CN105418632B

CN105418632B - Thienopyrimidine derivative, preparation method and medical application thereof

Info

Publication number: CN105418632B
Application number: CN201410483081.5A
Authority: CN
Inventors: 安晓霞; 别平彦; 刘俊; 庄戈诗
Original assignee: SHANGHAI ACEBRIGHT PHARMACEUTICALS GROUP Co Ltd
Current assignee: Jiangxi Desino Pharmaceutical Co ltd
Priority date: 2014-09-19
Filing date: 2014-09-19
Publication date: 2020-02-21
Anticipated expiration: 2034-09-19
Also published as: CN105418632A

Abstract

The invention provides a thienopyrimidine derivative, a preparation method thereof and application thereof in medicines. Specifically, the invention relates to a compound shown as a formula I, wherein each group is defined as the specification. The compounds are a potent class of tyrosine kinase inhibitors, and are particularly useful as EGFR and/or HER2 inhibitors.

Description

Thienopyrimidine derivative, preparation method and medical application thereof

Technical Field

The invention relates to the field of medicinal chemistry, and particularly provides a thienopyrimidine derivative and a preparation method and application thereof.

Background

Tumors are one of the most serious diseases threatening human health, and the treatment mainly comprises radiotherapy, chemotherapy and surgical treatment. With the development of cell biology and tumor pharmacology in recent years, the chemical drug therapy of tumors has changed greatly. The traditional chemotherapy drugs are gradually abandoned because cell division is nonspecifically blocked, so that normal cell death is caused while tumor cells are killed, and meanwhile, by taking key node proteins in abnormally activated signal channels in the tumor cells as targets, the discovery of high-efficiency, low-toxicity and strong-specificity micromolecule inhibitors becomes an important direction for the research and development of current antitumor drugs. Receptor Tyrosine Kinases (RTKs) which are abnormally expressed and activated in tumors play a key role in various links such as tumor occurrence and development, invasion and metastasis, chemotherapy resistance and the like, and become hot spots for research on antitumor drugs.

The Epidermal Growth Factor Receptor (EGFR), also known as HER1 or cerbB1, is the most widely expressed member of the HER family of tyrosine kinases in human cancers, the EGFR structure comprises three regions, an extracellular region, a transmembrane region and an intracellular region, the amino terminal end of the extracellular region consists of 622 amino acids, 2 cysteine-rich segments forming the ligand binding region, the transmembrane region is a single α helix, the intracellular region comprises a kinase region and a carboxy terminal tail with a number of tyrosine phosphorylation sites, tyrosine kinases (RTKs) transport the gamma phosphate of ATP to tyrosine residues, after binding to the ligand, the EGFR homodimerizes homomerizes or heterodimerizes to form a tight junction in the TK region, the phosphorylation sites are mediated at the carboxy terminal tail, binding sites for enzymes and connexin proteins are created (Y992, Y1068, Y1086, Y1148 and Y11730), thus enabling the initiation of intracellular signaling reactions, including proliferation, adhesion formation, and apoptosis inhibition.

Research shows that EGFR is expressed in non-small cell lung cancer, prostatic cancer, breast cancer, colorectal cancer, head and neck cancer, gastric cancer, ovarian cancer and pancreatic cancer, and EGFR activation triggers complex signaling reaction. In different types of solid tumors, EGFR is proliferated and overexpressed, leading to uncontrolled downstream signaling leading to the formation of various tumors. Mutations in the ATP-binding site of EGFR affect the RTK activity of the receptor, interfering with the formation of tumorigenic signals, while EGFR is also closely associated with tumor progression and poor prognosis.

Due to the unique role of EGFR and VEGFR in tumorigenesis, monoclonal antibodies and small molecule inhibitors thereof have become hot spots for the development of targeting antitumor drugs. Currently, there are several EGFR or VEGFR targeted inhibitors on the market, and nearly 20 drug candidates are in clinical stages of development. Among them, gefitinib and erlotinib represent the early marketed small molecule inhibitors targeting EGFR. Gefitinib (Gefitinib, also known as ZD1839 or Iressa) is used as a triple-line monotherapy for advanced non-small cell lung cancer (NSCLC). Erlotinib (also called OSI774 or Tarceva) is used as a second-line or third-line therapeutic agent for advanced NSCLC where standard treatment is ineffective.

However, with the clinical use of these drugs, it was found that not all patients with high expression of EGFR are effective against these drugs, and that some tumors that initially responded to Gefitinib (Gefitinib) developed disease after several months of treatment. These results indicate that the currently used EGFR inhibitor antitumor drugs have natural or secondary drug resistance phenomena, and therefore, the development of novel drugs having low drug resistance or capable of relieving early inhibitor drug resistance has become a new development direction of tyrosine kinase inhibitors.

In addition, the use of multi-targeted action in cancer therapy is advantageous, especially for cancer patients who develop resistance to single-targeted inhibitors. The development of tumors is a complex process of multi-step, multi-stage, in vitro and in vivo factor interaction involving multiple genes, and most tumors have 4 to 7 independent mutation sites, so that multi-target therapy is required to ensure the effectiveness and durability of the antitumor effect of the drug. In recent years, the FDA has approved multiple multi-target tyrosine kinase inhibitors in the marketplace, including sorafenib (sorafenib), dasatinib (dasatinib) in 2006, sunitinib (sunitinib) and lapatinib (lapatinib) in sequence. The action targets of lapatinib are epidermal growth factor receptor (EFGR) and human epidermal growth factor receptor 2 (HER-2), and double inhibition effects can be generated on the two targets.

In view of the above, there is an urgent need in the art to develop drugs having multi-target tyrosine kinase inhibitory activity.

Disclosure of Invention

The invention aims to provide a compound with multi-target tyrosine kinase inhibitory activity.

In a first aspect of the invention, there is provided a compound of formula I:

wherein:

R₁、R₂each independently selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, substituted or unsubstituted alkylacyl group having 1 to 6 carbon atoms, substituted or unsubstituted cycloalkylacyl group having 3 to 6 carbon atoms, substituted or unsubstituted alkenylacyl group having 2 to 6 carbon atoms, substituted or unsubstituted arylacyl group having 6 to 10 carbon atoms, sulfonyl group, substituted or unsubstituted amido-CONH having 2 to 6 carbon atoms, substituted or unsubstituted amido-acylnhco having 1 to 6 carbon atoms;

or R₁、R₂A substituted or unsubstituted monocyclic or polycyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms or a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms which is substituted at any position with a nitrogen atom, an oxygen atom or a sulfur atom;

or R₁、R₂And the adjacent carbon atoms of the nitrogen atoms form 5-7-membered nitrogen-containing heteroaryl;

R₃selected from the group consisting of: hydrogen, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkylacyl group having 1 to 6 carbon atoms, a substituted or unsubstituted acylamino group having 1 to 6 carbon atoms, a substituted or unsubstituted cycloalkylacyl group having 3 to 6 carbon atoms, a substituted or unsubstituted alkenylacyl group having 2 to 6 carbon atoms, a substituted or unsubstituted arylacyl group having 5 to 6 carbon atoms, a substituted or unsubstituted alkylphosphate group having 1 to 6 carbon atoms;

r is a substituted aromatic or heteroaromatic ring

One or more substituents selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 haloalkylA group, alkoxy, hydroxy, amino, cyano, hydroxy-C1-C4 alkyl, C2-C10 heterocycloalkyl, C2-C10 heterocycloalkyl-oxy, carboxy, or C2-C6 carboxylate;

Ar₂selected from the group consisting of: phenyl, halogen-substituted phenyl, C₁-C₆Alkyl-substituted phenyl, biphenyl, halogen-substituted biphenyl, naphthyl, pyridyl, thienyl, halogen-substituted thienyl, C₁-C₃Alkyl-substituted thienyl, furyl, halogen-substituted furyl, or C₁-C₃An alkyl-substituted furyl group;

