl ester;
4-[9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-benzenesulfonamide;
Biphenyl-4-ylmethyl- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
2- {4-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl}-acetamide;
{4-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl}-acetic acid methyl ester;
9-(lH-Indazol-6-yl)-2-[4-(2-methoxy-ethyl)-piperazin-l-yl]-9H-purine; 9-(lH-Indazol-6-yl)-2-piperazin-l-yl-9H-purine;
(3-Chloro-4-methoxy-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[3 -Chloro-4-(3 -fluoro-benzyloxy)-phenyl] - [9-(l H-indazol-6-yl)-9H-purin-2- yl]-amine;
[4-(3-Fluoro-benzyloxy)-3-methoxy-phenyl]-[9-(lH-indazol-6-yl)-9H-purin- 2-yl] -amine; [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]- [4-(4-methyl-piperazin- 1 -yl)-phenyl] -
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methyl-benzyl)-amine;
(3-Fluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-methoxy-benzyl)-amine;
(2,4-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(2,6-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(3 ,5 -Difluoro-benzyl)- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
Benzo [1,3] dioxol-5 -ylmethyl- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methoxy-benzyl)-methyl-amine;
4-(2-Chloro-purin-9-yl)-N-methyl-benzamide ;
N-Methyl-4-(2-thiomorpholin-4-yl-purin-9-yl)-benzamide;
N-Methyl-4-(2-piperidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Amino-purin-9-yl)-N-methyl-benzamide;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-((R)-l-phenyl-ethyl)-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-((S)-l-phenyl-ethyl)-amine;
N-Methyl-4-(2-morpholin-4-yl-purin-9-yl)-benzamide;
9-(lH-indazol-6-yl)-2-((3-(trifluoromethyl)benzyl)thio)-9H-purine;
4-(4-(9-( 1 H-indazol-6-yl)-9H-purin-2-yl)piperazin- 1 -yl)aniline;
4-((9-(lH-indazol-6-yl)-9H-purin-2-yl)amino)-2-methoxyphenol;
4-(9-(lH-indazol-6-yl)-9H-purin-2-yl)thiomorpholine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-N-methyl-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine;
9-(lH-indazol-5-yl)-2-(4-(2-methoxyethyl)piperazin-l-yl)-9H-purine;
N-((6-chloropyridin-3-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
2-(4-benzylpiperidin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-((4-methoxybenzyl)thio)-9H-purine;
2-((3 -fluorobenzyl)thio)-9-( 1 H-indazol-6-yl)-9H-purine;
2-((4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-(4-phenylpiperidin-l-yl)-9H-purine;
4-(9-(lH-indazol-6-yl)-9H-purin-2-yl)thiomorpholine 1,1-dioxide; N-((2,3-dihydrobenzo[b][l,4]dioxin-5-yl)methyl)-9-(lH-indazol-6-yl)-9H- purin-2-aniine;
N-(5-bromo-2-fluorobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-((2-methoxypyridin-4-yl)methyl)-9H-purin-2-amine; N-((2-fluoropyridin-4-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine; 2-((3-chloro-4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-5-yl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-2-amine; 9-(l H-indazol-6-yl)-2-(2 -methyl- 1 H-imidazol- 1 -yl)-9H-purine; and
N-methyl-4-(2-(pyrrolidin- 1 -yl)-9H-purin-9-yl)benzamide,
or a pharmaceutically acceptable salt thereof.
A second embodiment of the invention is a pharmaceutical composition comprising any one of the compounds of Formulas (I), (IA), (IB), (IA2), (IB2), one (IC), or a compound as described in any one of the specific embodiments 1 -44, or a pharmaceutically acceptable salt, hydrate, or ester thereof, and a pharmaceutically acceptable carrier or excipient.
A third embodiment of the invention is a method of treating an autoimmune disease or a symptom thereof comprising administering to a subject an effective amount of any one of the compounds of Formulas (I), (IA), (IB), (IA2), (IB2), one (IC), or a compound as described in any one of the specific embodiments 1-44, or a pharmaceutically acceptable salt thereof. Particularly, the autoimmune disease is selected from rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, seronegative spondyloarthropathies, and ankylosing spondylitis. Further, particularly, the autoimmune disease is rheumatoid arthritis, wherein the rheumatoid arthritis includes extra-articular manifestations selected from vasculitis, pericarditis, myocarditis, and Sjogren's syndrome. Further, particulary, the autoimmune disease is selected from inflammatory bowel disease, Crohn's disease, and ulcerative colitis. Further particularly, the autoimmune disease is inflammatory bowel disease, wherein the inflammatory bowel disease includes extraintestinal inflammatory conditions selected from peripheral arthritis, ankylosing spondylitis, sacroiliitis, uveitis, and primary sclerosing cholangitis. Further particularly, the autoimmune disease is selected from psoriasis, graft-versus-host disease, systemic lupus erythematosus, sarcoidosis, granulomatosis, vasculitis, asthma, Sjogren's syndrome, type I diabetes, peripheral arthritis, sacroiliitis, uveitis, primary sclerosing cholangitis, pericarditis, and myocarditis. Further particularly, the autoimmune disease is multiple sclerosis (MS).
Another specific embodiment of the present invention, is a method of treating an autoimmune disease or a symptom thereof as described in the third embodiment, wherein the compound is administered sequentially or concomitantly with an agent selected from non-steroidal anti-inflammatory drugs, corticosteroids, analgesics, and antibiotics
A fourth embodiment of the present invention is a method of modulating the activity of one or more kinases selected from the group of Flt3, CSF-1R, ACVR1 (ALK2), CDK2/CyclinA, CDK5/p25, CDK5p35, CLK4, EPHA1, FLT4 (VEGFR3), GSG2 (Haspin), KDR (VEGFR2), LRRK2, LRRK2 G2019S, MAP3K9 (MLK1), MAP3K10 (MLK2), MAPK3K11 (MLK3), MELK, MUSK, NLK, NTRKl (TRKA), NTRK3 (TRKC), PDGFRA, PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), RIPK2 and ROSl, comprising administering to a subject an effective amount of any one of the compounds of Formulas (I), (IA), (IB), (IA2), (IB2), one (IC), or a compound as described in any one of the specific embodiments 1-44, or a pharmaceutically acceptable salt thereof.
A fifth embodiment of the present invention is a method of treating cancer or bone metastases comprising administering to a subject an effective amount of any one of the compounds of Formulas (I), (IA), (IB), (IA2), (IB2), one (IC), or a compound as described in any one of the specific embodiments 1-44, or a pharmaceutically acceptable salt thereof. Particularly, the cancer is gliobastoma or astrocytoma.
Further particularly, the cancer is leukemia with Flt3 mutations.
A sixth embodiment of the present invention is a method for making a compound of Formula (I),
Figure imgf000047_0001
(I)
comprising: reacting a compound of formula (A)
Figure imgf000048_0001
(A)
with triethylformate in the presence of anhydrous magnesium sulfate to produce a compound of formula (I). The method can further include the step of of preparing the compound of formula (A) by reacting 5-amino-2,4-dichloro pyrimidine with sodium acetate b la (B)
Figure imgf000048_0002
(B)
to obtain a compound of formula (A).
As used herein, "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.
As used herein, "alkyl" refers to a straight-chain or branched saturated hydrocarbon group. As referred to herein, any "alkyl" or "(Ca-Cb)"alkyl (with "a" and "b" being whole numbers) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the alkyl group is substituted, or, it is specifically stated that the alkyl group is unsubstituted. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl, and isopropyl,), butyl (e.g., n- butyl, isobutyl, s-butyl, t-butyl), pentyl groups (e.g., n-pentyl, isopentyl, neopentyl), and the like. A lower alkyl group typically has up to 6 carbon atoms. In various embodiments, an alkyl group has 1 to 6 carbon atoms, and is referred to as a "Ci-6 alkyl group", "C1-C6 alkyl group", or (Cl-C6)alkyl group. Examples of C1-6 alkyl groups include, but are not limited to, methyl, ethyl, propyl (e.g., n-propyl and isopropyl), and butyl groups (e.g., n-butyl, isobutyl, s-butyl, t-butyl). A branched alkyl group has at least 3 carbon atoms (e.g., an isopropyl group) and up to 6 carbon atoms, e.g. it is a C3-6 alkyl group, i.e., a branched lower alkyl group. Examples of branched lower alkyl groups include, but are not limited to, isopropyl, isobutyl, sec- butyl, tert-butyl, isopentyl, neopentyl, and tert-pentyl. Substituents on alkyl groups include hydroxyl, oxo, amino, cyano, (Cl-C3)alkylamino, di(Cl-C3)alkylamino, halogen (typically, F, CI, and Br), (Cl-C3)alkoxy, (C3-C6)cycloalkyl, a 5- to 6- membered aryl or heteroaryl, -C(0)ORG2 or -C(0)NRH1RJ1, wherein RG2 is hydrogen or (Cl-C4)alkyl, and RH1 and RJ1 each independently are hydrogen or a (Cl-C3)alkyl.
As used herein, "alkenyl" refers to a straight-chain or branched alkyl group having one or more carbon-carbon double bonds. As referred to herein, any "alkenyl" or "(Ca-Cb)"alkenyl (with "a" and "b" being whole numbers, and "a" at least 2) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the alkenyl group is substituted, or, it is specifically stated that the alkenyl group is unsubstituted. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like. The one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene). A branched alkenyl group has at least 3 carbon atoms, and in various embodiments, has up to 6 carbon atoms, e.g. it is a C3-6 alkenyl or (C3-C6)alkenyl group.
The term "alkynyl" refers to a straight-chain or branched alkyl group having one or more carbon-carbon triple bonds. As referred to herein, any "alkynyl" or "(Ca- Cb)"alkynyl (with "a" and "b" being whole numbers, and "a" at least 2) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the alkynyl group is substituted, or, it is specifically stated that the alkynyl group is unsubstituted. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. The one or more carbon-carbon triple bonds can be internal (such as in 2-butyne) or terminal (such as in 1-butyne). The alkynyl group is suitably a C2-6 alkynyl group.
As used herein, "alkoxy" refers to an -O-alkyl group wherein the alkyl group may be a straight or branched chain. As referred to herein, any "alkoxy" or "(Ca- Cb)"alkoxy (with "a" and "b" being whole numbers) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the alkoxy group is substituted, or, it is specifically stated that the alkoxy group is unsubstituted.
Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy groups, and the like.
As used herein, "haloalkyl" refers to an alkyl group having one or more halogen substituents. Examples of haloalkyl groups include, but are not limited to, -CF3, -C2F5, -CHF2, -CH2F, -CC13, -CHC12, -CH2C1, -C2C15, and the like. Perhaloalkyl groups, i.e., alkyl groups wherein all of the hydrogen atoms are replaced with halogen atoms (e.g., CF3 and C2F5), are included within the definition of "haloalkyl."
As used herein, "heteroatom" refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, sulfur, phosphorus, and selenium.
As used herein, "non-aromatic heterocyclic group" or "heterocyclyl" refers to a non-aromatic cycle having 5-7 ring atoms (unless the number of "ring atoms" or "members" is specifically provided), among which 1 to 3 ring atoms (unless specifically provided) are heteroatoms independently selected from oxygen (O), nitrogen (N) and sulfur (S) (unless specifically provided), and that optionally contains one or more, e.g., two, double or triple bonds. One or more N or S atoms in a non- aromatic heterocyclic group ring can be oxidized (e.g., morpholine N-oxide, thiomorpholine S- oxide, thiomorpholine S,S-dioxide). Non-aromatic heterocyclic groups can also contain one or more oxo groups, such as piperidone, oxazolidinone, pyrimidine-2,4(lH,3H)-dione, pyridin-2(lH)-one, and the like. As referred to herein, any "non-aromatic heterocyclic group", "heterocyclyl" or a- to b-membered heterocyclyl (with "a" and "b" being whole numbers typically between 5 and 8 with b larger than a) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the heterocyclyl group is substituted, or, it is specifically stated that the heterocyclyl group is unsubstituted. A heterocyclyl can be substituted on any substitutable atoms including heteroatoms (e.g., N). Examples of non-aromatic heterocyclic groups and specifically for C1, C2, and C3, include, among others, morpholine, thiomorpholine, pyran, imidazolidine, imidazoline, oxazolidine, pyrazolidine, pyrazoline, pyrrolidine, pyrroline,
tetrahydrofuran, tetrahydrothiophene, piperidine, piperazine, and the like, all of which can be optionally substituted.
Particular examples for C1 include piperidinyl and
Figure imgf000051_0001
, all of which can be optionally substituted.
Particular examples for C2 and C3 include piperidinyl, piperazinyl, thiomorpholinyl, morpholinyl, pyrrolidinyl, and imidazolyl, all of which can be optionally substituted.
More particularly, examples for C include piperidinyl, morpholinyl and piperazinyl, all of which can be optionally substituted.
A non-aromatic heterocyclic group can be optionally substituted. The non- aromatic heterocyclic group may be C-attached or N-attached (where such is possible). Heterocyclyl groups (and, particularly, C1) can be substituted with C1-C3 alkyl, phenyl, benzyl or -C(0)ORE, wherein RE is hydrogen or a C1-C4 alkyl.
Heterocyclyl groups (and, particularly, C2) can further be substituted with one to three substituents R , wherein R , for each occurrence independently, is selected from hydroxyl, cyano, a halogen, a C1-C6 alkyl, -C(0)OR , or a 5- to 6-membered aryl or heteroaryl, wherein said C1-C6 alkyl is optionally substituted with RF and said 5- to 6-membered aryl or heteroaryl is optionally substituted with a hydroxyl, a C1-C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, -C(0)OR , or
-C(0)NRHRJ, wherein RF is hydroxyl, cyano, a C1-C3 alkyl, a C3-C6 cycloalkyl, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, a C1-C3 alkoxy, a 5- to 6- membered heteroaryl or phenyl, -C(0)ORG2 or -C(0)NRH1RJ1; RG, RG1, and RG2 are each independently hydrogen or a C1-C4 alkyl; and RH, RJ, RHI, RJ1 each
independently are selected from hydrogen or a C1-C3 alkyl. Heterocyclyl groups (and, particularly, C3) can further be substituted with a group R100 selected from a CI- C6 alkyl, a C3-C6 cycloalkyl, phenyl, cyano, hydroxyl and -C(0)ORlul, wherein R1U1 is methyl or ethyl.
As used herein, "oxo" refers to a double-bonded oxygen (i.e., =0).
As used herein, "heteroaryl" refers to an aromatic monocyclic ring system or a polycyclic ring system where at least one of the rings present in the ring system is aromatic, containing 5-7 or 5-9 ring atoms (unless specifically provided otherwise), among which 1 to 3 ring atoms (unless specifically provided otherwise) are heteroatoms independently selected from oxygen (O), nitrogen (N) and sulfur (S). Polycyclic heteroaryl groups include two or more heteroaryl rings fused together, and monocyclic heteroaryl rings fused to one or more aromatic carbocyclic rings, non- aromatic carbocyclic rings, and/or non-aromatic non-aromatic heterocyclic group rings. The heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain 0-0, S-S, or S-0 bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S, S -dioxide).
As referred to herein, "heteroaryl" or a- to b-membered heteroaryl (with "a" and "b" being whole numbers typically between 5 and 9 with b larger than a) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the heteroaryl group is substituted, or, it is specifically stated that the heteroaryl group is unsubstituted. A heteroaryl can be substituted on any substitutable atoms including heteroatoms (e.g., N).
Examples of heteroaryl groups and specifically for Ar1, Ar2, and Ar3, include, for example, the 5- and 6-membered monocyclic ring systems shown below:
Figure imgf000053_0001
where Z is O, S, or NH, wherein the solid line indicates connectivity to the remainder of the molecule. Examples of such heteroaryl rings include, but are not limited to, pyrrole, furan, thiophene, pyridine, pyrimidine, pyridazine, pyrazine, triazole, tetrazole, pyrazole, imidazole, isothiazole, thiazole, thiadiazole, isoxazole, oxazole, and oxadiazole. By way of example, the heteroaryl ring may be attached at C-2, C-3, or C-4; that is, be pyridin-2-yl, pyridine-3-yl, or pyridine-4-yl. The foregoing heteroaryl groups may be C-attached or N-attached (where such is possible). For instance, a group derived from pyrrole may be pyrrol-l-yl (N-attached) or pyrrol-3-yl (C-attached).
The term "aryl" means a carbocyclic aromatic ring system with five to fourteen carbon atoms (unless specifically stated otherwise). As referred to herein, "aryl" or "a- to b-membered aryl" (with "a" and "b" being whole numbers typically between 5 and 6 with b larger than a) can be optionally substituted (i.e., substituted or unsubstituted), unless it is specifically stated that the aryl group is substituted, or, it is specifically stated that the aryl group is unsubstituted. Examples for aryl groups and specifically for Ar1, Ar2, and Ar3 include, among others, phenyl, naphthyl, indanyl or tetrahydronaphthalene, all of which can be optionally substituted. Exemplary substituents include alkyl, alkoxy, alkylthio, alkylsulfonyl, halogen, trifluoromethyl, dialkylamino, nitro, cyano, C02H, CONH2, N-monoalkyl-substituted amido and N,N- dialkyl-substituted amido. The term "aryl" may be used interchangeably with the terms "aryl ring" "carbocyclic aromatic ring", "aryl group" and "carbocyclic aromatic group". Particular examples for Ar include optionally substituted phenyl and pyridyl.
A particular example for Ar2 is optionally substituted imidazolyl.
Particular examples for Ar3 include pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl, all of which can be optionally substituted.
Heteroaryl and aryl groups (and, particularly, Ar1) can be substituted with one to three substituents Rc selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl- C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two Rc groups taken together with the intervening atoms form a 1,3- dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
Heteroaryl and aryl groups (and, particularly, Ar2) can further be substituted with one to three substituents RC1 selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen, or, alternatively, two RC1 groups taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
Heteroaryl and aryl groups (and, particularly, Ar3) can further be substituted with one to three substituents R°, wherein each R° is independently selected from:
a halogen,
hydroxyl,
cyano,
- -S(02)NH2 or -S(02)N(CH3)2;
amino,
- a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally
substituted with a halogen or a 5- or 6-membered aryl or heteroaryl, a 5-6-membered aryl or heteroaryl;
a 5- to 7-membered non-aromatic heterocyclic ring including one or two heteroatoms selected form O, N and S;
- -C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl,
(C 1 -C3)alkylamino,
- di(Cl-C3)alkylamino, wherein, optionally, the alkyl portions of the di(Cl- C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl, optionally including one additional heteroatom selected from N, O or S; and
a C1-C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a 5- or 6- membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy, or, alternatively,
two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
As used herein, a "protecting group" ("PtG") refers to modification of a functional group that reduces the reactivity of the functional group in an unwanted reaction. Examples of protecting groups for amines include, but are not limited to, tert-butyloxycarbonyl (t-BOC), benzyl (Bn), and carbobenzyloxy (Cbz) groups.
Examples of protecting groups for carbonyls include, but are not limited to, acetals and ketals. Examples of protecting groups for carboxylic acids include, but are not limited to, methyl esters, benzyl esters, tert-butyl esters, and silyl esters. See Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, the entire disclosure of which is incorporated by reference herein for all purposes.
At various places in the present specification, substituents of compounds are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, the term "C1-6 alkyl" is specifically intended to individually disclose Q, C2, C3, C4, C5, C6, Ci-C6, Q-C5, Cj-C4, C1-C3, C C2, C2-C6, C2-Cs, C2-C4, C2-C3, C3- C6, C3-C5, C3-C , C4-C6, C -C5, and C5-C6 alkyl. By way of another example, the term "5-9 membered heteroaryl group" is specifically intended to individually disclose a heteroaryl group having 5, 6, 7, 8, 9, 5-9, 5-8, 5-7, 5-6, 6-9, 6- 8, 6-7, 7-9, 7-8, and 8- 9 ring atoms.
Representative compounds of Formula (I) in accordance with embodiments of the present invention include, but are not limited to, the compounds presented in Table 1 below. Table 1
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Pharmaceutically acceptable salts of the compounds of Formula (I), which can have an acidic moiety, can be formed using organic and inorganic bases. Both mono and polyanionic salts are contemplated, depending on the number of acidic hydrogens available for deprotonation. Suitable salts formed with bases include metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, or magnesium salts; ammonia salts and organic amine salts, such as those formed with morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine (e.g., ethyl-tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a mono-, di-, or trihydroxy lower alkylamine (e.g., mono-, di- or triethanolamine). Specific non-limiting examples of inorganic bases include NaHC03, Na2C03, KHC03, K2C03, Cs2C03, LiOH, NaOH, KOH, NaH2P04, Na2HP04, and Na3P04. Internal salts also can be formed. Similarly, when a compound disclosed herein contains a basic moiety, salts can be formed using organic and inorganic acids. For example, salts can be formed from the following acids: acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, dichloroacetic,
ethenesulfonic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, malonic, mandelic, methanesulfonic, mucic, napthalenesulfonic, nitric, oxalic, pamoic, pantothenic, phosphoric, phthalic, propionic, succinic, sulfuric, tartaric, toluenesulfonic, and as well as other known pharmaceutically acceptable acids.
Pharmaceutically acceptable esters in the present invention refer to non-toxic esters of the compounds of Formula (I), preferably the alkyl esters such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl esters, of which the methyl ester is preferred. However, other esters such as phenyl-Ci-5 alkyl may be employed if desired. Examples of pharmaceutically acceptable esters include, but are not limited to, C2-6 alkyl esters such as methyl esters and ethyl esters. Pharmaceutically acceptable esters include esters made with aliphatic carboxylic acids, preferably those with a linear chain of between two and six carbon atoms, preferably acetic acid, and made with aromatic carboxylic acids, e.g. C7-12 acids such as benzoic acid. The aliphatic and aromatic acids may optionally be substituted by one or more Cj4 alkyl groups.
Also provided in accordance with the present teachings are prodrugs of the compounds disclosed herein. As used herein, "prodrug" refers to a moiety that produces, generates or releases a compound of the present teachings when
administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either by routine manipulation or in vivo, from the parent compounds.
Examples of prodrugs include compounds as described herein that contain one or more molecular moieties appended to a hydroxyl, amino, sulfhydryl, or carboxyl group of the compound, and that when administered to a mammalian subject, is cleaved in vivo to form the free hydroxyl, amino, sulfhydryl, or carboxyl group, respectively. Examples of prodrugs can include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present teachings. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, the entire disclosures of which are incorporated by reference herein for all purposes.
Compounds of Formula (I) in accordance with the present invention can be prepared as outlined in the schemes below and as illustrated in the examples, from (a) commercially available starting materials, (b) compounds known in the literature, or readily prepared intermediates using literature procedures, or (c) new intermediates described in the schemes and experimental procedures herein.
Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but one skilled in the art can determine such conditions by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented may be varied for the purpose of optimizing the formation of the compounds described herein. Reactions are performed in a solvent appropriate to the reagents and materials employed and suitable for the transformation being effected. Suitable solvents typically are substantially nonreactive with the reactants, intermediates, and/or products at the temperatures at which the reactions are carried out, i.e., temperatures that can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected. One skilled in the art of organic synthesis can readily select suitable solvents.
