A small molecule inhibitor of NSD2, its preparation method and application

Abstract

This invention relates to a small molecule inhibitor of NSD2, its preparation method, and its applications. The NSD2 small molecule inhibitor is a compound with the structure shown in Formula (I) or a salt thereof: Formula (I). This NSD2 small molecule inhibitor exhibits high selectivity and potent efficacy against histone methyltransferase NDS2, and can be used to prepare drugs that inhibit NSD2, and drugs for the prevention and / or treatment of tumors or cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to an NSD2 small molecule inhibitor, its preparation method, and its application. Background Technology

[0002] NSD2, also known as the multiple myeloma SET domain (MMSET) or Wolf-Hirshhorn syndrome candidate 1 (WHSC1), is the shortest protein in the NSD family and is involved in various cellular processes, including DNA damage responses, epithelial-mesenchymal transition (EMT), cell cycle progression, and development. Current research has demonstrated that aberrant expression, amplification, or somatic mutations of NSD2 are associated with multiple types of cancer, including prostate cancer, gastric cancer, bladder cancer, breast cancer, lung cancer, colorectal cancer, kidney cancer, head and neck cancer, cervical cancer, osteosarcoma, neuroblastoma, and glioma. Nat. Rev. Drug Discov. 2021, 20, 265−286). Furthermore, the t(4;14)(p16;q32) chromosomal translocation leading to NSD2 overexpression has been observed in approximately 15–20% of multiple myeloma (MM) patients and is strongly associated with poor prognosis. Additionally, the overactivation mutation E1099K in the catalytic SET domain aberrantly enhances NSD2 methyltransferase activity and is associated with a subset of childhood acute lymphoblastic leukemia (ALL). Therefore, NSD2 has attracted increasing attention as a promising anticancer target over the past decade.

[0003] Currently, all developed NSD2 inhibitors exhibit weak or moderate potency and poor selectivity against NSD2-SET, highlighting the challenge of discovering highly effective and selective inhibitors of NSD2-SET and hindering the exploration of NSD2's biological functions and potential therapeutic applications. Therefore, there is a strong need to develop small-molecule inhibitors of NSD2 with high specificity and efficacy in cancer cells. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings of existing NSD2 inhibitors and provide a small molecule NSD2 inhibitor.

[0005] A further objective of this invention is to provide a method for preparing the above-mentioned NSD2 small molecule inhibitor.

[0006] A further object of the present invention is to provide the application of the above-mentioned NSD2 small molecule inhibitor in the preparation of drugs that inhibit NSD2.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] A small molecule inhibitor of NSD2 is a compound or a salt thereof with the structure shown in formula (I):

[0009]

[0010] Equation (I);

[0011] Where n1, n2, and n3 are independently selected from integers from 0 to 2;

[0012] X1 is CH or N;

[0013] R1 is selected from hydrogen, trifluoromethyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C5-C6 heteroaryl; the substitution refers to at least one site being substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, methylthio, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C1-C6 alkylamino, C3-C8 heterocyclic; C3-C8 cycloalkoxy, C3-C8 cycloalkylamine, aryl, C5-C6 heteroaryl, substituted or unsubstituted aryl or heteroaryl and their benzo[a] derivatives, 3- to 8-membered heterocyclic groups containing 1-2 heteroatoms selected from N, O or S, -COR a -CO2R a -CONR a R b -NR a C(O)R b -NR a SO2R b -SR a -SOR a -SO2R a -SO2NR a R b -OC(O)R a or -OC(O)NR a R b ;

[0014] R a R b Independently hydrogen, C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 alkynyl group, C 3-6 cycloalkyl, aryl, or heteroaryl;

[0015] R2 is selected from hydrogen, halogen, cyano, hydroxyl, C1-C6 alkoxy or trifluoromethoxy;

[0016] The whole is H; or X2 is CH2, O or NH, and n4 is an integer selected from 0 to 4.

[0017] R3 is selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted C5-C6 heteroaryl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylamino, substituted or unsubstituted C3-C8 cycloalkyloxy, substituted or unsubstituted C3-C8 cycloalkylamino, substituted or unsubstituted C3-C8 cycloamino, wherein the substitution means that at least one site is substituted by the following substituents: halogen, cyano, amino, nitro, hydroxyl, trifluoromethyl, methylthio, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C1-C6 alkylamino, C3-C8 heterocycloyl; C3-C8 cycloalkoxy, C3-C8 cycloalkylamino, aryl, C5-C6 heteroaryl.

[0018] Preferably, R1 is selected from hydrogen, C1-C6 alkyl, or C3-C8 cycloalkyl.

[0019] Preferably, R2 is selected from hydrogen or C1-C6 alkoxy.

[0020] Preferably, R3 is selected from hydrogen, C1-C6 alkyl, substituted or unsubstituted C3-C8 heterocyclic alkyl, aryl or C1-C6 alkoxy; the substitution means that at least one site is substituted by the following substituents: halogen.

[0021] Preferably, n1 is 0 or 1.

[0022] Preferably, n2 is 1.

[0023] Preferably, n3 is 1.

[0024] Preferably, n4 is 0, 1, 2 or 3.

[0025] Preferably, the salt is a pharmaceutically acceptable salt, including but not limited to hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, hydrogen sulfate, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylic acid, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, and o-acetyloxy The product contains at least one of the following: benzoyl benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannic acid salt, formate, stearate, ascorbate, palmitate, oleate, pyruvate, dihydroxynaphthyl salt, malonate, laurate, glutamate, propionate, estolate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate), or naphthalene-2-sulfonate.

[0026] The method for preparing the above-mentioned NSD2 small molecule inhibitor is characterized by comprising the following steps:

[0027] Includes the following steps:

[0028] The compound shown in formula (A) undergoes a coupling reaction with cyanide in the presence of a catalyst to obtain the NSD2 small molecule inhibitor.

[0029] Formula (A);

[0030] Alternatively, the compound shown in formula (B) and the compound shown in formula (C) may undergo a coupling reaction in the presence of a catalyst to obtain the NSD2 small molecule inhibitor.

[0031] Formula (B); Formula (C);

[0032] Or when When the whole is OH, the compound shown in formula (D) undergoes a reduction reaction under hydrogen atmosphere and palladium on carbon catalyst to obtain the NSD2 small molecule inhibitor.

[0033] Formula (D);

[0034] Alternatively, when X2 is O and R3 is not hydrogen, alkyl, cycloalkyl, or heterocycloalkyl, the compound shown in formula (E) reacts with the compound shown in formula (F) under DIAD and triphenylphosphine conditions to obtain the NSD2 small molecule inhibitor.

[0035] Equation (E); Formula (F).

[0036] Typically, the catalysts used for the coupling reaction of the compound shown in formula (A) with cyanide are tridibenzylacetone dipalladium and 1,1'-bis(diphenylphosphine)ferrocene; the reaction temperature of the coupling reaction is 140~160℃ and the reaction time is 2~4 hours.

[0037] Typically, the cyanide is zinc cyanide.

[0038] Typically, the catalysts used for the coupling reaction between the compound shown in formula (B) and the compound shown in formula (C) are cesium carbonate, tris(dibenzylacetone)palladium, and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine); the reaction temperature of the coupling reaction is 130~160℃, and the reaction time is 3~4 hours.

[0039] Typically, the reduction reaction is carried out at a temperature of 20-30°C; the Mitsunobu reaction is carried out at a temperature of 20-30°C.

[0040] Typically, the solvents used in each reaction are at least one of tetrahydrofuran, N,N-dimethylformamide, methanol, ethanol, and 1,4-dioxane.

[0041] The application of the above-mentioned NSD2 small molecule inhibitors in the preparation of drugs that inhibit NSD2 is also within the scope of protection of this invention.

[0042] Preferably, the drug is a drug for the prevention and / or treatment of tumors.

[0043] More preferably, the tumor is at least one of cancer or benign tumor. Cancer may include 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 adenoma, 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, oligodendroglioma, glioblastoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma, thyroid cancer, multiple myeloma (AML), mantle cell lymphoma, triple-negative breast cancer, or non-small cell lung cancer.

[0044] Preferably, the drug is a drug for the prevention and / or treatment of diseases related to NSD2, such as abnormal cell proliferation, morphological changes, or hypermotility.

[0045] A pharmaceutical composition comprising the above-mentioned NSD2 small molecule inhibitor.

[0046] Compared with the prior art, the beneficial effects of the present invention are:

[0047] This invention uses quinazoline and quinoline as the drug skeleton. Quinazoline and quinoline have good drug-like properties, and a cyano group is attached to the C-2 position. The cyano group allows the molecule to bind well to the cavity of NSD2, thereby making the NSD2 small molecule inhibitor highly selective and potent against histone methyltransferase NDS2. It can be used to prepare drugs that inhibit NSD2 and drugs for the prevention and / or treatment of tumors or cancer. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the process flow for preparing the NSD2 small molecule inhibitor of Example 1.

