4-substituted phenylacetamide compound as well as preparation method and application thereof

Abstract

The invention provides a 4-substituted phenylacetamide compound as well as a preparation method and application thereof. The structural formula of the compound is shown as a formula (I) or (II). The compound provided by the invention is a sphingosine kinase 2 (SphK2) targeting micromolecule, can effectively inhibit SphK2 enzyme activity and proliferation and migration of cancer cells such as human liver cancer (HepG2), mouse liver cancer (Hepa1-6), human colon cancer (HCT116), mouse colon cancer (MC-38), cervical cancer (MCF-7), gastric cancer (HGC-27) and the like, and has a better inhibition effect than that of a known inhibitor ABC294640; the polypeptide has the potential of being developed into a novel anti-cancer treatment drug. The synthesis steps of the compound are simple, the requirement on equipment is low, the operation is simple, the reaction yield can be greatly improved, and the production cost is further saved.

Description

Technical Field

[0001] The present invention relates to 4-substituted phenylacetamide compounds, preparation methods and applications thereof, in particular to compounds with substituted pyridine, substituted pyrazine and substituted pyrimidine skeletons, preparation methods and applications thereof. Background Art

[0002] The balance between sphingolipids in the human body is closely related to tumors, and targeting the sphingolipid pathway is a new approach to developing anti-cancer drugs. Ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) are all sphingolipid substances in the body. They are not only involved in the composition of cell membranes, but also can act as lipid signaling molecules to participate in processes such as cell proliferation, differentiation, and apoptosis. Studies have shown that Cer / Sph promotes cell apoptosis, while S1P promotes cell survival. When they are out of balance, they will promote the occurrence and development of tumors. Sphingolipid research usually uses the "sphingolipid-rheostat" to describe the balance and transformation between the three. Therefore, targeting the sphingolipid signaling pathway and regulating the "sphingolipid-rheostat" has become a new direction for the development of anti-tumor drugs.

[0003] SphK catalyzes the phosphorylation of Sph to produce S1P and is a key rate-limiting enzyme controlling the "sphingolipid rheostat." Inhibiting SphK can lead to an increase in Cer / Sph, which promotes cell apoptosis, and a decrease in S1P, which promotes cell survival, thereby producing an anti-tumor effect. SphKs have two subtypes, of which SphK1 has been widely reported as an oncogene. However, due to the contradictions in the key physiological functions of SphK2, related pathological research and drug development have been seriously hindered. Despite this, the important role of SphK2 in promoting tumorigenesis has been unanimously recognized by researchers in recent years. In particular, its selective inhibitor ABC294640 has now entered Phase II clinical development, which has strongly promoted the relevant mechanism research of SphK2.

[0004] Currently, few highly effective and selective SphK2 inhibitors have been reported. However, studies have shown that targeting SphK2 exhibits superior anti-tumor activity compared to targeting SphK1. However, the crystal structure of SphK2 remains unknown, and no SphK2 inhibitors have been approved for marketing worldwide. Furthermore, most reported SphK2 inhibitors are limited by low potency, low specificity, a single structure, and a small number of available drugs. This has significantly hampered research on sphingolipid signaling pathways and the development of related therapeutic drugs. Although ABC294640 has been widely used in biological research, it still suffers from issues such as low enzyme activity.

[0005] Therefore, it is urgent to develop new, efficient and highly selective SphK2 inhibitors to solve the above problems. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a 4-substituted phenylacetamide compound that can be used as a highly efficient and selective SphK2 inhibitor to solve the problems of low efficacy and single structure of existing SphK2 inhibitors.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned compound and its pharmaceutically acceptable salt.

[0008] A further object of the present invention is to provide the use of the above-mentioned compound and its pharmaceutically acceptable salt in the preparation of drugs for preventing and treating cancer.

[0009] To achieve the above object, the technical solution of the present invention is:

[0010] A 4-substituted phenylacetamide compound or a pharmaceutically acceptable salt thereof, characterized in that the general structural formula of the compound is as shown in formula (I) or (II):

[0011]

[0012] Wherein: in formula (I), X is C or N; when X=C, it is a pyridine ring; when X=N, it is a pyrazine ring;

[0013] R1 is any one of H, F, Cl or CF3; n=1, 2 or 3; when n=1, it is monosubstituted; when n=2, it is disubstituted, in which case R1 is the same or different; when n=3, it is trisubstituted, in which case R1 is the same or different;

[0014] R2 is Any one of .

[0015] Preferably, R1 is hydrogen, chlorine or trifluoromethyl, and R2 is any one of ethanolamine, glycylamino, L-prolinol, D-prolinamide or 4-hydroxypiperidinyl.

[0016] More preferably, the compound is selected from one of the following structures:

[0017]

[0018] Pharmaceutically acceptable salts of the compound include, but are not limited to, acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate, methanesulfonate, and the like.

[0019] The present invention also provides a method for preparing the above compound, and its synthetic route is as follows:

[0020]

[0021] The following steps are involved:

[0022] (1) Compound F reacts with pinacol borate to generate compound G.

[0023] (2) Compound A and compound B undergo a substitution reaction to generate compound C.

[0024] (3) Compound C and compound G undergo Suzuki coupling to generate compound D.

[0025] (4) Compound D undergoes hydrolysis to generate compound E.

[0026] (5) Compound E is condensed with different types of amine organic bases having an R2 structure, such as ethanolamine, glycylamine, L-prolinol, or L-prolinamide, to produce a compound represented by formula (I);

[0027] Specifically, in step (1), the conditions are as follows: Compound F and pinacol borate are reacted in a 1,4-dioxane solution under N2 protection and heated under reflux for 2 hours to generate Compound G; the molar ratio of Compound F to pinacol borate is 1:1.2;

[0028] Specifically, in step (2), the substitution conditions are as follows: Compound A and Compound B are heated under reflux at 65° C. in an acetone solution and reacted for 3 hours to generate Compound C; the molar ratio of Compound A to Compound B is 1:1.5.

[0029] Specifically, in step (3), the Suzuki coupling conditions are as follows: compound C and compound G undergo Suzuki coupling under anaerobic conditions, and are heated under reflux at 95° C. for 5 h to generate compound D; the molar ratio of compound C to G is 1:1.

[0030] Specifically, in step (4), the hydrolysis conditions are as follows: compound D and sodium hydroxide are heated under reflux at 70° C. for 1 to 1.5 hours in a methanol:water=4:1 system to generate compound E; the molar ratio of compound D to sodium hydroxide is 1:3.

[0031] Specifically, in step (5), the condensation conditions are: compound E reacts with an amine having an R2 structure in a dichloromethane solvent at room temperature of 25°C overnight to generate a compound represented by formula (I) or (II); the molar ratio of compound E to amine is 1:1.

