Use of purine derivatives as nsd2 and nsd3 inhibitors

Abstract

The present application relates to a kind of purine derivatives as the use of NSD2 and NSD3 inhibitor, the structural formula of the derivative is as shown in formula (I) or formula (II).The compound shows good NSD2, NSD3 inhibitory activity, can effectively reduce the methylation level of H3K36 in cell, and cause the reduction of cell proliferation activity of NSD2, NSD3 high expression tumor cell strain.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, in particular to the use of a purine derivative as an NSD2 and NSD3 inhibitor. BACKGROUND

[0002] Dynamic modification of histone methylation is one of the important mechanisms of epigenetic modification, which is regulated by various histone methyltransferases and demethylases, and has become a hot spot in the research and development of anti-tumor drugs in recent years. In 2020, the histone methyltransferase EZH2 inhibitor tazemetostat developed by EPZM company was officially approved for the treatment of epithelioid sarcoma, becoming the first anti-tumor drug targeting histone methyltransferase. At present, a variety of histone methyltransferase inhibitors in the research and development stage or clinical stage have been reported, and the development of drugs targeting this target is still in its infancy. The NSD subfamily belongs to the histone methyltransferase family, including three member proteins NSD1, NSD2 (WHSC1) and NSD3 (WHSC1L1), which are mainly responsible for the methylation modification of multiple lysine sites of histone H3 and are closely related to the process of cell proliferation, and play an important role in the occurrence and development of tumors. Specifically, the functions of NSD2 and NSD3 are to catalyze the dimethylation or trimethylation of the 36th LYS residue of the tail of histone H3, thereby changing the binding mode of histone and DNA. Studies have found that NSD2 and NSD3 appear abnormal gene amplification, protein expression up-regulation or gene fusion in various types of tumors. In lung cancer, breast cancer, liver cancer, bladder cancer, head and neck squamous cell carcinoma, colorectal cancer and other tumor cell lines or tissue samples, it is found that NSD3 protein exists abnormally high expression, which has the role of promoting tumor formation; in lung cancer, liver cancer, breast cancer, head and neck cancer, colorectal cancer, bladder cancer, pancreatic cancer, ovarian cancer, it is found that NSD3 gene or the 8p11-12 chromosomal interval where it is located appears abnormal gene amplification, which is considered to be a driver gene of tumor occurrence; in acute myeloid leukemia and small cell lung cancer, NSD3 gene is found to exist abnormal gene fusion with NUP98 and NUT, respectively; in osteosarcoma, knocking out NSD3 gene causes cell cycle arrest at G2 / M phase and induces apoptosis of tumor cells. NSD2 is highly expressed in bladder cancer, lung cancer, colon cancer, liver cancer, myeloma and other tumors. Therefore, NSD2 and NSD3 are considered as potential anti-tumor targets.

[0003] NSD2, NSD3 protein is composed of PWWP1, PHD1, PHD2, PHD3, PWWP2, Pre-SET, SET, Post-SET, PHD4 and other functional domains. Among them, the Pre-SET, SET and Post-SET domains form the catalytic domain of the protein, which is responsible for binding the methyl donor SAM and the histone substrate peptide, and completes the catalytic function of methyl transfer. The PWWP1 domain is responsible for binding BRD4 and CHD8, and is involved in regulating AML-related genes. Although the mechanism and molecular structure of NSD2, NSD3 have been understood to some extent, the research on small molecule compounds regulating the function of NSD2, NSD3 is relatively less.

[0004] For NSD3 inhibitors, in 2019 Coussens et al. reported a chemical probe BI-9321 binding to the PWWP1 domain of NSD3 protein, which can down-regulate Myc messenger RNA and inhibit the proliferation of MOLM-13 cells. Structural analysis further found that BI-9321 binds to the methylation lysine binding pocket of the PWWP1 domain.

[0005] For NSD2 inhibitors, there are 5-aminonaphthalene series compounds in the prior art that can target the NSD2-SET domain. In addition, Coussens et al. reported a number of compounds screened from the old drug library, including DA3003-1, PF-03882845, T C LPA5 4, ABT-199, DZNep, Chaetocin, which can bind to the NSD2 SET domain in the SPR experiment.

[0006] However, the research on small molecule compounds regulating the function of NSD2, NSD3 is relatively less, and there is no strong inhibitor that can target NSD2 and NSD3 at the same time. SUMMARY

[0007] The technical problem to be solved by the present application is to provide the use of a purine derivative as an NSD2 and NSD3 inhibitor to overcome the shortcomings and deficiencies of the prior art.

[0008] To solve the above technical problems, the present application adopts the following technical solutions:

[0009] The use of a purine derivative represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as an NSD2 and NSD3 inhibitor,

[0010]

[0011] Among them:

[0012] R1is selected from the group consisting of hydrogen, halogen, cyano, amino, -NHCOR7, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C

[0013] R2is selected from the group consisting of hydrogen, halogen or cyano;

[0014] R3is selected from the group consisting of hydrogen, halogen or cyano;

[0015] R4is selected from the group consisting of hydrogen or hydroxyl;

[0016] R5is selected from the group consisting of hydrogen, halogen, cyano, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C

[0017] R6is selected from the group consisting of hydrogen, halogen or cyano.