[0049] Figure 2 This is a schematic diagram of the process flow for preparing the NSD2 small molecule inhibitor of Example 18.

[0050] Figure 3 The figure shows the in vivo efficacy evaluation results of the NSD2 small molecule inhibitor in Example 13.

[0051] Figure 4 The image shows the results of a real-time quantitative PCR experiment on the NSD2 small molecule inhibitor in Example 13.

[0052] Figure 5 The figure shows the apoptosis assay results of the NSD2 small molecule inhibitor in Example 13.

[0053] Figure 6 This is a graph showing the cell cycle assay results of the NSD2 small molecule inhibitor in Example 13.

[0054] Figure 7 The figure shows the experimental results of the effect of the NSD2 small molecule inhibitor in Example 13 on the RS411 signaling pathway. Detailed Implementation

[0055] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0056] Example 1

[0057] This embodiment provides a small molecule inhibitor of NSD2 (6,7-dimethoxy-4-(1-methylpiperidine-4-amino)quinazolin-2-carboxynitrile), the process flow diagram of which is shown below. Figure 1 As shown, it includes the following steps:

[0058] 1) 2,4-Dichloro-6,7-dimethoxyquinazoline (200 mg, 0.77 mmol) was slowly added to ice-cold DMF (5 ml), followed by the slow addition of 4-amino-1-methylpiperidine (compound 1a, 0.3 ml, 1.16 mmol) and K2CO3 (318 mg, 2.31 mmol). The mixture was stirred at room temperature for 8 hours. The reaction mixture was then quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to obtain a white solid, compound 1b. 1 HNMR (500MHz, DMSO-d6) δ8.28 (s, 1H), 7.76 (s, 1H), 7.08 (s, 1H), 4.34 (s, 1H), 3.91 (s, 3H ), 3.89(s, 3H), 3.46(s, 2H), 2.75(s, 3H), 2.11(s, 2H), 1.97(s, 2H), 1.27-1.23(m, 2H).

[0059] 2) Compound 1b (77.7 mg, 0.30 mmol) was added to a microwave reaction tube. DMF was added first, followed by Zn(CN)₂ (104.24 mg, 0.89 mmol), dppf (22.94 mg, 0.04 mmol), and Pd₂dba₃ (27.1 mg, 0.03 mmol). The reaction was carried out under nitrogen protection and microwaved at 160 °C for 2 hours. The crude product was poured into an ammonium hydroxide solution (3 mL), filtered, and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, and the organic phase was evaporated to dryness. The crude product was purified by column chromatography to obtain the NSD₂ small molecule inhibitor (denoted as compound 1, structural formula: [insert structural formula here]). ). 1 HNMR (400MHz, DMSO-) d 6)δ8.10(d, J =7.7Hz, 1H), 7.71(s, 1H), 7.21(s, 1H), 4.15-4.06(m, 1H), 3.93(d, J =9.4Hz, 6H), 2.96-2.79(m, 2H), 2.22(s, 3H), 2.10-1.99(m, 2H), 1.96-1.88(m, 2H), 1.75-1.62(m, 2H).HRMS(ESI)calcdforC 17 H 21 N5O2(M+H + ):328.1768;found328.1771.

[0060] Example 2

[0061] This embodiment provides a small molecule inhibitor of NSD2 (6,7-dimethoxy-4-((1-propylpiperidin-4-yl)amino)quinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0062] 1) Replace 4-amino-1-methylpiperidine with an equimolar amount of 1-propylpiperidine-4-amine. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 1, to obtain a white solid (2-chloro-6,7-dimethoxy-N-(1-propylpiperidine-4-yl)quinazoline-4-amine). 1 HNMR (400MHz, DMSO-) d 6)δ8.03(d, J =7.7Hz, 1H), 7.67(s, 1H), 7.06(s, 1H), 4.14-4.07(m, 1H), 3.90(s, 3H), 3.89(s, 3H), 3.00-2.95(m, 2H), 2.34-2.30(m, 2H), 2.10-2.02(m, 2H), 1.94-1.90(m, 2H), 1.73-1.66(m, 2H), 1.50-1.45(m, 2H), 0.87(t, J =7.1Hz, 3H).

[0063] 2) An equimolar amount of 2-chloro-6,7-dimethoxy-N-(1-propylpiperidin-4-yl)quinazoline-4-amine, along with the other required raw materials, reagents, and process parameters, are used in step 2) of Example 1 to obtain the NSD2 small molecule inhibitor (denoted as compound 2, with the structural formula as follows). ). 1 HNMR (400MHz, DMSO-) d 6)δ8.07(d, J =7.6Hz, 1H), 7.69 (s, 1H), 7.20 (s, 1H), 4.17-4.08 (m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 2.93 (d, J =11.3Hz, 2H), 2.27(t, J =7.3Hz, 2H), 2.06-1.98(m, 2H), 1.96-1.90(m, 2H), 1.71-1.61(m, 2H), 1.48-1.41(m, 2H), 0.87(t, J =7.3Hz, 3H).HRMS(ESI)calcdforC 19 H 25 N5O2(M+H +):356.2081;found356.2073.

[0064] Example 3

[0065] This embodiment provides a small molecule inhibitor of NSD2 (6,7-dimethoxy-4-((1-isopropylpiperidin-4-yl)amino)quinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0066] 1) Replace 4-amino-1-methylpiperidine with an equimolar amount of 1-isopropylpiperidine-4-amine. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 1, to obtain a white solid (2-chloro-N-(1-isopropylpiperidine-4-yl)-6,7-dimethoxyquinazoline-4-amine). 1 HNMR (500MHz, DMSO-) d 6)δ8.00(d, J =7.7Hz, 1H), 7.65 (s, 1H), 7.06 (s, 1H), 4.08-4.02 (m, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 2.85 (d, J =11.1Hz, 2H), 2.77-2.71(m, 1H), 2.24(t, J =11.5Hz, 2H), 1.95-1.91(m, 2H), 1.64-1.57(m, 2H), 1.00(s, 3H), 0.98(s, 3H).

[0067] 2) Replace compound 1b with an equimolar amount of 2-chloro-N-(1-isopropylpiperidin-4-yl)-6,7-dimethoxyquinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 1, yielding the NSD2 small molecule inhibitor (denoted as compound 3, structural formula: [structural formula missing]). ). 1 HNMR (400MHz, DMSO-) d 6)δ8.09(d, J =7.5Hz, 1H), 7.71 (s, 1H), 7.20 (s, 1H), 4.14-4.03 (m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 2.85 (d, J =11.1Hz, 2H), 2.78-2.69(m, 1H), 2.24(d, J =11.8Hz, 2H), 2.05-1.89(m, 2H), 1.71-1.55(m, 2H), 1.00(s, 3H), 0.98(s, 3H).HRMS(ESI)calcdforC 19 H25 N5O2(M+H + ):356.2081;found356.2081..

[0068] Example 4

[0069] This embodiment provides a small molecule inhibitor of NSD2 (4-(1-cyclopropylpiperidin-4-yl)amino)-6,7-dimethoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0070] 1) Replace 4-amino-1-methylpiperidine with an equimolar amount of 4-amino-1-cyclopropylpiperidine. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 1, to obtain a white solid (2-chloro-N-(1-cyclopropylpiperidine-4-yl)-6,7-dimethoxyquinazoline-4-amine). 1 HNMR (400MHz, Chloroform- d )δ7.12(s, 1H), 6.79(s, 1H), 5.38(d, J =7.8Hz, 1H), 4.38-4.25(m, 1H), 3.98(s, 3H), 3.96(s, 3H), 3.05(dt, J =12.3, 3.8Hz, 2H), 2.44(td, J =11.9, 2.6Hz, 2H), 2.14(dt, J =13.0, 4.2Hz, 2H), 1.66(ddd, J =6.4, 4.2, 2.8Hz, 1H), 1.62-1.51(m, 2H), 0.53-0.46(m, 2H), 0.45-0.40(m, 2H).

[0071] 2) Replace compound 1b with an equimolar amount of 2-chloro-N-(1-cyclopropylpiperidin-4-yl)-6,7-dimethoxyquinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 1, yielding the NSD2 small molecule inhibitor (denoted as compound 4, structural formula: [structural formula missing]). ). 1 HNMR (400MHz, Chloroform- d )δ7.35(s, 1H), 7.16(s, 1H), 4.41-4.30(m, 1H), 4.04(s, 3H), 4.00(s, 3H), 3.15(d, J =11.9Hz, 2H), 2.45(t, J =12.2Hz, 2H), 2.07 (d, J=12.8Hz, 2H), 1.77-1.69(m, 1H), 1.62(t, J =12.4Hz, 2H), 0.63-0.55(m, 2H), 0.53-0.42(m, 2H).HRMS(ESI)calcdforC 19 H 23 N5O2(M+H + ):354.1925;found354.1921.

