4-substituted phenylacetamides, processes for their preparation and use thereof

Abstract

The application provides a 4-substituted phenylacetamide compound and a preparation method and application thereof, and a structural formula of the compound is shown as formula (I) or (II). The compound provided by the application is a kind of sphingosine kinase 2 (SphK2) targeted small molecule, can effectively inhibit SphK2 enzyme activity and human hepatoma (HepG2), murine hepatoma (Hepa1-6), human colon cancer (HCT116), murine colon cancer (MC-38), cervical cancer (MCF-7), gastric cancer (HGC-27) and other cancer cell proliferation and migration, and the inhibiting effect is better than that of a known inhibitor ABC294640, has the potential to be developed into a new anticancer drug. The synthesis steps of the compound are simple, the requirement for equipment is low, and the operation is simple, so that the yield of the reaction can be greatly improved, and the production cost is further saved.

Description

Technical Field

[0001] This invention relates to 4-substituted phenylacetamide compounds, their preparation methods and applications, and particularly to compounds with substituted pyridine, substituted pyrazine, and substituted pyrimidine skeletons, their preparation methods and applications. Background Technology

[0002] The balance of sphingolipids in the human body is closely related to tumors, and targeting the sphingolipid pathway is a novel approach to developing anticancer drugs. Ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphosine (S1P) are all sphingolipids in the body. They not only participate in the composition of cell membranes but also act as lipid signaling molecules involved in cell proliferation, differentiation, and apoptosis. Studies have shown that Cer / Sph promotes apoptosis, while S1P promotes cell survival; their imbalance promotes tumor development and progression. Sphingolipid research typically uses the term "sphingolipid-rheostat" to describe the balance and transformation among these three components. Therefore, targeting the sphingolipid signaling pathway and regulating the "sphingolipid-rheostat" has become a new direction for developing antitumor drugs.

[0003] SphK catalyzes the phosphorylation of Sph to S1P and is a key rate-limiting enzyme controlling the spholipid-variable rheostat. Inhibition of SphK can increase the Cer / Sph ratio, which promotes apoptosis, and decrease S1P, which promotes cell survival, thereby producing an anti-tumor effect. SphKs have two subtypes, with SphK1 being widely reported as an oncogene. However, due to the contradictory key physiological functions of SphK2, related pathological research and drug development have been severely hampered. Nevertheless, the important role of SphK2 in promoting tumorigenesis has been unanimously recognized by researchers in recent years, especially with its selective inhibitor ABC294640 currently in Phase II clinical development, which strongly promotes research on the related mechanisms of SphK2.

[0004] Currently, there are few reported highly effective and selective SphK2 inhibitors. However, studies have shown that targeting SphK2 exhibits superior antitumor activity compared to targeting SphK1. Nevertheless, the crystal structure of SphK2 remains unknown, and no SphK2 inhibitors have been approved for marketing globally. Furthermore, most reported SphK2 inhibitors are limited by low potency, low specificity, structural homogeneity, and limited availability, which significantly hinders research on the sphingolipid signaling pathway and the development of related therapeutic drugs. Although ABC294640 has been widely used in biological research, it still suffers from low enzyme activity.

[0005] Therefore, there is an urgent need to develop novel, highly efficient, and highly selective SphK2 inhibitors to solve the above problems. Summary of the Invention

[0006] One of the objectives of this invention is to provide a 4-substituted phenylacetamide compound that can serve as a highly efficient and selective SphK2 inhibitor, thereby addressing the problems of low efficacy and simple structure of existing SphK2 inhibitors.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned compounds and their pharmaceutical salts.

[0008] A further object of the present invention is to provide the use of the above-mentioned compounds and their pharmaceutical salts in the preparation of medicaments for the prevention and treatment of cancer.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

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

[0011]

[0012] In the formula: X in formula (I) 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 may be the same or different; when n = 3, it is trisubstituted, in which case R1 may be the same or different.