A. b, D are each independently selected from the group consisting of: carbon atom, nitrogen atom, oxygen atom, sulfur atom or none; and at most only one of A, B, D is absent;

n is 0, 1 or 2;

wherein, unless otherwise specified, said substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: halogen, hydroxy, oxo, nitro, C-C haloalkyl, C-C alkyl, C-C alkenyl, C-C alkynyl, C-C aryl, C-C arylalkyl, C-C alkoxy, C-C aryloxy, amino, C-C acylamino, C-C alkylcarbamoyl, C-C arylcarbamoyl, C-C aminoalkyl, C-C acyl, carboxy, C-C hydroxyalkyl, C-C alkylsulfonyl, C-C arylsulfonyl, C-C alkylsulfonylamino, C-C arylsulfonylamino, C-C aralkylsulfonylamino, C-C alkylcarbonyl, C-C alkenylacyl, C-C acyloxy, C-C hydroxy-acyl, cyano, ureido, C-C alkylacyl, C-C cycloalkylacyl, sulfonyl, ureido, C-C alkynoyl, C-C aryl, C-C alkoxycarbonyl, C-C aryl, C-C alkoxy, C-C aryloxy, C-C haloalkyl, C alkoxy, C-C alkoxy, a carbamate group;

the dotted line is a bond or nothing.

In another preferred embodiment, the heterocyclic ring is a saturated ring or an unsaturated ring.

In another preferred embodiment, R₁、R₂、R₃、R、Ar₂A, B, D are each a group corresponding to a particular compound described in Table 1.

It is to be understood that the above preferred groups may be combined with each other to form various preferred compounds of the present invention.

In another preferred embodiment, A, B, D are each independently selected from the group consisting of: carbon atom, nitrogen atom; and A, B, D one of them is a nitrogen atom.

In another preferred embodiment, only one of A, B, D is a nitrogen atom.

In another preferred embodiment:

A. b, D are all carbon atoms;

Ar₂is unsubstituted or halogen-substituted phenyl; and/or

R₁、R₂Each independently selected from the group consisting of: hydrogen, an alkyl group or a substituted alkyl group having 1 to 6 carbon atoms, an alkylacyl group having 1 to 6 carbon atoms, a cycloalkylacyl group having 3 to 6 carbon atoms, a substituted or unsubstituted alkenylacyl group having 2 to 6 carbon atoms, a substituted or unsubstituted arylacyl group having 6 to 10 carbon atoms, a sulfonyl group, a substituted or unsubstituted amido group having 2 to 6 carbon atoms, a substituted or unsubstituted amido-acyl group having 1 to 6 carbon atoms;

or R₁、R₂And a nitrogen atom to which they are bonded, to form a substituted or unsubstituted C3-C10 heterocyclic group, wherein the heterocyclic group has 1 to 3 hetero atoms selected from the group consisting of: o, S or N;

or R₁、R₂And the nitrogen atom and the adjacent carbon atom of the nitrogen atom form a 5-7-membered nitrogen-containing heteroaryl.

In another preferred embodiment, the compound of formula I is selected from the group consisting of:

(S) - (2-methoxy-1-phenyl-ethyl) - [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-yl ] -amine;

(S) - { 2-phenyl-2- [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] -methoxyethyl } -phosphoric acid diethyl ester;

(S) - (2-methoxy-phenyl-ethyl) - {6- [4- (5-methyl-isoxazol-3-yl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } -amine;

(S) -acetic acid-2-phenyl-2- [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] ethyl ester;

(S) -1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -ethanone;

(S) -1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -propenone;

(S) -2-hydroxy-1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -ethanone;

(S) -2-hydroxy-1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -propan-1-one;

(S) -8- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -8-azabicyclo [3.2.1] octan-3-ol;

(S) -2- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -1- (4-methyl-piperazin-1-yl) -ethanone;

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

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

(S) -1- (4-ethyl-piperazin-1-yl) -2- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -ethanone;

(S) - {6- [4- (4-ethyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] -amine;

(S) -8- (4- {4- [1- (4-fluoro-phenyl) -2-methoxy-ethylamine ] -thieno [3,2-d ] pyrimidin-6-yl } -benzyl) -8-azabicyclo [3.2.1] octan-3-ol;

(S) - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] - {6- [4- (4-methyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } -amine;

(S) -acetic acid-2-phenyl-2- [6- (4-piperidin-4-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] -ethyl ester;

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

(S) -acetic acid-2- {6- [6- (4-methyl-piperazin-1-yl) -pyridin-3-yl ] -thiophen [3,2-d ] pyrimidin-4-ylamine } -2-phenyl-ethyl ester;

(S) -acetic acid-2- {6- [6- (4-ethyl-piperazin-1-yl) -pyridin-3-yl ] -thiophen [3,2-d ] pyrimidin-4-ylamine } -2-phenyl-ethyl ester;

(S) -1- (4- {4- [4- (2-hydroxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -ethanone.

In a second aspect of the invention, there is provided a process for the preparation of a compound of formula I as described in the first aspect of the invention, comprising the steps of:

carrying out a coupling reaction of a compound of formula (1) with a compound of formula (1a) in the presence of a palladium catalyst to obtain a compound of formula I:

wherein each group is as defined in the first aspect of the invention.

In another preferred embodiment, the palladium catalyst is selected from the group consisting of: dichlorobis (triphenylphosphine) palladium, tetrakis (triphenylphosphine) palladium, [1, 1' -bis (diphenylphosphino) ferrocene ] dichloropalladium, or a combination thereof.

In another preferred example, the method further comprises the steps of: with compounds of formula (3) and R₃X to give a compound of formula I:

wherein X is selected from the group consisting of: cl, OTs.

In a third aspect of the invention, there is provided a tyrosine kinase inhibitor comprising an inhibitory effective amount of a compound of formula I as described in the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate thereof.

In another preferred embodiment, the tyrosine kinase inhibitor is an EGFR/HER2 dual inhibitor.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I according to the first aspect of the present invention, or a pharmaceutically acceptable salt, tautomer, optical isomer, pharmaceutically acceptable solvate thereof.

In another preferred embodiment, the pharmaceutical composition is used for treating diseases related to tyrosine kinase overexpression and/or tyrosine kinase activity, or the pharmaceutical composition is used for treating diseases related to epidermal growth factor receptor.

In another preferred embodiment, the epidermal growth factor receptor-related disease is selected from the group consisting of: abnormal cell proliferation, morphological changes, hyperkinesia, angiogenic diseases, tumor growth, metastatic diseases, or combinations thereof.

In a fifth aspect of the invention, there is provided the use of a compound of formula I as described in the first aspect of the invention for:

(a) preparing a tyrosine kinase inhibitor;

(b) for non-therapeutically inhibiting tyrosine kinase activity in vitro;

(c) for non-therapeutically inhibiting tumor cell growth in vitro;

(d) can be used for preparing medicines for treating diseases related to epidermal growth factor receptor activity.

In another preferred embodiment, the disease associated with epidermal growth factor receptor activity is selected from the group consisting of: abnormal cell proliferation, morphological changes, hyperkinesia, angiogenic diseases, tumor growth, metastatic diseases, or combinations thereof.

In another preferred embodiment, the tumor cell is a431 cell.

In another preferred embodiment, the tyrosine kinase inhibitor is a multi-target tyrosine kinase inhibitor.

In another preferred embodiment, the epidermal growth factor receptor is selected from the group consisting of: EGFR and/or HER 2.

In another preferred embodiment, the inhibited IC₅₀The value is less than or equal to 50 nM.

