HK40082485A - Treatment of proliferative diseases of the cns - Google Patents

Description

Treatment of proliferative diseases of the CNS

Technical Field

The present disclosure relates to methods and uses of a class of thiazole-pyrimidine compounds in the treatment of proliferative cell diseases or disorders of the Central Nervous System (CNS).

Priority file

The present application claims priority to australian provisional patent application No. 2020901435, entitled "Treatment of the productive disease of the CNS" and filed on 6/5/2020, the contents of which are hereby incorporated by reference in their entirety.

Background

Primary brain tumors consist of a diverse group of neoplasms that are derived from different cell lineages. Tumors of the Central Nervous System (CNS) are classified as astrocytic, oligodendroglial, or mixed tumors according to the World Health Organization (WHO) classification. These tumors were further classified by subtype and graded from I to IV according to histology, with grade IV being the most aggressive. In the united states alone, it is estimated that nearly 80,000 new 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 CNS tumor, accounting for 45% of malignant primary CNS tumors and 54% of all gliomas. Despite the increasing survival rates of most cancers in recent years, CNS cancers have not achieved the same level of success. For example, between 2009 and 2013 patients diagnosed with brain cancer have an approximately 25% chance of surviving five years. This is in sharp contrast to the survival rate of about 68% combined with all cancers at the same stage. For GBM patients, median survival is only 15-23 months and 5-year survival is about 4.6%, which is the lowest rate of all brain tumor types.

Aberrant cell cycle control leads to unrestricted cell cycle reentry and progression, a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are known to be associated with various cyclin subunits and play key roles in regulating a variety of important regulatory pathways in cells, including cell cycle control, apoptosis, neuronal physiology, differentiation and transcription. To date, at least 20 CDKs and 30 cyclins have been identified. They can be classified into two major groups reflecting their function: cell cycle regulators CDK and transcription regulators CDK (Wang S et al, trends Pharmacol Sci 29 (6): 302-313, 2008). The class of cell cycle regulators CDKs includes CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK7, and they function with their cyclin chaperones (e.g., cyclin A, B, C, D, D2, D3, E, F) to regulate the initiation of the cell cycle. The class of transcriptional regulators CDKs includes CDK7, CDK8, CDK9, and CDK11, which function with cyclin C, H, K, L, L2, T1, and T2, and tend to play a role in transcriptional regulation. It is not surprising that CDKs are involved in cell proliferative diseases and disorders, particularly cancer, in view of the function of each CDK class. Cell proliferation is the result of a direct or indirect deregulation of the cell division cycle, and CDKs play a key role in the regulation of the various stages of this cycle. Thus, CDK inhibitors and their associated cyclins are considered useful targets for cancer therapy.

CDK4/6 controls the cell cycle and is tightly regulated by the INK4 family of proteins. Numerous studies have shown that the CDK4/6 pathway is overactivated in the vast majority of cancers, including CNS tumors and gliomas (Xu G et al, J neuroncol 136, 445-452,2018 Parsons DW et al, science 321, 1807-1812,2008, bax D A et al, clin Cancer Res 16. Based on genome-scale analysis of gliomas in children and adults, the CDK4/6-Rb axis is deregulated in > 80% of GBMs, which results from: (ii) 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. Thus, inhibition of CDK4/6 may be an effective method for treating cancer, particularly glioblastoma. Three CDK4/6 inhibitors, named palbociclib (palbociclib), ribbociclib (ribociclib) and aberciib (abemaciclib), have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of breast cancer and have been tested in GBM patients. However, the results for palbociclib were disappointing and the test was terminated. The lack of efficacy may be due to their limited ability to cross the Blood Brain Barrier (BBB) and thus expose the drug to the brain (Karen E et al, in 2013 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics, vol.12 (suppl. 11 th edition) 2013).

Cell signaling through growth factor receptors and protein kinases is another important regulator of cell growth and proliferation. In normal cell growth, growth factors activate the MAP kinase pathway through receptor activation (i.e., PDGF or EGF, etc.). 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 results in activation and indirect phosphorylation of Raf kinase. Raf then phosphorylates MEK1 and MEK2 (Ahn et al, methods Enzymol 332. Activated MEK then phosphorylates ERK1 and ERK 2. Subsequently, phosphorylated ERK dimerizes and then migrates to the nucleus where it accumulates (Khokhlatchev et al, cell 93, 605-615,1998), and where it then participates in several important cellular functions including nuclear transport, signal transduction, DNA repair, nucleosome assembly and migration, and mRNA processing and translation (Ahn et al, molecular Cell 6 1343-1354,2000. In general, treatment with growth factors results in activation of ERK1/2, resulting in proliferation and resistance to therapy. Thus, targeting the MAP kinase pathway provides therapeutic opportunities for a range of cancer types, and recently, the MEK inhibitor, semetinib, has gained US Breakthrough Therapy identification (US Breakthrough Therapy Designation) for the treatment of patients with type 1 neurofibromatosis (NF 1) (symptomatic and/or progressive, inoperable plexiform neurofibroma, an incurable genetic disorder). Mutations in the NF1 gene may lead to dysregulation of RAS/RAF/MEK/ERK signaling, which may lead to cells growing, dividing and replicating themselves in an uncontrolled manner, and thus to tumor growth. Sematinib inhibits MEK enzymes, resulting in inhibition of tumor growth.

In cancer cells, the main consequence of the disruption of the signaling pathway is an imbalance in protein expression, which enables the cell to escape apoptosis, proliferation, and metastasis. Approximately 40% of GBM subtype tumors are characterized by dysfunctional EGFR, a cell surface receptor tyrosine kinase that activates a series of downstream intracellular signaling through the PI3K/AKT pathway. Its amplification and overexpression via mutation promotes glioma (assisted by angiogenesis) growth and survival, migration and metastasis. Therefore, EGFR has been proposed as an attractive therapeutic target. However, phase II studies of the EGFR inhibitor erlotinib (erlotinib) in patients with recurrent GBM showed no significant benefit. Other tyrosine kinase inhibitors tested in clinical phase II-III studies, such as the inhibitor enzastaurin, which targets PKC and PI3K/AKT, when used alone or in combination with chemotherapy, also did not show efficacy in GBM patients. The failure of these EGFR inhibitor compounds may be due to poor Pharmacokinetic (PK) and BBB permeability.

The approval rate of CNS drugs is generally much lower than non-CNS drugs, and the depletion rate of oncology CNS drug development is very high. Therefore, the discovery of drugs for the treatment of brain tumors is characterized by major obstacles and historical failures. For successful treatment, the drug must first be able to cross the BBB, a layer of endothelial cells intimately connected by endothelial cells unique to capillaries in the brain, which are intimately connected by tight junctions preventing paracellular movement. The passage of compounds through endothelial cells into the brain can be restricted by the action of ATP-binding cassette (ABC) transporters expressed on their apical membrane, that is, at the membrane in contact with the circulating blood. Of these, P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two major transporters that together limit brain penetration for many compounds (Wager et al, expert Opin Drug Discov 6. In fact, most chemotherapeutic agents are prevented from entering the brain by this barrier.

The location of the GBM and its extensive infiltration into the surrounding normal brain tissue means that surgical resection cannot completely remove the tumor. In addition, tumor cells that invade the surrounding normal brain are protected by the BBB from the therapeutic agents. Unfortunately, those therapeutic agents that do have access, such as temozolomide (TMZ, the only standard of care chemotherapy for glioblastoma), have limited efficacy and can only improve survival for a few months at best. To date, most clinical trials of GBM therapy have failed. Preclinical data show that the penetration of CDK4/6 inhibitors (i.e., palbociclib and abbeli) is limited by active efflux at the BBB. Abelix showed relatively higher exposure in rodent brain than palbociclib, however, its therapeutic potential remains to be revealed. Clearly, there is a need to identify drugs that target new targets such as CDK4/6 and also cross the BBB sufficiently to make them useful for the treatment of brain tumors and other CNS cancers.

Applicants have identified a class of thiazole-pyrimidine compounds for the treatment of proliferative cell diseases and disorders in the CNS, including glioblastomas. While not wishing to be bound by theory, it is believed that these compounds block tumor cell proliferation and are able to cross the BBB by inhibiting the activity of CDK4 and/or CDK 6.

