CN116535423A

CN116535423A - Compound for inhibiting/inducing degradation of EGFR kinase, and pharmaceutical composition and application thereof

Info

Publication number: CN116535423A
Application number: CN202210091311.8A
Authority: CN
Inventors: 方华祥; 杨乐武; 夏定
Original assignee: Wuhan Yuxiang Medical Technology Co ltd
Current assignee: Wuhan Yuxiang Medical Technology Co ltd
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2023-08-04

Abstract

The invention relates to a compound for inhibiting/inducing degradation of EGFR kinase, a pharmaceutical composition and application thereof. The compound is a compound shown in a formula I, or a pharmaceutically acceptable salt, or a stereoisomer, or a tautomer, or a hydrate, or a solvate, or a racemate, or a polymorphism, or an isotopic variant, or a metabolite, or a prodrug thereof, wherein R is ₁ ～R ₃ X is as follows ₁ 、L ₁ ～L ₄ 、Y ₁ 、Y ₂ 、Y ₃ The groups A, B, C, D and W are as defined in the specification. The compounds of the invention and pharmaceutical compositions comprising the same are useful for treating diseases associated with EGFR kinase, such as cancer; the invention also provides the preparation and the application of the pharmaceutical composition.

Description

Compound for inhibiting/inducing degradation of EGFR kinase, and pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of medicines, and in particular provides a compound capable of inhibiting EGFR kinase or inducing degradation of EGFR, a pharmaceutical composition and application thereof.

Background

Lung cancer is one of the most common malignant tumors, 210 thousands of new lung cancers worldwide account for 11.6% of all new tumor cases in 2018; 180 tens of thousands of deaths, accounting for 18.4% of all tumor deaths. Wherein, non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for about 80% -85% of the total lung cancer. The epidermal growth factor receptor (Epithelial Growth Factor Receptor, EGFR) is the most common non-small cell lung cancer driver gene, about 50% of Chinese non-small cell lung cancer patients and 11-16% of western non-small cell lung cancer patients have EGFR gene mutations, the most common types of mutations are the 19 exon deletion mutation (del E746-A750) and the 21 exon L858R point mutation, accounting for about 90% of all EGFR mutant populations.

EGFR small molecule inhibitors are standard therapies for the first-line treatment of EGFR gene mutated non-small cell lung cancer, have been widely used in the field of lung cancer treatment, and inhibit activation of tyrosine kinase by competing with endogenous ligands for EGFR, thereby blocking EGFR signaling pathway, inhibiting proliferation and metastasis of tumor cells, and promoting a series of biological effects such as apoptosis of tumor cells.

The first generation of EGFR small molecule inhibitors Gefitinib and Erlotinib have been used to treat advanced non-small cell lung cancer carrying an activated EGFR mutation (L858R.del E746-A750). However, patients develop resistance after 10-12 months of Gefitinib and Erlotinib, wherein more than 50% of patients with resistance are resistant due to the T790M secondary mutation of EGFR. The second-generation EGFR irreversible inhibitor Afatinib is effective on advanced non-small cell lung cancer patients carrying an activated EGFR mutation (L858R, del E746-A750), but cannot solve clinical resistance caused by EGFR T790M mutation, and Afatinib lacks selectivity to wild type EGFR and has high toxicity. The third generation irreversible inhibitor Osimertinib overcomes EGFR T790M resistance, and can be used for effectively treating EGFR T790M mutation-resistant advanced non-small cell lung cancer patients clinically. Although Osimertinib has achieved great success in the clinical treatment of EGFR T790M mutated non-small cell lung cancer, some patients who benefit from 9-14 months of treatment have developed drug resistance ((Nature Medicine,2015, 21 (6), 560-562)) studies have shown that up to 22% of drug resistant patients have drug resistance due to EGFR C797S mutation (JAMA Oncol.2018;4 (11):1527-1534). EGFR C797S mutation makes 797-position cysteine mutated to serine, osimertinib cannot be covalently bound to EGFR, eventually leading to drug resistance.

Ubiquitin-protease system (UPS) is a multicomponent system for intracellular protein degradation, and is involved in important physiological and biochemical processes such as cell growth, differentiation, DNA replication and repair, cell metabolism, immune reaction and the like. Ubiquitin-proteasome pathway mediated protein degradation is an important mechanism of body regulating intracellular protein level and function, playing an important role in maintaining protein homeostasis in vivo. Through the intracellular ubiquitin-proteasome approach, the degradation of EGFR is induced, and a new idea is provided for treating non-small cell lung cancer.

Disclosure of Invention

The present invention aims to overcome the problems in the prior art and provide compounds for inhibiting/inducing degradation of EGFR kinase, and pharmaceutical compositions and uses thereof.

In a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, racemate, polymorph, isotopic variant, metabolite or prodrug thereof,

wherein,,

Y ₁ and Y ₂ Independently selected from O, S, se, NR ₄ Or CR (CR) ₄ ；

Y ₃ Independently selected from C or N;

X ₁ independently selected from C or N;

ring a is independently selected from a 5-6 membered aromatic or saturated heterocyclic ring containing 1 to 3 heteroatoms independently selected from one or more of N, O and S, and said aromatic heterocyclic ring is optionally substituted with n R ₅ Substitution, wherein n is selected from any integer from 0 to 3;

the groups W and B are independently selected from 5-15 membered aromatic or heteroaromatic rings, heteroaromatic ring-fused heteroalkyl rings, heteroaromatic ring-fused spiro rings, or heteroaromatic ring-fused bridged rings, and wherein the aromatic, heteroaromatic ring-fused heteroalkyl rings, heteroaromatic ring-fused spiro rings, or heteroaromatic ring-fused bridged rings are each optionally substituted with at least 1R ₆ Substitution;

R ₁ 、R ₂ and R is ₃ Each independently selected from hydrogen, deuterium, halogen, cyano, amino, hydroxy, -NR ₇ R ₈ 、-OR ₇ 、C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₂ -C ₆ Alkenyl, C ₂ -C ₆ Alkynyl, C ₃ -C ₈ Cycloalkyl, C ₃ -C ₈ Heterocycloalkyl, C ₁ -C ₃ Alkoxy, C ₁ -C ₆ Haloalkoxy or C ₁ -C ₆ Haloalkyl, and wherein the alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, haloalkoxy, and haloalkyl are each optionally substituted with at least 1R ₉ Substitution;

R ₄ 、R ₅ and R is ₆ Independently selected from hydrogen, deuterium, halogen, cyano, amino, carbonyl, hydroxy, H, C ₃ -C ₈ Cycloalkyl, C ₃ -C ₈ Heterocycloalkyl, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₁ -C ₆ Alkoxy, C ₁ -C ₆ Haloalkoxy, -NR ₇ R ₈ OR-OR ₇ And wherein said cycloalkyl,Heterocycloalkyl, alkyl, heteroalkyl, alkoxy, and haloalkoxy are each optionally substituted with at least 1R ₉ Substitution;

R ₇ and R is ₈ Independently selected from hydrogen, deuterium, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₃ -C ₈ Cycloalkyl or C ₃ -C ₈ Heterocycloalkyl, and wherein said alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl are each optionally substituted with at least 1R ₉ Substitution;

L ₁ 、L ₂ 、L ₃ and L ₄ Independently selected from the group consisting of non-existent, self-chemical bond, -O-, - (CH) ₂ ) _m -C(O)-、-C(O)-C(H ₂ ) _p -、-C(O)-C(O)-、NR ₁₀ -C(O)-、-C(O)-NR ₁₀ 、-C(O)O-、-CH ₂ -CF ₂ -CH ₂ -、-CH ₂ -、 Wherein each m and p is independently any integer from 0 to 3;

the radicals C, D and E are each independently selected from the group consisting of absent, C ₆ -C ₁₀ Aryl, 5-15 membered heteroaryl, 4-8 membered monocycloalkyl, 6-15 membered spiroheterocycloalkyl, 6-15 membered bridged heterocycloalkyl or 6-15 membered fused heterocycloalkyl, and wherein said aryl, heteroaryl, monocycloalkyl, spiroheterocycloalkyl, bridged heterocycloalkyl and fused heterocycloalkyl are each optionally substituted with at least 1R ₁₁ Substitution;

R ₉ and R is ₁₀ Each independently selected from hydrogen, deuterium, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₃ -C ₈ Cycloalkyl or C ₃ -C ₈ Heterocycloalkyl, and wherein said alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl are each optionally substituted with at least 1R ₁₂ Substitution;

R ₁₁ selected from hydrogen, deuterium, halogen, cyano, amino, hydroxy, and C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₃ -C ₈ Cycloalkyl, C ₃ -C ₈ Heterocycloalkyl, C ₁ -C ₃ Alkoxy, C ₁ -C ₆ Haloalkoxy or C ₁ -C ₆ Haloalkyl, and wherein the alkyl, heteroalkyl, alkoxy, haloalkoxy, haloalkyl, cycloalkyl, and heterocycloalkyl are each optionally substituted with at least 1R ₁₂ Substitution;

R ₁ to R ₁₁ Wherein the hetero atoms or hetero atom groups contained in the hetero alkyl group, the hetero cycloalkyl group and the hetero aryl group are each independently selected from the group consisting of-C (=O) N (R) ₁₂ )-、-N(R ₁₂ )-、-NH-、-N＝、-O-、-S-、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)-、-S(＝O) ₂ -or-N (R) ₁₂ )C(＝O)N(R ₁₂ ) -and the number of heteroatoms or heteroatoms groups are each independently selected from 1, 2 and 3;

R ₁₂ each independently selected from the group consisting of hydrogen, chlorine, fluorine, cyano, hydroxy, amino, isopropyl, cyclopropyl, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy, 2-difluoroethoxy, 2-trifluoroethoxy and phenyl.

