CN111377935A - Selective CDK4/6 inhibitor and application thereof - Google Patents

Abstract

The invention discloses a selective CDK4/6 inhibitor and application thereof. The compound is a compound shown as a formula Ia, and pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof. The compound can be used for preparing medicaments for treating and/or preventing cancers.

Description

Selective CDK4/6 inhibitor and application thereof

Technical Field

The invention belongs to the field of biomedicine, and relates to a selective CDK4/6 inhibitor and application thereof.

Background

Tumorigenesis is associated with an imbalance of multiple oncogenes and tumor suppressor genes. Almost all functional effects of oncogenes and tumor suppressor genes eventually converge on the cell cycle. Thus, it can be said that tumors are a type of Cell Cycle Disease (CCD), and that modulating or blocking the cell cycle is one of the ways to treat tumors. Many molecules have been discovered to be involved in cell cycle regulation, among which Cyclin-Dependent Kinases (CDKs) are core molecules of cell cycle regulatory networks. CDKs are catalytic subunits, a class of serine (Ser)/threonine (Thr) kinases, which are important intracellular signaling molecules involved in different phases of the cell cycle. Research shows that cell cycle abnormality is caused by abnormality of any link of a cell cycle regulation network taking CDKs as a center, and finally tumor is caused. The CDK family currently has 21 subtypes, which act by binding to their regulatory subunits, cyclins (cyclins). The functions of various isoforms of CDKs include, in addition to their effects on the cell cycle, the regulation of transcription, DNA repair, differentiation and apoptosis. Based on the key role of CDKs in regulating the proliferation and death of tumor cells, the CDKs kinase family provides opportunities and new fields for the discovery and development of antitumor drugs.

Among the isoforms of CDKs involved in the cell cycle, CDK4/6 plays an irreplaceable role. The cell cycle mutation related to cancer is mainly present in the transformation processes of G1 stage and G1/S stage, CDK4/6 is combined with cyclinD to form a complex with kinase activity, and the complex is phosphorylated by an anti-cancer gene Rb product pRb, so that a combined transcription factor E2F is released, gene transcription related to the S stage is started, cells are promoted to pass through a check point, and the cells are transferred from the G1 stage to the S stage. CDK 4/6-specific activation is closely associated with proliferation in some tumors, with abnormalities in the cyclin D-CDK4/6-INK4-Rb pathway in approximately 80% of human tumors. The change of the pathway accelerates the G1 phase process, so that the tumor cell proliferation is accelerated to obtain the survival advantage. Thus, intervention in this pathway is a therapeutic strategy and CDK4/6 is a novel anti-tumor target. CDK4/6 has the advantages as an anti-tumor target: (1) most proliferating cells proliferate dependent on CDK2 or CDK4/6, but inhibitors of CDK4/6 do not exhibit the cytotoxicity of "pan-CDK inhibitors", such as myelosuppression and gut response. (2) Preclinical experiments show that if the cyclin D level of cells is increased or P16INK4a is inactivated, the sensitivity of the cells to drugs can be increased, and the targeting property of the drugs is increased to a certain extent due to the phenomenon of tumor cells relative to normal cells.

CDK inhibitor drugs that have been approved by the FDA to be marketed so far include: pfizer palbociclib was approved by FDA on 2/3 d 2015, Novartis ribociclib was approved by FDA on 3/13 d 2017, EliLilly abemaciclib was approved by FDA on 28 d 2017, all 3 indications are for the treatment of metastatic breast cancer, which plays a very positive role in the development of CDK4/6 inhibitors. In addition, some pharmaceutical companies such as Astex, Tolero, G1 and the like continuously report a series of CDK4/6 inhibitors with better selectivity, which are used for treating bone marrow diseases, blood tumors, breast tumors, lung cancers and the like, and are currently in clinical trial stages in different stages.

In order to achieve better tumor treatment effect and better meet the requirements of clinic and market, a new generation of selective CDK4/6 inhibitor with high efficiency and low toxicity is expected to be developed, and the selectivity of treatment and the damage of normal cells caused by some side effects are expected to be improved. Therefore, the development of a safer and more efficient novel CDK4/6 inhibitor drug has great social value and economic benefit.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the existing CDK4/6 medicaments, and provide a novel selective CDK4/6 inhibitor and application thereof.