In another preferred embodiment, in the third or fourth aspect of the invention, the pharmaceutically acceptable salt is a salt of a compound of formula I selected from the group consisting of: inorganic acid salts, organic acid salts, alkyl sulfonates, aryl sulfonates, or combinations thereof; preferably, the salt is selected from the group consisting of hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof;

the pharmaceutically acceptable solvate refers to a solvate formed by the compound shown in the formula I and a solvent selected from the following group: water, ethanol, isopropanol, diethyl ether, acetone, or combinations thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The inventor of the invention has long and intensive research and unexpectedly prepares a compound with a structure shown as a formula (I), and the compound is a potent tyrosine kinase inhibitor and is particularly suitable to be used as an EGFR and/or HER2 inhibitor. Based on the above findings, the inventors have completed the present invention.

Term(s) for

In the present invention, the alkyl group includes a linear or branched alkyl group, the alkenyl group includes a linear or branched alkenyl group, the alkynyl group includes a linear or branched alkynyl group, and the halogen is F, Cl, Br or I, preferably F or Br.

In the present invention, the term "substituted" means, unless otherwise specified, that one or more hydrogen atoms on a group are substituted with a substituent selected from the group consisting of: C1-C4 alkyl, C3-C7 cycloalkyl, C1-C4 alkoxy, halogen, hydroxyl, carboxyl (-COOH), C1-C4 aldehyde group, C2-C4 acyl, C2-C4 ester group, amino and phenyl; the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from: halogen, C1-C4 alkyl, cyano, OH, nitro, C3-C7 cycloalkyl, C1-C4 alkoxy and amino.

In particular, herein, unless otherwise specified, reference to an atom includes all isotopic forms thereof, for example, when referring to a "hydrogen atom," it is intended to refer to a hydrogen atom, a deuterium atom, a tritium atom, or a combination thereof. In the present invention, the abundance of each isotopic atom of a certain element may be a state in which the element naturally occurs in nature, or a state in which the element is isotopically enriched.

The term "C1-C4 alkyl" refers to a straight or branched chain alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

Specifically, in the present invention, when the number of carbon atoms of the group is not limited, it means a group having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms, unless otherwise specified.

The term "substituted or unsubstituted monocyclic or polycyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms" refers to a nitrogen-containing cyclic group having 3 to 10 carbon atoms, including monocyclic (e.g., piperazine, piperidine, tetrahydropyrrole, morpholine ring, etc.), bicyclic or polycyclic (including bridged ring, spiro ring) groups, wherein the above-mentioned monocyclic nitrogen-containing heterocyclic group is exemplified by (but not limited to): pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl, and the like, and the above-mentioned polycyclic nitrogen-containing heterocyclic groups include (but are not limited to): azabicyclo [3.2.1] octanyl, or a group as shown:

or the like.

The term "5-7 membered heteroaryl" refers to a heteroaryl group having 5-7 carbon atoms or heteroatoms (selected from N, O, S), such as pyrrolyl, pyridyl, furyl, or the like.

The term "C1-C4 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, or the like.

The term "alkanoyl" or "alkylcarbonyl" refers to a group having the structure "-CO-alkyl", such as, for example, methylacyl, ethylacyl, propylacyl, isopropylacyl, butylacyl, isobutylacyl, sec-butylacyl, tert-butylacyl, or the like.

The term "amido group having 2 to 6 carbon atoms" refers to a group having a structure represented by "C1-C5 alkyl-CONH-".

The term "amino-acyl group having 1 to 6 carbon atoms" or "amino-acyl group having 1 to 6 carbon atoms" means having, for example, "NH₂CO- "or" C1-C5 alkyl-NHCO- ", particularly when said group is" NH-₂When CO- "is used," amino-acyl "is written as" amino-acyl ".

As used herein, the term "oxo" refers to a group in which two or more hydrogen atoms are replaced by one or more oxygen atoms, e.g., -CH₂-forms the structure of-C (O) -after being oxo-substituted.

The term "pharmaceutically acceptable solvate" refers to a solvate of the corresponding compound with water, ethanol, isopropanol, diethyl ether, acetone.

A compound of formula I

The invention provides a compound shown as a formula I:

wherein:

R₁、R₂each independently selected from the group consisting of: hydrogen, substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, substituted or unsubstituted alkanoyl group having 1 to 6 carbon atoms, substituted or unsubstitutedSubstituted cycloalkylacyl containing 3-6 carbon atoms, substituted or unsubstituted alkenylacyl containing 2-6 carbon atoms, substituted or unsubstituted arylacyl containing 6-10 carbon atoms, sulfonyl, substituted or unsubstituted acylamino containing 2-6 carbon atoms, and substituted or unsubstituted aminoacyl containing 1-6 carbon atoms;

or R₁、R₂A substituted or unsubstituted monocyclic or bicyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms or a substituted or unsubstituted monocyclic or bicyclic nitrogen-containing heterocyclic group having 3 to 10 carbon atoms which is substituted at any position with a nitrogen atom, an oxygen atom or a sulfur atom;

R₃selected from the group consisting of: hydrogen, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkylacyl group having 1 to 6 carbon atoms, a substituted or unsubstituted acylamino group having 1 to 6 carbon atoms, a substituted or unsubstituted cycloalkylacyl group having 3 to 6 carbon atoms, a substituted or unsubstituted alkenylacyl group having 2 to 6 carbon atoms, a substituted or unsubstituted arylacyl group having 5 to 6 carbon atoms, a substituted or unsubstituted alkylphosphate group having 1 to 6 carbon atoms; preferably, R₃Is not a H atom;

r is a substituted aromatic or heteroaromatic ring

One or more substituents selected from the group consisting of: halogen, C1-C4 alkyl, C1-C4 haloalkyl, alkoxy, hydroxy, amino, cyano, hydroxy-C1-C4 alkyl, C2-C10 heterocycloalkyl, C2-C10 heterocycloalkyl-oxy, carboxy, or C2-C6 carboxylate;

Ar₂selected from the group consisting of: phenyl, halogen-substituted phenyl, C₁-C₆Alkyl-substituted phenyl, biphenyl, halogen-substituted biphenyl, naphthyl, pyridyl, thienyl, halogen-substituted thienyl, C₁-C₃Alkyl substitutedThienyl, furyl, halogen-substituted furyl, C₁-C₃Any one of alkyl-substituted furyl;

n is 0, 1 or 2;

wherein said substitution means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: halogen, hydroxy, oxo, nitro, C-C haloalkyl, C-C alkyl, C-C alkenyl, C-C alkynyl, C-C aryl, C-C arylalkyl, C-C alkoxy, C-C aryloxy, amino, C-C acylamino, C-C alkylcarbamoyl, C-C arylcarbamoyl, C-C aminoalkyl, C-C acyl, carboxy, C-C hydroxyalkyl, C-C alkylsulfonyl, C-C arylsulfonyl, C-C alkylsulfonylamino, C-C arylsulfonylamino, C-C aralkylsulfonylamino, C-C alkylcarbonyl, C-C alkenylacyl, C-C acyloxy, C-C hydroxy-acyl, cyano, ureido, C-C alkylacyl, C-C cycloalkylacyl, sulfonyl, ureido, C-C alkynoyl, C-C aryl, C-C alkoxycarbonyl, C-C aryl, C-C alkoxy, C-C aryloxy, C-C haloalkyl, C alkoxy, C-C alkoxy, a carbamate group;

the dotted line is a bond or nothing.

In preferred embodiments of the present invention, A, B, D are all carbon atoms;

Ar₂is phenyl; and/or

R₁、R₂Each independently selected from the group consisting of: hydrogen, an alkyl group or a substituted alkyl group having 1 to 6 carbon atoms, an alkylacyl group having 1 to 6 carbon atoms, a cycloalkylacyl group having 3 to 6 carbon atoms, a substituted or unsubstituted alkenylacyl group having 2 to 6 carbon atoms, a substituted or unsubstituted arylacyl group having 6 to 10 carbon atoms, a sulfonyl group, a substituted or unsubstituted amido group having 2 to 6 carbon atoms, a substituted or unsubstituted aminoacyl group having 1 to 6 carbon atoms;

or R₁、R₂Together with the nitrogen atom to which they are attached formA substituted or unsubstituted C3-C10 heterocyclic group, wherein the heterocyclic group has 1-3 heteroatoms selected from the group consisting of: o, S or N;

or R₁、R₂Together with the nitrogen atom to which they are attached and the carbon atom in the ortho position to the nitrogen atom form a 5-to 7-membered nitrogen-containing heteroaryl group (i.e., as in the case of compound 3 in Table 1).

