Cdk inhibitors

Abstract

To provide a compound that is useful in cancer therapy, and a method for treating cancer.SOLUTION: Provided is a compound represented by the structural formula below, or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to International Patent Application No. PCT / CN2020 / 088585, filed May 5, 2020. The entire contents of the aforementioned application are incorporated herein by reference. [Background technology]

[0002] Cyclin-dependent kinases (CDKs) are a group of protein kinases that were first discovered for their role in regulating the cell cycle. They have since been identified to play roles in regulating several other biological functions, such as transcription, mRNA processing, and neuronal differentiation.

[0003] CDKs are relatively small proteins with molecular weights of approximately 34-40 kDa. They contain almost exclusively a kinase domain and are essentially inactive when not complexed with a class of regulatory proteins called cyclins. CDK levels remain relatively constant throughout the cell cycle, and most regulation is post-translational, most notably through binding to cyclins.

[0004] As in all kinases, the active site, or ATP-binding site, of CDKs is a cleft between a small amino-terminal lobe and a larger carboxy-terminal lobe. The structure of human CDK2 revealed that CDKs possess a modified ATP-binding site that can be regulated by cyclin binding. Phosphorylation by CDK-activating kinase (CAK) at Thr161 on the T-loop increases complex activity. In the absence of cyclin, a flexible loop called the activation loop or T-loop occludes the cleft, rendering the position of several key amino acid residues suboptimal for ATP binding. In the presence of cyclin, two α-helices reposition to enable ATP binding. One of these, the L12 helix immediately preceding the T-loop in the primary sequence, becomes a β-strand and helps reposition the T-loop so that it no longer occludes the active site. The other α-helix, called the PSTAIRE helix, repositions and helps reposition key amino acid residues in the active site.

[0005] Thus, only cyclin-CDK complexes possess active kinase activity, and most known cyclin-CDK complexes regulate progression through the cell cycle. CDKs are ubiquitous in all known eukaryotes, and their regulatory function in the cell cycle is evolutionarily conserved. For example, yeast cells can grow normally when their CDK genes are replaced with homologous human genes. CDKs exert their regulatory function by phosphorylating their substrates on certain serine and threonine residues and on the consensus sequence [S / T]PX[K / R] (where S / T is the target for phosphorylation, Ser or Thr, P is proline, X is any amino acid, K is lysine, and R is arginine).

[0006] In animal cells, there are at least nine different CDKs, four of which (CDK1, 2, 3, and 4) are directly involved in regulating the cell cycle. In mammalian cells, CDK1, together with its binding partners cyclins A2 and B1, can drive the cell cycle alone. Cyclin-CDK complexes at early cell cycle stages can serve to activate cyclin-CDK complexes at later stages.

[0007] The same CDK can form complexes with different cyclins to regulate different phases of the cell cycle. For example, CDK2 can form complexes with cyclins D or E to regulate G1 phase, cyclins A or E to regulate S phase, and cyclins A to regulate G2 phase. Meanwhile, CDK4 and CDK6 can form complexes with cyclins D1, D2, and D3.

[0008] The highly homologous cyclin-dependent kinases (CDKs) CDK4 and CDK6, in combination with cyclin D, are key regulators of the transition through restriction point R between the G1 (growth) and S (DNA replication) phases of the cell cycle. CDK4 / 6 exert their effects through phosphorylation of the retinoblastoma protein (pRb). Once phosphorylated, pRb loses its inhibitory effect on the transcription of genes that promote S-phase entry.

[0009] In contrast, the endogenous protein modulator p16 INK4 Specific inhibition of CDK4 / 6 kinase activity by CDK4 kinases or by small molecule inhibitors results in hypophosphorylated pRb and cell arrest at the G1 restriction point. As the primary mechanism regulating the G1 restriction point, pathways regulated by these kinases are altered in a wide range of human tumors, and therefore, inhibition of CDK4 / CDK6 in these tumors has therapeutic benefit by preventing cell division.

[0010] There remains a need to provide CDK4 / 6 inhibitors that can be used in the treatment of cell proliferative disorders such as cancer. Summary of the Invention

[0011] Described herein are compounds, and pharmaceutically acceptable salts or stereoisomers thereof, that inhibit the activity of cyclin-dependent kinases (CDKs), such as CDK2, CDK4, and / or CDK6.

[0012] In one aspect, the present invention provides a compound represented by the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. Compound A has been discovered to not only actively inhibit CDK2, CDK4 and CDK6, but also exhibit potent antiproliferative activity.

[0013] In another aspect, the present invention provides a compound represented by the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. Compound B has been discovered to not only selectively inhibit CDK4 but also to have excellent brain penetrability.

[0014] Also provided are pharmaceutical compositions comprising a compound disclosed herein, or a pharmaceutically acceptable salt, or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0015] The present disclosure further provides methods for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of (1) a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or (2) a pharmaceutically acceptable composition comprising a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the cancer is selected from the group consisting of colorectal cancer, breast cancer (such as hormone receptor-positive, HER2 / neu-negative advanced or metastatic breast cancer in postmenopausal women), lung cancer, prostate cancer, glioblastoma, mantle cell lymphoma, chronic myeloid leukemia, and acute myeloid leukemia.

[0016] In certain embodiments of the methods of the present invention, cancer can be treated by inhibiting the activity of a cyclin-dependent kinase (CDK), such as CDK2, CDK4, and / or CDK6.

[0017] In certain embodiments of the methods of the present invention, the cancer is cancer of the bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, or skin, hematopoietic tumors of the lymphoid system, hematopoietic tumors of the myeloid system, follicular thyroid carcinoma, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoma, follicular thyroid carcinoma, or Kaposi's sarcoma.

[0018] In certain embodiments of the methods of the present invention, the compounds disclosed herein are administered together with any one of the second therapeutic agents described herein that also treat the same cancer.

[0019] The present disclosure also provides the use of a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising same, in any of the above-described methods of the present invention. In one embodiment, a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising same, is provided for use in any of the above-described methods of the present invention. In another embodiment, there is provided the use of a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or a pharmaceutical composition comprising same, for the manufacture of a medicament for any of the described methods of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] 1. Overview The present invention provides a compound of the present invention, or a pharmaceutically acceptable salt thereof, for use in therapy, such as cancer therapy.

[0021] The present invention also provides a pharmaceutical formulation comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0022] The present invention provides a compound of the present invention or a pharmaceutically acceptable salt thereof for use in treating cancer. In particular, these cancers include any of the cancers described herein below, such as colorectal cancer, breast cancer (including ER in adult or postmenopausal women), and the like. + HER2 - The cancer may be advanced or metastatic or recurrent breast cancer), lung cancer, particularly non-small cell lung cancer (NSCLC), prostate cancer, glioblastoma, mantle cell lymphoma (MCL), chronic myeloid leukemia (CML) and acute myeloid leukemia (AML).

[0023] The present invention further relates to the treatment of colorectal cancer, breast cancer (ER) in adult or postmenopausal women, and the like in mammals. + HER2 -and a method for treating cancer selected from the group consisting of: advanced, metastatic, or recurrent breast cancer), lung cancer, particularly non-small cell lung cancer (NSCLC), prostate cancer, glioblastoma, mantle cell lymphoma, chronic myeloid leukemia, and acute myeloid leukemia, which method comprises administering to a mammal in need of such treatment an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0024] Additionally, the present invention provides the use of a compound of the present invention or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of cancer. In particular, these cancers include colorectal cancer, breast cancer (ER cancer in adult or postmenopausal women), and the like. + HER2 - The cancer is selected from the group consisting of advanced, metastatic or recurrent breast cancer, lung cancer, particularly non-small cell lung cancer (NSCLC), prostate cancer, glioblastoma, mantle cell lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0025] Furthermore, the present invention provides a pharmaceutical formulation for use in a therapeutic method, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, or excipient. The present invention also provides a pharmaceutical formulation for use in a therapeutic method, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, or excipient, for the treatment of colorectal cancer, breast cancer (ER in adult women or postmenopausal women), or the like. + HER2 - Also provided are pharmaceutical formulations for treating advanced or metastatic or recurrent breast cancer, lung cancer, particularly non-small cell lung cancer (NSCLC), prostate cancer, glioblastoma, mantle cell lymphoma, chronic myeloid leukemia and acute myeloid leukemia.

[0026] Treatable disease indications and potential second therapeutic agents useful in combination therapy are described in further detail in the following sections.

[0027] It is understood that any embodiment described herein, including those described in only one of the following sections or only in the examples, may be combined with any one or more additional embodiments of the present invention, unless expressly disclaimed or otherwise inappropriate / inapplicable.

[0028] 2.Definition The compounds described herein may contain one or more asymmetric centers and therefore may exist in various stereoisomeric forms, e.g., enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of an individual enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers.

[0029] Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and diastereomers can also be obtained from diastereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. See, LL. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., University of Notre Dame Press, Notre Dame, IN 1972).

[0030] If a compound is designated by a name or structure that indicates a single enantiomer, unless otherwise specified, the compound is at least 60%, 70%, 80%, 90%, 99%, or 99.9% optically pure (also referred to as "enantiomerically pure"). Optical purity is the weight of the mixture of the named or depicted enantiomer divided by the total weight of the mixture of both enantiomers.

[0031] When the stereochemistry of a disclosed compound is named or depicted by a structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., as in the case of a diastereomeric pair), it is understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers is included. It is further understood that the stereoisomeric purity of the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% by weight. Stereoisomeric purity in this case is determined by dividing the total weight of the mixture of stereoisomers encompassed by the name or structure by the total weight of the mixture of all stereoisomers.

[0032] When a geometric isomer is depicted by name or structure, it is understood that the geometric isomer purity of the named or depicted isomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight. Geometric isomer purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of both geometric isomers in the mixture.

[0033] A racemic mixture means 50% of one enantiomer and 50% of its corresponding enantiomer. The present invention includes all enantiomerically pure, enantiomerically enriched, diastereomerically pure, diastereomerically enriched, and racemic mixtures, as well as diastereomeric mixtures, of the compounds of the present invention.

