CN107383019B - Pyrazolo [4,3-h] quinazoline compounds and application thereof - Google Patents

Abstract

The present invention relates to pyrazolos [4 shown in a kind of following general formula I, 3-h] quinazoline derivative and application thereof, such compound has the activity for inhibiting different subtype such as CDK4/6 in cyclin dependant enzyme family (CDKs), provides new means to prevent and treat cancer, metabolism and immunological diseases, cardiovascular disease and neurogenic disease.

Description

Pyrazolo [4,3-h] quinazoline compounds and application thereof

Technical field

The present invention relates to a kind of pyrazolo [4,3-h] quinazoline derivatives and application thereof.

Background technique

Cyclin dependant enzyme family (cyclin dependent kinases, CDKs) is the driving cell cycle The engine of operation is the core of entire regulated and control network.CDKs is that a kind of serine (serine)/threonine (threonine) swashs Enzyme plays an important role in cell cycle regulating, transcription, differentiation and process of cell death.13 have been had been acknowledged at present CDK member (CDK1-13) and corresponding thereto have 12 cyclins (cyclin) (A-L).It can be incited somebody to action according to function CDK family is divided into two major classes.A kind of CDK is to form complex in conjunction with corresponding cyclin and be activated and then participate in cell week The conversion of phase each phase.According to classical cell cycle model, D class cyclin and CDK4 or CDK6 combine Phase early stage G1, CDK2 and cyclin E can trigger the cell cycle and enter the S phase, and CDK2-cyclin A and CDK1-cyclin A tune The completion of S phase is controlled, CDK1-cyclin B is responsible for the division of cell.And in addition one kind CDK family member include CDK7, CDK8, CDK9, CDK10 and CDK11 etc. then take part in the transcriptional control of cell and play an important role in transcriptional regulatory.In addition, certain It is special that a little CDK also have the function of, as CDK5 has the function of adjusting nervous system.It is now recognized that playing tune in the cell cycle The mainly CDK1-4 and CDK6-7 of section effect.But many genetic evidences disclose the CDK of interphase in cell division phase such as recently CDK2, CDK4 and CDK6 be not necessary to normal cell proliferation, but what certain specific cells proliferation was relied on.CDK1 is then It is necessary in fission process.Meanwhile more and more researchs also indicate that, the proliferation of tumour cell relies on certain specific Interkinesis phase CDK.This provides theoretical basis without the research and development suspected of CDK inhibitor, i.e., by inhibiting tumour cell to increase The CDK for growing the certain specific phases relied on reaches antitumor action and does not influence the proliferation of normal cell.

Cyclin is a protein family greatly, is divided into four grades (A, B, D, E type cell cycle Albumen), the CDK- period holoenzyme of effect is as adjusting subunit.At present it has been confirmed that there are 25 kinds of cyclins (cyclin), mainly including CyclinB1, CyclinA, CyelinE, CyelinD1, CyclinD2 and CyclinD3 etc..CDKs Only its corresponding cyclin combines, and is just had its threonine residues phosphorylation by CAK (activity factor of CDK) Activity.CDKs with respective specific substrate (cyclin) mainly by combining the protein kinase for forming state of activation compound Object, so that catalysis substrate (RB albumen) phosphorylation, interferes and controls cell cycle progression, be sequentially completed the duplication synthesis of DNA with Mitosis causes the division growth of cell.

Generation, the development of malignant tumour are a complicated processes, and traditional therapeutic agent mainly divides alkylating agent, antimetabolic The shortcomings that several classes such as class drug, natural products and antibiotic, this few class drug is that curative effect is unobvious, and toxic side effect is big.It is small at present For molecule kinase inhibitor as potential cancer therapy, its development, which is one, allows the interested field of people, mutation and certain The unconventionality expression of CDKs and the corresponding position that they are controlled occur with uncontrolled tumour and are proliferated related.

Normal cell was replicated since G1 (DNA pre-synthesis phase), (thin by S (DNA synthesizes the phase), G2 (prophase), M Born of the same parents' division stage), mitotic stimulation is the switch for starting this process, and it is compound that these stimulations adjust CDK/ cyclin The formation of body and inducing expression in the G1 phase.Main in cell cycle there are two check points, one of them is located at the boundary G1-S Place, this is the key point that intraor extracellular information is transmitted, integrates, collected and regulated and controled to breeding.CDK4 and CDK6 have 71% amino acid identities have very big overlapping function, and the two biochemical property is similar, often plays a role jointly.Not With in degree, both can be used cooperatively with all three D type cyclins (D1, D2 and D3).Pass through mitosis After signal path activation, D type cyclin is in conjunction with CDK4 or CDK6.CDK activated protein kinase (CAK) promotes cdk4/6- CyclinD compound phosphorylation activation, inactivates tumor suppressor protein, leads to genetic transcription and cell cycle of the G1 phase to the S phase Process eventually leads to cell division.There are three the protein products of different relative molecular masses for Rb gene, wherein 110 be opposite Low phosphorylation type, 112,114 be high phosphorylation type, and low phosphorylation type is activated state, inhibits cell to grow in G0 the and G1 phase.

It is observed in the cancer of many types, the imbalance of CyclinD-CDK4/6-INK4-Rb approach causes proliferation to add Speed.Can be by various mechanism activation approach, including gene magnification or rearrangement, the forfeiture of negative regulator, epigenetic change and Point mutation in critical path ingredient.CyclinD-CDK4/6-INK4-Rb approach is the key that G1-S transition point of adjustment.CDK4/ 6 activity are (special by INK4 protein family (p16INK4A, p15INK4B, p18INK4C and p19INK4D) and Cip and Kip family Not p21CIP1 and p27KIP1) regulation.INK4 albumen inhibits the cyclinD-CDK4/6 living by direct in conjunction with CDK Property.P21CIP1 and p27KIP1 can stablize Cyclin D1-CDK4/6 compound, and all have under certain conditions CDK4/6 inhibitory activity.Have Rb in most cancer cells, these Rb+ cancers can by CDK4/6-cyclinD overexpression Lai CDK4/6-cyclinD feminine gender regulatory factor is activated, or promotes the oncogenic signals access of CDK4/6-cyclinD.Promote CDK4/6- The active approach of cyclinD has phosphatidyl-inositol 3-kinase (PI3K)/AKT/ rapamycin (mTOR), and mitrogen-activated protein swashs Enzyme (MAPK), wnt/b-catenin signal path, janus kinases (JAK)-signal transduction and activating transcription factor (STAT), it is living Nuclear factor kappa-light chain reinforcing agent and steroid hormone signal path (such as estrogen, the progesterone, and male of the B cell (NF-jB) of change Hormone).These approach work in CDK4/6-cyclinD signal transduction point.Encoding cyclin D1 (CCND1), CDK4 The gene of CDK6 amplification or encode p16INK4A (cell cycle protein dependent kinase inhibitor 2A [CDKN2A]) position The missing of point is to activate the main mechanism of cyclinD-CDK4/6-INK4-Rb approach.For example, some researches show that in breast cancer CCND1, CDK4 and CDK6 expand 17%, 4%, 3% and CDKN2A decline 21% respectively.Other research reports of breast cancer CDK4 expands 16%, CDK6 and expands 17%, p16INK4A loss 49%.CCND1 amplification is up to 35% in breast cancer case, And cyclinD is more than 50%.The amplification ratio of CCND1 is head and neck cancer (26-39%) respectively, non-small cell lung cancer (NSCLC；5- 30%), carcinoma of endometrium (26%) and cancer of pancreas (25%).

