Compounds for treatment of cancer

Abstract

The present invention relates to 4-((6-(2, 2-difluoroethyl)-8-(2-hydroxy-2-methylcyclopentyl)-7-oxo-7, 8-dihydropyrido [2, 3-d] pyrimidin-2-yl) amino) piperidine-1-sulfonamide or a pharmaceutically acceptable salt thereof, to compositions comprising them, to processes for their preparation, to intermediates used in such processes, and to methods for their preparation. And to methods of treating abnormal cell growth, including cancer, using such compounds, salts, and compositions. The present disclosure also relates to crystalline Form 1 thereof, to pharmaceutical compositions comprising Form 1, and to the use of Form 1 for the treatment of cancer.

Description

Technical Field

[0001] The present disclosure relates to novel 4-((6-(2,2-difluoroethyl)-8-(2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I) and its pharmaceutically acceptable salts, to pharmaceutical compositions comprising such compounds and salts, and to their use for treating CDK-related diseases such as cancer. The present disclosure also relates to a crystalline form of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide free base (referred to herein as "Form 1"), to pharmaceutical compositions comprising Form 1, and to the use of Form 1 for treating CDK-related diseases such as cancer. Background Art

[0002] Overcoming checkpoints that impede cell cycle progression is fundamental to tumor development. The cell cycle consists of a mitotic phase, during which DNA segregation and cell division are completed, and an interphase, during which G1 and G2 checkpoints occur before and after DNA synthesis, respectively (Choi et al., Signaling through cyclin D-dependent kinases. Oncogene 2014, 33(15):1890-903). Cells in G1 require the activity of cyclin-dependent kinase (CDK) 4 / 6-cyclin (CCN)-D to phosphorylate the retinoblastoma (Rb) tumor suppressor protein, which is further phosphorylated by CDK2-CCNE. Phosphorylation of Rb leads to its release from a pre-transcriptional complex containing the E2F transcription factor (TF) that regulates gene expression required for S-phase completion. Multiple genetic lesions that enhance this specific signaling event have been identified in multiple tumor types. For example, CCND1 and CCNE1 amplifications are common, as are deletions of Rb or the endogenous CDK4-CCND inhibitor p16. The prediction that pharmacological targeting of the CDK4 / 6-Rb axis is effective in cancer was confirmed by the clinical success of palbociclib in combination with anti-estrogens in hormone receptor (HR)+ breast cancer (Cristofanilli et al., Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): Final analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol 2016, 17(4):425-39).

[0003] Targeted therapies often result in initial clinical benefit, followed by acquired resistance through mutations or activation of orthologous pathways (Chong et al., Nat. Med. 2013, 19(11):1389 - 400). Resistance to palbociclib in preclinical cell models can occur through deletion of Rb or upregulation of CCNE1, while CCNE1 - amplified cell lines are sensitive to CDK2 / 4 / 6 inhibition (Herrera - Abreu et al., Cancer Res. 2016, 76(8):2301 - 13). In addition to ER - positive breast cancer patients refractory to palbociclib, several tumor types, including triple - negative breast cancer (TNBC), ovarian cancer, and others, have cyclin E - amplified alleles (CCNE1 or CCNE2) (The Cancer Genome Atlas Network, Nature 2012, 490(7418):61 - 70).

[0004] Based on this principle, developing potent CDK2 / 4 / 6 inhibitors has the potential to be an effective therapy for treating HER2 - negative advanced or metastatic breast cancer and other tumor types with increased cyclin E expression / CDK activity (including TNBC and ovarian cancer). CDK6 inhibition is known to be a safety burden to consider, which can lead to hematological adverse events in humans and has been observed for many CDK4 / 6 inhibitors (Desnoyers et al., Cancer Treat. Rev. 2020, 90:102086, PMID:32861975; Sun et al., J. Clin. Pharm. 2017, 57(9):1159 - 1173; Goel et al., Nat. Rev. Cancer 2022, 22:356 - 372). These adverse events (mainly neutropenia) can be dose - limiting and may affect the achievable efficacy of these molecules. However, although excessive CDK6 inhibition may lead to adverse events, it remains a potentially important CDK to inhibit in addition to CDK2 / 4 because it is also associated with CDK6 - driven resistance upon long - term use of CDK4 / 6 inhibitors (Yang et al., Oncogene 2017, 36:2255 - 2264).

[0005] Therefore, there remains a need for new CDK2 / 4 / 6 inhibitors with a reduced incidence and severity of hematological adverse events, which can allow for higher exposure and lead to more robust cell cycle inhibition and improved efficacy. SUMMARY OF THE INVENTION

[0006] Part of the present disclosure provides novel 4-((6-(2,2-difluoroethyl)-8-(2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I) and its pharmaceutically acceptable salts. The compounds of the present disclosure and their pharmaceutically acceptable salts can inhibit the activities of CDKs (including CDK2, CDK4, and / or CDK6), thereby achieving biological functions. Also provided are pharmaceutical compositions and medicaments that comprise a compound or salt of formula (I) alone or in combination with an additional anti-cancer therapeutic agent or palliative agent. Part of the present disclosure also provides methods for preparing the compounds or salts of formula (I), compositions comprising the compounds or salts of formula (I), and methods of using them.

[0007] According to an embodiment of the present disclosure, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0008]

[0009] In some aspects and embodiments, the present disclosure provides an anhydrous crystalline form of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide free base (referred to herein as "Form 1").

[0010] The following describes embodiments of the present disclosure, where for convenience, Embodiment 1 (E1) is the same as the embodiment of formula (I) provided above.

[0011] It should be understood that both the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit the present disclosure as claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 PXRD spectrum showing the anhydrous crystal (Form 1) of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide free base.

[0013] Figure 2 Histological images of mouse bone marrow biopsies showing treatment group 1 (75 mg / kg / dose BID compound 1), group 2 (150 mg / kg / dose BID PF-06873600), and vehicle control group 3.

[0014] Figure 3The displayed graph plots the change curve of plasma free (nM) over time (h) in the mouse exposure of treatment group 1 (75 mg / kg / dose BID compound 1) on day 14 and group 2 (150 mg / kg / dose BID PF-06873600) on day 8 in the toxicity study. Detailed Description of the Invention

[0015] The present disclosure can be more easily understood by referring to the following detailed description of the embodiments of the present disclosure and the examples included herein. It should be understood that the present disclosure is not limited to a specific synthetic preparation method, which may of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0016] The compound of formula (I) or a pharmaceutically acceptable salt thereof, as shown above.

[0017] Embodiment E2 The compound of Embodiment E1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I) has the absolute stereochemistry shown in formula (I-A), (I-B), (I-C) or (I-D):

[0018]

[0019] Embodiment E3 A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-A).

[0020] Embodiment E4 A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1S,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-B).

[0021] Embodiment E5 A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1R,2S)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-C).

[0022] Embodiment E6 A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1S,2S)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-D).

[0023] Compound according to any one of embodiments E1 - E3, which is 4 - ((6 - (2,2 - difluoroethyl)-8 - ((1R,2R)-2 - hydroxy - 2 - methylcyclopentyl)-7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide.

[0024] Compound according to any one of embodiments E1 - E3, which has the chemical structure

[0025]

[0026] Pharmaceutically acceptable salt of 4 - ((6 - (2,2 - difluoroethyl)-8 - ((1R,2R)-2 - hydroxy - 2 - methylcyclopentyl)-7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide.

[0027] Anhydrous crystalline form (Form 1) of the free base of 4 - ((6 - (2,2 - difluoroethyl)-8 - ((1R,2R)-2 - hydroxy - 2 - methylcyclopentyl)-7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide, which has a powder X - ray diffraction (PXRD) pattern comprising one, two, three, four, five or more than five peaks measured at °2θ ± 0.2°2θ in Table 1.

[0028] Anhydrous crystalline form (Form 1) of the free base of 4 - ((6 - (2,2 - difluoroethyl)-8 - ((1R,2R)-2 - hydroxy - 2 - methylcyclopentyl)-7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide, which has a powder X - ray diffraction (PXRD) pattern comprising peaks at the following 2θ values: 4.8, 14.3 and 19.7°2θ ± 0.2°2θ.

[0029] Anhydrous crystalline form according to any one of embodiments E10 - E11, which has a PXRD pattern further comprising a peak at the following 2θ value: 10.6°2θ ± 0.2°2θ.

[0030] Anhydrous crystalline form according to any one of embodiments E10 - E12, which has a PXRD pattern further comprising a peak at the following 2θ value: 19.1°2θ ± 0.2°2θ.

[0031] Anhydrous crystalline form according to any one of embodiments E10 - E13, which has a PXRD pattern comprising peaks at substantially the same 2θ values as Figure 1 shown.

[0032] E15 An anhydrous crystalline form according to any one of embodiments E10 - E14, wherein the crystalline form is substantially pure.

[0033] E16 A compound of formula (II)

[0034]

[0035] or a pharmaceutically acceptable salt thereof,

[0036] wherein Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y 10 , Y 11 and Y 12 are each independently hydrogen or deuterium.

[0037] E17 A compound of embodiment E16, wherein Y8 and Y9 are each deuterium.

[0038] E18 A compound according to any one of embodiments E16 - E17, wherein Y 10 and Y 11 are each deuterium.

[0039] E19 A compound according to any one of embodiments E16 - E18, wherein Y1 and Y2 are each deuterium.

[0040] E20 A compound according to any one of embodiments E16 - E19, wherein Y 12 is deuterium.

[0041] E21 A compound according to any one of embodiments E16 - E20, wherein Y7 is deuterium.

[0042] E22 A compound according to any one of embodiments E16 - E21, wherein Y 13 is deuterium.

[0043] E23 A compound according to any one of embodiments E16 - E22, wherein Y3 and Y4 are each deuterium.

[0044] E24 A compound according to any one of embodiments E16 - E23, wherein Y5 and Y6 are each deuterium.

[0045] E25 A pharmaceutical composition comprising a compound according to any one of embodiments E1 to E24 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0046] E26 A method of treating cancer, which comprises administering to an individual in need thereof a therapeutically effective amount of a compound according to any one of embodiments E1 to E25 or a pharmaceutically acceptable salt thereof.

[0047] E27 The method for treating cancer according to embodiment E26, further comprising administering an additional anti-cancer therapeutic agent.

[0048] E28 The method for treating cancer according to any one of embodiments E26 to E27, wherein the cancer is selected from breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, and thyroid cancer.

[0049] E29 The method for treating cancer according to embodiment E28, wherein the cancer is breast cancer or ovarian cancer.

[0050] E30 The method for treating cancer according to embodiment E29, wherein the breast cancer or ovarian cancer is characterized by amplification or overexpression of cyclin E1 (CCNE1) or cyclin E2 (CCNE2).

[0051] E31 The method for treating cancer according to any one of embodiments E28 or E29, wherein the breast cancer is ER-positive / HR-positive breast cancer, HER2-negative breast cancer, ER-positive / HR-positive breast cancer, HER2-positive breast cancer, triple-negative breast cancer (TNBC), or inflammatory breast cancer.

[0052] E32 The method for treating cancer according to any one of embodiments E28 to E31, wherein the breast cancer is advanced breast cancer or metastatic breast cancer.

[0053] E33 The compound or pharmaceutically acceptable salt according to any one of embodiments E1 to E24, used as a drug.

[0054] E34 The compound or pharmaceutically acceptable salt according to any one of embodiments E1 to E24, for treating cancer.

[0055] E35 Use of the compound or pharmaceutically acceptable salt according to any one of embodiments E1 to E24 for preparing a drug for treating cancer.

[0056] E36 A drug combination, comprising the compound or pharmaceutically acceptable salt according to any one of embodiments E1 to E24, at least one additional therapeutic agent, and at least one pharmaceutically acceptable excipient.

[0057] Each of the embodiments described herein can be combined with any other embodiment described herein, provided that the any other embodiment is not inconsistent with the embodiment with which it is combined.

[0058] Definition

[0059] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings that are commonly understood by one of ordinary skill in the art.

[0060] The disclosure described herein may be practiced appropriately without any element not specifically disclosed herein.

[0061] This disclosure provides compounds of formula (I), including stereoisomers of formula (I) having structures of formula (I-A), (I-B), (I-C) and (I-D), and intermediates used in their preparation, referred to as "compounds of this disclosure". One of ordinary skill in the art will understand that the compounds of this disclosure include conformational isomers (e.g., cis and trans isomers) and all optical isomers (e.g., enantiomers and diastereomers), racemic mixtures, mixtures of diastereomers and other mixtures of such isomers, their tautomers, where they may exist. One of ordinary skill in the art will also understand that the compounds of this disclosure include their solvates, hydrates, polymorphs, polycrystalline forms, esters, salt forms, prodrugs and isotopically labeled forms that they may form, where they may form.

[0062] As used herein, the singular forms "a", "an" and "the" include plural referents unless otherwise stated. For example, "a" substituent includes one or more substituents.

[0063] As used herein, the term "about", when used to modify a parameter that is a numerical limitation (e.g., a dose of 5 mg) means that the parameter can vary up to 10% below or above the stated value of the parameter. For example, a dose of about 5 mg means 5 mg ± 10%, i.e., it can vary between 4.5 mg and 5.5 mg.

[0064] If a substituent is described as "independently selected from" a group, the selection of each substituent is independent of the other substituents. Thus, each substituent can be the same as or different from the other substituents.

[0065] The disclosure described herein may be practiced appropriately without any element not specifically disclosed herein. Thus, for example, in each instance herein, any one of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with any one of the other two terms.

[0066] As used herein, the term "substantially the same" means taking into account the variability typical of a particular method. For example, with respect to the position of an X-ray diffraction peak, the term "substantially the same" means taking into account the typical variability in peak position and intensity. One of ordinary skill in the art will understand that the peak position (2θ) will exhibit some variability, typically up to ±0.2°. In addition, one of ordinary skill in the art will understand that relative peak intensities will show variability between devices, as well as variability due to crystallinity, preferred orientation, the surface of the sample prepared, and other factors known to one of ordinary skill in the art, and should only be used as a qualitative measure.

[0067] As used herein, the term "crystalline" means that the molecules or the external surface planes have a regular repeating arrangement. Crystalline forms can differ in terms of thermodynamic stability, physical parameters, x-ray structure, and the preparation process.

[0068] As used herein, the term "anhydrous" refers to a crystalline form that contains only the active pharmaceutical ingredient (API) as part of its crystal lattice.

[0069] As used herein, the term "solvate" describes a molecular complex that contains a compound (e.g., the API of a pharmaceutical product) and a stoichiometric or non-stoichiometric amount of one or more solvent molecules (e.g., water or ethanol). When the solvent is tightly bound to the compound, the resulting complex will have a well-defined stoichiometry independent of humidity. However, when the solvent is weakly bound, as in channel solvates and hygroscopic compounds, the solvent content depends on humidity and drying conditions. In such cases, the complex is typically non-stoichiometric.

[0070] As used herein, the term "hydrate" describes a solvate that contains a compound and a stoichiometric or non-stoichiometric amount of water. A "monohydrate" is a hydrate that contains one water molecule per molecule of the compound (i.e., a 1:1 stoichiometry of water to compound).

[0071] As used herein, the term "substantially pure" means that a crystalline or amorphous form described as substantially pure contains less than 5% by weight, preferably less than 3%, and more preferably less than 1% impurities, where the impurities include any other physical form of the compound. Alternatively, the crystalline or amorphous form described as substantially pure can be expressed as >95% pure, preferably >97% pure, and more preferably >99% pure, in each case by weight of impurities, where the impurities include any other physical form of the compound.

[0072] As used herein, "endocrine therapy" means an aromatase inhibitor, a selective estrogen receptor degrader (SERD), or a selective estrogen receptor modulator (SERM). In certain embodiments, endocrine therapy includes fulvestrant, tamoxifen, toremifene, anastrozole, exemestane, or letrozole.

[0073] As used herein, the term "pharmaceutically acceptable" means that a substance (e.g., a compound described herein), and any salt thereof, or a composition containing the substance or salt of the present disclosure, is suitable for administration to an individual or patient.