Disclosure of Invention

According to a first aspect, the present disclosure provides a method of treating a proliferative cell disease or disorder 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:

or a pharmaceutically acceptable salt, solvate or prodrug thereof,

optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient,

wherein:

R ¹ selected from H, alkyl, aryl, aralkyl, alicyclic, heterocyclic, halogen, NO ₂ 、CN、CF ₃ OH, O-alkyl, O-aryl, COOH, CO-alkyl, CO-aryl, CONH ₂ CONH-alkyl, CONH-aryl and CONH-alicyclic;

R ² selected from H, alkyl, halogen, NO ₂ 、CN、CF ₃ OH, O-alkyl and NH ₂ ；

R ³ Selected from the group consisting of heterocyclic radicals containing at least one N heteroatom, NH-alkyl, NH-aryl, N- (alkyl) ₂ N- (aryl) ₂ And N- (alkyl) (aryl); and is

n is an integer selected in the range of 0 to 3; and is

Wherein the 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 ₂ NH-alkyl, N (alkyl) ₂ COOH, COH, CO (alkyl), CONH ₂ And CF ₃ 。

Compounds of formula I have been found to have antiproliferative activity (e.g., these compounds are believed to block tumor cell proliferation by inhibiting the activity of CDK4 and/or CDK 6) and are further able to cross the BBB.

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 disorder of the Central Nervous System (CNS) in a subject, for example glioblastoma.

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 the treatment of a proliferative cell disease or disorder of the Central Nervous System (CNS) such as glioblastoma in a subject.

Drawings

FIG. 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 dose in a T98G GBM cell line in combination with (A) an inhibitor of mTOR (everolimus); denoted "Eve"), (B) an inhibitor of PI3K (apremilast (alpelisib); or (C) an inhibitor of MEK (Semtinib; "Sel");

FIG. 2 provides the results of annexin V/PI assays for GBM U87 cells at 48 hours after treatment with 5 μ M palbociclib ("Palb") or compound 1 alone or in combination with TMZ;

figure 3 shows that compound 1 inhibits GBM U87 cell colony formation by targeting CDK4/6 mediated Rb phosphorylation;

FIG. 4 provides 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) in Balb/C mice 24 hours after 2mg/kg intravenous administration (A) and after 10mg/kg oral administration (B); and (C) shows 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) in Balb/C mice after intravenous administration of 2 mg/kg;

figure 5 provides graphical results demonstrating the in vivo anti-tumor activity of compound 1 and compound 2 against subcutaneous GBM U87 cell xenografts; and is

Figure 6 provides graphical results demonstrating the in vivo anti-tumor activity of compound 1 and compound 2 against xenografts in situ in GBM U87 and G4T GBM patient-derived models, respectively.

Detailed Description

According to a first aspect, the present disclosure provides a method of treating a proliferative cell disease or disorder 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:

or a pharmaceutically acceptable salt, solvate or prodrug thereof,

optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient,

wherein:

R ¹ selected from H, alkyl, aryl, aralkyl, alicyclic, heterocyclic, halogen, NO ₂ 、CN、CF ₃ OH, O-alkyl, O-aryl, COOH, CO-alkyl, CO-aryl, CONH ₂ CONH-alkyl, CONH-aryl and CONH-cycloaliphatic;

R ² selected from H, alkyl, halogen, NO ₂ 、CN、CF ₃ OH, O-alkyl and NH ₂ ；

R ³ Selected from the group consisting of heterocyclic radicals containing at least one N heteroatom, NH-alkyl, NH-aryl, N- (alkyl) ₂ N- (aryl group)) ₂ And N- (alkyl) (aryl); and is

n is an integer selected in the range of 0 to 3; and is

Wherein the 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 ₂ NH-alkyl, N (alkyl) ₂ COOH, COH, CO (alkyl), CONH ₂ And CF ₃ 。

The compounds of formula I have been found to have antiproliferative activity (e.g., these compounds are believed to block tumor cell proliferation by inhibiting the activity of CDK4 and/or CDK 6) and are further able to cross the BBB. The compounds of formula I are therefore considered useful in the treatment of proliferative cell diseases and disorders of the CNS such as glioblastoma and other diseases and disorders of the CNS associated with uncontrolled cell proliferation (or, in other words, requiring control of the cell cycle). As used herein, an anti-proliferative effect within the scope of the present disclosure can be demonstrated by the ability to inhibit cell proliferation in an in vitro whole cell assay. Examples of such assays, including methods for performing, are described in more detail in the examples provided below.

Preferably, the compounds of formula I modulate (e.g. inhibit) the activity of one or more protein kinases selected from CDK4 and/or CDK 6. As described above, CDK4 and CDK6 promote cancer cell proliferation through their actions as cell cycle regulators. Thus, compounds of formula I that at least inhibit CDK4 and/or CDK6, and pharmaceutically acceptable salts, solvates, and prodrugs thereof, have utility in both in vitro and in vivo applications (e.g., in vitro cell-based assays) and as a basis for a therapeutic method of treating cancer or another proliferative disorder or condition in a subject.

The compounds of formula I may inhibit any step or stage in the cell cycle, for example, nuclear membrane formation, exit from the resting phase (G0) of the cell cycle, G1 progression, chromosome decondensation, nuclear membrane disruption, START, initiation of DNA replication, progression of DNA replication, termination of DNA replication, centrosome replication, G2 progression, activation of mitotic or meiotic functions, chromosome condensation, centrosome separation, microtubule nucleation, spindle formation and function, interaction with tubulin, separation and partitioning of chromatids, inactivation of mitotic function, formation of contractile loops and cytokinesis function. In particular, compounds of formula I may affect certain gene functions such as chromatin binding, formation of replication complexes, replication permissivity, phosphorylation or other secondary modification activity, proteolytic degradation, microtubule binding, actin binding, septin binding, microtubule tissue center nucleation activity and binding to components of cell cycle signaling pathways.

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 disorder of the Central Nervous System (CNS) in a subject, for example glioblastoma.

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 the treatment of a proliferative cell disease or disorder of the Central Nervous System (CNS) such as glioblastoma in a subject.

In this specification, a number of terms well known to those skilled in the art are used. However, for the sake of clarity, many of these terms are defined below.

As used herein, the term "treating" includes preventing as well as alleviating the established symptoms of a disease or disorder. Thus, the act of "treating" a disease or condition includes: (1) Preventing or delaying the onset of clinical symptoms of the disease or disorder developing in a subject suffering from or susceptible to the disease or disorder; (2) Inhibiting the disease or disorder (i.e., arresting, reducing, or delaying the development of the disease or disorder or its recurrence (in the case of maintenance therapy)) or at least one clinical or subclinical symptom thereof; (3) Alleviating or alleviating the disease or disorder (i.e., causing regression of the disease or disorder or at least one clinical or subclinical symptom thereof).

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

The term "aryl" as used herein refers to a substituted (mono or poly) or unsubstituted monoaromatic or polyaromatic group wherein the polyaromatic group may or may not be fused. The term thus includes groups having 6 to 10 carbon atoms (e.g., phenyl, naphthyl, etc.). It is also understood that the term "aryl" is synonymous with the term "aromatic".

As used herein, the term "aralkyl" is used as a combination of the terms alkyl and aryl as defined above.

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.

As used herein, the term "cycloaliphatic radical" refers to a cyclic aliphatic radical.

The term "halogen" refers to fluorine, chlorine, bromine and iodine.

As used herein, the term "heterocyclyl" refers to a saturated or unsaturated cyclic group that contains one or more heteroatoms (e.g., N) in the ring.

The term "derivative" as used herein includes any chemical modification of an entity. Examples of such chemical modifications are the replacement of hydrogen by halogen groups, alkyl groups, acyl groups or amino groups.

As used herein, the phrase "manufacture of a medicament" includes any stage of using one or more compounds of formula I directly as a medicament or in the manufacture of a medicament comprising one or more compounds of formula I.