It is understood by those skilled in the art of organic synthesis that the various functionalities present on the molecule must be consistent with the chemical transformation proposed. This may necessitate routine judgment as to the order of synthetic steps, and the need for protecting groups for remote functionalities. One skilled in the art can readily determine the need for protection and deprotection and select appropriate protecting groups. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, the entire disclosure of which is herein incorporated by reference.
The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., lH or 13C), infrared spectroscopy, spectrophotometry (e.g., UV -visible), or mass
spectrometry, or by chromatography such as high performance liquid chromatograpy (HPLC) or thin layer chromatography (tic or t.l.c).
In the schemes provided herein, unless expressed to the contrary, variables in chemical formulae are as defined in other formulae herein. Reference to R, Ra, Rb, Rc, and Rd in the following schemes are a generic representation, R, Ra, Rb, R°, and Rd do not have to be same at each occurrence, and R, Ra, Rb, R°, and Rd can be selected from, for example, R1, R2, R3, R4, R5, R6, A1, A2, among others as appropriate, and substituted in accordance with the teachings herein. The 9-aryl purine compounds of Formula (I) may be prepared via a number of routes as shown in Scheme 1. In one route shown in Scheme 1, the 4 position of Compound II can be substituted by reaction by a suitable aniline (step g) followed by amination of the 2-pyrimidine position in n-butanol (step j) to produce a compound of structure VII. Reduction of the nitro moiety (steps h or i) and subsequent purine ring formation (steps d or e) yields compounds of Formula (I) using methods as described in Cole et al., Tetrahedron Lett. 47:8897-8890, 2006; and Legraverend, Tetrahedron, 64:8585-8603, 2008.
Alternatively, also as shown in Scheme 1, 5-amino-2,4-dichloropyrimidine (III) is obtained by reduction of 2,4-dichloro-5-nitropyrimidine (II) with iron in acetic acid (step a) using procedures described in Inoue, Chem. Pharm. Bull. 6:343-346, 1958. Preparation of Compound IV is achieved by addition of a suitably substituted aniline to Compound III (steps b or c) and the subsequent purine ring formation can be performed in two steps using methodologies illustrated by Tanji, K.I. et al, Chem. Pharm. Bull. 35:4972-4976, 1987 or Aguado et al, J. Comb. Chem., 2009, 11 :210- 212. The final amination step at the 2-position of the purine ring is achieved in n- butanol with an organic base.
SCHEME 1
Figure imgf000084_0001
a) AcOH, Fe; b) EtOH, H20, HCI; c) NaOAc, AcOH, H20; d) A¾0, HC(OEt)3; e) HC(OEt)3l DMF, Mg2S04; f) n-BuOH, DMF, HR1; g) EtOH, NaC03; h) Na2S204, H20; i) SnCI2l EtOH; j) NaH, HR1
In particular, compounds of Formula (I) in accordance with aspects of the invention were prepared as shown in Scheme 2. Intermediate 5-amino-2,4-dichloro pyrimidine (III) was prepared by reduction of 2,4-dichloro-5-nitropyrimidine with iron powder in glacial acetic acid to yield (step a). The 5-aminopyrimidine derivative (III) was regio selectively coupled with an aniline with the appropriate substituents under acidic conditions (steps b or c) to yield Compound IV, which could be
converted to the purine compounds of Formula V using known methods (step d, acetic anhydride and triethylorthoformate).
Alternatively, compounds of Formula V can be prepared using novel reaction conditions in accordance with embodiments of the invention requiring condensation of triethylorthoformate in the presence of anhydrous magnesium sulfate (step e). Step e has the advantage of preparing high purity material in high yield. Compounds prepared via this reaction can be used without further purification in contrast to those prepared using the reaction conditions of step d. Compounds of Formula (I) were prepared via displacement of the 2- chloropurine moiety with alkylamines with the appropriate substitution pattern and substituents to obtain the desired compounds, such as those shown in Table 1.
SCHEME 2
Figure imgf000085_0001
Figure imgf000085_0002
a) AcOH, Fe; b) EtOH, H20, HCI; c) NaOAc, AcOH, H20; d) A¾0, HC(OEt)3; e) HC(OEt)3, DMF, Mg2S04; f) n-BuOH, DMF, HR1
Compounds in accordance with the invention can be evaluated by in vitro or in vivo assays. For example, kinase profiling of the compounds can be assessed using commercially available platforms such as the SelectScreen™ platform from
Invitrogen, Inc. (Carlsbad, CA, USA). Animal models for various diseases, including autoimmune diseases, are known in the art, and include, for example, Collagen- Induced Arthritis (CIA) and Collagen Antibody-Induced Arthritis (CAIA) for rheumatoid arthritis (RA), Experimental Autoimmune Encephalomyelitis (EAE) for multiple sclerosis (MS), dextran sodium sulfate (DSS)-induced murine colitis for inflammatory bowel disease (IBD) and 2,4,6-trinitrobenzene sulfonic acid (TNBS)- induced murine colitis for Crohn's Disease, Ova-Induced- Asthma for chronic obstructive pulmonary disease (COPD) or asthma, and granuloma and air pouch models for inflammation, among others. Various models can be used to study chronic or acute presentations of disease. The present teachings relate to in vitro or in vivo methods of modulating the activity of protein tyrosine kinases including, Flt3 and CSF-1 . Protein tyrosine kinases are major biological effectors which have become targets for drug
development due to their central role in regulatory signal transduction. The structures of all major kinases and their mutants are known, have been cloned and the functional domains, e.g. the juxtamembrane, ATP and catalytic components projecting from the cell membrane into the cytosol have been expressed through recombinant genetic techniques. The recombinant enzymes have been assembled into test panels to exploit the fact that in the presence of ATP they will phosphorylate an appropriate substrate and the phosphorylation rate and extent can then be measured via an optical reporter system. This testing stratagem and its application in drug development has been discussed in a review by M Vieth et al. (Drug Disc. Today 10: 839-846, 2005). The translation of in vitro results and the correlation to in vivo cellular assays is considered to be high and has been validated in cellular models, for example, in the work of J.S. Melnik et al. (Proc. Nat. Acad. Sci. 103:3153-3158, 2006).
Evaluation of representative compounds indicated that the compounds of the present teachings can modulate the activity of RTKs such as Flt3, CSF-1R, ACVR1 (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRK1 (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), and RIPK2, and ROS1. In certain
embodiments, the RTK is Flt3 or CSF-1R.
In certain embodiments, compounds of the invention selectively inhibit Flt3. As used herein, selectively means having a degree of specificity greater than about 90%, preferably greater than about 95%, and most preferably, greater than about 97%, where the degree of specificity can be quantified as a percent taken from the number of off-target leads expressed as a proportion of the total number of enzymes assayed, for example, using the panel of either 299 or 304 kinases according to the teachings herein.
In a novel and unprecedented finding within medicinal chemistry directed at Flt3 inhibition, we have discovered compounds of Formula (I) that selectively inhibit Flt3 over KIT, KDR, and INSR. In certain preferred embodiments, about 2 log units separate the corresponding median inhibitory effect concentration (IIC50) values for inhibition of KIT and KDR from the IC50 against Flt3. In certain preferred embodiments, the IC50 for compounds of Formula (I) for inhibition of the insulin receptor, INSR, is about 2000-fold greater than the IC50 for inhibition of Flt3.
Certain preferred compounds that are selective Flt3 inhibitors include those in Table 2:
Table 2
Figure imgf000087_0001
100 [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3,4,5-trimethoxy-phenyl)-amine
104 [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(l -methyl -piperidin-4-yl)-amine
110 [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl-amine
129 (3-Fluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine
140 [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -((R)- 1 -phenyl-ethyl)-amine
145 4-((9-(lH-indazol-6-yl)-9H-purin-2-yl)amino)-2-methoxyphenol
149 9-(lH-indazol-6-yl)-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine
Flt3 has been linked to the negative regulation of IL-10, a principal regulator of the immunogenic-tolerogenic balance toward tolerogenic Th2 and T-reg biology, wherein silencing Flt3 downregulates T-cell reactivity and T-reg signaling by upregulation of IL-10 (see, Astier et al, J.Immunology, 184:685 -693, 2010). IL-10 has a role in many diseases such as IBD, MS, and asthma, among others. In various animal models, IL-10-deficiency in mice leads to the development of colitis while IL- 10 treatments have been shown effective in inhibiting the development of type I diabetes and EAE. In arthritis, IL-10 may reduce Thl7 and macrophages that cause joint and bone erosion, while having only a limited impact on reducing joint inflammation. Compounds of the present invention can be used to modulate production of IL- 10.
In further embodiments, we have discovered that compounds of Formula (I) can modulate the activity of the following kinases: Flt3, CSF-IR, ACVRl (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRKl (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), ROS1, and RIPK2.
A variety of pathological conditions, states, disorders or diseases, including autoimmune diseases, can be treated by modulating the activity of one or more of these kinases. Compounds of Formula (I) can be used in the treatment of disorders mediated by one or more kinase selected from Flt3, CSF-IR, ACVRl (ALK2), GSG2 (Haspin), LRRK2, LRRK2 G2019S, MELK, MUSK, NLK, NTRKl (TRKA), NTRK3 (TRKC), PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pi 10 alpha/p85 alpha), PTK6 (Brk), ROS1, and RIPK2. In particular, ROS1 has been shown to be activated in gliobastomas and astrocytomas. As used herein, disorders mediated by one or more of these kinases are disorders in which one or more symptoms can be inhibited, alleviated, reduced or whose onset can be delayed by completely or partially inhibiting the protein kinase.
In certain specific aspects, the RTK is Flt3 or CSF-IR, and compounds of the present invention are useful in the treatment of a Flt3-mediated disorder or a CSF-1R- mediated disorder.
As used herein, the terms "Flt3 -mediated disorder" and "CSF-lR-mediated disorder" refer to disorders in which one or more symptoms can be inhibited, alleviated, reduced or whose onset can be delayed by completely or partially inhibiting the protein kinase Flt3 or the protein kinase CSF-IR, respectively.
Inhibition of Flt3 has been shown to suppress immune response, possibly via inhibition of DCs-induced stimulation of T cells, and may be considered for the treatment of autoimmune diseases (see, WO 2006/020145 A2; Whartenby, et al, Proc. Nat. Acad. Sci., 102: 16741-16746, 2005). Flt3-mediated disorders and conditions include autoimmune disorders such as ankylosing spondylitis, arthritis, aplastic anemia, Behcet's disease, type 1 diabetes mellitus, graft-versus-host disease, Graves' disease, autoimmune hemolytic anemia, Wegener's granulomatosis, hyper IgE syndrome, idiopathic thrombocytopenia purpura, rheumatoid arthritis, Crohn's disease, multiple sclerosis, Myasthenia gravis, psoriasis, and lupus, transverse myelitis, and amyotrophic lateral sclerosis; neurodegenerative diseases such as infantile spinal muscular atrophy and juvenile spinal muscular atrophy, Creutzfeldt- Jakob disease; and subacute sclerosing panencephalitis; and cancers such as leukemia including acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL),and hairy cell leukemia (HCL).
In particular, acute myeloid leukemia often presents with mutated Flt3. In particular, internal tandem duplications (ITD) in the juxtamembrane domain or mutations in the activating loop of Flt3 are observed in a proportion of patients with AML, and result in constitutive kinase activation leading to stimulation of multiple signaling pathways (see, Small, D. Hematology 2006 (1): 178); Flt3 kinase inhibitors are under clinical investigation for use in treating AML.
CSF-lR-mediated disorders and conditions include autoimmune disorders such as sarcoidosis, asthma, psoriasis, diabetes, Sjogren's syndrom, and uveitis;
cardiovascular disease involving chronic inflammation (e.g. atherosclerosis ); cancers such as osteolytic sarcoma, myeloma, breast cancer, tumor metastasis to bone, uterine cancer, stomach cancer, and hairy cell leukemia; diseases with an inflammatory component including glomerulonephritis, prosthesis failure, congestive obstructive pulmonary disease, pancreatitis, HIV infection, tumor related angiogenesis, age- related macular degeneration, diabetic retinopathy, restenosis, schizophrenia, skeletal pain caused by tumor metastasis or osteoarthritis, or visceral, inflammatory, and neurogenic pain, osteoporosis, Paget's disease, and prosthesis failure.
Evaluation of representative compounds according to embodiments of this invention indicated that the compounds of the present teachings can modulate dendritic cell maturation and activation.
Evaluation of representative compounds according to embodiments of this invention indicated that compounds of Formula (I) can be used in the treatment of autoimmune disorders or symptoms thereof.
Accordingly, in one embodiment, the present invention is a method of treating a patient suffering from an autoimmune disease by administering to a patient suffering from such disorders a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
In certain embodiments, compounds of Formula (I) are used in the treatment of an autoimmune disease or symptom thereof selected from multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, juvenile arthritis, ankylosing spondylitis (AS), inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis (UC), graft-versus-host disease (GvHD), systemic lupus erythematosus (SLE), sarcoidosis, granulomatosis, vasculitis, seronegative spondyloarthropathies, asthma, Sjogren's syndrome, and type I diabetes. The terms "inflammatory bowel disease" or "IBD" as used herein includes various inflammatory conditions of the colon and small intestine, and includes Crohn's disease and ulcerative colitis (UC). UC primarily affects the mucosal lining of the large intestine and rectum, while Crohn's disease may involve any part of the GI tract. Extraintestinal manifestations are often associated with IBD, and include inflammatory conditions such as peripheral arthritis, ankylosing spondylitis, sacroiliitis, uveitis, and primary sclerosing cholangitis, among others. As used herein, treatment of IBD and symptoms thereof includes treatment of both intestinal and extraintestinal aspects.
In accordance with some embodiments, preferred compounds useful for the treatment of a patient with IBD, UC, or Crohn's disease, or symptoms thereof, are represented by Structural Formula(IAl), (IA2), (IB1) or (IB2). In particular aspects, compounds represented by Structural Formula (IA1) and (IB2) useful for the treatment of a patient with IBD, UC, or Crohn's disease , or symptoms thereof, are Compounds 12, 36, 47, 129.
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by the chronic erosive inflammation in joints leading to the destruction of cartilage and bones. As used herein, the terms "rheumatoid arthritis" and "RA" include Juvenile Rheumatoid Arthritis (JRA) and Juvenile Idiopathic Arthritis (JIA). Extra-articular manifestations of RA include vasculitis, pericarditis, myocarditis, and Sjogren's syndrome, among others. Psoriatic arthritis, a chronic inflammatory arthritis, often accompanies psoriasis and is clinically treated similarly to RA. As used herein, treatment of RA and symptoms thereof includes treatment of both joint inflammatory and extra-articular aspects.
In accordance with some embodiments, compounds of Formula (I) useful for the treatment of a patient with RA or psoriatic arthritis , or symptoms thereof, have structure (IA1), (IA2), (IB2) or (IB1). In particular aspects, compounds of Formula (I) useful for the treatment of a patient with RA or psoriatic arthritis , or symptoms thereof, are Compounds 5, 36, 12, 47, 50, 54, 100, and 129. Multiple sclerosis (MS) is characterized by patches of demyelination in the brain and spinal cord, and is an example of a demyelinating condition. As used herein, a "demyelinating condition" is a condition that destroys, breaks the integrity of or damages a myelin sheath, the insulating layer surrounding vertebrate peripheral neurons that increases the speed of conduction and formed by Schwann cells in the peripheral or by oligodendrocytes in the central nervous system. In addition to MS, other demyelinating autoimmune conditions include Chronic Immune Demyelinating Polyneuropathy (CIDP); multifocal CIDP; inflammatory demylinating
polyneuropathy, autoimmune peripheral neuropathy; and POEMS syndrome
(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes).
In accordance with some embodiments, compounds useful for the treatment of a patient with MS or demyelinating autoimmune conditions have structure (IA1) or (IB1). In particular aspects, compounds of Formula (IA1) and (IB1) useful for the treatment of a patient with MS or demyelinating autoimmune conditions are
Compounds 12 and 37.
In some embodiments of this invention, compounds of Formula (I) can be used in the treatment of cancer. As used herein, the term "cancer" refers to the uncontrolled growth of abnormal cells that have mutated from normal tissues. A cancerous tumor (malignancy) is of potentially unlimited growth and expands locally by invasion and systemically by metastasis. Examples of cancers that can be treated by the compounds of the present invention include: breast cancer, colorectal cancer, non-small cell lung cancer, ovarian, renal, sarcoma, melanoma, head & neck, hepatocellular, thyroid, leukemia, lymphoma, multiple myeloma, esophageal, large bowel, pancreatic, mesothelioma, carcinoma (e.g. adenocarcinoma, including esophageal adenocarcinoma), sarcoma (e.g. spindle cell sarcoma, liposarcoma, leiomyosarcoma, abdominal leiomyosarcoma, sclerosing epithelioid sarcoma) and melanoma (e.g. metastatic malignant melanoma). In some embodiments, the patient can be treated for certain leukemias, including Flt3 -mediated leukemias such as acute myeloid leukemia characterized by one or more Flt3 mutations. "Treating a subject suffering from cancer" includes achieving, partially or substantially, one or more of the following: arresting the growth or spread of a cancer, reducing the extent of a cancer (e.g., reducing size of a tumor or reducing the number of affected sites), inhibiting the growth rate of a cancer, and ameliorating or improving a clinical symptom or indicator associated with a cancer (such as tissue or serum components).
In one embodiment, the patient can be treated for bone metastases. Bone is one of the most common metastatic sites for cancers, such as breast, lung and prostate cancers. In the process of bone metastasis, metastatic tumour cells in bone enhance bone resorption (osteolysis) by inducing and activating osteoclasts. This in turn releases growth factors, such as TGF-β and IGF-1 from the bone matrix that promote tumour growth. Osteoclasts are multinucleated cells formed by fusion of Flt3 -positive monocyte-macrophage cells, which can also differentiate to dendritic cells and macrophages. Therefore suppression of hyperactive osteoclasts (OCL), and thus osteolysis, may be effective against bone metastasis. "Treating bone metastases", as used herein, refers to reducing (partially or completely) the size of the bone metastases, slowing the growth of the metastases relative to the absence of treatment and reducing the extent of further spread of the cancer, and also includes pain reduction, decreased incidents of fractures, relief of spinal cord compression, control of hypercalcaemia, and/or restoration of normal blood cell counts.
The present teachings therefore include methods of administering to a mammal a therapeutically effective amount of a compound disclosed herein. As used herein, "administer" or "administering" refers to either directly administering a compound of the present teachings or a pharmaceutical composition containing the compound, or administering the compound or pharmaceutical composition indirectly via a prodrug derivative or analog which will form an equivalent amount of the active compound or substance within the body. The methods also can include identifying a mammal in need of such treatment, and administering a therapeutically effective amount of a compound disclosed herein to the mammal in need thereof. As used herein, "therapeutically effective" refers to a substance or an amount that elicits a desirable biological activity or effect. As used herein, "pharmaceutically effective" refers to an amount that can elicit an intended biological activity or effect.
In some embodiments, the method includes administering to a mammal a pharmaceutical composition that comprises a compound disclosed herein in combination or association with a pharmaceutically acceptable carrier. The term "mammal" refers to any warm blooded species, such as a human. The compound of the present teachings can be administered alone or in combination with other therapeutically effective compounds or therapies for the treatment of such
condition(s). For example, the other therapeutically effective compounds can include a cardiovascular disease agent and/or a nervous system disease agent. A nervous system disease agent can be a peripheral nervous system (PNS) disease agent and/or a central nervous (CNS) disease agent.
When administered for the treatment or inhibition of a particular disease state or disorder, it is understood that an effective dosage can vary depending upon the particular compound utilized, the mode of administration, and severity of the condition being treated, as well as the various physical factors related to the individual being treated. In therapeutic applications, a compound of the present teachings can be provided to a patient already suffering from a disease in an amount sufficient to treat the symptoms of the disease and its complications. The dosage to be used in the treatment of a specific individual typically must be subjectively determined by the attending physician. The variables involved include the specific condition and its state as well as the size, age and response pattern of the patient.
The present teachings also provide pharmaceutical compositions comprising at least one compound described herein and one or more pharmaceutically acceptable carriers, excipients, or diluents. Examples of such carriers are well known to those skilled in the art and can be prepared in accordance with acceptable pharmaceutical procedures, for example, those described in Remington's Pharmaceutical Sciences, 17th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, PA (1985), the entire disclosure of which is incorporated by reference herein for all purposes. As used herein, "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient. Accordingly, pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions. Compounds of the present teachings can be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers. Applicable solid carriers can include one or more substances which can also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents, or encapsulating materials. The compounds can be formulated in conventional manner, for example, in a manner similar to that used for known anti-inflammatory agents. Oral formulations containing an active compound disclosed herein can comprise any conventionally used oral form, including tablets, capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions. In powders, the carrier can be a finely divided solid, which is an admixture with a finely divided active compound. In tablets, an active compound can be mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets can contain up to about 99% or greater of the active compound.
Capsules can contain mixtures of active compound(s) with inert filler(s) and/or diluent(s) such as the pharmaceutically acceptable starches (e.g., corn, potato or tapioca starch), sugars, artificial sweetening agents, powdered celluloses (e.g., crystalline and microcrystalline celluloses), flours, gelatins, gums, and the like.
Useful tablet formulations can be made by conventional compression, wet granulation or dry granulation methods and utilize pharmaceutically acceptable diluents, binding agents, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents, including, but not limited to, magnesium stearate, stearic acid, sodium lauryl sulfate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, alginic acid, acacia gum, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, sucrose, sorbitol, dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, low melting waxes, and ion exchange resins.
Surface modifying agents can include nonionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidol silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. Oral formulations herein can utilize standard delay or time-release formulations to alter the absorption of the active compound(s). The oral formulation can also consist of administering an active compound in water or fruit juice, containing appropriate solubilizers or emulisifiers as needed.
Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups, and elixirs. An active compound described herein can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, or a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, and osmo-regulators.
Examples of liquid carriers for oral and parenteral administration include, but are not limited to, water (particularly containing additives as described above, e.g., cellulose derivatives such as a sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier can be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellants.
Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intrathecal, intramuscular,
intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. Compositions for oral administration can be in either liquid or solid form.
Preferably the pharmaceutical composition is in unit dosage form, for example, as tablets, capsules, powders, solutions, suspensions, emulsions, granules, or suppositories. In such form, the pharmaceutical composition can be sub-divided in unit dose(s) containing appropriate quantities of the active compound. The unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. Alternatively, the unit dosage form can be a capsule or tablet itself, or it can comprise the appropriate number of any such compositions in package form. Such unit dosage form may contain from about 1 mg/kg of active compound to about 500 mg/kg of active compound, and can be given in a single dose or in two or more doses. Such doses can be administered in any manner useful in directing the active compound(s) to the recipient's bloodstream, including orally, via implants, parenterally (including intravenous, intraperitoneal and subcutaneous injections), rectally, vaginally, and transdermally. Such administrations can be carried out using the compounds of the present teachings including pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (e.g., rectal and vaginal).