Since CDK4/6 activity is adjusting importance and the mechanism that is activated in cancer of the known approach in cell, CDK4/6 inhibitor becomes a kind of attractive treatment means.One main theoretical question is, CDKs normal cell with And play a crucial role in the proliferation of cancer cell, it may cause therapeutic window stenostomia, toxicity will affect clinical levels.So far Until, the most comprehensive general CDK inhibitor of research is flavopiridol, and clinical effectiveness is limited, and partly cause is its complexity Pharmacokinetics and side effect.In normal cell-cycle, cyclinD1 by acting synergistically with CDK4, by pRb approach and P53 approach joint effect cell cycle progression, P53-P21-pRb approach and P16-pRb approach are two known cell ageings Coherent signal approach.However, the overexpression of CCND1- gene is likely to result in cell cycle confusion and unregulated cell growth, into And lead to tumour.CyclinD1 is the important albumen of CCND1- gene coding.P16 gene is one of cell cycle base This gene, the albumen negative regulator cell Proliferation of expression, therefore it is a kind of intracorporal antioncogene.The albumen that it is encoded can be with CyclinD1 competitive binding CDK4/6 inhibits protein kinase activity, prevents pRb from phosphorylation and keep the state of activation, to thin Born of the same parents play negative regulation the period, and the variation of P16 gene or other protein inactivations will lead to cyclinD-CDK4/6-pRb-E2F tune Section approach it is out of control, lead to tumour.The research different from the past of CDK4/6 inhibitor acts on signal transduction molecules upstream Anti-tumor drugs targeting, not from the source situation monitoring cell cycle, but cell Proliferation is just prevented in the G1 phase, to reach Inhibit the purpose of tumor proliferation.INK4 belongs to one kind of CDK kinase inhibition albumen (CDIs), and CDIs can negative regulation CDK-Cyclin Compound, INK4 family include P16INK4a, P15INK4b, P18INK4c and P19INK4d, their main function is suppression The activity of CDK4 and CDK6 processed.There is Rb in most of tumours, and the exception of cdk4/6-cyclinD-INK4-Rb access is also general Store-through exists, the form of expression are as follows: (1) P16INK4a gene delection or DNA methylation cause P16INK4a activity to lack；(2)CDK4 Gene magnification or point mutation cause to lose with the binding ability of P16INK4a；(3) cyclinD1 is due to gene rearrangement or gene It expands and over-expresses.Due to the effect in cell Proliferation, research finds that it can be used as the native target for the treatment of of cancer, and And it has two big advantages: (1) most cells proliferation relies on cdk2 or cdk4/6, but cdk4/6 inhibitor will not be shown again The cytotoxicity of " general cdk4/6 inhibitor " institute's band.(2) studies have shown that cyclinD level increases, cell can be increased to drug Sensibility.So there is good prospect using cdk4/6 as target.

Many N- heterocyclic compounds show different bioactivity, are also successfully applied to the medicine based on pesticide Conduct industry.There are a large amount of heterocycles exploring at present, this is based on such as imidazoles, thiazole, piperidines, pyridine, pyrroles, indoles, purine, quinoline Compound of the heterocycles such as quinoline, isoquinolin, such as pyrazolopyrimidine, indazole, Pyrazolopyridine, Pyrazolopyridine etc..Pyrazoles and quinoline Oxazoline is condensed may to improve selectivity and sensibility.The newest report of Li et al. people has only compiled synthetic condensation agent pyrazoline derivative Including pyrazolo [1,5-c] quinazoline, the method for pyrazolo [4,3-h] quinazoline and pyrazolo [4,3-f] quinazoline.1978 Year, Vogt etc. (US4112098, publication date: on September 5th, 1978) is obtained about " pyrazolo [1,5-c] quinazoline derivant And related compound " patent.2009, Traquandi etc. (WO2004104007A1, publication date: on December 2nd, 2004) was mentioned Pyrazoloquinazolines derivative and its patent application as kinase inhibitor are handed over.They have synthesized pyrazolo [4,3-h] quinoline Oxazoline derivative, and disclose its anticancer and make the effect of the proliferation activity disorder of cell.2013, Caruso etc. (WO2008074788A1, publication date: on June 26th, 2008), which obtains substituted pyrazolecarboxylic and quinazoline derivant and its be used as kinases, to be pressed down The patent of preparation.

2009, Brasca etc. (US7482354B2, on January 27th, 2009) demonstrated pyrazolo [4,3-h] quinazoline- The CDK inhibitory activity of 3- formamide.The compound shows significant anti-A2780 Proliferation of Human Ovarian Cell proliferation activity, to CDKs It is active.SAR research emphasizes that the carboxylic acid amides (R1) of compound partially shows maximum inhibiting effect, and introduces in R1 larger Group then reduces the activity of CDK2.

Summary of the invention

It is an object of the present invention to design and synthesize a kind of novel pyrazole simultaneously [4,3-h] quinazoline analogs, such New small molecule reactive compound has the biological function for inhibiting cyclin dependant enzyme family (CDKs), to be It finds new treating cancer, metabolism and immunological diseases, cardiovascular disease and neurogenic disease etc. and opens up new way.

Therefore, on the one hand, the present invention provides one kind such as following general formula I compound represented or its is pharmaceutically acceptable Salt,

In general formula I,

R₁What the C1-C8 linear or branched alkyl group or unsubstituted or halogen replaced selected from H, unsubstituted or halogen replaced C3-C6 naphthenic base；Preferably, R₁The C1-C6 linear or branched alkyl group that replaces selected from H, unsubstituted or halogen or it is unsubstituted or The C3-C5 naphthenic base that halogen replaces；It is highly preferred that R₁The C1-C3 linear or branched alkyl group replaced selected from H, unsubstituted or halogen Or the C3-C5 naphthenic base that unsubstituted or halogen replaces；Wherein, halogen indicates fluorine, chlorine, bromine or iodine；

R₂And R₂' it is each independently selected from H, C1-C8 linear or branched alkyl group；Preferably, R₂And R₂' select each independently From H, C1-C6 linear or branched alkyl group；It is highly preferred that R₂And R₂' it is each independently selected from H, C1-C3 linear or branched alkyl group；

R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, C1-C8 alkyl or C1-C8 Alkoxy；Preferably, R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇Be each independently H, CN, C1-C6 alkyl or C1-C6 alkoxy；It is highly preferred that R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, first Base, ethyl, propyl, butyl, methoxyl group, ethyoxyl or propoxyl group；

A is 0 or 1；

X is straight key ,-CH₂-、-CH₂CH₂Or-CH₂CH₂CH₂-；

R₄For H or C_1-5Alkyl.

In specific example, R₁Selected from H, methyl, ethyl, isopropyl, cyclopenta or trifluoromethyl；

In specific example, R₂And R₂' it is each independently H or methyl；

In specific example, R₃Selected from carboxyl, methoxycarbonyl group, carbethoxyl group, carbamoyl, methyl-carbamoyl, two Methyl-carbamoyl, cyanoaminopyrimidine formoxyl or methoxyformamido base；

In a specific example, a 0；

In a specific example, a 1；

In a specific example, X is straight key；

In a specific example, X is-CH₂-；

In specific example, R₄For H, methyl or ethyl.

