CDK9 inhibitors

Abstract

Formula (I) or a salt (e.g., a pharmaceutically acceptable salt) or stereoisomer thereof wherein X, Y, R1a, R1b, R2, R3, R4s, R5, p and n are as defined herein. Also provided are uses of the compounds as components of pharmaceutical compositions and methods of using the same. # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 329,139, filed on April 8, 2022, entitled “CDK9 INHIBITORS,” the entire contents of which are hereby incorporated by reference. Technical Field

[0003] The present disclosure generally relates to compounds that inhibit the activity of protein kinases, such as cyclin-dependent protein kinases (CDKs), and to compositions and methods for treating cancer and other conditions associated with CDKs. Background Art

[0004] Cyclin-dependent kinases (CDKs) are serine-threonine kinases that function to coordinate multiple cellular functions and phosphorylate substrates necessary for cell cycle progression. The activity of specific CDKs at specific times is essential for both the initiation and coordinated progression of the cell cycle. For example, CDK7, CDK8, and CDK9 play a role in regulating transcription to further affect cell proliferation and survival by driving the expression of many target genes. The relevance of CDKs to cancer growth and survival has garnered widespread attention in the generation of CDK inhibitors.

[0005] A number of CDK9 inhibitors have been widely used to rapidly induce apoptosis in tumor cells. However, the complex toxicity profiles associated with activities other than CDK9 have hampered their development.

[0006] Therefore, there is a need for the rational design of specific and selective CDK9 inhibitors for the treatment of cancer and other mediated and / or related conditions. The present disclosure satisfies these needs and provides other related advantages. Summary of the invention

[0007] In short, the present disclosure provides CDK9 inhibitor compounds, including stereoisomers or salts thereof (e.g., pharmaceutically acceptable salts), which can be used alone or in combination with a pharmaceutically acceptable carrier. Also provided are methods of using CDK9 inhibitor compounds to treat various diseases or conditions such as bladder cancer, prostate cancer, and leukemia.

[0008] In one embodiment, a compound having the following structure (I) is provided:

[0009]

[0010] or a salt (eg, a pharmaceutically acceptable salt) or a stereoisomer thereof, wherein X, Y, R 1a , R 1b , R2 , R 3 , R 4s , R 5 , p and n are as defined herein. Also provided are uses of the compounds as components of pharmaceutical compositions and methods of use thereof. Also provided are pharmaceutical compositions comprising one or more of the compounds of the aforementioned structure (I) and a therapeutic agent.

[0011] In other embodiments, the disclosure provides a method for administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition comprising a compound of structure (I) and a therapeutic agent and delivering the composition to the patient.

[0012] These and other aspects of the disclosure will be apparent upon reference to the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In the drawings, the same reference numerals identify similar elements. The sizes and relative positions of the elements in the drawings are not necessarily drawn to scale, and some of these elements are enlarged and positioned to improve the legibility of the drawings. In addition, the specific shapes of these elements drawn are not intended to convey any information about the actual shapes of these specific elements, and they are selected only for ease of identification in the drawings.

[0014] Figure 1 is a graph showing the antiproliferative effects of compound 1-2 at different concentrations on six prostate cancer cell lines.

[0015] Figure 2 is a graph showing the cell antiproliferative efficacy of the benchmark compound AZD-4573 at different concentrations against six prostate cancer cell lines.

[0016] Figure 3 is a bar graph showing the antiproliferative effect of compound I-2 on cells of 22RV-1 colony.

[0017] Figure 4 is a bar graph showing the antiproliferative effect of compound I-2 on cells of LASCPC-1 colony.

[0018] Figure 5 is a series of images of LASCPC-01 cells.

[0019] Figure 6 is a series of graphs showing Annexin V / 7-AAD staining of LASCPC-01.

[0020] Figure 7 is a series of graphs showing Annexin V / 7-AAD staining of 22Rev1.

[0021] Figure 8 are images of Western blot analysis of N-MYC.

[0022] Fig. 9 are images of western blot analysis of N-MYC and C-MYC.

[0023] Fig.10 is a bar graph showing RT-PCR analysis of 22Rv1 and LASCPC-01.

[0024] Fig.11 is a series of images of LASCPC-01 cell staining.

[0025] Fig.12 are Western blot images of 22Rv1 with compound 1-2 and AZD-4573.

[0026] Fig.13 are Western blot images of LASCPC-01 with compound I-2 and AZD-4573.

[0027] Fig.14 is a graph showing a comparison of 22Rv1 mean tumor volumes.

[0028] Fig.15 is a graph showing a comparison of LASCPC-01 mean tumor volumes.

[0029] Fig.16 Relative body weights of treated and untreated mice are shown.

[0030] Fig.17 Shown are the mean tumor volumes of 22Rv1 at various doses of Compound 1-2 over the 21-day study.

[0031] Fig.18 Tumor growth inhibition plotted against dose and tumor weight is shown.

[0032] Fig.19 Relative body weights of treated and untreated mice are shown.

[0033] Fig. 20 Shown are the mean LASCPC-01 tumor volumes for different doses of Compound 1-2 over the 21-day study.

[0034] Fig.21 Tumor growth inhibition plotted against dose and tumor weight is shown.

[0035] Fig. 22Depicts the effect of compound I-2 (50 mg / kg, oral, daily) to (30 mg / kg, oral, once every two weeks) on tumor volume in C4-2 xenografts of male NOD.SCID mice. Statistical analysis was performed using GraphPad Prism software version 5.0. Two-way ANOVA was performed using Bonferroni's post-hoc test; all values ​​are expressed as mean ± SEM, 7-8 mice per group, *P<0.05, ***P<0.001 relative to vehicle control.

[0036] Fig.23 Shown is the effect of compound 1-2 (50 mg / kg, po, daily) modified to (30 mg / kg, po, biweekly) on tumor growth inhibition in C4-2 xenografts in male NOD.SCID mice.

[0037] Fig.24 The effect of compound I-2 (50 mg / kg, oral, daily) on tumor weight in C4-2 xenografts of male NOD.SCID mice was shown. Statistical analysis was performed using GraphPad Prism software version 5.0., t test; all values ​​are expressed as mean ± SEM, 8 mice per group, *P < 0.05 relative to vehicle control. DETAILED DESCRIPTION

[0038] In the following description, certain specific details are given in order to provide a thorough understanding of the various embodiments of the present disclosure. However, those of ordinary skill in the art will appreciate that the present disclosure can be practiced without these details. As used herein, unless otherwise indicated, the following terms have the meanings assigned to them.

[0039] Throughout this specification and claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted in an open and inclusive sense, ie, "including but not limited to."

[0040] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present disclosure. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. As used in the specification and claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise.

[0042] "Hydroxy" or "hydroxyl" refers to an -OH group.

[0043] "Amino" refers to a -NH2 group.

[0044] "Cyano" refers to a -CN group.

[0045] "Alkyl" refers to a saturated straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, having one to six carbon atoms (C1-C6 alkyl), which is attached to the rest of the molecule by a single bond. Hydrocarbon chain groups include, for example, methyl, ethyl, n-propyl, 1 methylethyl (isopropyl), n-butyl, n-pentyl, 1,1 dimethylethyl (tert-butyl), isopentyl, n-hexyl, etc. Unless otherwise specifically stated in the specification, alkyl groups are optionally substituted.

[0046] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group (C3-C8 cycloalkyl) having three to eight carbon atoms, which is attached to the rest of the molecule by a single bond. Saturated cyclic hydrocarbon groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. Unless specifically stated otherwise in this specification, cycloalkyl is optionally substituted.

[0047] "Bridged cycloalkyl" refers to a cycloalkyl as defined above that also includes an alkylene bridge between at least two carbon atoms of the ring. Bridged cycloalkyls include, for example, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[3.1.1]heptyl, (1R,5S)-bicyclo[3.2.1]octyl and bicyclo[2.2.2]octyl. Unless specifically stated otherwise in this specification, bridged cycloalkyls are optionally substituted.

[0048] "Halo" refers to fluoro, chloro, bromo or iodo. Halo belongs to Group 17 of the Periodic Table of Elements.

[0049] "Alkoxy" refers to a group of the formula -OR a A group in which R a is an alkyl group as defined above, containing from one to twelve carbon atoms (C1-C 12Alkoxy is optionally substituted with one to eight carbon atoms (C1-C8 alkoxy), or one to six carbon atoms (C1-C6 alkoxy), or any value within these ranges. Unless otherwise specifically stated in the specification, an alkoxy group is optionally substituted.

[0050] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. Unless stated otherwise specifically in the specification, a haloalkyl group is optionally substituted.

[0051] "Haloalkoxy" refers to a group having the formula: -O-haloalkyl, wherein haloalkyl is as defined above. Unless stated otherwise specifically in the specification, a haloalkoxy group is optionally substituted.

[0052] "Hydroxylalkyl" or "hydroxyalkyl" refers to an alkyl group as defined above substituted with one or more hydroxy groups. The hydroxyalkyl group is attached to the backbone via an alkyl carbon atom. Unless otherwise specifically stated in the specification, the hydroxyalkyl group is optionally substituted.

[0053] "Aryl" refers to a full carbon monocyclic or condensed polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group of 6 to 12 carbon atoms with a completely conjugated π electron system. Examples of aryl, but not limited to, are phenyl, naphthyl, and anthracenyl. Aryl may be substituted or unsubstituted. When substituted, as the term is defined below, aryl is substituted with one or more substituents, more preferably one, two or three, even more preferably one or two substituents independently selected from the group consisting of: alkyl (wherein the alkyl may be optionally substituted with one or two substituents), haloalkyl, halo, hydroxyl, alkoxy, sulfhydryl, alkylthio, cyano, acyl, nitro, phenoxy, heteroaryl, heteroaryloxy, haloalkyl, haloalkoxy, carboxyl, alkoxycarbonyl, amino, alkylaminodialkylamino, aryl, heteroaryl, carbocyclic or heterocyclic (wherein aryl, heteroaryl, carbocyclic or heterocyclic may be optionally substituted).

[0054] "Heterocyclyl" or "heterocycle" refers to a stable 3- to 18-membered non-aromatic ring group consisting of two to twelve carbon atoms and one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise specifically stated in the specification, the heterocyclyl may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused or bridged ring system; and the nitrogen, carbon or sulfur atoms in the heterocyclyl may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl may be partially or fully saturated. Examples of such heterocyclic groups include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithienyl, tetrahydropyranyl, thiomorpholinyl, thimorpholinyl, 1-oxo-thiomorpholinyl and 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise in the specification, the heterocyclic group may be optionally substituted.

[0055] "N-heterocyclyl" refers to a heterocyclyl as defined above, wherein at least one or more heteroatoms is nitrogen. Examples of N-heterocyclyl include azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, and the like. Unless specifically stated otherwise in this specification, N-heterocyclyl may be optionally substituted.

[0056] "Heteroaryl" refers to a 5- to 18-membered, e.g., 5- to 6-membered ring system radical containing one to thirteen ring carbon atoms, one to six ring heteroatoms, and at least one aromatic ring, wherein the ring heteroatoms are selected from the group consisting of nitrogen, oxygen, and sulfur. The heteroaryl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon, or sulfur atoms in the heteroaryl group may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples include, but are not limited to, azepanyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxazepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, isothiophene, Thiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 1-epoxypyridinyl, 1-epoxypyrimidinyl, 1-epoxypyrazinyl, 1-epoxypyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolyl, isoquinolinyl, tetrahydroquinolyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl and thienyl (i.e., thienyl). Unless otherwise specifically stated in the specification, heteroaryl is optionally substituted.

[0057] The term "substituted" as used herein refers to any of the above groups (e.g., alkyl, cycloalkyl, or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, such as, but not limited to, halogen atoms such as F, Cl, Br, and I; oxo (=O); hydroxyl (-OH); alkoxy (-OR a , where R a C1-C 12 alkyl or cycloalkyl); carboxyl (-OC(=O)R a OR -C(=O)OR a , where R a H, C1-C 12 Alkyl or cycloalkyl); amine (-NR a R b , where R a and R b Each independently is H, C1-C 12 alkyl or cycloalkyl); C1-C12 In some embodiments, the substituent is C1-C 12 In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluorine. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0058] "Optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description includes both substituted and unsubstituted alkyl radicals.

[0059] "Prodrug" is intended to refer to a compound that can be converted to a biologically active compound of the present disclosure under physiological conditions or by solvolysis. Thus, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of the present disclosure. A prodrug may be inactive when administered to a subject in need thereof, but is converted to an active compound of the present disclosure in vivo. Prodrugs are generally rapidly converted in vivo to produce the parent compound of the present disclosure, for example by hydrolysis in the blood. Prodrug compounds generally provide advantages of solubility, tissue compatibility or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam). A discussion of prodrugs is provided in Higuchi, T. et al., ACS Symposium Series, Vol. 14 and Bioreversible Carriers in Drug Design, Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987.

[0060] The term "prodrug" is also intended to include any covalently bonded carriers that release the active compound of the present disclosure in vivo when such prodrugs are administered to a mammalian subject. Prodrugs of compounds of the present disclosure can be prepared by modifying functional groups present in the compounds of the present disclosure in a manner that allows these modifications to be cleaved into the parent compound of the present disclosure in conventional manipulations or in vivo. Prodrugs include compounds of the present disclosure in which hydroxyl, amino or sulfhydryl groups are bonded to any group that, when the prodrugs of compounds of the present disclosure are administered to a mammalian subject, cleaves to form free hydroxyl, free amino or free sulfhydryl groups, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohols or amide derivatives of amine functional groups in compounds of the present disclosure, and the like.

[0061] The embodiments disclosed herein are also intended to encompass all pharmaceutically acceptable compounds of compounds of structure (I) that are isotopically labeled by replacing one or more atoms with atoms having a different atomic mass or mass number. Examples of isotopes that may be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, respectively. 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 36 Cl, 123 I and 125 I. These radiolabeled compounds can be used to help determine or measure the effectiveness of a compound, for example by characterizing the site or mode of action or binding affinity for a pharmacologically important site. Certain isotopically labeled compounds of structure (I), for example those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The radioactive isotope tritium (i.e., 3 H) and the radioactive isotope carbon-14 (i.e., 14 C) is particularly useful for this purpose due to its ease of incorporation and readily available detection means.

[0062] Deuterium 2 Substitution with heavier isotopes such as H may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances.

[0063] Use 11 C.18 F. 15 O and 13 Positron emitting isotope substitution such as N can be useful in positron emission tomography (PET) studies for examining substrate receptor occupancy. Isotopically labeled compounds of structure (I) can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the preparations and examples as shown below, using an appropriate isotopically labeled reagent in place of the unlabeled reagent previously employed.

[0064] Embodiments disclosed herein are also intended to encompass in vivo metabolites of disclosed compounds. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, esterification, etc. of the administered compound, mainly due to enzymatic processes. Therefore, the disclosure includes compounds produced by methods comprising administering a compound of the disclosure to a mammal for a period of time sufficient to produce its metabolites. Such products are generally identified in the following manner: a radiolabeled compound of the disclosure is administered to an animal (such as a rat, mouse, guinea pig, monkey or human) in a detectable dose, allowing sufficient time for metabolism to occur, and separating its conversion products from urine, blood or other biological samples.

[0065] "Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0066] "Mammal" includes humans as well as domestic animals such as laboratory animals and household pets (eg, cats, dogs, pigs, cows, sheep, goats, horses, rabbits) and non-domestic animals such as wild animals.

[0067] "Pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0068] "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts.

[0069] "Pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylsulfonic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, Formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid (TFA), undecylenic acid, etc.

[0070] "Pharmaceutically acceptable base addition salts" refer to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary amine salts, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0071] "Pharmaceutical composition" refers to a formulation of a compound of the present disclosure and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients.

[0072] "Effective amount" or "therapeutically effective amount" refers to an amount of a compound of the present disclosure that is sufficient to achieve treatment of a mammal (preferably a human) when administered to the compound. The amount of lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by a person of ordinary skill in the art based on his or her own knowledge and the present disclosure.

[0073] As used herein, "protein kinase-mediated condition" or "disease" refers to any disease or other deleterious condition in which a protein kinase is known to play a role and which is alleviated by treatment with a protein kinase inhibitor. In certain embodiments, the cancer is colon cancer, breast cancer, stomach cancer, prostate cancer, pancreatic cancer, or ovarian tissue cancer.

[0074] As used herein, "treating" or "treatment" encompasses the treatment of a disease or condition of interest in a mammal, preferably a human, suffering from the disease or condition of interest, and includes:

[0075] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the condition but has not yet been diagnosed with the condition;

[0076] (ii) inhibiting a disease or condition, i.e., preventing its development;

[0077] (iii) alleviate the disease or condition, i.e., cause regression of the disease or condition; or

[0078] (iv) relieving symptoms caused by the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms "disease" and "condition" may be used interchangeably or may be different, as a particular malady or condition may have no known causative agent (and thus the etiology has not been determined), and thus the particular malady or condition has not been identified as a disease, but only as an undesirable condition or syndrome in which clinicians have more or less identified a specific set of symptoms.

[0079] The compounds of the present disclosure or their salts (e.g., pharmaceutically acceptable salts) may contain one or more asymmetric centers, and thus may produce enantiomers, diastereomers, and other stereoisomeric forms, which may be defined in terms of absolute stereochemistry as (R)- or (S)- or (D)- or (L)- of an amino acid. The present disclosure is intended to include all such possible isomers and racemic and optically pure forms thereof. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise indicated, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0080] "Stereoisomers" refer to compounds composed of the same atoms connected by the same bonds but with different three-dimensional structures, which are not interchangeable. The present disclosure encompasses various stereoisomers and mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other. The present disclosure also contemplates "diastereomers," which refer to non-mirror images of non-identical stereoisomers. Diastereomers occur when two or more stereoisomers of a compound have different configurations at one or more of the equivalent stereocenters and are not mirror images of each other.

[0081] "Tautomer" refers to a proton shift from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the described compounds.

[0082] Compound

[0083] On the one hand, the present disclosure provides CDK9 inhibitor compounds, including stereoisomers or salts thereof (e.g., pharmaceutically acceptable salts), which can be used alone or in combination with a pharmaceutically acceptable carrier. Also provided are methods of using CDK9 inhibitor compounds to treat various diseases or conditions such as bladder cancer, prostate cancer, and leukemia.