In some cases, it may be desirable to administer a compound directly to the airways of the patient in the form of a dry powder or an aerosol. For administration by intranasal or intrabronchial inhalation, the compounds of the present teachings can be formulated, for example, into an aqueous or partially aqueous solution.
Compounds described herein can be administered enterally or parenterally (such as, without limitation, interperitoneal, intramuscular, intravascular, intrathecal, intra- articular or subcuteaneous injection or infusion). Solutions or suspensions of these active compounds or pharmaceutically acceptable salts thereof can be prepared in water suitably mixed with a surfactant such as hydroxyl-propylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations typically contain a preservative to inhibit the growth of microorganisms.
The pharmaceutical forms suitable for injection can include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In preferred embodiments, the form is sterile and its viscosity permits it to flow through a syringe. The form preferably is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
Compounds described herein can be administered transdermally, i.e., administered across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues. Such administration can be carried out using the compounds of the present teachings including pharmaceutically acceptable salts thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories (e.g., rectal and vaginal). Topical formulations that deliver active compound(s) through the epidermis can be useful for localized treatment of inflammation and arthritis.
Transdermal administration can be accomplished through the use of a transdermal patch containing an active compound and a carrier that can be inert to the active compound, can be non-toxic to the skin, and can allow delivery of the active compound for systemic absorption into the blood stream via the skin. The carrier can take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments can be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active compound can also be suitable. A variety of occlusive devices can be used to release the active compound into the blood stream, such as a semi-permeable membrane covering a reservoir containing the active compound with or without a carrier, or a matrix containing the active compound. Other occlusive devices are known in the literature.
Compounds described herein can be administered into a body cavity, (e.g., rectally or vaginally) in the form of a conventional suppository. Suppository formulations can be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water-soluble suppository bases, such as polyethylene glycols of various molecular weights, can also be used.
Lipid formulations or nanocapsules can be used to introduce compounds of the present teachings into host cells either in vitro or in vivo. Lipid formulations and nanocapsules can be prepared by methods known in the art. For example, the compounds described herein can be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any nontoxic, pharmacologically acceptable lipid capable of forming liposomes can be used.
To increase the effectiveness of compounds of the present teachings, it can be desirable to combine a compound with other agents effective in the treatment of the target disease. For inflammatory diseases, other active compounds (i.e., other active ingredients or agents) effective in their treatment, and particularly in the treatment of asthma and arthritis, can be administered with active compounds of the present teachings. The other agents can be administered at the same time or at different times than the compounds disclosed herein.
Drugs useful in the treatment of RA include anti-inflammatory agents such as non-steroidal anti-inflammatory drugs (NSAIDs), corticosteroids, disease modifying anti-rheumatic drugs (DMARDs), and analgesics. DMARDs include methotrexate, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, abatacept, rituximab, anakinra, antimalarials, gold salts, d-penicillamine, cyclosporin A, cyclophosphamide and azathioprine. NSAIDs include aspirin, ibuprofen, naproxen, meloxicam, etodolac, nabumetone, sulindac, tolementin, choline magnesium salicylate, diclofenac, diflusinal, indomethicin, ketoprofen, oxaprozin, and piroxicam, celecoxib, etoricoxib, lumiracoxib, rofecoxib, and valdecoxib. Corticosteroids include prednisone; methylprenisolone, triamcinolone and methylprednisolone.
Drugs useful in the treatment of IBD include 5-ASA compounds,
corticosteroids, and antibiotics. 5-ASA compounds include sulfasalazine and mesalamine. Antibiotics include metronidazole and ciprofloxacin.
In some embodiments, compounds of Formula (I) are used in combination with anti-inflammatory agents for the treatment of an autoimmune disease such as IBD, Crohn's disease, UC, or RA.
Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings also can consist essentially of, or consist of, the recited components, and that the processes of the present teachings also consist essentially of, or consist of, the recited processing steps.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.
The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise.
It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions may be conducted simultaneously. Compounds described herein can contain an asymmetric atom (also referred as a chiral center), and some of the compounds can contain one or more asymmetric atoms or centers, which can thus give rise to optical isomers (enantiomers) and diastereomers. The present teachings and compounds disclosed herein include such optical isomers (enantiomers) and diastereomers (geometric isomers), as well as the racemic and resolved, enantiomerically pure R and S stereoisomers, as well as other mixtures of the R and S stereoisomers and pharmaceutically acceptable salts thereof. Optical isomers can be obtained in pure form by standard procedures known to those skilled in the art, which include, but are not limited to, diastereomeric salt formation, kinetic resolution, and asymmetric synthesis. The present teachings also encompass cis and trans isomers of compounds containing alkenyl moieties (e.g., alkenes and imines). It is also understood that the present teachings encompass all possible regioisomers, and mixtures thereof, which can be obtained in pure form by standard separation procedures known to those skilled in the art, and include, but are not limited to, column chromatography, thin-layer chromatography, and high- performance liquid chromatography.
Throughout the specification, structures may or may not be presented with chemical names. Where any question arises as to nomenclature, the structure prevails.
Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
More specifically, the following examples illustrate various synthetic routes that can be used to prepare reagents and intermediates that can be used to prepare compounds of Formula (I).
EXAMPLE I: PREPARATION OF INVENTIVE COMPOUNDS
Compounds 1-142 can be prepared using the appropriate starting materials in accordance with the following examples. Selected compounds are shown in Table 3 below. It is understood by those skilled in the art of organic synthesis that the substitution patterns of the starting materials determines the substitution patterns of the products, and the skilled practitioner will be able to exercise routine judgment for the selection of suitable starting materials in order to prepare specific products, the order of synthetic steps, and the need for protecting groups for remote functionalities.
When reference is made to HPLC retention time, the following HPLC conditions were used:
HPLC Conditions A
Instrument: Waters 2695 series HPLC
Mobile Phase: A: 0.025M Ammonium formate pH 4
B: Acetonitrile
Gradient: %A %B
Initial: 90 10
15 min: 30 70
Flow Rate: 1.45 mL/min
UV: 254 nm
Column Temp: 30 °C
Column: Agilent Zorbax Eclipse RR C18, 4.6x75mm, 3.5μιη
Dilutions: in DMSO
HPLC Conditions B
Mobile Phase: A: 0.1% in TFA
B: 0.1% in Water
Gradient: %A %B
Initial: 10 90 (or 50/50)
15 min: 90 10 (or 90/10)
UV: 254 nm
Column Temp: 30 °C
Column: Agilent Eclipse XDB-C8, 4.6x150mm, 5 mm
Dilutions: in DMSO
EXAMPLE 1 : PREPARATION OF 5-ΑΜΓΝΟ-2,4- DICHLOROPYRIMIDINE
Figure imgf000103_0001
The title compound was prepared according to the methods in Inoue, Chem. Pharm. Bull. 6:343-346, 1958. 2,4-dichloro-5-nitro-pyrimidine (20 g, 0.103 mol) in acetic acid (400 mL) was agitated using an overhead stirrer and iron powder (20 g) added. The mixture was slowly heated until approximately 45-50 °C at which point an exotherm to 85-90 °C was observed. The mixture was removed from heating and stirred for a further 60 minutes or until no starting material is observed by t.l.c.
Once cooled, the reaction mixture was filtered through celite and the filtrate concentrated under vacuum. The residue was treated with a small amount of water and the solid filtered off. Whilst moist, the solid was re-crystallized from water, the solid filtered off and dried under vacuum overnight to yield the desired product
(12.512 g, 0.76 mol, 74%). 1H, 400MHz, δ (d6-DMSO): 6.07 (2H, s, NH2), 8.11 (1H, s, ArH); HPLC: 3.74 min; LRMS: m/z = 164.2 (M)
EXAMPLE 2: PREPARATION OF 2-CHLORO-N4-(lH-INDAZOL-5-YL)- PYRIMIDINE-4,5-DIAMiNE
Method 1
Figure imgf000103_0002
The title compound was prepared according to the methods in Tanji, K.I. et al., Chem. Pharm. Bull. 35:4972-4976, 1987: 5-Amino-2,4-dichloropyrimidine (9.3 g, 57.1 mmol) and 5-aminoindazole (7.5 g, 57.1 mmol) were combined and dissolved in a mixture of ethanol (30 mL), water (200 mL) and concentrated HC1 (3.0 mL). The reaction mixture was then heated to 90 °C for 20 hours at which point no starting materials remained by t.l.c. The reaction mixture was cooled and the precipitate filtered off washed with water (50 mL), saturated sodium bicarbonate solution (100 mL) and again with water (2 x 100 mL). The solid was dried under vacuum for 20 hours (10. 6g, 40.7 mmol, 71%).
Method 2 - Inventive Synthetic Route
Figure imgf000104_0001
5-Amino-2,4-dichloropyrimidine (1.33 g, 10 mmol) and 5-aminoindazole (1.64 g, 10 mmol) were slurried in a solution of sodium acetate (20 mL, 0.1 M buffered to pH 4 with acetic acid). The reaction mixture was then heated to 60-65 °C for 16 hours at which point no starting materials remained by t.l.c. The reaction mixture was cooled and the precipitate filtered off washed with water (20 mL), saturated sodium bicarbonate solution (10 mL) and again with water (2x20 mL). The solid was dried under vacuum for 12 hours (2.00 g, 7.6 mmol, 76% Yield). 1H,
400MHz, δ (d6-DMSO): 5.68 (3H, br s, NH), 7.52 (2H, s, ArH), 7.61 (IH, s, ArH), 8.05 (IH, s, ArH), 8.06 (IH, s, ArH), 9.09 (IH, s, NH); HPLC: 3.95 min; LRMS: m/z = 261.1 (M+H)
EXAMPLE 3: PREPARATION OF 2-CHLORO-9-(lH-INDAZOL-5-YL)- 9H-PURINE (Compound 1)
Method 1
Figure imgf000105_0001
The title compound was prepared according to the methods in Tanji, K.I. et al., Chem. Pharm. Bull. 35:4972-4976, 1987: 2-Chloro-N4-(lH-indazol-5-yl)- pyrimidine-4, 5 -diamine (7.43 g, 28.5 mmol), triethylorthoformate (40 mL) and acetic anhydride (40 mL) were combined and heated to 130 °C. After two hours no starting material was observed by t.l.c, the heat removed and the solvent removed under reduced pressure. Water (100 mL) and saturated sodium bicarbonate solution (100 mL) was added and the mixture stirred for 60 minutes. The resulting solid was filtered off and washed with water (100 mL) and dried in a vacuum oven overnight at 40 °C. The dried solid was then chromatographed over silica gel (CHCl3:MeOH) to yield the final product (4.86 g, 17.9 mmol, 63% yield).
Method 2 - Inventive Synthetic Route
Figure imgf000105_0002
2-Chloro-N4-(lH-indazol-5-yl)-pyrimidine-4,5-diamine (10.9 g, 41.8 mmol), dimethylformamide (200 mL), triethylorthoformate (69.8 mL, 418.1 mmol), and anhydrous magnesium sulfate (5.0 g, 41.8 mmol) were combined and heated together at 130 °C for 20 hours or until no starting materials were observed. After cooling, the solvent was removed under reduced pressure and the residue taken up in methanol (80 mL), water (40 mL) and concentrated HC1 (0.5 mL) and stirred for 2 hours. The resulting solid was filtered off and washed with water (100 mL), saturated sodium bicarbonate solution (60 mL) and water (100 mL) before drying in a vacuum oven at 40 °C overnight. The solid was shown by HPLC to be at least 95% pure and not purified further (10.6 g, 39.2 mmol, 94% yield). 1H, 400MHz, δ (d6-DMSO): 7.72 (IH, dd, ArH, J=8.8, 2.0), 7.73 (IH, d, ArH, J=8.8), 8.17 (IH, d, ArH=2.0), 8.24 (IH, s, ArH), 8.99 (IH, s, ArH), 9.19 (IH, s, ArH), 13.40 (IH, s, NH); HPLC: 4.88 min; LRMS: m/z = 271.1 (M+H)
EXAMPLE 4: PREPARATION OF 2-(N,N-DIMETHYL)-9-(lH-INDAZOL- 5-YL)-9H-PURINE (COMPOUND 4)
Figure imgf000106_0001
To a solution of 2-chloro-9-(lH-indazol-5-yl)-9H-purine (3.09 g, 11.43 mmol) in a 2:1 mixture of n-butanol:dimethylformamide (20 mL) was added dimethylamine (25.15 mmol, 12.57 mL of a 2.0M soution in methanol). The reaction mixture was sealed and stirred at room temperature for 3 days or until HPLC analysis showed no starting material present. The reaction mixture was poured into water (150 mL) and stirred for 5 hours with the resulting solid filtered off and washed with water. The crude solid was then dissolved in hot methanol, any insoluble impurities filtered off whilst hot and the product triturated with acetone, yielding the desired compound as a red/purple solid (1.726 g, 6.18 mmol, 54%). 1H, 400MHz, δ (d6-DMSO): 3.13 (6H, s, CH3), 7.72 (IH, d, ArH, J=9.2Hz), 7.82 (IH, dd, ArH, J=9.2, 2.0Hz), 8.18 (IH, s, ArH), 8.20 (IH, d, ArH, J=2.0Hz), 8.49 (IH, s, ArH), 8.79 (IH, s, ArH), 13.37 (IH, s, NH); HPLC: 5.82 min; LRMS: 282.2 (M+H)
EXAMPLE 5: PREPARATION OF 4- [2-(3 -DIETHYL ΑΜΓΝΟ- PROPYLAMINO)-PURIN-9-YL]-BENZAMIDE (COMPOUND 91)
Figure imgf000107_0001
A solution of 4-(2-chloro-purin-9-yl)-benzamide (0.672 g, 2.455 mmol) and N,N-diethyl-l,3-propyl-diamine (1.60 g, 12.3 mmol, 5.00 equiv) in n-butanol (25 mL) was stirred at 118 °C until the no starting material remained by HPLC. The mixture was filtered whilst hot and the filtrate reduced to dryness under reduced pressure. After concentration, the resulting solid was purified three times by chromatography on silica gel, slowly eluting with DMF, affording the title compound (0.108 g, yield 11.9%) as a purple solid. 1H, 400MHz, δ (d6-DMSO): 0.92 (6H, t, CH3, J=7.0Hz), 1.68 (2H, q, C¾, J=7.0Hz), 2.48 (5H, m, CH2), 2.80 (2H, broad, CH2), 3.21 (2H, dd, CH2, J=12.1, 6.2Hz), 7.32 (1H, s, ArH), 8.01 (2H, d, ArH, J=8.2Hz), 8.07 (2H, d, ArH, J=8.2 Hz), 8.59 (2H, m, ArH), 8.71 (1H, s, ArH); LRMS: 368.3 (M+H)
EXAMPLE 6: PREPARATION OF HCL SALT OF N,N-DIETHYL-N'-[9- (lH-rNDAZOL-5-YL)-9H-PURIN-2-YL]-N'-METHYL-PROPANE-l,3-DIAMINE (COMPOUND 5)
Figure imgf000107_0002
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-N'-methyl-propane-l ,3- diamine (1.8 g) was dissolved in hot methanol (30mL) and insoluble solids filtered off whilst hot. Solution heated to reflux and concentrated HC1 (0.5 mL) added. Mixture allowed to cool to around 40 °C and acetone added until solution becomes cloudy. After a few hours, the solids were filtered off and washed with acetone (0.44 g). Additional acetone added until new precipitate formed. The solids were filtered off and washed with acetone (0.35 g). The solids dissolved in methanol and combined before removing solvent to yield uniform batch of solid HCl salt material (0.78 g).
EXAMPLE 7: PREPARATION OF 9-(lH-TNDAZOL-5-YL)-2-METHOXY- 9H-PURINE (COMPOUND 24)
Figure imgf000108_0001
24
To an agitated suspension of 2-chloro-9-(lH-indazol-5-yl)-9H-purine (2.71 g, 10 mmol) in anhydrous methanol (50 mL) was added 0.5 M sodium methoxide in methanol (200 mL, 100 mmol). The resulting solution was heated under reflux for 5 hours before being concentrated to an approximate volume of 80 mL. The solution was then neutralized with 1 N aqueous HCl to pH 7 and stirred for 15 minutes. The resulting precipitate was collected by filtration. The filter cake was repeatedly washed with water to remove any inorganics. Vacuum-drying afforded the title product (2.28 g, yield 85%) as a reddish solid.
EXAMPLE 8: PREPARATION OF 9-(lH-INDAZOL-5-YL)-2-PHENOXY- 9H-PURINE (COMPOUND 24)
25
To a stirred solution of 2-chloro-9-(lH-indazol-5-yl)-9H-purine (2.71 g, 10 mmol) and phenol (1.88 g, 20 mmol) in DMF (100 mL) was added KF (0.581 g, 10 mmol). The mixture was heated at 130 °C for 21 hours, and then concentrated to dryness. A careful chromatography on silica gel, eluting first with 5% methanol in chloroform and then with DMF, afforded the title product (58 mg, yield 2%) as golden prisms.
EXAMPLE 9: PREPARATION OF 9-(lH-INDAZOL-5-YL)-2- PHENYLSULFANYL-9H-PURINE (COMPOUND 26)
Figure imgf000109_0001
To a stirred solution of thiophenol (2.54 mL, 24.82 mmol) in DMF (100 mL) was added potassium carbonate (3.431 g, 24.82 mmol). The mixture was heated at 80 °C for 30 min to ensure a complete formation of KSPh, at which point 2-chloro-9- (lH-indazol-5-yl)-9H-purine (3.36 g, 12.41 mmol) was added, and the resulting solution heated at 137 °C for 7 hours. The mixture was concentrated to give a powder, which was purified by chromatography on basic aluminum oxide, eluting with 1% triethyl amine and 5-10% methanol in chloroform, to afford the title product (2.65 g, yield 62%) as a reddish solid.
EXAMPLE 10: PREPARATION OF 9-(lH-INDAZOL-5-YL)-2- PrPERAZrN-l-YL-9H-PURINE (COMPOUND 27)
Figure imgf000109_0002
1 27 To a stirred solution of 2-chloro-9-(lH-indazol-5-yl)-9H-purine (2.71 g, 10 mmol) and piperazine (2.58 g, 30 mmol) in DMF (50 mL) was added sodium bicarbonate (1.68 g, 20 mmol). The mixture was heated at 75 °C for 3 hours and then concentrated to give a powder. To remove the excess of piperazine, the powder was placed in a filter and washed with hot water a few times. The resulting solid was purified by chromatography on basic aluminum oxide, eluting with 50:10:40 methanol :triethylamine: chloroform, to afford the title product (0.62 g, yield 19%) as a purple solid.
EXAMPLE 11 : PREPARATION OF 9-(lH-INDAZOL-5-YL)-2-(2- METHYL-IMID AZOL- 1 - YL)-9H-PURTNE (COMPOUND 29)
Figure imgf000110_0001
To a stirred solution of 2-chloro-9-(lH-indazol-5-yl)-9H-purine (5.41 g, 20 mmol) and 2-methylimidazole (2.05 g, 25 mmol) in DMF (120 mL) was added CsF (3.80 g, 25 mmol). The mixture was heated at 125 °C for 16 hours. Silica gel (30 g) was added and the whole was concentrated to give a powder, which was purified by chromatography on basic aluminum oxide, eluting with 20:1 :79
methanol:triethylamine:chloroform, to afford the title product (2.21 g, yield 35%) as a red solid.
EXAMPLE 12: PREPARATION OF 4-(2-Chloro-purin-9-yl)-phenol (COMPOUND 59)
Step 1. 4-(5-Amino-2-chloropyrimidin-4-ylamino phenol
II XII
A mixture of 4-aminophenol (25 g, 229.14 mmol), 2,4-dichloropyrimidin-5- amine (37.6 g, 229.14 mmol), ethanol (122 mL), water (811 mL), and concentrated HCI (12.1 mL) was heated at 90 C for 24 hours. The reaction was allowed to cool to ambient temperature and was neutralized with saturated NaHC03. Solids were filtered, washed with water, and dried under vacuum over P205 to afford 41 g of crude product. The aqueous layer was extracted with 10% MeOH/EtOAc to get another 6.0 g of crude product. The crude material (47 g) was used in the next step without further purification.
Step 2. 4-i2-Chloro-9H-purin-9-vnphenol (59)
Figure imgf000111_0002
XII 59
A mixture of 4-(5-Amino-2-chloropyrimidin-4-ylamino)phenol (46 g, 0.194 mol), triethyl orthoformate (324 mL, 1.94 mol), anhydrous MgS04 (23 g), and DMF (500 mL) was heated at 130 °C for 20 hours. The reaction was allowed to cool to r.t and the solvents were removed under reduced pressure. The residue was dissolved in a mixture of MeOH (350 mL), water (90 mL), and concentrated HCI (2.5 mL) and stirred for 2.5 hours at r.t. The mixture was neutralized with saturated NaHC03. Solids were filtered, washed with water, and dried under vacuum to afford 54 g of crude Compound 59 as a black solid. The aqueous layer was extracted with 10%
MeOH/acetone to get another 23.0 g of crude Compound 59. This combined crude material (77 g) was purified by flash chromatography eluting with MeOH/EtOAc to - I l l - give (14 g) still impure, and was further purified by flash chromatography eluting with 2-6% MeOH in CHCl3j to afford 3.16 g the title compound as a brown solid.
Table 3
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
HPLC: 7.07 min; LRMS: 323.2 (M+H)
EXAMPLE II: PHARMACOLOGICAL TESTING
Representative compounds of Formula (I) are screened for activity in several standard pharmacological test procedures. Based on the activity shown in the standard pharmacological test procedures, the compounds of the present teachings can be useful for treating chronic inflammatory and autoimmune diseases.
In various assays, Symadex is used for comparison. As used herein, "Symadex" and "C-1311" are both names for 5-(2-(diethylamino)ethylamino)-8- hydroxy-6H-imidazo[4,5,l-de]acridin-6-one. The use of Symadex as a Flt3 inhibitor and immune system modulator has been described previously by Ajami, A.M., Boss, M.A. and Paterson, J. in US Patent Appl. 2006/0189546A1. EXAMPLE 1 : DETERMINATION OF PROTEIN TYROSINE KINASE ACTIVITY TARGETING IN VITRO.
Representative compounds of Formula (I) were screened for activity against Flt3 and related protein tyrosine kinase using standard kinase screening platforms. The structures of all major kinases and their mutants are known, have been cloned and the functional domains, e.g. the juxtamembrane, ATP and catalytic components projecting from the cell membrane into the cytosol have been expressed through recombinant genetic techniques. The recombinant enzymes, restricted to their catalytic, intracellular domains, have been assembled into test panels to exploit the fact that in the presence of ATP they will phosphorylate an appropriate substrate and the phosphorylation rate and extent can then be measured via an optical reporter system. This testing stratagem and its application in drug development has been discussed in a review by M Vieth et al. (Drug Disc. Today 10: 839-846, 2005). The translation of in vitro results and the correlation to in vivo cellular assays is considered to be high and has been validated in cellular models, for example, in the work of J.S. Melnik et al. (Proc. Nat. Acad. Sci. 103:3153-3158, 2006).
