A purine derivative and its uses

Abstract

This invention relates to a purine derivative and its uses, the structural formula of which is shown in formula (I) or formula (II). This compound exhibits good NSD2 and NSD3 inhibitory activity, effectively reducing the methylation level of H3K36 within cells and causing a decrease in the cell proliferation activity of tumor cell lines with high NSD2 and NSD3 expression, thus serving as an NSD2 and NSD3 inhibitor.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical chemistry, and in particular to the use of a purine derivative as an inhibitor of NSD2 and NSD3. Background Technology

[0002] Dynamic methylation of histones is a crucial mechanism of epigenetic modification, regulated by various histone methyltransferases and demethyltransferases, and has become a hot topic in anti-tumor drug development in recent years. In 2020, EPZM's histone methyltransferase EZH2 inhibitor, tazestat, was officially approved for the treatment of epithelial sarcoma, becoming the first anti-tumor drug targeting histone methyltransferases. Currently, several histone methyltransferase inhibitors in the research and development or clinical stages have been reported, and drug development targeting this type of target is booming. The NSD subfamily belongs to the histone methyltransferase family and includes three member proteins: NSD1, NSD2 (WHSC1), and NSD3 (WHSC1L1). These proteins are primarily responsible for methylating multiple lysine residues of histone H3, which is closely related to cell proliferation and plays a vital role in tumorigenesis and development. Specifically, NSD2 and NSD3 catalyze the dimethylation or trimethylation of the LYS residue at the 36th residue of the histone H3 tail, thereby altering the binding pattern between histones and DNA. Studies have found that NSD2 and NSD3 exhibit abnormal gene amplification, upregulated protein expression, or gene fusion in various types of tumors. Abnormally high expression of NSD3 protein has been found in cell lines and tissue samples from various tumors, including lung cancer, breast cancer, liver cancer, bladder cancer, head and neck squamous cell carcinoma, and colorectal cancer, suggesting a role in promoting tumorigenesis. Abnormal gene amplification of the NSD3 gene or its 8p11-12 chromosomal region has been found in lung cancer, liver cancer, breast cancer, head and neck cancer, colorectal cancer, bladder cancer, pancreatic cancer, and ovarian cancer, and it is considered a driver gene for tumorigenesis. Abnormal gene fusion of the NSD3 gene with NUP98 and NUT has been found in acute myeloid leukemia and fibroblastic carcinoma, respectively. Knockout of the NSD3 gene in osteosarcoma leads to cell cycle arrest in the G2 / M phase and induces apoptosis in tumor cells. NSD2 is highly expressed in tumors such as bladder cancer, lung cancer, colon cancer, liver cancer, and myeloma. Therefore, NSD2 and NSD3 are considered potential anti-tumor targets.

[0003] The NSD2 and NSD3 proteins are composed of multiple functional domains, including PWWP1, PHD1, PHD2, PHD3, PWWP2, Pre-SET, SET, Post-SET, and PHD4. The Pre-SET, SET, and Post-SET domains form the catalytic domains, responsible for binding the methyl donor SAM and histone substrate peptides, thus catalyzing methyl transfer. The PWWP1 domain binds BRD4 and CHD8, participating in the regulation of AML-related genes. Although the mechanisms of action and molecular structures of NSD2 and NSD3 are somewhat understood, research on small molecule compounds that regulate their functions is relatively limited.

[0004] Regarding NSD3 inhibitors, in 2019 Researchers reported a chemical probe, BI-9321, that binds to the PWWP1 domain of the NSD3 protein. This compound downregulates Myc messenger RNA and inhibits the proliferation of MOLM-13 cells. Structural analysis further revealed that BI-9321 binds to the methylated lysine binding pocket of the PWWP1 domain.

[0005] For NSD2 inhibitors, existing technologies include 5-aminonaphthalene series compounds that can target the NSD2-SET domain. Additionally, Coussens et al. reported that several compounds screened from existing drug libraries, including DA3003-1, PF-03882845, TC LPA54, ABT-199, DZNep, and Chaetocin, can bind to the NSD2 SET domain in SPR experiments.