[0032] When the compound in step (2) is A2, the compound of formula (II) can be obtained according to the same synthetic route and the same reaction conditions, as follows:

[0033] (2') Compound A2 undergoes a substitution reaction with compound B2 to generate compound C2.

[0034] (3') Compound C2 and compound G undergo Suzuki coupling to generate compound D2.

[0035] (4') Compound D2 undergoes hydrolysis to generate compound E2.

[0036] (5') Compound E2 undergoes condensation with different types of amine organic bases having an R2 structure, such as ethanolamine, glycylamine, L-prolinol, or L-prolinamide, to generate a compound represented by formula (II);

[0037] Specifically, in step (2'), the substitution conditions are: Compound A2 and Compound B2 are heated to reflux at 65°C in an acetone solution for 3 hours to generate Compound C2; the molar ratio of Compound A2 to B2 is 1:1.5.

[0038] Specifically, in step (3'), the Suzuki coupling conditions are as follows: compound C2 and compound G undergo Suzuki coupling under anaerobic conditions, and are heated under reflux at 95°C for 5 hours to generate compound D2; the molar ratio of compound C2 to G is 1:1.

[0039] Specifically, in step (4'), the hydrolysis conditions are: compound D2 and sodium hydroxide are heated under reflux at 70°C in a methanol:water = 4:1 system for 1 to 1.5 hours to generate compound E2; the molar ratio of compound D2 to sodium hydroxide is 1:3.

[0040] Specifically, in step (5'), the condensation conditions are: compound E2 reacts with an amine having a structure R2 in a dichloromethane solvent at room temperature of 25°C overnight to generate a compound represented by formula (II); the molar ratio of compound E2 to amine is 1:1.

[0041] The present invention also provides the use of the biphenyl-like compound or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0042] The tumor is human histiocytic gastric cancer, cervical cancer, colon cancer, liver cancer or mouse liver cancer, colon cancer.

[0043] The present invention also provides a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

[0044] The present invention synthesized a new class of SphK2-targeted drug small molecules. The synthesized compounds exerted a very strong inhibitory effect in inhibiting SphK2 enzyme activity and cancer cell proliferation, which was better than the inhibitory effect of the existing inhibitor ABC294640. It has the potential to be developed into a new anti-cancer therapeutic drug.

[0045] The present invention has simple reaction steps for synthesizing SphK2-targeted drug molecules, has low equipment requirements and is easy to operate, can greatly improve the reaction yield, and further save production costs; at the same time, no dangerous flammable and explosive reagents are required, while maintaining a good catalytic effect and reducing costs, achieving the effects of simplifying the process, reducing costs, and facilitating post-processing procedures, and is suitable for industrial production. DETAILED DESCRIPTION

[0046] In the following examples, various processes and methods not described in detail are conventional methods known in the art, and all reagents used without indicating their sources and specifications are commercially available as analytical or chromatographically pure.

[0047] Example 1: Preparation of a series of compounds represented by formula (I): When X = C, it is a pyridine ring;

[0048] (1) 1.00 g (4.39 mmol) of methyl 4-bromophenylacetate (Compound F), 1.34 g (5.26 mmol) of pinacol borate, 1.29 g (13.16 mmol) of potassium acetate, and 0.06 g (0.09 mmol) of [1,1-bis(diphenylphosphino)ferrocene] palladium dichloride were placed in a 50 mL eggplant flask containing 20 mL of 1,4-dioxane. The mixture was refluxed at 95 °C for 2 h under argon protection. The reaction endpoint was detected by TLC. The solvent was removed under reduced pressure, and the reaction was quenched by adding water. The mixture was extracted with ethyl acetate three times, and the organic phases were combined and dried over anhydrous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: ethyl acetate: petroleum ether = 1:50) to obtain 1.05 g of white powder (Compound G) with a yield of approximately 86.78%. HR-MS-ESI m / z calcd for C 15 H 21 BO4,[M+Na] + 299.1425,found:299.1426.

[0049] (2) 0.87 g (5.00 mmol) of 2-hydroxy-5-bromopyridine (Compound A), 0.76 g (6.00 mmol) of benzyl chloride containing different substituents (Compound B), 2.07 g (15.00 mmol) of potassium carbonate, and 0.08 g (0.50 mmol) of potassium iodide were placed in a 50 mL eggplant flask containing 20 mL of acetone. The mixture was heated at 65°C for 2 h and the reaction endpoint was detected by TLC. The solvent was removed under reduced pressure, and the reaction was quenched by adding water. The organic phases were extracted three times with ethyl acetate, and the organic phases were combined and dried over anhydrous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: ethyl acetate: petroleum ether = 1:8) to obtain 1.22 g of a white powder (Compound C) with a yield of approximately 85.67-93.66%.

[0050] (3) 1.32 g (5.00 mmol) of compound C, 1.66 g (6.00 mmol) of compound G, 1.46 g (15.00 mmol) of potassium acetate, and 0.07 g (0.10 mmol) of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride were placed in a 50 mL eggplant flask containing 20 mL of a mixed solvent (ethanol:water = 1:1) under nitrogen protection. After heating and reflux at 85°C for 2 h, the reaction endpoint was detected by TLC. The ethanol in the mixed solvent was removed under reduced pressure, and the reaction was quenched by adding water. The mixture was extracted with ethyl acetate three times, and the organic phases were combined and dried over anhydrous Na2SO4. Purification was performed by silica gel chromatography (eluent: ethyl acetate: petroleum ether = 1:6) to obtain 1.49 g of a brown-red oil (compound D) with a yield of approximately 82.32-94.23%.

[0051] (4) 1.00 g (3.00 mmol) of compound D and 0.60 g (15.00 mmol) of sodium hydroxide were placed in a 50 mL eggplant flask containing 20 mL of a mixed solvent (methanol:water = 1:1). After heating at 50°C for 3 h, the reaction endpoint was detected by TLC. The methanol in the mixed solvent was removed under reduced pressure, and an appropriate amount of 1 mol·L -1 The pH of the reaction mixture was adjusted to neutral or acidic with hydrochloric acid solution until a light yellow solid precipitated, which was filtered and the filter cake was naturally dried to obtain 0.84 g of a brown solid (Compound E) with a yield of approximately 85.26-90.45%.

