CN105732615A

CN105732615A - CDK kinase inhibitor

Info

Publication number: CN105732615A
Application number: CN201510890859.9A
Authority: CN
Inventors: 吴永谦; 王统建
Original assignee: Shandong Xuanzhu Pharma Co Ltd
Current assignee: Xuanzhu Biopharmaceutical Co Ltd
Priority date: 2014-12-31
Filing date: 2015-12-04
Publication date: 2016-07-06
Anticipated expiration: 2035-12-04
Also published as: CN105732615B

Abstract

The invention belongs to the technical field of medicines and particularly relates to a CDK kinase inhibitor represented as the general formula (I), pharmaceutically-accepatable salt, ester and solvate thereof, and stereisomers thereof. The R1, R2, R3, R4, A1, A2, A3 and n are defined as the specification. The invention also relates to a preparation method of the compounds, a medicine preparation and a medicine composition comprising the compounds, and an application of the compounds, the pharmaceutically accepatable salt, ester and solvate thereof, and the stereisomers thereof in preparation of a medicine for treating and/or preventing cancer-related diseases induced by the CDK kinase.

Description

CDK inhibitors of kinases

Technical field

The invention belongs to pharmaceutical technology field, it is specifically related to CDK inhibitors of kinases, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, pharmaceutical preparation containing these compounds and pharmaceutical composition, and this compound, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, treat in preparation and/or prevent by the application in the medicine of cancer-related diseases kinase mediated for CDK.

Background technology

Tumor occur with multiple oncogene and antioncogene unbalance relevant.Nearly all oncogene, antioncogene functional effect, finally all can converge on cell cycle.Therefore, it can be said that tumor is a class cell cycle diseases (CellCycleDisease, CCD), regulating or block cell cycle is one of approach treating tumor.At present, it has been found that the molecule relevant with cell cycle regulating a lot, wherein cell cycle protein dependent kinase (Cyclin-Dependent-Kinases, CDKs) is cell cycle regulating server molecule.CDKs is catalytic subunit, is a class serine (Ser)/threonine (Thr) kinases, as signal transduction molecule important in cell, participates in the different times of cell cycle.Research shows, the cell cycle regulating network centered by CDKs, and the exception of any link all will cause cell cycle abnormal, ultimately result in the generation of tumor.CDK family has 21 hypotypes at present, plays a role by combining with its modulability subunit cyclins (cyclin).The function of the various hypotype of CDK, except acting on cell cycle, also includes transcribing, DNA repairs, the adjustment of differentiation and apoptosis.Based on the pivotal role risen in the CDKs propagation at modulate tumor cell and death, the discovery that CDKs kinase families is antitumor drug provides chance and new field with developing.

In research and development medicine, it is " pan-CDK " inhibitor with the first generation CDK inhibitor that flavopiridol, UCN-01 etc. are representative, they block all hypotypes of CDK family in the way of equivalence and show the toxicity that comparison is high in clinical trial, what have can not reach effective therapeutic dose, therefore people are excited to start to research and develop selectivity CDK inhibitor, it is desirable to improve the selectivity for the treatment of and prevent normal cell to be subject to the infringement of some side effect.

In the CDK hypotype participating in cell cycle, CDK4/6 plays irreplaceable effect.Suddenling change with the cell cycle of related to cancer is primarily present in G1 phase and G1/S phase conversion process, CDK4/6 and CyclinD combines the complex being formed with kinase activity, by study of tumor suppressor genes Rb product pRb phosphorylation, the transcription factor E2F that release combines, start the genetic transcription relevant with the S phase, promote cell to pass through check point, and shift to the S phase from the G1 phase.The specific activation of CDK4/6 is closely related with the propagation of some tumors, has the exception of cyclinD-CDK4/6-INK4-Rb path in the human tumor of about 80%.The change of this path, accelerates G1 phase process so that tumor cell proliferation is accelerated and obtained survival advantage.Therefore, the intervention of this path being become a kind of therapeutic strategy, CDK4/6 becomes a kind of new antitumor target spot.CDK4/6 is in that as the advantage of antitumor target spot: the cell of (1) great majority propagation relies on CDK2 or CDK4/6 propagation, but the inhibitor of CDK4/6 does not show the cytotoxicity of " pan-CDK inhibitor ", as bone marrow depression and intestinal react.(2) preclinical laboratory shows, if cell cyclinD level raises or P16INK4a inactivation, the cell sensitivity to medicine can be increased, owing to tumor cell exists above-mentioned phenomenon relative to normal cell, so adds somewhat to the targeting of medicine.

CDK9 is the same with other CDK, it it is a kind of serine/threonine kinases, being find in Human cDNA Library screens the earliest, it combines with corresponding cyclin (s) and forms positive transcriptional elongation factor b (Positivetranseriptionelongationfaetorb, p-TEFb).This complex can phosphorylation rna plymerase ii (RNApolymeraseII) and some negative transcription elongation factors so that transcribing and being extended from initial position.The unconventionality expression of CDK9/cyclinT and a lot of diseases have substantial connection.cyclinT₁/ CDK9 and estrogen receptor positive breast cancer cell, in this kind of cell, estrogen can lower the expression of HEXIMI, HEXIMI is also referred to as estrogen down-regulated gene (EDGI), and HEXIMI/EDG1 can be combined with estrogen receptor, again can with cyclinT₁In conjunction with.Cell proliferation and growth can be suppressed when mammary glandular cell process LAN HEXIMI/EDGI.Simultaneously take account of HEXIMI and can suppress the kinase activity of P-TEFb in cell, and hexa-methylene diethyl acid amide (HMBA) abduction delivering, cyclinT in cell can be utilized in multiple neoplastic cell₁/ CDK9 activity level have impact on the one-tenth tumor ability of tumor cell.At lymphoma cell, find cyclinT when T cell periodic activation₁/ CDK9 content raises, and cyclinT₁What/CDK9 expressed raises the activation and differentiation that affect this kind of cell.Except the function in tumor, cyclinT₁The complex that/CDK9 is formed is the major target class of HIV-1Tat albumen, and Tat can with the CyclinT of P-TEFb₁The combination of subunit and form Tat-cyclinT₁-CDK9 complex, by CDK9 kinase activity phosphorylation RNApolymeraseII and N-TEF, makes transcribing of HIV-1 be accomplished.Pharmaceutical research finds that the inhibition compound of CDK9 can effectively suppress the transcription and replication of HIV-1.In addition CDK9 has significant biological function in antiinflammatory, analgesia, myocardial hypertrophy etc..

Listing but without CDK inhibitor medicaments so far, including Pfizer, gift reports the good CDK4/6 inhibitor of a series of selectivity with Novartis etc. successively in some interior pharmaceuticals, just at clinical experimental stage.Wherein, it is worth noting the LEE-011 (PhaseIII) of the LY2835219 (PhaseIII) and Novartis of the Pfizer PD0332991 (palbociclib) developed and EliLilly especially.

In April, 2013, the PD0332991 of Pfizer obtains FDA important breakthrough medicine and assert, in August, 2014, submit new drug application (NDA) to FDA, seek the bent azoles (letrozole) of approval PD0332991 (palbociclib) associating for previously not accepting systematic treating to control the treatment of the postmenopausal women estrogen receptor positive (ER+) of the state of an illness in late period, negative (HER2-) Locally Advanced of human epidermal growth factor receptor 2 or metastatic breast cancer.The exploitation of CDK4/6 inhibitor is played a part very front by this.

In order to reach the purpose of better oncotherapy effect, better meet the market demand, it is therefore desirable to be able to develop the CDK inhibitors of kinases of the high-efficiency low-toxicity of a new generation.The present invention will provide for the selectivity CDK inhibitors of kinases of a kind of new structure, and finds that the compound of this type of structure has good drug effect, and can effectively pass through blood brain barrier, provides probability for CDK inhibitors of kinases as the treatment of the brain cancer.

