CN106831812B - Simultaneously pyrimidine or pyrazine compounds and its application of the heterocycle of the amide structure containing biaryl - Google Patents

Abstract

The invention discloses a kind of heterocycle of amide structure containing biaryl and pyrimidine or pyrazine compounds, as shown in general formula I or II:

Description

Simultaneously pyrimidine or pyrazine compounds and its application of the heterocycle of the amide structure containing biaryl

Technical field

The present invention relates to quinazoline compounds, in particular to the heterocycle and pyrimidine or pyrrole of a kind of amide structure containing biaryl Piperazine class compound and its application.

Background technique

Malignant tumour is a kind of disease for seriously endangering human life and health, and morbidity and mortality have been more than heart and brain Vascular diseases, occupy the first place of all diseases, and cancer is to threaten first big " killer " of human health.Expect the year two thousand thirty whole world There to be 21,400,000 new cases, death toll reaches 13,200,000 people, wherein 70% occurs the developing country in middle and low income. With the continuous social and economic development, municipal pollution is increasingly severe, aging of population development, in China, cancer mortality by Year rises, it has also become the major disease for seriously endangering China human mortality quality, hindering social development.China is three times in the whole country Coroner's inquest data show, nearly 30 years, composition of the Cancer in China in the cause of the death was by the 10.13% of 20 century 70s Rise to 22.32%.

Cancer be due to control growth and proliferation of cell mechanism it is not normal caused by disease, the essence of cell carcinogenesis be cell letter The imbalance of number conducting system, so as to cause the fast-growth and infinite multiplication of cancer cell.By phosphinositides -3- kinases (phosphoinositide3-kinase, PI3K) and protein kinase B (PKB/Akt), rapamycin target body albumen downstream (mTOR) the PI3K-Akt-mTOR access formed is referred to as PI3K access, has important work in the occurrence and development of tumour With, using key molecule in PI3K/Akt signal path as the micromolecular inhibitor of target spot have become current antineoplastic drug research Hot spot.

The cyclopean family that PI3K is made of lipid and serine/threonine kinases, including several phosphinositides kinases and Protein kinase such as ATM, ATR and DNA-PK etc. that DNA is relied on, it can make the third position di of phosphatidylinositols, generate Inositol lipid material with second messenger's effect --- phosphatidylinositols -3- phosphate ester (PIP3).Second messenger PIP3 can make PI3K is combined with the pairing of the effector (especially Akt) in downstream, so as to cause film recruitment and phosphorylation.Research shows that: PI3K family Numerous processes such as race and cell Proliferation, anti-apoptotic, cell migration, film bubble transhipment, cell cancerous transformation are related, these biological effects Mainly it is catalyzed what " anchor " molecule 3- phosphoinositide rouge (PIP, PIP2, PIP3) to be formed mediated by PI3K.The study found that PI3K access is generally lacked of proper care in extensive human tumor spectrum, dysfunction caused by certain gene mutations or missing meeting in the access Cause normal cell turnover, promote tumor cell proliferation and survival and the invasion and migration of mediate tumor cell, therefore is small point The favourable effects target position of sub- inhibitor provides chance for the treatment of cancer.

It was related to the patent (WO2009055730/WO2009036082) and relevant report of the inhibiting effect of PI3K in recent years (Journal ofMedicinal Chemistry,2008,51(18):5522-5532,Drugs ofthe Future,2007, 32 (6): 537-547.) it sharply increases, it has now been found that the micromolecular inhibitor of a variety of kinases in the signal path, such as day Right product wortmannin (Wortmannin) and Mongolian oak flavin compound L Y294002 are that two kinds of widely applied PI3K inhibit Agent, the former can be in conjunction with a variety of hypotypes of PI3K；The inhibitor 1L-6-hydroxymethyl-chiro-inositol 2- of Akt (R) -2-O-methyl-3-O-octadecylcarbonate, the IC of selective depression Akt₅₀Value is about 5 μM, hence it is evident that small In the IC that it inhibits PI3K₅₀90 μM of value；P70S6K is another feasibility target spot of PI3K-Akt signal path, immunosuppressor Rapamycin (RPM) is widely used in clinical organ transplant, the study found that RPM can make p70S6K dephosphorylation and inhibit The activity of the kinases, to inhibit the growth of tumour cell, at present in a variety of PTEN mutation or PI3K-Akt pathway activity The selective anti-tumor activity of RPM is observed in the human tumour cell line of up-regulation.

In PI3K family, I type PI3K can be activated by extracellular signal, therefore be ground so far in numerous PI3K hypotypes Study carefully most commonly used one kind.Currently, the compound of many targeting I type PI3K has entered clinical investigation phase, such as: natural products is wet Graceful penicillin, PX-866, LY-294002, TGX-115, TGX-155, PI-103, GDC-0941 etc., wherein most are PI3K-mTOR double inhibitor.

GDC-0941 (shown in following structural formula) reported in the literature belongs to thienopyrimidines, be by The oral PI3K inhibitor of Genentech company exploitation, is completed Phase I clinical trial at present.GDC-0941 is to p110 α and δ IC₅₀Value reaches 3nM, is p110 β, 10 times of γ and 25 times, has good selectivity.Preclinical study shows GDC- 0941 pair of a variety of human tumor cell line (including glioblastoma, breast cancer cell, prostate gland cancer cell etc.) shows significantly Inhibit proliferaton effect, IC₅₀Value reaches 0.009ug/mL.In nude mouse in Anticancer Activities, when oral dose is 75mg/ Kg reaches 80% or more to the growth inhibition rate of tumour.Double aryl urea structures are also widely used in anticancer small numerator inhibitor In research, Wyeth Venkatesan et al. reports a series of 1,3,5-triazines class PI3K inhibitor, wherein PKI-587 Growth of cancer cells and proliferation can be inhibited in active testing well in vitro and in vivo, and its apoptosis can be promoted；Object PKI-402 It is a substituted bisarylurea being transformed on the basis of PKI-587 and PKI-179 of Wyeth like object, compound tool There are good physicochemical property and pharmacokinetic property, in vitro and in vivo biomarker research discovery PKI-402 can be blocked PI3K-Akt-mTOR access and it can induce cancer cell-apoptosis.

Summary of the invention

It is an object of the invention to provide a kind of heterocycle of amide structure containing biaryl and pyrimidine or pyrazine compounds and Preparation method and application.