[0006] However, there is relatively little research on small molecule compounds that regulate the function of NSD2 and NSD3, and there are currently no potent inhibitors that can target both NSD2 and NSD3 simultaneously. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a purine derivative that can be used as an inhibitor of both NSD2 and NSD3, in order to address the shortcomings and deficiencies of the prior art.

[0008] To solve the above technical problems, the present invention adopts the following technical solution:

[0009] A purine derivative of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof.

[0010]

[0011] in,

[0012] R1 is selected from hydrogen, halogen, cyano, unsubstituted, or selected from halogen, cyano, and C.1-6 One or more substituents in the alkyl group replace the following groups: C 1-6 Alkyl, C 1-6 Alkoxy;

[0013] R2 is selected from hydrogen, halogen, or cyano;

[0014] R3 is selected from hydrogen, halogen, or cyano;

[0015] R4 is selected from hydrogen or hydroxyl groups;

[0016] R5 is selected from hydrogen, halogen, cyano, unsubstituted, or selected from halogen, cyano, and C. 1-6 One or more substituents in the alkyl group replace the following groups: C 1-6 Alkyl, C 1-6 Alkoxy;

[0017] R6 is selected from hydrogen, halogen, or cyano;

[0018] And exclude the following compounds:

[0019]

[0020] In some embodiments, R1 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 alkoxy or halogen-substituted C 1-6 alkyl.

[0021] In some embodiments, R1 is selected from Br, I, cyano, or CF3.

[0022] In some embodiments, R2 is selected from hydrogen, F, Cl, or cyano.

[0023] In some implementations, R3 is hydrogen or F.

[0024] In some embodiments, R4 is a hydroxyl group and R1 is a cyano group.

[0025] In some embodiments, R5 is selected from hydrogen, halogen, cyano, C 1-6 Alkyl, C 1-6 alkoxy or halogen-substituted C 1-6 alkyl.

[0026] In some implementations, R6 is hydrogen.

[0027] In some implementations, R5 is a cyano group.

[0028] In some embodiments, the purine derivatives are selected from compounds with the following structures:

[0029]

[0030] The present invention further relates to the use of purine derivatives of the structure shown in formula (I) or formula (II) above, or pharmaceutically acceptable salts thereof, as NSD2 and NSD3 inhibitors.

[0031] The present invention further relates to the use of purine derivatives of the structure shown in formula (I) or formula (II) above, or pharmaceutically acceptable salts thereof, in the preparation of medicaments for treating tumors.

[0032] In some embodiments, the tumor is selected from one or more combinations of lung cancer, liver cancer, breast cancer, head and neck cancer, colorectal cancer, bladder cancer, pancreatic cancer, ovarian cancer, prostate cancer, myeloma, and osteosarcoma.

[0033] Preferably, the tumor is lung cancer or breast cancer.

[0034] The present invention also provides several synthetic routes for purine derivatives with the structures shown in formula (I) or formula (II) above.

[0035] The first synthetic route is as follows:

[0036]

[0037] The second synthetic route is as follows:

[0038]

[0039] The third synthetic route is as follows:

[0040]

[0041] The fourth synthetic route is as follows:

[0042]

[0043]

[0044] The fifth synthetic route is as follows:

[0045]

[0046] Due to the implementation of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0047] The purine derivatives of formula (I) or formula (II) of this invention exhibit good inhibitory activity against NSD2 and NSD3 in biochemical experiments at the protein level. SPR experiments have demonstrated that the purine derivatives have micromolar affinity for NSD3. Intracellularly, the purine derivatives can effectively reduce the methylation level of H3K36 and reduce the cell proliferation activity of tumor cell lines that highly express NSD2 and NSD3. Therefore, they can be used as NSD2 and NSD3 inhibitors and for the preparation of antitumor drugs. Attached Figure Description

[0048] Figure 1 The image shows the results of Western blotting experiments on some of the compounds in this application. Detailed Implementation

[0049] Terminology Definition

[0050] In the compounds described in this invention, when any variable (e.g., R1, R2, etc.) appears more than once in any component, the definition of each occurrence is independent of the definitions of other occurrences. Similarly, combinations of substituents and variables are permitted, provided such combinations stabilize the compound. Lines drawn from substituents into the ring system indicate that the referred bond can be attached to any substituted ring atom. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic carbon and heteroatom substituents of organic compounds. It will be understood that those skilled in the art can select the substituents and substitution patterns of the compounds of this invention to provide chemically stable compounds that can be readily synthesized from readily available starting materials using techniques in the art and the methods described below. If a substituent itself is substituted by more than one group, it should be understood that these groups can be on the same carbon atom or different carbon atoms, as long as structural stability is achieved.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] The term "salt" refers to a pharmaceutically acceptable salt formed by the compound of the present invention with an acid, which may be an organic or inorganic acid, specifically selected from: phosphoric acid, sulfuric acid, hydrochloric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinic acid, fumaric acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, or analogs thereof.