[0072] Example 5

[0073] This embodiment provides a small molecule inhibitor of NSD2 (6,7-dimethoxy-4-((1-methylpiperidin-4-yl)methylamino)quinazolin-2-carboxynitrile), the preparation method of which includes the following steps:

[0074] 1) Replace 4-amino-1-methylpiperidine with an equimolar amount of 1-methylpiperidine-4-methylamine. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 1, to obtain a white solid (2-chloro-6,7-dimethoxy-N-(1-methylpiperidine-4-yl)methyl)quinazolin-4-amine). 1 HNMR (400MHz, Chloroform- d )δ7.12(s, 1H), 6.90(s, 1H), 5.85(s, 1H), 3.99(s, 3H), 3.96(s, 3H), 3.59(t, J =5.9Hz, 2H), 2.92(d, J =11.7Hz, 2H), 2.31(s, 3H), 2.01(t, J =10.7Hz, 2H), 1.80(p, J =5.8, 5.3Hz, 3H), 1.53-1.43(m, 2H).

[0075] 2) Replace compound 1b with an equimolar amount of 2-chloro-6,7-dimethoxy-N-(1-methylpiperidin-4-yl)methyl)quinazolin-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 1, yielding the NSD2 small molecule inhibitor (denoted as compound 5, structural formula: [structural formula missing]). ). 1 HNMR (500MHz, DMSO-) d 6)δ8.43(t, J =5.6Hz, 1H), 7.69(s, 1H), 7.20(s, 1H), 3.93(s, 3H), 3.92(s, 3H), 3.41(d, J=5.4Hz, 2H), 2.76 (d, J =10.9Hz, 2H), 2.14(s, 3H), 1.83(t, J =11.2Hz, 2H), 1.69(d, J =11.6Hz, 3H), 1.26(q, J =10.5Hz, 2H).HRMS(ESI)calcdforC 18 H 23 N5O2(M+H + ):342.1925;found342.1924.

[0076] Example 6

[0077] This embodiment provides a small molecule inhibitor of NSD2 (4-(1-isopropylpiperidin-4-yl)amino)-6-methoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0078] 1) Replace 2,4-dichloro-6,7-dimethoxyquinazoline with an equimolar amount of 2,4-dichloro-6-methoxyquinazoline. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 3, to obtain a white solid (2-chloro-N-(1-isopropylpiperidin-4-yl)-6-methoxyquinazoline-4-amine). 1 HNMR (400MHz, Chloroform- d )δ7.70(d, J =7.1Hz, 1H), 7.37 (d, J =9.1Hz, 1H), 6.88(s, 1H), 5.56(d, J =7.8Hz, 1H), 4.32-4.22(m, 1H), 3.91(s, 3H), 2.90(d, J =11.5Hz, 2H), 2.78 (p, J =6.7Hz, 1H), 2.40(t, J =11.6Hz, 2H), 2.18(d, J =12.3Hz, 2H), 1.66-1.55(m, 2H), 1.08(s, 3H), 1.06(s, 3H).

[0079] 2) Replace 2-chloro-N-(1-isopropylpiperidin-4-yl)-6,7-dimethoxyquinazoline-4-amine with an equimolar amount of 2-chloro-N-(1-isopropylpiperidin-4-yl)-6-methoxyquinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 3, yielding the NSD2 small molecule inhibitor (denoted as compound 6, structural formula: [structural formula missing]). ). 1 HNMR (400MHz, DMSO-) d 6)δ8.37(d, J =7.5Hz, 1H), 7.82(s, 1H), 7.73(d, J =9.1Hz, 1H), 7.52 (d, J =9.1Hz, 1H), 4.20-4.13 (m, 1H), 3.93 (s, 3H), 2.95 (s, 3H), 2.41 (s, 2H), 2.02-1 .97(m,2H),1.75-1.66(m,2H),1.06(s,3H),1.04(s,3H).HRMS(ESI)calcdforC 18 H 23 N5O(M+H + ):326.1975;found326.1974.

[0080] Example 7

[0081] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)-7-methoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0082] 1) Replace 2,4-dichloro-6,7-dimethoxyquinazoline with an equimolar amount of 2,4-dichloro-7-methoxyquinazoline. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 3, to obtain a white solid (2-chloro-N-(1-isopropylpiperidin-4-yl)-7-methoxyquinazoline-4-amine). 1 HNMR (400MHz, Chloroform- d )δ7.53(d, J =9.1Hz, 1H), 7.10(s, 1H), 7.04(d, J =9.1Hz, 1H), 5.55 (d, J =7.9Hz, 1H), 4.25(s, 1H), 3.89(s, 3H), 2.89(d, J =11.6Hz, 2H), 2.79(q, J =6.7Hz, 1H), 2.39(t, J =11.4Hz, 2H), 2.16(d, J =12.4Hz, 2H), 1.59(td, J =12.0, 5.9Hz, 2H), 1.08(s, 3H), 1.06(s, 3H).

[0083] 2) Replace 2-chloro-N-(1-isopropylpiperidin-4-yl)-6,7-dimethoxyquinazoline-4-amine with an equimolar amount of 2-chloro-N-(1-isopropylpiperidin-4-yl)-7-methoxyquinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 3, yielding the NSD2 small molecule inhibitor (denoted as compound 7, structural formula: [structural formula missing]). ). 1 HNMR (500MHz, DMSO-) d 6)δ8.39(dd, J =23.9, 8.3Hz, 2H), 7.27(dd, J =9.0, 2.6Hz, 1H), 7.19(d, J =2.6Hz, 1H), 4.15(td, J =12.5, 11.5, 5.9Hz, 1H), 3.90 (s, 3H), 2.97 (d, J =11.3Hz, 2H), 2.91(s, 1H), 1.99-1.93(m, 2H), 1.75(t, J =12.5Hz, 2H), 1.06(s, 3H), 1.05(s, 3H).HRMS(ESI)calcdforC 18 H 23 N5O(M+H + ):326.1975;found326.19.

[0084] Example 8

[0085] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)quinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0086] 1) Replace 2,4-dichloro-6,7-dimethoxyquinazoline with an equimolar amount of 2,4-dichloroquinazoline. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 3, to obtain a white solid (2-chloro-N-(1-isopropylpiperidin-4-yl)quinazoline-4-amine). 1 HNMR (400MHz, Chloroform- d )δ7.44-7.27(m, 5H), 7.14(s, 1H), 6.85(s, 1H), 5.53(d, J =7.9Hz, 1H), 5.23(s, 2H), 4.31-4.19(m, 1H), 3.97(s, 3H), 2.95-2.85(m, 2H), 2.78(p, J=6.5Hz, 1H), 2.38(td, J =11.8, 2.5Hz, 2H), 2.15(d, J =11.9Hz, 2H), 1.59(tt, J =11.9, 5.9Hz, 2H), 1.07(s, 3H), 1.06(s, 3H).

[0087] 2) Replace 2-chloro-N-(1-isopropylpiperidin-4-yl)-6,7-dimethoxyquinazoline-4-amine with an equimolar amount of 2-chloro-N-(1-isopropylpiperidin-4-yl)quinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 3, yielding the NSD2 small molecule inhibitor (denoted as compound 8, structural formula: [structural formula missing]). ). 1 HNMR (400MHz, Chloroform- d )δ7.90-7.76(m, 3H), 7.63-7.54(m, 1H), 6.32(d, J =8.0Hz, 1H), 4.42-4.28(m, 1H), 3.00(d, J =12.4Hz, 2H), 2.87 (d, J =6.6Hz, 1H), 2.47(t, J =10.6Hz, 2H), 2.23-2.13(m, 2H), 1.86-1.72(m, 2H), 1.14(s, 3H), 1.12(s, 3H).HRMS(ESI)calcdforC 17 H 21 N5(M+H + ):296.1870;found296.1879.

[0088] Example 9

[0089] This embodiment provides a small molecule inhibitor of NSD2 (7-(benzyloxy)-4-((1-isopropylpiperidin-4-yl)amino)-6-methoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0090] 1) Replace 2,4-dichloro-6,7-dimethoxyquinazoline with an equimolar amount of 7-benzyloxy-2,4-dichloro-6-methoxyquinazoline. The other required raw materials, reagents and process parameters are the same as in step 1) of Example 3, to obtain a white solid (7-benzyloxy-2-chloro-N-(1-isopropylpiperidin-4-yl)-6-methoxyquinazoline-4-amine). 1 HNMR (400MHz, Chloroform- d)δ7.44-7.27(m, 5H), 7.14(s, 1H), 6.85(s, 1H), 5.53(d, J =7.9Hz, 1H), 5.23(s, 2H), 4.31-4.19(m, 1H), 3.97(s, 3H), 2.95-2.85(m, 2H), 2.78(p, J =6.5Hz, 1H), 2.38(td, J =11.8, 2.5Hz, 2H), 2.15(d, J =11.9Hz, 2H), 1.59(tt, J =11.9, 5.9Hz, 2H), 1.07(s, 3H), 1.06(s, 3H).