[0018] In some embodiments, R1is selected from the group consisting of hydrogen, halogen, cyano, amino, -NHCOR7, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl.

[0019] In some embodiments, R1is selected from the group consisting of halogen, cyano, amino, -NHCOR7, C 1-6 alkyl.

[0020] In some embodiments, R1is selected from the group consisting of F, Cl, Br, I, cyano or CF3.

[0021] In some embodiments, R2is selected from the group consisting of hydrogen, F, Cl or cyano.

[0022] In some embodiments, R3is hydrogen or F.

[0023] In some embodiments, R4is hydroxyl and R1is cyano.

[0024] In some embodiments, R5is selected from the group consisting of hydrogen, halogen, cyano, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl.

[0025] In some embodiments, R6is hydrogen.

[0026] In some embodiments, R5is cyano.

[0027] In some embodiments, the purine derivatives are selected from compounds with the following structures:

[0028]

[0029]

[0030]

[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 derivative represented by the structure of formula (I) or formula (II) of the present application shows good inhibitory activity of NSD2 and NSD3 in the biochemical experiment at the protein level, the SPR experiment proves that the purine derivative has micromolar affinity with NSD3, the purine derivative can effectively reduce the methylation level of H3K36 in cells, and cause the reduction of cell proliferation activity of NSD2 and NSD3 high expression tumor cell strains, and therefore can be used as an NSD2 and NSD3 inhibitor, and used for preparing an anti-tumor drug. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The protein blotting experiment result graph of some compounds of the present application. DETAILED DESCRIPTION

[0049] TERMS

[0050] In the compounds of the present application, when any variable (e.g., R1, R2, etc.) occurs more than once in any constituent, its meaning is independent for each occurrence. Also, combinations of substituents and variables are permissible only if such combinations result in stable compounds. A dash ("-") that is not between two letters or symbols is used to indicate a point of attachment for a given chemical moiety. In a broad aspect, permissible substituents on the alkyl groups include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic, carbon- and heteroatom-containing groups. It is understood that one of ordinary skill in the art can select substituents and substitution patterns for compounds of the present application that result in stable compounds that are easily synthesized, chemically stable, and non-toxic for use as therapeutics. If a substituent is itself substituted with more than one group, the substituent groups can be on the same carbon atom or different carbon atoms, as long as the structure is stable.

[0051] Unless defined otherwise, 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 application belongs.

[0052] The term "salt" refers to a pharmaceutically acceptable salt of the compound of the present application formed with an acid, which can be an organic or inorganic acid, and can be selected from the group consisting of 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, methane sulfonic acid or the like.

[0053] The term "halogen" refers to fluorine, chlorine, bromine, iodine, preferably fluorine, chlorine, bromine.

[0054] The term "haloalkyl" refers to an alkyl group substituted with at least one halogen atom.

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

[0056] The compounds and synthetic methods described in the present application can be better understood in connection with the following synthetic schemes. The synthetic schemes described are illustrative schemes only and are not meant to limit the scope of the application.

[0057] The present application is further described in connection with the following examples which are not meant to limit the scope of the present application.

[0058] The technical features of the following examples can be combined in any manner, and for the sake of brevity, not all possible combinations are described in the following examples, however, any combination of the technical features is deemed to be within the scope of the present specification.

[0059] The following examples are only representative of several embodiments of the present application, which are described in more detail and specifically, but they are not meant to limit the scope of the patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these are all within the scope of the present application. Therefore, the scope of the patent should be determined by the appended claims.

[0060] The present application is further described in connection with the following examples which are not meant to limit the scope of the present application.

[0061] Example 1: Synthesis of 9-(2-(4-fluorophenoxy)ethyl)-9H-purin-6-amine

[0062]

[0063] a. To a solution of 2-(6-amino-9H-purin-9-yl)ethan-1-ol (10 g, 56 mmol) and triethylamine (17 g, 168 mmol) in tetrahydrofuran (100 mL) was added methanesulfonyl chloride (7.7 g, 67 mmol) dropwise while maintaining the temperature at no more than 5 °C. After the addition was complete, the reaction was stirred for an additional 2 h. The reaction was allowed to warm to room temperature and stirred overnight. TLC indicated that the reaction was complete. The reaction was diluted with ice water (200 mL) and dichloromethane (200 mL) and the layers were separated. The organic layer was washed with brine (300 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2-(6-amino-9H-purin-9-yl) ethyl methanesulfonate (55% yield).