[0074] As used herein, "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or pharmaceutically acceptable salts, solvates, hydrates or prodrugs thereof as active ingredients and at least one pharmaceutically acceptable excipient.

[0075] As used herein, "deuterium enrichment factor" refers to the ratio of deuterium abundance to the natural abundance of deuterium, each relative to hydrogen abundance. In certain embodiments, an atomic position designated as having deuterium typically has a deuterium enrichment factor of at least 1000 (15% deuterium incorporation), at least 2000 (30% deuterium incorporation), at least 3000 (45% deuterium incorporation), at least 3500 (52.5% deuterium incorporation), at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation) or at least 6633.3 (99.5% deuterium incorporation).

[0076] As used herein, "excipient" describes any ingredient other than the compounds of the invention. The choice of excipient will depend to a large extent on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The term "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, carriers, diluents, etc. Examples of excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof, and may include isotonic agents such as sugars, sodium chloride or polyols in the composition, such as mannitol or sorbitol. Examples of excipients also include a variety of organic solvents (such as hydrates and solvates). If desired, the pharmaceutical composition may contain additional excipients such as flavoring agents, binders / binders, lubricants, disintegrants, sweeteners or flavoring agents, coloring substances or dyes, etc. For example, for oral administration, tablets containing a variety of excipients such as citric acid can be used with a variety of disintegrants such as starch, alginic acid and certain complex silicates and with binders such as sucrose, gelatin and gum arabic. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, a variety of sugars and a variety of types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are commonly used for tableting purposes. Similar types of solid compositions can also be used in soft and hard filled gelatin capsules. Thus, non-limiting examples of excipients also include lactose (lactose) or milk sugar and high molecular weight polyethylene glycol. When an aqueous suspension or elixir is desired for oral administration, the active compound therein can be combined with a variety of sweeteners or flavoring agents, coloring substances or dyes and (if desired) emulsifying or suspending agents and additional excipients such as water, ethanol, propylene glycol, glycerol or combinations thereof.

[0077] Examples of excipients also include pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting agents or emulsifying agents, preservatives or buffering agents, which enhance the shelf life or effectiveness of the compound.

[0078] As used herein, the term "individual" refers to a human or animal individual. In certain preferred embodiments, the individual is a human.

[0079] As used herein, the term "abnormal cell growth" refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). Abnormal cell growth can be benign (non-cancerous) or malignant (cancerous). Abnormal cell growth includes the following abnormal growths: (1) tumor cells (tumors) that show increased CDK2 expression; (2) tumors that proliferate through abnormal CDK2 activation; (3) tumors characterized by amplification or overexpression of CCNE1 and / or CCNE2; and (4) tumors that are resistant to endocrine therapy, HER2 antagonists, or CDK4 / 6 inhibition.

[0080] As used herein, the term "additional anti-cancer therapeutic agent" means any one or more therapeutic agents other than the compounds of the present invention that are used or can be used to treat cancer, such as agents derived from the following classes: mitotic inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase I and II inhibitors, plant alkaloids, hormonal agents and antagonists, growth factor inhibitors, radiation, inhibitors of protein tyrosine kinases and / or serine / threonine kinases, cell cycle inhibitors, biologic response modifiers, enzyme inhibitors, antisense oligonucleotides or oligonucleotide derivatives, cytotoxic agents, and immuno-oncology agents.

[0081] As used herein, the term "cancer" refers to any malignant and / or invasive growth or tumor caused by abnormal cell growth. Cancer includes solid tumors named for the cell type from which they form, cancers of the blood, bone marrow, or lymphatic system. Examples of solid tumors include sarcomas and carcinomas. Cancers of the blood include, but are not limited to, leukemias, lymphomas, and myelomas. Cancer also includes primary cancers that originate at a specific site in the body, metastatic cancers that spread from their site of origin to other parts of the body, recurrences from the original primary cancer after remission, and second primary cancers, which are new primary cancers in individuals with a history of a previous cancer that is different from the latter cancer type.

[0082] As used herein, the term "treating" means reversing, alleviating, inhibiting the progression of a condition or disorder to which such terms are applied, or preventing the condition or disorder, or one or more symptoms of such condition or disorder. Unless otherwise specified, as used herein, the term "treatment" refers to the act of "treating" as just defined above. The term "treatment" also includes adjuvant and neoadjuvant treatment of an individual.

[0083] As used herein, the term "therapeutically effective amount" refers to an amount of a compound administered that will, to some extent, alleviate one or more symptoms of the disorder being treated. With respect to the treatment of cancer, a therapeutically effective amount is an amount that has one or more of the following effects: (1) reducing the size of a tumor, (2) inhibiting (i.e., slowing to some extent, preferably stopping) tumor metastasis, (3) inhibiting (i.e., slowing to some extent, preferably stopping) tumor growth or tumor invasion to some extent, and / or (4) alleviating (or preferably, eliminating) one or more signs or symptoms associated with cancer to some extent.

[0084] salt

[0085] The salts encompassed by the term "pharmaceutically acceptable salts" are salts of the compounds of the present disclosure, which are generally prepared by reacting the free base or free acid with a suitable organic or inorganic acid or a suitable organic or inorganic base, respectively, to provide salts of the compounds of the present disclosure suitable for administration to an individual or patient.

[0086] In addition, the compounds of formula (I) may also include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts, and which may be used as intermediates for one or more of the following: 1) preparing the compounds of formula (I); 2) purifying the compounds of formula (I); 3) separating the enantiomers of the compounds of formula (I); or 4) separating the diastereomers of the compounds of formula (I).

[0087] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclohexylsulfamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / monohydrogenphosphate / dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, 1,5-naphthalenedisulfonate, and xinofoate.

[0088] Suitable base salts are formed from bases that form non-toxic salts. Examples include, but are not limited to, aluminum salts, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts.

[0089] Hemisalts of acids and bases can also be formed, such as hemisulfates and hemicalcium salts.

[0090] For a review of suitable salts, see Paulekun, G.S. et al., Trends in Active Pharmaceutical Ingredient Salt Selection Based on Analysis of the Orange Book Database, J. Med. Chem. 2007; 50(26), 6665 - 6672.

[0091] Pharmaceutically acceptable salts of 4 - ((6 - (2,2 - difluoroethyl) - 8 - (2 - hydroxy - 2 - methylcyclopentyl) - 7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide can be prepared by methods well known to those skilled in the art, including but not limited to the following procedures:

[0092] (i) By reacting 4 - ((6 - (2,2 - difluoroethyl) - 8 - (2 - hydroxy - 2 - methylcyclopentyl) - 7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide with the desired acid or base;

[0093] (ii) Using the desired acid or base, by removing acid - labile or base - labile protecting groups from suitable precursors of the compounds disclosed herein, or by ring - opening of suitable cyclic precursors (such as lactones or lactams); or

[0094] (iii) By converting one salt of 4 - ((6 - (2,2 - difluoroethyl) - 8 - (2 - hydroxy - 2 - methylcyclopentyl) - 7 - oxo - 7,8 - dihydropyrido[2,3 - d]pyrimidin - 2 - yl)amino)piperidine - 1 - sulfonamide to another. This can be accomplished by reaction with an appropriate acid or base or by a suitable ion - exchange procedure.

[0095] These procedures are generally carried out in solution. The resulting salts can be precipitated and collected by filtration, or can be recovered by evaporation of the solvent.

[0096] Solvate

[0097] The compounds disclosed herein and their pharmaceutically acceptable salts can exist in non - solvated and solvated forms.

[0098] In addition, the compounds of the present disclosure may also include other solvates of such compounds, which are not necessarily pharmaceutically acceptable solvates, and which may be used as intermediates for one or more of the following: 1) preparing the compounds of the present disclosure; 2) purifying the compounds of the present disclosure; 3) separating the enantiomers of the compounds of the present disclosure; or 4) separating the diastereomers of the compounds of the present disclosure.

[0099] The currently recognized classification system for organic hydrates is the one that defines isolated-site hydrates, channel hydrates, or metal-ion coordinated hydrates, see Polymorphismin PharmaceuticalSolids by K.R. Morris (edited by H.G. Brittain, Marcel Dekker, 1995). Isolated-site hydrates are hydrates in which water molecules are separated from each other by intervening organic molecules and do not directly contact. In channel hydrates, water molecules are located in lattice channels and are adjacent to other water molecules. In metal-ion coordinated hydrates, water molecules are bonded to metal ions.

[0100] When the solvent or water is tightly bound, the complex can have a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water / solvent content can depend on humidity and drying conditions. In such cases, non-stoichiometry would be the norm.

[0101] Complex

[0102] Also within the scope of the present disclosure are multi-component complexes (in addition to salts and solvates), in which the compounds of the present disclosure and at least one other component are present in stoichiometric or non-stoichiometric amounts. This type of complex includes clathrates (drug-host inclusion complexes) and co-crystals. The latter are generally defined as crystalline complexes of neutral molecular components held together by non-covalent interactions, for example, hydrogen-bonded complexes (co-crystals) can be formed with neutral molecules or with salts. Co-crystals can be prepared by melt crystallization, by recrystallization from solvents, or by physically grinding the components together, see Chem Commun, 17; 1889-1896, O. Almarsson and M.J. Zaworotko (2004). For a general review of multi-component complexes, see J Pharm Sci, 64(8), 1269-1288, Haleblian (August 1975).

[0103] Solid form

[0104] The compounds of the present disclosure can exist in a continuous solid state ranging from amorphous to crystalline. The term "amorphous" refers to a state where the material lacks long-range order at the molecular level and may exhibit physical properties of a solid or a liquid depending on temperature. Typically, such materials do not exhibit a distinct X-ray diffraction pattern and, although showing solid properties, are more formally described as liquids. Upon heating, a change from solid to liquid properties occurs, characterized by a state change, typically a second-order (glass transition). The term "crystalline" refers to a solid phase in which the material has a regular ordered internal structure at the molecular level and exhibits a distinct X-ray diffraction pattern with well-defined peaks. Such materials also exhibit liquid properties upon sufficient heating, but the change from solid to liquid is characterized by a phase transition, typically a first-order ('melting point').

[0105] When subjected to suitable conditions, the compounds of the present disclosure can also exist in a mesomorphic state (mesophase or liquid crystal). The mesomorphic state lies between a truly crystalline state and a truly liquid state (melt or solution) and has two-dimensional order at the molecular level. Mesomorphism resulting from temperature changes is described as 'thermotropic', while that caused by the addition of a second component (such as water or another solvent) is described as 'lyotropic'. Compounds having the potential to form lyotropic mesophases are described as 'amphiphilic' and consist of a molecular composition with ionic (such as -COO - Na + 、-COO - K + or -SO3 - Na + ) or non-ionic (such as -N - N + (CH3)3) polar head groups. For more information, see Crystals and the Polarizing Microscope by N.H. Hartshorne and A. Stuart, 4th Edition (Edward Arnold, 1970).

[0106] In some embodiments, the present disclosure provides an anhydrous crystalline form of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide free base of formula (I-A) ("Form 1"). In some embodiments, Form 1 is substantially pure and free of alternative forms.

[0107] In some embodiments, Form 1 is characterized by powder X-ray diffraction (PXRD). Such crystalline forms can be further characterized by additional techniques, such as Raman spectroscopy and 13 C and 19F solid state NMR spectroscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), or differential thermal analysis (DTA).

[0108] In some embodiments, the present disclosure provides Form 1, characterized by having a PXRD pattern (2θ) that includes: (a) one, two, three, four, five, or more than five peaks selected from the peaks in Table 1 at °2θ ± 0.2°2θ; (b) one, two, three, four, or five peaks selected from the characteristic peaks in Table 1 at °2θ ± 0.2°2θ; or (c) peaks at 2θ values Figure 1 substantially the same as those shown;

[0109] In one embodiment, Form 1 has a PXRD pattern that includes one or more peaks selected from the following 2θ values: 4.8, 10.6, 14.3, 19.1, and 19.7°2θ ± 0.2°2θ. In another embodiment, Form 1 has a PXRD pattern that includes two or more peaks selected from the following 2θ values: 4.8, 10.6, 14.3, 19.1, and 19.7°2θ ± 0.2°2θ. In another embodiment, Form 1 has a PXRD pattern that includes three or more peaks selected from the following 2θ values: 4.8, 10.6, 14.3, 19.1, and 19.7°2θ ± 0.2°2θ. In another embodiment, Form 1 has a PXRD pattern that includes two or more peaks selected from the following 2θ values: 4.8, 10.6, 14.3, 19.1, and 19.7°2θ ± 0.2°2θ.

[0110] In one embodiment, Form 1 has a PXRD pattern that includes peaks at the following 2θ values: 4.8, 14.3, and 19.7°2θ ± 0.2°2θ. In one embodiment, Form 1 has a PXRD pattern that includes peaks at the following 2θ values: 4.8, 10.6, 14.3, and 19.7°2θ ± 0.2°2θ. In one embodiment, Form 1 has a PXRD pattern that includes peaks at the following 2θ values: 4.8, 10.6, 14.3, 19.1, and 19.7°2θ ± 0.2°2θ.

[0111] In one embodiment, Form 1 has a PXRD pattern that further comprises peaks at the following 2θ values: 4.8° 2θ ± 0.2° 2θ. In one embodiment, Form 1 has a PXRD pattern that further comprises peaks at the following 2θ values: 10.6° 2θ ± 0.2° 2θ. In one embodiment, Form 1 has a PXRD pattern that further comprises peaks at the following 2θ values: 14.3° 2θ ± 0.2° 2θ. In one embodiment, Form 1 has a PXRD pattern that further comprises peaks at the following 2θ values: 19.1° 2θ ± 0.2° 2θ. In one embodiment, Form 1 has a PXRD pattern that further comprises peaks at the following 2θ values: 19.7° 2θ ± 0.2° 2θ.

[0112] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the crystalline free base form of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide (Form 1) according to any of the embodiments described herein, and a pharmaceutically acceptable carrier or excipient.

[0113] In another aspect, the present disclosure provides a method of treating abnormal cell growth in a mammal, preferably a human, the method comprising administering to the mammal a therapeutically effective amount of Form 1 according to any of the embodiments described herein.

[0114] In another aspect, the present disclosure provides a method of treating abnormal cell growth in a mammal, preferably a human, the method comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising Form 1 according to any of the embodiments described herein.

[0115] In another aspect, the present disclosure provides the use of Form 1 according to any of the embodiments described herein for treating abnormal cell growth in a mammal, preferably a human.

[0116] In another aspect, the present disclosure provides the use of Form 1 according to any of the embodiments described herein for the preparation of a medicament for treating abnormal cell growth in a mammal, preferably a human.

[0117] In common embodiments of the methods, compositions, and uses described herein, the abnormal cell growth is cancer.

[0118] Stereoisomer

[0119] The compounds of the present disclosure can exist as two or more stereoisomers. The stereoisomers of the compounds can include cis and trans isomers (geometric isomers), optical isomers such as R and S enantiomers, diastereomers, rotamers, atropisomers, and conformational isomers. For example, the compounds of the present disclosure containing one or more asymmetric carbon atoms can exist as two or more stereoisomers.

[0120] The pharmaceutically acceptable salts of the compounds of the present disclosure can also contain optically active or racemic counterions.

[0121] Cis / trans isomers can be separated by conventional techniques well-known to those skilled in the art, such as chromatography and fractional crystallization.

[0122] Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from a suitable optically pure precursor, or resolution of a racemate (or a racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography (HPLC). Alternatively, a racemate (or a racemic precursor) can be reacted with a suitable optically active compound such as an alcohol, or in the case where the compound of the present disclosure contains an acidic or basic moiety, with a base or an acid such as 1-phenylethylamine or tartaric acid. The resulting diastereomeric mixture can be separated by chromatography, fractional crystallization, or by using both of these techniques, and one or both of the diastereomers can be converted into the corresponding pure enantiomers by methods well-known to the skilled person. The chiral compounds of the present disclosure (and their chiral precursors) can be obtained in enantiomerically enriched form using chromatography, typically HPLC. Concentration of the eluate provides an enriched mixture. Chiral chromatography using subcritical and supercritical fluids can be employed. Methods of chiral chromatography for some embodiments of the present disclosure are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography with Packed Columns), pages 223-249, and the references cited therein).