[0091] 2) Replace 2-chloro-N-(1-isopropylpiperidin-4-yl)-6,7-dimethoxyquinazoline-4-amine with an equimolar amount of 7-benzyloxy-2-chloro-N-(1-isopropylpiperidin-4-yl)-6-methoxyquinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 3, yielding the NSD2 small molecule inhibitor (denoted as compound 9, structural formula: [insert structural formula here]). ). 1 HNMR (500MHz, DMSO-) d 6)δ8.11(d, J =7.5Hz, 1H), 7.72(s, 1H), 7.48(d, J =7.3Hz, 2H), 7.42(t, J =7.4Hz, 2H), 7.36(t, J =7.2Hz, 1H), 7.30 (s, 1H), 5.26 (s, 2H), 4.12-4.05 (m, 1H), 3.94 (s, 3H), 2.85 (d, J =10.8Hz, 2H), 2.78-2.70(m, 1H), 2.25(t, J =11.2Hz, 2H), 1.94(d, J =10.5Hz, 2H), 1.67-1.55(m, 2H), 0.99(s, 3H), 0.98(s, 3H).HRMS(ESI)calcdforC 25 H 29 N5O2(M+H + ):432.2394;found432.2395.

[0092] Example 10

[0093] This embodiment provides a small molecule inhibitor of NSD2 (7-hydroxy-4-((1-isopropylpiperidin-4-yl)amino)-6-methoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0094] 200 mg (0.59 mmol) of 7-(benzyloxy)-4-((1-isopropylpiperidin-4-yl)amino)-6-methoxyquinazoline-2-carboxynitrile obtained in Example 9 was added to a reaction flask, followed by methanol (10 ml) and palladium on carbon (20 mg). The reaction was stirred at room temperature under a hydrogen atmosphere for 8 hours, and the reaction was monitored by TLC. The palladium on carbon was filtered, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain the NSD2 small molecule inhibitor (denoted as compound 10, structural formula: [insert structural formula here]). ). 1 HNMR (500MHz, DMSO-d6) δ8.25-8.13 (m, 1H), 7.79 (s, 1H), 7.05 (s, 1H), 4.21 (s, 1H), 3.95 (s, 3H), 3.17 (s, 2H), 3.10 (s, 2H), 2.64(s, 1H), 2.03(d, J=10.0Hz, 2H), 1.84(s, 2H), 1.13(d, J=3.8Hz, 6H).HRMS(ESI)calcdforC18H23N5O2(M+H+):342.1925;

[0095] found342.1926.

[0096] Example 11

[0097] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)-6-methoxy-7-(2-methoxyethoxy)quinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0098] 100 mg (0.293 mmol) of 7-hydroxy-4-((1-isopropylpiperidin-4-yl)amino)-6-methoxyquinazoline-2-carboxynitrile obtained in Example 10 was added to a reaction flask, followed by triphenylphosphine (422 mg, 1.172 mmol), then ultradry tetrahydrofuran (5 mL). While maintaining a N2 atmosphere in an ice bath, 37 µL (0.440 mmol) of ethylene glycol methyl ether and DIAD (290 µL, 1.162 mmol) were added, and then the ice bath was removed. The reaction was carried out at room temperature for 4 hours, and the reaction was monitored by TLC. The reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain the NSD2 small molecule inhibitor (denoted as compound 11, structural formula: [insert structural formula here]). ). 1 HNMR (500MHz, Chloroform- d)δ7.11(s, 1H), 6.81(s, 1H), 5.46(d, J =7.9Hz, 1H), 4.32-4.25(m, 1H), 4.25-4.23(m, 2H), 3.96(s, 3H), 3.87-3.81(m, 2H), 3.46(s, 3H), 2.95-2.89(m, 2H), 2.80(p, J =6.6Hz, 1H), 2.40(td, J =11.7, 2.5Hz, 2H), 2.20-2.15(m, 2H), 1.66-1.59(m, 2H), 1.08(s, 3H), 1.07(s, 3H).HRMS(ESI)calcdforC 21 H 29 N5O3(M+H + ):400.2343;found400.2343..

[0099] Example 12

[0100] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)-6-methoxy-7-(2-morpholinylethoxy)quinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0101] By replacing the ethylene glycol methyl ether in Example 11 with a molar amount of 2-morpholinoethanol, and using the same raw materials, reagents, and preparation method as in Example 11, a small molecule inhibitor of NSD2 (denoted as compound 12, structural formula: [insert structural formula here]) was obtained. ). 1 HNMR (400MHz, Chloroform- d )δ7.21(s, 1H), 6.98(s, 1H), 5.94-5.82(m, 1H), 4.32(d, J =15.0Hz, 2H), 4.27(s, 1H), 4.03(s, 3H), 3.78-3.73(m, 4H), 3.02(d, J =11.7Hz, 2H), 2.91(d, J =6.3Hz, 2H), 2.62(t, J =4.6Hz, 4H), 2.51(s, 1H), 2.43(s, 2H), 2.19(d, J =12.4Hz, 2H), 1.79(d, J =12.4Hz, 2H), 1.15(s, 3H), 1.13(s, 3H)HRMS(ESI)calcdforC 24 H34 N6O2(M+H + ):455.2765;found455.2762.

[0102] Example 13

[0103] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)-6-methoxy-7-(3-morpholinylpropoxy)quinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0104] By replacing the ethylene glycol methyl ether in Example 11 with a molar amount of 3-(4-morpholino)-1-propanol, and using the same raw materials, reagents, and preparation method as in Example 11, a small molecule inhibitor of NSD2 (denoted as compound 13, structural formula: [structural formula missing]) was obtained. ). 1 HNMR (400MHz, DMSO-) d 6)δ8.50(d, J =7.1Hz, 1H), 7.92(s, 1H), 7.24(s, 1H), 4.43-4.36(m, 1H), 4.19(t, J =6.4Hz, 2H), 3.97(s, 3H), 3.62(t, J =4.6Hz, 4H), 2.71-2.64(m, 1H), 2.45-2.28(m, 4H), 2.19-2.12(m, 4H), 1.99(t, J =7.8Hz, 2H), 1.34(s, 2H), 1.31(d, J =6.4Hz, 4H), 1.25(s, 3H), 1.23(s, 3H).HRMS(ESI)calcdforC 25 H 36 N6O2(M+H + ):469.2922;found469.2918.

[0105] Example 14

[0106] This embodiment provides a small molecule inhibitor of NSD2 (7-(3-(3,3-difluoropyrrolidone-1-yl)propoxy)-4-(1-isopropylpiperidin-4-ylamino)-6-methoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0107] Replacing the ethylene glycol methyl ether in Example 11 with a molar amount of 3-(3,3-difluoropyrrolidone-1-yl)propanol, with the remaining raw materials, reagents, and preparation methods the same as in Example 11, yielded the NSD2 small molecule inhibitor (denoted as Compound 14, structural formula: [insert structural formula here]). ). 1 HNMR (500MHz, Chloroform- d )δ7.42(s, 1H), 7.17(s, 1H), 6.95(d, J =6.0Hz, 1H), 4.45-4.38(m, 1H), 4.19(t, J =6.5Hz, 2H), 4.04(s, 3H), 3.24(d, J =11.6Hz, 2H), 3.15(dt, J =13.0, 6.5Hz, 1H), 2.92(t, J =13.3Hz, 2H), 2.80-2.62(m, 6H), 2.33-2.21(m, 6H), 2.07(p, J =6.7Hz, 2H), 1.27(d, J =6.6Hz, 6H).HRMS(ESI)calcdforC 25 H 34 N6O2F2(M+H + ):489.2784;found489.2782.

[0108] The synthesis of 3-(3,3-difluoropyrrolidone-1-yl)propanol was as follows: A solution of 3,3-difluoropyrrolidone (10.0 g, 93.37 mmol), triethylamine (3.0 mL, 21.7 mmol), and 3-bromopropane-1-ol (25.95 g, 186.7 mmol) in THF (10 mL) was stirred at 80°C for 12 hours. The solution was transferred to water, stirred, and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by rapid column chromatography on silica gel to give the white compound 3-(3,3-difluoropyrrolidone-1-yl)propanol. 1 H NMR (400 MHz, Chloroform- d ) δ 4.31 (s, 1H), 3.72 (t, 2H), 2.60 (t, J = 6.0 Hz, 4H), 1.95 (tt, J = 13.7, 5.8 Hz, 4H), 1.73 – 1.63 (m, 2H).