The present invention provide the heterocycle of the amide structure containing biaryl as shown in general formula I simultaneously pyrimidine or pyrazine compounds, its is several What isomers and its pharmaceutically acceptable salt, hydrate, solvate or prodrug, structure is as shown in the following general formula I or II:

Wherein:

X=S, Y=CH or X=CH, Y=S；

Z is phenyl ring, five yuan or hexa-atomic heteroaromatic；

A, B, D, E, G are respectively CH or N；

R¹Selected from H,

R²Selected from H or-CH₃；

Ar be phenyl, naphthalene, 5~10 unit's heteroaryls, 5~10 yuan saturation or fractional saturation heterocycle, the heteroaryl Contain 1~3 hetero atom for being selected from O, N and S, and optional 1~4 identical or different R of Ar with heterocycle³Replace；

R³Selected from hydrogen, halogen, hydroxyl, trifluoromethyl, trifluoromethoxy, amino, azido, nitro, cyano, sulfydryl, (C₁ ~C₄) alkyl, (C₃~C₆) naphthenic base, (C₂~C₄) alkenyl, (C₂~C₄) alkynyl, (C₁~C₄) alkoxy, (C₁~C₄) alkane sulphur Base, allyl, (2- methyl) allyl, (C₁~C₄) alkoxy methyl, (C₁~C₄) alkyl acyl or (C₁~C₃) alkylidene two The substituent group of oxygroup；

R⁴、R⁵It is identical or different, separately it is selected from (C₁~C₆) alkyl or (C₃~C₆) naphthenic base；Or R⁴And R⁵With The nitrogen-atoms connected with them is formed together 5~10 yuan of saturated heterocyclyls, the saturated heterocyclyl in addition to R⁴And R⁵Connection Outside nitrogen-atoms, the hetero atom of O, N and S are optionally selected from containing 1~3；

Present invention is preferably related to the heterocycle of such as above-mentioned general formula I or II amide structure containing biaryl and pyrimidine or Pyrazine chemical combination Object, its geometric isomer and its pharmaceutically acceptable salt, hydrate, solvate or prodrug, in which:

X=S, Y=CH or X=CH, Y=S；

Z is phenyl ring or pyridine ring；

A, B, D, E, G are respectively CH or N；

R¹Selected from H,

R²Selected from H or-CH₃；

Ar is phenyl, naphthalene, pyridyl group, pyrimidine radicals or thienyl, the phenyl, naphthalene, pyridyl group, pyrimidine radicals, thiophene Optional 1~4 identical or different R of base³Replace；

R³Selected from hydrogen, halogen, hydroxyl, trifluoromethyl, trifluoromethoxy, amino, azido, nitro, cyano, sulfydryl, (C₁ ~C₄) alkyl, (C₃~C₆) naphthenic base, (C₂~C₄) alkenyl, (C₂~C₄) alkynyl, (C₁~C₄) alkoxy, (C₁~C₄) alkane sulphur Base, allyl, (2- methyl) allyl, (C₁~C₄) alkoxy methyl, (C₁~C₄) alkyl acyl or (C₁~C₃) alkylidene two The substituent group of oxygroup；

In the present invention, the heterocycle and pyrimidine or pyrazine compounds of the amide structure containing biaryl of the general formula I or II are Selected from one of following compounds, but these compounds are not meant to any limitation of the invention:

4- (4- ethylphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide,

4- (2,4 difluorobenzene base)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide,

4- (3- fluorophenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide,

N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) -4- phenylpyridine amide,

4- (2,4- 3,5-dimethylphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) pyridine acyl Amine,

6- (4- methoxyphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) pyrimidine -4- first Amide,

N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) -6- (p-methylphenyl) pyrimidine -4- formamide,

N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) -6- (4- (trifluoromethyl) pyrimidine -4- Formamide,

5- (3- fluorophenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide,

5- (2,4 difluorobenzene base)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide,

N- (4- (- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -4- phenylpyridine amide,

4- (2,4 difluorobenzene base)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide,

4- (4- ethylphenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide,

4- (2,4- 3,5-dimethylphenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) pyridine acyl Amine,

N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -4- (p-methylphenyl) picolinamide,

5- (3- fluorophenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide,

5- (4- methoxyphenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide,

6- (4- methoxyphenyl)-N- (4- (8- morpholine imidazo [1,2-a] pyrazine -6- base) phenyl) pyrimidine -4- formyl Amine,

6- (4- bromophenyl)-N- (4- (8- morpholine imidazo [1,2-a] pyrazine -6- base) phenyl) pyrimidine -4- formamide,

N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -6- phenyl pyrimidine -4- formamide,

N- (5- (4- morpholino thieno [3,2-d] pyrimidine -2-base) pyridine -2- base) -4- phenylpyridine amide,

N- (5- (4- (3- methyl morpholine) thieno [3,2-d] pyrimidine -2-base) pyridine -2- base) -4- phenylpyridine amide,

N- (5- (6- ((dimethylamino) methyl) -4- (3- methyl morpholine) thieno [3,2-d] pyrimidine -2-base) pyridine - 2- yl) -4- (thiophene -2- base) picolinamide,

N- (5- (8- (3- methyl morpholine base) imidazo [1,2-a] pyrazine -6- base) pyridine -2- base) -4- (thiophene -2- base) Picolinamide,

N- (5- (8- (3- methyl morpholine base) imidazo [1,2-a] pyrazine -6- base) pyridine -2- base) -4- phenylpyridine acyl Amine,

N- (5- (6- (2- hydroxyl propyl- 2- yl) -4- morpholino thieno [3,2-d] pyrimidine -2-base) pyridine -2- base) -4- Phenylpyridine amide,

N- (5- (4- morpholino -6- (the fluoro- 2- hydroxyl propyl- 2- yl of 1,1,1- tri-) thieno [2,3-d] pyrimidine -2-base) pyrrole Pyridine -2- base) -4- phenylpyridine amide,

N- (5- (4,6- dimorpholino thieno [2,3-d] pyrimidine -2-base) pyridine -2- base) -4- phenylpyridine amide.

Following synthetic route describes the heterocycle and pyrimidine or pyrazine of general formula I or II amide structure containing biaryl of the present invention The preparation of class compound, all raw materials are all by mode described in synthetic route, by the common skill of organic chemistry filed It is the preparation of method known to art personnel or commercially available.All final derivative of the invention is all by describing in synthetic route Method or prepared by similar method, these methods are that organic chemistry filed is well-known to the ordinarily skilled artisan.It closes At the whole variable factors applied in route definition as follows.

The synthetic route of route 1 target compound I or II

Pass course 1 of the present invention synthetic intermediate 12 or 13 first, then with different substituents (e.g., pyridine acids or pyrimidine Acids) side chain reaction, obtain target compound.

According to some usual methods of the art, in the present invention, the heterocycle of the above-mentioned amide structure containing biaryl is simultaneously Pyrimidine or pyrazine compounds can generate pharmaceutically acceptable salt with acid.Pharmaceutically acceptable addition salts include inorganic acid and organic acid Addition salts, the salt with following sour addition is particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, to first Benzene sulfonic acid, benzene sulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, winestone Acid, benzoic acid etc..

In addition, the invention also includes the prodrugs of derivative of the present invention.The prodrug of derivative of the present invention is above-mentioned containing biaryl The heterocycle and pyrimidine or pyrazine compounds of amide structure, their own may have weaker activity even without activity, but It is that upon administration, (such as passing through metabolism, solvolysis or other mode) is converted to corresponding biology in physiological conditions Active form.

" halogen " refers to fluorine, chlorine, bromine or iodine generation in the present invention；" alkyl " refers to the alkyl of linear chain or branched chain；" alkylidene " Refer to the alkylidene of linear chain or branched chain；" naphthenic base " refers to substituted or unsubstituted naphthenic base；" the heterocycle of saturation or fractional saturation Base " refers to containing one or more heteroatomic monocycles or polycyclic cyclic annular system for being selected from N, O, S, such as pyrrolidinyl, morpholine Base, piperazinyl, piperidyl, pyrazolidinyl, imidazolidinyl and thiazolinyl etc..