[0034] Compounds described herein may also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0035] It will be recognized that some variation in natural isotope abundance will occur in synthesized compounds depending on the origin of the chemicals used in synthesis. Thus, preparations of the compounds disclosed herein will inherently contain small amounts of deuterated isotopes. Despite this variation, the concentrations of naturally abundant stable hydrogen and carbon isotopes are low and insignificant compared to the degree of stable isotope substitution in the compounds of the present invention. See, for example, Wada, E et al., Seikagaku, 1994, 66:15; Gannes, LZ et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119:725.

[0036] The compounds described herein may exist in various tautomeric forms. The term "tautomer" or "tautomeric" refers to two or more interconvertible compounds / substituents resulting from the formal migration of at least one hydrogen atom and at least one change in valence (e.g., from a single bond to a double bond, a triple bond to a single bond, or vice versa). Exemplary tautomerizations include keto to enol, amide to imide, lactam to lactim, enamine to imine, etc. Tautomerizations include tautomerization of enamines to (different enamines). The present teachings encompass tautomeric forms of compounds, including forms not structurally depicted. All such isomeric forms of such compounds are expressly included. If a tautomer of a compound is aromatic, then the compound is aromatic. Similarly, if a tautomer of a compound is heteroaryl, then the compound is heteroaryl.

[0037] In certain cases, tautomeric forms of the disclosed compounds exist, such as the tautomeric structures shown below. [ka]

[0038] When a compound herein is represented by a structural formula or designated by a chemical name herein, it is understood that all other tautomeric forms that may exist for that compound are encompassed by the structural formula.

[0039] The compounds of the present invention may exist for therapy in free form or, where appropriate, as a pharmaceutically acceptable salt form.

[0040] The term "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and that is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art; for example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glyceroate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobiolate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N-methyl-N ... + (C 1~4 Alkyl)4 -Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Additional pharmaceutically acceptable salts include those formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates, where appropriate. These include ammonium, quaternary ammonium, and amine cations.

[0041] Such pharmaceutically acceptable acid addition salts and general methodologies for preparing them are well known in the art (see, for example, Stahl et al., HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE, (VCHA / Wiley-VCH, 2002); Bighley et al., in "Encyclopedia of Pharmaceutical Technology," Eds. Swarbrick and Boylan, Vol. 13, Marcel Dekker, Inc., New York, Basel, Hong Kong 1995, pp. 453-499; Berge et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66(1):1977).

[0042] The terms "composition" and "formulation" are used interchangeably.

[0043] A "subject" is a mammal, preferably a human, but may also be an animal requiring veterinary treatment, such as pets (e.g., dogs, cats, and the like), livestock (e.g., cattle, sheep, pigs, horses, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

[0044] The terms "administer," "administering," or "administration" refer to a method of introducing a compound of the present invention or a composition thereof into or onto a subject. These methods include, but are not limited to, intra-articular (into a joint), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, subcutaneous, oral, topical, intrathecal, inhalation, transdermal, rectal, and the like. Administration techniques that can be employed with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon, and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.

[0045] The terms "treatment," "treat," and "treating" refer to reversing, alleviating, or inhibiting the progression of a disease as described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of a disease have occurred or are observed (i.e., therapeutic treatment). In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of symptom history and / or exposure to a pathogen) (i.e., prophylactic treatment). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence.

[0046] The terms "condition," "disease," and "disorder" are used interchangeably.

[0047] Generally, the effective amount of a compound taught herein will vary depending on a variety of factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one of ordinary skill in the art. The effective amount of a compound of the present teachings can be readily determined by one of ordinary skill in the art using routine methods known in the art.

[0048] The term "effective amount" means an amount that, when administered to a subject, produces beneficial or desired results, including, for example, clinical results, e.g., inhibits, suppresses, or reduces the symptoms of the condition being treated in the subject compared to a control. For example, an effective amount may be a single unit dosage form (e.g., For example, it can be administered at a dose of 1 mg to about 50 g per day, for example, 1 mg to about 5 g per day.

[0049] A "therapeutically effective amount" is an amount effective to detectably kill or inhibit the growth or spread of cancer cells, tumor size or number, or other measure of the level, stage, progression, or severity of cancer. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular anti-cancer agent, its mode of administration, concurrent treatment with other therapies, and the like.

[0050] General chemical terms used in the above formula have their usual meanings.

[0051] As used herein, "h" refers to one or several hours, "min" refers to minutes or minutes, "Cdk" or "CDK" refers to cyclin-dependent kinase, "pRb" refers to retinoblastoma protein, "MCL" refers to mantle cell lymphoma, "AML" refers to acute myeloid leukemia, "CML" refers to chronic myeloid leukemia, "Boc" refers to N-tert-butoxycarbonyl, "EA" refers to ethyl acetate, "DCM" refers to dichloromethane, "DMSO" refers to dimethyl sulfoxide, "DMA" refers to dimethylacetamide, "THF" refers to tetrahydrofuran, and "MtB "E" refers to methyl tert-butyl ether, "TEA" refers to triethylamine, "FBS" refers to fetal bovine serum, "PBS" refers to phosphate buffered saline, "BSA" refers to bovine serum albumin, "RT" refers to room temperature, "mpk" means milligrams per kilogram, "po" refers to oral (orally), "qd" means once daily administration, "HPLC" means high pressure liquid chromatography, "q2d" means a single dose every two days, "q2dx10" means a single dose every two days x 10, "VSMC" means vascular smooth muscle cells, and "XRD" refers to X-ray diffraction.

[0052] 3.Compound Another aspect of the present disclosure relates to labeled compounds (radiolabeled, fluorescently labeled, etc.) of the present invention that may be useful not only in imaging techniques but also in assays for localizing and quantifying CDKs in tissue samples, including humans, both in vitro and in vivo, and for identifying CDK ligands by inhibition of labeled compound binding. Accordingly, the present disclosure includes such labeled compounds.

[0053] The present disclosure further includes isotopically labeled compounds of the present invention. An "isotopically" or "radiolabeled" compound is a compound of the present invention in which one or more atoms have been replaced or substituted by an atom having an atomic mass or mass number different from that typically found in nature (i.e., occurring in nature). Suitable radionuclides that can be incorporated into the compounds of the present invention include: 2H (also written as D for deuterium), 3 H (also written as T in the case of tritium), 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I and 131 These include, but are not limited to, I. The radionuclide that is incorporated in the present radio-labeled compounds will depend on the particular application of that radio-labeled compound.

[0054] The present invention can further include synthetic methods for incorporating radioisotopes into the compounds of the present invention. Synthetic methods for incorporating radioisotopes into organic compounds are well known in the art, and one of ordinary skill in the art will readily recognize methods applicable to the compounds of the present invention.

[0055] The labeled compounds of the present invention can be used in screening assays to identify / evaluate compounds. For example, newly synthesized or identified compounds (i.e., test compounds) that are labeled can be used in screening assays to identify / evaluate compounds. A test compound (labeled) can be evaluated for its ability to bind to a CDK by monitoring its concentration fluctuations upon contact with the CDK through label tracking. For example, a test compound (labeled) can be evaluated for its ability to reduce the binding of another compound (i.e., a standard compound) known to bind to a CDK. Thus, the ability of a test compound to compete with a standard compound for binding to a CDK directly correlates with its binding affinity. Conversely, in some other screening assays, the standard compound is labeled, and the test compound is unlabeled. Thus, the concentration of the labeled standard compound is monitored to assess competition between the standard compound and the test compound, thereby determining the relative binding affinity of the test compound.

[0056] In one embodiment is a compound, or a pharmaceutically acceptable salt, or stereoisomer thereof, wherein one or more hydrogen atoms are replaced by deuterium.

[0057] 4. Treatable diseases and treatment methods Certain compounds of the present invention are selective inhibitors of CDK2, CDK4, and / or CDK6 and are therefore useful in the treatment of diseases or disorders characterized by abnormal cell proliferation that can be inhibited by reducing the activity of CDK-cyclin complexes that include CDK2, CDK4, and / or CDK6.

[0058] In certain embodiments, compounds of the invention selectively inhibit CDK4 / 6 over CDK2, with IC values ​​of the latter (CDK2) relative to the former (CDK4 / 6). 50 The ratio of values ​​is at least about 10, 20, 50, 100, 200, 300, 400, 500, 800, 1,000, 2,000 or more.

[0059] In certain embodiments, compounds of the invention selectively inhibit CDK4 over CDK6, with IC values ​​of the latter (CDK6) relative to the former (CDK4). 50 The ratio of values ​​is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50 or more.

[0060] In certain embodiments, compounds of the invention selectively inhibit CDK2 over CDK4, with IC of the latter (CDK4) relative to the former (CDK2). 50 The ratio of values ​​is at least about 2, 5, 10, 15, 20, 40, 50, 60, 80, 100 or more.

[0061] In certain embodiments, compounds of the invention have similar IC 50 value, e.g., within 10-fold, 5-fold, 3-fold, or 2-fold of the IC 50 Such compounds of the invention are useful for treating cancers associated with amplified or enhanced expression of cyclin D1 or E1 or E2.

[0062] CDK2 is a catalytic subunit of the CDK-cyclin complex whose activity is restricted to the G1-S phase of the cell cycle, allowing cells to make the proteins necessary for mitosis and replicate their DNA. CDK2 forms a complex with cyclin E or A. Cyclin E binds to CDK2 in G1, which is required for the transition from G1 to S phase. Meanwhile, CDK2 binding to cyclin A is required for progression through S phase.