The invention provides a compound shown as a formula Ia, and pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof,

wherein R is₁Selected from hydrogen, C₁-C₃Alkyl, or C₁-C₃A haloalkyl group;

R₂and R₃Each independently selected from unsubstituted C₁-C₃Alkyl, or R₂And R₃Each independently selected from C substituted by-OH, F, Cl or Br₁-C₃An alkyl group; r₂And R₃Or may be joined to each other to form a cyclic structure containing three to six atoms;

R₄selected from substituted or unsubstituted cyclohexyl, hydroxytetrahydrofuryl, morpholinyl, piperazinyl, piperidinyl, hydroxyphenyl, pyridinyl, or pyrimidinyl.

According to a particular embodiment of the invention, R is preferred₁Selected from hydrogen, or C₁-C₃An alkyl group;

according to a particular embodiment of the invention, R is preferred₂And R₃Each independently selected from H, -CH₃,-C₂H₅,-C₃H₇,-CH₂OH,-C₂H₄OH,-C₃H₆OH,-CHF₂,-CH₂F,-CH₃CF₃,-C₃H₆Cl,-CH₂-CH₂-,-(CH₂)₃-,-(CH₂)₄-,-(CH₂)₅-, or-CH (CH)₃)₂。

According to a particular embodiment of the invention, R is preferred₄Is selected from

Thus, throughout this specification, the skilled artisan can refer to R in the compounds of formula Ia₁～R₄And substituents thereof are selected to provide a stable compound of formula Ia, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof, as described in the examples of this invention.

It will be understood by those skilled in the art that, according to the convention used in the art, in the structural formulae of the present application,

for delineating chemical bonds, which are points of attachment of moieties or substituents, core structures, or backbone structures.

According to an embodiment of the present invention, the compound of formula Ia according to the present invention is any one of the following compounds:

the compound of formula Ia according to the present invention can be prepared according to conventional chemical synthesis methods in the art, and the procedures and conditions thereof can be referred to analogous reaction procedures and conditions in the art.

The compounds of the invention can be isolated and purified according to standard techniques well known to those skilled in the art. One particularly useful technique in purifying compounds is preparative liquid chromatography, which uses mass spectrometry as a means of detecting the pure compound flowing from a chromatographic column.

Preparative LC-MS is a standard efficient method for purifying small organic molecules, such as the compounds described herein. The Liquid Chromatography (LC) and Mass Spectrometry (MS) methods can be modified to allow better crude separation and to improve MS detection of the sample. Optimization of preparative gradient LC methods involves changing the column, volatile eluent and modulators and gradients. These methods are well known in the art of optimizing preparative LC-MS methods, which are employed to purify compounds. Such methods are described in the following documents: RosentreterU, huberu.; an Optimal fraction collecting and predicting LC/MS; j CombChem; 2004; 159-64 and leister W, Strauss K, Wisnoski D, ZHao Z, Lindsley C, Development of custom high-throughput predictive consistency/mass spectrometry for the predictive purification and analytical analysis of compounds; j Comb chem.; 2003; 5 (3); 322-9.

The reaction solvent used in each reaction step described in the present invention is not particularly limited, and any solvent that can dissolve the starting materials to some extent and does not inhibit the reaction is included in the present invention. Further, many equivalents, substitutions, or equivalents in the art to which this invention pertains, as well as different proportions of solvents, solvent combinations, and solvent combinations described herein, are deemed to be encompassed by the present invention.

The compounds of formula Ia described herein may exist in a number of different geometric isomeric and tautomeric forms, and reference to compounds of formula Ia includes all such forms. For the avoidance of doubt, when a compound may exist in one of a number of geometric or tautomeric forms and only one of these is specifically described or given, all other forms are still encompassed by formula Ia.

When a compound of formula Ia contains one or more chiral centers and may exist in two or more optically isomeric forms, reference to a compound of formula Ia includes all optically isomeric forms thereof (e.g., enantiomers, epimers, and diastereomers), either as a single optical isomer or as a mixture of two or more optical isomers (e.g., a racemic mixture), unless the context requires otherwise.

Optical isomers can be characterized and identified by their optical activity (i.e., the + and-isomers, or the d and l isomers), or they can be characterized by their absolute stereochemistry using the "R and S" nomenclature developed by Cahn, Ingold, and Prelog, see Advanced Organic Chemistry, JerryMarch, 4 th edition, John Wiley & Sons, New York, 1992, page 109-114, see also Cahn, Ingold & Prelog, Angew.Chem.int.Ed.Engl., 1966, 5, 385-415. Optical isomers can be separated by a number of techniques, including chiral chromatography (chromatography on a chiral support), such techniques being well known to those skilled in the art.