[0084] One embodiment provides a compound having the following structure (I):

[0085]

[0086] As a stereoisomer or salt thereof, wherein:

[0087] represents a double or single bond such that all valences are satisfied and a heteroaryl ring is formed;

[0088] X is N or CR 4b ;

[0089] Y is N or CR 4c ;

[0090] R 1a Not present or C1-C6 alkyl;

[0091] R 1b is hydrogen, halo, hydroxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 hydroxyalkyl;

[0092] R 2 is hydrogen or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl or C1-C6 hydroxyalkyl;

[0093] R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a substituted C3-C5 cycloalkyl, C6-C8 bridged cycloalkyl or 3-10 membered heterocyclic group, or

[0094] R 3 is optionally substituted by hydroxy, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3b Substituted C3-C8 cycloalkyl;

[0095] R 3a is an optionally substituted 3-10 membered heterocyclyl or an optionally substituted 3-10 membered heteroaryl;

[0096] R 3b is an optionally substituted 3-10 membered N-heterocyclyl or an optionally substituted 3-10 membered heteroaryl;

[0097] R 3c is hydrogen or optionally substituted C1-C6 alkyl;

[0098] R 4a , R 4b and R 4cEach is independently hydrogen, halo or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy;

[0099] R 5 is independently at each occurrence halo or optionally substituted C1-C6 haloalkyl;

[0100] n is 0, 1, 2, 3 or 4; and

[0101] p is 0, 1 or 2.

[0102] One embodiment provides a compound having the following structure (I'):

[0103]

[0104] As a stereoisomer or salt (e.g., a pharmaceutically acceptable salt) thereof, wherein:

[0105] X is N or CH;

[0106] R 1 is hydrogen, halo, hydroxy or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 hydroxyalkyl;

[0107] R 2 is hydrogen or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl or C1-C6 hydroxyalkyl;

[0108] R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a substituted C3-C5 cycloalkyl or 3-10 membered heterocyclic group, or

[0109] R 3 is optionally substituted by hydroxy, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3b Substituted C3-C8 cycloalkyl;

[0110] R 3a is an optionally substituted 3-10 membered heterocyclyl or an optionally substituted 3-10 membered heteroaryl;

[0111] R 3bis an optionally substituted 3-10 membered N-heterocyclyl or an optionally substituted 3-10 membered heteroaryl;

[0112] R 3c is hydrogen or optionally substituted C1-C6 alkyl;

[0113] R 4 is halogenated or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy;

[0114] m is 0, 1 or 2; and

[0115] n is 0, 1, 2, 3 or 4.

[0116] In some embodiments, the compound has one of the following structures (Ia) or (Ib):

[0117]

[0118] In certain embodiments, R 1a In some embodiments, R 1a In some embodiments, R 1a is C1-C6 alkyl. 1a It is methyl.

[0119] In some embodiments, R 1 is hydrogen, C1-C6 alkyl or C3-C8 cycloalkyl. 1 is hydrogen, cyclopropyl, cyclobutyl, cyclopentyl, isopropyl, methyl or ethyl. 1 is hydrogen, cyclopropyl, cyclobutyl, isopropyl or methyl. In certain embodiments, R 1 Not replaced.

[0120] In some embodiments, R 1b is hydrogen, C1-C6 alkyl or C3-C8 cycloalkyl. 1b is hydrogen, cyclopropyl, cyclobutyl, cyclopentyl, isopropyl, methyl or ethyl. 1b is hydrogen, cyclopropyl, cyclobutyl, isopropyl or methyl. In certain embodiments, R 1b Not replaced.

[0121] In some embodiments, R 2 is hydrogen or C1-C6 alkyl. In a more specific embodiment, R 2 is C1-C6 alkyl. 2In certain embodiments, R 2 is methyl, ethyl, isopropyl or n-propyl. 2 In certain embodiments, R 2 Not replaced.

[0122] In some embodiments, R 1 or R 2 In certain embodiments, at least one of R 1 and R 2 Both of them are hydrogen.

[0123] In some embodiments, R 1b or R 2 In certain embodiments, at least one of R 1b and R 2 Both of them are hydrogen.

[0124] In some embodiments, n is 0 or 1. In certain embodiments, n is 0. In some embodiments, n is 2, 3, or 4.

[0125] In some embodiments, R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a Substituted C3-C5 cycloalkyl or 3-10 membered heterocyclic group.

[0126] In some embodiments, R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a Substituted C3-C5 cycloalkyl, C6-C7 bridged cycloalkyl or 3-10 membered heterocyclic group.

[0127] In certain embodiments, R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a In some embodiments, R 3 is a C5 cycloalkyl group optionally substituted by hydroxyl or amino. 3 is a C5 cycloalkyl group optionally substituted by an amino group.3 is optionally substituted by hydroxy, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3b In some embodiments, R 3 is a 5-6 membered heterocyclic group. 3 In a more specific embodiment, R 3 Not replaced.

[0128] In certain embodiments, R 3 Has one of the following structures:

[0129]

[0130] In certain embodiments, R 3 Has one of the following structures:

[0131]

[0132] In some embodiments, R 3 Has one of the following structures:

[0133]

[0134] In some embodiments, R 3 Has one of the following structures:

[0135]

[0136] In some embodiments, R 3 Has the following structure:

[0137]

[0138] In certain embodiments, R 3 Has the following structure:

[0139]

[0140] In some embodiments, R 3 Has the following structure:

[0141]

[0142] In certain embodiments, R 3 Has the following structure:

[0143]

[0144] In some embodiments, R 3 Has the following structure:

[0145]

[0146] In certain embodiments, R 3 Has the following structure:

[0147]

[0148] In some embodiments, R 3 Has the following structure:

[0149]

[0150] In some embodiments, m is 1 or 2. In certain embodiments, R 4 is independently halo at each occurrence. In some embodiments, R 4 is independently fluorine, chlorine or bromine at each occurrence. In some more specific embodiments, R 4 In some embodiments, m is 0.

[0151] In some embodiments, R 4a In some embodiments, R 4a is halo (eg, chloro, fluoro or bromo).

[0152] In some embodiments, X is N. In some embodiments, X is CR 4b And R 4b In some embodiments, X is CR 4b And R 4b In some embodiments, X is CF. In some embodiments, X is CH.

[0153] In some embodiments, Y is N. In some embodiments, Y is CR 4b And R 4b In some embodiments, Y is CR 4b And R 4b In some embodiments, Y is CF. In some embodiments, Y is CH.

[0154] In some embodiments, p is 1. In some embodiments, p is 2. In certain embodiments, R 5 is halo (e.g., chloro, fluoro, or bromo) at each occurrence. In some embodiments, p is 0. In some embodiments, p is 1, and R 5 For chlorine.

[0155] In some embodiments, the compound is in free base form. In certain embodiments, the compound is a pharmaceutically acceptable salt. In some embodiments, the compound is a trifluoroacetate. In some embodiments, the compound is a trifluoroacetate, a hydrochloride, or a formate. In certain embodiments, the compound is a tautomer.

[0156] In various embodiments, the compound has one of the structures listed in Table 1 below (or a stereoisomer or salt thereof).

[0157] Table 1. Representative compounds of structure (I)

[0158]

[0159]

[0160]

[0161]

[0162] Also available as trifluoroacetate salt

[0163] Also available as the hydrochloride salt

[0164] # Also available as formate

[0165] IC 50 value:

[0166] ++++ indicates a value lower than 0.05 μM

[0167] +++ indicates values ​​between 0.05 and 0.10 μM

[0168] ++ indicates values ​​between 0.10 and 1.00 μM

[0169] + indicates a value greater than 1 μM

[0170] It should be understood that any embodiment of the compound of structure (I) as described above, and any specific substituent and / or variable in the compound of structure (I) as described above can be independently combined with other embodiments and / or substituents and / or variables of the compound of structure (I) to form embodiments of the present disclosure not specifically set forth above. In addition, in the case of listing substituents and / or variables for any specific R group or variable n or m in a specific embodiment and / or claim, it should be understood that each individual substituent and / or variable can be deleted from a specific embodiment and / or claim, and the remaining substituent and / or variable list will be considered to be within the scope of the present disclosure. It should be understood that in this specification, the combination of substituents and / or variables of the depicted formula is allowed only when such contributions produce stable compounds.

[0171] Pharmaceutical composition

[0172] Other embodiments relate to pharmaceutical compositions. The pharmaceutical composition comprises any one (or more) of the aforementioned compounds and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection. In still multiple embodiments, the pharmaceutical composition comprises a compound as disclosed herein and another therapeutic agent (e.g., an anticancer agent). Non-limiting examples of such therapeutic agents are described below.

[0173] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transmucosal, transdermal, vaginal, ear, nasal, and topical administration. In addition, parenteral delivery includes, by way of example only, intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injection.

[0174] In certain embodiments, the compounds as described herein are administered in a local rather than systemic manner, for example, via direct injection of the compound into an organ, typically in the form of a depot preparation or a sustained release formulation. In a specific embodiment, the long-acting formulation is administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In addition, in other embodiments, the compound is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ-specific antibody. In such embodiments, the liposome targets an organ and is selectively absorbed by the organ. In yet other embodiments, the compounds as described herein are provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. In yet other embodiments, the compounds described herein are administered topically.

[0175] In the treatment methods according to the embodiments of the present disclosure, an effective amount of at least one compound of structure (I) is administered to a subject suffering from or diagnosed as suffering from such a disease, disorder or medical condition. The effective amount or dose can be determined by methods such as modeling, dose escalation studies or clinical trials, such as the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder or condition, the subject's previous or ongoing therapy, the subject's health status and response to the drug, and the judgment of the treating physician.

[0176] Compounds according to the present disclosure are effective over a wide dosage range. For example, in the treatment of adults, dosages of 10 to 5000 mg, 100 to 5000 mg, 1000 mg to 4000 mg, and 1000 to 3000 mg per day are examples of dosages used in some embodiments. The exact dosage will depend on the route of administration, the form of administration of the compound, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

[0177] In some embodiments, the compounds of the present disclosure are administered in a single dose. Typically, such administration will be by injection, such as intravenous injection, to quickly introduce the agent. However, other routes are used when appropriate. A single dose of the compounds of the present disclosure can also be used to treat acute conditions.

[0178] In some embodiments, the compounds of the present disclosure are administered in multiple doses. In some embodiments, administration is about once, twice, three times, four times, five times, six times or more than six times a day. In other embodiments, administration is about once a month, once every two weeks, once a week or once every other day. In another embodiment, the compounds of the present disclosure and another agent (e.g., anticancer agent) are administered together about once a day to about 6 times a day. In another embodiment, the administration of the compounds of the present disclosure and the agent continues for less than about 7 days. In another embodiment, administration continues for more than about 6 days, 10 days, 14 days, 28 days, two months, six months or one year. In some cases, continuous administration is achieved and continuous administration continues for the required duration.

[0179] The administration of the disclosed compounds can continue for a desired period of time. In some embodiments, the disclosed compounds are administered for more than 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, or 28 days. In some embodiments, the disclosed compounds are administered for less than 28 days, 14 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the disclosed compounds are administered for a long period of time, for example, to treat chronic effects.

[0180] In some embodiments, the compounds of the present disclosure are administered in a single dosage form. It is known in the art that due to inter-subject variability in compound pharmacokinetics, individualization of dosing regimens is necessary for optimal therapy.

[0181] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. In specific embodiments, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate processing of the disclosed compounds into pharmaceutically usable preparations. Appropriate formulations depend on the selected route of administration. The pharmaceutical compositions described herein are formulated using any pharmaceutically acceptable techniques, carriers, and excipients suitable for use: Remington: The Science and Practice of Pharmacy, 19th ed. (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. 1975; Liberman, H. A. and Lachman, L., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. (Lippincott Williams & Wilkins 1999).

[0182] Provided herein are pharmaceutical compositions comprising one or more compounds of structure (I) and a pharmaceutically acceptable carrier.

[0183] Provided herein is a pharmaceutical composition comprising one or more compounds selected from compounds of structure (I) and a pharmaceutically acceptable diluent, excipient and carrier. In certain embodiments, the compound described is administered as a pharmaceutical composition, wherein one or more compounds selected from compounds of structure (I) are mixed with other active ingredients, such as in combination therapy. All combinations of active substances described in the following combination therapy sections and throughout the disclosure are contemplated herein. In a specific embodiment, the pharmaceutical composition includes one or more compounds of structure (I).

[0184] As used herein, pharmaceutical composition refers to a mixture of one or more compounds selected from the compound of structure (I) and other chemical components (such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients). In certain embodiments, pharmaceutical composition helps to apply the compound to an organism. In some embodiments, a therapeutically effective amount of one or more compounds selected from the compound of structure (I) provided herein is applied to a mammal suffering from a disease to be treated, a disease or a medical condition in the form of a pharmaceutical composition. In a specific embodiment, the mammal is a human. In certain embodiments, the therapeutically effective amount varies according to the severity of the disease, the age and relative health of the subject, the effectiveness of the compound used and other factors. The compounds described herein are used alone or in combination with one or more therapeutic agents as components of the mixture.

[0185] In one embodiment, one or more compounds selected from the compounds of structure (I) are formulated in an aqueous solution. In specific embodiments, by way of example only, the aqueous solution is selected from a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or a physiological saline buffer. In other embodiments, one or more compounds selected from the compounds of structure (I) are formulated for transmucosal administration. In specific embodiments, the transmucosal formulation includes a penetrant suitable for the barrier to be permeated. In still other embodiments in which the compounds described herein are formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and / or excipients.

[0186] In another embodiment, the compounds described herein are formulated for oral administration. The compounds described herein are formulated by combining the active compound with, for example, a pharmaceutically acceptable carrier or excipient. In various embodiments, the compounds described herein are formulated into oral dosage forms, which include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

[0187] In certain embodiments, pharmaceutical preparations for oral use are obtained by mixing one or more solid excipients with one or more compounds described herein, optionally grinding the resulting mixture, and, if desired, processing the mixture of particles after adding suitable auxiliary agents to obtain tablets or dragee cores. Suitable excipients are in particular fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; or others, such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In a specific embodiment, a disintegrant is optionally added. By way of example only, disintegrants include cross-linked sodium carboxymethylcellulose, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0188] In one embodiment, one or more suitable coatings are provided to dosage forms such as dragee cores and tablets. In a specific embodiment, concentrated sugar solution is used for coated dosage forms. Sugar solution optionally contains other components, such as only as an example, gum arabic, talcum, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture. Dye and / or pigment are also optionally added to the coating for identification purposes. In addition, dye and / or pigment are optionally used to characterize the different combinations of active compound dosage.

[0189] In certain embodiments, a therapeutically effective amount of at least one compound described herein is formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). In specific embodiments, push-fit capsules contain active ingredients mixed with one or more fillers. By way of example only, fillers include lactose, binders (such as starch) and / or lubricants (such as talc or magnesium stearate) and optionally stabilizers. In other embodiments, soft capsules contain one or more active compounds dissolved or suspended in a suitable liquid. By way of example only, suitable liquids include one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

[0190] In other embodiments, the compounds described herein are formulated for parenteral injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, the formulations for injection are presented in unit dosage form (e.g., in ampoules) or in multidose containers. Preservatives are optionally added to the injection formulations. In still other embodiments, the pharmaceutical composition is formulated into a form suitable for parenteral injection, such as a sterile suspension, solution or emulsion in an oily or aqueous vehicle. Parenteral injection formulations optionally contain formulation agents, such as suspending agents, stabilizers and / or dispersants. In specific embodiments, the pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds in water-soluble form. In additional embodiments, a suspension of one or more compounds selected from the compounds of structure (I) is prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. In certain specific embodiments, aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension contains a suitable stabilizer or increases the solubility of the compound to allow the preparation of a formulation of a high concentration solution. Alternatively, in other embodiments, the active ingredient is in powder form for reconstitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0191] The pharmaceutical composition includes at least one pharmaceutically acceptable carrier, diluent or excipient, and one or more compounds selected from the compounds of structure (I) described herein as active ingredients. The active ingredient is in the form of free acid or free base, or in the form of a pharmaceutically acceptable salt. In addition, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also referred to as polymorphs) and active metabolites of these compounds with the same type of activity. All tautomers of the compounds described herein are included within the scope of the compounds provided herein. In addition, the compounds described herein encompass non-solvated forms and solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. The solvated forms of the compounds presented herein are also considered to be disclosed herein. In addition, the pharmaceutical composition optionally includes other medical or pharmaceutical agents, carriers, adjuvants (such as preservatives, stabilizers, wetting agents or emulsifiers), dissolution promoters, salts for regulating osmotic pressure, buffers and / or other therapeutically valuable substances.

[0192] Methods for preparing compositions comprising the compounds described herein include formulating the compounds with one or more inert pharmaceutically acceptable excipients or carriers to form solids, semisolids or liquids. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets and suppositories. Liquid compositions include solutions in which the compounds are dissolved, emulsions containing the compounds, or solutions containing liposomes, micelles or nanoparticles containing the compounds disclosed herein. Semisolid compositions include, but are not limited to, gels, suspensions and creams. The pharmaceutical compositions described herein are in the form of liquid solutions or suspensions, solid forms suitable for dissolution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, and the like.

[0193] In some embodiments, the pharmaceutical composition comprising one or more compounds selected from the compounds of structure (I) is illustratively in liquid form, wherein the medicament is present in solution, suspension, or both. Typically, when the composition is administered as a suspension, the first portion of the medicament is present in solution, and the second portion of the medicament is present in a suspension in a liquid matrix in a granular form. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

[0194] In certain embodiments, the aqueous suspension contains one or more polymers as suspending agents. Polymers include water-soluble polymers such as cellulose polymers, such as hydroxypropyl methylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein contain mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0195] The pharmaceutical composition optionally also includes a solubilizing agent to help dissolving of one or more compounds selected from the compound of structure (I). The term "solubilizing agent" generally includes an agent that causes a micellar solution or a true solution of the agent to be formed. Some acceptable nonionic surfactants such as polysorbate 80 can be used as solubilizing agents, and ophthalmologically acceptable glycols, polyglycols (e.g., polyethylene glycol 400) and glycol ethers can also be used as solubilizing agents.

[0196] In addition, the pharmaceutical composition optionally includes one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, trishydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in amounts required to maintain the pH of the composition within an acceptable range.