[0064] b. To a solution of 2-(6-amino-9H-purin-9-yl)ethyl methanesulfonate (100 mg, 0.39 mmol) and 4-fluorophenol (65 mg, 0.58 mmol) in DMF (2 mL) was added potassium carbonate (1.45 eq). Nitrogen protection, heated to 50 °C, stirred for 16 hours. After the reaction mixture was cooled 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 obtain the crude product, which was purified by thin layer chromatography on silica gel plate (volume ratio: ethyl acetate:methanol = 10:1) to obtain compound 9-(2-(4-fluorophenoxy)ethyl)-9H-purin-6-amine (79% yield). The structure of this compound was confirmed by1H NMR (400 MHz, DMSO-d6) as: δ 4.34 (t, J = 5.2 Hz, 2H), 4.53 (t, J = 5.2 Hz, 2H), 6.91-6.94 (m, 2H), 7.06-7.11 (m, 2H), 7.21 (s, 2H), 8.15 (s, 1H), 8.17 (s, 1H), LC_MS: (ES): m / z 274.00 [M+H] 1 H NMR (400 MHz, DMSO-d6) as: δ 4.34 (t, J = 5.2 Hz, 2H), 4.53 (t, J = 5.2 Hz, 2H), 6.91-6.94 (m, 2H), 7.06-7.11 (m, 2H), 7.21 (s, 2H), 8.15 (s, 1H), 8.17 (s, 1H), LC_MS: (ES + ): m / z 274.00 [M+H] + . t R = 1.962 min. HPLC purity was 99.3%.

[0065] Example 2: Synthesis of 9-(2-(4-chlorophenoxy)ethyl)-9H-purin-6-amine

[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) and the mixture was heated to 50 °C and stirred under nitrogen atmosphere overnight. TLC showed that the reaction was complete. The reaction was cooled to room temperature and filtered. The filtrate was concentrated and recrystallized with ethanol / water in a volume ratio of 5:1 to obtain 9-(2-bromoethyl)-9H-purin-6-amine (35% yield).

[0068] b. 9-(2-bromoethyl)-9H-purine-6-amine (100 mg, 0.41 mmol), 4-chlorophenol (107 mg, 0.82 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 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 obtain the crude product. The crude product was purified by preparative chromatography to give compound 9-(2-(4-chlorophenoxy)ethyl)-9H-purine-6-amine (13% yield). 1 H NMR (400MHz, DMSO-d6) is: δ4.38(t,J=4.8Hz,2H), 4.53(t,J=5.2Hz,2H), 6.93(d ,J=8.8Hz,2H),7.32(d,J=8.8Hz,2H),8.41(s,1H),8.45(s,1H),8.60(br,2H). LC_MS:(ES + ):m / z 291.00,[M+H] + . t R = 2.132 min. HPLC purity is 98.3%.

[0069] Example 3: Synthesis of 9-(2-(4-bromophenoxy)ethyl)-9H-purine-6-amine

[0070]

[0071] The preparation of target compound 3 was similar to the synthesis of 9-(2-(4-fluorophenoxy)ethyl)-9H-purine-6-amine in Example 1. 9-(2-(4-bromophenoxy)ethyl)-9H-purine-6-amine was synthesized from 4-bromophenol (64% yield). This compound... 1 ¹H NMR (400 MHz, DMSO-d⁶): δ 4.36 (t, J = 5.2 Hz, 2H), 4.53 (t, J = 5.2 Hz, 2H), 6.89 (d, J = 7.2 Hz, 2H), 7.23 (s, 2H), 7.43 (d, J = 8.8 Hz, 2H), 8.15 (s, 1H), 8.18 (s, 1H). LC-MS (ES⁺): m / z 334.85, [M⁺H]⁺. tR = 2.247 min. HPLC purity was 99.9%.

[0072] Example 4: Synthesis of 9-(2-(4-iodophenoxy)ethyl)-9H-purine-6-amine

[0073]

[0074] The compound 4 was prepared in a similar manner as the synthesis of 9-(2-(4- chlorophenoxy)ethyl)-9H-purin-6-amine in Example 2. 9-(2-(4-Iodophenoxy)ethyl)- 9H-purin-6-amine was synthesized (8% yield) using 4-iodophenol (182 mg, 0.83 mmol) as starting material. The compound was characterized by1H NMR (400 MHz, DMSO-d6) as: δ 4.35 (t, J = 5.2 Hz, 2H), 4.52 (t, J = 5.2 Hz, 2H), 6.79 (d, J = 8.8.2 Hz, 2H), 7.22 (br, 2H), 7.56 (d, J = 8.8 Hz, 2H), 8.15 (s, 1H), 8.17 (s, 1H). LC_MS: (ES): m / z 382.05, [M+H] 1 H NMR (400 MHz, DMSO-d6) as: δ 4.35 (t, J = 5.2 Hz, 2H), 4.52 (t, J = 5.2 Hz, 2H), 6.79 (d, J = 8.8.2 Hz, 2H), 7.22 (br, 2H), 7.56 (d, J = 8.8 Hz, 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 was 97.7%.