When the compounds of formula Ia exist in two or more optically isomeric forms, one enantiomer of a pair of enantiomers may exhibit advantages over the other, for example in terms of biological activity. Thus, in some cases, it may be desirable to use only one of a pair of enantiomers or one of a large number of diastereomers as a therapeutic agent. Accordingly, the present invention provides compositions comprising a compound of formula Ia having one or more chiral centers, wherein at least 55% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the compound of formula Ia is present as a single optical isomer (e.g., enantiomer or diastereoisomer). In a general embodiment, 99% or more (e.g., substantially all) of the compound of formula Ia, based on the total amount of the compound of formula Ia, may be present as a single optical isomer (e.g., enantiomer or diastereomer).

The pharmaceutical preparation comprises the following components:

the invention also provides a pharmaceutical composition comprising the compound of formula Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof, and a pharmaceutical excipient.

While it is possible for a compound of formula Ia described herein to be administered as the active compound alone, it is preferred to present it as a pharmaceutical composition (e.g., formulation) comprising at least one active compound of the invention and one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilizers, preservatives, lubricants or other materials well known to those skilled in the art, and optionally other therapeutic or prophylactic agents. Thus, the present invention also provides a pharmaceutical composition as defined above and a process for the preparation of a pharmaceutical composition, which process comprises admixing at least one active compound as defined above with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers or other materials as described herein.

In the pharmaceutical composition, the compound of formula Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof may be used in a therapeutically effective amount.

The pharmaceutical excipients can be those widely used in the field of pharmaceutical production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrating agents, lubricants, antiadherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffering agents, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

The pharmaceutical compositions of the present invention may be prepared according to the disclosure using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions of the present invention may be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention may also be in a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry preparations which can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic formulations; aerosol: such as nasal sprays or inhalants; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

Oral administration of the compounds of the invention is preferred. Intravenous administration of the compounds of the invention is also preferred. Depending on the circumstances, other application routes may be applied or even preferred. For example, transdermal administration may be highly desirable for patients who are forgetful or whose oral medications are irritable. In particular cases, the compounds of the invention may also be administered by transdermal, intramuscular, intranasal or intrarectal routes. The route of administration may vary in any way, limited by the physical properties of the drug, the convenience of the patient and caregiver, and other relevant circumstances (Remington's Pharmaceutical Sciences, 18 th edition, mack publishing Co. (1990)).

And (3) biological activity:

the compounds of formula Ia of the present invention are inhibitors of CDKs. Preferred compounds are compounds that inhibit one or more CDK kinases selected from CDK1, CDK2, CDK4, CDK6, e.g. CDK4 and/or CDK 6.

As a result of their modulation or inhibition of CDK kinase activity, they are expected to be useful in providing a means of cell cycle arresting or restorative control over aberrantly differentiated cells. Thus, it is envisioned that these compounds will prove useful for treating or preventing proliferative disorders, such as cancer.

CDKs play a role in the regulation of cell cycle, apoptosis, transcription, differentiation and CNS function. Thus, CDK inhibitors may be useful in the treatment of diseases in which proliferative, apoptotic, or differentiation disorders are present, such as cancer. In particular, RB + ve tumors are particularly sensitive to CDK inhibitors. RB-ve tumors are also sensitive to CDK inhibitors.

Examples of cancers that can be inhibited include, but are not limited to, cancers such as bladder cancer, breast cancer, colon cancer (e.g., colorectal cancer, such as colon adenocarcinoma and colon adenoma), kidney cancer, epidermoid cancer, liver cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, and non-small cell lung cancer), esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer (e.g., exocrine pancreatic cancer), stomach cancer, cervical cancer, thyroid cancer, prostate cancer, or skin cancer (e.g., squamous cell carcinoma); hematopoietic tumors of lymphoid lineage, such as leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, Burkett's lymphoma, hematopoietic tumors of myeloid lineage, acute and chronic myelogenous leukemias, myelodysplastic syndrome, promyelocytic leukemia, thyroid follicular cancer, tumors of mesenchymal origin, fibrosarcoma, rhabdomyosarcoma, tumors of the central or peripheral nervous system, astrocytomas, neuroblastoma, glioma, schwannoma, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratocothinoma, thyroid follicular cancer, or Kaposi's sarcoma.

The cancer may be one that is sensitive to inhibition by any one or more cyclin dependent kinases selected from CDK1, CDK2, CDK4, and CDK6, for example one or more kinases selected from CDK1, CDK2, CDK4, and CDK6, for example CDK4 and/or CDK 6.

The activity of a compound of the invention as a CDK inhibitor may be measured using the assays described in the examples below, and the level of activity exhibited by a given compound may be measured by IC₅₀A value.