Specifically, compounds of formula I can be selected from compound in detail below:

On the other hand, the present invention provides a kind of method for preparing compounds of formula I, and the method passes through following formulas It carries out:

Wherein, in the above reaction equation, R₁、R₂And R₂’、R₃、R₄, a, X with it is defined above identical.

On the other hand, the present invention provides a kind of pharmaceutical composition, comprising the compounds of formula I of therapeutically effective amount or its Pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

On the other hand, the present invention provides above-mentioned compounds of formula I or its pharmaceutically acceptable salt is used as in preparation Purposes in the drug of CDK4/6 inhibitor.

On the other hand, the present invention provides above-mentioned compounds of formula I or its pharmaceutically acceptable salt to prepare for controlling Treat the purposes in the drug of cancer.

Particularly, the cancer be selected from bladder cancer, breast cancer, colon and rectum carcinoma, kidney, epidermal carcinoma, liver cancer, lung cancer, Cancer of the esophagus, gallbladder cancer, oophoroma, cancer of pancreas, gastric cancer, cervical carcinoma, thyroid cancer, rhinocarcinoma, head and neck cancer, prostate cancer, cutaneum carcinoma, The hematopoietic tumor of Lymphatic System, thyroid follcular carcinoma, is derived from stromal tumours, maincenter or week at medullary system hematopoietic tumor Enclose nervous system neoplasm, melanoma, glioma, seminoma, teratoma, osteosarcoma, xeroderma pitmentosum, angling spine Cytoma, thyroid follcular carcinoma or Kaposi sarcoma.

Particularly, it is thin to be selected from leukaemia, acute lymphatic leukemia, chronic lymphatic for the hematopoietic tumor of the Lymphatic System Born of the same parents' leukaemia, B- cell lymphoma, T- cell lymphoma, Huppert's disease, Hodgkin lymphoma, non-Hodgkin lymphoma, Hairy cell lymphoma or Burkitt's lymphoma.

Beneficial effect

It is right compared with for other cyclin dependent kinases that the compound of the present invention, which is particularly advantageously in them, The more selective inhibitor of CDK4/6.Claimed compound has very strong drug effect and the selection to CDK4/6 Property.This is advantageous in terms of the drug that exploitation is suitable as CDK4/6 inhibitor.

Specific embodiment

Term " pharmaceutically acceptable " refers to when feeding application pharmaceutical formulation and general does not generate allergy Or the molecular entity and composition of similar unsuitable reaction, such as digestive discomfort, dizziness etc..Preferably, art used herein Language " pharmaceutically acceptable " refers to federal regulator or national government approval or United States Pharmacopeia or other generally approve Pharmacopeia lift in animal, be more in particular in used in human body.

" alkyl " used herein refers to linear chain or branched chain saturated hydrocarbyl group.In some embodiments, alkyl group There can be 1 to 10 carbon atom (such as 1 to 8 carbon atom).The example of alkyl group includes methyl (Me), ethyl (Et), third Base (for example, n-propyl and isopropyl), butyl (for example, normal-butyl, isobutyl group, sec-butyl, tert-butyl), pentyl group (for example, N-pentyl, isopentyl, neopentyl), hexyl (for example, n-hexyl and its isomers) etc..Low-grade alkyl group is generally up to 4 Carbon atom.The example of low-grade alkyl group includes methyl, ethyl, propyl (such as n-propyl and isopropyl) and butyl group (example Such as normal-butyl, isobutyl group, sec-butyl, tert-butyl).An alkyl group or two or more alkyl bases in one embodiment Group can form the alkyl group of bridging.I.e. wherein alkyl group leads to through another group connection (being especially shown in cyclic group) It crosses alkyl chain bridging and forms ring, that is, form the fused rings of bridging.

As used herein, " naphthenic base " refers to non-aromatic carbocyclic groups, including cyclic alkyl, alkenyl and alkynyl group. Group of naphthene base can be monocycle (such as cyclohexyl) or polycyclic (for example, comprising condensed, bridging and/or spiro ring system), wherein Carbon atom is located inside or outside ring system.Group of naphthene base can have 3 to 14 annular atoms (for example, 3 to 8 as a whole Carbon atom is used for polycyclic naphthene base group for monocyclic cycloalkyl group and 7 to 14 carbon atoms).Any of group of naphthene base fits Position can be covalently attached with defined chemical structure on suitable ring.The example of group of naphthene base includes cyclopropyl, cyclobutyl, ring penta Base, cyclohexyl, suberyl, cyclopentenyl, cyclohexenyl group, cyclohexadienyl, cycloheptatriene base, bornyl, norpinyl, Norcaryl, adamantyl and spiral shell [4.5] decyl and its homologue, isomers etc..

The present invention includes the pharmaceutically acceptable compound isotopically labelled of whole of compound of Formula I of the invention, The atom replacement that middle one or more atom is had same atoms number, but atomic mass or mass number and be commonly found in nature Atomic mass or mass number are different.

The isotope example being suitably included in the compounds of this invention includes the isotope of hydrogen, such as²H and³H, carbon, example Such as¹¹C、¹³C and¹⁴C nitrogen is for example¹³N and¹⁵N, oxygen is for example¹⁵O、¹⁷O and¹⁸O。

It is with heavier isotope such as deuterium²H substitution can provide certain treatment advantages, there is better metabolic stability, For example, Half-life in vivo has increased or decreased volume requirements, and therefore preferred in some cases.

The synthetic method of the above compound 1-56 of embodiment detailed description of the present invention will be passed through below.

Prepare embodiment

The preparation of intermediate

1- ethyl -7- oxo -4,5,6,7- tetrahydro -1-H- indazole -3- carboxylic acid, ethyl ester

To (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate (10g, Ethyl hydrazine oxalates (6.225g, 41.7mmol) and sodium acetate is added dropwise in acetum (50ml) 41.7mmol) The acetum (50ml) of (6.81g, 83.4mmol), reacts 2h at room temperature.Water (18ml) and ethyl acetate (15ml) is added, point Liquid, aqueous layer with ethyl acetate (15 × 2ml) extraction, merges organic layer, and through saturated common salt water washing, anhydrous sodium sulfate is dry, filter Liquid is concentrated under reduced pressure, and residue is separated through column chromatography (eluant, eluent: methylene chloride: anhydrous methanol=20:1), obtains faint yellow solid (4.9g, 49.8%).MS(ESI)(m/z):237.4(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) δ: 4.62 (q, J=7.2Hz, 2H), 4.43 (q, J=7.12Hz, 2H), 3.04 (t, J=6.12Hz, 2H), 2.58 (t, J=6.16Hz, 2H), 2.15 (m, 2H), 1.43 (q, J=7.32,5H).