[0123] When any racemate crystallizes, two different types of crystals are possible. The first type is the racemic compound (true racemate) mentioned above, in which a homogeneous crystal form is produced that contains equimolar amounts of the two enantiomers. The second type is a racemic mixture or racemic conglomerate, in which equimolar amounts of two crystal forms are produced, each containing a single enantiomer. Although the two crystal forms present in a racemic mixture have the same physical properties, they may have different physical properties compared to a true racemate. Racemic mixtures can be separated by conventional techniques known to those skilled in the art; see, for example, Stereochemistry of Organic Compounds by E.L. Eliel and S.H. Wilen (Wiley, 1994).

[0124] Tautomerism

[0125] Tautomeric isomerism ('tautomerism') can occur where structural isomers can interconvert via a low energy barrier. This may take the form of proton tautomerism in the compounds of the present disclosure, which contain, for example, imino / amino, keto / enol or oxime / nitroso, lactam / lactim or so-called valence tautomerism in compounds containing an aromatic moiety. Thus, a single compound may exhibit more than one type of isomerism.

[0126] It must be emphasized that, although for the sake of brevity the compounds of the present disclosure are drawn herein in a single tautomeric form, all possible tautomeric forms are included within the scope of the present disclosure.

[0127] Isotope

[0128] The present disclosure includes all pharmaceutically acceptable isotopically labeled compounds of the present disclosure, wherein one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number predominant in nature.

[0129] Examples of isotopes suitable for inclusion in the compounds of the present disclosure may include isotopes of hydrogen, such as 2 H (D, deuterium) and 3 H (T, tritium); isotopes of carbon, such as 11 C, 13 C and 14 C; isotopes of chlorine, such as 36 Cl; isotopes of fluorine, such as 18 F; isotopes of iodine, such as 123 I and 125 I; isotopes of nitrogen, such as 13 N and15 N; an isotope of oxygen, such as 15 O, 17 O, and 18 O; an isotope of phosphorus, such as 32 P; and an isotope of sulfur, such as 35 S.

[0130] Certain isotopically labeled compounds of the present disclosure, such as those incorporating a radioactive isotope, can be used in one or both of drug or substrate tissue distribution studies. Radioactive isotopes, such as tritium and 14 C, are particularly suitable for this purpose in view of their ready availability for incorporation and detection. Isotopes emitting positrons, such as 11 C, 18 F, 15 O, and 13 N substitution can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Substitution with deuterium can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dose requirements, reduced CYP450 inhibition (competitive or time-dependent), or improved therapeutic index or tolerability.

[0131] In some embodiments, the present disclosure provides deuterium-labeled (or deuterated) compounds and salts, wherein the formulae and variables of such compounds and salts are each and independently as described herein. "Deuterated" means that at least one atom in the compound is deuterium at an abundance greater than the natural abundance of deuterium (typically about 0.015%). Those skilled in the art recognize that in a compound having a hydrogen atom, the hydrogen atom actually represents a mixture of H and D, where about 0.015% is D. The deuterium concentration incorporated into the deuterium-labeled compounds and salts of the present invention can be defined by the deuterium enrichment factor. It should be understood that one or more deuteriums can exchange with hydrogen under physiological conditions.

[0132] In some embodiments, the metabolic sites on the compounds of the present disclosure are deuterated.

[0133] The isotopically labeled compounds of the present disclosure can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the accompanying examples and preparations, using appropriate isotopically labeled reagents in place of the non-labeled reagents previously used.

[0134] Pharmaceutically acceptable solvates according to the present disclosure include those in which the solvent in which crystallization occurs can be isotopically substituted, such as those of D2O, d6-acetone, d6-DMSO.

[0135] Prodrug

[0136] The compounds of the present disclosure can be administered in the form of prodrugs. Thus, certain derivatives of the compounds of the present disclosure that may themselves have little or no pharmacological activity can be converted into the compounds of the present disclosure having the desired activity when administered into or onto the body, for example, by hydrolytic cleavage, particularly hydrolytic cleavage facilitated by esterases or peptidases. Such derivatives are referred to as 'prodrugs'. Further information on the use of prodrugs can be found in 'The Expanding Role of Prodrugs in Contemporary Drug Design and Development, Nature Reviews Drug Discovery, 17, 559 - 587 (2018) (J. Rautio et al.)'.

[0137] Prodrugs according to the present disclosure can be prepared, for example, by replacing appropriate functional groups present in the compounds of the present disclosure with certain moieties known to those skilled in the art as 'pro - moieties', as described, for example, in 'Design of Prodrugs' by H. Bundgaard (Elsevier, 1985).

[0138] Thus, prodrugs according to the present disclosure can be (a) ester or amide derivatives of carboxylic acids when a carboxylic acid is present in the compounds of the present disclosure; (b) ester, carbonate, carbamate, phosphate, or ether derivatives of hydroxyl groups when a hydroxyl group is present in the compounds of the present disclosure; (c) amide, imine, carbamate, or amine derivatives of amino groups when an amino group is present in the compounds of the present disclosure; (d) thioester, thiocarbonate, thiocarbamate, or sulfide derivatives of thiol groups when a thiol group is present in the compounds of the present disclosure; or (e) oxime or imine derivatives of carbonyl groups when a carbonyl group is present in the compounds of the present disclosure.

[0139] Some specific examples of prodrugs according to the present disclosure include:

[0140] (i) When the compound of the present disclosure contains a carboxylic acid functional group (-COOH), its esters, such as compounds in which the hydrogen of the carboxylic acid functional group of the compound is replaced by a C1 - C8 alkyl group (e.g., ethyl) or (C1 - C8 alkyl)C(=O)OCH2 - (e.g., tBuC(=O)OCH2 -);

[0141] (ii) When the compound of the present disclosure contains an alcohol functional group (-OH), its esters, such as compounds in which the hydrogen of the alcohol functional group of the compound is replaced by -CO(C1 - C8 alkyl) (e.g., methylcarbonyl) or the alcohol is esterified with an amino acid;

[0142] (iii) When the compounds of the present disclosure contain an alcohol functional group (-OH), their ethers, such as compounds in which the hydrogen of the alcohol functional group of the compound is replaced by (C1-C8 alkyl)C(=O)OCH2- or -CH2OP(=O)(OH)2;

[0143] (iv) When the compounds of the present disclosure contain an alcohol functional group (-OH), their phosphates, such as compounds in which the hydrogen of the alcohol functional group of the compound is replaced by -P(=O)(OH)2 or -P(=O)(O - Na + )2 or -P(=O)(O - )2Ca 2+ substituted compounds;

[0144] (v) When the compounds of the present disclosure contain a primary or secondary amine functional group (-NH2 or -NHR, where R≠H), their amides, such as compounds in which, as appropriate, one or two hydrogens of the amino functional group of the compound are replaced by (C1-C 10 ) alkanoyl, -COCH2NH2 substitution or the amino group is derivatized with an amino acid;

[0145] (vi) When the compounds of the present disclosure contain a primary or secondary amine functional group (-NH2 or -NHR, where R≠H), their amines, such as compounds in which, as appropriate, one or two hydrogens of the amino functional group of the compound are replaced by -CH2OP(=O)(OH)2.

[0146] Metabolite

[0147] Also included within the scope of the present disclosure are the active metabolites of the compounds of the present disclosure, i.e., compounds that are typically formed in vivo by oxidation or dealkylation upon administration. Some examples of metabolites according to the present disclosure include, but are not limited to:

[0148] (i) In the case where the compounds of the present disclosure contain an alkyl group, their hydroxyalkyl derivatives (-CH→-COH):

[0149] (ii) In the case where the compounds of the present disclosure contain an alkoxy group, their hydroxy derivatives (-OR→-OH);

[0150] (iii) In the case where the compounds of the present disclosure contain a tertiary amino group, their secondary amino derivatives (-NRR ' →-NHR or -NHR ' );

[0151] (iv) In the case where the compounds of the present disclosure contain a secondary amino group, their primary amino derivatives (-NHR→-NH2);

[0152] (v) Where the compound of the present disclosure contains a phenyl moiety, its phenol derivative (-Ph → -PhOH);

[0153] (vi) Where the compound of the present disclosure contains an amide group, its carboxylic acid derivative (-CONH2 → COOH); and

[0154] (vii) Where the compound contains a hydroxyl or carboxylic acid group, the compound can be metabolized, for example, by conjugation with glucuronic acid to form a glucuronide. There are other conjugation metabolic pathways. These pathways are generally referred to as phase II metabolism and include, for example, sulfation or acetylation. Other functional groups, such as NH groups, can also undergo conjugation.

[0155] Pharmaceutical composition

[0156] In another embodiment, the present disclosure includes a pharmaceutical composition.

[0157] "Pharmaceutical composition" means a mixture of a compound of the present disclosure or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof as an active ingredient and at least one pharmaceutically acceptable excipient.

[0158] The term "excipient" is used herein to describe any ingredient other than the compounds of the present disclosure. The choice of excipient will depend to a large extent on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The term "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, carriers, diluents, etc. Examples of excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof, and may include isotonic agents such as sugars, sodium chloride or polyols in the composition, such as mannitol or sorbitol. Examples of excipients also include a variety of organic solvents (such as hydrates and solvates). If desired, the pharmaceutical composition may contain additional excipients such as flavoring agents, binders / binders, lubricants, disintegrants, sweeteners or flavoring agents, coloring substances or dyes, etc. For example, for oral administration, tablets containing a variety of excipients such as citric acid can be used with a variety of disintegrants such as starch, alginic acid and certain complex silicates and with binders such as sucrose, gelatin and gum arabic. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, a variety of sugars and a variety of types of starches, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are commonly used for tableting purposes. Similar types of solid compositions can also be used in soft-fill and hard-fill gelatin capsules. Thus, non-limiting examples of excipients also include lactose or milk sugar and high molecular weight polyethylene glycol. When an aqueous suspension or elixir is desired for oral administration, the active compound therein can be combined with a variety of sweeteners or flavoring agents, coloring substances or dyes and (if desired) emulsifying or suspending agents and additional excipients such as water, ethanol, propylene glycol, glycerol or combinations thereof.

[0159] Examples of excipients also include pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting agents or emulsifying agents, preservatives or buffering agents, which enhance the shelf life or effectiveness of the compound.

[0160] The compositions of the present disclosure can be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms such as liquid solutions (such as injectable and infusible solutions), dispersions or suspensions, tablets, capsules, pills, powders, liposomes and suppositories. The form depends on the intended mode of administration and therapeutic application.

[0161] Typical compositions are in the form of injectable or infusible solutions, for example, compositions similar to those having antibodies for passive immunization in humans. One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the compound is administered by intravenous infusion or injection. In yet another embodiment, the compound is administered by intramuscular or subcutaneous injection.

[0162] Oral administration of solid dosage forms can be presented, for example, in discrete units such as hard or soft gelatin capsules, pills, cachets, lozenges or tablets, each containing a predetermined amount of at least one compound of the present disclosure. In another embodiment, the oral administration can be in the form of a powder or granules. In another embodiment, the oral dosage form is sublingual, such as a lozenge. In such solid dosage forms, the compounds of the present disclosure are generally combined with one or more excipients. Such capsules or tablets can contain controlled release formulations. In the case of capsules, tablets and pills, the dosage form can also contain buffering agents or can be prepared with enteric coatings.

[0163] In another embodiment, the oral administration can be in liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs, which contain inert diluents commonly used in the art (e.g., water). Such compositions can also contain excipients such as one or more of wetting agents, emulsifying agents, suspending agents, flavoring agents (e.g., sweeteners) or fragrances.

[0164] In another embodiment, the present disclosure includes parenteral dosage forms. "Parenteral administration" includes, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intracardiac injection and infusion. Injectable preparations (i.e., sterile injectable aqueous or oily suspensions) can be formulated according to known techniques using one or more suitable dispersing, wetting or suspending agents.

[0165] In another embodiment, the present disclosure includes topical dosage forms. "Topical administration" includes, for example, cutaneous and transdermal administration, such as by transdermal patches or iontophoresis devices, intraocular administration, or intranasal or inhaled administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments, and creams. Topical formulations may contain compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected area. When the compounds of the present disclosure are administered via a transdermal device, patches of the reservoir and porous membrane type or solid matrix type are used to effect administration. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages, and microemulsions. Liposomes may also be used. Typical excipients include alcohols, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be incorporated, see, for example, B.C. Finnin and T.M. Morgan, J. Pharm. Sci., vol. 88, pp. 955-958, 1999.

[0166] Formulations suitable for topical administration to the eye include, for example, eye drops, in which the compounds of the present disclosure are dissolved or suspended in a suitable excipient. Typical formulations suitable for ocular or otic administration may be in the form of drops of an isotonic, pH-adjusted, sterile saline micro-pulverized suspension or solution. Other formulations suitable for ocular and otic administration include ointments, biodegradable (i.e., absorbable gelfoams, collagen) and non-biodegradable (i.e., polysiloxane) implants, wafers, lenses, and particulate or vesicular systems, such as non-ionic surfactant vesicles (niosomes) or liposomes. Polymers such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulose polymers such as hydroxypropylmethylcellulose, hydroxyethylcellulose, or methylcellulose, or heteropolysaccharide polymers such as gellan gum, may be incorporated together with preservatives such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

[0167] For intranasal administration, the compounds of the present disclosure are conveniently delivered in the form of a solution or suspension from a pump spray container squeezed or pumped by the patient, or as an aerosol spray provided by a pressurized container or nebulizer, wherein a suitable propellant is used. Formulations suitable for intranasal administration are typically administered in dry powder form (alone, as a mixture, such as a dry mixture with lactose, or as blended component particles, such as blended with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably an atomizer that uses electrohydrodynamics to generate a fine mist) or nebulizer, with or without a suitable propellant, such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may contain a bioadhesive, such as chitosan or cyclodextrin.

[0168] In another embodiment, the present disclosure includes rectal dosage forms. Such rectal dosage forms may be, for example, in the form of suppositories. Cocoa butter is a traditional suppository base, but various alternatives may be suitably used.

[0169] Other excipients and modes of administration known in the pharmaceutical art may also be used. The pharmaceutical compositions of the present disclosure may be prepared by any well-known pharmaceutical technique, such as effective formulation and administration procedures. The above considerations regarding effective formulation and administration procedures are well known in the art and are described in standard textbooks. The formulation of drugs is discussed, for example, in Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Pharmaceutical Dosage Forms, edited by Liberman et al., Marcel Decker, New York, N.Y., 1980; and Handbook of Pharmaceutical Excipients (3rd Edition), edited by Kibbe et al., American Pharmaceutical Association, Washington, 1999.

[0170] Acceptable excipients are non-toxic to the individual at the dosages and concentrations employed and may include one or more of the following: 1) buffering agents, such as phosphates, citrates or other organic acids; 2) salts, such as sodium chloride; 3) antioxidants, such as ascorbic acid or methionine; 4) preservatives, such as cetyltrimethylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol; 5) alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol or m-cresol; 6) low molecular weight (less than about 10 residues) polypeptides; 7) proteins, such as serum albumin, gelatin or immunoglobulins; 8) hydrophilic polymers, such as polyvinylpyrrolidone; 9) amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; 10) monosaccharides, disaccharides or other carbohydrates, including glucose, mannose or dextrin; 11) chelating agents, such as EDTA; 12) sugars, such as sucrose, mannitol, trehalose or sorbitol; 13) salt-forming counterions, such as sodium, metal complexes (e.g., Zn-protein complexes), or 14) nonionic surfactants, such as polysorbates (e.g., polysorbate 20 or polysorbate 80), poloxamers or polyethylene glycol (PEG).