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

The term "pharmaceutically acceptable salt" as used herein refers to salts that retain the desired biological activity of the compounds of formula I and includes pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compounds of formula I may be prepared from inorganic acids or from organic acids. Examples of such mineral acids are hydrochloric acid, sulfuric acid and phosphoric acid. Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic and aryl sulfonic acids. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19 th edition, mack Publishing Co, easton PA 1995.

The term "solvate" refers to any form of the compound of formula I that results from solvation with a suitable solvent. Such forms may be, for example, crystalline solvates or complexes that may form between the solvent and the dissolved compound.

The term "prodrug" means a compound that undergoes conversion to a compound of formula I (typically by metabolic means, e.g., by hydrolysis, reduction or oxidation)) within a biological system. For example, ester prodrugs of compounds of formula I containing a hydroxy group may be converted to compounds of formula I by in vivo hydrolysis. Suitable esters of compounds of formula I containing a hydroxyl group may be, for example, acetate, citrate, lactate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-P-hydroxynapthenate, gentisate (gestisate), isethionate, P-methylbenzoyl tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, P-toluenesulfonate, cyclohexyl sulfamate and quinic acid ester. As another example, ester prodrugs of compounds of formula I containing a carboxyl group may be converted to compounds of formula I by in vivo hydrolysis. Examples of ester prodrugs include those described by leinwber FJ, drug meta Rev 18. Similarly, acyl prodrugs of compounds of formula I containing an amino group can be converted to compounds of formula I by in vivo hydrolysis. Examples of Prodrugs of these and other functional groups, including amines, are provided in produgs: gallenges and rewards, valentino J Stella (eds.), springer, 2007.

Where the compound of formula I is a solid, one skilled in the art will appreciate that the compound (or a pharmaceutically acceptable salt, solvate, or prodrug thereof) may exist in different crystalline or polymorphic forms, all of which are encompassed within the scope of the present disclosure.

The term "therapeutically effective amount" or "effective amount" is an amount sufficient to achieve a beneficial or desired clinical result. A therapeutically effective amount may be administered in one or more administrations. Generally, a therapeutically effective amount is sufficient to treat a disease or condition or otherwise alleviate, ameliorate, stabilize, reverse, slow or delay the progression of a disease or condition such as 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 between about 0.1 and about 250mg/kg body weight/day, more preferably between about 0.1 and about 100mg/kg body weight/day, and still more preferably between about 0.1 and about 25mg/kg body weight/day. Nonetheless, those skilled in the art will appreciate that a therapeutically effective amount may vary and will depend on a variety of factors, including the activity of the particular compound (or salt, solvate, or prodrug thereof), the metabolic stability and length of action of the particular compound (or salt, solvate, or prodrug thereof), the age, body weight, sex, health, route and time of administration, rate of excretion of the particular compound (or salt, solvate, or prodrug thereof), and, for example, the severity of the cancer or other proliferative cell disease or disorder being treated.

In some embodiments, R ¹ Is H, alkyl (e.g. C) _1-6 Alkyl radicals such as C _1-3 Alkyl radicals, such as methyl, ethyl and C (CH) ₃ ) ₂ Or C is _3-6 Cycloalkyl such as cyclopentyl) or heterocyclyl (e.g., saturated or unsaturated 5 or 6 membered cyclic groups containing one or two N, O or S heteroatoms). Most preferably, R ¹ Is H, C _1-3 Alkyl (e.g. methyl) or C _3-6 Cycloalkyl groups such as cyclopentyl.

In some embodiments, R ² Is H, alkyl (e.g. C) _1-6 Alkyl or preferably C _1-3 Alkyl radicals such as methylOr ethyl), CN or halogen (preferably F).

In some embodiments, R ³ Is a heterocyclyl group (preferably a saturated or unsaturated 5-or 6-membered cyclic group comprising one, but more preferably two N heteroatoms), which is optionally substituted with one or more groups selected from: alkyl (e.g. C) _1-6 Alkyl or preferably C _1-3 Alkyl radicals such as methyl, ethyl and CH (CH) ₃ ) ₂ )、NH ₂ NH-alkyl such as NH-methyl and NH-ethyl, N (alkyl) ₂ Such as N (C) _1-3 Alkyl radical) ₂ (e.g., N (CH) ₃ ) ₂ 、N(CH ₂ CH ₃ ) ₂ And N (CH) ₃ )(CH ₂ CH ₃ ) COH and CO (C) _1-3 Alkyl) (e.g. COCH) ₃ )。

In some embodiments, R ³ Selected from the following:

in some embodiments, n is 0, 1, or 2. When n is 1 or 2, the compound thus has an alkyl bridge to the carbon atom at position 4 of the pyridine ring (e.g. -CH) ₂ -or-CH ₂ CH ₂ -a bridge).

In a 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) 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-morpholinylpyridin-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.

In some preferred embodiments, the compounds of formula I exhibit antiproliferative activity in human cell lines, as measured by standard cytotoxicity assays. Preferably, the compounds exhibit an IC of less than 5 μ M, even more preferably less than 1 μ M ₅₀ Values as measured by standard cell viability assays. Still more preferably, the compound exhibits an IC of less than 0.5. Mu.M ₅₀ The value is obtained.

In some preferred embodiments, the compounds of formula I inhibit one or more protein kinases as measured by any standard assay well known to those skilled in the art. Preferably, the compound exhibits an IC of less than 1 μ Μ or less than 0.5 μ Μ (as measured by the kinase assay described in example 2 below), still more preferably less than 0.1 μ Μ ₅₀ The value is obtained.

Specific examples of compounds of formula I for use in the method of the first aspect are shown in table 1 below.

Table 1 chemical structures of selected compounds of the present disclosure

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 disorder. For example, the compounds may be used in combination with other anti-cancer agents to inhibit more than one cancer signaling pathway simultaneously, thereby rendering cancer cells more susceptible to anti-cancer therapy (e.g., treatment with other anti-cancer agents, chemotherapy, radiation therapy, or combinations thereof). Thus, the compounds of formula I may be used in combination with one or more of the following classes of anti-cancer agents, particularly when such anti-cancer agents are capable of crossing the BBB:

● Other antiproliferative/antineoplastic drugs and combinations thereof for medical oncology, such as alkylating agents (e.g., carmustine (carmustine), procarbazine, lomustine (lomustine), vincristine (vincristine), and TMZ); antimetabolites (e.g. gemcitabine and antifolates such as fluoropyrimidines, e.g. 5-fluorouracil and tegafur (tegafur), raltitrexed (raltitrexed), methotrexate (methotrexate), cytarabine (cytosine arabine), fludarabine (fludarabine) and hydroxyurea); antitumor antibiotics (e.g., anthracyclines such as doxorubicin (adriamycin), bleomycin (bleomycin), doxorubicin (doxorubicin), daunomycin (daunomycin), epirubicin (epirubicin), idarubicin (idarubicin), mitomycin-C (mitomycin-C), dactinomycin (dactinomycin), and mithramycin (mithramycin)); antimitotic agents (e.g., vinca alkaloid drugs such as vincristine, vinblastine (vinblastine), vindesine (vindesine), and vinorelbine (vinorelbine) and taxanes including paclitaxel (taxol) and taxotere (taxotere) and Polo-like kinase inhibitors); and topoisomerase inhibitors (e.g. podophyllotoxins such as etoposide and teniposide, amsacrine, topotecan and camptothecin);

● Cell growth inhibitors such as antiestrogens (e.g. tamoxifen (tamoxifen), fulvestrant (fulvestrant), toremifene (toremifene), raloxifene (raloxifene), droloxifene (droloxifene) and idoxifene (idoxyfene)), antiandrogens (e.g. bicalutamide (bicalutamide), flutamide (flutamide), nilutamide (nilutamide) and cyproterone acetate), LHRH antagonists or LHRH agonists (e.g. goserelin (goserelin), leuprolide (leuprorelin) and buserelin (buserelin)), agonists (e.g. megestrol acetate (megestrol acetate)), aromatase inhibitors (e.g. anastrozole), letrozole (letrozole), chlorazol (chlorazol) and vefluxofenamide (drox)), and inhibitors such as amantadine (isoxaflufenamide) and amantadine (e) such as amantadine reductase inhibitors (amantadine), and amantadine (e.g. 5-reductase);

