CN107383019A - Pyrazolo [4,3 h] quinazoline compounds and application thereof - Google Patents

Abstract

The present invention relates to the pyrazolo [4 shown in a kind of below formula I, 3 h] quinazoline derivative and application thereof, such compound has the activity for suppressing different subtype such as CDK4/6 in cyclin dependant enzyme family (CDKs), and new means are provided for prevention and treatment 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 derivative and application thereof.

Background technology

Cyclin dependant enzyme family (cyclin dependent kinases, CDKs) is the driving cell cycle The engine of operation, it is the core of whole regulated and control network.CDKs is that a kind of serine (serine)/threonine (threonine) swashs Enzyme, played an important role in cell cycle regulating, transcription, differentiation and process of cell death.13 are 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 families are divided into two major classes.A kind of CDK is to combine to form complex with corresponding cyclin and be activated and then participate in cell week The conversion of phase each phase.According to the cell cycle model of classics, D classes cyclin and CDK4 or CDK6 combine Phase early stage G1, CDK2 and cyclin E can trigger the cell cycle and enter the S phases, and CDK2-cyclin A and CDK1-cyclin A are adjusted The completion of S phases is controlled, CDK1-cyclin B are responsible for the division of cell.And in addition a kind of CDK family members include CDK7, CDK8, CDK9, CDK10 and CDK11 etc. then take part in the transcriptional control of cell and played an important role in transcriptional regulatory.In addition, certain A little CDK also have special function, as CDK5 has the function of regulation nervous system.It is now recognized that tune is played 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 Necessary to CDK2, CDK4 and CDK6 are not normal cell proliferation, but what some specific cells propagation was relied on.CDK1 is then It is necessary in fission process.Meanwhile increasing research also indicates that, the propagation dependence of tumour cell is some specific Interkinesis phase CDK.This provides theoretical foundation without the research and development suspected of CDK inhibitor, i.e., is increased by suppressing tumour cell The CDK for growing some specific phases relied on reaches antitumor action and does not influence the propagation of normal cell.

Cyclin is a protein family greatly, is divided into four grades (A, B, D, E type cell cycle Albumen), its CDK- cycles holoenzyme acted on is as regulation subunit.Have confirmed at present in the presence of 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 (CDK activity factor) Activity.CDKs is mainly compound by combining to form the protein kinase of state of activation with respective specific substrate (cyclin) Thing, so as to be catalyzed substrate (RB albumen) phosphorylation, disturb and control cell cycle progression, be sequentially completed DNA duplication synthesis with Mitosis, cause the division growth of cell.

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

Normal cell is replicated since G1 (DNA pre-synthesis phases), (thin by S (DNA synthesizes the phase), G2 (prophase), M Born of the same parents' division stage), mitotic stimulation is to start the switch of this process, and these stimulate regulation CDK/ cyclins compound The formation of body and the induced expression in the G1 phases.Mainly there are two check points in cell cycle, one of them is located at G1-S boundaries Place, this key point for being intraor extracellular information transmission, integrating, collecting and regulated and controled to breeding.CDK4 and CDK6 have 71% amino acid identities, there is very big overlapping function, both biochemical properties are similar, often play a role jointly.Not With in degree, both can be used cooperatively with all three D types cyclins (D1, D2 and D3).Pass through mitosis After signal path activation, D type cyclins are combined with CDK4 or CDK6.CDK activated protein kinases (CAK) promote cdk4/6- CyclinD compound phosphorylation activations, inactivate tumor suppressor protein, cause genetic transcription and cell cycle of the G1 phases to the S phases Process, ultimately result in cell division.Rb genes have the protein of three different relative molecular masses, wherein 110 be relative Low phosphorylation type, 112,114 be high phosphorylation type, and low phosphorylation type is activated state, suppresses cell growth in G0 the and G1 phases.

Observed in the cancer of many types, the imbalance of CyclinD-CDK4/6-INK4-Rb approach causes propagation to add Speed.Can be by various mechanism activation approach, including gene magnification or rearrangement, the forfeiture of down regulator, epigenetic change and Point mutation in critical path composition.CyclinD-CDK4/6-INK4-Rb approach is the crucial point of adjustment of G1-S transition.CDK4/ 6 activity are (special by INK4 protein families (p16INK4A, p15INK4B, p18INK4C and p19INK4D) and Cip and Kip families Not p21CIP1 and p27KIP1) regulation and control.INK4 albumen is lived by directly being combined with CDK to suppress cyclinD-CDK4/6 Property.P21CIP1 and p27KIP1 can stablize Cyclin D1-CDK4/6 compounds, and all have under certain conditions CDK4/6 inhibitory activity.Have Rb in most cancer cells, these Rb+ cancers can by CDK4/6-cyclinD overexpressions Lai CDK4/6-cyclinD feminine gender regulatory factors are activated, or promote CDK4/6-cyclinD oncogenic signals path.Promote CDK4/6- The approach of cyclinD activity has phosphatidyl-inositol 3-kinase (PI3K)/AKT/ rapamycins (mTOR), and mitrogen-activated protein swashs Enzyme (MAPK), wnt/b-catenin signal paths, 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 transductions point.Encoding cyclin D1 (CCND1), CDK4 Or the amplification of CDK6 gene or coding 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, there are some researches show in breast cancer CCND1, CDK4 and CDK6 expand 17%, 4%, 3% respectively, and CDKN2A declines 21%.Other research reports of breast cancer CDK4 expands 16%, CDK6 and expands 17%, p16INK4A losses 49%.CCND1 amplifications are up to 35% in breast cancer case, And cyclinD is more than 50%.CCND1 amplification ratio 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%).