[0052] (5) 0.32 g (1 mmol) of compound E, 0.29 g (1.5 mmol) of EDCI, and 0.03 g (0.2 mmol) of DMAP were dissolved in 5 mL of dichloromethane and reacted at room temperature for 1 h. Then, 1 mmol of different amines (see Table 1) were added and the mixture was allowed to react at room temperature overnight at 25°C. The reaction endpoint was detected by TLC, and the mixture was extracted with ethyl acetate and dried over aqueous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: dichloromethane:methanol = 60:1). The mixture was recrystallized from ethyl acetate to precipitate a white solid, which was then dried under vacuum to obtain the target product series (I) in a yield of approximately 75-88%.

[0053] Different types of amines are used in step (5), and the specific structures of the corresponding compounds obtained are shown in Table 1.

[0054] Table 1

[0055]

[0056]

[0057] The characterization data of compounds 1-8 are as follows:

[0058] Compound 1: 11H NMR (600 MHz, Chloroform-d) δ 7.60 (dd, J = 9.4, 2.6 Hz, 1H), 7.46 (d, J = 2.5 Hz, 1H), 7.37 - 7.26 (m, 10H), 6.99 (s, 1H), 6.71 (d, J = 9.4 Hz, 1H), 5.33 (s, 1H), 5.22 (s, 2H), 4.65 - 4.59 (m, 1H), 3.71 (s, 2H), 3.61 - 3.57 (m, 1H), 3.52 - 3.46 (m, 1H), 2.48 - 2.36 (m, 1H), 2.17 - 2.06 (m, 1H), 2.02 - 1.96 (m, 1H), 1.87 - 1.80 (m, 1H). 13 13C NMR (150 MHz, Chloroform-d) δ 173.13, 171.08, 161.93, 139.18, 136.27, 135.28, 134.47, 133.36, 129.86, 129.78, 128.97, 128.15, 128.13, 126.10, 126.01, 121.23, 120.00, 59.52, 52.23, 47.76, 41.36, 29.69, 25.04. HR-MS-ESI m / z calcd for C 25 H 25 N3O3, [M+Na] + 438.1788, found: 438.1788。

[0059] Compound 2: 1 1H NMR (600 MHz, DMSO-d6) δ 8.24 - 8.22 (m, 1H), 7.84 (d, J = 9.4, 1H), 7.51 - 7.47 (m, 2H), 7.37 - 7.32 (m, 4H), 7.29 - 7.25 (m, 3H), 6.52 (d, J = 9.4 Hz, 1H), 5.18 (s, 2H), 4.78 - 4.72 (m, 1H), 3.98 - 3.90 (m, 1H), 3.69 - 3.61 (m, 2H), 3.53 - 3.38 (m, 3H), 3.28 - 3.22 (m, 1H), 1.92 - 1.84 (m, 2H), 1.82 - 1.76 (m, 2H). 13C NMR(150MHz,DMSO-d6)δ169.09,139.06,137.36,136.14,129.81,129.79,128.51,127.72,127.4 8,125.07,124.99,119.93,118.08,58.81,58.64,47.05,40.77,26.67,23.42,21.34.HR-MS-ESI m / z calcd for C 25 H 26 N2O3,[M+Na] + 425.1835,found:452.1836.

[0060] Compound 3: 1 H NMR (600MHz, DMSO-d6) δ8.22(d,J=2.5Hz,1H),8.05(s,1H),7.83(dd,J=9.4,2.6Hz,1H),7.49-7.44(m,2H),7.37-7.32(m,4 H),7.31-7.26(m,3H),6.52(d,J=9.4Hz,1H),5.17(s,2H),4.67(t,J=5.4Hz,1H),3.42-3.38(m,4H),3.11(q,J=5.9Hz,2H). 13 C NMR(150MHz,DMSO-d6)δ170.07,160.62,139.08,137.35,136.10,135.27,133.88,129.54 ,128.51,127.72,127.48,125.07,119.93,118.12,59.83,51.50,41.86,41.60.HR-MS-ESI m / z calcd for C 22 H 22 N2O3,[M+Na] + 385.1532,found:385.1532.

[0061] Compound 4: 1 H NMR(600MHz,DMSO-d6)δ8.25(d,J=2.6Hz,1H),7.84(dd,J=9.4,2.6Hz,1H),7.52-7.47( m,2H),7.37-7.27(m,7H),6.53(d,J=9.4Hz,1H),5.18(s,2H),3.70(s,2H),3.61(s,3H). 13C NMR(150MHz,DMSO-d6)δ171.53,160.64,139.06,137.34,136.29,134.41,133.04,129 .90,128.51,127.73,127.48,125.24,119.93,117.95,51.68,51.50,50.40.HR-MS-ESI m / z calcd for C 22 H 21 N3O3,[M+Na] + 398.1475,found:398.1475.

[0062] Compound 5: 1 H NMR (600MHz, DMSO-d6) δ8.34–8.29(m,1H),7.90–7.86(m,1H),7.72(d,J=8.2Hz,2H),7.55(d,J=8.1Hz,2H),7.51–7.47(m,2H),7.30(d,J=8.2Hz,2H ),7.24(d,J=8.3Hz,1H),6.89(s,1H),6.56–6.51(m,1H),5.27(s,2H),4. 43–4.20(m,1H),3.71–3.67(m,1H),3.53–3.47(m,1H),2.03–1.77(m,4H). 13 C NMR(150MHz,DMSO-d6)δ173.95,173.74,168.94,168.83,160.62,142.01,139.3 5,136.24,134.42,133.81,133.73,130.01,129.93,128.37,128.15,127.94,125 .43,125.41,125.38,125.35,125.08,125.03,125.00,123.28,119.97,118.33,5 9.82,59.41,51.37,47.00,46.49,40.32,31.77,29.44,24.20,22.31.HR-MS-ESI m / z calcd for C 26 H 24 F3N3O3,[M+Na] + 506.1661,found:506.1662.

[0063] Compound 6: 11H NMR (600 MHz, DMSO-d6) δ 8.25 (d, J = 2.6 Hz, 1H), 8.05 (t, J = 5.4 Hz, 1H), 7.82 (dd, J = 9.5, 2.7 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.44 (dd, J = 8.5, 5.6 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.17 (t, J = 8.9 Hz, 2H), 6.52 (d, J = 9.4 Hz, 1H), 5.15 (s, 2H), 4.68 (t, J = 5.4 Hz, 1H), 3.43 (s, 2H), 3.40 (q, J = 5.9 Hz, 2H), 3.12 (q, J = 5.9 Hz, 2H). 13 13C NMR (150 MHz, DMSO-d6) δ 170.09, 162.35, 160.74, 160.59, 139.13, 135.99, 135.29, 133.87, 133.55, 133.53, 130.09, 130.04, 129.53, 125.09, 119.95, 118.24, 115.34, 115.20, 59.85, 50.89, 41.87, 41.60. HR-MS-ESI m / z calcd for C 22 17 21 14FN2O3, [M + H] + 381.1608, found: 381.1609.