6- [(dimethylamino) methylene] -1- ethyl -7- oxo -4,5,6,7- tetrahydro -1H- indazole -3- carboxylic acid, ethyl ester (in Mesosome B1)

By 1- ethyl -7- oxo -4,5,6,7- tetrahydro -1-H- indazole -3- carboxylic acid, ethyl esters (4g, 16.94mmol), dimethyl Formamide (40ml) be added 100ml reaction flask in, after stirring and dissolving be added n,N-Dimethylformamide dimethylacetal (7.6ml, 67.78mmol), 80 lower reaction 8h.Evaporating solvent under reduced pressure, through filtering, filter cake anhydrous methanol, petroleum ether obtain residue Faint yellow solid (4.4g, 89%).MS(ESI)(m/z):300.5(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) δ: 7.59 (s, 1H), 4.70 (m, 2H), 4.42 (q, J=7.08,2H), 3.14 (s, 6H), 2.95 (t, J=3.24,4H), 1.58 (s, 2H), 1.42(m,6H)。

6- [(dimethylamino) methylene] -1- ethyl -7- oxo -4,5,6,7- tetrahydro -1H- indazole -3- carboxylate methyl ester (in Mesosome B2)

In addition to using (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- base) (oxo) methyl acetate to replace Other than (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate, with 6- [(diformazan ammonia Base) methylene] -1- ethyl -7- oxo -4,5,6,7- tetrahydro -1H- indazole -3- carboxylic acid, ethyl ester the similar method of synthetic method Synthetic intermediate B2.

(3- methoxyl group -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate

Addition 3- methoxyl group -2- oxocyclohex -3- alkene (8.4g, 54.5mmol), anhydrous THF are molten in 250ml reaction flask The THF solution (33ml) of 1M LiHMDS is added in -50 DEG C for liquid (50ml), nitrogen protection, drips and finishes room temperature reaction 15min.It is added dropwise Anhydrous THF (50ml) solution of diethy-aceto oxalate (11ml, 81.8mmol) drips and finishes room temperature reaction 8h.Reaction solution is slowly added to In ice water (100ml), control temperature is no more than 20 DEG C, then the dilute hydrochloric acid (50ml) of 1M is added dropwise and is adjusted to pH=5, uses ethyl acetate (100ml × 2) extraction, liquid separation.Merge organic layer, is filtered after anhydrous magnesium sulfate is dry, filtrate decompression is concentrated to give crude product.Use second Acetoacetic ester: petroleum ether (1:1,50ml) recrystallizes crude product, and suction filtration obtains yellow solid (9.7g, 80%).MS-ESI(m/ z):226.2(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) δ: 7.58 (s, 1H), 4.4 (q, J=7.13Hz, 2H), 4.22 (s, 3H),3.12(s,6H),2.77(s,2H),1.40(t,3H)。

1- methyl -7- oxo -4,5,6,7- tetrahydro -1-H- indazole -3- carboxylic acid, ethyl ester

To the acetic acid of (3- methoxyl group -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate (10.3g, 46.0mmol) The acetum (20ml) of methyl hydrazine sulfate (2.44ml, 46.0mmol) is added dropwise in solution (65ml), reacts at room temperature 2h.Add Enter water (100ml) and ethyl acetate (100ml), liquid separation, aqueous layer with ethyl acetate (100 × 2ml) extraction merges organic layer, warp Filter after anhydrous magnesium sulfate is dry, filtrate decompression concentration, residue is through ethyl alcohol: water (1:1,50ml) recrystallization obtains yellow solid (6.85g, 60.2%).MS-ESI(m/z):250.3(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) δ: 4.43 (q, J=7.03Hz, 2H), 4.19 (s, 3H), 2.61 (t, J=6.45Hz, 2H), 1.98 (t, J=6.45Hz, 2H), 1.42 (t, J=7.18Hz, 3H)。

6- [(dimethylamino) methylene] -1- methyl -7- oxo -4,5,6,7- tetrahydro -1H- pyrazoles -3- carboxylic acid, ethyl ester (in Mesosome B3)

By 1- methyl -7- oxo -4,5,6,7- tetrahydro -1-H- indazole -3- carboxylic acid, ethyl esters (6.85g, 27.4mmol), dioxy Six rings ((30ml) be added 100ml reaction flask in, after stirring and dissolving be added n,N-Dimethylformamide dimethylacetal (13.1ml, 54.8mmol), 8h is reacted at room temperature.Evaporating solvent under reduced pressure, residue through column chromatography (eluant, eluent: petroleum ether: ethyl acetate=1: 1) it separates, obtains faint yellow solid (7.27g, 90%).MS-ESI(m/z):305.2(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) and δ: 7.60(s,1H),4.39-4.43(m,2H),4.24(s,3H),3.14(s,6H),2.96(s,1H),2.89(s,1H),2.78 (s,2H),1.42(s,6H),1.40-1.44(m,3H)。

6- [(dimethylamino) methylene] -1- methyl -7- oxo -4,5,6,7- tetrahydro -1H- pyrazoles -3- carboxylic acid, ethyl ester (in Mesosome B4)

In addition to using (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- base) (oxo) methyl acetate to replace Other than (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate, with 6- [(diformazan ammonia Base) methylene] -1- methyl -7- oxo -4,5,6,7- tetrahydro -1H- pyrazoles -3- carboxylic acid, ethyl ester the similar method of synthetic method Synthetic intermediate B4.

Bis- Boc- of 1,3- [5- (4- thyl-piperazin -1- base) methyl] pyridine -2- guanidine (intermediate A 1)

By [5- (4- thyl-piperazin -1- base) methyl] pyridine -2- amine (2g, 10.41mmol), methylene chloride (30ml), three Ethamine (1.26g, 12.50mmol) is added in 100ml reaction flask, and 1,3-, bis--Boc-2- (trifluoromethyl sulfonyl) guanidine is added Methylene chloride (10ml) solution of (4.48g, 11.45mmol), stirring and dissolving react at room temperature 3 days.Water (50ml), dichloro is added Methane (50ml), liquid separation, water layer use methylene chloride (15ml) to extract again, merge organic layer, through saturated common salt water washing, anhydrous sulphur After sour magnesium is dry, filtrate decompression concentration, residue is separated through column chromatography (eluant, eluent: petroleum ether: ethyl acetate=1:3).It obtains Intermediate A 1.

Bis- Boc- of 1,3- [5- (piperazine -1- base) methyl] pyridine -2- guanidine (intermediate A 2)

In addition to using [5- (piperazine -1- base) methyl] pyridine -2- amine to replace [5- (4- thyl-piperazin -1- base) methyl] pyrrole Other than pyridine -2- amine, with the method synthetic intermediate A2 similar with the synthetic method of intermediate A 1.

Bis- Boc-5- of 1,3- (4- ethyl-piperazin -1- base) pyridine -2- guanidine (intermediate A 3)

In addition to using 5- (4- ethyl-piperazin -1- base) pyridine -2- amine to replace [5- (4- thyl-piperazin -1- base) methyl] pyrrole Other than pyridine -2- amine, with the method synthetic intermediate A3 similar with the synthetic method of intermediate A 1.

Bis- Boc-5- of 1,3- (- piperazine -1- base) pyridine -2- guanidine (intermediate A 4)

In addition to using 5- (- piperazine -1- base) pyridine -2-- amine to replace [5- (4- thyl-piperazin -1- base) methyl] pyridine -2- Other than amine, with the method synthetic intermediate A4 similar with the synthetic method of intermediate A 1.