[0053] The term "halogen" refers to fluorine, chlorine, bromine, and iodine, with fluorine, chlorine, and bromine being preferred.

[0054] The term "halogenated alkyl" refers to an alkyl group that is substituted by at least one halogen atom.

[0055] In addition to standard methods known in the literature or illustrated in experimental procedures, the compounds of the present invention can be prepared using the following synthetic schemes.

[0056] A better understanding of the compounds and synthetic methods described in this invention can be achieved by referring to the following synthetic schemes. The synthetic schemes describe methods that can be used to prepare the compounds described in this invention. These methods are merely illustrative descriptions for explanatory purposes and do not constitute a limitation on the scope of this invention.

[0057] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of protection of the present invention.

[0058] The technical features of the embodiments described below can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the embodiments below are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0059] The following embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

[0060] The present invention will be further described below with reference to specific embodiments:

[0061] Example 1: Synthesis of compound 3

[0062]

[0063] a. Under nitrogen protection, methanesulfonyl chloride (7.7 g, 67 mmol) was added dropwise to a solution of 2-(6-amino-9H-purin-9-yl)ethane-1-ol (10 g, 56 mmol) and triethylamine (17 g, 168 mmol) in tetrahydrofuran (100 mL). The temperature was maintained below 5 °C during the addition, and the mixture was stirred at this temperature for 2 hours after the addition was complete. The reaction mixture was then heated to room temperature and stirred overnight. TLC showed that the reaction was complete. Ice water (200 mL) and dichloromethane (200 mL) were added to the reactants, and the mixture was separated. The organic layer was collected, washed with brine (300 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 5.4 g of ethyl 2-(6-amino-9H-purin-9-yl)methanesulfonate (55% yield).

[0064] b. Potassium carbonate (1.45 equivalents) was added to a DMF (2 mL) solution of ethyl 2-(6-amino-9H-purine-9-yl)methanesulfonate (100 mg, 0.39 mmol) and 4-bromophenol (0.58 mmol). Under nitrogen protection, the mixture was heated to 50 °C and stirred for 16 hours. After cooling the reaction mixture to room temperature, water (10 mL) and dichloromethane (10 mL) were added. The organic layer was collected, washed with brine (3 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product, and purified by thin-layer chromatography on silica gel plates (v / v: ethyl acetate: methanol = 10:1) to give compound 3 (64% yield). 1 ¹H NMR (400MHz, DMSO-d⁶): δ 4.36 (t, J = 5.2Hz, 2H), 4.53 (t, J = 5.2Hz, 2H), 6.89 (d, J = 7.2Hz, 2H), 7.23 (s, 2H), 7.43 (d, J = 8.8Hz, 2H), 8.15 (s, 1H), 8.18 (s, 1H). LC-MS (ES⁺): m / z 334.85, [M⁺H]⁺. tR = 2.247 min. HPLC purity: 99.9%.

[0065] Example 2: Synthesis of compound 4

[0066]

[0067] a. Adenine (10 g, 56 mmol), 1,2-dibromoethane (18.7 g, 100 mmol), and potassium carbonate (27.6 g, 150 mmol) were added to DMF (90 mL). The mixture was heated to 50 °C and stirred overnight under a nitrogen atmosphere. TLC showed that the reaction was complete. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and recrystallized from it with ethanol / water at a volume ratio of 5:1 to give 5.67 g of 9-(2-bromoethyl)-9H-purine-6-amine (35% yield).

