WO2024000763A1 - G9a/glp covalent inhibitor and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a G9a/GLP covalent inhibitor and a preparation method therefor and a use thereof. The G9a/GLP covalent inhibitor is a compound having a structure as shown in formula (I) and a salt thereof:

Description

A G9a/GLP covalent inhibitor and its preparation method and application Technical field

The invention relates to the field of medicine, and in particular to a G9a/GLP covalent inhibitor and its preparation method and application.

Background technique

Post-translational modification of histones plays an important role in the occurrence and development of various human diseases. Histone methyltransferase G9a (KMT1C or EHMT2) and GLP (KMT1D or EHMT1) are a type of closely related methyltransferases. . The SET domain of GLP has 80% sequence homology with G9a; GLP can also form heterodimers with G9a to jointly exert physiological functions. In addition to catalyzing the monomethylation and dimethylation of H3K9, G9a/GLP can also dimethylate the lysine 373 residue of the tumor suppressor gene p53, causing p53 transcriptional inactivation and increasing cancer cell proliferation. G9a/GLP GLP has been shown to be involved in many physiological and pathological processes in the body and is overexpressed in various human cancers including leukemia, prostate cancer, hepatocellular carcinoma and lung cancer. Therefore, in recent years, G9a/GLP has become a popular target for research on multiple diseases.

The currently developed G9a/GLP inhibitors generally suffer from low efficacy, and there are currently no candidate compounds that have entered the clinical research stage. Therefore, there is an urgent need to develop inhibitors with new modes of action to solve this problem. Up to now, all reported G9a/GLP inhibitors are non-covalent reversible inhibitors (Cao H, Li L, Yang D, et al. Recent progress in histone methyltransferase (G9a) inhibitors as anticancer agents. Eur J Med Chem.2019;179:537-546.), no covalent inhibitors have been reported. When covalent inhibitors bind to the target protein, they can form covalent bonds with the electrophilic amino acid residues on the target protein near the binding site. Therefore, compared with non-covalent inhibitors, covalent inhibitors have a longer action time. It has a series of advantages such as long life, strong efficacy and low dosage. There are electrophilic cysteine residues (G9a-Cys1098, GLP-1186) in the catalytic pocket of G9a/GLP, which meet the conditions for the development of covalent inhibitors.

Based on this, it is of great research significance and application value to design and develop a G9a/GLP covalent inhibitor with strong efficacy and druggability.

Contents of the invention

The purpose of the present invention is to overcome the lack of G9a/GLP covalent inhibitors and provide a G9a/GLP covalent inhibitor. The G9a/GLP covalent inhibitor provided by the invention has good specificity, strong drug effect, and high selectivity for histone methyltransferase G9a/GLP, and can be used to prepare drugs that inhibit G9a/GLP, prevent and/or treat tumors or Cancer drugs.

Another object of the present invention is to provide a method for preparing the above-mentioned G9a/GLP covalent inhibitor.

Another object of the present invention is to provide the use of the above-mentioned G9a/GLP covalent inhibitor in the preparation of drugs that inhibit G9a/GLP.

In order to achieve the above objects of the present invention, the present invention provides the following technical solutions:

A G9a/GLP covalent inhibitor, which is a compound with a structure shown in formula (I) and its salt:

Among them, n ₁ , n ₂ , n ₃ and n ₄ are independently selected from integers from 0 to 2;

n ₅ is an integer from 0 to 4;

X is CH or N;

R ₁ is selected from hydrogen, C ₁ -C ₆ alkyl and its deuterated products, C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ heterocycloalkyl; the alkyl and its deuterated products and cycloalkyl are any Optionally selected by halogen, cyano, hydroxyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₁ -C ₆ alkyl, amino, C ₁ -C ₆ alkylamino, bis C ₁ -C ₆ alkylamino, 4-12 membered heterocyclic group substituted by one or more groups;

R ₂ is

R ₃ is selected from hydrogen, trifluoromethyl, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₃ -C ₈ cycloalkyl, substituted or unsubstituted C ₃ -C ₈ Heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C ₅ -C ₆ heteroaryl; the substitution means that at least 1 position is substituted by the following substituents: halogen, cyano, amino, Nitro, hydroxyl, trifluoromethyl, methylthio, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkyl, C ₁ -C ₆ alkylamino, C ₃ -C ₈ heterocyclyl; C ₃ -C ₈ cycloalkoxy group, C ₃ -C ₈ cycloalkylamino group, aryl group, C ₅ -C ₆ heteroaryl group;

R ₄ is selected from hydrogen, halogen, cyano, hydroxyl, methoxy or trifluoromethoxy;

R ₅ is selected from substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₃ -C ₈ cycloalkyl, substituted or unsubstituted C ₃ -C ₈ heterocycloalkyl, substituted or Unsubstituted aryl, substituted or unsubstituted C ₅ -C ₆ heteroaryl, substituted or unsubstituted C ₁ -C ₆ alkoxy, substituted or unsubstituted C ₁ -C ₆ alkylamino, substituted or unsubstituted Substituted C ₃ -C ₈ cycloalkyloxy group, substituted or unsubstituted C ₃ -C ₈ cycloalkylamino group, substituted or unsubstituted C ₃ -C ₈ cycloalkylamino group, the substitution means at least 1 The position is substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, methylthio, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkyl, C ₁ -C ₆ alkylamino, C ₃ -C ₈ heterocyclyl; C ₃ -C ₈ cycloalkoxy, C ₃ -C ₈ cycloalkylamino, aryl, C ₅ -C ₆ heteroaryl; R _a is selected from hydrogen, halogen or R _d ;

R _b and R _c are independently selected from hydrogen or R _d ;

R _d is selected from substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₂ -C ₆ alkenyl, substituted or unsubstituted C ₂ -C ₆ alkynyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted 4-12 membered heterocyclyl;

Or R _a and R _b together with the carbon atoms to which they are connected form a 3-5-membered heterocyclyl group or a substituted 3-5-membered heterocyclyl group containing 0 or 1 additional heteroatom;

Y is selected from halogen.

The present invention uses quinazoline and quinoline as the drug skeleton. Quinazoline and quinoline have good drug properties, and the skeleton inhibitor has a cysteine residue near the binding pocket where it interacts with G9a. , is close to the C-2 position of quinazoline, and the direction and distance are suitable for adding an electrophilic active group, that is, a covalent warhead, which can undergo an electrophilic addition reaction with the cysteine residue near the binding pocket to form a covalent bond, thereby achieving a long-lasting inhibitory effect, and the G9a/GLP covalent inhibitor obtained after specific substitutions has good specificity, strong efficacy, and high selectivity for histone methyltransferase G9a/GLP, and can be used to prepare G9a inhibitors. /GLP drugs, drugs to prevent and/or treat tumors or cancer.

Preferably, n ₁ is 0 or 1.

Preferably, n ₂ is 0 or 1.

Preferably, n ₃ is 1.

Preferably, n ₄ is an integer from 0 to 3.

Preferably, _R1 is selected from hydrogen or _C1 - _C6 alkyl.

Preferably, R ₂ is selected from

in:

The substituents in R _d are one or more -JT groups; the substituents in the substituted 3-5-membered heterocyclic groups in R _a and R _b are one or more -J ₁ -T ₁ groups;

J is selected from a bond or substituted C ₁ -C ₆ alkylene;

T is selected from hydrogen, halogen, cyano, hydroxyl, -NR _f R _g , -C(O)R _f , -OR _f , -C(O)OR _f , -C(O)NR _f R _g , -NR _f C(O)R _g , -NR _h C(O)NR _f R _g , -NR _f C(O)OR _h or R _i ;

R _f , R _g , and Rh _h are each independently selected from hydrogen or R _j , and R _j is selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C _6- cycloalkyl, 4-12-membered heterocyclyl, 5- or 6-membered heteroaryl, aryl, R _j is substituted by one or more -J ₁ -T ₁ groups;

Or R _f and R _g together with the N atoms to which they are connected form a 4-12-membered heterocyclyl group containing 0 or 1 additional heteroatom, and the 4-12-membered heterocyclyl group is replaced by one or more -J ₁ -T ₁ group substitution;

R _i is selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₆ cycloalkyl, 4-12 membered heterocyclyl, 5-10 membered heterocyclic group Aryl, aryl, R _i is substituted by one or more -J ₁ -T ₁ groups;

J ₁ is selected from a bond or substituted C ₁ -C ₆ alkylene;

T ₁ is selected from hydrogen, halogen, cyano, hydroxyl, -NR _k R _l , -C(O)R _k , -OR _k , -C(O)OR _k , -C(O)NR _k R _l , - NR _k C(O)R _l , -NR _o C(O)NR _k R _l , -NR _k C(O)OR _o or R _p ;

