CN105418632A - Thienopyrimidine derivative, preparation method thereof and application thereof in medicines - Google Patents

Abstract

The invention provides a thienopyrimidine derivative, a preparation method thereof and an application thereof in medicines. Specifically, the invention relates to a compound represented by a formula I, wherein the radicals are defined in the description. The compound is effective tyrosine kinase inhibitors in one category, and is particularly suitable for being used as an EGFR and/or HER2 inhibitor.

Description

Thienopyrimidine analog derivative, its preparation method and in application pharmaceutically

Technical field

The present invention relates to medicinal chemistry art, particularly, the invention provides a kind of Thienopyrimidine analog derivative and its preparation method and application.

Background technology

Tumour is one of most serious disease threatening human health, and its treatment mainly comprises radiotherapy, chemotherapy and operative treatment.In recent years along with the development of cytobiology and tumor pharmacology, the chemotherapy of tumour there occurs huge change.Traditional chemotherapeutic agent is owing to non-specifically blocking cell fission thus also causing Normal cell death and abandoned gradually while killing tumour cell, simultaneously using the key node albumen in the signal path of abnormal activation in tumour cell as target spot, find that efficient, low toxicity, high specificity micromolecular inhibitor has become the important directions of current antitumor drug research and development.The receptor tyrosine kinase (RTK) that unconventionality expression activates in tumour has become the focus of antitumor drug research owing to all playing keying action at links such as tumor development, Invasion and Metastasis, chemoresistant.

EGF-R ELISA (EGFR, epidermalgrowthfactorreceptor, also known as HER1 or cerbB1) expresses Tyrosylprotein kinase HER family member the most widely in human cancer.EGFR structure comprises three regions: extracellular region, cross-film district and intracellular region.The N-terminal of extracellular region is made up of 622 amino acid, has 2 of formation ligand binding domain to be rich in halfcystine section; Cross-film district is a single α spiral; Intracellular region comprises kinases district and has the carboxyl terminal tail of many Tyr phosphorylation sites.Tyrosylprotein kinase (RTK) is that the γ phosphate transfection of ATP is transported to tyrosine residues.With ligand binding after, there is homology or heterodimer and make TK region form compact siro spinning technology in EGFR.Mediate Tyr phosphorylation site at carboxyl terminal tail RTK and carry out phosphorylation, create the binding site (Y992, Y1068, Y1086, Y1148 and Y11730) of enzyme and the sub-albumen of connection, thus Cellular Signaling Transduction Mediated reaction can be started.These intracellular signaling form different cell responses, comprise propagation, differentiation, adhere to and vascularization, transfer and apoptosis inhibit.

Research shows, EGFR has expression in nonsmall-cell lung cancer, prostate cancer, breast cancer, large bowel cancer, head and neck cancer, cancer of the stomach, ovarian cancer and carcinoma of the pancreas, and EGFR activation causes sophisticated signal conduction reaction.In dissimilar solid tumor, EGFR has propagation and overexpression, causes downstream signal conduct out of control and cause the formation of various tumour.In EGFR, to affect the RTK of acceptor active in the sudden change of ATP-binding site, the formation of interference tumorigenesis signal, meanwhile, EGFR also with the progress of tumour and poor prognosis closely related.

Due to the unique effect of EGFR and VEGFR in tumorigenesis, its monoclonal antibody and micromolecular inhibitor have become the focus of targeting antineoplastic medicine thing research and development.At present, had the inhibitor of several targeting EGFR or VEGFR to go on the market, nearly 20 drug candidates are in each development clinical.Wherein, the micromolecular inhibitor of Gefitinib and erlotinib representative listing targeting EGFR comparatively early.Gefitinib (Gefitinib, also known as ZD1839 or Iressa) is used for advanced Non-small cell lung (nonsmallcelllungcancer, NSCLC) as three line single therapy medicines.The two wires of the advanced NSCLC that erlotinib (Erlotinib, also known as OSI774 or Tarceva) is failed to respond to any medical treatment as standard scheme or three line medicines.

But, along with the clinical application of these medicines, it is found that, and not all high expression level EGFR patient can be effective to these medicines, some initially has the tumour of therapeutic response to occur progression of disease again after treatment some months to Gefitinib (Gefitinib).These results show, the EGFR inhibitor antitumor drug of current use has natural or secondary resistance phenomenon, therefore, Development of Novel has the new development direction that medicine that low resistance maybe can alleviate early stage inhibitor resistance has become tyrosine kinase inhibitor.

In addition, the application of multiple target effect in cancer therapy also has superiority, and especially produces the cancer patients of resistance for single target spot inhibitor.Tumour generation development be participated in by polygene multi-step, the multistage, inside and outside factor interaction complex process, and most tumors has 4 to 7 independently mutational sites, Mutiple Targets is therefore needed to treat validity and the persistence of guaranteeing drugs against tumor effect.In recent years, FDA successively have approved the listing of multiple multiple receptor tyrosine kinases inhibitor, comprises granted Xarelto (sorafenib) in 2005, granted Dasatinib (dasatinib) in 2006, granted Sutents (sunitinib) in 2007 and lapatinibditosylate (lapatinib).The action target spot of lapatinibditosylate is EGF-R ELISA (epidermalgrowthfactorreceptor, and human epidermal growth factor receptor 2 (humanepidermalgrowthfactorreceptor2 EFGR), HER-2), double inhibition effect can be produced to these two target spots.

In sum, this area has the medicine of Mutiple Targets tyrosine-kinase enzyme inhibition activity in the urgent need to exploitation.

Summary of the invention

The object of this invention is to provide the compound that a class has Mutiple Targets tyrosine-kinase enzyme inhibition activity.

A first aspect of the present invention, provides a kind of such as formula the compound shown in I:

Wherein:

R ₁, R ₂be selected from lower group independently of one another: hydrogen, the substituted or unsubstituted alkyl containing 1 ~ 6 carbon atom, the substituted or unsubstituted alkyl acyl containing 1 ~ 6 carbon atom, the substituted or unsubstituted cycloalkanoyl containing 3 ~ 6 carbon atoms, the substituted or unsubstituted alkenylacyl containing 2 ~ 6 carbon atoms, the substituted or unsubstituted aryl-acyl containing 6 ~ 10 carbon atoms, alkylsulfonyl, substituted or unsubstituted amide group-the CONH containing 2 ~ 6 carbon atoms, substituted or unsubstituted amido-acyl group the NHCO containing 1 ~ 6 carbon atom,

Or R ₁, R ₂form substituted or unsubstituted monocycle or many rings nitrogen heterocycle that carbonatoms is 3 to 10 together with the nitrogen-atoms be connected with them, or optional position is substituted or unsubstituted monocycle or the bicyclic nitrogen-containing heterocyclic base of 3 to 10 by the carbonatoms of nitrogen-atoms, Sauerstoffatom, sulphur atom displacement;

Or R ₁, R ₂5 ~ 7 membered nitrogen-containing heteroaryl bases are jointly formed with the ortho position carbon atom of the nitrogen-atoms be connected and nitrogen-atoms;

R ₃be selected from lower group: hydrogen, substituted or unsubstituted contain 1 ~ 6 carbon atom alkyl, substituted or unsubstituted contain 1 ~ 6 carbon atom alkyl acyl, substituted or unsubstituted contain 1 ~ 6 carbon atom amide group, substituted or unsubstituted contain 3 ~ 6 carbon atoms cycloalkanoyl, substituted or unsubstituted contain 2 ~ 6 carbon atoms alkenylacyl, substituted or unsubstitutedly contain the aryl-acyl of 5 ~ 6 carbon atoms, the substituted or unsubstituted alkylphosphonic acid carboxylic acid ester group containing 1 ~ 6 carbon atom;

R is for replacing aromatic ring or hetero-aromatic ring one or more substituting groups being selected from lower group of upper any hydrogen atom: halogen, C1-C4 alkyl, C1-C4 haloalkyl, alkoxyl group, hydroxyl, amino, cyano group, hydroxyl-C1-C4 alkyl, C2-C10 Heterocyclylalkyl, C2-C10 Heterocyclylalkyl-oxygen base, carboxyl, or C2-C6 carboxylic acid ester groups;

Ar ₂be selected from lower group: the phenyl of phenyl, halogen substiuted, C ₁-C ₆phenyl, xenyl, the xenyl of halogen substiuted, naphthyl, pyridyl, thienyl, the thienyl of halogen substiuted, C that alkyl replaces ₁-C ₃the furyl of the thienyl that alkyl replaces, furyl, halogen substiuted, or C ₁-C ₃the furyl that alkyl replaces;

A, B, D are selected from lower group independently of one another: carbon atom, nitrogen-atoms, Sauerstoffatom, sulphur atom or nothing; And only have one in A, B, D at the most for nothing;

N=0,1 or 2;

Wherein, unless stated otherwise, described replacement refers to that the one or more hydrogen atoms on group are selected from the substituting group replacement of lower group: halogen, hydroxyl, oxo, nitro, the haloalkyl of C1 ~ C6, the alkyl of C1 ~ C6, the thiazolinyl of C1 ~ C6, the alkynyl of C1 ~ C6, the aryl of C3 ~ C12, the arylalkyl of C3 ~ C12, the alkoxyl group of C1 ~ C6, the aryloxy of C3 ~ C12, amino, the acyl amino of C1 ~ C6, the alkyl-carbamoyl of C1 ~ C6, the aryl-amino-carbonyl of C3 ~ C12, the aminoalkyl group of C1 ~ C6, the acyl group of C1 ~ C6, carboxyl, the hydroxyalkyl of C1 ~ C6, the alkyl sulphonyl of C1 ~ C6, the aryl sulfonyl of C5 ~ C12, the alkyl sulfonyl of C1 ~ C6 is amino, the Arenesulfonyl amino of C5 ~ C12, the aralkylsulfonyl of C3 ~ C12 is amino, the alkyl-carbonyl of C1 ~ C6, the alkenylacyl of C1 ~ C6, the acyloxy of C1 ~ C6, the hydroxy-acyl of C1 ~ C6, cyano group, urea groups, the alkyl acyl of C1 ~ C6, the cycloalkanoyl of C1 ~ C6, alkylsulfonyl, carbamate groups,

Dotted line is chemical bond or nothing.

