CDK inhibitor

Abstract

The present invention provides compounds represented by the following structural formula: (I), or a pharmaceutically acceptable salt or stereoisomer thereof, which are useful for treating cancer. JPEG2023525005000016.jpg3974

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 on May 5, 2020. The entire content of the foregoing application is incorporated herein by reference.

Background Art

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

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

[0004] As with all kinases, the active site or ATP-binding site of CDK is a cleft between a small amino-terminal lobe and a larger carboxy-terminal lobe. From the structure of human CDK2, it has become clear that CDK has 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 and the positions of some important amino acid residues are not optimal for ATP binding. In the presence of cyclin, two α-helices reposition to allow ATP binding. One of them, the L12 helix immediately preceding the T-loop in the primary sequence, becomes a β-strand to assist in the repositioning of the T-loop, so that the T-loop no longer occludes the active site. Another α-helix, called the PSTAIRE helix, repositioned and helps to change the positions of important amino acid residues at the active site.

[0005] Therefore, only the cyclin-CDK complex has active kinase activity, and most of the known cyclin-CDK complexes regulate progression throughout the cell cycle. CDK is ubiquitous in all known eukaryotes, and their regulatory functions in the cell cycle are evolutionarily conserved. For example, yeast cells can grow normally when their CDK genes are replaced with homologous human genes. CDK exerts its regulatory functions by phosphorylating certain serine and threonine residues, as well as their substrates on the consensus sequence of [S / T]PX[K / R] (S / T is the target Ser or Thr for phosphorylation, 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 the regulation of the cell cycle. In mammalian cells, CDK1, together with its binding partners cyclin A2 and B1, can drive the cell cycle alone. Cyclin-CDK complexes in the early cell cycle stages can help activate cyclin-CDK complexes in later stages.

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

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

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

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

Summary of the Invention

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

[0012] In one aspect, the present invention provides a compound represented by the following structural formula:

Chemical formula

[0013] In another aspect, the present invention provides a compound represented by the following structural formula:

Chemical formula

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

[0015] The present disclosure further provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable composition comprising an effective amount of (1) a compound disclosed herein, or a pharmaceutically acceptable salt or stereoisomer thereof, or (2) 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 method of the invention, the 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 method of the 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, lymphoid hematopoietic tumors, myeloid hematopoietic tumors, thyroid follicular cancer, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, corneal tumor, thyroid follicular cancer, or Kaposi's sarcoma.

[0018] In certain embodiments of the method of the invention, the compound disclosed herein is administered together with any one of the second therapeutic agents described herein that similarly 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 the same, in any of the methods of the present invention described above. In one embodiment, there is provided a compound disclosed herein, or a pharmaceutically acceptable salt, or stereoisomer thereof, or a pharmaceutical composition comprising the same, for use in any of the methods of the present invention described above. 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 the same, for the manufacture of a medicament for any of the methods of the present invention described herein.

Embodiments for Carrying Out the Invention

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

[0021] The present invention also provides a pharmaceutical preparation 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 the treatment of cancer. In particular, these cancers can be any of the cancers described below in this specification, for example, colorectal cancer, breast cancer (including ER + HER2 - advanced or metastatic or recurrent breast cancer in adult women or postmenopausal women), 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 colorectal cancer, breast cancer (ER in adult women or postmenopausal women + HER2 -A method for treating cancer selected from the group consisting of breast cancer (including progressive, 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, comprising 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] In addition, 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 are colorectal cancer, breast cancer (ER in adult or postmenopausal women + HER2 - including progressive, 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, and are selected from the group consisting of.

[0025] Furthermore, the present invention provides a pharmaceutical formulation for use in a treatment 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 treating colorectal cancer, breast cancer (ER in adult or postmenopausal women + HER2 - including progressive, 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, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, or excipient.

[0026] The indications of treatable diseases and potential second therapeutic agents useful in combination therapies are described in more detail in the following section.

[0027] Any embodiment described herein that includes only one of the following sections or only the examples can be combined with any one or more additional embodiments of the invention, unless explicitly disclaimed or otherwise inappropriate / inapplicable.

[0028] 2. Definitions The compounds described herein can contain one or more chiral centers and, accordingly, can exist in various stereoisomers, such as enantiomers and / or diastereomers. For example, the compounds described herein can be in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers.

[0029] Mixtures of enantiomers and diastereomers 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 synthesis 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.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962), and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0030] When 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 defined as the weight of the named or depicted enantiomer in the mixture divided by the total weight of the mixture of both enantiomers.

[0031] If the stereochemistry of a disclosed compound is named or depicted by a structure and the named or depicted structure encompasses more than one stereoisomer (such as in the case of a pair of diastereomers), it should be understood that one of the included stereoisomers or any mixture of the included stereoisomers is included. It should be further understood that the stereoisomer purity of the named or depicted stereoisomer is at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 99 wt%, or 99.9 wt%. In this case, the stereoisomer purity is determined by dividing the total weight of the mixture of stereoisomers included in the name or structure by the total weight of the mixture of all stereoisomers.

[0032] When geometric isomers are depicted by a name or structure, it should be understood that the geometric isomer purity of the named or depicted isomer is at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 99 wt%, or 99.9% pure. 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 encompasses all enantiomerically pure, enantiomerically enriched, diastereomerically pure, diastereomerically enriched, and racemic mixtures of the compounds of the present invention, as well as diastereomeric mixtures.