Preferably, it is a compound represented by formula I-1 or I-2,

preferably, the present invention provides a specific compound represented by any one of formulas 1 to 3, which has the structural formula:

preferably, the stereoisomers include enantiomers or diastereomers.

In a second aspect, the present invention provides a pharmaceutical composition comprising an effective amount of one or more of a compound of the present invention, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a stereoisomer thereof, a tautomer thereof, a racemate thereof, a polymorph thereof, an isotopic variation thereof, a metabolite thereof, and a prodrug thereof.

Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

Preferably, the adjuvant comprises a pharmaceutically acceptable excipient or therapeutic agent.

In another aspect, the invention provides kits comprising a compound of the invention, and other therapeutic agent, together with a pharmaceutically acceptable carrier, adjuvant or vehicle.

In a third aspect, the present invention provides the use of one or more of the compounds of the present invention, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, stereoisomers thereof, tautomers thereof, racemates thereof, polymorphs thereof, isotopic variants thereof, metabolites thereof and prodrugs thereof in the manufacture of a medicament for the treatment and/or prevention of diseases mediated by EGER kinase.

In a fourth aspect, the present invention provides the use of one or more of the compounds of the present invention, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, stereoisomers thereof, tautomers thereof, racemates thereof, polymorphs thereof, isotopic variants thereof, metabolites thereof and prodrugs thereof in the manufacture of a medicament for the treatment and/or prophylaxis of cancer.

In another aspect, the invention provides a method of treating and/or preventing an EGFR kinase mediated disease in a subject comprising administering to the subject a compound of the invention or a composition of the invention.

In another aspect, the invention provides a compound of the invention or a composition of the invention for use in the treatment and/or prevention of EGFR kinase mediated diseases.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a compound, which is proved by relevant cell activity tests to have good activity in inhibiting the proliferation of Ba/F3 (EGFR L858R/T790M/C797S) cells, and the activities of a plurality of compounds are less than 100nM, thus the compound has extremely important antitumor potential. Meanwhile, as Protac molecules, on the premise of larger molecular weight, the Protac molecules still have good oral exposure, show good PK properties and can be orally administrated. Therefore, the compound provided by the invention has good clinical application prospect and can be used for preparing anticancer drugs.

Detailed Description

In particular embodiments, the diseases treated by the present invention include cancers, such as ovarian cancer, cervical cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, non-hodgkin's lymphoma, gastric cancer, lung cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumor (GIST), thyroid cancer, cholangiocarcinoma, endometrial cancer, renal cancer, anaplastic large cell lymphoma, acute Myelogenous Leukemia (AML), multiple myeloma, melanoma, mesothelioma.

General terms and definitions

Unless stated to the contrary, the terms used in the present invention have the following meanings.

"alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms, which may be, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present invention, "alkyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, and various branched isomers thereof, and the like. Non-limiting examples also include, but are not limited to, methylene, ethylene, propylene, butylene, and various branched isomers thereof. In addition, in the present invention, "alkyl" may be optionally substituted or unsubstituted.

"alkoxy" refers to an "-O-alkyl" group, where "alkyl" is defined above.

"alkenyl" refers to unsaturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms and at least 1 carbon-carbon double bond, and may be, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present invention, "alkenyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, vinyl (-ch=ch) ₂ ) Propen-1-yl (-ch=ch-CH) ₃ ) Propylene-2-yl (-C (CH) ₃ )＝CH ₂ ) Buten-1-yl (-ch=ch-CH) ₂ -CH ₃ ) Buten-2-yl (-C (C) ₂ H ₅ )＝CH ₂ ) 1-Methylpropen-1-yl (-C (CH) ₃ )＝CH-CH ₃ ) And various branched isomers thereof. Non-limiting examples also include, but are not limited to, 1-vinylidene (= c=ch ₂ ) 1, 2-ethenylene (-ch=ch-), 1-propenylene (=c=ch-CH) ₃ ) 1, 2-propenylidene (-Ch=c (CH) ₃ ) (-), 1, 3-propenylidene (-ch=ch-CH) ₂ (-) and various branched isomers thereof. In addition, in the present invention, "alkenyl" may be optionally substituted or unsubstituted.

"alkynyl" refers to unsaturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms and at least 1 carbon-carbon triple bond, and may be, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present invention, "alkynyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, ethynyl (-C.ident.CH), propynyl (C.ident.C-CH) ₃ ) Butynyl groupPentynyl->And various branched isomers thereof. Non-limiting examples also include, but are not limited to, ethynylene (C.ident.C-), propynylene->Sulbutylkynyl->And various branched isomers thereof. In addition, in the present invention, "alkynyl" may be optionally substituted or unsubstituted.

"Heteroalkyl" means a saturated aliphatic hydrocarbon group including straight and branched chain groups of 2 to 20 atoms, for example, straight and branched chain groups which may be 2 to 18 atoms, 2 to 12 atoms, 2 to 8 atoms, 2 to 6 atoms or 2 to 4 atoms, wherein one or more of the atoms is selected from nitrogen, oxygen or S (O) _m (wherein m is 0, 1 or 2) and the balance is carbon. In the present invention, "heteroalkyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, methoxymethyl (2-oxapropyl), methylthiomethyl (2-thiapropyl), methylaminomethyl (2-aza-propyl), and various branched isomers thereof, and the like. In addition, in the present invention, "heteroalkyl" may be optionally substituted or unsubstituted.

"cycloalkyl" refers to a saturated or partially unsaturated, mono-or polycyclic aliphatic hydrocarbon group comprising 3 to 12 ring atoms, which may be, for example, 3 to 12, 3 to 10, or 3 to 6 ring atoms (i.e., 3 to 6 membered rings). Non-limiting examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. In the present invention, "cycloalkyl" may be optionally substituted or unsubstituted.

"Heterocyclyl" means a saturated or partially unsaturated, mono-or polycyclic aliphatic hydrocarbon radical comprising 3 to 20 ring atoms, e.gMay be 3 to 16, 3 to 12, 3 to 10 or 3 to 6 ring atoms, wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O) _m (wherein m is 0, 1 or 2) and the remaining ring atoms are carbon. Preferably the heterocycloalkyl group comprises 3 to 12 ring atoms, of which 1 to 4 ring atoms are heteroatoms, more preferably 3 to 10 ring atoms, most preferably 5 or 6 ring atoms, of which 1 to 4, preferably 1 to 3, more preferably 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Non-limiting examples of polycyclic heterocycloalkyl groups include, but are not limited to, a fused, spiro, or bridged heterocycloalkyl group.

"halogen" means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.

"haloalkyl" or "haloalkoxy" refers to an alkyl or alkoxy group substituted with one or more halogen atoms, which may be the same or different, examples of preferred alkyl or alkoxy groups include, but are not limited to: trifluoromethyl, trifluoroethyl, trifluoromethoxy.

"cyano" refers to the "-CN" group.

"hydroxy" refers to an "-OH" group.

"amino" means "-NH- ₂ "group".

"carbamoyl" means "- (c=o) -NH ₂ "group".

"aryl" refers to monocyclic, bicyclic, and tricyclic carbocyclic ring systems containing 6 to 14 ring atoms, wherein at least one ring system is aromatic, wherein each ring system contains rings of 3 to 7 atoms and has one or more points of attachment to the remainder of the molecule. Examples include, but are not limited to: phenyl, naphthyl, anthracene, and the like. Preferably, the aryl group is a carbocyclic ring system of 6 to 10 or 6 to 7 ring atoms.

"heteroaryl" refers to monocyclic, bicyclic, and tricyclic ring systems containing 5 to 14 ring atoms, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms selected from nitrogen, oxygen, and sulfur, wherein each ring system contains a ring of 5 to 7 atoms and has one or more points of attachment to the remainder of the molecule. The term "heteroaryl" may be used interchangeably with the term "heteroaromatic ring" or "heteroaromatic compound". Examples include, but are not limited to: furyl, imidazolyl, 2-pyridyl, 3-pyridyl, thiazolyl, purinyl, and quinolinyl. Preferably, the heteroaryl group is a ring system of 5 to 10 ring atoms.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may be, but is not necessarily, present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

"substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents.