In another preference, described heterocycle is saturated rings or unsaturated ring.

In another preference, R ₁, R ₂, R ₃, R, Ar ₂, any one is respectively group corresponding in particular compound described in table 1 in A, B, D.

Should be understood that above-mentioned preferred group can combine to form various preferred compound of the present invention mutually.

In another preference, A, B, D are selected from lower group independently of one another: carbon atom, nitrogen-atoms; And have one in A, B, D for nitrogen-atoms.

In another preference, only there is one in A, B, D for nitrogen-atoms.

In another preference:

A, B, D are carbon atom;

Ar ₂for not replacing or the phenyl of halogen substiuted; And/or

R ₁, R ₂be selected from lower group independently of one another: hydrogen, alkyl containing the alkyl of 1 ~ 6 carbon atom or replacement, the alkyl acyl containing 1 ~ 6 carbon atom, the cycloalkanoyl containing 3 ~ 6 carbon atoms, substituted or unsubstituted contain 2 ~ 6 carbon atoms alkenylacyl, substituted or unsubstituted contain 6 ~ 10 carbon atoms aryl-acyl, alkylsulfonyl, substituted or unsubstitutedly contain the amide group of 2 ~ 6 carbon atoms, the substituted or unsubstituted amido-acyl group containing 1 ~ 6 carbon atom;

Or R ₁, R ₂form the heterocyclic radical of substituted or unsubstituted C3 ~ C10 together with the nitrogen-atoms be connected with them, wherein, described heterocyclic radical has the heteroatoms that 1 ~ 3 is selected from lower group: O, S or N;

Or R ₁, R ₂5 ~ 7 membered nitrogen-containing heteroaryl bases are jointly formed with the ortho position carbon atom of the nitrogen-atoms be connected and nitrogen-atoms.

In another preference, described formula I is selected from lower group:

(S)-(2-methoxyl group-1-phenyl-ethyl group)-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-yl]-amine;

(S)-{ 2-phenyl-2-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-is amino]-methoxyethyl }-diethyl phosphoric acid;

(S)-(2-methoxyl group-phenyl-ethyl group)-{ 6-[4-(5-methyl-isoxzzole-3-base)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-amine;

(S)-acetic acid-2-phenyl-2-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-is amino] ethyl ester;

(S)-1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-ethyl ketone;

(S)-1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-acrylketone;

(S)-2-hydroxyl-1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-ethyl ketone;

(S)-2-hydroxyl-1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-propane-1-ketone;

(S)-8-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-8-azabicyclo [3.2.1] octane-3-alcohol;

(S)-2-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl }-1-(4-thyl-piperazin-1-base)-ethyl ketone;

(S)-(2-methoxyl group-1-phenyl-ethyl group)-{ 6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-amine;

(S)-{ 6-[4-(4-ethyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-(2-methoxyl group-1-phenyl-ethyl group)-amine;

(S)-1-(4-ethyl-piperazin-1-base)-2-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl }-ethyl ketone;

(S)-{ 6-[4-(4-ethyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-amine;

(S)-8-(4-{4-[1-(the fluoro-phenyl of 4-)-2-methoxy-ethylamine]-thieno-[3,2-d] pyrimidine-6-base }-benzyl)-8-azabicyclo [3.2.1] octane-3-alcohol;

(S)-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-{ 6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-amine;

(S)-acetic acid-2-phenyl-2-[6-(4-piperidines-4-methylphenyl)-thieno-[3,2-d] pyrimidine-4-is amino]-ethyl ester;

(S)-acetic acid-2-{6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-is amino }-2-phenyl-ethyl ester;

(S)-acetic acid-2-{6-[6-(4-thyl-piperazin-1-base)-pyridin-3-yl]-thiophene [3,2-d] pyrimidine-4-yl amine }-2-phenyl-ethyl ester;

(S)-acetic acid-2-{6-[6-(4-ethyl-piperazin-1-base)-pyridin-3-yl]-thiophene [3,2-d] pyrimidine-4-yl amine }-2-phenyl-ethyl ester;

(S)-1-(4-{4-[4-(2-hydroxyl-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-ethyl ketone.

A second aspect of the present invention, provides a kind of preparation method of formula I as described in the first aspect of the invention, comprises step:

Under the existence of palladium catalyst, carry out linked reaction with formula (1) compound and formula (1a) compound, obtain formula I:

In formula, the definition of each group is as described in first aspect present invention.

In another preference, described palladium catalyst is selected from lower group: dichloro two (triphenylphosphine) palladium, tetrakis triphenylphosphine palladium, [1,1 '-bis-(diphenylphosphine) ferrocene] palladium chloride, or its combination.

In another preference, described method also comprises step: with formula (3) compound and R ₃x reacts, and obtains formula I:

Wherein, X is selected from lower group: Cl, OTs.

A third aspect of the present invention, provide a kind of tyrosine kinase inhibitor, described inhibitor contains the formula I as described in first aspect present invention suppressing significant quantity, or its pharmacy acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate.

In another preference, described tyrosine kinase inhibitor is EGFR/HER2 double inhibitor.

A fourth aspect of the present invention, provide a kind of pharmaceutical composition, described pharmaceutical composition contains the formula I as described in the first aspect of the invention for the treatment of significant quantity, or its pharmacy acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate.

In another preference, described pharmaceutical composition is used for the treatment of and Tyrosylprotein kinase process LAN and/or the too high relevant disease of tyrosine kinase activity, or described pharmaceutical composition is used for the treatment of the disease relevant to EGF-R ELISA.

In another preference, the disease that described EGF-R ELISA is relevant is selected from lower group: abnormal cell proliferation, metamorphosis, hypoerkinesia, angiogenesis disease, tumor growth, metastases disease, or its combination.

A fifth aspect of the present invention, provides a kind of purposes of formula I as described in the first aspect of the invention, for:

A () prepares tyrosine kinase inhibitor;

B () suppresses the activity of Tyrosylprotein kinase for external non-therapeutic;

C () is for the inhibition tumor cell growth of external non-therapeutic ground;

The medicine of d disease that () is correlated with for the preparation for the treatment of EGF-R ELISA activity.

In another preference, the disease that described EGF-R ELISA activity is relevant is selected from lower group: abnormal cell proliferation, metamorphosis, hypoerkinesia, angiogenesis disease, tumor growth, metastases disease, or its combination.

In another preference, described tumour cell is A431 cell.

In another preference, described tyrosine kinase inhibitor is multiple receptor tyrosine kinases inhibitor.

In another preference, described EGF-R ELISA is selected from lower group: EGFR and/or HER2.

In another preference, the IC of described suppression ₅₀value is≤50nM.

In another preference, in third aspect present invention or fourth aspect, described pharmacy acceptable salt is the salt of group under being selected from of formula I: inorganic acid salt, organic acid salt, alkylsulfonate, arylsulphonate, or its combination; Preferably, described salt is selected from lower group of hydrochloride, hydrobromate, nitrate, vitriol, phosphoric acid salt, formate, acetate, propionic salt, benzoate, maleate, fumarate, succinate, tartrate, Citrate trianion, metilsulfate, ethyl sulfonate, benzene sulfonate, tosilate, or its combination;

Described pharmaceutically acceptable solvate, refers to the solvate that formula I and the solvent being selected from lower group are formed: water, ethanol, Virahol, ether, acetone, or its combination.

Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.

Embodiment

The present inventor, through long-term and deep research, has unexpectedly prepared a class and had compound such as formula structure (I) Suo Shi, and described compound is the effective tyrosine kinase inhibitor of a class, is especially suitable for use as EGFR and/or HER2 inhibitor.Based on above-mentioned discovery, contriver completes the present invention.

Term

In the present invention, described alkyl comprises the alkyl of straight or branched, and described alkenyl comprises the alkenyl of straight or branched, and described alkynyl group comprises the alkynyl group of straight or branched, and described halogen is F, Cl, Br or I, is preferably F or Br.

Unless stated otherwise, in the present invention, term " replacement " refers to that the one or more hydrogen atoms on group are selected from the substituting group replacement of lower group: C1 ~ C4 alkyl, C3 ~ C7 cycloalkyl, C1 ~ C4 alkoxyl group, halogen, hydroxyl, carboxyl (-COOH), C1 ~ C4 aldehyde radical, C2 ~ C4 acyl group, C2 ~ C4 ester group, amino, phenyl; Described phenyl comprises unsubstituted phenyl or has 1-3 substituent substituted-phenyl, and described substituting group is selected from: halogen, C1-C4 alkyl, cyano group, OH, nitro, C3 ~ C7 cycloalkyl, C1 ~ C4 alkoxyl group, amino.

Especially, in this article, unless stated otherwise, the atom mentioned comprises its all isotopic form, such as, when mentioning " hydrogen atom ", refers to hydrogen atom, D atom, tritium atom or its combination.In the present invention, the abundance of the various isotope atom of certain element can be this element in the naturally occurring state of occurring in nature, also can be the state of certain isotopic enrichment.

Term " C1 ~ C4 alkyl " refers to the straight or branched alkyl with 1 ~ 4 carbon atom, such as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, sec-butyl, the tertiary butyl or similar group.

Especially, unless stated otherwise, in the present invention, when not limiting the carbon atom number of group, refer to that there is 1-10 carbon atom, the group of a preferred 1-4 carbon atom.

Term " carbonatoms is substituted or unsubstituted monocycle or many rings nitrogen heterocycle of 3 to 10 " refers to have the nitrogenous cyclic group of 3 ~ 10 carbon atoms, comprise monocycle (such as piperazine, piperidines, Pyrrolidine, morpholine ring etc.), two rings or many rings (comprising bridged ring, volution) base, wherein, above-mentioned monocycle nitrogen heterocycle such as (but being not limited to): pyrrolidyl, piperazinyl, piperidyl, morpholinyl etc., above-mentioned many rings nitrogen heterocycle comprises (but being not limited to): azabicyclo [3.2.1] octyl, or group as follows:

Or similar group.