Compounds that are active in inhibition or modulation of Flt3 activity can be used to treat inflammatory and autoimmune diseases.
As used herein, "IC50", the 50% inhibitory concentration, refers to the nanomolar concentration at median percent inhibition determined by dose response (DR) assay fitted to the four parameter Hill equation with a lower assay bound of 0% and an upper bound of 100% inhibition. As used herein, the term "Eiooo" refers to percent inhibition at 1000 nM determined by assay.
Testing of the compounds described in this invention was carried out using the SelectScreen® platform from Invitrogen, Inc. (Carlsbad, CA, USA), and component Z-Lyte®, Adapta®, and LanthaScreen® assays.
Briefly, the most common approach, exemplified by the Z-Lyte® assay is based on treating each specific kinase with a unique substrate and optical reporter system in the presence of ATP at apparent Km for each kinase, typically ranging from 5 to 500 μΜ. The biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non- phosphorylated peptides to proteolytic cleavage. In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET- peptides maintain FRET. A ratiometric method, which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress versus a baseline response obtained in controls. The related assays in the SelectScreen® are based on different detection principles but operate in a similar manner to afford a median inhibitory effect of a compound when graded amounts are used to treat a kinase reaction that in turn is monitored against the progress of a reagent blank.
Compounds were initially tested at 1000 nM concentrations and the percent inhibition of enzyme activity determined (Ε1000). The compounds were then re-tested by adding graded amounts of putative inhibitor in 5 separate increments to generate a dose response and yield the calculated IC5o value.
The IC50 value is a measure of potency. Preferably, compounds demonstrate inhibition in the low nM range. For the compounds of this invention, the in vitro kinase screens revealed high activity against wild type Flt3 and its constitutively activate mutant Flt3 D835Y. Inhibition of the D835Y point mutant in the activation loop is significant mechanistically. This mutant is believed to model the active conformation of the wild type receptor (Razumovskaya et al., Exp. Hematol. 37:979- 989, 2009), which in turn is associated with the conformational changes induced by the Flt3L growth factor, a key effector in the development and maturation of dendritic cells. Inhibitory assay (IC50) values for representative compounds of this invention against Flt3 and Flt3-D835Y, the primary target constructs, are shown in Table 4.
Table 4
Cpd.
Flt3-wt (nM) Flt3-D835Y (nM)
No.
1 113 53
2 48 63
3 134 79
4 20 10
5 41 26
6 201 36
7 91 25
8 36 9
9 52 14
10 53 19
11 54 19
12 44 13
13 37 11
14 135 50
15 123 46
16 322 203
17 14 6
18 286 237
19 28 13
20 122 94
21 48 24
22 87 29
23 98 37
24 214 81
. 25 57 14
26 15 1
27 93 26
28 135 56
29 192 123
31 188 24
32 91 8
33 240 17
34 257 20
35 81 9
36 2 0.3
37 21 1
38 302 41
39 10 1 Cpd.
Flt3-wt (nM) FIt3-D835Y (nM) No.
40 11 1
41 72 7
42 38 5
43 64 7
44 127 13
45 58 3
46 98 6
47 11 2
48 45 5
49 1227 362
50 250 22
51 455 41
52 46 5
53 25 2
54 31 2
55 4 7
56 469 47
57 56 5
58 65 5
59 419 5987
60 372 702
61 19 77
62 45 118
63 40 37
64 240 263
65 93 114
66 46 36
67 434 488
68 142 229
69 252 196
70 12 2
71 31 66
72 5851 4671
73 1066 766
74 83 39
75 310 91
76 555 181
77 669 335
78 108 73
79 69 18
80 525 216
81 2788 1652
82 244 99
83 170 61 Cpd.
Flt3- t (nM) FK3-D835Y (nM) No.
84 250 43
85 97 14
86 337 31
87 299 16
88 52 7
89 119 17
90 136 27
91 120 23
92 171 20
93 278 20
94 318 56
95 37 19
96 1786 116
97 243 29
98 42 4
99 44 3
100 5 2
101 311 17
102 3779 266
103 17234 643
104 39 2
105 169 7
106 2200 1200
107 3600 1400
108 131 4
109 678 113
110 25 2
111 21 1
112 67 3
113 49 3
114 17 1
115 26 1
116 67 4
117 102 6
118 11 1
119 3213 480
120 17 1
121 99 5
122 150 9
123 79 5
124 51 6
125 3399 222
127 20 3
128 147 8 Cpd.
Flt3-wt (nM) FK3-D835Y (nM)
No.
129 27 4
130 10 1
131 26 3
132 12 2
133 155 2
134 48 9
135 28 9
136 1647 1110
137 1225 1647
138 908 599
140 21 3
141 309 25
142 821 599
143 516 78
144 66 4
145 13 2
146 23 2
147 90 1
148 632 38
149 26 2
150 36 6
151 36 6
152 453 31
153 666 46
154 567 71
156 703 46
157 508 14
158 35 2
159 275 14
160 255 38
161 932 38
162 4627 427
163 9 1
164 314 5
165 1225 821
Example IB: Determination of kinase targeting specificity
Compounds which inhibit fewer kinases are considered more selective than those which inhibit greater numbers. To determine the specificity of the compounds of this invention, representative exemplars, with IC5o values <50 nM against the Flt3 isoforms, were tested against the kinases with the greatest structural homology to Flt3 and belonging to the Class III receptor tyrosine kinase grouping. These include CSF- 1R, PDGFRA, and wild-type KIT. These data are reported in Table 5 as percent (%) kinase inhibition at a 1 μΜ test compound concentration.
Testing for kinase inhibitory specificity was carried out using the
SelectScreen® platform from Invitrogen, Inc. (Carlsbad, CA, USA) and component Z- Lyte ®, Adapta®, and LanthaScreen® assays .
CSF-1R is the receptor for GM-CSF, an essential growth factor in the maturation of monocytic cell lineages during their differentiation into dendritic cells and macrophages. Inhibition of CSF-1R would be beneficial in autoimmune disease as a control point for the proliferation of potentially immunogenic cells and may be considered synergistic with the modulating effects accomplished by Flt3 inhibition in these types of cells, as described earlier.
PDGFR, the platelet derived growth factor, is involved in the signaling cascade that promotes vascularization as a conduit for inflammatory and
immunogenic cells, in the case of autoimmune disease, and for metastatic cells in cancer. To the extent that PDGFR inhibition would restrain the trafficking of activated lymphocytes, such an effect would be synergistic with the modulatory properties of Flt3 inhibitors.
In cancer, KIT is a proto-oncogene and a therapeutic target for the action of inhibitors considered chemotherapeutics. In normal immune system function, and by extension, during the recovery from autoimmune disease, however, KIT is essential to the growth and proliferation of stem cells, including those that serve as hematopoetic precursors. Inhibition of KIT outside of the cancer therapeutic setting is considered undesirable as it would impair the requisite regenerative processes that counterbalance the tissue destruction during the inflammatory phases of autoimmune disease.
Also tested in the same experiment were the insulin receptor, INSR, a tyrosine kinase considered essential to homeostasis and hence not a desirable target for inadvertent inhibition, and KDR, a closely related receptor tyrosine kinase involved in vascular endothelial growth, another homeostatic process that should not be affected by molecules deemed selective and specific towards Flt3.
Table 5
Flt3 Flt3 CSF-
Cpd No PDGFRa KDR KIT INSR wt D835Y 1R
2 77 96 34 14 14 3 6
4 97 100 83 45 31 58 1
5 96 99 82 39 20 33 3
6 81 94 57 17 24 9 2
7 89 95 65 40 23 13 3
8 95 100 96 39 21 47 6
9 93 100 92 34 25 40 4
10 96 100 69 45 14 43 3
1 1 95 100 45 45 19 38 0
12 96 99 96 47 29 54 8
13 95 99 95 41 23 47 5
84 74 92 55 25 33 8 0
85 94 98 57 30 42 23 5
86 74 95 50 6 27 7 5
87 81 97 57 19 26 10 0
88 97 100 91 35 32 21 0
89 91 100 74 25 29 16 0
90 85 100 68 25 27 38 0
91 84 98 57 21 22 15 0
92 73 97 73 16 22 21 0
93 74 97 86 16 20 16 0
31 278 24 45 10 25 1 0
32 87 101 79 48 26 31 3
14 92 97 72 38 7 15 12
74 94 95 47 32 38 51 7
33 79 100 55 16 16 20 7
15 87 94 56 23 1 49 4
95 95 99 63 53 21 37 0
34 69 96 60 28 29 9 0
17 94 100 76 83 24 37 3
19 97 98 82 48 17 41 10
63 94 95 64 42 29 35 7
79 83 98 52 24 18 19 7
21 94 100 39 21 11 15 24
35 91 100 34 32 7 0 21
36 100 100 88 86 26 2 18 37 96 100 48 47 15 6 14
38 89 99 41 30 13 27 5
39 97 100 70 69 19 24 27
40 99 100 73 83 21 23 3
41 91 100 72 43 10 2 5
42 95 100 100 95 33 40 0
43 97 100 81 79 23 28 0
44 97 100 88 80 32 17 0
45 95 100 89 61 14 0 3
46 95 100 49 38 4 1 0
47 98 100 85 85 14 2 0
48 95 100 84 86 25 24 2
66 96 100 63 100 26 15 8
70 100 100 93 100 84 37 12
22 94 103 58 87 25 34 13
23 93 103 65 87 3 47 11
25 93 100 63 42 18 16 0
26 100 100 96 96 72 31 14
50 80 100 67 43 13 6 8
51 70 100 56 26 25 21 9
52 97 100 82 86 27 29 10
53 94 100 90 69 1 23 8
54 96 100 86 63 21 19 7
55 97 98 77 66 21 68 1
56 61 91 26 10 11 15 1
57 85 99 64 38 15 14 2
58 87 98 52 52 5 29 0
27 96 99 62 36 15 53 4
71 93 76 54 24 19 20 5
97 74 98 12 12 0 17 0
98 92 100 37 48 3 0 0
99 95 100 31 54 11 18 0
100 99 100 100 101 97 45 4
101 70 97 55 22 15 9 0
104 96 100 24 50 2 0 0
105 70 95 63 32 17 25 0
163 98 100 90 98 57 36 0
110 100 100 38 76 18 28 11
1 12 96 100 32 65 12 25 7
1 13 96 100 16 42 4 13 9
1 16 93 100 14 43 2 10 8
1 11 100 100 28 67 7 26 6
108 94 102 50 52 49 28 5
114 98 100 76 90 87 51 8
1 15 100 100 65 78 78 46 8 122 83 100 33 19 1 9 0
117 90 100 34 32 4 14 6
1 18 98 103 94 91 89 35 10
120 99 102 54 63 15 17 6
121 91 102 24 36 10 18 10
123 89 101 28 25 13 3 6
130 99 75 90 38 21 11
124 93 101 87 62 66 20 8
128 90 101 68 46 18 9 11
129 95 100 80 52 21 8 8
127 97 100 100 90 98 50 19
131 93 101 66 41 12 5 5
132 98 102 78 76 16 2 3
133 75 95 70 7 11 14 0
134 89 99 71 39 26 18 5
135 93 101 63 57 14 4 5
140 95 103 74 44 17 5 6
141 70 99 66 9 16 8 4
144 87 102 11 10 44 1 1 3
145 99 100 96 92 87 57 12
146 96 100 65 58 26 26 4
149 95 100 69 63 27 16 4
151 95 100 65 48 17 8 4
158 94 100 49 64 19 19 4
160 78 98 66 8 8 0 2
161 71 98 51 7 14 8 1
Mean Inhibitory % 92 98 63 48 25 22 5
Corresponding IC5o
50 10 500 1,000 4,000 5,000 50,000
Range (nM)
The column means from this table provide strong evidence for the selectivity of the compounds as a class despite significant structural diversity. The selectivity becomes even more evident when considering that percent inhibition follows an inverse log linear relationship to IC50, the median inhibitory concentration, as would be predicted by solving the Hill Equation using the fitting constants, the screening concentration (1000 nM), and the observed inhibitory percent to backsolve for the ICso. By examining the correlation between the inhibitory capacity at 1 μΜ and the IC50 for a known set of tyrosine kinase inhibitors, as published by the assay provider, the midpoint IC50 for any set of mean inhibitory values at 1 μΜ can be predicted. Thus, inhibition of the Flt3 isoforms at 1 μΜ test article corresponds to an average IC50 of 50 nM for Flt3 and 10 nM for Flt3 D825Y.
For closely related CSF-1R, PDGFRa, and KDR, the representative inhibitory capacity translates into average IC50 values of 500, 1000, and 4000 nM, respectively, a 2-3 log unit separation. Notably, 3 log units separate the corresponding IC50 values for inhibition of KIT from the IC50 against Flt3. The insulin receptor, INSR, is unaffected for all practical purposes since the requisite IC50 would require a 4 log unit greater concentration of inhibitor than would suffice for the median effect on Flt3.
This specificity finding is novel and unprecedented within the corpus of the medicinal chemistry directed at Flt3 inhibition, particularly in view of the neglible activity against KIT.
The requisite ATP concentration for Flt3 (wt) inhibition is 470 μΜ, one of the highest reported values for any kinase. However, cellular ATP levels are
approximately 1000-5000 μΜ. Under these physiological conditions, a small molecule kinase inhibitor with the same inhibition constant for two kinases will cause greater inhibition of the kinase with the greater ATP km value, since ATP binding is weaker for this enzyme. In the case of the inventive compounds, a target like Flt3 with a high ATP requirement might be expected to be even more selectively inhibited in a cellular setting.
The individual inhibitory activity of compounds of the invention may vary; cf, Compounds 12 and 13 which are equipotent against Flt3 and CSF-1R but not against PDGFRA, KDR, while Compounds 70 and 100, in contrast, show equal inhibitory capacity against all four, although none are significant inhibitors of KIT or INSR. EXAMPLE 2: IN VITRO WHOLE KINOME SURVEYS.
Specificity can be further assessed by a survey of inhibitory capacity across a panel of kinases selected from each functional class of kinases, e.g. tyrosine, serine, threonine, receptor and non-receptor kinases, and encompassing approximately one third of the currently recognized kinome. These are listed in Table 6 by their common protein acronyms or designations. The degree of specificity can be quantified as a percent taken from the number of off-target leads expressed as a proportion of the total number of enzymes assayed.
Table 6
ABL1 CDK8/cyclin C EPHB1
ABL1 E255K CDK9/cyclin K EPHB2
ABL1 G250E CDK9/cyclin Tl EPHB3
ABL1 T315I CHEK1 (CHK1) EPHB4
ABL1 T315I CHEK2 (CHK2) ERBB2 (HER2)
ABL1 Y253F CHUK (IKK alpha) ERBB4 (HER4)
ABL2 (Arg) CLK1 FER
ACVR1 (ALK2) CLK2 FES (FPS)
ACVR1B (ALK4) CLK3 FGFR1
ADRBK1 (GRK2) CSFIR (FMS) FGFR2
ADRBK2 (GRK3) CSK FGFR3
AKT1 (PKB alpha) CSNK1A1 (CK1 alpha FGFR3 K650E
AKT2 (PKB beta) 1) FGFR4
AKT3 (PKB gamma) CSNK1D (CK1 delta) FGR
ALK CSNK1E (CK1 FLT1 (VEGFR1)
AMPK A1/B1/G1 epsilon) FLT3
AMPK A2/B1/G1 CSNK1G1 (CK1 FLT3 D835Y
AURKA (Aurora A) gamma 1) FLT4 (VEGFR3)
AURKB (Aurora B) CS K1G2 (CK1 FRAP1 (mTOR)
AURKC (Aurora C) gamma 2) FRK (PTK5)
AXL CSNK1G3 (CK1 FYN
BLK gamma 3) GRK4
BRAF CSNK2A1 (CK2 alpha GRK5
BRAF V599E 1) GRK6
BRSK1 (SAD1) CSNK2A2 (CK2 alpha GRK7
BTK 2) GSG2 (Haspin)
CAMK1 (CaMKl) DAPK1 GSK3A (GSK3 alpha)
CAMK1D (CaMKI DAPK3 (ZIPK) GSK3B (GSK3 beta) delta) DCAMKL2 (DCK2) HCK
CAMK2A (CaMKII DDR2 HIPK1 (Myak)
alpha) DMPK HIPK2
CAMK2B (CaMKII DYRK1A HIPK4
beta) DYRK1B IGF1R
CAMK2D (CaMKII DYRK3 IKBKB (IKK beta) delta) DYRK3 IKBKE (IKK epsilon)
CAMK4 (CaMKIV) DYRK4 INSR
CAMKK1 EEF2K INSRR (IRR)
(CAMKKA) EGFR (ErbBl) IRAKI
CAMKK2 (CaMKK EGFR (ErbBl) L858R IRAK4
beta) EGFR (ErbBl) L861Q ITK
CDC42 BPA EGFR (ErbBl) T790M JAK1
(MRCKA) EGFR (ErbBl) T790M JAK2
CDC42 BPB L858R JAK2 JH1 JH2
(MRCKB) EPHA1 JAK2 JH1 JH2 V617F
CDKl/cyclin B EPHA2 JAK3
CDK2/cyclin A EPHA3 KDR (VEGFR2)
CDK5/p25 EPHA4 KIT
CDK5/p35 EPHA5 KIT T670I
CDK7/cyclin EPHA7 KIT V654A
H/MNAT1 EPHA8 LCK LIMK1 MELK PIK3CD/PIK3R1
LIMK2 MERTK (cMER) (pl l0 delta/p85
LRRK2 MET (cMet) alpha)
LRRK2 G2019S MET M1250T PIK3CG (pl l0
LTK (TYKl) MINK1 gamma)
LYN A MLCK (MLCK2) PIM1
LYN B MST1R (RON) PKN1 (PRKl)
MAP2K1 (MEK1) MST4 PLK1
MAP2K1 (MEK1) MUSK PLK2
MAP2K1 (MEK1) MYLK (MLCK) PLK3
S218D S222D MYLK2 (skMLCK) PRKACA (PKA)
MAP2K2 (MEK2) NEKl PRKCA (PKC alpha)
MAP2K3 (MEK3) NEK2 PRKCB1 (PKC beta I)
MAP2K6 (MKK6) NEK4 PRKCB2 (PKC beta II)
MAP2K6 (MKK6) NEK6 PRKCD (PKC delta)
S207E T211E NEK7 PRKCE (PKC epsilon)
MAP3K10 (MLK2) NEK9 PRKCG (PKC gamma)
MAP3K11 (MLK3) NLK PRKCH (PKC eta)
MAP3K14 (NIK) NTRKl (TRKA) PRKCI (PKC iota)
MAP3K2 (MEKK2) NTRK2 (TRKB) PRKCN (PKD3)
MAP3K3 (MEKK3) NTRK3 (TRKC) PRKCQ (PKC theta)
MAP3K5 (ASK1) NUAK1 (ARK5) PRKCZ (PKC zeta)
MAP3K7/MAP3K7IP 1 PAK1 PRKDl (PKC mu)
(TAK1-TAB1) PAK2 (PAK65) PRKD2 (PKD2)
MAP3K8 (COT) PAK3 PRKGl
MAP3K9 (MLK1) PAK4 PRKG2 (PKG2)
MAP4K2 (GCK) PAK6 PRKX
MAP4K4 (HGK) PAK7 (KIAA1264) PTK2 (FAK)
MAP4K5 (KHSl) PASK PTK2B (FAK2)
MAPK1 (ERK2) PDGFRA (PDGFR PTK6 (Brk)
MAPK10 (ΓΝΚ3) alpha) RAF1 (cRAF) Y340D
MAPK11 (p38 beta) PDGFRA D842V Y341D
MAPK12 (p38 gamma) PDGFRA T674I RET
MAPK13 (p38 delta) PDGFRA V561D RET V804L
MAPK14 (p38 alpha) PDGFRB (PDGFR RET Y791F
MAPK14 (p38 alpha) beta) RIPK2
Direct PDK1 ROCK1
MAPK3 (ERK1) PDK1 Direct ROCK2
MAPK8 (JNK1) PHKG1 ROS1
MAPK9 (JNK2) PHKG2 RPS6KA1 (RSK1)
MAPKAPK2 PI4KA (PI4K alpha) RPS6KA2 (RSK3)
MAPKAPR3 PI4KB (PI4K beta) RPS6KA3 (RSK2)
MAPKAPK5 (PRAK) PIK3C2A (PI3K-C2 RPS6KA4 (MSK2)
MARKl (MARK) alpha) RPS6KA5 (MSK1)
MARK2 PIK3C2B (PI3K-C2 RPS6KA6 (RSK4)
MARK3 beta) RPS6KB1 (p70S6K)
MARK4 PIK3C3 (hVPS34) SGK (SGKl)
MATK (HYL) SGK2 SGKL (SGK3) SLK
SNF1LK2
SPHK1
SPHK2
SRC
SRC N1
SRMS (Sim)
SRPKl
SRPK2
STK16 (PKL12) STK17A (DRAK1) STK22B (TSSK2) STK22D (TSSKl) STK23 (MSSK1) STK24 (MST3) STK25 (YSK1) STK3 (MST2) STK33
STK4 (MST1) SYK
TAOK2 (TAOl) TAOK3 (UK) TBK1
TEC
TEK (Tie2) TNK2 (ACK) TTK TYK2
TYR03 (RSE)
WEE1
WNK2
YES1
ZAK
ZAP70
Example 2A
Three compounds, Compounds 61, 17, and 19 with IC50 values for Flt3 inhibition of between 14 to 28 nm Flt3, and three randomly selected analogs, Compounds 74, 33, and 15 with IC50 values for Flt3 inhibition of between 83 to 240 nm Flt3, were further assayed at a concentration of 1 μΜ (1000 nM), and using the apparent ATP km concentration for each kinase described in Table 6. The resulting percent inhibition was scored as follows: "++" for inhibitory activity > 80%, corresponding approximately to IC50 values of 200 nM or lower, "+" for inhibitory activity between 40 and 80%, corresponding to IC50 values between 200 and 2000 nM, and "-" for activity below 40%.
Accordingly, Tables 7-12 show the screening results for Compounds 74, 61, 33, 15, 17, and 19.
Only leads and hits are included; the kinases that were scored as not inhibited significantly, e.g. scored as "-" have been omitted from the tables. The degree of specificity can be quantified as a percent taken from the number of off-target leads expressed as a proportion of the total number of enzymes assayed.
Compound 74 (Table 7) significantly inhibits Flt3 and the point mutant kinase, Flt3- D835Y, the on-target leads, as well as the following four kinases: ACVRl, PDGFRA point mutant V561D, PIK3CA/PIK3R1, and RIPK2. Hence the specificity for Flt3 may be categorized as 100 - 100*4/(299-2) - 98.7%.
Table 7. Compound 74
Figure imgf000149_0001
Figure imgf000150_0001
Compound 61 (Table 8) significantly inhibits Flt3 and the point mutant kinase, Flt3- D835Y, but also three other kinases, PDGFRA point mutant V561D, PTK6, and RIPK2, thus showing a specificity for Flt3 of 99%
Table 8. Compound 61
Figure imgf000150_0002
Figure imgf000151_0001
Compound 33 (Table 9) significantly inhibits the Flt3 point mutant kinase, Flt3- D835Y, which, as described earlier, mimics the constitutively activate wild type enzyme. It also inhibits GSG2, thus showing a specificity for Flt3 of 99.7%.