Bis- Boc-5- of 1,3- (4- thyl-piperazin -1- base) pyridine -2- guanidine (intermediate A 5)

In addition to using 5- (4- thyl-piperazin -1- base) pyridine -2- amine to replace [5- (4- thyl-piperazin -1- base) methyl] pyrrole Other than pyridine -2- amine, with the method synthetic intermediate A5 (3.6g, 74.3%) similar with the synthetic method of intermediate A 1.MS(ESI) (m/z):435.9(M+H)⁺。¹H-NMR (CDCl3,400MHz) δ: 8.91 (s, 1H), 7.17 (s, 1H), 6.79 (d, J= 7.50Hz, 1H), 6.65 (d, J=7.10,1H), 2.0 (s, 1H), 3.15 (t, J=7.1Hz, 4H), 2.35 (t, J=7.1Hz, 4H),2.21(s,3H),1.42(s,18H)。

Embodiment 1

- 1- methyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- Ethyl formate (compound 2)

By intermediate A 1 (0.25g, 1.0mmol), intermediate B 3 (0.37g, 1.0mmol), sodium carbonate (0.136g, 1.0mmol) Isosorbide-5-Nitrae-dioxane (5ml) is added in 25ml reaction flask, is heated to reflux to 90 DEG C of reaction 2h.It is cooled to room temperature, it will Reaction solution is transferred to 100ml conical flask, and water (40ml) is slowly added dropwise, and gained precipitating is collected by filtration, dry, obtains faint yellow solid (0.41g, 90%).MS (ESI) m/z:463.9 (M+H)⁺。

Embodiment 2

- 1- methyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- methyl formate (compound 1)

Other than using intermediate B 4 to replace intermediate B 3, synthesized in method identical with the synthetic method of compound 2 Compound 1 (3.90g, 80%) (passes through the SiO of 0-10%MeOH/ dichloromethane eluent₂Chromatogram purification).MS(ESI)(m/z): 449.0(M+H)⁺。

Embodiment 3

- 1- methyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- formic acid sodium salt

By 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] -1- methyl -4,5- dihydro-1 h-pyrazole And 100ml is added in [4,3-h] quinazoline -3- Ethyl formate (compound 2) (1.5g, 3.2mmol), 95% dehydrated alcohol (40ml) In reaction flask, sodium hydroxide (0.4g, 1mmol) is added after stirring and dissolving, 90 DEG C of heating reflux reaction 3h.It is cooled to room temperature, analyses Solid out filters, and filter cake dehydrated alcohol (10ml), petroleum ether (1ml) wash, dry, obtains white solid.

Embodiment 4

- 1- methyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- formamide (compound 3)

By 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] -1- methyl -4,5- dihydro-1 h-pyrazole And 10ml reaction flask is added in [4,3-h] quinazoline -3- formic acid sodium salt (0.02g, 0.04mmol), dimethylformamide (1.6ml) In, methanol (0.4ml) solution of 50% sodium methoxide (0.024g, 0.44mmol) is added into reaction flask, stirs and evenly mixs, finally plus Enter formamide (0.06g, 1.33mmol), is stirred overnight at room temperature.Water (2ml) is slowly added dropwise into reaction solution, gained is collected by filtration Precipitating, it is dry, faint yellow solid (3.90g, 80%) is obtained (by the SiO of 0-10%MeOH/ dichloromethane eluent₂Chromatographically pure Change).MS(ESI)(m/z):434.0(M+H)⁺。

Embodiment 5

- 1- methyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- (N-METHYLFORMAMIDE) (compound 4)

By 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] -1- methyl -4,5- dihydro-1 h-pyrazole And [4,3-h] quinazoline -3- formic acid (9.5g, 19.561mmol) be added to anhydrous tetrahydro furan and dimethylformamide (1:1, In 50ml)；The THF solution (21.12ml, 42.24mmol) of 2M methylamine, I-hydroxybenzotriazole (5.332g, 39.458mmol) It is added sequentially in above-mentioned solution with methylene chloride (7.567g, 39.473mmol), reaction mixture reacts 10h at room temperature. Reaction mixture is poured into water (100ml) and is then extracted 4 times with methylene chloride (250ml).Organic layer is washed with saturated common salt, is used It is spin-dried for after sodium sulphate is dry.Crude product obtains product by column chromatographic purifying (polarity: methylene chloride: methanol=90:5) (3.90g, 80%) (passes through the SiO of 0-10%MeOH/ dichloromethane eluent₂Chromatogram purification).MS(ESI)(m/z):448.0(M+ H)⁺。

Embodiment 6

- 1- methyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- (N,N-dimethylformamide) (compound 5)

By 8- [[5- (4- methylpiperazine-1-yl) methyl] pyridine -2- base amino] -1- methyl -4,5- dihydro-1 h-pyrazole And 50ml reaction flask is added in [4,3-h] quinazoline -3- formic acid (0.2g, 0.44mmol), dimethylformamide (5ml), stirs molten Dichloroethanes (0.2mg, 1mmol) is added after solution, I-hydroxybenzotriazole (HOBT) (0.2g, 1.4mmol).After reacting 2h, to Dimethylamine (3ml) is added in bottle, the reaction was continued 3h.Water (5ml), methylene chloride (2ml) is added, liquid separation, water layer uses dichloromethane again Alkane (2ml) extraction, merges organic layer, and through saturated common salt water washing, crude product passes through column chromatographic purifying after anhydrous sodium sulfate is dry (polarity: methylene chloride: methanol=90:5) obtains product, obtains solid product (3.90g, 80%) and (passes through 0-10%MeOH/ bis- The SiO of chloromethanes elution₂Chromatogram purification).MS(ESI)(m/z):462.0(M+H)⁺。

The synthesis of compound 6-47

Different intermediates is respectively adopted, according to the synthetic method similar with above compound 1 or 2, synthesizes in following table 1 Compound.

Table 1

According to the synthetic method similar with above compound 3-5, amination is carried out to the compound in table 1 and obtains its correspondence Formamide and methyl replace or dimethyl replace carboxamide product (compound 8-10,13-15,18-20,22-24,26- 27,30-32,35-37,40-42 and 45-47).

Embodiment 7

8- [5- (4- methylpiperazine-1-yl)-pyridine -2- base amino] -1- ethyl -4,5- dihydro-1 h-pyrazole simultaneously [4,3- H] quinazoline -3- cyanamide (compound 48) synthesis

By 8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1- ethyl -4,5- dihydro-1 h-pyrazole simultaneously [4, 3-h] quinazoline -3- carboxylic acid sodium salt (0.2g, 0.44mmol), n,N-Dimethylformamide (2ml) addition 10ml reaction flask, stirring Methylene chloride (0.2mg, 0.10mmol) is added after dissolution, after HOBT (0.2g, 0.14mmol) reacts 1.2h, is added into bottle Cyanamide (0.028mg, 0.67mmol), the reaction was continued 3h.Water (2ml), methylene chloride (2ml) is added, liquid separation, water layer uses dichloro again Methane (2ml) extraction, merges organic layer, and through saturated common salt water washing, crude product is chromatographed pure by column after anhydrous sodium sulfate is dry Change (polarity: methylene chloride: methanol=90:5) and obtains product.It obtains product 11 (0.17g, 0.84% yield).MS (ESI) m/z: 459.6(M+H)⁺。