Term " 5 ~ 7 yuan of heteroaryls " refers to the heteroaryl with 5 ~ 7 carbon atoms or heteroatoms (being selected from N, O, S), such as pyrryl, pyridyl, furyl, or similar group.

In another preference, described heterocycle is saturated rings or unsaturated ring.

Term " C1 ~ C4 alkoxyl group " refers to the straight or branched alkoxyl group with 1-4 carbon atom, such as methoxyl group, oxyethyl group, propoxy-, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert.-butoxy or similar group.

Term " alkyl acyl " or " alkyl-carbonyl " refer to the group with "-CO-alkyl " structure, such as methylacyl, ethyl acyl group, Acryl, sec.-propyl acyl group, butyl acyl group, isobutyl-acyl group, sec-butyl acyl group, tertiary butyl acyl group or similar group.

Term " amide group containing 2 ~ 6 carbon atoms " refers to the group with structure as Suo Shi " C1-C5 alkyl-CONH-".

Term " amido-acyl group containing 1 ~ 6 carbon atom " or " the amido acyl group containing 1 ~ 6 carbon atom " refer to have as " NH ₂cO-" or " C1-C5 alkyl-NHCO-" shown in the group of structure, especially, when described group is " NH ₂cO-" time, " amido-acyl group " writing " amino-acyl ".

As used herein, term " oxo " refer to two or more hydrogen atoms on group replace by one or more Sauerstoffatoms, such as ,-CH ₂-by after oxo, formed-C (O)-structure.

Term " pharmaceutically acceptable solvate " refers to the solvate of corresponding compound and water, ethanol, Virahol, ether, acetone.

Formula I

The invention provides a kind of such as formula the compound shown in I:

Wherein:

R ₁, R ₂be selected from lower group independently of one another: hydrogen, the substituted or unsubstituted alkyl containing 1 ~ 6 carbon atom, the substituted or unsubstituted alkyl acyl containing 1 ~ 6 carbon atom, the substituted or unsubstituted cycloalkanoyl containing 3 ~ 6 carbon atoms, the substituted or unsubstituted alkenylacyl containing 2 ~ 6 carbon atoms, the substituted or unsubstituted aryl-acyl containing 6 ~ 10 carbon atoms, alkylsulfonyl, the substituted or unsubstituted amide group containing 2 ~ 6 carbon atoms, the substituted or unsubstituted amido acyl group containing 1 ~ 6 carbon atom,

Or R ₁, R ₂forming carbonatoms together with the nitrogen-atoms be connected with them is the substituted or unsubstituted monocycle of 3 to 10 or the nitrogen heterocycle of dicyclo, or optional position is substituted or unsubstituted monocycle or the bicyclic nitrogen-containing heterocyclic base of 3 to 10 by the carbonatoms of nitrogen-atoms, Sauerstoffatom, sulphur atom displacement;

Or R ₁, R ₂5 ~ 7 membered nitrogen-containing heteroaryl bases are jointly formed with the ortho position carbon atom of the nitrogen-atoms be connected and nitrogen-atoms;

R ₃be selected from lower group: hydrogen, substituted or unsubstituted contain 1 ~ 6 carbon atom alkyl, substituted or unsubstituted contain 1 ~ 6 carbon atom alkyl acyl, substituted or unsubstituted contain 1 ~ 6 carbon atom amide group, substituted or unsubstituted contain 3 ~ 6 carbon atoms cycloalkanoyl, substituted or unsubstituted contain 2 ~ 6 carbon atoms alkenylacyl, substituted or unsubstitutedly contain the aryl-acyl of 5 ~ 6 carbon atoms, the substituted or unsubstituted alkylphosphonic acid carboxylic acid ester group containing 1 ~ 6 carbon atom; Preferably, R ₃it is not H atom;

R is for replacing aromatic ring or hetero-aromatic ring one or more substituting groups being selected from lower group of upper any hydrogen atom: halogen, C1-C4 alkyl, C1-C4 haloalkyl, alkoxyl group, hydroxyl, amino, cyano group, hydroxyl-C1-C4 alkyl, C2-C10 Heterocyclylalkyl, C2-C10 Heterocyclylalkyl-oxygen base, carboxyl, or C2-C6 carboxylic acid ester groups;

Ar ₂be selected from lower group: the phenyl of phenyl, halogen substiuted, C ₁-C ₆phenyl, xenyl, the xenyl of halogen substiuted, naphthyl, pyridyl, thienyl, the thienyl of halogen substiuted, C that alkyl replaces ₁-C ₃thienyl, furyl, the furyl of halogen substiuted, C that alkyl replaces ₁-C ₃alkyl replace furyl in any one;

A, B, D are selected from lower group independently of one another: carbon atom, nitrogen-atoms, Sauerstoffatom, sulphur atom or nothing; And only have one in A, B, D at the most for nothing;

N=0,1 or 2;

Wherein, described replacement refers to that the one or more hydrogen atoms on group are selected from the substituting group replacement of lower group: halogen, hydroxyl, oxo, nitro, the haloalkyl of C1 ~ C6, the alkyl of C1 ~ C6, the thiazolinyl of C1 ~ C6, the alkynyl of C1 ~ C6, the aryl of C3 ~ C12, the arylalkyl of C3 ~ C12, the alkoxyl group of C1 ~ C6, the aryloxy of C3 ~ C12, amino, the acyl amino of C1 ~ C6, the alkyl-carbamoyl of C1 ~ C6, the aryl-amino-carbonyl of C3 ~ C12, the aminoalkyl group of C1 ~ C6, the acyl group of C1 ~ C6, carboxyl, the hydroxyalkyl of C1 ~ C6, the alkyl sulphonyl of C1 ~ C6, the aryl sulfonyl of C5 ~ C12, the alkyl sulfonyl of C1 ~ C6 is amino, the Arenesulfonyl amino of C5 ~ C12, the aralkylsulfonyl of C3 ~ C12 is amino, the alkyl-carbonyl of C1 ~ C6, the alkenylacyl of C1 ~ C6, the acyloxy of C1 ~ C6, the hydroxy-acyl of C1 ~ C6, cyano group, urea groups, the alkyl acyl of C1 ~ C6, the cycloalkanoyl of C1 ~ C6, alkylsulfonyl, carbamate groups,

Dotted line is chemical bond or nothing.

In a preferred embodiment of the invention, A, B, D are carbon atom;

Ar ₂for phenyl; And/or

R ₁, R ₂be selected from lower group independently of one another: hydrogen, alkyl containing the alkyl of 1 ~ 6 carbon atom or replacement, the alkyl acyl containing 1 ~ 6 carbon atom, the cycloalkanoyl containing 3 ~ 6 carbon atoms, substituted or unsubstituted contain 2 ~ 6 carbon atoms alkenylacyl, substituted or unsubstituted contain 6 ~ 10 carbon atoms aryl-acyl, alkylsulfonyl, substituted or unsubstitutedly contain the amide group of 2 ~ 6 carbon atoms, the substituted or unsubstituted amido acyl group containing 1 ~ 6 carbon atom;

Or R ₁, R ₂form the heterocyclic radical of substituted or unsubstituted C3 ~ C10 together with the nitrogen-atoms be connected with them, wherein, described heterocyclic radical has the heteroatoms that 1 ~ 3 is selected from lower group: O, S or N;

Or R ₁, R ₂5 ~ 7 membered nitrogen-containing heteroaryl bases (namely shape is as the situation of compound in table 1 3) are jointly formed with the ortho position carbon atom of the nitrogen-atoms be connected and nitrogen-atoms.

More preferably, described formula I is as shown in table 1.

Table 1

The preparation method of formula I

Present invention also offers a kind of preparation method of formula I, described method comprises step:

Under the existence of palladium catalyst; with formula (1) compound (i.e. 2-(the bromo-thiophene [3 of 6-of different substituents protection; 2-d] pyrimidine-4-is amino)-2-phenyl-ethanol) carry out linked reaction from formula (1a) compound (the different borate ester that replaces or phenylo boric acid), obtain formula I:

Wherein, the definition of each group is as noted before, and especially, in formula (1a), " B " in two assorted oxygen pentaborane structures represents boron atom.

In another preference, described linked reaction is Suzuki coupling.

In another preference, described palladium catalyst is selected from lower group: dichloro two (triphenylphosphine) palladium, tetrakis triphenylphosphine palladium, [1,1 '-bis-(diphenylphosphine) ferrocene] palladium chloride, or its combination.

Or the preparation method of described formula I comprises step: with formula (3) compound and R ₃x reacts, and obtains formula I:

Wherein, X is selected from lower group: Cl, OTs.

Pharmacy acceptable salt

The medicinal forms of the compounds of this invention can comprise compound itself, and other versions pharmaceutically acceptable, as optical isomer, and cis-trans-isomer etc., or pharmacy acceptable salt or solvate.

Preferably, described pharmacy acceptable salt comprises (but being not limited to): inorganic acid salt, example hydrochloric acid salt, hydrobromate, nitrate, vitriol, phosphoric acid salt etc.; Organic acid salt, as formate, acetate, propionic salt, benzoate, maleate, fumarate, succinate, tartrate, Citrate trianion etc.; Alkylsulfonate, as metilsulfate, ethyl sulfonate etc.; Arylsulphonate, as benzene sulfonate, tosilate etc.

Preferably, described pharmaceutically acceptable solvate comprises (but being not limited to): the solvate of described compound and water, ethanol, Virahol, ether, acetone etc.