Table 9. Compound 33
Figure imgf000151_0002
Compound 15 (Table 11) only inhibits Flt3 and the point mutant kinase, Flt3-D835Y, with no significant activity as a lead against all the other kinases assayed, thus showing a specificity for Flt3 of 100%.
Table 10. Compound 15
Kinase % Inhibition Score
CSFIR (FMS) +
FLT3 ++
Figure imgf000152_0001
Compound 17 (Table 11) inhibits both Flt3 isoforms and the intended secondary target CSF-IR but also may be identified as a lead for inhibition of 12 additional kinases: ACVRl, LRRK2, LRRK2 point mutant G2019S, MELK, MUSK, NLK, NTRKl, NTRK3, PDGFRA, PDGFRA point mutant D842V, PDGFRA point mutant V561D, and RIPK2, thus showing a specificity for Flt3 of 96%.
Table 11. Compound 17
Figure imgf000152_0002
Figure imgf000153_0001
Compound 19 (Table 12) inhibits both Flt3 isoforms plus the intended secondary target CSF-IR and three additional kinases: NTRK3, PDGFRA point mutant V561D, and PTK6, thus showing a specificity for Flt3 of 99%.
Table 12. Compound 19
Kinase % Inhibition Score
ACVR1 (ALK2) +
Figure imgf000154_0001
The average specificity for Flt3 of the six such exemplars in this study is 98.7%. It is noteworthy in this connection that a high proportion of the inhibited kinases, although unrelated to Flt3, play a role in the degenerative pathophysiology or the tissue
damagemechanisms that Flt3 inhibitors are intended to ameliorate . Compounds of Formula (I) in accordance with this invention are highly specific inhibitors for the Flt3 isoforms and closely related Class III receptor tyrosine kinases.
Example 2B
A more structurally diverse set of compounds— Compounds 12, 36, 39, 47, 54, 100, 110 and 129— all of them potent inhibitors of Flt3, were evaluated for target specificity and as the basis for discovery of off-target leads. The kinase panel described in Table 6, and previously used in the specificity screening for the compounds in Example 2A, was expanded to a count of 304 kinases by the addition of BMPRl A (ALK3), CLK4, DNA-PK, HIPK3 (YAK1) and MKNK1 (MNK1). The formula presented in Example 2A for calculation of Flt3 targeting specificity was adjusted accordingly.
Compound 12 (Table 13) inhibits both Flt3 isoforms plus the intended secondary target CSF-IR and four additional kinases: CLK2, FGR, NTRKl and NTRK3 showing a specificity for Flt3 of 99%.
Table 13. Compound 12
Figure imgf000155_0001
Compound 36 (Table 14) inhibits both Flt3 isoforms and twelve additional kinases: CDK2/cyclin A, CDK5/p25, CDK5/p35, FGR, MUSK, NTRKl, NTRK3, PDGFRA, PDGFRA D842V, PDGFRA V561D, ROSl, and SNF1LK2, showing a specificity for Flt3 of 96%.
Table 14. Compound 36
Figure imgf000156_0001
Compound 39 (Table 15) inhibits both Flt3 isoforms and one additional kinase:
PDGFRA V561D showing a specificity for Flt3 of 100%.
Table 15. Compound 39
Kinase % Inhibition
Score
CDK8/cyclin C +
CLK4 +
FGR +
FLT3 ++
FLT3 D835Y ++
Figure imgf000157_0001
Compound 47 (Table 16) inhibits both Flt3 isoforms and five additional kinases: CLK4, NTRKl, NTRK3, PDGFRA V561D and ROSl showing a specificity for Flt3 of 98%.
Table 16. Compound 47.
Figure imgf000157_0003
Compound 54 (Table 17) inhibits both Flt3 isoforms and three additional kinases: CAMK2D (CaMKII delta), MUSK, PDGFRA V561D showing a specificity for Flt3 of 99%.
Table 17. Compound 54
Figure imgf000157_0002
Figure imgf000158_0001
Compound 100 (Table 18) inhibits both Flt3 isoforms plus the intended secondary target CSF-IR and eighteen additional kinases: EPHAl, EPHBl , FLT4, JAK2, KDR (VEGFR2), LRRK2, LRRK2 G2019S, MAP3K10 (MLK2), MAP3K1 1 (MLK3), MAP3K9 (MLKl), PDGFRA, PDGFRA V561D, RET, RET V804L, RET Y791F, RPS6KA6 (RSK4), SRC Nl, and STK16 (PKL12) showing a specificity for Flt3 of 94%.
Table 18. Compound 100
Figure imgf000158_0002
Figure imgf000159_0001
Figure imgf000160_0001
Compound 110 (Table 19) inhibits both Flt3 isoforms and twelve additional kinases: ACVRl (ALK2), CDKl/cyclin B, CDK2/cyclinn A, CDK5/p25, CDK5/p35, CLK4, MAP3K7/MAP3K7IP 1 (TAKl-TABl), NTRK3, PDGFRA D842V, PDGFRA V561D, ROS1, and SNF1LK2 showing a specificity for Flt3 of 96%.
Table 19. Compound 110
Figure imgf000160_0002
Figure imgf000161_0001
Compound 129 (Table 20) inhibits both Flt3 isoforms and four additional kinases: ACVR1 (ALK2), CLK4, NTRK3 and ROS1 showing a specificity for Flt3 of 99%.
Table 20. Compound 129
Figure imgf000161_0002
The average specificity for Flt3 of the 8 such exemplars in this example is 97.6%, confirming the initial observations described in Example 2A. Again, it is noteworthy in this connection that a high proportion of the inhibited kinases, although unrelated to Flt3, play a role in the degenerative pathophysiology or the tissue damage mechanisms that Flt3 inhibitors are intended to ameliorate.
Table 21 lists the non-target kinases uncovered in the whole kinome screen which were inhibited with IC50 values predicted to fall in the 1-50 nM range.
Table 21
CDK 2/5 Cell cycle control; cdk5 in Alzheimer's disease
CLK4 RNA splicing control; regulates MAPT/TAU
CSF-1R Macrophage and dendritic cell stimulating factor receptor
EPHA1 Ephrin receptor; cell migration/invasion control
FLT4 Lymphatic endothelial growth regulator
KDR Vascular endothelial growth regulator
MAP3K 9/10/11 Mediator of neuronal inflammation and apoptosis MUSK Agrin receptor; neurite growth stop signal
NTRK3 Neuroinflammation and neurite growth modulator
RIPK2 Regulator of dendritic cell maturation and function
ROS1 Activated in glioblastomas and astrocytomas
STK16 Transcriptional factor in TGF-β pathway
EXAMPLE 3: EFFECT ON THE ACTIVATION AND MATURATION OF DENDRITIC CELLS
Representative compounds of this invention were tested by the following protocol, described here in terms of its operational components, performed under contract by ReachBio LLC (Seattle, WA; http://www.reachbio.com/). Human CD34+ bone marrow cells were obtained from a hematological products supplier were set up in 2 different culture systems (A and B), as described below. Prior to culture (day 0), CD34+ purity was determined to exceed 95% and the initial phenotypic profile was established by FACS assessment for the percentage and number of cells expressing CD la, CD1 lb, CD1 lc, CD40, CD54, CD58, CD74, CD80, CD83, CD86, CD123, CD142, CD209, CD275, CD303, and CD304.
The cells were then cultured as per Ratta et al. (Br. J. Haematol. 101 :756-765, 1998), with slight modifications, for a total of 15 days. Cultures were initiated at a cell concentration of 4 x 10"4 cells/mL in RPMI medium containing 10% FBS, L-glutamine and antibiotics ("complete medium") plus a cytokine cocktail consisting of GM-CSF (50 ng/ml), TNF (10 ng/ml), SCF (20 ng/ml), and Flt3L (50 ng/ml). On day 7 of culture, the media from all cultures was replaced with differentiation medium, consisting of complete medium plus the following cytokine combination: GM-CSF (50 ng/ml), TNF (10 ng/ml), and IL-4 (50 ng/ml). Cells were incubated in this differentiation medium for an additional 7 days. On day 14, one day prior to termination of the cultures, IFN-g (1000 U/mL) and LPS (1 μg/mL) were added to all the cultures as a final maturation step. This basic cell growth protocol was replicated both with and without test compounds, which were added to growth medium from day 0 and replenished at each change of culture medium.
Experiments to evaluate the temporal properties of dendritic cell phenotypes, as a function of test compound exposure, were carried out throughout the 15 day culture protocol described above at 5 different time points, days 3, 7, 10, 14 and 15. Samples from the cultures were assessed for total cell counts as well as the percentage of cells expressing each phenotypic surface marker of interest, separately as well as in combination with CD1 lc (an integrin, leukocyte adhesion protein), the hallmark for dendritic cells when differentiated from their CD34+ precursors. In all cases propidium iodide (1 μg/mL) was included in the final wash to allow exclusion gating of dead cells from analyses. Also, at each time point described above, new media with the appropriate cytokines, and replenished drug was added to the cultures so as to maintain a cell count equivalent to the cell density prior to sampling.
Three compounds were tested at a 1 μΜ concentration along with rapamycin, also at 1 μΜ, a known systemic immunosuppressant and dendritic cell differentiation and maturation inhibitor (Hackstein et al. Blood 101 :4457-4463, 2003). In vitro, rapamycin downregulates the IL-4 receptor complex, a key effector in the autoimmune and inflammatory T-cell stimulatory activity of DCs. In addition, rapamycin abrogates Flt3 signaling and steady state DCs generation normally triggered by Flt3 ligand. As a consequence, rapamycin also impairs the DC costimulatory molecule up-regulation, production of pro-inflammatory cytokines and T-cell allostimulatory capacity. These findings have prompted the re-evaluation of rapamycin as more than just an mTOR pathway inhibitor and, therefore, have repurposed it as a therapy for chronic autoimmune diseases.
Representative compounds of this invention, e.g, Compounds 4, 5, and 12, show similar activity patterns to those of rapamycin. However, in contrast to rapamycin, a total immunosuppressant, the compounds of this invention attenuate but do not completely abrogate the characteristics of dendritic cells, thereby leaving residual capacity for mounting a normal immune response, e.g. against pathogens. The effect on DCs proliferation versus untreated control is shown in Figure 1 : the time course of cell growth is presented in Figure 1 A, and a quantitative histogram of the cell counts at day 15, and the end of the DC differentiation and maturation cycle, is presented in Figure IB.
Figures 2A and 2B presents the previous result limited specifically to the
CD1 lc+CDl lb+ phenotype, which is characteristic of activated dendritic cells (CD1 lc) sharing the mixed lineage macrophage phenotype (CD1 lb) and hence combining the antigen presenting and phagocytic properties of the cell type responsible for tissue destruction in autoimmune disease settings. The presentation of this deleterious phenotype is demonstrably reduced versus control. In this regard, the compounds of this invention modulate dendritic cells in a similar manner to that of other kinase inhibitors, as shown in Figures 2C and 2D. These data show that the effect of Compound 12 falls between that of sunitinib and imatinib, both approved drugs that target Flt3, Csflr, cKit, and related kinase receptors, and which are known to downregulate dendritic cell differentiation and maturation, but not to completely suppress it.
Similarly, Figures 3A and 3B show the time course for the expression of the CD83 maturation marker coexpressed with the CDl lc dendritic cell hallmark. CD83 is also a strong indicator of DC maturation and activation and is understood to promote immunogenicity and the cellular interactions that follow lymphocyte activation. CD83 is also known as the B-cell activation protein. The compounds of this invention downregulated the presentation of this marker as well, thereby also downregulating the maturation and activation of DCs and leaving them in the more immature state associated with tolerogenic function.
Figures 4A and 4B present further significant evidence that the compounds of this invention exert their effects on lowering dendritic cell counts through mechanisms other than cell kill. As in the case of rapamycin, which is accepted as a non-cytotoxic agent, cells exposed to Compounds 4, 5, and 12 maintain a high level of viability, above 95% throughout the drug exposure period and indistinguishable from vehicle control.
Table 22, summarizes the modulatory effects, as shown in the preceding figures for the Day 15 cell growth and antigen presentation outcomes. Table 22 also includes data taken from the quantitation of additional related DCs markers of the immune response.
Table 22
Function of Marker
Day 15 Cell counts as Percent of Control coexpressed with CDl lc
Control Rap Cpd 4 Cpd 5 Cpd 12
Total DCs 100 2 23 37 24
CDl lc Surface glycoprotein
100 4 28 58 27 CDl lb (integrin) MAC-1
CDl lc CD40 Surface antigen CD40 100 4 29 59 30
Figure imgf000165_0001
Rap = Rapamycin
In summary, over a spectrum of indicators associated with the temporal proliferation, maturation and activation of DCs, the compounds of this invention alter the immunogenic properties of DCs in contrast to their matched vehicle controls, which continue along the path toward greater immunogenicity. Importantly, they downregulate the proliferation of dendritic cells under IL-4 and growth factor stimulation, prevailing circumstances that model autoimmune disease. They also decrement the expression of differentiation and maturation phenotypic cell surface markers. However, in contrast to rapamycin, these direct Flt3 inhibitors do not completely suppress the growth and differentiation of DCs, which would be an undesirable outcome given the homeostatic role of DCs in maintaining host immune competence. They attenuate rather than abrogate the progression of DCs towards the immunogenic phenotypes, in terms of total cell numbers and co-stimulatory phenotypic traits. Thus, the compounds of this invention serve as true modulators of DC mediated responses.
EXAMPLE 4: COLLAGEN-INDUCED ARTHRITIS (RA MODEL) The utility of a pharmaceutical compound for the treatment of various conditions associated with RA can be demonstrated by its ability to inhibit the induction of arthritis by collagen monoclonal antibodies (mABs). Collagen-Induced Arthritis (CIA) can be elicited in susceptible strains of rodents (such as rat and mouse) and nonhuman primates by
immunization with type II collagen, the major constituent protein of articular cartilage. CIA manifests as swelling and erythema in the limbs of the mouse, and shares several clinical and pathological features with rheumatoid arthritis (RA) and has become the most widely studied model of RA. CIA in the mouse as a model was first described by Courtenay et al. in 1980 (Courtnay, J.S., Dallman, M.J., Dayman, A.D., Martin A., and Mosedale, B. Nature 283:666- 668, 1980). Like RA, susceptibility to CIA is regulated by the class II molecules of the major histocompatibility complex (MHC), indicating the crucial role played by T cells. CIA can be primed following induction with type II collagen or type II collagen specific antibodies. The two methods differ in time before onset of disease.
Example 4A: Collagen Antibody-induced Arthritis (CAIA) Model Methods
Collagen-induced arthritis (CIA) relies on the generation of autoantibodies which bind to particular regions of type II collagen and complement. The disease model, in this form, requires immune cell activation and is restricted to the expression of certain class II major histocompatibility complex (MHC) alleles, indicating a dominant role for T cells in the initiation of disease. CIA can also be induced by administration of polyclonal antibodies, or a specific combination of monoclonal antibodies to type II collagen; for example, antibody- mediated CIA (also known as "CAIA") can be induced by i.v. injection of a combination of different monoclonal antibodies generated by mouse hybridoma cell lines. Improved antibody cocktails have been developed by selecting antibodies for their epitope specificity, which is critical to pathogenicity. Evidence suggests that antibodies directed against certain specific epitopes are better associated with the induction of arthritis than other epitopes. In the CAIA mouse model, the antibodies that correlate with arthritis are those associated with binding to the epitopes CI, Jl and Ul. Accordingly, the ArthritoMab™ cocktail (MD Biosciences, 2mg) used in this CAIA study is a mixture of four monoclonal antibodies that bind to the well-defined epitopes Cl lb, Jl, D3 and Ul, which are all major epitopes in mice immunized with CII that develop arthritis. These epitopes are also spread across the entire CII region (CB8, CB10, and CB11 fragments) possibly encouraging better immune complex formation on the cartilage surface for the initiation of arthritis. In this form of the disease, acute arthritis is generated by antibody binding and neutrophil infiltration, bypassing immune activation, but with many of the characteristics of traditional CIA. Thus, the CAIA model provides a faster time course for a therapeutic proof of concept, although in the case of autoimmune disease modulators and drugs that are not rapid acting anti-inflammatory or
immunosuppressive agents, the CAIA model requires careful evaluation by histopathological or immunohistochemical examination after necropsy as the principal demonstration for disease modifying effects, rather than simply focusing on clinical scores.
Representative compounds of this invention were tested by the following protocol, described here in terms of its operational components, performed under contract by Charles River (formerl MIR) Discovery and Imaging Sevices (Ann Arbor, MI). The course of disease induction entails injection of the antibody cocktail on day 0, with each animal receiving an LPS challenge on day 6. Then the following day, after disease had begun to develop, treatment was initiated on day 7. The animals were dosed according to individual body weight on the day of treatment through to day 20. Assessment of disease activity was achieved carried out by measurement of paw edema and clinical scoring (Takeshita M, Sugita et al Exp. Anim. 462:165-169, 1997). Animals that develop collagen antibody-induced arthritis are known to lose body weight over the course of the study due to disease burden, thus body weights, paw measurements, and clinical scores were also recorded.
The clinical scoring system is based on the Holmdahl scoring system. One point is awarded for each red or swollen digit, 5 points for each swollen footpad and 5 points for each swollen ankle. This gives a total of 15 per limb and 60 per animal.
On day 21, after the final measurements were taken, the animals were euthanized and whole blood was collected via cardiac puncture for plasma isolation. The hind limbs were removed from each animal at a level above the knee. The skin was removed and the right hind limb was placed in 10% neutral buffered formalin for subsequent immunohistochemistry and histopathological analysis. Toluidine blue and hematoxylin and eosin stain (H&E) staining were performed on the joints. The immunohistochemistry staining focused on macrophage markers (CDl lb/CDl lc and a macrophage marker such as Mac-2 or F4/80).
The arthritic scoring system for H&E or toluidine blue staining is:
Inflammation
0 = Normal
1 = Minimal infiltration of inflammatory cells in periarticular tissue
2 = Mild infiltration
3 = Moderate infiltration with moderate edema
4 = Marked infiltration with marked edema
5 = Severe infiltration with severe edema
Pannus
0 = Normal
1 = Minimal infiltration of pannus in cartilage and subchondral bone
2 = Mild infiltration
3 = Moderate infiltration
4 = Marked infiltration
5 = Severe infiltration
Cartilage Damage
0 = Normal
1 = Minimal to mild loss of staining density with no obvious chondrocyte loss or collagen disruption
2 = Mild loss of staining density with focal mild (superficial) chondrocyte loss and/or collagen disruption
3 = Moderate loss of staining density with multifocal moderate (depth to middle zone) chondrocyte loss and/or collagen disruption
4 = Marked loss of staining density with multifocal marked (depth to deep zone) chondrocyte loss and/or collagen disruption
5 = Severe diffuse loss of staining density with multifocal severe (depth to tide mark) chondrocyte loss and/or collagen disruption
Bone Resorption
0 = Normal
1 = Minimal; small areas of resorption, not readily apparent on low magnification, rare osteoclasts
2 = Mild; more numerous areas of resorption, not readily apparent on low
magnification, osteoclasts more numerous 3 = Moderate; obvious resorption of medullary trabecular and cortical bone without full thickness defects in cortex, loss of some medullary trabeculae, lesion apparent on low magnification, osteoclasts more numerous
4 = Marked; full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone numerous osteoclasts
5 = Severe - indistinct and irregular border between medullary and cortical bone, numerous osteoclasts
Periosteal Change/Exostotic Growth
0 = Normal
1 = Minimal; roughened periosteal surface with increase inflammatory cells,
periosteal cells, or osteoblasts
2 = Mild; numerous areas of exostosis extending from the joint to the boney shaft
3 = Moderate; bone thickening extending at least from the full length of one
longitudinal bone profile
4 = Marked; numerous longitudinal profiles of bone have exostotic growth and
include active marrow within the exostosis
5 = Severe; multiple exostosis, sometimes bridging across ankylosis or fibrotic joint; exostosis contains marrow elements and there is extensive bone deformity
CD1 lb/c Scoring System (counts per field)
0 - 0 counts
1 = 1-20 counts
2 = 21-50 counts
3 = 51-100 counts
4 = 101 -150 counts
5 = 151+
Mac-2 Scoring System (cells per field)
0 = 0 cells
1 = 1-5 cells
2 = 6-15 cells
3 = 16-30 cells
4 = 31-45 cells
5 = 46+ cells
In the final experimental step, the left hind limb was flash frozen in liquid nitrogen, and the limbs are pulverized and homogenized in phosphate buffered saline containing protease and phosphatase inhibitors, submitted for biomarker analysis against the
RodentMAP version 2.0 Antigens panel (Rules Based Medicine). Results
Representative compounds were evaluated in the CAIA model using Symadex dosed daily at 10 mg/kg for 14 days as a comparator. Compounds of Formula (I) were dosed daily at 30 mg/kg for 14 days. Test articles were dissolved in Pharmatek HRC© Formulation 6 (Pharmatek Laboratories, Inc., San Diego, CA) to obtain a stock concentration of 20 mg/ml, and dosing was by means of oral gavage. Treatment was initiated on day 7. A reduction of approximately 23% in total clinical score was observed for Compound 5 as a representative example in comparison to a reduction of approximately 10% in overall clinical score observed for Symadex (Figure 5).
The clinical effect observed was matched with corresponding histology results based on H&E or toiuidine blue staining. The averaged histology score is presented in Figure 6. All percentages are represented as percentage change of vehicle control. The inflammation score was reduced by approximately 38% (p = 0.05) and 26% (p = 0.08) for Compound 5 and Symadex, respectively. This result was significant in the case of Compound 5 and
approached significance for Symadex. However, and given their mode of action, neither compound achieved complete suppression of inflammation as compared to treatment with corticosteroids in this model.
In contrast, histopathological examination revealed a more impressive effect on disease modification than implied at first glance by the modest decrement in inflammatory symptoms. A 58% reduction in pannus was achieved by Compound 5. Symadex reduced the pannus score by 42%. Consistently, cartilage damage was lessened by approximately 52% and 17%) for Compound 5 and Symadex, respectively. Bone resorption showed a reduction of 55% for Compound 5 and about 22% for Symadex. Finally, periosteal and exostotic changes yielded a similar result with a decrease of 50% for Compound 5 and almost 29% for
Symadex.
In each case, disease modification was highly significant, with average p values scores for Compound 5 below 0.01 and for Symadex below 0.03. These findings are consistent with reported observations in the CAIA model on the efficacy of methotrexate, a drug that is a staple in the clinical treatment of arthritis, and which provides significant disease modification but without a pronounced anti-inflammatory effect. Likewise, representative compounds of the invention were shown to provide significant disease modification , although without a pronounced anti-inflammatory effect.
Further evidence of disease modification by immune modulation emerged from immunohistochemical and biomarker evaluations.