[0034] The compounds described herein may also contain all isotopes of atoms that occur 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] Depending on the origin of the chemical substances used in the synthesis, it will be recognized that some variation in the natural isotope abundances will occur in the synthesized compounds. Thus, preparations of the compounds disclosed herein will essentially contain minor amounts of deuterated isotopologs. Despite this variation, the concentrations of the naturally abundant and stable hydrogen and carbon isotopes are low and not significant when compared to the degree of stable isotope substitution of 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 terms "tautomer" or "tautomeric" refer to two or more interconvertible compounds / substituents resulting from at least one formal shift of a hydrogen atom and at least one change in valence (e.g., from a single bond to a double bond, from a triple bond to a single bond, or vice versa). Exemplary tautomerizations include the tautomerization from keto to enol, from amide to imide, from lactam to lactim, from enamine to imine, and from enamine to (a different enamine). The present teachings encompass compounds in tautomeric forms, including forms not structurally depicted. All such isomeric forms of such compounds are explicitly included. If the tautomer of a compound is aromatic, the compound is aromatic. Similarly, if the tautomer of a compound is heteroaryl, the compound is heteroaryl.

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

Chemical Structure

[0038] It should be understood that if the compounds of this specification are represented by a structural formula or designated by a chemical name herein, all other tautomeric forms that may exist for that compound are encompassed by the structural formula.

[0039] The compounds of the present invention can exist in free form for therapeutic use or, where appropriate, as pharmaceutically acceptable salt forms.

[0040] The term "pharmaceutically acceptable salt" refers to salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and that have a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art, and for example, Berge et al., in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference, describe pharmaceutically acceptable salts in detail. The 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, glycerate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1~4 alkyl) 4 -It contains salts. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0041] Such pharmaceutically acceptable acid addition salts and general methodologies for their preparation 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 synonymously.

[0043] The “subject” is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, such as a pet (e.g., dog, cat, and the like), livestock (e.g., cow, sheep, pig, horse, and the like), and laboratory animals (e.g., rat, mouse, guinea pig, and the like).

[0044] The terms "administer", "administering", or "administration" refer to methods of introducing the compounds of the invention or compositions thereof into or onto a subject. These methods include, but are not limited to, intra-articular (within the 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 "treat", "treating", and "treatment" refer to reversing, alleviating, or inhibiting the progression of a disease described herein. In some embodiments, treatment can be administered after one or more signs or symptoms of a disease have occurred or been observed (i.e., therapeutic treatment). In other embodiments, treatment can be administered in the absence of signs or symptoms of a disease. For example, treatment can be administered to a susceptible subject prior to the onset of symptoms (i.e., prophylactic treatment) (e.g., taking into account the symptom history and / or exposure to a pathogen). Treatment can also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

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

[0047] Generally, the effective amount of the compounds taught herein will vary depending on various factors such as the particular 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 nonetheless can be routinely determined by one of ordinary skill in the art. The effective amount of the compounds 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 a beneficial or desired result, including, for example, clinical results, such as inhibiting, suppressing, or alleviating the symptoms of a condition being treated in the subject as compared to a control. For example, an effective amount can be administered in a unit dosage form (e.g., from 1 mg to about 50 g per day, such as from 1 mg to about 5 grams per day).

[0049] A "therapeutically effective amount" is an amount effective for the detectable killing or inhibition of cancer cell growth or spread, tumor size or number, or other measures of cancer level, stage, progression, or severity. 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 anticancer agent, its mode of administration, combination therapy with other treatments, and the like.

[0050] The general chemical terms used in the above formulas have their ordinary meanings.

[0051] As used herein, "h" refers to one or several hours, "min" refers to several or a few 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, "MtBE" 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 orally (by mouth), "qd" means once daily administration, "HPLC" means high performance liquid chromatography, "q2d" means single administration every two days, "q2dx10" means single administration every two days × 10 times, "VSMC" means vascular smooth muscle cells, and "XRD" refers to X-ray diffraction.

[0052] 3. Compounds Another aspect of the disclosure relates to the labeled compounds (radioactive labels, fluorescent labels, etc.) of the present invention that would be useful in assays for localizing and quantifying CDK in tissue samples, including human, both in vitro and in vivo, as well as for identifying CDK ligands by inhibition of the binding of the labeled compound. Accordingly, the disclosure includes such labeled compounds.

[0053] The disclosure further includes the isotope-labeled compounds of the present invention. An "isotope" or "radioactive label" compound is a compound of the present invention in which one or more atoms are replaced or substituted by atoms having an atomic weight or mass number different from that typically found in nature (i.e., naturally occurring). Suitable radionuclides that can be incorporated into the compounds of the present invention are 2H (also denoted as D in the case of deuterium), 3 H (also denoted 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 I, including but not limited to these. The radionuclide incorporated into this radiolabeled compound will depend on the specific use of the radiolabeled compound.

[0054] The present invention can further include a synthesis method for incorporating a radioisotope into the compounds of the present invention. Synthesis methods for incorporating radioisotopes into organic compounds are well known in the art, and those skilled 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 for identifying / evaluating compounds. For example, a newly synthesized or identified compound to be labeled (i.e., a test compound) can be evaluated for its ability to bind to CDK by monitoring its concentration fluctuations when in contact with CDK through tracking the label. For example, a test compound (labeled) can be evaluated for its ability to reduce the binding of another compound known to bind to CDK (i.e., a standard compound). Thus, the ability of a test compound to compete with a standard compound for binding to CDK is directly correlated with its binding affinity. Conversely, in some other screening assays, the standard compound is labeled and the test compound is not labeled. Therefore, the concentration of the labeled standard compound is monitored to evaluate the competition between the standard compound and the test compound, and thus the relative binding affinity of the test compound is confirmed.

