Treatment of proliferative diseases of the CNS

Abstract

Thiazole-pyrimidine compounds are provided for use in the treatment of proliferative cell diseases and conditions in the CNS, including glioblastoma.SOLUTION: Provided is the use of a compound having the structure I: or a pharmaceutically acceptable salt, or solvate thereof. The compound can block the proliferation of tumor cells by inhibiting the activity of CDK4 and / or CDK6, and can cross the blood brain barrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods and uses of thiazole-pyrimidine compounds in treating proliferative cell diseases or conditions of the central nervous system (CNS).

[0002] Priority document This application claims priority to Australian Provisional Patent Application No. 2020901435, filed May 6, 2020, entitled "Treatment of Proliferative Disorders of the CNS," the entire contents of which are incorporated herein by reference. [Background technology]

[0003] Primary brain tumors comprise a diverse group of neoplasms, originating from various cell lineages. According to the World Health Organization (WHO) classification, tumors of the central nervous system (CNS) are classified as astrocytic, oligodendroglial, or mixed. These tumors are further classified by subtype and graded from I to IV based on histology, with grade IV being the most aggressive. In the United States alone, it is estimated that nearly 80,000 new cases of primary brain tumors and other CNS tumors are diagnosed each year. Unfortunately, these cancers are characterized by poor prognosis and low survival rates; glioblastoma multiforme (GBM) is the most aggressive primary tumor of the CNS, accounting for 45% of malignant primary CNS tumors and 54% of all gliomas. While survival rates for many cancers have continued to improve in recent years, CNS cancers have not yet experienced the same level of success. For example, patients diagnosed with brain cancer between 2009 and 2013 had an approximately 25% chance of surviving five years. This contrasts sharply with the approximately 68% survival rate for all cancers combined over the same period. For patients with GBM, median survival was only 15-23 months, with a five-year survival rate of approximately 4.6%, the lowest of all brain tumor types.

[0004] Abnormal cell cycle regulation, resulting in unlimited cell cycle reentry and progression, is a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are known to associate with various cyclin subunits and play a pivotal role in regulating various important regulatory pathways in cells, including cell cycle control, apoptosis, neurophysiology, differentiation, and transcription. To date, at least 20 CDKs and 30 cyclins have been identified. They can be classified into two major groups based on their functions: cell cycle regulator CDKs and transcription regulator CDKs (Wang S et al., Trends Pharmacol Sci 29(6):302-313, 2008). Cell cycle regulator CDKs include CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, and CDK7, which function together with their cyclin partners (e.g., cyclins A, B, C, D1, D2, D3, E, and F) to regulate cell cycle progression. Transcriptional regulator CDKs include CDK7, CDK8, CDK9, and CDK11, which tend to play a role in transcriptional regulation by acting together with cyclins C, H, K, L1, L2, T1, and T2. Considering the function of the CDK class, it is not surprising that CDKs are involved in cell proliferation diseases and conditions, especially cancer. Cell proliferation is the result of direct or indirect deregulation of the cell division cycle, and CDKs play an important role in regulating various phases of this cycle. Therefore, inhibitors of CDKs and their associated cyclins are considered to be useful targets for cancer therapy.

[0005] CDK4 / 6 controls the cell cycle and is tightly regulated by the INK4 family of proteins. Numerous studies have demonstrated that the CDK4 / 6 pathway is hyperactivated in a significant proportion of cancers, including CNS tumors and gliomas (Xu G et al. al., J Neurooncol 136: 445-452, 2018; Parsons DW et al., Science 321:1807-1812, 2008; Bax DA et al., Clin Cancer Res 16:3368-3377, 2010; and Cancer Genome Atlas Research, N. Nature 455:1061-1068, 2008). Genome-wide profiling of pediatric and adult gliomas has revealed that the CDK4 / 6-Rb axis is deregulated in over 80% of GBMs, resulting from (i) deletion of the CDKN2A / B genes encoding p16INK4a and p15INK4b, (ii) amplification / overexpression of CDK4 / 6, and (iii) deletion / mutation of Rb (Schmidt EE et al., Cancer Res 54:6321-6324, 1994). CDK4, p16INK4a, and Rb are independent predictors of poor survival (Aoki K et al., Neuro Oncol 20:66-77, 2018). Therefore, inhibition of CDK4 / 6 may be an effective approach for treating cancer, particularly glioblastoma. Three CDK4 / 6 inhibitors, namely palbociclib, ribociclib, and abemaciclib, have been approved by the US Food and Drug Administration (FDA) for the treatment of breast cancer and have been studied in patients with GBM. However, outcomes with palbociclib were disappointing, and the study was discontinued. The lack of efficacy may be due to its limited ability to cross the blood-brain barrier (BBB) ​​and expose the drug in the brain (Karen E et al. al., In 2013 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, Vol. 12(11 Suppl) 2013).

[0006] Cell signaling via growth factor receptors and protein kinases is another important regulator of cell growth and proliferation. In normal cell growth, growth factors (e.g., PDGF or EGF) activate the MAP kinase pathway through receptor activation. One of the most important MAP kinase pathways involved in normal and uncontrolled cell growth is the Ras / Raf kinase pathway. Active GTP-bound Res leads to the activation and indirect phosphorylation of Raf kinase. Raf then phosphorylates MEK1 and MEK2 (Ahn et al., Methods Enzymol 332:417-431, 2001). Activated MEK then phosphorylates ERK1 and ERK2. Subsequently, phosphorylated ERK dimerizes and then translocates to the nucleus, where it accumulates (Khokhlatchev et al., Cell 93:605-615, 1998), where it is then involved in several important cellular functions, including nuclear transport, signal transduction, DNA repair, nucleosome assembly and translocation, and mRNA processing and translation (Ahn et al., Molecular Cell 6:1343-1354, 2000). Overall, growth factor treatment leads to activation of ERK1 / 2, which leads to proliferation and resistance to therapy. Therefore, targeting the MAP kinase pathway offers therapeutic opportunities for a wide range of cancer types, and recently, the MEK inhibitor, selumetinib, was granted US Breakthrough Therapy Designation for the treatment of patients with neurofibromatosis type 1 (NF1), symptomatic and / or progressive, inoperable plexiform neurofibromas, an incurable genetic condition. NF1 gene mutations can lead to dysregulation of RAS / RAF / MEK / ERK signaling, which can cause cells to grow, differentiate, and copy uncontrollably, thus causing tumor growth. Selumetinib inhibits the MEK enzyme, which leads to inhibition of tumor growth.

[0007] In cancer cells, the main consequence of disrupted signaling pathways is an imbalance in protein expression that allows cells to avoid apoptosis, proliferation, and metastasis. Approximately 40% of GBM subtype tumors are characterized by dysfunctional EGFR, a cell surface receptor tyrosine kinase that activates a cascade of downstream intracellular signaling via the PI3K / AKT pathway. Its amplification and overexpression via mutations disrupts angiogenesis, migration, and metastasis. EGFR promotes glioma growth and survival, aided by EGFR. Therefore, EGFR has been proposed as an attractive therapeutic target. However, a phase II trial of the EGFR inhibitor erlotinib in patients with recurrent GBM failed to demonstrate significant benefit. Other tyrosine kinase inhibitors tested in phase II-III clinical trials, such as enzastaurin, an inhibitor targeting PKC and PI3K / AKT, failed to demonstrate efficacy in GBM patients when used alone or in combination with chemotherapy. The failure of these EGFR inhibitor compounds may be due to poor pharmacokinetics (PK) and BBB permeability.

[0008] Approval rates for CNS drugs are typically much lower than for non-CNS drugs, and the attrition rate in oncology CNS drug development is very high. Thus, drug discovery for treating brain tumors has been characterized by significant obstacles and historical failures. For successful treatment, drugs must first be able to cross the BBB, a layer of closely connected endothelial cells unique to capillaries in the brain that are tightly joined by tight junctions, preventing paracellular movement. The passage of compounds through endothelial cells to the brain can be limited by the action of ATP-binding cassette (ABC) transporters expressed on their apical membrane; i.e., the membrane in contact with circulating blood. Among these, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two major transporters that together limit the brain penetration of many compounds (Wager et al., Expert Opin Drug Discov 6:371-381, 2011; Agarwal et al., J Pharmacol Exp Ther 336:223-233, 2011). Indeed, many chemotherapeutic drugs are prevented from accessing the brain by this barrier.

[0009] The location of GBM and its extensive infiltration of normal surrounding brain tissue means that surgical resection cannot completely remove the tumor. Furthermore, tumor cells that invade the surrounding normal brain are protected from therapeutic agents by the BBB. Unfortunately, even accessible drugs, such as temozolomide (TMZ, the only standard-of-care chemotherapy for glioblastoma), have limited efficacy, improving survival by only a few months at best. To date, the majority of clinical trials for GBM treatment have failed. Preclinical data have shown that penetration of CDK4 / 6 inhibitors (i.e., palbociclib and abemaciclib) is limited by active efflux at the BBB. Abemaciclib demonstrated relatively higher exposure than palbociclib in rodent brains; however, its therapeutic potential remains to be uncovered. Clearly, there is a need to identify drugs directed at new targets, such as CDK4 / 6, that can sufficiently cross the BBB to be effective in treating brain tumors and other cancers of the CNS.