Macrophage populations were stained with the Mac-2 antibody to assess macrophage infiltration in the joint tissue. Overall, Compound 5 reduced macrophage infiltration by 54% (p = 0.02) and Symadex reduced macrophage infiltration by approximately 57% (p = 0.02), both with respect to vehicle control (Figure 7).
Biomarker analysis was performed on homogenized joint tissue that was frozen immediately after the necropsy. The homogenate was analyzed against the rodent biomarker panel from Rules Based Medicine. Plasma samples were analyzed in conjunction with the tissue homogenate to compare systemic biomarker changes compared to local effects.
Figures 8A and 8B shows representative biomarkers (Figure 8A shows data for IL-4 and IL-6, Figure 8B shows data for IL-10, IL-12p70) that demonstrated marked change in this study. These cytokines were decreased locally in the joint at the site of arthritis following treatment with Compound 5, although little effect was observed on the systemic concentrations.
Figure 9 shows representative biomarkers that demonstrated marked change in this study. MCP-3 and MIP-lb have roles as pro-inflammatory chemokines in arthritis. Little effect in changing systemic concentrations of these cytokines was observed, however, levels of these cytokines decreased locally in the joint at the site of arthritis consistent with an antiinflammatory cytokine modulating effect following treatment with Compound 5.
Example 4B: Collagen-induced Arthritis (CIA) Model Methods
In a separate study, representative compounds of this invention were tested in the CIA model, performed under contract by Bolder Biopath, Inc. (Boulder, CO; http://www.bolderbiopath.com). CIA was induced following immunization of type II collagen. Male DBA/1 mice at approximately 7 weeks old were anesthetized with isoflurane and given 150 μΐ of Bovine Type II collagen in Freund's complete adjuvant injections (day 0 and day 21). On days 21-35, onset of arthritis was observedoccur and mice were randomized into treatment groups after swelling was obviously established in at least one paw; and approximately equal mean scores were distributed across the groups at time of enrollment. Treatment was initiated after enrollment and continued every day, followed by recording of clinical scores for each of the paws (right front, left front, right rear, left rear). Mice were weighed on arthritis days 1, 3, 5, 7, 9, 11, 13, 15, 17 and prior to tissue collection on day 18 (final day).
Clinical Scoring Criteria for Fore and Hind Paws
0 = normal
1 = 1 hind or fore paw joint affected or minimal diffuse erythema and swelling
2 = 2 hind or fore paw joints affected or mild diffuse erythema and swelling
3 = 3 hind or fore paw joints affected or moderate diffuse erythema and swelling
4 = Marked diffuse erythema and swelling, or =4 digit joints affected
5 = Severe diffuse erythema and severe swelling entire paw, unable to flex digits
At necropsy, animals were be bled via cardiac puncture for plasma collection; whole blood was spun at 13,000 rpm for 8 minutes and stored at -80 °C for subsequent biomarker analysis (Rules Based Medicine, RodentMAP v.2.0 Antigens). The left hind paw from all animals will was homogenized in buffer containing protease and phosphatase inhibitors. The right hind paw, fore paws, and knees were be removed and placed in 10% NBF for histology.
The arthritic scoring system for H&E and/or toluidine blue staining is:
Inflammation
0 = Normal
1 = Minimal infiltration of inflammatory cells in periarticular tissue
2 = Mild infiltration
3 = Moderate infiltration with moderate edema
4 = Marked infiltration with marked edema
5 = Severe infiltration with severe edema
Pannus 0 = Normal
1 = Minimal infiltration of pannus in cartilage and subchondral bone
2 = Mild infiltration
3 = Moderate infiltration
4 = Marked infiltration
5 = Severe infiltration
Cartilage Damage
0 = Normal
1 = Minimal to mild loss of staining density with no obvious chondrocyte loss or collagen disruption
2 = Mild loss of staining density with focal mild (superficial) chondrocyte loss and/or collagen disruption
3 = Moderate loss of staining density with multifocal moderate (depth to middle zone) chondrocyte loss and/or collagen disruption
4 = Marked loss of staining density with multifocal marked (depth to deep zone) chondrocyte loss and/or collagen disruption
5 = Severe diffuse loss of staining density with multifocal severe (depth to tide mark) chondrocyte loss and/or collagen disruption
Bone Resorption
0 = Normal
1 = Minimal; small areas of resorption, not readily apparent on low magnification, rare osteoclasts
2 = Mild; more numerous areas of resorption, not readily apparent on low
magnification, osteoclasts more numerous
3 = Moderate; obvious resorption of medullary trabecular and cortical bone without full thickness defects in cortex, loss of some medullary trabeculae, lesion apparent on low magnification, osteoclasts more numerous
4 = Marked; full thickness defects in cortical bone, often with distortion of profile of remaining cortical surface, marked loss of medullary bone numerous osteoclasts
5 = Severe - indistinct and irregular border between medullary and cortical bone, numerous osteoclasts
Periosteal Change/Exostotic Growth
0 = Normal
1 = Minimal; roughened periosteal surface with increase inflammatory cells,
periosteal cells, or osteoblasts
2 = Mild; numerous areas of exostosis extending from the joint to the boney shaft
3 = Moderate; bone thickening extending at least from the full length of one
longitudinal bone profile
4 = Marked; numerous longitudinal profiles of bone have exostotic growth and
include active marrow within the exostosis 5 = Severe; multiple exostosis, sometimes bridging across ankylosis or fibrotic joint; exostosis contains marrow elements and there is extensive bone deformity
CD1 lb/c Scoring System (counts per field)
0 = 0 counts
1 = 1-20 counts
2 = 21-50 counts
3 = 51-100 counts
4 = 101 -150 counts
5 = 151+
Statistical Analysis
Clinical data were calculated by first scoring each of four paws individually and then reporting the average score per animal per day for days 1-18 to afford the "Arthritis Score (All Paws)". Paws not showing disease at the time of enrollment were separately scored individually and the average per animal is reported as the "Arthritis Score (Non-Enrollment Paws)". Means for each group were determined and percent inhibition from arthritis controls was then calculated by comparing values for treated and normal animals. Animal body weights were analyzed for differences using a One- Way Analysis of Variance (ANOVA) with significance is set at p<0.05. Paw scores and histologic parameters (mean ± S.E.M.) for each group were analyzed for differences from control using the Kruskal-Wallis test appliedto the individual animal scores for the last seven days of the in-life time course with significance set at p<0.05. The mean drug effect was determined from the average daily animal score, taken as the increment above the entry score on enrollment. The corresponding values for each cohort over the last seven days were then compared to control by the
Wilcoxon Rank-Sum test on the daily medians with significance set at p<0.05.
Results
Representative compounds of the invention— Compounds 12, 36, 47, 50, 54, 100, and 129— and two immunomodulating positive controls, imatinib and methotrexate, were evaluated in the CIA model. Oral dosing of all test articles was achieved by gavage. The test compounds were dispensed at 50 mg/kg in Pharmatek Formulation 6, which was also used as the vehicle control for all experiments. Imatinib was dosed at 100 mg/kg in phosphate buffered saline (PBS) and methotrexate at 1.5 mg/kg in 1.5% aqueous carboxymethylcellulose (CMC). Collagen induced arthritis was primed in mice by injecting with collagen type II. After boosting the immunization with a second injection of collagen type II, disease onset became observable shortly thereafter. Animals were enrolled into cohorts when at least one paw showed a clinical score of 0.5. Dosing began at this stage of disease and continued for 18 days thereafter with periodic monitoring of body weight and clinical scores.
During the in-life portion of the study, all test cohorts and control mice tolerated the dosing without mortality. Progressive weight gain was observed within the normal variation of mice treated with vehicle alone. A representative plot for Compounds 12, 36, 47 and 129 is shown in Figure 10. This finding is considered a general confirmation of safety and rules out the possibility that any biological effect on disease may have resulted from failure to grow owing to drug or vehicle toxicity.
All the compounds tested in the CIA model, including the positive controls, significantly (p <= 0.05) reduced the disease burden in the treatment cohorts. These disease modifying effects were manifested either as a reduction in the clinical scores, indicating a decrement in paw swelling, or in the underlying histopathological hallmarks of disease, such as inflammation, pannus, bone resorption and exostotic bone growth. Representative results on disease modification ("All Paws" scoring) and on disease prevention ("Non-enrollment Paws" scoring), respectively, are charted in Figures 1 1 A and 1 IB for Compound 36 in comparison with imatinib, a proven immunomodulator in the CIA model, and in Figures 12A and 12B for Compound 129 in comparison with methotrexate, a standard therapy for arthritis.
A summary of the disease reduction scores as a percent of control is presented in Table 23. It includes the preceding data as well as Compounds 12, 47, 50, 54, and 100.
Table 23
Reduction in Clinical Score as % Control
Test Disease Modification Disease Prevention
Compound All Paws Non-enrollment Paws
12 6% 39%
36 30% 28%
47 0 % 0% 50 27% 13%
54 33% 41%
100 17% 24%
129 50% 73%
Imatinib 29% 28%
Methotrexate 37% 53%
As observed in the CAIA model presented in Example 4A, the CIA clinical scores address paw swelling and hence the symptoms, not the causes, of the underlying pathology. A more comprehensive estimate of efficacy relies on histopathological analysis and quantification of the reduction in anatomical aberrations that are fundamental to the disease. Figure 13 is illustrative of this point. The grayscale photomicrograph shows a comparison of the toluidine-blue stained tibio-tar sal joint slice of a vehicle treated animal in the CIA model to that of an animal treated with Compound 12. The image on the left is the low
magnification image (20x) and the inset box in each is shown at the right at higher magnification (lOOx). The black arrow in the Vehicle micrograph on the left points to a large area of inflammation, seen as a dark grey shadow, surrounding the joint. The matched area in the lower left panel shows the result of treatment with Compound 12 and appears clear of severe inflammation, as evidenced by the light gray, almost transparent staining. Similarly, in the left panels, the area of the inset in the Vehicle slide shows a degenerate joint, while the section from a treated animal shows that the anatomic integrity of the joint has been effectively repaired.
Greater detail to this effect is provided in the right hand, higher magnification panels. The arrows marked "a", "e" show areas of inflammation in the Vehicle sample, which are largely resolved upon treatment, and the mild residual inflammation in the treatment sample is pointed to by the arrow marked "h". The arrow marked "c" shows pannus, a granulomatous growth invading the joint. In contrast, pannus is not observable after treatment with
Compound 12. The black bracket shows bone resorption in the vehicle, which is not evident in the treatment sample. The arrow marked "b" shows exostitic bone growth, an aberration caused in response to bone degradation, but the same situation does not occur in the treatment sample (lower right panel). Lastly, the arrows marked "d", "f ', and "g" show cartilage degeneration. Note should be made of the decreased severity of these changes in the
Compound 12 treated sample.
The qualitative contrast between the disease status of the untreated Vehicle control and the outcome upon treatment with Compound 12 is a stark one and belies the conclusion that might have been arrived at from the clinical scores shown in Table 15. The clinical scores would suggest that efficacy on disease modification ("All Paws" score") based solely on paw swelling is 6%, yet the histopathological evidence is far more dramatic, showing no pannus, very little bone resorption or exostotic bone growth in the image.
Because visual inspection of a single image is subject to sampling errors, multiple sections from each joint and each mouse in the experimental cohorts were evaluated and quantified by a blinded observer. The resulting analysis for Compounds 12, 36, 47, and 129 is presented in Figures 14-17.
Figure 14 shows a quantitative measurement of inflammation based on the
characteristic staining of invasive inflammatory cells in tissues of the joint taken at necropsy, rather than based on gross physical measurements of joint swelling during the in-life clinical course. The representative compounds of this invention all reduced the severity of the inflammatory score to at least 50% of control (p < 0.05 in all cases) and in step with the known immune modulators used as positive controls.
Figure 15 shows a quantitative measurement of pannus in joints in the presence of a representative compound of the present invention as well as controls.
Figure 16 shows a quantitative measurement of cartilage erosion in joints in the presence of a representative compound of the present invention as well as controls.
Figure 17 shows a quantitative measurement of exostotic growth in joints in the presence of a representative compound of the present invention as well as controls.
To illustrate that symptom modification may not always correlate with disease modification, the more desirable outcome in the longer term, the histopathological record indicates a 50% average reduction in the titer of inflammatory cells for both Compound 12 (a 6% reduction in the clinical score in Table 15), and Compound 47 (no response in clinical score in Table 15).
Compound 12 showed the sharpest reduction in both pannus (inflammatory granulomatous tissue) to 25% of control as shown in Figure 15, and cartilage erosion to 40% of control as shown in Figure 16, while Compound 47 decremented the level of exostotic growth to approximately 10% of control as shown in Figure 17.
The results for Compound 36 by histopathological assessment closely coincided with the effects predicted by the clinical score, matching the 30% reduction in disease burden across the board.
Compound 129, the best performer in clinical score reduction according to Table 15, showed consistent performance in disease modification, with levels of disease modification close to 50% in severity reduction across the spectrum of objective criteria, as shown in Figures 15-17, and matching the activity of the positive controls.
Further evidence of disease modification by immune modulation in the CIA model emerged from biomarker evaluations, for which Compound 36 was used as an illustrative example, given the consistency of it efficacy profile measure both in terms of the clinical and histopathological disease reduction assessments. As previously described in Example 4A, the biomarker analysis was performed on homogenized joint tissue that was frozen immediately after the necropsy. The homogenate was analyzed against the rodent biomarker panel from Rules Based Medicine. Plasma samples were analyzed in conjunction with the tissue homogenate to compare systemic biomarker changes compared to local effects.
Figure 18 shows a comparison between the mean IL-6 titers per cohort in a nai've animals unexposed to CIA, the vehicle control, and the treatment group. Compound 36 elicited a significant reduction of IL-6, to less than 50% of control and approaching the levels in normal animals. This response was characteristic of the inflammatory milieu in the joint, where IL-6 is a key effector after immunogenic antigen presentation. Reduction of IL-6 is consistent with the tolerogenic mode of action of the compounds of this invention and their effect on dendritic cells. The reverse scenario is described in Figure 19, where it can be seen that titers of IL- 10 were significantly increased over control by almost twofold. IL-10 is a counter-regulatory cytokine associated with the mechanism of action of immunomodulatory therapies that reduce the more destructive Th-17 phenotypes and promote regulatory T-lymphocytes, which in turn attenuate inflammatory and tissue destructive autoimmune responses. Lower concentrations of IL-10 are secreted by normal tissues, as seen in the response of naive animals which had not been subjected to an autoimmune insult.
As seen in Figure 20, treatment with Compound 36 achieved a twofold reduction in the titer of the pro-inflammatory chemokine biomarker, MIP-2, characteristic of activated, invasive macrophages, again demonstrating that the overall autoimmune response in the CIA model had been attenuated versus vehicle control, consistent with the clinical and
histopathological record.
Notably, each of these (IL-6, IL-10, and MIP-2) biomarker responses were observed in the joint homogenates, while no change in titer was observed in plasma. This observation is significant because it confirms that the effect of compounds of this invention, as exemplified by Compound 36, exert their therapeutic action at the site of disease without the kind of systemic impact that is observed with global immunosuppressants.
EXAMPLE 5: DSS-INDUCED COLITIS (IBD MODEL)
Inflammatory bowel disease (IBD) is a condition that results from a combination of environmental stimuli (the presence of bacterial flora), genetic factors, and a predisposition toward elevated immune response that cannot be replicated in one-dimensional systems such as cell or tissue culture. Because of the genetic complexity of this disease, it is not amenable to computer modeling and requires an in vivo system. Mice represent an excellent in vivo system as they can be tested in high numbers (increasing the statistical power of any study).
The colitis model induced by dextran sulfate sodium (DSS) DSS is one possible model to examine IBD characteristics. DSS chemically "strips" the epithelium of the colon to induce inflammation. The acute DSS model differs from the chronic DSS model in that only one cycle of DSS is given to the mice with DSS concentrations ranging from 3-10% in the drinking water. As a result, it allows for monitoring of inflammatory reduction and mucosal restitution as a consequence of drug treatments. The chronic model usually entails several on/off cycles of DSS, ranging from 1-3%, to mimic the disease recurrence in a person with IBD.
Acute DSS-Induced Colitis Model
Representative compounds of this invention were tested in the DSS-Induced Colitis, performed under contract by Dr. Hans-Christian Reinecker of the Center for the Study of Inflammatory Bowel Diseases, the Massachusetts General Hospital (Boston, MA). Female C57BL/6 mice weighing 18 g ± 0.5 g were treated with 4% DSS for five days (day 1-5). Dosing of drugs occurred on day 3 and day 6. Body weights were monitored through the course of the study through day 12. Substantial decrease in body weight is suggestive of disease progression. Mice are necropsied on day 12. Colons, spleens and mesenteric lymph nodes were removed for gross assessment. Enlarged colons, spleens, and mesenteric lymph nodes are indicative of inflammation. The colons were subsequently frozen for histological assessment. The mRNA from the spleens and mesenteric lymph nodes were isolated for subsequent cytokine assessment. Hematoxylin and eosin stain (H&E) staining was performed on the colons for histology.
Histology Scoring
Inflammation was scored from 0-4 using the following criteria:
0 = No change from normal tissue
1 = Patchy mononuclear cell infiltrates in the lamina propria
2 = More uniform mononuclear cell inflammation involving both the epithelium and lamina propria accompanied by minimal epithelial hyperplasia and slight-to-no depletion of mucus from goblet cells
3 = Some epithelial and muscle hypertrophy with patchy lymphocytic infiltrates extending into the muscle layers, mucus depletion and occasional crypt abscesses and epithelial erosions
4 = Lesions involving most of the intestine, inflammation is composed mostly of lymphocytes and some neutrophils, is transmural and severe with prominent thickening of both the epithelial and muscle layers, mucus depletion and more frequent crypt abscesses.
Chronic DSS-Induced Colitis Model Seven week old C57BL/6 mice were used for this study. They were housed on IL-10 KO-bedding containing natural flora for the entire study, to ensure the transfer of enteric flora required to accurately model colitis. Mice were given a 4% DSS treatment in acidified water ad libitum for three cycles of 5 days on and 7 days off in between; the last non-DSS cycle consisted of 4 days. Dosing began at the beginning of the second DSS treatment. Terminal blood was drawn. Colons were harvested, flushed, weighed, and lengths were recorded. The colons were subsequently frozen in OCT. Throughout the course of the study body weights and disease activity index scoring was monitored. Disease Activity Index (DAI) represents non-invasive observations of disease severity. Animals were individually examined at different times throughout the course of disease (three times a week)
Disease Activity Index (DAI) Scoring System
Prolapse Stool Consistency Fecal Occult Blood
0 = Normal Anus 0 = Solid Pellet 0 = None
1 = Partial 1 = Semi-Solid 1 = Occult Blood
2 = Moderate 2 = Soft Stool 2 = Gross Blood
3 = Full Prolapse 3 = Diarrhea
Histology Scoring
For each colon section, submucosal edema was quantitated by measuring the distance from the muscularis mucosa to the internal border of the outer muscle layer. The extent of inflammation (foamy macrophage, lymphocyte and polymorphonuclear cell infiltrate) was assigned severity scores according to the following ranking:
0 = Normal
1 = Minimal
2 = Mild
3 = Moderate
4 = Marked
5 = Severe
For each of the sections, the parameters reflecting epithelial cell loss/damage were scored individually using a percent area involved scoring method:
0 = None
1 = 1-10% of the mucosa affected
2 = 11-25% of the mucosa affected 3 = 26-50% of the mucosa affected
4 = 51-75% of the mucosa affected
5 = 76-100% of the mucosa affected
Specific parameters scored using the above percent involvement scale include:
Colon erosion - this reflects loss of surface epithelium and generally is associated with mucosal hemorrhage (reflective of the bleeding seen clinically and at necropsy).
Colon glandular epithelial loss - this includes crypt epithelial as well as remaining gland epithelial loss.
The three important scored parameters (i.e., inflammation, glandular epithelial loss, and surface epithelial erosion) were be combined to arrive at a sum of overall histopathology score for each section of the colon. This summation indicates the overall damage in the distal or proximal colon and shows a maximum score of 15.
Example 5A; Acute Colitis Model with Compounds 12, 36, 39, and 47
Representative compounds were evaluated, and acute colitis was induced in C57BL/6 mice with 5 day treatment of DSS (day 1-5). Dosing of compounds of Formula (I) was administered on day 3 and day 6. Body weight was measured at the initiation of the study through day 12. As shown in Figure 21, Compound 12 and Symadex prevented weight loss due to DSS treatment. As shown in Figure 22, less weight loss was observed for animals treated with Compounds 12, 36, and 47 compared to the vehicle control; these compounds appear to have mucosal protective properties.
Colons, mesenteric lymph nodes, and spleens were harvested from the mice at the end of the study (day 12). The vehicle treated mice show thickened colonic walls, enlarged mesenteric lymph nodes and spleens. The mice treated with Compound 12 have
morphologically normal colons; this is apparent at regions containing no fecal matter where the colon wall is relatively thin. The mice treated with Symadex appear to have a mixed morphology with regions of inflamed and normal colonic walls.
Histopathology was subsequently performed on the colons. Colons from animals after treatment with vehicle, Symadex, and representative Compound 12 were compared to a nai've colon. The DSS treatment (vehicle) caused severe inflammation in the colon leading to epithelial hyperplasia, thickening of the muscle wall, partial transmural inflammation, crypt abscesses, and mononuclear cell infiltration. Mice treated with Compound 12 on the other hand, demonstrated almost complete restoration of the colonic epithelium with uniform epithelial cell morphology. In addition, the colon from animals treated with Compound 12 showed excess mucus-forming goblet cell presence, indicating a prior inflammation.
Mucosal restitution becomes evident following treatment with Compound 12.
The inflammation score as determined from histopathological assessment is presented in Figure 23; treatment with Symadex and Compound 12 shows significant reduction in inflammation of the colon as compared to vehicle.
Example 5B: Acute Colitis Model with Compounds 47 and 129
DSS colitis was induced in female C57B1/6 mice by adding 4% DSS to their drinking water for a period of 5 days. Normal drinking water was continued thereafter. Mice were dosed orally with test compounds (50 mg/kg) on Day 3 and Day 6 of the study. On Day 5, a few mice from each group were sacrificed to harvest the mesenteric lymph nodes (MLNs) for cytokine quantitative PCR (qPCR). MLNs were also harvested at the end of the study (day 12) for cytokine expression qPCR. Colons were harvested for histology and small intestines were harvested to characterize the dendritic cell population after treatment.
As shown in Figure 24, compounds 47 and 129 were able to reduce the weight loss associated with severe colitis in mice. More significantly, the compounds protected the mice from colitis and induced mucosal healing and recovery from colitis. Comparatively, the percent weight loss in mice treated with vehicle control alone was higher and recovery from colitis was delayed.
Histological analysis of colon sections harvested from mice treated with compounds 47 and 129 presented with improved colon morphology with little or no signs of mucosal inflammation after two oral doses. Photomicrographs of representative sections of colons harvested from mice treated with representative compounds and the vehicle control and stained with H&E stain, are shown in Figures 25A, 25B, and 25C. Based on these stained sections and the histological examination, three independent parameters were measured in a blinded fashion: severity of inflammation, depth of injury/inflammation, and crypt damage. The resulting histograms, shown in Figure 26, confirm that all three parameters were significantly reduced in treated mice versus vehicle control, reaffirming the anatomical findings.