R _k , R _l , and R _o are each independently selected from hydrogen or R _q , and R _q is selected from the following substituted groups: C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkyne Base, C ₃ -C ₆ cycloalkyl group, 4-12 membered heterocyclyl group, 5-membered or 6-membered heteroaryl group, aryl group;

Or R _k and R _l together with the N atom to which they are connected form a 4-12 membered heterocyclyl group containing 0 or 1 additional heteroatom, and the heterocyclyl group is optionally selected from halogen, hydroxyl, oxo, C ₁ -C ₆ alkyl, OR _x , -NR _x R _y , -C(O)R _x , -O(CH ₂ ) _n OR _x is substituted by one or more groups;

R _p is selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₆ cycloalkyl, 4-12 membered heterocyclyl, 5 to 6 membered heterocyclic group Aryl, aryl;

R _x and R _y are each independently selected from hydrogen or R _z , and R _z is selected from the following groups or substituted groups: C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ Alkynyl, C ₃ -C ₆ cycloalkyl, 4-12-membered heterocyclyl, aryl, 5- or 6-membered heteroaryl; _Rz is replaced by halogen, hydroxyl, 5- or 6-membered arylheteroyl, aryl or one or more substituted 5- or 6-membered heteroaryl groups,

Or R _x , R _y and the N atom to which they are connected together form a 4-12 membered heterocyclyl group containing 0 or 1 additional heteroatom;

or -J ₁ -T ₁ is an oxo group;

or -J-T is oxo;

Preferably, R ₃ is selected from hydrogen, aryl, C ₁ -C ₆ alkyl, C ₃ -C ₈ heterocycloalkyl or C ₁ -C ₆ alkyl substituted by C ₃ -C ₈ heterocycloalkyl base.

Preferably, R ₅ is selected from C ₁ -C ₆ alkyl or C ₃ -C ₈ heterocycloalkyl.

Preferably, the G9a/GLP covalent inhibitor is the structure shown in the following numbering and its salt:

The salts in the present invention are pharmaceutically acceptable salts.

Preferably, the salt is a hydrochloride, a hydrobromide, a nitrate, a methyl nitrate, a sulfate, a hydrogen sulfate, an aminosulfate, a phosphate, an acetate, a glycolate, or a phenyl ethyl salt. Propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate Acid, p-aminosalicylate, glycolate, lactate, enanthate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate , chlorobenzoate, methylbenzoate, dinitrobenzoate, paraben, methoxybenzoate, mandelate, tannin, formate, stearate Acid, ascorbate, palmitate, oleate, pyruvate, pasate, malonate, laurate, glutarate, glutamate, propionate lauryl sulfate ( estolate), methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate) or naphthalene-2-sulfonate Acid.

The preparation method of the above-mentioned G9a/GLP covalent inhibitor includes the following steps:

The compound represented by formula (1) and acid undergo a condensation reaction under the conditions of a condensing agent and an organic base to obtain a compound with a structure represented by formula (I).

Preferably, the molar ratio of the compound represented by formula (1) to acid, condensing agent and organic base is 1:(1.2~1.5):(1.2~1.4):(2~3).

Preferably, the acid is propionic acid, acrylic acid, methacrylic acid, 2-butenoic acid, 2-butynoic acid, chloroacetic acid, cyanoacetic acid, 1-cyano-1-cyclopropanecarboxylic acid or (E)- One or more of 4-(dimethylamino)but-2-enoic acid.

Preferably, the condensing agent is acid chloride.

Preferably, the organic base is DIPEA.

Preferably, the solvent is ultradry methylene chloride.

Preferably, the reaction temperature of the condensation reaction is 0°C to room temperature (for example, 0°C to 26°C), and the reaction time is 1 to 2 hours.

Preferably, the compound represented by formula (1) is prepared by the following process: the compound represented by formula (2) and the compound represented by formula (3) undergo a substitution reaction in the presence of basic substances and solvents to generate formula (4) The intermediate shown in formula (4) then undergoes a substitution reaction with methylamine under heating conditions to obtain the compound shown in formula (1);

Preferably, the molar ratio of the compound represented by the formula (2) to the compound represented by the formula (3) and the basic substance is 1:(1.5~2):(2.5~3).

Preferably, the alkaline substance is K ₂ CO ₃ .

Preferably, the solvent is N,N-dimethylformamide.

Preferably, the reaction temperature for the substitution reaction between the compound represented by formula (2) and the compound represented by formula (3) is 0°C to room temperature (for example, 0°C to 26°C), and the reaction time is 3 to 4 hours.

Preferably, the conditions for the substitution reaction between the intermediate represented by formula (4) and methylamine are: methylamine solution is the solvent, the reaction temperature is 120°C, and the reaction time is 8 hours.

The application of the above-mentioned G9a/GLP covalent inhibitor in preparing drugs that inhibit G9a/GLP is also within the protection scope of the present invention.

Studies have shown that G9a/GLP covalent inhibitors can inhibit the expression of G9a/GLP, prevent and/or treat abnormal cell proliferation, morphological changes, and hypermotility related to G9a/GLP, and can treat and/or prevent tumor growth. and transfer.

Preferably, the drug is a drug for preventing and/or treating diseases involving abnormal cell proliferation, morphological changes, and hypermotility related to G9a/GLP.

Preferably, the drug is a drug for treating and/or preventing tumor growth and metastasis.

More preferably, the tumor is one or more of cancer or benign tumors. The cancer can be pancreatic cancer, breast cancer, lung cancer, bone cancer, stomach cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, brain cancer, pituitary gland cancer Adenomas, epidermoid carcinoma, T-cell lymphoma, chronic and acute leukemia, colorectal cancer, kidney cancer, esophageal cancer, breast cancer, cervical cancer, bladder cancer, fibrosarcoma, esophageal cancer, bladder cancer, hematopoietic system cancer, lymphoma , medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, head and neck squamous cell carcinoma, brain cancer, hepatocellular carcinoma, melanoma, oligodendroblastoma Glioma, glioblastoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, multiple myeloma (AML), mantle cell lymphoma, triple-negative breast cancer, non-small cell lung cancer.

Compared with the existing technology, the present invention has the following advantages and effects:

The G9a/GLP covalent inhibitor provided by the invention has good specificity, strong drug effect, and high selectivity for histone methyltransferase G9a/GLP, and can be used to prepare drugs that inhibit G9a/GLP, prevent and/or treat tumors or Cancer drugs.

Description of drawings

Figure 1 shows the mass spectrum verification of covalent binding of G9a to compound 14.

Figure 2(A) shows the predicted binding mode of compound 14 and G9a protein, and Figure 2(B) shows the predicted binding mode of compound 14 and GLP protein;

Figure 3 shows the methylation inhibition elution experiment of compounds 14 and 26;

Figure 4 shows that compounds 14 and 26 inhibit the clonogenesis of MDA-MB-231 and PANC1;

Figure 5 shows the methylation inhibition experiments of compounds 14 and 26.

Figure 6 shows the in vivo anti-tumor activity experiment of compound 14.

Detailed ways

The present invention will be further explained below with reference to the examples and drawings, but the examples do not limit the invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

Unless otherwise stated, the reagents and materials used in the present invention are all commercially available.

Example 1 N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)meth)acrylamide

Add raw material 1a (100mg, 0.23mmol) into the reaction bottle, add 10ml of ultra-dry dichlorohexane, add HATU (114.07mg, 0.30mmol), acrylic acid (19.20ul, 0.28mmol) and DIPEA (120.19ul) at 0°C , 0.69mmol), then stir at room temperature, and detect the reaction progress with TLC. After 2 hours of reaction, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, then filter and spin the solvent dry, and the crude product is separated and purified by column chromatography to obtain The target compound 1 is a white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.23 (s, 1H), 7.16 (s, 1H), 6.91 (s, 1H), 6.35–6.31 (m, 1H), 5.69 (dd, J＝9.2, 2.5Hz,1H),5.47(d,J＝7.4Hz,1H),4.59(d,J＝4.3Hz,2H),4.22(t,J＝6.7Hz,3H),3.98(s,3H),2.93 (d,J＝11.5Hz,2H),2.71(d,J＝7.4Hz,2H),2.59(d,J＝5.9Hz,4H),2.35(s,3H),2.24–2.12(m,6H) ,1.84–1.80(m,4H),1.69(dd,J＝11.9,3.6Hz,2H).HRMS(ESI)calcd for C ₂₆ H ₃₈ N ₆ O ₃ (M+H ⁺ ):483.3078; found483.3078 .