[0197] The composition also optionally includes one or more salts in an amount required to bring the osmotic pressure of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0198] Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as phenylmercuric borate and thimerosal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0199] The composition may include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxyl alcohol 10, octoxyl alcohol 40.

[0200] The composition may include one or more antioxidants to enhance chemical stability where desired. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0201] In certain embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case a preservative is generally included in the composition.

[0202] In alternative embodiments, other delivery systems for hydrophobic drug compounds are used. Liposomes and emulsions are examples of delivery vehicles or carriers that can be used herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also used. In other embodiments, sustained release systems such as semipermeable matrices of solid hydrophobic polymers containing therapeutic agents are used to deliver the compounds described herein. Various sustained release materials can be used herein. In some embodiments, sustained release capsules release compounds for several weeks to more than 100 days. Depending on the chemical properties and biological stability of the therapeutic agent, additional protein stabilization strategies are used.

[0203] In certain embodiments, the formulations described herein comprise one or more antioxidants, metal chelators, thiol-containing compounds, and / or other general stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / w ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparins, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0204] In some embodiments, the concentration of one or more compounds selected from the compounds of structure (I) provided in the pharmaceutical compositions of the present disclosure is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%. %, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14.25%, 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0. 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w / w, w / v or v / v.

[0205] In some embodiments, the concentration of one or more compounds selected from the compounds of structure (I) provided in the pharmaceutical compositions of the present disclosure is between about 0.0001% and about 50%, about 0.001% and about 40%, about 0.01% and about 30%, about 0.02% and about 29%, about 0.03% and about 28%, about 0.04% and about 27%, about 0.05% and about 26%, about 0.06% and about 25%, about 0.07% and about 24%. %, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, about 1% to about 10% w / w, w / v or v / v range.

[0206] In some embodiments, the amount of one or more compounds selected from the compounds of structure (I) provided in the pharmaceutical composition of the present disclosure is equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g , 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g or 0.0001g.

[0207] In some embodiments, the amount of one or more compounds selected from the compounds of structure (I) provided in the pharmaceutical compositions of the present disclosure is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g or 1-3 g.

[0208] Packaging materials for packaging pharmaceutical compositions described herein include packaging materials found in, for example, U.S. Pat. Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging materials suitable for selected formulations and intended modes of administration and treatment. For example, the container includes one or more compounds described herein, optionally in a composition or in combination with another medicament disclosed herein. The container optionally has a sterile inlet (e.g., the container is an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). Such kits optionally include compounds with identification descriptions or labels or instructions related to their use in the methods described herein.

[0209] For example, the kit typically includes one or more additional containers, each container having one or more various materials (such as reagents, optionally in concentrated form, and / or devices) required for the use of the compounds described herein from a commercial and user perspective. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carriers, packaging, containers, vials and / or tube labels listing the contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also generally be included. The label is optionally on or associated with the container. For example, when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, the label is located on the container; when the label is present in a receiver or carrier that also holds the container, such as as a package insert, the label is associated with the container. In addition, the label is used to indicate that the contents will be used for a specific therapeutic application. In addition, the label indicates instructions for use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is present in a package or dispenser device containing one or more unit dosage forms containing the compounds provided herein. The package, for example, contains metal or plastic foil, such as a blister pack. Alternatively, the package or dispenser device is accompanied by instructions for administration. Alternatively, the package or dispenser is accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of drugs, which reflects the agency's approval of the form of the drug for human or veterinary use. For example, such a notice is a prescription drug label approved by the U.S. Food and Drug Administration, or an approved product insert. In some embodiments, a composition containing a compound provided herein formulated in a compatible pharmaceutical carrier is prepared, placed in an appropriate container, and labeled for treatment of a designated condition.

[0210] One embodiment provides a method for treating neuroendocrine prostate cancer (NEPC). Some embodiments provide a method for treating N-MYC or MCL-1 overexpression. Some embodiments provide a method for treating metastatic castration-resistant prostate cancer or CRPC. In some embodiments, the condition or disease includes treating a tumor with drug resistance. In some embodiments, NEPC is caused by prostate adenocarcinoma, androgen deprivation therapy (ADT), or abnormal expression and activation of various kinases. Some embodiments provide a method for treating hematological tumors or solid tumors.

[0211] In some embodiments, the method comprises inhibiting or silencing the transcriptional activity of pCDK9, pSer2, P-TEFb, MYC oncogenes, or inhibiting an active super enhancer complex.

[0212] In some embodiments, the methods include promoting prostate cancer cell death and overcoming drug resistance (eg, resistance to chemotherapy and other current targeted therapeutics).

[0213] In certain embodiments, the method comprises inhibiting CDK9, N-MYC, C-MYC and its related super enhancer gene expression profile. In some embodiments, the method comprises increasing the (median) survival time to greater than 12, 13, 13.5, 13.6, 13.7, 14, 18, 20 or 36 months. In some embodiments, the method further comprises administering a compound of structure (I) in combination with another chemotherapeutic agent (e.g., docetaxel, abiraterone, enzalutamide).

[0214] In some embodiments, the method includes inhibiting CDK9 as a treatment for prostate tumors. In some embodiments, the method includes increasing prostate cancer cell death and overcoming resistance due to chemotherapeutics and current targeted therapeutics. In some embodiments, the method includes inhibiting the CDK9-cyclin T complex, which phosphorylates Ser2 of the CTD of the negative elongation factor (NELF) complex, DRB sensitivity inducing factor (DSIF), and RNAPII, thereby affecting the removal of elongation blockade.

[0215] In some embodiments, the method includes reversibly binding and inhibiting CDK9. In some embodiments, the method includes treating malignant prostate cancer cells, especially CRPC, mCRPC, NEPC and malignant prostate cancer cells derived from treatment-resistant subtypes, while also showing no toxicity to normal prostate cells. In some embodiments, the method includes regulating N-MYC, C-MYC and MCL-1 transcription (for example, in NEPC cells, such as 22RV1, LASCPC-01, C4-2 and C4-2B from prostate cancer patients). In certain embodiments, the method includes oral administration of a compound of structure (I). In some embodiments, the method includes global regulation of transcription, silencing and inhibition of CDK9. In some embodiments, the method includes dual inhibition of CDK9, MYC and related super enhancer genes. In some embodiments, the method includes inhibiting tumor growth.

[0216] In certain embodiments, the methods comprise binding or targeting residues within the CDK9 ATP binding site, such as the gate keeper Phe103, hinge residues Asp104, Phe105, Cys106, and the DFG loop (167, 168, 169 residues), including the back pocket K48 and the carbohydrate binding pocket residues Glu107, His108, and Asp109.

[0217] As described above, the compounds and compositions of the present disclosure will be useful for a wide range of diseases and conditions mediated by protein kinases, including diseases and conditions mediated by kinases. Such diseases may include, for example, but are not limited to, cancers such as lung cancer, NSCLC (non-small cell lung cancer), oat cell carcinoma, bone cancer, pancreatic cancer, skin cancer, dermatofibrosarcoma protuberans, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, anal cancer, gastric cancer, colon cancer, breast cancer, gynecological tumors (e.g., uterine sarcoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer or vulvar cancer), Hodgkin's Disease, hepatocellular carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, systemic cancers (e.g. thyroid, pancreatic, parathyroid or adrenal), soft tissue sarcomas, urethral cancer, penile cancer, prostate cancer (especially hormone refractory), chronic or acute leukemias, solid tumors in children, hypereosinophilia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer (e.g. renal cell carcinoma, renal pelvic cancer), pediatric malignancies, central nervous system tumors (e.g. primary CNS lymphoma, spinal axis tumors, medulloblastoma, brain stem glioma or pituitary adenoma), Barrett's esophagus (precancerous syndrome), neoplastic skin diseases, psoriasis, mycosis fungoides and benign prostatic hypertrophy, diabetes-related diseases such as diabetic retinopathy, retinal ischemia and retinal neovascularization, cirrhosis, angiogenesis, cardiovascular diseases such as atherosclerosis, immune diseases such as autoimmune diseases and kidney disease.

[0218] In some embodiments, the pharmaceutical composition has the above-described compound and a pharmaceutically acceptable carrier, including, for example, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0219] In some embodiments, a method of treating a disease or disorder comprises administering to a subject in need thereof an effective amount of a compound or pharmaceutical composition described herein.

[0220] In some embodiments, the disease or condition is a cancer that expresses a kinase. In some specific embodiments, the cancer is bladder cancer. In some other specific embodiments, the cancer is prostate cancer. In some other specific embodiments, the cancer is a hematological malignancy, such as acute myeloid leukemia. In some other specific embodiments, the disease or condition is an autoimmune or inflammatory disease.

[0221] Preparation of compounds

[0222] The preparation methods of the above compounds and compositions are described below and / or known in the art. It will be appreciated by those skilled in the art that in the process described herein, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, sulfhydryl and carboxylic acid. Suitable protecting groups for hydroxyl include trialkylsilyl or diarylalkylsilyl (e.g., tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, etc. Suitable protecting groups for amino, amidino and guanidino include tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable protecting groups for sulfhydryl include -C (O) -R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl, etc. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley. As will be appreciated by those skilled in the art, the protecting group may also be a polymer resin, such as Wang resin, Rink resin or 2-chlorotrityl-chloride resin.

[0223] Those skilled in the art will also understand that, although such protected derivatives of compounds of the present disclosure may not themselves have pharmacological activity, the derivatives can be administered to mammals and then metabolized in vivo to form compounds of the present disclosure that have pharmacological activity. Such derivatives can therefore be described as "prodrugs". All prodrugs of compounds of the present disclosure are included within the scope of the present disclosure.

[0224] In addition, all compounds in the present disclosure that exist in free base or acid form can be converted into their pharmaceutically acceptable salts by methods known to those skilled in the art, by treatment with appropriate inorganic or organic bases or acids. Salts of compounds of the present disclosure can be converted into free base or acid forms by standard techniques.

[0225] The following reaction schemes illustrate methods for preparing compounds of the present disclosure, i.e., compounds of structure (I):

[0226]

[0227] or a salt (eg, a pharmaceutically acceptable salt) or a stereoisomer thereof, wherein X, Y, R 1a , R 1b , R 2 , R 3 , R 4s , R 5, p and n are as defined herein. It should be understood that those skilled in the art can prepare these compounds by similar methods or by combining other methods known to those skilled in the art. It should also be understood that those skilled in the art can prepare other compounds of structure (I) not specifically described below in a similar manner as described below, by using appropriate starting components and changing synthesis parameters as required. Usually, starting components can be obtained from sources (such as SigmaAldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI and Fluorochem, Inc., USA), or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley Press, December 2000)), or prepared as described in the present disclosure.

[0228] The following examples are offered for purposes of illustration and not limitation.

[0229] Abbreviations:

[0230] ℃(Celsius); 1 H NMR (proton nuclear magnetic resonance); DCM (dichloromethane); DMSO (dimethyl sulfoxide); eq (equivalent); EtOAc (ethyl acetate); g (gram); h (hour); MeOH (methanol); mg (milligram); min (minute); mL (milliliter); mmol (millimole); TFA (trifluoroacetic acid); THF (tetrahydrofuran); TLC (thin layer chromatography); LDA (lithium diisopropylamide); AcOH (acetic acid); mCPBA (3-chloroperbenzoic acid).

[0231] Example 1

[0232] Synthesis of Compound I-1

[0233]

[0234]

[0235] Synthesis of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (20)

[0236]

[0237] To a stirred solution of 5-bromo-2methyl-3-(propan-2-yl-2H-indazole) (10) (127 mg, 0.50 mmol, 1.0 eq) and bis(pinacolato)diborane (152 mg, 0.60 mmol, 1.2 eq) in 1,4-dioxane (8 mL) in a microwave vial were added potassium acetate (119 mg, 1.25 mmol, 2.5 eq), Pd(dppf)Cl 2. DCM (20 mg, 0.025 mmol, 0.05 equiv). Then, the vial was sealed with a cap and degassed with argon for 5 minutes. The reaction mixture was stirred at 90 ° C for 16 hours. After the reaction was completed by TLC, the reaction mixture was diluted with water and ethyl acetate, and the resulting slurry was filtered by The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated to provide the crude product 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (20) (150 mg, yield: 100%) as a yellow solid.

[0238] Synthesis of 3-isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]-1H-indazole (30)

[0239]

[0240] To a stirred solution of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (20) (150 mg, 0.50 mmol, 1 eq) in 1,4-dioxane and water (3:1) (8 mL) was added 4-chloro-2-(methylthio)pyrimidine (96 mg, 0.60 mmol, 1.2 eq), KCO (173 mg, 0.25 mmol, 2.5 eq), Pd(dppf)ClDCM (20 mg, 0.025 mmol, 0.05 eq) in a microwave vial and degassed with argon for 5 min. The reaction mixture was then stirred at 100 °C for 16 h. The reaction mixture was diluted with water and ethyl acetate and the resulting slurry was filtered through The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated to provide a crude product, which was purified by Combiflash chromatography (4 g column) to give 3-isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]-1H-indazole (30) (130 mg, yield: 87%) as a yellow solid. TLC system: Hexane:EtOAc (2:1), R f Value: ~0.2.

[0241] Synthesis of a mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)indazole and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)indazole (40)

[0242]

[0243] 3-Isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]-1H-indazole (30) (98 mg, 0.33 mmol, 1.0 equiv) was added to a stirred solution of DCM (4 mL) cooled to 0°C with 3-chloroperbenzoic acid (mCPBA) (purity, 77%) (96 mg, 0.43 mmol, 1.3 equiv). The reaction mixture was stirred at 0°C for 4 hours. After completion of the reaction by TLC, the reaction mixture was quenched with saturated aqueous NaHCO3 (10 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine solution, dried over sodium sulfate and concentrated to give a crude mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)indazole and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)indazole (40) (104 mg, yield: 100%) as a yellow solid. TLC system: EtOAc (100%) R f Values: ~0.01 and 0.2.

[0244] Synthesis of tert-butyl (1S,3R)-[3-[4-(3-isopropyl-2-methylindazol-5-yl)pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate (50a)

[0245]

[0246] To a stirred solution of a mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)indazole and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)indazole (40) (30 mg, 0.10 mmol, 1 eq) in DMSO (5 mL) at room temperature was added (1S, 3S)-3-amino-1-(BOC-amino)cyclopentane (28 mg, 0.14 mmol, 1.5 eq) and cesium fluoride (21 mg, 0.14 mmol, 1.5 eq). The reaction mixture was then stirred at 95 °C for 16 hours. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature and extracted with ethyl acetate. The organic layer was washed with brine solution, dried over anhydrous sodium sulfate, and then concentrated to provide the product, which was purified by Combiflash chromatography (4 g column) to give tert-butyl (1S,3R)-[3-[4-(3-isopropyl-2-methylindazol-5-yl)pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate (50a) (23 mg, 53%) as a yellow solid. TLC system: Hexane:EtOAc (1:1), R f Value: ~0.2.

[0247] Synthesis of (1S,3R)-[3-[4-(3-isopropyl-2-methylindazol-5-yl)pyrimidin-2-yl]aminocyclopentane-1-yl]-1,3-trifluoroacetic acid diamine (Compound I-1)

[0248]

[0249] To a stirred solution of (1S, 3R)-[3-[4-(3-isopropyl-2-methylindazole-5-yl)pyrimidin-2-yl]aminocyclopentane-1-yl] tert-butyl carbamate (50a) (22 mg, 0.05 mmol, 1 equivalent) in DCM (4 mL) at room temperature, trifluoroacetic acid (TFA) (2 mL) was added, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the solvent was removed in vacuo. Diethyl ether was then added to the reaction solution, and a large amount of yellow solids appeared in the reaction solution. The mixture was centrifuged, washed with diethyl ether and dried at room temperature to provide a product (1S, 3R)-[3-[4-(3-isopropyl-2-methylindazole-5-yl)pyrimidin-2-yl]aminocyclopentane-1-yl]-1,3-trifluoroacetic acid diamine (Compound I-1) (25 mg, 100%) as a yellow solid. 1HNMR(400MHz,MeOD-d4)δ8.82(s,1H),8.29(d,J=6.0Hz,1H),8.14(d,J=9.0Hz,1H),7.64(d,J=9.0Hz,1H),754(d,J=6.0Hz,1H),4.59(width,1H),4.1 9(s,3H),3.76-3.74(m,2H),2.79-2.73(m,1H),2.30-2.20(m,2H),2.00 -1.87(m,2H),1.80-1.72(m,1H),1.60(d,J=6.8Hz,6H); HRMS(ESI)m / z:C 20 H 26 N6's [M+H] + Calculated value, 351.2292, experimental value, 351.2270.

[0250] Example 2

[0251] Synthesis of Compound I-2

[0252] The precursors 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)indazole and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)indazole (40) were prepared according to the synthesis described in Example 1 following steps 1-3.

[0253] Synthesis of tert-butyl (1S,3S)-[3-[4-(3-isopropyl-2-methylindazol-5-yl)pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate (50b)

[0254]

[0255] To a stirred solution of a mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)indazole and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)indazole (40) (30 mg, 0.10 mmol, 1 eq) in DMSO (5 mL) at room temperature was added (1S, 3S)-3-amino-1-(BOC-amino)cyclopentane (28 mg, 0.14 mmol, 1.5 eq) and cesium fluoride (21 mg, 0.14 mmol, 1.5 eq). The reaction mixture was then stirred at 95 °C for 16 hours. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature and extracted with ethyl acetate. The organic layer was washed with brine solution, dried over anhydrous sodium sulfate, and then concentrated to provide the product, which was purified by Combiflash chromatography (4 g column) to give tert-butyl (1S,3S)-[3-[4-(3-isopropyl-2-methylindazol-5-yl)pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate (50b) (25 mg, 58%) as a yellow solid. TLC system: Hexane:EtOAc (1:1), R f Value: ~0.2.