[0064] Compound 7: 1 1H NMR (600 MHz, Chloroform-d) δ 7.56–7.51 (m, 1H), 7.37 (s, 2H), 7.35 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.12–7.08 (m, 1H), 6.67–6.63 (m, 1H), 5.07 (s, 2H), 4.18 (d, J = 8.1 Hz, 1H), 3.63 (s, 2H), 3.60 (s, 1H), 3.55–3.50 (m, 2H), 3.43–3.39 (m, 1H), 2.01–1.95 (m, 1H), 1.90–1.84 (m, 1H), 1.82–1.76 (m, 1H), 1.54–1.50 (m, 1H). 13C NMR(150MHz,Chloroform-d)δ172.06,161.72,139.65,136.51,134.90,134.16,133.81,133.03,132.38,130.90,12 9.98,129.87,127.42,126.14,125.93,121.43,120.52,67.34,61.57,51.50,48.38,41.80,28.32,24.45.HR-MS-ESI m / z calcd forC 25 H 24 Cl2N2O3,[M+Na] + 493.1056,found:493.1056.

[0065] Compound 8: 1 H NMR(600MHz,Chloroform-d)δ7.56(dd,J=9.4,2.6Hz,1H),7.37(d,J=2.2Hz,2H),7.35(d, J=8.3Hz,1H),7.25(d,J=8.2Hz,2H),7.21(d,J=8.2Hz,2H),7.12(dd,J=8.3,1.8Hz,1H),6. 64(d,J=9.4Hz,1H),5.07(s,2H),4.04–3.98(m,1H),3.85–3.79(m,1H),3.67(s,3H),3.19 –3.11(m,2H),1.81–1.75(m,1H),1.71–1.67(m,1H),1.44–1.37(m,1H),1.31–1.25(m,1H). 13 C NMR(150MHz,Chloroform-d)δ169.08,139.70,136.49,134.65,134.13,133.04,132.39,130.91,129.97 ,129.48,127.39,126.13,121.43,120.58,66.95,51.55,43.33,40.44,39.17,34.31,29.68.HR-MS-ESI m / z calcd forC 25 H 24 Cl2N3O3,[M+Na] + 493.1056,found:493.1056.

[0066] Example 2: Preparation of a series of compounds represented by formula (I): When X = N, it is a pyrazine ring;

[0067] Step (1) is the same as in Example 1.

[0068] (2) 0.88 g (5.00 mmol) of 2-hydroxy-5-bromopyrazine (Compound A1), 0.76 g (6.00 mmol) of benzyl chloride containing different substituents (Compound B1), 2.07 g (15.00 mmol) of potassium carbonate, and 0.08 g (0.50 mmol) of potassium iodide were placed in a 50 mL eggplant flask containing 20 mL of acetone. The mixture was heated to reflux at 65°C for 2 h, and the reaction endpoint was detected by TLC. The solvent was removed under reduced pressure, and the reaction was quenched with water. The organic phases were extracted three times with ethyl acetate, and the combined organic phases were dried over anhydrous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: ethyl acetate: petroleum ether = 1:16) to obtain 1.23 g of a light yellow powder (Compound C1) with a yield of approximately 80.45-94.34%. (3) 1.32 g (5.00 mmol) of compound C1, 1.66 g (6.00 mmol) of compound G, 1.46 g (15.00 mmol) of potassium acetate, and 0.07 g (0.10 mmol) of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride were placed in a 50 mL eggplant flask containing 20 mL of a mixed solvent (ethanol:water = 1:1). The mixture was refluxed at 85°C for 2 h under nitrogen protection, and the reaction endpoint was detected by TLC. The ethanol in the mixed solvent was removed under reduced pressure, and the reaction was quenched by adding water. The mixture was extracted three times with ethyl acetate, and the organic phases were combined and dried over anhydrous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: ethyl acetate:petroleum ether = 1:6) to obtain 1.48 g of a light yellow powder (compound D1) with a yield of approximately 81.33-94.56%.

[0069] (4) Weigh 1.67 g (5.00 mmol) of compound D1 and 1.00 g (25.00 mmol) of sodium hydroxide into a 50 mL eggplant flask containing 20 mL of a mixed solvent (methanol:water = 1:1). Heat at 50°C and reflux for 3 h. The reaction endpoint is detected by TLC. Methanol is removed from the mixed solvent under reduced pressure, and an appropriate amount of 1 mol·L -1 The pH of the solution was adjusted to neutral or acidic with hydrochloric acid until a light yellow solid precipitated, which was filtered and the filter cake was naturally dried to obtain 1.36 g of a light yellow solid (Compound E1) with a yield of about 71.14-84.98%.

[0070] (5) 0.32 g (1 mmol) of compound E1, 0.29 g (1.5 mmol) of EDCI, and 0.03 g (0.2 mmol) of DMAP were dissolved in 5 mL of dichloromethane and reacted at room temperature for 1 h. 1 mmol of different amines (see Table 2) were then added and the mixture was allowed to react at room temperature overnight at 25°C. The reaction endpoint was detected by TLC, and the mixture was extracted with ethyl acetate and dried over aqueous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: dichloromethane:methanol = 40:1). A white solid was precipitated by recrystallization from ethyl acetate and dried in vacuo to obtain the target product series (I) in a yield of approximately 65-85%.

[0071] Different types of amines are used in step (5), and the specific structures of the corresponding compounds obtained are shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075]

[0076] The characterization data of compounds 9-16 are as follows:

[0077] Compound 9: 1 H NMR(600MHz,Chloroform-d)δ8.30(s,1H),7.63(d,J=8.0Hz,2H),7.45(s,1H),7.41-7.34(m,5H),7.29(d,J=8.0Hz,2H),5.16(s,2H),4.65- 4.61(m,1H),3.71(s,2H),3.60-3.55(m,1H),3.50-3.44(m,1H),2.42- 2.36(m,1H),2.11-2.05(m,1H),1.99-1.93(m,1H),1.87-1.81(m,1H). 13 C NMR(150MHz,Chloroform-d)δ173.23,171.06,155.37,148.81,134.70,134.34,134.12,133.67,129.66,129 .55,129.23,128.73,128.48,125.36,125.27,124.15,59.55,52.13,47.76,41.56,27.16,25.01.HR-MS-ESI m / z calcdfor C 24 H 24 N4O3,[M+Na] +439.1740, found: 439.1741。