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

[0057] 4. Treatable Diseases and Methods of Treatment Certain compounds of the present invention are selective inhibitors of CDK2, CDK4, and / or CDK6, and are thus useful for the treatment of diseases or disorders characterized by abnormal cell proliferation that can be inhibited by a decrease in the activity of CDK-cyclin complexes including CDK2, CDK4, and / or CDK6.

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

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

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

[0061] In certain embodiments, the compounds of the present invention inhibit CDK2 / 4 / 6 with similar IC 50 values, e.g., within 10-fold, 5-fold, 3-fold, or 2-fold of the IC 50 value. Such compounds of the present invention are useful for treating cancers associated with amplified or enhanced expression of cyclin D1 or E1 or E2.

[0062] CDK2 is the catalytic subunit of a CDK-cyclin complex whose activity is restricted to the G1-S phase of the cell cycle, and the cell makes the proteins necessary for mitosis and replicates their DNA. CDK2 forms a complex with cyclin E or A. Cyclin E binds to CDK2 in the G1 phase, which is necessary for the transition from the G1 phase to the S phase. On the other hand, CDK2 that binds to cyclin A is necessary to proceed through the S phase.

[0063] CDK2 is almost unnecessary in the cell cycle of normal functioning cells, but is important in the abnormal proliferation process of cancer cells. Overexpression of cyclin E occurs in many tumor cells, causing the cells to be dependent on CDK2 and cyclin E. Abnormal cyclin E activity is observed in breast cancer, lung cancer, colorectal cancer, gastric cancer, and bone cancer, as well as leukemia and lymphoma. Similarly, abnormal expression of cyclin A2 is associated with chromosomal instability and tumor growth, and inhibition leads to a decrease in tumor growth. Therefore, CDK2 and its cyclin binding partners represent possible therapeutic targets for new cancer therapeutics. Preclinical models have shown preliminary success in restricting tumor growth and have also been observed to reduce the side effects of current chemotherapeutic 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 that predict disease progression in either tamoxifen-resistant or metastatic breast cancer, that high expression of CycE2 is characteristic of the luminal B and HER2 subtypes of breast cancer, and that it strongly predicts a shortened survival without distant metastasis 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 anti-estrogen resistance in tamoxifen-resistant cells. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Moreover, ectopic expression of cyclin E1 or E2 also decreased sensitivity to inhibition of CDK4 but not CDK2. Furthermore, inhibition of CDK2 in E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition.

[0065] These data demonstrate that overexpression of cyclin E2 is a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition, that CDK2 inhibitors can effectively inhibit cyclin E1 and E2 overexpressing cells, overcome such resistance, and may be beneficial as a component of combination therapy in endocrine-resistant diseases to enhance the efficacy of other therapeutic agents. Similarly, the compounds of the subject having potent inhibitory activity against both CDK2 and CDK4 are expected to be effective against cancer cells that are either non-resistant or resistant to endocrine therapy or CDK4 inhibition.

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

[0067] The IC of the compound of the present invention against CDK2 / 4 / 6 50 value can be measured, for example, using the methods described in Examples 1 to 3 (incorporated herein by reference).

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

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

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

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

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

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

[0074] Thus, in certain embodiments, the compounds of the invention are useful for the treatment of pRb in mammals + cancer, 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 invention are also expected to be useful for the treatment of rhabdomyosarcoma in mammals (e.g., humans) (Saab et al., Mol. Cancer. Ther. 5(5):1299, 2006), and multiple myeloma including relapsed and refractory multiple myeloma (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) reported that inhibition of CDK4 / 6 in vivo can lead to a decrease in phosphorylation and thus an increase in the degradation of Cullin 3 Cdh1 by (APC / C SPOP ), which in turn leads to an increase in the level of PD-L1 on the tumor cell surface and a reduction in the number of tumor-infiltrating lymphocytes (TIL) in mouse tumors and primary human prostate cancer specimens. In other words, inhibition of CDK4 / 6 in vivo increases the PD-L1 protein level and contributes to an increase in resistance to immune checkpoint therapy targeting PD-1 (programmed cell death protein 1) and PD-L1 (ligand of PD-1). On the other hand, combining CDK4 / 6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumor regression and dramatically improves the overall survival rate in mouse tumor models.

[0076] Thus, in certain embodiments, the compounds of the present invention are used in combination with PD-1 / PD-L1 immune checkpoint inhibitors and can enhance the therapeutic effect on human cancer.

[0077] The 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 can be used with the compounds of the present invention to enhance the therapeutic effect on human cancer include monoclonal antibodies specific for CTLA-4, such as ipilimumab (Yervoy), or antigen-binding fragments thereof.

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

[0080] In certain embodiments, the compounds of the invention are used in combination with a Tyr kinase inhibitor, e.g., a receptor Tyr kinase (RTK) inhibitor, to enhance the therapeutic effect against human cancers. Exemplary Tyr kinase inhibitors include ALK inhibitors (such as crizotinib, ceritinib, alectinib, brigatinib, etc.), Bcr-Abl inhibitors (such as bosutinib, dasatinib, imatinib, nilotinib, ponatinib, etc.), BTK inhibitors (such as ibrutinib, etc.), c-Met inhibitors (such as crizotinib, cabozantinib, etc.), EGFR inhibitors (such as gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, etc.), JAK inhibitors (such as ruxolitinib, tofacitinib, etc.), MEK1 / 2 inhibitors (such as trametinib, etc.), PDGFR inhibitors (such as axitinib, gefitinib, imatinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib, sunitinib, etc.), RET inhibitors (such as vandetanib, etc.), Src family kinase inhibitors (such as bosutinib, dasatinib, ponatinib, vandetanib, etc.), and VEGFR family inhibitors (such as 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 (which is 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 (effective, e.g., in the treatment of lung cancer, colorectal cancer, and head and neck cancer), and anti-HER2 mAbs such as trastuzumab (effective, e.g., in the treatment of breast cancer).