Summary of the invention

It is an object of the invention to provide a class targeting CDK inhibitors of kinases, concrete technical scheme is as follows:

1, logical compound shown in formula I, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer,

Wherein

A¹、A²And A³It is independently selected from nitrogen or carbon；

R¹Selected from C_1-6Alkyl, C_1-6Alkoxyl or optionally by Q¹3～8 yuan of cycloalkyl replaced, Q¹Selected from C_1-6Alkyl, C_1-6Alkoxyl；

R²Selected from C_1-6Alkyl, C_1-6Alkoxyl, cyano group, carbamoyl or C_1-6Alkyl-carbonyl-amino；

R⁴Selected from halogen or hydrogen；

R³Selected from optionally by Q²3～8 yuan of heterocyclic radicals replaced, 6～14 yuan of condensed hetero ring bases, 5～8 yuan of heteroaryls, 6～14 yuan of thick heteroaryls, phenyl, naphthyl, 6～12 yuan of bridge heterocyclic radicals or 6～12 yuan of spiro heterocyclic radicals；Described " 5～8 yuan of heteroaryls " is preferably " 5～7 yuan of heteroaryls ", it is more preferably " 5～6 yuan of heteroaryls ", more preferably " 5～6 membered nitrogen-containing heteroaryl base " etc., described " 6～14 yuan of thick heteroaryls " is preferably " 6～10 yuan of thick heteroaryls ", it is more preferably " 7～10 yuan of thick heteroaryls ", it is more preferably " 9～10 yuan of thick heteroaryls ", more preferably " 9～10 yuan of nitrogenous thick heteroaryls " etc.；

Q²Selected from amino, hydroxyl, halogen, trifluoromethyl, cyano group, C_1-6Alkyl, C_1-6Alkoxyl, C_1-6Alkyl sulphonyl, C_1-6Alkyl sulfonyl amino, 3～8 yuan of heterocyclic radicals or 6～9 yuan of bridge heterocyclic radicals；

N is selected from the integer of 0～3.

2, compound as described in scheme 1, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

A¹、A²And A³It is independently selected from nitrogen or carbon；

R¹Selected from C_1-4Alkyl or C_1-4Alkoxyl；

R²Selected from C_1-4Alkyl, C_1-4Alkoxyl, cyano group, carbamoyl or C_1-4Alkyl-carbonyl-amino；

R⁴Selected from halogen；

R³Selected from optionally by Q²5～7 yuan of heterocyclic radicals replaced, 6～11 yuan of condensed hetero ring bases, 6～11 yuan of bridge heterocyclic radicals or 6～11 yuan of spiro heterocyclic radicals；nullDescribed " 6～11 yuan of condensed hetero ring bases " is preferably " 6～10 yuan of condensed hetero ring bases "，It is more preferably " 7～10 yuan of condensed hetero ring bases "，It is more preferably " 9～10 yuan of condensed hetero ring bases "，More preferably " 9～10 yuan of nitrogenous condensed hetero ring bases " etc.，Described " 6～11 yuan of bridge heterocyclic radicals " is preferably " 6～9 yuan of bridge heterocyclic radicals "，It is more preferably " 7～10 yuan of bridge heterocyclic radicals "，It is more preferably " 7～9 yuan of bridge heterocyclic radicals "，It is more preferably " 7～8 yuan of bridge heterocyclic radicals "，It is more preferably " 8 yuan of bridge heterocyclic radicals "，More preferably " 8 yuan containing nitrogen bridge heterocyclic radical " etc.，Described " 6～11 yuan of spiro heterocyclic radicals " is preferably " 7～11 yuan of spiro heterocyclic radicals "，It is more preferably " 7～10 yuan of spiro heterocyclic radicals "，It is more preferably " 7～9 yuan of spiro heterocyclic radicals "，It is more preferably " 7～8 yuan of spiro heterocyclic radicals "，More preferably " 7～8 yuan of nitrogenous spiro heterocyclic radicals " etc.；

Q²Selected from amino, hydroxyl, trifluoromethyl, cyano group, C_1-4Alkyl, C_1-4Alkoxyl, C_1-4Alkyl sulphonyl, C_1-4Alkyl sulfonyl amino, 5～6 yuan of heterocyclic radicals or 7～9 yuan of bridge heterocyclic radicals；Described " 5～6 yuan of heterocyclic radicals " is preferably " 5～6 member heterocyclic ring containing nitrogen base ", described " 7～9 yuan of bridge heterocyclic radicals ", it is preferred to " 7～8 yuan of bridge heterocyclic radicals ", more preferably " 8 yuan of bridge heterocyclic radicals ", more preferably " 8 yuan containing nitrogen bridge heterocyclic radical " etc.；

N is selected from the integer of 0～3.

3, compound as described in scheme 2, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

A¹、A²And A³It is independently selected from nitrogen or carbon；

R¹It it is isopropyl；

R²Selected from methyl, methoxyl group, cyano group, carbamoyl or acetylamino；

R⁴It it is fluorine；

R³Selected from optionally by Q²5～6 yuan of heterocyclic radicals replaced, Q²Selected from amino, hydroxyl, trifluoromethyl, cyano group, C_1-4Alkyl, C_1-4Alkoxyl, C_1-4Alkyl sulphonyl, C_1-4Alkyl sulfonyl amino, 5～6 yuan of heterocyclic radicals or 7～9 yuan of bridge heterocyclic radicals；Described " 5～6 yuan of heterocyclic radicals " is preferably " 5～6 member heterocyclic ring containing nitrogen base ", described " 7～9 yuan of bridge heterocyclic radicals ", it is preferred to " 7～8 yuan of bridge heterocyclic radicals ", more preferably " 8 yuan of bridge heterocyclic radicals ", more preferably " 8 yuan containing nitrogen bridge heterocyclic radical " etc.；

N is selected from the integer of 0～2.

4, compound as described in scheme 3, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

R³Selected from optionally by Q²5～6 member heterocyclic ring containing nitrogen bases replaced, Q²Selected from amino, hydroxyl, trifluoromethyl, cyano group, C_1-4Alkyl or C_1-4Alkoxyl, described " 5～6 member heterocyclic ring containing nitrogen base " is preferably " 6 member heterocyclic ring containing nitrogen base "；

N is 1.

5, compound as described in scheme 4, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

R³Selected from optionally by Q²Pyrrolidinyl, piperidyl or piperazinyl, the Q replaced²Selected from trifluoromethyl, C_1-4Alkyl or C_1-4Alkoxyl；

N is 1.

The part of compounds of the present invention

Detailed Description Of The Invention

" halogen atom " of the present invention includes fluorine atom, chlorine atom, bromine atoms and atomic iodine.

" C of the present invention_1-6Alkyl " can be straight or branched, including such as " C_1-4Alkyl ", " C_1-3Alkyl " etc.; instantiation includes but not limited to: methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, 2-methyl-propyl, 1-methyl-propyl, 1; 1-dimethyl ethyl, n-pentyl, 3-methyl butyl, 2-methyl butyl, 1-methyl butyl, 1-ethyl propyl, n-hexyl, 4-methyl amyl, 3-methyl amyl, 2-methyl amyl, 1-methyl amyl, 3; 3-dimethylbutyl, 2; 2-dimethylbutyl, 1; 1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethyl-butyl, 1,2-dimethyl propyl etc..

" C of the present invention_1-6Alkoxyl, C_1-6Alkyl-carbonyl-amino, C_1-6Alkyl sulphonyl, C_1-6Alkyl sulfonyl amino " refer to C_1-6Alkyl-O-, C_1-6Alkyl-C (O) NH-, C_1-6Alkyl-SO₂-、C_1-6Alkyl-SO₂The group that NH-mode is formed, wherein " C_1-6Alkyl " literary composition as defined above described in.

" C of the present invention_1-4Alkoxyl, C_1-4Alkyl-carbonyl-amino, C_1-4Alkyl sulphonyl, C_1-4Alkyl sulfonyl amino " refer to C_1-4Alkyl-O-, C_1-4Alkyl-C (O) NH-, C_1-4Alkyl-SO₂-、C_1-4Alkyl-SO₂The group that NH-mode is formed, wherein " C_1-4Alkyl " literary composition as defined above described in.

" 3～8 yuan of cycloalkyl " of the present invention, refers to that the paraffin section of 3～8 carbon atoms removes the cyclic alkyl that a hydrogen atom is derivative, including such as " 3～6 yuan of cycloalkyl ", " 4～6 yuan of cycloalkyl " etc..The example includes but not limited to: cyclopropane base, Tetramethylene. base, Pentamethylene. base, cyclohexyl, cycloheptane base, cyclooctane base etc..