Example 2 N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)methyl)methacrylamide

Acrylic acid was replaced with an equal molar amount of methacrylic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR(400MHz,Chloroform-d)δ7.49(s,1H),7.15(s,1H),6.89(s,1H),5.90(s,1H),5.40(s,2H),4.58(d ,J＝4.2Hz,2H),4.22(t,J＝6.8Hz,3H),3.97(s,3H),2.90(d,J＝11.2Hz,2H),2.66(t,J＝7.3Hz,2H ),2.55(d,J＝5.8Hz,4H),2.33(s,3H),2.22–2.14(m,6H),2.09(s,3H),1.84–1.76(m,4H),1.71–1.61( m,2H).HRMS(ESI)calcd for C ₂₇ H ₄₀ N ₆ O ₃ (M+H ⁺ ):497.3235; found 497.3235.

Example 3 6-methoxy-4-(1-methylpiperidin-4-amino)-7-(3-pyrrolidin-1-yl)propoxyquinazolin-2-yl)methylbutan -2-enamide

Acrylic acid was replaced with an equal molar amount of 2-butenoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR (500MHz, Chloroform-d) δ7.17 (s, 1H), 7.06 (d, J = 4.4Hz, 1H), 6.89 (q, J = 7.6, 7.0Hz, 2H), 6.01 (dd, J ＝15.2,2.0Hz,1H),5.42(d,J＝7.3Hz,1H),4.58(d,J＝4.3Hz,2H),4.23(t,J＝6.8Hz,3H),3.97(s,3H ),2.90(d,J＝11.5Hz,2H),2.66(t,J＝7.3Hz,2H),2.54(d,J＝5.9Hz,4H),2.33(s,3H),2.16(dd,J ＝14.6,7.0Hz,6H),1.89(dd,J＝6.8,1.6Hz,3H),1.83–1.77(m,4H),1.65(dt,J＝11.3,5.6Hz,2H).HRMS(ESI) calcd for C ₂₇ H ₄₀ N ₆ O ₃ (M+H ⁺ ):497.3235; found 497.3234.

Example 4 N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)methyl)but-2-ynamide

Acrylic acid was replaced with an equimolar amount of 2-butynoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR(400MHz,Chloroform-d)δ7.34(t,J＝4.6Hz,1H),7.20(s,1H),6.89(s,1H),5.40(d,J＝7.4Hz,1H), 4.55(d,J＝4.5Hz,2H),4.24(t,J＝6.7Hz,3H),4.00(s,3H),2.92(d,J＝11.3Hz,2H),2.67(t,J＝7.3 Hz,2H),2.56(q,J＝3.2Hz,4H),2.34(s,3H),2.22–2.14(m,6H),2.00(s,3H),1.83–1.79(m,4H),1.67 (dd,J＝11.8,3.8Hz,2H).HRMS(ESI)calcd for C ₂₇ H ₃₈ N ₆ O ₃ (M+H ⁺ ): 495.3078; found 495.3078.

Example 5 4-(dimethylamino)-N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidine-1-yl) )propoxy)quinazolin-2-yl)methyl)but-2-enamide

Acrylic acid was replaced with an equal molar amount of 4-dimethylaminobutenoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR(500MHz,Chloroform-d)δ7.20(t,J＝4.3Hz,1H),7.17(s,1H),6.93–6.81(m,2H),6.16(d,J＝15.5Hz,1H ),5.37(d,J＝7.5Hz,1H),4.58(d,J＝4.3Hz,2H),4.22(q,J＝6.7Hz,3H),3.99(s,3H),3.10(d,J ＝6.1Hz,2H),2.90(d,J＝11.5Hz,2H),2.67(t,J＝7.3Hz,2H),2.55(d,J＝5.9Hz,4H),2.33(s,3H), 2.28(s,6H),2.19–2.13(m,6H),1.82–1.78(m,4H),1.69–1.61(m,2H).HRMS(ESI)calcd for C ₂₉ H ₄₅ N ₇ O ₃ (M +H ⁺ ):540.3657; found540.3657.

Example 6 2-Chloro-N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-pyrrolidin-1-yl)propoxyquinazole Phin-2-ylmethyl)acetamide

Acrylic acid was replaced with an equal molar amount of chloroacetic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR (400MHz, Chloroform-d) δ8.23 (s, 1H), 7.16 (s, 1H), 6.84 (s, 1H), 5.29 (d, J = 7.5Hz, 1H), 4.56 (d, J ＝4.2Hz,2H),4.27(dd,J＝7.5,3.9Hz,1H),4.23(t,J＝6.7Hz,2H),4.18(s,2H),4.00(s,3H),2.92(d ,J＝11.3Hz,2H),2.69–2.64(m,2H),2.59–2.52(m,4H),2.34(s,3H),2.23–2.12(m,6H),1.83–1.77(m,4H ),1.71–1.62(m,2H).HRMS(ESI)calcd for C ₂₅ H ₃₇ N ₆ O ₃ Cl(M+H ⁺ ):505.2688; found 505.2688.

Example 7 2-cyano-N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-pyrrolidin-1-yl)propoxyquin oxazolin-2-yl)methyl)acetamide

Acrylic acid was replaced with an equal molar amount of cyanoacetic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.86 (s, 1H), 7.18 (s, 1H), 6.87 (s, 1H), 5.37 (d, J = 7.7Hz, 1H), 4.55 (d, J ＝3.7Hz,2H),4.32(s,1H),4.24(t,J＝6.6Hz,2H),4.01(s,3H),3.51(s,2H),2.91(d,J＝11.6Hz,2H ),2.68(t,J＝7.3Hz,2H),2.58(d,J＝5.9Hz,4H),2.35(s,3H),2.20(d,J＝19.4Hz,6H),1.97–1.77(m ,4H),1.70(d,J＝12.4Hz,2H).HRMS(ESI)calcd for C ₂₆ H ₃₇ N ₇ O ₃ (M+H ⁺ ): 496.3031; found 496.3032.

Example 8 1-cyano-N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy )quinazolin-2-yl)methyl)cyclopropane-1-carboxamide

Acrylic acid was replaced with an equal molar amount of 1-cyano-1-cyclopropanecarboxylic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 1 to obtain a white solid. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.32 (t, J = 5.3 Hz, 1H), 7.63 (s, 1H), 7.61 (s, 1H), 7.01 (s, 1H), 4.29 (d, J＝5.3Hz,2H),4.23–4.17(m,1H),4.13(t,J＝6.4Hz,2H),3.90(s,3H),2.84(d,J＝10.8Hz,2H),2.57( t,J＝7.2Hz,2H),2.46(d,J＝5.8Hz,4H),2.20(s,3H),2.09–2.02(m,2H),1.98–1.91(m,4H),1.72–1.64 (m,6H),1.62(d,J＝3.6Hz,2H),1.55(t,J＝3.6Hz,2H).HRMS(ESI)calcd for C ₂₈ H ₃₉ N ₇ O ₃ (M+H ⁺ ) :522.3187; found 522.3186.

Example 9 N-((6-methoxy-4-((1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinoline -2-yl)meth)acrylamide

Add raw material 9a (100mg, 0.23mmol) into the reaction bottle, add 10ml of ultra-dry dichlorohexane, add HATU (114.07mg, 0.30mmol), acrylic acid (19.20ul, 0.28mmol) and DIPEA (120.19ul) at 0°C , 0.69mmol), then stir at room temperature, and detect the reaction progress with TLC. After 2 hours of reaction, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, then filter and spin the solvent dry, and the crude product is separated and purified by column chromatography to obtain Target compound 9, white solid. ¹ H NMR (400MHz, CDCl ₃ ) δ7.43 (t, J = 4.7Hz, 1H), 7.31 (s, 1H), 6.92 (s, 1H), 6.37–6.29 (m, 3H), 5.66 (dd, J＝9.5,2.2Hz,1H),4.80(d,J＝7.2Hz,1H),4.62(d,J＝4.6Hz,2H),4.23(d,J＝6.9Hz,2H),3.97(s, 3H),3.52–3.46(m,1H),2.88(d,J＝11.4Hz,2H),2.66(d,J＝7.3Hz,2H),2.54(d,J＝5.8Hz,4H),2.33( s,3H),2.17(d,J＝14.2Hz,6H),1.79(q,J＝3.8,3.3Hz,4H),1.70–1.63(m,2H).HRMS(ESI)calcd for C ₂₇ H ₃₉ N ₅ O ₃ (M+H ⁺ ):482.3126; found 482.3125.