More preferably, the compound of formula I is as shown in table 1.

TABLE 1

Process for the preparation of compounds of formula I

The present invention also provides a process for the preparation of a compound of formula I, said process comprising the steps of:

coupling a compound of formula (1) (i.e. a differently substituted 2- (6-bromo-thieno [3,2-d ] pyrimidin-4-amino) -2-phenyl-ethanol) with a compound of formula (1a) (a differently substituted phenylboronic acid ester or phenylboronic acid) in the presence of a palladium catalyst to give a compound of formula I:

wherein each group is as defined above, particularly in formula (1a), "B" in the structure of the dioxaborolan represents a boron atom.

In another preferred embodiment, the coupling reaction is a Suzuki coupling.

Or the preparation method of the compound of the formula I comprises the following steps: with compounds of formula (3) and R₃X to give a compound of formula I:

wherein X is selected from the group consisting of: cl, OTs.

Pharmaceutically acceptable salts

The pharmaceutical forms of the compounds of the invention may include the compounds themselves, as well as other pharmaceutically acceptable variations, such as optical isomers, cis-trans isomers, and the like, or pharmaceutically acceptable salts or solvates.

Preferably, the pharmaceutically acceptable salts include (but are not limited to): inorganic acid salts such as hydrochloride, hydrobromide, nitrate, sulfate, phosphate and the like; organic acid salts such as formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, and the like; alkyl sulfonates such as methylsulfonate, ethylsulfonate, and the like; aryl sulfonates such as benzenesulfonate, p-toluenesulfonate, and the like.

Preferably, the pharmaceutically acceptable solvates include (but are not limited to): the compound is a solvate of water, ethanol, isopropanol, ether, acetone and the like.

Use of compounds of formula I

According to research, the thienopyrimidine derivative has inhibitory activity of Epidermal Growth Factor Receptor (EGFR) and/or human epidermal growth factor receptor 2 (HER 2), so that the thienopyrimidine derivative or any one or a mixture of several of tautomers, racemates, enantiomers, diastereomers, pharmaceutically acceptable salts and pharmaceutically acceptable solvates of the thienopyrimidine derivative can be applied to preparation of tyrosine kinase inhibitors, and particularly can be applied to preparation of EGFR and/or HER2 inhibitors.

Meanwhile, the inhibitor can be applied to the preparation of medicaments for preventing or treating diseases related to epidermal growth factor receptors EGFR and/or HER 2. In particular to the application of the compound in preparing the medicine for preventing or treating abnormal cell proliferation, morphological change, hyperkinesia, angiogenesis and tumor metastasis diseases related to epidermal growth factor receptors EGFR and/or HER 2.

In addition, the inhibitor can be applied to the preparation of medicaments for treating or preventing tumor growth and metastasis related to epidermal growth factor receptors EGFR and/or HER 2.

The active ingredient of the inhibitor disclosed by the patent is preferably any one or a mixture of several of compounds shown in the table 1, or tautomers, racemes, enantiomers, diastereoisomers, pharmaceutically acceptable salts and pharmaceutically acceptable solvates of the compounds shown in the table.

The main advantages of the invention include:

compared with the prior art, the thienopyrimidine derivatives provided by the invention have novel structures, obvious EGFR inhibitory activity and obvious inhibitory activity to VEGFR of partial compounds, are expected to be developed into tyrosine kinase EGFR or/and VEGFR inhibitors, are used for preparing medicines for preventing or treating related diseases such as abnormal cell proliferation, morphological change and hyperkinesia related to epidermal growth factor receptor EGFR and/or vascular growth factor receptor VEGFR and related diseases related to angiogenesis or tumor metastasis, are especially expected to be used for preparing medicines for preventing or treating tumor growth and metastasis related to epidermal growth factor receptor EGFR and/or vascular growth factor receptor VEGFR, provide new development directions and ways for developing novel tyrosine kinase inhibitor medicines with low drug resistance or capable of relieving early inhibitor drug resistance, has wide application prospect and medicinal value.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

The structures of the compounds prepared in the following examples are by nuclear magnetic resonance (¹HNMR) and Mass Spectrometry (MS).

¹HNMR shift (δ) is given in units of parts per million (ppm).¹HNMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Internal standard is Tetramethylsilane (TMS), and chemical shift is 10^-6Given as a unit.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Therm, model: Finnigan LCQadvantage MAX).

IC₅₀The values were determined with a NovoStar microplate reader (BMG, Germany).

The thin silica gel layer is prepared from HSGF254 or GF254 silica gel plate.

The silica gel column chromatography uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.

HPLC tests were carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

The microwave reaction was carried out using a CEM Discover-S908860 type microwave reactor.

In the following examples, the reaction was carried out in a nitrogen atmosphere unless otherwise specified.

The argon atmosphere means that the reaction flask is connected with an argon balloon having a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

In the following examples, the solution in the reaction is an aqueous solution, unless otherwise specified.

Example 1

The first step is as follows:

60% sodium hydride (30g, 0.75mol) was dissolved in tetrahydrofuran (300ml) under ice-bath, then a solution of L-phenylalaninol (50g, 0.36mol) in tetrahydrofuran (600ml) was added, the ice-bath was removed, and the mixture was stirred at room temperature for 2 hours, then methyl iodide (24ml) was added dropwise under nitrogen protection, stirred at room temperature for 1 hour, and refluxed at 70 ℃ for 2 hours. The reaction was completed until TLC monitored the starting material reaction, cooled, the system was added to ice brine, extracted with methyl tert-butyl ether, the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2-methoxy-1-phenyl-ethylamine (51g, colorless oil) in yield: 92 percent.

The second step is that:

6-bromo-4-chlorothieno [3,2-d ] pyrimidine (40g, 0.16mol) and 2-methoxy-1-phenyl-ethylamine (29g, 0.19mol) were dissolved in N, N-dimethylformamide (250ml), and then N, N-diisopropylethylamine (40ml) was added while cooling on ice, and the ice bath was removed and the temperature was raised to 55 ℃ for reaction. Reaction until TLC monitored the reaction of the starting material was complete, water was added, ethyl acetate was extracted, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, then petroleum ether was added for recrystallization, suction filtered, and the filter cake was washed with n-hexane to give (6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (32g, white solid), yield: and 55 percent.

The third step:

the compound (6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (18.2g, 50mmol) and 4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxolan-2-yl) -phenyl ] -piperazine-1-carboxylic acid tert-butyl ester (22.1g, 55mmol) were dissolved in N, N-dimethylformamide (400ml) at room temperature, tetrakis (triphenylphosphine) palladium (1.2g, 1mmol), sodium carbonate solution (1mol/L, 80ml) were added in sequence, nitrogen protected, heated to 90 ℃ until TLC monitored that the starting material had reacted completely, cooled to room temperature, the reaction solution was added to ice water, suction filtered, the filter cake was washed with water, dried, tert-butyl 4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -piperazine-1-carboxylate (27g, light pink solid) was obtained in yield: 96 percent.