[0109] Example 15

[0110] This embodiment provides a small molecule inhibitor of NSD2 (7-(3-(4,4-difluoropiperidin-1-yl)propoxy)-4-(1-isopropylpiperidin-4-yl)amino)-6-methoxyquinazoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0111] By replacing the ethylene glycol methyl ether in Example 11 with a molar amount of 3-(4,4-difluoropiperidin-1-yl)propanol, and using the same raw materials, reagents, and preparation method as in Example 11, a small molecule inhibitor of NSD2 (denoted as compound 15, structural formula: [insert structural formula here]) was obtained. ). 1 HNMR (400MHz, Chloroform- d )δ8.00(d, J =7.6Hz, 1H), 7.83(s, 1H), 7.17(s, 1H), 4.62-4.50(m, 1H), 4.19(t, J =6.6Hz, 2H), 4.04(s, 3H), 3.56-3.43(m, 3H), 3.02(t, J =11.7Hz, 2H), 2.70(q, J =12.0, 11.5Hz, 2H), 2.58(q, J =6.3, 5.5Hz, 6H), 2.36(d, J =12.2Hz, 2H), 2.02(ddq, J =33.2, 13.4, 6.2, 5.7Hz, 6H), 1.45(d, J =6.7Hz, 6H).HRMS(ESI)calcdforC 26 H 36 N6O2F2(M+H + ):503.2941;found503.2936.

[0112] Example 16

[0113] This embodiment provides a small molecule inhibitor of NSD2 (6-methoxy-4-((1-propylpiperidin-4-yl)amino)-7-(3-(pyrrolidone-1-yl)propoxy)quinazoline-2-carboxylon), the preparation method of which includes the following steps:

[0114] 1) Replace 2,4-dichloro-6,7-dimethoxyquinazoline with an equimolar amount of 2,4-dichloro-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline, replace compound 1a with an equal amount of 1-propylpiperidin-4-amine, and use the same raw materials, reagents and process parameters as in step 1) of Example 1 to obtain a white solid (2-chloro-6-methoxy-N-(1-propylpiperidin-4-yl)-7-(3-(pyrrolidin-1-yl)propoxy)quinazoline-4-amine). 1 HNMR (500MHz, DMSO-) d 6) δ8.35 (s, 1H), 7.85 (s, 1H), 7.07 (s, 1H), 4.19 (t, J =6.2Hz, 2H), 3.93(s, 3H), 3.25(s, 2H), 3.09(s, 6H), 2.63(s, 2H), 2.19-2.13(m, 2H), 1.94(d, J =56.0Hz, 10H), 1.62(s, 2H), 0.89(t, J =7.4Hz, 3H).

[0115] 2) Replace compound 1b with an equimolar amount of 2-chloro-6-methoxy-N-(1-propylpiperidin-4-yl)-7-(3-(pyrrolidone-1-yl)propoxy)quinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 1, yielding the NSD2 small molecule inhibitor (denoted as compound 16, structural formula: [structural formula missing]). ). 1 HNMR (500MHz, DMSO-) d 6)δ8.08(d, J =7.7Hz, 1H), 7.70(s, 1H), 7.18(s, 1H), 4.14(dt, J =17.5, 6.8Hz, 3H), 3.93(s, 3H), 2.92(d, J =11.7Hz, 2H), 2.56(t, J =7.2Hz, 2H), 2.47(t, J =6.2Hz, 4H), 2.29-2.25(m, 2H), 2.01(t, J =11.3Hz, 2H), 1.96-1.90(m, 4H), 1.70-1.67(m, 4H), 1.67-1.61(m, 2H), 1.45(h, J =7.3Hz, 2H), 0.87(t, J =7.4Hz, 3H).

[0116] Example 17

[0117] This embodiment provides a small molecule inhibitor of NSD2 (4-(1-cyclopropylpiperidin-4-yl)amino)-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinazoline-2-carboxylonite), the preparation method of which includes the following steps:

[0118] 1) Replace 2,4-dichloro-6,7-dimethoxyquinazoline with an equimolar amount of 2,4-dichloro-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinazoline, replace compound 1a with an equal amount of 4-amino-1-cyclopropylpiperidine, and use the same raw materials, reagents and process parameters as in step 1) of Example 1 to obtain a white solid (2-chloro-N-(1-cyclopropylpiperidine-4-yl)-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinazoline-4-amine). 1 HNMR (500MHz, DMSO-) d 6) δ8.35 (s, 1H), 7.85 (s, 1H), 7.07 (s, 1H), 4.19 (t, J =6.2Hz, 2H), 3.93(s, 3H), 3.25(s, 2H), 3.09(s, 6H), 2.63(s, 2H), 2.19-2.13(m, 2H), 1.94(d, J =56.0Hz, 10H), 1.62(s, 2H), 0.89(t, J =7.4Hz, 3H).

[0119] 2) Replace compound 1b with an equimolar amount of 2-chloro-N-(1-cyclopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinazoline-4-amine. The remaining raw materials, reagents, and process parameters are the same as in step 2) of Example 1, yielding the NSD2 small molecule inhibitor (denoted as compound 17, structural formula: [structural formula missing]). ). 1 HNMR (500MHz, DMSO-) d 6)δ8.05(d, J =7.6Hz, 1H), 7.68(s, 1H), 7.18(s, 1H), 4.17-4.10(m, 3H), 3.93(s, 3H), 3.00(d, J =11.7Hz, 2H), 2.56(t, J =7.2Hz, 2H), 2.46(t, J =6.3Hz, 4H), 2.34-2.28(m, 2H), 1.94(dq, J=15.3, 9.2, 7.9Hz, 4H), 1.69(p, J =3.1Hz, 4H), 1.64(dt, J =6.5, 3.1Hz, 1H), 1.57(td, J =12.1, 3.7Hz, 2H), 0.42(dt, J =6.1, 3.0Hz, 2H), 0.32-0.28(m, 2H).

[0120] Example 18

[0121] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)-6,7-dimethoxyquinoline-2-carboxynitrile), the process flow diagram of which is shown below. Figure 2 As shown, it includes the following steps:

[0122] 1) 2,4-Dichloro-6,7-dimethoxyquinoline (2.0 g, 5.63 mmol) was added to DMF, followed by Zn(CN)2 (330.7 mg, 2.81 mmol), dppf (330.7 mg, 2.81 mmol) and pd2(dba)3 (515 mg, 0.56 mmol). The mixture was heated in a microwave oven at 150 °C for 2 h under nitrogen protection to obtain compound 2b. 1 HNMR (400MHz, Chloroform- d ) δ7.50 (s, 1H), 7.41 (s, 1H), 7.37 (s, 1H), 4.09 (s, 3H), 4.06 (s, 3H).

[0123] 2) Cs₂CO₃ (1.41 g, 4.34 mmol), 1-isopropylpiperidin-4-amine (1.73 mmol), Pd₂(dba)₃ (132.7 mg, 0.14 mmol), and BINAP (180.5 mg, 0.29 mmol) were added to a 1,4-dioxane solution of compound 31b (500 mg, 1.45 mmol). The reaction was carried out under nitrogen protection by microwave heating at 130°C for 3 h to obtain a small molecule inhibitor of NSD₂ (denoted as compound 18, structural formula: [insert structural formula here]). ). 1 HNMR (500MHz, DMSO-) d 6) δ7.62 (s, 1H), 7.24 (s, 1H), 7.12 (s, 1H), 6.96 (s, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 3.66 (s, 1H), 2.98 (s, 4H), 2.02 (d, J=12.1Hz, 2H), 1.73(s, 3H), 1.08(s, 6H).

[0124] Example 19

[0125] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-cyclopropylpiperidin-4-yl)amino)-6,7-dimethoxyquinoline-2-carboxynitrile), the preparation method of which includes the following steps:

[0126] By replacing the 1-isopropylpiperidine-4-amine in Example 18 with a molar amount of 4-amino-1-cyclopropylpiperidine, and using the same raw materials, reagents, and preparation method as in Example 18, a small molecule inhibitor of NSD2 (denoted as compound 19, structural formula: [insert structural formula here]) was obtained. ). 1 HNMR (400MHz, DMSO-) d 6) δ7.55(s, 1H), 7.20(s, 1H), 6.95(d, J =7.8Hz, 1H), 6.91 (s, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 3.61 (s, 1H), 2.97 (s, 2H), 2.34 (s, 2H), 1.92 (d, J =12.0Hz, 2H), 1.67-1.50 (m, 3H), 0.42 (s, 2H), 0.28 (s, 2H).