" 3～8 yuan of heterocyclic radicals " of the present invention, including such as " 3～7 yuan of heterocyclic radicals ", " 3～6 yuan of heterocyclic radicals ", " 4～7 yuan of heterocyclic radicals ", " 4～6 yuan of heterocyclic radicals ", " 5～7 yuan of heterocyclic radicals ", " 5～6 yuan of heterocyclic radicals ", " 5～6 member heterocyclic ring containing nitrogen base ", " 6 member heterocyclic ring containing nitrogen base " etc..nullInstantiation includes but are not limited to: aziridine base、2H-aziridine base、Diazacyclo propyl、3H-diazacyclo acrylic、Azetidinyl、1,4-dioxane base、1,3-dioxane base、1,3-dioxolane base、1,4-Dioxin base、Tetrahydrofuran base、Pyrrolin base、Pyrrolidinyl、Imidazolidinyl、4,5-glyoxalidine base、Pyrazolidinyl、4,5-pyrazoline base、2,5-dihydro-thiophene base、Tetrahydro-thienyl、4,5-dihydro-thiazolyl、Piperidyl、Piperazinyl、Morpholinyl base、4,5-dihydro azoles base、4,5-dihydro isoxazole base、2,3-dihydro isoxazole base、2H-1,2-piperazine base、6H-1,3-piperazine base、4H-1,3-thiazinyl、6H-1,3-thiazinyl、2H-pyranose、2H-pyran-2-one base、3,4-dihydro-2H-pyranose etc.，It is preferably " 5～6 member heterocyclic ring containing nitrogen base ".

Rule according to IUPAC Compound nomenclature, condensed ring of the present invention refers to that being shared two adjacent atoms (namely sharing a key) each other by two or more circuluses couples together the condensed cyclic structure formed.Bridged ring of the present invention refers to that being shared two non-adjacent carbon atoms each other by two or more circuluses couples together the caged scaffold formed.Volution of the present invention refers to that being shared a carbon atom each other by two or more circuluses couples together the spirane structure formed.

" 6～14 yuan of condensed hetero ring bases " of the present invention, refer to that two or more circuluses share two adjacent atoms (namely sharing a key) each other and couple together the condensed cyclic structure at least containing heteroatomic 6～14 annular atomses formed, including such as " 6～11 yuan of condensed hetero ring bases ", " 6～10 yuan of condensed hetero ring bases ", " 7～10 yuan of condensed hetero ring bases ", " 9～10 yuan of condensed hetero ring bases " etc..nullInstantiation includes but are not limited to: imidazolidine also [4,5-c] pyridine radicals、3,4-dihydroquinazoline base、1,2-dihydro-quinoxaline base、Benzo [d] [1,3] dioxa cyclopentenyl、1,3-dihydroisobenzofuran base、2H-chromogen thiazolinyl、2H-chromogen alkene-2-ketone group、4H-chromenyl、4H-chromene-4-ketone group、Chromanyl、4H-1,3-benzimidazole dihydrochloride base、4,6-dihydro-1H-furo [3,4-d] imidazole radicals、3a,4,6,6a-tetrahydrochysene-1H-furo [3,4-d] imidazole radicals、4,6-dihydro-1H-thieno [3,4-d] imidazole radicals、4,6-dihydro-1H-pyrrolo-[3,4-d] imidazole radicals、4,5,6,7-tetrahydrochysene-1H-benzo [d] imidazole radicals etc..

" 5～8 yuan of heteroaryls " of the present invention, including such as " 5～7 yuan of heteroaryls ", " 5～6 yuan of heteroaryls ", " 5～6 membered nitrogen-containing heteroaryl base " etc..nullInstantiation includes but are not limited to furyl、Thienyl、Pyrrole radicals、Thiazolyl、Isothiazolyl、Thiadiazolyl group、Azoles base、Isoxazole base、Di azoly、Imidazole radicals、Pyrazolyl、1,2,3-triazolyl、1,2,4-triazolyl、1,2,3-di azoly、1,2,4-di azoly、1,2,5-di azoly、1,3,4-di azoly、Pyridine radicals、2-pyridone、4-pyridone、Pyrimidine radicals、1,4-Dioxin base、2H-1,2-piperazine base、4H-1,2-piperazine base、6H-1,2-piperazine base、4H-1,3-piperazine base、6H-1,3-piperazine base、4H-1,4-piperazine base、Pyridazinyl、Pyrazinyl、1,2,3-triazine radical、1,3,5-triazine radical、1,2,4,5-tetrazine base、Azacyclo-heptantriene base、1,3-diazacyclo heptantriene base、Azepine cyclooctatetraenyl etc.，It is preferably " 5～6 membered nitrogen-containing heteroaryl base ".

" 6～14 yuan of thick heteroaryls " of the present invention, refer to two or more circuluses share each other two adjacent atoms (namely sharing a key) couple together formed at least contain heteroatomic 6～14 annular atomses, the condensed cyclic structure with armaticity, including such as " 6～10 yuan of thick heteroaryls ", " 7～10 yuan of thick heteroaryls ", " 9～10 yuan of thick heteroaryls ", " 9～10 yuan of nitrogenous thick heteroaryls " etc..Instantiation includes but not limited to: benzofuranyl, benzisoxa furyl, benzothienyl, indyl, iso-indoles, benzoxazolyl group, benzimidazolyl, indazolyl, benzotriazole base, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolines, isoquinolyl, acridinyl, phenanthridinyl, benzo pyridazinyl, phthalazinyl, quinazolyl, quinoxalinyl, phenol piperazine base, pteridine radicals, purine radicals, naphthyridinyl, azophenlyene, phenothiazine etc..

" 6～12 yuan of bridge heterocyclic radicals " of the present invention, refer to that any two ring shares the caged scaffold at least containing heteroatomic 6～12 annular atomses that two non-conterminous atoms are formed, referring to that any two ring shares the circulus at least containing heteroatomic 6～12 annular atomses that two non-conterminous atoms are formed, described hetero atom is selected from N, S, O, CO, SO and/or SO₂Deng.Including such as " 6～11 yuan of bridge heterocyclic radicals ", " 6～9 yuan of bridge heterocyclic radicals ", " 7～10 yuan of bridge heterocyclic radicals ", " 7～9 yuan of bridge heterocyclic radicals ", " 7～8 yuan of bridge heterocyclic radicals ", " 8 yuan of bridge heterocyclic radicals ", " 8 yuan containing nitrogen bridge heterocyclic radical " etc..The example includes but not limited to such as: Deng.

" 6～12 yuan of spiro heterocyclic radicals " of the present invention, refers to and has at least two rings to share the circulus at least containing heteroatomic 6～12 annular atomses that an atom is formed, described hetero atom is selected from N, S, O, CO, SO and/or SO₂Deng.Including such as " 6～11 yuan of spiro heterocyclic radicals ", " 7～11 yuan of spiro heterocyclic radicals ", " 7～10 yuan of spiro heterocyclic radicals ", " 7～9 yuan of spiro heterocyclic radicals ", " 7～8 yuan of spiro heterocyclic radicals ", " 7～8 yuan of nitrogenous spiro heterocyclic radicals " etc..The example includes but are not limited to such as: Deng.

The preparation method that present invention also offers formula (I) compound, it includes but not limited to following process route, and (definition that wherein, each abbreviation is representative is as follows: DCM: dichloromethane；DIPEA:N, N-diisopropylethylamine；DMSO: dimethyl sulfoxide；EA: ethyl acetate；HATU:2-(7-azo BTA)-N, N, N', N'-tetramethylurea hexafluorophosphoric acid ester；MeOH: methanol；NBS:N-bromo-succinimide；PE: petroleum ether；THF: oxolane；Double; two diphenylphosphine-9,9-dimethyl the xanthene of Xant-phos:4,5-；X-phos:2-dicyclohexyl phosphorus-2', 4', 6'-tri isopropyl biphenyl):

Process route:

R¹、R²、R³、R⁴、n、A¹、A²、A³As it was noted above, X represents halogen, selected from fluorine, chlorine, bromine, iodine.

Concrete illustrative steps is as follows:

1, the preparation of intermediate 1

Being dissolved in organic solvent by raw material 1 and organic base, be slowly added dropwise raw material 2 under cryogenic conditions, after stirring reaction terminates, extract, organic layer dries, and obtains intermediate 1, the wherein preferred DCM of organic solvent or Isosorbide-5-Nitrae-dioxane after concentration, and organic base is preferably triethylamine.

2, the preparation of intermediate 2

Intermediate 1, raw material 3 and organic base are dissolved in organic solvent, drip phosphorus oxychloride, after reaction terminates, add alkali, it is neutral for adjusting pH value, extracts, separates organic facies and dry, concentrate to obtain intermediate 2, the wherein preferred dichloromethane of organic solvent or 1,2-dichloroethanes, organic base selects triethylamine.

3, the preparation of intermediate 3

Being dissolved in organic solvent by intermediate 2, add potassium tert-butoxide, be warming up to 100 DEG C and react 2 hours, after reaction terminates, add water cancellation, extracts, and dry, after concentration, residue column chromatography obtains intermediate 3, wherein the preferred DCM of organic solvent.