[0010] Applicants have identified a class of thiazole-pyrimidine compounds for use in treating proliferative cell diseases and conditions in the CNS, including glioblastoma. Without wishing to be bound by theory, it is believed that these compounds block tumor cell proliferation by inhibiting the activity of CDK4 and / or CDK6 and are able to cross the BBB. Summary of the Invention

[0011] In a first aspect, the disclosure provides a method of treating a proliferative cell disease or condition of the central nervous system (CNS) in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I, shown below: [ka] [In formula: R 1is selected from H, alkyl, aryl, aralkyl, alicyclic, heterocyclic, halogen, NO2, CN, CF3, OH, O-alkyl, O-aryl, COOH, CO-alkyl, CO-aryl, CONH2, CONH-alkyl, CONH-aryl, and CONH-alicyclic; R 2 is selected from H, alkyl, halogen, NO2, CN, CF3, OH, O-alkyl, and NH2; R 3 is selected from heterocyclic containing at least one N heteroatom, NH-alkyl, NH-aryl, N-(alkyl), N-(aryl), and N-(alkyl)(aryl); and n is an integer selected from the range of 0 to 3; and wherein said alkyl, aryl, aralkyl, alicyclic and heterocyclic groups may be optionally substituted with one or more groups selected from alkyl, halogen, CN, OH, O-methyl, NH, NH-alkyl, N(alkyl), COOH, COH, CO(alkyl), CONH, and CF. or a pharmaceutically acceptable salt, solvate or prodrug thereof, Optionally, the method comprises administering in combination with a pharmaceutically acceptable carrier, diluent and / or excipient.

[0012] Compounds of formula I have been found to have antiproliferative activity (e.g., they are believed to block tumor cell proliferation by inhibiting the activity of CDK4 and / or CDK6) and are further able to cross the BBB.

[0013] In a second aspect, the present disclosure provides the use of a compound as defined in the first aspect, or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for treating a proliferative cell disease or condition of the central nervous system (CNS), such as glioblastoma, in a subject.

[0014] In a third aspect, the present disclosure provides the use of a compound as defined in the first aspect, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for treating a proliferative cell disease or condition of the central nervous system (CNS), such as glioblastoma, in a subject. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the antiproliferative activity of compound 1 (5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine) as a single agent and in combination with inhibitors of (A) mTOR (everolimus; shown in the figures as "Eve"), (B) PI3K (alpelisib; "Alp"), or (C) MEK (selumetinib; "Sel") in the T98G GBM cell line. [Figure 2] 1 shows the results of an Annexin V / PI assay of GBM U87 cells 48 hours after treatment with 5 μM palbociclib (“Palb”) or Compound 1, alone or in combination with TMZ. [Figure 3] 1 shows inhibition of GBM U87 cell colony formation by Compound 1 through targeting CDK4 / 6-mediated Rb phosphorylation. [Figure 4] 1 shows graphical results showing the brain uptake of compound 1 and compound 2 (N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-methylthiazol-2-amine) after intravenous administration of 2 mg / kg in Balb / C mice (A), and (B) 24 hours after oral administration of 10 mg / kg; while (C) shows the brain uptake of compound 6 (N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine) after intravenous administration of 2 mg / kg in Balb / C mice. [Figure 5]1 shows graphical results demonstrating the in vivo antitumor activity of Compound 1 and Compound 2 in subcutaneous GBM U87 cell xenografts. [Figure 6] 1 shows graphical results demonstrating the in vivo antitumor activity of Compound 1 and Compound 2 in orthotopic xenografts of GBM U87 and G4T GBM patient-derived models, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0016] In a first aspect, the disclosure provides a method of treating a proliferative cell disease or condition of the central nervous system (CNS) in a subject, comprising administering to the subject a therapeutically effective amount of a compound of Formula I, shown below: [ka] [In formula: R 1 is selected from H, alkyl, aryl, aralkyl, alicyclic, heterocyclic, halogen, NO2, CN, CF3, OH, O-alkyl, O-aryl, COOH, CO-alkyl, CO-aryl, CONH2, CONH-alkyl, CONH-aryl, and CONH-alicyclic; R 2 is selected from H, alkyl, halogen, NO2, CN, CF3, OH, O-alkyl, and NH2; R 3 is selected from heterocyclic containing at least one N heteroatom, NH-alkyl, NH-aryl, N-(alkyl), N-(aryl), and N-(alkyl)(aryl); and n is an integer selected from the range of 0 to 3; and wherein said alkyl, aryl, aralkyl, alicyclic and heterocyclic groups may be optionally substituted with one or more groups selected from alkyl, halogen, CN, OH, O-methyl, NH, NH-alkyl, N(alkyl), COOH, COH, CO(alkyl), CONH, and CF. or a pharmaceutically acceptable salt, solvate or prodrug thereof, Optionally, the method comprises administering in combination with a pharmaceutically acceptable carrier, diluent and / or excipient.

[0017] The compounds of Formula I have been found to have antiproliferative activity (e.g., they are believed to block tumor cell growth by inhibiting the activity of CDK4 and / or CDK6), and furthermore, are able to cross the BBB. Thus, the compounds of Formula I are believed to be useful in treating proliferative cell diseases and conditions of the CNS, such as glioblastoma, as well as other diseases and conditions of the CNS associated with uncontrolled cell proliferation (or, in other words, requiring cell cycle control). As used herein, antiproliferative activity within the scope of the present disclosure can be demonstrated by the ability to inhibit cell proliferation in an in vitro whole cell assay. Example(s) of such assays, including methods of performance, are described in more detail in the Examples provided herein below.

[0018] Preferably, the compounds of Formula I modulate (e.g., inhibit) the activity of one or more protein kinases selected from CDK4 and / or CDK6. As discussed above, CDK4 and CDK6 promote cancer cell proliferation through their roles as cell cycle regulators. Accordingly, compounds of Formula I, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, that inhibit at least CDK4 and / or CDK6 have utility in both in vitro and in vivo applications (e.g., in vitro cell-based assays) and as the basis of therapeutic methods for treating cancer or another proliferative disorder or condition in a subject.

[0019] Compounds of Formula I may inhibit any step or stage in the cell cycle, such as nuclear envelope formation, exit from the cell cycle resting phase (G0), G1 progression, chromosome disaggregation, nuclear envelope breakdown, START, DNA replication initiation, DNA replication progression, DNA replication arrest, centrosome duplication, G2 progression, activation of mitotic or meiotic functions, chromosome condensation, centrosome separation, microtubule nucleation, spindle formation and function, interaction with microtubule motor proteins, chromatid separation and segregation, inactivation of mitotic functions, contractile ring formation, and cytokinesis functions. In particular, compounds of Formula I may affect certain gene functions, such as chromatin binding, replication complex formation, replication licensing, phosphorylation or other secondary modification activity, proteolytic degradation, microtubule binding, actin binding, septin binding, microtubule nucleation activity, and binding to components of cell cycle signaling pathways.

[0020] In a second aspect, the present disclosure provides the use of a compound as defined in the first aspect, or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for treating a proliferative cell disease or condition of the central nervous system (CNS), such as glioblastoma, in a subject.

[0021] In a third aspect, the present disclosure provides the use of a compound as defined in the first aspect, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for treating a proliferative cell disease or condition of the central nervous system (CNS), such as glioblastoma, in a subject.

[0022] This specification uses a number of terms that are well known to those of ordinary skill in the art. Nevertheless, for the sake of clarity, some of these terms are defined hereinafter.

[0023] As used herein, the term "treating" includes the prevention and even alleviation of established symptoms of a disease or condition. Thus, the act of "treating" a disease or condition includes (1) preventing or delaying the appearance of clinical symptoms of the onset of a disease or condition in a subject suffering from or susceptible to the disease or condition; (2) inhibiting the disease or condition or at least one clinical or subclinical symptom thereof (i.e., arresting, reducing, or delaying the onset of the disease or condition or its recurrence (in the case of maintenance treatment)); and (3) alleviating or attenuating the disease or condition (i.e., causing regression of the disease or condition or at least one clinical or subclinical symptom thereof).

[0024] As used herein, the term "alkyl" includes straight-chain, branched-chain, and cyclic alkyl groups having 1 to 8 carbon atoms (e.g., methyl, ethylpropyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, and the like).