[0171] For oral administration, the composition may be provided in the form of tablets or capsules containing 1.0, 5, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, 250, 500 or 1000 milligrams of the active ingredient for symptomatic regulation of the dosage in a patient. The medicament generally contains from about 1 mg to about 1000 mg of the active ingredient, or in another embodiment, from about 1 mg to about 100 mg of the active ingredient. The dosing regimen may depend on the route of administration, the dosing schedule and the use of flat-dose, body surface area or weight-based dosing. For example, for weight-based dosing, the intravenous dose may be from about 0.01 to about 10 mg / kg / minute during a constant rate infusion.

[0172] Liposomes containing the compounds of the present disclosure can be prepared by methods known in the art (see, e.g., Chang, H.I.; Yeh, M.K.; Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy; Int J Nanomedicine 2012; 7; 49-60). Particularly useful liposomes can be produced by the reverse evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to produce liposomes with the desired diameter.

[0173] The compounds of the present disclosure can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxyethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000).

[0174] Sustained-release formulations can be used. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the compounds of the present disclosure, which matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as those used for depot suspensions of leuprolide acetate (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0175] Formulations for intravenous administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. The compounds of the present disclosure are generally placed in a container with a sterile inlet, e.g., an intravenous solution bag or bottle with a stopper pierceable by a hypodermic injection needle.

[0176] Suitable emulsions can be prepared using commercially available fat emulsions, such as lipid emulsions containing soybean oil, fat emulsions for intravenous administration (e.g., containing safflower oil, soybean oil, lecithin, and glycerol in water), emulsions containing soybean oil and medium-chain triglycerides, and lipid emulsions of cottonseed oil. The active ingredient can be dissolved in the premixed emulsion composition, or alternatively, it can be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and form an emulsion when mixed with phospholipids (e.g., lecithin, soy phospholipid, or soy lecithin) and water. It should be understood that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions generally contain up to 20% oil, such as 5% to 20%. The fat emulsion can contain fat droplets of 0.1 μm to 1.0 μm, particularly 0.1 μm to 0.5 μm, and have a pH of 5.5 to 8.0.

[0177] For example, the emulsion compositions can be those prepared by mixing a compound of the present disclosure with a lipid emulsion containing soybean oil or its components (soybean oil, lecithin, glycerol, and water).

[0178] Compositions for inhalation or insufflation include solutions and suspensions, or mixtures thereof, in pharmaceutically acceptable aqueous or organic solvents, as well as powders. The liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered via the oral or nasal respiratory route for local or systemic effects. Compositions in a preferably sterile pharmaceutically acceptable solvent can be atomized using a gas. The atomized solution can be breathed directly from the atomizing device, or the atomizing device can be connected to a face mask, tent, or intermittent positive pressure ventilator. The solution, suspension, or powder composition can preferably be administered orally or nasally from a device that delivers the formulation in a suitable manner.

[0179] A drug product intermediate (DPI) is a partially processed material that must undergo further processing steps before becoming a bulk drug product. The compounds of the present disclosure can be formulated into a drug product intermediate DPI containing an active ingredient in a form having a higher free energy than the crystalline form. One reason for using a DPI is to improve oral absorption characteristics due to low solubility, slow dissolution, improved transport of the substance through the mucus layer of adjacent epithelial cells, and in some cases due to limitations of biological barriers such as metabolism and transport proteins. Other reasons can include improved solid state stability and downstream manufacturability. In one embodiment, the drug product intermediate contains a compound of the present disclosure isolated and stabilized in an amorphous state (e.g., an amorphous solid dispersion (ASD)). There are many techniques known in the art for manufacturing an ASD that produce a material suitable for integration into a bulk drug product, such as spray dried dispersions (SDD), melt extrudates (commonly referred to as HME), co-precipitates, amorphous drug nanoparticles, and nano-adsorbates. In one embodiment, the amorphous solid dispersion contains a compound of the present disclosure and a polymeric excipient. Other excipients and the concentrations of the excipient and the compound of the present disclosure are well known in the art and are described in standard textbooks. See, for example, Navnit Shah et al., “Amorphous Solid Dispersions Theory and Practice”.

[0180] Administration and dosing

[0181] As used herein, the term “treatment” includes prophylactic, i.e., preventive, and palliative treatment, i.e., alleviating, reducing, or slowing the progression of a disease (or condition) or any tissue damage associated with the disease in a patient.

[0182] As used herein, the terms “subject”, “individual”, or “patient” are used interchangeably and refer to any animal, including mammals. Mammals according to the present disclosure include dogs, cats, cows, goats, horses, sheep, pigs, rodents, lagomorphs, primates, humans, etc., and encompass mammals in utero. In one embodiment, a human is a suitable subject. A human subject can be of any gender and at any stage of development.

[0183] As used herein, the phrase “therapeutically effective amount” refers to the amount of an active compound or agent that elicits a biological or medical response sought by a researcher, veterinarian, physician, or other clinician in a tissue, system, animal, individual, or human, which can include one or more of the following:

[0184] (1) Preventing the disease; for example, preventing a disease, disorder, or condition in an individual who may be predisposed to the disease, disorder, or condition but has not yet experienced or shown the pathology or symptoms of the disease;

[0185] (2) Inhibiting the disease; for example, inhibiting a disease, disorder, or condition in an individual who is experiencing or showing the pathology or symptoms of the disease, disorder, or condition (i.e., preventing (or slowing) the further development of the pathology or symptoms or both); and

[0186] (3) Alleviating the disease; for example, alleviating a disease, disorder, or condition in an individual who is experiencing or showing the pathology or symptoms of the disease, disorder, or condition (i.e., reversing the pathology or symptoms or both).

[0187] Generally, the compounds of the present disclosure are administered in an amount effective to treat the conditions described herein. The compounds of the present disclosure can be administered as the compound itself or as a pharmaceutically acceptable salt.

[0188] The compounds of the present disclosure are administered by any suitable route in the form of a pharmaceutical composition adapted to such route and in a dose effective for the intended treatment. The compounds of the present disclosure can be administered orally, rectally, vaginally, parenterally, topically, intranasally, or by inhalation.

[0189] The compounds of the present disclosure can be administered orally. Oral administration can involve swallowing such that the compound enters the gastrointestinal tract, or buccal or sublingual administration can be used, whereby the compound enters the bloodstream directly from the mouth.

[0190] In another embodiment, the compounds of the present disclosure can also be administered parenterally, for example, directly into the bloodstream, into a muscle, or into an internal organ. Suitable modes for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) syringes, needleless syringes, and infusion techniques.

[0191] In another embodiment, the compounds of the present disclosure can also be administered topically to the skin or mucosa, i.e., cutaneous or transdermal administration. In another embodiment, the compounds of the present disclosure can also be administered intranasally or by inhalation. In another embodiment, the compounds of the present disclosure can be administered rectally or vaginally. In another embodiment, the compounds of the present disclosure can also be administered directly to the eye or ear.

[0192] The dosing regimen of the compounds of the present disclosure or the compositions containing said compounds is based on a variety of factors, including the type, age, weight, sex and medical condition of the patient; the severity of the disorder; the route of administration; and the activity of the specific compound used. Accordingly, the dosing regimen can vary widely. In one embodiment, the total daily dose of the compounds of the present disclosure is generally from about 0.01 mg / kg to about 100 mg / kg (i.e., mg of the compound of the present disclosure / kg body weight) for the treatment of the specified disorders discussed herein. In another embodiment, the total daily dose of the compounds of the present disclosure is from about 0.1 mg / kg to about 50 mg / kg, and in another embodiment from about 0.5 mg / kg to about 30 mg / kg. It is not uncommon for the administration of the compounds of the present disclosure to be repeated multiple times (usually not more than 4 times) during the day. If desired, multiple doses per day can generally be used to increase the total daily dose.

[0193] Therapeutic methods and uses

[0194] The compounds of the present disclosure can inhibit the activity of CDKs (including CDK2, CDK4, and / or CDK6), thereby achieving biological functions. Accordingly, the compounds of the present disclosure can be used to treat, prevent, inhibit, and mitigate diseases such as cancers, disorders, and conditions mediated by CDK2, CDK4, and / or CDK6 or combinations thereof.

[0195] In one aspect, the present disclosure provides a method for treating abnormal cell growth in an individual, the method comprising administering to the individual a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof. In common embodiments, the abnormal cell growth is cancer.

[0196] In another aspect, the present disclosure provides a method for inhibiting cancer cell proliferation in an individual, the method comprising administering to the individual an amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof effective to inhibit cell proliferation.

[0197] In another aspect, the present disclosure provides a method for inhibiting cancer cell invasion in an individual, the method comprising administering to the individual an amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof effective to inhibit cell invasion.

[0198] In another aspect, the present disclosure provides a method for inducing apoptosis of cancer cells in an individual, the method comprising administering to the individual an amount of a compound of the present disclosure or a pharmaceutically acceptable salt thereof effective to induce apoptosis.

[0199] In some embodiments of the methods provided herein, the abnormal cell growth is cancer, where the cancer is selected from breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer (including NSCLC, SCLC, squamous cell carcinoma, or adenocarcinoma), esophageal cancer, head and neck cancer, colorectal cancer, kidney cancer (including RCC), liver cancer (including HCC), pancreatic cancer, stomach cancer (i.e., gastric cancer), and thyroid cancer. In additional embodiments of the methods provided herein, the cancer is selected from breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, liver cancer, pancreatic cancer, and stomach cancer. In some such embodiments, the cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2.

[0200] In some embodiments, the cancer is selected from breast cancer and ovarian cancer. In some such embodiments, the cancer is breast cancer or ovarian cancer, which is characterized by amplification or overexpression of CCNE1 and / or CCNE2. In some such embodiments, the cancer is (a) breast cancer or ovarian cancer; (b) characterized by amplification or overexpression of cyclin E1 (CCNE1) or cyclin E2 (CCNE2); or (c) both (a) and (b).

[0201] In some embodiments, the cancer is ovarian cancer. In some such embodiments, the ovarian cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2. In some embodiments, the ovarian cancer is advanced or metastatic breast cancer.

[0202] In other embodiments, the cancer is breast cancer.

[0203] In some embodiments, the breast cancer is estrogen receptor (ER) positive (ER+) / hormone receptor (HR) positive (HR+).

[0204] In some embodiments, the breast cancer is human epidermal growth factor receptor 2 (HER2) negative (HER2-).

[0205] In some embodiments, the breast cancer is ER positive / HR positive.

[0206] In some embodiments, the breast cancer is HER2 positive.

[0207] In some embodiments, the breast cancer is triple-negative breast cancer (TNBC).

[0208] In some embodiments, the breast cancer is inflammatory breast cancer.

[0209] In some embodiments, the breast cancer is endocrine-resistant breast cancer, trastuzumab-resistant breast cancer, or breast cancer that exhibits primary or acquired resistance to CDK4 / CDK6 inhibition.

[0210] In some embodiments, the breast cancer is advanced breast cancer or metastatic breast cancer. In some embodiments of each of the foregoing, the breast cancer is characterized by amplification or overexpression of CCNE1 and / or CCNE2.

[0211] In some embodiments, the compounds of the present disclosure are administered as a first-line therapy. In other embodiments, the compounds of the present disclosure are administered as a second (or later)-line therapy. In some embodiments, the compounds of the present disclosure are administered as a second (or later)-line therapy after treatment with an endocrine therapeutic agent and / or a CDK4 / CDK6 inhibitor. In some embodiments, the compounds of the present disclosure are administered as a second (or later)-line therapy after treatment with an endocrine therapeutic agent. In some embodiments, the compounds of the present disclosure are administered as a second (or later)-line therapy after treatment with a CDK4 / CDK6 inhibitor. In some embodiments, the compounds of the present disclosure are administered as a second (or later)-line therapy after treatment with one or more chemotherapy regimens such as those including taxanes or platinum agents. In some embodiments, the compounds of the present disclosure are administered as a second (or later)-line therapy after treatment with a HER2-targeting agent such as trastuzumab. In some embodiments, the compounds of the present disclosure are administered after failure of treatment with an endocrine therapeutic agent.

[0212] Co - administration

[0213] The compounds of the present disclosure can be used alone or in combination with one or more other therapeutic agents. The present disclosure provides any use, method, or composition as defined herein, wherein the compound of formula (I) or a pharmaceutically acceptable salt thereof is used in combination with one or more other therapeutic agents discussed herein.

[0214] "Combination" administration of two or more compounds means that all of the compounds are administered close enough in time to affect the treatment of the individual. Depending on the treatment regimen, the two or more compounds can be administered simultaneously or sequentially via the same or different routes of administration, on the same or different dosing schedules, and with or without a specific time limit. Additionally, simultaneous administration can be effected by mixing the compounds prior to administration or by administering the compounds at the same time point but as separate dosage forms at the same or different administration sites. Examples of "combination" include, but are not limited to, "concurrent administration", "co-administration", "simultaneous administration", "sequential administration", and "simultaneous application".

[0215] The compounds of formula (I) and said one or more other therapeutic agents can be administered as a fixed or non-fixed combination of active ingredients. The term "fixed combination" means that the compound of formula (I) or a pharmaceutically acceptable salt thereof and said one or more therapeutic agents are both administered to an individual simultaneously in a single composition or dose. The term "non-fixed combination" means that the compound of formula (I) or a pharmaceutically acceptable salt thereof and said one or more therapeutic agents are formulated as separate compositions or doses such that they can be administered to an individual in need thereof simultaneously or at different times with variable intervention time intervals, wherein such administration provides effective levels of two or more compounds in the individual's body.

[0216] The classes of additional chemotherapeutic agents that can be administered in combination with the compounds of the present disclosure include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, antiestrogens and selective estrogen receptor modulators (SERMs), antiprogesterones, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists; IL-2 receptor agonists (recombinant cytokines or cytokine receptor agonists); and antisense oligonucleotides or oligonucleotide derivatives that inhibit gene expression involved in abnormal cell proliferation or tumor growth.

[0217] Other additional chemotherapeutic agents include not only taxanes or platinum-based agents, but also HER2-targeting agents such as trastuzumab.

[0218] In another embodiment, such additional anticancer therapeutic agents include compounds derived from the following classes: mitotic inhibitors, alkylating agents, antimetabolites, antitumor antibiotics, antiangiogenic agents, topoisomerase I and II inhibitors, plant alkaloids, spindle poison plant alkaloids, MCT4 inhibitors; MAT2a inhibitors; alk / c-Met / ROS inhibitors (including crizotinib or lorlatinib); mTOR inhibitors (including temsirolimus or gedatolisib); src / abl inhibitors (including bosutinib); cyclin-dependent kinase (CDK) inhibitors (including palbociclib); erb inhibitors (including dacomitinib); PARP inhibitors (including talazoparib); SMO inhibitors (including glasdegib); EGFR T790M inhibitors; PRMT5 inhibitors; TGFβR1 inhibitors; growth factor inhibitors; cell cycle inhibitors, biological response modifiers; enzyme inhibitors; and cytotoxic agents.