[0068] b. 9-(2-Bromoethyl)-9H-purine-6-amine (100 mg, 0.41 mmol), 4-iodophenol (182 mg, 0.83 mmol), and potassium carbonate (172 mg, 1.23 mmol) were added to acetonitrile, heated to 50 °C under nitrogen protection, and stirred for 16 hours. The reaction mixture was cooled to room temperature, and water and ethyl acetate were added. The organic layer was collected, washed with brine (3 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. This crude product was purified by preparative chromatography to give compound 4 (8% yield). 1H NMR (400MHz, DMSO-d6) is: δ4.35(t,J=5.2Hz,2H), 4.52(t,J=5.2Hz,2H), 6.79(d, J=8.8.2Hz,2H),7.22(br,2H),7.56(d,J=8.8Hz,2H),8.15(s,1H),8.17(s,1H). LC_MS:(ES + ):m / z 382.05,[M+H] + . t R =1.997 min. HPLC purity is 97.7%.

[0069] Example 3: Synthesis of Compound 5

[0070]

[0071] Under nitrogen protection and ice bath cooling, diisopropyl azodicarbonate (226 mg, 1.12 mmol) was added dropwise to a solution of 2-(6-amino-9H-purin-9-yl)ethane-1-ol (100 mg, 0.56 mmol), 4-trifluoromethylphenol (109 mg, 0.67 mmol), and triphenylphosphine (293 mg, 1.12 mmol) in tetrahydrofuran (5 mL). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. The reaction mixture was poured into ice water (5 mL) and ethyl acetate (20 mL). The organic layer was collected, washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. Purification by silica gel column chromatography (dichloromethane containing 5% methanol) gave the compound 9-(2-(4-(trifluoromethyl)phenoxy)ethyl)-9H-purin-6-amine (24% yield). 1 HNMR (400MHz, DMSO-d6) is: δ4.46(t,J=4.8Hz,2H), 4.57(t,J=5.2Hz,2H), 7.11( d,J=8.4Hz,2H),7.21(s,2H),7.62(d,J=8.8Hz,2H),8.15(s,1H),8.19(s,1H). LC_MS:(ES + ):m / z324.55,[M+H] + . t R = 2.284 min. HPLC purity is 97.5%.

[0072] Comparative Example 1: Synthetic Compound 6

[0073]

[0074] Compound 6 was prepared in a manner similar to the synthesis of compound 5 in Example 3. 9-(2-(4-methoxyphenoxy)ethyl)-9H-purine-6-amine (41% yield) was synthesized from 4-methoxyphenol (166 mg, 1.34 mmol). The compound... 1 HNMR (400MHz, DMSO-d6) is: δ3.67(s,3H),4.29-4.50(m,4H),6.80-6.86(m,4H),7.23(s,2H),8.15(s,1H),8.17(s,1H). LC_MS:(ES + ):m / z 286.05[M+H] + . t R =1.849min.

[0075] Comparative Example 2: Synthetic Compound 9

[0076]

[0077]

[0078] a. Under nitrogen protection, a DMF solution of p-cresol (6 g, 55.5 mmol) and cesium carbonate (27.11 g, 83.2 mmol) in 60 mL was stirred for 30 min. Then, tert-butyl (2-bromoethyl)carbamate (12.44 g, 55.5 mmol) was added, and the mixture was heated to 100 °C and stirred overnight. TLC showed that the reaction was complete. The reaction mixture was cooled to room temperature, ethyl acetate (100 mL) was added, and the mixture was stirred for 30 min and then filtered. Water (250 mL) and ethyl acetate (150 mL) were added to the filtrate. The organic layer was collected, washed with brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the crude product. Purification was achieved by silica gel column chromatography (using n-hexane containing 2.4% ethyl acetate) to give the oily tert-butyl (2-(p-tolyloxy)ethyl)carbamate (39% yield). LC_MS: (ES+): m / z 252[M+H]+. tR=3.178min.

[0079] b. 5.47 g of 2-(p-toluoxy)ethyl)carbamate tert-butyl ester was added to 50 mL of 4 mol HCl / 1,4-dioxane and stirred overnight at room temperature. TLC showed that the reaction was complete. The mixture was filtered, and the filter cake was dried under reduced pressure to give a white solid 2-(p-toluoxy)ethane-1-amine hydrochloride (65.6% yield). 1H NMR (400MHz, CD3OD): 2.22 (s, 3H), 3.358 (t, J = 4.6 Hz, 2H), 4.20 (t, J = 4.4 Hz, 2H), 6.89 (d, J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H). LC_MS:(ES + ):m / z 152.10[M+H] + tR = 1.706 min.