[0025] As used herein, the term "aryl" refers to a substituted (mono- or poly-) or unsubstituted monoaromatic or polyaromatic group, wherein said polyaromatic group may be fused or unfused. Thus, the term includes groups having 6 to 10 carbon atoms (e.g., phenyl, naphthyl, etc.). It should also be understood that the term "aryl" is synonymous with the term "aromatic."

[0026] As used herein, the term "aralkyl" is used as a link between the terms alkyl and aryl, as defined above.

[0027] The term "aliphatic" has its ordinary meaning in the art and includes non-aromatic groups such as alkanes, alkenes and alkynes and substituted derivatives thereof.

[0028] As used herein, the term "alicyclic" refers to a cyclic aliphatic group.

[0029] The term "halogen" refers to fluoro, chloro, bromo and iodo.

[0030] As used herein, the term "heterocyclic" refers to a saturated or unsaturated cyclic group that contains one or more heteroatoms (eg, N) in the ring.

[0031] The term "derivative" as used herein includes any chemical modification of an entity. Examples of such chemical modifications are replacement of hydrogen with a halogen, alkyl, acyl or amino group.

[0032] As used herein, the phrase "manufacturing a medicament" includes the use of one or more compounds of Formula I either directly as a medicament or in any stage of the manufacture of a medicament containing one or more compounds of Formula I.

[0033] Some compounds of Formula I may exist as single stereoisomers, racemates, and / or mixtures of enantiomers and / or diastereoisomers. All such single stereoisomers, racemates, and mixtures thereof are encompassed within the scope of the present disclosure. Isomeric forms, such as diastereoisomers, enantiomers, and geometric isomers, can be separated by physical and / or chemical methods known to those skilled in the art.

[0034] The term "pharmaceutically acceptable salt" as used herein refers to a compound of Formula I It refers to a salt that maintains the desired biological activity, and includes pharmaceutically acceptable acid addition salts and base addition salts.Suitable pharmaceutically acceptable acid addition salts of the compound of Formula I can be prepared from inorganic acids or organic acids.Examples of such inorganic acids are hydrochloric acid, sulfuric acid and phosphoric acid.Suitable organic acids can be selected from the aliphatic, alicyclic, aromatic, heterocyclic carboxylic and sulfonic acid groups of organic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkylsulfonic acid and arylsulfonic acid.Additional information about pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co, Easton PA 1995.

[0035] The term "solvate" refers to any form of a compound of Formula I resulting from solvation with a suitable solvent. Such a form may be, for example, a crystalline solvate or a complex that may be formed between a solvent and a dissolved compound.

[0036] The term "prodrug" refers to a compound that is converted to a compound of Formula I in a biological system, usually by metabolic means (e.g., by hydrolysis, reduction, or oxidation). For example, an ester prodrug of a compound of Formula I containing a hydroxyl group can be converted to a compound of Formula I by hydrolysis in vivo. Suitable esters of a compound of Formula I containing a hydroxyl group can be, for example, acetate, citrate, lactate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-p-hydroxynaphthoate, gentisate, gentisate isethionate, di-p-toluoyltartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. As another example, an ester prodrug of a compound of Formula I containing a carboxy group may be convertible by in vivo hydrolysis to the compound of Formula I. Examples of ester prodrugs include those described by Leinweber FJ, Drug Metab Rev 18:379-439 (1987). Similarly, acyl prodrugs of compounds of formula I containing an amino group can be converted by in vivo hydrolysis to compounds of formula I. Examples of prodrugs for these and other functional groups, including amines, are provided in Prodrugs: challenges and rewards, Valentino J Stella (ed), Springer, 2007.

[0037] In the case of compounds of Formula I that are solid, it will be understood by those of skill in the art that the compound (or its pharmaceutically acceptable salt, solvate or prodrug) may exist in various crystalline or polymorphic forms, all of which are encompassed within the scope of this disclosure.

[0038] The term "therapeutically effective amount" or "effective amount" refers to an amount sufficient to produce beneficial or desired clinical results. A therapeutically effective amount can be administered in one or more administrations. Typically, a therapeutically effective amount is sufficient to treat a disease or condition, or otherwise alleviate, ameliorate, stabilize, reverse, slow the progression of, or delay a disease or condition, such as, for example, cancer or another proliferative cell disease or condition. By way of example only, a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, may comprise from about 0.1 to about 250 mg / kg of body weight per day, more preferably from about 0.1 to about 100 mg / kg of body weight per day, and even more preferably from about 0.1 to about 25 mg / kg of body weight per day. Notwithstanding the foregoing, however, a therapeutically effective amount may vary and may vary depending on the activity of a particular compound (or salt, solvate, or prodrug thereof), the effect of a particular compound (or salt, solvate, or prodrug thereof), and the like. It will be understood by those skilled in the art that the administration of a compound will depend on a variety of factors, including metabolic stability and length, age, weight, sex, health, route and time of administration, excretion rate of the particular compound (or salt, solvate or prodrug thereof), and the severity of, for example, cancer or other proliferative cell disease or condition being treated.

[0039] In some embodiments, R 1 is H, alkyl (e.g., C 1~6 Alkyl, e.g., C 1~3 Alkyl, for example, methyl, ethyl, and C(CH3)2 or C 3~6 cycloalkyl, e.g., cyclopentyl), or heterocyclic (e.g., a saturated or unsaturated 5- or 6-membered ring group containing 1 or 2 N, O, or S heteroatoms). Most preferably, R 1 is H, C 1~3 Alkyl (e.g., methyl) or C 3~6 Cycloalkyl, for example, cyclopentyl.

[0040] In some embodiments, R 2 is H, alkyl (e.g., C 1~6Alkyl or, preferably, C 1~3 alkyl, for example, methyl or ethyl), CN or halogen (preferably F).

[0041] In some embodiments, R 3 is an alkyl (e.g., C 1~6 Alkyl or, preferably, C 1~3 alkyl, for example, methyl, ethyl, and CH(CH3)2), NH2, NH-alkyl, for example, NH-methyl and NH-ethyl, N(alkyl)2, for example, N(C 1~3 alkyl)2 (e.g., N(CH3)2, N(CH2CH3)2 and N(CH3)(CH2CH3)), COH and CO(C 1~3 Heterocyclic groups (preferably saturated or unsaturated 5- or 6-membered ring groups containing one, but more preferably two, N heteroatoms) optionally substituted with one or more groups selected from alkyl (e.g., COCH).

[0042] In certain embodiments, R 3 is as follows: [ka] is selected from.

[0043] In some embodiments, n is 0, 1, or 2. When n is 1 or 2, the compound thereby includes an alkyl bridge (e.g., a —CH— or —CHCH— bridge) to the carbon atom at the 4-position of the pyridine ring.

[0044] In one particularly preferred embodiment, the compound is: 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-methylthiazol-2-amine; N-Cyclopentyl-4-methyl-5-(2-((5-(piperazin-1-yl)pyridine) pyrimidin-2-yl)amino)pyrimidin-4-yl)thiazol-2-amine; N-cyclopentyl-5-(2-((5-(4-ethylpiperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine; 2-((5-(4-acetylpiperazin-1-yl)pyridin-2-yl)amino)-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile; N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine; 5-(2-((5-(4-aminopiperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(2-((5-(4-aminopiperidin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-N-cyclopentyl-4-methylthiazol-2-amine; N-cyclopentyl-5-(5-fluoro-2-((5-morpholinopyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine; 5-(2-((5-(4-(ethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(2-((5-(4-(ethyl(methyl)amino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(5-fluoro-2-((5-((4-methylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(5-fluoro-2-((5-((4-isopropylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; or 5-(2-((5-(4-(diethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine is.

[0045] In some preferred embodiments, compounds of Formula I exhibit antiproliferative activity in human cell lines as measured by standard cytotoxicity assays. Preferably, the compounds have an IC of less than 5 μM, and even more preferably less than 1 μM, as measured by standard cell viability assays. 50 More preferably still, the compound has an IC value of less than 0.5 μM. 50 Indicates the value.

[0046] In some preferred embodiments, compounds of Formula I inhibit one or more protein kinases as measured by any standard assay known to those of skill in the art. Preferably, the compounds have an IC50 of less than 1 μM or less than 0.5 μM, and more preferably even less than 0.1 μM, as measured by the kinase assay described in Example 2 herein below. 50 Indicates the value.