Due to the mechanism that importance of the CDK4/6 activity in cell is adjusted and the known approach are activated in cancer, CDK4/6 inhibitor turns into a kind of attractive treatment means.One main theoretical question is, CDKs normal cell with And played a crucial role in the propagation of cancer cell, therapeutic window stenostomia may be caused, its toxicity will influence clinical levels.So far Untill, the most comprehensive general CDK inhibitor of research is flavopiridol, and its clinical effectiveness is limited, and partly cause is that it is complicated Pharmacokinetics and side effect.In normal cell-cycle, cyclinD1 by being acted synergistically with CDK4, by pRb approach and P53 approach joint effect cell cycle progressions, P53-P21-pRb approach and P16-pRb approach are two known cell ageings Coherent signal approach.However, the overexpression of CCND1- genes is likely to result in cell cycle confusion and unregulated cell growth, enter And cause tumour.CyclinD1 is the important albumen of CCND1- gene codes.P16 genes are a kind of bases in the cell cycle This gene, the albumen negative regulator cell propagation of expression, therefore it is a kind of internal antioncogene.The albumen that it is encoded can be with CyclinD1 competitive binding CDK4/6, suppress protein kinase activity, prevent pRb from phosphorylation and keep the state of activation, to thin Born of the same parents play negative regulation the cycle, and other protein inactivations of the change XOR of P16 genes can cause cyclinD-CDK4/6-pRb-E2F to adjust Section approach it is out of control, cause 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 just prevent cell from breeding in the G1 phases, so as to reach Suppress the purpose of tumor proliferation.INK4 belongs to one kind of CDK kinase inhibitions albumen (CDIs), and CDIs can negative regulation CDK-Cyclin Compound, INK4 families include P16INK4a, P15INK4b, P18INK4c and P19INK4d, and their main function is suppression CDK4 and CDK6 processed activity.There is Rb in most of tumours, and the exception of cdk4/6-cyclinD-INK4-Rb paths is also general Store-through exists, and the form of expression is：(1) P16INK4a gene delections, or DNA methylation cause P16INK4a activity missings；(2)CDK4 Gene magnification or point mutation, cause to lose with P16INK4a binding ability；(3) cyclinD1 is due to gene rearrangement or gene Expand and over-express.Due to the effect in breeding in cell, research find it can as the native target for the treatment of of cancer, and And it has two big advantages：(1) most cells propagation 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) research shows, the horizontal rises of cyclinD, by increasing capacitance it is possible to increase cell is to medicine Sensitiveness.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 agricultural chemicals Conduct industry.There are a large amount of heterocycles to explore at present, this is to be based on such as imidazoles, thiazole, piperidines, pyridine, pyrroles, indoles, purine, quinoline The compound of the heterocycles such as quinoline, isoquinolin, such as pyrazolopyrimidine, indazole, Pyrazolopyridine, Pyrazolopyridine etc..Pyrazoles and quinoline Oxazoline fusion may improve selectivity and sensitiveness.Report newest Li et al. 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, (US4112098, the publication date such as Vogt：On September 5th, 1978) obtain on " pyrazolo [1,5-c] quinazoline derivant And related compound " patent.2009, (WO2004104007A1, the publication date such as Traquandi：On December 2nd, 2004) carry 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 disorderly effect of the proliferation activity of cell.2013, Caruso etc. (WO2008074788A1, publication date：On June 26th, 2008) obtain substituted pyrazolecarboxylic and quinazoline derivant and its press down as kinases The patent of preparation.

2009, Brasca etc. (US7482354B2, on January 27th, 2009) demonstrate pyrazolo [4,3-h] quinazoline- The CDK inhibitory activity of 3- formamides.The compound shows significant anti-A2780 Proliferation of Human Ovarian Cell proliferation activity, to CDKs It is active.SAR researchs emphasize that the carboxylic acid amides (R1) of compound partly shows the inhibitory action of maximum, and is introduced in R1 larger Group then reduces CDK2 activity.

The content of the invention

It is an object of the present invention to design with synthesizing a kind of novel pyrazole simultaneously [4,3-h] quinazoline analogs, such New small molecule reactive compound have suppress cyclin dependant enzyme family (CDKs) biological function, so as to for Find new treating cancer, metabolism and immunological diseases, cardiovascular disease and neurogenic disease etc. and open up new way.

Therefore, on the one hand, the present invention provides a kind of compound as shown in below general formula I or its is pharmaceutically acceptable Salt,

In formula I,

R₁Selected from H, the C1-C8 straight or branched alkyls of unsubstituted or halogen substitution or the substitution of unsubstituted or halogen C3-C6 cycloalkyl；Preferably, R₁Selected from H, the substitution of unsubstituted or halogen C1-C6 straight or branched alkyls or it is unsubstituted or The C3-C5 cycloalkyl of halogen substitution；It is highly preferred that R₁C1-C3 straight or branched alkyls selected from H, the substitution of unsubstituted or halogen Or the C3-C5 cycloalkyl of unsubstituted or halogen substitution；Wherein, halogen represents fluorine, chlorine, bromine or iodine；

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

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 alkoxies；It is highly preferred that R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, first Base, ethyl, propyl group, butyl, methoxyl group, ethyoxyl or propoxyl group；

A is 0 or 1；

X is straight key ,-CH₂-、-CH₂CH₂- or-CH₂CH₂CH₂-；

R₄For H or C_1-5Alkyl.