The purposes of formula I

After deliberation, Thienopyrimidine analog derivative of the present invention has EGF-R ELISA (EGFR) and/or human epidermal growth factor receptor 2 (humanepidermalgrowthfactorreceptor2, HER2) inhibit activities, therefore, the tautomer of Thienopyrimidine analog derivative of the present invention or described derivative, racemic modification, enantiomer, diastereomer, pharmacy acceptable salt, any one or a few mixture in pharmaceutically acceptable solvate, can be applicable to prepare tyrosine kinase inhibitor, especially can be applicable to preparation EGFR and/or HER2 inhibitor.

Meanwhile, described inhibitor can be applicable to the medicine preparing prevention or treatment and EGF-R ELISA EGFR and/or HER2 relative disease.Specifically, can be applicable to the medicine prepared prevention or treat abnormal cell proliferation, metamorphosis, hypoerkinesia, angiogenesis and the metastases disease relevant to EGF-R ELISA EGFR and/or HER2.

In addition, described inhibitor can be applicable to the medicine prepared treatment or prevent the growth and metastasis of tumours relevant to EGF-R ELISA EGFR and/or HER2.

Described in this patent, the activeconstituents of inhibitor is preferably compound shown in table 1, or the tautomer of shown compound, racemic modification, enantiomer, diastereomer, pharmacy acceptable salt, any one or a few mixture in pharmaceutically acceptable solvate.

Major advantage of the present invention comprises:

Compared with prior art, the novel structure of Thienopyrimidine analog derivative provided by the invention, there is obvious EGFR inhibit activities, and part of compounds also has obvious inhibit activities to VEGFR, be expected to be developed as Tyrosylprotein kinase EGFR or/and VEGFR inhibitor, for the preparation of the abnormal cell proliferation prevented or treatment is relevant to EGF-R ELISA EGFR and/or Angiogenesis factor receptors VEGFR, the relative disease such as metamorphosis and hypoerkinesia and the medicine with angiogenesis or metastases relative disease, especially be expected for the preparation for the treatment of or the medicine preventing the growth and metastasis of tumours relevant to EGF-R ELISA EGFR and/or Angiogenesis factor receptors VEGFR, new developmental direction and approach is provided for Development of Novel has the tyrosine kinase inhibitor medicine that low resistance maybe can alleviate early stage inhibitor resistance, have broad application prospects and pharmaceutical use.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

The structure of compound obtained in following embodiment be by nucleus magnetic resonance ( ¹hNMR) and mass spectrum (MS) determined.

¹hNMR displacement (δ) provides with the unit of 1,000,000/(ppm). ¹the mensuration of HNMR uses BrukerAVANCE-400 nuclear magnetic resonance spectrometer, and the solvent of mensuration is deuterated dimethyl sulfoxide (DMSO-d ₆), deuterochloroform (CDCl ₃), be inside designated as tetramethylsilane (TMS), chemical shift is with 10 ^-6provide as unit.

The mensuration of MS is with FINNIGANLCQAd (ESI) mass spectrograph (manufacturer: Therm, model: FinniganLCQadvantageMAX).

IC ₅₀the mensuration of value is with NovoStar microplate reader (German BMG company).

Thin layer silica gel uses Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica-gel plate.

Silica gel column chromatography uses Yantai Huanghai Sea silica gel 200 ~ 300 order silica gel to be carrier.

HPLC test uses Agilent 1200DAD high pressure liquid chromatograph (SunfireC18150 × 4.6mm chromatographic column) and Waters2695-2996 high pressure liquid chromatograph (GiminiC18150 × 4.6mm chromatographic column).

Microwave reaction uses CEMDiscover-S908860 type microwave reactor.

In addition, without specified otherwise in following examples, reaction is carried out all under nitrogen atmosphere.

Argon atmospher refers to that reaction flask connects the argon gas balloon of an about 1L volume.

Nitrogen atmosphere refers to that reaction flask connects the hydrogen balloon of an about 1L volume.

Without specified otherwise in following examples, the solution in reaction refers to the aqueous solution.

Embodiment 1

The first step:

Under ice bath; by 60% sodium hydride (30g; 0.75mol) be dissolved in tetrahydrofuran (THF) (300ml), then add tetrahydrofuran (THF) (600ml) solution being dissolved with L-phthalic anhydride ammonia alcohol (50g, 0.36mol); remove ice bath; stirring at room temperature 2 hours, then under nitrogen protection, drips methyl iodide (24ml); stirring at room temperature 1 hour, then 70 DEG C are refluxed 2 hours.Reaction is until TLC monitors raw material reaction completely, and cool, system adds in icy salt solution, methyl tertiary butyl ether extracts, organic phase anhydrous sodium sulfate drying, concentrating under reduced pressure, obtain 2-methoxyl group-1-phenyl-ethylamine (51g, colorless oil), yield: 92%.

Second step:

By bromo-for 6-4-chlorothiophene also [3,2-d] pyrimidine (40g, 0.16mol) with 2-methoxyl group-1-phenyl-ethylamine (29g, 0.19mol) be dissolved in N, in dinethylformamide (250ml), then under ice bath, add DIPEA (40ml), remove ice bath, be warming up to 55 DEG C of reactions.Reaction is until TLC monitoring raw material reaction is complete, add water, extraction into ethyl acetate, organic phase anhydrous sodium sulfate drying, concentrating under reduced pressure, then sherwood oil recrystallization is added, suction filtration, filter cake n-hexane, obtain (the bromo-thieno-[3 of 6-, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (32g, white solid), yield: 55%.

3rd step:

By compound (the bromo-thieno-[3 of 6-under room temperature, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (18.2g, 50mmol) with 4-[4-(4, 4, 5, 5-tetramethyl--[1, 3, 2] dioxy boron penta ring-2-base)-phenyl]-piperazine-1-carboxylic acid tert-butyl ester (22.1g, 55mmol) be dissolved in N, in dinethylformamide (400ml), add tetrakis triphenylphosphine palladium (1.2g successively, 1mmol), sodium carbonate solution (1mol/L, 80ml), nitrogen protection, be heated to 90 DEG C until TLC monitoring raw material reaction is complete, be cooled to room temperature, reaction solution adds in frozen water, suction filtration, filter cake washes with water, dry, obtain 4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3, 2-d] pyrimidine-6-base]-phenyl }-piperazine-1-carboxylic acid tert-butyl ester (27g, light pink solid), yield: 96%.

4th step:

By 4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl }-piperazine-1-carboxylic acid tert-butyl ester (5.6g, 10mmol) be dissolved in methylene dichloride (50ml), trifluoroacetic acid (15ml is added under ice bath, 200mmol), stir 10 minutes, then remove ice bath, stirring at room temperature is complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, gained residue is purified with silica gel column chromatography, obtain (2-methoxyl group-1-phenyl-ethyl group)-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-yl]-amine (5.4g, white solid).MSm/z(ESI):460.58[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.38(s,1H),8.33(d,1H),7.82(t,3H),7.46(m,4H),7.31(m,2H),7.22(m,1H),5.67(m,1H),3.80(m,1H),3.62(m,1H),3.52(s,2H),3.32(s,3H),3.17(s,1H),2.41(m,4H),2.40(m,4H).

Embodiment 2

By 2-phenyl-2-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-amino] ethanol (500mg, 1.12mmol) be dissolved in DMF, add magnesium isopropoxide (128mg, 0.90mmol), be warming up to 65 DEG C to stir 30 minutes, be then cooled to 50 DEG C, add tolysulfonyl methyl-phosphorous acid diethyl ester (0.43ml, 1.68mmol), TLC monitoring raw material reaction is stirred to complete.Then water is added, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, { 2-phenyl-2-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-is amino]-methoxyethyl }-diethyl phosphoric acid (260mg, white solid), yield: 40%.MSm/z(ESI):596.70[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.36(s,1H),8.18(d,1H),7.80(t,3H),7.43(d,4H),7.31(t,2H),7.22(t,1H),5.45(m,1H),4.98(t,1H),4.02(m,4H),3.75(m,2H),3.50(s,2H),2.77(d,2H),2.59(s,4H),2.40(s,4H),1.23(t,6H).

Embodiment 3

The first step:

By oxammonium hydrochloride (7.2g, 86mmol) be dissolved in methyl alcohol (30ml) and water (20ml), add magnesium oxide (5.1g again, 129mmol), stir 10 minutes, then add tetrahydrofuran (THF) (300ml) solution of Tosyl chloride (8g, 43mmol), room temperature vigorous stirring, reacts complete to TLC monitoring raw material reaction.After suction filtered through kieselguhr, anhydrous magnesium sulfate drying, concentrating under reduced pressure, obtains N-hydroxy-4-methyl-benzsulfamide (7g, white solid), yield: 87.5%.

Second step:

By N-hydroxy-4-methyl-benzsulfamide (2.8g, 15mmol) be dissolved in methyl alcohol (12ml) and water (2ml), add salt of wormwood (3.33g successively, 24mmol) with 4-(the bromo-phenyl of 4-)-Ding-3-alkene-2 ketone (450mg, methyl alcohol (6ml) solution 2mmol), complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, organic phase anhydrous sodium sulfate drying, concentrating under reduced pressure, purifies gained residue with silica gel column chromatography, obtains 3-(the bromo-phenyl of 4-)-5-methyl-isoxzzole (220mg, yellow solid), yield: 46%.

3rd step:

3-(the bromo-phenyl of 4-)-5-methyl-isoxzzole (100mg, 0.42mmol) is dissolved in Isosorbide-5-Nitrae-dioxane, add connection boric acid pinacol ester (127mg successively, 0.5mmol), 1,1 '-bis-Diphenyl phosphino ferrocene palladium chloride (18mg, 0.021mmol), Potassium ethanoate (42mg, 0.42mmol), nitrogen replacement, be warming up to 95 DEG C of stirrings, complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, purifies gained residue with silica gel column chromatography, obtains 5-methyl-3-[4-(4,4,5,5-tetramethyl--[1,3,2] two assorted oxygen pentaborane-2-bases)-phenyl] isoxzzole (98mg, white solid), yield: 81.6%.