[0075] Example 5: Synthesis of 9-(2-(4-(trifluoromethyl)phenoxy)ethyl)-9H-purin-6- amine

[0076]

[0077] To a solution of 2-(6-amino-9H-purin-9-yl)ethan-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) was added dropwise diisopropyl azodicarboxylate (226 mg, 1.12 mmol) under nitrogen protection and ice bath cooling. After the addition was completed, it was raised 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 obtain a crude product. Purification by silica gel column chromatography (5% methanol in dichloromethane) to obtain compound 9-(2-(4-(trifluoromethyl)phenoxy)ethyl)-9H-purin-6-amine (24% yield). The compound was characterized by1H NMR (400 MHz, DMSO-d6) as: δ 4.46 (t, J = 4.8 Hz, 2H), 4.57 (t, J = 5.2 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 7.21 (s, 2H), 7.62 (d, J = 8.8 Hz, 2H), 8.15 (s, 1H), 8.19 (s, 1H). LC_MS: (ES): m / z 324.55, [M+H] 1 H NMR (400 MHz, DMSO-d6) as: δ 4.35 (t, J = 5.2 Hz, 2H), 4.52 (t, J = 5.2 Hz, 2H), 6.79 (d, J = 8.8.2 Hz, 2H), 7.22 (br, 2H), 7.56 (d, J = 8.8 Hz, 2H), 8.15 (s, 1H), 8.17 (s, 1H). LC_MS: (ES + ): m / z 382.05, [M+H] + . t R= 2.284 min. HPLC purity was 97.5%.

[0078] Example 6: Synthesis of 9-(2-(4-aminophenoxy)ethyl)-9H-purin-6-amine

[0079]

[0080] The target compound 9-(2-(4-methoxyphenoxy)ethyl)-9H-purin-6-amine was synthesized in a similar manner as 9-(2-(4-(trifluoromethyl)phenoxy)ethyl)-9H-purin-6-amine in Example 5. 9-(2-(4- Methoxyphenoxy)ethyl)-9H-purin-6-amine was synthesized (41% yield) using 4- methoxyphenol (166 mg, 1.34 mmol) as starting material. The compound had H NMR (400 MHz, DMSO-d6) δ 3.67 (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] 1 + + t R = 1.849 min.

[0081] Example 7: Synthesis of 9-(2-(4-aminophenoxy)ethyl)-9H-purin-6-amine

[0082]

[0083] a.The target compound 9-(2-(4-nitrophenoxy)ethyl)-9H-purin-6-amine was synthesized in a similar manner as 9-(2-(4-fluorophenoxy)ethyl)-9H-purin-6-amine in Example 1. 9-(2-(4-Nitrophenoxy)ethyl)-9H-purin-6-amine was synthesized (41% yield) using 4-nitrophenol (736 mg, 5.29 mmol) as starting material. The compound had H NMR (400 MHz, DMSO-d6) δ 4.53 (d, J = 4.4 Hz, 2H), 4.57 (d, J = 4.8 Hz, 2H), 7.13 (d, J = 9.2 Hz, 2H), 7.24 (s, 2H), 8.15 (d, J = 9.2 Hz 2H), 8.15 (s, 1H), 8.20 (s, 1H). LC_MS: (ES): m / z 301.15 [M+H] 1 + + t R = 1.882 min. HPLC purity was 93.4%.

[0084] ​​​​b. 9-(2-(4-nitrophenoxy)ethyl)-9H-purin-6-amine (66 mg, 0.24 mmol), ammonium chloride (50.8 mg, 0.95 mmol) and iron powder (134.4 mg, 2.4 mmol) were added to isopropanol (3 mL) and water (1 mL) and stirred at 90 °C overnight under nitrogen protection. TLC showed the reaction was complete. The reaction mixture was poured into water (10 mL) and dichloromethane (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, which was purified by silica gel column chromatography (5% methanol in dichloromethane) to give 9-(2-(4- aminophenoxy)ethyl)-9H-purin-6-amine (17% yield) as a white solid. The structure of this compound was confirmed by1H NMR. 1 H NMR (400 MHz, DMSO-d6) δ 4.20 (t, J = 5.2 Hz, 2H), 4.55 (t, J = 5.2 Hz, 2H), 6.49 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 7.20 (s, 2H), 8.14 (s, 2H). LC_MS: (ES + ): m / z 270.65, [M+H] + tR= 0.798 min. HPLC purity was 96.3%. R

[0085] Example 8: Synthesis of N-(4-(2-(6-amino-9H-purin-9-yl)ethoxy)phenyl)acetamide

[0086] The target compound 8 was prepared in a similar manner as the synthesis of 9-(2-(4- (trifluoromethyl)phenoxy)ethyl)-9H-purin-6-amine in Example 5. N-(4-(2-(6-amino-9H- purin-9-yl)ethoxy)phenyl)acetamide was synthesized (25% yield) starting from 4N-(4- hydroxyphenyl)acetamide (405 mg, 2.68 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 1.98 (s, 3H), 4.31 (t, J = 5.2 Hz, 2H), 4.51 (t, J = 5.2 Hz, 2H), 6.84 (d, J = 9.2 Hz, 2H), 7.21 (s, 2H), 7.44 (d, J = 8.8 Hz, 2H), 8.14 (s, 1H), 8.16 (s, 1H), 9.76 (s, 1H). LC_MS: (ES + ): m / z 313.15 [M+H] + tR= 1.071 min.