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

[0084] The cancers treatable with the compounds of the present invention include non-Hodgkin lymphoma, malignant mesothelioma, non-small cell lung cancer, cholangiocarcinoma, soft tissue sarcoma, glioblastoma, (recurrent) brain tumor, hormone receptor-positive breast cancer, non-small cell lung cancer, brain metastases secondary to melanoma (including melanoma with positive 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, the treatable cancers include cancers 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, lymphoid hematopoietic tumors, myeloid hematopoietic tumors, thyroid follicular cancer, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, familial melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, corneal tumors, thyroid follicular cancer, Kaposi's sarcoma, squamous cell carcinoma, sarcoma, or tumors of mesenchymal origin.

[0086] In certain embodiments, the lymphoid hematopoietic tumors are leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, or Burkitt lymphoma.

[0087] In certain embodiments, the tumors of the central or peripheral nervous system are 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 invention and gemcitabine HCl.

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

[0092] In certain embodiments, the compound of the invention can be used in combination with other agents for the treatment of NSCLC, pancreatic cancer, ovarian cancer and metastatic breast cancer. For example, the compound of the invention can be used in a concurrent, 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. The compound of the invention can be administered concomitantly with, separately from, or sequentially to temozolomide.

[0095] Treatment of breast cancer In certain embodiments, the compound of the invention can be used to treat breast cancer.

[0096] Breast cancer is a major health burden worldwide, accounting for approximately 7% of all cancer-related deaths in the United States in 2016 alone. Of all breast cancers, approximately 75% are diagnosed as hormone receptor-positive (HR + ) breast cancer, which expresses estrogen receptor (ER) and / or progesterone receptor (PgR) and typically relies on the ER signaling pathway for growth and survival. That is, HR + breast cancer utilizes the biological functions of the ER pathway to promote the growth, development, and progression of breast cancer. On the other hand, because HR + breast cancer relies on ER signaling, such breast cancer is a therapeutic target 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 constitutes the treatment backbone for HR + breast cancer, but the effectiveness of endocrine therapy is limited by a high percentage of both existing and newly acquired resistance during treatment due to the existence of alternative survival or "escape" pathways. Many of the ER pathway and known escape pathways act through cyclin D-CDK4 / 6 inhibitors of the CDK4(INK4)-retinoblastoma (Rb) pathway to promote tumor growth. Thus, targeting both the ER pathway and the cyclin D-CDK4 / 6-INK4-Rb pathway usually leads to broader inhibition of tumor growth, prevents activation of escape pathways, and inhibits 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 - It is a HER2 / neu negative cancer, including progressive or metastatic or recurrent breast cancer. In certain embodiments, HR + HER2 - or ER + HER2 - Progressive or metastatic or recurrent breast cancer is present in adult women, or postmenopausal women.

[0099] In certain embodiments, the compounds of the invention are used either alone or in combination with an aromatase inhibitor (which inhibits estrogen production) to treat HR positive, HER2 negative progressive or 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, the compounds of the invention are used in combination with a compound that interferes with the ability of estrogen to stimulate the growth of breast cancer cells, for example, a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor and prevents estrogen binding, such as tamoxifen (NOLVADEX®) and toremifene (FARESTON®). Tamoxifen has been used for over 30 years to treat HR + breast cancer.

[0101] In certain embodiments, the compounds of the invention are used in combination with a pure anti - estrogen that does not have estrogen agonist activity, such as fulvestrant (FASLODEX®).

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

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

[0104] In certain embodiments, the compounds of the invention are used in combination with a compound that temporarily suppresses 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 used together with 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, dronedarone, fluvoxamine, imatinib, valerian, buprenorphine, caffeic acid phenethyl ester, cilostazol, fosaprepitant, gabapentin, lomitapide, orphenadrine, ranitidine, ranolazine, tacrolimus, ticagrelor, valproic acid, amlodipine, cannabidiol, dithiocarbamate, mifepristone, norfloxacin, delavirdine, gestodene, mibefradil, starfruit, thistle, niacinamide, ginkgo, piperine, isoniazid, and quercetin.

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

[0107] In certain embodiments, the compounds of the 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, abrogate pRb phosphorylation, and inhibit the activation of the S-phase transcriptional program. Representative PI3K inhibitors for use with the compounds of the invention include idelalisib, copanlisib, duvelisib, taselisib, perifosine, buparlisib, alpelisib, umbralisib, copanlisib, ductilisib, and buparlisib.

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

[0109] In addition, certain compounds of the present invention exhibit the advantageous property that they can cross the blood-brain barrier. Thus, such compounds can penetrate the brain and are therefore useful for the treatment of primary and metastatic brain tumors in which cells are proliferating and contain a functional and intact Rb1 gene. Such pRb + 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 in the treatment of 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 and causes chemical modification / damage (Marchesi et al., Pharmacol. Res. 56(4):275-287, 2007). Thus, in some embodiments, the compounds of the present invention are for primary and metastatic pRb such as glioblastoma and astrocytoma + For the treatment of brain tumors, for example, such metastases can be used in combination with temozolomide when such metastases are derived from melanoma, breast cancer, or NSCLC.