[0078] Compound 10: 1 H NMR (600 MHz, Chloroform-d) δ 8.29 (d, J = 1.3 Hz, 1H), 7.63 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 1.3 Hz, 1H), 7.42 - 7.37 (m, 2H), 7.36 - 7,32 (m, 3H), 7.29 (d, J = 8.0 Hz, 2H), 5.15 (s, 2H), 4.98 - 4.94 (m, 1H), 4.26 - 4.20 (m, 1H), 3.70 (s, 2H), 3.68 - 3.64 (m, 1H), 3.59 - 3.53 (m, 2H), 3.47 - 3.41 (m, 1H), 2.06 - 2.00 (m, 1H), 1.93 - 1.81 (m, 1H), 1.84 - 1.81 (m, 1H), 1.62 -.52 (m, 1H). 13 C NMR (150 MHz, Chloroform-d) δ 172.11, 155.38, 148.77, 134.71, 134.31, 134.28, 133.73, 129.53, 129.23, 128.73, 128.49, 125.36, 125.14, 124.18, 67.27, 61.55, 52.13, 48.37, 42.06, 28.31, 24.42. HR-MS-ESI m / z calcd for C 24 H 25 N3O3, [M + Na] + 426.1788, found: 426.1788。

[0079] Compound 11: 1 H NMR (600 MHz, Chloroform-d) δ 8.30 (d, J = 1.2 Hz, 1H), 7.65 - 7.60 (m, 2H), 7.46 (d, J = 1.2 Hz, 1H), 7.41 - 7.37 (m, 2H), 7.37 - 7.34 (m, 3H), 7.30 - 7.27 (m, 2H), 5.16 (s, 2H), 4.10 - 4.05 (m, 1H), 3.89 - 3.84 (m, 1H), 3.75 (s, 2H), 3.73 - 3.68 (m, 1H), 3.25 - 3.16 (m, 2H), 1.86 - 1.80 (m, 1H), 1.72 - 1.68 (m, 1H), 1.49 - 1.43 (m, 1H), 1.32 - 1.27 (m, 1H). 13C NMR(150MHz,Chloroform-d)δ169.13,155.38,148.69,135.14,134.72,134.02,133.74,132.38,129.20 ,129.12,128.69,128.43,125.33,124.21,66.79,52.19,43.37,40.70,39.17,34.25,33.73.HR-MS-ESI m / z calcd for C 24 H 25 N3O3,[M+Na] + 426.1788,found:426.1788.

[0080] Compound 12: 1 H NMR(600MHz,Chloroform-d)δ8.32(s,1H),7.73–7.66(m,4H),7.50–7.45(m,3H),7.32(d,J=8.1Hz,2H),5.21(s,2H),4.91(d,J=7.6Hz,1H),4.2 6–4.21(m,1H),3.71(s,2H),3.69–3.64(m,1H),3.60–3.55(m,2H),3.49 –3.42(m,1H),2.08–2.03(m,1H),1.95–1.81(m,2H),1.61–1.56(m,1H). 13 C NMR(150MHz,Chloroform-d)δ172.08,155.17,149.11,138.72,134.55,134.02,129.61,128.55,126.20,12 6.17,126.15,126.12,125.40,123.95,67.34,61.56,51.92,48.37,42.03,29.68,28.31,24.43.HR-MS-ESI m / zcalcd for C 25 H 24 F3N3O3,[M+Na] + 494.1661,found:494.1662.

[0081] Compound 13: 11H NMR (600 MHz, Chloroform-d) δ 8.33–8.29 (m, 1H), 7.67–7.62 (m, 4H), 7.50–7.46 (m, 3H), 7.34–7.29 (m, 2H), 5.21 (s, 2H), 4.11–4.05 (m, 1H), 3.89–3.85 (m, 1H), 3.75 (s, 2H), 3.74–3.69 (m, 1H), 3.24–3.16 (m, 2H), 1.88–1.80 (m, 1H), 1.73–1.68 (m, 1H), 1.44–1.40 (m, 1H), 1.35–1.28 (m, 1H). 13 13C NMR (150 MHz, Chloroform-d) δ 169.07, 155.18, 149.08, 138.73, 135.38, 134.01, 133.79, 129.22, 128.53, 126.19, 126.16, 126.14, 126.11, 125.38, 123.94, 66.97, 51.96, 43.34, 40.71, 39.15, 34.27, 33.79, 29.68. HR-MS-ESI m / z calcd for C 25 18 24 15F3N3O3, [M+Na]+ + 494.1661, found: 494.1662。

[0082] Compound 14: 1 1H NMR (600 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.18 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 7.31 (s, 1H), 7.02 (s, 1H), 5.25 (s, 2H), 3.67–3.63 (m, 2H), 3.52 (s, 2H). 13 13C NMR (150 MHz, DMSO-d6) δ 170.79, 170.21, 154.55, 147.75, 140.62, 135.81, 133.52, 132.07, 129.51, 128.62, 126.66, 125.53, 125.50, 125.47, 125.45, 124.32, 51.51, 48.56, 41.91, 41.72. HR-MS-ESI m / z calcd for C 22 20 19 15F3N4O3, [M+Na]++ 467.1301, found: 467.1301。

[0083] Compound 15: 1 H NMR (600 MHz, Chloroform-d) δ 8.29 (d, J = 1.2 Hz, 1H), 7.66–7.61 (m, 2H), 7.44 (d, J = 1.3 Hz, 1H), 7.38–7.34 (m, 2H), 7.30 (dd, J = 8.5, 2.9 Hz, 4H), 6.95 (s, 1H), 5.37 (s, 1H), 5.11 (s, 2H), 4.61 (dd, J = 8.1, 2.1 Hz, 1H), 3.72 (s, 2H), 3.60–3.55 (m, 1H), 3.52–3.44 (m, 1H), 2.43–2.38 (m, 1H), 2.14–2.05 (m, 1H), 1.99–1.96 (m, 1H), 1.88–1.84 (m, 1H). 13 C NMR (150 MHz, Chloroform-d) δ 172.10, 155.23, 148.95, 134.81, 134.44, 134.13, 133.91, 133.26, 129.80, 129.57, 129.40, 125.38, 123.92, 67.31, 61.55, 48.37, 29.67, 28.31, 24.42. HR-MS-ESI m / z calcd for C 24 H 23 ClN4O3, [M+Na] + 473.1350, found: 473.1351。

[0084] Compound 16: 1 H NMR (600 MHz, Methanol-d4) δ 8.18–8.14 (m, 2H), 7.79–7.74 (m, 2H), 7.50–7.44 (m, 2H), 7.38–7.33 (m, 2H), 7.15–7.05 (m, 2H), 5.20 (s, 2H), 4.57 (s, 1H), 3.16–3.12 (m, 2H), \3.55 (s, 2H), 3.29 (s, 2H). 13C NMR(150MHz,Methanol-d4)δ174.15,157.18,148.81,137.01,131.57,131.52,1 30.65,126.97,126.34,116.76,116.62,61.56,53.02,43.48,43.16.HR-MS-ESI m / z calcd for C 21 H 20 FN3O3,[M+Na] + 404.1380,found:404.1381.