[0063] CDK2 is largely dispensable for the cell cycle of normally functioning cells, but is important for the abnormal proliferation process of cancer cells. Overexpression of cyclin E occurs in many tumor cells, causing the cells to become dependent on CDK2 and cyclin E. Aberrant cyclin E activity is observed in breast, lung, colorectal, gastric, and bone cancers, as well as in leukemias and lymphomas. Similarly, aberrant expression of cyclin A2 is associated with chromosomal instability and tumor growth, while inhibition leads to reduced tumor growth. Therefore, CDK2 and its cyclin-binding partners represent possible therapeutic targets for novel cancer therapeutics. Preclinical models have demonstrated that CDK2 limits tumor growth. It has shown preliminary success in reducing the side effects of current chemotherapy drugs.

[0064] For example, Caldon et al. (Mol Cancer Ther 11(7):1488-1499, 2012) reported that cyclin E2 is included in several gene signatures predicting disease progression in either tamoxifen-resistant or metastatic breast cancer, and that high expression of cyclin E2 is characteristic of luminal B and HER2 subtypes of breast cancer and strongly predicts shorter metastasis-free survival after endocrine therapy. Furthermore, tamoxifen-resistant (MCF-7 TAMR) breast cancer cells overexpress cyclin E2, and expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute anti-estrogen resistance, suggesting that overexpression of cyclin E contributes to the anti-estrogen resistance of tamoxifen-resistant cells. Growth of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, ectopic expression of cyclin E1 or E2 also reduced sensitivity to inhibition of CDK4, but not CDK2, and CDK2 inhibition in E-cyclin-overexpressing and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition.

[0065] These data demonstrate that cyclin E2 overexpression is a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition, and that CDK2 inhibitors may overcome such resistance by effectively inhibiting cyclin E1 and E2-overexpressing cells and enhancing the efficacy of other therapeutic agents, and may be useful as components of combination therapies in endocrine-resistant disease. Similarly, subject compounds with potent inhibitory activity against both CDK2 and CDK4 are expected to be effective against cancer cells that are both non-resistant and resistant to endocrine therapy or CDK4 inhibition.

[0066] Thus, in certain embodiments, compounds of the invention may have potent inhibitory effects on both CDK2 and CDK4 (e.g., independently, IC<10 nM, <5 nM, <1 nM levels). 50value), and therefore is effective for treating tamoxifen-resistant or metastatic breast cancer, such as tamoxifen-resistant or metastatic breast cancer with CycE overexpression.

[0067] IC of the compounds of the present invention against CDK2 / 4 / 6 50 The values ​​can be measured, for example, using the methods described in Examples 1-3 (hereby incorporated by reference).

[0068] In particular, the compounds of the present invention are useful in the treatment of cancer. In other embodiments, the compounds of the present invention are useful in the treatment of chronic inflammatory diseases such as arthritis and cystic fibrosis.

[0069] Thus, in one aspect, the present invention provides a method of treating cancer, particularly the cancers described herein, in a mammal, comprising administering to a mammal in need of such treatment an effective amount of a compound of the invention.

[0070] In a related aspect, the invention is directed to the use of the compounds of the invention in the manufacture of a medicament for treating cancer, particularly the cancers described herein.

[0071] In another related aspect, the compounds of the invention can be used in the manufacture of a medicament for the treatment of cancer, particularly the cancers described herein.

[0072] In another related aspect, the invention provides a compound of the invention for use in the treatment of cancer, particularly the cancers described herein.

[0073] According to any of the above-described related aspects of the present invention, CDK4 and CDK6 can regulate their effects on the cell cycle, in part, through pRb phosphorylation. Thus, certain compounds of the present invention can inhibit pRb phosphorylation, and thus cell proliferation and / or tumor growth, through inhibition of CDK4 / 6 activity in any cancer type in which the cells are proliferating and contain a functional, intact Rb1 gene encoding pRb.

[0074] Thus, in certain embodiments, the compounds of the present invention inhibit pRb in mammals. + Cancers, such as colorectal cancer, breast cancer, lung cancer, prostate cancer, chronic myeloid leukemia, acute myeloid leukemia (Fry et al., Mol. Cancer Ther. 3(11):1427, 2004), mantle cell lymphoma (Marzec et al., Blood 108(5):1744, 2006), ovarian cancer (Kim et al., Cancer Research 54:605, 1994), pancreatic cancer (Schutte et al., Cancer Research 57:3126, 1997), malignant melanoma and metastatic malignant melanoma (Maelandsmo et al., British Journal of Cancer 73:909, 1996). The compounds of the present invention are also expected to be useful in the treatment of rhabdomyosarcoma (Saab et al., Mol. Cancer. Ther. 5(5):1299, 2006) and multiple myeloma, including relapsed and refractory multiple myeloma, in mammals (e.g., humans) (Baughn et al., Cancer Res. 66(15):7661, 2006).

[0075] On the other hand, Zhang et al. (Nature dx.doi.org / 10.1038 / nature25015, 2017) showed that inhibition of CDK4 / 6 in vivo resulted in decreased phosphorylation and therefore (APC / C Cdh1 Cullin 3 SPOPWe reported that CDK4 / 6 inhibition can lead to increased degradation of E3 ligases, which in turn leads to increased PD-L1 levels on the tumor cell surface and reduced numbers of tumor-infiltrating lymphocytes (TILs) in mouse tumors and primary human prostate cancer specimens. In other words, CDK4 / 6 inhibition in vivo increases PD-L1 protein levels and contributes to increased resistance to immune checkpoint therapy targeting PD-1 (programmed cell death protein 1) and PD-L1 (PD-1 ligand). Meanwhile, combining CDK4 / 6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumor regression and dramatically improves overall survival in mouse tumor models.

[0076] Thus, in certain embodiments, compounds of the invention can be used in combination with PD-1 / PD-L1 immune checkpoint inhibitors to enhance the treatment of human cancers.

[0077] PD-1 and PD-L1 inhibitors that can be used with the compounds of the present invention are known in the art. PD-1 inhibitors include monoclonal antibodies specific for PD-1 or antigen-binding fragments thereof. Exemplary PD-1 inhibitors include pembrolizumab (Keytruda), nivolumab (Opdivo), and cemiplimab (Libtayo). PD-L1 inhibitors include monoclonal antibodies specific for PD-L1 or antigen-binding fragments thereof. Exemplary PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi).

[0078] Additional immune checkpoint inhibitors that may be used together with the compounds of the invention to enhance the treatment of human cancers include monoclonal antibodies or antigen-binding fragments thereof specific for CTLA-4, such as ipilimumab (Yervoy).

[0079] Additional immune checkpoint inhibitors that can be used together with the compounds of the present invention to enhance the therapeutic effect of human cancers include bispecific monoclonal antibodies or antigen-binding fragments thereof specific for PD-1 and PD-L1, or combinations of monoclonal antibodies or antigen-binding fragments thereof specific for PD-1 and PD-L1, or PD-1 and CTLA-4, etc.

[0080] In certain embodiments, the compounds of the present invention can be used in combination with Tyr kinase inhibitors, such as receptor Tyr kinase (RTK) inhibitors, to enhance the therapeutic effect on human cancer. Exemplary Tyr kinase inhibitors include ALK inhibitors (such as crizotinib, ceritinib, alectinib, and brigatinib), Bcr-Abl inhibitors (such as bosutinib, dasatinib, imatinib, nilotinib, and ponatinib), BTK inhibitors (such as ibrutinib), c-Met inhibitors (such as crizotinib and cabozantinib), EGFR inhibitors (such as gefitinib, erlotinib, lapatinib, vandetanib, afatinib, and osimertinib), JAK inhibitors (such as ruxolitinib and tofacitinib), and MEK inhibitors. 1 / 2 inhibitors (trametinib, etc.), PDGFR inhibitors (axitinib, gefitinib, imatinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib, sunitinib, etc.), RET inhibitors (vandetanib, etc.), Src family kinase inhibitors (bosutinib, dasatinib, ponatinib, vandetanib, etc.), and VEGFR family inhibitors (axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, sunitinib, etc.).

[0081] Additional suitable kinase inhibitors that can be used in combination with the subject compounds, as well as treatable cancer indications, are described in Bhullar et al., Molecular Cancer 17:48, 2018, incorporated herein by reference in its entirety.

[0082] Further additional RTK inhibitors include monoclonal antibodies and antigen-binding fragments thereof, including anti-EGFR mAbs such as cetuximab (e.g., effective in treating lung cancer, colorectal cancer, and head and neck cancer), and anti-HER2 mAbs such as trastuzumab (e.g., effective in treating breast cancer).

[0083] In certain embodiments, the compounds of the invention can be used in combination with antagonists of hormone receptor signaling, such as those previously described for the treatment of breast cancer.

[0084] Cancers that may be treated with the compounds of the invention include non-Hodgkin's lymphoma, malignant mesothelioma, non-small cell lung cancer, cholangiocarcinoma, soft tissue sarcoma, glioblastoma, (recurrent) brain tumors, hormone receptor positive breast cancer, non-small cell lung cancer, brain metastases secondary to melanoma (including melanoma positive for cyclin D1 expression), (recurrent or persistent) endometrial cancer, (recurrent or metastatic) head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma, esophageal squamous cell carcinoma (SCC), esophageal adenocarcinoma (ADC), renal cell carcinoma, and urothelial carcinoma.

[0085] In certain embodiments, treatable cancers include cancer of the bladder, breast, colon, kidney, epidermis, liver, lung (including SCLC and NSCLC), esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, or skin, hematopoietic malignancies of the lymphoid system, hematopoietic malignancies of the myeloid system, follicular thyroid carcinoma, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, familial melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoma, follicular thyroid carcinoma, Kaposi's sarcoma, squamous cell carcinoma, sarcoma, or tumors of mesenchymal origin.

[0086] In certain embodiments, the hematopoietic tumor of the lymphoid system is leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, or leukemia. lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkitt's lymphoma.

[0087] In certain embodiments, the tumor of the central or peripheral nervous system is an astrocytoma, neuroblastoma, glioma, or schwannoma.