[0127] Example 20

[0128] This embodiment provides a small molecule inhibitor of NSD2 (6-methoxy-4-(1-propylpiperidine-4-amino)-7-(3-(pyrrolidine-1-propoxy)quinoline-2-carboxylon), the preparation method of which includes the following steps:

[0129] 1) Replace 2,4-dichloro-6,7-dimethoxyquinoline with an equimolar amount of 2,4-dichloro-6-methoxy-7-(3-(pyrrolidine-1-propoxy)quinoline, and use the same raw materials, reagents and process parameters as in Example 18 to obtain a white solid (4-chloro-6-methoxy-7-(3-(pyrrolidine-1-yl)propoxy)quinoline-2-carboxynitrile). 1 HNMR (400MHz, Methanol-) d 4) δ7.94(s, 1H), 7.55(s, 1H), 7.52(s, 1H), 4.40(t, J =5.6Hz, 2H), 4.11(s, 3H), 3.59-3.44(m, 6H), 2.44-2.37(m, 2H), 2.19-2.13(m, 4H).

[0130] 2) Replace 1-isopropylpiperidine-4-amine with an equimolar amount of 1-propylpiperidine-4-amine, and replace compound 2b with an equimolar amount of 4-chloro-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinoline-2-carboxynitrile. The remaining raw materials, reagents, and preparation methods are the same as in Example 18, yielding the NSD2 small molecule inhibitor (denoted as compound 20, structural formula: [structural formula missing]). ). 1 HNMR (400MHz, DMSO-) d 6) δ7.58(s, 1H), 7.21(s, 1H), 7.00(d, J =7.9Hz, 1H), 6.92(s, 1H), 4.13(t, J =6.4Hz, 2H), 3.94(s, 3H), 3.64-3.57(m, 1H), 2.91(d, J =11.1Hz, 2H), 2.58(t, J =7.1Hz, 2H), 2.49-2.47(m, 2H), 2.27(t, J =7.4Hz, 2H), 2.07(t, J =11.4Hz, 2H), 2.00-1.90(m, 4H), 1.74-1.58(m, 6H), 1.49-1.40(m, 2H), 1.32-1.17(m, 2H), 0.87(t, J =7.4Hz, 3H).

[0131] Example 21

[0132] This embodiment provides a small molecule inhibitor of NSD2 (4-((1-isopropylpiperidin-4-yl)amino)-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinoline-2-carboxylon), the preparation method of which includes the following steps:

[0133] By replacing 1-propylpiperidin-4-amine with an equimolar amount of 1-isopropylpiperidin-4-amine, and using the same raw materials, reagents, and preparation method as in Example 20, a small molecule inhibitor of NSD2 (denoted as compound 21, structural formula: [insert structural formula here]) was obtained. ). 1 HNMR (500MHz, DMSO-) d 6) δ7.58(s, 1H), 7.21(s, 1H), 7.00(d, J =7.9Hz, 1H), 6.93(s, 1H), 4.13(t, J =6.4Hz, 2H), 3.94(s, 3H), 3.62-3.49(m, 1H), 2.83(d,J =11.0Hz, 2H), 2.80-2.69(m, 1H), 2.56(t, J =7.2Hz, 2H), 2.49-2.43(m, 6H), 2.31(t, J =11.5Hz, 2H), 2.04-1.89(m, 3H), 1.75-1.66(m, 4H), 1.66-1.55(m, 2H), 1.00(d, J =6.6Hz, 6H).

[0134] Example 22

[0135] This embodiment provides a small molecule inhibitor of NSD2 (4-(1-cyclopropylpiperidin-4-yl)amino)-6-methoxy-7-(3-(pyrrolidone-1-yl)propoxy)quinoline-2-carboxylonite), the preparation method of which includes the following steps:

[0136] By replacing 1-propylpiperidine-4-amine with an equimolar amount of 4-amino-1-cyclopropylpiperidine, and using the same raw materials, reagents, and preparation method as in Example 20, a small molecule inhibitor of NSD2 (denoted as compound 22, structural formula: [insert structural formula here]) was obtained. ). 1 HNMR (500MHz, DMSO-) d 6) δ7.56 (s, 1H), 7.21 (s, 1H), 6.96 (d, J =7.9Hz, 1H), 6.93(s, 1H), 4.13(t, J =6.4Hz, 2H), 3.93(s, 3H), 3.67-3.57(m, 1H), 2.99(dt, J =10.9, 2.9Hz, 2H), 2.55(t, J =7.2Hz, 2H), 2.48-2.42(m, 4H), 2.35(td, J =11.9, 2.4Hz, 2H), 1.99-1.91(m, 4H), 1.72-1.62(m, 5H), 1.62-1.51(m, 2H), 0.47-0.40(m, 2H), 0.33-0.27(m, 2H).

[0137] Performance testing

[0138] Experiment 1: Molecular-level inhibition assay of the protein DNA methyltransferase NSD2 activity

[0139] Compound preparation: Take the NSD2 small molecule inhibitors (compounds 1-22) from each example, centrifuge at 12000g for 5 min, add DMSO to prepare a 10 mM stock solution, vortex to homogenize, sonicate for 10 min, and store at -20℃. For testing, dilute the compounds from the stock solution to the required concentration using the appropriate reaction buffer.

[0140] 1) Dilute NSD-mutation enzyme, SAM, inhibitors, and recombinant polynucleosomes (H3.1) with AssayBuffer. 2) Add the following reagents sequentially to a 384-well microplate: 5 μL of inhibitor (2X) or AssayBuffer, 2.5 μL of NSD-mutation enzyme (4X), and 2.5 μL of recombinant polynucleosomes (H3.1) / SAM mix (4X). 3) Seal the microplate with a topseal and incubate at room temperature for 60 min. 4) Prepare 1X HighSalt Buffer. 5) Add 5 μL of 1X HighSalt Buffer to terminate the reaction and incubate at room temperature for 15 min. 6) Prepare 1X Detection Buffer. 7. Add 5 μL of Biotin-anti-H3 (final concentration 1 nM) / anti-H3K36me2AcceptorBeadsmix (final concentration 20 ng / μL) prepared with 1X Detection Buffer and react for 1 h. 8. Prepare Streptavidin Donorbeads (final concentration 20 ng / μL) using 1X Detection Buffer. 9. Add 5 μL of Streptavidin Donorbeads, seal, and incubate in the dark for 30 mins. 10. Use EnVision to read the values.

[0141] Each experiment consisted of two wells and a blank control group. The inhibition rate (%) of the compound on enzyme activity was calculated using the following formula:

[0142] Inhibition rate (%) = (Fluor control wells - Fluor drug administration wells) / Fluor control wells × 100%

[0143] The corresponding IC50 was calculated based on the inhibition rate at each concentration. The in vitro enzyme activity results of the NSD2 small molecule inhibitors of each example are shown in Table 1 below.

[0144] Table 1. Inhibitory level of NSD2 small molecule inhibitors on the activity of DNA methyltransferase NSD2.

[0145]

[0146] The structure of compound X is as follows: The compound, which is derived from the

[0051] section of patent CN 115160294 A, was subjected to the same test as the test one of the present invention.

[0147] As shown in Table 1, the NSD2 small molecule inhibitor of the present invention has high inhibitory activity against DNA methyltransferase NSD2.

[0148] Experiment 2: Pharmacokinetics in Mice

[0149] Experimental objective: After a single intravenous injection or oral gavage administration of compound 13 to ICR mice, blood samples were collected at different time points to detect the concentration of the test substance and calculate relevant parameters, thereby investigating the pharmacokinetic characteristics of the test substance in mice.

[0150] Experimental Methods: Six healthy male ICR mice, weighing 25±5g, were provided by Zhejiang Vital River Laboratory Animal Technology Co., Ltd. For the intravenous administration group, the dose was 1 mg / kg, and blood samples (approximately 0.030 mL) were collected at the following time points after administration: 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 h. For the oral administration group, the dose was 50 mg / kg, and blood samples were collected at 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h after administration. Heparin sodium was pre-added to the blood collection tubes as an anticoagulant. Blood samples were centrifuged at 6800g for 6 minutes, and plasma was collected and stored at -80℃. The concentration of compound 13 in plasma was determined by LC-MS / MS. Pharmacokinetic parameters such as AUC(0-∞), T1 / 2, Cmax, Tmax, and oral bioavailability were calculated using blood drug concentration data at different time points using WinNonlin.

[0151] Sample preparation for LC-MS / MS assay: 10 µL of plasma sample was precipitated for protein using 200 µL of methanol containing 100 ng / mL IS (IS is tolbutamide). The mixture was vortexed for 1 min and then centrifuged at 18000 g for 7 min. 150 µL of the supernatant was transferred to a 96-well plate and diluted with 150 µL of purified water. 6 µL of the supernatant was analyzed by LC-MS / MS.

[0152] The experimental results are shown in Table 2 below:

[0153] Table 2 Pharmacokinetic data of compound 13

[0154]

[0155] As shown in Table 2, the NSD2 small molecule inhibitor of the present invention has good pharmacokinetic properties and good drug-likeness.