4, the preparation of intermediate 4

Intermediate 3 and pinacol borate are dissolved in organic solvent, palladium, thricyclohexyl phosphorus and potassium acetate, under nitrogen protection, reacting by heating.After reaction terminates, adding water and organic solvent extraction, organic layer dries, and concentration obtains intermediate 4 through column chromatography for separation, wherein the preferred DMF of organic solvent or Isosorbide-5-Nitrae-dioxane.

5, the preparation of intermediate 5

Being dissolved in organic solvent by raw material 4 and intermediate 4, add inorganic base and tetrakis triphenylphosphine palladium, under nitrogen protection, heating terminates to reaction, adds water; extract, dry, concentration; intermediate 5 is obtained, wherein the preferred Isosorbide-5-Nitrae dioxane of organic solvent or DMF through column chromatography for separation.

6, the preparation of intermediate 6

Being dissolved in organic solvent by raw material 5, add benzoyl peroxide and NBS heating terminates to reaction, filter, concentration, residue obtains intermediate 6 through column chromatography for separation, wherein the preferred carbon tetrachloride of organic solvent.

7, the preparation of intermediate 7

Being dissolved in organic solvent by intermediate 6 and raw material 6, add inorganic base room temperature reaction, terminate to reaction, filter, filtrate concentrates, and residue column chromatography for separation obtains intermediate 7, wherein the preferred acetonitrile of organic solvent.

8, the preparation of formula (I) compound

Intermediate 5 and intermediate 7 are dissolved in organic solvent, add reacting by heating under three (dibenzalacetone) two palladium, 2-dicyclohexyl phosphorus-2', 4', 6'-tri isopropyl biphenyl, cesium carbonate, nitrogen protection.After reaction terminates, adding water, extract, dried residue column chromatography for separation obtains formula (I) compound, wherein the preferred Isosorbide-5-Nitrae dioxane of organic solvent or DMF.

" pharmaceutically acceptable salt " of compound shown in formula (I) refers to the salt that the acidic functionality existed in formula (I) compound and suitable inorganic or organic cation (alkali) are formed, including the salt formed with alkali metal or alkaline-earth metal, ammonium salt, and the salt formed with nitrogenous organic base；And the basic functionality (such as-NH existed in formula (I) compound₂Deng) salt that formed with suitable inorganic or organic anion (acid), including with mineral acid and organic carboxyl acid.

Shown in formula (I), " ester " of compound refers to, when there is carboxyl in formula (I) compound, the ester that can be formed with alcohol generation esterification, when there is hydroxyl in formula (I) compound, it is possible to organic acid, mineral acid, acylate etc. esterification occurs and the ester that formed.Ester is under acid or alkali existent condition, it is possible to occur hydrolysis to generate corresponding acid or alcohol.

" solvate " of compound shown in formula (I) refers to its material formed with solvent molecule association.Described solvent can be organic solvent (such as methanol, ethanol, propanol, acetonitrile etc.), water etc..Such as formula (I) compound can form alcoholate with ethanol, forms hydrate with water.

" stereoisomerism " of the compounds of this invention is divided into conformation and configuration isomery, and configuration isomery is also divided into cis-trans isomerism and optical isomerism.Conformational isomerism refer to the organic molecule with certain configuration due to carbon, carbon single bond rotate or distortion and make each atom of molecule or atomic group produce a kind of stereo-isomerism of different arrangement mode in space, common are the structure of alkane and naphthene-based compounds, in cyclohexane structure occur chair conformation and boat conformation." stereoisomer ", refers to that working as the compounds of this invention contains one or more asymmetric center, thus can as racemic modification and racemic mixture, single enantiomer, non-enantiomer mixture and single diastereomer.The compounds of this invention has asymmetric center, and this kind of asymmetric center respectively will produce two optical isomers independently, and the scope of the present invention includes all possible optical isomer and non-enantiomer mixture and pure or partial-purified compound.If compound of the present invention contains olefinic double bonds, unless stated otherwise, the present invention includes cis-isomer and transisomer.Compound of the present invention can exist with tautomeric forms, and it has the junction point of different hydrogen by one or more double-bond shifts.Such as, ketone and its Enol forms are ketoenol tautomerization bodies.Each tautomer and mixture thereof all include in the compound of the present invention.The enantiomer of all formulas (I) compound, diastereomer, racemization isomer, cis-trans-isomer, tautomer, geometric isomer, epimer and mixture thereof, be included in the scope of the invention.

Further requirement of the present invention protection includes the pharmaceutical composition of the compound shown in formula (I), its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer and one or more pharmaceutical carriers and/or diluent, it is possible to make pharmaceutically acceptable arbitrary dosage form.It is applied to the patient needing this treatment in modes such as oral, parenteral, rectum or transpulmonary administration.During for oral administration, can be made into the solid preparation of routine, such as tablet, capsule, pill, granule etc.；May be made as oral liquid, such as oral solution, oral suspensions, syrup etc..When making oral formulations, it is possible to add suitable filler, binding agent, disintegrating agent, lubricant etc..During for parenteral, can be made into injection, including injection, injectable sterile powder and concentrated solution for injection.When making injection, the conventional method in existing pharmaceutical field can be adopted to produce, during preparation injection, it is possible to be added without additives, it is possible to add suitable additives according to the character of medicine.During for rectally, can be made into suppository etc..During for transpulmonary administration, can be made into inhalant or spray etc..

Further requirement of the present invention protection includes the pharmaceutical composition of the compound shown in formula (I), its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer and other one or more antitumor agents and immunosuppressant.nullDescribed antitumor agent and immunosuppressant，Include but not limited to methotrexate、Capecitabine、Gemcitabine、Doxifluridine、Pemetrexed disodium、Pazopanib、Imatinib、Erlotinib、Lapatinib、Gefitinib、ZD6474、Trastuzumab、Bevacizumab、Rituximab、Herceptin、Paclitaxel、Vinorelbine、Docetaxel、Doxorubicin、Hydroxy camptothecin、Mitomycin、Epirubicin、Pirarubicin、Bleomycin、Letrozole、Tamoxifen、Fulvestrant、Music score of Chinese operas Rayleigh、Flutamide、Leuprorelin、Anastrozole、Ifosfamide、Busulfan、Cyclophosphamide、Carmustine、Nimustine、Semustine、Chlormethine、L-Sarcolysinum、Chlorambucil、Carboplatin、Cisplatin、Oxaliplatin、Network platinum、Topotecan、Camptothecine、Topotecan、Everolimus、Sirolimus、Special cancer is fitted、Ismipur、6-thioguanine、Azathioprine、Rhzomorph D、Daunorubicin、Amycin、Mitoxantrone、Bleomycin A5、Plicamycin or aminoglutethimide.

Present invention also offers the compound shown in formula (I), its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer in preparation treatment and/or to prevent by the application in the medicine of cancer-related diseases kinase mediated for CDK.The disease that described cancer is relevant is selected from cerebroma, pulmonary carcinoma, squamous cell cancer, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, cancer of pancreas, breast carcinoma, head and neck cancer, cervical cancer, carcinoma of endometrium, rectal cancer, hepatocarcinoma, renal carcinoma, adenocarcinoma of esophagus, esophageal squamous cell carcinoma, carcinoma of prostate, female reproductive tract cancer, cancer in situ, lymphoma, neurofibroma, thyroid carcinoma, osteocarcinoma, skin carcinoma, colon cancer, carcinoma of testis, gastrointestinal stromal tumor, tumor of prostate, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma.

The compounds of this invention has the advantage that

(1) formula (I) compound, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer have the CDK kinase inhibiting activity of excellence；

(2) formula (I) compound, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer demonstrate good biological stability, there is good pharmacokinetic property, effect is more lasting, and bioavailability is high.

(3) formula (I) compound, its pharmaceutically acceptable salt, its ester, its solvate or their stereoisomer demonstrate good blood brain barrier by property, provide probability for CDK inhibitor as the treatment of the brain cancer.

(4) formula (I) compound, its pharmaceutically acceptable salt, its ester, its solvate or their stereoisomer demonstrate relatively low toxicity, and drug resistance is good, and safety is high.

Test below by way of external zymetology and cytology's inhibitory activity and the compounds of this invention beneficial effect is expanded on further, but this should not being interpreted as, the compounds of this invention only has following beneficial effect.

The external zymetology activity experiment of experimental example 1 the compounds of this invention

Tester: part of compounds 1,3 of the present invention, its chemical name and preparation method are shown in the preparation embodiment of each compound.