[0219] In another embodiment, such additional anti-cancer therapeutic agents include compounds derived from anti-angiogenic agents, including, for example, tyrosine kinase / vascular endothelial growth factor (VEGF) receptor (VEGFR) inhibitors (including sunitinib, axitinib, sorafenib, and tivozanib), TIE-2 inhibitors, PDGFR inhibitors, angiopoietin inhibitors, PKCβ inhibitors, COX-2 (cyclooxygenase II) inhibitors, integrin (α-v / β-3), MMP-2 (matrix-metalloproteinase 2) inhibitors, and MMP-9 (matrix-metalloproteinase 9) inhibitors. Preferred anti-angiogenic agents include sunitinib (Sutent TM ), bevacizumab (Avastin TM ), axitinib (Inlyta TM ), SU 14813 (Pfizer), and AG13958 (Pfizer). Additional anti-angiogenic agents include vatalanib (CGP 79787), pegaptanib octasodium (Macugen TM ), vandetanib (Zactima TM ), PF-0337210 (Pfizer), SU14843 (Pfizer), AZD 2171 (AstraZeneca), ranibizumab (Lucentis TM ), Neovastat TM (AE 941), tetrathiomolybdate (Coprexa TM ), AMG 706 (Amgen), VEGF Trap (AVE 0005), CEP 7055 (Sanofi-Aventis), XL 880 (Exelixis), telatinib (BAY 57-9352), and CP-868,596 (Pfizer). Other anti-angiogenic agents include enzastaurin (LY 317615), midostaurin (CGP 41251), perifosine (KRX 0401), teprenone (Selbex TM ), and UCN 01 (Kyowa Hakko). Other examples of anti-angiogenic agents include celecoxib (Celebrex TM ), parecoxib (Dynastat TM ), deracoxib (SC 59046), lumiracoxib (Preige TM) valdecoxib (Bextra TM ) rofecoxib (Vioxx TM ) iguratimod (Careram TM ) IP 751 (Invedus), SC-58125 (Pharmacia), and etoricoxib (Arcoxia TM )。Additional anti-angiogenic agents include exisulind (Aptosyn TM ) salsalate (Amigesic TM ) diflunisal (Dolobid TM ) ibuprofen (Motrin TM ) ketoprofen (Orudis TM ) nabumetone (Relafen TM ) piroxicam (Feldene TM ) naproxen (Aleve TM , Naprosyn TM ) diclofenac (Voltaren TM ) indomethacin (Indocin TM ) sulindac (Clinoril TM ) tolmetin (Tolectin TM ) etodolac (Lodine TM ) ketorolac (Toradol TM ) and oxaprozin (Daypro TM )。Additional anti-angiogenic agents include ABT 510 (Abbott), apratastat (TMI 005), AZD8955 (AstraZeneca), incyclinide (Metastat TM ) and PCK3145 (Procyon)。Additional anti-angiogenic agents include acitretin (Neotigason TM ) plitidepsin (aplidine TM ) cilengtide (EMD 121974), combretastatin A4 (CA4P), fenretinide (4HPR), halofuginone (Tempostatin TM ) Panzem TM(2-Methoxyestradiol), PF-03446962 (Pfizer), rebimastat (BMS275291), catumaxomab (Removab TM ), lenalidomide (Revlimid TM ), evizon (EVIZON TM ), thalidomide (Thalomid TM ), Ukrain TM (NSC 631570), Vitaxin TM (MEDI 522), and zoledronic acid (Zometa TM ).

[0220] In another embodiment, such additional anti-cancer therapeutic agents include compounds derived from hormonal agents and antagonists. Examples include anti-hormonal agents that are used to modulate or inhibit the action of hormones on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), and selective estrogen receptor degraders (SERDs), including tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, toremifene (Fareston), and fulvestrant. Examples also include aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal gland, and include compounds such as 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprorelin, fluridil, apalutamide, enzalutamide, cimetidine, and goserelin.

[0221] In another embodiment, such additional anti-cancer therapeutic agents include compounds derived from signal transduction inhibitors, such as inhibitors of protein tyrosine kinases and / or serine / threonine kinases: signal transduction inhibitors (e.g., inhibit the way in which regulatory molecules that control cell growth, differentiation, and survival communicate within cells). Signal transduction inhibitors include small molecules, antibodies, and antisense molecules. Signal transduction inhibitors include, for example, kinase inhibitors (e.g., tyrosine kinase inhibitors or serine / threonine kinase inhibitors) and cell cycle inhibitors. More specifically, signal transduction inhibitors include, for example, farnesyl protein transferase inhibitors, EGF inhibitors, ErbB-1 (EGFR), ErbB-2, pan-Erb, IGF1R inhibitors, MEK (including binimetinib (Mektovi TM), c-Kit inhibitors, FLT-3 inhibitors, K-Ras inhibitors, PI3 kinase inhibitors, JAK inhibitors, STAT inhibitors, Raf kinase inhibitors, BRAF (including encorafenib (Braftovi TM ), Akt inhibitors, mTOR inhibitors, P70S6 kinase inhibitors, inhibitors of the WNT pathway, and multi-target kinase inhibitors.

[0222] In another embodiment, such additional anti-cancer therapeutic agents include docetaxel, paclitaxel, paclitaxel protein-bound particles, cisplatin, carboplatin, oxaliplatin, capecitabine, gemcitabine, or vinorelbine.

[0223] In another embodiment, such additional anti-cancer therapeutic agents include compounds derived from epigenetic regulators, examples of which include inhibitors of EZH2 (including PF-06821497), SMARCA4, PBRM1, ARID1A, ARID2, ARID1B, DNMT3A, TET2, MLL1 / 2 / 3, NSD1 / 2, SETD2, BRD4, DOT1L, HKMTsanti, PRMT1-9, LSD1, UTX, IDH1 / 2, or BCL6.

[0224] In another embodiment, such additional anti-cancer therapeutic agents include compounds that are immuno-oncology agents, including immunomodulators.

[0225] In another embodiment, combinations with pattern recognition receptors (PRRs) are contemplated. A PRR is a receptor expressed by cells of the immune system that recognizes a variety of molecules associated with pathogens and / or cell damage or death. PRRs are involved in both innate and adaptive immune responses. PRR agonists can be used to stimulate an individual's immune response. There are multiple classes of PRR molecules, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and stimulator of interferon genes (STING) proteins.

[0226] The STING protein functions as a cytoplasmic DNA sensor and adaptor protein in type I interferon signaling. The terms "STING" and "stimulator of interferon genes" refer to any form of the STING protein, as well as variants, isoforms, and species homologs that retain at least a portion of STING activity. Unless otherwise specified, such as by specific reference to human STING, STING includes all mammalian species of the native sequence STING, such as human, monkey, and mouse, and STING is also known as TMEM173.

[0227] As used herein, "STING agonist" means any molecule which, when bound to STING, (1) stimulates or activates STING, (2) enhances, increases, promotes, induces or prolongs the activity, function or presence of STING, or (3) enhances, increases, promotes or induces the expression of STING. STING agonists that can be used in any of the therapeutic methods, drugs and uses of the present disclosure include, for example, nucleic acid ligands that bind to STING.

[0228] Examples of STING agonists that can be used in the therapeutic methods, drugs and uses of the present disclosure include a variety of immunostimulatory nucleic acids such as synthetic double-stranded DNA, cyclic di-GMP, cyclic GMP-AMP (cGAMP), synthetic cyclic dinucleotides (CDNs) such as MK-1454 and ADU-S100 (MIW815) and small molecules such as WO2019027858, WO20180093964, WO2017175156, WO2017175147.

[0229] Therapeutic antibodies can be specific for a variety of different antigens. For example, a therapeutic antibody can be specific for a tumor-associated antigen such that binding of the antibody to the antigen promotes the death of cells expressing the antigen. In other examples, a therapeutic antibody can be specific for an antigen on an immune cell such that binding of the antibody prevents downregulation of the activity of cells expressing the antigen (and thus promotes the activity of cells expressing the antigen). In some cases, a therapeutic antibody can act by a variety of different mechanisms (e.g., it can i) promote the death of cells expressing the antigen, and ii) prevent the antigen from causing downregulation of the activity of immune cells in contact with cells expressing the antigen).

[0230] In another embodiment, such additional anti-cancer therapeutic agents include antibodies that block or inhibit at the target: CTLA-4 (including ipilimumab or tremelimumab), PD-1 or PD-L1 (including atezolizumab, avelumab, cemiplimab, durvalumab, nivolumab, sasanlimab or pembrolizumab), LAG-3, TIM-3 or TIGIT.

[0231] In another embodiment, such additional anti-cancer therapeutic agents include antibodies that are agonists of 4-1BB, OX40, GITR, ICOS or CD40.

[0232] In another embodiment, the anti-cancer therapy can be CAR-T cell therapy.

[0233] Examples of therapeutic antibodies include: anti-OX40 antibody, anti-4-1BB antibody, anti-HER2 antibody (including anti-HER2 antibody-drug conjugate (ADC)), bispecific anti-CD47 / anti-PD-L1 antibody, and bispecific anti-P-cadherin / anti-CD3 antibody. Examples of cytotoxic agents that can be incorporated into ADCs include anthracyclines, auristatin, dolastatin, combretastatin, duocarmycin, pyrrolobenzodiazepine dimers, indolinobenzodiazepine (indolino-benzodiazepine) dimers, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, camptothecin, tubulysin, hemiasterlin, spliceostatin, pladienolide, and their stereoisomers, bioisosteres, analogs or derivatives. Exemplary immunomodulators that can be incorporated into ADCs include ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-15, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factor designated "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0234] Other examples of therapeutic antibodies can include the following antigens, where exemplary antibodies against the antigens are also included below (in parentheses / brackets after the antigen). The following antigens may also be referred to herein as "target antigens" and the like. Target antigens for therapeutic antibodies herein include, for example: 4-1BB (e.g., utomilumab); 5T4; A33; alpha-folate receptor 1 (e.g., mirvetuximab soravtansine); Alk-1; BCMA [see, e.g., US9969809]; BTN1A1 (see, e.g., WO2018222689); CA-125 (e.g., abagovomab); carbonic anhydrase IX; CCR2; CCR4 (e.g., mogamulizumab); CCR5 (e.g., leronlimab); CCR8; CD3 [e.g., blinatumomab (CD3 / CD19 bispecific), CD3 / P-cadherin bispecific, CD3 / BCMA bispecific] CD19 (e.g., blinatumomab, MOR208); CD20 (e.g., ibritumomab tiuxetan, obinutuzumab, ofatumumab, rituximab, ublituximab); CD22 (inotuzumab ozogamicin, moxetumomab pasudotox); CD25; CD28; CD30 (e.g., brentuximab vedotin); CD33 (e.g., gemtuzumab ozogamicin); CD38 (e.g., daratumumab, isatuximab), CD40; CD-40L; CD44v6; CD47 (e.g., Hu5F9-G4, CC-90002, SRF231, B6H12); CD52 (e.g., alemtuzumab); CD56; CD63; CD79 (e.g., polatuzumab vedotin); CD80; CD123; CD276 / B7-H3 (e.g., omburtamab); CDH17; CEA; ClhCG; CTLA-4 (e.g., ipilimumab, tremelimumab), CXCR4; desmoglein 4; DLL3 (e.g., rovalpituzumab tesirine); DLL4; E-cadherin; EDA; EDB; EFNA4;EGFR (such as cetuximab, depatuxizumab mafodotin, necitumumab, panitumumab); EGFRvIII; endosialin; EpCAM (such as oportuzumab monatox); FAP; fetal acetylcholine receptor; FLT3 (see, for example, WO2018 / 220584); GD2 (such as dinutuximab, 3F8); GD3; GITR; GloboH; GM1; GM2; HER2 / neu [such as margetuximab, pertuzumab, trastuzumab; ado-trastuzumab emtansine, trastuzumab duocarmazine, [see US8828401]; HER3; HER4; ICOS; IL-10; ITG-AvB6; LAG-3 (such as relatlimab); Lewis-Y; LG; Ly-6; M-CSF [see US7326414]; MCSP; mesothelin; MUC1; MUC2; MUC3; MUC4; MUC5AC; MUC5B; MUC7; MUC16; Notch1; Notch3; connexin-4 (such as enfortumab vedotin); OX40 [see US7960515]; P-Cadherin [see WO2016 / 001810]; PCDHB2; PDGFRA (such as olaratumab); plasma cell antigen; PolySA; PSCA; PSMA; PTK7 [see US9409995]; Ror1; SAS; SCRx6; SLAMF7 (such as elotuzumab); SHH; SIRPa (such as ED9, Effi-DEM); STEAP; TGF-β; TIGIT; TIM-3; TMPRSS3; TNF-α precursor; TROP-2 (such as sacituzumab govitecan); TSPAN8; VEGF (such as bevacizumab, brolucizumab); VEGFR1 (such as ranibizumab); VEGFR2 (such as ramucirumab, ranibizumab); Wue-1.;

[0235] Exemplary imaging agents that can be included in an ADC include fluorescein, rhodamine, lanthanide phosphors, and their derivatives, or radioisotopes conjugated to chelating agents. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosin, Alexa (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelating agents include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-pentanedioic acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA).

[0236] Exemplary therapeutic proteins that can be included in an ADC include toxins, hormones, enzymes, and growth factors.

[0237] Exemplary biocompatible polymers that can be incorporated into an ADC include water-soluble polymers such as polyethylene glycol (PEG) or its derivatives, and zwitterionic-containing biocompatible polymers (e.g., polymers containing phosphocholine).

[0238] Exemplary biocompatible polymers that can be incorporated into an ADC include antisense oligonucleotides.

[0239] The present disclosure also relates to the use of radiation in combination with any anti-cancer therapeutic agent administered herein. More specifically, the compounds of the present disclosure can be administered in combination with additional therapies such as, for example, radiotherapy and / or chemotherapy.

[0240] These agents and compounds of the present disclosure can be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, glucose solution, etc. Specific dosing regimens, i.e., dose, timing, and repetition, will depend on the particular individual and the individual's medical history.

[0241] Kit

[0242] Another aspect of the present disclosure provides a kit comprising a compound of formula (I) or a pharmaceutical composition comprising the compound of formula (I). In addition to the compound of formula (I) or its pharmaceutical composition, the kit may further include a diagnostic agent or a therapeutic agent. The kit may also include instructions for a diagnostic or therapeutic method. In some embodiments, the kit comprises the compound or its pharmaceutical composition and a diagnostic agent. In other embodiments, the kit comprises the compound or its pharmaceutical composition and one or more therapeutic agents.

[0243] In yet another embodiment, the present disclosure includes a kit suitable for performing the therapeutic methods described herein. In one embodiment, the kit comprises a first dosage form comprising an amount of one or more compounds of the present disclosure sufficient to perform the methods of the present disclosure. In another embodiment, the kit comprises an amount of one or more compounds of the present disclosure sufficient to perform the methods of the present disclosure and a container for the dosage and a container for the dosage.

[0244] Synthetic method

[0245] The compounds of formula (I) can be synthesized by synthetic routes including methods similar to those well-known in the chemical art, particularly according to the descriptions contained herein. The starting materials are generally commercially available or can be prepared using methods well-known to those skilled in the art. Many of the compounds used herein relate to or can be derived from compounds in which one or more scientific interests or commercial needs have occurred. Thus, such compounds can be one or more of the following: 1) commercially available; 2) reported in the literature; or 3) prepared by those skilled in the art from other common substances using materials reported in the literature.

[0246] For illustrative purposes, the reaction schemes described below provide possible routes for synthesizing the compounds of the present disclosure as well as key intermediates. For a more detailed description of each reaction step, see the Examples section below. Those skilled in the art will understand that other synthetic routes can be used to synthesize the compounds of the present invention. Although specific starting materials and reagents are discussed below, other starting materials and reagents can be substituted to provide one or more of a variety of derivatives or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified using conventional chemistry well-known to those skilled in the art in accordance with the present disclosure.

[0247] Those skilled in the art will understand that the experimental conditions set forth in the following schemes illustrate suitable conditions for achieving the indicated transformations, and that the exact conditions for preparing the compounds of the present disclosure may need to or be desired to be changed. It should also be understood that the transformations may need to or be desired to be carried out in a different order than described in the schemes, or that one or more of the transformations may be modified to provide the desired compound of formula (I).

[0248] In the preparation of the compounds of the present disclosure, it should be noted that some of the preparation methods that can be used to prepare the compounds described herein may require protection of remote functional groups (e.g., primary amines, secondary amines, carboxyl groups, etc. in the precursors of formula (I)). The need for such protection will vary depending on the nature of the remote functional group and the conditions of the preparation method. It is readily determined by those skilled in the art whether such protection is needed. The use of such protection / deprotection methods is also within the skills of those skilled in the art. For a general description of protecting groups and their use, see March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 8th Edition.

