Heterocyclic pyrazole derivatives as type III receptor tyrosine kinase inhibitors

Abstract

For the treatment of diseases or disorders mediated by protein kinases, especially class III RTKs such as FLT3, PDGFRα, c-KIT and / or CSF-1R kinase, compounds that can act as inhibitors of such kinases are represented by formula (I): [Formula 1] JPEG2022545019000083.jpg26149 or a stereoisomer, tautomer, pharmaceutically acceptable salt thereof, proteolysis-triggered chimeric molecule (PROTAC) binder. The compound may be used in the treatment of diseases or conditions mediated by FLT3, PDGFR, c-KIT, and / or CSF-1R kinases, or by mutant kinases of FLT3, PDGFR, c-KIT, and / or CSF-1R kinases. Such diseases or conditions may include cancer, autoimmune diseases, and bone resorption diseases.

Description

FIELD OF THEINVENTION

[0001]

[0001] The present invention relates to novel chemical compounds and methods for the use of such compounds in therapy and preparation. In particular, the present invention relates to certain substituted heterocyclic pyrazole compounds and their use in the inhibition, regulation and / or modulation of type III receptor tyrosine kinases, such as FLT3, PDGFR, c-KIT and / or CSF-1R and their associated signaling pathways. [Background technology]

[0002] Protein kinases (PKs) play important roles in cell signaling pathways that regulate various cellular functions such as differentiation, proliferation, migration, survival, and apoptosis. These enzymes catalyze the transfer of a phosphate group from ATP to tyrosine, serine, or threonine residues of protein substrates. Phosphorylation by kinases and dephosphorylation by phosphatases are involved in a myriad of cellular processes in response to diverse intracellular or extracellular signals, regulation of cellular functions, and activation or inactivation of cellular operations. Receptor tyrosine kinases (RTKs) are a subfamily of protein kinases, and a class of RTKs known as the type III receptor tyrosine kinase family, including FLT3, PDGFR, c-KIT, and CSF-1R, has been implicated in various proliferative and inflammatory diseases.

[0003]

[0003] Small molecule inhibitors of type III receptor tyrosine kinases provide a rational approach to new therapies for the treatment of autoimmune diseases and specifically for blocking chronic tissue destruction mediated by the innate immune system. Moreover, inhibition of type III receptor tyrosine kinases also provides a rational approach to new therapies for the treatment of cancer, particularly for the treatment of cancer invasiveness, cancer angiogenesis or vasculogenesis, cancer metastasis, and cancer immune tolerance.

[0004]

[0004] FLT3 (also called Fms-like tyrosine kinase 3, Flk2) and its ligand (FL) are one of the regulators of normal hematopoiesis, and activating mutations or overexpression of FLT3 are frequently found in AML. More than a dozen known FLT3 inhibitors have been developed, some of which have shown promising clinical efficacy and have been approved for the treatment of FLT3 mutant-positive AML. FLT3 receptors are also expressed in the majority of dendritic cell precursors, and stimulation of FLT3 receptors leads to the proliferation and differentiation of these precursors into dendritic cells (DCs). Dendritic cells are the primary initiators of T cell-mediated immune responses, including autoreactive immune responses, so inhibition of FLT3 provides a mechanism for down-regulating DC-mediated inflammatory and autoimmune responses. One study shows that the FLT3 inhibitor CEP-701 is effective in reducing myelin loss in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. High levels of FLT3 ligand are found in the sera of patients with Langerhans cell histiocytosis and systemic lupus erythematosus, an observation further implicating FLT3 signaling in the dysregulation of dendritic cell precursors in these autoimmune diseases.

[0005]

[0005] PDGFR (platelet-derived growth factor receptor) and its ligand (PDGF) regulate cell proliferation, survival, differentiation and migration, mainly of cells of mesenchymal origin. Abnormalities in the PDGF pathway, including overexpression or amplification of PDGF receptor (PDGFR), gain-of-function point mutations, or activating chromosomal translocations, have been observed in a wide range of pathologies, such as cancer, fibrosis, neurological conditions and atherosclerosis, making them potential targets for disease treatment.

[0006]

[0006] c-KIT (also known as stem cell growth factor receptor, SCFR) and its ligand (SCF) induce cell proliferation, differentiation or migration within the hematopoietic, gametogenic and melanogenic lineages at various developmental stages. The presence of c-KIT mutations is a key diagnostic marker for gastrointestinal stromal tumors (GISTs). Gleevec (imatinib mesylate or STI571), the first FDA-approved receptor tyrosine kinase (RTK) inhibitor, originally approved for c-Abl-mediated chronic myeloid leukemia, gained FDA approval for c-KIT-mediated GISTs in 2002, validating the molecular-based approach of c-KIT inhibition for the treatment of GISTs. Gain-of-function mutations in c-KIT are also associated with mast cell / myeloid leukemia and seminoma / dysgerminoma. c-KIT mutations have also been identified in certain melanomas and are recognized as potential therapeutic targets for melanomas.

[0007]

[0007] CSF-1R (also known as macrophage colony-stimulating factor receptor (M-CSFR) or Fms) is the receptor for macrophage colony-stimulating factor (M-CSF or CSF-1). CSF-1 / CSF-1R is the primary growth factor that regulates the survival, proliferation and differentiation of cells of the mononuclear phagocyte lineage. Several studies have demonstrated that CSF-1 / CSF-1R plays a specific role in tumor tissues. Elevated expression or activation of CSF-1R and / or its ligands has been found in various cancers, and elevated levels of M-CSF are associated with poor prognosis in certain cancers. M-CSF is one of several cytokines involved in the recruitment of tumor-associated macrophages (TAMs), which contribute to tumor angiogenesis and tumor progression to metastasis. Activation of CSF-1R also leads to the proliferation and differentiation of osteoclast precursors, thereby mediating the process of bone resorption. Thus, inhibition of CSF-1R provides a treatment for cancer, particularly cancer invasion, angiogenesis, metastasis, immune tolerance and bone metastasis. Due to its role in osteoclast biology, CSF-1R is also an important therapeutic target for osteoporosis, inflammatory arthritis and other inflammatory bone erosions.

[0008]

[0008] Although various tyrosine kinase inhibitors have been shown to be useful therapeutic agents, there remains a need for type III kinase inhibitors. Summary of the Invention

[0009] The present invention relates to novel inhibitors that inhibit kinases, particularly class III RTKs such as FLT3, PDGFRα, c-KIT and / or CSF-1R kinases, for the treatment of diseases or disorders mediated by such kinases. The inhibitors of the present invention also find utility in the treatment of other mammalian diseases, including human diseases, mediated by FLT3, PDGFR, c-KIT or CSF-1R kinases. Such diseases include, but are not limited to, cancer, autoimmune diseases and bone resorption diseases.

[0010]

[0010] Embodiments of the present invention are based on the unexpected finding that certain heterocyclic pyrazole compounds can inhibit the activity of members of the type III receptor tyrosine kinase family (e.g., FLT3, PDGFR, c-KIT and CSF-1R). The compounds are useful in medical treatments, pharmaceutical compositions and methods for modulating the activity of FLT3, PDGFR, c-KIT and / or CSF-1R kinases, including wild-type and / or mutant forms of FLT3, PDGFR, c-KIT and / or CSF-1R kinases. These properties allow these heterocyclic pyrazole compounds to be used in treating protein tyrosine kinase-related diseases and / or conditions.