[0080] c. A solution of 2-(p-tolyloxy)ethyl-1-amine hydrochloride (1 g, 5.35 mmol), 4,6-dichloropyrimidin-5-amine (587 mg, 3.58 mmol), and diisopropylethylamine (1.73 g, 13.4 mmol) in n-butanol (14 mL) was stirred overnight at 110 °C. TLC showed the reaction was complete. The reaction mixture was cooled to room temperature, and water (30 mL) and dichloromethane (30 mL) were added. The organic layer was collected, washed with brine (50 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (using n-hexane containing 33% ethyl acetate) to give 6-chloro-N4-(2-(p-tolyloxy)ethyl)pyrimidin-4,5-diamine (68.5% yield). LC-MS:(ES) + ):m / z 280.15,[M+H] + tR = 2.689 min.

[0081] d. A solution of 6-chloro-N4-(2-(p-tolyloxy)ethyl)pyrimidin-4,5-diamine (500 mg, 1.79 mmol) and p-toluenesulfonic acid (34 mg, 0.179 mmol) in triethyl orthoformate (2.65 g, 17.9 mmol) was stirred overnight at 115 °C. TLC showed that the reaction was complete. The reaction mixture was cooled to room temperature and filtered. The filter cake was dried under reduced pressure to give 6-chloro-9-(2-(p-tolyloxy)ethyl)-9H-purine (68% yield). 1 H NMR (400MHz, DMSO-d6): 2.21 (s, 3H), 4.37 (t, J = 5.2Hz, 2H), 4.68 (t, J = 5.2Hz ,2H),6.77(d,J=8.4Hz,2H),7.05(d,J=8.0Hz,2H),8.75(s,1H),8.80(s,1H). LC_MS:(ES + ):m / z 290.00[M+H] + tR = 2.721 min.

[0082] e. 6-Chloro-9-(2-(p-tolyloxy)ethyl)-9H-purine (100 mg, 0.35 mmol) was added to ammonia water (3 mL), heated to 100 °C, and stirred overnight. TLC showed that the reaction was complete. Water (10 mL), dichloromethane (30 mL), and methanol (3 mL) were added. The organic layer was collected, washed with brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 9-(2-(p-tolyloxy)ethyl)-9H-purine-6-amine (96% yield). 1 H NMR (400MHz, DMSO-d6): δ2.20(s,3H).4.31(t,J=5.4Hz,2H),4.51(t,J=5.2Hz,2H), 6.79(d,J=8.4Hz,2H),7.05(d,J=8.0Hz,2H),7.19(s,2H),8.14(s,1H),8.15(s,1H). LC_MS:(ES + ):m / z 270.40[M+H] + tR = 2.079 min. HPLC purity was 95.8%.

[0083] Example 4: Synthesis of compound 10

[0084]

[0085] The preparation of target compound 10 was similar to the synthesis of compound 9 in Comparative Example 2. 4-(2-(6-amino-9H-purine-9-yl)ethoxy)benzonitrile (78% yield) was synthesized from 4-hydroxybenzonitrile (3 g, 25.18 mmol) via a 5-step reaction. 1 H NMR (400MHz, DMSO-d6): δ4.47(t,J=5.2Hz,2H),4.56(t,J=5.2Hz,2H),7.09(d ,J=8.8Hz,2H),7.19(s,2H),7.40(d,J=8.8Hz,2H),8.14(s,1H),8.18(s,1H). LC_MS:(ES + ):m / z281.40[M+H] + tR = 1.601 min. HPLC purity was 98.5%.

[0086] Example 5: Synthesis of compound 14

[0087]

[0088] The preparation of target compound 14 was similar to the synthesis of compound 3 in Example 1. 4-(2-(6-amino-9H-purine-9-yl)ethoxy)-2-chlorobenzyl nitrile (16% yield) was synthesized from 4-hydroxy-2-chlorobenzonitrile (101 mg, 0.661 mmol). 1 H NMR (400MHz, DMSO-d6): δ4.51-4.55(m,4H),7.07-7.10(m,1H),7.26(s,2H) ,7.36-7.37(m,1H),7.84-7.87(d,J=8.8Hz,1H),8.15(s,1H),8.18(s,1H). LC_MS:(ES + ):m / z356.3[M+H+MeCN] + tR = 0.997 min. HPLC purity is 99.4%.