By "pharmaceutically acceptable salts" is meant salts prepared from the compounds of the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups (e.g., carboxyl or sulfonic groups), the base addition salts may be obtained by contacting the free form thereof with a sufficient amount of a base in pure solution or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, organic amine salts, or the like. When the compounds of the present invention contain relatively basic functional groups (e.g., amino or guanidino), the acid addition salts may be obtained by contacting the free form with a sufficient amount of an acid in a pure solution or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable acid addition salts include, but are not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, carbonate, bicarbonate, phosphate, monohydrogen phosphate, dihydrogen phosphate, phosphite, sulfate, bisulfate, etc.), organic acid salts (e.g., acetate, propionate, isobutyrate, malonate, succinate, suberate, maleate, fumarate, citrate, tartrate, lactate, mandelate, benzoate, phthalate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, glucuronate, etc.), and amino acid salts (e.g., arginate, etc.). Specific forms of pharmaceutically acceptable salts can also be found in Berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science,1977, 66:1-19). Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner to isolate the parent compound, thereby regenerating the neutral form of the compound. The parent form of a compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents. According to an embodiment of the present invention, the pharmaceutically acceptable salt of the compound of formula I is preferably an acid addition salt, preferably a hydrochloride, hydrobromide, phosphate or sulfate salt, more preferably a hydrochloride salt.

"pharmaceutical composition" means a pharmaceutically acceptable composition comprising one or more compounds of formula I or a pharmaceutically acceptable form thereof (e.g., salts, hydrates, solvates, stereoisomers, tautomers, racemates, polymorphs, isotopic variations, metabolites, prodrugs, etc.), and other components (e.g., pharmaceutically acceptable excipients).

In the present invention, "pharmaceutically acceptable excipients" refers to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of the use of auxiliary substances is to provide a pharmaceutical composition which is safe to use, stable in nature and/or has specific functionalities, and to provide a method so that the active ingredient can be dissolved at a desired rate after administration of the drug to a subject, or so that the active ingredient is effectively absorbed in the subject to whom it is administered. Pharmaceutically acceptable excipients may be inert fillers or may be functional ingredients that provide some function to the pharmaceutical composition (e.g., to stabilize the overall pH of the composition or to prevent degradation of the active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, binders, disintegrants, lubricants, anti-adherent agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweetening agents, and the like.

The pharmaceutical compositions of the present invention may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.

In the present invention, the purpose of the pharmaceutical composition is to promote the administration to a living body, facilitate the absorption of an active ingredient, and further exert biological activity. The pharmaceutical compositions of the present invention may be administered by any form including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid, oral liquid), rectal, inhalation, implantation, topical (e.g. ocular) administration, and the like. Non-limiting examples of oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like. Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Non-limiting examples of parenteral formulations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, and the like. The pharmaceutical compositions of the invention may also be formulated in controlled-or delayed-release dosage forms (e.g. liposomes or microspheres).

Preferably, the compounds of the present invention or pharmaceutical compositions comprising the same are administered orally or intravenously to an individual in need thereof. Depending on the specific circumstances of the subject, other routes of administration may also be employed or even preferred. For example, transdermal administration would be a very important mode of administration for patients with amnesia or irritability to oral medications. In the present invention, the route of administration can be varied or adjusted in any suitable manner to meet the nature of the drug, the convenience of the patient and medical personnel, and other related factors.

The compound or pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof or the pharmaceutical composition containing the compound has excellent PLK1 enzyme inhibition activity and cell proliferation inhibition activity, can be used as a PLK inhibitor, is used for preventing and/or treating diseases or symptoms caused by PLK over-expression, and has good clinical application and medical application. Preferably, non-limiting examples of diseases or conditions caused by PLK1 overexpression are cancers, including but not limited to hematological tumors, pancreatic cancers, colorectal cancers and lung cancers.

The following examples are provided to further illustrate the invention and are not intended to limit the scope of the invention. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.

The preparation of the compounds of the present invention may be accomplished by synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed in combination with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention. The known starting materials used in the present invention may be synthesized by methods known in the art or purchased through conventional commercial means (e.g., from Shaohuan chemical technology, beijing coupling technology, etc.). Unless otherwise indicated, the reactions were carried out under argon or nitrogen atmosphere. The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 3 times. The reaction temperature is room temperature and the temperature range is 20-30 ℃. Monitoring of the progress of the reaction may be accomplished by synthetic methods well known to those skilled in the art, including but not limited to Thin Layer Chromatography (TLC). Thin layer chromatography silica gel plates using Qingdao ocean GF254 silica gel plates, the developer system includes but is not limited to A: methylene chloride and methanol systems; b: petroleum ether and ethyl acetate system, and the volume ratio of the solvent can be adjusted according to the polarity of the compound.

The isolation and purification of the compounds of the present invention may be accomplished by synthetic methods well known to those skilled in the art, including, but not limited to, column Chromatography (CC), high Performance Liquid Chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), and the like. Column chromatography typically uses Qingdao ocean 200-300 mesh silica gel as a carrier, and eluent systems include, but are not limited to, A: methylene chloride and methanol systems; b: the volume ratio of the petroleum ether to the ethyl acetate can be adjusted according to the polarity of the compound, and a small amount of acidic or alkaline tailing-preventing agent can be added for adjustment. HPLC spectra were determined using an Agilent 1200DAD HPLC chromatograph (column: sunfire C18,150X 4.6mm,5 μm) or a Waters2695-2996HPLC chromatograph (column: gimini C18,150X 4.6mm,5 μm).

Structural identification of the compounds of the present invention may be accomplished by methods well known to those skilled in the art, including but not limited to Nuclear Magnetic Resonance (NMR), mass Spectrometry (MS), and the like. NMR spectra were measured using Bruker AVANCE-400 or Varian Oxford-300 nuclear magnetic instruments using deuterated dimethyl sulfoxide (DMSO-d) ₆ ) Deuterated chloroform (CDC 1) ₃ ) Or deuterated methanol (CD) ₃ OD), internal standard Tetramethylsilane (TMS), chemical shift of 10 ^-6 (ppm). MS spectra were determined using an Agilent SQD (ESI) mass spectrometer (model: 6110) or a Shimadzu SQD (ESI) mass spectrometer (model: 2020).

Preparation of intermediates

Preparation of intermediate INT-1

The preparation method comprises the following steps:

the first step: synthesis of Compound INT-1B

Compound INT-1A (100.0 g,457 mmol) was dissolved in 1, 4-dioxane (1000 mL), and 85% hydrazine hydrate (51.6 g, 284 mmol) was added to the reaction mixture, which was then heated to 95℃and stirred for 16 hours. After TLC showed that the reaction solution was cooled to room temperature, water (3L) was added to the reaction solution under stirring, a large amount of white solid was precipitated, after stirring for 30 minutes, filtration was performed, the cake was washed with water (500 mL), and then the cake was dried to give Compound INT-1B (88.0 g, white solid, yield 89.8%).

MS(ESI):m/z 215.1[M+1] ⁺ /217.1[M+3] ⁺ 。

And a second step of: synthesis of Compound INT-1C

Compound INT-1B (70.0 g,327 mmol) was dissolved in N, N-dimethylacetamide (700 mL), ethyl bromoacetate (108.6 g, 254 mmol) was added to the reaction, and the reaction was heated to 100deg.C and stirred for 40 hours. After TLC showed that the reaction was completed, the reaction solution was poured into a mixed solvent of ethyl acetate (1000 mL) and water (800 mL), an organic phase was separated, an aqueous phase was extracted with ethyl acetate (300 ml×2), the combined organic phases were washed with a saturated sodium chloride solution (500 mL), the organic phase was separated, concentrated, and the residue was purified by chromatography on a silica gel column (eluent: petroleum ether/ethyl acetate=10/1 (volume ratio)) to give compound INT-1C (50.0 g, white solid, yield 51.0%).

MS(ESI):m/z 301.1[M+1] ⁺ /303.1[M+3] ⁺ 。

And a third step of: synthesis of Compound INT-1E

The compound pyrrolidine-1, 2-dicarboxylic acid 1-tert-butyl ester (22.2 g,103 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL), N' -carbonyldiimidazole (16.7 g,103 mmol) was added in portions at 0deg.C, and after the addition was transferred to 25deg.C and stirred for 16 hours to give a solution of INT-1D. The compound INT-1C (20.0 g,66.7 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL), cooled to-70℃and 2M lithium diisopropylamide tetrahydrofuran solution (51.5 mL,103 mmol) was added dropwise at-60℃under controlled temperature, reacted at-70℃for 30 minutes after the completion of the addition, then the INT-1D solution was added dropwise to the reaction solution under controlled temperature below-60℃and the mixture was stirred for 2 hours at 25℃naturally. After TLC showed that saturated ammonium chloride solution (100 mL) and ethyl acetate (100 mL) were added to the reaction solution, the aqueous phase was extracted with ethyl acetate (50 ml×3), then the organic phases were combined, concentrated, and the residue was flash-purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give crude compound INT-1E (33.1 g, brown oil, yield 100%).