The present invention can contain the heterocycle and pyrimidine or pyrazine compounds of the above-mentioned amide structure containing biaryl, and pharmaceutically Acceptable carrier or excipients are prepared by mixing into composition, and are prepared into clinically acceptable dosage form, above-mentioned pharmaceutically The excipients of receiving refer to any diluent that can be used for pharmaceutical field, adjuvant and/or carrier.Derivative of the invention can be with It is applied in combination with other active ingredients, as long as they do not generate other unfavorable effects, such as allergic reaction.

The heterocycle and pyrimidine or pyrazine compounds of the above-mentioned amide structure containing biaryl of the present invention are used for the clinical agent of patient Amount can basis: active constituent therapeutic efficiency in vivo and bioavilability, their metabolism and discharge rate and patient's Age, gender, disease phase carry out appropriate adjustment, but adult daily dosage generally should be 10~500mg, preferably 50 ~300mg.Therefore, when pharmaceutical composition of the invention is made into unit dosage forms, it is contemplated that above-mentioned effective dose, per unit system Agent should the heterocycle containing the amide structure containing biaryl of 10~500mg above formula I and pyrimidine or pyrazine compounds, preferably 50 ~300mg.According to the guidance of doctor or pharmacist, these preparations can divide at certain intervals is administered (preferably one to six several times It is secondary).

The present invention also provides a kind of Pharmaceutical compositions, above-mentioned general formula I or II acyl containing biaryl including therapeutically effective amount It is the heterocycle of amine structure and pyrimidine or pyrazine compounds, its geometric isomer and its pharmaceutically acceptable salt, hydrate, molten Agent compound or prodrug are as active constituent and pharmaceutically acceptable excipients.If Pharmaceutical composition of the invention can be configured to Dry kind of dosage form, wherein containing some common excipient in drug field.Several dosage form as described above can be using injection The drug forms such as agent, tablet, capsule, aerosol, suppository, film, pill, externally-applied liniment, ointment.

Carrier for pharmaceutical composition of the present invention is available common type in drug field, comprising: adhesive, profit Lubrication prescription, disintegrating agent, cosolvent, diluent, stabilizer, suspending agent, non-pigment, corrigent, preservative, solubilizer and matrix etc.. Pharmaceutical preparation can by oral administration or parenteral (such as in intravenous, subcutaneous, peritonaeum or part) is administered, if some drugs It is unstable under the conditions of stomach, enteric coated tablets can be configured to.

It has also been found that the heterocycle of the above-mentioned amide structure containing biaryl simultaneously treat in preparation by pyrimidine or pyrazine compounds And/or the application in prevention proliferative disease drug.Reactive compound of the invention or its officinal salt and its solvate can It is used alone, or can be used for the anti-proliferate Drug combination listed as unique anti-proliferate drug Treatment and/or prevention proliferative disease, such as psoriasis, benign prostatauxe, atherosclerosis and restenosis.

It has also been found that the quinazoline compounds of the above-mentioned structure containing acylhydrazone are in preparation treatment and/or the medicine of pre- anti-cancer Application in object.The compounds of this invention has inhibition tumor cell growth activity in vitro, and therefore, it may be used as preparation treatment And/or the drug of pre- anti-cancer, such as mammary gland, lung, liver, kidney, colon, rectum, stomach, prostate, bladder, uterus, pancreas, bone Marrow, testis, ovary, lymph, soft tissue, neck, thyroid gland, the cancer of esophagus and leukaemia, neuroblastoma etc..

It has also been found that the heterocycle of the above-mentioned amide structure containing biaryl and pyrimidine or pyrazine compounds, are treated in preparation And/or the application in the drug of prevention lung cancer, liver cancer, gastric cancer, colon cancer, breast cancer.

By inhibiting lung carcinoma cell H460, Human Prostate Cancer Cells PC-3 and hepatoma cell strain SGC-7901 activity in vitro Test, people malignant glioblastoma cell U87MG, the compounds of this invention is to lung carcinoma cell, prostate gland cancer cell and liver cancer Cell, which has, significantly inhibits effect, it is especially useful in the drug of preparation treatment and/or prevention prostate cancer, lung cancer and liver cancer.

It is found by testing mTOR enzymatic activity, the compounds of this invention has significant inhibition mTOR kinase activity, right The highly expressed lung carcinoma cell of mTOR, Human Prostate Cancer Cells, glioblast etc. have stronger inhibiting effect, it is especially useful in preparation The drug for the treatment of and/or prevention lung cancer.

It is independent that reactive compound of the invention or its officinal salt and its solvate can be used as unique anti-tumor drug It uses, or can be with the anti-tumor drug (such as the platinum medicine cis-platinum, camptothecine Irinotecan, Changchun that have listed Flower bases drug Noviburn, deoxidation born of the same parents' former times class drug gemcitabine, etoposide, taxol etc.) it is used in combination.Combination therapy is logical Cross each therapeutic component simultaneously, sequence or separate administration to realize.

Present invention design has synthesized a series of new heterocycles and pyrimidine (pyrazine) analog derivative, is inhibited in vitro to mTOR Agent overexpression cell line carries out antitumor activity screening, the results showed that has stronger anti-tumor activity and selectivity.

Specific embodiment

In order to better explain the present invention, below in conjunction with specific embodiment, the present invention is described in further detail, but Limiting the invention for they.

Embodiment is intended to illustrate and be not intended to limit the scope of the invention.The nuclear magnetic resonance spectroscopy of derivative is used BrukerARX-400 measurement, mass spectrum are measured with 1100 LC/MSD of Agilent；Agents useful for same is that analysis is pure or chemical pure.

The heterocycle of general formula I or II amide structure containing biaryl and pyrimidine or pyrazine compounds:

The structural formula of the embodiment of the present invention 1~28 is as shown in table 1 below.

The structural formula of 1 Examples 1 to 28 of table

The preparation of step A thieno [2,3-d] pyrimidine -2,4 (1H, 3H)-diketone (1)

Under nitrogen protection, 2- aminothiophene -3- methyl formate (10g) is uniformly mixed with urea (30g), mechanical stirring Under be warming up to 180 DEG C, react 2h.End of reaction is cooled to 100 DEG C hereinafter, reactant is poured into the 1mol/L's equipped with 500mL It in sodium hydroxide solution beaker, is sufficiently stirred, filters；With concentrated hydrochloric acid tune filtrate pH to 6, pureed solid is precipitated；It filters, filtrate is cold Solid is precipitated after hiding 12h, filters to obtain faint yellow product, i.e. thieno [2,3-d] pyrimidine -2,4 (1H, 3H)-diketone, yield: 56.1%.

1H NMR (400MHz, DMSO) δ 11.94 (s, 1H, CONH), 11.18 (s, 1H, CONH), 7.11 (d, J= 16.0,5.6Hz,2H,2Ar-H)；ESI-MS[M-H]+m/z:167.2.