[0047] Specific examples of compounds of formula I for use in the method of the first aspect are set out in Table 1 below. [Table 1-1] [Table 1-2]

[0048] The compounds of Formula I (and pharmaceutically acceptable salts, solvates and prodrugs thereof) may be administered in combination with one or more additional agents for the treatment of cancer or another proliferative disease or condition. The compounds of formula I can be administered in combination with one or more anti-cancer agents. For example, the compounds can be used in combination with other anti-cancer agents to simultaneously inhibit more than one cancer signaling pathway, making cancer cells more sensitive to anti-cancer treatment (e.g., treatment with other anti-cancer agents, chemotherapy, radiation therapy, or a combination thereof). Thus, the compounds of formula I can be used in combination with one or more of the following categories of anti-cancer agents, especially when such anti-cancer agents can penetrate the BBB: Other antiproliferative / antineoplastic drugs and combinations thereof as used in clinical oncology, such as alkylating agents (e.g., carmustine, procarbazine, lomustine, vincristine, and TMZ); antimetabolites (e.g., gemcitabine and antifolates, e.g., fluoropyrimidines such as 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, fludarabine, and hydroxyurea); antitumor antibiotics (e.g., anthracyclines, e.g., adriamycin, bleomycin, mycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, and mithramycin; cytostatics (e.g., vinca alkaloids, e.g., vincristine, vinblastine, vindesine, and vinorelbine, and taxoids including taxol and taxotere, and polo kinase inhibitors); and topoisomerase inhibitors (e.g., epipodophyllotoxins, e.g., etoposide and teniposide, amsacrine, topotecan, and camptothecin); Cytostatic agents, such as antiestrogens (e.g. tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxifene), antiandrogens (e.g. bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or agonists (e.g. goserelin, leuprorelin and buserelin), progestogens (e.g. megestrol acetate), aromatase inhibitors (e.g. anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase, such as finasteride; anti-invasive agents (e.g., c-Src kinase family inhibitors, e.g., 4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline (AZD0530; International Patent Publication No. WO 01 / 94341), N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide (dasatinib) and bosutinib (SKI-606)), and metalloproteinase inhibitors including marimastat, inhibitors of urokinase plasminogen activator receptor function, or antibodies against heparanase; Inhibitors of growth factor function (e.g., growth factor antibodies and growth factor receptor antibodies, such as the anti-erbB2 antibody trastuzumab (Herceptin™), the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab (Erbitux, C225) and any of the growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology / haematology, 2005, Vol. 54, pp11-29). Such inhibitors include tyrosine kinase inhibitors, such as inhibitors of the epidermal growth factor family (e.g., EGFR family tyrosine kinase inhibitors, such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors, such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; inhibitors of the platelet-derived growth factor family, such as imatinib and / or nilotinib. inhibitors of serine / threonine kinases (e.g., Ras / Raf signaling inhibitors, e.g., sorafenib (BAY 43-9006), farnesyltransferase inhibitors including tipifarnib (R115777), and lonafarnib (SCH66336), inhibitors of cell signaling mediated by MEK and / or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; Aurora kinase inhibitors (e.g., AZD1152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528, and AX39459) and cyclin-dependent kinase inhibitors, for example, CDK4 and / or CDK6 inhibitors (e.g., palbociclib, ribociclib, and abemaciclib); Anti-angiogenic agents, such as those that inhibit the action of vascular endothelial growth factor (e.g., the anti-vascular endothelial growth factor antibody bevacizumab (Avastin™) and VEGF receptor tyrosine kinase inhibitors, such as vandetanib (ZD6474), vatalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736), pazopanib (GW786034) and 4-(4-fluoro-2-methylindole-5-yl)-2-oxo ... compounds such as those disclosed in International Patent Publication Nos. WO 97 / 22596, WO 97 / 30035, WO 97 / 32856 and WO 98 / 13354, as well as compounds that act by other mechanisms (e.g., linomide, an inhibitor of integrin αvβ3 function and angiostatin); vascular damaging agents, such as combrestatin A4 and compounds disclosed in International Patent Publication Nos. WO99 / 02166, WO00 / 40529, WO00 / 41669, WO01 / 92224, WO02 / 04434 and WO02 / 08213; Endothelin receptor antagonists, such as zibotentan (ZD4054) or atrasentan; Antisense therapy, e.g., directed to the targets listed above, e.g., ISIS2503, anti-ras antisense; gene therapy approaches, including, for example, approaches to replace abnormal genes, e.g., abnormal p53 or abnormal BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches, e.g., those using cytosine deaminase, thymidine kinase, or bacterial nitroreductase enzymes, and approaches to increase patient tolerance to chemotherapy or radiation therapy, e.g., multidrug resistance gene therapy; and Immunotherapeutic approaches, including ex vivo and in vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines, e.g., interleukin 2, interleukin 4, or granulocyte-macrophage colony-stimulating factor, approaches to reduce T-cell anergy, approaches using transfected immune cells, e.g., cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines, and approaches using anti-idiotypic antibodies.

[0049] In some embodiments, the compound of Formula I (and its pharmaceutically acceptable salts, solvates, and prodrugs) can be administered in combination with TMZ (and / or radiation). Using glioblastoma xenografts, it has been demonstrated that the use of abemaciclib or palbociclib (both CDK4 / 6 inhibitors) in combination with TMZ or radiation inhibits DNA double-strand break repair and increases apoptosis (Raub TJ et al. Drug Metab Dispos 43:1360-1371, 2015; and Michaud K et al. Cancer Res 8:70, 2010). Palbociclib can be combined with the mTOR inhibitor everolimus. Palbociclib synergizes with glioblastoma xenografts when administered in combination with other agents (Olmez I et al. Clinic Cancer Res DOI: 10.1158 / 1078-0432.CCR-17-0803, 2018). Palbociclib has also been shown to enhance tumor cell antigen and anti-tumor T cell responses, suggesting the immunological effects of CDK4 and / or CDK6 inhibition (Deng J et al. Cancer Discovery DOI: 10.1158 / 2159-8290.CD-17-0915, 2018). In other embodiments, the compound of Formula I (and pharmaceutically acceptable salts, solvates, and prodrugs thereof) can be administered in combination with a kinase inhibitor selected from inhibitors of PI3K, mTOR, and / or MEK.

[0050] When used in combination with other anti-cancer agents, the compound of Formula I and the other anti-cancer agent can be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. If administered in separate pharmaceutical compositions, the compound and the other anti-cancer agent can be administered simultaneously or sequentially in any order (e.g., within seconds or minutes or even hours (e.g., 2-48 hours)).

[0051] The compounds of Formula I are typically applied to the treatment of cancer or another proliferative cell disease or condition in human subjects, however, the subject may also be selected from, for example, livestock animals (e.g., cows, horses, pigs, sheep, and goats), companion animals (e.g., dogs and cats), and exotic animals (e.g., non-human primates, tigers, elephants, etc.).

[0052] Cancers of the CNS and other proliferative cell diseases and conditions that can be treated according to the present disclosure include brain and spinal cord cancers, including glioblastoma (e.g., GBM), medulloblastoma, primary central nervous system (CNS) lymphoma, other malignant CNS tumors, such as astrocytomas, ependymonas, oligodendrogliomas, or metastatic brain tumors, and benign neoplasms of the CNS, such as schwannomas, pituitary adenomas, meningiomas, and craniopharyngiomas.

[0053] In some preferred embodiments, compounds of Formula I are used to treat cancers of the CNS and other proliferative cell diseases and conditions selected from those characterized by overexpression of CDK4 and / or cyclin D, including, for example, GBM. CDK4 and / or cyclin D overexpression can be determined, for example, by assessing the amount of mRNA encoding CDK4 and / or cyclin D in an appropriate sample using any of the techniques well known to those of skill in the art (e.g., quantitative amplification techniques such as qPCR).

[0054] In some preferred embodiments, compounds of Formula I are administered to treat cancers of the CNS selected from those characterized by overexpression of CDK6 and / or cyclin D, including, for example, medulloblastoma, and other proliferative cell diseases and conditions (Tadesse et al., Targeting CDK6 in Cancer: State of the Art and CDK6 and / or cyclin D overexpression can be determined by assessing the amount of mRNA encoding CDK6 and / or cyclin D in a suitable sample, for example, using any of the techniques well known to those skilled in the art (e.g., quantitative amplification techniques such as qPCR).

[0055] The compounds of Formula I can be formulated into pharmaceutical compositions using pharmaceutically acceptable carriers, diluents and / or excipients. Examples of suitable carriers and diluents are well known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 1995. Examples of excipients suitable for the various different forms of pharmaceutical compositions described herein can be found in Handbook of Pharmaceutical Excipients, 2 ndEdition, (1994), Edited by A. Wade and P. J. Weller. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerol, and water. Carriers, diluents, and / or excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0056] Pharmaceutical compositions containing a compound of Formula I may further contain any suitable binders, lubricants, suspending agents, coating agents, and solubilizing agents. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, fluid lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and flavoring agents may also be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0057] Pharmaceutical compositions containing compounds of Formula I can be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal, or sublingual administration. For oral administration, compressed tablets, pills, tablets, gels, drops, and capsules can be particularly used. In other administration forms, pharmaceutical compositions can be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermal, intraperitoneally, or intramuscularly, and can include solutions or emulsions prepared from sterile or sterilizable solutions. Pharmaceutical compositions containing compounds of Formula I can also be in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions, or dusting powders. Pharmaceutical compositions can be formulated in unit dosage form (i.e., in the form of discrete portions containing a unit dose or a multiple or subunit thereof).