Embodiment 8

8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1- ethyl -4,5- dihydro-1 h-pyrazole simultaneously [4,3- H] quinazoline -3- formic acid methoxy amide (compound 49) synthesis

By 8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1- ethyl -4,5- dihydro-1 h-pyrazole simultaneously [4, 3-h] quinazoline -3- carboxylic acid sodium salt (0.2g, 0.44mmol), dimethylformamide (2ml) addition 10ml reaction flask, stirring and dissolving Dichloroethanes (0.2mg, 1.0mmol) is added afterwards, after HOBT (0.2g, 1.4mmol) reacts 1,2h, after alkali tune is added into bottle Methoxamine hydrochloride (22mg, 26mmol), the reaction was continued 3h.Water (2ml), methylene chloride (2ml) is added, liquid separation, water layer is again It is extracted with methylene chloride (2ml), merges organic layer, through saturated common salt water washing, crude product passes through column after anhydrous sodium sulfate is dry Chromatographic purifying (polarity: methylene chloride: methanol=90:5) obtains product and obtains product (0.014g, 69%).MS(ESI) 464.9m/z:(M+H)⁺。

Embodiment 9

8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1- ethyl -4,5- dihydro-1 h-pyrazole simultaneously [4,3- H] quinazoline -3- acetamide (compound 50) synthesis

By 8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1- ethyl -4,5- dihydro-1 h-pyrazole simultaneously [4, 3-h] quinazoline -3- carboxylic acid sodium salt (0.2g, 0.44mmol), dimethylformamide (5ml) addition 10ml reaction flask, stirring and dissolving Dichloroethanes (0.2mg, 0.10mmol) is added afterwards, after HOBT (0.2g, 1.4mmol) reacts 1,2h, ethamine is added into bottle (3ml), the reaction was continued 3h.Water (5ml), methylene chloride (5ml), liquid separation is added, water layer uses methylene chloride (2ml) to extract again, closes And organic layer, through saturated common salt water washing, crude product analyses purifying (polarity: methylene chloride: first by column after anhydrous sodium sulfate is dry Alcohol=90:5) obtain product (0.015g, 74%).MS (ESI) 464.9m/z:(M+H)⁺。

Embodiment 10

- 1,4- dimethyl -4,5- dihydro-1 h-pyrazole is simultaneously by 8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] The synthesis of [4,3-h] quinazoline -3- Ethyl formate (compound 54)

3- methoxyl group -5- methyl-cyclohexyl -2- ketenes

By 5- methyl-1, hydroresorcinol (50g, 0.36mol), methanol (400ml) are added in 1000ml three-necked bottle, stirring The dichloromethane solution (10.75ml) of the titanium tetrachloride (2.0g, 0.01mol) of 1M is added dropwise after dissolution, drop finishes, and reacts at room temperature 8h. Slowly add water (300ml) quenching reaction, then 5% NaHCO is added dropwise₃Solution (about 80ml) is extracted with ethyl acetate (200ml × 3) It takes, liquid separation.Merge organic layer, filtered after anhydrous magnesium sulfate is dry, filtrate decompression concentration obtains pale yellow oily liquid (49.8g, 93%), is directly used in the next step.MS-ESI(m/z):140.2(M+H)⁺。¹H-NMR(CDCl₃,400MHz)δ: 5.34(s,1H),3.67(s,3H),2.25(s,2H),2.18(s,2H),1.05(d,3H)。

5- methyl-cyclohexyl -2- ketenes

500ml tri- is added in 3- methoxyl group -5- methyl-cyclohexyl -2- ketenes (40g, 0.26mol), anhydrous THF (135ml) In neck bottle, the LiAlH of 1M is slowly added dropwise under ice bath for nitrogen protection₄The anhydrous THF solution (91ml) of (3.4g, 0.09mol) is protected Temperature is held at 0-5 DEG C, drop, which finishes, is warmed to room temperature reaction 2h.It is diluted with ethyl acetate (100ml), is slowly added dropwise 2M's under condition of ice bath Dilute hydrochloric acid (about 150ml) quenching reaction controls ph between 8-9, addition ethyl acetate (100ml) and water (100ml), liquid separation, Aqueous layer with ethyl acetate (100 × 2ml) extraction, merges organic layer, filters after anhydrous magnesium sulfate is dry, filtrate decompression concentration, It obtains pale yellow oily liquid (27.1g, 85%), is directly used in the next step.MS-ESI(m/z):111.2(M+H)⁺。¹H-NMR (CDCl₃, 400MHz) and δ: 6.85 (dt, J=9.96Hz, J=4.10Hz, 1H), 6.01 (dt, J=9.96Hz, J=2.05Hz, 1H), 2.26 (s, 2H), 2.22 (dd, J=4.10Hz, J=2.05Hz, 2H), 1.05 (d, 3H).

4- methyl -7- oxa--two ring [4.1.0] heptane -2- ketone

5- methyl-cyclohexyl -2- ketenes (25g, 0.20mol), methanol (200ml) are added in 500ml three-necked bottle, under ice bath 30% hydrogen peroxide (100ml, 1.0mol) is added dropwise, stirs 30min.Be slowly added dropwise again 2% NaOH solution (55ml, 0.027mol), drop finishes reacts overnight at 4 DEG C.Methyl tertiary butyl ether(MTBE) (100ml) and water (100ml) (100 × 2ml) is added, point From organic layer.Evaporating solvent under reduced pressure, the Na of the middle addition 5% of residue (about 80ml)₂S₂O₅Solution (250ml, 0.80mol) is in room Temperature stirring 30min.Be added methyl tertiary butyl ether(MTBE) (100ml) and water (100ml), liquid separation, water layer with methyl tertiary butyl ether(MTBE) (100 × It 2ml) extracts, merges organic layer.Organic layer filters after anhydrous magnesium sulfate is dry, and filtrate decompression concentration obtains colourless oil liquid (22g, 78.5%).MS-ESI(m/z):126.1(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) δ: 3.48 (t, J=4.10Hz, 1H), 3.21 (dt, J=3.74Hz, J=0.92Hz, 1H), 2.63 (d, J=13.77Hz, 1H), 2.02 (d, J=15.53Hz, 1H), 1.81 (m, 2H), 1.11 (s, 3H), 0.92 (d, 3H).

2- methoxyl group -5- methyl-cyclohexyl -2- ketenes

- two ring [4.1.0] heptane -2- ketone (20g, 0.14mol) of 4- methyl -7- oxa- is dissolved in methanol (65ml), is added dropwise Into methanol (10ml) solution of 85% anhydrous potassium hydroxide (7.84g, 0.14mol), 8h is reacted at room temperature, it is anti-to reheat reflux Answer 30min.It is cooled to room temperature, water (100ml) and methyl tertiary butyl ether(MTBE) (50ml), liquid separation, water layer methyl tertiary butyl ether(MTBE) is added (100 × 2ml) extraction, merges organic layer.It is filtered after anhydrous magnesium sulfate is dry, filtrate decompression concentration obtains colourless oil liquid (15.8g, 75% yield).MS-ESI(m/z):141.2(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) and δ: 5.67 (t, J= 4.54Hz, 1H), 3.59 (t, 3H), 2.35 (s, 2H), 2.30 (d, J=4.69Hz, 2H), 1.05 (d, 3H).