The invention also provides application of the compound shown in the formula Ia, pharmaceutically acceptable salts, hydrates, solvates, metabolites, stereoisomers, tautomers or prodrugs thereof in preparation of CDK4/6 inhibitors.

The CDK4/6 inhibitor can be used in vivo; also useful in vitro, primarily for experimental purposes, for example: the comparison is provided as a standard or control, or a kit is prepared according to methods conventional in the art, to provide a rapid test for the inhibitory effect of CDK 4/6.

The invention also provides application of the compound shown in the formula Ia, pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof in preparation of medicines for treating and/or preventing cancers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is standard in the art to which the claimed subject matter belongs. In case there are multiple definitions for a term, the definitions herein control. When referring to a URL or other identifier or address, it should be understood that such identifier may change and that particular information on the internet may change, but equivalent information may be found by searching the internet. The reference demonstrates that such information is available and publicly disseminated.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Furthermore, the term "comprising" is open-ended and not closed-ended.

The present invention employs, unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques or pharmacological detection, and the various steps and conditions may be referred to those conventional in the art. Unless otherwise indicated, the present invention employs standard nomenclature for analytical chemistry, organic synthetic chemistry, and medicinal chemistry, as well as standard laboratory procedures and techniques. In some cases, standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and drug delivery, and treatment of patients.

The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic ammonia or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids (e.g., arginine, etc.), and salts of organic acids such as glucuronic acid (see Berge et al, "Pharmaceutical salts," Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt. Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms by certain physical properties, such as solubility in polar solvents.

The term "pharmaceutically acceptable salts" as used herein pertains to derivatives of the compounds of the present invention wherein the parent compound is modified by salification with an acid or by salification with a base. Examples of pharmaceutically acceptable salts include, but are not limited to: inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acid groups such as carboxylic acids, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound, for example, salts formed with non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic or organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, glycolic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannin, tartaric acid, and p-toluenesulfonic acid.

The "pharmaceutically acceptable salts" of the present invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent or a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

In addition to salt forms, the compounds provided herein also exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the present invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound of formula Ia) is a prodrug within the scope and spirit of the present invention. For example, compounds containing carboxyl groups may form physiologically hydrolyzable esters that act as prodrugs by hydrolyzing in vivo to give the compounds of formula Ia themselves. The prodrugs are preferably administered orally, since hydrolysis in many cases takes place mainly under the influence of digestive enzymes. Parenteral administration may be used when the ester itself is active or hydrolysis occurs in the blood. In addition, prodrugs can be converted to the compounds of the present invention in an in vivo environment by chemical or biochemical means.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polycrystalline or amorphous form.

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labelled with radioactive isotopes, such as tritium (A), (B), (C³H) Iodine-125 (¹²⁵I) Or C-14(¹⁴C) In that respect All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "effective amount" or "therapeutically effective amount" with respect to a drug or pharmacologically active agent refers to a sufficient amount of the drug or agent that is non-toxic but achieves the desired effect. For oral dosage forms of the invention, an "effective amount" of one active agent in a composition is the amount required to achieve the desired effect when combined with another active agent in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient and also on the particular active substance, and an appropriate effective amount in an individual case can be determined by a person skilled in the art according to routine tests.

The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

The term "comprising" is open-ended, i.e. includes the elements indicated in the present invention, but does not exclude other elements.

The CDK inhibitors described herein may be used as a single agent, or in combination with other therapeutic agents, to enhance the effect of these therapeutic agents. It has been found that certain cyclin-dependent kinase inhibitors may be used in combination with other anti-cancer agents. For example, the cyclin-dependent kinase inhibitor alvocidib has been used in combination therapy with other anti-cancer agents.

The compound shown in the formula Ia has good solubility, is stable in liver microsomes and plasma, and has good pharmacokinetic characteristic and good stability. Compared with the Palbociclib which is a CDK4/6 inhibitor on the market at present, the compound disclosed by the invention shows better in-vivo pharmacodynamic characteristics, is more efficient and low-toxic, and has better tumor treatment effect.

The compounds of formula Ia according to the present invention provide a new commercial choice for novel CDK inhibitors, useful in the treatment and/or prevention of tumors and other uncontrolled cell proliferative disorders.

The positive progress effects of the invention are as follows:

(1) the novel compounds described in the present invention as selective inhibitors of CDK4/6, which are novel structures, have better biological activity (IC of kinase activity on CDK 4)₅₀All within 16nM, IC for kinase activity of CDK6₅₀All within 18 nM). Further tests show that the compound disclosed by the invention has no inhibitory activity on CDK1 and CDK2, the compounds disclosed by the formulas Ia-1-Ia-6 disclosed by the invention have good kinase inhibitory activity on CDK4 and CDK6, and the compound prepared by the invention can be effectively used as a selective CDK4/6 inhibitor.