[0087] ​Example 9: Synthesis of 9-(2-(p-tolyloxy)ethyl)-9H-purin-6-amine

[0088]

[0089] a. A solution of p-methylphenol (6 g, 55.5 mmol) and cesium carbonate (27.11 g, 83.2 mmol) in DMF (60 mL) was stirred under nitrogen for 30 min. Then tert-butyl (2-bromoethyl)carbamate (12.44 g, 55.5 mmol) was added and the reaction mixture was warmed to 100 °C and stirred overnight. TLC showed the reaction to be complete. The reaction mixture was cooled to room temperature, ethyl acetate (100 mL) was added and after stirring for 30 min, it was filtered. To the filtrate, water (250 mL) and ethyl acetate (150 mL) were added. The organic layer was collected, washed with brine (100 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to get the crude product. Purification by silica gel column chromatography (2.4% ethyl acetate in n-hexane) gave tert-butyl (2-(p-tolyloxy)ethyl)carbamate as an oil (39% yield). LC_MS: (ES+): m / z 252 [M+H]+. tR= 3.178 min.

[0090] b. tert-Butyl (2-(p-tolyloxy)ethyl)carbamate (5.47 g) was added to 4 mol HCl / 1,4-dioxane (50 mL) and stirred at room temperature overnight. TLC showed the reaction to be complete. It was filtered and the filter cake was dried under reduced pressure to get 2-(p-tolyloxy)ethan-1-amine hydrochloride as a white solid (65.6% yield). 1 H NMR (400 MHz, 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. +

[0091] ​c. A solution of 2-(p-tolyloxy)ethyl-l-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 at 110 °C overnight. TLC showed the reaction was complete. The reaction mixture was cooled to room temperature, 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 (with 33% ethyl acetate in n-hexane) to give 6-chloro-N4-(2-(p-tolyloxy)ethyl)pyrimidine-4,5-diamine (68.5% yield). LC_MS: (ES + ): m / z 280.15, [M+H] + . tR= 2.689 min.

[0092] d. A solution of 6-chloro-N4-(2-(p-tolyloxy)ethyl)pyrimidine-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 at 115 °C overnight. TLC showed 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 (400 MHz, DMSO-d6): 2.21 (s, 3H), 4.37 (t, J = 5.2 Hz, 2H), 4.68 (t, J = 5.2 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.0 Hz, 2H), 8.75 (s, 1H), 8.80 (s, 1H). LC_MS: (ES + ): m / z 290.00 [M+H] + . tR= 2.721 min.

[0093] e. 6-chloro-9-(2-(p-tolyloxy)ethyl)-9H-purine (100 mg, 0.35 mmol) was added to aqueous ammonia (3 mL) and heated to 100 °C with stirring overnight. TLC showed 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, concentrated under reduced pressure to give 9-(2-(p-tolyloxy)ethyl)-9H-purin-6-amine (96% yield). 1H NMR (400 MHz, DMSO-d6): δ 2.20 (s, 3H). 4.31 (t, J = 5.4 Hz, 2H), 4.51 (t, J = 5.2 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.0 Hz, 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 95.8%.

[0094] Example 10: Synthesis of 4-(2-(6-amino-9H-purin-9-yl)ethoxy)benzonitrile

[0095]

[0096] The target compound 10 was synthesized in a manner similar to the synthesis of 9-(2-(p- methoxyphenoxy)ethyl)-9H-purin-6-amine in Example 9. 4-Hydroxybenzonitrile (3 g, 25.18 mmol) was used as the starting material to synthesize 4-(2-(6-amino-9H-purin-9-yl)ethoxy)benzonitrile (78% yield) through 5 steps. 1 H NMR (400 MHz, DMSO-d6): δ 4.47 (t, J = 5.2 Hz, 2H), 4.56 (t, J = 5.2 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 7.19 (s, 2H), 7.40 (d, J = 8.8 Hz, 2H), 8.14 (s, 1H), 8.18 (s, 1H). LC_MS: (ES + ): m / z 281.40 [M+H] + . tR= 1.601 min. HPLC purity 98.5%.

[0097] Example 11: Synthesis of 9-(2-(2-chlorophenoxy)ethyl)-9H-purin-6-amine

[0098]

[0099] The target compound 11 was synthesized in a manner similar to the synthesis of 9-(2-(4- chlorophenoxy)ethyl)-9H-purin-6-amine in Example 2. 2-Chlorophenol (53 mg, 0.41 mmol) was used as the starting material to synthesize 9-(2-(2-chlorophenoxy)ethyl)-9H-purin-6-amine (25% yield). 1H NMR (400 MHz, DMSO-d6): δ 4.43 (t, J = 5.2 Hz, 2H), 4.65 (t, J = 5.2 Hz, 2H), 6.95 (dt, J = 1.2, 7.6 Hz, 1H), 7.15 (dd, J = 1.2, 8.0 Hz, 1H), 7.27 (dt, J = 1.6, 8.8 Hz, 1H), 7.39 (dd, J = 1.6, 7.6 Hz, 1H), 8.32 (s, 1H), 8.35 (s, 1H). LC_MS: (ES + ): m / z 290.15 [M+H] + . tR= 1.736 min. HPLC purity 99.6%.