[0111] 5. Pharmaceutical Compositions The present invention provides a pharmaceutical composition 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 excipients" and "pharmaceutically acceptable carriers" mean substances that assist in the formulation and / or administration of the active agent to a subject and / or absorption by the subject and can be included in the compositions of the present disclosure without causing significant adverse toxic effects to the subject. Non-limiting examples of pharmaceutically acceptable carriers and excipients include water, NaCl, physiological saline, lactated Ringer's solution, ordinary sucrose, ordinary 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 adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for affecting osmotic pressure, buffering agents, coloring agents, and / or aromatic substances, and the like, that do not react detrimentally with the compounds provided herein or interfere with their activity. 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 can be administered to patients 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 transplantation, farnesyl transferase inhibitors, bisphosphonates, thalidomide, cancer vaccines, hormone therapy, antibodies, radiation, etc.). For example, the additional therapeutic agent for combined administration or inclusion in a pharmaceutical composition with a compound of the present invention can be one or more other anti-cancer agents.

[0114] As described herein, the compositions of the present invention, as used herein, include the compounds of the present invention together with a pharmaceutically acceptable carrier which includes any solvent, diluent, or other vehicle, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, suitable 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 the formulation of pharmaceutical compositions and known techniques for their preparation. The use thereof is contemplated to be within the scope of the present invention, except when any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effect or otherwise interacting in a detrimental manner with any other component of the pharmaceutical composition. Some examples of materials which 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 such as sodium carboxymethyl cellulose, 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, sesame oil, castor oil, olive oil, corn oil and soybean oil, glycols such as propylene glycol, esters such as ethyl oleate and ethyl laurate, agar, buffering agents such as magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and likewise, coloring agents, release agents, coating agents, sweetening agents, flavoring agents and perfuming agents, preservatives and antioxidants may also be present in the composition.

[0115] 6. Formulation The present invention also encompasses a class of compositions comprising the active compounds of the present invention, together 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 together with a pharmaceutically acceptable carrier.

[0117] In certain embodiments, the present invention provides a pharmaceutical formulation for treating cancers selected from the group consisting of colorectal cancer, mantle cell lymphoma, breast cancer (including ER + HER2 - progressive or metastatic or recurrent breast cancer in adult or post-menopausal women), glioblastoma, acute myeloid leukemia, and lung cancer, particularly NSCLC, comprising a compound of the present invention or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier.

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

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

[0121] In certain embodiments, the invention also provides a pharmaceutical formulation comprising a compound of the 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 invention can preferably be administered by such routes in the form of a pharmaceutical composition adapted to any suitable route and in an amount effective for the intended treatment. The compounds and compositions of the invention are in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles, and can be administered, for example, orally, to the mucosa, topically, rectally, to the lungs by inhalation spray, etc., or parenterally, including intravascularly, intravenously, intraperitoneally, subcutaneously, intramuscularly, intrasternal, and by injection techniques.

[0123] The pharmaceutically active compounds of the invention can be processed according to conventional pharmaceutical methods to produce agents for administration to patients including humans and other mammals.

[0124] For oral administration, the pharmaceutical composition can be, for example, in the form of tablets, capsules, suspensions, or liquids. The pharmaceutical composition is preferably prepared in the form of dosage units containing a specific amount of the active ingredient.

[0125] Examples of such dosage units are tablets or capsules. For example, a suitable daily dose for a human or other mammal can vary depending on the condition of the patient and other factors, but can also be determined in a routine manner in this case.

[0126] The amount of the compound administered and the dosing schedule for treating a disease state with the compounds and / or compositions of the invention depend on various factors including the age, weight, sex, and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the particular compound employed. Accordingly, the dosing schedule can vary widely but can be determined routinely using standard methods. As described above, the daily dose can be administered in a single dose or divided into two, three, four, or more administrations.

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

[0128] In the case of skin conditions, it may be preferable to apply the topical preparation of the compounds of the invention to the affected area two to four times a day. 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 be present in the formulation in an amount of from 0.001% to 10% w / w, for example, 1% to 2% by weight, although amounts of up to 10% w / w of the formulation may be used, preferably 5% w / w or less, more preferably 0.1% to 1%.

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

[0130] The phase may contain only an emulsifier, but may contain a mixture of at least one emulsifier and a fat or an oil, or a mixture with both a fat and an oil. Preferably, the 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, with or without a stabilizer, the emulsifier constitutes a so-called emulsifying wax, and the wax, together with the oil and the fat, constitutes a 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 containing wax, or other materials well-known in the art.

[0131] Due to the very low solubility of the active compound in most oils that are likely to be used in pharmaceutical emulsion formulations, the selection of a suitable oil or fat for the formulation is based on achieving the desired aesthetic properties. Thus, the cream should preferably be a non-greasy, non-staining, washable product with a suitable consistency to avoid leakage from a tube or other container. Straight-chain or branched-chain monobasic or dibasic alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate, or a blend of branched-chain esters can be used. These can be used alone or in combination, depending on the required properties.

[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 in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent for the active ingredient.

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

[0135] Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions can 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 can 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 buffer solutions. Other adjuvants and modes of administration are well and widely known in the pharmaceutical field. The active ingredient can also be administered by injection as a composition comprising a suitable carrier containing physiological saline, dextrose, or water, or cyclodextrin (i.e., Captisol), cosolvent solubilization (i.e., propylene glycol) or micellar solubilization (i.e., Tween80).

[0136] Sterile injectable preparations can also be sterile injection solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils have conventionally been employed as a solvent or suspending medium. For this purpose, any bland fixed oil containing synthetic monoglycerides or diglycerides can be employed. In addition, fatty acids such as oleic acid have been 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 a suitable non-irritating excipient such as cocoa butter and polyethylene glycol, which is solid at room temperature but liquid at rectal temperature, and melts in the rectum to release the drug, with the drug.