The fourth step:

tert-butyl 4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -piperazine-1-carboxylate (5.6g, 10mmol) was dissolved in dichloromethane (50ml), trifluoroacetic acid (15ml, 200mmol) was added while cooling on ice, stirred for 10 min, then the ice bath was removed and stirred at room temperature until TLC monitored that the starting material was completely reacted. Concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography to give (2-methoxy-1-phenyl-ethyl) - [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-yl ] -amine (5.4g, white solid). MS M/z (ESI) 460.58[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 2

2-phenyl-2- [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] ethanol (500mg, 1.12mmol) was dissolved in N, N-dimethylformamide, magnesium isopropoxide (128mg, 0.90mmol) was added, the temperature was raised to 65 ℃ and stirred for 30 minutes, then cooled to 50 ℃, diethyl p-toluenesulfonylmethylphosphonate (0.43ml, 1.68mmol) was added and stirred until TLC monitored that the starting material was completely reacted. Then, water was added, extraction was performed with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give { 2-phenyl-2- [6- (4-piperazine-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] -methoxyethyl } -phosphoric acid diethyl ester (260mg, white solid), yield: 40 percent. MS M/z (ESI) 596.70[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 3

The first step is as follows:

hydroxylamine hydrochloride (7.2g, 86mmol) was dissolved in methanol (30ml) and water (20ml), magnesium oxide (5.1g, 129mmol) was added and stirred for 10 min, then p-toluenesulfonyl chloride (8g, 43mmol) in tetrahydrofuran (300ml) was added and stirred vigorously at room temperature until TLC monitored that the starting material was completely reacted. After suction filtration over celite, drying over anhydrous magnesium sulfate and concentration under reduced pressure, N-hydroxy-4-methyl-benzenesulfonamide (7g, white solid) was obtained in yield: 87.5 percent.

The second step is that:

n-hydroxy-4-methyl-benzenesulfonamide (2.8g, 15mmol) was dissolved in methanol (12ml) and water (2ml), and potassium carbonate (3.33g, 24mmol) and a solution of 4- (4-bromo-phenyl) -but-3-en-2-one (450mg, 2mmol) in methanol (6ml) were added sequentially until TLC monitored that the starting material was completely reacted. Water was added, extraction was performed with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 3- (4-bromo-phenyl) -5-methyl-isoxazole (220mg, yellow solid), yield: 46 percent.

The third step:

3- (4-bromo-phenyl) -5-methyl-isoxazole (100mg, 0.42mmol) was dissolved in 1, 4-dioxane, pinacol diboron (127mg, 0.5mmol), 1, 1' -bis diphenylphosphinoferrocene palladium dichloride (18mg, 0.021mmol), potassium acetate (42mg, 0.42mmol) were added in sequence, replaced with nitrogen, warmed to 95 ℃ and stirred until the starting material reaction was monitored by TLC to completion. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 5-methyl-3- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -phenyl ] isoxazole (98mg, white solid) in yield: 81.6 percent.

The fourth step:

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (47mg, 0.13mmol) and 3(40mg, 0.14mmol) were dissolved in N, N-dimethylformamide, followed by the sequential addition of tetrakis (triphenylphosphine) palladium (3mg, 0.0026mmol), 1N sodium carbonate solution (1ml), nitrogen displacement, warming to 90 ℃ and stirring until the starting material reaction was complete as monitored by TLC. Water was added, extraction was performed with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give (2-methoxy-phenyl-ethyl) - {6- [4- (5-methyl-isoxazol-3-yl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } -amine (50mg, white solid) in yield: 87.7 percent. MS M/z (ESI) 443.62[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 4

The first step is as follows:

tert-butyl 4- {4- [4- (2-hydroxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -piperazine-1-carboxylate (200mg, 0.37mmol) was dissolved in tetrahydrofuran, acetic anhydride (0.17ml, 1.83mmol), triethylamine (0.26ml, 1.83mmol) were added in that order, and the mixture was stirred at room temperature until TLC monitored that the starting material was completely reacted. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 4- {4- [4- (2-acetoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -piperazine-1-carboxylic acid tert-butyl ester (189mg, white solid), yield: 87 percent.

The second step is that:

tert-butyl 4- {4- [4- (2-acetoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -piperazine-1-carboxylate (189mg, 0.32mmol) was dissolved in dichloromethane and trifluoroacetic acid (0.24ml, 3.2mmol) was added and stirred at room temperature until TLC monitored that the starting material reaction was complete. A few drops of ammonia were added dropwise thereto, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give acetic acid-2-phenyl-2- [6- (4-piperazine-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] ethyl ester (120mg, white solid), yield: 77 percent. MS M/z (ESI) 488.62[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 5

(2-methoxy-1-phenyl-ethyl) - [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-yl ] -amine (150mg, 0.33mmol) was dissolved in methanol (10ml), acetic anhydride (33mg, 0.33mmol) was added, stirred at room temperature until TLC monitored the starting material reaction complete. Concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography to give 1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -ethanone (75mg, white solid) in yield: 45.3 percent. MS M/z (ESI) 502.65[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 6

(2-methoxy-1-phenyl-ethyl) - [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-yl ] -amine (200mg, 0.44mmol) was dissolved in N, N-dimethylformamide (10ml), acrylic acid (0.04ml, 0.52mmol), O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (166mg, 0.52mmol) and N, N-diisopropylethylamine (0.15ml, 0.88mmol) were added and the reaction was allowed to proceed at room temperature until TLC monitored that the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -propenone (85mg, white solid) in yield: 37.6 percent. MS M/z (ESI) 514.74[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 7

(2-methoxy-1-phenyl-ethyl) - [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-yl ] -amine (120mg, 0.26mmol) was dissolved in N, N-dimethylformamide (5ml), glycolic acid (24mg, 0.31mmol), O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (100mg, 0.31mmol) and N, N-diisopropylethylamine (0.09ml, 0.52mmol) were added and the reaction was allowed to proceed at room temperature until TLC monitored for complete reaction of starting materials. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 2-hydroxy-1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -ethanone (50mg, white solid) in yield: 37.1 percent.

¹HNMR(400Hz,DMSO-d₆):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).MS m/z(ESI):518.65[M+1]

Example 8

(2-methoxy-1-phenyl-ethyl) - [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-yl ] -amine (250mg, 0.54mmol) was dissolved in N, N-dimethylformamide (10ml), 2-hydroxypropionic acid (59mg, 0.65mmol), O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate (208mg, 0.65mmol) and N, N-diisopropylethylamine (0.19ml, 1.08mmol) were added and the reaction was monitored at room temperature by TLC for complete reaction of starting materials. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 2-hydroxy-1- (4- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -propan-1-one (85mg, white solid) in yield: 29.6 percent. MS M/z (ESI) 532.65[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 9

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (100mg, 0.27mmol) was dissolved in N, N-dimethylformamide (5ml), 8- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -8-azabicyclo [3.2.1] octan-3-ol (141mg, 0.41mmol), tetrakis (triphenylphosphine) palladium (32mg, 0.028mmol), 1N sodium carbonate (0.4ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and the reaction was followed by TLC until the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 8- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -8-azabicyclo [3.2.1] octan-3-ol (80mg, yellow solid) in yield: 59 percent. MS M/z (ESI) 501.68[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 10

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (350mg, 1mmol) was dissolved in N, N-dimethylformamide (10ml), and 1- (4-methyl-piperazin-1-yl) -2- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaoxopentaborane-2-yl) -phenyl ] -ethanone (516.4mg, 1.5mmol), tetrakis (triphenylphosphine) palladium (120mg, 0.1mmol), 1N sodium carbonate (1ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and the reaction was monitored by TLC for the completion of the starting material reaction. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 2- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -1- (4-methyl-piperazin-1-yl) -ethanone (290mg, white solid) in yield: 58 percent. MS M/z (ESI) 502.66[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 11

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (100mg, 0.27mmol) was dissolved in N, N-dimethylformamide (5ml), and 1-methyl-4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -piperazine (174mg, 0.55mmol), tetrakis (triphenylphosphine) palladium (31mg, 0.027mmol), 1N sodium carbonate (0.4ml) were sequentially added thereto, replaced with nitrogen, warmed to 85 ℃ and stirred, reacted until TLC monitored that the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give (2-methoxy-1-phenyl-ethyl) - {6- [4- (4-methyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } -amine (30mg, white solid), yield: 23 percent. MS M/z (ESI) 474.65[ M +1]