[0014] R2 is , , , , Any one of them.

[0015] Preferably, R1 is hydrogen, chlorine or trifluoromethyl, and R2 is any one of ethanolamine, glycineamido, L-prolyl, D-prolylamide or 4-hydroxypiperidinyl.

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

[0017]

[0018] The pharmaceutical salts of the compounds include, but are not limited to, acetates, ascorbic acid salts, benzoates, benzenesulfonates, citrates, fumarates, hydrochlorides, hydrobroms, maleates, and mesylates.

[0019] The present invention also provides a method for preparing the above-mentioned compound, the synthetic route of which is as follows:

[0020] or

[0021] Includes the following steps:

[0022] (1) Compound F reacts with pinacol boronic acid ester to produce compound G.

[0023] (2) Compound A undergoes a substitution reaction with compound B to produce compound C.

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

[0025] (4) Compound D undergoes a hydrolysis reaction to produce compound E.

[0026] (5) Compound E undergoes condensation with different types of amine organic bases with R2 structures, such as ethanolamine, glycine, L-prolyl or L-prolylamide, to produce the compound shown in formula (I);

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

[0028] Specifically, in step (2), the substitution conditions are: compound A and compound B are heated to reflux at 65°C in acetone solution and reacted for 3 hours to generate compound C; the molar ratio of compound A to 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 react under reflux at 95°C for 5 hours 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 react in a methanol:water = 4:1 system, heated under reflux at 70°C for 1 to 1.5 h 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 as follows: compound E reacts with an amine having an R2 structure in dichloromethane solvent overnight at room temperature (25°C) to generate the compound shown in formula (I) or (II); the molar ratio of compound E to the amine is 1:1.

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

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

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

[0035] (4') Compound D2 undergoes a hydrolysis reaction to produce compound E2.

[0036] (5') Compound E2 undergoes condensation with different types of amine organic bases with the R2 structure, such as ethanolamine, glycine, L-prolyl or L-prolylamide, to produce the compound shown in formula (II);

[0037] Specifically, in step (2'), the substitution conditions are as follows: compound A2 and compound B2 are heated to reflux at 65°C in acetone solution and reacted 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 the reaction is carried out 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 as follows: compound D2 and sodium hydroxide react in a methanol:water = 4:1 system, heated under reflux at 70°C for 1 to 1.5 h 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 as follows: compound E2 reacts with an amine having an R2 structure in dichloromethane solvent overnight at room temperature (25°C) to produce the compound shown in formula (II); the molar ratio of compound E2 to the amine is 1:1.

[0041] The present invention also provides the use of the aforementioned biphenyl compounds or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0042] The tumor is a human tissue cell gastric cancer, cervical cancer, colon cancer, liver cancer, or murine liver cancer or colon cancer.

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

[0044] This invention synthesizes a novel class of SphK2-targeting small drug molecules. The synthesized compounds exhibit a very strong inhibitory effect on SphK2 enzyme activity and cancer cell proliferation, showing better inhibitory effects than the existing inhibitor ABC294640, and have the potential to be developed into novel anticancer therapeutic drugs.

[0045] The present invention provides a simple reaction procedure for synthesizing SphK2-targeted drug molecules, with low equipment requirements and easy operation, which can significantly improve the reaction yield and further save production costs. At the same time, it eliminates the need for dangerous, flammable and explosive reagents, achieving the effects of simplifying the process, reducing costs and facilitating post-processing while maintaining good catalytic effect and reducing costs, making it suitable for industrial production. Detailed Implementation

[0046] In the following embodiments, the various processes and methods not described in detail are conventional methods known in the art, and the reagents used, unless otherwise specified, are commercially available analytical grade or chromatographic grade.