[0058] The compounds of Formula I can be provided as pharmaceutically acceptable salts, including, for example, appropriate acid addition or base salts thereof. Reviews of suitable pharmaceutical salts are found in Berge et al., J Pharm Sci 66:1-19 (1977). Salts are formed, for example, with strong inorganic acids, such as mineral acids (e.g., sulfuric acid, phosphoric acid, or hydrohalic acids), strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms that are unsubstituted or substituted (e.g., by halogens), for example, acetic acid, saturated or unsaturated dicarboxylic acids (e.g., oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, or terephthalic acid), hydroxycarboxylic acids (e.g., ascorbic acid, glycolic acid, lactic acid, malic acid, tartaric acid, or citric acid), amino acids (e.g., aspartic acid or glutamic acid), benzoic acid, or organic sulfonic acids (e.g., (C1-C4)-alkyl- or aryl-sulfonic acids that are unsubstituted or substituted (e.g., by halogens), for example, methane- or p-toluenesulfonic acid).

[0059] The compounds of formula I can be supplied in their various crystalline forms, polymorphs and anhydrous / hydrous forms. In this regard, it is well known to those skilled in the art that chemical compounds can be isolated in any of such forms by slightly varying the purification and / or isolation methods from solvents used in the synthetic preparation of such compounds.

[0060] Methods for synthesizing compounds of Formula I have been previously described (see, for example, WO2017 / 020065). In some embodiments, compounds of Formula I can be prepared according to the following general synthetic scheme: : [ka] where the general reaction conditions are: (a) DMF-DMA or Bredereck reagent, reflux; (b) Select Fluor, MeOH; (c) EtN, HgCl, DCM; (d) TFA / DCM (1:1), reflux; (e) A, B, NaOH, 2-methoxyethanol, microwave; and (f) Pddba, Xantphos, t-BuONa, dioxane, microwave.

[0061] In connection with the description of the synthetic methodology of Scheme 1 above, it will be understood by those skilled in the art that all of the reaction conditions presented, including the choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and work-up procedures, can be readily selected. Furthermore, it will be understood by those skilled in the art that the functional groups present on various portions of the molecule must be compatible with the reagents and reaction conditions utilized.

[0062] The necessary starting materials can be obtained by standard procedures of organic chemistry. The preparation of such starting materials is described in the Examples hereinafter, along with the following representative process variants. Alternatively, the necessary starting materials can be obtained by procedures analogous to those exemplified, which are within the ordinary skill of one skilled in the art. Furthermore, it will be appreciated that during the synthesis of a compound, or of a particular starting material, it may be desirable to protect certain substituents to prevent their undesired reactions. Those skilled in the art will readily understand when such protection is required and how such protecting groups can be introduced and subsequently removed. Examples of protecting groups can be found, for example, in Protective Groups in Organic Chemistry, Vol. 1, No. 1, pp. 111-114, 1997. The protecting groups can be removed by any convenient method known to those skilled in the art as being suitable for removing the protecting group in question, with such method being selected to remove the protecting group with minimal disruption to groups elsewhere in the molecule. Thus, if the reactant contains, for example, an amino, carboxyl, or hydroxyl group, the present invention In some of the reactions mentioned herein it may be desirable to use protecting groups.

[0063] In addition, one skilled in the art would be able to select suitable reaction conditions for use in the coupling reaction of a compound of Formula A or Formula B shown in Scheme 1. Typically, however, the reaction is carried out under anhydrous conditions and in the presence of an inert atmosphere, such as argon or nitrogen. The reaction can also be carried out at elevated temperatures, for example, within the range of 80-180°C, for a suitable time, for example, from 20 minutes to 48 hours. Suitably, the reaction is carried out under microwave heating, for example, at 80-180°C, for 20 minutes to 1.5 hours.

[0064] The resulting compounds can be isolated and purified using techniques well known to those skilled in the art.

[0065] The methods and uses of the present disclosure are further described herein below with reference to the following non-limiting examples and accompanying drawings. [Example]

[0066] Example 1 Synthesis of Representative Compounds General 1 H and 13 C NMR spectra were obtained at 300 K on a Bruker AVANCE III 500 spectrometer (500 MHz 1 H). 1 The H NMR spectrum shows the presence of residual non-deuterated solvent (or tetramethylsilane). 1 The H signal was used as the reference. High-resolution mass spectra were recorded on an AB SCIEX TripleTOF® 5600 mass spectrometer, and ionization of all samples was performed using ESI. The purity of the compounds was determined by analytical HPLC to be greater than 95%. CBM-20A Communication Bus Module, DGU-20A 5R Degassing unit, LC-20AD liquid chromatograph pump, SIL-20A HT Analytical HPLC was performed on a Shimadzu Prominence UFLC (UltraFast Liquid Chromatograph) system equipped with an autosampler, an SPD-M20A photodiode array detector, a CTO-20A column oven, and a Phenomenex Kinetex 5u C18 100A 250 mm × 4.60 mm column using Method A (5% to 95% MeOH containing 0.1% FA over 7 min at a flow rate of 1 mL / min, followed by 95% MeOH containing 0.1% FA over 13 min) and Method B (5% to 95% MeCN containing 0.1% FA over 7 min at a flow rate of 1 mL / min, followed by 95% MeCN containing 0.1% FA over 13 min).

[0067] 5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (1): To a solution of crude 1-(5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (524 mg, 2.00 mmol) in 2-methoxyethanol (3 mL) was added ((E)-3-(dimethylamino)-2-fluoro-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one (243 mg, 1.00 mmol) and NaOH (80.0 mg, 2.00 mmol). The reaction mixture was heated at 180° C. for 1 h under microwave irradiation, cooled to room temperature, and then concentrated under reduced pressure. The residue was purified by chromatography (silica gel, gradient from DCM to DCM:MeOH=90:10 with constant addition of 0.5 mL of 32% ammonia) to give 1 as a brown solid (76 mg, 17.2%). 1 H NMR (DMSO-d6) δ 1.50 (q, 2H, J 11.0), 1.84 (d, 3H, J 11.0), 2.21 (s, 7H), 2.47 (s, 3H, thiazole-CH3), 2.64 (t, 2H, J 11. 0), 2.86 (t, 3H, J 3.5), 3.63 (d, 1H, J 11.0), 7.39 (app d, 1H, J 7.0), 7.92 (d, HRMS (ESI): m / z 443.2136 [M+H] + .

[0068] N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-methylthiazol-2-amine (2): To a solution of 5-(2-amino-5-fluoropyrimidin-4-yl)-N-cyclopentyl-4-methylthiazol-2-amine (200 mg, 0.68 mmol) in dioxane (3 mL), 1-((6-bromopyridin-3-yl)methyl)-4-ethylpiperazine (233 mg, 0.82 mmol), Pd2dba3 (31 mg, 0.034 mmol), Xantphos (41 mg, 0.07 mmol), and t-BuONa (98 mg, 1.02 mmol) were added and heated at 150 °C for 1 h under microwave irradiation. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by chromatography (silica gel, gradient DCM to DCM:MeOH = 93:7) to give 2 as an orange solid (100 mg, 29%). 1 H NMR (DMSO-d6) δ 0.99 (t, 3H, J 7.0), 1.49-1.59 (m, 4H), 1.64 - 1.72 (m, 2H), 1.90 - 1.97 (m, 2H), 2.38 (s br, 10H), 2.48 (d, 3H, J 2.5), 3.42 (s, 2H), 3.95 - 3.98 (m, 1H), 7.64 (dd, 1H, J 8.5 & 2.0), 8.10 (d, 1H, J 8.5), 8.16 (d, 1H, J 2.0), 8.27 (d, 1H, J 7.0), 8.46 (d, 1H, J 3.5), 9.77 (s, 1H). HRMS (ESI): m / z 497.2601 [M+H] + .

[0069] N-cyclopentyl-4-methyl-5-(2-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)thiazol-2-amine (3): To a mixture of crude 1-(5-(piperazin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (441 mg, 2.00 mmol) and (E)-1-(2-(cyclopentylamino)-4-methylthiazol-5-yl)-3-(dimethylamino)prop-2-en-1-one (279 mg, 1.00 mmol) in 2-methoxyethanol (3 mL) was added NaOH (80.0 mg, 2.00 mmol). The reaction mixture was heated at 180 °C under microwave irradiation for 1 h, cooled to room temperature, and concentrated under reduced pressure. The residue was purified by chromatography (silica gel, gradient from DCM to DCM:MeOH = 92:8) and recrystallized from DCM and MeOH to give 3 as a dark yellow solid (70.0 mg, 16%). mp 210-213 °C. 1 H NMR (DMSO-d6) 1.49-1.68 (m, 7H), 1.89-1.94 (m, 2H), 2.46 (s, 3H), 2.85 (t, 4H, J 4.5), 3.02 (t, 4H, J 5.0), 3.98 (m, 1H), 6.90 (d, 1H, J 5.5), 7.36 (dd, 1H, J 9.0 & 3.0), 7.98 (d, 1H, J 3.0), 8.07 (d, 1H, J 9.0), 8.18 (d, 1H, J 7.0), 8.33 (d, 1H, J 5.5), 9.33 (s, 1H). HRMS (ESI): m / z 437.2222 [M+H] + .