[0100] Example 12: Synthesis of 9-(2-(3-chlorophenoxy)ethyl)-9H-purin-6-amine

[0101]

[0102] The target compound 12 was prepared in a similar manner as 9-(2-(4- chlorophenoxy)ethyl)-9H-purin-6-amine synthesized in Example 2. 9-(2-(3- chlorophenoxy)ethyl)-9H-purin-6-amine was synthesized (14% yield) using 3- chlorophenol (107 mg, 0.82 mmol) as starting material. 1 H NMR (400 MHz, DMSO-d6): δ 4.43 (t, J = 5.2 Hz, 2H), 4.65 (t, J = 5.2 Hz, 2H), 6.95 (dt, J = 1.2, 7.6 Hz, 1H), 7.15 (dd, J = 1.2, 8.0 Hz, 1H), 7.27 (dt, J = 1.6, 8.8 Hz, 1H), 7.39 (dd, J = 1.6, 7.6 Hz, 1H), 8.32 (s, 1H), 8.35 (s, 1H). LC_MS: (ES + ): m / z 290.10 [M+H] + . tR= 2.121 min. HPLC purity 99.9%.

[0103] Example 13: Synthesis of 2-(2-(6-amino-9H-purin-9-yl)ethoxy)benzonitrile

[0104]

[0105] The target compound 13 was prepared in a similar manner as 9-(2-(4- (trifluoromethyl)phenoxy)ethyl)-9H-purin-6-amine synthesized in Example 5. 2-(2- (6-amino-9H-purin-9-yl)ethoxy)benzonitrile was synthesized (20% yield) using o- hydroxybenzonitrile (80 mg, 0.67 mmol) as starting material. 1H NMR (400 MHz, DMSO-d6): δ 4.50 (t, J = 5.2 Hz, 2H), 4.61 (t, J = 5.2 Hz, 2H), 7.09 (t, J = 7.6 Hz, 1H), 7.22 (s, 2H), 7.25 (t, J = 8.4 Hz, 1H), 7.63 (dt, J = 2.0, 7.6 Hz, 1H), 7.70 (dd, J = 1.6, 7.6 Hz, 1H), 8.14 (s, 1H), 8.15 (s, 1H). LC_MS: (ES + ): m / z 281.60, [M+H] + . tR= 1.632 min. HPLC purity 97.3%.

[0106] Example 14: Synthesis of 4-(2-(6-amino-9H-purin-9-yl)ethoxy)-2- chlorobenzonitrile

[0107]

[0108] The target compound 14 was prepared in a similar manner as 9-(2-(4- fluorophenoxy)ethyl)-9H-purin-6-amine in Example 1. 4-(2-(6-amino-9H-purin-9- yl)ethoxy)-2-chlorobenzonitrile was synthesized (16% yield) starting from 4- hydroxy-2-chlorobenzonitrile (101 mg, 0.661 mmol). 1 H NMR (400 MHz, DMSO-d6): δ 4.50 (t, J = 5.2 Hz, 2H), 4.61 (t, J = 5.2 Hz, 2H), 7.09 (t, J = 7.6 Hz, 1H), 7.22 (s, 2H), 7.25 (t, J = 8.4 Hz, 1H), 7.63 (dt, J = 2.0, 7.6 Hz, 1H), 7.70 (dd, J = 1.6, 7.6 Hz, 1H), 8.14 (s, 1H), 8.15 (s, 1H). LC_MS: (ES + ): m / z 356.3 [M+H+MeCN] + . tR= 0.997 min. HPLC purity 99.4%.

[0109] Example 15: Synthesis of 4-(2-(6-amino-9H-purin-9-yl)ethoxy)phthalonitrile

[0110]

[0111] The target compound 15 was prepared in a similar manner as 9-(2-(4- fluorophenoxy)ethyl)-9H-purin-6-amine in Example 1. 4-(2-(6-amino-9H-purin-9- yl)ethoxy)phthalonitrile was synthesized (14% yield) starting from 3,4- dicyanophenol (101 mg, 0.7 mmol). 1H NMR (400 MHz, DMSO-d6): δ 4.56 (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 99.2%.