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

[0140] An alternative composition of the present invention comprises a compound of the formula described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle. Such a composition can optionally contain one or more additional therapeutic agents, including, for example, a kinase inhibitor (small molecule, polypeptide, antibody, etc.), an immunosuppressant, an anticancer agent, an antiviral agent, an anti-inflammatory agent, an antifungal agent, an antibiotic, or an anti-angiogenic compound.

[0141] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a patient together with a compound of the present invention, does not destroy its pharmacological activity, and is non-toxic when administered in a dosage sufficient to deliver a therapeutically effective amount of the compound. Pharmaceutically acceptable carriers, adjuvants, or vehicles that can be used in the pharmaceutical compositions of the present invention include ion exchange agents, 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, buffering 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 carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin, but are not limited thereto. Cyclodextrins such as u-, P-, and y-cyclodextrins, or chemically modified derivatives such as hydroxyalkyl cyclodextrins containing 2 and 3-hydroxypropyl-cyclodextrin, or other solubilizing derivatives can also be advantageously used to enhance the delivery of the compounds of the formulas 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. In the case of 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 the oil phase and combined with emulsifying and / or suspending agents.

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

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

[0145] 7. Therapeutic Kit One aspect of the present invention relates to a kit for conveniently and effectively practicing a method or use according to the present invention. Generally, a pharmaceutical pack or kit includes 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 include several unit dosage forms and may also include a card having dosages oriented in the order of their intended use. If desired, a memory aid can be provided, for example, in the form of numbers, letters, or other markings, or with a calendar insert specifying the dates of the treatment schedule on which the dosages can be administered. Optionally associated with such containers may be a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval of the agency for manufacture, use, or sale for human administration.

[0146] The following representative examples include important additional information, illustrations, and guidance that may be adapted to the practice of the invention in its various embodiments and their equivalents. The following examples are intended to assist in illustrating the invention and are not intended, nor should they be construed, to limit 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 review of the following examples, as well as the references to scientific and patent literature cited herein.

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

[0148] In addition, for the purposes of the present invention, chemical elements are those defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th thIt is identified according to the periodic table of elements on the front and back covers of the Ed. In addition, the 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. Synthetic Scheme The compounds of Formula I can be prepared by one of ordinary skill in the art according to techniques and procedures recognized in the art. More specifically, the compounds of Formula I can be prepared as described in the schemes, methods, and examples set forth below. Those skilled in the art will recognize that the individual steps in the following schemes can be varied to provide the compounds of Formula I. Reagents and starting materials are readily available to one of ordinary skill in the art. Unless otherwise specified, all substituents are as previously defined.

Examples

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

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

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

[0153] Intermediate Intermediate 1 [Chemical formula] 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 hours. The mixture was concentrated under reduced pressure to obtain 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 Cs 2 CO 3 (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 hours. 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) and dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The residue was purified by column chromatography (eluting with PE / EA = 10 / 1) to obtain 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) at N 2It was added dropwise at -20 °C below. The reaction product was stirred at -20 °C for 1 hour. Subsequently, 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 below. The reaction mixture was stirred at 10 °C for 3 hours. 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 obtain the desired crude product (41 mg) as a yellow solid. LC-MS: m / z 265 [M+H] + .

[0156] Step 4 Pd / C (60 mg) was added to a solution of 5-benzyloxy-3-isopropenyl-pyrazolo[1,5-a]pyridine (600 mg, 2.3 mmol) in methanol (50 mL). The reaction mixture was stirred at 30 °C for 48 hours under H 2 below. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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), Tf 2 O (1.1 g, 4.1 mmol) was added at 0 °C below. The reaction mixture was stirred at 0 °C for 2 hours. The reaction mixture was washed with brine (15 mL) and dried over Na 2 SO 2 above. The organic layer was filtered and the filtrate was concentrated to obtain the desired product (1.1 g) as a colorless oil. LC-MS: m / z 309 [M+H] 4 . + .

[0158] Step 6 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 with Pd(dppf)Cl 2 (249 mg, 340 μmol) and KOAc (1.0 g, 10.2 mmol). The reaction mixture was stirred at 110 °C for 2 h under N 2 . The mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the desired crude product (950 mg) as a dark solid. LC-MS: m / z 287 [M+H] + .

[0159] Step 7 H 2 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 H 2 CO 3 (1 mL) and 1,4-dioxane (15 mL) was added with Na 2 CO 2 (1.2 g, 10 mmol) and Pd(dppf)Cl + (242 mg, 332 μmol). The mixture was stirred at 110 °C for 6 h under N

[0160] Intermediate 2 [Chemical formula] A solution of 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 (2.7 g, 18.4 mmol) in water (3 mL) and 1,4-dioxane (60 mL) was added with Pd(dppf)Cl 2 (0.9 g, 1.2 mmol) and Na 2 CO 3 (1.52 g, 14 mmol). The reaction mixture was stirred at 110 °C for 6 h under N 2 . The mixture was concentrated under reduced pressure and the residue was purified by flash column chromatography (PE containing 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 [Chemical Structure Diagram] 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 ethanol (45 L) and water (3 L) was added dropwise HCl (1.5 L, 12 M in H 2 O) in water (6.5 L) at 0 °C over 1 h, 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 hydrogen carbonate (solid). The mixture was filtered and washed with ethyl acetate (500 mL). The filtrate was concentrated to remove the solvent. Then the solution was 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 h under a nitrogen atmosphere. The reaction mixture was concentrated. Formic acid (1.5 L) was added. The resulting mixture was stirred at 120 °C for 2 h. 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), K 2 CO 3 (5680 g, 17.4 mol) and 2-iodopropane (3951 g, 23.2 mol) was dissolved in DMF (5 L) and stirred at 20 °C for 24 h under a nitrogen atmosphere. Ethyl acetate (40 L) was added to the reaction mixture and the mixture was filtered. The filtrate was concentrated to give a residue, which was purified by silica gel column (petroleum ether / ethyl acetate 3:1 - 1:1) 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 then the mixture was stirred at 140 °C for 1.5 h. The resulting mixture was poured into water (100 mL) and extracted with EA (3 × 30 mL). The combined organic layers were dried over Na 2 SO 4 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 - 2 / 1) to give the desired product (210 mg, 75% yield) as an off-white solid. LC-MS: m / z 214.1 [M+H] + 。