The preparation of step B 2,4- dichloro-thiophene simultaneously [2,3-d] pyrimidine (2)

10g phosphorus oxychloride is added to 1g dry thieno [2,3-d] pyrimidine -2,4 (1H, 3H)-diketone, 1 drop DMF is added Catalysis is warming up to 115 DEG C, reacts 5h, and reactant is slowly added in trash ice, is vigorously stirred simultaneously by end of reaction, obtains brown color Solid filters, and filter residue is dissolved in 50mL methylene chloride, and 1g active carbon and 2g silica gel are added into solution, and it is de- to be warming up to 45 DEG C of reflux Color 1h, filters while hot, and solvent is evaporated off, and obtains faint yellow solid, i.e. 2,4- dichloro-thiophene simultaneously [2,3-d] pyrimidine, yield: 56.0%.

¹H NMR (400MHz, DMSO) δ 8.16 (d, J=3.4Hz, 1H, Ar-H), 7.62 (d, J=3.4Hz, 1H, Ar- H).ESI-MS[M+H]+m/z:205.1。

The preparation of step C 4- (2- chlorothiophene simultaneously [2,3-d] pyrimidine-4-yl) morpholine (3)

By 2, the 4- dichloro-thiophene of 1g, simultaneously [2,3-d] pyrimidine is dissolved in 20mL methanol, 1mL morpholine is added dropwise under ice bath, gradually It is warmed to room temperature, reacts 0.5h, solid is precipitated, filter, obtain white solid, i.e. 4- (2- chlorothiophene simultaneously [2,3-d] pyrimidine-4-yl) Morpholine, yield: 86.1%.

¹H NMR(400MHz,DMSO)δ7.70(s,1H,Ar-H),7.69–7.65(m,1H, Ar-H),3.96–3.86 (m,4H,OCH₂), 3.75 (d, J=4.4Hz, 4H, NCH₂).ESI-MS [M+H]⁺m/z:256.1。

The preparation of step D 4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) aniline (12)

By para-bromoaniline (2g, 0.01mol), bis- (pinacol combined) two boron (3.8g, 0.015mol), potassium acetate (2.9g, 0.03mol), catalyst bi triphenyl phosphorus palladium chloride (0.18g, 0.00025mol) is dissolved in three equipped with dioxane 50mL In neck flask, it is continually fed into nitrogen 10min, is warming up to 80 DEG C, reacts 3h, 20mL water is added into reaction system for solution blackening, React 2min, sequentially add sodium carbonate (2.1g, 0.02mol), catalyst bi triphenyl phosphorus palladium chloride (0.18g, 0.00025mol) and 4- (2- chlorothiophene simultaneously [2,3-d] pyrimidine-4-yl) morpholine (1.2g, 0.006mol).100 DEG C are warming up to, React 8h.End of reaction, reaction solution vacuum distillation, removes partial solvent, and 100mL water is added into residual reaction liquid, is precipitated black Color solid filters, and filter residue is dissolved in methylene chloride: in methanol=5:1 solution 100mL, 2g active carbon and 5g being added into solution Silica gel is heated to reflux decoloration 1h, filters while hot, filtrate is evaporated to obtain faint yellow solid, i.e. 4- (4- morpholino thieno [2,3-d] Pyrimidine -2-base) aniline.

Step E 4- (4- ethylphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) pyridine Amide

Pyridine acids side chain compound is dissolved in 8mL methylene chloride, the dry DMF of 1 drip-dry is added, oxalyl chloride is added dropwise dropwise (1.5 times of equivalents), the DIPEA of the methylene chloride of 4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) aniline and 1mL is mixed It closes solution to be added dropwise in the dichloromethane solution of side chain, finishes reaction 10min, TLC stops reaction after detecting fully reacting.

Reaction solution is washed 2 times with saturated sodium carbonate solution, takes organic layer, and solvent is evaporated off, and isopropanol is added and stirs 30min, It filters, filter residue is dissolved in 15mL methylene chloride, and a small amount of silica gel is added, and stirs 10min, is filtered, is taken filtrate, solvent is evaporated off, and is added The washing of 10mL isopropanol, filters to obtain neat solid product.

Embodiment 1

4- (4- ethylphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide

First according to step A~D synthetic intermediate 12, last intermediate 12 passes through step E and pyridine acids side chain compound Reaction, obtains 4- (4- ethylphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide.

m.p.282-284℃；ESI-MS[M+H]⁺m/z:522.1；¹H NMR(400MHz,DMSO) δ10.88(s,1H), 8.78 (d, J=4.7Hz, 1H), 8.40 (d, J=7.2Hz, 3H), 8.08 (d, J=8.4Hz, 2H), 8.00 (s, 1H), 7.83 (d, J=7.6Hz, 2H), 7.64 (dd, J=18.7,6.0Hz, 2H), 7.41 (d, J=7.5Hz, 2H), 3.96 (s, 4H), 3.79 (s, 4H), 2.68 (d, J=7.4Hz, 2H), 1.23 (d, J=7.7Hz, 3H)

According to the method for embodiment 1, first according to step A~D synthetic intermediate 12, last intermediate 12 by step E with The side chain of different substituents (e.g., pyridine acids or pyrimidine acids) reacts, and 2~10 compound of embodiment is made respectively.

Embodiment 2

4- (2,4 difluorobenzene base)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide

m.p.255-256℃；ESI-MS[M+H]⁺m/z:530.1；¹H NMR(400MHz,DMSO) δ10.90(s,1H), 8.86 (s, 1H), 8.40 (d, J=7.7Hz, 2H), 8.31 (s, 1H), 8.07 (d, J=7.1Hz, 2H), 7.89 (s, 2H), 7.64 (d, J=13.0Hz, 2H), 7.51 (s, 1H), 7.31 (s, 1H), 3.97 (s, 4H), 3.79 (s, 4H)

Embodiment 3

4- (3- fluorophenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide

m.p.281-283℃；ESI-MS[M+H]⁺m/z:512.1；¹H NMR(400MHz,DMSO) δ10.90(s,1H), 8.83 (d, J=4.5Hz, 1H), 8.47-8.36 (m, 2H), 8.08 (d, J=8.6 Hz, 3H), 7.85-7.71 (m, 2H), 7.71-7.54 (m, 3H), 7.48 (d, J=6.0Hz, 1H), 7.37 (s, 1H), 6.60 (d, J=8.3Hz, 1H), 3.97 (s, 4H),3.78(s,4H).

Embodiment 4

N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) -4- phenylpyridine amide

m.p.223-225℃；ESI-MS[M+H]⁺m/z:494.1；¹H NMR(400MHz,DMSO) δ10.87(s,1H), 8.79 (d, J=12.7Hz, 2H), 8.41 (d, J=9.2Hz, 2H), 8.08 (d, J=7.7Hz, 2H), 7.77 (d, J= 7.0Hz, 2H), 7.64 (d, J=13.0Hz, 2H), 7.35 (dd, J=20.2,7.3Hz, 3H), 6.60 (d, J=8.2Hz, 1H),3.97(s,4H),3.79(s,4H).