Example 10 2-Chloro-N-((6-methoxy-4-((1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy yl)quinolin-2-yl)methyl)acetamide

Acrylic acid was replaced with an equal molar amount of chloroacetic acid, and the remaining required raw materials, reagents, and preparation methods were the same as in Example 9 to obtain a light yellow solid. ¹ H NMR (400MHz, CDCl ₃ ) δ8.12 (s, 1H), 7.33 (s, 1H), 6.85 (s, 1H), 6.33 (s, 1H), 4.58 (dd, J = 10.9, 5.9Hz, 3H),4.27(t,J＝6.7Hz,2H),4.16(s,2H),4.02(s,3H),3.52(s,1H),2.91(d,J＝11.5Hz,2H),2.71( d,J＝7.4Hz,2H),2.57(d,J＝5.9Hz,4H),2.36(s,3H),2.25–2.16(m,6H),1.86–1.65(m,6H).HRMS(ESI )calcd for C ₂₆ H ₃₈ N ₅ O ₃ Cl(M+H ⁺ ):504.2736; found 504.2736.

Example 11 (E)-N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy) Quinolin-2-yl)methyl)but-2-enamide

Acrylic acid was replaced with an equal molar amount of 2-butenoic acid, and the remaining required raw materials, reagents, and preparation methods were the same as in Example 9 to obtain a light yellow solid. ¹ H NMR (400MHz, CDCl ₃ ) δ7.33 (s, 1H), 7.18 (t, J = 4.8Hz, 1H), 6.89 (q, J = 7.6, 7.0Hz, 2H), 6.33 (s, 1H) ,6.00(dd,J＝15.2,1.9Hz,1H),4.74(d,J＝7.3Hz,1H),4.61(d,J＝4.7Hz,2H),4.24(t,J＝6.8Hz,2H) ,3.99(s,3H),3.54–3.41(m,1H),2.96–2.85(m,2H),2.67(t,J＝7.4Hz,2H),2.58–2.50(m,4H),2.34(s ,3H),2.22–2.11(m,6H),1.88(dd,J＝6.8,1.7Hz,3H),1.81(p,J＝3.0Hz,4H),1.66(q,J＝10.1Hz,2H) .HRMS(ESI)calcd for C ₂₈ H ₄₁ N ₅ O ₃ (M+H ⁺ ):496.3282; found 496.3282.

Example 12 N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinoline-2 -Methyl)methacrylamide

Acrylic acid was replaced by an equal molar amount of methacrylic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 9 to obtain a white solid. ¹ H NMR (500MHz, CDCl ₃ ) δ7.52(s,1H),6.90(s,1H),6.33(s,1H),5.86(s,1H),5.38(s,1H),4.73(d, J＝7.7Hz,1H),4.60(d,J＝4.6Hz,2H),4.23(t,J＝6.8Hz,2H),3.98(s,3H),3.56–3.47(m,1H),2.89( d,J＝11.5Hz,2H),2.66(t,J＝7.3Hz,2H),2.54(d,J＝5.1Hz,4H),2.33(s,3H),2.22–2.14(m,6H), 2.06(s,3H),1.79(q,J＝4.1,3.6Hz,4H),1.69–1.63(m,2H).HRMS(ESI)calcd for C ₂₈ H ₄₁ N ₅ O ₃ (M+H ⁺ ) :496.3282; found 496.3282.

Example 13 N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinoline-2 -yl)methyl)but-2-ynamide

Acrylic acid was replaced with an equal molar amount of 2-butynoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 9 to obtain a white solid. ¹ H NMR (500MHz, CDCl ₃ ) δ7.41 (t, J＝4.9Hz, 1H), 7.33 (s, 1H), 6.85 (s, 1H), 6.29 (s, 1H), 4.59 (dd, J＝ 26.7,6.0Hz,3H),4.24(t,J＝6.7Hz,2H),4.00(s,3H),3.50(d,J＝11.1Hz,1H),2.89(d,J＝11.5Hz,2H) ,2.68(t,J＝7.4Hz,2H),2.57(d,J＝6.3Hz,4H),2.34(s,3H),2.23–2.13(m,6H),1.98(s,3H),1.81( t,J＝3.8Hz,4H),1.70–1.62(m,2H).HRMS(ESI)calcd for C ₂₈ H ₃₉ N ₅ O ₃ (M+H ⁺ ):494.3126; found 494.3125.

Example 14 N-(6-methoxy-4-(1-methylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)-N-methacrylamide

Step 1: Add compound 14a (300mg, 0.84mmol) into the reaction bottle, add DMF solvent to dissolve it, add 1-methylpiperidine-4-amino (158.11ul, 1.26mmol) and slowly add K at 0°C. ₂ CO ₃ (348.29mg, 2.52mmol), then stirred at room temperature, TLC detected the reaction progress, the 3h reaction was completed, extracted with ethyl acetate, the organic layer was washed with brine, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and vortexed The solvent was dried, and the crude product was separated and purified by column chromatography to obtain compound 14b. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.00 (d, J = 7.6 Hz, 1H), 7.66 (s, 1H), 7.03 (s, 1H), 4.13 (t, J = 6.4 Hz, 2H) ,4.07–4.01(m,1H),3.90(s,3H),2.86–2.80(m,2H),2.54(d,J＝7.2Hz,2H),2.43(td,J＝4.8,4.2,2.0Hz ,4H),2.19(s,3H),2.00–1.87(m,6H),1.68(td,J＝6.8,3.5Hz,6H).

Step 2: Add the product from the previous step (200 mg, 0.46 mmol) into a sealed tube, add methylamine solution (3 ml), and heat at 120°C overnight. After the reaction is completed, spin the solvent dry, extract with ethyl acetate, and use saline for the organic layer. Wash, combine the organic phases, dry with anhydrous sodium sulfate, then filter and spin dry the solvent. No purification is required. Directly proceed to the next reaction. Add the crude product to the reaction bottle, add 10 ml of ultra-dry DMF, lower the temperature to 0°C, and quickly Add acryloyl chloride (119.90ul, 1.38mmol) and triethylamine (191.82ul, 1.38mmol), stir at room temperature, and detect the reaction progress with TLC. After 2 hours of reaction, extract with ethyl acetate, combine the organic phases, and use anhydrous sodium sulfate. After drying, filtering and spinning the solvent to dryness, the crude product was separated and purified by column chromatography to obtain target compound 14 as a light yellow solid. ¹ H NMR (400MHz, Methanol-d ₄ ) δ7.61 (s, 1H), 7.05 (s, 1H), 4.23 (q, J = 4.3, 3.7Hz, 1H), 4.18 (t, J = 6.0Hz, 2H),3.97(s,3H),3.36(s,3H),3.00–2.93(m,2H),2.78–2.73(m,2H),2.68–2.62(m,4H),2.54(q,J＝ 7.5Hz,2H),2.33(s,3H),2.24–2.17(m,2H),2.15–2.04(m,4H),1.84(d,J＝3.5Hz,4H),1.78(dd,J＝12.1 ,3.4Hz,2H),1.11(t,J＝7.5Hz,3H).HRMS(ESI)calcd for C ₂₆ H ₃₈ N ₆ O ₃ (M+H ⁺ ):483.3078; found483.3077.

Example 15 N-(6-methoxy-4-(1-propylpiperidin-4-yl)amino)-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)-N-methacrylamide

Replace 4-amino-1-methylpiperidine with an equal molar amount of 4-amino-1-propylpiperidine. The remaining required raw materials, reagents and preparation methods are the same as in Example 14 to obtain a white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.12 (s, 1H), 6.91 (s, 1H), 6.76 (dd, J = 16.8, 10.3Hz, 1H), 6.36 (dd, J = 16.9, 2.0Hz ,1H),5.59–5.47(m,2H),4.23(t,J＝6.7Hz,2H),4.18(dd,J＝7.4,3.8Hz,1H),4.00(s,3H),3.56(s, 3H),3.02(d,J＝11.8Hz,2H),2.70(t,J＝7.4Hz,2H),2.62–2.54(m,4H),2.40–2.35(m,2H),2.15(q,J ＝6.9,6.4Hz,6H),1.82(t,J＝3.6Hz,4H),1.71(dd,J＝12.0,3.5Hz,2H),1.58–1.52(m,2H),0.93(t,J＝ 7.4Hz,3H).HRMS(ESI)calcd for C ₂₈ H ₄₂ N ₆ O ₃ (M+H ⁺ ):511.3391; found 511.3391.