● Anti-invasive agents (e.g., c-Src kinase family inhibitors such as 4- (6-chloro-2,3-methylenedioxyanilino) -7- [2- (4-methylpiperazin-1-yl) ethoxy ] -5-tetrahydropyran-4-yloxyquinazoline (AZD 0530; 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)), as well as metalloproteinase inhibitors including marimastat (marimastat), inhibitors of urokinase plasminogen activator receptor function, or antibodies to heparanase;

● Inhibitors of growth factor function (e.g., growth factor antibodies and growth factor receptor antibodies, such as the anti-erbB 2 antibody trastuzumab (Herceptin) ^TM ) anti-EGFR antibody panitumumab (panitumumab), anti-erbB 1 antibody cetuximab (cetuximab) (Erbitux, C225) and Critical reviews in environmental/Haematology, 20 by Stern et al05, volume 54, pages 11-29). Such inhibitors also 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, ZD 1839), N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib, OSI-774) and 6-propylacylamido-N- (3-chloro-4-fluorophenyl) -7- (3-morpholinopropoxy) -quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib (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 (imatinib) and/or nilotinib (nilotinib) (AMN 107); inhibitors of serine/threonine kinases (e.g., ras/Raf signaling inhibitors such as farnesyltransferase inhibitors, including sorafenib (BAY 43-9006), tipifarnib (R115777), and lonafarnib (SCH 66336)); an inhibitor of cell signaling through MEK and/or AKT kinase; c-kit inhibitors; an abl kinase inhibitor; a PI3 kinase inhibitor; a Plt3 kinase inhibitor; 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 AX 39459) and cyclin dependent kinase inhibitors such as CDK4 and/or CDK6 inhibitors (e.g., palbociclib, ribociclib, and abbeli);

● Anti-angiogenic agents, such as those that inhibit the action of vascular endothelial growth factor (e.g., anti-vascular endothelial growth factor antibody bevacizumab (Avastin)) ^TM ) And VEGF receptor tyrosine kinase inhibitors such as vandetanib (Zd 6474), vatalanib (vatalanib) (PTK 787), sunitinib (sunitinib) (SU 11248), axitinib (axitinib) (AG-013736), pazopanib (pazopanib) (GW 786034) and 4- (4-fluoro-2-methylindol-5-yloxy) -6-methoxy-7- (3-pyrrolidin-1-ylpropoxy) quinazoline (AZD 2171; implementation in International patent publication No. WO 00/47212Example 240); compounds such as those disclosed in international patent publication nos. WO97/22596, WO 97/30035, WO 97/32856 and WO 98/13354, and compounds that act by other mechanisms (e.g., linoamine (linomide), inhibitors of integrin α v β 3 function and angiostatin);

● Vascular disrupting agents such as Combretastatin A4 and the compounds disclosed in International patent publication Nos. WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

● Endothelin receptor antagonists such as zibotentan (zibotentan) (ZD 4054) or atrasentan (atrasentan);

● Antisense therapies, such as those directed against the above-listed targets, such as ISIS 2503, anti-ras antisense;

● Gene therapy methods, including, for example, methods of replacing abnormal genes such as abnormal p53 or abnormal BRCA1 or BRCA 2; GDEPT (gene-directed enzyme prodrug therapy) methods, such as those using cytosine deaminase, thymidine kinase, or bacterial nitroreductase enzymes, and methods of increasing patient tolerance to chemotherapy or radiation therapy, such as multi-drug resistance gene therapy; and

● Immunotherapeutic approaches, including, for example, ex vivo and in vivo approaches to increase the immunogenicity of patient tumor cells (such as transfection with cytokines such as interleukin 2, interleukin 4, or granulocyte-macrophage colony stimulating factor), approaches to reduce T cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines, and approaches using anti-idiotypic antibodies.

In some embodiments, the compounds of formula I (and pharmaceutically acceptable salts, solvates, and prodrugs thereof) may be administered in combination with TMZ (and/or used with irradiation). Using glioblastoma xenografts, it has been demonstrated that the use of abelmoschus or palbociclib (both CDK4/6 inhibitors) with TMZ or irradiation inhibits DNA double strand break repair and increases apoptosis (Raub TJ et al Drug meta-Dispos 43 1360-1371,2015; and Michaud K et al Cancer Res 8, 70, 2010. When combined with the mTOR inhibitor everolimus, palbociclib synergizes with everolimus against glioblastoma xenografts (Olmez I et al clinical Cancer Res DOI:10.1158/1078-0432.CCR-17-0803,2018). Pabociclib has also been shown to increase tumor cell antigens and anti-tumor T cell responses, suggesting that CDK4 and/or CDK6 inhibition has an immune effect (Deng J et al Cancer Discovery DOI:10.1158/2159-8290.CD-17-0915,2018). In other embodiments, the compounds of formula I (and pharmaceutically acceptable salts, solvates, and prodrugs thereof) can be administered in combination with a kinase inhibitor selected from an inhibitor of PI3K, mTOR and/or MEK.

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

The compounds of formula I are typically used to treat cancer or another proliferative cell disease or disorder in a human subject. However, the subject may also be selected from, for example, livestock (e.g., cattle, horses, pigs, sheep, and goats), companion animals (e.g., dogs and cats), and exotic animals (e.g., non-human primates, tigers, elephants, and the like).

Cancers and other proliferative cell diseases and disorders of the CNS 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 astrocytoma, ependymoma, oligodendroglioma, or metastatic brain tumors), and benign neoplasms of the CNS, such as schwannoma, pituitary adenoma, meningioma, and craniopharyngioma.

In some preferred embodiments, the compounds of formula I are used to treat cancers and other proliferative cellular diseases and disorders of the CNS selected from those characterized by overexpression of CDK4 and/or cyclin D, including, for example, GBM. CDK4 and/or cyclin D overexpression may be determined by assessing the amount of CDK4 and/or cyclin D encoding mRNA in a suitable sample, for example using any technique well known to those skilled in the art (e.g., quantitative amplification techniques such as qPCR).

In some preferred embodiments, the compounds of formula I are used to treat cancers of the CNS and other proliferative cell diseases and disorders selected from those characterized by overexpression of CDK6 and/or cyclin D, including, for example, medulloblastomas (reviewed in Tardesse et al, targeting CDK 6in Cancer: state of the Art and New instruments in press). CDK6 and/or cyclin D overexpression may be determined by assessing the amount of mRNA encoding CDK6 and/or cyclin D in a suitable sample, for example using any technique well known to those skilled in the art (e.g. quantitative amplification techniques such as qPCR).

The compounds of formula I may be formulated into pharmaceutical compositions with 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 suitable Excipients for use in the various forms of Pharmaceutical compositions described herein can be found in Handbook of Pharmaceutical Excipients, 2 nd edition, (1994), edited by a Wade and PJ Weller. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of carrier, diluent and/or excipient may be made with respect to the intended route of administration and standard pharmaceutical practice.

Pharmaceutical compositions comprising a compound of formula I may further comprise 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, free-flowing lactose, beta-lactose), corn sweeteners, natural and synthetic gums (such as gum arabic, tragacanth gum, or sodium alginate), carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical compositions. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

Pharmaceutical compositions comprising a compound of formula I may be suitable for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration. For oral administration, compressed tablets, pills, tablets, gels, drops and capsules may be used in particular. For other forms of administration, the pharmaceutical composition may comprise a solution or emulsion, which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally, or intramuscularly, and which is prepared from a sterile or sterilizable solution. Pharmaceutical compositions comprising a compound of formula I may also be in the form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders. The pharmaceutical compositions may be formulated in unit dosage form (i.e., in the form of discrete portions of a unit dose containing a unit dose or multiple unit doses or subunits).