[0011] According to an embodiment of the present invention, the compound has the general formula I: [ka] or a stereoisomer, tautomer, pharma- ceutically acceptable salt thereof, a binding agent for a proteolysis-inducing chimeric molecule (PROTAC), X is CR 2 and N.R. 3 is selected from the group consisting of Y is CR 2 and N.R. 3 is selected from the group consisting of G is selected from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, alkynyl, and a direct bond; L is -CH=, -CHR 4 -, -(CH2) q -, -NR 5 -, -O-, -O(CH2) q -, -C(O)-, -C(O)(CH2) q selected from the group consisting of -, -C(O)NH-, -SO2-, and a direct bond, each q is individually and independently an integer from 1 to 4; Z is selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkylamine, C1-C4 dialkylamine, and optionally substituted heterocyclyl, and heterocyclic spiro compounds; R 1 is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, amino, cyano, trifluoromethyl, trifluoromethoxy, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C1-C6 dialkylamino; R 2is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C5-C6 cycloalkenyl, C1-C6 alkylamino, C1-C6 dialkylamino, C3-C6 cycloalkylamino, C1-C6 alkoxy, C3-C6 cycloalkoxy, aryl, 3-6 membered heterocyclyl, and 5-6 membered heteroaryl, and is selected from the group consisting of alkylamino, dialkylamino, alkoxy, alkyl, alkenyl, alkynyl, cycloalkylamino, and alkylamino. cycloalkenyl, cycloalkylamino, cycloalkoxy, heterocyclyl, aryl and heteroaryl are optionally substituted by halogen, amino, hydroxyl, cyano, nitro, acyl, acyloxy, C1-C4 alkyl, hydroxylC1-C4 alkyl, alkoxyC1-C4 alkyl, acyloxyC1-C4 alkyl, C1-C4 alkoxy, C1-C4 dialkylamino, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, aryl and 5-6 membered heteroaryl; R 3 is selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl, C1-C6 alkylcarbonyl, C2-C6 alkenylcarbonyl, C1-C6 alkoxycarbonyl, aminocarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 dialkylaminocarbonyl; R 4 is selected from the group consisting of hydrogen, deuterium, and C1-C6 alkyl; R 5 is selected from the group consisting of hydrogen and C1-C6 alkyl. may have:

[0012]

[0012] In another aspect, an embodiment of the present invention provides a pharmaceutical composition comprising a compound of formula I, its stereoisomer, tautomer, solvate, prodrug or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, excipient or additive, respectively.

[0013] In another aspect, embodiments of the present invention provide a method for inhibiting a type III receptor tyrosine kinase, such as FLT3, PDGFR, c-KIT and / or CSF-1R kinase, in a mammal, comprising administering to said mammal in need thereof a therapeutically effective amount of a compound of formula I.

[0014] In another aspect of the present invention, an embodiment of the present invention provides a binder of proteolysis-inducing chimeric molecule (PROTAC) for degradation of type III receptor tyrosine kinases, such as FLT3, PDGFR, c-KIT and / or CSF-1R kinase in mammals via the ubiquitin pathway. Proteolysis-inducing chimeric molecule (PROTAC) is a type of dibasic molecule that can remove unwanted proteins induced by proteolytic selectivity in cells. PROTAC consists of two protein binding moieties, one for E3 ubiquitin ligase and the other for the target protein (the binder described herein). By binding the two proteins, PROTAC transfers the target protein to E3 ligase, resulting in the tagging (i.e., ubiquitination) of the target protein for subsequent degradation by the proteasome. Many molecules are known to bind to various E3 ubiquitin ligases, such as nutlin, methyl-bestatin, VHL ligand, thalidomide, pomalidomide and lenalidomide, as disclosed in WO 2019 / 140003A1, the disclosure of which is incorporated by reference. Any of these E3 ligase binding agents may be coupled to the compounds of the present invention. A linker may be used between the two binding moieties to achieve spacing and ease of the coupling reaction. Some examples of linkers can be found in WO 2019 / 140003. In addition, Burslem et al. were inspired by the ability of receptor tyrosine kinases to target protein degradation via low nanomolar concentrations of proteolysis-inducing chimeric molecules (PROTACs). Burslem et al. developed a FLT3 PROTAC using the known FLT3 inhibitor quizartinib as the mobilizing element and a von Hippel-Lindau (VHL) ligand via an optimized linker. The compound FLT3 PROTAC effectively induced FLT3-ITD protein degradation at low nanomolar concentrations in MOLM-14 and MV4-11 cells (J. Am. Chem. Soc. 2018;140:16428-16432).

[0015] In another aspect, embodiments of the present invention provide a method for treating a disease or disorder selected from fibrosis, bone-related disease, cancer, an autoimmune disorder, an inflammatory disease, and a cardiovascular disease in a mammal, comprising administering to said mammal in need thereof a therapeutically effective amount of a compound of formula I or a pharma- ceutically acceptable salt thereof.

[0016]

[0016] In another aspect, an embodiment of the present invention provides the use of a compound of formula I in the manufacture of a medicament for the treatment of a disease or disorder selected from fibrosis, bone-related diseases, cancer, autoimmune disorders, inflammatory diseases and cardiovascular diseases in a mammal.

[0017]

[0017] In another aspect, an embodiment of the present invention provides the use of a compound of formula I in the treatment of a disease or disorder selected from fibrosis, bone-related diseases, cancer, autoimmune disorders, inflammatory diseases and cardiovascular diseases in a mammal.

[0018] In another aspect, embodiments of the present invention provide intermediates for preparing compounds of formula I. In one embodiment, certain compounds of formula I may be used as intermediates for the preparation of other compounds of formula I.

[0019] In another aspect, embodiments of the present invention provide processes for preparing the compounds described herein, methods of isolating and methods of purifying the compounds.

[0020]

[0020] Other aspects and advantages of the present invention will become apparent from the following description and appended claims.

[0021] definition

[0021] Reference will now be made in detail to certain specific embodiments, examples of which are illustrated in the accompanying structures and formulas. Although the enumerated embodiments are described, it should be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the invention. The invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated literature and similar materials differs or contradicts with this application, including but not limited to defined terms, usage of terms, techniques described, etc., this application shall control.

[0022]

[0022] The term "alkyl" refers to a straight or branched chain monovalent saturated hydrocarbon containing 1 to 20 carbon atoms, unless otherwise specified. Numeric ranges herein are intended to include any number or numbers within the defined range as if each number were disclosed separately. For example, an alkyl group of 1 to 20 carbons would include C1, C2, . . . C20, and C1-C20, C1-C15, C1-C10, C1-C6, C1-C4, etc. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl.

[0023]

[0023] The term "alkenyl" refers to a straight or branched monovalent hydrocarbon containing 2 to 20 carbon atoms (e.g., C2-C10) and one or more double bonds. Examples of alkenyl include, but are not limited to, ethenyl, propenyl, allyl, and 1,4-butadienyl.

[0024]

[0024] The term "alkynyl" refers to a straight or branched monovalent hydrocarbon containing 2 to 20 carbon atoms (e.g., C2-C10) and one or more triple bonds. Examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 1- and 2-butynyl, and 1-methyl-2-butynyl.

[0025] The term "alkoxy" refers to an -O-alkyl radical, where the alkyl portion is as defined above. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy.

[0026] The term "acyloxy" refers to the radical -OC(O)-R, where R can be H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl.

[0027] The term "amino" refers to NH2. The term "alkylamino" refers to an -N(R)-alkyl radical, where R can be H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl.

[0028]

[0028] The term "cycloalkyl" refers to a monovalent saturated hydrocarbon ring system having 3 to 30 carbon atoms (e.g., C3-C6 or C3-C12). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and adamantanyl.

[0029]

[0029] The term "cycloalkenyl" refers to a monovalent non-aromatic hydrocarbon ring system having 3 to 30 carbons (e.g., C3-C6 or C3-C12) and one or more double bonds. Examples include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0030] The term "heterocycloalkyl" refers to a monovalent non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, or Se). Examples of heterocycloalkyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, piperidinyl, dioxanyl, morpholinyl, and tetrahydrofuranyl.

[0031] The term "heterocycloalkenyl" refers to a monovalent non-aromatic 5- to 8-membered monocyclic, 8- to 12-membered bicyclic, or 11- to 14-membered tricyclic ring system having one or more heteroatoms (such as O, N, S, or Se) and one or more double bonds.

[0032] The term "aryl" refers to a monovalent 6-carbon monocyclic, 10-carbon bicyclic, or 14-carbon tricyclic aromatic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.

[0033] The term "aryloxy" refers to -O-aryl. The term "arylamino" refers to -N(R)-aryl, where R can be H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl. The term "heteroaryl" refers to a monovalent aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, or Se). Examples of heteroaryl groups include, but are not limited to, pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, thiazolyl, pyrrolyl, isoquinolinyl, purinyl, oxazolyl, pyrazolyl, and carbazolyl. In all these terms, the "aryl" moiety is as defined above.