¹HNMR(400Hz,DMSO-d₆):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)

Example 12

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (200mg, 0.55mmol) was dissolved in N, N-dimethylformamide (10ml), and 1-ethyl-4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -piperazine (399mg, 1.1mmol), tetrakis (triphenylphosphine) palladium (64mg, 0.055mmol), 1N sodium carbonate (0.9ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and reacted until TLC monitored that the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give {6- [4- (4-ethyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } - (2-methoxy-1-phenyl-ethyl) -amine (85mg, white solid), yield: 31.7 percent. MS M/z (ESI) 487.69[ M +1]

¹HNMR(400Hz,DMSO-d₆):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)

Example 13

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - (2-methoxy-1-phenyl-ethyl) amine (100mg, 0.27mmol) was dissolved in N, N-dimethylformamide (5ml), and 1- (4-ethyl-piperazin-1-yl) -2- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaoxopentaborane-2-yl) -phenyl ] -ethanone (143mg, 0.41mmol), tetrakis (triphenylphosphine) palladium (32mg, 0.028mmol), 1N sodium carbonate (0.4ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and the reaction was followed by TLC until the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 1- (4-ethyl-piperazin-1-yl) -2- {4- [4- (2-methoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -phenyl } -ethanone (30mg, white solid) in yield: 21.6 percent. MS M/z (ESI) 516.68[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 14

The first step is as follows:

1- (4-fluoro-phenyl) -2-methoxy-ethylamine (1g, 4.862mmol) was dissolved in N, N-dimethylformamide (15ml), 6-bromo-4-chlorothieno [3,2-d ] pyrimidine (809mg, 3.24mmol) and triethylamine (1.8ml, 12.97mmol) were added, the temperature was raised to 60 ℃ and stirring was carried out until TLC monitored that the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give (6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] -amine (1.08g, yellow solid), yield: 87.1 percent.

The second step is that:

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] -amine (100mg, 0.26mmol) was dissolved in N, N-dimethylformamide (5ml), and 1-ethyl-4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -piperazine (129mg, 0.39mmol), tetrakis (triphenylphosphine) palladium (30mg, 0.026mmol), 1N sodium carbonate (0.4ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and the reaction was monitored by TLC for completion of the starting material reaction. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give {6- [4- (4-ethyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] -amine (65mg, white solid) yield: 49.2% MS M/z (ESI) 506.67[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 15

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] -amine (100mg, 0.26mmol) was dissolved in N, N-dimethylformamide (5ml), and 8- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -8-azabicyclo [3.2.1] octan-3-ol (135mg, 0.39mmol), tetrakis (triphenylphosphine) palladium (30mg, 0.026mmol), and 1N sodium carbonate (0.4ml) were sequentially added thereto, replaced with nitrogen, heated to 85 ℃ and stirred, and reacted until TLC monitored that the starting material reaction was complete. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 8- (4- {4- [1- (4-fluoro-phenyl) -2-methoxy-ethylamine ] -thieno [3,2-d ] pyrimidin-6-yl } -benzyl) -8-azabicyclo [3.2.1] octan-3-ol (60mg, white solid) in yield: 44.4 percent. MS M/z (ESI) 519.65[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 16

(6-bromo-thieno [3,2-d ] pyrimidin-4-yl) - [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] -amine (100mg, 0.26mmol) was dissolved in N, N-dimethylformamide (5ml), and 1-methyl-4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -piperazine (124mg, 0.39mmol), tetrakis (triphenylphosphine) palladium (30mg, 0.026mmol), 1N sodium carbonate (0.4ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and the reaction was monitored by TLC for completion of the starting material reaction. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give [1- (4-fluoro-phenyl) -2-methoxy-ethyl ] - {6- [4- (4-methyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-yl } -amine (70mg, white solid) in yield: 54.7 percent. MS M/z (ESI) 492.65[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 17

The first step is as follows:

2- (6-bromo-thieno [3,2-d ] pyrimidin-4-amino) -2-phenyl-ethanol (767mg, 2.19mmol) was dissolved in N, N-dimethylformamide (15ml), 4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -piperidine-1-carboxylic acid tert-butyl ester (970mg, 2.41mmol), tetrakis (triphenylphosphine) palladium (253mg, 0.219mmol), 1N sodium carbonate (5ml) were added sequentially, replaced with nitrogen, warmed to 85 ℃ and stirred until TLC monitored that the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 4- {4- [4- (2-hydroxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperidine-1-carboxylic acid tert-butyl ester (1.1g, yellow solid) in yield: 92 percent.

The second step is that:

tert-butyl 4- {4- [4- (2-hydroxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperidine-1-carboxylate (163mg, 0.3mmol) was dissolved in tetrahydrofuran (10ml), acetic anhydride (153mg, 1.5mmol) and triethylamine (152mg, 1.5mmol) were added in that order, stirred at 30 ℃ and reacted until TLC monitored that the starting material was completely reacted. Concentrated under reduced pressure and the resulting residue was purified by silica gel column chromatography to give 4- {4- [4- (2-acetoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperidine-1-carboxylic acid tert-butyl ester (130mg, yellow solid) in yield: 73.9 percent.

The third step:

4- {4- [4- (2-acetoxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperidine-1-carboxylic acid tert-butyl ester (130mg, 0.22mmol) was dissolved in hydrochloric acid in ethyl acetate (2N) and stirred at room temperature until TLC monitored that the starting material was completely reacted. Concentrated under reduced pressure, added with water, adjusted to PH 12 with 2N sodium hydroxide solution under ice bath, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give acetic acid-2-phenyl-2- [6- (4-piperidine-4-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] -ethyl ester (20mg, light yellow solid), yield: 28 percent. MSm/z (ESI) 487.65[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 18

2- (6-bromo-thieno [3,2-d ] pyrimidin-4-amino) -2-phenyl-ethanol (350mg, 1mmol) was dissolved in N, N-dimethylformamide (10ml), 1-methyl-4- [4- (4,4,5, 5-tetramethyl- [1,3,2] dioxaborolan-2-yl) -benzyl ] -piperazine (348mg, 1.1mmol), tetrakis (triphenylphosphine) palladium (58mg, 0.05mmol), 1N sodium carbonate (2ml) were added thereto in this order, replaced with nitrogen, warmed to 85 ℃ and stirred, and the reaction was followed by TLC until the starting material was completely reacted. Water was added, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give 2- {6- [4- (4-methyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-amino } -2-phenyl-ethanol (287mg, yellow solid) in yield: 62.4 percent.

2- {6- [4- (4-methyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-amino } -2-phenyl-ethanol (143mg, 0.3mmol) was dissolved in tetrahydrofuran (10ml), acetic anhydride (153mg, 1.5mmol) and triethylamine (152mg, 1.5mmol) were added in sequence, stirred at 30 ℃ and reacted until TLC monitored that the starting material had reacted completely. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give acetic acid-2- {6- [4- (4-methyl-piperazin-1-methyl) -phenyl ] -thieno [3,2-d ] pyrimidin-4-amino } -2-phenyl-ethyl ester (140mg, yellow solid), yield: 89.7 percent. MS M/z (ESI) 502.76[ M +1]

¹HNMR(400Hz,DMSO-d₆):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).