Embodiment 5

4- (2,4- 3,5-dimethylphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide m.p.277-279℃；ESI-MS[M+H]⁺m/z:522.2；¹H NMR(400MHz,DMSO)δ 10.87(s,1H),8.78(d,J =4.7Hz, 1H), 8.66 (s, 1H), 8.06 (d, J=8.3Hz, 3H), 7.75-7.63 (m, 2H), 7.20 (dd, J=20.4, 9.6Hz, 5H), 3.97 (s, 4H), 3.79 (s, 4H), 2.25 (d, J=7.9Hz, 6H)

Embodiment 6

6- (4- methoxyphenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) pyrimidine -4- first Amide

m.p.251-252℃；ESI-MS[M+H]⁺m/z:525.2；¹H NMR(400MHz,CDCl₃) δ10.50(s,1H), 10.06 (s, 1H), 9.26 (s, 1H), 8.48 (s, 2H), 8.22 (d, J=8.7Hz, 3H), 7.90 (d, J=8.5Hz, 1H), 7.69 (d, J=8.2Hz, 1H), 7.07 (s, 3H), 3.91 (s, 8H)

Embodiment 7

N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) -6- (p-methylphenyl) pyrimidine -4- formamide

m.p.244-245℃；ESI-MS[M+H]⁺m/z:509.2；¹H NMR(400MHz,DMSO) δ9.38(s,1H), 9.33 (s, 1H), 8.53 (s, 1H), 8.37 (d, J=8.4Hz, 2H), 8.19 (d, J=8.1Hz, 3H), 8.03 (d, J= 7.9Hz, 2H), 7.37 (d, J=7.0Hz, 3H), 3.93 (s, 4H), 3.75 (s, 4H), 2.36 (s, 3H)

Embodiment 8

N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) -6- (4- (trifluoromethyl) pyrimidine -4- Formamide

m.p.266-267℃；ESI-MS[M+H]⁺m/z:563.1；¹H NMR(400MHz,DMSO) δ9.50(s,1H), 9.13 (s, 1H), 8.68 (s, 1H), 8.50 (d, J=8.1Hz, 2H), 8.37 (t, J=7.9Hz, 2H), 8.04 (d, J= 8.5Hz, 1H), 7.92 (d, J=7.5Hz, 1H), 7.85 (d, J=8.3 Hz, 1H), 7.72 (s, 1H), 7.62 (s, 1H), 7.59 (s,1H),3.93(s,4H),3.75(s,4H).

Embodiment 9

5- (3- fluorophenyl)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide

m.p.262-263℃；ESI-MS[M+H]⁺m/z:512；¹H NMR(400MHz,DMSO) δ10.75(s,1H), 8.79 (d, J=5.0Hz, 1H), 8.55 (s, 1H), 8.40 (s, 1H), 8.00 (s, 2H), 7.98 (s, 2H), 7.92 (s, 1H), 7.88 (d, J=7.3Hz, 2H), 7.56 (d, J=3.1Hz, 2H), 7.53 (s, 1H), 4.27 (s, 4H), 3.80-3.74 (m, 4H).

Embodiment 10

5- (2,4 difluorobenzene base)-N- (4- (4- morpholino thieno [2,3-d] pyrimidine -2-base) phenyl) picolinamide

m.p.287-289℃；ESI-MS[M+H]⁺m/z:530.1；¹H NMR(400MHz,DMSO) δ10.77(s,1H), 8.80 (d, J=4.6Hz, 1H), 8.55 (s, 1H), 8.40 (s, 1H), 8.00 (s, 2H), 7.98 (s, 2H), 7.92 (s, 2H), 7.62(s,2H),7.56(s,1H),4.27(s,4H),3.77(s,4H).

The preparation of the bromo- 2- Aminopyrazine (9) of step F 3,5- bis-

Methylene chloride (200mL) and pyridine are added into the three-necked bottle equipped with 2- Aminopyrazine (14.27g, 0.15mol) The mixed solution of (25.3mL, 0.315mol)；Be protected from light, 40 DEG C under the conditions of flow back, be slowly added dropwise bromine (16.2mL, Methylene chloride (100mL) solution 0.315mol), solution is become orange and is eventually become Chinese red from orange in reaction process, about 1h is added dropwise；Continue the 30min that flows back at 40 DEG C；It is cooled to room temperature, distilled water (50mL) is added into reaction system, acutely Stir 10min, stratification；Lower liquid is collected, the liquid being collected into is washed 2 times with distilled water (100mL)；It will be organic It mutually moves in the flask equipped with silica gel (10g) and active carbon (1g), boiling reflux 30min；It filters, collect filtrate and depressurizes steaming It evaporates, the solid obtained after distillation is moved in the flask equipped with n-hexane (45mL), flow back 2h at 80 DEG C；It filters while hot, it will It is weighed after obtained solid product is dry, obtains pale yellow solid 18.15g, the i.e. bromo- 2- Aminopyrazine of 3,5- bis-, yield 47.8%.

¹H NMR(400MHz,DMSO)δ:8.14(s,1H),7.01(s,2H).

The preparation of the bromo- 2- Aminopyrazine (10) of step G 3- morpholine -5-

Morpholine (50mL) is added in the three-neck flask of the bromo- 2- Aminopyrazine of 3,5- bis- equipped with 12.50g, is kept for 80 DEG C Lower back flow reaction 1h, TLC detection reaction are completed.System after reflux is cooled to room temperature, the burning equipped with ice water (300mL) is added to It is stirred continuously in cup, solid is precipitated；It filters, obtain the yellow solid 12.30g, i.e. 3- morpholine -5- with metallic luster after drying Bromo- 2- Aminopyrazine, yield 96.1%.

¹H NMR(400MHz,DMSO)δ:7.70(s,1H),6.28(s,2H),3.85-3.62(m, 4H),3.04(d,J =4.0Hz, 4H).

The preparation of step H 4- (6- bromine imidazo [1,2-a] pyrazine -8- base) morpholine (11)

4.4g chloroacetaldehyde is mixed with 15mL isopropanol, takes 10mL mixed solution that the round bottom that 1.7g intermediate 10 is housed is added In flask, it is warming up to 45 DEG C and starts to react 1h；The mixed solution of remaining chloroacetaldehyde and isopropanol is added in flask, is warming up to 65 DEG C reaction, TLC detection react, stop react.It is cooled to room temperature, is added in the beaker equipped with 300mL ice water, brown color is precipitated Solid filters, is dry, obtaining yellow-brown solid product, i.e. 4- (6- bromine imidazo [1,2-a] pyrazine -8- base) morpholine.

The preparation of step I 4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) aniline (13)

It referring to step D method, is reacted, is made with para-bromoaniline by 4- (6- bromine imidazo [1,2-a] pyrazine -8- base) morpholine 4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) aniline.

Step J N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -4- phenylpyridine amide

Pyridine acids side chain compound is dissolved in 8mL methylene chloride, the dry DMF of 1 drip-dry is added, oxalyl chloride is added dropwise dropwise (1.5 times of equivalents) the DIPEA mixed solution of 4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) aniline and 1mL are added dropwise Into the dichloromethane solution of side chain, reaction 10min is finished, TLC stops reaction after detecting fully reacting.

Reaction solution is washed 2 times with saturated sodium carbonate solution, takes organic layer, and solvent is evaporated off, and isopropanol is added and stirs 30min, It filters, filter residue is dissolved in 15mL methylene chloride, and a small amount of silica gel is added, and stirs 10min, is filtered, is taken filtrate, solvent is evaporated off, and is added The washing of 10mL isopropanol, filters to obtain neat solid product, i.e. N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) benzene Base) -4- phenylpyridine amide.