Comparison medicine: LY2835219, structural formula is shown in background section, is purchased from Yong Can bio tech ltd, Wuhan.The implication representated by abbreviation in following experiment is as follows:

Experimental technique: adopt CaliperMobilityShift method to carry out the kinase whose inhibitory activity of CDK and measure

1.1 times of kinase buffer liquid preparations:

1) preparation of 1 times of CDK4/9 kinase buffer liquid

Take the HEPES of the pH7.5 that mother liquid concentration is 1000mM respectively, mother liquid concentration is the TritonX-100 of 10%, add ultra-pure water mixing, make HEPES final concentration of 20mM, TritonX-100 final concentration of 0.01%.

2. the preparation of stop buffer

Take respectively mother liquid concentration be 4% be coated liquid CoatingReagent#3 (carry in the 12-sipperchip that Caliper instrument uses and be coated liquid), mother liquid concentration is the HEPES of 1000mMpH7.5, mother liquid concentration is the EDTA of 1M, mother liquid concentration is the Brij-35 of 30%, add ultra-pure water mixing, make CoatingReagent#3 final concentration of 0.2%, the final concentration of 100mM of HEPES, EDTA final concentration of 50mM, Brij-35 final concentration of 0.015%.The wherein CDK1 of compound 3,2,7, the EDTA mother liquid concentration of use is 0.5M, and other conditions are constant.

3.2.5 the preparation of times kinase solution

1) preparation of 2.5 times of CDK4/D3 kinase solution

Take CDK4/D3 enzymatic solution that mother liquid concentration is 6.421 μMs respectively, mother liquid concentration is the DTT of 1M, adds 1 times of CDK4 kinase buffer liquid mixing, makes the CDK4/D3 enzyme final concentration of 5mM of final concentration of 25nM, DTT.

2) preparation of 2.5 times of CDK9/T1 kinase solution

Take CDK9/T1 enzymatic solution that mother liquid concentration is 7.096 μMs respectively, mother liquid concentration is the DTT of 1M, adds 1 times of CDK9 kinase buffer liquid mixing, makes the CDK9/T1 enzyme final concentration of 5mM of final concentration of 37.5nM, DTT.

4.2.5 the preparation of times polypeptide solution

1) preparation of 2.5 times of CDK4/D3 polypeptide solutions

Take ATP solution that mother liquid concentration is 100mM respectively, mother liquid concentration is the MgCl of 1M₂, mother liquid concentration is the polypeptide 8 of 300 μMs of FAM labellings, adds 1 times of CDK4 kinase buffer liquid mixing, makes final concentration of 552.5 μMs of ATP, MgCl₂Final concentration of 25mM, final concentration of 7.5 μMs of polypeptide 8.

3) preparation of 2.5 times of CDK9/T1 polypeptide solutions

Take ATP solution that mother liquid concentration is 10mM respectively, mother liquid concentration is the MgCl of 1M₂, mother liquid concentration is 300 μMs of CTD3 polypeptide, adds 1 times of CDK9 kinase buffer liquid mixing, makes final concentration of 57.5 μMs of ATP, MgCl₂Final concentration of 7.5 μMs of final concentration of 25mM, CTD3 polypeptide.

5.5 times of tester solution preparations:

Take the DMSO storing solution of 10mM tester, make, with DMSO dilution, the solution that concentration is 50 μMs, as mother solution.With DMSO by four times of stepwise dilutions of above-mentioned mother solution, then each concentration dilutes 10 times with 1 times of kinase buffer liquid respectively, makes 5 times of compound solutions.Compound 1 is the DMSO storing solution using 5mM tester, and other conditions are constant.

6.CDK/4//9 zymetology reaction:

1) hole corresponding in 384 orifice plates is separately added into 5 times of tester solution that 5 μ L prepare, 2.5 times of kinase solution that 10 μ L prepare, incubated at room 10 minutes.

2) corresponding Kong Zhongzai is separately added into 10 μ L, the 2.5 times of polypeptide solutions prepared, and makes tester final concentration of 1000nM, 250nM, 63nM, 16nM, 4nM, 1nM, 0.2nM, 0.1nM, 0.02nM, 0.004nM.Hatching startup enzyme reaction for 28 DEG C, CDK4 reacts 5 hours, and CDK9 reacts 90 minutes.

7. zymetology detection:

Each corresponding hole is separately added into 25 μ L stop buffers, terminates reaction.

8.Caliper instrument reads data, and calculates suppression ratio by data:

Suppression ratio=(maximum-sample value)/(maximum-minima) × 100, adopt XLfit software to carry out curve fitting, draw IC₅₀Value.

Maximum: be not added with the positive control of tester, minima: not enzyme-added negative control.

Experimental result and conclusion:

The external zymetology inhibitory activity of table 1 the compounds of this invention

The external zymetology inhibitory activity of table 2 the compounds of this invention

By table 1,2 it can be seen that CDK kinases is demonstrated enough good inhibitory activity by compound 1 and compound 3, especially for the activity of CDK9/T1 is significantly better than comparison medicine activity.

The cell in vitro inhibitory activity of experimental example 2 the compounds of this invention

Comparison medicine: LY2835219, structural formula is shown in background section, is purchased from Yong Can bio tech ltd, Wuhan.

The implication that the abbreviation of following experiment is representative is as follows:

Experimental technique: adopt BrdU method (BrdU cell proliferation test test kit, CellSignalingTechnology company) to carry out cell proliferation detection

1. reagent and compound preparation

1) 1 times of washing liquid preparation:

The washing liquid ultra-pure water that mother liquid concentration is 20 times is diluted to 1 times of washing liquid.

2) 1 times of detection antibody-solutions preparation:

The BrdU that mother liquid concentration is 100 times is detected antibody detection antibody diluent and is diluted to 1 times of detection antibody-solutions.

3) the two of 1 times of HRP labelling anti-solution preparation:

Anti-mouse IgG, the HRP traget antibody HRP traget antibody diluted that mother liquid concentration is 100 times is become two anti-solution of 1 times of HRP labelling.

4) 10 times of BrdU solution:

Culture medium corresponding for the BrdU solution cell that mother liquid concentration is 1000 times is diluted to 10 times of BrdU solution.

5) preparation test compound:

Preparation test compound stock solutions: be configured to the mother solution of 10mM with 100%DMSO.

Preparation test compound gradient dilution solution: taking DMSO4 times of continuous gradient dilution of test Compound Stock solution of 10mM, concentration is 2.5mM respectively, 625 μMs, 156 μMs, 39 μMs, 9.8 μMs, 2.5 μMs.The compound of the DMSO dilution taking 2 μ L respectively is added in the 198 μ L culture fluid containing 10%FBS and is configured to 10 times of testers, and tester maximum concentration is 100 μMs, and DMSO concentration is 1%, totally 7 Concentraton gradient.

6) culture medium preparation:

MDA-MB-435S culture medium: L-15+10%FBS+0.01mg/mL insulin

MCF-7 culture medium: DMEM+10%FBS+0.01mg/mL insulin

U87MG culture medium: MEM+10%FBS

Colo205, K562 culture medium: RPMI-1640+10%FBS

MDA-MB-468 culture medium: L-15+10%FBS

2. test procedure

1) trypsinization grows to the cell (exponential phase) of 80%, centrifugal collecting cell.With the culture medium re-suspended cell without FBS, count and adjust inoculation 96 orifice plates, the U87MG cell inoculation 4000/hole/81 μ L of compound 1, the MDA-MB-468 cell inoculation 4000/hole/81 μ L of compound 3, other all inoculate 3000/hole/81 μ L, are placed in 37 DEG C of cell culture incubators and cultivate；

2) after 24 hours, 9 μ LFBS are added in every hole, make FBS final concentration of 10%；

3) every hole adds 10 times of testers of 10 μ L variable concentrations, makes tester final concentration respectively 10 μMs, 2.5 μMs, 625nM, 156nM, 39nM, 9.8nM, 2.5nM, 3 multiple hole/groups, cultivates 72 hours for 37 DEG C；

Solvent control: 0.1%DMSO

Blank: only add culture medium, it does not have cell

Normal cell controls: do not have the normal cell of any process

4) every hole adds L10 times of BrdU solution of 10 μ, discards culture medium after hatching 4 hours in cell culture incubator；K562 cell 1000rpm discards culture medium after centrifugal 5 minutes；

5) every hole adds 100 μ L and fixes/degeneration liquid, incubated at room 30 minutes, discards solution；

6) every hole adds 100 μ L1 times and detects antibody-solutions, incubated at room 1 hour, discards solution, washes 3 times with 1 times of washing liquid 200 μ L/ hole；

7) every hole adds two anti-solution of L1 times of HRP labelling of 100 μ, incubated at room 30 minutes, discards solution, washes 3 times with 1 times of washing liquid 200 μ L/ hole；

8) every hole adds 100 μ LTMB substrate solutions, incubated at room 30 minutes；

9) every hole adds 100 μ L stop buffers, and microplate reader 450nm detects OD value.