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

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

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

In an instantiation, a 0；

In an instantiation, a 1；

In an instantiation, X is straight key；

In an instantiation, X is-CH₂-；

In instantiation, 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 methods described passes through formulas below Carry out：

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

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

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

Especially, the cancer be selected from carcinoma of urinary bladder, breast cancer, colon and rectum carcinoma, kidney, epidermal carcinoma, liver cancer, lung cancer, Cancer of the esophagus, gallbladder cancer, oophoroma, cancer of pancreas, stomach 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, come from stromal tumours, maincenter or week Enclose nervous system neoplasm, melanoma, glioma, seminoma, teratoma, osteosarcoma, xeroderma pitmentosum, angling spine Cytoma, thyroid follcular carcinoma or Kaposi sarcoma.

Especially, 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 lymphomas, T- cell lymphomas, Huppert's disease, Hodgkin lymphoma, NHL, 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 favourable in terms of exploitation is suitable for use as the medicine of CDK4/6 inhibitor.

Embodiment

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

" alkyl " used herein refers to straight or branched 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 group), pentyl group (for example, N-pentyl, isopentyl, neopentyl), hexyl (for example, n-hexyl and its isomers) etc..Low-grade alkyl group typically be up to 4 Carbon atom.The example of low-grade alkyl group includes methyl, ethyl, propyl group (such as n-propyl and isopropyl) and butyl group (example Such as normal-butyl, isobutyl group, sec-butyl, the tert-butyl group).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 connects (being especially shown in cyclic group) through another group, leads to Cross alkyl chain bridging and form ring, i.e. form the fused rings of bridging.

As used herein, " cycloalkyl " refers to non-aromatic carbocyclic groups, including cyclic alkyl, alkenyl and alkynyl group. Group of naphthene base can be monocyclic (such as cyclohexyl) or polycyclic (for example, comprising fusion, bridging and/or spiro ring system), wherein Carbon atom is located inside or outside member ring systems.Group of naphthene base can have 3 to 14 annular atoms (for example, 3 to 8 as overall Carbon atom is used for monocyclic cycloalkyl group and 7 to 14 carbon atoms are used for polycyclic naphthene base group).Any of group of naphthene base fits Position can be covalently attached with defined chemical constitution 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 the compound of Formula I of the present invention, its The atom that middle one or more atoms are had same atoms number is replaced, but atomic mass or mass number are with being 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 substitutions can provide some treatment advantages, and it has more preferable metabolic stability, For example, Half-life in vivo has increased or decreased volume requirements, and it is therefore preferred in some cases.

The above compound 1-56 of present invention synthetic method will be described in detail by embodiment below.

Prepare embodiment

The preparation of intermediate

1- ethyl -7- oxo -4,5,6,7- tetrahydrochysene -1-H- indazole -3- carboxylic acid, ethyl esters

To (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- bases) (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) are added, point Liquid, aqueous layer with ethyl acetate (15 × 2ml) extraction, merges organic layer, through saturated common salt water washing, anhydrous sodium sulfate drying, filter Liquid is concentrated under reduced pressure, and residue is through column chromatography (eluant, eluent：Dichloromethane：Absolute methanol=20：1) separate, obtain faint yellow solid (4.9g, 49.8%).MS(ESI)(m/z):237.4(M+H)⁺。¹H-NMR(CDCl₃, 400MHz) and 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- tetrahydrochysene -1H- indazole -3- carboxylic acid, ethyl esters (in Mesosome B1)

By 1- ethyl -7- oxos -4,5,6,7- tetrahydrochysene -1-H- indazole -3- carboxylic acid, ethyl esters (4g, 16.94mmol), dimethyl Formamide (40ml) is added in 100ml reaction bulbs, added after stirring and dissolving DMF dimethylacetal (7.6ml, 67.78mmol), 80 times reaction 8h.Remove solvent under reduced pressure, residue filter cake absolute methanol, petroleum ether, obtains through filtering 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- tetrahydrochysene -1H- indazole -3- carboxylate methyl esters (in Mesosome B2)

Except using (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- bases) (oxo) methyl acetate to replace Beyond (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- bases) (oxo) ethyl acetate, with 6- [(diformazan ammonia Base) methylene] -1- ethyl -7- oxo -4,5,6,7- tetrahydrochysene -1H- indazole -3- carboxylic acid, ethyl esters the similar method of synthetic method Synthetic intermediate B2.