[0088] In certain embodiments, the cancer is small cell lung cancer, non-small cell lung cancer, pancreatic cancer, breast cancer, glioblastoma multiforme, T-cell ALL, and mantle cell lymphoma.

[0089] In certain embodiments, the cancer is selected from the group consisting of colorectal cancer, mantle cell lymphoma, breast cancer (including advanced or metastatic or recurrent breast cancer), pancreatic cancer, ovarian cancer, glioblastoma, acute myeloid leukemia, and lung cancer, particularly NSCLC.

[0090] In certain embodiments, the cancer is NSCLC, pancreatic cancer, ovarian cancer, or metastatic breast cancer, and the treatment comprises administering to a mammal in need thereof a therapeutically effective combination of a compound of the present invention and gemcitabine HCl.

[0091] In certain embodiments, the cancer is NSCLC, pancreatic cancer, ovarian cancer, or metastatic breast cancer, and the medicament comprising the compound of the present invention also comprises gemcitabine HCl or is administered simultaneously, separately, or sequentially with gemcitabine HCl.

[0092] In certain embodiments, the compounds of the present invention can be used in combination with other drugs for the treatment of NSCLC, pancreatic cancer, ovarian cancer, and metastatic breast cancer. For example, the compounds of the present invention can be used in simultaneous, separate, or sequential combination with gemcitabine HCl in the treatment of NSCLC, pancreatic cancer, ovarian cancer, or metastatic breast cancer.

[0093] In certain embodiments, the cancer is selected from the group consisting of colorectal cancer, glioblastoma, acute myeloid leukemia, and lung cancer.

[0094] In certain embodiments, the cancer is glioblastoma or astrocytoma, and the treatment utilizes a therapeutically effective combination of a compound of the invention and temozolomide, which may be administered simultaneously, separately, or sequentially with temozolomide.

[0095] Breast cancer treatment In certain embodiments, the compounds of the present invention can be used to treat breast cancer.

[0096] Breast cancer represents a significant health burden worldwide, accounting for approximately 7% of all cancer-related deaths in the United States in 2016. Approximately 75% of all breast cancers are hormone receptor-positive (HR + ) breast cancer, which expresses estrogen receptors (ER) and / or progesterone receptors (PgR) and typically depends on the ER signaling pathway for growth and survival. + Breast cancer utilizes the biological functions of the ER pathway to promote breast cancer growth, development, and progression. + Because breast cancer relies on ER signaling, such cancers are therapeutic targets for endocrine therapy agents that target the estrogen signaling pathway, such as aromatase inhibitors (AIs; including letrozole, anastrozole, and exemestane), selective ER modulators (tamoxifen), and selective ER downregulators (fulvestrant).

[0097] Endocrine therapy is HR + Although it constitutes the therapeutic backbone of breast cancer, the effectiveness of endocrine therapy remains limited by the existence of alternative survival or "escape" pathways, both pre-existing and new resistance and acquired resistance during treatment. Endocrine therapy is limited by high rates of both ER and cyclin D-mediated resistance. The ER pathway and many of the known escape pathways act through cyclin D-CDK4 / 6-inhibitors of the CDK4 (INK4)-retinoblastoma (Rb) pathway, promoting tumor growth. Therefore, combined targeting of both the ER pathway and the cyclin D-CDK4 / 6-INK4-Rb pathway typically leads to more widespread inhibition of tumor growth, preventing the activation of escape pathways and the development of endocrine therapy resistance. See Sammons et al., Current Cancer Drug Targets 17:637-649, 2017.

[0098] Thus, in certain embodiments, the breast cancer is pRb+ breast cancer. In certain embodiments, the breast cancer is hormone receptor (HR) positive (e.g., estrogen receptor positive (ER)). + ), progesterone receptor positive (PR + ), or ER + PR + ), HR + HER2 - or ER + HER2 - and HER2 / neu negative cancers, including advanced, metastatic, or recurrent breast cancer. + HER2 - or ER + HER2 - Advanced, metastatic or recurrent breast cancer occurs in adult or postmenopausal women.

[0099] In certain embodiments, the compounds of the present invention are used either alone or in combination with an aromatase inhibitor (which inhibits estrogen production) to treat HR-positive, HER2-negative advanced, metastatic, or recurrent breast cancer. In certain embodiments, the aromatase inhibitor temporarily inactivates aromatase (such as anastrozole (ARIMIDEX®) and letrozole (FEMARA®)). In certain embodiments, the aromatase inhibitor permanently inactivates aromatase (such as exemestane (AROMASIN®)).

[0100] In certain embodiments, compounds of the invention are used with compounds that interfere with the ability of estrogen to stimulate the growth of breast cancer cells, such as selective estrogen receptor modulators (SERMs), which bind to the estrogen receptor and prevent estrogen binding, such as tamoxifen (NOLVADEX®) and toremifene (FARESTON®). Tamoxifen inhibits HR + It has been used to treat breast cancer for over 30 years.

[0101] In certain embodiments, compounds of the invention are used with a pure anti-estrogen that lacks estrogen agonist activity, such as fulvestrant (FASLODEX®).

[0102] In certain embodiments, the HR-positive, HER2-negative advanced, metastatic, or recurrent breast cancer is present in a postmenopausal woman, hi certain embodiments, the HR-positive, HER2-negative advanced, metastatic, or recurrent breast cancer has progressed after treatment that alters the patient's hormones (e.g., estrogen and / or progesterone) or has worsened after treatment with another hormone therapy.

[0103] In certain embodiments, the compounds of the present invention are used in patients who are undergoing or have undergone oophorectomy, hi certain embodiments, oophorectomy is through oophorectomy or radiation therapy.

[0104] In certain embodiments, the compounds of the invention are used in conjunction with compounds that temporarily suppress ovarian function (e.g., estrogen and / or progesterone production). Such compounds include gonadotropin-releasing hormone (GnRH) agonists or luteinizing hormone-releasing hormone (LH-RH) agonists, including goserelin (ZOLADEX®) and leuprolide (LUPRON®).

[0105] In certain embodiments, the compounds of the invention are compounds that inhibit CYP3A4, such as ritonavir, indinavir, nelfinavir, saquinavir, clarithromycin, telithromycin, chloramphenicol, ketoconazole, itraconazole, posaconazole, voriconazole, nefazodone, cobicistat, amiodarone, aprepitant, verapamil, diltiazem, erythromycin, fluconazole, miconazole, bergamottin, cimetidine, ciprofloxacin, cyclosporine, and donedarone. , fluvoxamine, imatinib, valerian, buprenorphine, cafestol, cilostazol, fosaprepitant, gabapentin, lomitapide, orphenadrine, ranitidine, ranolazine, tacrolimus, ticagrelor, valproic acid, amlodipine, cannabidiol, dithiocarbamate, mifepristone, norfloxacin, delavirdine, gestodene, mibefradil, star fruit, milk thistle, niacinamide, ginkgo biloba, piperine, isoniazid, and quercetin.

[0106] In certain embodiments, the compounds of the invention are used in conjunction with inhibitors of IGF-1 / IGF-2, such as monoclonal antibodies against IGF-1 / IGF-2 or antigen-binding fragments thereof. Exemplary antibodies include the humanized IgG1 mAb xentuzumab.

[0107] In certain embodiments, the compounds of the present invention are used together with compounds that inhibit PI3K. Inhibition of PI3K is thought to reduce the levels of cyclin D1 and other G1-S cyclins, abolish pRb phosphorylation, and inhibit the activation of the S-phase transcription program. Representative PI3K inhibitors for use together with the compounds of the present invention include idelalisib, copanlisib, duvelisib, taselisib, perifosine, buparlisib, alpelisib, umbralisib, copanlisib, dactolisib, and voxtalisib.

[0108] In certain embodiments, the mammal being treated is a human, such as an adult female with breast cancer (e.g., a postmenopausal woman or adult female with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, advanced, metastatic, or recurrent breast cancer that has progressed after receiving a treatment that alters the patient's hormones).

[0109] In addition, certain compounds of the present invention exhibit the advantageous property of being able to cross the blood-brain barrier. Thus, such compounds can penetrate the brain and are therefore useful for treating primary and metastatic brain tumors in which the cells are proliferating and contain a functional and intact Rb1 gene. + Examples of brain tumors include glioblastoma, as well as medulloblastoma and astrocytoma (Lee et al., Science 235:1394, 1987).

[0110] Temozolomide is a cytotoxic DNA alkylating agent used to treat brain tumors, including glioblastoma and astrocytoma (Friedman et al., Clin. Cancer Res. 6(7):2585-2597, 2000), including brain metastases from melanoma, breast cancer, and NSCLC (Siena et al., Annals of Oncology, doi:10.1093 / annonc / mdp343, 2009). Temozolomide interacts with DNA, causing chemical modification / damage (Marchesi et al., Pharmacol. Res. 56(4):275-287, 2007). Thus, in some embodiments, compounds of the present invention inhibit primary and metastatic pRb tumors, such as glioblastoma and astrocytoma. + For the treatment of brain tumors, for example, when such metastases are from melanoma, breast cancer, or NSCLC, it can be used in combination with temozolomide.

[0111] 5. Pharmaceutical Compositions The present invention provides pharmaceutical compositions comprising any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0112] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the formulation and / or administration of and / or absorption by a subject of an active agent and that can be included in the compositions of the present disclosure without causing significant adverse toxicological effects to the subject. Non-limiting examples of pharmaceutically acceptable carriers and excipients include water, NaCl, saline, lactated Ringer's solution, regular sucrose, regular glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, coloring agents, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and / or flavoring substances, and the like, which do not deleteriously react with or interfere with the activity of the compounds provided herein. Those skilled in the art will recognize that other pharmaceutical carriers and excipients are suitable for use with the disclosed compounds.

[0113] These compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the compounds of the present invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic regimens (e.g., Gleevec or other kinase inhibitors, interferon, bone marrow transplant, farnesyltransferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormone therapy, antibodies, radiation, etc.). For example, the additional therapeutic agent for co-administration or inclusion in a pharmaceutical composition with the compounds of the present invention may be another one or more anti-cancer agents.