[0085] Example 3: Preparation of a series of compounds represented by formula (II)

[0086] Step (1) is the same as in Example 1

[0087] (2) 0.66 g (5.00 mmol) of 4-chloro-6-hydroxypyrimidine (Compound A2), 0.76 g (6.00 mmol) of benzyl chloride containing different substituents (Compound B2), 2.07 g (15.00 mmol) of potassium carbonate, and 0.08 g (0.50 mmol) of potassium iodide were placed in a 50 mL eggplant flask containing 20 mL of acetone. The mixture was heated to reflux at 65°C for 2 h, and the reaction endpoint was detected by TLC. The solvent was removed under reduced pressure, and the reaction was quenched with water. The mixture was extracted three times with ethyl acetate, and the organic phases were combined and dried over anhydrous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: ethyl acetate: petroleum ether = 1:6) to obtain 1.03 g of a white powder (Compound C2) with a yield of approximately 82.14-96.34%.

[0088] (3) 1.10 g (5.00 mmol) of compound C2, 1.66 g (6.00 mmol) of compound G, 1.46 g (15.00 mmol) of potassium acetate, and 0.07 g (0.10 mmol) of [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride were placed in a 50 mL eggplant flask containing 20 mL of a mixed solvent (ethanol:water = 1:1). Under argon protection, the mixture was heated at 85°C for 2 h and the reaction endpoint was detected by TLC. The ethanol in the mixed solvent was removed under reduced pressure, and the reaction was quenched by adding water. The mixture was extracted three times with ethyl acetate, and the organic phases were combined and dried over anhydrous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: ethyl acetate: petroleum ether = 1:4) to obtain 1.48 g of a white powder (compound D2) with a yield of approximately 75.98-88.67%.

[0089] (4) 1.67 g (5.00 mmol) of compound D2 and 1.00 g (25.00 mmol) of sodium hydroxide were placed in a 50 mL eggplant flask containing 20 mL of a mixed solvent (methanol:water = 1:1). After heating at 50°C for 3 h, the reaction endpoint was detected by TLC. The methanol in the mixed solvent was removed under reduced pressure, and an appropriate amount of 1 mol·L -1 The pH of the solution was adjusted to neutral or acidic with hydrochloric acid until a light yellow solid precipitated, which was filtered and the filter cake was dried naturally to obtain 1.40 g of a light yellow solid (Compound E2) with a yield of about 75.66-89.45%.

[0090] (5) 0.32 g (1 mmol) of compound E2, 0.29 g (1.5 mmol) of EDCI, and 0.03 g (0.2 mmol) of DMAP were dissolved in 5 ml of dichloromethane and reacted at room temperature for 1 h. 1 mmol of different amines (see Table 1) were then added and the mixture was allowed to react at room temperature overnight at 25°C. The reaction endpoint was detected by TLC, and the mixture was extracted with ethyl acetate and dried over aqueous Na2SO4. The mixture was separated and purified by silica gel chromatography (eluent: dichloromethane:methanol = 60:1). A white solid was precipitated by recrystallization from ethyl acetate and dried in vacuo to obtain the target product series (II) in a yield of approximately 65-86%.

[0091] Different types of amines are used in step (5), and the specific structures of the corresponding compounds obtained are shown in Table 3.

[0092] Table 3

[0093] Table 3

[0094]

[0095]

[0096] The characterization data of compounds 17-23 are as follows:

[0097] Compound 17: 1 H NMR(600MHz,Methanol-d4)δ8.42(s,1H),7.95-7.90(m,2H),7.42-7.36(m,6H),7.36-7.34(m,1H),6.89( d,J=2.9Hz,1H),5.20(s,2H),4.46-4.42(m,1H),3.83-3.64(m,3H),3.58-3.55(m,1H),2.19-1.92(m,4H). 13C NMR(150MHz,Methanol-d4)δ176.92,172.11,163.86,162.90,152.43,138.97,136.80,135.75,131.08,130.99,13 0.25,129.62,129.32,128.58,128.51,110.40,79.17,78.96,78.74,61.33,50.91,49.18,42.58,30.85.HR-MS-ESI m / z calcd for C 24 H 24 N4O3,[M+Na] + 439.1740,found:439.1741.

[0098] Compound 18: 1 H NMR(600MHz,Chloroform-d)δ8.21(s,1H),7.93-7.89(m,2H),7.39-32(m,6H),6.88(s,1H),5.15(s,2H),4.91-4.88(m,1H),4.26-4.22 (m,1H),3.74(s,2H),3.68-3.63(m,1H),3.62-3.54(m,2H),3.49-3.46(m,1H),2.07-2.01(m,1H),1.96-1.80(m,2H),1.61-1.57(m,1H). 13 C NMR(150MHz,Chloroform-d)δ171.74,161.65,160.64,150.24,137.22,135.27,134.74,129.55,129.47,12 9.10,128.45,128.19,127.31,127.10,109.59,67.19,61.55,49.44,48.39,42.22,29.63,24.41.HR-MS-ESI m / z calcd for C 24 H 25 N3O3,[M+Na] + 426.1788,found:426.1788.

[0099] Compound 19: 11H NMR (600 MHz, Methanol-d4) δ 8.57 (s, 1H), 8.03 - 7.98 (m, 2H), 7.43 - 7.28 (m, 7H), 6.92 (s, 1H), 5.21 (s, 2H), 4.57 (s, 2H), 4.11 - 4.09 (m, 1H), 3.87 - 3.82 (m, 2H), 3.81 - 3.77 (m, 1H), 3.34 (s, 2H), 3.29 - 3.26 (m, 1H), 3.18 - 3.15 (m, 1H), 1.85 - 1.79 (m, 1H), 1.70 (s, 0H), 1.41 - 1.38 (m, 1H), 1.29 - 1.26 (m, 1H). 13 13C NMR (150 MHz, Methanol-d4) δ 171.46, 163.90, 162.86, 152.81, 139.84, 137.33, 130.34, 129.97, 129.27, 129.05, 128.55, 109.86, 67.59, 50.65, 44.90, 41.24, 40.68, 35.27, 34.66. HR-MS-ESI m / z calcd for C 24 H 25 N3O3, [M+Na] + 426.1788, found: 426.1788。