[0156] Experiment 3: Enzyme Selectivity Test

[0157] Experimental objective: To test the selectivity of compound 13 for NSD2.

[0158] Enzyme types tested: PRMT1, PRMT4, PRMT8, PRMT5, EZH2, NSD3

[0159] Experimental methods: a) The established AlphaLISA and HTRF methods were used to further investigate the inhibitory effect of compound 13 on several other histone-modifying enzymes at the molecular level, in order to determine the selectivity of compound 13 for histone-modifying enzymes. The inhibitory activity of compound 13 on histone-modifying enzymes PRMT1, PRMT4, PRMT8, PRMT5, and NSD3 at the molecular level was detected using AlphaLISA. Compound 13, enzymes (PRMT1, PRMT4, PRMT8, PRMT5, NSD3), and substrate solutions were all prepared in 1× assay buffer; b) The following reagents were added sequentially to a 384-well microplate: 5 μL inhibitor, 2.5 μL enzyme solution, incubation at 37°C for 10 min, and 2.5 μL substrate / S-adenosine-L-methionine mixture; c) The microplate was sealed and incubated at room temperature; d) A 1x concentration assay solution was prepared; e) A 1x concentration assay solution was prepared. f) Add 5 μL of anti-histone methylation antibody-conjugated receptor beads (100 μg / mL) to a final concentration of 20 μg / mL; g) Prepare streptavidin donor beads (50 μg / mL) with a 1x concentration of detection solution to a final concentration of 20 μg / mL; h) Add 10 μL of diluted streptavidin donor beads, seal, and incubate in the dark for 30 minutes; i) Read the values ​​using a microplate reader.

[0160] Each experiment group has two sets of wells, and a blank control group is also included.

[0161] Inhibition rate (%) = {[(positive control signal value - blank control signal value) - (test compound signal value - blank control signal value)] / (positive control signal value - blank control signal value)}.

[0162] Selectivity for EZH2 was assessed using an HTFR assay. In the HTFR assay, the enzyme, SAM, compound, and peptide substrate in the assay buffer were diluted in 1× assay buffer before use. 4 μL of the compound and 2 μL of the enzyme were added to a 384-well microplate and incubated at room temperature for 10 minutes. Subsequently, 4 μL of substrate / SAM was mixed with the reaction mixture and incubated at room temperature for 4 hours. Antibody and SA-XL665 were prepared separately using 1× Detection Buffer. These were mixed to form a 2× mixture, and 10 μL of the mixture was added to each well and incubated at room temperature in the dark for 1 hour. Readings were taken using a microplate reader. Two wells were used for each experiment, and a blank control group was included. Inhibition rate (%) = {[(positive control signal value - blank control signal value) - (test compound signal value - blank control signal value)] / (positive control signal value - blank control signal value)}. Competitive inhibition curves were plotted using GraphPadprism software, with inhibition rate (%) as the ordinate and compound concentration as the abscissa. The concentration (IC50) at which the compound and protein binding rate reached 50% was calculated using the resulting regression equation. 50 ).

[0163] Experimental results: The results of the enzyme selectivity test are shown in Table 3 below.

[0164] Table 3. Selectivity determination of compound 13 for NSD2

[0165]

[0166] As shown in Table 3, the NSD2 small molecule inhibitor of the present invention has high selectivity for DMA methyltransferase NSD2 and is a class of inhibitors that specifically target NSD2.

[0167] In vivo efficacy evaluation test of test four

[0168] Experimental objective: To test the antitumor efficacy of compound 13 in the RS411 nude mouse xenograft model.

[0169] Experimental Methods: NU / NU nude mice (4 weeks old, female, purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were purchased and acclimatized for one week before the experiment. RS411 cells were expanded in vitro. Logarithmic growth phase cells were suspended in serum-free RPMI 1640 medium and mixed with Metrigel (1:1). 100 μL (1.0*10⁻¹¹) of the cell suspension was injected subaxillarily. 7 (Number of mice). Animal experimental procedures were conducted in accordance with animal experimental ethics guidelines. The average tumor volume in the mice to be induced to bear tumors reached 50-100 mm. 3Mice were randomly divided into two groups: a solvent control group and a 50 mg / kg compound group (n=6 per group). After grouping, the mice were administered the compound once daily for 21 consecutive days, with tumor volume measured and weighed twice weekly. Tumor volume (TV) was calculated using the formula: TV = 1 / 2 * a * b 2 Where a and b represent length and width, respectively. The tumor inhibition rate (TGI) is calculated based on the measurement results using the formula: TGI = [TV(control group) - TV(experimental group) / TV(control group)] * 100%.

[0170] Experimental Results: The antitumor efficacy of compound 13 in the A375RS411 nude mouse xenograft model is as follows: Figure 3 As shown. From Figure 3 It is known that compound 13 has a TGI of 52.69% at a dose of 50 mg / kg, indicating that it has good antitumor activity, which shows that the NSD2 small molecule inhibitor of the present invention has good antitumor activity.

[0171] Experiment 5: Cell proliferation inhibition experiment

[0172] Experimental objective: To evaluate the effect of compound 13 on cell proliferation.

[0173] Test cell lines: RS4;11, KMS-11, MOLM-13, MOLT-4, MV-4-11 cell lines

[0174] Observation index: Inhibition rate of compound 13 on the proliferation of various cells

[0175] Detection method: CCK-8 cell counting kit (Liji Biotechnology)

[0176] Test Principle: Cell growth can be manifested as an increase in cell number and cell volume. Cell volume is usually within a certain range, while the number of cells can increase through continuous division. The CCK-8 chromogenic reagent utilizes the dehydrogenases present in the cells themselves to reduce the tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfo-phenyl)-2H-tetrazolium] to produce a water-soluble yellow product—formazan. The number of viable cells is directly proportional to the amount of formazan dye produced. It can be used to detect the number of viable cells in cell proliferation and toxicity analysis experiments.

[0177] Duration of action: 6 days.

[0178] Reagents, consumables and instruments: The water used in the experiment was distilled water produced by Sinopharm Group; all reagents used were purchased from Sinopharm Chemical Reagent Co., Ltd.; the full-wavelength microplate reader used for the experiment was a product of MolecularDevice, model: SpectraMax190.

[0179] Compound preparation: Centrifuge 12000g of the compound for 5 min, add DMSO to prepare a 10 mM stock solution, vortex until homogenized, and sonicate for 10 min before use. Store at -40℃ (for compounds with special storage requirements, modify according to specific conditions). During testing, dilute the compound from the stock solution to the test concentration with physiological saline (DMSO concentration in the system should not exceed 0.5%).

[0180] Experimental Methods: The inhibitory effect of the compound on the proliferation of RS4;11, KMS-11, MOLM-13, MOLT-4, and MV-4-11 cell lines was detected using a CCK-8 cell counting kit (Liji Biotechnology). The specific steps are as follows: Cells in the logarithmic growth phase were seeded at an appropriate density into 96-well culture plates, 190 μL per well. After overnight culture, different concentrations of the compound (10 μL) were added and incubated for 6 days. A solvent control group (negative control) was set up. After 6 days of compound treatment, the effect of the compound on cell proliferation was detected using a CCK-8 cell counting kit. 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37℃ for 2-4 hours. Readings were then performed using a SpectraMax 190 microplate reader at a wavelength of 450 nm.

[0181] The inhibition rate (%) of the compound on tumor cell growth was calculated using the following formula:

[0182] Inhibition rate (%) = (OD control well - OD drug administration well) / OD control well × 100%

[0183] The corresponding IC50 was calculated based on the inhibition rate at each concentration.

[0184] Experimental results: The inhibitory effects of compound 13 on the cell lines RS4;11, KMS-11, MOLM-13, MOLT-4, and MV-4-11 are shown in Table 4 below.

[0185] Table 4. Determination of the inhibitory effects of compound 13 on different cell lines.

[0186]

[0187] The data in Table 4 show that the NSD2 small molecule inhibitor of the present invention has high inhibitory activity against RS4;11 at the cellular level.

[0188] Experiment 6: Real-time Quantitative PCR

[0189] Cells in 6-well plates were treated with the drug for 72 hours and then collected. RNA was extracted using an RNA purification kit (B0004DP) purchased from EZBscience. The specific procedure was as follows: Centrifuge and discard the supernatant to collect cells; add 500 μL of Lysis Buffer to each well, mix thoroughly by pipetting 30 times, and then transfer the cells to a 1.5 mL EP tube; add 500 μL of anhydrous ethanol, mix thoroughly, and then transfer to a centrifuge column; centrifuge at 4000g for 1 min and discard the waste liquid; add 500 μL of washing buffer to the centrifuge column, centrifuge at 12000g for 1 min, discard the waste liquid, and dry the tube; centrifuge at 12000g for 1 min; transfer the collection column to a new 1.5 mL EP tube and allow it to evaporate at room temperature for 2 min; add 25-30 μL of RNA elution solution to the center of the collection column, incubate at room temperature for 2 min, and centrifuge at 12000g for 1 min to obtain RNA; the RNA can be added back to the center of the collection column and centrifuged again to increase the RNA concentration.