[0114] As described herein, the compositions of the present invention, as used herein, comprise the compounds of the present invention together with a pharmaceutically acceptable carrier, including any and all solvents, diluents, or other vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present invention. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; excipients such as powdered tragacanth, malt, gelatin, talc, cocoa butter, and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar, buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer; and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; as well as coloring agents, releasing agents, coating agents, sweetening agents, flavorings, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0115] 6. Preparation The present invention also encompasses a class of compositions comprising the active compounds of the present invention in association with one or more pharmaceutically acceptable carriers and / or diluents and / or adjuvants (collectively referred to herein as "carrier" materials), and, if desired, other active ingredients.

[0116] In certain embodiments, the present invention provides a pharmaceutical formulation for treating cancer, particularly the cancers described herein, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier.

[0117] In certain embodiments, the present invention provides a method for treating colorectal cancer, mantle cell lymphoma, breast cancer (ERG in adult or postmenopausal women), or ovarian cancer, comprising administering to the patient a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier. + HER2 - The present invention provides a pharmaceutical formulation for treating cancer selected from the group consisting of advanced, metastatic or recurrent breast cancer, glioblastoma, acute myeloid leukemia and lung cancer, particularly NSCLC.

[0118] In certain embodiments, the present invention provides a pharmaceutical formulation for treating glioblastoma or astrocytoma, comprising a compound of the present invention and temozolomide, together with a pharmaceutically acceptable carrier.

[0119] In certain embodiments, the present invention also provides a pharmaceutical formulation comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and temozolomide, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0120] In certain embodiments, the present invention provides a method for treating NSCLC, pancreatic cancer, ovarian cancer, or metastatic breast cancer (including ER in adult or postmenopausal women) comprising administering a compound of the present invention and gemcitabine HCl together with a pharmaceutically acceptable carrier. + HER2 - The present invention provides a pharmaceutical formulation for treating breast cancer (including advanced, metastatic or recurrent breast cancer).

[0121] In certain embodiments, the present invention also provides a pharmaceutical formulation comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and gemcitabine HCl, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0122] The active compounds of the present invention can be preferably administered by any suitable route in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the intended treatment. The compounds and compositions of the present invention can be administered in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles, for example, orally, mucosally, topically, rectally, pulmonary by inhalation spray, etc., or parenterally, including intravascular, intravenous, intraperitoneal, subcutaneous, intramuscular, intrasternal, and infusion techniques.

[0123] The pharmaceutically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.

[0124] For oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension, or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient.

[0125] Examples of such dosage units are tablets or capsules. A suitable daily dose for, for example, a human or other mammal may vary depending on the condition of the patient and other factors, but, again, can be determined using routine methods.

[0126] Amounts of compounds and compositions administered for treating disease states with the compounds and / or compositions of the present invention The dosage and administration schedule depend on various factors, including the age, weight, sex and condition of the patient, the type of disease, the severity of disease, the route and frequency of administration, and the specific compound used.Therefore, the dosage schedule can vary widely, but can be routinely determined using standard methods.As mentioned above, the daily dose can be administered in one administration, or can be divided into 2, 3, 4 or more administrations.

[0127] For therapeutic purposes, the active compound of the present invention is usually combined with one or more adjuvants, excipients or carriers suitable for the indicated administration route.When administered orally, the compound can be mixed with lactose, sucrose, starch powder, cellulose ester of alkanoic acid, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.Such capsules or tablets can contain controlled-release formulations, such as those provided by a dispersion of the active compound in hydroxypropylmethylcellulose.

[0128] For skin conditions, it may be preferable to apply a topical preparation of the compounds of the invention to the affected area two to four times daily. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes), and drops suitable for administration to the eye, ear, or nose. For topical administration, the active ingredient may comprise as much as 10% w / w of the formulation, but preferably not more than 5% w / w, more preferably 0.1% to 1%, although it may comprise 0.001% to 10% w / w, e.g., 1% to 2% by weight, of the formulation.

[0129] The compounds of the present invention can also be administered by transdermal devices.Preferably, transdermal administration will be achieved using a patch of either a reservoir and porous membrane type or a solid matrix type.In either case, the active agent is continuously delivered from the reservoir or microcapsules through a membrane into the active agent permeable adhesive, which is in contact with the skin or mucosa of the recipient.When the active agent is absorbed through the skin, a controlled and predetermined flow of the active agent is administered to the recipient.In the case of microcapsules, the encapsulating agent can also function as a membrane.The oil phase of the emulsion of the present invention can be composed of known ingredients in a known manner.

[0130] The phase may contain only an emulsifier, or may contain a mixture of at least one emulsifier with a fat or oil, or a mixture of both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier, with or without a stabilizer, constitutes the so-called emulsifying wax, and the wax, together with the oil and fat, constitutes the so-called emulsifying ointment base that forms the oily dispersed phase of the cream formulation. Suitable emulsifiers and emulsion stabilizers for use in the formulations of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate, glyceryl distearate alone or with a wax, or other materials known in the art.

[0131] Since the solubility of active compounds in most oils likely to be used in pharmaceutical emulsion formulations is very low, the selection of a suitable oil or fat for the formulation is based on achieving the desired aesthetic properties. Therefore, the cream should preferably be a non-sticky, non-staining, and washable product with a suitable consistency to avoid leakage from tubes or other containers. Diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate. Linear or branched chain mono- or dibasic alkyl esters, such as methyl esters, ...

[0132] Alternatively, high melting point lipids such as white soft paraffin and / or liquid paraffin or other mineral oils can be used.

[0133] Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.

[0134] The active ingredient is preferably present in such formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%, especially about 1.5% w / w.

[0135] Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules using one or more of the carriers or diluents mentioned for use in formulations for oral administration, or by using other suitable dispersing or wetting agents and suspending agents. These compounds may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, tragacanth gum, and / or various buffers. Other adjuvants and modes of administration are well and widely known in the pharmaceutical art. The active ingredient may also be administered by injection in a composition containing a suitable carrier, including saline, dextrose, or water, or a cyclodextrin (i.e., Captisol), cosolvent solubilization (i.e., propylene glycol), or micelle solubilization (i.e., Tween 80).

[0136] Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

[0137] For pulmonary administration, the pharmaceutical composition can be administered in the form of an aerosol or using an inhaler containing a dry powder aerosol.

[0138] Suppositories for rectal administration of drugs can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycol, which is solid at room temperature but liquid at rectal temperature and will melt in the rectum to release the drug.

[0139] The pharmaceutical compositions may be subjected to conventional pharmaceutical processes such as sterilization and / or may contain conventional adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Tablets and pills can further be prepared with enteric coatings. Such compositions may also contain auxiliary agents such as wetting agents, sweeteners, flavoring agents, and perfuming agents. The pharmaceutical compositions of the present invention comprise a compound of a formula described herein or a pharmaceutically acceptable salt thereof, an additional agent selected from a kinase inhibitor (such as a small molecule, polypeptide, antibody), an immunosuppressant, an anti-cancer agent, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an anti-vascular hyperproliferation compound, and any pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0140] Alternative compositions of the present invention include compounds of the formulae described herein or pharmaceutically acceptable salts thereof. and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Such compositions may optionally include one or more additional therapeutic agents, including, for example, a kinase inhibitor (small molecule, polypeptide, antibody, etc.), an immunosuppressant, an anti-cancer agent, an anti-viral agent, an anti-inflammatory agent, an anti-fungal agent, an antibiotic, or an anti-vascular hyperproliferation compound.

[0141] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a patient, together with a compound of the invention, which does not destroy its pharmacological activity and which is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound. Pharmaceutically acceptable carriers, adjuvants, or vehicles that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as D-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tween or other similar polymer delivery matrices, serum albumin such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Cyclodextrins, such as u-, P-, and y-cyclodextrin, or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-cyclodextrin, or other solubilizing derivatives, may also be advantageously used to enhance delivery of compounds of the formulae described herein.

[0142] The pharmaceutical composition can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.For tablets for oral use, commonly used carriers include lactose and corn starch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dried corn starch.When aqueous suspensions and / or emulsions are orally administered, the active ingredient can be suspended or dissolved in an oily phase and combined with an emulsifying agent and / or suspending agent.

[0143] If desired, certain sweetening, flavoring and / or coloring agents may be added. The pharmaceutical compositions may include formulations utilizing liposome or microencapsulation techniques, various examples of which are known in the art.

[0144] Pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents, examples of which are also well known in the art.

[0145] 7. Medical Kit One aspect of the present invention relates to a kit for conveniently and effectively carrying out the method or use according to the present invention. Typically, a pharmaceutical pack or kit comprises one or more containers filled with one or more of the components of the pharmaceutical composition of the present invention. Such kits are particularly suitable for the delivery of solid oral dosage forms such as tablets or capsules. Such kits preferably contain several unit dosage forms and may also include a card with the dosages oriented in the order of their intended use. If desired, they may also include, for example, in the form of numbers, letters, or other markings, or a calendar insert designating the days of the treatment schedule on which the dosages can be administered. Optionally, associated with such container may be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting approval by the agency of the manufacture, use, or sale for human administration.

[0146] The following representative examples contain important additional information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. The following examples are intended to help illustrate the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art upon consideration of this document, including the following examples and reference to the scientific and patent literature cited herein.

[0147] The contents of the cited references are incorporated herein by reference to help illustrate the state of the art.

[0148] Additionally, for purposes of the present invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "Organic Chemistry," Morrison & Boyd (3d Ed.), the entire contents of both of which are incorporated herein by reference.