3. data process

1) cell survival rate (%)=(OD_Tester-OD_Blank)/(OD_{Normal cell controls}-OD_Blank) × 100%, OD_Blank: blank value, OD_{Normal cell controls}: normal cell controls value；

2) data use the mapping of GraphPadPrism5 software, obtain curve and IC₅₀Value.

Experimental result:

The cell in vitro activity of table 3 the compounds of this invention

The cell in vitro activity of table 4 the compounds of this invention

By table 3,4 it can be seen that compound 1 and 3 pairs of cancerous cell all have good inhibiting effect；The cell in vitro activity of compound 1 is with to compare medicine suitable；The cell in vitro activity of compound 3 is significantly better than comparison medicine.

Detailed description of the invention

The detailed description of the invention of form by the following examples, is described in further detail the foregoing of the present invention.But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention belong to the scope of the present invention.

The preparation of preparation example 1:5-bromo-3-fluorine pyridine-2-amine

3-fluorine pyridine-2-amine (10.0g, 89.3mmol) is dissolved in acetic acid (100mL), when ice-water bath, bromine (14.3g, 89.3mmol) is dropped in solution.React 2h, TLC detection reaction under room temperature to complete, add ethyl acetate (80mL), be evaporated to the 1/3 of original volume, sucking filtration, weigh after the solid drying obtained (12.0g, productivity 70.6%).

The preparation of the preparation fluoro-2-nitropyridine of the bromo-3-of example 2:5-

Concentrated sulphuric acid (50mL) and hydrogen peroxide (50mL) are mixed, 5-bromo-3-fluorine pyridine-2-amine (10.0g, 52.36mmol) be dissolved in concentrated sulphuric acid (20mL) after drop in mixed solution, under room temperature react 24h, LC-MS detection reaction.Reactant liquor is poured in trash ice, adjust pH=7-8 with sodium carbonate, extract by ethyl acetate (3 × 200mL), concentration, residue obtains title compound (5.6g, productivity 48.4%) through silica gel column chromatography (petroleum ether: ethyl acetate=3:1).

The preparation of preparation example 3:5-bromo-N-isopropyl-2-nitropyridine-3-amine

By fluoro-for bromo-for 5-3-2-nitropyridine (5.6g, 25.34mmol) it is dissolved in N, in dinethylformamide (50mL), add potassium carbonate (7.0g, 50.68mmol), it is added dropwise over 2-aminopropane. (7.5g, 127.0mmol), react 3h, LC-MS detection reaction under room temperature to complete.Being poured into by reactant liquor in water (200mL), extract by ethyl acetate (2 × 100mL), organic facies saturated sodium-chloride washs, and anhydrous sodium sulfate dries, concentration, and residue is directly used in next step reaction.

The preparation bromo-N of example 4:5-³The preparation of-isopropyl-2,3-diamidogen

Bromo-for 5-N-isopropyl-2-nitropyridine-3-amine (6.0g, 23.1mmol) is dissolved in ethanol (50mL), adds stannous chloride dihydrate (15.6g, 69.2mmol), be heated to reflux 7h, LC-MS detection reaction and complete.Solution is concentrated, adds ethyl acetate (100mL), adjust pH=7-8 with sodium bicarbonate solution, filter, separatory, concentration, residue obtains title compound (2.5g, productivity 47.1%) through silica gel column chromatography (petroleum ether: ethyl acetate=1:1).

The preparation of preparation example 5:6-bromo-1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine

By bromo-for 5-N³-isopropyl-2,3-diamidogen (2.5g, 10.87mmol), acetic acid (0.652g, 10.87mmol) and polyphosphoric acids (30mL) mix, and heating is to 170 DEG C, and reaction 2h, LC-MS detect reaction and complete.Reactant liquor is poured in trash ice (100g), pH=7-8 is adjusted with saturated sodium bicarbonate solution, extract by ethyl acetate (2 × 200mL), organic facies saturated nacl aqueous solution washs, concentration, residue obtains title compound (1.5g, productivity 54.3%) through silica gel column chromatography (petroleum ether: ethyl acetate=1:2).

The preparation of preparation example 6:1-isopropyl-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxy boron penta ring-2-base)-1H-imidazo [4,5-b] pyridine

By bromo-for 6-1-isopropyl-2-methyl isophthalic acid H-imidazo [4, 5-b] pyridine (200mg, 0.787mmol), connection boric acid pinacol ester (400mg, 1.575mmol) it is dissolved in 1, in 4-dioxane (20mL), add [1, double, two (diphenylphosphine) ferrocene of 1'-] palladium chloride (57.6mg, 0.0787mmol) with potassium acetate (231.4mg, 2.361mmol), nitrogen is replaced, heating is to 120 DEG C, reaction 12h, LC-MS detects reaction and completes, concentration, add ethyl acetate (100mL), kieselguhr filters, concentration, residue is directly used in next step reaction.

The preparation of the preparation fluoro-4-of example 7:5-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-amine

By 1-isopropyl-2-methyl-6 (4,4,5,5-tetramethyl-1,3,2-dioxy boron penta ring-2-bases)-1H-imidazo [4,5-b] pyridine (593mg, 1.97mmol), 2-amino-4-iodo-5-fluorine pyridine (469mg, 1.97mmol) is dissolved in 1, in 4-dioxane (20mL), add tetra-triphenylphosphine palladium (25mg, 0.197mmol) and potassium carbonate (815.6mg, 5.91mmol), nitrogen is replaced, heating, to 100 DEG C, is reacted 2 hours, and reaction completes.Concentration, adds ethyl acetate (50mL), filters, and concentration, residue obtains title compound (400mg, productivity 71.3%) through silica gel column chromatography (dichloromethane: methanol=50:1).

Embodiment 1N-(5-((4-ethyl piperazidine-1-base) methyl) pyridine-2-base) the fluoro-4-of-5-(1-isopropyl-2-first Base-1H-imidazo [4,5-b] pyridine-6-base) preparation of pyrimidine-2-amine (compound 1)

(1) preparation of 1-[(6-aminopyridine-3-base) carbonyl]-4-ethyl piperazidine

By 6-amino-nicotinic acid (13.8g, 0.1mol) with NEP (11.4g, 0.1mol) it is dissolved in DCM (100mL), HATU (57g, 0.15mol) and DIPEA (38.7g, 0.3mol) is added under stirring condition, add water after room temperature reaction 1h (20mL), being layered to obtain organic facies, silica gel column chromatography (DCM:MeOH=20:1) obtains title compound (18.6g, productivity 79.5%).

(2) preparation of 1-((6-aminopyridine-3-base) methyl)-4-ethyl piperazidine

By 1-[(6-aminopyridine-3-base) carbonyl]-4-ethyl piperazidine (18.6g, 0.08mol) it is dissolved in oxolane (70mL), is cooled to 0 degree, be slowly added to Lithium Aluminium Hydride (6.08g, 0.16mol), rise to after feeding in raw material and 12h is stirred at room temperature.TLC detects raw material and disappears, and adds water (1mL) cancellation reaction, and sucking filtration, filtrate decompression dries to obtain title compound (15g, productivity: 85.7%).

(3) preparation of 6-(2-chloro-5-FU-4-base)-1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine

By 1-isopropyl-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxy boron penta ring-2-base)-1H-imidazo [4,5-b] pyridine (236.9mg, 0.787mmol), 2, chloro-5-FU (the 131.4mg of 4-bis-, 0.787mmol) it is dissolved in glycol dimethyl ether (20mL), adds two triphenylphosphine palladium (27.6mg, 0.039mmol) and sodium carbonate liquor (1.18mL2M), nitrogen is replaced, heating is to 80 DEG C, and reaction 12h, LC-MS detect reaction and complete.Concentration, adds 50mL ethyl acetate, filters, and concentration, residue obtains title compound (150mg, productivity 62.3%) through silica gel column chromatography (dichloromethane: methanol=100:1).