[0100] Compound 20: 1 1H NMR (600 MHz, DMSO-d6) δ 8.75 (s, 1H), 8.09 (t, J = 5.8 Hz, 1H), 7.99 - 7.93 (m, 2H), 7.39 - 7.34 (m, 6H), 7.33 - 7.28 (m, 1H), 6.96 (s, 1H), 5.13 (s, 2H), 4.68 (t, J = 5.4 Hz, 1H), 3.48 (s, 2H), 3.40 (q, J = 5.8 Hz, 2H), 3.12 (q, J = 5.9 Hz, 2H). 13 13C NMR (150 MHz, DMSO-d6) δ 169.76, 160.68, 159.80, 152.07, 139.30, 136.52, 133.74, 129.39, 128.61, 127.72, 127.71, 126.69, 108.33, 59.83, 48.65, 42.08, 41.62, 39.94. HR-MS-ESI m / z calcd for C 21 H 21 N3O3, [M+Na] +386.1475, found: 386.1475。

[0101] Compound 21: 1H NMR (600 MHz, Chloroform-d) δ 8.22 (d, J = 0.9 Hz, 1H), 7.94–7.88 (m, 2H), 7.38–7.33 (m, 4H), 7.10–7.01 (m, 2H), 6.87 (d, J = 0.9 Hz, 1H), 5.11 (s, 2H), 4.27–4.23 (m, 1H), 3.74 (s, 2H), 3.67 (d, J = 11.4 Hz, 1H), 3.61–3.54 (m, 2H), 3.46 (d, J = 10.3 Hz, 1H), 2.06–2.02 (m, 1H), 1.94–1.91 (m, 1H), 1.86–1.82 (m, 1H), 1.61–1.58 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 168.81, 168.66, 162.46, 160.84, 160.67, 159.86, 159.83, 151.99, 139.19, 138.59, 130.14, 130.09, 129.72, 126.68, 126.60, 115.45, 115.31, 108.35, 108.32, 62.78, 61.08, 58.85, 58.67, 48.06, 45.42, 40.94. HR-MS-ESI m / z calcd for C 24 H 24 FN3O3, [M+Na]+ 444.1693, found: 444.1694。

[0102] [[ID=IO]]Compound 22: 1 1H NMR (600 MHz, DMSO-d6) δ 8.68–8.6� (m, 1H), 8.11–8.08 (m, 1H), 8.03–7.99 (m, 2H), 7.54–7.50 (m, 1H), ⁷.⁴¹–⁷.³⁸ (m, 2H), 7.24–7.20 (m, 2H), 7.02–6.97 (m, 1H), 5.17 (s, 2H), 4.71–4.67 (m, 1H), 3.49 (s, 2H), 3.41 (q, J = 5.8 Hz, 2H), 3.13 (q, J = 5.9 Hz, 2H). 1313C NMR (150 MHz, DMSO-d6) δ 169.77, 162.13, 160.69, 160.49, 159.90, 152.27, 139.40, 133.66, 132.95, 132.88, 130.87, 130.81, 129.77, 129.75, 129.42, 126.74, 116.88, 116.71, 114.60, 114.46, 108.28, 59.83, 46.70, 42.09, 41.63. HR-MS-ESI m / z calcd for C 21 H 19 ClFN3O3, [M+Na] + 438.0991, found: 438.0991。

[0103] Compound 23: 1 1H NMR (600 MHz, DMSO-d6) δ 8.75 (d, J = 0.9 Hz, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.66 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.34 (dd, J = 8.4, 2.0 Hz, 1H), 7.32 (d, J = 8.1 Hz, 2H), 6.95 (d, J = 0.9 Hz, 1H), 5.10 (s, 2H), 4.69–4.66 (m, 1H), 3.92–3.87 (m, 1H), 3.75 (d, J = 2.5 Hz, 2H), 3.75–3.71 (m, 1H), 3.64–3.61 (m, 1H), 3.17–3.12 (m, 1H), 2.96–2.92 (m, 1H), 1.66–1.61 (m, 1H), 1.61–1.58 (m, 1H), 1.22–1.17 (m, 1H), 1.15–1.12 (m, 1H). 13 13C NMR (150 MHz, DMSO-d6) δ 168.16, 160.67, 159.87, 152.03, 138.97, 137.49, 133.64, 131.10, 130.76, 130.45, 130.07, 129.32, 128.25, 126.83, 108.35, 65.45, 54.84, 47.91, 43.03, 38.89, 34.49, 33.79. HR-MS-ESI m / z calcd for C 24 H 23 Cl2N3O3, [M+Na] + 494.1008, found: 494.1009。

[0104] Example 4: Study on the activity of the series of compounds of the present invention on SphK2

[0105] This example studies the activities of all the above compounds against SphK2.

[0106] (1) Experimental materials: target compound and positive control ABC294640, recombinant human SphK2 protein kinase, substrate SPH+ATP, microplate reader (Bio-Stack) and 384-well plate, ADP-Glo TM Kinase Assay Kit

[0107] (2) Experimental methods:

[0108] The inhibition rates of all the above samples on SphK2 were measured at different concentrations of 10 μM and 100 μM (n=3, three replicate wells). The experimental results are shown in Table 4.

[0109] Table 4 Inhibition rate of the present invention series of compounds on SphK2 at different concentrations of 10μM and 100μM

[0110]

[0111]

[0112] Note: a Standard deviation, b Inactive

[0113] Compounds 7, 8, and 12 with good inhibition rates were selected, and different concentration gradients were set for compounds with different inhibition rates. Three replicate wells were used to perform nonlinear curve fitting analysis on the inhibition rate of each concentration and calculate the half-maximal inhibition concentration (IC) 50 value.

[0114] The 384-well plate contained four systems: blank group, negative group, positive control group, and sample group. Among them, the blank group: 3 μL complete buffer, 2 μL substrate SPH + ATP; negative group: 1 μL complete buffer, 2 μL substrate SPH + ATP, 2 μL SphK2 protein kinase; sample group: 1 μL different concentrations of compound, 2 μL substrate SPH + ATP, 2 μL SphK2 protein kinase; positive control group: 1 μL different concentrations of ABC294640, 2 μL substrate SPH + ATP, 2 μL SphK2 protein kinase; then incubated at room temperature 25 ° C for 60 minutes, ADP-Glo was added TMKinase detection reagent. The fluorescence intensity value (F value) was measured using a Bio-Stack microplate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm, and the inhibition rate of compounds at different concentrations on the SphK2 enzyme was calculated. The negative group and the drug-treated group minus the blank group are the respective correction values. Inhibition rate = (negative group correction value - drug-treated group correction value) / negative group correction value x 100%. Select compounds with an inhibition rate greater than the best, set 6 different concentration gradients and 3 replicates, perform nonlinear curve fitting analysis on the inhibition rate of each concentration, and calculate the half-maximal inhibitory concentration IC 50 Value. Half maximal inhibitory concentration IC 50 The lower the value, the stronger the inhibitory ability of the compound on SphK2. The experimental results are shown in Table 5.