[0149] 8.Synthesis Scheme Compounds of formula I can be prepared by those skilled in the art according to techniques and procedures recognized in the art. More specifically, compounds of formula I can be prepared as described in the schemes, methods, and examples described below. Those skilled in the art will recognize that individual steps in the following schemes can be modified to provide compounds of formula I. Reagents and starting materials are readily available to those skilled in the art. Unless otherwise specified, all substituents are as previously defined. [Example]

[0150] Synthesis Example Equipment Description 1 H NMR spectra were recorded on a Bruker Ascend 400 spectrometer. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants are in hertz (Hz). Splitting patterns indicate apparent multiplicity and are expressed as s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), and br (broad).

[0151] Analytical low-resolution mass spectra (MS) were recorded on a Waters ACQUITY UPLC equipped with an SQ Detector using a Waters CORTECS C18+, 2.7 μm 4.6×30 mm, using gradient elution.

[0152] Solvent A: 0.1% formic acid (FA) in water Solvent B: 0.1% FA in acetonitrile 5% ACN to 95% ACN in 1.0 min, hold for 1.0 min; Total time: 2.5 minutes, flow rate: 1.8 mL / min, column temperature: 40 degrees.

[0153] Intermediates Intermediate 1 [ka] Step 1 To a solution of 4-benzyloxypyridine (185 mg, 998 μmol) in DCM (10 mL) was added amino 2,4,6-trimethylbenzenesulfonate (236 mg, 1.1 mmol) at 25° C. The reaction mixture was stirred at 25° C. for 14 h. The mixture was concentrated under reduced pressure to give the desired crude product (400 mg) as a colorless oil. LC-MS: m / z 202[M+H] + .

[0154] Step 2 To a solution of the above product (187 mg, 487 μmol) in DMF (10 mL) was added CsCO (192 mg, 1.4 mmol) and but-3-yn-2-one (94 mg, 1.4 mmol). The reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was quenched with water (50 mL) and extracted with DCM (2 × 25 mL). The organic layer was washed with brine (20 mL), dried over anhydrous NaSO, and concentrated under reduced pressure. The residue was purified by column chromatography (eluted with PE / EA = 10 / 1) to give the desired product (90 mg, 35% yield) as a yellow solid. LC-MS: m / z 267 [M+H] + .

[0155] Step 3 To a solution of methyl(triphenyl)phosphonium bromide (241 mg, 675 μmol) in THF (10 ml) was added butyllithium (43.3 mg, 675 μmol) dropwise at −20° C. under N2. The reaction was stirred at −20° C. for 1 h. Then, a solution of 1-(5-benzyloxypyrazolo[1,5-a]pyridin-3-yl)ethanone (90 mg, 338 μmol) in THF (15 ml) was added dropwise at −20° C. The reaction mixture was stirred at 10° C. for 3 h. The reaction mixture was quenched with MeOH (3 ml) and concentrated under reduced pressure. The residue was purified by preparative HPLC (eluted with PE:EA=1 / 1) to give the desired crude product (41 mg) as a yellow solid. LC-MS: m / z 265 [M+H] + .

[0156] Step 4 To a solution of 5-benzyloxy-3-isopropenyl-pyrazolo[1,5-a]pyridine (600 mg, 2.3 mmol) in methanol (50 mL) was added Pd / C (60 mg). The reaction mixture was stirred under H at 30° C. for 48 hours. The reaction mixture was filtered and concentrated under reduced pressure to give the desired product (380 mg) as a yellow solid. LC-MS: m / z 177 [M+H] + .

[0157] Step 5 To a solution of 3-isopropylpyrazolo[1,5-a]pyridin-5-ol (650 mg, 3.7 mmol) and DIPEA (410 mg, 4.1 mmol) in DCM (15 mL) was added TfO (1.1 g, 4.1 mmol) under N at 0 °C. The reaction mixture was stirred at 0 °C for 2 h. The reaction mixture was washed with brine (15 mL) and dried over NaSO. The organic layer was filtered and the filtrate was concentrated to give the desired product (1.1 g) as a colorless oil. LC-MS: m / z 309 [M+H] + .

[0158] Step 6 To a solution of (3-isopropylpyrazolo[1,5-a]pyridin-5-yl)trifluoromethanesulfonate (1.1 g, 3.4 mmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (1.3 g, 5.1 mmol) in dioxane (10 mL) was added Pd(dppf)Cl (249 mg, 340 μmol) and KOAc (1.0 g, 10.2 mmol). The reaction mixture was stirred at 110° C. under N for 2 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give the desired crude product (950 mg) as a dark solid. LC-MS: m / z 287 [M+H] + .

[0159] Step 7 To a solution of 3-isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyridine (950 mg, 3.3 mmol) and 2,4-dichloro-5-fluoro-pyrimidine (665 mg, 4.0 mmol) in HO (1 mL) and 1,4-dioxane (15 mL) was added NaCO (1.2 g, 10 mmol) and Pd(dppf)Cl (242 mg, 332 μmol). The mixture was stirred at 110 °C under N for 6 h. The mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (PE / EA with 0-50% EA) to give the desired product (650 mg, 67% yield) as a yellow solid. LC-MS: m / z 291[M+H] + .

[0160] Intermediate 2 [ka] 3-Isopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazolo[1,5-a]pyridine (3.5 g, 12.2 mmol) and 2,4-dichloropyrimidine (2. To a solution of Pd(dppf)Cl (0.7 g, 18.4 mmol) was added Pd(dppf)Cl (0.9 g, 1.2 mmol) and NaCO (1.52 g, 14 mmol). The reaction mixture was stirred at 110 °C under N for 6 h. The mixture was concentrated under reduced pressure, and the residue was purified by flash column chromatography (PE with 0-50% EA) to give the desired product (2.1 g, 62% yield) as a yellow solid. LC-MS: m / z 273 [M+H] + .

[0161] Intermediate 3 [ka] Step 1 To a mixture of 2,6-dichloro-3-nitropyridin-4-amine (1500 g, 7.2 mol) and iron powder (1933 g, 34.6 mmol) in ethyl alcohol (45 L) and water (3 L), HCl (1.5 L, 12 M in HO) in water (6.5 L) was added dropwise over 1 h at 0 °C, and the resulting mixture was stirred at 95 °C for 16 h. The mixture was cooled to room temperature and then neutralized to pH = 9 with sodium bicarbonate (solid). The mixture was filtered and washed with ethyl acetate (500 mL). The filtrate was concentrated to remove the solvent. The solution was then extracted with ethyl acetate (9 L). The combined organic layers were washed with brine (1 L), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give 2,6-dichloropyridine-3,4-diamine (1200 g) as a yellow solid.

[0162] Step 2 A solution of 2,6-dichloropyridine-3,4-diamine (1200 g, 6.74 mol) in triethoxymethane (3 L) was stirred at 140° C. for 28 hours under a nitrogen atmosphere. The reaction was concentrated. Formic acid (1.5 L) was added. The resulting mixture was stirred at 120° C. for 2 hours. The solution was concentrated to give a residue, which was triturated with petroleum ether / ethyl acetate (1 / 1, 400 mL) to give 4,6-dichloro-1H-imidazo[4,5-c]pyridine (1360 g) as a yellow solid.

[0163] Step 3 A mixture of 4,6-dichloro-1H-imidazo[4,5-c]pyridine (1360 g, 5.8 mol), KCO (5680 g, 17.4 mol), and 2-iodopropane (3951 g, 23.2 mol) was dissolved in DMF (5 L) and stirred at 20 °C under a nitrogen atmosphere for 24 h. Ethyl acetate (40 L) was added to the reaction, and the mixture was filtered. The filtrate was concentrated to give a residue, which was purified on a silica gel column (3:1 to 1:1 petroleum ether / ethyl acetate) to give the desired product (710 g) as a yellow solid.

[0164] Step 4 To a mixture of 4,6-dichloro-1-isopropyl-imidazo[4,5-c]pyridine (250 mg, 1.1 mmol) in DMSO (10 mL) was added CsF (510 mg, 3.4 mmol), and the mixture was then stirred at 140° C. for 1.5 h. The mixture was poured into water (100 mL) and extracted with EA (3 × 30 mL). The combined organic layers were dried over NaSO and filtered. The filtrate was concentrated. The residue was purified by flash column (80 g of 200-300 mesh silica gel, PE / EA = 5 / 1 to 2 / 1) to give the desired product (210 mg, 75% yield) as a milky white solid. LC-MS: m / z 214.1 [M+H] + .

[0165] Step 5 To a solution of 6-chloro-4-fluoro-1-isopropyl-imidazo[4,5-c]pyridine (30 mg, 140 μmol) and 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (35.6 mg, 140 μmol) in dioxane (5 mL) was added potassium acetate (41.3 mg, 421 μmol) and cyclopentyl(diphenyl)phosphane dichloropalladium iron (15.4 mg, 21.1 μmol). The mixture was degassed with N and stirred at 110° C. for 16 hours. The mixture was filtered through a Celite pad. The filtrate was concentrated under reduced pressure to the desired crude product (50 mg) as a black oil, which was used directly in the next step. LC-MS: m / z 224.2 [M+H] + .

[0166] Step 6 To a solution of (4-fluoro-1-isopropyl-imidazo[4,5-c]pyridin-6-yl)boronic acid (50 mg, 224 μmol) and 2-chloro-4-iodo-pyrimidine (53.9 mg, 224 μmol) in dioxane (3 mL) was added Pd(dppf)Cl (24.6 mg, 33.6 μmol) and KOAc (66 mg, 672 μmol). The mixture was degassed with N and stirred at 110 °C for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography eluting with 0-60% ethyl acetate in petroleum ether to give the desired product (30 mg, 46% yield) as a white solid. LC-MS: m / z) 292.1 [M+H] + .

[0167] Intermediate 4 [ka] Step 1 To a solution of 5-bromo-2-nitro-pyridine (1 g, 4.9 mmol) and 1-isopropylpiperazine (631.6 mg, 4.9 mmol) in dioxane (40 mL) was added tris(dibenzylideneacetone)dipalladium(0) (451.1 mg, 492 μmol), (5-diphenylphosphanyl-9,9-dimethyl-xanthen-4-yl)-diphenylphosphane (570 mg, 985 μmol), and cesium carbonate (4.8 g, 14.8 mmol). The reaction mixture was then stirred at 110 °C under N for 3 h. The reaction mixture was concentrated under reduced pressure and purified by silica gel chromatography eluting with 1-100% ethyl acetate in petroleum ether to give the desired product (850 mg, 68% yield) as a yellow solid. LC-MS: m / z 251.1 [M+H] + .