[0249] For example, if a compound contains an amine or carboxylic acid functional group, such functional groups may interfere with reactions at other sites of the molecule if not protected. Therefore, such functional groups can be protected by appropriate protecting groups (PGs), which can be removed in subsequent steps. Suitable protecting groups for amine and carboxylic acid protection include those commonly used in peptide synthesis (e.g., N-tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and 9-fluorenylmethoxycarbonyl (Fmoc) for amines and lower alkyl or benzyl esters for carboxylic acids), which are generally not chemically reactive under the reaction conditions described and can generally be removed without chemically altering other functional groups in the compounds of formula (I).

[0250] General experimental details

[0251] Recorded on a Bruker XWIN-NMR (400 or 700 MHz) spectrometer 1 1H nuclear magnetic resonance (NMR) spectra. Report the 1H resonances at low field in parts per million (ppm) relative to tetramethylsilane 1 1H resonances. 1 1H NMR data are reported as multiplicity (e.g., s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; dd, doublet of doublets; dt, doublet of triplets; br s, broad singlet). For spectra obtained in CDCl3, DMSO-d6, and CD3OD, use the residual protons (7.27, 2.50, and 3.31 ppm, respectively) as internal references. All observed coupling constants J are reported in Hertz (Hz). Exchangeable protons are not always observable.

[0252] Determine the optical rotation on a Jasco P-2000 or Rudolph Autopol IV polarimeter. Unless otherwise stated, all final compounds are purified to ≥95% purity.

[0253] Mass spectrometry MS (m / z) was recorded using electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). Where relevant, the m / z data provided is for isotopes 19 F, 35 Cl, 79 Br and 127 I.

[0254] Nomenclature was written as described by IUPAC (International Union of Pure and Applied Chemistry, generated within Perkin Elmers Chemdraw 18.0.0.231). The naming conventions provided by Perkin Elmers Chemdraw 18.0.0.231 are well known to those skilled in the art and are believed to generally be in accordance with the Recommendations of the Nomenclature of Organic Chemistry of IUPAC (International Union of Pure and Applied Chemistry) and the CAS Index Rules.

[0255] The following abbreviations were used throughout the examples: "Ac" represents acetyl, "OAc" represents acetoxy, "aq" represents aqueous, "DCM" (CH2Cl2) represents dichloromethane, "d.i." represents deionized, "DIEA" represents diisopropylethylamine, "DMSO" represents dimethyl sulfoxide, "EtOAc" represents ethyl acetate, "EtOH" represents ethanol, "HOAc" or "AcOH" represents acetic acid, "i-Pr" or "iPr" represents isopropyl, "LiHMDS" represents lithium hexamethyldisilazide (lithium bis(trimethylsilyl)amide), "Me" represents methyl, "MeOH" represents methanol, "MS" represents mass spectrometry, "MTBE" represents methyl tert-butyl ether, "THF" represents tetrahydrofuran, "2-MeTHF" represents 2-methyltetrahydrofuran, "PXRD" represents powder X-ray diffraction, "oxone" is the potassium salt of peroxymonosulfuric acid, "SFC" represents supercritical fluid chromatography, "TLC" represents thin layer chromatography, "r.b." represents round bottom, "Rf" represents retention factor, "~" represents approximately, "rt" represents room temperature, "h" represents hour, "min" represents minute, "equiv" represents equivalent, "sat." represents saturated.

[0256] Compounds 1-4 are potent inhibitors of CDK2, CDK4, and CDK6 having the following formula:

[0257]

[0258] Compound 1, R = CH2CHF2, R' = NH2

[0259] Compound 2 (PF-06873600), R = CHF2, R' = CH3

[0260] Compound 3, R = CH2CHF2, R' = CH3

[0261] Compound 4, R = CHF2, R' = NH2

[0262] Scheme 1: Synthesis of Compound 1

[0263]

[0264] Compound 1: 4-({6-(2,2-difluoroethyl)-8-[(1R,2R)-2-hydroxy-2-methylcyclopentyl]-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl}amino)piperidine-1-sulfonamide.

[0265] Intermediate 1a (112 g, 420 mmol) and iPrOH (240 mL) were added to a 2 L reaction flask equipped with a top stirrer. Intermediate 1a was prepared according to the procedure described by Duan, S et al. in Organic Process Research & Development 2020, 24(11), 2734 - 2744. The reaction was stirred at room temperature for about 10 minutes, and DIEA (244 g, 1890 mmol) was added dropwise. After stirring, CAS 1044145-59-6 (80 g, 420 mmol) was added, and the reaction headspace was purged with nitrogen. The reaction temperature was raised to 82 °C and maintained at this temperature with stirring for 27 h. After cooling to room temperature, the reaction was concentrated to a total volume of ∼150 mL. Water (200 mL) was added, and the aqueous layer was extracted with 2-MeTHF (500 mL x 3). The combined organic extracts were washed with 30% aqueous K2CO3 solution (280 mL x 2). The organic layer was concentrated to a total volume of ∼150 mL, and MTBE (500 mL) was added. The resulting slurry was stirred at 15 - 25 °C for 14 h and then cooled to 0 °C. The resulting solid was filtered. After drying the wet filter cake in a vacuum oven at 45 °C, intermediate 1b (1R,2R)-2-{[5-(hydroxymethyl)-2-(methylthio)pyrimidin-4-yl]amino}-1-methylcyclopentanol (86 g, 76%) was obtained as an off-white solid. 11H NMR (400 MHz, CDCl3) δ = 7.76 (s, 1H), 6.01 (d, J = 4.6 Hz, 1H), 5.31 (br s, 1H), 4.55 (s, 2H), 4.26 (ddd, J = 5.7, 8.2, 10.5 Hz, 1H), 2.50 (s, 3H), 2.21 (ddd, J = 3.5, 8.2, 12.1 Hz, 1H), 1.97 (dt, J = 3.5, 7.7 Hz, 1H), 1.89 - 1.76 (m, 2H), 1.75 - 1.63 (m, 1H), 1.60 - 1.50 (m, 2H), 1.11 (s, 3H). MS: 270 [M+H] + . Optical rotation: [α] D 22 +37.7 (c 1.0, MeOH).

[0266] Oxidation with MnO2 to obtain intermediate 1c

[0267] Dissolve intermediate 1b (133 g, 495 mmol) in THF (670 mL). Add activated MnO2 (151 g, 1740 mmol), and stir the reaction at 55 °C for 26 h. Filter off the MnO2 by - product using diatomaceous earth, and wash the diatomaceous earth layer with THF (1340 mL). Concentrate the filtrate to ~150 mL, and add heptane (670 mL). Concentrate the heptane to ~150 mL, and repeat the heptane treatment two more times, each time concentrating to ~150 mL. Wash the filtered solid with heptane (~200 mL), and dry the wet filter cake in a vacuum oven at 45 °C for 20 h to obtain intermediate 1c 4-{[(1R,2R)-2 - hydroxy - 2 - methylcyclopentyl]amino}-2-(methylthio)pyrimidine - 5 - carbaldehyde (132 g, 95%), as a solid. 1 1H NMR (400 MHz, CDCl3) δ = 9.73 (s, 1H), 8.66 (br s, 1H), 8.35 (s, 1H), 4.39 (ddd, J = 6.5, 8.2, 9.6 Hz, 1H), 4.16 (s, 1H), 2.57 (s, 3H), 2.33 - 2.22 (m, 1H), 2.03 - 1.92 (m, 1H), 1.89 - 1.68 (m, 3H), 1.68 - 1.56 (m, 1H), 1.17 (s, 3H), MS: 268 [M+H] + . Optical rotation [α] D 22 +12.7 (c 1.0, CHCl3).

[0268] The aldol reaction to obtain intermediate 1d using a flow reactor

[0269] Intermediate 1c (80.0 g, 299 mmol), CAS 1866071-82-0 (70.3 g, 509 mmol), THF (1.44 L), and toluene (320 mL) were added to Reactor #1. LiHMDS (1.05 L, 1 M in THF, 1.05 mol) and toluene (184 mL) were added to Reactor #2. Water (100 mL) was added to Reactor #3. A peristaltic pump fed the solutions from Reactors #1 and #2 into a pre-cooled coil maintained at a temperature of 15–25 °C. The flow rate from Reactor #1 was set at 2.82 mL / min, the flow rate from Reactor #2 was set at 1.84 mL / min, and the flow rate from Reactor #3 was set at 0.6 mL / min. After mixing the effluents from Reactors #1 and #2 at a temperature of 15–25 °C, the mixture flowed into a stirred tank where the effluent from Reactor #3 was also added. The stirred tank was the location where the reaction quenching occurred. The temperature of the stirred tank was maintained at 0–10 °C. The quenched mixture was stirred at 0–10 °C for 0.5 - 3 h. 0.718 mol of the quenched reaction mixture was obtained from two effluent batches of the above reaction on a comparable scale and was worked up as follows. First, the aqueous lower layer was drained from the mixture. Then, 2N HCl (80 mL) was added and the mixture was stirred for 20 minutes. After separating the layers, the lower layer was drained. The resulting organic layer was concentrated to ~720 mL. Solvent exchange to MTBE was carried out by adding 400 mL of MTBE and concentrating to 720 mL twice. After the solvent exchange, 400 mL of MTBE was added, followed by 1N HCl (160 mL). The mixture was stirred for 30 minutes and the layers were separated. The lower layer was drained and 1N HCl (160 mL) was added again. The mixture was stirred for 30 minutes and the layers were separated. The lower layer was drained. 7% aqueous NaHCO3 (160 mL) was added, the mixture was stirred for 30 minutes, and the layers were separated. The organic layer was concentrated to ~720 mL and 2-MeTHF (400 mL) was added. The resulting mixture was concentrated to ~720 mL and 2-MeTHF (400 mL) was added again. After concentrating to ~720 mL, 2-MeTHF (400 mL) was added. The solid formed was allowed to stand for 2–5 h and Intermediate 1d 6-(2,2-difluoroethyl)-8-[(1R,2R)-2-hydroxy-2-methylcyclopentyl]-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (240 g, 94%) was collected as a solid. 11H NMR (400 MHz, chloroform-d) δ 8.63 (s, 1H), 7.59 (s, 1H), 6.31 - 5.97 (m, 1H), 5.88 (t, J = 8.6 Hz, 1H), 3.15 (dt, J = 4.5, 16.3 Hz, 2H), 2.87 - 2.72 (m, 1H), 2.64 (s, 3H), 2.32 - 2.21 (m, 2H), 2.12 - 2.06 (m, 1H), 2.04 - 2.01 (m, 1H), 2.00 - 1.91 (m, 1H), 1.91 - 1.82 (m, 1H), 1.14 (s, 3H), MS: 356.1 [M+H] + .

[0270] Oxidation with Oxone to obtain intermediate 1d

[0271] Intermediate 1d (300 g, 0.844 mol) was dissolved in 2-MeTHF (2.7 L) and water (1.05 L) was added. The temperature was cooled to 0–10 °C. Oxone (1.3 kg, 2.1 mol) was added and the reaction was heated to 20–30 °C. Stirring was continued at this temperature for 16 h. Water (1.5 L) was added and stirring was continued for 15 minutes. After separating the layers, the lower aqueous layer was drained. Then, 5% aqueous NaHSO3 solution (900 mL) was added and stirring was continued for 15 minutes. The lower aqueous layer was drained and 5% aqueous Na2SO4 solution (900 mL) was added and stirred for 15 minutes. The lower aqueous layer was drained. 30 g of activated carbon was added to the organic phase and stirring was continued for 16 h. The organic layer was filtered through diatomaceous earth and the filter cake was washed with 2-MeTHF (600 mL). The organic layer was concentrated and MTBE (1.2 L) was added. The organic layer was concentrated and MTBE (1.2 L) was added again. The organic layer was concentrated and MTBE (1.2 L) was added again. The mixture was heated to 30–40 °C and stirred for 1 h. While stirring, the reaction was cooled to 10–20 °C and stirred at this temperature for 2 h. The solid formed was collected, washed with MTBE (600 mL), and dried in a vacuum oven at 40 °C for 16 h. Recrystallization was carried out by dissolving the obtained solid in 2-MeTHF (600 mL), concentrating, and adding MTBE (1.2 L). MTBE was removed in vacuo at 40 °C. MTBE (1.2 L) was added and the organic layer was concentrated. MTBE (1.2 L) was added again, the mixture was heated to 30–40 °C and stirred for 1 h. While stirring, the reaction was cooled to 10–20 °C and stirred at this temperature for 2 h. The solid formed was collected, washed with MTBE (600 mL), and dried in a vacuum oven at 40 °C for 16 h to give intermediate 1e 6-(2,2-difluoroethyl)-8-[(1R,2R)-2-hydroxy-2-methylcyclopentyl]-2-(methylsulfonyl)pyrido[2,3-d]pyrimidin-7(8H)-one (211 g, 64%) as an off-white solid.

[0272] The SnAr reaction gave compound 1

[0273] At 20 °C, CAS 1016818-01-1 (19.7 g, 110 mmol) was added to a stirred solution of compound intermediate 1e (17 g, 44 mmol) in 2-MeTHF (350 mL). The reaction was heated to 60 °C and stirred for 16 h. TLC (DCM:MeOH = 10:1) showed completion of the reaction. The mixture was diluted with water (400 mL) and extracted with EtOAc (4 x 300 mL). The combined organic layers were washed with brine (100 mL), dried over MgSO4, filtered, and concentrated to give the crude product, which was purified by flash chromatography using a 0–10% MeOH gradient in DCM. This gave 18 g of crude compound 1 as a yellow solid, which was further purified by preparative-HPLC using an Xtimate C18 (150 mm * 40 mm * 5 mm) column and a gradient elution of CH3CN in water containing 0.2% formic acid. After evaporation of most of the solvent from the fractions, the remaining solution was dissolved in EtOAc (750 mL) and washed with saturated aqueous NaHCO3. The EtOAc layer was washed with brine (100 mL), dried over MgSO4, filtered, and concentrated to give compound 1, 4-({6-(2,2-difluoroethyl)-8-[(1R,2R)-2-hydroxy-2-methylcyclopentyl]-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl}amino)piperidine-1-sulfonamide (13.5 g, 78%), as a white EtOAc solvate.

[0274] The solvate of compound 1 was converted to the anhydrous solid form

[0275] To obtain the anhydrous solid form, a 1 L three-necked r.b. flask was equipped with an internal thermometer, an OH stirrer, and a heating source (oil bath). The solvated compound 1 (10.9 g, 22.4 mmol) was added to the r.b. followed by 250 mL of d.i. water. Stirring was started while heating the oil bath to obtain an internal temperature of 48 °C. Stirring was continued at approximately 600 rpm for 4 days while maintaining the internal temperature at 45 - 48 °C. On the 4th day, a sample was taken for PXRD, and PXRD showed conversion to the desired anhydrous form. Due to the drying time prior to PXRD, the total stirring time for this batch was 5 days at 45 - 48 °C. On the 5th day, the entire batch was filtered, rinsed with water to transfer all the material to the Buchner funnel. Filtration was carried out slowly to obtain a wet filter cake. The wet filter cake was compressed and suction filtration was continued for 1 h. The material was placed under H-vac overnight. Anhydrous compound 1 (9.6 g, 88%) was obtained. The identification of the anhydrous form was also supported by elemental analysis and the absence of residual solvents observed by 1 1H NMR. 11H NMR (DMSO-d6, 400 MHz, 80 °C) δ 8.57 (s, 1H), 7.69 (s, 1H), 7.40 (br d, 1H, J = 6.8 Hz), 6.47 (s, 2H), 6.19 (tt, 1H, J = 4.6, 57.2 Hz), 5.8 - 5.9 (m, 1H), 4.01 (s, 1H), 3.9 - 4.0 (m, 1H), 3.5 - 3.6 (m, 2H), 3.0 - 3.1 (m, 2H), 2.75 (tt, 2H, J = 2.4, 11.7 Hz), 2.2 - 2.3 (m, 1H), 1.8 - 2.1 (m, 5H), 1.5 - 1.8 (m, 3H), 1.01 (s, 3H), MS: 487.2 [M+H] + , [α] D 22 -20.3 (c 0.2, MeOH), C 20 H 28 Anal. Calcd for C19H21F2N6O4S: C, 49.37; H, 5.80; N, 17.27; Found: C, 49.49; H, 5.65; N, 17.09; qNMR = 98.0 + / - 1.8%, HPLC purity = 98.9%.