4th step:

By (the bromo-thieno-of 6-[3,2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (47mg, 0.13mmol) He 3 (40mg, 0.14mmol) are dissolved in DMF, then tetrakis triphenylphosphine palladium (3mg is added successively, 0.0026mmol), 1N sodium carbonate solution (1ml), nitrogen replacement, be warming up to 90 DEG C of stirrings, complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, organic phase anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain (2-methoxyl group-phenyl-ethyl group)-{ 6-[4-(5-methyl-isoxzzole-3-base)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-amine (50mg, white solid), yield: 87.7%.MSm/z(ESI):443.62[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.35(d,1H),7.99(s,4H),7.85(s,1H),7.52(m,2H),7.30(m,2H),7.25(m,1H),6.86(s,1H),5.65(m,1H),3.80(m,1H),3.65(m,1H),3.32(s,3H),2.49(s,3H).

Embodiment 4

The first step:

By 4-{4-[4-(2-hydroxyl-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl }-piperazine-1-carboxylic acid tert-butyl ester (200mg, 0.37mmol) be dissolved in tetrahydrofuran (THF), add acetic anhydride (0.17ml successively, 1.83mmol), triethylamine (0.26ml, 1.83mmol), stirring at room temperature, complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 4-{4-[4-(2-acetoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl-piperazine-1-carboxylic acid tert-butyl ester (189mg, white solid), yield: 87%.

Second step:

By 4-{4-[4-(2-acetoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl }-piperazine-1-carboxylic acid tert-butyl ester (189mg, 0.32mmol) be dissolved in methylene dichloride, add trifluoroacetic acid (0.24ml, 3.2mmol), stirring at room temperature, complete to TLC monitoring raw material reaction.Drip several ammoniacal liquor inwards, concentrating under reduced pressure, gained residue is purified with silica gel column chromatography, obtain acetic acid-2-phenyl-2-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-amino] ethyl ester (120mg, white solid), yield: 77%.MSm/z(ESI):488.62[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.38(s,1H),8.33(d,1H),7.82(t,3H),7.46(m,4H),7.31(m,2H),7.22(m,1H),5.67(m,1H),3.80(m,1H),3.62(m,1H),3.52(s,2H),2.41(m,4H),2.40(m,4H),2.01(s,3H).

Embodiment 5

By (2-methoxyl group-1-phenyl-ethyl group)-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-yl]-amine (150mg, 0.33mmol) be dissolved in methyl alcohol (10ml), add acetic anhydride (33mg, 0.33mmol), stirring at room temperature, complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, gained residue is purified with silica gel column chromatography, obtain 1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-ethyl ketone (75mg, white solid), yield: 45.3%.MSm/z(ESI):502.65[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.31(d,1H),7.82(t,3H),7.48(d,4H),7.32(t,2H),7.24(t,1H),5.67(m,1H),3.80(t,1H),3.56(m,3H),3.45(s,4H),3.36(s,2H),3.31(s,3H),2.41(d,4H),1.98(s,3H).

Embodiment 6

By (2-methoxyl group-1-phenyl-ethyl group)-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-yl]-amine (200mg, 0.44mmol) be dissolved in N, in dinethylformamide (10ml), add vinylformic acid (0.04ml, 0.52mmol), O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid (166mg, 0.52mmol) and DIPEA (0.15ml, 0.88mmol), room temperature reaction is complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-acrylketone (85mg, white solid), yield: 37.6%.MSm/z(ESI):514.74[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.32(d,1H),7.82(t,3H),7.48(d,4H),7.34(t,2H),7.26(t,1H),6.80(m,1H),6.12(d,1H),5.70(d,2H),3.80(t,1H),3.50-3.66(m,7H),3.32(s,3H),2.40(s,4H).

Embodiment 7

By (2-methoxyl group-1-phenyl-ethyl group)-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-yl]-amine (120mg, 0.26mmol) be dissolved in N, in dinethylformamide (5ml), add oxyacetic acid (24mg, 0.31mmol), O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid (100mg, 0.31mmol) and DIPEA (0.09ml, 0.52mmol), room temperature reaction is complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 2-hydroxyl-1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-ethyl ketone (50mg, white solid), yield: 37.1%.

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.31(d,1H),7.82(t,3H),7.48(d,4H),7.32(t,2H),7.24(t,1H),5.67(m,1H),4.52(t,1H),4.08(d,2H),3.80(t,1H),3.57(m,3H),3.48(s,2H),3.35(s,2H),3.31(s,3H),2.40(s,4H).MSm/z(ESI):518.65[M+1]

Embodiment 8

By (2-methoxyl group-1-phenyl-ethyl group)-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-yl]-amine (250mg, 0.54mmol) be dissolved in N, in dinethylformamide (10ml), add 2 hydroxy propanoic acid (59mg, 0.65mmol), O-benzotriazole-N, N, N ', N '-tetramethyl-urea Tetrafluoroboric acid (208mg, 0.65mmol) and DIPEA (0.19ml, 1.08mmol), room temperature reaction is complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 2-hydroxyl-1-(4-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-propane-1-ketone (85mg, white solid), yield: 29.6%.MSm/z(ESI):532.65[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.34(s,1H),8.32(d,1H),7.82(t,3H),7.50(d,4H),7.35(t,2H),7.25(t,1H),5.65(m,1H),4.86(d,1H),4.40(t,1H),3.62(m,2H),3.55(s,3H),3.50(d,4H),3.32(s,3H),2.40(s,4H).

Embodiment 9

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (100mg, 0.27mmol) be dissolved in N, in dinethylformamide (5ml), add 8-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-benzyl]-8-azabicyclo [3.2.1] octane-3-alcohol (141mg, 0.41mmol), tetrakis triphenylphosphine palladium (32mg, 0.028mmol), 1N sodium carbonate (0.4ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 8-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl-8-azabicyclo [3.2.1] octane-3-alcohol (80mg, yellow solid), yield: 59%.MSm/z(ESI):501.68[M+1]

¹HNMR(400Hz,DMSO-d ₆):10.89(s,1H),8.45(d,2H),7.95(d,5H),7.55(d,2H),7.39(t,2H),7.30(t,1H),5.74(m,1H),5.00(s,1H),4.22(m,2H),3.86(m,3H),3.68(m,3H),3.39(s,3H),3.23(d,1H),2.45(s,3H),2.31(s,2H).

Embodiment 10

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (350mg, 1mmol) be dissolved in N, in dinethylformamide (10ml), add 1-(4-thyl-piperazin-1-base)-2-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-phenyl]-ethyl ketone (516.4mg, 1.5mmol), tetrakis triphenylphosphine palladium (120mg, 0.1mmol), 1N sodium carbonate (1ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 2-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl-1-(4-thyl-piperazin-1-base)-ethyl ketone (290mg, white solid), yield: 58%.MSm/z(ESI):502.66[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.49(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.67(m,1H),3.80(m,1H),3.78(s,2H),3.62(m,1H),3.49(m,4H),3.31(s,3H),2.25(m,4H),2.16(s,3H).

Embodiment 11

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (100mg, 0.27mmol) be dissolved in N, in dinethylformamide (5ml), add 1-methyl-4-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-benzyl]-piperazine (174mg, 0.55mmol), tetrakis triphenylphosphine palladium (31mg, 0.027mmol), 1N sodium carbonate (0.4ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain (2-methoxyl group-1-phenyl-ethyl group)-{ 6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-amine (30mg, white solid), yield: 23%.MSm/z(ESI):474.65[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.68(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),3.32(s,3H),2.78(m,4H),2.55(m,4H),2.46(s,3H)

Embodiment 12

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (200mg, 0.55mmol) be dissolved in N, in dinethylformamide (10ml), add 1-ethyl-4-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-benzyl]-piperazine (399mg, 1.1mmol), tetrakis triphenylphosphine palladium (64mg, 0.055mmol), 1N sodium carbonate (0.9ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain { 6-[4-(4-ethyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-(2-methoxyl group-1-phenyl-ethyl group)-amine (85mg, white solid), yield: 31.7%.MSm/z(ESI):487.69[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.49(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.70(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),3.32(s,3H),2.78(m,4H),2.55(m,4H),2.45(m,2H),1.27(t,3H)

Embodiment 13

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-(2-methoxyl group-1-phenyl-ethyl group) amine (100mg, 0.27mmol) be dissolved in N, in dinethylformamide (5ml), add 1-(4-ethyl-piperazin-1-base)-2-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-phenyl]-ethyl ketone (143mg, 0.41mmol), tetrakis triphenylphosphine palladium (32mg, 0.028mmol), 1N sodium carbonate (0.4ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 1-(4-ethyl-piperazin-1-base)-2-{4-[4-(2-methoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-phenyl-ethyl ketone (30mg, white solid), yield: 21.6%.MSm/z(ESI):516.68[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.49(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.70(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),3.32(s,3H),2.78(m,4H),2.55(m,4H),2.45(m,2H),1.27(t,3H).

Embodiment 14

The first step:

By 1-(the fluoro-phenyl of 4-)-2-methoxy-ethylamine (1g, 4.862mmol) be dissolved in N, in dinethylformamide (15ml), add the bromo-4-chlorothiophene of 6-also [3,2-d] pyrimidine (809mg, 3.24mmol) and triethylamine (1.8ml, 12.97mmol), be warming up to 60 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain (the bromo-thieno-of 6-[3,2-d] pyrimidine-4-yl)-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-amine (1.08g, yellow solid), yield: 87.1%.

Second step:

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-amine (100mg, 0.26mmol) be dissolved in N, in dinethylformamide (5ml), add 1-ethyl-4-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-benzyl]-piperazine (129mg, 0.39mmol), tetrakis triphenylphosphine palladium (30mg, 0.026mmol), 1N sodium carbonate (0.4ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain { 6-[4-(4-ethyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-amine (65mg, white solid), yield: 49.2%MSm/z (ESI): 506.67 [M+1]

¹HNMR(400Hz,DMSO-d ₆):8.37(s,1H),8.27(d,1H),7.77(t,3H),7.50(t,2H),7.47(d,2H),7.15(t,2H),5.62(m,1H),3.78(t,1H),3.58(m,1H),3.49(s,2H),3.30(s,3H),2.37(s,8H),2.31(m,2H),0.92(t,3H).