[0168] Step 2 1-Isopropyl-4-(6-nitro-3-pyridyl)pyridinone in methanol (30 mL) To a solution of perazine (850 mg, 3.4 mmol) was added Pd / C (412 mg, 10%). The reaction mixture was then degassed with H2 three times and stirred at 25°C for 3 hours. The reaction mixture was filtered and then washed with methanol (20 mL). The combined solvents were concentrated under reduced pressure to give the desired product (620 mg, 82% yield) as a brown solid. LC-MS: m / z 221.2 [M+H] + .

[0169] Intermediate 5 [ka] Step 1 To a mixture of piperidine-2,4-dione (2.1 g, 18.5 mmol) and N-methylmethanamine (3.4 g, 74.2 mmol) in DCM (36 mL) and THF (18 mL) was added CHCOOH (10 mL), and the resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 3 hours. Sodium triacetoxyborohydride (7.8 g, 37.1 mmol) was added to the mixture, and the resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 12 hours. The reaction was quenched with water (50 mL) and concentrated in vacuo to remove DCM and THF. The mixture was extracted with DCM (3 × 100 mL). The organic solution was washed with brine (20 mL). The organic phase was dried over NaSO, filtered, and concentrated under reduced pressure to give the desired product (2.6 g), which was used in the next step without purification. LC-MS: m / z 141.2 [M+H] + .

[0170] Step 2 To a mixture of 4-(dimethylamino)-2,3-dihydro-1H-pyridin-6-one (1.0 g, 7.1 mmol) in methanol (15 mL) was added sodium borohydride (539 mg, 14.2 mmol), and the resulting mixture was stirred at 25 °C under a nitrogen atmosphere for 12 hours. The reaction was quenched with saturated aqueous NH4Cl (10 mL) and then concentrated in vacuo to remove MeOH. The aqueous solution was purified by reverse-phase column (C18, 40 g) eluting with (MeCN / water (0.1% NH4OH) = 1 / 10) to give the desired product (0.3 g, 32% yield) as a pale yellow solid. LC-MS: m / z 143.2 [M+H] + .

[0171] Step 3 To a mixture of 4-(dimethylamino)piperidin-2-one (270 mg, 1.9 mmol), 5-iodopyridin-2-amine (1.0 g, 4.7 mmol), and potassium phosphate (1.2 g, 5.7 mmol) in dioxane (26 mL) was added (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (162 mg, 1.1 mmol) and CuI (108 mg, 569 μmol), and the resulting mixture was stirred at 110 °C under a nitrogen atmosphere for 12 hours. The reaction was filtered. The filtrate was concentrated in vacuo to give a residue, which was purified by reverse-phase column (C18, 20 g) eluting with MeCN / water (0.1% NH4OH) = 1 / 10 to give the desired product (272 mg, 61% yield) as a pale yellow solid. LC-MS: m / z 235.2 [M+H] + .

[0172] Synthesis Example 1 [ka] To a mixture of 1-(6-amino-3-pyridyl)-4-(dimethylamino)piperidin-2-one (30 mg, 128 μmol) and 5-(2-chloropyrimidin-4-yl)-3-isopropyl-pyrazolo[1,5-a]pyridine (38.4 mg, 140.8 μmol) in dioxane (5 mL) was added cesium carbonate (125.1 mg, 384.1 μmol), tris(dibenzylideneacetone)dipalladium(0) (11.7 mg, 12.8 μmol), and RuPhos (11.9 mg, 25.6 μmol). The resulting mixture was stirred at 110° C. under a nitrogen atmosphere for 4 hours. The reaction mixture was extracted with EA (20 mL). The organic phase was washed with water (3 × 20 mL), brine (3 × 20 mL), and dried over NaSO. The mixture was concentrated under reduced pressure and purified by flash column chromatography (DCM / MeOH=10:1) to give the desired product (23.8 mg, 39% yield) as a yellow solid. LC-MS: m / z 471.2 [M+H] + .

[0173] Synthesis Examples 2 and 3 [ka] N-[5-[4-(dimethylamino)-1-piperidyl]-2-pyridyl]-4-(3-isopropylpyrazolo[1,5-a]pyridin-5-yl)pyrimidin-2-amine (210 mg, 459.9 μmol) was chiral separated by SFC using a mobile phase (hexane / EtOH / DEA=60 / 40 / 0.1) (wavelength: UV 214 nm, column: CHIRALCEL OD-H id 5.0 cm x length 25 cm, flow rate: 60 mL / min) to give Synthesis Example 2 (44.2 mg, 21% yield) as a yellow solid (LC-MS: m / z 471.2[M+H] + , ee value >99%) to give Synthesis Example 3 (41 mg, 19% yield) as a yellow solid (LC-MS: m / z 471.2 [M+H] + , ee value = 97%).

[0174] Synthesis Example 4 [ka] To a solution of 5-(4-isopropylpiperazin-1-yl)pyridin-2-amine (124.6 mg, 565 μmol) and 6-(2-chloropyrimidin-4-yl)-4-fluoro-1-isopropyl-imidazo[4,5-c]pyridine (150 mg, 514 μmol) in dioxane (15 mL) was added Pd(dba) (47.1 mg, 51 μmol), RuPhos (47.9 mg, 102 μmol), and CsCO (502.6 mg, 1.5 mmol). The mixture was stirred at 110 °C under N for 3 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with 0–10% MeOH in DCM to give the desired product (107.2 mg, 43% yield) as a yellow solid. LC-MS: m / z 476.2 [M+H] + .

[0175] Biological Example 1. Assay for Inhibition of CDK4 / Cyclin D1 I C 50 The CDK4 enzyme assay for determination was performed as follows. Phosphorylation of a peptide substrate by CDK4 / cyclin D1 was monitored using microfluidic kinase detection technology (Caliper). The total reaction volume was 15 μL, containing Buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl, 10 μM sodium orthovanadate, 10 μM beta-glycerophosphate), 200 μM ATP, 1 nM CDK4 / cyclin D1 (Thermofisher, PR8064A), 1 μM FL-34 (5-FAM-RRRFRPASPLRGPPK), and test compound at the appropriate dilution in DMSO. All components were added to a 384-well plate (Corning, 4514) and incubated at room temperature for 3 hours. The reaction was stopped by adding 15 μL of stop buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 Reagent (PerkinElmer, 760050)). The plate was then transferred to a Caliper EZ The reaction mixture containing the substrate and product was loaded onto a reader (EZ Reader II, PerkinElmer, HD-4HYSG2772) and introduced into the microfluidic chip for separation and detection. The IC of the test compound was calculated by fitting the inhibition curve with a four-parameter sigmoidal dose-response model using Xlfit5 / GraphPad Prism 5 software. 50 value was determined.

[0176] Biological Example 2. Assay for CDK6 / Cyclin D3 Inhibition I C 50 The CDK6 enzyme assay for determination was performed as follows. Microfluidic kinase detection technology (Caliper) was used to monitor the phosphorylation of a peptide substrate by CDK6 / cyclin D3. The total reaction volume was 15 μL, containing Buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 μM sodium orthovanadate, 10 μM beta-glycerophosphate), 300 μM ATP, 2 nM CDK6 / cyclin D3 (Carna, 04-107), 1 μM FL-34 (5-FAM-RRRFRPASPLRGPPK), and test compound at the appropriate dilution in DMSO. All components were added to a 384-well plate (Corning, 4514) and incubated at room temperature for 3 hours. The reaction was stopped by adding 15 μL of stop buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent (PerkinElmer, 760050)). The plate was then loaded onto a Caliper EZ Reader (EZ Reader II, PerkinElmer, HD-4HYSG2772), and the reaction mixture containing the substrate and product was introduced into a microfluidic chip for separation and detection. The IC values ​​of the test compounds were determined by fitting the inhibition curves with a four-parameter sigmoidal dose-response model using Xlfit5 / GraphPad Prism 5 software. 50 value was determined.

[0177] Biological Example 3. Assay for Inhibition of CDK2 / Cyclin E1 I C 50 The CDK2 enzyme assay for determination was performed as follows. Phosphorylation of a peptide substrate by CDK2 / cyclin E1 was monitored using microfluidic kinase detection technology (Caliper). The total reaction volume was 15 μL, containing Buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl2, 10 μM sodium orthovanadate, 10 μM beta-glycerophosphate), 100 μM ATP, 5 nM CDK2 / cyclin E1 (SignalChem, C29-18G), 5 μM FL-18 (5-FAM-QSPKKG-NH2), and test compound at the appropriate dilution in DMSO. All components were added to a 384-well plate (Corning, 4514) and incubated at room temperature for 3 hours. The reaction was stopped by adding 15 μL of stop buffer (180 mM HEPES (pH 7.5), 20 mM EDTA, Coating-3 reagent (PerkinElmer, 760050)). The plate was loaded onto a Caliper EZ Reader (EZ Reader II, PerkinElmer, HD-4HYSG2772), and the reaction mixture containing the substrate and product was introduced into a microfluidic chip for separation and detection. The IC of the test compound was determined by fitting the inhibition curve with a four-parameter sigmoidal dose-response model using Xlfit5 / GraphPad Prism 5 software. 50 value was determined.

[0178] IC of each example compound against CDK2, CDK4, and CDK6 50 Values ​​are provided in the synthesis examples below. IC 50 Values ​​are designated as "A," "B," "C," and "D" for values ​​below 10 nM, below 100 nM, below 1 μM, and above 1 μM, respectively.