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

It is molten that 3- methoxyl group -2- oxocyclohex -3- alkene (8.4g, 54.5mmol), anhydrous THF are added in 250ml reaction bulbs Liquid (50ml), nitrogen protection, 1M LiHMDS THF solution (33ml) is added in -50 DEG C, drips and finishes room temperature reaction 15min.It is added dropwise Anhydrous THF (50ml) solution of diethy-aceto oxalate (11ml, 81.8mmol), drip and finish room temperature reaction 8h.Reaction solution is slowly added to In frozen water (100ml), control temperature is no more than 20 DEG C, then 1M watery hydrochloric acid (50ml) is added dropwise and is adjusted to pH=5, uses ethyl acetate (100ml × 2) extract, liquid separation.Merge organic layer, filtered after anhydrous magnesium sulfate is dried, filtrate decompression is concentrated to give crude product.Use second Acetoacetic ester：Petroleum ether (1:1,50ml) crude product is recrystallized, 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- tetrahydrochysene -1-H- indazole -3- carboxylic acid, ethyl esters

To the acetic acid of (3- methoxyl group -2- oxocyclohex -3- alkene -1- bases) (oxo) ethyl acetate (10.3g, 46.0mmol) Methyl hydrazine sulfate (2.44ml, 46.0mmol) acetum (20ml) 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, merge organic layer, warp Anhydrous magnesium sulfate filters after drying, and filtrate decompression concentration, residue is through ethanol：Water (1:1,50ml) recrystallize, obtain yellow solid (6.85g, 60.2%).MS-ESI(m/z):250.3(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- methyl -7- oxo -4,5,6,7- tetrahydrochysene -1H- pyrazoles -3- carboxylic acid, ethyl esters (in Mesosome B3)

By 1- methyl -7- oxos -4,5,6,7- tetrahydrochysene -1-H- indazole -3- carboxylic acid, ethyl esters (6.85g, 27.4mmol), dioxy Six rings ((30ml) is added in 100ml reaction bulbs, added after stirring and dissolving DMF dimethylacetal (13.1ml, 54.8mmol), 8h is reacted at room temperature.Remove solvent under reduced pressure, residue is through column chromatography (eluant, eluent：Petroleum ether：Ethyl acetate=1： 1) separate, obtain faint yellow solid (7.27g, 90%).MS-ESI(m/z):305.2(M+H)⁺。¹H-NMR(CDCl₃,400MHz)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- tetrahydrochysene -1H- pyrazoles -3- carboxylic acid, ethyl esters (in Mesosome B4)

Except using (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- bases) (oxo) methyl acetate to replace Beyond (3- methoxyl group -6,6- dimethyl -2- oxocyclohex -3- alkene -1- bases) (oxo) ethyl acetate, with 6- [(diformazan ammonia Base) methylene] -1- methyl -7- oxo -4,5,6,7- tetrahydrochysene -1H- pyrazoles -3- carboxylic acid, ethyl esters the similar method of synthetic method Synthetic intermediate B4.

The Boc- of 1,3- bis- [5- (4- thyl-piperazin -1- bases) methyl] pyridine -2- guanidines (intermediate A 1)

By [5- (4- thyl-piperazin -1- bases) methyl] pyridine -2- amine (2g, 10.41mmol), dichloromethane (30ml), three Ethamine (1.26g, 12.50mmol) is added in 100ml reaction bulbs, adds 1,3- bis--Boc-2- (trifluoromethyl sulfonyl) guanidine Dichloromethane (10ml) solution of (4.48g, 11.45mmol), stirring and dissolving, react at room temperature 3 days.Add water (50ml), dichloro Methane (50ml), liquid separation, water layer are extracted with dichloromethane (15ml) again, merge organic layer, through saturated common salt water washing, anhydrous sulphur After sour magnesium is dried, filtrate decompression concentration, residue is through column chromatography (eluant, eluent：Petroleum ether：Ethyl acetate=1：3) separate.Obtain Intermediate A 1.

The Boc- of 1,3- bis- [5- (piperazine -1- bases) methyl] pyridine -2- guanidines (intermediate A 2)

Except using [5- (piperazine -1- bases) methyl] pyridine -2- amine to replace [5- (4- thyl-piperazin -1- bases) methyl] pyrrole Beyond pyridine -2- amine, with the method synthetic intermediate A2 similar with the synthetic method of intermediate A 1.

The Boc-5- of 1,3- bis- (4- ethyl-piperazin -1- bases) pyridine -2- guanidines (intermediate A 3)

Except replacing [5- (4- thyl-piperazin -1- bases) methyl] pyrrole using 5- (4- ethyl-piperazin -1- bases) pyridine -2- amine Beyond pyridine -2- amine, with the method synthetic intermediate A3 similar with the synthetic method of intermediate A 1.

The Boc-5- of 1,3- bis- (- piperazine -1- bases) pyridine -2- guanidines (intermediate A 4)

Except replacing [5- (4- thyl-piperazin -1- bases) methyl] pyridine -2- using 5- (- piperazine -1- bases) pyridine -2-- amine Beyond amine, with the method synthetic intermediate A4 similar with the synthetic method of intermediate A 1.