(2) Compared with the currently marketed CDK4/6 inhibitor Palbociclib, the compound disclosed by the invention shows better in-vivo pharmacodynamic characteristics, higher efficiency and lower toxicity and better tumor treatment effect in an in-vivo pharmacodynamic model test of human breast cancer and leukemia cell lines.

(3) The invention has convenient preparation and lower production cost.

Drawings

FIG. 1 is a graph showing the effect of compounds of the present invention on a model of pharmacodynamics in human breast cancer cells (MDA-MB-231) in vivo.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The embodiment of the invention provides a compound shown as a formula Ia or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof, a method and an intermediate for preparing the compound shown as the formula I or the pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer or prodrug thereof, a pharmaceutical composition and application of the compound in preparing medicines.

EXAMPLE 1 preparation of Compound Ia-1

Tert-amyl alcohol (160 mL) was added to the first reactor followed by compound Ia-1A (22.9g, 0.1mol), compound Ia-1B (21.2g, 0.1mol), potassium carbonate (41.5g, 0.3 mol). 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (1.3g) was charged into the first reactor, and nitrogen was added thereto for nitrogen blanketing, followed by stirring the first reactor. Tris (dibenzylideneacetone) dipalladium (1.0g) was added to the mixture and heated to 90 ℃ to 95 ℃. The reaction was stirred and monitored by HPLC until completion. It was cooled to room temperature, 100 ml of dichloromethane were added, stirred well and the solid was removed by filtration. The filtrate was washed with brine three times. The combined organic layers were dried over anhydrous magnesium sulfate. The solvent was removed in vacuo and purified by silica gel chromatography to give the compound represented by formula Ia-1 (22.6g) in 55.9% yield and 98.9% HPLC purity.

m/z:405(M+H)⁺.

EXAMPLE 2 preparation of Compound Ia-2

Preparation of Compound Ia-2 an experiment was carried out in a similar manner to example 1, Compound Ia-2A (23.1g, 0.1mol) and Compound Ia-2B (24.0g, 0.1 mol). Purification by silica gel chromatography gave the compound of formula Ia-2 (29.2g) in 67.2% yield and 99.3% HPLC purity.

m/z:435(M+H)⁺.

EXAMPLE 3 preparation of Compounds Ia-3

Preparation of Compound Ia-3 an experiment was carried out in a similar manner to example 1, Compound Ia-3A (24.3g, 0.1mol) and Compound Ia-1B (21.1g, 0.1 mol). Purification by silica gel chromatography gave the compound represented by formula Ia-3 (33.9g) in 81.0% yield and 97.7% HPLC purity.

m/z:419(M+H)⁺.

EXAMPLE 4 preparation of Compounds Ia-4

Preparation of Compound Ia-4 an experiment was carried out in a similar manner to example 1, Compound Ia-4A (27.1g, 0.1mol) and Compound Ia-1B (21.1g, 0.1 mol). Purification by silica gel chromatography gave the compound of formula Ia-4 (29.2g) in 67.2% yield and 99.3% HPLC purity.

m/z:447(M+H)⁺.

EXAMPLE 5 preparation of Compounds Ia-5

Preparation of Compound Ia-5 an experiment was carried out in a similar manner to example 1, Compound Ia-5A (26.7g, 0.1mol) and Compound Ia-5B (19.9g, 0.1 mol). Purification by silica gel chromatography gave the compound represented by formula Ia-5 (15.7g) in 36.6% yield and 97.0% HPLC purity.

m/z:430(M+H)⁺.

EXAMPLE 6 preparation of Compounds Ia-6

Preparation of Compound Ia-6 an experiment was carried out in a similar manner to example 1, Compound Ia-6A (24.5g, 0.1mol) and Compound Ia-5B (19.9g, 0.1 mol). Purification by silica gel chromatography gave the compound represented by formula Ia-6 (28.6g) in 70.3% yield and 98.2% HPLC purity.

m/z:408(M+H)⁺.

Effect example 1 biological assay-inhibition assay of CDK4 by Compounds of the invention

The results of the following analyses confirm that the compounds listed herein are useful as particular CDK4/6 inhibitors and as anti-cancer agents. As used herein, "IC₅₀"denotes the concentration of the active agent that produces 50% of the maximum inhibitory response possible for the active agent, and" EC₅₀"refers to the concentration of the active agent that produces 50% of the maximum response possible for the active agent.