[0034] The term "halogen" refers to F, Cl, Br or I. Preferably, the halogen is F, Cl or Br.

[0035]

[0035] The above alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, amino, alkylamino, arylamino, alkoxy, aryloxy, aryl and heteroaryl may be substituted or unsubstituted moieties. Possible substituents on amino, alkylamino, arylamino, alkoxy, aryloxy, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl and heteroaryl are C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C10 alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, ... 0 alkylamino, arylamino, hydroxy, halo, oxo (O=), thioxo (S=), thio, C1-C10 alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amidino, mercapto, amido, thioureido, thiocyanato, sulfonamido, guanidine, ureido, cyano, nitro, acyl, thioacyl, acyloxy, carbamido, carbamyl (-C(O)NH2), carboxyl (-COOH), and carboxylic acid ester. On the other hand, possible substituents on alkyl, alkenyl, or alkynyl include all of the above listed substituents except C1-C10 alkyl. Cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl can also be fused to each other.

[0036]

[0036] Provided herein are compounds and pharmaceutical compositions thereof that are useful in the treatment or prevention of diseases, conditions and / or disorders modulated / mediated by (or associated with) type III receptor tyrosine kinases such as FLT3, PDGFR, c-KIT and / or CSF-1R kinase.

[0037] Some embodiments of the present invention relate to a compound of formula I: [ka] (In the formula, X is CR 2 and N.R. 3 is selected from the group consisting of Y is CR 2 and N.R. 3 is selected from the group consisting of G is selected from the group consisting of optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, alkynyl, and a direct bond; L is -CHR 4 -, -(CH2) q -, -NR 5 -, -O-, -O(CH2) q -, -C(O)-, -C(O)(CH2) q selected from the group consisting of -, -C(O)NH-, -SO2-, and a direct bond, each q is individually and independently an integer from 1 to 4; Z is selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 alkylamine, C1-C4 dialkylamine, optionally substituted 4-, 5-, and 6-membered cyclic amines, and optionally substituted heterocyclyl, and heterocyclic spiro compounds; R 1 is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl, amino, cyano, trifluoromethyl, trifluoromethoxy, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, and C1-C6 dialkylamino; R 2is selected from the group consisting of hydrogen, deuterium, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C5-C6 cycloalkenyl, C1-C6 alkylamino, C1-C6 dialkylamino, C3-C6 cycloalkylamino, C1-C6 alkoxy, C3-C6 cycloalkoxy, aryl, 3-6 membered heterocyclyl, and 5-6 membered heteroaryl, and is selected from the group consisting of alkylamino, dialkylamino, alkoxy, alkyl, alkenyl, alkynyl, cycloalkylamino, and alkylamino. cycloalkenyl, cycloalkylamino, cycloalkoxy, heterocyclyl, aryl and heteroaryl are optionally substituted by halogen, amino, hydroxyl, cyano, nitro, acyl, acyloxy, C1-C4 alkyl, hydroxylC1-C4 alkyl, alkoxyC1-C4 alkyl, acyloxyC1-C4 alkyl, C1-C4 alkoxy, C1-C4 dialkylamino, C3-C6 cycloalkyl, 3-6 membered heterocyclyl, aryl and 5-6 membered heteroaryl; R 3 is selected from the group consisting of hydrogen, deuterium, C1-C6 alkyl, C1-C6 alkylcarbonyl, C2-C6 alkenylcarbonyl, C1-C6 alkoxycarbonyl, aminocarbonyl, C1-C6 alkylaminocarbonyl, and C1-C6 dialkylaminocarbonyl; R 4 is selected from the group consisting of hydrogen, deuterium, and C1-C6 alkyl; R 5 is selected from the group consisting of hydrogen and C1-C6 alkyl), or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof, or a binding agent for a proteolysis-inducing chimeric molecule (PROTAC) to provide.

[0038]

[0038] Those skilled in the art will understand that all possible combinations or permutations of different substituents in the above formula (I) are within the scope of the present invention. These compounds can be prepared using readily available materials / reagents and known chemical reactions. Based on the general knowledge in the art and the teachings in this disclosure, those skilled in the art should be able to prepare and use these compounds without undue experimentation.

[0039] The following reaction schemes, Scheme 1 to Scheme 6, provide exemplary procedures that can be used to prepare compounds of formula (I). However, one skilled in the art will recognize that these examples are illustrative only and that modifications or variations are possible without departing from the scope of the present invention. The heterocyclic pyrazole compounds synthesized according to embodiments of the present invention may be purified using any known technique, such as by flash column chromatography, high performance liquid chromatography, crystallization, or any other suitable method. [ka]

[0040] A boronic acid or boronate (1.2 equiv.) was added to a solution of 2-bromo-5,6-dihydro-4H-cyclopenta[b]thiophen-4-one (1.0 equiv.) in 1,4-dioxane / water (5:1). K2CO3 (2 equiv.) and Suzuki coupling Pd(II) or Pd(0) catalyst (5-8 mol%) (e.g., Pd(dppf)Cl2, Pd(PPh3)4 or Pd(OAc)2, etc.) were added to the solution. The resulting mixture was then heated under reflux. After the reaction was complete, water was added and the solution was extracted with ethyl acetate. The organic layer was dried over MgSO4 and evaporated in vacuum. The final compound was obtained after purification by chromatography. [ka]

[0041]

[0041] 60% sodium hydride (NaH, 1.3 equiv.) was added to a suspension of imidazole active ester (1.1 equiv.) and dihydro-cyclopenta[b]thiophen-4-one derivative (1.0 equiv.) in tetrahydrofuran (THF, 0.2M) under nitrogen atmosphere. The reaction was then heated under reflux for 4 hours. After completion of the reaction, the reaction was quenched with acetic acid (HOAc, 1.5 equiv.) at 0°C. The resulting yellow precipitate was then filtered and washed with water and cold methanol. The solid was then dried under vacuum to yield the desired 1,3-diketone compound. [ka]

[0042] Hydrazine hydrate (3 eq.) was added to a suspension of diketone (1 eq.) and acetic acid (HOAc, 5 eq.) in ethanol (EtOH, 0.2 M). The reaction was then heated under reflux for 6 hours. After the reaction was complete, the resulting yellow precipitate was then filtered and washed with water and cold methanol. The solid was then dried under vacuum to yield the desired pyrazole compound. [ka]

[0043]

[0043] 60% sodium hydride (NaH, 1.3 eq.) was added to a suspension of imidazole active ester (1.1 eq.) and dihydro-cyclopenta[b]thiophen-4-one derivative (1.0 eq.) in tetrahydrofuran (THF, 0.2 M) under nitrogen atmosphere. The reaction was then heated under reflux for 4 hours. After completion of the reaction, the reaction was quenched with acetic acid (AcOH, 1.5 eq.) at 0°C. The resulting precipitate was then filtered and washed with water and cold methanol. The solid was then dried under vacuum to yield the desired 1,3-diketone compound. [ka]

[0044] Hydrazine hydrate (3 eq.) was added to a suspension of diketone (1 eq.) and acetic acid (HOAc, 5 eq.) in ethanol (EtOH, 0.2 M). The reaction was then heated under reflux for 6 hours. After the reaction was complete, the resulting precipitate was then filtered and washed with water and cold methanol. The solid was then dried under vacuum to yield the desired pyrazole compound. [ka]

[0045] A boronic acid or boronate (1.2 equiv.) was added to a solution of a pyrazole derivative (1.0 equiv.) in 1,4-dioxane / water (5:1). Potassium carbonate (K2CO3, 2 equiv.) and a regular Suzuki coupling Pd(II) or Pd(0) catalyst (5-8 mol%) (e.g., Pd(dppf)Cl2, Pd(PPh3)4 or Pd(OAc)2, etc.) were added to the solution. The resulting mixture was then heated under reflux. After the reaction was complete, water was added and the solution was extracted with ethyl acetate. The organic layer was dried over MgSO4 and evaporated in vacuum. The final compound was obtained after purification by chromatography.