[0070] N-cyclopentyl-5-(2-((5-(4-ethylpiperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine (4): To a mixture of crude 1-(5-(4-ethylpiperazin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (496 mg, 2.00 mmol) and (E)-1-(2-(cyclopentylamino)-4-methylthiazol-5-yl)-3-(dimethylamino)prop-2-en-1-one (279 mg, 1.00 mmol) in 2-methoxyethanol (3 mL) was added NaOH (80.0 mg, 2.00 mmol). The reaction mixture was heated at 180 °C under microwave irradiation for 1 h, cooled to room temperature, and concentrated under reduced pressure. The residue was purified by chromatography (silica gel, gradient DCM to DCM:MeOH = 96:4) and recrystallized from MeOH to give 4 as a yellow solid (117 mg, 25%). 1 H NMR (CDCl3) δ 1.14 (t, 3H, J 7.0), 1.56-1.76 (m, 6H), 2.06-2.12 (m, 2H), 2.49 (q, 2H, J 7.5), 2.54 (s, 3H), 2.64 (s, 3H), 3.19 (t, 4H, J 4.5), 3.14 (t, 4H, J 5.0), 3.86 (app s, 1H), 5.77 (s, 1H), 6.84 (d, 1H, J 5.0), 7.34 (dd, 1H, J 9.0 & 3.0), 7.94 (d, 1H, J 3.0), 7.94 (s, 1H), 8.01 (d, 1H, J 3.0), 8.26 (d, 1H, J 9.0), 8.33 (d, 1H, J 5.5). HRMS (ESI): m / z 465.2541 [M+H] + .

[0071] 2-((5-(4-acetylpiperazin-1-yl)pyridin-2-yl)amino)-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile (5): To a solution of crude 1-(5-(4-acetylpiperazin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (315 mg, 1.20 mmol) in 2-methoxyethanol (4 mL) was added tert-butyl (E)-(5-(2-cyano-3-(dimethylamino)acryloyl)-4-methylthiazol-2-yl)(methyl)carbamate (350 mg, 1.00 mmol) and NaOH (82.0 mg, 2.40 mmol). The reaction mixture was heated at 180° C. for 90 min under microwave irradiation, cooled to room temperature, and then concentrated under reduced pressure. The residue was purified by chromatography (silica gel, gradient from DCM to DCM:MeOH=90:10 with successive addition of 32% aqueous ammonia up to 3%). The solid was washed with DCM and MeOH and then filtered to give 5 as a pale yellow solid (157 mg, 35%). 1 H NMR (DMSO-d6) δ 2.04 (s, 3H), 2.40 (s, 3H), 2.87 (s, 3H), 3.10 (t, 2H, J 5.0), 3.16 (t, 2H, J 5.0), 3.58 (t, 4H, J 5.0), 7.46 (dd, 1H, J 9.5 & 3.0), 7.90 (d, 1H, J 9.0), 8.06 (d, 1H, J 3.0), 8.26 (q, 1H, J 3.0), 8.75 (s, 1H), 10.33 (s, 1H). HRMS (ESI): m / z 450.1844 [M+H] + .

[0072] N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine (6): To a solution of 5-(2-aminopyrimidin-4-yl)-N-cyclopentyl-4-methylthiazol-2-amine (275 mg, 1.00 mmol) in dioxane (3 mL), 1-((6-bromopyridin-3-yl)methyl)-4-ethylpiperazine (341 mg, 1.2 mmol), Pd2dba3 (45.8 mg, 0.05 mmol), Xantphos (58 mg, 0.1 mmol), and t-BuONa (144 mg, 1.5 mmol) were added and heated at 150° C. for 1 h under microwave irradiation. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by chromatography (silica gel, DCM to DC Purification by elution with a gradient of M:MeOH:NH4OH=9:1:0.3 and recrystallization with DCM and MeOH gave 6 as a white solid (200 mg, 42%). 1 H NMR (CDCl3) δ 1.09 (t, 3H, J 7.0), 1.58 - 1.76 (m, 6H), 2.08 - 2.14 (m, 2H), 2.43 (q, 2H, J 7.0, CH2CH3), 2.55 (s br, 11H), 3.48 (s, 2H), 3.86 - 3.92 (m, 1H), 5.42 (d, 2H, J 7.0), 6.90(d, 1H, J 5.5), 7.68 (dd, 1H, J 9.0 & 2.5), 7.89 (s, 1H), 8.19 (d, 1H, J 2.0), 8.35 - 8.38 (m, 2H). HRMS (ESI): m / z 479.2703[M+H] + .

[0073] 5-(2-((5-(4-aminopiperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine (7): Compound 7 was obtained as an orange solid (40.0 mg, 10%) by reacting 1-(5-(4-aminopiperidin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (702 mg, 3.00 mmol) and (469 mg, 2.00 mmol) with ((E)-3-(dimethylamino)-2-fluoro-1-(4-methyl-2-(methylamino)thiazol-5-yl)prop-2-en-1-one (243 mg, 1.00 mmol). 1 H NMR (DMSO-d6) δ 1.75-1.80 (m, 2H), 2.45 (d, 3H, J 2.0), 2.62 (t, 2H, J 6.0), 2.85 (t, 2H, J 5.5), 3.45 (t, 2H, J 5.0), 3.53 (t, 2H, J 6.0), 7.13 (dd, 1H, J 9.0 & 3.0), 7.78 (s, 1H), 7.79 (d, 1H, J 4.5), 8.08 (q, 1H, J 4.5), 8.37 (d, 1H, J 3.5), 9.21 (s, 1H). HRMS (ESI): m / z 415.1821 [M+H] + .

[0074] 5-(2-((5-(4-aminopiperidin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-N-cyclopentyl-4-methylthiazol-2-amine (8): To a mixture of 1-(5-(4-aminopiperidin-1-yl)pyridin-2-yl)guanidine trifluoroacetate (702 mg, 3.00 mmol) and (E)-1-(2-(cyclopentylamino)-4-methylthiazol-5-yl)-3-(dimethylamino)prop-2-en-1-one (558 mg, 2.00 mmol) in 2-methoxyethanol (5 mL) was added NaOH (160.0 mg, 4.00 mmol). The reaction mixture was heated at 180° C. under microwave irradiation for 2 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was purified by chromatography to give 8 as a yellow solid (90 mg, 10%). 1H NMR (CDCl3) δ 1.50-1.77 (m, 10H), 1.95 (d, 2H, J 10.5), 2.07-2.13 (m, 2H), 2.54 (s, 3H), 2.75-2.85 (m, 3H), 3.53 - 3.56 (m, 2H), 3.85 - 3.91 (m, 1H), 5.43 (d, J 5.0, 1H), 6.84 (d, 1H, J 5.5), 7.34 (dd, 1H, J 9.0 & 3.0), 7.75 (s, 1H), 8.00 (d, 1H, J 3.0), 8.25 (d, 1H, J 9.0), 8.32 (d, 1H, J 5.5). HRMS (ESI): m / z 451.2415 [M+H] + .

[0075] N-cyclopentyl-5-(2-((5-morpholinopyridin-2-yl)amino)pyrimidin-4-yl)-4-(trifluoromethyl)thiazol-2-amine (9): To a mixture of 1-(5-morpholinopyridin-2-yl)guanidine trifluoroacetate (442 mg, 2.00 mmol) and (E)-1-(2-(cyclopentylamino)-4-methylthiazol-5-yl)-3-(dimethylamino)-2-fluoroprop-2-en-1-one (297 mg, 1.00 mmol) in 2-methoxyethanol (3 mL) was added NaOH (80.0 mg, 2.00 mmol). The reaction mixture was heated at 180° C. under microwave irradiation for 1 h, cooled to room temperature, and concentrated under reduced pressure. The residue was purified by chromatography to give 9 as a brown solid (120 mg, 26%). 1 H NMR (DMSO-d6) δ 1.50-1.57 (m, 4H), 1.66 - 1.69 (m, 2H), 1.90 - 1.95 (m, 2H), 2.47 (d, 1H, J 2.5), 3.09 (t, 4H, J 5.0), 3.75 (t, 4H, J 5.0), 3.96 (m, 1H), 7.42 (dd, 1H, J 9.0 & 3.0), 7.96 (d, 1H, , J 9.0), 7.98 (d, 1H, J 3.0), 8.24 (d, 1H, J 7.0), 8.41 (d, 1H, J 7.0), 9.52(s, 1H). HRMS (ESI): m / z 456.1976 [M+H] + .