[0256] Synthesis of (1S,3S)-[3-[4-(3-isopropyl-2-methylindazol-5-yl)pyrimidin-2-yl]aminocyclopentane-1-yl]-1,3-trifluoroacetic acid diamine (Compound I-2)

[0257]

[0258] To a stirred solution of (1S, 3S)-[3-[4-(3-isopropyl-2-methylindazole-5-yl) pyrimidin-2-yl] aminocyclopentane-1-yl] tert-butyl carbamate (50b) (25 mg, 0.056 mmol, 1 equivalent) in DCM (4 mL) at room temperature, trifluoroacetic acid (TFA) (2 mL) was added, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the solvent was removed in vacuo. Diethyl ether was then added to the reaction solution, and a large amount of yellow solids will appear in the reaction solution. The mixture was centrifuged, washed with diethyl ether and dried at room temperature to provide a product (1S, 3S)-[3-[4-(3-isopropyl-2-methylindazole-5-yl) pyrimidin-2-yl] aminocyclopentane-1-yl]-1,3-trifluoroacetic acid diamine (compound I-2) (22 mg, 69%) as a yellow solid. 1HNMR(400MHz,MeOD-d4)δ8.79(s,1H),8.28(d,J=6.2Hz,1H),8.15(d,J=9.1 Hz,1H),7.64(d,J=9.1Hz,1H),7.52(s,width,1H),4.71(s,width,1H),4.19(s,3H),3 .89-3.82(m,1H),3.70-3.62(m,1H),2.44-2.34(m,2H),2.30-2.18(m,2H),1 .92-1.84(m,1H),1.79-1.70(m,1H),1.60(d,J=7.0Hz,6H); HRMS(ESI)m / z:C 20 H 26 N6's [M+H] + Calculated value, 351.2292, experimental value, 351.2265.

[0259] Example 3

[0260] Synthesis of Compound I-3

[0261]

[0262]

[0263] Synthesis of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[4,3-b]pyridine (twenty one)

[0265]

[0266] To a stirred solution of 5-bromo-2methyl-3-propylpyrazolo[4,3-b]pyridine (11) (45 mg, 0.18 mmol, 1.0 equiv) and bis(pinacolato)diborane (56 mg, 0.22 mmol, 1.2 equiv) in 1,4-dioxane (8 mL) in a microwave vial were added potassium acetate (44 mg, 0.45 mmol, 2.5 equiv), Pd(dppf)Cl 2. DCM (8 mg, 0.01 mmol, 0.05 equiv). Then, the vial was sealed with a cap and degassed with argon for 5 minutes. The reaction mixture was stirred at 90 ° C for 16 hours. After the reaction was completed by TLC, the reaction mixture was diluted with water and ethyl acetate, and the resulting slurry was filtered through The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated to provide the crude product 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[4,3-b]pyridine (21) (54 mg, yield: 100%) as a brown oil.

[0267] Synthesis of 3-isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]pyrazolo[4,3-b]pyridine (31)

[0268]

[0269] To a stirred solution of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[4,3-b]pyridine (21) (54 mg, 0.18 mmol, 1 eq) in 1,4-dioxane and water (3:1) (8 mL) was added 4-chloro-2-(methylthio)pyrimidine (43 mg, 0.27 mmol, 1.5 eq), KCO (62 mg, 0.45 mmol, 2.5 eq), Pd(dppf)ClDCM (8 mg, 0.01 mmol, 0.05 eq) in a microwave vial and degassed with argon for 5 min. The reaction mixture was then stirred at 100 °C for 16 h. The reaction mixture was diluted with water and ethyl acetate and the resulting slurry was filtered through The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated to provide a crude product, which was purified by Combiflash chromatography (4 g column) to give 3-isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]pyrazolo[4,3-b]pyridine (31) (10 mg, yield: 18%) as a yellow solid. TLC system: Hexane:EtOAc (2:1), R f Value: ~0.3.

[0270] Synthesis of a mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine (41)

[0271]

[0272] 3-Isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]pyrazolo[4,3-b]pyridine (31) (10 mg, 0.033 mmol, 1.0 equiv) was added to a stirred solution of DCM (4 mL) cooled to 0°C with 3-chloroperbenzoic acid (mCPBA) (purity, 77%) (10 mg, 0.043 mmol, 1.3 equiv). The reaction mixture was stirred at 0°C for 4 hours. After completion of the reaction by TLC, the reaction mixture was quenched with saturated aqueous NaHCO3 (10 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine solution, dried over sodium sulfate and concentrated to give a crude mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine (41) (10 mg, yield: 100%) as a yellow solid. TLC system: EtOAc (100%) R f Values: ~0.01 and 0.2.

[0273] Synthesis of (1S,3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentane-1-yl

[0274] tert-Butyl]carbamate (51)

[0275]

[0276] To a stirred solution of a mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine (41) (10 mg, 0.033 mmol, 1 eq) in DMSO (5 mL) was added (1S, 3S)-3-amino-1-(BOC-amino)cyclopentane (10 mg, 0.05 mmol, 1.5 eq) and cesium fluoride (8 mg, 0.05 mmol, 1.5 eq) at room temperature. The reaction mixture was then stirred at 95 °C for 16 hours. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature and extracted with ethyl acetate. The organic layer was washed with brine solution, dried over anhydrous sodium sulfate, and then concentrated to provide the product, which was purified by Combiflash chromatography (4 g column) to give tert-butyl (1S,3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate (51) (10 mg, 67%) as a yellow solid. TLC system: Hexane:EtOAc (1:1), R f Value: ~0.2.

[0277] Synthesis of (1S,3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentane-1-yl

[0278] [4-( ...2-(trifluoroacetic acid)-1-yl)-1,3-diamine trifluoroacetate (Compound I-17)

[0279]

[0280] To a stirred solution of (1S, 3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentane-1-yl]t-butyl carbamate (51) (10 mg, 0.022 mmol, 1 equivalent) in DCM (4 mL) at room temperature, trifluoroacetic acid (TFA) (2 mL) was added, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the solvent was removed in vacuo. Diethyl ether was then added to the reaction solution, and a large amount of yellow solids were present in the reaction solution. The mixture was centrifuged, washed with diethyl ether and dried at room temperature to afford the product (1S,3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentan-1-yl]-1,3-trifluoroacetic acid diamine (Compound I-3) (5 mg, 38%) as a yellow solid. 1HNMR (400 MHz, MeOD-d4) δ8.53 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.6 Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.86 (J = 5.6 Hz, 1H), 4.68 (s, wide, 1H), 4.07 (s, 3H), 3.87-3.81 (m, 1H), 3.64-3.57 (m, 1H), 2.41-2.33 (m, 2H), 2.23-2.19 (m, 2H), 1.85-1.72 (m, 2H), 1.54 (d, J = 7.0 Hz, 6H); HRMS data: C 19 H 25 HRMS (ESI) m / z [M+H]+ calcd for N7, 352.2244, found: 352.2217.

[0281] Example 4

[0282] Synthesis of Compound I-4

[0283] Compound I-4 was prepared from the fluorine-substituted derivative of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (20) and then the same synthesis as described in Example 2 was performed.

[0284] Example 5

[0285] Synthesis of Compound I-5

[0286] Compound I-21 was prepared from the fluorine-substituted derivative of 5-bromo-2methyl-3-propylpyrazolo[4,3-b]pyridine (11) and then the same synthesis as described in Example 3 was performed.

[0287] Example 6

[0288] Synthesis of Compound I-6

[0289]

[0290] Overall reaction scheme:

[0291]

[0292] Synthesis of 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0293]

[0294] A solution of 5-bromo-2-methyl-2H-indazole (500 mg, 2.36 mmol, 1.0 eq.), bis(pinacolato)diborane (722 mg, 2.84 mmol, 1.2 eq.) and potassium acetate (582 mg, 5.92 mmol, 2.5 eq.) in 1,4-dioxane (10 mL) was degassed for 5 minutes, and Pd(dppf)Cl2.DCM (97 mg, 0.118 mmol, 0.05 eq.) was added. Degassed with nitrogen for another 5 minutes and stirred at 80 ° C for 16 hours. After the reaction was completed by TLC, it was diluted with water (50 mL) and extracted with ethyl acetate (2×50 mL). The combined organic layers were washed with brine solution (20 mL), dried over sodium sulfate and concentrated to give 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (630 mg, crude product) as a black semi-solid. TLC system: EtOAc / petroleum ether (40:60), R f Value: -0.45 (single spot and expected to be the boronate).Cr: LC / MS: 74.5% of 177.1 [M+H] as boronic acid and 18% of 259.2 [M+H] as the boronate.

[0295] Synthesis of 5-(2-chloropyrimidin-4-yl)-2-methyl-2H-indazole

[0296]

[0297] In a sealed tube, 2,4-dichloropyrimidine (381 mg, 2.58 mmol, 1.2 eq) and KCO (735 mg, 5.32 mmol, 2.5 eq) were added to a stirred solution of 2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (550 mg, 2.13 mmol, 1 eq) in 1,4-dioxane and water (3:1) (11 mL) at room temperature. Degassing was performed for 5 minutes, and then Pd(dppf)Cl was added. 2· DCM (87 mg, 0.106 mmol, 0.05 eq). The reaction mixture was stirred at 80 ° C for 16 hours. After completion of the reaction by TLC, it was diluted with water and extracted with ethyl acetate (2×50 mL). The combined organic layer was dried over sodium sulfate, concentrated and purified by silica gel column (60-120 mesh, eluent: 50% ethyl acetate / petroleum ether) to give 5-(2-chloropyrimidin-4-yl)-2-methyl-2H-indazole (450 mg, 88%) as a light yellow solid. TLC system: EtOAc (100%), R f Value: ~0.2. LC / MS retention time = 2.92 minutes, 245.0 [M+H]+ .

[0298] 1 H NMR(400MHz, CDCl3)δ8.6(d,J=5.2Hz,1H),8.60–8.59(m,1H),8.06(s,1H),7.94(dd ,J=9.2&1.6Hz,1H),7.79(dd,J=8.8&0.8Hz,1H),7.67(d,J=5.2Hz,1H),4.27(s,3H).

[0299] Synthesis of tert-butyl ((1S,3S)-3-((4-(2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate

[0300]

[0301] To a stirred solution of 5-(2-chloropyrimidin-4-yl)-2-methyl-2H-indazole (450 mg, 1.84 mmol, 1 eq) in IPA (5 mL) was added tert-butyl ((1S, 3S)-3-aminocyclopentyl)carbamate (443 mg, 2.21 mmol, 1.2 eq) at room temperature and stirred at 110 ° C for 60 hours. TLC showed polar spots, and the reaction mixture was allowed to reach room temperature. The reaction mixture was evaporated and diluted with water (30 mL) and extracted with EtOAc (2×30 ml). The organic layer was dried over Na2SO4, filtered, evaporated and purified by reverse phase column (eluting in 25% to 35% acetonitrile and 0.01% FA in water) to give tert-butyl ((1S,3S)-3-((4-(2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (235 mg, 32%) as an off-white solid. TLC system: EtOAc / petroleum ether (70:30), R f Value: ~0.40; LC / MS retention time = 3.0 minutes, 409.2 [M+H] + .

[0302] 1H NMR (400MHz, CDCl3): δ8.39(s,1H),8.31(d,J=5.2Hz,1H),8.01(s,1H),7.95(d,J=8.0Hz,1H),7.75(d,J=9.2Hz,1H),7.01(d,J=5.6Hz,1H),5.21(d,J =6.8Hz,1H),4.59(brs,1H),4.53–4.48(m,1H),4.25(s,3H),4.18–4.11(m, 1H),2.34–2.20(m,2H),2.04–1.98(m,2H),1.77–1.48(m,2H),1.45(s,9H).

[0303] Synthesis of (1S,3S)-N1-(4-(2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt

[0304]

[0305] To a stirred solution of tert-butyl ((1S,3S)-3-((4-(2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (230 mg, 0.56 mmol, 1 eq) in 1,4-dioxane (2 mL) was added 4M HCl in 1,4-dioxane (0.4 mL) at 0°C and stirred at room temperature for 2 hours. After completion of the starting material, the reaction mixture was evaporated and triturated with diethyl ether (2×3 mL) and then lyophilized to afford (1S,3S)-N1-(4-(2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt (120 mg, 56%) as a yellow solid. TLC system: MeOH / DCM (20:80), R f Value: ~0.1; LC / MS retention time = 1.88 minutes, 309.3 [M+H] + .

[0306] 1 H NMR (400MHz, CD3OD) δ8.90(brs,1H),8.62(s,1H),8.31–8.28(m,2H),7.77(d,J=9.2Hz,1H),7.67(d,J=6.8Hz,1 H),4.93–4.89(m,1H),4.31(s,3H),3.92–3.87(m,1H),2.47–2.36(m,2H),2.32–2.28(m,2H),1.94–1.86(m,2H).

[0307] Example 7

[0308] Synthesis of Compound I-7

[0309]

[0310] Overall reaction scheme:

[0311]

[0312] Synthesis of 5-bromo-3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole

[0313]

[0314] To a stirred solution of 5-bromo-3-isopropyl-2H-indazole (200 mg, 0.84 mmol, 1.0 equiv) in DCM (4 mL) was added dihydropyran (0.2 mL, 1.67 mmol, 2.0 equiv), p-TSA (8 mg, 0.04 mmol, 0.05 equiv) at room temperature and stirred for 16 hours. After completion of the reaction by TLC, it was diluted with cold water and DCM (2 × 30 mL) (30 mL). The organic layer was dried over sodium sulfate, filtered, concentrated and purified by silica gel column (60-120 mesh, eluent: 15% EtOAc / petroleum ether) to obtain 5-bromo-3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (260 mg, yield: 96%) as a yellow liquid. TLC system: EtOAc / petroleum ether (20: 80), R f Value: ~0.8. LC / MS retention time = 4.48 minutes, 323.0 [M+H] + .

[0315] 1 H NMR(400MHz, CDCl3)δ7.86(t,J=1.2Hz,1H),7.42–7.41(m,2H),5.61(dd,J=9.6&2.4Hz,1H),4.06–4.03(m,1H),3.75–3.69(m,1H),3.3 7–3.30(m,1H),2.53–2.49(m,1H),2.14–2.12(m,1H),2.04–2.00(m,1H),1.77–1.71(m,2H),1.65–1.62(m,1H),1.44(d,J=7.6Hz,6H).

[0316] Synthesis of 3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0317]

[0318] A stirred solution of 5-bromo-3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (260 mg, 0.87 mmol, 1.0 eq.), bis(pinacolato)diborane (224 mg, 1.04 mmol, 1.2 eq.) and potassium acetate (213 mg, 2.17 mmol, 2.5 eq.) in 1,4-dioxane (6 mL) was degassed for 5 minutes, and Pd(dppf)Cl2.DCM (36 mg, 0.043 mmol, 0.05 eq.) was added. The reaction mixture was stirred at 90 °C for 16 hours. After completion of the reaction by TLC, it was diluted with water (50 mL) and extracted with ethyl acetate (2×50 mL). The organic layer was dried over sodium sulfate and concentrated to give 3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (325 mg, crude product) as a black semi-solid. TLC system: EtOAc / petroleum ether (30:70), R f Value: ~0.5. LC / MS retention time = 4.68 minutes, 371.2 [M+H] + Cr: LC / MS: 62.1% boronate ester m / z and 10% boronic acid m / z observed, the material was used in the next step without purification.

[0319] Synthesis of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole

[0320]

[0321] To a degassed stirred solution of 3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (320 mg, 0.86 mmol, 1.0 eq) in 1,4-dioxane and water (3:1) (6 mL) was added 2,4-dichloropyrimidine (154 mg, 1.04 mmol, 1.2 eq), K2CO3 (298 mg, 2.16 mmol, 2.5 eq) followed by Pd(dppf)Cl2DCM (35 mg, 0.043 mmol, 0.05 eq) and stirred at 90° C. for 16 h. After completion of the reaction by TLC, it was diluted with water and extracted with ethyl acetate (2×50 mL). The combined organic layers were dried over sodium sulfate, concentrated and purified by silica gel column (60-120 mesh, eluent: 25% EtOAc / petroleum ether) to give 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (280 mg, 70% relative purity) as a white solid. TLC system: EtOAc / petroleum ether (30:70), R f Value: ~0.2; LC / MS retention time = 4.77 minutes, 357.2 [M+H] + ; 76% purity

[0322] Synthesis of tert-butyl ((1S,3S)-3-((4-(3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate

[0323]

[0324] To a stirred solution of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (280 mg, 0.79 mmol, 1 eq) in IPA (4.5 mL) was added tert-butyl ((1S, 3S)-3-aminocyclopentyl)carbamate (190 mg, 0.94 mmol, 1.2 eq) and stirred at 110 °C for 36 hours. TLC showed polar spots and the reaction mixture was allowed to reach room temperature. The reaction mixture was evaporated and diluted with water (30 mL) and extracted with EtOAc (3×20 ml). The organic layer was dried over Na2SO4, filtered, evaporated and purified by silica gel column (60-120 mesh, eluent: 15% to 70% EtOAc / petroleum ether) to give tert-butyl ((1S,3S)-3-((4-(3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (150 mg, yield: 37%) as a yellow viscous liquid. TLC system: EtOAc / petroleum ether (70:30), R f Value: ~0.20; LC / MS retention time = 4.67 minutes, 521.3 [M+H] + .

[0325] Synthesis of (1S,3S)-N1-(4-(3-isopropyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt

[0326]

[0327] To a stirred solution of ((1S,3S)-3-((4-(3-isopropyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (150 mg, 0.29 mmol, 1 eq) in MeOH (0.7 mL) at 0°C was added concentrated HCl (0.7 mL) and stirred at room temperature for 18 hours. After completion of the starting material, the volatiles were removed, triturated with diethyl ether (2×5 mL) and purified by reverse phase column (eluent: 8% ACN and 0.01% FA in water) to afford (1S,3S)-N1-(4-(3-isopropyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt (28 mg, yield: 29%) as a yellow sticky solid. TLC system: MeOH / DCM (30:70), R f Value: ~0.1; LC / MS retention time = 2.32 minutes, 337.4 [M+H] + .

[0328] 1H NMR (400MHz, CD3OD) δ8.84(s,1H),8.38(brs,1H),8.29(d,J=6.8Hz,1H),7.73(d,J=6.4Hz,1H),7.65(d,J=9.2Hz,1H),3.91–3.8 7(m,1H),3.59–3.53(m,1H),3.35–3.33(m,1H),2.48–2.36(m,2H),2.32–2.27(m,2H),1.93–1.80(m,2H),1.49(d,J=6.8Hz,6H).