[0046]

[0046] The above reaction scheme illustrates how the heterocyclic pyrazole compounds of the present invention can be prepared. Those skilled in the art will understand that the reactions involved and the reagents used in these reactions are known in the art. Therefore, based on the above teachings and the general knowledge in the art, the heterocyclic pyrazole compounds having various substituents as defined herein can be prepared by those skilled in the art without inventive efforts.

[0047]

[0047] The compounds of formula I represent novel potent inhibitors of type III receptor tyrosine kinases, such as FLT3, PDGFR, c-KIT and / or CSF-1R kinases, and may be useful in the prevention and treatment of disorders resulting from the action of these kinases.

[0048] The compounds of formula I may be of therapeutic value in the treatment of a disease or disorder selected from fibrosis, bone-related diseases, cancer, autoimmune disorders, inflammatory diseases and cardiovascular diseases.

[0049] According to some embodiments of the present invention, the compound of formula I is useful for treating fibrotic diseases. Examples of fibrosis include idiopathic pulmonary fibrosis (IPF), nephrogenic systemic fibrosis (NSF), liver cirrhosis, diabetes-induced nephropathy, myocardial fibrosis (e.g., endomyocardial fibrosis), mediastinal fibrosis, bone marrow fibrosis, retroperitoneal fibrosis, Crohn's disease, keloid formation, scleroderma and systemic sclerosis. Additional examples of fibrotic diseases include focal segmental glomerulosclerosis (FSGS), systemic scleroderma-associated interstitial lung disease (SSc-ILD), primary biliary cirrhosis, ethanol cirrhosis, interstitial fibrosis and tubular atrophy (CAD), proliferative vitreoretinopathy and scarring (hypertrophy and keloid).

[0050] According to some embodiments of the present invention, the compounds of formula I are useful for treating bone-related diseases, including metastatic bone disease, treatment-induced bone loss, osteoporosis, rheumatoid arthritis, ankylosing spondylitis, Paget's disease, and periodontal disease. Osteoporosis can result from bone loss secondary to (1) menopause in women, (2) aging in men or women, (3) suboptimal bone growth during childhood and adolescence resulting in failure to reach peak bone mass, and / or (4) other pathologies, eating disorders, drugs, and / or medical treatments (e.g., as a result of glucocorticoid treatment, aromatase inhibitor therapy, or antiandrogen therapy).

[0051]

[0051] Other osteolytic diseases that can be treated according to embodiments of the present invention are more localized. A particular example is metastatic tumor-induced osteolysis. In the state of metastatic tumor-induced osteolysis, bone cancer or bone metastases induce localized osteolysis, which causes pain, bone weakness and fractures. Such localized osteolysis also allows tumors to grow larger by creating space in the bone for larger tumors and releasing growth factors from the bone matrix. Cancers currently known to cause tumor-induced osteolysis include hematological malignancies (e.g., myeloma and lymphoma) and solid tumors (e.g., breast, prostate, lung, kidney and thyroid), all of which are contemplated for treatment by the present invention.

[0052] According to some embodiments of the present invention, the compounds of formula I are useful for the treatment of cancer and proliferative disorders. Examples include multiple myeloma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), prostate cancer, breast cancer, ovarian cancer, melanoma, glioblastoma multiforme, giant cell tumor of bone (also known as osteoblastoma), giant cell tumor of tendon sheath (also known as tenosynovial giant cell tumor or TGCT), tumor metastasis to other tissues, myelofibrosis and other chronic myeloproliferative disorders such as pigmented villonodular synovitis (PVNS).

[0053] According to some embodiments of the present invention, the compounds of formula I are useful for treating autoimmune disorders and inflammatory diseases, including, but not limited to, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, ankylosing spondylitis, adult Still's disease, glomerulonephritis, osteoporosis, Sjogren's syndrome, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Langerhans cell histiocytosis, hemophagocytic syndrome, multicentric reticulohistiocytosis, and Paget's disease. Additional examples of autoimmune diseases and disorders include primary sclerosing cholangitis and transplant rejection (including liver, kidney, and heart / lung transplantation).

[0054] According to some embodiments of the present invention, the compounds of formula I are useful for treating cardiovascular disease. Examples of cardiovascular disease include atherosclerosis, peripheral vascular disease, coronary artery disease, ischemia / reperfusion, hypertension, restenosis, pulmonary arterial hypertension, and arteritis. Additional examples of cardiovascular disease include acute respiratory distress syndrome (ARDS), patent arteriovenous (AV) fistula, and veno-occlusive disease (post-HSC / BMT).

[0055] The heterocyclic pyrazole compounds described herein may contain non-aromatic double bonds and one or more asymmetric centers, for example, in the substituents attached to the core aromatic ring. Thus, these compounds may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis or trans isomeric forms. All such isomeric forms are within the scope of the present invention. The heterocyclic pyrazole compounds of the present invention may have acidic or basic functional groups (e.g., in the substituent groups) capable of forming salts, particularly pharma-ceutically acceptable salts. The formation of such salts is a common practice in the pharmaceutical industry. Examples of salts that may be used with the heterocyclic pyrazole compounds of the present invention include, for example, chlorides, sulfates, mesylate, besylate, tosylate, formate, acetate, malate, succinate, and the like, for basic functional groups. Such heterocyclic pyrazole salts are within the scope of the present invention. Similarly, acidic or basic groups may be functionalized, for example, to esters. Such functionalized derivatives will be hydrolyzed in vivo. Thus, such derivatives can function as prodrugs of the heterocyclic pyrazole compounds of the present invention. Formation of the prodrug involves no more than routine skill, and one of ordinary skill in the art would know how to prepare and use such prodrugs without undue experimentation.

[0056]

[0056] Also within the scope of the present invention are (1) a pharmaceutical composition containing an effective amount of at least one of the heterocyclic pyrazole compounds of the present invention and a pharma- ceutically acceptable carrier, (2) a method for treating a protein kinase-associated disease (e.g., cancer) by administering an effective amount of such a heterocyclic pyrazole compound to a subject in need of such treatment, and (3) a method for reducing the activity of at least one protein kinase by contacting at least one protein kinase with at least one of the heterocyclic pyrazole compounds of the present invention.

[0057] As used herein, the term "protein kinase-associated disease / disorder" or "disease / disorder associated with protein kinase" or "disease / disorder modulated by protein kinase" refers to a disease or condition characterized by abnormal protein kinase (PK) activity or a disease or condition that can be treated by a change in the activity of at least one PK. Abnormal PK activity can result from an elevated level of PK expression or the presence of PK expression that does not occur under normal conditions. PK-associated diseases / disorders described herein include, but are not limited to, cancer, diabetes, hyperproliferative disorders, hyperproliferative disorders of the kidney, renal disease, von Hippel-Lindau disease, restenosis, fibrosis, psoriasis, osteoarthritis, rheumatoid arthritis, inflammatory disorders, immune disorders such as autoimmune diseases (e.g., AIDS, lupus, etc.), cardiovascular disorders (e.g., atherosclerosis), and vascular proliferative disorders such as abnormal angiogenesis.

[0058] The term "treating" refers to administering a heterocyclic pyrazole compound of the present invention to a subject having a protein kinase-associated disease / disorder or a symptom of or a predisposition to the disease / disorder, for the purpose of curing, relieving, alleviating, altering, correcting, ameliorating, affecting, or reducing the risk of the disorder, the symptoms of the disorder, or the predisposition thereto. For example, treating cancer refers to the treatment resulting in inhibition of cancer growth or cancer cell growth, regression in cancer growth (i.e., reducing the size of the detectable cancer), or disappearance of the cancer.

[0059]

[0059] The term "effective amount" refers to the amount of active agent required to impart the intended therapeutic effect in a subject. The effective amount may vary depending on the route of administration, the use of additives, and the possibility of co-use with other agents, as recognized by those skilled in the art. Determining an effective amount requires only routine skill, and one of ordinary skill in the art would be able to determine such an effective amount for the intended use without undue experimentation. The subject requiring treatment may be a mammal. The term "mammal" refers to a human or non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat, or mouse.