Example 19

2- {6- [6- (4-methyl-piperazin-1-yl) -pyridin-3-yl ] -thiophen [3,2-d ] pyrimidin-4-ylamine } -2-phenyl-ethanol (134mg, 0.3mmol) was dissolved in tetrahydrofuran (10ml), acetic anhydride (153mg, 1.5mmol) and triethylamine (152mg, 1.5mmol) were added sequentially, warmed to 30 ℃ and stirred, and the reaction was followed by TLC to monitor completion of the starting material reaction. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give (S) -acetic acid-2- {6- [6- (4-methyl-piperazin-1-yl) -pyridin-3-yl ] -thiophen [3,2-d ] pyrimidin-4-ylamine } -2-phenyl-ethyl ester (131mg, yellow solid), yield: 90 percent. MS M/z (ESI) 489.6[ M +1]

¹HNMR(400Hz，DMSO-d₆)：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).

Example 20

2- {6- [6- (4-ethyl-piperazin-1-yl) -pyridin-3-yl ] -thiophen [3,2-d ] pyrimidin-4-ylamine } -2-phenyl-ethanol (138mg, 0.3mmol) was dissolved in tetrahydrofuran (10ml), acetic anhydride (153mg, 1.5mmol) and triethylamine (152mg, 1.5mmol) were added sequentially, warmed to 30 ℃ and stirred, and the reaction was followed by TLC to monitor completion of the starting material reaction. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give (S) -acetic acid-2- {6- [6- (4-ethyl-piperazin-1-yl) -pyridin-3-yl ] -thiophen [3,2-d ] pyrimidin-4-ylamine } -2-phenyl-ethyl ester (129mg, yellow solid), yield: 86 percent. MS M/z (ESI) 503.6[ M +1]

¹HNMR(400Hz，DMSO-d₆)：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).

Example 21

2-phenyl-2- [6- (4-piperazin-1-methyl-phenyl) -thieno [3,2-d ] pyrimidin-4-amino ] -ethanol (250mg, 0.56mmol) was dissolved in methanol (10ml), acetic anhydride (57mg, 0.56mmol) was added, stirring at room temperature, until TLC monitored that the starting material was completely reacted. Concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography to give (S) -1- (4- {4- [4- (2-hydroxy-1-phenyl-ethylamine) -thieno [3,2-d ] pyrimidin-6-yl ] -benzyl } -piperazin-1-yl) -ethanone (200mg, white solid) yield: 73.26 percent. MS M/z (ESI) 488.65[ M +1]

¹HNMR(400Hz，DMSO-d₆)：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 experiments

1. Tyrosine kinase EGFR enzyme activity inhibition IC₅₀Evaluation experiment

In the experiment, an HTRF kinEASE TKkit (Cat.62TK0PEB, Cisbio) kit of a CISBIO company is adopted to test the inhibitory effect of the candidate compound on the activity of the EGFR enzyme, and the test method is carried out according to a standard method provided by a manufacturer. The general experimental procedure is as follows: in a 10. mu.L enzyme reaction system, enzyme reaction substrates TK-biotin, ATP, EGFR enzyme (Cat. PV3872, Invitrogen) and a certain concentration of compound at 50mM Hepes/NaOH pH 7.5, 10mM MgCl were added₂1mM EGTA, 0.01% BRIJ-35 in enzyme reaction buffer for 30 minutes at room temperature. And (4) taking a plurality of wells for testing each screening concentration of the compound to be tested, and setting a negative control well without adding EGFR kinase and a positive control well without adding the compound in each experiment. After completion of the reaction, 10. mu.l of a mixed assay solution of Streptavidin-XL665 and TK antibody europedium cryptate (1:100) diluted with 50mM Hepes/NaOH pH 7.0, 0.1% BSA, 0.8 mM KF, 20mM EDTA was added to all reaction wells, and after 1 hour of reaction at room temperature, they were reacted with 2104

Fluorescence signals (320nm stimulation, 665nm, 615nm emission) were detected with a Multilabel Reader (Perkinelmer) instrument. Inhibition rates of each well were calculated from full activity and background signal wells, and the mean values were taken for duplicate wells, while half inhibitory activity (IC50) was fitted to each test compound using professional graphical analysis software GraphPad PRISM 5.0.

2. HER2 kinase activity test method

In the experiment, an HTRF kinEASE TKkit (Cat.62TK0PEB, Cisbio) kit of a CISBIO company is adopted to test the inhibitory effect of the candidate compound on the activity of HER2 enzyme, and the test method is carried out according to a standard method provided by a manufacturer. The general experimental procedure is as follows: in a 10. mu.L enzyme reaction system, enzyme reaction substrates TK-biotin, ATP, HER2 enzyme (Cat: PV3366, Invitrogen) and a concentration of compound at 50mM Hepes/NaOH pH 7.5, 5mM MgCl₂1mM DTT, 50nM SEB, 1mM MnCl2 in enzyme reaction buffer for 30 minutes at room temperature. For each concentration of test compound, duplicate wells were tested, with negative control wells without HER2 enzyme and positive control wells without compound for each experiment. After completion of the reaction, 10. mu.l of a mixed assay solution of Streptavidin-XL665 and TK antibody europedium cryptate (1:100) diluted with 50mM Hepes/NaOH pH 7.0, 0.1% BSA, 0.8 mM KF, 20mM EDTA was added to all reaction wells, and after 1 hour of reaction at room temperature, they were reacted with 2104

3. Detection of cytotoxicity inhibition IC of compound on human skin squamous cell carcinoma cell line A431 by CCK-8 detection kit₅₀The value is obtained.

Cell lines:

a431 human skin squamous cell carcinoma cell line (Shanghai cell bank of Chinese academy of sciences)

Reagents and consumables:

Cell Counting Kit-8(Cat#CK04-13，Dojindo)

96-well culture plate (Cat #3599, Corning Costar)

Fetal bovine serum (Cat #10099-141, GIBCO)

Culture medium (Invitrogen)

Desktop Microplate reader SpectraMax M5Microplate reader (molecular devices)

Preparation of culture Medium

Cell lines	Culture medium
		A431	DMEM+10％FBS

Preparation of the compound: the compounds were diluted with DMSO to a final concentration of 10 mM.

Cell culture

a) Collecting cells in logarithmic growth phase, counting, resuspending the cells with complete medium,

b) the cell concentration was adjusted to the appropriate concentration and seeded in 96-well plates, 100. mu.l of cell suspension per well.

c) Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 24 hours.

IC₅₀Experiment of

a) Cells in the logarithmic growth phase were collected, counted, resuspended in complete medium, adjusted to the appropriate concentration (as determined by the cell density optimization assay) and seeded into 96-well plates with 100. mu.l of cell suspension per well. Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 24 hours.

b) The test compound was diluted to 500. mu.M with the medium and then diluted 8 times in gradient. Cells were added at 25. mu.l/well. The final concentration of the compound was diluted in 5-fold gradient from 100. mu.M to 0. mu.M for 10 concentration points.

c) Cells were incubated at 37 ℃ and 100% relative humidity, 5% CO₂Incubate in incubator for 72 hours.

d) The medium was aspirated off, complete medium containing 10% CCK-8 was added and incubated in an incubator at 37 ℃ for 2-4 hours.

e) After gentle shaking, absorbance at a wavelength of 450nm was measured on a SpectraMax M5Microplate Reader, and the inhibition rate was calculated with the absorbance at 650nm as a reference.

Data processing

The inhibition rate of the drug on the growth of tumor cells was calculated according to the following formula: the inhibition rate of tumor cell growth [ (% A)_c-A_s)/(A_c-A_b)]×100％

A_sOA of the sample (cell + CCK-8+ test Compound)

A_cOA of negative control (cell + CCK-8+ DMSO)

A_bOA of positive control (Medium + CCK-8+ DMSO)

IC50 curves were fitted and IC calculated using the software Graphpad Prism 5₅₀The value is obtained.

Table 2 partial example compound inhibits tyrosine kinase EGFR, HER2 enzyme activity and A431 cell experimental result

"-" indicates that the activity was not tested

The biological activity results show that part of the compounds included in the invention have better tyrosine kinase EGFR inhibitory activity and HER2 inhibitory activity, and the examples 1,4 and 21 show obvious A431 cell inhibitory activity on a cellular level.