The compounds of formula I may be provided as pharmaceutically acceptable salts (including, for example, suitable acid addition or base salts thereof). An overview of suitable pharmaceutically acceptable salts can be found in Berge et al, J Pharm Sci 66 (1977). Salts may be formed, for example, with the following acids: strong mineral acids such as mineral acids (e.g., sulfuric acid, phosphoric acid, or hydrohalic acids); strong organic carboxylic acids such as unsubstituted or (e.g. halogen-substituted) alkane carboxylic acids having 1 to 4 carbon atoms, such as acetic acid; saturated or unsaturated dicarboxylic acids (e.g., oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid, or tetraphthalic 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. unsubstituted or substituted, e.g. by halogen (C) ₁ -C ₄ ) -alkyl or aryl sulphonic acids) such as methane sulphonic acid or p-toluene sulphonic acid).

The compounds of formula I may be provided in their various crystalline, polymorphic and anhydrous/aqueous forms. In this regard, it is well known to those skilled in the art that chemical compounds can be isolated in any such form by slightly varying the method of purification and/or isolation from the solvent used for the synthetic preparation of such compounds.

Methods for synthesizing compounds of formula I have been previously described (see, e.g., WO 2017/020065). In some embodiments, compounds of formula I may be synthesized by employing the following general synthetic scheme:

scheme 1

Wherein the general reaction conditions are: (a) DMF-DMA or Braidenesk's Reagent (Bredereck's Reagent), reflux; (b) Select Fluor, meOH; (c) Et (ethyl acetate) ₃ N、HgCl ₂ DCM; (d) TFA/DCM (1:1), reflux; (e) A, B, naOH, 2-methoxyethanol, microwave; and (f) Pd ₂ dba ₃ Xanthphose, t-Buona, dioxane, microwave.

With respect to the description of the synthetic method of scheme 1 above, those skilled in the art will appreciate that all suggested reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, experimental duration and procedure (workup procedure), can be readily selected. Furthermore, one skilled in the art will appreciate that the functionalities present on different parts of the molecule must be compatible with the reagents and reaction conditions used.

The necessary starting materials are obtainable by standard procedures of organic chemistry. The preparation of such starting materials is described in connection with the following representative process variations and within the examples below. Alternatively, the necessary starting materials may be obtained by procedures analogous to those described as being within the ordinary skill of the person skilled in the art. Furthermore, it will be appreciated that during the synthesis of compounds, or during the synthesis of certain starting materials, certain substituents may need to be protected to prevent their unwanted reactions. One skilled in the art will readily recognize when such protection is required and how to put such protecting groups in place and then remove them. Examples of protecting Groups are described, for example, in Protective Groups in Organic Synthesis by Therodora Green (publisher: john Wiley & Sons). The protecting group may be removed by any convenient method known to those skilled in the art to be suitable for removal of the protecting group in question, such method being selected so as to effect removal of the protecting group with minimal interference with groups elsewhere in the molecule. Thus, if a reactant includes a group such as an amino, carboxyl, or hydroxyl group, it may be desirable to protect that group in some of the reactions mentioned herein.

Furthermore, the skilled person will be able to select suitable reaction conditions for the coupling reaction of the compound of formula a or formula B shown in scheme 1. However, the reaction will generally be carried out under anhydrous conditions and in the presence of an inert atmosphere such as argon or nitrogen. The reaction may also be carried out at elevated temperature, such as for example in the range of 80 to 180 ℃ for a suitable period of time, for example 20 minutes to 48 hours. Suitably, the reaction is carried out under microwave heating, for example at 80 to 180 ℃ for 20 minutes to 1.5 hours.

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

The methods and uses of the present disclosure are further described below with reference to the following non-limiting examples and figures.

Examples

EXAMPLE 1 Synthesis of representative Compounds

Summary of the invention

Recording at 300K on a Bruker AVANCE III 500 spectrometer ¹ H and ¹³ c NMR spectrum (C: (C NMR) ¹ H at 500 MHz). ¹ H NMR spectroscopy with reference to residual non-deuterated solvents (or tetramethylsilane) ¹ The H signal. At AB SCIEXHigh resolution mass spectra were recorded on a 5600 mass spectrometer and all samples were ionized using ESI. The purity of the compound was determined by analytical HPLC and was greater than 95%. Analytical HPLC used method A (gradient: 5 to 95% MeOH with 0.1% FA over 7min, then 95% with 0.1% FAMeOH over 13min, flow rate 1 mL/min), method B (gradient: 5 to 95% MeCN with 0.1% FA within 7min, then 95% MeCN with 0.1% FA within 13min at a flow rate of 1 mL/min) on a Shimadzu research UFLC (ultra fast liquid chromatography) system with the following apparatus: CBM-20A communication bus module, DGU-20A _5R Degassing unit, LC-20AD liquid chromatography pump, SIL-20A _HT Autosampler, SPD-M20A photodiode array detector, CTO-20A column oven and Phenomenex Kinetex 5u C18 100A 250mm x 4.60mm column.

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 (524mg, 2.00mmol) in 2-methoxyethanol (3 mL) were added ((E) -3- (dimethylamino) -2-fluoro-1- (4-methyl-2- (methylamino) thiazol-5-yl) prop-2-en-1-one (243mg, 1.00mmol) and NaOH (80.0mg, 2.00mmol), the reaction mixture was heated at 180 ℃ under microwave irradiation for 1h, cooled to room temperature, then concentrated under reduced pressure, the residue was purified by chromatography (silica gel, DCM was increased linearly to DCM: meOH =90, with the addition of 0.5ml 32% ammonia) to give 1 as a brown solid (76mg, 17.2%). ¹ H NMR(DMSO-d ₆ )δ1.50(q,2H,J11.0),1.84(d,3H,J11.0),2.21(s,7H),2.47(s,3H,thiazole-CH ₃ ),2.64(t,2H,J11.0),2.86(t,3H,J3.5),3.63(d,1H,J11.0),7.39(app d,1H,J7.0),7.92(d,1H,J9.0),7.98(s,1H),8.10(1H,J4.0),8.41(s,1H),9.43(s,1H)。HRMS(ESI):m/z443.2136[M+H] ⁺ 。

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 (200mg, 0.68mmol) in dioxane (3 mL) was added 1- ((6-bromopyridin-3-yl) methyl) -4-ethylpiperazine (233.mg, 0.82mmol), pd2dba3 (31mg, 0.034mmol), xantphos (41mg, 0.07mmol) and t-BuONa (98mg, 1.02mmol) and heated at 150 ℃ for 1h under microwave irradiation. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. Will remainThe material was purified by chromatography (silica gel, DCM increased linearly to DCM: meOH = 93) to give 2 as an orange solid (100mg, 29%). ¹ H NMR(DMSO-d ₆ )δ0.99(t,3H,J7.0),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,J2.5),3.42(s,2H),3.95-3.98(m,1H),7.64(dd,1H,J8.5&2.0),8.10(d,1H,J8.5),8.16(d,1H,J2.0),8.27(d,1H,J7.0),8.46(d,1H,J3.5),9.77(s,1H)。HRMS(ESI):m/z 497.2601[M+H] ⁺ 。

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 (441mg, 2.00mmol) and (E) -1- (2- (cyclopentylamino) -4-methylthiazol-5-yl) -3- (dimethylamino) prop-2-en-1-one (279mg, 1.00mmol) in 2-methoxyethanol (3 mL) was added NaOH (80.0mg, 2.00mmol). The reaction mixture was heated at 180 ℃ for 1h under microwave irradiation, cooled to room temperature and concentrated under reduced pressure. The residue was purified by chromatography (silica gel, DCM increased linearly to DCM: meOH = 92) and recrystallized with DCM and MeOH to give 3 as a dark yellow solid (70.0 mg, 16%). m.p.210-213 ℃. ¹ H NMR(DMSO-d ₆ )1.49-1.68(m,7H),1.89-1.94(m,2H),2.46(s,3H),2.85(t,4H,J4.5),3.02(t,4H,J5.0),3.98(m,1H),6.90(d,1H,J5.5),7.36(dd,1H,J9.0&3.0),7.98(d,1H,J3.0),8.07(d,1H,J9.0),8.18(d,1H,J7.0),8.33(d,1H,J5.5),9.33(s,1H)。HRMS(ESI):m/z 437.2222[M+H] ⁺ 。