[0089] Example 6: Synthesis of compound 15

[0090]

[0091] The preparation of target compound 15 was similar to the synthesis of compound 3 in Example 1. 4-(2-(6-amino-9H-purine-9-yl)ethoxy)phthalonitrile (14% yield) was synthesized from 3,4-dicyanophenol (101 mg, 0.7 mmol). 1 H NMR (400MHz, DMSO-d6): δ4.56(s,4H),7.23(s,2H),7.43-7.46(m,1H),7.79-7.80(m,1H),8.01-8.04(m,1H),8.14(s,1H),8.18(s,1H). LC_MS:(ES + ):m / z 306.30[M+H] + tR = 0.893 min. HPLC purity is 99.2%.

[0092] Example 7: Synthesis of compound 17

[0093]

[0094] The preparation of target compound 17 was similar to the synthesis of compound 4 in Example 2. 9-(2-(4-bromo-2-fluorophenoxy)ethyl)-9H-purine-6-amine (11% yield) was synthesized from 4-bromo-2-fluorophenol (79 mg, 0.413 mmol). 1H NMR (400MHz, DMSO-d6): δ4.46(t,J=5.2Hz,2H), 4.55(t,J=5.2Hz,2H), 7.14(t,J=9.2Hz,1H), 7.21(s,2H),7.28-7.32(m,1H),7.48-7.52(dd,J=2.4,11.2Hz,1H),8.13(s,1H),8.14(s,1H). LC_MS:(ES + ):m / z 352.05[M+H] + tR = 1.950 min. HPLC purity was 94.9%.

[0095] Example 8: Synthesis of compound 18

[0096]

[0097] The preparation of target compound 18 was similar to the synthesis of compound 4 in Example 2. 9-(2-(4-bromo-3-fluorophenol)ethyl)-9H-purine-6-amine (17% yield) was synthesized from 4-bromo-3-fluorophenol (158 mg, 0.826 mmol). 1 H NMR (400MHz, DMSO-d6): δ4.39(t,J=5.2Hz,2H), 4.53(t,J=5.2Hz,2H), 6.76(dd,J=2.0,8.8Hz, 1H),7.7(dd,J=2.8,10.8Hz,1H),7.19(s,2H),7.55(t,J=8.4Hz,1H),8.14(s,1H),8.17(s,1H). LC_MS:(ES + ):m / z 352.00[M+H] + tR = 1.957 min. HPLC purity is 99.8%.

[0098] Example 9: Synthesis of compound 19

[0099]

[0100] The preparation of target compound 19 was similar to the synthesis of compound 3 in Example 1. 5-(2-(6-amino-9H-purine-9-yl)ethoxy)pyridinium (21% yield) was synthesized from 2-cyano-5-hydroxypyridine (101 mg, 0.7 mmol). 1HNMR (400MHz, DMSO-d6): δ4.57(s,4H),7.23(s,2H),7.60(t,J=5.6Hz,1H),7.98(d,J=8.4Hz,1H),8.14(s,1H),8.20(s,1H),8.41(d,J=2.8Hz,1H). LC_MS:(ES + ):m / z 282.10[M+H] + tR = 0.839 min. HPLC purity was 97.0%.

[0101] Comparative Example 3: Synthetic Compound 20

[0102]

[0103] a. 4-(2-(6-amino-9H-purin-9-yl)ethoxy)benzonitrile (200 mg, 0.7 mmol) and N-bromosuccinimide (255.97 mg, 1.4 mmol) were added to N,N-dimethylformamide (8 mL) and stirred overnight at room temperature. TLC showed that the starting material remained. N-bromosuccinimide (255.97 mg, 1.4 mmol) was added, and stirring continued for 24 hours. TLC showed that the reaction was complete. Water (30 mL) and dichloromethane (30 mL) were added, the organic layer was collected, washed with brine (30 mL), and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure, preliminarily purified by silica gel column chromatography (using dichloromethane containing 5% methanol), and then purified by preparative HPLC to give 4-(2-(6-amino-8-bromo-9H-purin-9-yl)ethoxy)benzonitrile (40% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6): 4.48 (t, J = 4.8 Hz, 2H), 4.54 (t, J = 4.8 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 7.39 (s, 2H), 7.72 (d, J = 8.8 Hz, 2H), 8.14 (s, 1H). LC_MS:(ES + ):m / z360.30[M+H] + tR = 2.142 min. HPLC purity was 97.6%.