Example 16 N-(4-(1-isopropylpiperidin-4-yl)amino)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline -2-yl)-N-methacrylamide

Replace 4-amino-1-methylpiperidine with an equal molar amount of 4-amino-1-isopropylpiperidine. The remaining required raw materials, reagents and preparation methods are the same as in Example 14 to obtain a white solid. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.79 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 6.99 (s, 1H), 6.75 (dd, J = 16.8, 10.2 Hz, 1H),6.11(dd,J＝16.9,2.3Hz,1H),5.53(dd,J＝10.2,2.3Hz,1H),4.13(t,J＝6.5Hz,2H),4.05–3.99(m,1H ),3.89(s,3H),3.37(s,3H),2.85(d,J＝11.0Hz,2H),2.72(p,J＝6.6Hz,2H),2.55(d,J＝7.1Hz,1H ),2.44(d,J＝5.5Hz,4H),2.19–2.13(m,2H),1.96–1.89(m,4H),1.69(t,J＝3.6Hz,4H),1.62–1.55(m, 2H),0.98(d,J=6.5Hz,6H).HRMS(ESI)calcd for C ₂₈ H ₄₂ N ₆ O ₃ (M+H ⁺ ): 511.3391; found 511.3391.

Example 17 N-(4-(1-cyclohexylpiperidin-4-yl)amino)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)-N-methacrylamide

Replace 4-amino-1-methylpiperidine with an equal molar amount of 4-amino-1-cyclohexylpiperidine. The remaining required raw materials, reagents and preparation methods are the same as in Example 14 to obtain a white solid. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.85 (d, J = 7.6 Hz, 1H), 7.66 (s, 1H), 6.99 (s, 1H), 6.75 (dd, J = 16.9, 10.2 Hz, 1H),6.11(d,J＝16.8Hz,1H),5.53(d,J＝10.5Hz,1H),4.14(t,J＝6.4Hz,2H),4.03(s,1H),3.89(s, 3H),3.44(s,3H),3.36(s,2H),3.17(s,1H),2.91(d,J＝11.4Hz,2H),2.68–2.55(m,4H),2.25(d,J ＝44.4Hz,4H),1.94(t,J＝13.9Hz,4H),1.82–1.67(m,8H),1.58(d,J＝13.6Hz,2H),1.27–1.18(m,4H).HRMS (ESI)calcd for C ₃₁ H ₄₆ N ₆ O ₃ (M+H ⁺ ):551.3704; found 551.3703.

Example 18 N-(4-(1-benzylpiperidin-4-yl)amino)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline- 2-yl)-N-methacrylamide

Replace 4-amino-1-methylpiperidine with an equal molar amount of 4-amino-1-benzylpiperidine. The remaining required raw materials, reagents and preparation methods are the same as in Example 14 to obtain a light yellow solid. ¹ H NMR (400MHz, Chloroform-d) δ7.35 (d, J＝4.3Hz, 5H), 7.13 (s, 1H), 6.85 (s, 1H), 6.76 (dd, J＝16.8, 10.3Hz, 1H ),6.36(dd,J＝16.9,2.0Hz,1H),5.54(dd,J＝10.3,2.0Hz,1H),5.37(d,J＝7.6Hz,1H),4.23(t,J＝6.7Hz ,2H),4.17(d,J＝4.2Hz,1H),4.00(s,3H),3.57(s,2H),3.56(s,3H),2.95(d,J＝11.6Hz,2H),2.71 (t,J＝7.4Hz,2H),2.61(p,J＝3.8Hz,4H),2.16(ddd,J＝19.8,16.1,11.1Hz,6H),1.88–1.78(m,4H),1.67( dd, J＝11.9, 3.7Hz, 2H).HRMS(ESI)calcd for C ₂₆ H ₃₈ N ₆ O ₃ (M+H ⁺ ): 559.3391; found 559.3391.

Example 19 N-(6-methoxy-4-(1-methylpiperidin-4-yl)methylamino)-7-(3-(pyrrolidin-1-yl)propoxy)quinazole Phin-2-yl)-N-methacrylamide

Replace 4-amino-1-methylpiperidine with an equal molar amount of (1-methyl-4-piperidine-)methylamine. The remaining raw materials, reagents and preparation methods are the same as in Example 14 to obtain a light yellow solid. . ¹ H NMR (400MHz, Chloroform-d) δ7.11 (s, 1H), 7.00 (s, 1H), 6.77 (dd, J = 16.9, 10.3Hz, 1H), 6.37 (d, J = 2.0Hz, 1H ),6.04(t,J＝6.0Hz,1H),5.55(dd,J＝10.3,2.0Hz,1H),4.21(t,J＝6.7Hz,2H),3.97(s,3H),3.53(d ,J＝9.9Hz,5H),2.87(d,J＝11.1Hz,2H),2.64(d,J＝7.4Hz,2H),2.56–2.51(m,4H),2.27(s,3H),2.13 (t,J＝7.1Hz,2H),1.95–1.89(m,2H),1.79(t,J＝3.7Hz,4H),1.76–1.75(m,1H),1.39(dt,J＝18.3,11.1 Hz,4H).HRMS(ESI)calcd for C ₂₇ H ₄₀ N ₆ O ₃ (M+H ⁺ ):497.3235; found 497.3235.

Example 20 N-(6,7-dimethoxy-4-(1-methylpiperidin-4-yl)amino)quinazolin-2-ylmethyl)acrylamide

Add compound 20a (100 mg, 0.30 mmol) into the reaction bottle, add 10 ml of ultra-dry dichlorohexane, and add HATU (88.31 mg, 0.23 mmol), acrylic acid (24.68 ul, 0.36 mmol), and DIPEA (157.88 ul) at 0°C. , 0.91mmol), then stir at room temperature, and detect the reaction progress with TLC. After 2 hours of reaction, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, then filter and spin the solvent dry, and the crude product is separated and purified by column chromatography to obtain Target compound 20. ¹ H NMR (400MHz, DMSO-d6) δ8.38 (s, 1H), 7.57 (d, J = 29.8Hz, 2H), 7.04 (s, 1H), 6.40 (t, J = 9.0Hz, 1H), 6.12(d,J＝17.0Hz,1H),5.62(d,J＝10.2Hz,1H),4.42–4.25(m,2H),4.12(s,1H),3.97–3.81(m,6H),2.82 (d,J＝11.2Hz,3H),2.19(s,3H),2.04–1.88(m,4H),1.61(d,J＝12.6Hz,2H).HRMS(ESI)calcd for C ₂₀ H ₂₇ N ₅ O ₃ (M+H ⁺ ):386.2187; found 386.2187.

Example 21 2-Chloro-N-(6,7-dimethoxy-4-(1-methylpiperidin-4-yl)amino)quinazolin-2-yl)methyl)acetamide

Replace acrylic acid with an equal molar amount of chloroacetic acid, and other required raw materials, reagents and preparation methods are the same as in Example 20. ¹ H NMR (400MHz, Chloroform-d) δ8.22 (s, 1H), 7.16 (s, 1H) ,6.89(s,1H),5.42(d,J＝7.6Hz,1H),4.58(d,J＝4.3Hz,2H),4.29(dtd,J＝11.3,7.2,3.8Hz,1H),4.19( s,2H),4.02(d,J＝1.3Hz,6H),2.92(d,J＝11.4Hz,2H),2.34(s,3H),2.18(d,J＝11.1Hz,4H),1.74– 1.63(m,2H).HRMS(ESI)calcd for C ₁₉ H ₂₆ N ₅ O ₃ Cl(M+H ⁺ ):408.1797; found 408.1797.

Example 22 N-(6,7-dimethoxy-4-(1-methylpiperidin-4-yl)amino)quinazolin-2-ylmethyl)methacrylamide

Acrylic acid was replaced with an equal molar amount of methacrylic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 20 to obtain a white solid. ¹ H NMR(400MHz,Chloroform-d)δ7.12(s,1H),6.97(s,1H),5.91(s,1H),5.41(s,1H),4.58(s,2H),4.23(t ,J＝10.7Hz,1H),3.99(d,J＝5.5Hz,6H),2.92(d,J＝11.2Hz,2H),2.33(s,3H),2.17(t,J＝12.3Hz,4H ),2.09–1.93(m,3H),1.70(q,J＝12.8Hz,2H).HRMS(ESI)calcd for C ₂₁ H ₂₉ N ₅ O ₃ (M+H ⁺ ):400.2343; found 400.2342.