[0190] RNA concentration was measured using a nucleic acid concentration testing instrument, and then reverse transcribed into cDNA using the Novizan reverse transcription kit (#R222-01, Vazyme). The resulting cDNA was stored at -20℃ for later use.

[0191] qPCR was performed using Novizan (#Q121-02, Vazyme) real-time quantitative PCR. The system was as follows: 10 μL SYBR Green mixture per well, 0.5 μL forward primer, 0.5 μL reverse primer, 7 μL ddH2O, and 2 μL cDNA sample. Each sample was tested in 2 or 3 replicates. After PCR, the relative expression percentage of the target gene was calculated using the Delta-deltaCT method, with the expression of the internal standard GAPDH gene as 100% based on the Ct value. At least two independent replicates were performed, and the results are expressed as mean ± SEM. The primers used for PCR are shown below:

[0192] GAPDH:

[0193] 5'-GAGTCCACTGGCGTCTTCAC-3'

[0194] 5'-TTCACACCCATGACGAACAT-3'

[0195] PAK1:

[0196] 5'-GGTTTCAAGTGTTTAGTAACTTTTCCA-3'

[0197] 5'-TTAGCTGCAGCAATCAGTGG-3'

[0198] TGFA:

[0199] 5'-CCTGGCTGTCCTTATCATCAC-3'

[0200] 5'-GGCACCACTCACAGTGTTTTC-3'

[0201] TREML2:

[0202] 5'-CCACAGGGTTGCGTCTCAG-3'

[0203] 5'-GGAGCACTGCACAGACAGAG-3'

[0204] HSPG2:

[0205] 5'-CCAAATGCGCTGGACACATTC-3'

[0206] 5'-CGGACACCTCTCGGAACTCT-3'

[0207] Experimental results: The results of real-time quantitative PCR experiments for compound 13 are as follows: Figure 4 As shown. From Figure 4 It can be seen that after treatment with compound 13, the downstream genes of NSD2 were inhibited, indicating that the NSD2 small molecule inhibitor of the present invention acts on NSD2.

[0208] Experiment 7: Apoptosis and Cell Cycle Experiment

[0209] Experimental objective: To investigate the specific mechanism of proliferation inhibition by compound 13.

[0210] Test cell line: RS411 cell line

[0211] Test method:

[0212] 1) Apoptosis detection:

[0213] Apoptosis was detected using Annexin-FITC (A211-01, Vazyme) staining. The specific procedure was as follows: Cells were seeded at an appropriate density in 6-well plates, and the required concentration gradient of drugs was added the next day for 6 days. Cells were collected in 2 mL EP tubes and centrifuged at 500 g, 4 °C for 5 min. The culture medium was aspirated, and the cells were washed twice with pre-cooled PBS and centrifuged at 500 g, 4 °C for 5 min. 100 μL of 1× Binding Buffer, 5 μL of Annexin V-FITC, and 5 μL of LPI were added to each sample to resuspend the cells. The cells were stained at room temperature in the dark for 15 min, and then 400 μL of 1× Binding Buffer was added. The cells were filtered through a filter and transferred to flow cytometry tubes for analysis using a CytoFLEX (BECKMANCOULTER) flow cytometer. Cell populations are distinguished by drawing a cross based on Annexin V-FITC and PI fluorescence values. Cells that are negative for both parameters (Annexin V- / PI-) are viable cells, cells that are positive for Annexin V (Annexin V+ / PI-) are early apoptotic cells, and cells that are positive for both parameters (Annexin V+ / PI+) are late apoptotic cells. The total number of cells in both ranges is the number of apoptotic cells.

[0214] 2) Cell cycle detection:

[0215] Cell cycle analysis was performed using PI (ST511, Beyotime) and RNase A (ST579, Beyotime). The specific procedure was as follows: Cells were seeded at an appropriate density in 6-well plates, and then treated with different concentrations of the compound. After 48 hours of treatment, the cells were collected. Cells were centrifuged at 500g, 4℃, for 5 minutes to collect the cells. The cells were washed with pre-chilled PBS and centrifuged again. Finally, the cells were gently resuspended in 300 μL of pre-chilled PBS and mixed thoroughly. EP tubes were placed on a vortex mixer, and 700 μL of pre-chilled anhydrous ethanol was added dropwise while vortexing. After thorough mixing, the samples were fixed overnight at -20℃. The next day, the cells were centrifuged and the liquid discarded. The cells were then washed with 1 mL of pre-chilled PBS, centrifuged again, and the supernatant discarded. 500 μL of pre-chilled PBS, 5 μL of PI (10 mg / mL), and 5 μL of RNase A (dissolved in PBS, 10 mg / mL) were added to each sample, and the cells were thoroughly mixed and resuspended. The cells were stained at room temperature in the dark for 30 minutes. Finally, the cells were filtered into single cells using a filter and placed into flow cytometry tubes for immediate cell cycle analysis.

[0216] The results of the apoptosis detection experiment are as follows: Figure 5 As shown, the experimental results for cell cycle detection are as follows: Figure 6 As shown, from Figure 5 , Figure 6 It is known that cytotoxicity is partly dependent on apoptosis and not on the cell cycle.

[0217] Experiment 8: RNA-seq Validation

[0218] Experimental objective: To investigate the effects of compound 13 on downstream transcriptome pathways.

[0219] Test cell line: RS411 cell line

[0220] Experimental Methods: Cells in the logarithmic growth phase were seeded in 10cm cell culture dishes. The experiment consisted of a DMSO treatment group and a 5μM drug treatment group, with two replicates for each group. After 72 hours of drug treatment, cells were collected using 1.5mL of TRIzol and then stored at -80℃ before being sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for transcriptome sequencing analysis.

[0221] The test results are as follows Figure 7 As shown, Figure 7 Figure A in the diagram is a GSEA GO analysis of differentially expressed genes of compound 13 after treatment with 5 μM for 72 hours in RS411 cells using RNA-seq. Figure 7 B in the diagram is a gene volcano plot showing differentially expressed genes in RS411 cells treated with compound 13; from Figure 7 It is known that the NSD2 small molecule inhibitor of the present invention can affect multiple key biological processes in RS411, including mitotic nucleus and DNA replication.

[0222] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A NSD2 small molecule inhibitor, characterized in that, A compound as shown in formula (I) or a salt thereof: Formula (I); wherein n1, n2, n3 are independently selected from an integer from 0 to 2; X1 is CH or N; R1 is selected from hydrogen, C1-C6 alkyl or C3-C8 cycloalkyl; R2 is selected from hydrogen or C1-C6 alkoxy; X2is H; or X2is CH2, O or NH, n4is an integer selected from 0 to 4, R3 is selected from hydrogen, C1-C6 alkyl, substituted or unsubstituted C3-C8 heterocycloalkyl, aryl or C1-C6 alkoxy; the aryl is phenyl; the substitution means at least one site is substituted by halogen.

2. The NSD2 small molecule inhibitor of claim 1, wherein, The NSD2 small molecule inhibitor is any one of the following structures or a salt thereof: , , , , , , , , , , , , , , , , , , , , or .

3. A method of preparing the NSD2 small molecule inhibitor of any one of claims 1-2, characterized in that, The method comprises the following steps: The compound shown in formula (A) is coupled with cyanide in the presence of a catalyst to obtain the NSD2 small molecule inhibitor; Formula (A); or the compound shown in formula (B) is coupled with the compound shown in formula (C) in the presence of a catalyst to obtain the NSD2 small molecule inhibitor; Formula (B); Formula (C); or when When the whole is OH, the compound of formula (D) is reduced under the condition of hydrogen atmosphere and palladium-carbon catalyst to obtain the NSD2 small molecule inhibitor. Formula (D); or when X2 is O and R3 is not hydrogen, alkyl, cycloalkyl and heterocycloalkyl, the compound shown in formula (E) is subjected to Mitsunobu reaction with the compound shown in formula (F) in the presence of DIAD and triphenyl phosphine to obtain the NSD2 small molecule inhibitor; Formula (E); Formula (F).

4. Use of the NSD2 small molecule inhibitor according to any one of claims 1-3 in the preparation of a medicament for inhibiting NSD2 related tumors, which are chronic and acute leukemia or multiple myeloma.

5. Use according to claim 4, characterized in that, The medicament is a medicament for preventing and / or treating tumors with abnormal cell proliferation, morphological changes or hyperfunction of movement related to NSD2. The medicament is a medicament for preventing and / or treating tumors with abnormal cell proliferation, morphological changes or hyperfunction of movement related to NSD2.

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