[0179] Biological Example 4. Antiproliferative Assay in T47D Cells T47D is a human breast cancer cell line commonly used in biomedical research involving hormone expression in cancer cells.T47D cells are different from other human breast cancer cells in that their progesterone receptor (PR) is not regulated by estradiol, a hormone abundant in the cells themselves.T47D cells have been used to study the effects of progesterone on breast cancer and the corresponding transcriptional regulation caused by introduced drugs.These cells have been found to be highly resistant to estrogen and anti-estrogen.

[0180] T47D breast cancer cells (ATCC, HTB-133) from the American Type Culture Collection were seeded at 3000 cells / well in 96-well plates and incubated at 37°C and 5% CO2 in RPMI 1640 medium (Gibco, 31800105) containing 10% fetal bovine serum (FBS, Biowest, FB-1058). After overnight incubation, baseline values ​​of samples from one plate were measured using Cyquant reagent (Invitrogen, C35011) according to the manufacturer's recommendations. Cells were incubated with the detection reagent for 1 hour at 37°C, and then fluorescence was measured using a Spectra Max M5 (Molecular Devices, HD-4HYSG3196) with excitation at 485 nm and emission at 535 nm. The other plate was dosed with compounds at 10 dose concentrations ranging from 10 μM to 0.51 nM in a 3-fold dilution scheme. Six days after compound addition, Cyquant reagent was added and fluorescence was measured using a Spectra Max M5. IC values ​​of the antiproliferative activity of test compounds were calculated using Xlfit5 / GraphPad Prism 5 software. 50 Values ​​were determined from baseline-subtracted viability readout curves.

[0181] Biological Example 5. Inhibition of retinoblastoma protein (pRb) phosphorylation in T47D cells T47D breast cancer cells (ATCC, HTB-133) from the American Type Culture Collection were seeded at 40,000 cells / well in 96-well plates and incubated in RPMI 1640 medium (Gibco, 31800105) containing 10% fetal bovine serum (FBS, Biowest, FB-1058). Cells were then allowed to adhere overnight at 37°C and 5% CO2. The following day, compounds were titrated using a 3-fold dilution scheme, with the highest compound concentration tested being 10 μM. After 24 hours of incubation with compounds, cells were lysed in ice-cold lysis buffer containing a phosphatase inhibitor cocktail and 1 mM PMSF. Cell lysates (50 μL / well) were then transferred to ELISA plates (pRb Ser807 / 811 ELISA kit, Cell Signaling, 13152 or pRb Ser780 ELISA kit, Cell Signaling, 13016). The plate was incubated overnight at 4°C with constant, slow shaking. After incubation, the plate was washed according to the manufacturer's recommendations, and then 100 μL of reconstituted detection antibody was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed, and then 100 μL of reconstituted HRP-conjugated secondary antibody was added to each well and incubated at 37°C for 30 minutes. After incubation, the plate was washed again. 100 μL of TMB substrate was then added to each well and incubated at 37°C for 10 minutes or 25°C for 30 minutes. Finally, 100 μL of stop solution was added to each well and gently mixed for a few seconds. The plate was read on an Envision plate reader (PerkinElmer, 2104-0010) using the 96-well luminescence mode. The IC was calculated using a four-parameter sigmoidal dose-response model in Xlfit5 / GraphPad Prism 5 software. 50 values ​​were calculated.

[0182] The cell data obtained from Biological Examples 4 and 5 are listed below in Table A. IC 50Values ​​are indicated as "++++" for values ​​below 100 nM, "+++" for values ​​below 500 nM, "++" for values ​​below 1 μM, and "+" for values ​​above 1 μM, respectively.

[0183] [Table 1]

Claims

1. The following structural formula: 【Chemical 1】 A compound represented by:

2. 2. The compound according to claim 1, having an optical purity of 60% or more.

3. The compound according to claim 2, having an optical purity of 90% or more.

4. The compound according to claim 3, having an optical purity of 99% or more.

5. A pharmaceutical composition comprising an effective amount of the compound of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

6. 6. The pharmaceutical composition according to claim 5 for treating cancer, wherein the cancer is cancer of the bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, or skin; hematopoietic tumor of the lymphoid system; hematopoietic tumor of the myeloid system; thyroid follicular carcinoma; tumor of mesenchymal origin; tumor of the central nervous system or peripheral nervous system; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoma; thyroid follicular carcinoma; or Kaposi's sarcoma.

7. 6. The pharmaceutical composition of claim 5 for inhibiting the activity of a cyclin-dependent kinase (CDK) in a subject.

8. The pharmaceutical composition of claim 7 , wherein the subject has cancer.

9. 9. The pharmaceutical composition of claim 8, wherein the cancer comprises cancer of the bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, or skin, hematopoietic tumors of the lymphoid system, hematopoietic tumors of the myeloid system, follicular thyroid carcinoma, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, or Kaposi's sarcoma.

10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the lymphatic hematopoietic tumor is leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkitt's lymphoma.

11. The cancer is pRb ＋ The pharmaceutical composition according to any one of claims 7 to 9, wherein the cancer is breast cancer or hormone receptor (HR)-positive, HER2 / neu-negative breast cancer.

12. The cancer is estrogen receptor positive (ER) ＋ ), progesterone receptor positive (PR ＋ ), or ER ＋ PR ＋ The pharmaceutical composition of claim 11, wherein

13. The pharmaceutical composition according to claim 11 or 12, wherein the cancer is advanced, metastatic or recurrent breast cancer.

14. The pharmaceutical composition of claim 13, wherein the breast cancer is present in an adult woman or a postmenopausal woman.

15. 15. The pharmaceutical composition of any one of claims 8 to 14, for use in combination with a second agent selected from an aromatase inhibitor, a selective estrogen receptor modulator (SERM), a pure anti-estrogen without estrogen agonist activity, a compound that temporarily suppresses ovarian function, a compound that inhibits CYP3A4, or a monoclonal antibody against IGF-1 / IGF-2 or an antigen-binding fragment thereof.

16. 16. The pharmaceutical composition of claim 15, wherein the second agent comprises a compound that temporarily suppresses estrogen and / or progesterone production.

17. 17. The pharmaceutical composition of claim 16, wherein the second agent comprises a gonadotropin-releasing hormone (GnRH) agonist or a luteinizing hormone-releasing hormone (LH-RH) agonist.

18. 18. The pharmaceutical composition according to any one of claims 6 to 17, for use in combination with an immune checkpoint inhibitor, a receptor Tyr kinase inhibitor, and / or an antagonist of a hormone receptor.

19. 19. The pharmaceutical composition of claim 18, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor, or the hormone receptor antagonist comprises an estrogen receptor antagonist.

20. The following structural formula: 【Chemistry 2】 or a pharmaceutically acceptable salt, or stereoisomer thereof.

21. 21. The compound according to claim 20, wherein the optical purity of the compound, or a pharmaceutically acceptable salt thereof, is 60% or greater.

22. 22. The compound according to claim 21, wherein the optical purity of the compound, or a pharmaceutically acceptable salt thereof, is 90% or greater.

23. 23. The compound according to claim 22, wherein the optical purity of the compound, or a pharmaceutically acceptable salt thereof, is 99% or greater.

24. A pharmaceutical composition comprising an effective amount of the compound according to any one of claims 20 to 23, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

25. 25. The pharmaceutical composition according to claim 24 for treating cancer, wherein the cancer is cancer of the bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, or skin, hematopoietic tumors of the lymphoid system, hematopoietic tumors of the myeloid system, thyroid follicular carcinoma, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoma, thyroid follicular carcinoma, or Kaposi's sarcoma.

26. 25. The pharmaceutical composition of claim 24 for inhibiting the activity of a cyclin-dependent kinase (CDK) in a subject.

27. 27. The pharmaceutical composition of claim 26, wherein the subject has cancer.

28. 28. The pharmaceutical composition of claim 27, wherein the cancer comprises cancer of the bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, nose, head and neck, prostate, or skin, hematopoietic tumors of the lymphoid system, hematopoietic tumors of the myeloid system, thyroid follicular carcinoma, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoacanthoma, or Kaposi's sarcoma.

29. The pharmaceutical composition according to any one of claims 26 to 28, wherein the lymphatic hematopoietic tumor is leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkitt's lymphoma.

30. The cancer is pRb ＋ The pharmaceutical composition according to any one of claims 26 to 28, wherein the cancer is breast cancer or hormone receptor (HR)-positive, HER2 / neu-negative breast cancer.

31. The cancer is estrogen receptor positive (ER) ＋ ), progesterone receptor positive (PR ＋ ), or ER ＋ PR ＋ 31. The pharmaceutical composition of claim 30, wherein

32. 32. The pharmaceutical composition of claim 30 or 31, wherein the cancer is advanced, metastatic, or recurrent breast cancer.

33. 33. The pharmaceutical composition of claim 32, wherein the breast cancer is in an adult or postmenopausal woman.

34. 34. The pharmaceutical composition of any one of claims 27 to 33, for use in combination with a second agent selected from an aromatase inhibitor, a selective estrogen receptor modulator (SERM), a pure anti-estrogen without estrogen agonist activity, a compound that temporarily suppresses ovarian function, a compound that inhibits CYP3A4, or a monoclonal antibody against IGF-1 / IGF-2 or an antigen-binding fragment thereof.

35. 35. The pharmaceutical composition of claim 34, wherein the second agent comprises a compound that temporarily suppresses estrogen and / or progesterone production.

36. 36. The pharmaceutical composition of claim 35, wherein the second agent comprises a gonadotropin-releasing hormone (GnRH) agonist or a luteinizing hormone-releasing hormone (LH-RH) agonist.

37. 37. The pharmaceutical composition of any one of claims 25 to 36 for use in combination with an immune checkpoint inhibitor, a receptor Tyr kinase inhibitor, and / or an antagonist of a hormone receptor.

38. 38. The pharmaceutical composition of claim 37, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor, or the hormone receptor antagonist comprises an estrogen receptor antagonist.

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