MS(ESI):m/z 498.1[M+1] ⁺ /500.1[M+3] ⁺ 。

Fourth step: synthesis of Compound INT-1F

Compound INT-1E (33.1 g,66.7 mmol) was dissolved in ethyl acetate (300 mL), the reaction cooled to 0deg.C, 2M hydrochloric acid/ethyl acetate solution (600 mL) was added dropwise, and after the addition was stirred at 40deg.C for 16 hours. TLC showed that after the reaction was completed, the reaction solution was concentrated to dryness to give compound INT-1F (26.5 g, brown oil, yield 100%).

MS(ESI):m/z 398.0[M+1] ⁺ /400.0[M+3] ⁺ 。

Fifth step: synthesis of Compound INT-1G

Compound INT-1F (26.5 g,66.7 mmol) was dissolved in water (300 mL) and t-butanol (100 mL), and potassium thiocyanate (9.8 g,100 mmol) was added to the reaction mixture, and after the addition, the temperature was raised to 90℃for 3 hours. TLC showed that after the reaction was completed, ethyl acetate (300 mL) was poured into the reaction solution, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (60 ml×2), then the organic phases were combined, concentrated, and the residue was purified by chromatography on a silica gel column (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give compound INT-1G (6.8G, brown foamy solid, three-step yield 23.1%).

MS(ESI):m/z 439.0[M+1] ⁺ /441.0[M+3] ⁺ 。

Sixth step: synthesis of Compound INT-1H

Compound INT-1G (6.8G, 15.5 mmol) was dissolved in glacial acetic acid (68 mL), the reaction solution was cooled to 0℃and 30% hydrogen peroxide (7.03G, 62 mmol) was added dropwise at a reaction temperature below 10℃and after the addition was completed, the reaction solution was transferred to 25℃and stirred for 1 hour. After TLC showed the reaction was completed, ethyl acetate (100 mL) was added, then pH was adjusted to 7-8 with saturated sodium carbonate solution, the organic layer was separated, the aqueous layer was extracted with ethyl acetate (50 ml×2), the organic phases were combined, concentrated, and the residue was purified by chromatography on a silica gel column (eluent: ethyl acetate=1/1 (volume ratio)) to give compound INT-1H (4.2 g, brown foamy solid, yield 66.7%).

MS(ESI):m/z 407.0[M+1] ⁺ /409.0[M+3] ⁺ 。

Seventh step: synthesis of Compound INT-1J

Compound INT-1H (4.2 g,10.3 mmol) was dissolved in tetrahydrofuran (40 mL) and water (5 mL), the reaction solution was cooled to 0℃and lithium hydroxide monohydrate (0.32 g,13.4 mmol) was added to the reaction solution, and after the addition, the reaction solution was transferred to 25℃for 1 hour. TLC showed that after the reaction had been completed, pH was adjusted to 6-7 with 4M hydrochloric acid, the reaction solution was concentrated to dryness to give compound INT-1J (4.0 g, brown foamy solid, yield 100%).

MS(ESI):m/z 379.0[M+1] ⁺ /381.0[M+3] ⁺ 。

Eighth step: synthesis of Compound INT-1

Compound INT-1J (4.0 g,10.3 mmol) was dissolved in N, N-dimethylformamide (50 mL), and 2-aminothiazole (1.34 g,13.4 mmol) and N, N-diisopropylethylamine (4.0 g,30.9 mmol) were sequentially added to the reaction solution, and the reaction solution was cooled to 0℃and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (5.9 g,15.4 mmol) was added in portions, and after the addition, the reaction solution was transferred to 25℃and stirred for 2 hours. After TLC showed that the reaction was completed, the reaction solution was poured into ethyl acetate (100 mL) and water (120 mL), the organic phase was separated, the aqueous phase was extracted with ethyl acetate (50 ml×2), then the organic phases were combined, washed with saturated sodium chloride solution (40 ml×2), the organic phase was separated, concentrated, and the residue was purified by chromatography on a silica gel column (eluent: ethyl acetate/methanol=10/1 (volume ratio)) to give compound INT-1 (2.8 g, off-white solid, yield 58.9%).

MS(ESI):m/z 461.0[M+1] ⁺ /463.0[M+3] ⁺ 。

¹ H NMR(300MHz,DMSO-d ₆ )δ12.83(s,1H),8.36(s,1H),7.76(t,J＝1.0Hz,1H),7.67(s,1H),7.50(d,J＝3.6Hz,1H),7.28(d,J＝3.6Hz,1H),7.07(dd,J＝9.9,1.2Hz,1H),6.68(s,1H),4.02-3.96(m,2H),2.87(s,2H),2.71(s,2H).

Preparation of intermediate INT-2

The preparation method comprises the following steps:

the first step: synthesis of Compound INT-2B

Concentrated sulfuric acid (103.0 g,1.03 mol) was added dropwise to water (19 mL) at 0deg.C, stirred for 5 minutes, then warmed to room temperature, and after completion of the addition, 4-chloroacetoacetate (12.4 g,75.1 mmol) was added dropwise under ice bath, and after completion of the addition, the reaction solution was reacted at room temperature for 16 hours. After TLC showed that the reaction was slowly poured into ice water (500 mL), a large amount of solid was precipitated, filtered, and the cake was washed with water (50 mL) and dried to give compound INT-2B (10.0 g, white solid, yield 62.8%).

MS(ESI):m/z 274.9[M+1] ⁺ /276.9[M+3] ⁺ 。

And a second step of: synthesis of Compound INT-2C

Compound INT-2B (10.0 g,36.3 mmol) was dissolved in water (100 mL) and then sodium hydroxide (4.79 g,120 mmol) was added in portions and heated to 80℃after the addition was completed and the reaction was stirred for 5 hours. TLC showed that after completion of the reaction, cooled to room temperature, then pH was adjusted to 5-6 with 1M hydrochloric acid in ice bath, a large amount of solid was precipitated, filtered, and the cake was washed with water (50 mL) and dried to give Compound INT-2C (6.0 g, white solid, yield 64.8%).

MS(ESI):m/z 255.0[M+1] ⁺ /257.0[M+3] ⁺ 。

And a third step of: synthesis of Compound INT-2D

Compound INT-2C (6.0 g,23.5 mmol) was dissolved in absolute ethanol (60 mL), and then concentrated sulfuric acid (1.2 g,11.8 mmol) was added dropwise under ice bath, and the reaction mixture was heated to 80℃and stirred for 2 hours. TLC showed that after the reaction was completed, ethanol was distilled off under reduced pressure, the residue was dissolved with ethyl acetate (200 mL), then water (200 mL) was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (100 mL), the organic phases were combined, dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1 (volume ratio)), to give compound INT-2D (6.0 g, colorless oil, yield 90.1%).

MS(ESI):m/z 283.0[M+1] ⁺ /285.0[M+3] ⁺ 。

Fourth step: synthesis of Compound INT-2E

Compound INT-2D (3.0 g,10.6 mmol) was dissolved in anhydrous tetrahydrofuran (30 mL), and acrylamide (7.53 g,106 mmol) and potassium tert-butoxide (2.97 g,26.5 mmol) were added in portions under an ice bath, and reacted at 25℃for 1 hour after the addition. TLC showed that after the reaction was completed, water (100 mL) was added to the reaction solution to dilute, followed by extraction with ethyl acetate (100 ml×4), the organic phases were combined, dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give compound INT-2E (1.3 g, yellow solid, yield 39.8%).

MS(ESI):m/z 308.0[M+1] ⁺ /310.0[M+3] ⁺ 。

¹ H NMR(300MHz,DMSO-d ₆ )δ10.92(s,1H),7.92(d,J＝14.0Hz,2H),7.56(d,J＝8.3Hz,1H),7.41(d,J＝8.2Hz,1H),4.14(dd,J＝12.0,4.7Hz,1H),2.77-2.67(m,1H),2.59-2.53(m,1H),2.38-2.24(m,1H),2.12-2.06(m,1H).

Fifth step: synthesis of Compound INT-2F

The compound INT-2E (1.0 g,3.25 mmol) was dissolved in a mixed solvent of 1, 4-dioxane (8 mL) and water (2 mL), and N-benzyloxycarbonyl-3, 6-dihydro-2H-pyridine-4-boronic acid pinacol ester (1.67 g,4.87 mmol), cesium fluoride (1.0 g,6.50 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (100 mg,0.163 mmol) were added, respectively, and after the addition, the reaction solution was replaced 3 times with nitrogen, and then heated to 90℃under nitrogen protection for reaction for 16 hours. After TLC showed that the reaction was cooled to room temperature, diluted with water (80 mL), extracted with ethyl acetate (70 ml×3), the organic phases were combined, dried, and the residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give compound INT-2F (800 mg, yellow solid, yield 55.5%).