[0104] b. 4-(2-(6-amino-8-bromo-9H-purin-9-yl)ethoxy)benzonitrile (50 mg, 0.13 mmol) and thiourea (53 mg, 0.69 mmol) were added to n-butanol (5 mL) and stirred overnight at room temperature. TLC showed that the reaction was complete. The reaction mixture was diluted with a mixed solution (dichloromethane:methanol 4:1, 12.5 mL). The resulting mixture was washed with brine (25 mL * 3). The organic layer was collected, dried over anhydrous sodium sulfate, concentrated under reduced pressure to give a crude product, which was purified by preparative HPLC to give 4-(2-(6-amino-8-mercapto-9H-purin-9-yl)ethoxy)benzonitrile (94% yield) as a white solid. 1 H NMR (400MHz, DMSO-d6): 4.51 (s, 4H), 6.89 (s, 2H), 7.05 (d, J = 8.8Hz, 2H), 7.72 (d, J = 8.8Hz, 2H), 8.16 (s, 1H), 12.41 (s, 1H). LC_MS:(ES + ):m / z 313.20[M+H] + tR = 2.186 min. HPLC purity was 97.2%.

[0105] Example 10: Synthesis of compound 21

[0106]

[0107] a. 4-(2-((5-amino-6-chloropyrimidin-4-yl)amino)ethoxy)benzonitrile (210 mg, 0.72 mmol) and 1,1'-carbonyldiimidazole (235 mg, 1.4 mmol) were added to N,N-dimethylformamide (3 mL) and stirred overnight at 90 °C under nitrogen protection. TLC showed the reaction was complete. The reaction mixture was poured into water (200 mL) and extracted with dichloromethane (30 mL). The crude product was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane containing 5% methanol) to give a yellow solid 4-(2-(6-chloro-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile (43% yield). LC-MS:(ES) + ):m / z 315.90[M+H] + tR = 2.291 min.

[0108] b. 4-(2-(6-chloro-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile (30 mg, 0.095 mmol), (2,4-dimethoxyphenyl)methylamine (26.47 mg, 0.158 mmol), and diisopropylethylamine (30.69 mg, 0.24 mmol) were added to n-butanol (1 mL), heated to 110 °C, and stirred overnight. TLC showed that the reaction was complete. After concentration under reduced pressure, water (10 mL) and ethyl acetate (10 mL) were added, and the organic phase was collected and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (n-hexane containing 10% ethyl acetate) to give a yellow solid 4-(2-(6-((2,4-dimethoxybenzyl)amino)-8-oxo-7,8-dihydro-9H-purin-9-yl)ethoxy)benzonitrile (40% yield). LC_MS:(ES) + ):m / z 447.55[M+H] + tR = 2.579 min.

[0109] c. 4-(2-(6-((2,4-dimethoxybenzyl)amino)-8-oxo-7,8-dihydro-9H-purin-9-yl)ethoxy)benzonitrile (49.7 mg, 0.11 mmol) and trifluoroacetic acid (2 mL) were added to dichloromethane (2 mL) and stirred overnight at room temperature. TLC showed that the reaction was complete. The dichloromethane and excess trifluoroacetic acid were removed by concentration under reduced pressure. The residue was poured into water (20 mL) and alkalized to pH 8 with aqueous sodium hydroxide solution (2N). The product was extracted with dichloromethane, the organic layer was washed with brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to give crude product, and purified by preparative HPLC to give 4-(2-(6-amino-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile (37% yield). 1 H NMR (400MHz, DMSO-d6: 4.14 (t, J = 5.6 Hz, 2H), 4.41 (t, J = 5.6 Hz, 2H), 7.10-7.02 (m, 2H), 7.77-7.69 (m, 2H), 8.08 (s, 1H), 10.28 (s, 1H). LC_MS: (ES + ):m / z 297.35[M+H] + tR = 1.793 min. HPLC purity was 97.7%.