N-cyclopentyl-5- (2- ((5- (4-ethylpiperazin-1-yl) pyridin-2-yl) amino) pyrimidin-4-yl) -4-methylthia- -ne Oxazol-2-amine (4):to a mixture of crude 1- (5- (4-ethylpiperazin-1-yl) pyridin-2-yl) guanidine trifluoroacetate (496mg, 2.00mmol) and (E) -1- (2- (cyclopentylamino) -4-methylthiazol-5-yl) -3- (dimethylamino) prop-2-en-1-one (279mg, 1.00mmol) in 2-methoxyethanol (3 mL) was added NaOH (80.0 mg, 2.00mmol). The reaction mixture was heated at 180 ℃ for 1h under microwave irradiation, cooled to room temperature and concentrated under reduced pressure. The residue was purified by chromatography (silica gel, DCM increased linearly to DCM: meOH = 96) and recrystallized from MeOH to give a yellow solidFormula 4 (117mg, 25%). ¹ H NMR(CDCl ₃ )δ1.14(t,3H,J7.0),1.56-1.76(m,6H),2.06-2.12(m,2H),2.49(q,2H,J7.5),2.54(s,3H),2.64(s,3H),3.19(t,4H,J4.5),3.14(t,4H,J5.0),3.86(app s,1H),5.77(s,1H),6.84(d,1H,J5.0),7.34(dd,1H,J9.0&3.0),7.94(d,1H,J3.0),7.94(s,1H),8.01(d,1H,J3.0),8.26(d,1H,J9.0),8.33(d,1H,J5.5)。HRMS(ESI):m/z 465.2541[M+H] ⁺ 。

2- ((5- (4-acetylpiperazin-1-yl) pyridin-2-yl) amino) -4- (4-methyl-2- (methylamino) thiazole- 5-yl) pyrimidine-5-carbonitrile (5):to a solution of crude 1- (5- (4-acetylpiperazin-1-yl) pyridin-2-yl) guanidine trifluoroacetate (315mg, 1.20mmol) in 2-methoxyethanol (4 mL) were added tert-butyl (E) - (5- (2-cyano-3- (dimethylamino) acryloyl) -4-methylthiazol-2-yl) (methyl) carbamate (350mg, 1.00mmol) and NaOH (82.0 mg, 2.40mmol). The reaction mixture was heated at 180 ℃ for 90min under microwave irradiation, cooled to room temperature and then concentrated under reduced pressure. The residue was purified by chromatography (silica gel, DCM increased linearly to DCM: meOH =90, 32% aqueous ammonia was added continuously, up to 3%). The solid was washed with DCM and MeOH, then filtered to give 5 as a pale yellow solid (157mg, 35%). ¹ H NMR(DMSO-d ₆ )δ2.04(s,3H),2.40(s,3H),2.87(s,3H),3.10(t,2H,J5.0),3.16(t,2H,J5.0),3.58(t,4H,J5.0),7.46(dd,1H,J9.5&3.0),7.90(d,1H,J9.0),8.06(d,1H,J3.0),8.26(q,1H,J3.0),8.75(s,1H),10.33(s,1H)。HRMS(ESI):m/z 450.1844[M+H]+。

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 (275mg, 1.00mmol) in dioxane (3 mL) was added 1- ((6-bromopyridin-3-yl) methyl) -4-ethylpiperazine (341mg, 1.2mmol), pd ₂ dba ₃ (45.8mg, 0.05mmol), xantphos (58mg, 0.1mmol) and t-BuONa (144mg, 1.5mmol) and heated at 150 ℃ under microwave irradiation for 1h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was chromatographed (silica, DCM was increased linearly to DCM: meOH: NH) ₄ OH =9And recrystallized from DCM and MeOH to give 6 as a white solid (200mg, 42%). ¹ H NMR(CDCl ₃ )δ1.09(t,3H,J7.0),1.58-1.76(m,6H),2.08-2.14(m,2H),2.43(q,2H,J7.0,CH ₂ CH ₃ ),2.55(sbr,11H),3.48(s,2H),3.86-3.92(m,1H),5.42(d,2H,J7.0),6.90(d,1H,J5.5),7.68(dd,1H,J9.0&2.5),7.89(s,1H),8.19(d,1H,J2.0),8.35-8.38(m,2H)。HRMS(ESI):m/z479.2703[M+H] ⁺ 。

5- (2- ((5- (4-aminopiperidin-1-yl) pyridin-2-yl) amino) -5-fluoropyrimidin-4-yl) -N, 4-dimethylthia-ne Oxazol-2-amine (7):compound 7 (40.0mg, 10%) was obtained as an orange solid by reacting 1- (5- (4-aminopiperidin-1-yl) pyridin-2-yl) guanidine trifluoroacetate (702mg, 3.00mmol) with (469mg, 2.00mmol) and ((E) -3- (dimethylamino) -2-fluoro-1- (4-methyl-2- (methylamino) thiazol-5-yl) prop-2-en-1-one (243mg, 1.00mmol). ¹ H NMR(DMSO-d6)δ1.75-1.80(m,2H),2.45(d,3H,J2.0),2.62(t,2H,J6.0),2.85(t,2H,J5.5),3.45(t,2H,J5.0),3.53(t,2H,J6.0),7.13(dd,1H,J9.0&3.0),7.78(s,1H),7.79(d,1H,J4.5),8.08(q,1H,J4.5),8.37(d,1H,J3.5),9.21(s,1H)。HRMS(ESI):m/z415.1821[M+H]+。

5- (2- ((5- (4-aminopiperidin-1-yl) pyridin-2-yl) amino) pyrimidin-4-yl) -N-cyclopentyl-4-methylthioi-ne Oxazol-2-amine (8):to a mixture of 1- (5- (4-aminopiperidin-1-yl) pyridin-2-yl) guanidine trifluoroacetate (702mg, 3.00mmol) and (E) -1- (2- (cyclopentylamino) -4-methylthiazol-5-yl) -3- (dimethylamino) prop-2-en-1-one (558mg, 2.00mmol) in 2-methoxyethanol (5 mL) was added NaOH (160.0mg, 4.00mmol). The reaction mixture was heated at 180 ℃ for 2h under microwave irradiation, cooled to room temperature and concentrated under reduced pressure. The residue was purified by chromatography to give 8 as a yellow solid (90mg, 10%). ¹ H NMR(CDCl ₃ )δ1.50-1.77(m,10H),1.95(d,2H,J10.5),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,J5.0,1H),6.84(d,1H,J5.5),7.34(dd,1H,J9.0&3.0),7.75(s,1H),8.00(d,1H,J3.0),8.25(d,1H,J9.0),8.32(d,1H,J5.5)。HRMS(ESI):m/z451.2415[M+H] ⁺ 。

N-cyclopentyl-5- (2- ((5-morpholinylpyridin-2-yl) amino) pyrimidin-4-yl) -4- (trifluoromethyl) thiazol-2- Amine (9):to a mixture of 1- (5-morpholinylpyridin-2-yl) guanidine trifluoroacetate (442mg, 2.00mmol) and (E) -1- (2- (cyclopentylamino) -4-methylthiazol-5-yl) -3- (dimethylamino) -2-fluoroprop-2-en-1-one (297mg, 1.00mmol) in 2-methoxyethanol (3 mL) was added NaOH (80.0mg, 2.00mmol). The reaction mixture was heated at 180 ℃ for 1h under microwave irradiation, cooled to room temperature and concentrated under reduced pressure. The residue was purified by chromatography to give 9 as a brown solid (120mg, 26%). ¹ H NMR(DMSO-d ₆ )δ1.50-1.57(m,4H),1.66–1.69(m,2H),1.90–1.95(m,2H),2.47(d,1H,J2.5),3.09(t,4H,J5.0),3.75(t,4H,J5.0),3.96(m,1H),7.42(dd,1H,J9.0&3.0),7.96(d,1H,,J9.0),7.98(d,1H,J3.0),8.24(d,1H,J7.0),8.41(d,1H,J7.0),9.52(s,1H)。HRMS(ESI):m/z456.1976[M+H] ⁺ 。