The Boc-5- of 1,3- bis- (4- thyl-piperazin -1- bases) pyridine -2- guanidines (intermediate A 5)

Except replacing [5- (4- thyl-piperazin -1- bases) methyl] pyrrole using 5- (4- thyl-piperazin -1- bases) pyridine -2- amine Beyond 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-pyrazoles are simultaneously by 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] The synthesis of [4,3-h] quinazoline -3- Ethyl formates (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 bulbs, is heated to reflux to 90 DEG C of reaction 2h.Room temperature is cooled to, will Reaction solution is transferred to 100ml conical flasks, and water (40ml) is slowly added dropwise, and gained is collected by filtration and precipitates, dries, obtains faint yellow solid (0.41g, 90%).MS(ESI)m/z：463.9(M+H)⁺。

Embodiment 2

- 1- methyl -4,5- dihydro-1 h-pyrazoles are simultaneously by 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] The synthesis of [4,3-h] quinazoline -3- methyl formates (compound 1)

In addition to replacing intermediate B 3 using intermediate B 4, synthesized in the synthetic method identical method with compound 2 Compound 1 (3.90g, 80%) (passes through the SiO of 0-10%MeOH/ dichloromethane eluents₂Chromatogram purification).MS(ESI)(m/z): 449.0(M+H)⁺。

Embodiment 3

- 1- methyl -4,5- dihydro-1 h-pyrazoles are simultaneously by 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] The synthesis of [4,3-h] quinazoline -3- formic acid sodium salts

By 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] -1- methyl -4,5- dihydro-1 h-pyrazoles And [4,3-h] quinazoline -3- Ethyl formates (compound 2) (1.5g, 3.2mmol), 95% absolute ethyl alcohol (40ml) add 100ml In reaction bulb, sodium hydroxide (0.4g, 1mmol), 90 DEG C of heating reflux reaction 3h are added after stirring and dissolving.Room temperature is cooled to, is analysed Go out solid, filter, filter cake is washed with absolute ethyl alcohol (10ml), petroleum ether (1ml), is dried, is obtained white solid.

Embodiment 4

- 1- methyl -4,5- dihydro-1 h-pyrazoles are simultaneously by 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] The synthesis of [4,3-h] quinazoline -3- formamides (compound 3)

By 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] -1- methyl -4,5- dihydro-1 h-pyrazoles And [4,3-h] quinazoline -3- formic acid sodium salt (0.02g, 0.04mmol), dimethylformamide (1.6ml) add 10ml reaction bulbs In, 50% sodium methoxide (0.024g, 0.44mmol) methanol (0.4ml) solution is added into reaction bulb, is stirred and evenly mixed, is finally added Enter formamide (0.06g, 1.33mmol), be stirred overnight at room temperature.Water (2ml) is slowly added dropwise into reaction solution, gained is collected by filtration Precipitation, dry, obtain faint yellow solid (3.90g, 80%) (by the SiO of 0-10%MeOH/ dichloromethane eluents₂Chromatographically pure Change).MS(ESI)(m/z):434.0(M+H)⁺。

Embodiment 5

- 1- methyl -4,5- dihydro-1 h-pyrazoles are simultaneously by 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] [4,3-h] quinazoline -3- (N-METHYLFORMAMIDE) (compound 4) synthesis

By 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] -1- methyl -4,5- dihydro-1 h-pyrazoles And [4,3-h] quinazoline -3- formic acid (9.5g, 19.561mmol) is added to anhydrous tetrahydro furan and dimethylformamide (1:1, In 50ml)；The THF solution (21.12ml, 42.24mmol) of 2M methylamines, I-hydroxybenzotriazole (5.332g, 39.458mmol) It is added sequentially to dichloromethane (7.567g, 39.473mmol) in above-mentioned solution, reactant mixture reacts 10h at room temperature. Reactant mixture is poured into water (100ml) and then extracted 4 times with dichloromethane (250ml).Organic layer is washed with saturated common salt, is used Sodium sulphate is spin-dried for after drying.Crude product purifies (polarity by column chromatography：Dichloromethane:Methanol=90:5) product is obtained (3.90g, 80%) (passes through the SiO of 0-10%MeOH/ dichloromethane eluents₂Chromatogram purification).MS(ESI)(m/z):448.0(M+ H)⁺。

Embodiment 6

- 1- methyl -4,5- dihydro-1 h-pyrazoles are simultaneously by 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] [4,3-h] quinazoline -3- (N,N-dimethylformamide) (compound 5) synthesis

By 8- [[5- (4- methylpiperazine-1-yls) methyl] pyridine -2- bases amino] -1- methyl -4,5- dihydro-1 h-pyrazoles And [4,3-h] quinazoline -3- formic acid (0.2g, 0.44mmol), dimethylformamide (5ml) add 50ml reaction bulbs, stirring is molten Dichloroethanes (0.2mg, 1mmol), I-hydroxybenzotriazole (HOBT) (0.2g, 1.4mmol) are added after solution.After reacting 2h, to Dimethylamine (3ml) is added in bottle, continues to react 3h.Water (5ml), dichloromethane (2ml) are added, liquid separation, water layer uses dichloromethane again Alkane (2ml) extracts, and merges organic layer, through saturated common salt water washing, crude product is purified by column chromatography after anhydrous sodium sulfate drying (polarity：Dichloromethane:Methanol=90:5) product is obtained, solid product (3.90g, 80%) is obtained and (passes through 0-10%MeOH/ bis- The SiO of chloromethanes elution₂Chromatogram purification).MS(ESI)(m/z):462.0(M+H)⁺。

Compound 6-47 synthesis

Different intermediates is respectively adopted, according to the synthetic method similar with above-claimed cpd 1 or 2, synthesizes in table 1 below Compound.