[0112] Example 16: Synthesis of 9-(2-(2-chloro-4-methylphenoxy)ethyl)-9H-purin-6-amine

[0113]

[0114] The target compound 16 was prepared in a similar manner as 9-(2-(4- chlorophenoxy)ethyl)-9H-purin-6-amine synthesized in example 2. 9-(2-(2-chloro-4- methylphenoxy)ethyl)-9H-purin-6-amine was synthesized (8% yield) starting from 2- chloro-4-methylphenol (59 mg, 0.413 mmol). 1 H NMR (400 MHz, DMSO-d6): δ 2.21 (s, 3H), 4.38 (t, J = 5.2 Hz, 2H), 4.63 (t, J = 5.2 Hz, 2H), 7.01-7.08 (m, 2H), 7.21 (d, J = 1.2 Hz, 1H), 8.35 s, 1H), 8.38 (s, 1H). LC_MS: (ES + ): m / z 304.10 [M+H] + . tR= 1.923 min. HPLC purity 95.8%.

[0115] Example 17: Synthesis of 9-(2-(4-bromo-2-fluorophenoxy)ethyl)-9H-purin-6-amine

[0116]

[0117] The target compound 17 was prepared in a similar manner as 9-(2-(4- chlorophenoxy)ethyl)-9H-purin-6-amine synthesized in example 2. 9-(2-(4-bromo-2- fluorophenoxy)ethyl)-9H-purin-6-amine was synthesized (11% yield) starting from 4- bromo-2-fluorophenol (79 mg, 0.413 mmol). 1H NMR (400 MHz, DMSO-d6): δ 4.46 (t, J = 5.2 Hz, 2H), 4.55 (t, J = 5.2 Hz, 2H), 7.14 (t, J = 9.2 Hz, 1H), 7.21 (s, 2H), 7.28-7.32 (m, 1H), 7.48-7.52 (dd, J = 2.4, 11.2 Hz, 1H), 8.13 (s, 1H), 8.14 (s, 1H). LC_MS: (ES + ): m / z 352.05 [M+H] + . tR= 1.950 min. HPLC purity 94.9%.

[0118] Example 18: Synthesis of 9-(2-(4-bromo-3-fluorophenoxy)ethyl)-9H-purin-6-amine

[0119]

[0120] The target compound 18 was prepared in a similar manner as 9-(2-(4- chlorophenoxy)ethyl)-9H-purin-6-amine synthesized in Example 2. 9-(2-(4-bromo-3- fluorophenoxy)ethyl)-9H-purin-6-amine was synthesized (17% yield) starting from 4- bromo-3-fluorophenol (158 mg, 0.826 mmol). 1 H NMR (400 MHz, DMSO-d6): δ 4.39 (t, J = 5.2 Hz, 2H), 4.53 (t, J = 5.2 Hz, 2H), 6.76 (dd, J = 2.0, 8.8 Hz, 1H), 7.7 (dd, J = 2.8, 10.8 Hz, 1H), 7.19 (s, 2H), 7.55 (t, J = 8.4 Hz, 1H), 8.14 (s, 1H), 8.17 (s, 1H). LC_MS: (ES + ): m / z 352.00 [M+H] + . tR= 1.957 min. HPLC purity 99.8%.

[0121] Example 19: Synthesis of 5-(2-(6-amino-9H-purin-9-yl)ethoxy)picolinonitrile

[0122]

[0123] The target compound 19 was prepared in a similar manner as 9-(2-(4- fluorophenoxy)ethyl)-9H-purin-6-amine synthesized in Example 1. 5-(2-(6-amino-9H- purin-9-yl)ethoxy)picolinonitrile was synthesized (21% yield) starting from 2-cyano-5- hydroxypyridine (101 mg, 0.7 mmol). 1H NMR (400 MHz, DMSO-d6): δ 4.57 (s, 4H), 7.23 (s, 2H), 7.60 (t, J = 5.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 8.14 (s, 1H), 8.20 (s, 1H), 8.41 (d, J = 2.8 Hz, 1H). LC_MS: (ES + ): m / z 282.10 [M+H] + . tR= 0.839 min. HPLC purity 97.0%.

[0124] Example 20: Synthesis of 4-(2-(6-amino-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile

[0125]

[0126] 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 at 90 °C overnight under nitrogen protection. TLC showed the reaction was complete. The reaction mixture was poured into water (200 mL) and extracted with dichloromethane (30 mL). Concentrated under reduced pressure to get the crude product, which was purified by silica gel column chromatography (5% methanol in dichloromethane) to get 4-(2-(6-chloro-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile as a yellow solid (43% yield). LC_MS: (ES + ): m / z 315.90 [M+H] + . tR= 2.291 min.

[0127] b. 4-(2-(6-chloro-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile (30 mg, 0.095 mmol), (2,4-dimethoxyphenyl)methanamine (26.47 mg, 0.158 mmol) and diisopropylethylamine (30.69 mg, 0.24 mmol) were added to n-butanol (1 mL) and heated to 110 °C and stirred overnight. TLC showed the reaction was complete. After concentrated under reduced pressure, water (10 mL) and ethyl acetate (10 mL) were added, the organic phase was collected and concentrated under reduced pressure to get the crude product, which was purified by silica gel column chromatography (10% ethyl acetate in n-hexane) to get 4-(2-(6-((2,4-dimethoxybenzyl)amino)-8-oxo-7,8-dihydro-9H-purin-9-yl)ethoxy)benzonitrile as a yellow solid (40% yield). LC_MS: (ES + ): m / z 447.55 [M+H] + . tR= 2.579 min.