Example 23 N-((6,7-dimethoxy-4-(1-methylpiperidin-4-yl)amino)quinazolin-2-yl)methyl)butan-2-ynamide

Acrylic acid was replaced with an equal molar amount of 2-butenoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 20 to obtain a white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.15 (s, 1H), 7.01 (d, J = 4.7Hz, 1H), 6.91 (s, 1H), 6.00 (dd, J = 15.1, 1.8Hz, 1H ),5.47(d,J＝7.3Hz,1H),4.59(d,J＝4.4Hz,2H),4.20(tt,J＝7.1,3.6Hz,1H),4.00(d,J＝5.0Hz,6H ),2.91(d,J＝12.2Hz,2H),2.34(s,3H),2.19(ddd,J＝17.8,12.9,5.6Hz,4H),1.89(dd,J＝6.8,1.7Hz,3H) ,1.71–1.59(m,2H).HRMS(ESI)calcd for C ₂₁ H ₂₇ N ₅ O ₃ (M+H ⁺ ):398.2187; found 398.2187.

Example 24 6,7-dimethoxy-4-(1-methylpiperidin-4-ylamino)quinazolin-2-ylmethyl)-4-dimethylaminobut-2-enamide

Acrylic acid was replaced with an equal molar amount of 4-dimethylaminobutenoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 20 to obtain a white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.30 (s, 1H), 7.11 (d, J = 1.5Hz, 1H), 4.51 (d, J = 1.5Hz, 2H), 4.28 (dd, J = 10.6 ,5.7Hz,1H),4.01(d,J＝3.9Hz,6H),2.97(d,J＝11.6Hz,2H),2.37(d,J＝3.1Hz,3H),2.26–2.19(m,2H ),2.13–2.03(m,3H),1.66(dd,J＝12.2,3.8Hz,2H).HRMS(ESI)calcd for C ₂₃ H ₃₄ N ₆ O ₃ (M+H ⁺ ):443.2765; found443. 2765.

Example 25 6,7-dimethoxy-4-(1-methylpiperidin-4-ylamino)quinazoline-2-methylbut-2-enamide

Acrylic acid was replaced with an equal molar amount of 2-butenoic acid, and the remaining required raw materials, reagents and preparation methods were the same as in Example 20 to obtain a white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.16 (s, 1H), 6.92 (s, 1H), 6.16 (dt, J = 15.4, 1.6Hz, 1H), 5.49 (d, J = 7.4Hz, 1H ),4.60(d,J＝4.4Hz,2H),4.27–4.15(m,1H),4.02(d,J＝3.4Hz,6H),3.11(dd,J＝6.1,1.6Hz,2H),2.92 (d,J＝12.2Hz,2H),2.35(s,3H),2.29(s,6H),2.18(s,2H),1.67(tt,J＝12.0,6.2Hz,2H),1.27(t, J＝5.9Hz,2H).HRMS(ESI)calcd for C ₂₁ H ₂₉ N ₅ O ₃ (M+H ⁺ ): 400.2343; found 400.2342.

Covalent verification experiment and biological activity evaluation part

The final target products prepared in Examples 1 to 25 are respectively designated as compounds 1 to 15.

In order to facilitate more intuitive covalent verification, we designed and synthesized the non-covalent comparison compound 26 of compound 14. The two have similar physical and chemical properties, but compound 26 does not have the ability to covalently bind.

Compound 26 was prepared by the following process: replacing acryloyl chloride with an equal molar amount of propionyl chloride, and the remaining required raw materials, reagents and preparation methods were the same as in Example 14 to obtain a white solid. ¹ H NMR (400MHz, Methanol-d ₄ ) δ7.61 (s, 1H), 7.05 (s, 1H), 4.23 (q, J = 4.3, 3.7Hz, 1H), 4.18 (t, J = 6.0Hz, 2H),3.97(s,3H),3.36(s,3H),3.00–2.93(m,2H),2.78–2.73(m,2H),2.68–2.62(m,4H),2.54(q,J＝ 7.5Hz,2H),2.33(s,3H),2.24–2.17(m,2H),2.15–2.04(m,4H),1.84(d,J＝3.5Hz,4H),1.78(dd,J＝12.1 ,3.4Hz,2H),1.11(t,J＝7.5Hz,3H).HRMS(ESI)calcd for C ₂₆ H ₄₀ N ₆ O ₃ (M+H ⁺ ):485.3235; found 485.3280.

1. Covalent verification: mass spectrometry

The experimental method is as follows: G9a protein is incubated with compound 14, and then electrospray time-of-flight mass spectrometry is used for verification. The known amino acid sequence and mass spectrum of G9a protein, the molecular weight of G9a is 59.62KD, after incubation of compound and protein G9a, The mass spectrometry data shows that compared with the G9a protein blank mass spectrometry data, the small molecule processed spectrum has a new peak near 59.62KD. The molecular weight is equal to the molecular weight of the G9a protein and Compound 14 complex. By comparing the G9a-Compound 14 complex peak corresponding The difference data between the molecular weight and the molecular weight corresponding to the peak of G9a protein (60397-59915=482, the molecular weight of compound 14 is 482.63). The mass spectrum directly proves that compound 14 and G9a are covalently bound, as shown in Figure 1.

2. Covalent verification: molecular docking experiment

In order to further study the mode of action of the compound, a molecular docking simulation experiment was performed to simulate docking compound 14 into the G9a complex (PDBcode: 3K5K) and the GLP complex (PDBcode: 5TUZ). The binding mode diagrams are shown in Figure 2(A). and Figure 2(B). It can be seen from the binding mode diagram that the acryl group on the 2-position side chain of quinazoline is very close to the cysteine residue Cys1098 in the G9a complex and Cys1186 in the GLP complex, and can co-exist with cysteine. valence combines to form a C-S bond. These results indicate that compound 14 can enter this binding pocket and covalently bind to the cysteine residue, further validating covalency.

The C-2 position of the compounds represented by formula (I) all contain electrophilic active groups, that is, covalent warheads, so they can all undergo Michael addition reactions with cysteine residues near the binding pocket of the target protein. Generate covalent bonds to achieve the purpose of covalent binding.

3. Covalent verification: histone methylation elution experiment

The experimental method is as follows: After the MDA-MB-231 cell density reaches about 90% and the cells are in good condition, the cells are digested and counted, then seeded into a six-well plate at a density of 120,000/well, and placed in an incubator for overnight culture. On the second day after seeding the plate, compound 14 and non-covalent control compound 26 were added into the wells at a final concentration of 10 μM, and a well containing the same DMSO as the 10 μM well was set as a control, and the culture was continued for 48 h. Take out the six-well plate, remove the culture medium, and wash it twice with PBS. Collect the cells treated for 48 hours and freeze them at -80°C. Add complete culture medium to the remaining wells to continue culturing, and continue culturing at 24 hours, 48 hours, and 72 hours. The correspondingly treated cells were collected at 24h, 48h, and 72h after elution. Add all collected cells to 60-200μL RIPA cell lysis buffer (containing phosphatase inhibitor A, phosphatase inhibitor B and PMSF at a volume ratio of 100:1) according to the number of cells. After the cells are fully lysed on ice, Centrifuge at 15000rpm and 4℃ for 15min. Take the supernatant and perform protein quantification using the BCA method. Adjust the protein concentration according to the quantitative results. Add 5x loading buffer and boil in a metal bath at 100°C for 10 minutes for denaturation. Prepare the corresponding SDS-PAGE gel according to the protein molecular weight, load the prepared protein samples in sequence, and perform electrophoresis separation. The electrophoresis parameters are set as follows: run the stacking gel at a constant voltage of 70V for 40 minutes; run the separation gel at 120V for 60-80 minutes until the desired bands are fully separated. Then perform wet transfer with a constant current of 235mA for 90-150 minutes. After the transfer was completed, the PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. After washing the membrane with TBST, incubate it with the corresponding primary antibody overnight. The next day, wash the membrane 5 times with TBST, 4 minutes each time, incubate with the secondary antibody at room temperature for 1 hour, and then wash the membrane 5 times with TBST, 4 minutes each time. Finally, the film was treated with chemiluminescence liquid to protect it from light, and then exposed and imaged on a Bio-Rad chemiluminescence imager.

Analysis of experimental results: As shown in Figure 3, after 48 hours of compound 26 and 14 acting, H3K9me2 was significantly inhibited compared to the DMSO group. However, 24 hours after the compounds were washed away, the covalent effect of compound 14 inhibited H3K9me2. has continued, while that of compound 26 has recovered. In addition, the inhibitory effect of compounds 14 and 26 on H3K9me2 does not depend on the inhibition of G9a protein expression, but changes its enzymatic activity.