Pharmacokinetic studies in rats of example 1 (code No. CDDD-000261) and example 21 (code No. CDDD-000257) given individually either intravenously or orally in a single session

The purpose of this experiment was to administer test samples to SD rats via a single Intravenous (IV) and oral (PO) administration of an effective amount of the compound of example 21 and the compound of example 1, to collect blood samples at different time points, to determine the concentration of the test sample in the plasma of rats after administration of the test sample by LC/MS/MS and to calculate the relevant parameters and bioavailability.

Preparation of test article

The test article, the compound of example 21 and the compound of example 1 were dissolved in water for injection to give solutions each having a concentration of 5mg/mL for intravenous and intragastric administration.

Animal dispensing

20 male SD rats (male body weight 130-150g) of approximately 6-8 weeks of age were purchased from Shanghai Sphere-BiKai laboratory animals Co., Ltd and 12 animals were used for the experiment. All groups of animals fasted overnight (10-14 hours) prior to intravenous and oral administration and were fed 4 hours after administration.

Administration of drugs

Test articles the compound of example 21 and the compound of example 1 were administered in a single dose, either intravenously or orally. The dosing information is given in the following table

Sample collection and processing

The blood sampling time points of the animals in the groups 1 to 8 are as follows: before administration, 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h and 24 h. Approximately 0.25mL of blood was collected per animal per heart puncture and anticoagulated with K2 EDTA. Blood samples were collected and placed on ice and plasma was centrifuged (centrifugation conditions: 8000 rpm, 6 minutes, 2-8 ℃). The collected plasma was stored at-80 ℃ before analysis. In addition, the remaining animals, which were not used in the experimental study, were used for the collection of blank blood. The centrifuged blank plasma was used for bioanalytical method development and biological sample analysis of the test articles throughout the study.

Pharmacokinetic analysis

According to the data of the blood concentration of the drug, pharmacokinetic parameters of the test sample are respectively calculated by using a pharmacokinetic calculation software WinNonlin5.2 non-atrioventricular model. Any data less than the lower limit of quantitation (LLOQ ═ 1ng/mL) was replaced with a 0 value, and the mean and standard deviation were calculated from the replacement values. Any values below the lower limit of quantitation when calculating individual animal PK parameters will be rejected.

Conclusion of the experiment

Pharmacokinetics of the compound of example 21 in male SD rat plasma:

animals received an intravenous dose of 5mg/kg of the compound of example 21, with an average total clearance of 2.60L/hr/kg. Half life (T)¹/₂) Average value of (1) was 1.67 hr. After administration, the mean Cmax was 3218.39 μ g/L and the mean Tmax was 0.083 hr. AUC (0-t) values were 1903.30 hr. mu.g/L. The volume average of the apparent distribution of the final phase was 6.22L/kg.

Animals orally administered a dose of 30mg/mL of the compound of example 21 with a mean Cmax of 1528.74 μ g/L, a mean Tmax of 0.50hr, a mean AUC (0-T) of 3736.13hr μ g/L, and a half-life (T¹/₂) The average value was 2.51 hr. The average bioavailability of the compound of example 21 was 33.01%.

Pharmacokinetics of the compound of example 1 in male SD rat plasma:

animals received an intravenous dose of 5mg/kg of the compound of example 1, with an average total clearance of 3.74L/hr/kg. Half life (T)¹/₂) Average value of (5) was 5.11 hr. After administration, the mean Cmax was 616.65 μ g/L and the mean Tmax was 0.083 hr. AUC (0-t) values were 1309.41 hr. mu.g/L. The volume average of the apparent distribution of the final phase was 27.66L/kg.

Animals orally administered a dose of 30mg/mL of the compound of example 1, with a mean Cmax of 296.20 μ g/L, a mean Tmax of 5.33hr, a mean AUC (0-T) of 3593.95hr μ g/L, and a half-life (T¹/₂) The average value was 5.67 hr. The compound of example 1 had an average bioavailability of 47.71%.

Comparison of therapeutic effects on subcutaneous transplantation tumor of human epidermoid carcinoma A431 nude mouse

Drug name and lot number

CDDD-000257 (i.e., the compound of example 21) is a white powder with a content of 99.7%, run Nos. 4010057-78; CDDD-000261 (i.e., the compound of example 1) was a pale yellow powder with a content of 99.1%, run No. 4010071-08; the positive control CDDD-000275 (i.e., the positive control gefitinib) was a white powder with a content of 99% and a lot number R020-06-130619.

The preparation method comprises the following steps: all were made up with 20% PEG400 distilled water.

Laboratory animal

BALB/cA-nude mice, 6-7 weeks old, purchased from Shanghai Spirol-bikai laboratory animals, Inc. Certificate number: SCXK (Shanghai) 2008 + 0016 breeding environment: SPF grade.

Experimental procedure

Inoculating human epidermoid carcinoma A431 cells subcutaneously to the nude mouse until the tumor grows to 100-³Thereafter, the animals were randomly assigned (D0). The dosage and schedule of administration are shown in table 1. Tumor volumes were measured 2-3 times a week, mice weighed, and data recorded. Tumor volume (V) was calculated as:

V＝1/2×a×b²wherein a and b represent length and width, respectively.

T/C (%) - (T-T0)/(C-C0) 100 where T, C is the tumor volume at the end of the experiment; t0, C0 are tumor volumes at the beginning of the experiment.

The compound of example 21, the compound of example 1, the positive control gefitinib (3, 10, 30mg/kg, PO, QD21) all dose-dependently inhibited the growth of subcutaneous transplantable tumors of EGFR-overexpressing human epidermoid carcinoma a431 nude mice; wherein the compound of example 21 and the high dose group of the compound of example 1 each caused partial regression of 3/6 mouse tumors. The tumor-bearing mice can well tolerate the medicaments, and symptoms such as weight loss and the like do not occur. Overall comparison, the antitumor activities of 3 compounds against human epidermoid carcinoma a431 nude mice subcutaneous transplantation tumor were ranked as: example 21 compound > example 1 compound > positive control gefitinib.

TABLE 1 curative effect of CDDD-000257, CDDD-000261, CDDD-000275 on naked mouse transplanted tumor of human epidermoid carcinoma A431.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound of formula I:

wherein the compound is selected from the group consisting of:

2. A process for the preparation of a compound of formula I according to claim 1, characterized in that it comprises the steps of:

wherein each group is defined as the corresponding group of the compound as set forth in claim 1.

3. A process for the preparation of a compound of formula I according to claim 1, characterized in that it comprises the steps of: with compounds of formula (3) and R₃X to give a compound of formula I:

wherein X is selected from the group consisting of: cl, OTs.

4. A tyrosine kinase inhibitor comprising an inhibitory effective amount of a compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is for the treatment of a disease associated with overexpression and/or hyperactivity of tyrosine kinase or for the treatment of a disease associated with epidermal growth factor receptor.

7. The inhibitor according to claim 4 or the pharmaceutical composition according to claim 5, characterized in that:

the pharmaceutically acceptable salt is a salt of a compound of formula I selected from the group consisting of: inorganic acid salts, organic acid salts, or combinations thereof.

8. The inhibitor of claim 4 or the pharmaceutical composition of claim 6, wherein the pharmaceutically acceptable salt is a salt of a compound of formula I selected from the group consisting of: hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof.

9. Use of a compound according to claim 1 for:

(a) preparing a tyrosine kinase inhibitor;

(b) for use in vitro in the non-diagnostic, non-therapeutic and non-prophylactic inhibition of tyrosine kinase activity;

(c) for non-diagnostic, non-therapeutic and non-prophylactic inhibition of tumor cell growth in vitro;

10. The use according to claim 9, wherein the epidermal growth factor receptor is selected from the group consisting of: EGFR and/or HER 2.