Embodiment 11

N- (4- (- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -4- phenylpyridine amide

First according to step F synthetic intermediate 9, intermediate 9 and morpholine by step G synthetic intermediate 10, intermediate 10 with Chloroethene aldehyde reaction is reacted with para-bromoaniline through step I synthetic intermediate by step H synthetic intermediate 11, then by intermediate 11 13, last intermediate 13 is reacted by step J with pyridine acids side chain compound, obtains N- (4- (- morpholino imidazo [1,2- A] pyrazine -6- base) phenyl) -4- phenylpyridine amide.

m.p.261-262℃；ESI-MS[M+H]⁺m/z:477.2；¹H NMR(400MHz,DMSO) δ9.55(s,1H), 7.59 (d, J=5.0Hz, 1H), 7.34 (s, 1H), 7.20 (s, 1H), 6.79 (d, J=7.8Hz, 5H), 6.74-6.65 (m, 3H), 6.34 (dd, J=8.4,4.3Hz, 4H), 3.07 (s, 4H), 2.61-2.51 (m, 4H)

According to the method for embodiment 11, first according to step F synthetic intermediate 9, intermediate 9 and R²Substituted morpholine passes through step Rapid G synthetic intermediate 10, intermediate 10 and chloroethene aldehyde reaction pass through step H synthetic intermediate 11, then by intermediate 11 and to bromine By step I synthetic intermediate 13, last intermediate 13 passes through step J different substituents for aniline or the reaction of -2 amine of 5- bromopyridine The side chain of (e.g., pyridine acids or pyrimidine acids) reacts, and 12~20 compound of embodiment is made respectively.

Embodiment 12

4- (2,4 difluorobenzene base)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide

m.p.266-267℃；ESI-MS[M+H]⁺m/z:513.2；¹H NMR(400MHz,DMSO) δ10.76(s,1H), 8.79 (d, J=5.1Hz, 1H), 8.52 (s, 1H), 8.26 (s, 1H), 7.96 (d, J=7.3Hz, 4H), 7.89 (s, 1H), 7.85-7.74 (m, 2H), 7.52 (s, 1H), 7.43 (d, J=9.6Hz, 1H), 7.26 (t, J=8.3Hz, 1H), 4.23 (s, 4H), 3.74 (d, J=4.3Hz, 4H)

Embodiment 13

4- (4- ethylphenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide

m.p.261-262℃；ESI-MS[M+H]⁺m/z:505.2；¹H NMR(400MHz,DMSO) δ10.88(s,1H), 8.78 (d, J=4.7Hz, 1H), 8.40 (d, J=7.2Hz, 3H), 8.08 (d, J=8.4Hz, 2H), 8.03 (s, 1H), 7.74 (d, J=7.6Hz, 2H), 7.54 (dd, J=18.7,6.0Hz, 2H), 7.50 (d, J=7.5Hz, 1H, 7.45 (d, J= 7.5Hz, 2H), 3.91 (s, 4H), 3.77 (s, 4H), 2.68 (d, J=7.4Hz, 2H), 1.23 (d, J=7.7Hz, 3H)

Embodiment 14

4- (2,4- 3,5-dimethylphenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide

m.p.234-235℃；ESI-MS[M+H]⁺m/z:505.2；¹H NMR(400MHz,DMSO) δ10.83(s,1H), 8.77 (d, J=5.0Hz, 1H), 8.62 (s, 1H), 8.00 (dd, J=17.9,9.6Hz, 6H), 7.69-7.63 (m, 2H), 7.25-7.11 (m, 3H), 4.20 (s, 4H), 3.77 (s, 4H), 2.28 (d, J=29.3Hz, 6H)

Embodiment 15

N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -4- (p-methylphenyl) picolinamide

m.p.271-273℃；ESI-MS[M+H]⁺m/z:491.2；¹H NMR(400MHz,DMSO) δ9.87(s,1H), 7.90 (d, J=5.2Hz, 1H), 7.56 (s, 1H), 7.44 (s, 1H), 7.22-7.15 (m, 3H), 6.95 (d, J=8.5Hz, 3H),6.73(s,1H),6.54(s,4H),3.45(s,4H),3.15– 2.68(m,4H),0.68–0.16(m,3H).

Embodiment 16

5- (3- fluorophenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide

m.p.261-264℃；ESI-MS[M+H]⁺m/z:495.2；¹H NMR(400MHz,CDCl₃) δ10.18(s,1H), 8.85 (s, 2H), 8.40 (d, J=8.1Hz, 2H), 8.29 (d, J=8.1Hz, 1H), 8.09 (s, 3H), 7.88 (s, 1H), 7.75 (s, 1H), 7.52 (d, J=5.1Hz, 1H), 7.45 (s, 1H), 7.36 (d, J=9.2Hz, 1H), 7.20 (d, J= 9.0Hz,1H),4.17(s,4H),4.03(s,4H).

Embodiment 17

5- (4- methoxyphenyl)-N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) picolinamide

m.p.255-257℃；ESI-MS[M+H]⁺m/z:507.2；¹H NMR(400MHz,DMSO) δ11.05(s,1H), 8.74 (d, J=5.1Hz, 1H), 8.57 (s, 1H), 8.47 (s, 1H), 8.03 (d, J=8.6Hz, 2H), 8.02 (s, 2H), 7.87 (d, J=9.8Hz, 2H), 7.86 (d, J=8.6Hz, 2H), 7.51 (s, 1H), 7.14 (d, J=8.6Hz, 2H), 4.32 (s, 4H), 4.19 (s, 2H), 3.85 (d, J=3.9Hz, 3H)

Embodiment 18

6- (4- methoxyphenyl)-N- (4- (8- morpholine imidazo [1,2-a] pyrazine -6- base) phenyl) pyrimidine -4- formyl Amine

m.p.269-271℃；ESI-MS[M+H]⁺m/z:508.2；¹H NMR(400MHz,DMSO) δ10.96(s,1H), 9.38 (s, 1H), 8.57 (d, J=22.1Hz, 2H), 8.46 (s, 1H), 8.32 (d, J=8.0Hz, 2H), 8.03 (dd, J= 14.7,8.4Hz, 4H), 7.96 (s, 1H), 7.59 (s, 1H), 7.14 (d, J=8.1Hz, 2H), 4.29 (s, 4H), 3.87 (s, 3H),3.80(s,4H).