[0165] Step 5 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 with 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 2 and stirred at 110 °C for 16 h. The mixture was filtered through a pad of Celite. The filtrate was concentrated under reduced pressure until the desired crude product (50 mg) was obtained 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) were added Pd(dppf)Cl 2 (24.6 mg, 33.6 μmol) and KOAc (66 mg, 672 μmol). The mixture was degassed with N 2 and stirred at 110 °C for 16 h. The mixture was concentrated under reduced pressure. The residue was purified by flash chromatography eluting with ethyl acetate in 0 - 60% 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 [Chemical formula] Step 1 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 with 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). Then, the reaction mixture was stirred at 110 °C for 3 h under N 2 and concentrated under reduced pressure, and purified by silica gel chromatography eluting with ethyl acetate in 1 - 100% petroleum ether to obtain the desired product (850 mg, 68% yield) as a yellow solid. LC-MS: m / z 251.1 [M+H] + .

[0168] Step 2 Pd / C (412 mg, 10%) was added to a solution of 1-isopropyl-4-(6-nitro-3-pyridyl)piperazine (850 mg, 3.4 mmol) in methanol (30 mL). Then, the reaction mixture was degassed three times with H 2 and stirred at 25 °C for 3 h. 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

Chemical Structure

[0170] Step 2 Sodium borohydride (539 mg, 14.2 mmol) was added to a mixture of 4-(dimethylamino)-2,3-dihydro-1H-pyridin-6-one (1.0 g, 7.1 mmol) in methanol (15 mL), and the resulting mixture was stirred at 25 °C for 12 h under a nitrogen atmosphere. The reaction was quenched with saturated NH 4 Cl aqueous solution (10 mL), 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% NH 4 OH) = 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 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 to (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (162 mg, 1.1 mmol) and CuI (108 mg, 569 μmol). The resulting mixture was stirred at 110 °C for 12 h under a nitrogen atmosphere. The reaction mixture was filtered. The filtrate was concentrated in vacuo to give a residue, which was purified by a reverse-phase column (C18, 20 g) eluting with (MeCN / water (0.1% NH 4 OH) = 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

Chemical formula

[0173] Synthesis Examples 2 and 3 [Chemical Formula] 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 subjected to chiral separation by SFC using a mobile phase (hexane / EtOH / DEA = 60 / 40 / 0.1) (wavelength: UV 214 nm, column: CHIRALCEL OD-H inner diameter 5.0 cm × length 25 cm, flow rate: 60 mL / min), yielding Synthesis Example 2 (44.2 mg, 21% yield) as a yellow solid (LC-MS: m / z 471.2 [M+H] + , ee value > 99%), and Synthesis Example 3 (41 mg, 19% yield) was obtained as a yellow solid (LC-MS: m / z 471.2 [M+H] + , ee value = 97%).

[0174] Synthesis Example 4 [Chemical Formula] 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), Pd 2 (dba) 3 (47.1 mg, 51 μmol), RuPhos (47.9 mg, 102 μmol) and Cs 2 CO 3 (502.6 mg, 1.5 mmol) were added. The mixture was stirred at 110 °C for 3 hours under N 2 . The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography eluting with MeOH in 0 - 10% 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 IC 50 The CDK4 enzyme assay for determination was performed as follows. Using microfluidic kinase detection technology (Caliper), phosphorylation of a peptide substrate by CDK4 / Cyclin D1 was monitored. 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 2 , 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 the test compound at an 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 the addition of 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 50 value of the test compound was determined by fitting an inhibition curve using a 4-parameter sigmoid dose-response model with Xlfit5 / GraphPad Prism 5 software.

[0176] Biological Example 2. Assay for Inhibition of CDK6 / Cyclin D3 IC 50The CDK6 enzyme assay for determination was performed as follows. Using the microfluidic kinase detection technology (Caliper), phosphorylation of the peptide substrate by CDK6 / cyclin D3 was monitored. 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 2 , 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 the test compound at an 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 the addition of 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 the microfluidic chip for separation and detection. The IC 50 value of the test compound was determined by fitting the inhibition curve using the 4-parameter sigmoid dose-response model with Xlfit5 / GraphPad Prism 5 software.

[0177] Biological Example 3. Assay for Inhibition of CDK2 / Cyclin E1 IC 50 The CDK2 enzyme assay for determination was performed as follows. Using the microfluidic kinase detection technology (Caliper), phosphorylation of the peptide substrate by CDK2 / cyclin E1 was monitored. The total reaction volume was buffer A (100 mM HEPES (pH 7.5), 0.1% BSA, 0.01% Triton X-100, 1 mM DTT, 10 mM MgCl 2, 15 μL, containing 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 the test compound at an 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 the addition of 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 50 value of the test compound was determined by fitting an inhibition curve to a 4-parameter sigmoid dose-response model using Xlfit5 / GraphPad Prism 5 software.