(4) preparation of N-(5-((4-ethyl piperazidine-1-base) methyl) pyridine-2-base) the fluoro-4-of-5-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyrimidine-2-amine

nullBy 6-(2-chloro-5-FU-4-base)-1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine (150.0mg，0.49mmol)，5-((4-ethyl piperazidine-1-base) methyl) pyridine-2-amine (107.8mg，0.49mmol) it is dissolved in 1,In 4-dioxane (20mL)，Add three (dibenzalacetone) two palladium (22.4mg，0.0245mmol)，2-dicyclohexylphosphontetrafluoroborate-2,4,6-tri isopropyl biphenyl (23.4mg，0.049mmol) with cesium carbonate (318.5mg，0.98mmol)，Nitrogen is replaced，Heating is to 120 DEG C，Reaction 12h，LC-MS detects reaction and completes，Concentration，Add ethyl acetate (100mL)，Kieselguhr filters，Concentration，Residue obtains title compound (40mg through silica gel column chromatography (dichloromethane: methanol=10:1)，Productivity 16.7%).

Molecular formula: C₂₆H₃₂FN₉Molecular weight: 489.6LC-MS (m/z): 490.3 (M+H⁺)

¹H-NMR(400MHz,DMSO-d₆)(10.13 s, 1H), 9.00 (s, 1H), 8.72 (s, 1H), 8.71 (s, 1H), 8.22 (s, 1H), 8.19 (s, 1H), 7.65-7.68 (m, 1H), 4.83-4.87 (m, 1H), 3.39 (s, 2H), 2.67 (s, 3H), 2.28-2.49 (m, 10H), 1.61 (d, J=6.8Hz, 6H), 0.92-0.98 (m, 3H).

Embodiment 25-((4-ethyl piperazidine-1-base) methyl) the fluoro-4-of-N-5-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-base) preparation of pyrimidine-2-amine (compound 2)

The preparation of (1) 5 (bromomethyl)-2-chloropyrimide

By chloro-for 2-5-methylpyrimidine (5.0g, 38.9mmol) be dissolved in carbon tetrachloride (40mL), add the benzoyl peroxide (100mg) of N-bromo-succinimide (6.92g, 38.9mmol) and catalytic amount, heated overnight at reflux, it is cooled to room temperature, filtering, filtrate concentrates, and residue is through column chromatography for separation (petroleum ether: ethyl acetate=1:1), obtain title compound (3.0g, productivity 37.5%).(2) preparation of the chloro-5-of 2-((4-ethyl piperazidine-1-base) methyl) pyrimidine

At NEP (276mg, in tetrahydrofuran solution (15mL) 2.42mmol), add diisopropylethylamine (624mg, 4.84mmol) He 5 (bromomethyl)-2-chloropyrimide (500mg, 2.42mmol), continuing reaction 4 hours under room temperature, reaction completes.Concentration, add ethyl acetate (50mL) and water (30mL), separatory, aqueous phase ethyl acetate (30mL) extracts, merging organic facies, concentration, residue is through column chromatography for separation (petroleum ether: ethyl acetate=1:1), obtain title compound (300mg, productivity 51.6%).

(3) 5-((4-ethyl piperazidine-1-base) methyl) the fluoro-4-of-N-5-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-base) preparation of pyrimidine-2-amine

nullBy fluoro-for 5-4-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-amine (100.0mg，0.351mmol)，The chloro-5-of 2-((4-ethyl piperazidine-1-base) methyl) pyrimidine (84.2mg，0.351mmol) it is dissolved in 1,In 4-dioxane (15mL)，Add three (dibenzalacetone) two palladium (32mg，0.035mmol)，2-dicyclohexylphosphontetrafluoroborate-2,4,6-tri isopropyl biphenyl (33.5mg，0.07mmol) with cesium carbonate (342mg，1.05mmol)，Nitrogen is replaced，Heating is to 110 DEG C，Reaction is overnight，LC-MS detects reaction and completes，By reacting liquid filtering，Concentration，Residue obtains title compound (31mg through silica gel column chromatography (dichloromethane: methanol=15:1)，Productivity 18.2%).

Molecular formula: C₂₆H₃₂FN₉Molecular weight: 489.6LC-MS (m/z): 490.4 (M+H⁺)

¹H-NMR(400MHz,CDCl₃)8.80 (d, J=1.6Hz, 1H), 8.71 (d, J=6.0Hz, 1H), 8.44 (s, 2H), 8.27 (d, J=2.4Hz, 1H), 8.09-8.11 (m, 2H), 4.71-4.78 (m, 1H), 3.50 (s, 2H), 2.74 (s, 3H), 2.42-2.70 (m, 9H), 1.69 (d, J=6.8Hz, 6H), 1.09 (t, J=7.2Hz, 3H).

Embodiment 3N-(5-((4-ethyl piperazidine-1-base) methyl) pyridine-2-base) the fluoro-4-of-5-(1-isopropyl-2-first Base-1H-imidazo [4,5-b] pyridine-6-base) preparation of pyridine-2-amine (compound 3)

The preparation of (1) 5 (bromomethyl)-2-chloropyridine

By chloro--methylpyridine (3.0g, 23.51mmol) be dissolved in carbon tetrachloride (15mL), add N-bromo-succinimide (4.185g, 23.51mmol) and the benzoyl peroxide (100mg) of catalytic amount, heated overnight at reflux, is cooled to room temperature, filters, filtrate concentrates, and residue is directly used in next step.

(2) preparation of 1-((6-chloropyridine-3-base) methyl)-4-ethyl piperazidine

At NEP (2.685g, 23.51mmol) tetrahydrofuran solution (50mL) in, add diisopropylethylamine (6.065g, 47.02mmol) and 5 (bromomethyl)-2-chloropyridine (4.854g, 23.51mmol), continuing reaction 4 hours under room temperature, reaction completes.Concentration, add ethyl acetate (50mL) and water (30mL), separatory, aqueous phase ethyl acetate (30mL) extracts, merging organic facies, concentration, residue is through column chromatography for separation (petroleum ether: ethyl acetate=1:1), obtain title compound (4.0g, productivity 71.4%).

(3) preparation of N-(5-((4-ethyl piperazidine-1-base) methyl) pyridine-2-base) the fluoro-4-of-5-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-amine

nullBy fluoro-for 5-4-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-amine (100.0mg，0.351mmol)，1-((6-chloropyridine-3-base) methyl)-4-ethyl piperazidine (83.5mg，0.351mmol) it is dissolved in 1,In 4-dioxane (15mL)，Add three (dibenzalacetone) two palladium (32mg，0.035mmol)，2-dicyclohexylphosphontetrafluoroborate-2,4,6-tri isopropyl biphenyl (33.5mg，0.07mmol) with cesium carbonate (342mg，1.05mmol)，Nitrogen is replaced，Heating is to 110 DEG C，Reaction is overnight，LC-MS detects reaction and completes，By reacting liquid filtering，Concentration，Residue obtains title compound (28mg through silica gel column chromatography (dichloromethane: methanol=15:1)，Productivity 16.4%).

Molecular formula: C₂₇H₃₃FN₈Molecular weight: 488.6LC-MS (m/z): 489.4 (M+H⁺)

¹H-NMR(400MHz,CDCl₃)8.75 (s, 1H), 8.21 (s, 1H), 8.16 (s, 1H), 8.04-8.07 (m, 2H), 7.60 (d, J=8.4Hz, 1H), 7.38 (s, 1H), 7.27-7.29 (m, 1H), 4.70-4.77 (m, 1H), 3.49 (s, 2H), 2.77 (s, 3H), 2.36-2.74 (m, 10H), 1.69 (d, J=6.8Hz, 6H), 1.10-1.16 (m, 3H).

Embodiment 4N-(4-((4-ethyl piperazidine-1-base) methyl) phenyl) the fluoro-4-of-5-(1-isopropyl-2-methyl isophthalic acid Hydrogen-imidazo [4,5-b] pyridine-6-base) preparation of pyrimidine-2-amine (compound 4)

(1) preparation of the fluoro-4-iodine pyridine of 2,5-bis-

Measure diisopropylamine (17mL; 122mmol) join in THF (220mL); it is cooled to-20 DEG C, under nitrogen protection, is slowly added to n-BuLi (49mL, 122.5mmol); after reinforced;-20 DEG C of stirring 0.5h, are cooled to-78 DEG C, are slowly added dropwise 2; 4h is stirred at this temperature after the tetrahydrofuran solution (30mL) of 5-difluoro pyridine (13.3g, 115mmol).Weigh iodine (32g, 126mmol) and be dissolved in THF (100mL), be slowly added dropwise in above-mentioned reactant liquor at-78 DEG C, after dropwising, stir 1h.Room temperature is risen to after adding water (10mL) and THF (30mL), add saturated sodium thiosulfite, it is layered to obtain organic facies, aqueous phase ethyl acetate (3 × 100mL) extracts, merge organic facies, anhydrous sodium sulfate dries, and silica gel column chromatography (PE:EA=50:1) obtains title compound (13.5g, productivity 48%).