[0115] Table 5 Inhibitory activity of some compounds of the present invention on SphK2

[0116] Compound <![CDATA[IC 50 ]]> 7 3.62±1.42 8 10.54±1.24 12 10.95±1.47 ABC294640 11.40±1.33

[0117] Compounds 7, 8, and 12 had significant inhibitory effects on SphK2, and their effects were significantly better than the positive control drug ABC294640.

[0118] Example 5: Study on the effects of the compounds of the present invention on cancer cell proliferation

[0119] (1) Experimental materials: target compound and positive control ABC294640, trypsin, washing solution PBS, fetal bovine serum (Gemini), human hepatocellular carcinoma (HepG2), mouse hepatocellular carcinoma (Hepa1-6), human colon cancer (HCT116), mouse colon cancer (MC-38), cervical cancer (MCF-7), gastric cancer (HGC-27) cells (Shanghai Cell Bank, Chinese Academy of Sciences), microplate reader (thermoscientific), 96-well plates, DMEM medium and 1640 medium (Solarbio, where the final concentrations of penicillin and streptomycin were 100 U / mL and 100 U / mL, respectively).

[0120] (2) Experimental methods:

[0121] Cells grown to the logarithmic phase were prepared into a uniformly dispersed single cell suspension, with 90 μL of single cell suspension per well containing approximately (2-5) x10 3Cells were seeded into 96-well plates and incubated in a 5% CO2, 37°C incubator for 48 hours. After the cells adhered, 10 μL of 6 samples of different concentrations were added, and 3-5 parallels were set for each concentration. After incubation for 48 hours, 10 μL of CCK8 was added to each well and incubated for 1 to 2 hours. The absorbance (OD value) of each well was measured using a microplate reader at a wavelength of 450 nm. Each group of experiments was independently paralleled three times. Inhibition rate = 1-[(OD experimental group-OD blank group) / (OD negative control-OD blank group)]×100%. Graphpad 7.0 software was used for analysis, and the logarithmic value of the sample concentration was linearly regressed with the cell inhibition rate to calculate the half-maximal inhibitory concentration IC of the compound for each cell. 50 Values (mean ± standard deviation).

[0122] Table 6 Comparison of experimental results of half-maximal inhibitory concentration of some compounds of the present invention on cancer cells

[0123]

[0124] The experimental results showed that compound 7 had a significant inhibitory effect on six cancer cell lines, and its effect was significantly better than the positive control drug ABC294640; compound 8 had a significant inhibitory effect on MC-38 mouse colon cancer cells, which was better than the positive control ABC294640; compound 12 had a significant inhibitory effect on human liver cancer (HepG2), mouse liver cancer (Hepa1-6), and cervical cancer (MCF-7) cells, which was better than the positive control ABC294640.

[0125] In summary, the above examples demonstrate that according to the present invention, a series of compounds with higher enzyme inhibition and anticancer activity than the positive control ABC294640 were discovered, among which compound 7 showed the best enzyme inhibition and anticancer cell proliferation effects, indicating that it can be further developed into a new generation of anticancer drugs.

[0126] The above description of the embodiments is intended to facilitate understanding and application of the invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any minor variations in the formulations and processes described herein are considered within the scope of the present invention.

Claims

1. A 4-substituted phenylacetamide compound or a pharmaceutically acceptable salt thereof, characterized in that: The general structural formula of the compound is shown in formula (I) or (II): Wherein: X is C or N; R1 is any one of H, F, Cl or CF3; n=1, 2 or 3; when n=1, it is monosubstituted; when n=2, it is disubstituted, in which case R1 is the same or different; when n=3, it is trisubstituted, in which case R1 is the same or different; R2 is Any one of .

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that R1 is hydrogen, chlorine or trifluoromethyl, and R2 is any one of ethanolamine, glycylamino, L-prolinol, D-prolinamide or 4-hydroxypiperidinyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that Selected from the following structures:

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The salt is any one of acetate, ascorbate, benzoate, benzenesulfonate, citrate, fumarate, hydrochloride, hydrobromide, maleate or methanesulfonate.

5. A method for preparing the compound according to claim 1, characterized in that: The synthetic route is as follows: Wherein, X, R1 and R2 are as defined in claim 1.

6. The preparation method according to claim 5, characterized in that The following steps are involved: (1) Compound F and pinacol borate are reacted in a 1,4-dioxane solution under N2 protection and heated under reflux for 2 h to generate compound G; the molar ratio of compound F to pinacol borate is 1:1.2; (2) Compound A and compound B are heated to reflux at 65°C in an acetone solution for 3 h to produce compound C; The molar ratio of compound A to compound B is 1:1.5; (3) Compound C and compound G undergo Suzuki coupling under anaerobic conditions, and the reaction is heated under reflux at 95°C for 5 hours to generate compound D; the molar ratio of compound C to compound G is 1:1; (4) Compound D and sodium hydroxide are reacted in a methanol:water = 4:1 system, heated under reflux at 70°C for 1 to 1.5 hours to generate compound E, wherein the molar ratio of compound D to sodium hydroxide is 1:3; (5) Compound E reacts with an amine having a structure R2 in a dichloromethane solvent at room temperature (25° C.) overnight to produce a compound represented by formula (I); the molar ratio of compound E to amine is 1:1; or, (2') Compound A2 and compound B2 were heated to reflux at 65°C in an acetone solution for 3 hours to produce compound C2; the molar ratio of compound A2 to compound B2 was 1:1.5; (3') Compound C2 and compound G underwent Suzuki coupling under anaerobic conditions, and the reaction was heated under reflux at 95°C for 5 h to produce compound D2; the molar ratio of compound C2 to G was 1:1; (4') Compound D2 and sodium hydroxide are reacted in a methanol:water system of 4:1, heated under reflux at 70°C for 1-1.5 hours to produce compound E2, wherein the molar ratio of compound D2 to sodium hydroxide is 1:3; (5') Compound E2 reacts with an amine having a structure R2 in a dichloromethane solvent at room temperature (25°C) overnight to generate a compound represented by formula (II); the molar ratio of compound E2 to amine is 1:

1.

7. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug.

8. The use according to claim 7, characterized in that The tumor is human histiocytic gastric cancer, cervical cancer, human colon cancer, human liver cancer, mouse liver cancer or mouse colon cancer.

9. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

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