Example 2 biological Activity

Kinase assay

The Eurofins Pharma Discovery or Reaction Biology Corporation Kinase Profile service was used to measure the inhibition of CDKs and other kinases by radiometric assay (RIA). The inhibition of CDK 4/cyclin D1, CDK 6/cyclin D3 and CDK9/T1 was also determined internally using the ADP Glo kinase assay (Promega Corporation, madison WI, united States of America). Briefly, the kinase reactions of CDK 4/cyclin D1 and CDK 6/cyclin D3 used kinase reaction buffer (40 nM Tris base pH 7.5, 20mM MgCl) ₂ 0.4mM DTT), 0.1mg/ml BSA and RB-CTF substrate (retinoblastoma protein 1C-terminal portion). For CDK 9/cyclin T1, the kinase reaction was performed using standard assay buffer and kinase dilution buffer and RBER-irstride substrate. Serial dilutions of 1:3 were prepared for 10 concentrations (10 μ M to 0.5 nM) of test compound. The kinase reaction was started by addition of ATP, incubated at 37 ℃ for 40min, and then stopped by addition of 10. Mu.L of ADP Glo reagent. After incubation for 40min at Room Temperature (RT) protected from light, 20 μ L of kinase detection reagent per well was added and incubated for 40min. Using an EnVision Multi-Label microplate reader (PerkinElmer, buckinghamshire, united)Kingdom) measures luminescence. Positive and negative controls were performed in the presence and absence of CDK kinase, respectively. Calculation of semi-Inhibitory Concentration (IC) Using a 4-parameter logistic non-linear regression model of Graphpad prism (version 6.0) ₅₀ ) The value is obtained. Apparent inhibition constant (K) _i ) Values according to K of the corresponding kinase _m (ATP) and IC ₅₀ A value is calculated. The results for representative compounds are shown in table 2.

TABLE 2 inhibition of cyclin-dependent kinases

Cell culture

All three GBM cell lines used in this example (i.e., U87, U251 and T98G) were at 37 ℃ and 5% CO ₂ Lower at 75cm ² Cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum in a culture flask. All cell lines were confirmed to be free of mycoplasma prior to any experimental setup.

MTT proliferation assay and combinatorial studies

The standard MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) and Resazurin assays were performed on representative compounds from example 1 on solid tumor cell lines (including GBM cell lines) and leukemia cells, respectively, as previously reported (Wang S et al, J Med Chem 47 1662-1675,2004; and Diab S. Et al, chemMed Chem 9 962-972,2014. Concentration of compound required to inhibit 50% of cell Growth (GI) ₅₀ ) The samples were analyzed using GraphPad Prism 8 (GraphPad Software, inc; san Diego, CA, united States of America). The results are shown in table 3.

TABLE 3 antiproliferative activity (72h ₅₀ μM)

For the combination study, cells were treated with different concentrations of 10 μ L of either everolimus (inhibitor of mTOR), abacteriol (inhibitor of PI 3K), or semetinib (inhibitor of MEK) and 10 μ L of one of the CDK4/6 inhibitors of the present disclosure for 72h. At the same time, 50. Mu.L of MTT was added to each well and incubated for 3.5h, followed byThe absorbance was measured at 550nm with a multi-label microplate reader (Buckinghamshire, UK). The results were determined by the Chou-Talalay method using CompuSyn v 1.0 (Combosyn, NJ, united States of America). Combination Index (CI) values < 1, 1 and > 1 are considered synergistic, additive and antagonistic drug interactions, respectively (Chou TC et al, trends Pharmacol Sci 4. The results are provided in figure 1. The combined treatment provided a higher level of inhibitory activity on proliferation relative to the single dose treatment. There is evidence that synergistic levels of inhibition are indicated by CI < 1.

Apoptosis assay

The ability of compound 1 to induce apoptosis in GBM cells was studied using annexin V-FITC apoptosis assay kit I (BD Pharmingen Inc.; san Diego, CA, united States of America) according to the manufacturer's protocol. The cell pellet was washed twice with 1mL cold PBS and centrifuged at 300x g for 5min. After removal of the supernatant, the cell pellet was stained with 100 μ L of 1 × annexin V binding buffer, 3 μ L of annexin V-FITC and 3 μ L of propidium iodide staining solution and incubated at room temperature for 15min in the dark. The percentage of apoptotic cells was measured by CytoFLEX flow cytometry. GBM U87 cells were treated with 5 μ M palbociclib ("Palb") or compound 1 (with and without 5 μ M Temozolomide (TMZ)), and then the cells were assayed after 48 hours. The results in figure 2 show that compound 1 (with or without TMZ) is able to induce apoptosis in GBM cells at higher levels than the control anti-cancer agent palbociclib (with or without TMZ).

Western blot analysis

Prior to compound treatment, cells were plated on sterile petri dishes with 10mL of fresh medium at 3 × 10 ⁵ And 5% CO at 37 deg.C ₂ Incubate overnight. After 24h, cells were harvested with PBS, centrifuged at 300X g for 5min, and lysed with 100 μ L of lysis buffer (25mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 300mM sodium chloride, 1.5mM magnesium chloride, 0.5% sodium deoxycholate, 20mM β -glycerophosphate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.2mM EDTA, 0.5mM dithiothreitol, 1mM sodium orthovanadate, and 25 Xprotease inhibitor mix). Protein lysates were collected after centrifugation at maximum speed for 10min at 4 ℃. Protein concentration of cell lysates was determined by performing a BSA standard curve using Bio-Rad DC protein assay kit II (Bio-Rad, sydney, NSW, australia) according to the manufacturer's protocol. 30 μ g of protein was measured, mixed with 3 XLoading buffer, and denatured at 95 ℃ for 5min 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 120V for 1h. The resolved proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk in Tris buffered saline and Tween 20 (TBST) for 1h at room temperature. Antibodies were diluted in TBST with 5% skim milk. Membranes were incubated overnight with primary antibody at-20 ℃, washed four times with TBST, treated one hour with secondary antibody at room temperature, and washed four more times with TBST. Chemiluminescence on the membrane was then visualized using Amersham ECL prime/select Western blot detection reagent (GE Healthcare, sydney, NSW, australia). Band intensities were determined using a ChemiDocTM multiplexed imaging system (Bio-Rad). Antibodies used were purchased from Cell Signalling Technology (Danvers, MA, united States of America): 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-linked anti-mouse IgG #7076, and HRP-linked anti-rabbit IgG #7074. FIG. 3A shows that Compound 1 (and Pabociclib, positive control) is reducedLevels of phosphorylated Rb proteins (pRb Ser780, pRbSer795, pRb 807/811) confirm their cellular CDK4/6 inhibition.

In vitro tumorigenicity assay

In twelve well plates, seeded at a density of 200 to 400 cells/well and incubated overnight prior to 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 staining solution (0.05% crystal violet, 1% formaldehyde, 1% PBS, 1% ethanol); the results are shown in fig. 3B. Compound 1 inhibited growth and proliferation of all three GBM cell lines.

Brain uptake assay

The propensity of the compounds from example 1 to be taken up by the brain (thereby indicating their ability to cross the BBB) was investigated using the method as described (T J Raub et al, supra). Briefly, compound 1 or compound 2 was administered intravenously to Balb/C mice. At 5min and 1h post-dose, heart blood was collected and cerebral hemispheres were excised. For oral dosing experiments, mice were dosed with drug and blood plus cerebral hemispheres were collected at 0.5, 1,2, 4, 6 and 24h post-dose. Measuring the drug concentration in the homogenized brain and plasma by LC-MS/MS; those in the brain homogenate were converted to brain concentrations (expressed per g of brain tissue) and corrected to an average measured plasma volume of 16 μ L/g brain tissue. Use ofThe ultrafiltration device measures the unbound fraction in plasma and brain. Adding K of each drug _p,u Values were compared to a set of guidelines for neurotherapeutics (Kulkarni AD et al Expert Opin Drug Deliv 13, 85-92,2016. Control experiments were performed using equivalent doses of palbociclib and/or abbeli. The results shown in FIG. 4 are expressed as K _p,u : the brain to plasma ratio of unbound drug concentration is generally divided into the following sections: k is _p,u Less than 0.1, unable to cross the BBB; k is _p,u 0.3-0.5, sufficient to enter the BBB; k _p,u > 1.0, freely cross the BBB (Kulkarni et al, supra). Compounds 1,2 and 6 showed significantly higher penetration than the control anticancer agentThe propensity of the BBB.