Cytokine expression in MLNs was assessed at day 5 (acute inflammatory response) and day 12 (recovery phase).
MLNs harvested from treated mice at day 5 of the study were suspended in Trizol for RNA isolation. The RNA was isolated from the MLNs using Trizol-chloroform method. The cDNA was synthesized using cDNA synthesis kit and further used for the quantitative PCR (qPCR) assay. The AAC (t) values were normalized against GAPDH as an internal control. The cytokine gene expression values were plotted as normalized gene expression. qPCR performed on RNA isolated from the MLNs harvested on Day 5 (following a first dose administered on day 3) indicated that mice treated with compounds 47 and 129 showed lower expression of pro-inflammatory cytokines such as IL-Ιβ and TNF-D (Figure 27) after one oral dose.
Cytokine gene expression in MLNs was also assessed during the recovery phase. qPCR was performed on MLNs harvested on Day 12 from mice treated with compounds 47 and 129 and vehicle control. By Day 12, expression of IL-10 and IL-27 (Figure 28) was higher in the MLNs of the groups treated with compounds 47.
EXAMPLE 6: EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (MS MODEL)
Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of multiple sclerosis. Depending on the species, allergen, and methodology used, animals tested by the EAE model may experience a single (acute EAE) or several (chronic relapsing EAE) attacks. The animals are injected with the whole or parts of various proteins that make up myelin, the insulating sheath that surrounds nerve cells (neurons). These proteins induce an autoimmune response in the animals in which their immune system mounts an attack on its own myelin as a result of exposure to the injection. The animals develop a disease process that closely resembles MS in humans. The EAE model is an appropriate method for studying the inflammation of the brain and spinal cord associated with MS (Bolton, C. Mult. Scler. 1 : 143- 9, 1995), and the utility of a pharmaceutical compound for the treatment of MS can be demonstrated by its effectiveness in the EAE model. Treatment can be prophylactic or preventative, whereby the therapeutic composition is administered before immunization; treatment can be initiated during the first week of induction; and treatment can be
interventious, initiated after clinical symptoms are extant (acute or chronic).
For this model, C57BL/6 mice develop chronic paralysis after immunization with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) emulsified in Complete Freund's Adjuvant (CFA) with a pertussis toxin injection. Mice develop EAE 8-14 days after immunization and stay chronically paralyzed for the duration of the experiment (typically, mice are observed for 30-40 days). Around 25% of the mice show exacerbation of EAE symptoms after initial partial recovery. This usually occurs 20-27 days after immunization.
Methods
In a representative experiment, female 10 week old C57BL/6 mice will be immunized with MOG3 -55 in Complete Freund's Adjuvant on day 0, two hours later the mice are injected with pertussis toxin. Twenty four hours after immunization, the mice will receive a second injection of pertussis toxin. One day after the first clinical signs of EAE have been detected in each mouse, the mice will be assigned to an experimental group in a balanced manner, such that disease is even among the groups. Treatment is initiated for that mouse on the day of assignment and will continue for 14 days. Mice will be monitored for clinical signs for an additional 2 weeks posts treatment. Mice are scored daily from initiation of disease onset until the end of the study. Body weights are measured three times/week (e.g., Monday, Wednesday and Friday), starting on day -1.
Mice which develop EAE last or which develop unusual signs of EAE such as head tilting will not be assigned to any treatment group. Clinical Score
EAE is scored using the following scale ranging from 0-5 in 0.5 unit increments
0 = No obvious changes in motor functions of the mouse in comparison to non- immunized mice. When picked up by the tail, the tail has tension and is erect. Hind legs are usually spread apart. When the mouse is walking, there is no gait or head tilting.
1 = Limp tail. When the mouse is picked up by the tail, instead of being erect, the whole tail drapes over your finger.
2 = Limp tail and weakness of hind legs. When mouse is picked up by tail, legs are not spread apart, but held closer together. When the mouse is observed when walking, it has a clearly apparent wobbly walk.
3 = Limp tail and complete paralysis of hind legs (most common) OR Limp tail with paralysis of one front and one hind leg OR ALL of these characteristics: severe head tilting, walking only along the edges of the cage, pushing against the cage wall, spinning when picked up by the tail.
4 = Limp tail, complete hind leg and partial front leg paralysis. Mouse is minimally moving around the cage but appears alert and feeding. Usually, euthanasia is recommended after the mouse scores level 4 for 2 days. When the mouse is euthanized because of severe paralysis, score of 5 is entered for that mouse for the rest of the experiment.
5 = Complete hind and complete front leg paralysis, no movement around the cage
OR mouse is spontaneously rolling in the cage OR mouse is found dead due to paralysis.
If mouse is alive, euthanize the mouse immediately if it scores 5. Once mouse is scored 5, the same score is entered for all the days for the rest of the experiment.
Histology
The spines from the mice will be harvested and fixed in 10% neutral buffered formalin for about 24-48 hours. The samples are subsequently transferred into ethanol until histological assessment can be performed. The spines are stained with H&E stain and CDl lb and CDl lc antibodies.
Scoring System for Inflammation
0 = No inflammation
1 = Inflammation in less than 10% of the organ
2 = Inflammation that occurs in 10 - 20%> of the organ
3 = Inflammation that occurs in 21 - 30% of the organ
4 = Inflammation that occurs in 31 - 40% of the organ
5 = Inflammation that occurs in over 40% of the organ CD1 lb/c Scoring System (counts per field)
0 = 0 counts
1 = 1-20 counts
2 = 21-50 counts
3 = 51-100 counts
4 = 101 -150 counts
5 = 151+
Results
Representative compounds will be evaluated in the EAE model concurrently with FTY720 (fingolimod), an approved drug for the treatment of multiple sclerosis, as a positive control. Daily dosing will begin after disease induction occurs and/or reaches a clinical score of approximately 2.5 to 3. Treatment continues for a 3 week period, after which the drug is removed and the daily clinical scores monitored for another 2-3 weeks. In model validation studies, it is notable that after, after the drug withdrawal, treatment ceased, animals treated with FTY720 rebound to full disease after a week.
In conjunction with clinical score, body weight is measured three times a week throughout the course of the study after immunization with the MOG peptide. The days are approximated since these mice body weights are not synchronized similarly to the clinical scores. In general, the effects observed from clinical score are consistent with the loss and recovery of body weight associated with disease and drug administration. For FTY720, body weight loss due to onset of disease is observed; and, as drug is administered, gain of body weight is observed. After drug treatment is discontinued, FTY720 cohorts lose body weight, which is consistent with rebounding of disease.
Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and the essential characteristics of the present teachings. It is not intended that the present invention be limited to the illustrated embodiments but rather by the following claims, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS impound of Formula (I)
Figure imgf000189_0001
(I)
or a pharmaceutically acceptable salt thereof, wherein:
A1 is selected from H, hydroxyl, a C1 -C6 alkoxy, or -C(0)NRARB, wherein RA and R each independently are hydrogen or a C 1 -C3 alkyl,
A is selected from H, hydroxyl, a C1-C6 alkoxy or a C1-C6 alkyl, optionally substituted with a halogen;
or
A1 and A2 taken together with the intervening atoms form a lH-pyrazole ring or a pyrrol-2-one ring;
provided that A1 and A2 are not simultaneously hydrogens;
R1 is a halogen, -OR2, -SR3, or -NR4R5;
R2 and R3 each independently are hydrogen or -L^-R6;
L1 is -(CH2)X-, wherein x is 0, 1, 2, 3, or 4;
R6 is independently for each occurrence selected from hydrogen, C1-C4 alkyl, or a 5- to 6-membered aryl or heteroaryl Ar1;
R4 is hydrogen or a C1-C4 alkyl;
R5 is selected from:
- hydrogen,
- -L2-R7,
- -C(0)RD, and
wherein
RD is hydrogen or a C1-C3 alkyl, optionally substituted with amino, a (Cl- C3)alkylamino, or a di(Cl-C3)alkylamino, and further wherein, optionally, the alkyl portions of the di(Cl-C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl;
or, alternatively,
R4 and R5, taken together with the nitrogen atom to which they are attached, form a 5-membered heteroaryl Ar2, optionally including one or two additional heteroatoms selected from N, S or O;
or, alternatively,
R4 and R5, taken together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocyclyl C2, optionally including an additional heteroatom selected from N, O or S, wherein S is optionally oxidized into S(02);
L2 is -(CHRK)y-, wherein y is 0, 1, 2, 3 or 4, and each RK is independently hydrogen or a C1-C4 alkyl;
R7 is:
- hydrogen,
- a Cl-C4 alkyl,
- -C(0)ORL, wherein RL is hydrogen or a C 1 -C4 alkyl,
- a C1-C4 alkoxy,
- -NRMRN,
a 5- to 6-membered heterocyclyl C1 including one or two heteroatoms independently selected from N, O or S, or
- a 5- to 6-membered aryl or heteroaryl Ar ,
wherein
RM and RN each independently are hydrogen or a C1-C4 alkyl,
or, alternatively,
RM and RN, taken together with the nitrogen to which they are attached, form a 5- to 7-membered heterocyclyl C3, optionally including one additional heteroatom selected from N, O or S.
2. The compound of Claim 1, wherein Ar1 is substituted with one to three substituents Rc selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said CI -C3 alkyl is optionally substituted with a halogen,
or, alternatively, two R groups taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
3. The compound of Claim 1 , wherein C1 is substituted with a substituent selected from a C1-C3 alkyl, phenyl, benzyl or -C(0)ORE, wherein RE is hydrogen or a C1-C4 alkyl.
4. The compound of Claim 1, wherein Ar2 is substituted with one to three substituents
C 1
R selected from a halogen, C1-C3 alkyl, hydroxyl, and a (Cl-C3)alkoxy, wherein said C1-C3 alkyl is optionally substituted with a halogen,
or, alternatively,
two RCI groups taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l ,4-dioxine ring.
5. The compound of Claim 1 , wherein C2 is substituted with one to three substituents R8, wherein
R8, for each occurrence independently, is selected from hydroxyl, cyano, a halogen, a C1-C6 alkyl, -C(0)ORG1, or a 5- to 6-membered aryl or heteroaryl, wherein said C1-C6 alkyl is optionally substituted with RF and said 5- to 6-membered aryl or heteroaryl is optionally substituted with a hydroxyl, a C1 -C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, -C(0)ORG, or -C(0)NRHRJ, wherein RF is hydroxyl, cyano, a C1-C3 alkyl, a C3-C6 cycloalkyl, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino, a C1-C3 alkoxy, a 5- to 6-membered heteroaryl or phenyl, -C(0)ORG2 or -C(0)NRH1RJ1;
RG, RG1, and RG2 are each independently hydrogen or a C1-C4 alkyl; and RH, RJ, RH1, RJ1 each independently are selected from hydrogen or a C1-C3 alkyl.
6. The compound of Claim 1, wherein Ar3 is substituted with one to three substituents R°
7. The compound of Claim 6, wherein each R is independently selected from: a halogen,
- hydroxyl,
cyano,
- -S(02)NH2 or ~S(02)N(CH3)2;
amino,
- a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, each optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl,
- a 5 -6-membered aryl or heteroaryl;
a 5- to 7-membered non-aromatic heterocyclic ring including one or two heteroatoms selected form 0, N and S;
- -C(0)ORp, wherein Rp is hydrogen or a C 1 -C4 alkyl,
- (Cl-C3)alkylamino,
di(Cl-C3)alkylamino, wherein, optionally, the alkyl portions of the di(Cl- C3)alkylamino, taken together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclyl, optionally including one additional heteroatom selected from N, O or S; and
a C 1 -C6 alkoxy group, wherein
said C1-C6 alkoxy group is optionally substituted with a 5- or 6-membered aryl or heteroaryl, said 5- or 6-membered aryl or heteroaryl further optionally substituted with a halogen, hydroxyl or a C1-C6 alkoxy,
or, alternatively,
two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
8. The compound of Claim 1, wherein C3 includes an additional heteroatom selected from O or N, and wherein
if said additional heteroatom is nitrogen, then said additional heteroatom is optionally substituted with a group R100 selected from a C1-C6 alkyl, a C3-C6 cycloalkyl, phenyl, cyano, hydroxyl and -C(0)OR101, wherein R101 is methyl or ethyl.
9. The compound of Claim 1, wherein A1 and A2 each independently are a C1-C6 alkoxy, optionally substituted with a halogen or a 5- or 6-membered aryl or heteroaryl.
The compound of Claim 9, wherein A1 and A2 each independently are selected from -O-benzyl,- O-tButyl, -O-Methyl, -O-Ethyl, -O-iPropyl and -0-CF3.
The compound of any one of claim 1 or 3 to 10, wherein R1 is -NR4R5.
The compound of any one claim 1 to 8, or a pharmaceutically acceptable salt thereof, wherein A1 and A2 taken together with the carbon atoms to which they are bonded form a lH-pyrazole ring to form chemical formula (IA) or (IB)
Figure imgf000193_0001
(IA) (IB).
13. The compound of any one claim 1, 6 or 7, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by chemical formula (IA2) or (IB2):
Figure imgf000193_0002
(IA2) (IB2) wherein:
R4 is H or Ci-6 alkyl,
R5 is -L2-R7, R7 is a 5- to 6-membered aryl or heteroaryl Ar3; andAr3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl.
14. The compound of claim 13, wherein y is 1.
15. The compound of claims 13 or 14, wherein
R4 is H;
y is 1 , and
Ar3 is phenyl; and
wherein Ar3 is optionally substituted with R°, and, for each occurrence, R* independently is selected from a halogen, Ci-6 alkyl, C1-6 haloalkyl, amino, (Cl- C3)alkylamino, di(Cl-C3)alkylamino, phenyl, and a C1-C6 alkoxy group,
or, alternatively, two groups R° taken together with the intervening atoms form a 1,3-dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
16. The compound of claims 13 or 14, wherein
R4 is H;
Ar3 is pyridyl;
y is 1 ; and
RK is H or methyl.
17. The compound of claim 13 or 14, wherein
R4 is H;
Ar3 is thiophen-2-yl;
y is 1 ; and
RK is H or methyl.
18. The compound of claim 13, wherein
R4 is H;
Ar3 is pyridyl;
y is 2; and R is H or methyl.
19. The compound of claim 13, wherein
R4 is H;
Ar is phenyl;
y is 2;
R is H or methyl; and
the optional substituent R is, for each occurrence independently, selected from a halogen and a C 1 -C6 alkoxy group,
or, alternatively, two groups R° taken together with the intervening atoms form a 1,3- dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
The compound of any one of claims 1 , 6 and 7 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by chemical formula (IA2) or (IB2)
Figure imgf000195_0001
(IA2) (IB2) wherein
R4 is H or Ci-6 alkyl;
R5 is -L2-R7;
y is 0;
R7 is a 5- to 6-membered aryl or heteroaryl Ar3; and
Ar3 is selected from pyridyl, thiophenyl, phenyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and furyl. The compound of claim 20, wherein
R4 is H;
Ar3 is phenyl,
wherein the optional substitutent R°, for each occurrence independently, is selected from halogen, C2-6 alkynyl, a 5 to 7 membered non-aromatic heterocyclic ring including one or two heteroatoms selected from N, O and S, phenyl, benzyl; -S02NH2 , -S02N(CH3)2 or a C1-C6 alkoxy group,
or, alternatively, two groups R° taken together with the intervening atoms form a 1,3- dioxole ring or a 2,3-dihydro-l,4-dioxine ring.
The compound of any one of claims 1 or 5, or a pharmaceutically acceptable salt thereof, wherein
Figure imgf000196_0001
wherein Q1 is O, S, S02, CH2, CHR8A, NH, or NR8B;
R8A is Ci-6 alkyl, phenyl, benzyl, CN, or -C(0)ORtjl, wherein said C1-C6 alkyl optionally substituted with a C1-C2 alkoxy; and
R8B is Ci-6 alkyl, phenyl, or -C(0)ORG1, wherein said C1-6 alkyl is optionally substituted with -CN, hydroxyl, a C1-C3 alkoxy, or cyclopropyl, and wherein said phenyl is optionally substituted with C1-C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(Cl-C3)alkylamino.
The compound of claim 22, wherein R4 and R5, taken together with the nitrogen to which they are attached, form N-morpholinyl, N-thiomorpholinyl, N-piperidinyl, N-piperazinyl, or N-pyrrolidinyl.
The compound of any one of claims 1, 3, 6, 7 and 8, or a pharmaceutically acceptable salt thereof, wherein
the compounds are presented by structural formal (IA2) or (IB2)
Figure imgf000197_0001
The compound of claim 24, wherein
R7 is -NRMRN,
RM and RN, taken together with the nitrogen to which they are attached, form 5- to 7-membered heterocyclyl C3, and further wherein C3 isselected from the group consisting of
Figure imgf000197_0002
wherein
Q2 is O, C¾, NH, or NR100;R100 is -C(0)OEt or a d.6 alkyl optionally substituted with -CN, -OH, phenyl, or cyclopropyl. The compound of claim 25, wherein
y is 2; and
Q2 is O, NR100, or C¾.
The compound of claim 25, wherein C3 is N-morpholinyl, N-piperidinyl, or
N-piperazinyl.
The compound of any one of claims 1 and 3, or a pharmaceutically acceptable salt thereof, wherein the compound is presented by structural formla (IA2) or (IB2)
Figure imgf000198_0001
(IA2) (IB2), wherein R5 is a 5- to 6-membered heterocyclyl C1 including one or two heteroatoms independently selected from N, O or S.
29. The compound of claim 28, wherein y is 0, C is
Figure imgf000198_0002
, wherein
R50 is benzyl, methyl, or -C(0)OEt.
30. The compound of Claim 28,
wherein R5 is L2-R7, y is 2, R7 is C1, and R5 is represented by the following structural formula:
Figure imgf000198_0003
The compound of claim 28, wherein y is 0, and C1 is
Figure imgf000199_0001
The compound of any one claims 3 to 8, wherein
R1 is NR4R5;
A1 is hydroxyl, a C1-C6 alkoxy or H; and
A2 is selected from hydroxyl, a C1-C6 alkoxy, C1-6 alkyl, C1-6 haloalkyl, anc
H.
The compound of claim 32, wherein
R4 and R5, taken together with the nitrogen atom to which they are attached d form the group consisting of
Figure imgf000199_0002
wherein Q1 is 0, S, S02, CH2, CHR8A, NH, or NR8B;
R8A is C1-6 alkyl, phenyl, benzyl, CN, or -C(0)ORQ1, wherein said C1-C6 alkyl optionally substituted with a C1-C2 alkoxy; and
R8B is Ci-6 alkyl, phenyl, or -C(0)ORG1, wherein said Ci-6 alkyl is optionally substituted with -CN, hydroxyl, a C1-C3 alkoxy, or cyclopropyl, and wherein said phenyl is optionally substituted with C1-C3 alkoxy, amino, a (Cl-C3)alkylamino, a di(C 1 -C3 )alkylamino .
34. The compound of any of claims 1 and 2, or a pharmaceutically acceptable salt thereof wherein the compound is represented by formula (IA) or (IB)
Figure imgf000199_0003
wherein R1 is -OR2 or -SR3.
35. The compound of claim 34, wherein R2 and R3 each independently are hydrogen or
36. The compound of any of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by structural formula (IC)
Figure imgf000200_0001
(IC).
37. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the following structural formula