[0060] To practice the method of the present invention, any of the above-mentioned pharmaceutical compositions can be administered parenterally, orally, by inhalation spray, topically, rectally, intranasally, bucally, vaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. According to some embodiments of the present invention, the heterocyclic pyrazole compound of the present invention may be administered intravenously, and suitable carriers may include, but are not limited to, solutions containing saline or phosphate buffered saline (PBS), as well as viscosity enhancing and solubilizing agents such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

[0061]

[0061] Sterile injectable compositions, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated using suitable dispersing or wetting agents (such as Tween 80) and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable excipient or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media (for example, synthetic mono- or diglycerides). Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, especially in polyoxyethylated form, as are natural pharma- ceutical acceptable oils such as olive oil or castor oil. These oil solutions or suspensions can also contain long-chain alcohol excipients or dispersion media, or carboxymethylcellulose or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans, or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0062]

[0062] The composition for oral administration may be any orally acceptable dosage form, including, but not limited to, capsules, tablets, emulsions, and aqueous suspensions, dispersions, and solutions. In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful excipients include lactose and dried cornstarch. When aqueous suspensions or emulsions are administered orally, the active ingredient may be suspended or dissolved in an oily phase combined with an emulsifying or suspending agent. If desired, certain sweeteners, flavors, or colorants may be added. Nasal aerosol or inhalant compositions may be prepared according to techniques well known in the art of pharmaceutical formulation. Heterocyclic pyrazole compound-containing compositions may also be administered in the form of suppositories for rectal administration.

[0063]

[0063] The carrier in the pharmaceutical composition should be "acceptable" in the sense that it is compatible with the active ingredient of the formulation (and preferably can stabilize the active ingredient) and is not harmful to the subject being treated. One or more solubilizers (e.g., cyclodextrins) that form a more soluble complex with the active heterocyclic pyrazole compound can be utilized as pharmaceutical carriers for the delivery of the active compound. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, and sodium lauryl sulfate.

[0064]

[0064] The above reaction scheme illustrates how the heterocyclic pyrazole compounds of the present invention can be prepared. Those skilled in the art will understand that the reactions involved and the reagents used in these reactions are known in the art. Therefore, based on the above teachings and the general knowledge in the art, the heterocyclic pyrazole compounds having various substituents as defined herein can be prepared by those skilled in the art without inventive efforts.

[0065]

[0065] Without further elaboration, it is believed that the above description has fully enabled the present invention. Accordingly, the following examples are to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever. EXAMPLES

[0066] Example 1: Synthesis of compounds of formula (I)

[0066] Exemplary heterocyclic pyrazole compounds are listed in Table 1. 1 1 H NMR and mass data are provided in Table 2.

[0067] [Table 1] TIFF0007651557000010.tif85149

[0068] [Table 2] JPEG0007651557000012.jpg194149 JPEG0007651557000013.jpg189149 JPEG0007651557000014.jpg194149 JPEG0007651557000015.jpg189149 JPEG0007651557000016.jpg212149 JPEG0007651557000017.jpg189149 JPEG0007651557000018.jpg194149 JPEG0007651557000019.jpg194149 JPEG0007651557000020.jpg200149 JPEG0007651557000021.jpg194149 JPEG0007651557000022.jpg194149 JPEG0007651557000023.jpg212149 JPEG0007651557000024.jpg189149 JPEG0007651557000025.jpg189149 JPEG0007651557000026.jpg189149 JPEG0007651557000027.jpg194149

[0069]

Table 3

[0070]

Table 4

[0071] Example 2: Evaluation of the inhibitory activity of compounds of formula (I) in in vitro biochemical assays Various compounds of formula I were tested for inhibitory activity against various receptor tyrosine kinases. A brief description of the different assays is given below.

[0072] FLT3 kinase assay

[0068] The inhibition of FLT3 kinase activity by the test compounds disclosed herein was estimated by AlphaScreen (PerkinElmer, Akron, Ohio, USA). Standard assay conditions were 1.5 ng of recombinant FLT3 kinase (SignalChem, Richmond, BC, Canada) plus 6 ng of biotin-conjugated poly-(Glu4:Tyr1) (Cisbio, Bedford, MA, USA) in assay buffer (10 μM ATP, 10 mM MOPs, pH 7.0, 0.21 mM EDTA, 0.5% glycerol, 1 mg / mL BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35, 10 mM MgCl2) in a final volume of 25 μL. The reaction was incubated at 30° C. for 30 minutes and stopped by adding 5 μL of 50 mM EDTA. The resulting products were analyzed using an AlphaScreen kit and counted in Enspire Alpha (PerkinElmer, Waltham, MA, USA). The readout from the control reaction (complete reaction mixture) was designated as 0% inhibition, and the readout for the reaction without enzyme was designated as 100% inhibition. The IC50 values ​​of the inhibitors were determined after performing assays in duplicate with serially diluted concentrations of each compound. The results were analyzed using linear regression software (GraphPad Prism 5; GraphPad Software Inc.).

[0073] FLT3-D835Y mutant kinase assay

[0069] The inhibition of FLT3-D835Y mutant kinase activity by the test compounds disclosed herein was estimated by AlphaScreen (PerkinElmer). Standard assay conditions were 1 ng of recombinant FLT3-D835Y mutant kinase (SignalChem) plus 6 ng of biotin-conjugated poly-(Glu4:Tyr1) (Cisbio) in assay buffer (10 μM ATP, 10 mM MOPs, pH 7.0, 0.21 mM EDTA, 0.5% glycerol, 1 mg / mL BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35, 10 mM MgCl2) in a final volume of 25 μL. The reaction was incubated at 30°C for 30 minutes and stopped by adding 5 μL of 50 mM EDTA. The resulting products were analyzed using AlphaScreen kit and counted in Enspire Alpha (PerkinElmer). The readout from the control reaction (complete reaction mixture) was assigned as 0% inhibition, and the readout for the reaction without enzyme was assigned as 100% inhibition. IC of inhibitors 50 Values ​​were determined after assays using serially diluted concentrations of each compound performed in duplicate. Results were analyzed using linear regression software (GraphPad Prism 5; GraphPad Software Inc.).

[0074] PDGFRα kinase assay

[0070] The inhibition of PDGFRα kinase activity by the test compounds disclosed herein was estimated by AlphaScreen (PerkinElmer). Standard assay conditions were 1.5ng of recombinant PDGFRα kinase (SignalChem) plus 6ng of biotin-conjugated poly-(Glu4:Tyr1) (Cisbio) in assay buffer (10μM ATP, 10mM MOPs, pH7.0, 0.21mM EDTA, 0.5% glycerol, 1mg / mL BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35, 10mM MgCl2) in a final volume of 25μL. The reaction was incubated at 30℃ for 30 minutes and stopped by adding 5μL of 50mM EDTA. The resulting products were analyzed using AlphaScreen kit and counted in Enspire Alpha (PerkinElmer). The readout from the control reaction (complete reaction mixture) was assigned as 0% inhibition, and the readout for the reaction without enzyme was assigned as 100% inhibition. IC of inhibitors 50 Values ​​were determined after assays using serially diluted concentrations of each compound performed in duplicate. Results were analyzed using linear regression software (GraphPad Prism 5; GraphPad Software Inc.).

[0075] MET kinase assay

[0071] The compounds disclosed herein were tested in the inhibition of MET kinase activity predicted by AlphaScreen (PerkinElmer) as a counterscreen (comparison) to demonstrate selective kinase inhibitory activity against class III RTKs. MET kinase, which belongs to the class VIII RTK family, has one transmembrane α chain linked to one extracellular β chain, which is different from the class III RTK family that has five immunoglobulin-like extracellular domains. Standard assay conditions were 1 ng recombinant MET kinase (SignalChem) plus 3 ng biotin-conjugated poly-(Glu4:Tyr1) (Cisbio) in assay buffer (10 μM ATP, 10 mM MOPs, pH 7.0, 0.21 mM EDTA, 0.5% glycerol, 1 mg / mL BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35, 10 mM MgCl2) in a final volume of 25 μL. Reactions were incubated at 30° C. for 45 min and stopped by adding 5 μL of 50 mM EDTA. The resulting products were analyzed using an AlphaScreen kit and counted in an Enspire Alpha (PerkinElmer). The readout from the control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme was designated as 100% inhibition. The IC50 values ​​of inhibitors were determined after performing assays in duplicate with serially diluted concentrations of each compound. Results were analyzed using linear regression software (GraphPad Prism 5; GraphPad Software Inc.).