In vivo antitumor efficacy

Compound 1 and compound 2 were also investigated for in vivo anti-tumor activity in a subcutaneous xenograft model. Briefly, U87 GBM cells were harvested and resuspended in 1:1 mixture of serum-free medium and matrigel, and 5x10 cells were resuspended ⁶ Individual cells were injected subcutaneously into the posterior flank of 5-6 week old CD1 nu/nu female mice. When the average tumor volume reaches 150-200mm ³ At times, animals were randomly assigned to treatment groups (n =10 per group) and orally treated with vehicle (water containing 0.5% carboxymethylcellulose), compound 1 or compound 2 daily for 21 days, with the doses shown in fig. 5A and 5B. Mice were observed daily for signs of toxicity and weight loss, and tumor volumes were measured every other day. Both compounds significantly reduced tumor growth compared to vehicle treatment (T/C =31% and 6% at day 21 for compound 1 and compound 2, respectively; p < 0.001, as determined by the two-tailed T-test) without any significant toxicity.

In another study, U87 xenografted 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 50mg/kg, PO,5 days/week), (5) treatment first with compound 2 (25 mg/kg, PO, x10 per day), then TMZ (5 mg/kg, PO,5 days/week x 2), (6) treatment first with TMZ (5 mg/kg, PO,5 days/week x 2), then compound 2 (25 mg/kg, PO, x10 per day), and (7) were concurrent with compound 2 and TMZ, respectively. All treatment groups were completed on day 21, followed by a 7-day drug holiday, after which a second treatment cycle was started from day 29 (fig. 5B). Compound 2 exhibited significant anti-tumor efficacy (p < 0.001) as a single dose and in combination with TMZ without any significant toxicity when compared to the vehicle treated group.

The in vivo anti-tumor efficacy of compound 1 and compound 2 was also studied in a GBM orthotopic mouse xenograft model. For example, compound 2 was orally administered once daily to NSG mice (n = 8) implanted with U87 GBM cells per treatment group (The Jackson Laboratory, ben Harbor, ME, united States of America) for 21 days. TMZ was orally administered once daily for 5 days and used as a positive control. Disease burden is measured by non-invasive bioluminescence whole body imaging techniques. Treatment with compound 2 resulted in significant inhibition of tumor growth at day 21 (TGI =81.4%, p < 0.001) compared to vehicle-treated mice. An increase of 154.8% in the lifespan ratio [ ILS = (days T-days C)/days C, where days C = control survival days and days T = treatment survival days ] of compound 2-treated mice was observed when compared to vehicle control (fig. 6A).

In an independent study, NSG mice (n = 8) originally engrafted with GBM patient-derived G4T cells were administered vehicle, compound 1, TMZ, and concurrent treatment of compound 1 and TMZ, respectively, as shown in fig. 6B. Disease burden was measured for each treatment group by bioluminescence whole body imaging. Compound 1 showed significant inhibition of tumor growth either as a single dose or in combination with TMZ (p < 0.001, fig. 6B).

Conclusion

The compounds of formula I are demonstrated to inhibit CDK4/6 and have antiproliferative activity against GBM cell lines in both in vitro and in vivo. Furthermore, the compounds represented, 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 very effective in animal models carrying GBM tumors, suggesting that the compound of formula I has excellent prospects, providing an effective therapeutic agent for the treatment of proliferative cell diseases and disorders of the CNS such as GBM.

Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment of any form of suggestion that such prior art forms part of the common general knowledge.

Those skilled in the art will appreciate that the present disclosure does not limit its use to the particular applications described. The present disclosure is not limited in its application to the details of the preferred embodiments of the particular elements and/or features described or depicted herein. It should also be understood that the present disclosure is not limited to the embodiment(s) disclosed, but is capable of numerous rearrangements, modifications, and substitutions without departing from the scope of the disclosure as set forth and defined by the following claims.

It is noted that the following claims are only provisional claims and are 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 the present application. The entireties may be added to or omitted from the exemplary claims at a later time in order to further define or redefine various aspects of the disclosure.

Claims (15)

1. A method of treating a proliferative cell disease or disorder 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:

or a pharmaceutically acceptable salt, solvate or prodrug thereof,

optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient,

wherein:

R ¹ selected from H, alkyl, aryl, aralkyl, alicyclic, heterocyclic, halogen, NO ₂ 、CN、CF ₃ OH, O-alkyl, O-aryl, COOH, CO-alkyl, CO-aryl, CONH ₂ CONH-alkyl, CONH-aryl and CONH-cycloaliphatic;

R ² selected from H, alkyl, halogen, NO ₂ 、CN、CF ₃ OH, O-alkyl and NH ₂ ；

R ³ Selected from the group consisting of heterocyclic radicals containing at least one N heteroatom, NH-alkyl, NH-aryl, N- (alkyl) ₂ N- (aryl) ₂ And N- (alkyl) (aryl); and is provided with

n is an integer selected in the range of 0 to 3; and is

Wherein the 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 ₂ NH-alkyl, N (alkyl) ₂ COOH, COH, CO (alkyl), CONH ₂ And CF ₃ 。

2. The method of claim 1, wherein R ¹ Is H, alkyl (e.g. C) _1-6 Alkyl radicals such as C _1-3 Alkyl radicals such as methyl, ethyl and C (CH) ₃ ) ₂ Or C is _3-6 Cycloalkyl such as cyclopentyl) or heterocyclyl (e.g., saturated or unsaturated 5 or 6 membered cyclic groups containing one or two N, O or S heteroatoms).

3. The method of claim 2, wherein R ¹ Is H, methyl, ethyl, cyclopropyl or cyclopentyl.

4. The method of any one of claims 1 to 3, wherein R ² Is H, alkyl (e.g. C) _1-6 Alkyl or preferably C _1-3 Alkyl such as methyl or ethyl), CN or halogen (preferably F).

5. The method of claim 4, wherein R ² Is H, methyl, ethyl, CN or F.

6. The method of any one of claims 1 to 5, wherein R ³ Is heterocyclyl (preferably a saturated or unsaturated 5-or 6-membered cyclic group containing one, but more preferably two, N heteroatoms), optionally substituted by one or moreSubstituted with one or more groups selected from: alkyl (e.g. C) _1-6 Alkyl or preferably C _1-3 Alkyl radicals such as methyl, ethyl and CH (CH) ₃ ) ₂ )、NH ₂ NH-alkyl such as NH-methyl and NH-ethyl, N (alkyl) ₂ Such as N (C) _1-3 Alkyl radical) ₂ (e.g., N (CH) ₃ ) ₂ 、N(CH ₂ CH ₃ ) And N (CH) ₃ )(CH ₂ CH ₃ ) COH and CO (C) _1-3 Alkyl) (e.g. COCH) ₃ )。

7. The method of claim 6, wherein R ³ Selected from the following:

8. the method of any one of claims 1 to 7, wherein n is 0, 1 or 2.

9. The method of claim 1, wherein 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) 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-morpholinylpyridin-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.

10. The method of any one of claims 1-9, wherein the compound, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is formulated for intravenous and/or oral administration.

11. The method of any one of claims 1-10, wherein the compound, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, is administered to the subject in combination with Temozolomide (TMZ) or other kinase inhibitors.

12. The method of claim 11, wherein the other kinase inhibitor is selected from inhibitors of PI3K, mTOR and/or MEK.

13. The method of any one of claims 1 to 12, wherein the proliferative cell disease or disorder is Glioblastoma (GBM), medulloblastoma, primary Central Nervous System (CNS) lymphoma, astrocytoma, ependymoma, oligodendroglioma, or metastatic brain tumor.

14. Use of a compound as defined in any one of claims 1 to 9, or a pharmaceutically acceptable salt, solvate or prodrug thereof, in the manufacture of a medicament for treating a proliferative cell disease or disorder of the Central Nervous System (CNS) in a subject.

15. Use of a compound as defined in any one of claims 1 to 9, or a pharmaceutically acceptable salt, solvate or prodrug thereof, for treating a proliferative cell disease or disorder of the Central Nervous System (CNS) in a subject.