Embodiment 15

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-amine (100mg, 0.26mmol) be dissolved in N, in dinethylformamide (5ml), add 8-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-benzyl]-8-azabicyclo [3.2.1] octane-3-alcohol (135mg, 0.39mmol), tetrakis triphenylphosphine palladium (30mg, 0.026mmol), 1N sodium carbonate (0.4ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 8-(4-{4-[1-(the fluoro-phenyl of 4-)-2-methoxy-ethylamine]-thieno-[3,2-d] pyrimidine-6-base }-benzyl)-8-azabicyclo [3.2.1] octane-3-alcohol (60mg, white solid), yield: 44.4%.MSm/z(ESI):519.65[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.37(s,1H),8.28(d,1H),7.76(t,3H),7.48(m,4H),7.15(t,2H),5.64(m,1H),4.30(s,1H),3.78(m,2H),3.58(m,2H),3.31(s,3H),3.04(s,2H),2.08(d,2H),1.90(s,4H),1.59(d,2H).

Embodiment 16

By (the bromo-thieno-of 6-[3, 2-d] pyrimidine-4-yl)-[1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-amine (100mg, 0.26mmol) be dissolved in N, in dinethylformamide (5ml), add 1-methyl-4-[4-(4 inwards successively, 4, 5, 5-tetramethyl--[1, 3, 2] two assorted oxygen pentaborane-2-bases)-benzyl]-piperazine (124mg, 0.39mmol), tetrakis triphenylphosphine palladium (30mg, 0.026mmol), 1N sodium carbonate (0.4ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain [1-(the fluoro-phenyl of 4-)-2-methox-etlayl]-{ 6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-yl }-amine (70mg, white solid), yield: 54.7%.MSm/z(ESI):492.65[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.36(s,1H),8.28(d,1H),7.77(t,3H),7.48(m,4H),7.13(t,2H),5.62(m,1H),3.75(t,1H),3.58(m,1H),3.48(s,2H),3.29(s,3H),3.37(s,8H),2.15(s,3H).

Embodiment 17

The first step:

By 2-(the bromo-thieno-[3 of 6-, 2-d] pyrimidine-4-amino)-2-phenyl-ethanol (767mg, 2.19mmol) be dissolved in N, in dinethylformamide (15ml), add 4-[4-(4 inwards successively, 4,5,5-tetramethyl--[1,3,2] two assorted oxygen pentaborane-2-bases)-benzyl]-piperidines-1-t-butyl formate (970mg, 2.41mmol), tetrakis triphenylphosphine palladium (253mg, 0.219mmol), 1N sodium carbonate (5ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 4-{4-[4-(2-hydroxyl-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl-piperidines-1-t-butyl formate (1.1g, yellow solid), yield: 92%.

Second step:

By 4-{4-[4-(2-hydroxyl-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperidines-1-t-butyl formate (163mg, 0.3mmol) be dissolved in tetrahydrofuran (THF) (10ml), add acetic anhydride (153mg successively, 1.5mmol), triethylamine (152mg, 1.5mmol), be warming up to 30 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 4-{4-[4-(2-acetoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl-piperidines-1-t-butyl formate (130mg, yellow solid), yield: 73.9%.

3rd step:

By 4-{4-[4-(2-acetoxyl group-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperidines-1-t-butyl formate (130mg, 0.22mmol) be dissolved in the ethyl acetate solution (2N) of hydrochloric acid, stirring at room temperature, complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, add water, PH to 12 is adjusted, extraction into ethyl acetate, anhydrous sodium sulfate drying with 2N sodium hydroxide solution under ice bath, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain acetic acid-2-phenyl-2-[6-(4-piperidines-4-methylphenyl)-thieno-[3,2-d] pyrimidine-4-is amino]-ethyl ester (20mg, faint yellow solid), yield: 28%.MSm/z(ESI):487.65[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.72(m,1H),4.37(d,2H),3.25(m,2H),2.78(m,4H),1.99(s,3H),1.55～1.30(m,5H).

Embodiment 18

By 2-(the bromo-thieno-[3 of 6-, 2-d] pyrimidine-4-amino)-2-phenyl-ethanol (350mg, 1mmol) be dissolved in N, in dinethylformamide (10ml), add 1-methyl-4-[4-(4 inwards successively, 4,5,5-tetramethyl--[1,3,2] two assorted oxygen pentaborane-2-bases)-benzyl]-piperazine (348mg, 1.1mmol), tetrakis triphenylphosphine palladium (58mg, 0.05mmol), 1N sodium carbonate (2ml), nitrogen replacement, be warming up to 85 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Add water, extraction into ethyl acetate, anhydrous sodium sulfate drying, concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain 2-{6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-amino-2-phenyl-ethanol (287mg, yellow solid), yield: 62.4%.

By 2-{6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-amino }-2-phenyl-ethanol (143mg, 0.3mmol) be dissolved in tetrahydrofuran (THF) (10ml), add acetic anhydride (153mg successively, 1.5mmol), triethylamine (152mg, 1.5mmol), be warming up to 30 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, purify gained residue with silica gel column chromatography, obtain acetic acid-2-{6-[4-(4-thyl-piperazin-1-methyl)-phenyl]-thieno-[3,2-d] pyrimidine-4-amino-2-phenyl-ethyl ester (140mg, yellow solid), yield: 89.7%.MSm/z(ESI):502.76[M+1]

¹HNMR(400Hz,DMSO-d ₆):8.39(s,1H),8.32(d,1H),7.83(t,3H),7.49(d,4H),7.32(m,2H),7.24(m,1H),5.68(m,1H),3.80(m,1H),3.62(m,1H),3.58(s,2H),2.78(m,4H),2.55(m,4H),2.46(s,3H),1.99(s,3H).

Embodiment 19

By 2-{6-[6-(4-thyl-piperazin-1-base)-pyridin-3-yl]-thiophene [3,2-d] pyrimidine-4-yl amine }-2-phenyl-ethanol (134mg, 0.3mmol) be dissolved in tetrahydrofuran (THF) (10ml), add acetic anhydride (153mg successively, 1.5mmol), triethylamine (152mg, 1.5mmol), be warming up to 30 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, gained residue is purified with silica gel column chromatography, obtain (S)-acetic acid-2-{6-[6-(4-thyl-piperazin-1-base)-pyridin-3-yl]-thiophene [3,2-d] pyrimidine-4-yl amine }-2-phenyl-ethyl ester (131mg, yellow solid), yield: 90%.MSm/z(ESI):489.6[M+1]

¹HNMR(400Hz，DMSO-d ₆)：8.60(d,1H),8.35(s,1H),8.08(d,1H),8.01(dd,1H),7.66(s,1H),7.46(d,2H),7.29(m,3H)，6.96(d,1H),5.44(m,1H),3.78(m,2H),3.62(m,4H)，2.41(m,4H),2.23(s,3H),2.05(s,3H).

Embodiment 20

By 2-{6-[6-(4-ethyl-piperazin-1-base)-pyridin-3-yl]-thiophene [3,2-d] pyrimidine-4-yl amine }-2-phenyl-ethanol (138mg, 0.3mmol) be dissolved in tetrahydrofuran (THF) (10ml), add acetic anhydride (153mg successively, 1.5mmol), triethylamine (152mg, 1.5mmol), be warming up to 30 DEG C of stirrings, react complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, gained residue is purified with silica gel column chromatography, obtain (S)-acetic acid-2-{6-[6-(4-ethyl-piperazin-1-base)-pyridin-3-yl]-thiophene [3,2-d] pyrimidine-4-yl amine }-2-phenyl-ethyl ester (129mg, yellow solid), yield: 86%.MSm/z(ESI):503.6[M+1]

¹HNMR(400Hz，DMSO-d ₆)：8.60(s,1H),8.34(s,1H),8.09(d,1H),8.01(d,1H),7.66(s,1H),7.40(d,2H),7.24(m,3H)，6.98(d,1H),5.44(m,1H),3.79(m,2H),3.64(m,4H)，2.47(m,6H),2.07(s,3H),1.07(t,3H).

Embodiment 21

By 2-phenyl-2-[6-(4-piperazine-1-methylphenyl)-thieno-[3,2-d] pyrimidine-4-amino]-ethanol (250mg, 0.56mmol) be dissolved in methyl alcohol (10ml), add acetic anhydride (57mg, 0.56mmol), stirring at room temperature, complete to TLC monitoring raw material reaction.Concentrating under reduced pressure, gained residue is purified with silica gel column chromatography, obtain (S)-1-(4-{4-[4-(2-hydroxyl-1-phenyl-ethylamine)-thieno-[3,2-d] pyrimidine-6-base]-benzyl }-piperazine-1-base)-ethyl ketone (200mg, white solid), yield: 73.26%.MSm/z(ESI):488.65[M+1]

¹HNMR(400Hz，DMSO-d ₆)：8.37(s,1H),8.20(d,1H),7.82(t,3H),7.45(t,4H),7.32(t,2H),7.22(t,1H),5.44(m,1H),4.99(t,1H),3.74(m,1H),3.56(s,2H),3.45(d,4H),2.33-2.40(m,4H),1.99(s,3H).