MS(ESI):m/z 445.2[M+1] ⁺ 。

Sixth step: synthesis of Compound INT-2G

Compound INT-2F (700 mg,1.57 mmol) was dissolved in glacial acetic acid (15 mL), 10% wet Pd/C (150 mg) was added, and after the addition was completed, the reaction system was replaced three times with hydrogen, and then heated to 50℃under a hydrogen pressure of 50psi (hydrogen balloon) and reacted for 2 hours with stirring. After TLC showed that the reaction was completed, the reaction solution was cooled to room temperature, filtered, the filter cake was washed with methanol, and the filtrate was collected and dried by spin to give compound INT-2G (450 mg, yellow oil, yield 91.5%).

MS(ESI):m/z 313.1[M+1] ⁺ 。

Seventh step: synthesis of Compound INT-2H

Compound INT-2G (450 mg,1.44 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of tert-butyl bromoacetate (365 mg,1.87 mmol) and N, N-diisopropylethylamine (945 mg,4.32 mmol), and the reaction was stirred at 25℃for 1 hour after the addition. After TLC showed that the reaction was completed, water (50 mL) was added to the reaction solution to dilute, followed by extraction with ethyl acetate (50 ml×2), and the organic phases were combined, washed with saturated brine (30 ml×2), dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3/1 (volume ratio)) to give compound INT-2H (400 mg, off-white solid, yield 65.1%).

MS(ESI):m/z 427.2[M+1] ⁺ 。

¹ H NMR(300MHz,DMSO-d ₆ )δ10.88(s,1H),7.82(s,1H),7.51-7.37(m,2H),7.13(d,J＝8.2Hz,1H),4.09(dd,J＝11.9,4.9Hz,1H),3.12(s,2H),2.92(d,J＝11.1Hz,2H),2.76-2.67(m,1H),2.58-2.52(m,2H),2.33-2.24(m,3H),2.14-2.03(m,1H),1.73-1.70(m,4H),1.41(s,9H).

Eighth step: synthesis of Compound INT-2

Compound INT-2H (100 mg,0.234 mmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (0.33 mL) was added, and after the addition was completed, the temperature was raised to 40℃for reaction for 5 hours. After TLC showed that the reaction was completed, the reaction solution was dried by spin-drying to give compound INT-2 (80 mg,0.216mmol, pale yellow foamy solid, yield 92.1%) which was used directly in the next reaction.

MS(ESI):m/z 371.1[M+1] ⁺ 。

Intermediate INT-3 preparation of

The preparation method comprises the following steps:

the first step: synthesis of Compound INT-3H

Diisopropylamine (2.2 mL,15.6 mmol) was dissolved in anhydrous tetrahydrofuran (12.5 mL), then the reaction solution was cooled to 0 ℃, 2.5M N-butyllithium solution (6.25 mL,15.6 mmol) was dropwise added at a temperature of 0-5℃to the reaction solution, after the completion of the dropwise addition, the reaction solution was kept at a temperature of 0-5℃for 15 minutes, then the reaction solution was cooled to-78℃and a temperature of the reaction solution was controlled to be below-60℃to the reaction solution, and the compound INT-3G (2.83G, 12.0 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) and dropwise added to the reaction solution, then reacted at-78℃for 1 hour, finally the reaction solution was controlled to be at a temperature of below-60℃to the N-phenylbis (trifluoromethanesulfonyl imide) and dropwise added to the reaction solution, after the completion of the dropwise addition, reacted at-78℃for 2 hours and then heated to 25℃for 16 hours. After TLC showed that the reaction was completed, the reaction solution was concentrated, then methyl t-butyl ether (100 mL) was added and dissolved, and washed successively with water (50 mL), 2M sodium hydroxide solution (50 mL. Times.3), water (50 mL) and saturated brine solution (50 mL), the organic phase was concentrated, dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1 (volume ratio)), to give compound INT-3H (1.3 g, pale yellow oil, yield 29.7%).

MS(ESI):m/z 368[M+1] ⁺ 。

And a second step of: synthesis of Compound 3B

Compound INT-2E (800 mg,2.60 mmol) was dissolved in 1, 4-dioxane (1 mL), then pinacol biboronate (1.0 g,3.90 mmol), potassium acetate (780 mg,7.80 mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (160 mg,0.13 mmol) were added sequentially, after the addition, the reaction solution was replaced with nitrogen 3 times, and then heated to 90℃under nitrogen protection for reaction for 5 hours. TLC showed that after completion of the reaction, water (100 mL) was added to the reaction system for dilution, extraction with ethyl acetate (50 ml×3), the organic phases were combined, dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give compound INT-3B (800 mg, tan oil, yield 86.8%).

MS(ESI):m/z 356.2[M+1] ⁺ 。

And a third step of: synthesis of Compound INT-3C

Compound INT-3B (500 mg,1.41 mmol) was dissolved in water (100 mL), then compound INT-3H (1.5 g,4.23 mmol), sodium carbonate (350 mg,4.23 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (100 mg,0.071 mmol) were added sequentially, after the addition was replaced 3 times with nitrogen, and then heated to 55℃under nitrogen protection for reaction for 5 hours. TLC showed that after the reaction was completed, cooled to room temperature, then diluted with water (100 mL), extracted with ethyl acetate (40 ml×3), the organic phases were combined, dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give compound INT-3C (500 mg, yellow solid, yield 79.6%).

MS(ESI):m/z 447.2[M+1] ⁺ 。

Fourth step: synthesis of the Compound INT-3D

Compound INT-3C (500 mg,1.12 mmol) was dissolved in methanol (10 mL), then 10% wet Pd/C (50 mg) was added, and after the addition was completed, the reaction system was replaced three times with hydrogen, and then reacted at a hydrogen pressure of 50psi (hydrogen balloon) for 16 hours at 25 ℃. After TLC showed the reaction was completed, the reaction solution was filtered and dried by spin to give compound INT-3D (500 mg, yellow oil, 99.6% yield).

MS(ESI):m/z 449.2[M+1] ⁺ 。

Fifth step: synthesis of Compound INT-3E

To compound INT-3D (500 mg,1.11 mmol) was added 8% hydrochloric acid/ethyl acetate solution (10 mL), and after the addition was completed, the mixture was reacted at 25℃for 2 hours. TLC showed that after the reaction was completed, the reaction solution was directly spin-dried to give crude compound INT-3E which was directly used in the next reaction (500 mg, yellow solid, yield 100.0%).

MS(ESI):m/z 349.1[M+1] ⁺ 。

Sixth step: synthesis of Compound INT-3F

Compound INT-3E (500 mg,1.44 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by addition of tert-butyl bromoacetate (264 mg,1.87 mmol) and N, N-diisopropylethylamine (945 mg,4.32 mmol), and reaction at 25℃for 1 hour after the addition. After TLC showed that the reaction was completed, water (30 mL) was added to the reaction solution to dilute, followed by extraction with ethyl acetate (30 ml×2), and the organic phases were combined, washed with saturated brine (20 ml×2), dried, and spin-dried, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3/1 (volume ratio)) to give compound INT-3F (600 mg, off-white solid, yield 96.2%).

MS(ESI):m/z 463.2[M+1] ⁺ 。

¹ H NMR(300MHz,DMSO-d ₆ )δ10.89(s,1H),7.87(s,1H),7.51(d,J＝8.2Hz,2H),7.19(d,J＝8.3Hz,1H),4.11(dd,J＝11.9,4.8Hz,1H),3.30-3.26(m,2H),3.23-3.13(m,2H),2.97-2.93(m,1H),2.87(s,1H),2.77-2.68(m,2H),2.60-2.53(m,2H),2.39-2.25(m,1H),2.15-2.08(m,2H),1.43(s,9H)。

Seventh step: synthesis of Compound INT-3

Compound INT-3F (100 mg,0.234 mmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (0.33 mL) was added, and after the addition was completed, the temperature was raised to 40℃for reaction for 5 hours. TLC showed that after the reaction was completed, the reaction solution was dried by spin-drying to give compound INT-3 (80 mg, pale yellow foamy solid, yield 85.5%).

MS(ESI):m/z 407.1[M+1] ⁺ 。

Preparation and functional verification of target compounds

Example 1: preparation of Compound 1

The structural formula of the compound 1 is as follows:

the synthetic route of compound 1 is:

the specific preparation method of the compound 1 comprises the following steps:

the preparation method comprises the following steps:

the first step: synthesis of Compound 1B

Compound 1A (2 g,6.94 mmol) was dissolved in toluene (60 mL) and 4 bromoiodobenzene (4.92 g,17.36 mmol), sodium t-butoxide (6.6 g,69.4 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (340 mg,0.42 mmol) were added sequentially at room temperature (20deg.C). The reaction system was replaced with nitrogen three times, and then heated to 90℃for 16 hours. After TLC showed that the reaction was completed, the reaction solution was poured into 100mL of saturated aqueous ammonium chloride, ethyl acetate (70 ml×3) was extracted, the organic layers were separated, the combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the organic solvent was dried by filtration, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=5:1 (volume ratio)) to give compound 1B (2 g, pale yellow solid, yield 81.9%).