4. Molecular level histone methyltransferase G9a activity inhibition experiment

The experimental method is as follows: Prepare 1x detection buffer (modified Tris buffer). Compound serial dilutions: Transfer compounds to assay plates via Echo in 100% DMSO. Prepare enzyme solution: Prepare enzyme solution in 1x Assay Buffer. Prepare substrate mix solution: Prepare substrate mix solution in 1x Assay Buffer, transfer 5 µL of enzyme solution to assay plate or for low control transfer 5 µL of 1x Assay Buffer, incubate at room temperature for 15 minutes, add to each well Start the reaction with 5 μL of substrate mixed solution, and incubate G9a for 60 minutes at room temperature. Then prepare 1x Alphalisa buffer and prepare acceptor and donor bead mixed solution in 1x Alphalisa buffer. Add 15 μL of acceptor and donor bead mixed solution, incubate at room temperature for 60 minutes under low light conditions, and read the endpoint using Envision or EnSpire using Alpha mode. Then perform data processing, use equation (1) to fit the data in Excel to obtain the inhibition value, equation (1): Inh%=(Max-Signal)/(Max-Min)*100, use equation (2) ) Fit the data in XL-Fit to obtain the IC50 value, Equation (2): Y=Bottom+(Top-Bottom)/(1+(IC _50/ X)*HillSlope), Y is the inhibition percentage, and X is the compound concentration , the in vitro enzyme activity results of the inhibitor G9a of the compound of the present invention are shown in Table 1 below.

Table 1 Compounds have inhibitory activity against G9a enzyme

5. Experiment on compound inhibiting the proliferation of human pancreatic cancer and breast cancer cells

The compound's inhibitory effect on cell activity was tested using cell viability assay. The experimental method is as follows: until the cell density reaches about 90% and the cells are in good condition, digest the cells, count them, and inoculate them into a 96-well plate at a concentration of 1500 cells/100 μL per well. , put in the incubator overnight. Observe whether the cell status is good the next day. If it is good, prepare the drug first, dilute the compound in equal proportions with complete culture medium, and then add three duplicate wells of each concentration into the wells, 50 μL per well, and return the plate to the incubator. Continue culturing for 96h. After 96 hours, observe the cells under a microscope, add 10 μL CCK-8 to each well in the dark, and place it in an incubator for 1-2 hours to incubate. Remove the air bubbles in the wells and measure the OD value at 450nm with a microplate reader, so that the OD value of the control group is between 0.7-1.5. Calculate cell viability according to the following formula: Cell viability (%) = (OD value of the experimental group - OD value of the blank control group) / (OD value of the control group - OD value of the blank control group) * 100%. Finally, nonlinear regression was performed using GraphPad Prism 8 software to obtain the corresponding half inhibitory concentration (IC ₅₀ ). Among them, the inhibitory activity of the non-covalent control compound on Panc-1 cells is 14.78±0.07μm, the inhibitory activity on Mda-mb-231 cells is 9.734±0.04μm, and the inhibitory activity of the covalent compound 14 on Panc-1 cells is 2.68±0.15μm, and the inhibitory activity against Mda-mb-231 cells is 2.88±0.64μm. Compared with compound 26, compound 14 has a more significant medicinal effect, which can also prove that compound 14 and G9a achieve covalent binding. .

6. Clone formation experiment of compounds

The experimental method is as follows: After digesting and counting MDA-MB-231 and PANC-1 cells, they were seeded into a six-well plate at a density of 1200 cells/well and 800 cells/well respectively, and placed in an incubator to culture overnight. On the second day after seeding the plate, compound 14 and its non-covalent control compound 26 were added into the wells at final concentrations of 1.25, 2.5, and 5 μM, and a well containing the same DMSO as the 5 μM well was set as a control. Each treatment setting Make 3 multiple wells, put the plate back into the incubator and continue culturing for 15 days. Change the complete medium every three days, and add corresponding concentrations of compound 14 and non-covalent control compound 26. After 15 days, the size of the cell clones in the control group has grown to the size visible to the naked eye. The culture medium is removed, washed three times with PBS, and 800 μL of 4% paraformaldehyde is added to each well and fixed for 15 minutes. Remove the fixative, wash 3 times with PBS, add 800 μL of crystal violet dye to each well, and stain in the dark for 30 minutes. Recover the crystal violet dye solution, wash away the excess dye solution with ultrapure water, and place the 6-well plate in a fume hood to dry. Use a printer to scan the 6-well plate, and use Image J to count the number of cell clones.

Analysis of experimental results: The results are shown in Figure 4. Compounds 14 and 26 reduce the cloning ability of PANC-1 cells and MDA-MB-231 cells in a concentration-dependent manner. Compared with the non-covalent control compound 26, the covalent compound 14 can more significantly inhibit the colony formation of cells. The colony formation rate reflects two important characteristics: cell population dependence and proliferation ability.

7. Evaluation of H3K9 methylation inhibitory activity of compounds

The experimental method is as follows: After the MDA-MB-231 cell density reaches about 90% and the cells are in good condition, the cells are digested and counted, then seeded into a six-well plate at a density of 120,000/well, and placed in an incubator for overnight culture. On the 2nd, 3rd, 4th, and 5th days of the seeding plate, compound 14 was added into the wells at a final concentration of 10 μM, and the drug was allowed to act for 96h, 72h, 48h, and 24h respectively, and the same well containing DMSO as the 10 μM well was set as a control. . On the 6th day after plating, take out the six-well plate, remove the culture medium, and wash gently twice with PBS. Extract histones according to the literature, and use the Bradford kit for protein quantification. Adjust the protein concentration according to the quantitative results, add 5x loading buffer, and cook in a metal bath at 100°C for 10 minutes for denaturation. Prepare a 15% SDS-PAGE gel, load the prepared protein samples one by one, and perform electrophoresis separation. The electrophoresis parameters are set as follows: run the stacking gel at a constant voltage of 70V for 40 minutes; run the separation gel at 120V for 60-80 minutes until the desired bands are fully separated. Afterwards, wet transfer was performed with a constant current of 235mA for 90 minutes. After the transfer was completed, the PVDF membrane was blocked with 5% skim milk at room temperature for 1 hour. After washing the membrane with TBST, incubate it with the corresponding primary antibody overnight. The next day, wash the membrane 5 times with TBST, 4 minutes each time, incubate with the secondary antibody at room temperature for 1 hour, and then wash the membrane 5 times with TBST, 4 minutes each time. Finally, the film was treated with chemiluminescence liquid to protect it from light, and then exposed and imaged on a Bio-Rad chemiluminescence imager.

Analysis of experimental results: As shown in Figure 5, in MDA-MB-231 cells, compound 14 has a stronger effect on inhibiting G9a methylation than compound 26. At an action concentration of 10 μM, it can be inhibited for 24 hours. Significantly inhibits H3K9me2 in a time- and concentration-dependent manner.

8. In vivo anti-tumor activity experiments of compounds

The experimental method is as follows: First, digest, centrifuge and count the amplified PANC-1 cells in good condition and in the logarithmic growth phase, resuspend and wash twice with pre-cooled PBS, and finally wash with PBS: Matrigel = 1:1 The mixture was resuspended to obtain a cell suspension of 3×10 ⁶ cells/100 μL, which was placed on ice for later use. Then, 100 μL of cell suspension was subcutaneously injected into the ventral and dorsal sides of each Balb/c nu/nu until the tumor volume reached ^50mm3 or so, were randomly divided into 2 groups (n=5). The control group (recorded as Vehicle) was given drug cosolvent, and the experimental group (recorded as 14-2mg/kg) was given 2mg/kg of compound 14. The administration method was Intraperitoneal injection, 5 times a week, for three consecutive weeks. The weight and tumor volume of the mice were recorded every two days. The calculation formula for tumor volume was: V (mm ³ ) = π/6 × (length × width ² ). After the administration, the mice were euthanized and the subcutaneous tumors were peeled off. Weigh and take photos, and take photos of major organs. Some tumors and organs are stored in tissue fixative, and the other part is frozen in liquid nitrogen for subsequent experiments.

The experimental results are shown in Figure 6: In animal experiments on the pancreatic cancer cell PANC-1 subcutaneous transplanted tumor model, the covalent inhibitor 14 can significantly inhibit the growth of PANC-1 subcutaneous transplanted tumors without obvious toxicity to the major organs of mice.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. For those of ordinary skill in the art, based on the above descriptions and ideas, they can also make There are other variations or modifications in different forms, and it is not necessary and impossible to exhaustively enumerate all implementations here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

9. Enzyme selectivity test

The established AlphaLISA and HTRF methods were used to further investigate the inhibitory effect of compound 14 on multiple other histone-modifying enzymes at the molecular level to determine the selectivity of the compound against histone-modifying enzymes. AlphaLISA technology was used to detect the molecular-level inhibitory activities of compounds against histone-modifying enzymes PRMT1, PRMT4, and PRMT5. Selectivity to EZH2, MLL1, MLL4, DNMT1 was assessed using the HTRF assay.