[0076] result

[0072] In comparison with the MET kinase inhibitory activity, the compounds of the present invention were found to have unexpected and selective inhibitory activity against class III RTKs. More specifically, all of the compounds of the present invention had IC values ​​of less than 10 nM against FLT3 kinase and FLT3-D835Y mutant kinase. 50 values, with 57 of the 105 compounds of the invention having IC values ​​of less than 10 nM for PDGFR kinase. 50The IC values ​​are shown, demonstrating the high potency of the compounds of the present invention against class III RTKs. 50 The values ​​are summarized in Table 3. In the table, the designation "A+" denotes an IC of less than 1 nM. 50 "A" stands for IC 50 "B" means that the IC is between 1 and 10 nM. 50 is between 10 and 30 nM, and "C" is the IC 50 "D" means that the IC is between 30 and 100 nM. 50 is between 100 and 300 nM, and "E" is the IC 50 "F" means that the IC is between 300 and 1000 nM 50 means that the concentration is greater than 1,000 nM.

[0077] [Table 5] JPEG0007651557000057.jpg210147 JPEG0007651557000058.jpg209147 JPEG0007651557000059.jpg208147 JPEG0007651557000060.jpg209147 JPEG0007651557000061.jpg208147 JPEG0007651557000062.jpg144147

[0078] Example 3: Evaluation of the antiproliferative activity of compounds of formula (I) against a panel of human cancer cell lines with different signaling dependencies As noted above, the compounds of the present invention can be used to treat protein kinase-related diseases or disorders. The protein kinase-related disease can be cancer, autoimmune disease or vascular proliferation disorder. The cancer can be lung cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, stomach cancer, kidney cancer, salivary gland cancer, ovarian cancer, uterine cancer, cervical cancer, oral cancer, skin cancer, brain cancer, lymphoma or leukemia.

[0079] The inhibition of cell growth by the compounds was measured using CellTiter™-96 assay. The cytotoxicity of the compounds of the present invention was evaluated in a panel of cell lines with different signaling pathway dependencies, including MV4-11 human acute myeloid leukemia cells with FLT3-ITD mutation, Molm-14 human acute myeloid leukemia cells with FLT3-ITD mutation, THP-1 human acute myeloid leukemia cells with NRAS G12D mutation, A375 melanoma cells with BRAF V600E mutation, and HCC827 NSCLC cells with EGFR exon 19 deletion mutation.

[0080] MV4-11 cell culture

[0075] The compounds disclosed herein were tested in MV4-11 cell proliferation assay to determine cellular potency on FLT3 signal pathway dependent cell growth. MV4-11 cells are human acute myeloid leukemia (AML) cells that harbor a ligand-independent FLT3-ITD activating mutation, making the abnormal growth of AML cells dependent on FLT3 signal pathway. Inhibition of FLT3 kinase activity will result in reduced growth and / or cell death. MV4-11 cells (catalog number CRL-9591) were obtained from American Type Culture Collection (ATCC, Manassas, VA). Briefly, cells were grown in IMDM medium supplemented with 10% heat-inactivated fetal bovine serum and incubated at 37°C in a 5% CO2 atmosphere. Cells were expanded until saturation was reached, at which point cells were subcultured or harvested for assay use.

[0081] Molm-14 cell culture

[0076] The compounds disclosed herein were tested in Molm-14 cell proliferation assay to determine cellular potency on FLT3 signal pathway dependent cell growth. Molm-14 cells are human acute myeloid leukemia (AML) cells that harbor a ligand-independent FLT3-ITD activating mutation, making the abnormal growth of AML cells dependent on FLT3 signal pathway. Inhibition of FLT3 kinase activity will result in reduced growth and / or cell death. Molm-14 cells (catalog number ACC-777) were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Briefly, cells were grown in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum and incubated at 37°C in a 5% CO2 atmosphere. Cells were expanded until saturation was reached, at which point cells were subcultured or harvested for assay use.

[0082] THP-1 cell culture

[0077] The compounds disclosed herein were tested in a THP-1 cell proliferation assay as a counterscreen (comparison) to demonstrate selective antiproliferative activity against cell growth dependent on class III RTK signaling. THP-1 cells are human acute myeloid leukemia cells that carry the NRAS G12D mutation, rendering cell growth independent of class III RTK signaling. THP-1 cells (catalog number TIB-202) were obtained from ATCC. Briefly, cells were grown in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 0.05 mM 2-mercaptoethanol and incubated at 37°C in a 5% CO2 atmosphere. Cells were expanded until saturation was reached, at which point cells were subcultured or harvested for assay use.

[0083] A375 cell culture

[0078] The compounds disclosed herein were tested in A375 cell proliferation assay as a counter screen to demonstrate selective antiproliferative activity against cell growth dependent on class III RTK signaling. A375 cells are melanoma cells encoding the BRFA V600E mutation, which makes cell growth dependent on BRAF signaling instead of class III RTK signaling. A375 cells (catalog number CRL-1619) were obtained from ATCC. Briefly, cells were cultured in DMEM containing 10% FBS and incubated at 37°C in a 5% CO2 atmosphere. Cells were expanded until saturation was reached, at which point cells were subcultured or harvested for assay use.

[0084] HCC827 cell culture

[0079] The compounds disclosed herein were tested in HCC827 cell proliferation assay as a counter screen to demonstrate selective antiproliferative activity against cell growth dependent on class III RTK signaling. HCC827 cells are non-small cell lung cancer (NSCLC) cells encoding EGFR exon 19 deletion mutations, making cell growth dependent on EGFR signaling instead of class III RTK signaling. HCC827 cells (catalog number CRL-2868) were obtained from ATCC. Briefly, cells were cultured in RPMI1640 medium supplemented with 10% FBS and incubated at 37°C in a 5% CO2 atmosphere. Cells were expanded until saturation was reached, at which point cells were subcultured or harvested for assay use.

[0085] MV4-11, Molm-14, THP-1, A375 and HCC827 cells were seeded at 6500, 25000, 5000, 2000 and 8000 cells / well, respectively, in 96-well plates and incubated overnight at 37°C, 5% CO2. These seeded cells were then treated with increasing concentrations of test compounds and incubated for another 72 hours. At the end of the incubation, CellTiter96® Aquos One Solution Reagent (Promega, San Luis Obispo, CA, USA) was added and incubated for an additional 2-4 hours. Cell viability was determined by measuring absorbance at 490 nm using an EMax® microplate reader (Molecular Devices, San Jose, CA, USA).

[0086] result

[0081] Compared to the antiproliferative activity in THP-1, A375 or HCC827 cells, the compounds of the present invention exhibited unexpected and selective cytotoxicity against cell growth dependent on class III RTK signaling. More specifically, 68 compounds of the present invention showed IC<30 nM for leukemia cell growth dependent on FLT3 signaling. 50 values ​​are shown, demonstrating the high cellular potency of the compounds of the invention.

[0087]

[0082] 50% inhibitory concentration (IC 50 IC values ​​were calculated by plotting the percentage of growth inhibition versus compound concentration using GraphPad Prism 5 software for curve fitting. The antiproliferative activity of the compounds of the invention is summarized in Table 4. In the table, the symbol "A" represents the IC 50 "B" indicates that the IC is less than 0.03 μM. 50 is between 0.03 and 0.1 μM, and "C" is the IC 50 is between 0.1 and 0.3 μM, and "D" indicates the IC 50 is between 0.3 and 1 μM, and "E" is the IC 50is between 1 and 10 μM, and "F" indicates the IC 50 is higher than 10 μM.