[0047] Example 1: Preparation of the series of compounds shown in 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 pinacolborate, 1.29 g (13.16 mmol) of potassium acetate, and 0.06 g (0.09 mmol) of [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride were placed in a 50 mL flask containing 20 mL of 1,4-dioxane. The mixture was heated under argon protection and refluxed at 95 °C for 2 h. The reaction endpoint was determined by TCL. The solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column 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 with 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 round-bottom 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 TCL. The solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:8) to give 1.22 g of 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(diphenylphosphine)ferrocene]palladium dichloride were placed in a 50 mL round-bottom flask containing 20 mL of mixed solvent (ethanol:water = 1:1). Under nitrogen protection, the mixture was heated to reflux at 85 °C for 2 h, and the reaction endpoint was detected by TCL. The ethanol in the mixed solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate:petroleum ether = 1:6) to give 1.49 g of a brownish-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 round-bottom flask containing 20 mL of mixed solvent (methanol:water = 1:1). The mixture was heated under reflux at 50 °C for 3 h, and the reaction endpoint was detected by TCL. The methanol in the mixed solvent was removed under reduced pressure, and an appropriate amount of 1 mol·L⁻¹ sodium hydroxide solution was added. -1 The pH was adjusted to neutral or acidic with hydrochloric acid solution until a pale yellow solid precipitated. The solid was filtered and the filter cake was dried naturally to give 0.84 g of brown solid (compound E), with a yield of approximately 85.26-90.45%.

[0052] (5) Dissolve 0.32 g (1 mmol) of compound E in 5 mL of dichloromethane, add 0.29 g (1.5 mmol) of EDCI and 0.03 g (0.2 mmol) of DMAP, and react at room temperature for 1 h. Then add (1 mmol) of different types of amines (see Table 1), and react overnight at room temperature (25 °C). Detect the reaction endpoint by TLC, extract with ethyl acetate, and dry with water and Na2SO4. Separate and purify by silica gel column chromatography (eluent: dichloromethane:methanol = 60:1), recrystallize from ethyl acetate, and a white solid precipitates. Dry under vacuum to obtain the target product series (I), with a yield of approximately 75-88%.

[0053] The specific structures of the corresponding compounds obtained by using different types of amines in step (5) are shown in Table 1.

[0054] Table 1

[0055]

[0056] The characterization data of compounds 1-8 are as follows:

[0057] Compound 1: 1 H NMR (600 MHz, Chloroform- d ) d 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 C NMR (150 MHz, Chloroform- d ) d173.13, 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。

[0058] Compound 2: 1 H NMR (600 MHz, DMSO- d 6) d 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 = 9.4Hz, 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). 13 C NMR (150 MHz, DMSO- d 6) d 169.09, 139.06, 137.36, 136.14, 129.81, 129.79, 128.51, 127.72, 127.48, 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。

[0059] Compound 3: 1 H NMR (600 MHz, DMSO- d 6) d 8.22 (d, J = 2.5 Hz, 1H), 8.05 (s,1H), 7.83 (dd, J = 9.4, 2.6 Hz, 1H), 7.49-7.44 (m, 2H), 7.37-7.32 (m, 4H), 7.31-7.26 (m, 3H), 6.52 (d, J = 9.4 Hz, 1H), 5.17 (s, 2H), 4.67 (t, J = 5.4 Hz,1H), 3.42-3.38 (m, 4H), 3.11 (q, J = 5.9 Hz, 2H). 13 C NMR (150 MHz, DMSO- d 6) d 170.07, 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-ESIm / z calcd for C 22 H 22 N₂O₃, [M+ Na] + 385.1532, found: 385.1532.

[0060] Compound 4: 1 H NMR (600 MHz, DMSO- d 6) d 8.25 (d, J = 2.6 Hz, 1H), 7.84 (dd, J =9.4, 2.6 Hz, 1H), 7.52-7.47 (m,2H), 7.37-7.27 (m, 7H), 6.53 (d, J = 9.4 Hz,1H), 5.18 (s, 2H), 3.70 (s, 2H), 3.61 (s, 3H). 13 C NMR (150 MHz, DMSO- d 6) d171.53, 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。

[0061] Compound 5: 1 H NMR (600 MHz, DMSO- d 6) d 8.34 – 8.29 (m, 1H), 7.90 – 7.86 (m, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.51 – 7.47 (m, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.3 Hz, 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 (150 MHz, DMSO- d 6) d173.95, 173.74, 168.94, 168.83, 160.62, 142.01, 139.35, 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, 59.82, 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.