The enzyme selectivity test results (Table 2) show that compound 14 has good selectivity for protein G9a/GLP and is a type of inhibitor that specifically targets G9a/GLP.

Table 2 Compound 14 has inhibitory activity against G9a enzyme.

Claims (10)

1. A G9a/GLP covalent inhibitor, characterized in that it is a compound with a structure shown in formula (I) and its salt:

   

   Among them, n ₁ , n ₂ , n ₃ and n ₄ are independently selected from integers from 0 to 2;

   n ₅ is an integer from 0 to 4;

   X is CH or N;

   R ₁ is selected from hydrogen, C ₁ -C ₆ alkyl and its deuterated products, C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ heterocycloalkyl; the alkyl and its deuterated products and cycloalkyl are any Optionally selected by halogen, cyano, hydroxyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkylthio, C ₁ -C ₆ alkyl, amino, C ₁ -C ₆ alkylamino, bis C ₁ -C ₆ alkylamino, 4-12 membered heterocyclic group substituted by one or more groups;

   R ₂ is

   

   R ₃ is selected from hydrogen, trifluoromethyl, substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₃ -C ₈ cycloalkyl, substituted or unsubstituted C ₃ -C ₈ Heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C ₅ -C ₆ heteroaryl; the substitution means that at least 1 position is substituted by the following substituents: halogen, cyano, amino, Nitro, hydroxyl, trifluoromethyl, methylthio, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkyl, C ₁ -C ₆ alkylamino, C ₃ -C ₈ heterocyclyl; C ₃ -C ₈ cycloalkoxy group, C ₃ -C ₈ cycloalkylamino group, aryl group, C ₅ -C ₆ heteroaryl group;

   R ₄ is selected from hydrogen, halogen, cyano, hydroxyl, methoxy or trifluoromethoxy;

   R ₅ is selected from substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₃ -C ₈ cycloalkyl, substituted or unsubstituted C ₃ -C ₈ heterocycloalkyl, substituted or Unsubstituted aryl, substituted or unsubstituted C ₅ -C ₆ heteroaryl, substituted or unsubstituted C ₁ -C ₆ alkoxy, substituted or unsubstituted C ₁ -C ₆ alkylamino, substituted or unsubstituted Substituted C ₃ -C ₈ cycloalkyloxy group, substituted or unsubstituted C ₃ -C ₈ cycloalkylamino group, substituted or unsubstituted C ₃ -C ₈ cycloalkylamino group, the substitution means at least 1 The position is substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, methylthio, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₈ cycloalkyl, C ₁ -C ₆ alkylamino, C ₃ -C ₈ heterocyclyl; C ₃ -C ₈ cycloalkoxy, C ₃ -C ₈ cycloalkylamino, aryl, C ₅ -C ₆ heteroaryl; R _a is selected from hydrogen, halogen or R _d ;

   R _b and R _c are independently selected from hydrogen or R _d ;

   R _d is selected from substituted or unsubstituted C ₁ -C ₆ alkyl, substituted or unsubstituted C ₂ -C ₆ alkenyl, substituted or unsubstituted C ₂ -C ₆ alkynyl, substituted or unsubstituted C ₃ -C ₆ cycloalkyl, substituted or unsubstituted 4-12 membered heterocyclyl;

   Or R _a and R _b together with the carbon atoms to which they are connected form a 3-5-membered heterocyclyl group or a substituted 3-5-membered heterocyclyl group containing 0 or 1 additional heteroatom;

   Y is selected from halogen.
2. The G9a/GLP covalent inhibitor according to claim 1, wherein R ₂ is:

   

   in:

   The substituents in R _d are one or more -JT groups; the substituents in the substituted 3-5-membered heterocyclic groups in R _a and R _b are one or more -J ₁ -T ₁ groups;

   J is selected from a bond or substituted C ₁ -C ₆ alkylene;

   T is selected from hydrogen, halogen, cyano, hydroxyl, -NR _f R _g , -C(O)R _f , -OR _f , -C(O)OR _f , -C(O)NR _f R _g , -NR _f C(O)R _g , -NR _h C(O)NR _f R _g , -NR _f C(O)OR _h or R _i ;

   R _f , R _g , and Rh _h are each independently selected from hydrogen or R _j , and R _j is selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C _6- cycloalkyl, 4-12-membered heterocyclyl, aryl, 5- or 6-membered heteroaryl, R _j is substituted by one or more -J ₁ -T ₁ groups;

   Or R _f and R _g together with the N atoms to which they are connected form a 4-12-membered heterocyclyl group containing 0 or 1 additional heteroatom, and the 4-12-membered heterocyclyl group is replaced by one or more -J ₁ -T ₁ group substitution;

   R _i is selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₆ cycloalkyl, 4-12 membered heterocyclyl, aryl, 5- 10-membered heteroaryl or, R _i is substituted by one or more -J ₁ -T ₁ groups;

   J ₁ is selected from a bond or substituted C ₁ -C ₆ alkylene;

   T ₁ is selected from hydrogen, halogen, cyano, hydroxyl, -NR _k R _l , -C(O)R _k , -OR _k , -C(O)OR _k , -C(O)NR _k R _l , - NR _k C(O)R _l , -NR _o C(O)NR _k R _l , -NR _k C(O)OR _o or R _p ;

   R _k , R _l , and R _o are each independently selected from hydrogen or R _q , and R _q is selected from the following substituted groups: C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkyne Base, C ₃ -C ₆ cycloalkyl, 4-12 membered heterocyclyl, aryl, 5- or 6-membered heteroaryl;

   Or R _k and R _l together with the N atom to which they are connected form a 4-12 membered heterocyclyl group containing 0 or 1 additional heteroatom, and the heterocyclyl group is optionally selected from halogen, hydroxyl, oxo, C ₁ -C ₆ alkyl, OR _x , -NR _x R _y , -C(O)R _x , -O(CH ₂ ) _n OR _x is substituted by one or more groups;

   R _p is selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₆ cycloalkyl, 4-12 membered heterocyclyl, aryl, 5 to 6-membered heteroaryl;

   R _x and R _y are each independently selected from hydrogen or R _z , and R _z is selected from the following groups or substituted groups: C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ Alkynyl, C ₃ -C ₆ cycloalkyl, 4-12-membered heterocyclyl, aryl, 5- or 6-membered heteroaryl; _Rz is replaced by halogen, hydroxyl, aryl or 5- or 6-membered arylheteroyl or one or more substituted aryl groups or 5- or 6-membered heteroaryl groups,

   Or R _x , R _y and the N atom to which they are connected together form a 4-12 membered heterocyclyl group containing 0 or 1 additional heteroatom;

   or -J ₁ -T ₁ is an oxo group;

   or -J-T is oxo;

   R ₁ is selected from hydrogen or C ₁ -C ₆ alkyl.
3. The G9a/GLP covalent inhibitor according to claim 1, characterized in that the substituents in the C ₁ -C ₆ alkylene substituted in J or J ₁ are independently selected from halogen, cyano, hydroxyl or C ₁ -One or more groups in C ₆ alkoxy.
4. The G9a/GLP covalent inhibitor according to claim 1, characterized in that R ₃ is selected from hydrogen, aryl, C ₁ -C ₆ alkyl, C ₃ -C ₈ heterocycloalkyl or C ₃ - C ₈ heterocycloalkyl substituted C ₁ -C ₆ alkyl.
5. The G9a/GLP covalent inhibitor according to claim 1, characterized in that R ₅ is selected from C ₁ -C ₆ alkyl or C ₃ -C ₈ heterocycloalkyl.
6. The G9a/GLP covalent inhibitor according to claim 1, characterized in that the G9a/GLP covalent inhibitor is the structure shown in the following numbering and its salt:

   

   
7. The preparation method of G9a/GLP covalent inhibitor according to any one of claims 1 to 6, characterized in that it includes the following steps:

   

   The compound represented by formula (1) and acid undergo a condensation reaction under the conditions of a condensing agent and an organic base to obtain a compound with a structure represented by formula (I).
8. Use of the G9a/GLP covalent inhibitor described in any one of claims 1 to 6 in the preparation of a drug that inhibits G9a/GLP.
9. The application according to claim 8, characterized in that the drug is a drug for preventing and/or treating diseases involving abnormal cell proliferation, morphological changes and hypermotility related to G9a/GLP.
10. The application according to claim 8, characterized in that the drug is a drug for treating and/or preventing tumor growth and metastasis.

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