[0076] Example 2 Biological activity キナーゼアッセイ Inhibition of CDKs and other kinases was measured by radiometric assay (RIA) using Eurofins Pharma Discovery or Reaction Biology Corporation Kinase Profiler services. Inhibition of CDK4 / cyclin D1, CDK6 / cyclin D3, and CDK9 / T1 was also determined in-house using the ADP Glo Kinase Assay (Promega Corporation, Madison, WI, USA). Briefly, kinase reactions for CDK4 / cyclin D1 and CDK6 / cyclin D3 were performed using kinase reaction buffer (40 nM Tris base pH 7.5, 20 mM MgCl2, 0.4 mM DTT), 0.1 mg / ml BSA, and RB-CTF substrate (retinoblastoma protein 1 C-terminal fragment). For CDK9 / cyclin T1, kinase reactions were performed using standard assay buffer, Kinase Dilution Buffer, and RB-IRStide substrate. Serial 1:3 dilutions of test compounds were prepared for 10 concentrations (10 μM to 0.5 nM). Kinase reactions were initiated by the addition of ATP and incubated for 40 minutes at 37°C, then stopped by adding 10 μL of ADP Glo reagent. After 40 minutes of incubation at room temperature (RT) in the dark, 20 μL of kinase detection reagent was added per well and incubated for 40 minutes. Luminescence was measured using an EnVision Multilabel plate reader (PerkinElmer, Buckinghamshire, UK). Positive and negative controls were performed in the presence and absence of CDK kinases, respectively. A four-parameter logistic nonlinear regression model was used in Graphpad Prism (Version 6.0) to calculate 50% inhibition (IC 50 ) values ​​were calculated for each kinase. m (ATP) and IC 50 From the values, the apparent inhibition constant (K i ) values ​​were calculated. The results for representative compounds are shown in Table 2. [Table 2]

[0077] cell culture All three GBM cell lines used in this example, U87, U251, and T98G, were cultured at 75 cm in Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum. 2 They were cultured in flasks at 37°C and 5% CO. All cell lines were confirmed to be mycoplasma-free before any experimental setup.

[0078] MTT proliferation assay and combination studies As previously reported (Wang S et al., J Med Chem 47:1662-1675, 2004; and Diab S. et al. CheMedChem 9:962-972, 2014), representative compounds from Example 1 were subjected to standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and resazurin assays in solid tumor cell lines (including GBM cell lines) and leukemia cell lines, respectively. The compound concentration required to inhibit 50% of cell proliferation (GI 50 ) was determined using GraphPad Prism 8 (GraphPad Software, Inc.; San Diego, CA, USA). The results are shown in Table 3. [Table 3]

[0079] For combination studies, cells were treated with 10 μL of everolimus (an mTOR inhibitor), alpelisib (a PI3K inhibitor), or selumetinib (a MEK inhibitor) and 10 μL of one of the disclosed CDK4 / 6 inhibitors at various concentrations for 72 hours. During this time, 50 μL of MTT was added to each well. After 3.5 hours of incubation, absorbance was measured at 550 nm using an EnVision® multilabel plate reader (Buckinghamshire, UK). Results were determined using CompuSyn v 1.0 (ComboSyn, NJ, USA) according to the Chou-Talalay method. Combination index (CI) values ​​of less than 1, 1, and greater than 1 were considered synergistic, additive, and antagonistic drug interactions, respectively (Chou TC et al., Trends Pharmacol Sci 4:450-454, 1983). The results are shown in Figure 1. The combination treatments resulted in higher levels of inhibitory activity against proliferation compared to the single agent treatments. There was evidence of synergistic levels of inhibition, as indicated by a CI of less than 1.

[0080] Apoptosis assay The ability of Compound 1 to induce apoptosis in GBM cells was investigated using the Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen Inc.; San Diego, CA, USA) according to the manufacturer's protocol. The cell pellet was washed twice with 1 mL of cold PBS and centrifuged at 300 × g for 5 minutes. After removing the supernatant, the cell pellet was stained with 100 μL of 1× Annexin V binding buffer, 3 μL of Annexin V-FITC, and 3 μL of propidium iodide staining solution, and incubated in the dark at room temperature for 15 minutes. The percentage of apoptotic cells was measured using a CytoFLEX flow cytometer. GBM U87 cells were treated with 5 μM palbociclib ("Palb") or Compound 1 (with or without 5 μM temozolomide (TMZ)), and then assayed 48 hours later. The results in Figure 2 showed that Compound 1 (with and without TMZ) could induce apoptosis in GBM cells at higher levels than the control anticancer agent, palbociclib (with and without TMZ).

[0081] Western blot analysis 3 x 10 cells 5 The cells were seeded at a density of 1000 x g / ml onto sterile culture dishes with 10 mL of fresh medium and incubated overnight at 37°C, 5% CO2 before compound treatment. After 24 hours, the cells were resuspended in PBS Cells were collected at 4°C, centrifuged at 300 × g for 5 minutes, and lysed in 100 μL of lysis buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 300 mM sodium chloride, 1.5 mM magnesium chloride, 0.5% sodium deoxycholate, 20 mM β-glycerophosphate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, and 25× protease inhibitor cocktail). Protein lysates were collected after 10 minutes of maximum speed centrifugation at 4°C. Protein concentrations of cell lysates were determined by running a BSA standard curve using the Bio-Rad DC Protein Assay Kit II (Bio-Rad, Sydney, NSW, Australia) according to the manufacturer's protocol. Thirty micrograms of protein was measured, mixed with 3x loading buffer, and denatured at 95°C for 5 minutes using a T100 thermal cycler (Bio-Rad). The molecular mass of each sample was then separated by gel electrophoresis on a 4-20% polyacrylamide gel at 120 V for 1 hour. The dissolved proteins were transferred to a nitrocellulose membrane and blocked with 5% nonfat milk in Tris-buffered saline and Tween 20 (TBST) for 1 hour at room temperature. Antibodies were diluted in 5% nonfat milk in TBST. The membrane was incubated with primary antibody overnight at -20°C, washed four times with TBST, treated with secondary antibody for 1 hour at room temperature, and washed four times with TBST again. Chemiluminescence on the membrane was then developed using Amersham ECL Prime / Select Western Blotting Detection Reagent (GE Healthcare, Sydney, NSW, Australia). Band intensities were determined using a ChemiDoc™ multiplexed imaging system (Bio-Rad).The antibodies used were obtained from Cell Signaling Technology (Danvers, MA, USA): p-Rb(S780)#9307, p-Rb(S795)#9301, p-Rb(S807 / 811)#8516, Rb #9309, cyclin D1 #2978, cyclin E #4132, CDK4 #12790, CDK6 #13331, β-actin #4970, HRP-conjugated anti-mouse IgG #7076, and HRP-conjugated anti-rabbit IgG #7074. Figure 3A shows that compound 1 (and palbociclib, a positive control) reduced the levels of phosphorylated Rb proteins (pRbSer780, pRbSer795, and pRb807 / 811), confirming its cellular CDK4 / 6 inhibition.

[0082] In vitro tumorigenicity assay Cells were seeded at a density of 200–400 cells / well in 12-well plates and incubated overnight before compound treatment. Each well was replaced with fresh medium containing Compound 1 (or the positive control, palbociclib) every 2–3 days. After 10 days, cells were washed with PBS, fixed, and stained with crystal violet stain (0.05% crystal violet, 1% formaldehyde, 1% PBS, 1% ethanol); the results are shown in Figure 3B. Compound 1 inhibited the growth and proliferation of all three GBM cell lines.

[0083] Brain uptake assay The propensity of the compound from Example 1 to be taken up by the brain (thereby indicating its ability to cross the BBB) was investigated using a method as described (TJ Raub et al., supra). Briefly, Balb / C mice were intravenously administered Compound 1 or Compound 2. Five minutes and one hour after administration, cardiac blood was collected, and the cerebral hemispheres were excised. For oral administration experiments, mice were given the drug, and blood and cerebral hemispheres were collected 0.5, 1, 2, 4, 6, and 24 hours after administration. Drug concentrations in homogenized brain and plasma were measured by LC-MS / MS; concentrations in brain homogenates were converted to brain concentrations (expressed per gram of brain tissue) and corrected for an average measured plasma volume of 16 μL / gram of brain tissue. The unbound fraction in plasma and brain was determined using a Centrifree® Ultrafiltration Device. The K for each drug was 0.5, 1, 2, 4, 6, and 24 hours after administration. p,u The value of The results were compared with set guidelines for neurotherapeutic agents (Kulkarni AD et al. Expert Opin Drug Deliv 13:85-92, 2016). Control experiments were performed using equivalent doses of palbociclib and / or abemaciclib. The results, shown in Figure 4, indicate that K p,u : Expressed as the brain-to-plasma ratio of unbound drug concentration, this is usually divided into the following bands: K p,u <0.1, unable to cross the BBB; K p,u 0.3–0.5, sufficient access to the BBB; K p,u >1.0, freely cross the BBB (Kulkarni et al., supra). Compounds 1, 2 and 6 demonstrated a significantly higher propensity to cross the BBB than control anti-cancer agents.