[0329] Example 8

[0330] Synthesis of Compound I-3

[0331]

[0332] Overall reaction scheme:

[0333]

[0334] Synthesis of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[4,3-b]pyridine

[0335]

[0336] To a stirred solution of 5-bromo-2methyl-3-propylpyrazolo[4,3-b]pyridine (45 mg, 0.18 mmol, 1.0 equiv) and bis(pinacolato)diborane (56 mg, 0.22 mmol, 1.2 equiv) in 1,4-dioxane (8 mL) in a microwave vial were added potassium acetate (44 mg, 0.45 mmol, 2.5 equiv), Pd(dppf)Cl 2. DCM (8 mg, 0.01 mmol, 0.05 equiv). Then, the vial was sealed with a cap and degassed with argon for 5 minutes. The reaction mixture was stirred at 90 ° C for 16 hours. After the reaction was completed by TLC, the reaction mixture was diluted with water and ethyl acetate, and the resulting slurry was filtered through The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated to provide the crude product 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[4,3-b]pyridine (54 mg, yield: 100%) as a brown oil.

[0337] Synthesis of 3-isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]pyrazolo[4,3-b]pyridine

[0338]

[0339] To a stirred solution of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[4,3-b]pyridine (54 mg, 0.18 mmol, 1 eq) in 1,4-dioxane and water (3:1) (8 mL) was added 4-chloro-2-(methylthio)pyrimidine (43 mg, 0.27 mmol, 1.5 eq), KCO (62 mg, 0.45 mmol, 2.5 eq), Pd(dppf)ClDCM (8 mg, 0.01 mmol, 0.05 eq) in a microwave vial and degassed with argon for 5 minutes. The reaction mixture was then stirred at 100 °C for 16 hours. The reaction mixture was diluted with water and ethyl acetate and the resulting slurry was filtered through The organic layer was separated, washed with brine solution, dried over sodium sulfate and concentrated to provide a crude product, which was purified by Combiflash chromatography (4 g column) to give 3-isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]pyrazolo[4,3-b]pyridine (10 mg, yield: 18%) as a yellow solid. TLC system: Hexane:EtOAc (2:1), R f Value: ~0.3;

[0340] Synthesis of a mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine

[0341]

[0342] 3-Isopropyl-2-methyl-5-[2-(methylthio)pyrimidin-4-yl]pyrazolo[4,3-b]pyridine (10 mg, 0.033 mmol, 1.0 eq) was added to a stirred solution of DCM (4 ml) cooled to 0°C with 3-chloroperbenzoic acid (mCPBA) (purity, 77%) (10 mg, 0.043 mmol, 1.3 eq). The reaction mixture was stirred at 0°C for 4 hours. After the reaction was completed by TLC, the reaction mixture was quenched with saturated NaHCO3 aqueous solution (10 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine solution, dried over sodium sulfate and concentrated to give a crude mixture of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine as a yellow solid (10 mg, yield: 100%). TLC system: EtOAc (100%) R f Values: ~0.01 and 0.2.

[0343] Synthesis of tert-butyl (1S,3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate

[0344]

[0345] To a stirred solution of 3-isopropyl-2-methyl-5-(2-methylsulfinylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine and 3-isopropyl-2-methyl-5-(2-methylsulfonylpyrimidin-4-yl)pyrazolo[4,3-b]pyridine (10 mg, 0.033 mmol, 1 eq) in DMSO (5 mL) at room temperature, (1S, 3S)-3-amino-1-(BOC-amino)cyclopentane (10 mg, 0.05 mmol, 1.5 eq) and cesium fluoride (8 mg, 0.05 mmol, 1.5 eq) were added. The reaction mixture was then stirred at 95 ° C for 16 hours. After the reaction was completed by TLC, the reaction mixture was cooled to room temperature and extracted with ethyl acetate. The organic layer was washed with brine solution, dried over anhydrous sodium sulfate, and then concentrated to provide the product, which was purified by Combiflash chromatography (4 g column) to give tert-butyl (1S, 3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentan-1-yl]carbamate (10 mg, 67%) as a yellow solid. TLC system: Hexane:EtOAc (1:1), Rf value: ~0.2.

[0346] Synthesis of (1S,3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentan-1-yl]-1,3-trifluoroacetic acid diamine

[0347]

[0348] To a stirred solution of (1S, 3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentane-1-yl]t-butyl carbamate (10mg, 0.022mmol, 1 equivalent) in DCM (4mL) at room temperature, trifluoroacetic acid (TFA) (2ml) was added, and the reaction mixture was stirred at room temperature for 24 hours. After the reaction was completed by TLC, the solvent was removed in vacuo. Diethyl ether was then added to the reaction solution, and a large amount of yellow solids will appear in the reaction solution. The mixture was centrifuged, washed with diethyl ether and dried at room temperature to provide a product (1S, 3S)-[3-[4-[3-isopropyl-2-methylpyrazolo[4,3-b]pyridin-5-yl]pyrimidin-2-yl]aminocyclopentane-1-yl]-1,3-trifluoroacetic acid diamine (5mg, 38%) as a yellow solid. 1 HNMR (400 MHz, MeOD-d4) δ8.53 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 5.6 Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 7.86 (J = 5.6 Hz, 1H), 4.68 (s, wide, 1H), 4.07 (s, 3H), 3.87-3.81 (m, 1H), 3.64-3.57 (m, 1H), 2.41-2.33 (m, 2H), 2.23-2.19 (m, 2H), 1.85-1.72 (m, 2H), 1.54 (d, J = 7.0 Hz, 6H); HRMS data: C 19 H 25 HRMS (ESI) m / z [M+H]+ calculated for N7, 352.2244, found: 352.2217

[0349] Example 9

[0350] Synthesis of Compound I-9

[0351]

[0352] Overall reaction plan

[0353]

[0354] Synthesis of 5-bromo-3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole

[0355]

[0356] To a stirred solution of 5-bromo-3-methyl-2H-indazole (500mg, 2.36mmol, 1.0 equivalent) in DCM (5mL) was added dihydropyran (0.43mL, 4.73mmol, 2.0 equivalent), p-TSA (22mg, 0.04mmol, 0.05 equivalent) at room temperature and stirred for 16 hours. After completion of the reaction by TLC, it was diluted with cold water and DCM (2×30mL) (30mL). The organic layer was dried over sodium sulfate, filtered, concentrated and purified by silica gel column (60-120 mesh, eluent: 10% EtOAc / petroleum ether) to obtain 5-bromo-3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (550mg, yield: 79%) as a yellow liquid. TLC system: EtOAc / petroleum ether (40:60), R f Value: ~0.8.

[0357] 1 H NMR(400MHz, CDCl3) δ7.78(dd,J=0.8&2Hz,1H),7.46–7.39(m,2H),5.59(dd,J=10&2.8Hz,1H),4.07–4.04(m,1H), 3.75–3.69(m,1H),2.56–2.50(m,4H),2.15–2.12(m,1H),2.05–2.00(m,1H),1.77–1.71(m,2H),1.65–1.62(m,1H).

[0358] Synthesis of 3-methyl-2-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0359]

[0360] A stirred solution of 5-bromo-3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (550 mg, 1.86 mmol, 1.0 eq.), bis(pinacolato)diborane (570 mg, 2.23 mmol, 1.2 eq.) and potassium acetate (460 mg, 4.66 mmol, 2.5 eq.) in 1,4-dioxane (11 mL) was degassed for 5 minutes, and Pd(dppf)Cl2.DCM (76 mg, 0.093 mmol, 0.05 eq.) was added. The reaction mixture was stirred at 90 °C for 16 hours. After completion of the reaction by TLC, it was diluted with water (50 mL) and extracted with ethyl acetate (2×50 mL). The organic layer was dried over sodium sulfate and concentrated to give 3-methyl-2-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (630 mg, crude product) as a black semi-solid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.3. LC / MS retention time = 4.19 minutes, 343.2 [M+H] + The material was used in the next step.

[0361] Synthesis of 5-(2-chloropyrimidin-4-yl)-3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole

[0362]

[0363] 2,4-Dichloropyrimidine (329 mg, 2.21 mmol, 1.2 eq) and K2CO3 (635 mg, 4.60 mmol, 2.5 eq) were added to a degassed, stirred solution of 3-methyl-2-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (630 mg, 1.84 mmol, 1.0 eq) in 1,4-dioxane and water (3:1) (12 mL). The reaction mixture was degassed for 5 minutes and Pd(dppf)Cl2DCM (75 mg, 0.092 mmol, 0.05 eq) was added. The reaction mixture was stirred at 90 °C for 16 hours. After completion of the reaction by TLC, it was diluted with water and extracted with ethyl acetate (2×50 mL). The combined organic layers were dried over sodium sulfate, concentrated and purified by silica gel column (60-120 mesh, eluent: 50% EtOAc / petroleum ether) to give 5-(2-chloropyrimidin-4-yl)-3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (550 mg, semi-pure) as an off-white solid. TLC system: EtOAc / petroleum ether (30:70), R fValue: ~0.3; LC / MS retention time = 3.76 minutes, 329.1 [M+H] + ; 69% purity. The material was used in the next step.

[0364] Synthesis of tert-butyl ((1S,3S)-3-((4-(3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate

[0365]

[0366] To a stirred solution of 5-(2-chloropyrimidin-4-yl)-3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazole (550 mg, 1.67 mmol, 1 eq) in IPA (8 mL) was added tert-butyl ((1S, 3S)-3-aminocyclopentyl)carbamate (335 mg, 2.01 mmol, 1.2 eq) and stirred at 110 °C for 24 h. TLC showed polar spots and the RM was allowed to reach room temperature. The reaction mixture was evaporated and diluted with water (30 mL) and extracted with EtOAc (3×20 ml). The organic layer was dried over Na2SO4, filtered, evaporated and purified by silica gel column (60-120 mesh, eluent: 15% to 60% EtOAc / petroleum ether) to give tert-butyl ((1S,3S)-3-((4-(3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (240 mg, semi-pure) as an off-white solid. TLC system: EtOAc / petroleum ether (70:30), R f Value: ~0.20; LC / MS retention time = 4.06 minutes, 493.3 [M+H] + ; 58% purity. The material was used in the next step.

[0367] Synthesis of (1S,3S)-N1-(4-(3-methyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt

[0368]

[0369] To a stirred solution of tert-butyl ((1S,3S)-3-((4-(3-methyl-2-(tetrahydro-2H-pyran-2-yl)-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (170 mg, 0.29 mmol, 1 eq) in MeOH (1 mL) at 0 °C was added concentrated HCl (1 mL) and stirred at room temperature for 12 hours. After completion of the starting material, the volatiles were removed, triturated with diethyl ether (2×5 mL) and purified by preparative HPLC (ABC method). Ammonium salts were avoided by treatment and lyophilized by addition of aqueous HCl to give (1S,3S)-N1-(4-(3-methyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt (32 mg, yield: 5% in 4 steps) as a yellow solid. TLC system: MeOH / DCM (30:70), R f Value: ~0.1; LC / MS retention time = 1.78 minutes, 309.2 [M+H] + .

[0370] 1 H NMR (400MHz, CD3OD) δ8.83(s,1H),8.42(d,J=6.4Hz,1H),8.31(d,J=6.8Hz,1H),7.73(d,J=6.8Hz,1H),7.67(d,J=8.8H z,1H),3.91–3.88(m,1H),3.29–3.28(m,1H),2.69(s,3H),2.48–2.38(m,2H),2.30(t,J=6.8Hz,2H),1.93–1.82(m,2H).

[0371] Example 10

[0372] Synthesis of Compound I-4

[0373]

[0374] Overall reaction plan

[0375]

[0376] Synthesis of 5-bromo-2,3-difluorobenzaldehyde

[0377]

[0378] To a stirred solution of 2,3-difluorobenzaldehyde (5g, 35.2mmol, 1.0 equivalent) in sulfuric acid (38mL) was added NBS (31.3mL, 52.78mmol, 1.5 equivalents) at room temperature and stirred at 60°C for 16 hours. After completion of the reaction by TLC, it was diluted with cold water (50mL) and extracted with EtOAc (2×100mL). The organic layer was washed with NaHCO3, dried over sodium sulfate and concentrated to provide 7g of material. The crude compound was purified by silica gel column (100-200 mesh, eluent: 2% EtOAc / hexane) to obtain 5-bromo-2,3-difluorobenzaldehyde (3.5g, 45%) as a yellow liquid. TLC system: EtOAc / petroleum ether (05:95), R f Value: ~0.8.

[0379] 1 H NMR (400MHz, CDCl3) δ10.30(s,1H),7.79–7.77(m,1H),7.63–7.58(m,1H).

[0380] Synthesis of 5-bromo-7-fluoro-2H-indazole

[0381]

[0382] To a stirred solution of 5-bromo-2,3-difluorobenzaldehyde (2) (3.4 g, 15.5 mmol, 1.0 equiv) in EtOH (34 mL, 10 volumes) was added 85% hydrazine hydrate (17 mL, 5 volumes) at room temperature and stirred at 100 ° C for 16 hours. After completion of the reaction by TLC, the RM was evaporated, diluted with water (100 mL), and extracted with ethyl acetate (3×50 mL). The organic layer was separated, dried over sodium sulfate, and concentrated to provide 4.1 g of material. The crude compound was purified by silica gel column (60-120 mesh, eluent: 25% to 35% EtOAc / hexane) to obtain 5-bromo-7-fluoro-2H-indazole (1.7 g, 51%) as an off-white solid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.2. LC / MS retention time = 3.10 min, 214.9 [M+H] + .

[0383] Synthesis of 5-bromo-7-fluoro-2-methyl-2H-indazole

[0384]

[0385] To a stirred solution of 5-bromo-7-fluoro-2H-indazole (1.7 g, 7.94 mmol, 1.0 equiv) in EtOAc (54 mL, 30 volumes) at room temperature, trimethyloxonium tetrafluoroborate (7.05 g, 47.7 mmol, 6.0 equiv) was added, and stirring was continued for 16 hours. After completion of the reaction by TLC, the reaction mixture was evaporated and purified by a silica gel column (60-120 mesh, eluent: 15% to 18% EtOAc / hexane) to obtain 5-bromo-7-fluoro-2-methyl-2H-indazole (750 mg, 42%) as an off-white solid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.5.

[0386] 1 H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 2.8 Hz, 1H), 7.61 (d, J = 1.6 Hz, 1H), 7.08 (dd, J = 10.4 & 1.6 Hz, 1H), 4.26 (s, 3H). Confirmed by nOe.

[0387] Synthesis of 5-bromo-7-fluoro-3-iodo-2-methyl-2H-indazole

[0388]

[0389] Pyridine (1.7 g, 4.93 mmol, 1.5 eq) and PIFA (1.7 g, 3.95 mmol, 1.2 eq) were added to a stirred solution of 5-bromo-7-fluoro-2-methyl-2H-indazole (0.75 g, 3.28 mmol, 1.0 eq) in DCM (15 mL, 20 volumes) at 0 ° C. The reaction mixture was stirred at 0 ° C for 30 minutes, then iodine (0.5 g, 3.95 mmol, 1.2 eq) was added and allowed to stir at room temperature for 18 hours. After completion of the reaction by TLC, the reaction mixture was diluted with saturated NaHCO (50 mL) and extracted with DCM (2 × 20 mL). The organic layer was separated, dried over sodium sulfate, and concentrated to provide a crude product. Purification by silica gel column (60-120 mesh, eluent: 5% EtOAc / hexane) gave 5-bromo-7-fluoro-3-iodo-2-methyl-2H-indazole (700 mg, 60%) as a yellow liquid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.8.

[0390] 1 H NMR (400MHz, CDCl3) δ7.38 (d, J=1.6Hz, 1H), 7.10 (dd, J=10.4&1.6Hz, 1H), 4.27 (s, 3H).

[0391] Synthesis of 5-bromo-7-fluoro-2-methyl-3-(prop-1-en-2-yl)-2H-indazole

[0392]

[0393] To a stirred solution of 5-bromo-7-fluoro-3-iodo-2-methyl-2H-indazole (700 mg, 1.98 mmol, 1.0 equiv) in 1,4-dioxane (14 mL) was added 4,4,5,5-tetramethyl-2-(prop-1-ene-2-yl)-1,3,2-dioxaborolane (398 mg, 2.37 mmol, 1.2 equiv) and potassium carbonate (682 mg, 4.94 mmol, 2.5 equiv) at room temperature and degassed for 5 minutes. Pd(dppf)Cl2.DCM (80 mg, 0.098 mmol, 0.05 equiv) was then added. The reaction mixture was stirred at 90 ° C for 16 hours. After the reaction was completed by TLC. The volatiles were evaporated, the residue was diluted with water (80 mL), and extracted with ethyl acetate (2×50 mL). The organic layer was separated, washed with brine solution (30 mL), dried over sodium sulfate and concentrated. The crude compound was purified by silica gel column (60-120 mesh, eluent: 10% to 12% EtOAc / hexane) to afford 5-bromo-7-fluoro-2-methyl-3-(prop-1-en-2-yl)-2H-indazole (450 mg, 85%) as a yellow liquid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.50. LC / MS retention time = 3.99 minutes, 269.0 [M+H] + .

[0394] 1 H NMR (400MHz, CDCl3) δ7.54 (s, 1H), 7.04 (d, J = 10Hz, 1H), 5.63 (s, 1H), 5.28 (s, 1H), 4.16 (s, 3H), 2.22 (s, 3H).

[0395] Synthesis of 5-bromo-7-fluoro-3-isopropyl-2-methyl-2H-indazole

[0396]

[0397] To a stirred solution of 5-bromo-7-fluoro-2-methyl-3-(prop-1-en-2-yl)-2H-indazole (400 mg, 1.49 mmol, 1.0 eq) in EtOAc (8 mL, 20 vols) was added PtO (11.3 mg, 0.036 mmol, 0.05 eq) and stirred at room temperature under H atmosphere for 7 h. After completion of the reaction by TLC, the reaction mixture was purified by The organic layer was evaporated and purified by silica gel column (100-200 mesh, eluent: 50% EtOAc / hexane) to give 5-bromo-7-fluoro-3-isopropyl-2-methyl-2H-indazole (360 mg, 70%) as a brown solid. TLC system: EtOAc / petroleum ether (50:50), R f Value: ~0.25; LC / MS retention time = 3.87 minutes, 271.0 [M+H] + .