[0110] Comparative Example 4: Synthetic Compound 22

[0111]

[0112] 4-(2-(6-amino-9H-purin-9-yl)ethoxy)benzonitrile (200 mg, 0.7 mmol) and N-bromosuccinimide (255.97 mg, 1.4 mmol) were added to N,N-dimethylformamide (8 mL) and stirred overnight at room temperature. TLC showed that the starting material remained. N-bromosuccinimide (255.97 mg, 1.4 mmol) was added, and stirring continued for 24 hours. TLC showed that the reaction was complete. Water (30 mL) and dichloromethane (30 mL) were added, the organic layer was collected, washed with brine (30 mL), and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure, preliminarily purified by silica gel column chromatography (using dichloromethane containing 5% methanol), and then purified by preparative HPLC to give 4-(2-(6-amino-8-bromo-9H-purin-9-yl)ethoxy)benzonitrile (40% yield) as a white solid. 1 ¹H NMR (400MHz, DMSO-d⁶): 4.48 (t, J = 4.8Hz, 2H), 4.54 (t, J = 4.8Hz, 2H), 7.05 (d, J = 8.8Hz, 2H), 7.39 (s, 2H), 7.72 (d, J = 8.8Hz, 2H), 8.14 (s, 1H). LC-MS (ES⁺): m / z 360.30 [M⁺H]⁺. tR = 2.142 min. HPLC purity: 97.6%.

[0113] The bioactivity of the above compounds was determined by an enzyme activity assay at the NSD2 and NSD3 protein levels. The results were the average of at least three independent tests, and are shown in Table 1 below. NA (non-detectable activity) and ND (not tested) were also included.

[0114] Table 1. Inhibitory activities of compounds on NSD2 and NSD3 protein-level enzymes.

[0115]

[0116]

[0117] The above compound 3 and the control compound SAM (whose structural formula is...)

[0118] The bioactivity of NSD3-SET was determined using the SPR assay, which measured the binding affinity of both NSD3-SET to the protein. The results are shown in Table 2 below.

[0119] Table 2. Kd values ​​of compounds and NSD3-SET protein

[0120] compound <![CDATA[K d (μM)]]> 3 5.51 SAM 25

[0121] The bioactivity of compound 3 was determined by an epigenetic modifying enzyme selectivity assay. The IC50 values ​​of its inhibitory activity against the epigenetic modifying enzymes NSD2, NSD3, SETD2, SMYD3, G9a, PRMT1, and PRMT4 were measured, and the results are shown in Table 3 below.

[0122] Table 3 Selectivity of epigenetic modifying enzymes of compound 3

[0123] enzymes IC50 (μM) NSD2 0.81±0.04 NSD3 0.84±0.06 SETD2 >20 SMYD3 >20 G9a >20 PRMT1 >20 PRMT4 >20

[0124] As shown in Table 3, compound 3 specifically inhibits NSD2 and NSD3, but does not show significant activity against other epigenetic modifying enzymes.

[0125] Cellular activity assays were performed on compounds 2, 3, 10, 14, 15, 18, 19, and 20. Western blotting was used to determine the effects of these compounds on the NSDs substrate H3K36me2 within cells. The results are as follows: Figure 1 As shown, compounds 2, 3, 10, 14, 15, 18, 19, and 21 can effectively reduce the levels of NSD2 and NSD3 substrate H3K36me2 in the H460 and H1299 non-small cell lung cancer cell lines.

[0126] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. The use of a purine derivative of formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a drug for treating tumors, wherein the tumor is lung cancer. （I）， in: R1 is selected from Br or cyano; R2 is selected from hydrogen, chlorine, fluorine, or cyano; R3 is selected from hydrogen; R4 is selected from hydrogen.

2. The use of purine derivatives or pharmaceutically acceptable salts thereof in the preparation of drugs for treating tumors, characterized in that: The purine derivatives are selected from compounds with the following structures: ； The tumor is non-small cell lung cancer.

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