MS(ESI):m/z 353.1[M+H] ⁺ 。

And a second step of: synthesis of Compound 1C

Compound 1B (1.7 g,4.83 mmol) was dissolved in dioxane (60 mL) and pinacol borate (2.0 g,7.73 mmol), potassium acetate (1.4 g,14.5 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (350 mg,0.44 mmol) were added sequentially at room temperature (20deg.C). The reaction system was replaced with nitrogen three times, and then heated to 90℃for 16 hours. After TLC showed that the reaction was completed, the reaction solution was poured into 100mL of saturated aqueous ammonium chloride, extracted with ethyl acetate (60 ml×3), the organic layers were separated, the combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the organic solvent was dried by spin-drying, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=5:1 (volume ratio)), to give compound 1C (1.3 g, off-white solid, yield 67.3%).

MS(ESI):m/z 401.3[M+H] ⁺ 。

And a third step of: synthesis of Compound 1D

Compound 1C (566.8 mg,1.42 mmol) and intermediate INT-1 (500.0 mg,1.09 mmol) were added to a mixed solvent of 1,4 dioxane (6 mL) and water (1 mL), and sodium carbonate (231.1 mg,2.18 mmol), 2-di-tert-butyl phosphino-2 ',4',6 '-triisopropyl biphenyl (93.4 mg,0.22 mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (94.3 mg,0.13 mmol) were added sequentially at room temperature (20 ℃ C.). The reaction system was subjected to microwave reaction at 80℃for 2 hours under nitrogen atmosphere. After TLC monitored complete reaction of the starting materials, saturated aqueous ammonium chloride (50 mL), ethyl acetate (40 mL. Times.3) was added to the reaction mixture, the organic phases were combined, washed with saturated brine (2X 20 mL), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and spun-dried under reduced pressure, the residue was purified by HPLC (Waters Sunfire OBD 100X30 mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 1D (30 mg, white solid, yield 4.2%).

MS(ESI):m/z:328.1[1/2M+H] ⁺ 。

Fourth step: synthesis of Compound 1E

Compound 1D (30 mg,0.046 mmol) was dissolved in dichloromethane (2 mL) and trifluoroacetic acid (0.4 mL) was added and reacted at 25℃for 30 min after the addition. TLC showed that after the reaction was completed, the reaction solution was directly spin-dried to give crude compound 1E which was directly used in the next reaction (30 mg, yellow oil, 100.0%).

MS(ESI):m/z 555.2[M+1] ⁺ 。

Fifth step: synthesis of Compound 1

Intermediate INT-3 (28 mg,0.071 mmol) was dissolved in N, N-dimethylformamide (0.5 mL), N, N-diisopropylethylamine (30.3 mg,0.24 mmol) and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (29.8 mg,0.078 mmol) were sequentially added to the reaction solution, reacted at 25℃for 15 minutes after the addition was completed, and then 1E (30 mg,0.047 mmol) was dissolved in N, N-dimethylformamide (0.5 mL) and then added dropwise to the reaction solution, followed by continuing the stirring reaction at 25℃for 1 hour after the addition was completed. TLC showed that after the reaction was completed, the reaction solution was directly purified by HPLC (Waters Sunfire OBD 100X30 mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 1 (18 mg, off-white solid, yield 41.3%).

MS(ESI):m/z 472.2[1/2M+1] ⁺ 。

¹ H NMR(600MHz,dmso)δ12.79(s,1H),10.87(s,1H),8.22(s,1H),7.86(s,1H),7.65(s,1H),7.57(d,J＝8.5Hz,2H),7.49(dd,J＝11.2,5.9Hz,2H),7.25(d,J＝3.2Hz,1H),7.18(d,J＝8.3Hz,1H),7.08(d,J＝12.3Hz,1H),6.66(s,1H),6.51(d,J＝8.7Hz,2H),4.40(s,2H),4.09(dt,J＝16.4,6.4Hz,2H),4.05-3.90(m,4H),3.27(s,2H),3.16(dt,J＝25.7,8.4Hz,3H),2.94(d,J＝10.0Hz,1H),2.83-2.77(m,1H),2.76-2.64(m,2H),2.63-2.50(m,3H),2.45-2.38(m,2H),2.34-2.26(m,1H),2.20-2.12(m,1H),2.11-2.04(m,1H),2.00-1.92(m,1H),1.79(ddd,J＝11.8,8.8,7.0Hz,1H),1.47-1.39(m,1H),1.27(ddd,J＝11.1,7.8,5.2Hz,1H)。

Example 2: preparation of Compound 2

The structural formula of the compound 2 is as follows:

the synthetic route for compound 2 is:

the specific preparation method of the compound 2 comprises the following steps:

the preparation method comprises the following steps:

the first step: synthesis of Compound 2B

Compound 2A (3 g,13.95 mmol) was dissolved in a mixed solvent of tetrahydrofuran and triethylamine (75 mL/7.5 mL), and trimethylsilylacetylene (4.1 g,41.85 mmol), bis (triphenylphosphine) palladium dichloride (369.6 mg,0.84 mmol), and cuprous iodide (159.6 mg,0.84 mmol) were added sequentially at room temperature (25 ℃ C.). The reaction system was replaced with nitrogen three times, and then heated to 60℃to react for 16 hours. After TLC showed that the reaction was completed, the reaction solution was poured into 100mL of saturated aqueous ammonium chloride, extracted with ethyl acetate (60 ml×3), the organic layers were separated, the combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the organic solvent was dried by filtration, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3:1 (volume ratio)), to give compound 2B (2.6 g, yellow solid, yield 80.0%).

MS(ESI):m/z:234.1[M+H] ⁺ 。

And a second step of: synthesis of Compound 2C

2B (2.5 g,10.72 mmol) was dissolved in a mixed solvent of methanol and methylene chloride (30 mL/30 mL), potassium fluoride (1.86 g,32.16 mmol) was added at room temperature (25 ℃ C.), and the reaction system was reacted at room temperature for 2 hours. After TLC showed that the reaction was completed, the reaction solution was poured into 100mL of water, dichloromethane (40 ml×3) was extracted, the organic layers were separated, the combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the organic solvent was spin-dried, and the obtained residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3:1 (volume ratio)) to give 2C (1.5 g, yellow solid, yield 86.9%).

MS(ESI):m/z:162.1[M+H] ⁺ 。

And a third step of: synthesis of Compound 2D

Compound 2C (700 mg,4.35 mmol) was dissolved in a mixed solvent of tetrahydrofuran and water (5 mL/5 mL), lithium hydroxide monohydrate (365 mg,8.7 mmol) was added at room temperature (20 ℃ C.) and stirred at room temperature for 5 hours. After TLC showed complete reaction, the reaction was concentrated to remove the organic phase, diluted with 10mL of water, the aqueous phase was adjusted to ph=4 with 4M hydrochloric acid, extracted with ethyl acetate (50 ml×3), the organic layers were separated, the combined organic phases were washed with saturated brine (2×30 mL), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the organic solvent was dried to give crude 2D (500 mg, white solid, yield 78.2%).

MS(ESI):m/z:148.0[M+H] ⁺ 。

Fourth step: synthesis of Compound 2E

Compound 2D (450 mg,3.06 mmol) was added to N, N dimethylformamide (10 mL), N, N-diisopropyl-N-ethyl (792.0 mg,6.12 mmol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (1.4 g,3.67 mmol), compound 2D-1 (730 mg,3.67 mmol) was added at 0deg.C, and stirred for 16 hours. After TLC monitored complete reaction of the starting materials, saturated aqueous ammonium chloride (50 mL) was added to the reaction solution, extracted with ethyl acetate (40 ml×3), and the organic phases were combined, washed with saturated brine (2×20 mL), dried over anhydrous sodium sulfate, filtered to remove the drying agent, dried under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3:1 (volume ratio)) to give compound 2E (800 mg, pale yellow solid, yield 79.5%).

MS(ESI):m/z:330.2[M+H] ⁺ 。

Fifth step: synthesis of Compound 2F

Compound 2E (429.1 mg,1.30 mmol) and intermediate INT-1 (500.0 mg,1.09 mmol) were added to N, N dimethylformamide (10 mL), and N, N-diisopropyl-N-ethyl (154.6 mg,1.20 mmol), cuprous iodide (10.3 mg,0.055 mmol), triphenylphosphine (28.8 mg,0.11 mmol), and diphenylphosphine palladium dichloride (34.6 mg,0.055 mmol) were added sequentially at room temperature (20 ℃). The reaction system was subjected to microwave reaction at 80℃for 2 hours under nitrogen atmosphere. After TLC monitored complete reaction of the starting materials, saturated aqueous ammonium chloride (50 mL), ethyl acetate (40 mL. Times.3) was added to the reaction mixture, the organic phases were combined, washed with saturated brine (2X 20 mL), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and spun-dried under reduced pressure, the residue was purified by HPLC (Waters Sunfire OBD 100X30 mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 2F (30 mg, white solid, 3.9% yield).