To confirm that the compounds comprised by the present invention show affinity for CDK4 kinase, CDK4 assays were performed. Functional analysis provides support for: the compounds shown in the formula I show good capability of inhibiting CDK4 kinase activity. All ligands, radiolabels, solvents and reagents used in the following assays are readily available from commercial sources or can be readily synthesized by one skilled in the art.

mu.L of test compound in 20% DMSO, 20. mu.L of adenosine 5' -triphosphate (ATP) and C-terminal retinoblastoma fragment (CTRF) (Upstate cat #12-439) solution, and 10. mu.L of enzyme solution were mixed in a 96-well plate. ATP and CRTF solution was prepared by diluting 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) at 68mM pH7.4, 6.72mM MgCl2. 6.72mM Dithiothreitol (DTT) and 0.013% TRITON^TM40 μ MATP, 0.16 μ Ci [ mu ] Ci in X-100 kinase buffer³³P]-a mixture of ATP and 1 μ M CTRF. The enzyme solution was prepared from 8ng CDK4 enzyme (Proqinase cat #0142-0373-1) diluted in the above kinase buffer. Test compounds were serially diluted 1: 3 in 20% DMSO to generate a 10 point curve starting at 20. mu.M. 20% DMSO buffer alone without test compound was used as a control, 500mM ethylenediaminetetraacetic acid (EDTA) was used to measure background in the absence of enzyme activity³³The level of P. The reagents were mixed and incubated at 20 ℃ for 90 minutes. By adding 80. mu.L of 10% (v/v) H₃PO₄The reaction was terminated and the material was precipitated onto a glass fiber filter plate (Millipore, MAFC N0B 50). The wells were washed with 0.5% H₃PO₄Wash 4 times and measure incorporated radioactivity with a microplate scintillation counter (microbeta trilux, Wallac).

The difference between the median values of the high and low controls was considered to be 100% activity. Applying ActivityBase^TMA4-parameter logistic curve fit obtained by software (IDBS, Alameda CA) was used to generate IC₅₀The value is obtained. The compounds of formulae Ia-1 to Ia-6 of the present invention all showed IC in the above assays₅₀< 16 nM. See table 1 for details.

Effect example 2 biological assay-inhibition assay of CDK6 by Compounds of the invention

mu.L of test compound in 20% DMSO, 20. mu. LATP and CTRF (Upstate cat #12-439) solution, and 10. mu.L of enzyme solution were mixed in a 96-well plate. ATP and CRTF solutions were prepared, diluted at 68mM HEPES pH7.4, 6.72mM MgCl₂2.64mM DTT and 0.004% TRITON^TMThe final concentration of the kinase buffer solution of X-100 is 100. mu. MATP, 0.5. mu. Ci³³P]ATP and 0.8. mu.M CTRF. Enzyme solutions were prepared to obtain a final concentration of 1.7 ng/. mu.L CDK6 enzyme (Proqinase cat #7533) diluted in CDK4 inhibition assay in the above kinase buffer. Test compounds were mixed at a ratio of 1: serial 3 dilutions in 20% DMSO resulted in 10 point curves starting at 20 μ M. 20% DMSO buffer alone without test compound was used as a control, 500mM EDTA for absence measurementBackground at enzyme Activity³³The level of P. The reagents were mixed and incubated at 20 ℃ for 90 minutes. By adding 80. mu.L of 10% (v/v) H₃PO₄The reaction was terminated and the material was precipitated onto a glass fiber filter plate (Millipore, MAFC N0B 50). The wells were washed with 0.5% H₃PO₄Wash 4 times and measure incorporated radioactivity with a microplate scintillation counter (Microbeta Trilux, Wallac).

Data were analyzed in the same manner as CDK 4. Compounds of formulae Ia-1 to Ia-6 of the present invention, the majority of which exhibit their IC for CDK 6in the above assay₅₀< 18 nM. See table 1 for details.

Effect example 3 biological assay-inhibition assay of CDK1 by Compounds of the invention

CDK 1/cyclin B kinase Activity assay

A384 well microplate MAP-FPTM (molecular Devices Trade Mark technology) endpoint assay was used for CDK 1/cyclin B kinase activity assay. The same assay was used to determine the IC of the compounds of formulae Ia-1 to Ia-6 according to the invention₅₀. Typically, the kinase reaction is carried out in a volume of 20 μ L of a reaction solution containing: mu.L of compound (in 20% DMSO), 8. mu.L CDK 1/cyclin B in 1 Xreaction buffer (Molecular Devices), 10. mu.L Tamra histone-H1 substrate mix (Molecular Devices) and ATP in 1 Xreaction buffer, fresh 1mM DTT. The final reaction mixture contained 0.005-10 μ M compound (inhibitor), 2% DMSO, 0.25nM CDK 1/cyclin B, 100nM Tamra histone-H1 peptide and 20 μ MATP. All reactions were performed in black 384-well plate Costar plates (Corning) at room temperature for 120 min, and then 60 μ L of 400-fold diluted 1 × progressive binding buffer a (molecular devices) was added to stop the reaction. After 2 hours incubation at room temperature, the fluorescence polarization signal was read on an Evison Multilabel reader.