Embodiment 19

6- (4- bromophenyl)-N- (4- (8- morpholine imidazo [1,2-a] pyrazine -6- base) phenyl) pyrimidine -4- formamide

m.p.221-223℃；ESI-MS[M+H]⁺m/z:557.1；¹H NMR(400MHz,DMSO) δ10.91(s,1H), 9.49 (s, 1H), 8.65 (s, 1H), 8.59 (d, J=11.9Hz, 2H), 8.38 (d, J=8.3Hz, 2H), 8.00-7.94 (m, 3H), 7.85 (d, J=8.3Hz, 2H), 7.59 (d, J=2.7Hz, 2H), 4.30 (s, 4H), 3.79 (d, J=4.0Hz, 4H)

Embodiment 20

N- (4- (8- morpholino imidazo [1,2-a] pyrazine -6- base) phenyl) -6- phenyl pyrimidine -4- formamide

m.p.272-275℃；ESI-MS[M+H]⁺m/z:478.1；¹H NMR(400MHz,DMSO) δ10.79(s,1H), 8.75 (d, J=5.1Hz, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 8.04 (d, J=8.7Hz, 2H), 8.01 (s, 1H), 7.98 (d, J=6.3Hz, 2H), 7.95 (s, 1H), 7.89 (d, J=8.5 Hz, 2H), 7.58 (s, 1H), 7.13 (d, J= 8.5Hz, 2H), 4.29 (s, 4H), 3.80 (d, J=4.3Hz, 4H)

Embodiment 21

N- (5- (4- morpholino thieno [3,2-d] pyrimidine -2-base) pyridine -2- base) -4- phenylpyridine amide

ESI-MS[M+H]+m/z:494.2

Embodiment 22

N- (5- (4- (3- methyl morpholine) thieno [3,2-d] pyrimidine -2-base) pyridine -2- base) -4- phenylpyridine amide

ESI-MS[M+H]+m/z:509.2

Embodiment 23

N- (5- (6- ((dimethylamino) methyl) -4- (3- methyl morpholine) thieno [3,2-d] pyrimidine -2-base) pyridine - 2- yl) -4- (thiophene -2- base) picolinamide

ESI-MS[M+H]⁺m/z:572.2

Embodiment 24

N- (5- (8- (3- methyl morpholine base) imidazo [1,2-a] pyrazine -6- base) pyridine -2- base) -4- (thiophene -2- base) Picolinamide

ESI-MS[M+H]⁺m/z:498.2

Embodiment 25

N- (5- (8- (3- methyl morpholine base) imidazo [1,2-a] pyrazine -6- base) pyridine -2- base) -4- phenylpyridine acyl Amine

ESI-MS[M+H]+m/z:492.2

Embodiment 26

N- (5- (6- (2- hydroxyl propyl- 2- yl) -4- morpholino thieno [3,2-d] pyrimidine -2-base) pyridine -2- base) -4- Phenylpyridine amide

ESI-MS[M+H]⁺m/z:553.2

Embodiment 27

N- (5- (4- morpholino -6- (the fluoro- 2- hydroxyl propyl- 2- yl of 1,1,1- tri-) thieno [2,3-d] pyrimidine -2-base) pyrrole Pyridine -2- base) -4- phenylpyridine amide

ESI-MS[M+H]⁺m/z:607.2

Embodiment 28

N- (5- (4,6- dimorpholino thieno [2,3-d] pyrimidine -2-base) pyridine -2- base) -4- phenylpyridine amide

ESI-MS[M+H]⁺m/z:580.2

In vitro cytotoxic effect

Heterocycle and pyrimidine or pyrazine compounds to the amide structure containing biaryl according to general formula I or II of the present invention carry out External inhibition lung carcinoma cell H460, colon cancer cell HT-29, human breast cancer cell MDA-MB-231, people's glioblastoma are female thin Born of the same parents' oncocyte U87MG, human lung carcinoma cell H1975 and Liver cancer cell SMMC-7721 screening active ingredients, reference substance GDC-0941 according to Document (J.Med.Chem., 2008,51 (18), pp 5522-5532) the method is prepared.

1) after 2~3 stabilizations of cell recovery and passage, it is made to disappear from culture bottle bottom with trypsin solution (0.25%) Change is got off.After cell dissociation buffer is poured into centrifuge tube, culture solution is added later to terminate digestion.By centrifuge tube in 800r/min 5mL culture solution is added after discarding supernatant liquid in lower centrifugation 10min, and piping and druming mixes cell, draws 10 μ L cell suspensions and cell is added It is counted in tally, adjustment cell concentration is 10⁴A/hole.Except the hole A1 is that blank well is not added extracellularly in 96 orifice plates, remaining all adds Enter 100 μ L cell suspensions.96 orifice plates are put into incubator and are cultivated for 24 hours.

2) with 50 μ L dmso solution given the test agent, appropriate culture solution is then added, sample is made to be dissolved into 2mg/mL Then sample is diluted to 20,4,0.8,0.16,0.032 μ g/mL in 24 orifice plates by medical fluid.

3 holes are added in each concentration, wherein surrounding two rows, two column cell growing way is affected by environment larger, it only and is blanc cell Hole uses.96 orifice plates are put into incubator and cultivate 72h.

3) band medicine culture solution in 96 orifice plates is discarded, is rinsed cell twice with phosphate buffer solution (PBS), in every hole MTT (tetrazole) (0.5mg/mL) 100 μ L is added to be put into incubator after 4h, discards MTT solution, 100 μ of dimethyl sulfoxide is added L.Oscillation dissolves survivaling cell sufficiently with MTT reaction product on magnetic force oscillator, is put into measurement result in microplate reader.It is logical Drug IC can be found out by crossing Bliss method₅₀Value.

Inhibition lung carcinoma cell H460, colon cancer cell HT-29, human breast cancer cell MDA-MB-231, the people of compound dislike Property glioblastoma cells U87MG, human lung carcinoma cell H1975 and Liver cancer cell SMMC-7721 Activity Results are shown in Table 2.

The experiment of PI3K α enzymatic activity

1, solution is prepared

1) untested compound adds 1mL DMSO, is made into 10mM storage solutions.Positive compound GDC-0941 storing liquid concentration For 10mM (being dissolved in DMSO), the storing liquid concentration of positive compound cis-platinum is 2mM (being dissolved in DMSO).

2) DMSO diluted compounds storing liquid is used, 2mM solution (100X) is made into.

3) 2 μ L2mM solution are taken, 18 μ L reaction solution diluted compounds are added to 200 μM of (10X) solution.

4) the above-mentioned solution of 2 μ L and 18 μ L reaction solutions are added in working plate, are made into 10X solution.

5) it takes with 1 μ L of solution in upper plate to detection plate.

6) 1 μ L kinase reaction liquid is added in the full inhibition control and null suppression control wells of detection plate, so that the concentration of DMSO It is 10%.

2, experimental procedure

1) layout of orifice plate according to experiment needs 384 orifice plates to arrange, in which:

A) HPE (complete to inhibit control): kinases and compound is not added, adds ATP, substrate and 1%DMSO.

B) ZPE (null suppression control): compound is not added, adds kinases, ATP, substrate and 1%DMSO.

C) positive reference compound hole: add kinases, ATP, substrate and various concentration positive compound.

D) untested compound hole: add kinases, ATP, substrate and untested compound.

2) agents useful for same is prepared

4XATP: ATP is diluted to 4X with reaction solution.

4X substrate solution: substrate is diluted to 4X with reaction solution.

2.5X kinase solution: with reaction solution by kinase dilution to 2.5X.

3) kinase reaction

A) 1 μ L10X compound (positive control of untested compound or various kinases) solution is added according to the every hole of arrangement, It is complete to inhibit control and null suppression control wells that 1 μ L reaction solution is added.

B) 4 μ L2.5X kinase solutions are added according to the every hole of arrangement.It is complete to inhibit control wells that 4 μ L reaction solutions are added.

C) it will test plate 1000rpm centrifugation to mix.

D) 4XATP solution is mixed in equal volume with 4X substrate solution, obtains 2XATP- substrate solution.

E) the above-mentioned 2XATP- substrate solution of 5 μ L is added according to the every hole of arrangement.