Table 1

According to the synthetic method similar with above-claimed cpd 3-5, it is corresponding that it is obtained to the compound progress amination in table 1 Formamide and methyl substitution or dimethyl substitution 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-yls)-pyridine -2- bases amino] -1- ethyl -4,5- dihydro-1 h-pyrazoles simultaneously [4,3- H] quinazoline -3- cyanamides (compound 48) synthesis

By 8- [5- (4- thyl-piperazin -1- bases)-pyridine -2- bases amino] -1- ethyl -4,5- dihydro-1 h-pyrazoles simultaneously [4, 3-h] quinazoline -3- carboxylic acid sodium salts (0.2g, 0.44mmol), DMF (2ml) add 10ml reaction bulbs, stirring Dichloromethane (0.2mg, 0.10mmol) is added after dissolving, after HOBT (0.2g, 0.14mmol) reacts 1.2h, is added into bottle Cyanamide (0.028mg, 0.67mmol), continue to react 3h.Water (2ml), dichloromethane (2ml) are added, liquid separation, water layer uses dichloro again Methane (2ml) extracts, and merges organic layer, through saturated common salt water washing, crude product is pure by column chromatography after anhydrous sodium sulfate drying Change (polarity：Dichloromethane:Methanol=90:5) product is obtained.Obtain product 11 (0.17g, 0.84% yield).MS(ESI)m/z： 459.6(M+H)⁺。

Embodiment 8

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

By 8- [5- (4- thyl-piperazin -1- bases)-pyridine -2- bases amino] -1- ethyl -4,5- dihydro-1 h-pyrazoles simultaneously [4, 3-h] quinazoline -3- carboxylic acid sodium salts (0.2g, 0.44mmol), dimethylformamide (2ml) add 10ml reaction bulbs, stirring and dissolving Dichloroethanes (0.2mg, 1.0mmol) is added afterwards, after HOBT (0.2g, 1.4mmol) reacts 1,2h, after adding alkali tune into bottle Methoxamine hydrochloride (22mg, 26mmol), continue react 3h.Water (2ml), dichloromethane (2ml) are added, liquid separation, water layer is again Extracted with dichloromethane (2ml), merge organic layer, through saturated common salt water washing, crude product passes through post after anhydrous sodium sulfate drying Chromatographic purifying (polarity：Dichloromethane:Methanol=90:5) obtain product and obtain product (0.014g, 69%).MS(ESI) 464.9m/z：(M+H)⁺。

Embodiment 9

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

By 8- [5- (4- thyl-piperazin -1- bases)-pyridine -2- bases amino] -1- ethyl -4,5- dihydro-1 h-pyrazoles simultaneously [4, 3-h] quinazoline -3- carboxylic acid sodium salts (0.2g, 0.44mmol), dimethylformamide (5ml) add 10ml reaction bulbs, stirring and dissolving Dichloroethanes (0.2mg, 0.10mmol) is added afterwards, and after HOBT (0.2g, 1.4mmol) reacts 1,2h, ethamine is added into bottle (3ml), continue to react 3h.Water (5ml), dichloromethane (5ml), liquid separation are added, water layer is extracted with dichloromethane (2ml) again, is closed And organic layer, through saturated common salt water washing, crude product analyses purifying (polarity by post after anhydrous sodium sulfate drying：Dichloromethane:First Alcohol=90:5) product (0.015g, 74%) is obtained.MS(ESI)464.9m/z：(M+H)⁺。

Embodiment 10

- 1,4- dimethyl -4,5- dihydro-1 h-pyrazoles are simultaneously by 8- [5- (4- thyl-piperazin -1- bases)-pyridine -2- bases amino] The synthesis of [4,3-h] quinazoline -3- Ethyl formates (compound 54)

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

By 5- methyl isophthalic acids, hydroresorcinol (50g, 0.36mol), methanol (400ml) are added in 1000ml three-necked bottles, stirring The dichloromethane solution (10.75ml) of 1M titanium tetrachloride (2.0g, 0.01mol) is added dropwise after dissolving, drop finishes, and reacts at room temperature 8h. Slowly plus reaction is quenched in water (300ml), then 5% NaHCO is added dropwise₃Solution (about 80ml), extracted with ethyl acetate (200ml × 3) Take, liquid separation.Merge organic layer, filtered after anhydrous magnesium sulfate is dried, filtrate decompression concentration, obtain 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

3- methoxyl group -5- methyl-cyclohexyl -2- ketenes (40g, 0.26mol), anhydrous THF (135ml) are added into 500ml tri- In neck bottle, nitrogen is protected, and 1M LiAlH is slowly added dropwise under ice bath₄The anhydrous THF solution (91ml) of (3.4g, 0.09mol), protect Temperature is held at 0-5 DEG C, drop, which finishes, is warmed to room temperature reaction 2h.Diluted with ethyl acetate (100ml), be slowly added dropwise 2M's under condition of ice bath Reaction is quenched in watery hydrochloric acid (about 150ml), and ph is between 8-9 for control, addition ethyl acetate (100ml) and water (100ml), liquid separation, Aqueous layer with ethyl acetate (100 × 2ml) extracts, and merges organic layer, is filtered after anhydrous magnesium sulfate is dried, filtrate decompression concentration, Pale yellow oily liquid (27.1g, 85%) is obtained, is directly used in the next step.MS-ESI(m/z):111.2(M+H)⁺。¹H-NMR (CDCl₃,400MHz)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-s-two ring [4.1.0] heptane -2- ketone