Effect example 4 biological assay-inhibition assay of CDK2 by Compounds of the invention

CDK 2/cyclin A kinase Activity assay

Cyclin a kinase activity was performed under the same conditions as CDK 1/cyclin B.

Effects the results of examples 1 to 4 are shown in table 1 below.

Table 1:

the results show that: the compounds of formulae Ia-1 to Ia-6 of the present invention all showed IC in the CDK4 activity assay described above₅₀< 16nM, wherein the compounds of formula Ia-1, formula Ia-2, formula Ia-5, formula Ia-6 all show IC₅₀< 10 nM. This demonstrates that the compounds of the present invention have good CDK4 kinase inhibitory activity and are potent CDK4 inhibitors.

The compounds of formulae Ia-1 to Ia-6 of the present invention, for the most part, showed IC in the CDK6 activity assay described above₅₀Less than 18nM, wherein the compounds of formula Ia-1, formula Ia-2, formula Ia-5, formula Ia-6 all show IC₅₀< 18 nM. This demonstrates that the compounds of the present invention have good CDK6 kinase inhibitory activity and are potent CDK6 inhibitors.

Compounds of formulae Ia-1 to Ia-6 of the present invention, both of which show IC in the CDK1 and CDK2 activity assays described above₅₀> 10. mu.M. This demonstrates that the compounds described in the present invention have no inhibitory activity against CDK1 and CDK 2. The compounds shown in the formulas Ia-1-Ia-6 have good kinase inhibition activity on CDK4 and CDK6, and the kinase inhibition activity of the compounds shown in the invention on CDK4 and CDK6 is better than that of a control product Palbociclib. The compound prepared by the invention can be effectively used as a selective CDK4/6 inhibitor.

Effect example 5 Effect of Compounds of the invention on in vivo pharmacodynamic model of human Breast cancer cells (MDA-MB-231)

Human breast cancer cells (MDA-MB-231) (MDA-MB-231 cells were cultured in L-15 medium containing 10% fetal bovine serum FBSAt 5% CO₂Culturing in a 37 deg.C incubator, collecting tumor cells in logarithmic growth phase after passage growth, counting, re-suspending in L-15 culture medium, and adjusting cell suspension concentration to 5 × 10⁷Perml (1:1with matrigel) was used for inoculation).

The mean tumor volume was about 170mm ^3 21 days after MDA-MB-231 tumor cell inoculation, and the groups were divided into 10 animals per group. Day of grouping was regarded as Day0, and administration was performed on Day of grouping. The body weight and tumor size of the animals were measured twice weekly during the experiment, while the clinical symptoms of the animals were observed and recorded daily, with reference to the body weight of the animal measured the last time each administration. The experiment was carried out for 28 days, animals were euthanized, tumor-bearing animals were photographed by group after the animals were sacrificed, then tumors were collected and weighed and tumor weight was recorded, and finally tumors were photographed by group. And carrying out pollution-free treatment on animal carcasses and tumor samples after the experiment is finished.

Drawing a tumor growth curve by taking the time point as an X axis and the tumor volume as a Y axis; and drawing a weight increase change curve by taking the time point as an X axis and the animal weight as a Y axis. Comparison between groups by t-test, p<0.05 is a significant difference, p<0.01 is a very significant difference. The evaluation index of the antitumor activity was the relative tumor proliferation rate T/C (%), T/C (%)>40% is ineffective, T/C (%) is less than or equal to 40%, and P is treated by statistics<0.05 is effective, and T/C (%) is calculated as: T/C (%) ═ T_RTV/C_RTV)×100％。T_RTVRelative tumor volume for treatment group, C_RTVThe relative tumor volume of the negative control group, and the tumor inhibition rate (%) of TGI (mean tumor volume or weight of the negative control group-mean tumor volume or weight of the administered group)/mean tumor volume or weight of the negative control group × 100%.