(3- methoxyl group -6- methyl -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate

2- methoxyl group -5- methyl-cyclohexyl -2- ketenes (8.4g, 54.5mmol), anhydrous four are added in 250ml reaction flask The THF solution (33ml) of 1M lithium hexamethyldisilazide is added in -50 DEG C for hydrogen furans (THF) solution (50ml) nitrogen protection, Drop finishes room temperature reaction 15min.Anhydrous THF (50ml) solution of diethy-aceto oxalate (11ml, 81.8mmol) is added dropwise, it is anti-to drip complete room temperature Answer 8h.Reaction solution is slowly added in ice water (100ml), control temperature is no more than 20 DEG C, then the dilute hydrochloric acid (50ml) of 1M is added dropwise It is adjusted to pH=5, is extracted with ethyl acetate (100ml × 2), liquid separation.Merge organic layer, filters, filter after anhydrous magnesium sulfate is dry Crude product is concentrated under reduced pressure to obtain in liquid.With ethyl acetate: petroleum ether (1:1,50ml) recrystallizes crude product, and suction filtration obtains pale yellow colored solid Body (9.7g, 80%).MS-ESI(m/z):241.2(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) and δ: 7.58 (s, 1H), 4.43 (q, J=7.13Hz, 2H), 4.22 (s, 3H), 3.12 (s, 6H), 2.77 (s, 2H), 1.40 (t, 3H).

1,4- dimethyl -7- oxo -4,5,6,7- tetrahydro -1-H- indazole -3- carboxylic acid, ethyl ester

To (3- methoxyl group -6- methyl -2- oxocyclohex -3- alkene -1- base) (oxo) ethyl acetate (10.3g, The acetum (20ml) of methyl hydrazine (2.44ml, 46.0mmol), room are added dropwise in acetum (65ml) 46.0mmol) Temperature reaction 2h.Water (100ml) and ethyl acetate (100ml), liquid separation is added, aqueous layer with ethyl acetate (100 × 2ml) extraction is closed And organic layer, it is filtered after anhydrous magnesium sulfate is dry, filtrate decompression concentration, residue is through ethyl alcohol: water (1:1,50ml) recrystallization, It obtains faint yellow solid (6.85g, 60.2%).MS-ESI(m/z):237.3(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) and δ: 4.43 (q, J=7.03Hz, 2H), 4.19 (s, 3H), 2.61 (t, J=6.45Hz, 2H), 1.98 (t, J=6.45Hz, 2H), 1.42 (t, J=7.18Hz, 3H).

6- [(dimethylamino) methylene] -1,4- dimethyl -7- oxo -4,5,6,7- tetrahydro -1H- indazole -3- carboxylic acid second Ester

By Isosorbide-5-Nitrae-dimethyl -7- oxo -4,5,6,7- tetrahydro -1-H- indazole -3- carboxylic acid, ethyl esters (6.85g, 27.4mmol), Tetrahydrofuran (30ml) is added in 100ml reaction flask, and n,N-Dimethylformamide dimethylacetal is added after stirring and dissolving (13.1ml, 54.8mmol), reacts 8h at room temperature.Vacuum distillation remove solvent, residue through column chromatography (eluant, eluent: petroleum ether: Ethyl acetate=1:1) separation, obtain faint yellow solid (7.27g, 90%).MS-ESI(m/z):292.2(M+H)⁺。¹H-NMR (CDCl₃, 400MHz) and δ: 7.60 (s, 1H), 4.39-4.43 (m, 2H), 4.24 (s, 3H), 3.14 (s, 6H), 2.96 (s, 1H), 2.89(s,1H),2.78(s,2H),1.42(d,3H),1.40-1.44(m,3H)。

The synthesis of final product (compound 54)

By 6- [(dimethylamino) methylene] -1,4- dimethyl -7- oxo -4,5,6,7- tetrahydro -1H- indazole -3- carboxylic acid 100ml reaction flask is added in ethyl ester (5.01g, 16.4mmol), intermediate A 5 (3.82g, 16.4mmol), dioxane (40ml) In, it is heated to 90 DEG C of reaction 4h.It is cooled to room temperature, water (50ml) is slowly added dropwise into reaction solution, gained precipitating is collected by filtration, does It is dry, obtain faint yellow solid (6.75g, 90%).MS (ESI) m/z:463.6 (M+H)⁺。

Embodiment 11

8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1,4,4- trimethyl -4,5- dihydro-1 h-pyrazole And the synthesis of [4,3-h] quinazoline -3- Ethyl formate (compound 51)

In addition to using dimethyl -1 5,5-, hydroresorcinol replaces 5- methyl-1, other than hydroresorcinol, with compound The identical method of 54 synthetic method is synthesized.

Embodiment 12

8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1- isopropyl -4,4- dimethyl -4,5- dihydro - The synthesis of 1H- pyrazolo [4,3-h] quinazoline -3- Ethyl formate (compound 52)

In addition to using 5,5- dimethyl-hydroresorcinol instead of 5- methyl-1, hydroresorcinol and using isopropyl Hydrazine replaces being synthesized other than methyl hydrazine in method identical with the synthetic method of compound 54.

Embodiment 13

8- [5- (4- thyl-piperazin -1- base)-pyridine -2- base amino] -1,4,4- trimethyl -4,5- dihydro-1 h-pyrazole And the synthesis of [4,3-h] quinazoline -3- formic acid (compound 53)

Compound 51 (6.25g, 13.2mmol), dehydrated alcohol (40ml) are added in 100ml reaction flask, after stirring and dissolving 95% ethanol solution (13.2ml) of 1.5M potassium hydroxide, heating reflux reaction 3h is added.It is cooled to room temperature, solid is precipitated, take out Filter, filter cake are washed with cold 95% ethyl alcohol (5ml × 3), filter cake are dissolved in 50ml water, and the hydrochloric acid of 1M, pH 3-4, stirring is added dropwise 1h has white solid precipitation, filters, and filter cake is washed with water 3 times, and drying obtains target compound (4.5.0g, 85.3%).MS (ESI) m/z:448.8 (M+H)⁺。

Embodiment 14

The synthesis of compound 55

In addition to using 1,3- cyclopentanedione that 5- methyl-1, hydroresorcinol, and use isopropyl hydrazine is replaced to replace methyl Other than hydrazine, the synthetic method of the synthesis reference aforesaid compound 54 of compound 55.

Embodiment 15

The synthesis of compound 56

Other than using cyclopenta hydrazine to replace isopropyl hydrazine, the synthesis of the synthesis reference aforesaid compound 55 of compound 56 Method.Finally obtain faint yellow solid (6.75g, 90%).MS (ESI) m/z:489.26 (M+H)⁺。

The hydrogen modal data of the compound prepared by above method is listed in table 2 below.