Biological experiment

1, Tyrosylprotein kinase EGFR enzyme is lived and is suppressed IC ₅₀evaluation experimental

This experiment adopts HTRFkinEASETKkit (Cat.62TK0PEB, the Cisbio) test kit of CISBIO company to carry out the test of candidate compound to EGFR inhibition of enzyme activity effect, and the standard method that testing method provides according to manufacturer is carried out.Experiment flow is roughly as follows: in the enzyme reaction system of 10 μ L, adds enzyme reaction substrate TK-biotin, and ATP, EGFR enzyme (Cat.PV3872, Invitrogen) and certain density compound are at 50mMHepes/NaOHpH7.5,10mMMgCl ₂, room temperature reaction 30 minutes in the enzyme reaction buffered soln of 1mMEGTA, 0.01%BRIJ-35.Each screening concentration of testing compound gets the test of multiple hole, and the Positive control wells not adding the kinase whose negative control hole of EGFR and do not add compound is all established in each experiment.After having reacted, in all reacting holes, add 10 μ l through 50mMHepes/NaOHpH7.0,0.1%BSA, 0.8MKF, Streptavidin-XL665 and TKantibodyeuropiumcryptate (1:100) the hybrid detection liquid of 20mMEDTA dilution, after room temperature reaction 1h, with 2104 multilabelReader (Perkinelmer) instrument detects fluorescent signal (320nm stimulates, and 665nm, 615nm launch).Calculated the inhibiting rate in each hole by complete active hole and background signal hole, average in multiple hole, with professional picture analysis software GraphPadPRISM5.0, each testing compound is carried out to the matching of half inhibit activities (IC50) simultaneously.

2, HER2 kinase activity testing method

This experiment adopts HTRFkinEASETKkit (Cat.62TK0PEB, the Cisbio) test kit of CISBIO company to carry out the test of candidate compound to HER2 inhibition of enzyme activity effect, and the standard method that testing method provides according to manufacturer is carried out.Experiment flow is roughly as follows: in the enzyme reaction system of 10 μ L, adds enzyme reaction substrate TK-biotin, and ATP, HER2 enzyme (Cat:PV3366, Invitrogen) and certain density compound are at 50mMHepes/NaOHpH7.5,5mMMgCl ₂, room temperature reaction 30 minutes in the enzyme reaction buffered soln of 1mMDTT, 50nMSEB, 1mMMnCl2.Each screening concentration of testing compound gets the test of multiple hole, and the negative control hole not adding HER2 enzyme and the Positive control wells not adding compound are all established in each experiment.After having reacted, in all reacting holes, add 10 μ l through 50mMHepes/NaOHpH7.0,0.1%BSA, 0.8MKF, Streptavidin-XL665 and TKantibodyeuropiumcryptate (1:100) the hybrid detection liquid of 20mMEDTA dilution, after room temperature reaction 1h, with 2104 multilabelReader (Perkinelmer) instrument detects fluorescent signal (320nm stimulates, and 665nm, 615nm launch).Calculated the inhibiting rate in each hole by complete active hole and background signal hole, average in multiple hole, with professional picture analysis software GraphPadPRISM5.0, each testing compound is carried out to the matching of half inhibit activities (IC50) simultaneously.

3, by CCK-8 detection kit detection compound, IC is suppressed to the cytotoxicity of human skin epidermoid carcinoma cell strain A431 ₅₀value.

Cell strain:

A431 human skin epidermoid carcinoma cell strain (Chinese Academy of Sciences's Shanghai cell bank)

Reagent and consumptive material:

CellCountingKit-8(Cat#CK04-13，Dojindo)

96 well culture plates (Cat#3599, CorningCostar)

Foetal calf serum (Cat#10099-141, GIBCO)

Substratum (Invitrogen)

Desk-top microplate reader SpectraMaxM5MicroplateReader (MolecularDevices)

The preparation of substratum

Clone	Substratum
		A431	DMEM+10％FBS

The preparation of compound: make final concentration be 10mM by DMSO diluted compounds.

Cell cultures

A) logarithmic phase cell is collected, counting, with perfect medium Eddy diffusion cell,

B) adjust cell concn to suitable concn, inoculate 96 orifice plates, 100 μ l cell suspensions are inoculated in every hole.

C) cell is at 37 DEG C, 100% relative humidity, 5%CO ₂24 hours are hatched in incubator.

IC ₅₀experiment

A) collect logarithmic phase cell, counting, with perfect medium Eddy diffusion cell, adjustment cell concn is to suitable concn (determining according to cell density optimization Test result), and inoculate 96 orifice plates, every hole adds 100 μ l cell suspensions.Cell at 37 DEG C, 100% relative humidity, 5%CO ₂24 hours are hatched in incubator.

B) gradient dilution 8 times after testing compound being diluted to 500 μMs with substratum.Cell is added by 25 μ l/ holes.Compound effects final concentration from 100 μMs to 0 μM, 5 times of gradient dilutions, totally 10 concentration point.

C) cell is placed in 37 DEG C, 100% relative humidity, 5%CO ₂72 hours are hatched in incubator.

D) inhale and abandon substratum, the perfect medium added containing 10%CCK-8 is placed in 37 DEG C of incubators and hatches 2-4 hour.

E) on SpectraMaxM5MicroplateReader, measure the absorbancy at 450nm wavelength place after shaking gently, using 650nm place absorbancy as reference, calculate inhibiting rate.

Data processing

Be calculated as follows the inhibiting rate of drug on tumor Growth of Cells: growth of tumour cell inhibiting rate %=[(A _c-A _s)/(A _c-A _b)] × 100%

A _s: the OA (cell+CCK-8+ testing compound) of sample

A _c: the OA (cell+CCK-8+DMSO) of negative control

A _b: the OA (substratum+CCK-8+DMSO) of positive control

Adopt software GraphpadPrism5 matching IC50 curve and calculate IC ₅₀value.

Table 2 section Example compound is lived to Tyrosylprotein kinase EGFR, HER2 enzyme and is suppressed and A431 cell experiment result

"-" represents that activity is not tested

Above-mentioned biological activity result shows, the part of compounds that the present invention includes all has good Tyrosylprotein kinase EGFR inhibit activities and HER2 inhibit activities, and embodiment 1,4,21 also demonstrates significantly suppression A431 cytoactive on a cellular level.

Single dose intravenous or orally give embodiment 1 (code name CDDD-000261) and embodiment 21 (code name CDDD-000257) pharmacokinetic in rat body respectively

This experiment purpose is embodiment 21 compound and embodiment 1 compound that are given SD donor rat test product significant quantity by single dose intravenous (IV) and oral (PO), in different time points blood sample collection, after LC/MS/MS measures and gives trial-product in rat plasma trial-product concentration and calculate correlation parameter and bioavailability.

The preparation of trial-product

Trial-product, embodiment 21 compound and embodiment 1 compound are dissolved in water for injection, obtain the solution that concentration is 5mg/mL, for vein and gastric infusion.

Animal is distributed

Buy 20 male SD rats (male Body Weight 130-150g) in about 6-8 age in week from Shanghai western pul-Bi Kai laboratory animal company limited, 12 animals are used for test.Before vein and oral administration, all Group Animals overnight fastings (10-14 hour), administration is feeding after 4 hours.

Administration

Trial-product embodiment 21 compound and embodiment 1 compound are by vein or oral single-dose.Drug administration information sees the following form

Sample collecting and process

1-8 treated animal blood sampling time point is: before administration, 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24h.Every each careful dirty puncture of animal adopts about 0.25mL blood, K2EDTA anti-freezing.Blood specimen collection is placed on ice, centrifugal separation plasma (centrifugal condition: 8000 revs/min, 6 minutes, 2-8 DEG C).Cun Fang Yu – 80 DEG C before the plasma analysis collected.In addition, unnecessary animal not used for experimental study is remained for the collection of blank blood.Blank plasma after centrifugal is used for bioanalytical method exploitation and the biological sample analysis of trial-product in whole research.

Pharmacokinetic analyses

According to the plasma drug concentration data of medicine, the non-compartment model of pharmacokinetics software for calculation WinNonlin5.2 is used to calculate the pharmacokinetic parameter of trial-product respectively.Any data being less than lower limit of quantitation (LLOQ=1ng/mL) are replaced by 0 value, and its mean value and standard deviation calculate according to replacement values.When calculating individual animals PK parameter, any numerical value being less than lower limit of quantitation is by disallowable.

Experiment conclusion

Embodiment 21 compound pharmacokinetics in male SD rat blood plasma:

Animals iv injected dose is after embodiment 21 compound of 5mg/kg, total body clearance mean value 2.60L/hr/kg.Transformation period (T ¹/ ₂) mean value be 1.67hr.After administration, Cmax mean value is 3218.39 μ g/L, and Tmax mean value is 0.083hr.AUC (0-t) value is 1903.30hr* μ g/L.Final phase apparent volume of distribution mean value is 6.22L/kg.

Animal administered oral dose is after embodiment 21 compound of 30mg/mL, and Cmax mean value is 1528.74 μ g/L, and Tmax mean value is 0.50hr, AUC (0-t) mean value is 3736.13hr* μ g/L, transformation period (T ¹/ ₂) mean value is 2.51hr.Embodiment 21 compound mean bioavailability is 33.01%.

Embodiment 1 compound pharmacokinetics in male SD rat blood plasma:

Animals iv injected dose is after embodiment 1 compound of 5mg/kg, total body clearance mean value 3.74L/hr/kg.Transformation period (T ¹/ ₂) mean value be 5.11hr.After administration, Cmax mean value is 616.65 μ g/L, and Tmax mean value is 0.083hr.AUC (0-t) value is 1309.41hr* μ g/L.Final phase apparent volume of distribution mean value is 27.66L/kg.

Animal administered oral dose is after embodiment 1 compound of 30mg/mL, and Cmax mean value is 296.20 μ g/L, and Tmax mean value is 5.33hr, AUC (0-t) mean value is 3593.95hr* μ g/L, transformation period (T ¹/ ₂) mean value is 5.67hr.Embodiment 1 compound mean bioavailability is 47.71%.

To the comparitive study of people epidermoid carcinoma A431 nude mouse subcutaneous transplantation knurl

Medicine name and lot number

CDDD-000257 (i.e. embodiment 21 compound) is white powder, content 99.7%, lot number 4010057-78; CDDD-000261 (i.e. embodiment 1 compound) is pale yellow powder, content 99.1%, lot number 4010071-08; Positive control CDDD-000275 (i.e. positive control gefitinib) is white powder, content 99%, lot number R020-06-130619.

Compound method: all prepare with 20%PEG400 distilled water.