Figure imgf000200_0002
38. The compound of any claim 1 to 10, wherein R1 is a halogen.
A compound of claim 1 , or a pharmaceutically acceptable salt thereof, selected from: 2-Chloro-9-(lH-indazol-5-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-propane-l,3-diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(3-morpholin-4-yl-propyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-dimethyl-amine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-N'-methyl-ethane-l ,2-diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(2-methoxy-ethyl)-amine;
[9-(l H-Indazol-5-yl)-9H-purin-2-yl]-phenethyl-amine; Diethyl-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-amine;
9-(lH-Indazol-5-yl)-2-morpholin-4-yl-9H-purine;
9-(lH-Indazol-5-yl)-2-(4-methyl-piperazin-l -yl)-9H-purine;
9-( 1 H-Indazol-5-yl)-2-piperidin- 1 -yl-9H-purine;
9-(lH-Indazol-5-yl)-2-pyrrolidin-l-yl-9H-purine;
9-( 1 H-Indazol-5 -yl)-9H-purin-2-ylamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[3-(4-methyl-piperazin-l-yl)-propyl]-amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
2-(4-tert-Butyl-piperidin- 1 -yl)-9-(l H-indazol-5-yl)-9H-purine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[2-(l-methyl-piperidin-4-yl)-ethyl]-amine;
2-{4-[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperazin-l-yl}-ethanol;
2-(4-Cyclopropylmethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
3 - { 4- [9-( 1 H-Indazol-5 -yl)-9H-purin-2-yl] -piperazin- 1 -yl } -propionitrile;
9-(lH-Indazol-5-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
9-(lH-Indazol-5-yl)-2-[4-(2-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
9-(lH-Indazol-5-yl)-2-methoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenylsulfanyl~9H-purine;
9-(lH-Indazol-5-yl)-2-piperazin-l-yl-9H-purine;
1- [9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid ethyl ester; 9-(lH-Indazol-5-yl)-2-(2-methyl-imidazol- 1 -yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-(pyridin-4-ylsulfanyl)-9H-purine;
2- Chloro-9-(lH-indazol-6-yl)-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-dimethyl-amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-N'-methyl-ethane-l,2-diamine; N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-N,N',N'-trimethyl-propane-l,3-diamine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-3-ylmethyl-amine;
9-(lH-Indazol-6-yl)-2-morpholin-4-yl-9H-purine;
N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-N,N',N'-trimethyl-ethane-l ,2-diarnine; 9-(lH-Indazol-6-yl)-2-piperidin-l-yI-9H-purine; [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(4-methoxy-phenyl)-methyl-amine;
(3-Chloro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-phenethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-[2-(3-methoxy-phenyl)-ethyl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-2-yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-methyl-amine;
2- {4-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl}-ethanol;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methoxy-benzyl)-amine;
[2-(3-Chloro-phenyl)-ethyl]-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(3,4-Dichloro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
3- {[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-methyl-amino}-benzoic acid;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-3-yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridm-2-ylmethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-4-yl-ethyl)-amine;
9-(lH-Indazol-6-yl)-2-[4-(2-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
4- [9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-piperidine-l-carboxylic acid ethyl ester; (4-Dimethylamino-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
4-(2-Chloro-purin-9-yl)-phenol;
4-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
4-(2-Dimethylamino-purin-9-yl)-phenol;
4-(2-Pyrrolidin-l-yl-purin-9-yl)-phenol;
4-(2-Piperidin-l-yl-purin-9-yl)-phenol;
4-[2-(4-Ethyl-piperazin- 1 -yl)-purin-9-yl]-phenol;
4-(2-Morpholin-4-yl-purin-9-yl)-phenol;
4-{2-[(3-Dimethylamino-propyl)-methyl-amino]-purin-9-yl}-phenol;
4-{2-[(2-Dimethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
4- {2-[4-(2-Hydroxy-ethyl)-piperazin-l -yl]-purin-9-yl} -phenol;
4-[2-(4-Methyl-piperazin- 1 -yl)-purin-9-yl]-phenol;
4- {2-[(Pyridin-3-ylmethyl)-amino]-purin-9-yl} -phenol;
4-[2-(3,4-Dichloro-benzylamino)-purin-9-yl]-phenol; 4-(2-Chloro-purin-9-yl)-2-methyl-phenol;
4-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-2-methyl-phenol; 4-(2-Dimethylamino-purin-9-yl)-2 -methyl-phenol;
2-Methyl-4-[2-(3-morpholin-4-yl-propylamino)-purin-9-yl]-phenol;
4-[2-(4-Ethyl-piperazin- 1 -yl)-purin-9-yl]-2 -methyl -phenol;
2-Methyl-4-{2-[3-(4-methyl-piperazin-l-yl)-propylamino]-purin-9-yl}-phenol; 2-Methyl-4-(2-pyrrolidin-l-yl-purin-9-yl)-phenol;
2- Methyl-4-(2-piperidin- 1 -yl-purin-9-yl)-phenol;
4-[2-(3-Diethylamino-propylamino)-purin-9-yl]-2 -methyl-phenol;
3- (2-Chloro-purin-9-yl)-phenol;
3-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
3-(2-Dimethylamino-purin-9-yl)-phenol;
3-(2-Chloro-purin-9-yl)-benzamide;
3-(2-Dimethylamino-purin-9-yl)-benzamide;
3- {2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-benzamide;
4- (2-Methylamino-purin-9-yl)-benzamide;
4-(2-Piperidin-l -yl-purin-9-yl)-benzamide;
4-(2-Pyrrolidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Diethylamino-purin-9-yl)-benzamide;
4-[2-(3-Diethylamino-propylamino)-purin-9-yl]-benzamide;
4-(2-Isobutylamino-purin-9-yl)-benzamide;
4-[2-(3-Methyl-butylamino)-purin-9-yl]-benzamide;
4- [2-(4-Ethyl-piperazin-l -yl)-purin-9-yl]-benzamide;
5- (2-Chloro-purin-9-yl)-2,3-dihydro-isoindol-l-one;
N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-3-piperidin-l-yl-propionamide;
9-(lH-Indazol-6-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine; (l-Benzyl-piperidin-4-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
9-(lH-Indazol-6-yl)-2-pyrrolidin-l-yl-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3,4,5-trimethoxy-phenyl)-amine;
(4-Chloro-3-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
{ [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -methyl-amino } -acetic acid;
(S)-l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyrrolidine-2-carboxylic acid; [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(l-methyl-piperidin-4-yl)-amine;
(3-Chloro-4-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
4-(2-Dimethylamino-purin-9-yl)-N-methyl-benzamide;
4-[2-(3-Diethylarnino-propylamino)-purin-9-yl]-N-methyl-benzamide;
(3-Ethynyl-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-trifluoromethyl-phenyl)-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl-amine;
{ l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl}-methanol;
9-(lH-Indazol-6-yl)-2-phenylsulfanyl-9H-purine;
l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carbonitrile;
(2,3-Dihydro-benzo[l,4]dioxin-6-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
Benzo[l,3]dioxol-5-yl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl-amine;
1- [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid methyl ester; 4-[9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-benzenesulfonamide;
Biphenyl-4-ylmethyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
2- {4- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -piperazin- 1 -yl} -acetamide;
{ 4- [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl} -acetic acid methyl ester;
9-(lH-Indazol-6-yl)-2-[4-(2-methoxy-ethyl)-piperazin-l-yl]-9H-purine;
9-(lH-Indazol-6-yl)-2-piperazin-l-yl-9H-purine;
(3-Chloro-4-methoxy-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[3-Chloro-4-(3-fluoro-benzyloxy)-phenyl]-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-
[4-(3 -Fluoro-benzyloxy)-3 -methoxy-phenyl] - [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-[4-(4-methyl-piperazin-l-yl)-phenyl]-amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -(3 -methyl-benzyl)-amine;
(3-Fluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-methoxy-benzyl)-amine;
(2,4-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(2,6-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(3,5-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine; Benzo [ 1 ,3 ]dioxol-5-ylmethyl- [9-( 1 H-indazol-6-yl)-9H-purin-2 -yl] -amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -(3 -methoxy-benzyl)-methyl-amine;
4-(2-Chloro-purin-9-yl)-N-methyl-benzamide ;
N-Methyl-4-(2-thiomorpholin-4-yl-purin-9-yl)-benzamide;
N-Met yl-4-(2-piperidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Amino-purin-9-yl)-N-methyl-benzamide;
[9-(l H-Indazol-6-yl)-9H-purin-2-yl] -((R)- 1 -phenyl-ethyl)-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-((S)-l-phenyl-ethyl)-amine;
N-Methyl-4-(2-morpholin-4-yl-purin-9-yl)-benzamide;
9-(lH-indazol-6-yl)-2-((3-(trifluoromethyl)benzyl)thio)-9H-purine;
4-(4-(9-( 1 H-indazol-6-yl)-9H-purin-2-yl)piperazin- 1 -yl)aniline;
4-((9-(lH-indazol-6-yl)-9H-purin-2-yl)amino)-2-methoxyphenol;
4-(9-( 1 H-indazol-6-yl)-9H-purin-2-yl)thiomorpholine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-N-methyl-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine;
9-(lH-indazol-5-yl)-2-(4-(2-methoxyethyl)piperazin-l-yl)-9H-purine;
N-((6-chloropyridin-3-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
2-(4-benzylpiperidin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-((4-methoxybenzyl)thio)-9H-purine;
2-((3-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
2-((4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-(4-phenylpiperidin-l-yl)-9H-purine;
4-(9-(l H-indazol-6-yl)-9H-purin-2-yl)thiomorpholine 1 , 1 -dioxide;
N-((2,3-dihydrobenzo[b][l,4]dioxin-5-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-
N-(5-bromo-2-fluorobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-((2-methoxypyridin-4-yl)methyl)-9H-purin-2 -amine;
N-((2-fluoropyridin-4-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
2-((3-chloro-4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-5-yl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-2-amine;
9-( 1 H-indazol-6-yl)-2-(2-methyl- 1 H-imidazol- 1 -yl)-9H-purine; and N-methyl-4-(2-(pyri lidin-l-yl)-9H-purin-9-yl)benzamide.
40. A pharmaceutical composition comprising the compound of any one claim 1 to 39, or a pharmaceutically acceptable salt, hydrate, or ester thereof, and a pharmaceutically acceptable carrier or excipient.
41. A method of treating an autoimmune disease or a symptom thereof comprising
administering to a subject an effective amount of a compound of any one claim 1 to 39.
42. The method of claim 41 wherein the autoimmune disease is selected from rheumatoid arthritis, psoriatic arthritis, juvenile arthritis, seronegative spondyloarthropathies, and ankylosing spondylitis.
43. The method of claim 42 wherein the autoimmune disease is rheumatoid arthritis, wherein the rheumatoid arthritis includes extra-articular manifestations selected from vasculitis, pericarditis, myocarditis, and Sjogren's syndrome.
44. The method of claim 41 wherein the autoimmune disease is selected from
inflammatory bowel disease, Crohn's disease, and ulcerative colitis.
45. The method of claim 41 wherein the autoimmune disease is inflammatory bowel disease, wherein the inflammatory bowel disease includes extraintestinal
inflammatory conditions selected from peripheral arthritis, ankylosing spondylitis, sacroiliitis, uveitis, and primary sclerosing cholangitis.
46. The method of claim 41 wherein the autoimmune disease is selected from psoriasis, graft-versus-host disease, systemic lupus erythematosus, sarcoidosis, granulomatosis, vasculitis, asthma, Sjogren's syndrome, type I diabetes, peripheral arthritis, sacroiliitis, uveitis, primary sclerosing cholangitis, pericarditis, and myocarditis.
47. The method of claim 41 wherein the autoimmune disease is multiple sclerosis (MS).
48. The method of claim 41 wherein the compound is administered sequentially or concomitantly with an agent selected from non-steroidal anti-inflammatory drugs, corticosteroids, analgesics, and antibiotics.
49. A method of modulating the activity of one or more kinases selected from the group of Flt3, CSF-1R, ACVR1 (ALK2), CDK2/CyclinA, CDK5/p25, CDK5p35, CLK4, EPHA1, FLT4 (VEGFR3), GSG2 (Haspin), KDR (VEGFR2), LRRK2, LRRK2 G2019S, MAP3K9 (MLK1), MAP3K10 (MLK2), MAPK3K11 (MLK3), MELK, MUSK, NLK, NTRK1 (TRKA), NTRK3 (TRKC), PDGFRA, PDGFRA D842V, PDGFRA V561D, PIK3CA/PIK3R1 (pl l O alpha/p85 alpha), PTK6 (Brk), RIPK2 and ROSl , comprising administering to a subject an effective amount of the compound of any of claim 1 to 39.
50. A method for making a compound of formula (I) according to any of claim 1 to 39,
Figure imgf000207_0001
(I)
comprising:
reacting a compound of formula (A)
Figure imgf000207_0002
The method of claim 50, further including the step of preparing the compound of formula (A) by reacting 5-amino-2,4-dichloro pyrimidine with sodium acetate buffer and an amine of formula (B)
Figure imgf000208_0001
(B)
to obtain a compound of formula (A).
52. A method of treating cancer or bone metastases comprising administering to a subject an effective amount of a compound of any of claim 1 through 39.
53. The method of claim 52 wherein the cancer is gliobastoma or astrocytoma. 54. The method of claim 53 wherein the cancer is leukemia with Flt3 mutations. 55. A compound selected from the groups consisting of:
9-(l H-Indazol-5-yl)-2-pyrrolidin- 1 -yl-9H-purine, [9-( 1 H-Indazol-6-yl)-9H- purin-2-yl]-pyridin-3-ylmethyl-amine, 9-(lH-Indazol-6-yl)-2-morpholin-4-yl-9H- purine, 9-(lH-Indazol-6-yl)-2-piperidin-l-yl-9H-purine, [9-(lH-Indazol-6-yl)-9H- purin-2-yl] -(3 -methoxy-benzyl)-amine, [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -pyridin- 2-ylmethyl-amine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-4-yl-ethyl)-amine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3,4,5-trimethoxy-phenyl)-amine, [9-(lH- Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl-amine, (2,3-Dihydro- benzo[l ,4]dioxin-6-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine, 4-[9-(lH-Indazol- 6-yl)-9H-purin-2-ylamino]-benzenesulfonamide, (3-Fluoro-benzyl)-[9-(lH-indazol-6- yl)-9H-purin-2-yl]-amine. (2,6-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]- amine, [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-((R)-l-phenyl-ethyl)-amine, and 4-((9- (lH-indazol-6-yl)-9H-purin-2-yl)amino)-2-methoxyphenol,
or a pharmaceutically acceptable salt thereof. A compound selected from the group consisting of:
2-Chloro-9-(lH-indazol-5-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-propane-l,3-diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(3-morpholin-4-yl-propyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-dimethyl-amine;
N,N-Diethyl-N'-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-N'-methyl-ethane-l,2-diamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-(2-methoxy-ethyl)-amine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-phenethyl-amine;
Diethyl-[9-(lH-indazol-5-yl)-9H-purin-2-yl]-amine;
9-(lH-Indazol-5-yl)-2-morpholin-4-yl-9H-purine;
9-( 1 H-Indazol-5-yl)-2-(4-methyl-piperazin- 1 -yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-piperidin-l-yl-9H-purine;
9-(lH-Indazol-5-yl)-2-pyrrolidin-l-yl-9H-purine;
9-(lH-Indazol-5-yl)-9H-purin-2-ylamine;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[3-(4-rnethyl-piperazin-l-yl)-propyl]-amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
2-(4-tert-Butyl-piperidin-l -yl)-9-(lH-indazol-5-yl)-9H-purine;
[9-( 1 H-Indazol-5 -yl)-9H-purin-2-yl] -pyridin-4-ylmethyl-amine ;
[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-[2-(l-methyl-piperidin-4-yl)-ethyl]-amine;
2- {4- [9-( 1 H-Indazol-5-yl)-9H-purin-2-yl] -piperazin- 1 -yl } -ethanol;
2- (4-Cyclopropylmethyl-piperazin-l-yl)-9-(lH-indazol-5-yl)-9H-purine;
3- {4-[9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperazin-l-yl}-propionitrile;
9-(lH-Indazol-5-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
9-(lH-Indazol-5-yl)-2-[4-(2-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
9-(lH-Indazol-5-yl)-2-methoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenoxy-9H-purine;
9-(lH-Indazol-5-yl)-2-phenylsulfanyl-9H-purine;
9-(l H-Indazol-5-yl)-2-piperazin- 1 -yl-9H-purine;
1 - [9-(lH-Indazol-5-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid ethyl ester; 9-(lH-Indazol-5-yl)-2-(2-methyl-imidazol-l-yl)-9H-purine;
9-(lH-Indazol-5-yl)-2-(pyridin-4-ylsulfanyl)-9H-purine;
2- Chloro-9-(lH-indazol-6-yl)-9H-purine; [9-(lH-Indazol-6-yi)-9H-purin-2-yl]-dimethyl-amine;
2-(4-Ethyl-piperazin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
N,N-Diethyl-N'-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-N'-methyl-ethane-l,2-diamine; N-[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]-N,N',N'-trimethyl-propane- 1 ,3 -diamine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-3-ylmethyl-amine;
9-(lH-Indazol-6-yl)-2-morpholin-4-yl-9H-purine;
N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-N,N',N'-trimethyl-ethane-l,2-diamine;
9-( 1 H-Indazol-6-yl)-2-piperidin- 1 -yl-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(4-methoxy-phenyl)-methyl-amine;
(3-Chloro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]-phenethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-[2-(3-methoxy-phenyl)-ethyl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-2-yl-ethyl)-amine;
Benzyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-methyl-amine;
2- {4- [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -piperazin- 1 -yl } -ethanol;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methoxy-benzyl)-amine;
[2-(3-Chloro-phenyl)-ethyl]-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(3,4-Dichloro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-4-ylmethyl-amine;
3- {[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-methyl-amino}-benzoic acid;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-3-yl-ethyl)-amine;
Benzyl- [9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyridin-2-ylmethyl-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-pyridin-4-yl-ethyl)-amine;
9-(l H-Indazol-6-yl)-2-[4-(2-methoxy-phenyl)-piperazin- 1 -yl]-9H-purine;
4- [9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-piperidine-l-carboxylic acid ethyl ester; (4-Dimethylamino-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
4-(2-Chloro-purin-9-yl)-phenol;
4-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
4-(2-Dimethylamino-purin-9-yl)-phenol;
4-(2-Pyrrolidin-l -yl-purin-9-yl)-phenol;
4-(2-Piperidin-l-yl-purin-9-yl)-phenol; 4-[2-(4-Ethyl-piperazin-l -yl)-purin-9-yl]-phenol;
4-(2-Morpholin-4-yl-purin-9-yl)-phenol;
4- {2- [(3 -Dimethylamino-propyl)-methyl-amino] -purin-9-yl } -phenol;
4- {2- [(2-Dimethylamino-ethyl)-methyl-amino] -purin-9-yl } -phenol;
4-{2-[4-(2-Hydroxy-ethyl)-piperazin-l-yl]-purin-9-yl}-phenol;
4-[2-(4-Methyl-piperazin-l-yl)-purin-9-yl]-phenol;
4-{2-[(Pyridin-3-ylmethyl)-amino]-purin-9-yl}-phenol;
4-[2-(3,4-Dichloro-benzylamino)-purin-9-yl]-phenol;
4-(2-Chloro-purin-9-yl)-2 -methyl-phenol;
4- {2- [(2-Diethylamino-ethyl)-methyl-amino] -purin-9-yl } -2-methyl-phenol;
4-(2-Dimethylamino-purin-9-yl)-2-methyl-phenol;
2-Methyl-4-[2-(3-morpholin-4-yl-propylamino)-purin-9-yl]-phenol;
4-[2-(4-Ethyl-piperazin-l -yl)-purin-9-yl]-2-methyl-phenol;
2-Methyl-4-{2-[3-(4-methyl-piperazin-l-yl)-propylamino]-purin-9-yl}-phenol;
2-Methyl-4-(2-pyrrolidin- 1 -yl-purin-9-yl)-phenol;
2- Methyl-4-(2-piperidin- 1 -yl-purin-9-yl)-phenol;
4-[2-(3-Diethylamino-propylamino)-purin-9-yl]-2-methyl-phenol;
3- (2-Chloro-purin-9-yl)-phenol;
3-{2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-phenol;
3-(2-Dimethylamino-purin-9-yl)-phenol;
3-(2-Chloro-purin-9-yl)-benzamide;
3-(2-Dimethylamino-purin-9-yl)-benzamide;
3- {2-[(2-Diethylamino-ethyl)-methyl-amino]-purin-9-yl}-benzamide;
4- (2-Methylamino-purin-9-yl)-benzamide;
4-(2-Piperidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Pyrrolidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Diethylamino-purin-9-yl)-benzamide;
4-[2-(3-Diethylamino-propylamino)-purin-9-yl]-benzamide;
4-(2-Isobutylamino-purin-9-yl)-benzamide;
4-[2-(3-Methyl-butylamino)-purin-9-yl]-benzamide;
4- [2-(4-Ethyl-piperazin- 1 -yl)-purin-9-yl]-benzamide;
5- (2-Chloro-purin-9-yl)-2,3-dihydro-isoindol-l-one; N-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-3-piperidm-l -yl-propionamide;
9-(lH-Indazol-6-yl)-2-[4-(3-methoxy-phenyl)-piperazin-l-yl]-9H-purine;
(l-Benzyl-piperidin-4-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
9-(lH-Indazol-6-yl)-2-pyrrolidin-l-yl-9H-purine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3,4,5-trimethoxy-phenyl)-amine;
(4-Chloro-3-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
{ [9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -methyl-amino } -acetic acid;
(S)-l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-pyrrolidine-2-carboxylic acid;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]-( 1 -methyl -piperidin-4-yl)-amine;
(3-Chloro-4-fluoro-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
4-(2-Dimethylamino-purin-9-yl)-N-methyl-benzamide;
4-[2-(3-Diethylamino-propylamino)-purin-9-yl]-N-methyl-benzamide;
(3-Ethynyl-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-trifluoromethyl-phenyl)-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-thiophen-2-ylmethyl-amine;
{l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl}-methanol;
9-(lH-Indazol-6-yl)-2-phenylsulfanyl-9H-purine;
l-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carbonitrile;
(2,3-Dihydro-berizo[l,4]dioxin-6-yl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
Benzo[l,3]dioxol-5-yl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidin-4-yl-amine;
1- [9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperidine-4-carboxylic acid methyl ester; 4-[9-(lH-Indazol-6-yl)-9H-purin-2-ylamino]-benzenesulfonamide;
Biphenyl-4-ylmethyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
2- {4-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl}-acetamide;
{4-[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-piperazin-l-yl} -acetic acid methyl ester; 9-(lH-Indazol-6-yl)-2-[4-(2-methoxy-ethyl)-piperazin-l-yl]-9H-purine;
9-(l H-Indazol-6-yl)-2-piperazin- 1 -yl-9H-purine;
(3-Chloro-4-methoxy-phenyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[3-Chloro-4-(3-fluoro-benzyloxy)-phenyl]-[9-(lH-indazol-6-yl)-9H-purin-2-yl]- [4-(3 -Fluoro-benzyloxy)-3 -methoxy-phenyl] -[9-( 1 H-indazol-6-yl)-9H-purin-2-yl] -
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-[4-(4-methyl-piperazin-l-yl)-phenyl] -amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(3-methyl-benzyl)-amine;
(3-Fluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-(2-methoxy-benzyl)-amine;
(2,4-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(2,6-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
(3,5-Difluoro-benzyl)-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
Benzo[l,3]dioxol-5-ylmethyl-[9-(lH-indazol-6-yl)-9H-purin-2-yl]-amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl] -(3 -methoxy-benzyl)-methyl-amine;
4-(2-Chloro-purin-9-yl)-N-methyl-benzamide ;
N-Methyl-4-(2-thiomorpholin-4-yl-purin-9-yl)-benzamide;
N-Methyl-4-(2-piperidin- 1 -yl-purin-9-yl)-benzamide;
4-(2-Amino-purin-9-yl)-N-methyl-benzamide;
[9-(lH-Indazol-6-yl)-9H-purin-2-yl]-((R)-l-phenyl-ethyl)-amine;
[9-( 1 H-Indazol-6-yl)-9H-purin-2-yl]-((S)- 1 -phenyl-ethyl)-amine;
N-Methyl-4-(2-morpholin-4-yl-purin-9-yl)-benzamide;
9-(lH-indazol-6-yl)-2-((3-(trifluoromethyl)benzyl)thio)-9H-purine;
4-(4-(9-(lH-indazol-6-yl)-9H-purin-2-yl)piperazin-l-yl)aniline;
4-((9-(lH-indazol-6-yl)-9H-purin-2-yl)amino)-2-methoxyphenol;
4-(9-(lH-indazol-6-yl)-9H-purin-2-yl)thiomorpholine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
N-(3-bromobenzyl)-9-(lH-indazol-6-yl)-N-methyl-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-(tetrahydro-2H-pyran-4-yl)-9H-purin-2-amine;
9-(lH-indazol-5-yl)-2-(4-(2-methoxyethyl)piperazin-l-yl)-9H-purine;
N-((6-chloropyridin-3-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
2-(4-benzylpiperidin-l-yl)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-((4-methoxybenzyl)thio)-9H-purine;
2-((3-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
2-((4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-6-yl)-2-(4-phenylpiperidin-l-yl)-9H-purine; 4-(9-( 1 H-indazol-6-yl)-9H-purin-2-yl)thiomorpholine 1 , 1 -dioxide;
N-((2,3-dihydrobenzo[b][l,4]dioxin-5-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-
N-(5-bromo-2-fluorobenzyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
9-(lH-indazol-6-yl)-N-((2-methoxypyridin-4-yl)methyl)-9H-purin-2-amine;
N-((2-fluoropyridin-4-yl)methyl)-9-(lH-indazol-6-yl)-9H-purin-2-amine;
2-((3-chloro-4-fluorobenzyl)thio)-9-(lH-indazol-6-yl)-9H-purine;
9-(lH-indazol-5-yl)-N-(4-methoxyphenyl)-N-methyl-9H-purin-2-amine;
9-( 1 H-indazol-6-yl)-2-(2-methyl- 1 H-imidazol- 1 -yl)-9H-purine ; and
N-methyl-4-(2-(pyrrolidin- 1 -yl)-9H-purin-9-yl)benzamide,
or a pharmaceutically acceptable salt thereof.
PCT/US2011/024404 2010-02-24 2011-02-10 Purine compounds for treating autoimmune and demyelinating diseases WO2011106168A1 (en)

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