[0084] In vivo antitumor efficacy Compounds 1 and 2 were also investigated for in vivo antitumor activity in a subcutaneous xenograft model. Briefly, U87 GBM cells were harvested and resuspended in a 1:1 mixture of serum-free medium and Matrigel, and 5 × 10 6Cells were injected subcutaneously into the flanks of 5-6 week-old female CD1 nu / nu mice. The average tumor volume was 150-200 mm. 3 Once the tumor size reached 0.05 mg / kg, animals were randomized into treatment groups (n=10 per group) and treated orally daily with vehicle (0.5% carboxymethylcellulose in water), Compound 1, or Compound 2 at the doses indicated in Figures 5A and 5B for 21 days. Mice were observed daily for signs of toxicity and weight loss, and tumor volumes were measured every other day. Compared to vehicle treatment, both compounds significantly reduced tumor growth (T / C=31% and 6% at day 21 for Compound 1 and Compound 2, respectively; p<0.001 as determined by two-tailed t-test) without any overt toxicity.

[0085] In a separate study, U87 xenograft mice (n=8 per group) were treated with (1) vehicle, (2) Compound 2 (25 mg / kg PO, daily), (3 and 4) TMZ (5 mg / kg or 50 mg / kg PO, 5 days / week), (5) Compound 2 pretreatment (25 mg / kg PO, daily x 10) followed by TMZ (5 mg / kg PO, 5 days / week x 2), (6) TMZ pretreatment (5 mg / kg PO, 5 days / week x 2) followed by Compound 2 (25 mg / kg PO, daily x 10), and (7) Compound 2 and TMZ cotreatment. All treatment groups were completed by day 21, followed by a 7-day washout, after which a second cycle of treatment began on day 29 (Figure 5B). Compound 2 demonstrated significant antitumor efficacy both as a single agent and in combination with TMZ without any apparent toxicity when compared to the vehicle-treated group (p<0.001).

[0086] The in vivo antitumor efficacy of Compound 1 and Compound 2 was also investigated in a GBM orthotopic mouse xenograft model. For example, Compound 2 was orally administered once daily for 21 days to NSG mice (n = 8) (The Jackson Laboratory, Ben Harbor, ME, USA) orthotopically implanted with U87 GBM cells for each treatment cohort. TMZ was orally administered once daily for 5 days and used as a positive control. Disease burden was measured using non-invasive bioluminescence whole-body imaging technology. Treatment with Compound 2 resulted in a significant inhibition of tumor growth (TGI = 81.4%, p < 0.001) at day 21 compared with vehicle-treated mice. Compared with vehicle controls, a 154.8% increase in the lifespan ratio [ILS = (T day - C day) / C day, where C day = survival days of the control group and T day = survival days of the treatment group] was observed in Compound 2-treated mice (Figure 6A).

[0087] In another study, as shown in Figure 6B, NSG mice (n = 8) orthotopically implanted with GBM patient-derived G4T cells were administered vehicle, compound 1, TMZ, and co-treatment with compound 1 and TMZ, respectively. Disease burden in each treatment group was measured by bioluminescence whole-body imaging. Compound 1 demonstrated significant inhibition of tumor growth, both as a single agent and in combination with TMZ (p < 0.001, Figure 6B).

[0088] conclusion The compounds of formula I have been shown to inhibit CDK4 / 6 and thus have antiproliferative activity against GBM cell lines in both in vitro and in vivo settings. Further representative compounds include Compound 1 (5-(2-((5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine), Compound 2 (N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-methylthiazol-2-amine), and compound 6 (N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine) were found to be able to cross the blood-brain barrier and to be highly effective in GBM tumor-bearing animal models, indicating that compounds of formula I have great potential to provide effective therapeutic agents for treating proliferative cell diseases and conditions of the CNS, such as GBM.

[0089] Throughout this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations thereof, such as "comprising" and "comprising," will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0090] The reference herein to any prior art is not, and should not be taken as, any form of admission of an suggestion that such prior art forms part of the common general knowledge.

[0091] Those skilled in the art will appreciate that the present disclosure is not limited in its use to the particular applications described. Nor is the present disclosure limited to its preferred embodiments with respect to the specific elements and / or features described or illustrated herein. It will also be appreciated that the present disclosure is not limited to one or more disclosed embodiments, but that numerous rearrangements, modifications, and substitutions are possible without departing from the scope of the present disclosure as described and defined by the following claims.

[0092] Please note that the following claims are provisional claims only, provided as examples of possible claims, and are not intended to limit the scope of what may be claimed in any future patent application based on this application. Integer numbers may be added to or deleted from the example claims at a later date to further define or redefine various aspects of the present disclosure.

Claims

1. 1. Use of a compound of formula (I) as shown below, or a pharmaceutically acceptable salt, or solvate thereof, in the manufacture of a medicament for treating a proliferative cell disease or condition of the central nervous system (CNS) in a subject. 【Chemistry 1】 [In the formula: R １ is H, alkyl, aryl, aralkyl, alicyclic, heterocyclic, halogen, NO ２ , C.N., C.F. ３ , OH, O-alkyl, O-aryl, COOH, CO-alkyl, CO-aryl, CONH ２ , CONH-alkyl, CONH-aryl, and CONH-alicyclic; R ２ is H, alkyl, halogen, NO ２ , C.N., C.F. ３ , OH, O-alkyl, and NH ２ Selected from: R ３ is a heterocyclic group containing at least one N heteroatom, NH-alkyl, NH-aryl, N-(alkyl) ２ , N-(aryl) ２ and N-(alkyl)(aryl); and n is an integer selected from the range of 0 to 3; and Here, the alkyl, aryl, aralkyl, alicyclic and heterocyclic groups are alkyl, halogen, CN, OH, O-methyl, NH ２ , NH-alkyl, N(alkyl) ２ , COOH, COH, CO(alkyl), CONH ２ and CF ３ and optionally substituted with one or more groups selected from

2. 1. Use of a compound of formula (I) as shown below, or a pharmaceutically acceptable salt, or solvate thereof, in the manufacture of a medicament formulated for intravenous and / or oral administration for treating a proliferative cell disease or condition of the central nervous system (CNS) in a subject, comprising: 【Chemistry 2】 [In the formula: R １ is selected from methyl and cyclopentyl; R ２ is selected from H, F, and CN; R ３ is selected from: 【change】 and n is an integer selected from 0 and 3; Optionally, in combination with a pharmaceutically acceptable carrier, diluent and / or excipient.

3. 3. The use of claim 2, wherein the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a subject in combination with temozolamide or another kinase inhibitor.

4. 4. The use according to claim 3, wherein the other kinase inhibitor is selected from inhibitors of PI3K, mTOR and / or MEK.

5. 5. The use of claim 4, wherein the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a subject in combination with everolimus, alpelisib, or selumetinib.

6. 6. The use of any one of claims 2 to 5, wherein the proliferative cell disease or condition is glioblastoma (GBM), medulloblastoma, primary central nervous system (CNS) lymphoma, astrocytoma, ependymoma, oligodendroglioma, or metastatic brain tumor.

7. The compound is: N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-4-methylthiazol-2-amine; N-cyclopentyl-4-methyl-5-(2-((5-(piperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)thiazol-2-amine; N-cyclopentyl-5-(2-((5-(4-ethylpiperazin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine; 2-((5-(4-acetylpiperazin-1-yl)pyridin-2-yl)amino)-4-(4-methyl-2-(methylamino)thiazol-5-yl)pyrimidine-5-carbonitrile; N-cyclopentyl-5-(2-((5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine; 5-(2-((5-(4-aminopiperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(2-((5-(4-aminopiperidin-1-yl)pyridin-2-yl)amino)pyrimidin-4-yl)-N-cyclopentyl-4-methylthiazol-2-amine N-cyclopentyl-5-(5-fluoro-2-((5-morpholinopyridin-2-yl)amino)pyrimidin-4-yl)-4-methylthiazol-2-amine 5-(2-((5-(4-(ethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(2-((5-(4-(ethyl(methyl)amino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(5-fluoro-2-((5-((4-methylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; 5-(5-fluoro-2-((5-((4-isopropylpiperazin-1-yl)methyl)pyridin-2-yl)amino)pyrimidin-4-yl)-N,4-dimethylthiazol-2-amine; or 5-(2-((5-(4-(diethylamino)piperidin-1-yl)pyridin-2-yl)amino)-5-fluoropyrimidin-4-yl)-N,4-dimethylthiazol-2-amine The use according to claims 2 to 6, wherein

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