5- methyl-cyclohexyl -2- ketenes (25g, 0.20mol), methanol (200ml) are added in 500ml three-necked bottles, 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) are added, point From organic layer.Remove solvent under reduced pressure, 5% Na is added in residue (about 80ml)₂S₂O₅Solution (250ml, 0.80mol) is in room Temperature stirring 30min.Add methyl tertiary butyl ether(MTBE) (100ml) and water (100ml), liquid separation, water layer with methyl tertiary butyl ether(MTBE) (100 × 2ml) extract, merge organic layer.Organic layer filters after anhydrous magnesium sulfate is dried, 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

Ring [4.1.0] heptane -2- ketone (20g, 0.14mol) of 4- methyl -7- oxa-s-two 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 backflow Answer 30min.Room temperature is cooled to, adds water (100ml) and methyl tertiary butyl ether(MTBE) (50ml), liquid separation, water layer methyl tertiary butyl ether(MTBE) (100 × 2ml) is extracted, and merges organic layer.Filtered after anhydrous magnesium sulfate is dried, filtrate decompression concentration, obtain colourless oil liquid (15.8g, 75% yield).MS-ESI(m/z):141.2(M+H)⁺。¹H-NMR(CDCl₃,400MHz)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- bases) (oxo) ethyl acetate

2- methoxyl group -5- methyl-cyclohexyl -2- ketenes (8.4g, 54.5mmol), anhydrous four are added in 250ml reaction bulbs Hydrogen furans (THF) solution (50ml) nitrogen is protected, and the THF solution (33ml) of 1M lithium hexamethyldisilazides is added in -50 DEG C, 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 frozen water (100ml), control temperature is no more than 20 DEG C, then 1M watery hydrochloric acid (50ml) is added dropwise PH=5 is adjusted to, is extracted with ethyl acetate (100ml × 2), liquid separation.Merge organic layer, filtered after anhydrous magnesium sulfate is dried, filtered Liquid is concentrated under reduced pressure to obtain crude product.Use ethyl acetate：Petroleum ether (1:1,50ml) crude product is recrystallized, suction filtration obtains pale yellow colored solid Body (9.7g, 80%).MS-ESI(m/z):241.2(M+H)⁺。¹H-NMR(CDCl₃,400MHz)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- tetrahydrochysene -1-H- indazole -3- carboxylic acid, ethyl esters

To (3- methoxyl group -6- methyl -2- oxocyclohex -3- alkene -1- bases) (oxo) ethyl acetate (10.3g, Methyl hydrazine (2.44ml, 46.0mmol) acetum (20ml), room are added dropwise in acetum (65ml) 46.0mmol) Temperature reaction 2h.Water (100ml) and ethyl acetate (100ml) are added, liquid separation, aqueous layer with ethyl acetate (100 × 2ml) extraction, is closed And organic layer, filtered after anhydrous magnesium sulfate is dried, filtrate decompression concentration, residue is through ethanol：Water (1:1,50ml) recrystallize, Obtain 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- tetrahydrochysene -1H- indazole -3- carboxylic acid second Ester

By Isosorbide-5-Nitrae-dimethyl -7- oxos -4,5,6,7- tetrahydrochysene -1-H- indazole -3- carboxylic acid, ethyl esters (6.85g, 27.4mmol), Tetrahydrofuran (30ml) is added in 100ml reaction bulbs, and DMF dimethylacetal is added after stirring and dissolving (13.1ml, 54.8mmol), reacts 8h at room temperature.It is evaporated under reduced pressure and removes solvent, residue is through column chromatography (eluant, eluent：Petroleum ether： Ethyl acetate=1：1) separate, obtain faint yellow solid (7.27g, 90%).MS-ESI(m/z):292.2(M+H)⁺。¹H-NMR (CDCl₃,400MHz)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 end-product (compound 54)

By 6- [(dimethylamino) methylene] -1,4- dimethyl -7- oxo -4,5,6,7- tetrahydrochysene -1H- indazole -3- carboxylic acids Ethyl ester (5.01g, 16.4mmol), intermediate A 5 (3.82g, 16.4mmol), dioxane (40ml) add 100ml reaction bulbs In, it is heated to 90 DEG C of reaction 4h.Room temperature is cooled to, water (50ml) is slowly added dropwise into reaction solution, gained is collected by filtration and precipitates, 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- bases)-pyridine -2- bases amino] -1,4,4- trimethyl -4,5- dihydro-1 h-pyrazoles And the synthesis of [4,3-h] quinazoline -3- Ethyl formates (compound 51)

Except using 5,5- dimethyl -1, hydroresorcinol replaces 5- methyl isophthalic acids, beyond hydroresorcinol, with compound 54 synthetic method identical method is synthesized.

Embodiment 12

8- [5- (4- thyl-piperazin -1- bases)-pyridine -2- bases amino] -1- isopropyl -4,4- dimethyl -4,5- dihydros - The synthesis of 1H- pyrazolos [4,3-h] quinazoline -3- Ethyl formates (compound 52)

Except replacing 5- methyl isophthalic acids using 5,5- dimethyl-hydroresorcinol, hydroresorcinol and isopropyl is used Hydrazine is replaced beyond methyl hydrazine, is synthesized in the synthetic method identical method with compound 54.