F) it will test plate 1000rpm centrifugation to mix.

G) it will test plate and be placed in 30 DEG C of reactions 1 hour.

H) 10 μ LKinase glo plus or ADP-Glo reaction reagents are added in every hole, and 27 DEG C are placed 20 minutes.

I) 20 μ LKinase Detection reagents are added in every hole, and 27 DEG C are placed 30 minutes.

J) Envision reads fluorescence values.

Note: needing preset room temperature before the use of Kinase glo plus, ADP-Glo and Kinase Detection reagent Half an hour.

4) primary data analysis

Prism5.0 analyzes initial data.

The IC of compound is calculated according to Bliss method₅₀

Experimental result is as shown in table 2.IC in table 1₅₀>=80%, is indicated with " +++ ", 80% > IC₅₀>=60%, with "+ + " indicate, 60% > IC₅₀>=40%, is indicated with "+", IC₅₀≤ 40%, it is indicated with "-", " NA " indicates inactive, " ND " table Show and does not test.

2 target compound anti tumor activity in vitro of table and enzymatic activity

From above-mentioned test result it can be clearly seen that the amide containing biaryl of the claimed general formula I or II of the present invention The heterocycle and pyrimidine or pyrazine compounds of structure, have good anti tumor activity in vitro.

The heterocycle and pyrimidine or pyrazine compounds of the amide structure containing biaryl of formula of I or II of the present invention can be applied individually With, but usually given with pharmaceutical carrier mixture, the selection of the pharmaceutical carrier will be according to required route of administration and standard medicine Object practice, separately below with the various pharmaceutical dosage forms of such compound, such as tablet, capsule, injection, aerosol, suppository, The preparation method of film, pill, externally-applied liniment and ointment illustrates its new opplication in pharmaceutical field.

Application examples 1: tablet

100 are pressed into, often after adding auxiliary material 12g to mix according to the general pressed disc method of pharmacy with 2 compound 6g of embodiment Slice weight 180mg.

Application examples 2: capsule

With 8 compound 10g of embodiment be packed into hollow glue after auxiliary material 20g being mixed according to the requirement of pharmacy capsule Capsule, each capsule weight 150mg.

Application examples 3: injection

Activated carbon adsorption is carried out according to pharmacy conventional method with 9 compound 10g of embodiment, is filtered through 0.65 μm of micropore After film filtering, hydro-acupuncture preparation is made in filling nitrogen gas tank, and every dress 2mL is filling 100 bottles total.

Application examples 4: aerosol

It after distilled water and other spoke material are added, is made after the dissolution of appropriate propylene glycol with 12 compound 20g of embodiment The clear solution of 100mL to obtain the final product.

Application examples 5: suppository

With 15 compound 5g of embodiment, by finely ground addition glycerol it is appropriate, the glycerin gelatine melted is added after grinding well, grinds Mill uniformly, is poured into the model for having applied lubricant, and suppository 25 is made

Application examples 6: film

With 17 compound 5g of embodiment, will be dissolved by heating after the stirrings such as polyvinyl alcohol, medicinal glycerin, water expansion, 80 meshes Net filtration, then 12 compound of embodiment is added to stirring and dissolving in filtrate, film applicator is film-made 50.

Application examples 7: pill

It is instilled in cryogenic liquid paraffin after being mixed with matrix 50g heating fusings such as gelatin with 18 compound 15g of embodiment, 1000 ball of dripping pill is made altogether.

Application examples 8: externally-applied liniment

With 20 compound 20g of embodiment, according to the auxiliary materials 5g mixed grinding such as conventional dose method and emulsifier, then plus steam Distilled water is obtained to 400mL.

Application examples 9: ointment

With 24 compound 5g of embodiment, ground well after finely ground with oleaginous bases 250g such as vaseline obtained.

Although describing the present invention by specific embodiment, modification and equivalent variations are for being proficient in this field It will be apparent from for technical staff, and they are included within the scope of the invention.

Claims (5)

1. a kind of heterocycle of amide structure containing biaryl and pyrimidine or pyrazine compounds, which is characterized in that structure is for example following logical Shown in Formulas I or II:

Wherein:

X=S, Y=CH or X=CH, Y=S；

Z is phenyl ring, five yuan or hexa-atomic heteroaromatic；

A, B, D, E, G are respectively CH or N；

R¹Selected from H,

R²Selected from H or-CH₃；

Ar is phenyl, naphthalene, 5~10 unit's heteroaryls, and the heteroaryl contains 1~3 hetero atom for being selected from O, N and S, and Ar appoints Select 1~4 identical or different R³Replace；

R³Selected from hydrogen, halogen, hydroxyl, trifluoromethyl, trifluoromethoxy, amino, azido, nitro, cyano, sulfydryl, (C₁~C₄) Alkyl, (C₃~C₆) naphthenic base, (C₂~C₄) alkenyl, (C₂~C₄) alkynyl, (C₁~C₄) alkoxy, (C₁~C₄) alkylthio group, allyl Base, (2- methyl) allyl, (C₁~C₄) alkoxy methyl or (C₁~C₄) alkyl acyl；

R⁴、R⁵It is identical or different, separately it is selected from (C₁~C₆) alkyl or (C₃~C₆) naphthenic base；Or R⁴And R⁵With and it The nitrogen-atoms that is connected be formed together 5~10 yuan of saturated heterocyclyls, the saturated heterocyclyl in addition to R⁴And R⁵The nitrogen of connection is former It is sub outer, the hetero atom of O, N and S are optionally selected from containing 1~3.

2. the heterocycle of the amide structure containing biaryl and pyrimidine or pyrazine compounds according to claim 1, it is characterised in that:

Wherein:

X=S, Y=CH or X=CH, Y=S；

Z is phenyl ring, pyridine ring；

A, B, D, E, G are respectively CH or N；

R¹Selected from H,

R²Selected from H or-CH₃；

Ar is phenyl, naphthalene, pyridyl group, pyrimidine radicals or thienyl, and the phenyl, naphthalene, pyridyl group, pyrimidine radicals, thienyl are appointed Select 1~4 identical or different R³Replace；

R³Selected from hydrogen, halogen, hydroxyl, trifluoromethyl, trifluoromethoxy, amino, azido, nitro, cyano, sulfydryl, (C₁~C₄) Alkyl, (C₃~C₆) naphthenic base, (C₂~C₄) alkenyl, (C₂~C₄) alkynyl, (C₁~C₄) alkoxy, (C₁~C₄) alkylthio group, allyl Base, (2- methyl) allyl, (C₁~C₄) alkoxy methyl or (C₁~C₄) alkyl acyl.

3. a kind of heterocycle of amide structure containing biaryl and pyrimidine or pyrazine compounds, it is characterised in that: the compound is Selected from one of following compounds:

4. a kind of Pharmaceutical composition, the amide structure containing biaryl described in any one of claims 1 to 3 including therapeutically effective amount Heterocycle and pyrimidine or pyrazine compounds and its pharmaceutically acceptable salt are active constituent and pharmaceutically acceptable excipient Agent.

5. the heterocycle and pyrimidine or pyrazine compounds of a kind of amide structure containing biaryl as described in claim 1 are treated in preparation And/or prevention prostate cancer and cervical carcinoma drug in application.