Laboratory animal

BALB/cA-nude nude mouse, in 6-7 week, ♀, purchased from Shanghai western pul-Bi Kai laboratory animal company limited.Conformity certification number: SCXK (Shanghai) 2008-0016 feeding environment: SPF level.

Experimental procedure

Nude mouse subcutaneous vaccination people epidermoid carcinoma A431 cell, treats that tumor growth is to 100-200mm ³after, by animal random packet (D0).Dosage and dosage regimen are in table 1.Survey 2-3 knurl volume weekly, claim mouse heavy, record data.Gross tumor volume (V) calculation formula is:

V=1/2 × a × b ²wherein a, b represent length and width respectively.

T/C (%)=(T-T0)/(C-C0) 100 wherein T, C is the gross tumor volume at the end of experiment; Gross tumor volume when T0, C0 are experiment beginning.

Embodiment 21 compound, embodiment 1 compound, positive control gefitinib (3,10,30mg/kg, PO, QD21) all dose-dependently suppress the growth of the people epidermoid carcinoma A431 nude mouse subcutaneous transplantation knurl of high expression level EGFR; Wherein embodiment 21 compound and embodiment 1 compound high dose group cause 3/6 mouse tumor partial remission respectively.Tumor-bearing mice all can tolerate very well to above medicine, and the symptom such as not lose weight occurs.Totally compare, 3 compounds to the sequence of people epidermoid carcinoma A431 nude mouse subcutaneous transplantation knurl antitumor activity are: embodiment 21 compound > embodiment 1 compound > positive control gefitinib.

Table 1.CDDD-000257, CDDD-000261, CDDD-000275 are to the curative effect of people epidermoid carcinoma A431 Naked mice-transplanted tumor.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Claims (10)

1. one kind such as formula the compound shown in I:

Wherein:

R ₁, R ₂be selected from lower group independently of one another: hydrogen, the substituted or unsubstituted alkyl containing 1 ~ 6 carbon atom, the substituted or unsubstituted alkyl acyl containing 1 ~ 6 carbon atom, the substituted or unsubstituted cycloalkanoyl containing 3 ~ 6 carbon atoms, the substituted or unsubstituted alkenylacyl containing 2 ~ 6 carbon atoms, the substituted or unsubstituted aryl-acyl containing 6 ~ 10 carbon atoms, alkylsulfonyl, the substituted or unsubstituted amide group containing 2 ~ 6 carbon atoms, substituted or unsubstituted amido-the acyl group containing 1 ~ 6 carbon atom,

Or R ₁, R ₂form substituted or unsubstituted monocycle or many rings nitrogen heterocycle that carbonatoms is 3 to 10 together with the nitrogen-atoms be connected with them, or optional position is substituted or unsubstituted monocycle or the bicyclic nitrogen-containing heterocyclic base of 3 to 10 by the carbonatoms of nitrogen-atoms, Sauerstoffatom, sulphur atom displacement;

Or R ₁, R ₂5 ~ 7 membered nitrogen-containing heteroaryl bases are jointly formed with the ortho position carbon atom of the nitrogen-atoms be connected and nitrogen-atoms;

R ₃be selected from lower group: hydrogen, substituted or unsubstituted contain 1 ~ 6 carbon atom alkyl, substituted or unsubstituted contain 1 ~ 6 carbon atom alkyl acyl, substituted or unsubstituted contain 1 ~ 6 carbon atom amide group, substituted or unsubstituted contain 3 ~ 6 carbon atoms cycloalkanoyl, substituted or unsubstituted contain 2 ~ 6 carbon atoms alkenylacyl, substituted or unsubstitutedly contain the aryl-acyl of 5 ~ 6 carbon atoms, the substituted or unsubstituted alkylphosphonic acid carboxylic acid ester group containing 1 ~ 6 carbon atom;

R is for replacing aromatic ring or hetero-aromatic ring one or more substituting groups being selected from lower group of upper any hydrogen atom: halogen, C1-C4 alkyl, C1-C4 haloalkyl, alkoxyl group, hydroxyl, amino, cyano group, hydroxyl-C1-C4 alkyl, C2-C10 Heterocyclylalkyl, C2-C10 Heterocyclylalkyl-oxygen base, carboxyl, or C2-C6 carboxylic acid ester groups;

Ar ₂be selected from lower group: the phenyl of phenyl, halogen substiuted, C ₁-C ₆phenyl, xenyl, the xenyl of halogen substiuted, naphthyl, pyridyl, thienyl, the thienyl of halogen substiuted, C that alkyl replaces ₁-C ₃the furyl of the thienyl that alkyl replaces, furyl, halogen substiuted, or C ₁-C ₃the furyl that alkyl replaces;

A, B, D are selected from lower group independently of one another: carbon atom, nitrogen-atoms, Sauerstoffatom, sulphur atom or nothing; And only have one in A, B, D at the most for nothing;

N=0,1 or 2;

Wherein, unless stated otherwise, described replacement refers to that the one or more hydrogen atoms on group are selected from the substituting group replacement of lower group: halogen, hydroxyl, oxo, nitro, the haloalkyl of C1 ~ C6, the alkyl of C1 ~ C6, the thiazolinyl of C1 ~ C6, the alkynyl of C1 ~ C6, the aryl of C3 ~ C12, the arylalkyl of C3 ~ C12, the alkoxyl group of C1 ~ C6, the aryloxy of C3 ~ C12, amino, the acyl amino of C1 ~ C6, the alkyl-carbamoyl of C1 ~ C6, the aryl-amino-carbonyl of C3 ~ C12, the aminoalkyl group of C1 ~ C6, the acyl group of C1 ~ C6, carboxyl, the hydroxyalkyl of C1 ~ C6, the alkyl sulphonyl of C1 ~ C6, the aryl sulfonyl of C5 ~ C12, the alkyl sulfonyl of C1 ~ C6 is amino, the Arenesulfonyl amino of C5 ~ C12, the aralkylsulfonyl of C3 ~ C12 is amino, the alkyl-carbonyl of C1 ~ C6, the alkenyl carbonyl of C1 ~ C6, the acyloxy of C1 ~ C6, the hydroxy-acyl of C1 ~ C6, cyano group, urea groups, the alkyl acyl of C1 ~ C6, the cycloalkanoyl of C1 ~ C6, alkylsulfonyl, carbamate groups,

Dotted line is chemical bond or nothing.

2. compound according to claim 1, is characterized in that:

A, B, D are carbon atom;

Ar ₂for not replacing or the phenyl of halogen substiuted; And/or

R ₁, R ₂be selected from lower group independently of one another: hydrogen, alkyl containing the alkyl of 1 ~ 6 carbon atom or replacement, the alkyl acyl containing 1 ~ 6 carbon atom, the cycloalkanoyl containing 3 ~ 6 carbon atoms, substituted or unsubstituted contain 2 ~ 6 carbon atoms alkenylacyl, substituted or unsubstituted contain 6 ~ 10 carbon atoms aryl-acyl, alkylsulfonyl, substituted or unsubstitutedly contain the amide group of 2 ~ 6 carbon atoms, the substituted or unsubstituted amido-acyl group containing 1 ~ 6 carbon atom;

Or R ₁, R ₂form the heterocyclic radical of substituted or unsubstituted C3 ~ C10 together with the nitrogen-atoms be connected with them, wherein, described heterocyclic radical has the heteroatoms that 1 ~ 3 is selected from lower group: O, S or N;

Or R ₁, R ₂5 ~ 7 membered nitrogen-containing heteroaryl bases are jointly formed with the ortho position carbon atom of the nitrogen-atoms be connected and nitrogen-atoms.

3. the preparation method of formula I as claimed in claim 1, it is characterized in that, described method comprises step:

Under the existence of palladium catalyst, carry out linked reaction with formula (1) compound and formula (1a) compound, obtain formula I:

In formula, the definition of each group as described in the appended claim 1.

4. the preparation method of formula I as claimed in claim 1, it is characterized in that, described method comprises step: with formula (3) compound and R ₃x reacts, and obtains formula I:

Wherein, X is selected from lower group: Cl, OTs.

5. a tyrosine kinase inhibitor, it is characterized in that, described inhibitor contains the formula I as described in claim 1 or 2 suppressing significant quantity, or its pharmacy acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate.

6. a pharmaceutical composition, it is characterized in that, described pharmaceutical composition contains the formula I as described in claim 1 or 2 for the treatment of significant quantity, or its pharmacy acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate.

7. pharmaceutical composition as claimed in claim 6, it is characterized in that, described pharmaceutical composition is used for the treatment of and Tyrosylprotein kinase process LAN and/or the too high relevant disease of tyrosine kinase activity, or described pharmaceutical composition is used for the treatment of the disease relevant to EGF-R ELISA.

8. inhibitor as claimed in claim 5 or as described in claim 6 pharmaceutical composition, is characterized in that:

Described pharmacy acceptable salt is the salt of group under being selected from of formula I: inorganic acid salt, organic acid salt, alkylsulfonate, arylsulphonate, or its combination; Preferably, described salt is selected from lower group of hydrochloride, hydrobromate, nitrate, vitriol, phosphoric acid salt, formate, acetate, propionic salt, benzoate, maleate, fumarate, succinate, tartrate, Citrate trianion, metilsulfate, ethyl sulfonate, benzene sulfonate, tosilate, or its combination;

Described pharmaceutically acceptable solvate, refers to the solvate that formula I and the solvent being selected from lower group are formed: water, ethanol, Virahol, ether, acetone, or its combination.

9. the purposes of the formula I as described in as arbitrary in claim 1 or 2, is characterized in that, for:

A () prepares tyrosine kinase inhibitor;

B () suppresses the activity of Tyrosylprotein kinase for external non-therapeutic;

C () is for the inhibition tumor cell growth of external non-therapeutic ground;

The medicine of d disease that () is correlated with for the preparation for the treatment of EGF-R ELISA activity.

10. purposes as claimed in claim 9, it is characterized in that, described EGF-R ELISA is selected from lower group: EGFR and/or HER2.