Embodiment 13

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

Compound 51 (6.25g, 13.2mmol), absolute ethyl alcohol (40ml) are added in 100ml reaction bulbs, after stirring and dissolving Add 95% ethanol solution (13.2ml) of 1.5M potassium hydroxide, heating reflux reaction 3h.Room temperature is cooled to, solid is separated out, takes out Filter, filter cake are washed with cold 95% ethanol (5ml × 3), filter cake are dissolved in 50ml water, and 1M hydrochloric acid, 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

Except replacing 5- methyl isophthalic acids, hydroresorcinol using 1,3- cyclopentanediones, and methyl is replaced using isopropyl hydrazine Beyond hydrazine, the synthetic method of the synthesis reference aforesaid compound 54 of compound 55.

Embodiment 15

The synthesis of compound 56

In addition to replacing isopropyl hydrazine using cyclopenta hydrazine, the synthesis of the synthesis reference aforesaid compound 55 of compound 56 Method.Finally give 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

Experiment 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. prepare 1x kinases base buffer and stop buffer

1) it is used for CDK2, CDK6 1x kinases base buffers

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 CDK4 1x kinases base buffers

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

5ug cdk kinases is added in 2.5ml 1x kinases base buffers 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 buffers.

3) the DMSO buffer solns of 500nM testing compound are prepared

The DMSO solution of 25uM compounds is first prepared, takes the DMSO solution of 10ul compound, adds 90ul's thereto 1x kinases base buffers.Mixing 10min obtains solution of the compound in 10%DMSO.5 μ l change is added on 96 orifice plates Solution of the compound 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 10 minutes at room temperature.

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

7) kinase reaction terminates

1h is incubated in 28 DEG C of baking ovens.Add 25 μ l stop buffer terminating reactions.Detected with Caliper instruments.

Inhibiting rates of the compound 1-56 (concentration 500nM) to CDK2, CDK4 and CDK6 is shown in table 3 below.

Table 3

More than 90% is reached to CDK4/6 inhibitory activity from most compounds that the present invention is can be seen that with upper table 3, because This, good CDK4/6 inhibitory activity is presented in compound of the invention, is used as CDK4/6 inhibitor.

Above example is only the exemplary embodiment of the present invention, is 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 make respectively in the essence and protection domain of the present invention to the present invention Kind modification or equivalent substitution, this modification or equivalent substitution also should be regarded as being within the scope of the present invention.

Claims (10)

1. a kind of compound or its pharmaceutically acceptable salt as shown in below general formula I,

In formula I,

R₁C3-C6 rings selected from H, the C1-C8 straight or branched alkyls of unsubstituted or halogen substitution or the substitution of unsubstituted or halogen Alkyl；Wherein, halogen represents fluorine, chlorine, bromine or iodine；

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

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

A is 0 or 1；

X is straight key ,-CH₂-、-CH₂CH₂- or-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 Substituted C1-C6 straight or branched alkyls or the C3-C5 cycloalkyl of unsubstituted or halogen substitution；It is highly preferred that R₁Selected from H, The C1-C3 straight or branched alkyls of unsubstituted or halogen substitution or the C3-C5 cycloalkyl of unsubstituted or halogen substitution.

3. compound according to claim 1 or its pharmaceutically acceptable salt, wherein, R₂And R₂' be each independently selected from H, C1-C6 straight or branched alkyls；It is highly preferred that R₂And R₂' it is each independently selected from H, C1-C3 straight or branched alkyl；

R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, C1-C6 alkyl or C1-C6 alcoxyls Base；It is highly preferred that R₃Selected from-C (O) OR₅Or-CONR₆R₇, wherein R₅、R₆、R₇It is each independently H, CN, methyl, ethyl, third Base, butyl, methoxyl group, ethyoxyl or propoxyl group.

4. 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 group ammonia Base formoxyl or methoxyformamido base；

A is 0 or 1；

X is straight key or-CH₂-；

R₄For H, methyl or ethyl.

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

6. a kind of pharmaceutical composition, the compound according to any one of claim 1-5 comprising therapeutically effective amount or its Pharmaceutically acceptable salt and at least one pharmaceutically acceptable excipient, carrier and/or diluent.

7. the compound or its pharmaceutically acceptable salt according to any one of claim 1-5 are being prepared as CDK4/6 suppressions Purposes in the medicine of preparation.

8. the compound or its pharmaceutically acceptable salt according to any one of claim 1-5 are used for treating cancer in preparation Medicine in purposes.

9. purposes according to claim 8, wherein, the cancer is selected from carcinoma of urinary bladder, breast cancer, colon and rectum carcinoma, kidney Cancer, epidermal carcinoma, liver cancer, lung cancer, cancer of the esophagus, gallbladder cancer, oophoroma, cancer of pancreas, stomach cancer, cervical carcinoma, thyroid cancer, rhinocarcinoma, head Neck cancer, prostate cancer, cutaneum carcinoma, the hematopoietic tumor of Lymphatic System, medullary system hematopoietic tumor, thyroid follcular carcinoma, come from Stromal tumours, maincenter or peripheral nervous system neoplasms, melanoma, glioma, seminoma, teratoma, bone and flesh Knurl, xeroderma pitmentosum, angling prickle cell knurl, thyroid follcular carcinoma or Kaposi sarcoma.

10. purposes according to claim 9, wherein, the hematopoietic tumor of the Lymphatic System is selected from leukaemia, acute leaching Bar property leukaemia, chronic lymphocytic leukemia, B- cell lymphomas, T- cell lymphomas, Huppert's disease, Huo Qijin leaching Bar knurl, NHL, hairy cell lymphoma or Burkitt's lymphoma.