[0398] 1 H NMR (400MHz, CDCl3) δ7.70(d,J=1.6Hz,1H),7.02(dd,J=10.4&1.6Hz,1H),4.16(s,3H),3.45–3.38(m,1H),1.51(d,J=6.8Hz,6H).

[0399] Synthesis of 7-fluoro-3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0400]

[0401] To a stirred solution of 5-bromo-7-fluoro-3-isopropyl-2-methyl-2H-indazole (300 mg, 1.11 mmol, 1.0 equiv) in 1,4-dioxane (6 mL) was added bis(pinacol)diborane (335 mg, 1.33 mmol, 1.2 equiv) and potassium acetate (272 mg, 2.77 mmol, 2.5 equiv) at room temperature, degassed for 5 minutes, followed by the addition of Pd(dppf)Cl2.DCM (45 mg, 0.055 mmol, 0.05 equiv). The reaction mixture was stirred at 100 ° C for 16 hours. After completion of the reaction by TLC. The volatiles were removed under vacuum, diluted with water (50 mL), and extracted with ethyl acetate (3×30 mL). The organic layer was separated, washed with brine solution (30 mL), dried over sodium sulfate and concentrated to give 7-fluoro-3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (350 mg, crude product) as a black semi-solid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.45. Cr: LC / MS showed 44% boronic ester and 16.3% boronic acid m / z. LC / MS retention time = 5.39 minutes, 319.2 [M+H] + The crude material was used as such in the next step.

[0402] Synthesis of 5-(2-chloropyrimidin-4-yl)-7-fluoro-3-isopropyl-2-methyl-2H-indazole

[0403]

[0404] To a stirred solution of 7-fluoro-3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (350 mg, 1.10 mmol, 1 eq) in 1,4-dioxane and water (3:1) (6 mL) was added 4-chloro-2-(methylthio)pyrimidine (208 mg, 1.43 mmol, 1.3 eq) and K2CO3 (380 mg, 2.75 mmol, 2.5 eq). The reaction mixture was degassed for 5 minutes and Pd(dppf)Cl2DCM (45 mg, 0.055 mmol, 0.05 eq) was added. The reaction mixture was stirred at 100 °C for 16 hours. After completion of the reaction by TLC, it was diluted with water and extracted with ethyl acetate (3×20 mL). The organic layer was dried over Na2SO4, evaporated and purified by silica gel column (100-200 mesh, eluent: 25% to 30% EtOAc / hexanes) to give 5-(2-chloropyrimidin-4-yl)-7-fluoro-3-isopropyl-2-methyl-2H-indazole (180 mg, 54%) as a yellow solid. TLC system: EtOAc / hexanes (50:50), R f Value: ~0.25. LC / MS retention time = 3.53 minutes, 305.0 [M+H] + .

[0405] 1 H NMR (400MHz, CDCl3) δ8.61(d,J=5.6Hz,1H),8.41(s,1H),7.61–7.57(m,2H),4.20(s,3H),3.56–3.48(m,1H),1.58(d,J=7.2Hz,6H).

[0406] Synthesis of tert-butyl ((1S,3S)-3-((4-(7-fluoro-3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate

[0407]

[0408] To a stirred solution of 5-(2-chloropyrimidin-4-yl)-7-fluoro-3-isopropyl-2-methyl-2H-indazole (150 mg, 0.493 mmol, 1 eq) in isopropanol (2.5 mL) was added tert-butyl ((1S, 3S)-3-aminocyclopentyl)carbamate (120 mg, 0.64 mmol, 1.3 eq) and heated at 120 °C for 16 hours. TLC showed polar spots and the reaction mixture was allowed to reach room temperature. The reaction mixture was evaporated, diluted with water (20 mL) and extracted with EtOAc (2×20 mL). The organic layer was dried over Na2SO4, filtered, evaporated and purified by silica gel column (40% EtOAc / hexanes eluent) to give tert-butyl((1S,3S)-3-((4-(7-fluoro-3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (130 mg, 56%) as a yellow solid. TLC system: MeOH / DCM (10:90), R f Value: ~0.30; LC / MS retention time = 3.50 minutes, 469.2 [M+H] + .

[0409] Synthesis of (1S,3S)-N1-(4-(7-fluoro-3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine TFA salt

[0410]

[0411] To a solution of tert-butyl ((1S,3S)-3-((4-(3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (130 mg, 0.277 mmol, 1 eq) in 1,4-dioxane (1.3 mL) at 0°C was added 4M HCl in 1,4-dioxane (0.3 mL) and stirred at room temperature for 16 hours. After completion of the starting material, the reaction mixture was concentrated and purified by preparative HPLC (32% acetonitrile and 0.1% TFA in water eluent) to give (1S,3S)-N1-(4-(7-fluoro-3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine TFA salt (40 mg, 39%) as a yellow solid. TLC system: MeOH / DCM (10:90), R f Value: ~0.1; LC / MS retention time = 2.67 minutes, 369.3 [M+H] + .

[0412] 1H NMR (400MHz, CD3OD) δ8.55(s,1H),8.29(d,J=6.4Hz,1H),7.85(d,J=13.2Hz,1H),7.47(d,J=5.6Hz,1H),4.71–4.68(m,1H),4.20(s,3H), 3.86–3.83(m,1H),3.69–3.61(m,1H),2.41–2.35(m,2H),2.25–2.19(m,2H),1.88–1.82(m,1H),1.80–1.75(m,1H),1.59(d,J=7.2Hz,6H).

[0413] Embodiment 11

[0414] Synthesis of Compound I-10

[0415]

[0416] Overall reaction scheme:

[0417]

[0418] Synthesis of 5-bromo-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine

[0419]

[0420] To a stirred solution of 5-bromo-3-isopropyl-2H-pyrazolo[3,4-b]pyridine (0.8 g, 3.33 mmol, 1.0 equiv) in THF (24 mL, 30 volumes) at 0 ° C., 2M NaHMDS in THF (2.4 mL, 4.99 mmol, 1.5 equiv) was added, followed by MeI (0.8 mL, 9.99 mmol, 3 equiv) and stirred at room temperature for 4 hours. After completion of the reaction by TLC, it was diluted with cold water (100 mL) and extracted with EtOAc (2 × 80 mL). The organic layer was separated, dried over sodium sulfate and concentrated to give 5-bromo-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine (600 mg, yield: 71%) as a yellow solid. TLC system: EtOAc / petroleum ether (1: 1), R f Value: ~0.4;

[0421] 1 H NMR (400MHz, CDCl3) δ8.61 (d, J = 2.4Hz, 1H), 8.25 (d, J = 2.0Hz, 1H), 4.13 (s, 3H), 3.43–3.39 (m, 1H), 1.49 (d, J = 7.2Hz, 6H).

[0422] Synthesis of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-pyrazolo[3,4-b]pyridine

[0423]

[0424] To a stirred solution of 5-bromo-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine (600 mg, 2.36 mmol, 1.0 equiv) in 1,4-dioxane (12 mL) was added bis(pinacol)diborane (722 mg, 2.83 mmol, 1.2 equiv), potassium acetate (581 mg, 5.90 mmol, 2.5 equiv) and degassed for 5 minutes. Pd(dppf)Cl2.DCM (96 mg, 0.12 mmol, 0.05 equiv) was then added and stirred at 90 °C for 16 hours. After the reaction was completed by TLC. The volatiles were removed under vacuum, diluted with water (70 mL), and extracted with ethyl acetate (3×30 mL). The organic layer was separated, washed with brine solution (30 mL), dried over sodium sulfate and concentrated to provide 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-pyrazolo[3,4-b]pyridine (700 mg, crude product) as a sticky material. TLC system: EtOAc (100%), R f Value: ~0.1; Cr: LC / MS: 55% boric acid m / z, 220.1 [M+H] + .

[0425] Synthesis of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine

[0426]

[0427] To a stirred solution of 3-isopropyl-2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-pyrazolo[3,4-b]pyridine (700 mg, 2.32 mmol, 1 eq) in 1,4-dioxane and water (3:1) (14 mL) was added 4-chloro-2-(methylthio)pyrimidine (572 mg, 2.78 mmol, 1.2 eq), K2CO3 (1.1 g, 5.80 mmol, 2.5 eq) and degassed for 5 minutes. Then Pd(dppf)Cl2DCM (131 mg, 0.12 mmol, 0.05 eq) was added and stirred at 80°C for 16 hours. After completion of the reaction by TLC, the reaction mixture was evaporated, diluted with water (80 mL) and extracted with ethyl acetate (2×70 mL). The organic layer was separated, dried over sodium sulfate and concentrated to provide 2.6 g of crude product, which was purified by silica gel column (100-200 mesh, eluent: 80% EtOAc / petroleum ether) to give 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine (180 mg, yield: 26%) as a viscous liquid. TLC system: EtOAc (100%), R f Value: ~0.2;

[0428] 1 H NMR (400MHz, CDCl3) δ9.28(d,J=2.4Hz,1H),9.04(d,J=2.4Hz,1H),8.69(d,J=5.2Hz ,1H),7.75(d,J=5.2Hz,1H),4.26(s,3H),3.58–3.53(m,1H),1.63(d,J=6.8Hz,6H).

[0429] Synthesis of tert-butyl ((1S,3S)-3-((4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate

[0430]

[0431] To a stirred solution of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine (180 mg, 0.63 mmol, 1 eq) in IPA (1.8 mL) was added tert-butyl ((1S, 3S)-3-aminocyclopentyl)carbamate (163 mg, 0.82 mmol, 1.3 eq) and stirred at 110 °C for 48 hours. TLC showed a new polar spot and traces of starting material. The volatiles were removed under vacuum, diluted with water (20 mL), and extracted with EtOAc (2×20 ml). The organic layer was dried over Na2SO4, filtered, concentrated and purified by silica gel column (100-200 mesh, eluent: 70-80% EtOAc / petroleum ether) to give tert-butyl ((1S,3S)-3-((4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (200 mg, 70%) as a yellow sticky solid. TLC system: MeOH / DCM (5:95), R f Value: ~0.4; LC / MS: retention time = 3.49 minutes, 452.2 [M+H] + .

[0432] 1 H NMR (400MHz, CDCl3): δ9.31(brs,1H),8.83(s,1H),8.38(d,J=5.2Hz,1H),7.06(d,J=5.2Hz,1H),5.26(d,J=6.8Hz,1H),4.62(brs,1H),4.55–4 .50(m,1H),4.24–4.18(m,4H),3.57–3.50(m,1H),2.38–2.25(m,2H),2. 08–1.99(m,2H),1.61(d,J=7.2Hz,6H),1.58–1.52(m,2H),1.49(s,9H).

[0433] Synthesis of (1S,3S)-N1-(4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt

[0434]

[0435] To a stirred solution of tert-butyl ((1S,3S)-3-((4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (6) (200 mg, 0.44 mmol, 1 eq) in DCM (2 mL) at 0°C was added 2M HCl in EtO (1 mL) and stirred at room temperature for 16 h. After completion of the starting material, the volatiles were removed under vacuum, triturated with diethyl ether (2×3 mL) and lyophilized to afford ((1S,3S)-N1-(4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt (94 mg, 50%) as a brown solid. TLC system: MeOH / DCM (10:90), R f Value: ~0.1; LC / MS retention time = 2.29 minutes, 352.2 [M+H] + .

[0436] 1 H NMR (400MHz, CD3OD) δ9.79–9.74(m,2H),8.48(d,J=6.8Hz,1H),7.77(d,J=6.4Hz,1H),5.11–5.05(br,1H),4.34(s,3H) ,3.94–3.87(m,1H),3.84–3.77(m,1H),2.49–2.36(m,2H),2.33–2.29(m,2H),1.94–1.81(m,2H),1.66(d,J=6.8Hz,6H).

[0437] Example 12

[0438] Synthesis of Compound I-11

[0439]

[0440] Overall reaction scheme:

[0441]

[0442] Synthesis of 1-(5-bromo-2-fluoropyridin-3-yl)-2-methylpropan-1-ol

[0443]

[0444] To a stirred solution of 5-bromo-2-fluoronicotinaldehyde (8 g, 39.2 mmol, 1.0 eq.) and dry THF (80 mL) was added 1.5 M isopropylmagnesium bromide in THF (31.3 mL, 47.1 mmol, 1.2 eq.) at 0 ° C and stirred at room temperature for 3 hours. After completion of the reaction by TLC, the reaction mixture was diluted with saturated NH4Cl (50 mL) and extracted with EtOAc (2×100 mL). The organic layer was dried over sodium sulfate, concentrated and purified by silica gel column (60-120 mesh, eluent: 12% EtOAc / petroleum ether) to give 1-(5-bromo-2-fluoropyridin-3-yl)-2-methylpropan-1-ol (2.71 g, yield: 28%) as a yellow liquid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.4. 1 H NMR(400MHz, CDCl3)δ8.16(dd,J=2.4&1.2Hz,1H),8.04–8.01(m,1H),4.72(t, J=4.8Hz,1H),1.99–1.97(m,2H),0.93(d,J=6.8Hz,3H),0.87(d,J=6.8Hz,3H).

[0445] Synthesis of 1-(5-bromo-2-fluoropyridin-3-yl)-2-methylpropan-1-one

[0446]

[0447] To a stirred solution of 1-(5-bromo-2-fluoropyridin-3-yl)-2-methylpropan-1-ol (3) (2.7 g, 10.9 mmol, 1.0 eq.) in DCM (54 mL) was added DMP (6.95 g, 16.4 mmol, 1.5 eq.) at 0 °C and stirred at room temperature for 2 hours. After completion of the reaction by TLC, it was diluted with saturated NaHCO3 solution (30 mL) and extracted with DCM (2×50 mL). The organic layer was separated, dried over sodium sulfate and concentrated to provide 3 g of crude product, which was purified by silica gel column (60-120 mesh, eluent: 4% EtOAc / petroleum ether) to give 1-(5-bromo-2-fluoropyridin-3-yl)-2-methylpropan-1-one (2.3 g, yield: 86%) as a yellow liquid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.85.

[0448] 1H NMR (400MHz, CDCl3) δ8.41(dd,J=2.4&1.2Hz,1H),8.35(dd,J=8.0&2.4Hz,1H),3.44–3.40(m,1H),1.20(d,J=6.8Hz,6H).

[0449] Synthesis of 5-bromo-3-isopropyl-2H-pyrazolo[3,4-b]pyridine

[0450]

[0451] 85% hydrazine hydrate (12mL, 5 volumes) was added to a stirred solution of 1-(5-bromo-2-fluoropyridin-3-yl)-2-methylpropan-1-one (2.3g, 9.38mmol, 1.0 equivalent) in EtOH (23mL, 10 volumes) and stirred at 100°C for 16 hours. After completion of the reaction by TLC, the reaction mixture was evaporated, diluted with water (80mL) and extracted with ethyl acetate (3×50mL). The organic layer was separated, dried over sodium sulfate and concentrated to provide 2.6g of crude product, which was purified by silica gel column (60-120 mesh, eluent: 9% to 12% EtOAc / petroleum ether) to obtain 5-bromo-3-isopropyl-2H-pyrazolo[3,4-b]pyridine (1.1g, yield: 49%) as an off-white solid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.30. LC / MS retention time = 3.25 minutes, 240.0 [M+H] + .

[0452] 1 H NMR (400MHz, CDCl3) δ11.05 (brs, 1H), 8.59 (d, J = 2.0Hz, 1H), 8.27 (d, J = 2.0Hz, 1H), 3.41–3.36 (m, 1H), 1.48 (d, J = 6.8Hz, 6H).

[0453] Synthesis of 5-bromo-3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine

[0454]

[0455] To a stirred solution of 5-bromo-3-isopropyl-2H-pyrazolo[3,4-b]pyridine (1.0 g, 4.18 mmol, 1.0 equivalent) in EtOAc (40 mL, 40 volumes) trimethyloxonium tetrafluoroborate (3.71 g, 16.4 mmol, 4 equivalents) was added and stirred at room temperature for 12 hours. After completion of the reaction by TLC, it was diluted with cold water (100 mL) and extracted with EtOAc (2 × 80 mL). The organic layer was separated, dried over sodium sulfate and concentrated to provide 1.5 g of crude product, which was purified by a silica gel column (60-120 mesh, eluent: 8% to 11% EtOAc / petroleum ether) to obtain 5-bromo-3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine (280 mg, yield: 27%) as a yellow liquid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.7;

[0456] 1 H NMR (400MHz, CDCl3) δ8.51 (d, J = 2.0Hz, 1H), 8.18 (d, J = 2.0Hz, 1H), 4.06 (s, 3H), 3.36–3.29 (m, 1H), 1.43 (d, J = 7.2Hz, 6H).

[0457] Synthesis of 3-isopropyl-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine

[0458]

[0459] To a stirred solution of 5-bromo-3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine (280 mg, 1.11 mmol, 1.0 eq) in 1,4-dioxane (6 mL) was added bis(pinacol)diborane (337 mg, 1.32 mmol, 1.2 eq), potassium acetate (271 mg, 2.77 mmol, 2.5 eq) and degassed for 5 minutes. Pd(dppf)Cl2.DCM (45 mg, 0.055 mmol, 0.05 eq) was then added and stirred at 90 °C for 16 hours. After the reaction was completed by TLC. The volatiles were removed under vacuum, diluted with water (50 mL), and extracted with ethyl acetate (3×20 mL). The organic layer was separated, washed with brine solution (10 mL), dried over sodium sulfate and concentrated to provide 3-isopropyl-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (360 mg, crude product) as a black semi-solid. TLC system: EtOAc / petroleum ether (20:80), R f Value: ~0.45. Cr: LC / MS: 56% boric acid m / z, 220.1 [M+H] + .