[0128] 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 at room temperature overnight. TLC showed the reaction to be complete. The dichloromethane and excess trifluoroacetic acid were removed by concentration under reduced pressure, the residue was poured into water (20 mL) and basified to pH 8 with aqueous sodium hydroxide (2N). The product was extracted with dichloromethane, the organic layer was washed with brine (20 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product which was purified by preparative HPLC to give 4-(2-(6-amino-8-hydroxy-9H-purin-9-yl)ethoxy)benzonitrile (37% yield). 1 H NMR (400 MHz, 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 97.7%.

[0129] Comparative Example 1 : Synthesis of compound 20

[0130]

[0131] 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 at room temperature overnight. TLC showed the presence of starting material. After additional addition of N-bromosuccinimide (255.97 mg, 1.4 mmol) stirring was continued for 24 h. TLC showed the reaction to be 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. Concentration under reduced pressure gave the crude product which was first purified by silica gel column chromatography (using 5% methanol in dichloromethane) followed by purification by preparative HPLC to give 4-(2-(6-amino-8-bromo-9H-purin-9-yl)ethoxy)benzonitrile as a white solid (40% yield). 1H NMR (400 MHz, 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 / z 360.30 [M+H] + tR= 2.142 min. HPLC purity was 97.6%.

[0132] 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 at room temperature overnight. TLC showed the reaction was complete. The reaction mixture was diluted with a mixture 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 and 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 as a white solid (94% yield). 1 H NMR (400 MHz, DMSO-d6): 4.51 (s, 4H), 6.89 (s, 2H), 7.05 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 8.8 Hz, 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%.

[0133] Comparative Example 2: Synthesis of compound 22

[0134]

[0135] To a solution of 4-(2-(6-amino-9H-purin-9-yl)ethoxy)benzonitrile (200 mg, 0.7 mmol) and N-bromosuccinimide (255.97 mg, 1.4 mmol) in N,N-dimethylformamide (8 ml) was stirred at room temperature overnight. TLC showed some starting material remained. After additional N-bromosuccinimide (255.97 mg, 1.4 mmol) was added and stirred for another 24 hours. TLC showed 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. After concentration under reduced pressure, the crude product was purified by silica gel column chromatography (5% methanol in dichloromethane) first and then by preparative HPLC to give 4-(2-(6-amino-8-bromo-9H-purin-9-yl)ethoxy)benzonitrile as a white solid (40% yield). 1 H NMR (400 MHz, 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 / z 360.30 [M+H]+. tR= 2.142 min. HPLC purity 97.6%.

[0136] The biological activity of the above compounds was determined by NSD2, NSD3 protein level enzyme activity experiment, and the experimental results were from the average of at least three independent tests, and the results were shown in Table 1, wherein NA indicated no activity detected, and ND indicated no test.

[0137] Table 1 Inhibitory activity of compounds on NSD2, NSD3 protein level enzyme

[0138]

[0139]

[0140] The biological activity of the above compound 3 and the control compound SAM (the structural formula of which is

[0141] The biological activity of the above compound 3 and the control compound SAM (the structural formula of which is

[0142] Table 2 Kd value of compound and NSD3-SET protein

[0143] Compounds K d (μM) 3 5.51 SAM 25

[0144] The above compound 3 was subjected to bioactivity determination, i.e. epigenetic modification enzyme selectivity determination, and the IC50 values of the compound 3 for the enzyme activities of epigenetic modification enzymes NSD2, NSD3, SETD2, SMYD3, G9a, PRMT1 and PRMT4 were determined, respectively, and the results are shown in Table 3 below.

[0145] Table 3: Compound 3 epigenetic modification enzyme selectivity

[0146] Enzyme IC50 (pM) NSD2 0.81±0.04 NSD3 0.84±0.06 SETD2 >20 SMYD3 >20 G9a >20 PRMT1 >20 PRMT4 >20

[0147] As shown in Table 3, the compound 3 specifically inhibits NSD2 and NSD3, and does not show obvious activity for other epigenetic modification enzymes.

[0148] The above compounds 2, 3, 10, 14, 15, 18, 19 and 20 were subjected to cell activity test, and the effects of the compounds on NSDs substrate H3K36me2 in cells were determined by Western blot experiment, and the results are shown in Table 4 below. Figure 1 As shown in Table 4, the compounds 2, 3, 10, 14, 15, 18, 19 and 21 can effectively reduce the level of NSD2, NSD3 substrate H3K36me2 in H460 and H1299 non-small cell lung cancer cell lines.

[0149] The above examples are only used to help understand the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. Use of a purine derivative or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a neoplasm, characterized in that: The purine derivative is selected from the group consisting of compounds represented by the following structures: The tumor is lung cancer.

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