In the study, the animals can tolerate the human breast cancer MDA-MB-231 xenograft tumor female nude mice after the administration of each component, and the change rate of the animal body weight (BWC%) is as follows by day 28: 6.6%, 2.7%, -1.0%, -5.1%, -3.5%, and-2.6%, no toxic death associated with drug toxicity was found.

The experimental result shows that compared with the Palbociclib serving as a control drug CDK4/6 inhibitor on the market, the compounds disclosed in the embodiments 1, 2, 5 and 6 of the invention have better tumor inhibition effect and better anti-tumor activity than Palbociclib in a human breast cancer MDA-MB-231 xenograft tumor model, and all the compounds reach the effective judgment standard of T/C% < 40%. See figure 1 for details.

In addition, in vivo pharmacodynamic studies of Acute Myeloid Leukemia (AML) cells (MV4-11) were performed, wherein MV4-11 was grown in Iscove's modified Dulbecco's medium containing L-glutamine, 25mM HEPES, 10% FBS. In AMLMV4-11 xenograft, the compound of the invention in the embodiment 1 and 6 is used, and the tumor regression is observed at the dosage of 100mg/kg (mpk), which shows that the compound of the invention in the embodiment 1 and 6 has good anti-tumor activity and can be used as the treatment drug of human acute granulocytic leukemia.

Accordingly, the compounds of the present invention are useful as inhibitors of CDK4/6 and in the treatment of proliferative disorders caused by CDKs. The compounds of the present invention are useful for the treatment and/or prevention of cancer diseases, for example by inhibiting CDK4/6 kinase.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example" or "some examples" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims (10)

1. A compound of formula Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof;

wherein the content of the first and second substances,

the R is₁Selected from hydrogen, C₁-C₃Alkyl, or C₁-C₃A haloalkyl group;

the R is₂And R₃Each independently selected from unsubstituted C₁-C₃Alkyl, or R₂And R₃Each independently selected from C substituted by-OH, F, Cl or Br₁-C₃An alkyl group; r₂And R₃Or may be joined to each other to form a cyclic structure containing three to six atoms;

the R is₄Selected from substituted or unsubstituted cyclohexyl, hydroxytetrahydrofuryl, morpholinyl, piperazinyl, piperidinyl, hydroxyphenyl, pyridinyl, or pyrimidinyl.

2. A compound of formula Ia, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof according to claim 1,

the R is₁Selected from hydrogen, or C₁-C₃An alkyl group;

the R is₂And R₃Each independently selected from H, -CH₃,-C₂H₅,-C₃H₇,-CH₂OH,-C₂H₄OH,-C₃H₆OH,-CHF₂,-CH₂F,-CH₃CF₃,-C₃H₆Cl,-CH₂-CH₂-,-(CH₂)₃-,-(CH₂)₄-,-(CH₂)₅-, or-CH (CH)₃)₂。

3. Such asA compound of formula Ia, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof, according to claim 1, wherein R is₄Is selected from

4. A compound of formula Ia, or a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof, according to claim 1,

the compound shown in the formula Ia is any one of the following compounds:

5. a pharmaceutical composition comprising a compound Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable adjuvant; the compound Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer, or prodrug thereof can be used in a therapeutically effective amount.

6. Use of a compound Ia according to any one of claims 1 to 4, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof, or a pharmaceutical composition according to claim 5 for the preparation of an inhibitor of CDK 4/6.

7. The use of claim 6, wherein:

the CDK4/6 inhibitor is used in vivo; or in vitro, as experimental uses, for example: a kit.

8. Use of a compound Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of a disease in which a disturbance of proliferation, apoptosis or differentiation is present.

9. Use of a compound Ia, a pharmaceutically acceptable salt, hydrate, solvate, metabolite, stereoisomer, tautomer or prodrug thereof according to any one of claims 8 for the preparation of a medicament for the treatment and/or prophylaxis of cancer which is sensitive to inhibition of any one or more cyclin-dependent kinases.

10. The use of claim 9, wherein the cancer is bladder, breast, colon, kidney, epidermis, liver, lung, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, or skin; hematopoietic tumors of lymphoid lineage, such as leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma, Burkett's lymphoma, hematopoietic tumors of myeloid lineage, acute and chronic myelogenous leukemias, myelodysplastic syndrome, promyelocytic leukemia, thyroid follicular cancer, tumors of mesenchymal origin, fibrosarcoma, rhabdomyosarcoma, tumors of the central or peripheral nervous system, astrocytomas, neuroblastoma, glioma, schwannoma, melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratocothinoma, thyroid follicular cancer, or kaposi's sarcoma;

optionally, the cancer is breast cancer;

optionally, the cancer is acute and chronic myeloid leukemia.

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