MS(ESI):m/z:355.6[1/2M+H] ⁺ 。

Sixth step: synthesis of Compound 2G

Compound 2F (30 mg,0.042 mmol) was dissolved in ethanol (0.5 mL) and then 8% hydrochloric acid/ethyl acetate solution (1 mL) was added, and after the addition was completed, the mixture was heated to 40℃and reacted for 2 hours. TLC showed that after the reaction was completed, the reaction solution was directly spin-dried to give crude compound 2G which was directly used in the next reaction (30 mg, yellow solid, 100.0%).

MS(ESI):m/z 610.1[M+1] ⁺ 。

Seventh step: synthesis of Compound 2

Intermediate INT-2 (27 mg,0.071 mmol) was dissolved in N, N-dimethylformamide (0.5 mL), N, N-diisopropylethylamine (30.3 mg,0.24 mmol) and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (29.8 mg,0.078 mmol) were sequentially added to the reaction solution, reacted at 25℃for 15 minutes after the addition was completed, then compound 2G (30 mg,0.047 mmol) was dissolved in N, N-dimethylformamide (0.5 mL) and then added dropwise to the reaction solution, and the reaction was continued with stirring at 25℃for 1 hour after the addition was completed. TLC showed that after the reaction was completed, the reaction solution was directly purified by HPLC (Waters Sunfire OBD 100X30 mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 2 (12 mg, off-white solid, yield 29.5%).

MS(ESI):m/z 481.7[1/2M+1] ⁺ 。

¹ H NMR(400MHz,dmso)δ12.83(s,1H),10.87(s,1H),8.86-8.76(m,2H),8.38(s,1H),8.18(dd,J＝8.1,2.0Hz,1H),8.08(d,J＝8.1Hz,1H),7.82(d,J＝2.4Hz,2H),7.67(s,1H),7.50(d,J＝3.6Hz,1H),7.48(d,J＝8.1Hz,1H),7.43(s,1H),7.28(d,J＝3.6Hz,1H),7.15(d,J＝8.1Hz,1H),7.04(d,J＝10.8Hz,1H),6.73(s,1H),4.37(d,J＝12.4Hz,1H),4.22-3.93(m,5H),3.18-3.05(m,2H),2.98(br.s,2H),2.83(t,J＝8.7Hz,1H),2.76-2.61(m,3H),2.60-2.56(m,1H),2.55-2.50(m,2H),2.46-2.42(m,1H),2.37-2.24(m,2H),2.23-2.01(m,3H),1.88-1.64(m,6H),1.57-1.46(m,1H)。

Example 3: preparation of Compound 3

The structural formula of the compound 3 is as follows:

the synthetic route for compound 3 is:

the specific preparation method of the compound 3 comprises the following steps:

the preparation method comprises the following steps:

the first step: synthesis of Compound 3B

Compound 3A (3 g,16.13 mmol) and p-bromoiodobenzene (10 g,35.48 mmol) were dissolved in toluene (82 mL), sodium t-butoxide (3.41 g,35.48 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (1.57 g,1.61 mmol) were added with stirring at room temperature, and the reaction solution was replaced with nitrogen for 3 times and stirred at 65℃for 16 hours. After TLC showed that the reaction solution was poured into water (200 mL) and extracted with dichloromethane (100 mL. Times.3). The combined organic phases were washed with saturated brine (80 ml×2), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, desolventized under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=5:1 (volume ratio)) to give compound 3B (1.64 g, yellow solid, yield 29.9%).

MS(ESI):m/z 341.08[M+1] ⁺ 。

And a second step of: synthesis of Compound 3C

Compound 3B (1 g,2.94 mmol) was dissolved in 1, 4-dioxane (10 mL), then pinacol biborate (1.49 g,5.88 mmol), potassium acetate (0.86 g,8.82 mmol) and [1,1' -bis (diphenylphosphine) ferrocene ] palladium dichloride dichloromethane complex (0.29 g,0.29 mmol) were added sequentially under stirring at room temperature, and after the addition was completed, the reaction solution was replaced with nitrogen 3 times, and then stirred at 80℃for 4 hours. TLC showed that after the reaction was completed, the reaction solution was diluted with water (50 mL) and then extracted with ethyl acetate (40 ml×3). The combined organic phases were washed with saturated brine (30 ml×2), dried over anhydrous sodium sulfate, the drying agent was removed by filtration, desolventized under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=5:1 (volume ratio)) to give compound 3C (1.01 g, white solid, yield 88.5%).

MS(ESI):m/z 389.25[M+1] ⁺ 。

And a third step of: synthesis of Compound 3D

Compound 3C (551.0 mg,1.42 mmol) and intermediate INT-1 (500.0 mg,1.09 mmol) were added to a mixed solvent of 1,4 dioxane (6 mL) and water (1 mL), and sodium carbonate (231.1 mg,2.18 mmol), 2-di-tert-butyl phosphino-2 ',4',6 '-triisopropyl biphenyl (93.4 mg,0.22 mmol) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex (94.3 mg,0.13 mmol) were added sequentially at room temperature (20 ℃ C.). The reaction system was subjected to microwave reaction at 80℃for 2 hours under nitrogen atmosphere. After TLC monitored complete reaction of the starting materials, saturated aqueous ammonium chloride (50 mL), ethyl acetate (40 mL. Times.3) was added to the reaction solution, the organic phases were combined, washed with saturated brine (2X 20 mL), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and spun-dried under reduced pressure, the residue was purified by HPLC (Waters Sunfire OBD 100X30 mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 3D (30 mg, white solid, yield 4.3%).

MS(ESI):m/z 322.1[1/2M+1] ⁺ 。

Fourth step: synthesis of Compound 3E

Compound 3D (30 mg,0.047 mmol) was dissolved in ethanol (0.5 mL) and then 8% hydrochloric acid/ethyl acetate solution (1 mL) was added, after which the reaction was carried out by heating to 40℃for 2 hours. TLC showed that after the reaction was completed, the reaction solution was directly spin-dried to give crude compound 3E which was directly used in the next reaction (30 mg, yellow solid, 100.0%).

MS(ESI):m/z 543.2[M+1] ⁺ 。

Fifth step: synthesis of Compound 3

Compound INT-2 (27 mg,0.071 mmol) was dissolved in N, N-dimethylformamide (0.5 mL), N, N-diisopropylethylamine (30.3 mg,0.24 mmol) and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (29.8 mg,0.078 mmol) were sequentially added to the reaction solution, reacted at 25℃for 15 minutes after the addition was completed, and then 3E (30 mg,0.047 mmol) was dissolved in N, N-dimethylformamide (0.5 mL) and then added dropwise to the reaction solution, followed by continuing the reaction at 25℃with stirring for 1 hour after the addition was completed. TLC showed that after the reaction was completed, the reaction solution was directly purified by HPLC (Waters Sunfire OBD 100X30 mm,5 μm, mobile phase A: 0.1% TFAin water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 3 (14 mg, off-white solid, 34.0% yield).

MS(ESI):m/z 448.2[1/2M+1] ⁺ 。

¹ H NMR(400MHz,dmso)δ12.79(s,1H),10.88(s,1H),9.52(s,1H),8.25(s,1H),7.86(s,1H),7.66(dd,J＝15.8,6.8Hz,3H),7.53(d,J＝8.1Hz,1H),7.50(d,J＝3.6Hz,1H),7.44(s,1H),7.27(d,J＝3.6Hz,1H),7.19-7.11(m,2H),7.07(d,J＝8.6Hz,2H),6.69(s,1H),4.44-4.30(m,2H),4.10(dd,J＝11.7,4.5Hz,1H),4.06-3.96(m,2H),3.73-3.65(m,2H),3.63-3.50(m,3H),3.29-3.19(m,4H),3.17-3.06(m,2H),3.00-2.89(m,2H),2.85-2.68(m,3H),2.61-2.50(m,3H),2.31-2.24(m,1H),2.16-1.81(m,6H)。

Experimental example 1: cell antiproliferative activity assay

(1) Experimental materials:

RPMI1640, glutamine, interleukin 3, pancreatin was purchased from Life Technology.

Fetal bovine serum, diabody, was purchased from Hyclone.

Blasticidin was purchased from Merck.

Phosphate buffer was purchased from Corning.

CellTiter-Glo kit was purchased from Promega.

Ba/F3 cells were purchased from Riken.

Ba/F3 (EGFR L858R/T790M/C797S) cells were constructed from well-established science.

Ba/F3 cell culture Medium: 88% RPMI1640, 10% fetal bovine serum, 10ng/mL interleukin 3,1% glutamine, 1% diabody.

Ba/F3 (EGFR L858R/T790M/C797S) cell culture medium: 87.9% RPMI1640, 10% fetal bovine serum, 0.1% blasticidin, 1% glutamine, 1% diabody.