[0088] [Table 6] JPEG0007651557000064.jpg211147 JPEG0007651557000065.jpg212147 JPEG0007651557000066.jpg212147 JPEG0007651557000067.jpg213147 JPEG0007651557000068.jpg212147 JPEG0007651557000069.jpg134147

[0089] The data in Table 4 clearly show that the compounds of the present invention have unexpected and selective antiproliferative activity against cancer cell growth that depends on the class III RTK signaling pathway. Therefore, these compounds may be used in the treatment of protein kinase-associated diseases, such as diseases or disorders mediated by FLT3, PDGFR, c-KIT and / or CSF-1R kinases, or mutant kinases of FLT3, PDGFR, c-KIT and / or CSF-1R kinases. A method according to one embodiment of the present invention comprises administering to a subject in need thereof an effective amount of a compound of the present invention.

[0090] Example 4: Identification of heterocyclic pyrazole derivatives (Formula I) conjugated with 6,5-fused bicyclic heteroaryl at the 5- or 6-position, with the 2-position being a nitrogen atom, that exhibit unexpected and superior potency against FLT3 kinase and its mutants. [ka]

[0091]

[0084] As shown in Table 5, Compound 1 and Compound 2 are almost identical in structure to Examples A to G, respectively, except that the 6,5-fused bicyclic heteroaryl ring in Compound 1 and Compound 2 each contains two nitrogen atoms at the 2-position.

[0092] [Table 7]

[0093] Unexpectedly, Compound 1 and Compound 2 inhibit FLT3 kinase and the FLT3-D835Y mutant: Comparative Example It was observed that the potency was superior to that of A to G, and was about 5 times more potent. Based on the above results, it was unexpectedly found that 6,5-fused bicyclic heteroaryls containing two nitrogen atoms at the 2-position exhibit extremely strong inhibitory activity against FLT3 kinase and its mutants.

[0094] Example 5: Comparison of efficacy of compounds of formula (I) with structurally related compounds in the prior art

[0086] Comparative Example Compounds H-K are found or covered in the prior art (US Pat. No. 8,853,207 B2). Compounds 40 and 3 have been shown to have in vitro potency of: Comparative Example Compounds 1 and 2 were selected for comparison with the potency of compounds H. Compounds 1 and 2 were selected for comparison with the potency of compounds H. Comparative Example Compounds 36 and 30 were selected for comparison with the potency of compounds I and J. Comparative Example was chosen for comparison with the efficacy of K.

[0095] As shown in Table 6, Compound 40, Compound 3, Compound 1, Compound 2, Compound 36 and Compound 30 are Comparative Example Compared to the prior art compounds H to K, the compounds of the present invention each have a substituted indazole moiety directly bonded to a pyrazole, Comparative Example The structure is almost identical to that of compounds H~K.

[0096] [Table 8] TIFF0007651557000073.tif67149

[0097]

[0088] Unexpectedly, Comparative Example Compared with the prior art compounds HK, the compounds of the present invention exhibit superior potency in inhibiting FLT3 kinase and its mutants, being approximately 10-fold more potent.

[0098] Example 6: Evaluation of the cellular potency of compounds of formula (I) on phosphorylation of FLT3 and its down-stream effector STAT5 in MV4-11 leukemia cell line with constitutive activation of FLT3 signaling

[0089] FLT3 is expressed in AML cells of most patients, and FLT3 is mutated in approximately 30% of AML cells. Mutations include internal tandem duplications (ITDs), which are present in AML cells in approximately 25% of patients, and point mutations in the tyrosine kinase domain (TKD), which are present in approximately 5% of patients. Both ITD and TKD mutations are activating, leading to ligand-independent or constitutive, FLT3 receptor signaling, thereby promoting cytokine-independent AML cell survival and proliferation (Blood 2002;100:1532-42.). Both FLT3-ITD and FLT3 TKD mutations result in constitutive activation of FLT3 signaling, involving FLT3 kinase and downstream proliferative signaling pathways of FLT3 kinase, including the Ras / MAPK kinase (MEK) / extracellular signal-regulated kinase (ERK) pathway and the PI3K / Akt pathway. In addition, in contrast to wild-type FLT3 signaling, FLT3-ITD strongly activates the STAT5 pathway. STAT5 induces STAT5 target genes, such as cyclin D1, c-myc, and the anti-apoptotic gene p21, which are important for cell growth. These effects may indicate a role for FLT3-ITD in the aberrant cell growth of leukemia cells. FLT3-ITD constitutive activation of STAT5 can accelerate AML cell growth (J Hematol Oncol.2011;4:13.).

[0099]

[0090] The potent compound of formula (I) in Example 2 was evaluated by Western blotting assay for its cellular potency on phosphorylation of FLT3 and its downstream effector STAT5 in MV4-11 leukemia cell line with constitutive activation of FLT3 signaling. For Western blotting experiments, MV4-11 cells were cultured overnight in IMDM medium containing 0.1% FBS. On the day of assay, 3 million cells were treated with or without compound for 3 hours in 15 mL tubes. Then, whole cell lysates were collected after extraction by adding 2xSDS sample buffer and boiling on a hot plate at 105°C for 15 minutes. The obtained cell lysates were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane. Protein expression was detected using immunoblot with corresponding primary and secondary antibodies according to standard procedures. Antibodies against p-FLT3 (Tyr-591), anti-rabbit IgG HRP-linked secondary antibody, and anti-mouse IgG HRP-linked secondary antibody were purchased from Cell Signaling Technology. Antibodies against p-STAT5 (Tyr-694) were purchased from BD biosciences. Antibodies against actin were purchased from R&D system. Immunoblots were revealed by chemiluminescence (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo) and detected by ChemiDoc™ MP Imaging System (Bio-Rad). Band intensities of Western blots were also quantified by ChemiDoc™ MP Imaging System. The relative intensity of bands corresponding to drug treatment at a single concentration (30 nM) was compared with the DMSO control group. The inhibition percentage (%) at each drug concentration was calculated using the formula: Percentage of inhibition (%) = (1-relative potency) x 100 was used to calculate.

[0100] Selected compounds of Formula I that exhibit greater than 50% inhibition of phosphorylation of FLT3 and its downstream effector STAT5 at 30 nM treatment are listed in Table 7 below.

[0101] [Table 9]

[0102] Some embodiments of the present invention relate to a method for treating a protein kinase-associated disease. The method according to one embodiment of the present invention comprises administering to a subject in need thereof an effective amount of a compound of the present invention.

[0103]

[0093] While the present invention has been described with respect to a limited number of embodiments, those skilled in the art having the benefit of this disclosure will recognize that other embodiments may be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the present invention should be limited only by the appended claims.

Claims

1. A compound represented by formula (II), (III), (IV) or (V): 【Chemistry 1】 (In the formula, G is selected from the group consisting of C 6 aryl, 5-6 membered heteroaryl having one or more heteroatoms selected from N and S, 5-8 membered heterocyclyl having one or more heteroatoms selected from O and N, and C 2 -C 10 alkynyl, wherein said C 6 aryl, 5-6 membered heteroaryl, and 5-8 membered heterocyclyl are optionally substituted with halogen, trifluoromethyl, trifluoromethoxy, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy; L is -CH=, -CHR ４ -, -(CH ２ ) ｑ --, --NR ５ -, -O-, -O(CH ２ ) ｑ -, -C(O)-, -C(O)(CH ２ ) ｑ - and - SO ２ -, each q is individually and independently an integer from 1 to 4; Z is hydrogen, deuterium, halogen, C １ ~C ４ Alkyl, C １ ~C ４ Alkylamines, C １ ~C ４ dialkylamines, optionally substituted 4-7 membered heterocyclyl or heterocyclic spiro compounds; R １ is selected from the group consisting of hydrogen and deuterium; R ２ is hydrogen, deuterium, halogen and C １ ~C ６ is selected from the group consisting of alkyl, R3 is hydrogen, deuterium and C １ ~C ６ is selected from the group consisting of alkyl, R ４ is hydrogen, deuterium and C １ ~C ６ is selected from the group consisting of alkyl, R ５ is hydrogen and C １ ~C ６ alkyl) Compounds of 4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)morpholine, 4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)morpholin-3-one, 4-(5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)morpholine, 3-(1-methyl-1H-indazol-6-yl)-6-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 6-(2-chloropyridin-4-yl)-3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 3-(1-methyl-1H-indazol-6-yl)-6-(2-(4-methylpiperazin-1-yl)pyridin-4-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, or 3-(1-methyl-1H-indazol-6-yl)-6-(piperidin-4-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, or a stereoisomer, tautomer, or pharma- ceutically acceptable salt thereof.