[0178] The IC 50 values for each exemplary compound against CDK2, CDK4, and CDK6 are provided in the following synthesis examples. The IC 50 values are shown as "A", "B", "C", and "D" for values of 10 nM or less, 100 nM or less, 1 μM or less, and greater than 1 μM, respectively.

[0179] Biological Example 4. Anti-proliferation assay in T47D cells T47D is a human breast cancer cell line commonly used in biomedical research involving hormonal expression in cancer cells. T47D cells differ from other human breast cancer cells in that their progesterone receptor (PR) is not regulated by estradiol, a hormone that is abundant within the cell itself. T47D cells have been employed in the study of the effects of progesterone on breast cancer and the corresponding transcriptional regulation induced by introduced drugs. The cells have been found to be highly resistant to estrogen and anti-estrogens.

[0180] T47D breast cancer cells (ATCC, HTB-133) from the American Type Culture Collection were seeded into 96-well plates at 3000 cells / well and incubated in RPMI 1640 medium (Gibco, 31800105) containing 10% fetal bovine serum (FBS, Biowest, FB-1058) at 37 °C, 5% CO 2 2. After overnight incubation, the baseline values of the samples from one plate were measured using the Cyquant reagent (Invitrogen, C35011) according to the manufacturer's recommendations. The cells were incubated with the detection reagent at 37 °C for 1 hour and then fluorescence was measured at an excitation of 485 nm and an emission of 535 nm using a Spectra Max M5 (Molecular Devices, HD-4HYSG3196). Compounds were added to other plates at 10 dose concentrations from 10 μM to 0.51 nM in a 3-fold dilution scheme. On the 6th day after compound addition, the Cyquant reagent was added and fluorescence was measured using a Spectra Max M5. The IC 50 value of the test compound's anti-proliferative activity was determined from the survival reading curve with the baseline subtracted.

[0181] Biological Example 5. Inhibition of phosphorylation of retinoblastoma protein (pRb) in T47D cells T47D breast cancer cells (ATCC, HTB-133) from the American Type Culture Collection were seeded into 96-well plates at 40,000 cells / well and incubated in RPMI 1640 medium (Gibco, 31800105) containing 10% fetal bovine serum (FBS, Biowest, FB-1058). Then the cells were incubated at 37 °C, 5% CO 2It was allowed to adhere overnight. The next day, the compound was titrated in a 3-fold dilution scheme, and the highest compound concentration tested was 10 μM. After 24-hour incubation with the compound, the cells were lysed in ice-cold lysis buffer containing a phosphatase inhibitor cocktail and 1 mM PMSF. Then, the cell lysate (50 μL / well) was transferred to an ELISA plate (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 shaking at a constant low speed. After incubation, the plate was washed according to the manufacturer's recommendation, 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. Then, 100 μL of TMB substrate was added to each well and incubated at 37 °C for 10 minutes or at 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 50 value was calculated using the 4-parameter sigmoid dose-response model of Xlfit5 / GraphPad Prism 5 software.

[0182] The cell data obtained from Biological Examples 4 and 5 are listed in Table A below. The IC 50 values are shown as "++++" for values of 100 nM or less, "+++" for values of 500 nM or less, "++" for values of 1 μM or less, and "+" for values exceeding 1 μM, respectively.

[0183] [Table 1]

Claims

1. A compound represented by the following structural formula: 【Chemical 1】 or a pharmaceutically acceptable salt or stereoisomer thereof.

2. A pharmaceutical composition comprising an effective amount of the compound according to claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2 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, lymphoid hematopoietic tumors, myeloid hematopoietic tumors, thyroid follicular cancer, tumors of mesenchymal origin, tumors of the central or peripheral nervous system, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, corneal tumor, thyroid follicular cancer, or Kaposi's sarcoma.

4. The pharmaceutical composition according to claim 2 for inhibiting the activity of cyclin-dependent kinase (CDK) in a subject.

5. The pharmaceutical composition according to claim 4, wherein the subject has cancer.

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

7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the lymphoid hematopoietic tumor is leukemia, acute lymphoblastic 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.

8. wherein the cancer is pRb ＋ The pharmaceutical composition according to any one of claims 4 to 6, wherein the cancer is breast cancer, or hormone receptor (HR)-positive and HER2 / neu-negative breast cancer.

9. wherein the cancer is estrogen receptor positive (ER ＋ ), progesterone receptor positive (PR ＋ ), or ER ＋ PR ＋ ; the pharmaceutical composition according to claim 8.

10. The pharmaceutical composition according to claim 8 or 9, wherein the cancer is advanced or metastatic or recurrent breast cancer.

11. The pharmaceutical composition according to claim 10, wherein the breast cancer is present in an adult female or a postmenopausal female.

12. The pharmaceutical composition according to any one of claims 5 to 11 for use in combination with a second agent selected from an aromatase inhibitor, a selective estrogen receptor modulator (SERM), a pure anti-estrogen having no 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.

13. The pharmaceutical composition according to claim 12, wherein the second agent comprises a compound that temporarily suppresses estrogen and / or progesterone production.

14. The pharmaceutical composition according to claim 13, wherein the second agent comprises a gonadotropin-releasing hormone (GnRH) agonist or a luteinizing hormone-releasing hormone (LH-RH) agonist.

15. The pharmaceutical composition according to any one of claims 3 to 14 for use in combination with an immune checkpoint inhibitor, a receptor Tyr kinase inhibitor, and / or an antagonist of a hormone receptor.

16. The pharmaceutical composition according to claim 15, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor, or the antagonist of the hormone receptor comprises an antagonist of the estrogen receptor.

Images