Experiment plate: 781091 (Greiner)

Plate reading instrument: envision (PerkinElmer)

(2) The experimental method comprises the following steps:

the test compounds were 3-fold gradient diluted with DMSO at 10 points on ECHO, transferring 250nL of the compound to 384 well plates, respectively, at a final concentration of 5-0.00025 μm, positive and negative controls transferring 250nL of DMSO; cells in a uniform T75 flask were blown, counted, and the Ba/F3 (EGFR L858R/T790M/C797S) cell concentration was adjusted to 4X 10 with medium ⁴ mu.L of phosphate buffer was added to the peripheral well of 384-well plate, 50. Mu.L of cell suspension was added to the other well, and the mixture was allowed to stand at room temperature for 15min, and then was allowed to stand at 37℃with 5% CO ₂ Incubating for 72 hours in an incubator; to each well of 384 well plates, 25. Mu.L CellTiter-Glo was added, centrifuged at 1000rpm for 15s, and after shaking at room temperature for 15min, read with Envision.

(3) Data analysis:

the reading was converted to inhibition (%) (Sample value-HC)/(LC-HC) 100. Parametric curve fitting (XL-fit software) to measure IC by the following equation ₅₀ Data.

(4) Test results:

the data for the inhibition activity of Ba/F3 (EGFR L858R/T790M/C797S) cells of the compounds of the present invention are shown in Table 1 below.

Table 1 provides the inhibitory activity of the compounds of the present invention on Ba/F3 (EGFR L858R/T790M/C797S) cell proliferation.

TABLE 1 anti-cell proliferation Activity data (IC) for the compounds of the invention ₅₀ )

As can be seen from the experimental results in Table 1, the compounds of the present invention have good activity in inhibiting the proliferation of Ba/F3 (EGFR L858R/T790M/C797S) cells. The activity of each of the plurality of compounds was less than 100nM. Shows extremely important antitumor potential and has good clinical application prospect.

Experimental example 2: pharmacokinetic experiments

1. Experimental materials

Using the compounds prepared in the above examples, oral drug formulations were formulated as 4.76mg/mL clear solutions (5% DMSO+30% PEG300+2% Tween80+63% H) ₂ O)。

2. Experimental animal

Male ICR mice, grade SPF, weighing 3, 27-28g, were supplied by Shanghai Laike laboratory animal liability Co. The subject mice were given an environmental adaptation period of 2-4 days prior to the experiment, and the animals were fed normally prior to the administration.

3. Experimental method

1) After normal feeding, the mice took 0 time blank plasma;

2) Taking the mice in the step 1), and orally (PO) administering 50mg/kg of the compound to be tested;

3) After oral administration by gavage, 0.083h,0.25h,0.5h,1h,2h,4h,6h,8h,24h. Collecting blood through cheek, collecting about 0.05mL of each sample, anticoagulating heparin sodium, and placing on wet ice after collection; blood samples were collected and placed on ice and the plasma was centrifuged within 1 hour (centrifugation conditions: 6000g,3 minutes, 2-8 ℃). The plasma samples were stored in a-80 ℃ freezer prior to analysis.

4) Pharmacokinetic parameters were calculated from the blood concentration data at different time points according to step 3) using Phoenix WinNonlin8.2.0 to provide parameters such as AUC0-T, AUC0- +.infinity, MRT0- +.infinity, cmax, tmax, and T1/2, as well as their mean and standard deviation, and the test results are shown in Table 2 below.

TABLE 2 pharmacokinetic data for the compounds of the invention

As shown in table 2, the compounds of the present invention, when administered orally or intravenously to mice or rats, have a longer half-life and higher exposure in animal plasma and can be administered orally.

While embodiments of the present invention have been illustrated and described above, it will be appreciated that the above described embodiments are illustrative and should not be construed as limiting the invention. Variations, modifications, substitutions, and alterations are also possible in the above described embodiments without departing from the principles and spirit of the invention, and such variations, modifications, substitutions, and alterations are to be within the scope of this disclosure.

Claims

1. A compound for inhibiting/inducing degradation of EGFR kinase, wherein the compound is a compound shown in formula I or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, racemate, polymorph, isotopic variant, metabolite or prodrug thereof,

wherein,,

Y ₁ and Y ₂ Independently selected from O, S, se, NR ₄ Or CR (CR) ₄ ；

Y ₃ Independently selected from C or N;

X ₁ independently selected from C or N;

R ₁ 、R ₂ and R is ₃ Each independently selected from hydrogen, deuterium, halogen, cyano, aminoHydroxy, -NR ₇ R ₈ 、-OR ₇ 、C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₂ -C ₆ Alkenyl, C ₂ -C ₆ Alkynyl, C ₃ -C ₈ Cycloalkyl, C ₃ -C ₈ Heterocycloalkyl, C ₁ -C ₃ Alkoxy, C ₁ -C ₆ Haloalkoxy or C ₁ -C ₆ Haloalkyl, and wherein the alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkoxy, haloalkoxy, and haloalkyl are each optionally substituted with at least 1R ₉ Substitution;

R ₄ 、R ₅ and R is ₆ Independently selected from hydrogen, deuterium, halogen, cyano, amino, carbonyl, hydroxy, H, C ₃ -C ₈ Cycloalkyl, C ₃ -C ₈ Heterocycloalkyl, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₁ -C ₆ Alkoxy, C ₁ -C ₆ Haloalkoxy, -NR ₇ R ₈ OR-OR ₇ And wherein the alkyl, cycloalkyl, heterocycloalkyl, heteroalkyl, alkoxy, and haloalkoxy are each optionally substituted with at least 1R ₉ Substitution;

R ₇ and R is ₈ Independently selected from hydrogen, deuterium, C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₃ -C ₈ Cycloalkyl, or C ₃ -C ₈ Heterocycloalkyl, and wherein said alkyl, heteroalkyl, cycloalkyl, and heterocycloalkyl are each optionally substituted with at least 1R ₉ Substitution;

L ₁ 、L ₂ 、L ₃ and L ₄ Independently selected from the group consisting of absence, chemical bond, -O-, - (CH) ₂ ) _m -C(O)-、-C(O)-C(H ₂ ) _p -、-C(O)-C(O)-、NR ₁₀ -C(O)-、-C(O)-NR ₁₀ 、-C(O)O-、-CH ₂ -CF ₂ -CH ₂ -、-CH ₂ -、 Wherein each m and p is independently any integer from 0 to 3;

R ₁₁ selected from hydrogen, deuterium, halogen, cyano, amino, hydroxy, and C ₁ -C ₆ Alkyl, C ₁ -C ₆ Heteroalkyl, C ₃ -C ₈ Cycloalkyl, C ₃ -C ₈ Heterocycloalkyl, C ₁ -C ₃ Alkoxy, C ₁ -C ₆ Haloalkoxy or C ₁ -C ₆ Haloalkyl, and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, haloalkoxy, and haloalkyl are each optionally substituted with at least 1R ₁₂ Substitution;

R ₁₂ each independently selected from hydrogen, chlorine, fluorine, cyano, hydroxy, amino, isopropylCyclopropyl, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy, 2-difluoroethoxy, 2-trifluoroethoxy and phenyl.

2. A compound according to claim 1, wherein the compound is a compound of formula I-1 or I-2,

3. a compound according to claim 2, characterized in that it is a compound as shown in any one of formulae 1 to 3:

4. the compound of claim 1, wherein the stereoisomers comprise enantiomers or diastereomers.

5. A pharmaceutical composition comprising an effective amount of one or more of the compounds of any one of claims 1-3, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, stereoisomers thereof, tautomers thereof, racemates thereof, polymorphs thereof, isotopic variations thereof, metabolites thereof and prodrugs thereof.

6. The pharmaceutical composition of claim 5, further comprising at least one pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, wherein the adjuvant comprises a pharmaceutically acceptable excipient or therapeutic agent.

8. Use of one or more of the compounds of any one of claims 1-3, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, stereoisomers thereof, tautomers thereof, racemates thereof, polymorphs thereof, isotopic variants thereof, metabolites thereof and prodrugs thereof for the manufacture of a medicament for the treatment and/or prevention of EGFR kinase mediated diseases.

9. The use of one or more of the compounds of any one of claims 1-3, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, stereoisomers thereof, tautomers thereof, racemates thereof, polymorphs thereof, isotopic variants thereof, metabolites thereof and prodrugs thereof for the manufacture of a medicament for the treatment and/or prevention of cancer.

10. The use of claim 9, wherein the cancer comprises ovarian cancer, cervical cancer, colorectal cancer, breast cancer, pancreatic cancer, glioma, glioblastoma, melanoma, prostate cancer, leukemia, lymphoma, non-hodgkin's lymphoma, gastric cancer, lung cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal stromal tumor, thyroid cancer, cholangiocarcinoma, endometrial cancer, renal cancer, anaplastic large cell lymphoma, acute myelogenous leukemia, multiple myeloma, melanoma, or mesothelioma.