[0460] Synthesis of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine

[0461]

[0462] To a stirred solution of 3-isopropyl-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolo[3,4-b]pyridine (360 mg, 1.19 mmol, 1 eq) in 1,4-dioxane and water (3:1) (7 mL) was added 4-chloro-2-(methylthio)pyrimidine (213 mg, 1.43 mmol, 1.2 eq), K2CO3 (412 mg, 2.99 mmol, 2.5 eq) and degassed for 5 min. Pd(dppf)Cl2DCM (48 mg, 0.06 mmol, 0.05 eq) was then added and stirred at 80 °C for 16 h. After completion of the reaction by TLC, the RM was evaporated, diluted with water (30 mL), and extracted with ethyl acetate (2×40 mL). The organic layer was separated, dried over sodium sulfate and concentrated to provide 2.6 g of crude product, which was purified by silica gel column (100-200 mesh, eluent: 18-22% EtOAc / petroleum ether) to give 5-(2-chloropyrimidin-4-yl)-3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine (230 mg, semi-pure) as a light yellow liquid. TLC system: EtOAc / petroleum ether (30:70), R f Value: ~0.2. LC / MS: retention time = 3.56 minutes, 288.1 [M+H] + ; 68% purity.

[0463] Synthesis of tert-butyl ((1S,3S)-3-((4-(3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate

[0464]

[0465] To a stirred solution of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridine (230 mg, 0.80 mmol, 1 eq) in IPA (4 mL) was added tert-butyl ((1S, 3S)-3-aminocyclopentyl)carbamate (240 mg, 1.20 mmol, 1.5 eq) and stirred at 110 ° C for 60 hours. TLC showed a new polar spot and no starting material was present. The volatiles were removed under vacuum, diluted with water (20 mL), and extracted with EtOAc (2×20 ml). The organic layer was dried over Na2SO4, filtered, concentrated and purified by reverse phase column (eluent: 32-59% acetonitrile and water containing 0.01% formic acid) to give tert-butyl ((1S,3S)-3-((4-(3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (120 mg, 33%) as a brown solid. TLC system: EtOAc / petroleum ether (50:50), R f Value: ~0.40; LC / MS retention time = 3.65 minutes, 452.2 [M+H] + .

[0466] 1 H NMR (400MHz, CDCl3): δ9.17(brs,1H),8.70(s,1H),8.34(d,J=4.8Hz,1H),7.02(d,J=5.2Hz,1H),5.32(bs,1H),4.57(brs ,1H),4.52–4.47(m,1H),4.15–4.12(m,4H),3.49–3.40(m,1H),2.34–2.23(m,2H),2.05–1.95(m,2H),1.50–1.45(m,17H).

[0467] Synthesis of (1S,3S)-N1-(4-(3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt

[0468]

[0469] To a stirred solution of tert-butyl ((1S,3S)-3-((4-(3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)amino)cyclopentyl)carbamate (120 mg, 0.27 mmol, 1 eq) in 1,4-dioxane (1.2 mL) was added 4M HCl in 1,4-dioxane (0.6 mL) at 0°C and stirred at room temperature for 2 hours. After completion of the starting material, the volatiles were removed under vacuum, triturated with diethyl ether (2 x 3 mL) and lyophilized. The obtained material was purified by reverse phase column (eluted with 32% acetonitrile and water containing 0.01% formic acid) to give (1S,3S)-N1-(4-(3-isopropyl-1-methyl-1H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-yl)cyclopentane-1,3-diamine HCl salt (40 mg, 43%) as a brown sticky solid. TLC system: MeOH / DCM (10:90), R f Value: ~0.1; LC / MS retention time = 2.27 minutes, 352.4 [M+H] + .

[0470] 1 H NMR (400MHz, CD3OD) δ9.47(brs,1H),9.15(s,1H),8.36(d,J=6.4Hz,1H),7.77(d,J=6.4Hz,1H),4.55–4.49(br,1H),4.10(s ,3H),3.92–3.88(m,1H),3.54–3.50(m,1H),2.47–2.38(m,2H),2.31–2.28(m,2H),1.94–1.80(m,2H),1.48(d,J=6.8Hz,6H).

[0471] Embodiment 13

[0472] Synthesis of Compound I-13

[0473]

[0474] Synthesis of N-((1R,2S,4R)-bicyclo[2.2.1]hept-2-yl)-4-(3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-amine

[0475]

[0476] In a sealed tube, a solution of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-methyl-2H-indazole (300 mg, 1.04 mmol, 1 eq) and (1S,2S,4R)-bicyclo[2.2.1]hept-2-amine (250 mg, 1.36 mmol, 1.3 eq) in isopropanol (3 mL) was stirred at 110° C. for 16 hours. After completion of the reaction by TLC, the reaction mixture was evaporated and purified by reverse phase column [eluted in 56% acetonitrile / water containing 0.1% formic acid] to give N-((1R,2S,4R)-bicyclo[2.2.1]hept-2-yl)-4-(3-isopropyl-2-methyl-2H-indazol-5-yl)pyrimidin-2-amine (77 mg, yield: 20%) as an off-white solid. TLC system: EtOAc / hexane (1:1), R f Value: ~0.2; LC / MS(m / z):362.3(M+H) +

[0477] 1 H NMR (400MHz, CDCl3) δ: 8.60 (brs, 1H), 8.30 (d, J = 4.8Hz, 1H), 7.88 (d, J = 9.2Hz, 1 H),7.67(d,J=9.2Hz,1H),6.99(d,J=5.2Hz,1H),5.08–5.06(m,1H),4.16(s,3H), 3.89(brs,1H),3.53–3.46(m,1H),2.43(brs,1H),2.33(brs,1H),1.95–1.89(m, 1H),1.60–1.57(m,6H),1.54–1.49(m,2H),1.38–1.33(m,2H),1.32–1.28(m,2H).

[0478] Embodiment 14

[0479] Synthesis of Compound I-14

[0480]

[0481] Synthesis of N-((1R,2S,4R)-bicyclo[2.2.1]hept-2-yl)-4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-amine TFA salt (BLX-3051)

[0482]

[0483] In a sealed tube, a solution of 5-(2-chloropyrimidin-4-yl)-3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridine (300 mg, 1.04 mmol, 1 eq) and (1S,2S,4R)-bicyclo[2.2.1]hept-2-amine (150 mg, 1.35 mmol, 1.3 eq) in IPA (5 mL) was stirred at 110 °C for 36 h. After completion of the reaction by TLC, the reaction mixture was evaporated and purified by reverse phase column [eluted in 45% acetonitrile / water containing 0.1% formic acid] to give N-((1R,2S,4R)-bicyclo[2.2.1]hept-2-yl)-4-(3-isopropyl-2-methyl-2H-pyrazolo[3,4-b]pyridin-5-yl)pyrimidin-2-amine TFA salt (89m, yield: 24%) as a pale yellow solid. TLC system: EtOAc / hexane (60:40), R f Value: ~0.2; LC / MS(m / z):363.3(M+H) +

[0484] 1 H NMR(400MHz, DMSO-d6)δ:9.32(s,1H),9.10(brs,1H),8.39(d,J=5.6Hz,1H),7.72(brs,1H),7.48(d,J=4.0Hz,1H),4.17(s,3H),3.79(brs, 1H),3.64–3.57(m,1H),2.48–2.45(m,1H),2.33–2.27(m,2H),1.76–1 .71(m,1H),1.56–1.48(m,10H),1.29–1.26(m,1H),1.16–1.12(m,2H).

[0485] Biological Example 1

[0486] Cytotoxicity assay

[0487] Multiplexed LDH-Glo cytotoxicity assay and CellTiter-Glo 2D cell viability assay. Compound I-2 and AZD-4573 were used at 10 doses of IC 50 RWPE-1 normal prostate cells were treated in duplicate with 3-fold serial dilutions starting from 100 μM, 5000 cells / well in a 96-well format for 72 hours. Changes in toxicity were measured using the LDH-Glo cytotoxicity assay by combining 50 μL of diluted sample with 50 μL of LDH detection reagent and recording luminescence after 1 hour of incubation. After removing the sample for LDH measurement, an equal volume of CellTiter Glo 2D reagent was added to the remaining cell suspension and luminescence was recorded after a 30-minute incubation period.

[0488] The cytotoxicity of Compound I-2 was tested in RWPE-1 normal healthy donor prostate cells using a very sensitive assay that measures the release of lactate dehydrogenase (LDH), a cytosolic enzyme that is released into the cell culture medium when plasma membrane integrity is lost. Data from the LDH-Glo assay and a cell viability assay using CellTiter-Glo indicated that Compound I-2 did not kill prostate cancer cells until concentrations greater than 15-fold that required to kill most prostate cancer cells (the IC in these normal cells) were reached. 50 The reference compound AZD-4573 is highly toxic to normal prostate cells.

[0489] Table 2. Cytotoxicity results of compound I-2 and AZD-4573

[0490]

[0491] Biological Example 2

[0492] C ELL T ITER -G LO Determination

[0493] 2.0 Luminescent Cell Viability Assay Reagent was purchased from Promega, and 22Rv1 and LASCPC-01 cell lines were purchased from ATCC. The test compound, i.e., compound I-2, was incubated at 10 IC 50 The model was dosed in duplicate with 3-fold serial dilutions starting from 100 μM, 4000 cells / well (adherent) in a 96-well format, and a treatment time of 72 hours was used for all selected cell lines. An equal volume of CellTiter-Glo 2D reagent was added to the cell suspension, and luminescence was recorded after a 30-minute incubation period. The results are shown in Figure 1 and 2 middle. Figure 1 The antiproliferative efficacy of compound 1-2 at different concentrations against six prostate cancer cell lines was demonstrated. Figure 2 Demonstrated the antiproliferative efficacy of the benchmark CDK9 inhibitor AD-4573 at different concentrations against six prostate cancer cell lines. Experimental Data Figure 1 and 2 The results are summarized in Table 3.

[0494] Table 3. Cell Titer-Glo assay results

[0495]

[0496] Biological Example 3

[0497] Colony formation assay

[0498] Colony formation assays were performed on 2 different NEPC cell lines. Cells were seeded in 6-well plates at 800 cells per well and adhered for 24 hours in complete growth medium. The range of treatment with compound I-2 was NT to 10.0 μM for 14 days. Cells were fixed with a 3:1 methanol / acetic acid mixture and stained with methylene blue. SEM from two independent experiments. (*P<0.05, **P<0.01, ***P<0.005 relative to DMSO control). The results showed that compound I-2 significantly inhibited the proliferation and colony formation of 22Rv1 more than LASCPC-01, which was shown in Figure 3 and 4 middle.

[0499] Biological Example 4

[0500] Annexin V / 7-AAD apoptosis assay

[0501] Apoptosis of 2 NEPC cells by Annexin V / 7-AAD staining: Apoptosis induced by CDK9 inhibitor Compound I-2 was assessed by Annexin V / 7-AAD double staining and FACS analysis using a GUAVA flow cytometer. Compound I-2 treatment increased apoptosis in a dose-dependent manner. Real-time measurements of apoptosis induced by Compound I-2 treatment in prostate cancer cells were determined using the IncuCyte S3 Live Cell Analysis System. Representative images and bar graphs of LASCPC-01 cells are shown in Figure 5 In addition, cell apoptosis induced by CDK9 inhibitor compound I-2 was evaluated by annexin V / 7-AAD dual staining and FACS analysis. Compound I-2 treatment increased cell apoptosis in a dose-dependent manner, and the annexin V / 7-AAD staining results are shown in Figure 6 and 7 middle.

[0502] Treatment with compound 1-2 increased apoptosis in a dose-dependent manner and was comparable to the results obtained by the CellTiter-Glo assay described herein. NEPC cells were treated for 72 hours and showed that LASCPC-01, 22Rv1, and C4-2 cells were very sensitive to treatment with compound 1-2. The treatment resulted in 69, 71, and 84% cell death, respectively, comparable to the 2D CellTiter-Glo assay results.

[0503] Biological Example 5

[0504] Western blotting

[0505] Two NEPC cell lines (22Rv1 and LASCPC-01) were treated with compound I-2 for 6 and 24 hours. Cytoplasmic and nuclear extracts from these cells were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher). Cell extracts were separated on 4-12% Bis-Tris NuPAGE gels and then transferred to 0.45 nitrocellulose membranes, followed by blocking with Pierce fast blocking buffer for 30 minutes and then incubated with the designated antibodies at 4°C overnight. The relative protein expression of CDK9, N-Myc and other downstream signaling was quantified by RT-PCR genes using GraphPad Prism 8 and ImageJ software. The results are shown in Figure 8-10 middle.

[0506] Biological Example 6

[0507] Express

[0508] Total RNA was extracted using RNeasy micro kit (Qiagen). cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative RT-PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad) using SsoAdvanced SYBR Green Supermix (Bio-Rad). Data were normalized to GUS, and the percentage of gene expression was determined by the ΔCt method.

[0509] Biological Example 7

[0510] ADP-G LO Kinase assay

[0511] ADP-Glo ​​assay kit was purchased from Promega, and CDK9 / CycT1 protein and PDKtide substrate were purchased from SignalChem. 50The model is administered in duplicate, wherein 3 times of serial dilutions are performed starting from 100 μM. The reaction is started by pre-incubating 30 minutes in 1X assay buffer (50 mM HEPES, 3 mM MgCl2, 3 mM MnCl2 and 1 mM DTT) with 4 μL protein (20 nM), 2 μL substrate (100 μM) and 2 μL serial dilution drugs. After pre-incubation, 4 μL ATP (1 μM) is added to each well and incubated for 90 minutes in addition. ADP-Glo ​​reagent (10 μL) and kinase detection reagent (20 μL) are added to each well, and incubated according to the suggestion of Promega. The reaction is quantitatively measured by measuring luminescence and IC is calculated using GraphPad Prism software 50 .

[0512] Biological Example 8

[0513] Immunofluorescence staining

[0514] LASCPC-01NEPC cells were treated with compound I-2 starting from NT, 1.1, 3.3, 10 and 30 μM for 6 hours. After 6 hours, the cells were fixed with 4% formaldehyde and probed with primary N-Myc antibody overnight. The cells were then incubated with secondary antibodies conjugated with Alexa Fluor 488 and counterstained with Hoechst 33342. Images were taken with a Nikon automated widefield CCD camera and quantified using ImageJ and GraphPad Prism 8 software, which are shown in Fig.11 middle

[0515] Biological Example 9

[0516] Cell cycle arrest assay

[0517] NEPC cells were treated with compound I-2 starting from NT, 1 and 3.16 μM for 8 hours. The cells were then harvested with PBS and fixed with ice-cold 70% ethanol at -20°C overnight. The cells were then washed 2× with ice-cold PBS and then resuspended in DAPI staining buffer and incubated in the dark for 30 minutes. Cells were analyzed using a BD Fortessa flow cytometer, and cell cycle distribution was analyzed using FlowJo and GraphPad Prism 8 software. Western blot analysis of nuclear extracts showed that compound I-2 inhibited CDK9 downstream targets. Representative blots of 22Rv1 and LASCPC-01 are shown in Figure 12-13 middle.

[0518] Biological Example 10

[0519] Cell cycle arrest assay

[0520] This article describes a pilot in vivo xenograft transplantation study of 22Rv1 and LASCPC-01. Animals: Male BALB / c nude mice (8 weeks old). NEPC cell lines: 22Rv1 and LASCPC-01. Cell number: 22Rv1 (1.2 × 10 6 ) and LASCPC-01(10 6 Tumor volume was measured twice a week by caliper and by length × (width 2 ) / 2. When the tumor reaches about 100-500mm 3 At 1 , mice were randomized and selected to be treated with Compound 1-2 as a TFA salt at an oral dose of 18 mg / kg. The data from this experiment are presented in Figure 14-15 middle.

[0521] Biological Example 11

[0522] Kinase profiling

[0523] Using a miniaturized radioisotope-based assay platform ( 33 P), CDK family kinase profiling assays were performed using Kds KINOMEscan (KdELECT) and DiscoveRx Matrix from Reaction Biology. IC 50 Values ​​are estimated based on the best available curve fit. Compound I-2 and AZD4573 (CAS#2057509-72-3) show IC relative to CDK9 50 The values ​​were 4.2 and <5.0 nM, respectively.

[0524] In summary, compound 1-2 exhibited promising selectivity, ranging from 3 to 50-fold over the closest CDK homologs tested.

[0525] Table 4. 10-dose IC of Compound I-2 relative to CDK9 homologues 50 Value (nM / Kd).

[0526] Kinase CDK9 CDK2 / Cyclin A <![CDATA[17.60IC 50 / 30Kd]]> CDK3 / Cyclin E <h2 style=";text-align:left;direction:ltr"><![CDATA[115IC <h2 style=";text-align:left;direction:ltr"> 50 <h2 style=";text-align:left;direction:ltr"> / 31Kd]]><h2 style=";text-align:left;direction:ltr"> CDK4 / Cyclin D <![CDATA[22.80IC 50 / 22Kd]]> CDK5 / P21 245 CDK7 / Cyclin H 119 CDK9 / Cyclin T <5 / 4.2 Staurosporine <1

[0527] Biological Example 12

[0528] Off-target inhibition test

[0529] Compound I-2 had potent activity in a cell-free assay, with IC 50 The IC value in the NanoBRET target engagement assay is 4.2 nM in cell-based assays. 50 The IC of 22Rv1, LASCPC-01, C4-2 and C4-2B cells derived from NEPC was 15.58 nM. 50The values ​​were 132, 58, 28 and 31 nM. Compound I-2 reduced pCDK9, pSer2 and its downstream N-MYC, C-MYC and MCL-1 targets in newly diagnosed and resistant prostate cancer patient samples, indicating its response to overcoming drug resistance and anti-tumor efficacy. The data show that compound I-2 has strong development potential as a candidate drug for NEPC therapy.

[0530] The scanMAX panel was used to analyze off-target inhibition of other kinases. The scanMAX panel contains 468 kinases, including lipid kinases and atypical kinases. Compound I-2 was tested at 1 mM concentration, with 2.7% activity against the target of interest, CDK9, which is consistent with its IC of 4.2 nM. 50 At 1 mM concentration, it was found that each kinase exhibited 0-1% of the control activity relative to the activity of 9 other kinases (CLK / 2.9Kd), DYRKA1 (9Kd), ERK8 (5.5Kd), FLT3 / ITD (D835V) (1.2Kd), Haspin (2.8Kd), PCTK1 (8.2Kd), PRKD3 (230Kd), TAOK1 (19Kd), TAOK3 (9.2Kd). The Kd and IC of these 9 off-target kinases were further tested. 50 values, and are shown in Table 3 above. Based on this selectivity profiler screen, the key off-target kinase is FLT3. The FDA has approved 48 kinase inhibitors, 11 of which bear 2,4-disubstituted pyrimidines, such as compound I-2, and exhibit similar off-target profiles. The structure of compound I-2 includes a unique chemotype with high cell permeability, which has excellent overall selectivity and efficacy compared to the other 11 approved kinase inhibitors.