[0276] The anhydrous compound 1 prepared above was further characterized by powder X-ray diffraction (PXRD).

[0277] Powder X-ray diffraction:

[0278] Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavor diffractometer equipped with a Cu radiation source. The divergence slit was set to 15 mm continuous illumination. The diffracted radiation was detected by a PSD-Lynx Eye detector, where the detector PSD opening was set to 4.107 degrees. The X-ray tube voltage and amperage were set to 40 kV and 40 mA, respectively. Using a step size of 0.01 degrees and a time step of 1.0 second, in a Theta-Theta goniometer at the Cu wavelength Data was collected from 3.0 degrees to 40.0 degrees 2θ. The anti-scatter screen was set at a fixed distance of 1.5 mm. The sample was rotated at 15 / min during data collection. The sample was prepared by placing it in a silicon low-background sample holder and rotating it during collection. Data was collected using Bruker DIFFRACPlus software and analyzed using EVA diffract plus software. The PXRD data file was not processed before peak search. In the EVA software, the peak search algorithm was used, and a peak search with a threshold of 1 was selected for preliminary peak assignment. To ensure validity, manual adjustment was performed; the output of the automatic assignment was visually inspected, and the peak position was adjusted to the peak maximum. Peaks with a relative intensity ≥ 3% were generally selected. Generally, peaks that were not resolved or consistent with noise were not selected. The typical error associated with the peak position of PXRD is specified in the USP as + / −0.2° 2θ (USP-941).

[0279] The PXRD pattern of compound 1 free base form 1 is shown in Figure 1 Table 1 below provides the PXRD peak list and relative intensity data (2θ°) for compound 1 free base form 1.

[0280] Table 1. PXRD peak list for anhydrous crystalline free base form 1. The asterisk peak positions indicate the characteristic peaks of form 1

[0281] Angle (2θ) Relative intensity (%) 4.8* 100.0 10.6* 38.0 14.3* 84.5 17.0 11.4 17.2 5.2 17.4 4.5 18.4 15.4 19.1* 41.9 19.7* 66.8 20.2 10.8 21.8 3.1 22.9 3.4 23.9 10.8 25.8 3.9 28.8 4.4 34.4 3.6 35.2 4.3

[0282] Synthesis of Compounds 2-4

[0283] The preparation of compounds 2-4 is disclosed in International Patent Publication No. WO2018 / 033815 and U.S. Patent Application No. 2018 / 0044344. Improved methods for preparing compound 2 (PF-06873600) have been disclosed in several publications. Freeman-Cook et al., J. Med. Chem., 2021, 64(13), 9056-9077; Meng, D. et al., Cell Reports Physical Science, 2021, 2(4), 100394. The content of each of the foregoing documents is incorporated herein by reference in its entirety.

[0284] Deuterated analogs of compound 1

[0285] The compounds shown in Table 2 are predictive deuterated analogs (PDAs) of compound 1. The PDAs were predicted based on the metabolic profile of compound 1 (obtained from metabolic assessment and the MetaSite database).

[0286] Table 2. Deuterated analogs of compound 1

[0287]

[0288] PDA <![CDATA[Y1]]> <![CDATA[Y2]]> <![CDATA[Y3]]> <![CDATA[Y4]]> <![CDATA[Y5]]> <![CDATA[Y6]]> <![CDATA[Y7]]> <![CDATA[Y8]]> <![CDATA[Y9]]> <![CDATA[Y 10 > <![CDATA[Y 11 > <![CDATA[Y 12 > <![CDATA[Y 13 > 1 D D H H H H H H H H H H H 2 H H D D H H H H H H H H H 3 H H H H D D H H H H H H H 4 H H H H H H D H H H H H H 5 H H H H H H H D D H H H H 6 H H H H H H H H H D D H H 7 H H H H H H H H H H H D H 8 H H H H H H H H H H H H D

[0289] The preliminary assessment of the metabolism of Compound 1 was carried out using mouse, rat, rabbit, dog, monkey and human hepatocytes, human liver microsomes, and plasma from monkey, dog and mouse studies following oral administration. The major metabolic pathway of Compound 1 is oxidation.

[0290] General methods for obtaining metabolite profiles of compounds and identifying metabolites / reviews are described in: Dalvie et al., “Assessment of Three Human in Vitro Systems in the Generation of Major Human Excretory and Circulating Metabolites,” Chemical Research in Toxicology, 2009, 22, 2, 357 - 368, tx8004357(acs.org); King, R., “Biotransformations in Drug Metabolism,” Chapter 3, Drug Metabolism Handbook Introduction, https: / / doi.org / 10.1002 / 9781119851042.ch3; Wu, Y. et al., “Metabolite Identification in the Preclinical and Clinical Phase of Drug Development,” Current Drug Metabolish, 2021, 22, 11, 838 - 857, 10.2174 / 1389200222666211006104502; Godzien, J. et al., “Chapter Fifteen - Metabolite Annotation and Identification”.

[0291] Many publicly available and commercially available software tools can be used to assist in predicting the metabolic pathways and metabolites of compounds. Examples of such tools include BioTransformer 3.0 (biotransformer.ca / new), which uses a database of known metabolic reactions to predict the metabolic biotransformation of small molecules; MetaSite (moldiscovery.com / software / metasite / ), which predicts metabolic transformations related to reactions mediated by cytochrome P450 and flavin-containing monooxygenases in phase I metabolism; and Lhasa Meteor Nexus (lhasalimited.org / products / meteor-nexus.htm) which uses a series of machine learning models to provide predictions of metabolic pathways and metabolite structures, covering phase I and phase II biotransformations of small molecules.

[0292] PDAs 1 - 6 in Table 2 can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dosage requirements, reduced CYP450 inhibition (competitive or time-dependent), or improved therapeutic index or tolerability.

[0293] One of ordinary skill in the art can prepare additional deuterated analogs of Compound 1 having different combinations of Y1 - Y as provided in Table 2. Such additional deuterated analogs can provide similar therapeutic advantages that can be achieved by deuterated analogs. 13 The additional deuterated analogs of Compound 1 having different combinations of Y1 - Y as provided in Table 2 can provide similar therapeutic advantages that can be achieved by deuterated analogs.

[0294] Example 1: Biochemical assay

[0295] Compound 1 and PF-06873600 were tested against highly purified, unlabeled CDK complexes using mobility shift assays (MSA).

[0296] CDK2 / Cyclin E1 mobility shift assay

[0297] The purpose of the CDK2 / cyclin E1 assay was to evaluate the inhibition (percent inhibition, K iapp and K iValue). CDK2 / cyclin E1 catalyzes the production of ADP from ATP, accompanied by the transfer of a phosphoryl group to the substrate peptide FL-peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, CA). Mobility shift assays electrophoretically separate the fluorescently labeled peptides (substrate and phosphorylated product) after the kinase reaction. Both the substrate and the product are measured, and the ratio of these values is used to generate the conversion percentage of substrate to product by the LabChip EZReader. The wild-type full-length CDK2 / wild-type full-length cyclin E1 enzyme complex was prepared in-house (baculovirus expression, LJIC-2080 / LJIC-2103), and in the presence of 10 mM MgCl2 and 5 mM ATP, the CDK2:CDK7 ratio was 50:1 (concentration mg / mL), and it was phosphorylated for 1 hour at room temperature by the CDK7 / cyclin H1 / Mat1 enzyme complex. A typical reaction solution (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP K m = 67.4 μM), 0.005% Tween-20, 3 μM FL-peptide-18, and 0.36 nM (active site with catalytic activity) phosphorylated wild-type full-length CDK2 / cyclin E1 enzyme complex. After pre-incubating the enzyme and inhibitor in the reaction mixture at room temperature for 15 minutes, the assay was initiated by adding ATP. The reaction was stopped after 45 minutes at room temperature by adding 50 μL of 80 mM EDTA (pH 7.5). The K i value was determined from the fit of the data to the Morrison tightly bound competitive inhibition equation, where the enzyme concentration was the variable.

[0298] CDK4 / Cyclin D1 mobility shift assay

[0299] The purpose of the CDK4 / cyclin D1 assay was to evaluate the inhibition (inhibition %, K iapp and K iValues). CDK4 / cyclin D1 catalyzes the production of ADP from ATP, accompanied by the transfer of a phosphoryl group to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:1). Mobility shift assays electrophoretically separate the fluorescently labeled peptides (substrate and phosphorylated product) after the kinase reaction. Both the substrate and product are assayed, and the ratio of these values is used to generate the conversion percentage of substrate to product by the LabChip EZReader. A typical reaction solution contains 2% DMSO (± inhibitor), 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% TW-20, 3 μM 5-FAM-Dyrktide, 3 nM (active site) activated CDK4 / cyclin D1 in 40 mM HEPES buffer at pH 7.5.

[0300] After pre-incubating the enzyme and inhibitor in the reaction mixture at 22 °C for 18 minutes, the inhibitor K of activated CDK4 / cyclin D1 (2007E1 / 2008+PO4) was initiated by adding ATP (50 μL final reaction volume). i The assay. The reaction was stopped after 195 minutes by adding 50 μL of 30 mM EDTA. K i Values were determined based on the curve of fractional velocity versus inhibitor concentration and were fit by the Morrison equation, with enzyme concentration as the variable.

[0301] CDK6 / Cyclin D3 mobility shift assay

[0302] The purpose of the CDK6 / cyclin D3 assay was to evaluate the inhibition (inhibition %, K iapp and K iValue). CDK6 / cyclin D3 catalyzes the production of ADP from ATP, accompanied by the transfer of a phosphoryl group to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:1). Mobility shift assays electrophoretically separate the fluorescently labeled peptides (substrate and phosphorylated product) after the kinase reaction. Both the substrate and product are assayed, and the ratio of these values is used to generate the conversion percentage of substrate to product by the LabChip EZReader. A typical reaction solution contains 2% DMSO (± inhibitor), 2% glycerol, 10 mM MgCl2, 1 mM DTT, 3.5 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 4 nM (active site) activated CDK6 / cyclin D3 in 40 mM HEPES buffer at pH 7.5.

[0303] After pre-incubating the enzyme and inhibitor in the reaction mixture at 22 °C for 18 minutes, the inhibitor K of activated CDK6 / cyclin D3 (LJIC-2009G1 / 2010+PO4) was assayed by adding ATP (50 μL final reaction volume). i The reaction was stopped after 95 minutes by adding 50 μL of 30 mM EDTA. The determination of the K i value was based on the curve of the fractional velocity versus inhibitor concentration and was fitted by the Morrison equation, with the enzyme concentration as a variable.

[0304] For CDK4 and CDK6 mobility shift assays, also see Morrison, J.F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D.J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.

[0305] Results of biochemical assay:

[0306] The biological activity data of Compound 1 and PF-06873600 in CDK2, CDK6, and CDK4 mobility shift assays are provided as Ki (nM) in Table 3.

[0307] Table 3. Biochemical potencies of Compounds 1 and 2 against CDK2, CDK6, and CDK4 as measured in mobility shift.

[0308]

[0309] The Ki values (geometric mean, nM), 95% confidence intervals (95% CI), and number of independent replicates (n) are shown.

[0310] Example 2: Cellular pharmacology

[0311] Cellular CDK activities of Compound 1 and PF-06873600 were evaluated in the human CCNE1-amplified ovarian cancer cell line model OVCAR3 and the human ER+ breast cancer cell line model MCF7 cells. OVCAR3 and MCF7 cells were treated with the highest dose of 10 μM inhibitor diluted in DMSO for 1 h and 24 h, respectively, and a 1:3 dilution dose curve was used to determine the IC 50 values. OVCAR3 cells were treated with 1 mM hydroxyurea overnight to enrich G1 / S phase cells prior to treatment with the inhibitor. Functional effects were measured in a 7-day antiproliferation assay in one human ovarian cancer (OVCAR3), two human ER+ breast cancer (MCF7 and T47D), and three human non-small cell lung cancer (NCIH2087, NCIH358, and A549) cell line models. A 1:3 dilution dose curve with the highest dose of 10 μM inhibitor in DMSO was used in triplicate to determine the IC 50 values.

[0312] Cell proliferation assay

[0313] OVCAR3, MCF7, T47D, HCC1428, NCIH2087, NCIH358, or A549 cells were seeded at 1 - 3000 cells / well in growth medium containing 10% FBS in 96 - well plates and cultured overnight at 37 °C, 5% CO₂. The next day, the compounds were serially diluted from the highest dose of 10 mM to obtain an 11 - point 3 - fold dilution curve in DMSO. Compounds 1 and PF - 06873600 were serially diluted 1:200 into the growth medium and then 1:5 on the cells to give final concentrations on the cells of 10 μM to 0.1 nM in 0.1% DMSO. The cells were incubated for 7 days at 37 °C, 5% CO₂. Then, the CYQUANT Direct cell proliferation assay (Molecular Probes, Eugene, OR) was performed according to the manufacturer's recommendations to determine the relative number of viable cells at an excitation wavelength of 508 nM and an emission wavelength of 527 nM on a Perkin Elmer Envision 2104 multimode microplate reader. The IC 50 values were calculated by fitting the concentration - response curve using a four - parameter analysis method with GraphPad Prism software.

[0314] Phosphorylated serine 807 / 811 Rb ELISA

[0315] OVCAR3 or MCF7 cells were seeded at 25000 cells / well in 100 μL of growth medium and allowed to adhere overnight at 37 °C, 5% CO₂. OVCAR3 cells were treated with 1 mM hydroxyurea overnight to enrich G1 / S - phase cells before treatment with the inhibitor. The next day, the compounds were serially diluted from the highest dose of 10 mM to obtain an 11 - point 3 - fold dilution curve in DMSO. Compounds 1 and PF - 06873600 were serially diluted 1:200 into the growth medium and then 1:5 on the cells to give final concentrations on the cells of 10 μM to 0.1 nM in 0.1% DMSO. OVCAR3 cells were treated for 1 hour, while MCF7 cells were treated overnight at 37 °C with 5% CO₂. The cells were lysed on ice in 100 μL / well of CST lysis buffer and transferred to pre - coated and blocked anti - phosphorylated Ser807 / 811 Rb ELISA plates for overnight incubation at 4 °C. The plates were washed to remove residual, unbound cellular proteins and total Rb detection antibody was added at 37 °C for 90 minutes. After washing to remove unbound total Rb antibody, HRP - labeled antibody was allowed to bind at 37 °C for 30 minutes. After washing to remove unbound HRP antibody, Glo substrate reagent was added and incubated in the dark for 5 to 10 minutes. The plates were read in luminescence mode and the IC 50 values were calculated.

[0316] Results:

[0317] Compound 1 inhibits pRb in MCF7 ER + breast cancer and OVCAR ovarian cancer cells

[0318] The cellular activity of Compound 1 was evaluated in ER+, HER2− MCF7 cells and CCNE1-amplified ovarian cancers. Activity was determined by inhibition of phosphorylation of the Rb protein at serine 807 / 811 following treatment with Compound 1 or PF-06873600. In a palbociclib-sensitive MCF7 breast cancer cell model, Compound 1 exhibited an average IC 50 of 147.8 nM, while the average IC 50 of PF-06873600 was 70.6 nM (Table 4). In OVCAR3 cells, the average IC 50 for Compound 1 treatment was 16.2 nM, while the average IC 50 of PF-06873600 was 21.8 nM (Error! Reference source not found.4).

[0319] Compound 1 inhibits the proliferation of ER + human breast cancer cells, palbociclib - resistant variants, ovarian cancer cells and NSCLC cells Example 3: Prediction of human elimination half - life

[0320] The effect of Compound 1 on cell proliferation was evaluated in multiple ER+ breast cancer cell lines, including parental and palbociclib-resistant cell lines.