(2) preparation of the iodo-PA of the fluoro-4-of 5-

2,5-bis-fluoro-4-iodine pyridines (12.05g, 50mmol) are dissolved in DMSO (40mL), under stirring condition, add ammonia (40mL), when 90 DEG C of tube sealings, stir 12h.Adding ethyl acetate (150mL), be layered to obtain organic facies, anhydrous sodium sulfate dries, and silica gel column chromatography (DCM:MeOH=30:1) obtains title compound (5.95g, productivity 50%).

(3) preparation of 4-bromobenzyl bromine

Being dissolved in carbon tetrachloride (20mL) by 4-methyl bromobenzene (2.0g, 11.7mmol), add N-bromo-succinimide (2.08g, 11.7mmol), heating, to 70 DEG C, is reacted 4 hours, and concentration, residue is directly used in next step.

(4) preparation of 1-(4-bromophenyl)-4-ethyl piperazidine

By 4-bromobenzyl bromine (2.93g, 11.7mmol), NEP (3.34g, 29.2mmol) is dissolved in 50mL oxolane, drips diisopropylethylamine (3.02g, 23.4mmol), react overnight under room temperature under ice-water bath.Concentration, adds 100mL ethyl acetate and 50mL water, separatory, concentration, residue column chromatography for separation (petroleum ether: ethyl acetate=2:1), obtains title compound (1.5g, productivity 45.28%).

(5) preparation of N-(4-((4-ethyl piperazidine-1-base) methyl) phenyl) the fluoro-4-of-5-(1-isopropyl-2-methyl isophthalic acid hydrogen-imidazo [4,5-b] pyridine-6-base) pyrimidine-2-amine

nullThe fluoro-4-of 5-(1-isopropyl-2-methyl isophthalic acid H-imidazo [4,5-b] pyridine-6-base) pyridine-2-amine (100.0mg，0.351mmol)，1-(4-bromophenyl)-4-ethyl piperazidine (99mg，0.351mmol) it is dissolved in 1,In 4-dioxane (15mL)，Add three (dibenzalacetone) two palladium (32mg，0.035mmol)，2-dicyclohexylphosphontetrafluoroborate-2,4,6-tri isopropyl biphenyl (33.5mg，0.07mmol) with cesium carbonate (342mg，1.05mmol)，Nitrogen is replaced，Heating is to 110 DEG C，Reaction is overnight，LC-MS detects reaction and completes，By reacting liquid filtering，Concentration，Residue obtains title compound (44mg through silica gel column chromatography (dichloromethane: methanol=15:1)，Productivity 25.7%).

Molecular formula: C₂₈H₃₄FN₇Molecular weight: 487.6LC-MS (m/z): 488.3 (M+H⁺)

¹H-NMR(400MHz,DMSO-d₆)9.15 (s, 1H), 8.51 (s, 1H), 8.31 (s, 1H), 8.23 (s, 1H), 7.60 (d, J=8.4Hz, 2H), 7.16 (d, J=8.0Hz, 2H), 7.01 (d, J=4.2Hz, 1H), 4.80-4.84 (m, 1H), 3.37 (s, 2H), 2.65 (s, 3H), 2.32-2.49 (m, 10H), 1.58 (d, J=6.8Hz, 6H), 0.92-0.98 (m, 3H).

Claims

1. logical compound shown in formula I, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer,

Wherein

A¹、A²And A³It is independently selected from nitrogen or carbon；

R⁴Selected from halogen or hydrogen；

R³Selected from optionally by Q²3～8 yuan of heterocyclic radicals replaced, 6～14 yuan of condensed hetero ring bases, 5～8 yuan of heteroaryls, 6～14 yuan of thick heteroaryls, phenyl, naphthyl, 6～12 yuan of bridge heterocyclic radicals or 6～12 yuan of spiro heterocyclic radicals；

N is selected from the integer of 0～3.

2. the compound of claim 1, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

A¹、A²And A³It is independently selected from nitrogen or carbon；

R¹Selected from C_1-4Alkyl or C_1-4Alkoxyl；

R⁴Selected from halogen；

R³Selected from optionally by Q²5～7 yuan of heterocyclic radicals replaced, 6～11 yuan of condensed hetero ring bases, 6～11 yuan of bridge heterocyclic radicals or 6～11 yuan of spiro heterocyclic radicals；

Q²Selected from amino, hydroxyl, trifluoromethyl, cyano group, C_1-4Alkyl, C_1-4Alkoxyl, C_1-4Alkyl sulphonyl, C_1-4Alkyl sulfonyl amino, 5～6 yuan of heterocyclic radicals or 7～9 yuan of bridge heterocyclic radicals；

N is selected from the integer of 0～3.

3. compound as claimed in claim 2, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

A¹、A²And A³It is independently selected from nitrogen or carbon；

R¹It it is isopropyl；

R⁴It it is fluorine；

R³Selected from optionally by Q²5～6 yuan of heterocyclic radicals replaced, Q²Selected from amino, hydroxyl, trifluoromethyl, cyano group, C_1-4Alkyl, C_1-4Alkoxyl, C_1-4Alkyl sulphonyl, C_1-4Alkyl sulfonyl amino, 5～6 yuan of heterocyclic radicals or 7～9 yuan of bridge heterocyclic radicals；

N is selected from the integer of 0～2.

4. compound as claimed in claim 3, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

R³Selected from optionally by Q²5～6 member heterocyclic ring containing nitrogen bases replaced, Q²Selected from amino, hydroxyl, trifluoromethyl, cyano group, C_1-4Alkyl or C_1-4Alkoxyl；

N is 1.

5. compound as claimed in claim 4, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, wherein

N is 1.

6. compound as claimed in claim 1, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer, described compound is selected from:

7. pharmaceutical composition, comprises the compound described in claim 1-6 any claim, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer and one or more pharmaceutical carriers.

null8. pharmaceutical composition as claimed in claim 7，Also can contain one or more antitumor agents and immunosuppressant，Described antitumor agent and immunosuppressant，Include but not limited to methotrexate、Capecitabine、Gemcitabine、Doxifluridine、Pemetrexed disodium、Pazopanib、Imatinib、Erlotinib、Lapatinib、Gefitinib、ZD6474、Trastuzumab、Bevacizumab、Trastuzumab、Bevacizumab、Rituximab、Herceptin、Paclitaxel、Vinorelbine、Docetaxel、Doxorubicin、Hydroxy camptothecin、Mitomycin、Epirubicin、Pirarubicin、Bleomycin、Letrozole、Tamoxifen、Fulvestrant、Music score of Chinese operas Rayleigh、Flutamide、Leuprorelin、Anastrozole、Ifosfamide、Busulfan、Cyclophosphamide、Carmustine、Nimustine、Semustine、Chlormethine、L-Sarcolysinum、Chlorambucil、Carboplatin、Cisplatin、Oxaliplatin、Network platinum、Topotecan、Camptothecine、Topotecan、Everolimus、Sirolimus、Special cancer is fitted、6 purinethols、6 thioguanine、Azathioprine、Rhzomorph D、Daunorubicin、Amycin、Mitoxantrone、Bleomycin A5、Plicamycin or aminoglutethimide.

9. the compound described in claim 1-6 any claim, its pharmaceutically acceptable salt, its ester, its solvate and their stereoisomer are used for the application treated and/or prevent in the medicine of the cancer-related diseases kinase mediated by CDK in preparation, the disease that described cancer is relevant is selected from cerebroma, pulmonary carcinoma, squamous cell cancer, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, cancer of pancreas, breast carcinoma, head and neck cancer, cervical cancer, carcinoma of endometrium, rectal cancer, hepatocarcinoma, renal carcinoma, adenocarcinoma of esophagus, esophageal squamous cell carcinoma, carcinoma of prostate, female reproductive tract cancer, cancer in situ, lymphoma, neurofibroma, thyroid carcinoma, osteocarcinoma, skin carcinoma, colon cancer, carcinoma of testis, gastrointestinal stromal tumor, tumor of prostate, mast cell tumor, multiple myeloma, melanoma, glioma or sarcoma.