[0531] Biological Example 13

[0532] Target engagement assay in HEK293 cells

[0533] HEK293 cells were transfected with 1 μg CDK9 and 9 μg cyclin T1 DNA, and the transfected cells were treated with compound I-2 (starting from 100 μM, 10 doses, 3-fold dilutions) and reference compound Dinaciclib (starting from 10 μM, 10 doses, 3-fold dilutions). Curve fitting was performed only when the NanoBRET signal% at the highest compound concentration was less than 55%. Fresh HEK293 cells were purchased from ATCC, and the compound treatment time was 1 hour. The results were plotted and IC 50 The data were calculated as shown in Table 4 below.

[0534] Table 5. NanoBRET target engagement assay results

[0535] Compound <![CDATA[CDK9 + Cyclin T1 IC 50 Value (nM)]]> Compound I-2 15.58nM Denasili (reference) 5.05nM

[0536] Biological Example 14

[0537] Anti-proliferative effect

[0538] 2.0 Luminescent Cell Viability Assay reagents were purchased from Promega, and 22Rv1, LASCPC-01, C4-2, C4-2B, NCI-H660, DU-145, PC-3, and LNCap cell lines were obtained from ATCC and the Clinical Core Facility of the Huntsman Cancer Institute. 50 Format Compound I-2 and reference compound AZD-4573 were tested in duplicate with 3-fold serial dilutions starting from 100 μM, 2500 cells / well (adherent) in 96-well format, and a treatment time of 72 hours was used for all selected cell lines. Results for cell viability were obtained and IC 50 Values ​​(see Table 5 below), showing submicromolar cell killing of these prostate cancer cells. Compound 1-2 was particularly sensitive to all NEPC cells (see, e.g., results for 22Rv1, LASCPC-01, NCI-H660, C4-2, C4-2B).

[0539] Table 6. 2D CellTiter-Glo assay results; IC 50 Values ​​are shown in nM

[0540]

[0541]

[0542] Biological Example 15

[0543] Colony formation assay 2

[0544] Prostate cancer cells were seeded in 6-well plates at 400 cells per well and allowed to adhere for 24 hours in complete growth medium, followed by treatment with compound 1-2 at concentrations ranging from 0.123, 0.37, 1.1, 3.0, and 10 μM for 72 hours. Treatment resulted in complete inhibition of colony formation of all prostate cancer cells.

[0545] Biological Example 16

[0546] Fluorescence polarization assay

[0547] Predictor TMThe hERG Fluorescence Polarization Assay Kit is intended for biochemical binding studies of the hERG channel in the absence of radioligand. Assay performance has been validated using a panel of established hERG channel blockers. TM The results of the assay showed a high correlation with the results obtained by the patch clamp technique. Compound I-2 was found to have no hERG activity, and the measured IC 50 It is 57.8mM.

[0548] Biological Example 17

[0549] P450 isozyme inhibition assay

[0550] P450 rod-shaped body reagent (ie, microsome) is prepared by insect cells infected with recombinant baculovirus containing human P450 isozymes and rabbit NAPDH-P450 reductase, because only a single P450 isozyme is expressed on human liver microsomes (HLM). Compound 1-2 is incubated with rod-shaped body plus substrate, and the reaction is initiated by adding NAPDH-450. At the end of the incubation time, a stop solution is added to terminate the reaction, and a stable fluorescent signal is generated compared to compound 1-2 and solvent controls. The inhibitory effect of compound 1-2 relative to five P450 isozymes was evaluated, and the results are provided as follows.

[0551] Table 7. P450 determination results

[0552] P540 isozyme <![CDATA[IC of Compound I-2 50 (μM)]]> 1A2 29.4 2C19 28.3 2C9 50.8 2D6 100 3A4 31.1

[0553] Biological Example 17

[0554] Pharmacokinetic Studies-Bioavailability

[0555] 1mg / kg compound I-2 was administered intravenously to male beagles, showing a moderate plasma clearance compared to normal liver blood flow (ie, 40 ml / min / kg). It was found that the steady-state volume distribution was higher, and this may be one of the reasons for tissue distribution. The last average concentration time was 24 hours after intravenous bolus injection. The half-life of compound I-2 in canine plasma was shown to be 4.26 hours. Compound I-2 was orally administered to male beagles at 3mg / kg to demonstrate that absorption had no lag phase and the median time to peak concentration was 1 hour. Compound I-2 showed higher systemic availability. It was found that the oral bioavailability was 83 ± 22%. Overall, the average residence time and plasma half-life in plasma after intravenous and oral administration were comparable.

[0556] Table 8: Mean pharmacokinetic parameters of Compound 1-2 in male beagle dogs following single intravenous and oral gavage administration at doses of 1 mg / kg and 3 mg / kg

[0557]

[0558] C0: extrapolated concentration at time zero; C0 reported for intravenous route; C reported for oral route max ;

[0559] a Reported median (min-max)

[0560] b Nominal dose and AUC for intravenous and oral administration inf To calculate bioavailability

[0561] %F b Oral bioavailability

[0562] Compound I-2 obtains good systemic exposure after oral administration. The high steady state of distribution volume indicates distribution to tissues. The higher distribution volume helps to maintain plasma concentration until 24 hours after administration, which may be one of the reasons for the high plasma half-life of 4.26 and 5.81 hours in intravenous and oral modes, respectively. In the case of moderate clearance of compound I-2, oral bioavailability is shown to be as high as 83% compared with the intravenous route.

[0563] Pharmacokinetic studies in mice and rats have been completed and compound I-2 achieved excellent systemic exposure, with a body weight-dependent exposure pattern. The oral bioavailability was 28% in mice and 28% in rats. Utilization rate is 38% .

[0564] Biological Example 18

[0565] In vivo tumor efficacy studies

[0566] Antitumor efficacy of compound I-2 in 22RV1-induced neuroendocrine prostate cancer (NEPC) CDX male Nu / Nu (Balb / c background)

[0567] 22Rv1 cells were cultured in RPMI supplemented with 10% FBS and 1% P / S at 37°C and 5% CO2 in a humidified incubator. Nu / nu Balb / c homozygous mice were obtained by Jackson Laboratory (strain 002019). Mice were fed Teklad irradiation (sterilization) mouse diets and pads with Teklad irradiation (sterilization) corn cob litter from Envigo (Indianapolis, IN, Indiana). Mice were placed in the Optimice turntable sterile area and filtered air was provided in disposable cages from Animal Care Systems, Inc. (Centenaire, Colorado). 12-hour light / 12-hour dark light cycle was observed, with animal treatment only during the light cycle.

[0568] 22Rv1 transplantation: On the day of implantation, trypsinize the cells and allow them to detach from the flask. Then neutralize the trypsin with complete medium and spin the cells at 300 × g. Aspirate the medium and centrifuge the cells at 5 x 10 7 Resuspend in 50:50 Cultrex RPMI (no supplement) at a concentration of 10 cells / mL. Inject 100 μL into the right hind flank of each animal (a total of 5 x 10 cells / mL per mouse). 6 cells). Cell viability was checked using trypan blue exclusion and was found to be >98%.

[0569] Study Groups and Treatments: When mean tumor volume reached ∼225 mm 3 At 4 hr, mice were stratified and divided into (5) treatment groups as described below.

[0570] Table 9: 22Rv1 Study Group - Compound I-2 Efficacy Study in Nu / Nu BALB / c Mice

[0571]

[0572] All mice were orally dosed, and the observation duration was 21 days.

[0573] Treatments were administered by oral gavage (10 μL / gram adjusted volume). Dosing solutions were prepared daily using molecular biology grade water as vehicle (TFA salts were adjusted using a salt correction factor of 1.65).

[0574] Animal Weight: Changes in animal weight during the study are another indicator of tolerability. All animal weights were recorded (see e.g. Fig.16 ).

[0575] Relative body weight of each group in the study: Mice in the 50 and 60 mg / kg groups and the single dose 65 mg / kg group were withdrawn from the study starting on day 5. Tumor growth inhibition (TGI) data were analyzed according to dose and tumor weight.

[0576] Tumor volume: Tumor volume was measured by digital calipers and the formula [width 2 The volume was calculated as [x length] / 2. All tumor volume measurements were recorded.

[0577] The 22Rv1 prostate cancer model is a model originally developed for the growth of human prostate cancer in immunocompromised mice. In this embodiment, tumors were grown subcutaneously for efficacy studies (4 groups of studies comparing vehicle and 30mg / kg, 50mg / kg and 65mg / kg of compound I-2; oral administration once a day, with another group of 4 mice given a single dose of 60mg / kg). It was noted that there were tolerance issues at dose levels of ≥50mg / kg, causing some mice to withdraw from the study, and some mice required a dosing holiday for recovery. Compound I-2 appears to inhibit tumor growth in a dose-dependent manner, with tumor growth inhibition observed in all treatment groups.

[0578] Plasma, tumor, spleen, femur, heart, kidney, liver, lung, and brain were collected 6 hours after the last dose and processed for PK / PD studies.

[0579] The results relative to the mean weight on day 0, the mean tumor volume change during the 21-day study period, and the mean tumor weight are shown in Figure 16-18 middle.

[0580] Biological Example 19

[0581] In vivo tumor efficacy study 2

[0582] Antitumor efficacy of compound 1-2 in LASCPC-01-induced neuroendocrine prostate cancer (NEPC) CDX male Nu / Nu (Balb / c background): A similar protocol to that of the 22Rv1 study was performed using 30, 50, 65 and 80 mg / kg doses of LASCPC-01 study.

[0583] Changes in animal weight during the study were another indicator of tolerability. All animal weights were recorded. Tumor volume was measured by digital calipers and the formula [width 2 x length] / 2 to calculate volume. All tumor volume measurements were recorded. LASCPC-01 tumors showed robust growth (from 180 mm in 19 days) 3 Grow to 2700mm 3 ). By observation, dose-dependent tumor growth inhibition (TGI) was evident.

[0584] Mice in the 50 and 80 mg / kg groups were withdrawn from the study starting on day 5. Vehicle had large tumors resected on day 19, therefore day 21 does not reflect the average size of all mice in the group.

[0585] Compound I-2 appears to inhibit tumor growth in a dose-dependent manner, with the 80 mg / kg group having a maximum growth inhibition of 117% on day 5. In the mid-dose group (50 mg / kg), a maximum tumor growth inhibition of ∼100% was noted early in the study, and >80% inhibition was maintained throughout the study. Plasma, tumors, and bones were collected for PK / PD studies.

[0586] The results relative to the mean weight on day 0, the mean tumor volume change during the 21-day study period, and the mean tumor weight are shown in Figure 19-21 middle.

[0587] Biological Example 20

[0588] In vivo tumor efficacy study 3

[0589] This example demonstrates the evaluation and efficacy testing of Compound 1-2 in the C4-2 human neuroendocrine prostate cancer xenograft model using male NOD.SCID mice.

[0590] When compound I-2 (50 mg / kg, oral, daily) doses were administered from day 1 to day 4, weight loss was observed. Starting from day 8, compound I-2 was administered at a dose of 30 mg / kg once every two weeks. From day 13, compound I-2 was orally administered at 30 mg / kg every day. Compared with the vehicle control group, from day 8 to day 18, compound I-2 (50-30 mg / kg) showed a decrease in tumor volume. Compared with the vehicle control group, from day 18 to day 21, 50-30 mg / kg of compound I-2 showed a significant decrease in tumor volume (see, e.g. Fig. 22 Compared with the vehicle control group, compound I-2 at a dose of 50-30 mg / kg showed an increase in tumor growth inhibition from day 8 to day 12 (see, e.g. Fig.23 ). At the final 21st day, 50-30 mg / kg of compound I-2 showed 43.25% tumor growth inhibition compared to the vehicle control group. On day 21, 50-30 mg / kg of compound I-2 showed a significant decrease in tumor weight by 48.72% compared to the vehicle control group (see, e.g. Fig.24). Starting from day 4, 50-30 mg / kg of compound I-2 showed significant weight loss compared to the vehicle control group. Death was observed in one mouse in the compound I-2 treated group, which may be due to severe weight loss (i.e., ~15%). Blood was collected and processed 1.5 hours after the last administration, and plasma and tumor samples were processed for bioanalysis LC-MS / MS and showed promising PK / PD related efficacy.

[0591] If necessary, various aspects of the embodiments may be modified to adopt the concepts of various patents, applications, and publications to provide yet further embodiments. These and other changes may be made to the embodiments in view of the above detailed description. Generally, in the following claims, the terms used should not be interpreted as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted as including all possible embodiments together with the full range of equivalents required by such claims. Therefore, the claims are not limited by the present disclosure.

Claims

1. A compound having the following structure (I): As a stereoisomer or salt thereof, wherein: represents a double or single bond such that all valences are satisfied and a heteroaryl ring is formed; X is N or CR 4b ; Y is N or CR 4c ; R 1a Not present or C1-C6 alkyl; R 1b is hydrogen, halo, hydroxy, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 hydroxyalkyl; R 2 is hydrogen or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl or C1-C6 hydroxyalkyl; R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a substituted C3-C5 cycloalkyl, C6-C8 bridged cycloalkyl or 3-10 membered heterocyclic group, or R 3 is optionally substituted by hydroxy, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3b Substituted C3-C8 cycloalkyl; R 3a is an optionally substituted 3-10 membered heterocyclyl or an optionally substituted 3-10 membered heteroaryl; R 3b is an optionally substituted 3-10 membered N-heterocyclyl or an optionally substituted 3-10 membered heteroaryl; R 3c is hydrogen or optionally substituted C1-C6 alkyl; R 4a , R 4b and R 4c Each is independently hydrogen, halo or optionally substituted: C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy; R 5 is independently at each occurrence halo or optionally substituted C1-C6 haloalkyl; n is 0, 1, 2, 3 or 4; and p is 0, 1 or 2.

2. The compound according to claim 1, wherein the compound has one of the following structures (Ia) or (Ib):

3. A compound according to any one of claims 1 to 2, wherein R 1a Does not exist.

4. A compound according to any one of claims 1 to 2, wherein R 1a It is methyl.

5. A compound according to any one of claims 1 to 4, wherein R 1b It is hydrogen, C1-C6 alkyl or C3-C8 cycloalkyl.

6. A compound according to any one of claims 1 to 5, wherein R 1b is hydrogen, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, isopropyl, methyl or ethyl.

7. A compound according to any one of claims 1 to 6, wherein R 1b is hydrogen, cyclopropyl, cyclobutyl, isopropyl or methyl.

8. A compound according to any one of claims 1 to 7, wherein R 1b Not replaced.

9. A compound according to any one of claims 1 to 8, wherein R 2 It is hydrogen or C1-C6 alkyl.

10. A compound according to any one of claims 1 to 9, wherein R 2 It is a C1-C6 alkyl group.

11. A compound according to any one of claims 1 to 10, wherein R 2 It is -CH3.

12. A compound according to any one of claims 1 to 9, wherein R 2 For hydrogen.

13. A compound according to any one of claims 1 to 9, wherein R 2 Not replaced.

14. A compound according to any one of claims 1 to 13, wherein R 1b or R 2 At least one of them is hydrogen.

15. A compound according to any one of claims 1 to 9, wherein R 1b and R 2 Both are hydrogen.

16. The compound according to any one of claims 1 to 15, wherein n is 0 or 1.

17. The compound according to any one of claims 1 to 16, wherein n is 0.

18. A compound according to any one of claims 1 to 17, wherein R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a Substituted C3-C5 cycloalkyl, C6-C7 bridged cycloalkyl or 3-10 membered heterocyclic group.

19. The compound according to claim 18, wherein R 3 is optionally substituted by hydroxy, amino, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R 3c )R 3a Substituted C5 cycloalkyl.

20. The compound according to any one of claims 18 or 19, wherein R 3 is a C5 cycloalkyl group optionally substituted by a hydroxy group or an amino group.

21. A compound according to any one of claims 1 to 17, wherein R 3 is optionally substituted by hydroxy, cyano, halo, C1-C6 alkyl, C3-C8 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or -N(R3c )R 3b Substituted C3-C8 cycloalkyl.

22. A compound according to any one of claims 1 to 17, wherein R 3 It is a 5-6 membered heterocyclic group.

23. A compound according to any one of claims 1 to 22, wherein R 3 was replaced.

24. A compound according to any one of claims 1 to 22, wherein R 3 Not replaced.

25. A compound according to any one of claims 1 to 24, wherein R 3 Has one of the following structures:

26. A compound according to any one of claims 1 to 25, wherein R 3 Has one of the following structures:

27. A compound according to any one of claims 1 to 26, wherein R 4a For hydrogen.

28. A compound according to any one of claims 1 to 27, wherein X is N.

29. A compound according to any one of claims 1 to 27, wherein X is CH or CF.

30. A compound according to any one of claims 1 to 29, wherein Y is N.

31. A compound according to any one of claims 1 to 29, wherein Y is CR 4c .

32. A compound according to any one of claims 1 to 29, wherein Y is CH or CF.

33. A compound according to any one of claims 1 to 32, wherein p is 1, and R 5 For chlorine.

34. A compound according to any one of claims 1 to 32, wherein p is 0.

35. The compound of claim 1, wherein the compound has one of the following structures: or a stereoisomer or a salt thereof.

36. A compound according to any one of claims 1 to 35, wherein the compound is in free base form.

37. The compound according to any one of claims 1 to 35, wherein the compound is a pharmaceutically acceptable salt.

38. The compound of any one of claims 1 to 35, wherein the compound is a trifluoroacetate salt, a hydrochloride salt or a formate salt.

39. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 38 and a pharmaceutically acceptable carrier.

40. A method of treating a disease or condition, the method comprising administering to a subject in need thereof an effective amount of a compound or salt according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39.

41. The method of claim 40, wherein the disease or disorder is a kinase-expressing cancer.

42. The method of claim 41, wherein the cancer is bladder cancer.

43. The method of claim 41, wherein the cancer is prostate cancer.

44. The method of claim 41, wherein the cancer is a hematological malignancy.

45. The method of claim 44, wherein the hematological malignancy is acute myeloid leukemia.

46. ​​The method of claim 41, wherein the disease or disorder is an autoimmune or inflammatory disease.

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