2. Ring G is 【Chemistry 2】 (In the formula, R ６ is hydrogen, deuterium, halogen, trifluoromethyl, trifluoromethoxy, C １ ~C ６ Alkyl and C １ ~C ６ alkoxy; each n is individually and independently an integer from 0 to 4; R ７ is C1 to C ６ is alkyl) 2. The compound of claim 1 selected from the group consisting of:

3. 3. The compound of claim 1 or 2, wherein Z is fluorine, chlorine, bromine, dimethylamino, or an optionally substituted 4, 5, 6 or 7 membered heterocyclyl or heterocyclic spiro compound.

4. Z, 【Chemistry 3】 (In the formula, R ８ is hydrogen, deuterium, halogen, nitro, cyano, trifluoromethyl, trifluoromethoxy, 4-morpholinyl, C １ ~C ６ Alkyl and C １ ~C ６ alkoxy; each n is individually and independently an integer from 0 to 4; R ９ is hydrogen, deuterium and C １ ~C ６ alkyl) 4. The compound of claim 3 selected from the group consisting of:

5. 3-(1H-indazol-5-yl)-6-(6-methoxypyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 3-(1H-indazol-6-yl)-6-(6-methoxypyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 5-(3-(1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-amine, 6-(6-methoxypyridin-3-yl)-3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, N,N-dimethyl-5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-amine, 4-(4-(3-(1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, (2S,6R)-4-(4-(3-(1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)-2,6-dimethylmorpholine, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, (2S,6R)-2,6-dimethyl-4-((5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)methyl)morpholine, 4-((5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)methyl)morpholine, 3-(1-methyl-1H-indazol-6-yl)-6-(6-methylpyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, (2S,6R)-2,6-dimethyl-4-(3-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(3-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 3-(1-methyl-1H-indazol-6-yl)-6-(3-((4-methylpiperazin-1-yl)methyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(4-methylpiperazin-1-yl)methanone, (2R,6S)-4-((5-(3-(1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)methyl)-2,6-dimethylmorpholine, 3-(1-methyl-1H-indazol-6-yl)-6-(2-methylpyridin-4-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 4-((5-(3-(1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)methyl)morpholine, ((2S,6R)-2,6-dimethylmorpholino)(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)methanone, 4-(1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)ethyl)morpholine, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(morpholino)methanone, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenethyl)morpholine, (5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)(morpholino)methanone, 4-(2-methoxy-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(2-fluoro-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(2-methyl-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)-2-(trifluoromethyl)benzyl)morpholine, 4-(1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)propyl)morpholine, 4-(3-methoxy-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 3-(1H-indazol-6-yl)-6-(6-methylpyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, (5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-3-yl)(morpholino)methanone, 4-(3-fluoro-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 3-(1-methyl-1H-indazol-6-yl)-6-(6-methylpyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(piperidin-1-yl)methanone, 4-(4-(3-(1-isobutyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 3-(1H-indazol-5-yl)-6-(6-methylpyridin-3-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, (3-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(morpholino)methanone, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(pyrrolidin-1-yl)methanone, 3-(1-methyl-1H-indazol-6-yl)-6-(4-(thiomorpholinomethyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 5-(3-(1H-indazol-5-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-amine, (5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyrimidin-2-yl)(morpholino)methanone, 4-(4-(3-(1-isopropyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, (2-methoxy-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(morpholino)methanone, (2-fluoro-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)(morpholino)methanone, 5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyrimidin-2-amine, methyl-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)-1H-pyrrol-2-yl)(morpholino)methanone, 4-((4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)sulfonyl)morpholine, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)thiophen-2-yl)(morpholino)methanone, N,N-dimethyl-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzamide, N,N-dimethyl-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzenesulfonamide, 4-((5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)thiophen-2-yl)methyl)morpholine, 4-((4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)thiophen-2-yl)methyl)morpholine, 4-(2,6-difluoro-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, (2S,6R)-2,6-dimethyl-4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-((6-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-3-yl)methyl)morpholine, (2-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-4-yl)(morpholino)methanone, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)-3,6-dihydropyridin-1(2H)-yl)(morpholino)methanone, tert-butyl-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)piperidine-1-carboxylate, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholin-3-one, (4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)thiazol-2-yl)(morpholino)methanone, 1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)piperidin-2-one, 3-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)oxazolidin-2-one, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)piperazin-2-one, 1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)pyrrolidin-2-one, 1,3-dimethyl-4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)piperazin-2-one, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenethyl)morpholin-3-one, 6-(4-((3,3-difluoropyrrolidin-1-yl)methyl)phenyl)-3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 4-(4-(3-(3-bromo-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(2-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenoxy)ethyl)morpholine, 4-(2-(3-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenoxy)ethyl)morpholine, 4-(3-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)prop-2-yn-1-yl)morpholine, N-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)tetrahydro-2H-pyran-4-amine, N-methyl-N-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)tetrahydro-2H-pyran-4-amine, (R)-3-methyl-4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, (S)-3-methyl-4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 3-(1-methyl-1H-indazol-6-yl)-6-(4-((tetrahydro-4H-pyran-4-ylidene)methyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 3-(1-methyl-1H-indazol-6-yl)-6-(4-((tetrahydro-2H-pyran-4-yl)methyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 4-(1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)piperidin-4-yl)morpholine, 4-(4-(3-(3-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 3-(1H-indazol-6-yl)-6-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 3-(1-methyl-1H-indazol-6-yl)-6-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 4-(2,3-difluoro-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(4-(3-(1-methyl-1H-benzo[d][1,2,3]triazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)-3-(trifluoromethoxy)benzyl)morpholine, 4-(3-methyl-4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)morpholine, 5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-amine, N,N-dimethyl-1-(5-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-2-yl)methanamine, N,N-dimethyl-1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)phenyl)methanamine, 4-((6-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)pyridin-3-yl)methyl)morpholine, 4-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)-1,4-oxazepane, 1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)piperidin-1-yl)-2-morpholinoethan-1-one, 3-(1-methyl-1H-indazol-6-yl)-6-(4-(pyrrolidin-1-ylmethyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 3-(1-methyl-1H-indazol-6-yl)-6-(4-(piperidin-1-ylmethyl)phenyl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 1-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)piperazin-2-one, 6-(4-(3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazol-6-yl)benzyl)-2-oxa-6-azaspiro[3.3]heptane, 6-(4-(azetidin-1-ylmethyl)phenyl)-3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, 6-(4-((3,3-difluoroazetidin-1-yl)methyl)phenyl)-3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole, or 6-(4-((3-methoxyazetidin-1-yl)methyl)phenyl)-3-(1-methyl-1H-indazol-6-yl)-1,4-dihydrothieno[2',3':4,5]cyclopenta[1,2-c]pyrazole 2. The compound of claim 1 ,

6. A pharmaceutical composition comprising the compound according to any one of claims 1 to 5, its stereoisomer, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient or carrier.

7. 7. The pharmaceutical composition of claim 6 for the treatment of a disease or disorder mediated by FLT3, PDGFR, c-KIT and / or CSF-1R kinases or by mutant kinases of FLT3, PDGFR, c-KIT and / or CSF-1R kinases.

8. The pharmaceutical composition of claim 7 , wherein the disease or disorder is cancer.

9. 9. The pharmaceutical composition of claim 8, wherein the cancer is selected from the group consisting of multiple myeloma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), prostate cancer, breast cancer, ovarian cancer, melanoma, glioblastoma multiforme, giant cell tumor of bone, non-small cell lung cancer, giant cell tumor of tendon sheath, tumor metastasis to other tissues, myelofibrosis, pigmented villonodular synovitis, or gastrointestinal stromal tumor.

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