[0321] Treatment with Compound 1 resulted in anti-proliferative IC 50 values of 14.3 nM to 17.6 nM in parental ER+ breast cancer cell lines MCF7, T47D, and HCC1428 and 37.3 nM to 65.7 nM in palbociclib-resistant (PalboR) derivative cell lines of these cell lines (Error! Reference source not found.4). Treatment of the CCNE1-amplified ovarian cancer cell line OVCAR3 with Compound 1 also effectively inhibited cell proliferation with an IC 50 of 11.4 nM. Similar results were shown with NCIH2087, NCIH358, and A549 NSCLC cell lines (Error! Reference source not found.4).

[0322] The average IC 50 values for CDK2 / 4 / 6 cellular activity inhibition and anti-proliferative activity of Compound 1 and PF-06873600 are summarized in Table 4 below.

[0323] Table 4. Phosphorylated Rb inhibition and anti-proliferative activity of Compound 1 and PF-06873600 in human cancer cell lines.

[0324]

[0325] The IC50 values (mean, nM), standard error of the mean (SEM), and number of independent replicates (n) are shown.

[0326] Human hepatocyte stability

[0327] This example shows that Compound 1 exhibits a high blood-to-plasma ratio, resulting in high blood binding and an unexpectedly long predicted human elimination half-life.

[0328] The ability of Compound 1 to partition between human plasma and blood has been evaluated according to methods well known in the art. Based on the observed partitioning and plasma protein binding, blood binding was estimated based on the unbound fraction in human blood (fu b ) = unbound fraction in human plasma (fu p ) / blood-to-plasma partition ratio (BPR) in humans.

[0329] To determine and compare the predicted human elimination half-lives, the blood-to-plasma partition ratios (BPRs) of Compounds 1-4 and other key in vitro ADME data, including metabolic stability in human hepatocytes (CL int HHEP), were evaluated.

[0330] First, based on the in vitro human metabolism data shown below, the predicted total human clearance (CL b ) was determined using the following equation:

[0331] CL b = (Q·fu b ·CL int HHEP) / [(Q + fu b )·CL int HHEP]

[0332] (Based on hepatocellularity (120x10 6 / g liver), grams of liver per Kg body weight (21 g / Kg), Cl int HHEP was converted from μl / min / M to ml / min / kg, assuming negligible hepatocyte binding, and Q = 20 ml / min / kg.)

[0333] Then, the predicted volume of distribution at steady state in human blood (V dss_人 ) was calculated based on animal (rat and / or dog) PK data. The differences in blood binding between compounds were considered in the calculation of V dss_人 . Due to the high blood-to-plasma partition ratios in Compounds 1 and 4, the corresponding V dss_人 was limited to the blood volume, where V dss_人 was estimated to be 0.07 L / Kg.

[0334] Using the CL b and V dss_人 estimates, the predicted human elimination half-lives of Compounds 1-4 were calculated according to the following equation:

[0335] Half-life = 0.693·V dss_人 / CL b

[0336] Due to the unexpectedly much higher BPR and lower CL int of Compound 1, a significantly longer human half-life was predicted (i.e., predicted t 1 / 2 of Compound 1 = 74 hours vs. predicted t 1 / 2 of PF-06873600 = 6 hours). However, because the observed human t 1 / 2 of PF-06873600 was 3 hours (~2-fold the prediction), the predicted t 1 / 2 of Compounds 1, 3, and 4 was adjusted accordingly. The adjusted predicted t 1 / 2 of Compound 1 was 34 hours, which was significantly higher than the observed t 1 / 2 of PF-06873600 and the adjusted predicted t 1 / 2 of Compounds 3 and 4. This longer half-life of Compound 1 may provide QD or less frequent dosing, as well as minimized C max concentration.

[0337] Table 5

[0338]

[0339] *V dss_人 = (average V dss_未结合 )·(fu b );Lower limit of V dss_人 = 0.07 L / kg Human plasma protein binding

[0340] The test compound (i.e., Compound 1, 2, 3, or 4) (1 μM) was incubated with human hepatocytes at 0.5 million cells / mL at 37 °C in an incubator (relative humidity ≥90%, 5% CO2 / air) for 4 hours. At different time points, samples were taken and analyzed by LC-MS / MS. CL int HHEP was calculated based on the loss of the parent compound over time using the equation discussed in “A novel relay method for determining low-clearance values,” Drug Metab Dispos., 2012;40(9):1860–5.

[0341] Human blood - to - plasma partition ratio (BPR)

[0342] Frozen plasma in K3EDTA was purchased from BioIVT, and Dulbecco's phosphate-buffered saline (DPBS) and HCl were purchased from Sigma.

[0343] The unbound fraction (fu) of human plasma was determined by equilibrium dialysis using an HTD 96 apparatus (HTDialysis, LLC, Gales Ferry, CT) assembled with a 12-14k MWCO membrane. p ). Plasma was thawed and adjusted to pH 7.4 with 1N HCl before use. The dialysis chambers were loaded with 150 μL plasma and 150 μL PBS in the donor and receptor chambers, respectively. The dialysis plates were sealed with a gas-permeable membrane and stored in a 37°C water-jacketed incubator maintained at 75% relative humidity and 5% CO2, on a 100 rpm plate shaker. After 6 h of incubation, the samples were matrix matched and quenched by protein precipitation, followed by LC-MS analysis. A set of satellite samples was included to determine stability after the 6 h incubation. Incubations were performed 4 to 12 times. The fu was calculated by dividing the analyte to internal standard peak area ratio or the analyte concentration in the buffer sample by the signal in the donor sample. p , corrected for any dilution factor. All incubations had >70% analyte recovery and >70% stability over 6 hours.

[0344] Human blood binding

[0345] According to the method described in Novak, JJ et al., "Effects of low temperature on blood-to-plasmaratio measurement," Biopharm Drug Dispos. 2021, 42(5): 234–41, the distribution of the compound between plasma and whole blood was determined by adding the test compound (i.e., compound 1, 2, 3, or 4) (1 mM) to fresh blood and incubating on an oscillator (450 rpm) at 37°C for 1 hour in an incubator (90% humidity, 5% CO2 / air). At the end of the incubation, the blood and plasma samples were matrix matched and analyzed by LC-MS / MS. The BPR was calculated by dividing the peak areas measured in blood and plasma.

[0346] Pre - clinical animal pharmacokinetic studies

[0347] By putting fu p Divide by BPR to calculate the unbound fraction of human blood (fu b ).

[0348] Figure 2

[0349] All activities involving animals were conducted in accordance with federal, state, local, and institutional guidelines that govern the use of laboratory animals in research in facilities accredited by the Accreditation of Laboratory Animal Care (AAALAC) and were reviewed and approved by Pfizer's Institutional Animal Care and Use Committee.

[0350] Rat PK studies were conducted at Pfizer (Groton, CT) or BioDuro Pharmaceutical Product Development Inc. (Shanghai, China); male Wistar-Hannover rats with jugular vein cannulas were purchased from Charles River Laboratories, Inc. (Wilmington, MA) or Vital River (Beijing, China) and were typically 7–10 weeks old at the time of dosing. During the pharmacokinetic studies, all animals were housed individually. Access to food and water was provided ad libitum.

[0351] The compound was administered i.v. via the tail vein (n = 2 or 3), dosed at 1 mL / kg as a 1 mg / mL solution using a standard compatible excipient to achieve a dose of 2 mg / kg. Serial blood samples were collected via the jugular vein cannula at predetermined time points after dosing. Animals were monitored for pain or distress throughout the study and at least daily during normal housing prior to the start of the study. At the end of the study, animals were euthanized by overdose inhalation anesthesia followed by exsanguination. Blood samples were collected into tubes containing K3EDTA and stored on ice until centrifuged to obtain plasma, which was then frozen and stored at −20 °C or lower. Urine samples were collected at room temperature and frozen at 20 °C or lower at the end of each time interval.

[0352] Dog PK studies were conducted at Pfizer (Groton, CT); animal care and in-life procedures were conducted in accordance with guidelines from Pfizer's Institutional Animal Care and Use Committee. Male beagle dogs were purchased from Marshall BioResources (North Rose, New York) and were typically 1–5 years old at the time of dosing.

[0353] The compound was administered i.v. via the cephalic vein (n = 2), dosed at 0.5 mL / kg as a solution using a standard compatible excipient to obtain a dose of 0.1 mg / kg. Serial blood samples were collected via the jugular vein at predetermined time points after dosing. The animals were monitored for pain or distress throughout the study, and at least daily during the normal housing period prior to the start of the study. Blood samples were collected into tubes containing K3EDTA and stored on ice until centrifuged to obtain plasma, which was then frozen and stored at -20 °C or lower. Urine samples were collected at room temperature and frozen at -20 °C or lower at the end of each time interval.

[0354] Plasma samples from the animal PK study were processed for protein precipitation with acetonitrile:methanol (1:1) containing the internal standard verapamil and then quantified against a standard curve prepared in blank plasma by LC-MS / MS. Pharmacokinetic parameters were calculated using non-compartmental analysis (Watson v.7.5, Thermo Scientific).

[0355] Example 4: Mouse Early Toxicology Screening (ETS) Study

[0356] In the early toxicology screening study, three groups of ten-week-old male CD1 mice were evaluated separately. Group 1 (n = 5 / group) was administered oral doses of Compound 1 at 25, 50, and 75 mg / kg BID for 14 days. Group 2 (n = 5 / group) was administered oral doses of PF-06873600 at 25, 75, and 150 mg / kg BID for 14 days. A vehicle control group 3 (n = 5 / group) was also included, in which the vehicle was administered BID for 14 days. Study evaluations included clinical observations, body weight, clinical pathology, toxicokinetics, gross pathology, and microscopic pathology. Neutropenia was not observed at any of the dosing levels in Group 1 (i.e., treated with Compound 1).

[0357] These results were unexpected because, as described in the background art, previous studies have shown that treatment with CDK4 / 6 inhibitors that inhibit CDK6 can result in hematological adverse events, but here, although Compound 1 exhibited CDK6 activity as shown in the biochemical assay (see Table 3), neutropenia was not observed in Group 1.

[0358] At the end of the ETS study on Day 14, the mice were sacrificed and images of the bone marrow of the mice from each study group (n = 5) were obtained. Figure 3Display the histological images of mouse bone marrow biopsies from (1) Group 1 of 75 mg / kg BID Compound 1, (2) Group 2 of 150 mg / kg BID PF-06873600, and (3) Group 3 of vehicle control. Imaging assessment of mouse bone marrow confirmed that Compound 1 had no effect on mouse bone marrow. The vehicle and Group 1 images looked very similar, having dense healthy cells, whereas in contrast, PF-06873600 showed vacuoles and large gaps between cells in the bone marrow image, indicating cell death / apoptosis.

[0359] The systemic exposures of Compound 1 and PF-06873600 were also compared.

[0360] Exposures from the mouse toxicity study were determined using plasma samples collected at time points on Day 8 or Day 14 of the study, and protein precipitation treatment was performed using a solvent mixture of acetonitrile:methanol (1:1) containing the internal standard verapamil, followed by quantification against a standard curve prepared in blank plasma by LC-MS / MS. Free plasma concentrations were calculated using mouse plasma protein binding of Compound 1 (fu p = 0.182) and PF-06873600 (fu p = 0.277). ( ​ )

[0361] Surprisingly, although both Compound 1 and PF-06873600 showed CDK6 activity and achieved similar free systemic exposures in the mouse toxicology study (BID oral administration within 14 days), neutropenia was not observed in Group 1 treated with Compound 1. These observed differences between Treatment Groups 1 and 2 cannot be explained currently. However, as described above, the predicted (adjusted) human half-life of Compound 1 is much longer than the observed human half-life of PF-06873600, and Compound 1 shows a reduced effect on circulating neutrophils, which would provide improved characteristics for Compound 1 compared to PF-06873600.

[0362] It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the scope or spirit of the present disclosure. Other embodiments of the present disclosure will be apparent to those skilled in the art by considering the specification and practice of the present disclosure disclosed herein. The specification and examples are intended to be considered merely exemplary, and the true scope and spirit of the present disclosure are indicated by the appended claims.

[0363] All references cited herein, including patents, patent applications, papers, textbooks, etc., as well as the references cited therein, to the extent that they have not been incorporated into this text as a whole, are incorporated herein by reference in their entirety. In the event of one or more differences or contradictions between the incorporated documents and similar materials and this application, including but not limited to the defined terms, term usage, the described technologies, etc., this application shall prevail.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of formula (I) has the absolute stereochemistry shown in formula (I-A), (I-B), (I-C) or (I-D):

3. A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-A).

4. A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1S,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-B).

5. A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1R,2S)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-C).

6. A compound or a pharmaceutically acceptable salt thereof, which is 4-((6-(2,2-difluoroethyl)-8-((1S,2S)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide of formula (I-D).

7. The compound according to any one of claims 1-3, which is 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide.

8. The compound according to any one of claims 1-3, which has the chemical structure 9. A pharmaceutically acceptable salt of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide.

10. The anhydrous crystalline form (Form 1) of the free base of 4-((6-(2,2-difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide, which has a powder X-ray diffraction (PXRD) pattern comprising one, two, three, four, five or more than five peaks measured at °2θ ± 0.2°2θ in Table 1.

11. 4-((6-(2,2-Difluoroethyl)-8-((1R,2R)-2-hydroxy-2-methylcyclopentyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-sulfonamide free base anhydrous crystalline form (Form 1), which has a powder X-ray diffraction (PXRD) pattern comprising peaks at the following 2θ values: 4.8, 14.3, and 19.7° 2θ ± 0.2° 2θ.

12. The anhydrous crystalline form according to any one of claims 10-11, which has a PXRD pattern further comprising a peak at the following 2θ value: 10.6° 2θ ± 0.2° 2θ.

13. The anhydrous crystalline form according to any one of claims 10-12, which has a PXRD pattern further comprising a peak at the following 2θ value: 19.1° 2θ ± 0.2° 2θ.

14. The anhydrous crystalline form according to any one of claims 10-13, which has a PXRD pattern comprising peaks at substantially the same 2θ values as shown in Figure 1.

15. The anhydrous crystalline form according to any one of claims 10-14, wherein the crystalline form is substantially pure.

16. A pharmaceutical composition comprising a compound according to any one of claims 1-15 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient.

17. A method of treating cancer, the method comprising administering to an individual in need thereof a therapeutically effective amount of a compound according to any one of claims 1-16 or a pharmaceutically acceptable salt thereof.

18. The method of treating cancer according to claim 17, the method further comprising administering an additional anti-cancer therapeutic agent.

19. The method of treating cancer according to any one of claims 17-18, wherein the cancer is selected from breast cancer, ovarian cancer, bladder cancer, uterine cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, kidney cancer, liver cancer, pancreatic cancer, gastric cancer, and thyroid cancer.

20. The method of treating cancer according to any one of claims 17-19, wherein the cancer is breast cancer or ovarian cancer.

21. The method of treating cancer according to claim 20, wherein the breast cancer or ovarian cancer is characterized by amplification or overexpression of cyclin E1 (CCNE1) or cyclin E2 (CCNE2).

22. The method of treating cancer according to any one of claims 19-21, wherein the breast cancer is ER-positive / HR-positive breast cancer, HER2-negative breast cancer, ER-positive / HR-positive breast cancer, HER2-positive breast cancer, triple-negative breast cancer (TNBC), or inflammatory breast cancer.

23. The method of treating cancer according to any one of claims 19-22, wherein the breast cancer is advanced breast cancer or metastatic breast cancer.

24. A compound or pharmaceutically acceptable salt according to any one of claims 1-15, for use as a medicament.

25. A compound or pharmaceutically acceptable salt according to any one of claims 1-15, for use in the treatment of cancer.

26. Use of a compound or a pharmaceutically acceptable salt according to any one of claims 1 - 15 for the preparation of a medicament for treating cancer.

27. A pharmaceutical combination comprising a compound or a pharmaceutically acceptable salt according to any one of claims 1 - 15, at least one additional therapeutic agent, and at least one pharmaceutically acceptable excipient.

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