Table 2

EXPERIMENTAL EXAMPLE

Experimental material

CDK2/CycA2(Carna,Cat.No.04-103,Lot.No 06CBS-3024,GST-CDK2(1-298 (end)))

CDK4/CycD3(Carna,Cat.No.04-103,Lot.No 06CBS-3024,GST-CDK2(1-298 (end)))

CDK6/cycD3(eurofins,Cat.No.14-957M,Lot.No.D14SP004NB,GST-CDK4(1- 303end)/GST-CycD3(1-292end))

Peptide FAM-P8(GL Biochem,Cat.No.112396,Lot.No.P100804-XZ112396)

Peptide FAM-P18(GL Biochem,Cat.No.114202,Lot.No.P080319-XY114202)

ATP(Sigma,Cat.No.A7699-1G,CAS No.987-65-5)

DMSO(Sigma,Cat.No.D2650,Lot.No.474382)

EDTA(Sigma,Cat.No.E5134,CAS No.60-00-4)

96 orifice plates (Corning, Cat.No.3365, Lot.No.22008026)

384 orifice plates (Corning, Cat.No.3573, Lot.No.12608008)

Staurosporine(MCE,Cat.No.HY-15141,Lot.No.19340)

Experimental implementation

1. preparing 1x kinases base buffer and stop buffer

1) the 1x kinases base buffer of CDK2, CDK6 are used for

10ml solution, including 50mM HEPES, pH 7.5,0.0015%Brij-35,10mM MgCl are prepared with purified water₂ And 2mM DTT.

2) it is used for the 1x kinases base buffer of CDK4

10ml solution, including 20mM HEPES, pH 7.5,0.01%Triton X-100,10mM are prepared with purified water MgCl₂And 2mM DTT.

3) stop buffer

10ml solution, including 100mM HEPES, pH 7.5,0.015%Brij-35 and 50mM are prepared with purified water EDTA。

2. kinase reaction

1) 2.5x enzyme solutions are prepared

The cdk kinases of 5ug is added in the 1x kinases base buffer of 2.5ml and is configured to enzyme solutions.

2) 2.5x enzyme solutions are prepared

50ug Rb and 10mg ATP is taken to add in 5.0ml 1x kinases base buffer.

3) the DMSO buffer soln of the untested compound of 500nM is prepared

The DMSO solution for first preparing 25uM compound, takes the DMSO solution of the compound of 10ul, is added 90ul's thereto 1x kinases base buffer.Mixing 10min obtains solution of the compound in 10%DMSO.The change of 5 μ l is added on 96 orifice plates Close solution of the object in 10%DMSO.

4) 2.5x enzyme solutions are transferred in assay plate

10 μ l 2.5x enzyme solutions are added into each hole of 96 hole analysis plates.

5) it is incubated at room temperature 10 minutes.

6) 2.5x peptide solution is transferred to assay plate.The 2.5x peptide that 10 μ l are added into each hole of 96 hole assay plates is molten Liquid.

7) kinase reaction terminates

1h is incubated in 28 DEG C of baking ovens.25 μ l stop buffers are added and terminate reaction.It is detected with Caliper instrument.

Show compound 1-56 (concentration 500nM) to the inhibiting rate of CDK2, CDK4 and CDK6 in following table 3.

Table 3

From the above table 3 as can be seen that most compounds of the invention reach 90% or more to the inhibitory activity of CDK4/6, because This, the inhibitory activity of good CDK4/6 is presented in the compound of the present invention, is used as the inhibitor of CDK4/6.

Above embodiments are only exemplary embodiment of the present invention, are not used in the limitation present invention, protection scope of the present invention It is defined by the claims.Those skilled in the art can within the spirit and scope of the present invention make respectively the present invention Kind modification or equivalent replacement, this modification or equivalent replacement also should be regarded as being within the scope of the present invention.

Claims (13)

1. a kind of such as following general formula I compound represented or its pharmaceutically acceptable salt,

In general formula I,

R₁The C3-C6 ring that the C1-C8 linear or branched alkyl group or unsubstituted or halogen replaced selected from H, unsubstituted or halogen replaces Alkyl；Wherein, halogen indicates fluorine, chlorine, bromine or iodine；

R₂And R₂' it is each independently selected from H, C1-C8 linear or branched alkyl group；

R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, C1-C8 alkyl or C1-C8 alcoxyl Base；

A is 0 or 1；

X is straight key ,-CH₂-、-CH₂CH₂Or-CH₂CH₂CH₂-；

R₄For H or C_1-5Alkyl.

2. compound according to claim 1 or its pharmaceutically acceptable salt, wherein R₁Selected from H, unsubstituted or halogen The C3-C5 naphthenic base that substituted C1-C6 linear or branched alkyl group or unsubstituted or halogen replace.

3. compound according to claim 2 or its pharmaceutically acceptable salt, wherein R₁It is taken selected from unsubstituted or halogen The C1-C3 linear or branched alkyl group in generation.

4. compound according to claim 1 or its pharmaceutically acceptable salt, wherein R₂And R₂' be each independently selected from H, C1-C6 linear or branched alkyl group；

R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, C1-C6 alkyl or C1-C6 alcoxyl Base.

5. compound according to claim 4 or its pharmaceutically acceptable salt, wherein R₂And R₂' be each independently selected from C1-C3 linear or branched alkyl group；R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇Be each independently methyl, ethyl, Propyl, butyl, methoxyl group, ethyoxyl or propoxyl group.

6. compound according to claim 1 or its pharmaceutically acceptable salt, wherein

R₁Selected from H, methyl, ethyl, isopropyl, cyclopenta or trifluoromethyl；

R₂And R₂' it is each independently H or methyl；

R₃Selected from carboxyl, methoxycarbonyl group, carbethoxyl group, carbamoyl, methyl-carbamoyl, dimethylamino formoxyl, cyano ammonia Base formoxyl or methoxyformamido base；

A is 0 or 1；

X is straight key or-CH₂-；

R₄For H, methyl or ethyl.

7. compound according to claim 1 or its pharmaceutically acceptable salt, wherein compounds of formula I be selected from Lower compound:

8. a kind of pharmaceutical composition, the compound described in any one of -7 according to claim 1 comprising therapeutically effective amount or its Pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

9. compound described in any one of -7 or its pharmaceutically acceptable salt are used as CDK4/6 in preparation and press down according to claim 1 Purposes in the drug of preparation.

10. compound described in any one of -7 or its pharmaceutically acceptable salt are being prepared for treating cancer according to claim 1 Purposes in the drug of disease.

11. purposes according to claim 10, wherein the cancer be selected from bladder cancer, breast cancer, colon and rectum carcinoma, Kidney, epidermal carcinoma, liver cancer, lung cancer, cancer of the esophagus, gallbladder cancer, oophoroma, cancer of pancreas, gastric cancer, cervical carcinoma, thyroid cancer, rhinocarcinoma, Head and neck cancer, prostate cancer, cutaneum carcinoma, the hematopoietic tumor of Lymphatic System, medullary system hematopoietic tumor, thyroid follcular carcinoma, source In stromal tumours, maincenter or peripheral nervous system neoplasms, melanoma, glioma, seminoma, teratoma, bone Sarcoma, xeroderma pitmentosum, angling prickle cell tumor or Kaposi sarcoma.

12. purposes according to claim 11, wherein it is thin that the hematopoietic tumor of the Lymphatic System is selected from leukaemia, B- Born of the same parents' lymthoma, T- cell lymphoma, Huppert's disease, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma or Burkitt's lymphoma.

13. purposes according to claim 12, wherein the leukaemia is selected from acute lymphatic leukemia or chronic lymphatic Chronic myeloid leukemia.