[0062] Compound 6: 1 H NMR (600 MHz, DMSO- d 6) d 8.25 (d, J J = 2.6 Hz, 1H), 8.05 (t, J J = 5.4 Hz, 1H), 7.82 (dd, J J = 9.5, 2.7 Hz, 1H), 7.49 (d, J J = 8.2 Hz, 2H), 7.44 (dd, J J = 8.5, 5.6 Hz, 2H), 7.30 (d, J J = 8.2 Hz, 2H), 7.17 (t, J J = 8.9 Hz, 2H), 6.52 (d, J J = 9.4 Hz, 1H), 5.15 (s, 2H), 4.68 (t, J J = 5.4 Hz, 1H), 3.43 (s, 2H), 3.40 (q, J J = 5.9 Hz, 2H), 3.12 (q, J J = 5.9 Hz, 2H). 13 C NMR (150 MHz, DMSO- d 6) d170.09, 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 H 21 FN2O3, [M+H] + 381.1608, found: 381.1609.

[0063] Compound 7: 1 H NMR (600 MHz, Chloroform- d ) d 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). 13 C NMR (150 MHz, Chloroform- d ) d172.06, 161.72, 139.65, 136.51, 134.90, 134.16, 133.81, 133.03, 132.38, 130.90, 129.98, 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 for C 25 H 24 Cl2N2O3, [M+Na] + 493.1056, found: 493.1056.

[0064] Compound 8: 1 H NMR (600 MHz, Chloroform- d ) d 7.56 (dd, J J = 9.4, 2.6 Hz, 1H), 7.37 (d, J J = 2.2 Hz, 2H), 7.35 (d, J J = 8.3 Hz, 1H), 7.25 (d, J J = 8.2 Hz, 2H), 7.21 (d, J J = 8.2 Hz, 2H), 7.12 (dd, J J = 8.3, 1.8 Hz, 1H), 6.64 (d, J J = 9.4 Hz, 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 (150 MHz, Chloroform- d ) d169.08, 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 calcdfor C 25 H 24 Cl2N3O3, [M+Na] + 493.1056, found: 493.1056.

[0065] Example 2: Preparation of the series of compounds shown in formula (I): When X=N, it is a pyrazine ring;

[0066] Step (1) is the same as in Example 1.

[0067] (2) 0.88 g (5.00 mmol) of 2-hydroxy-5-bromopyrazine (compound A1), 0.76 g (6.00 mmol) of benzyl chloride with 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 round-bottom 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 TCL. The solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column chromatography (eluent: ethyl acetate: petroleum ether = 1:16) to give 1.23 g of pale 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(diphenylphosphine)ferrocene]palladium dichloride were placed in a 50 mL round-bottom flask containing 20 mL of mixed solvent (ethanol:water = 1:1). Under nitrogen protection, the mixture was heated to reflux at 85 °C for 2 h, and the reaction endpoint was detected by TCL. The ethanol in the mixed solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate:petroleum ether = 1:6) to give 1.48 g of pale yellow powder (compound D1), with a yield of approximately 81.33-94.56%.

[0068] (4) Weigh 1.67 g (5.00 mmol) of compound D1 and 1.00 g (25.00 mmol) of sodium hydroxide, place them in a 50 mL round-bottom flask containing 20 mL of mixed solvent (methanol:water = 1:1), heat under reflux at 50 °C for 3 h, and then use a TCL to detect the reaction endpoint. Remove methanol from the mixed solvent under reduced pressure, and add an appropriate amount of 1 mol·L⁻¹. -1 The pH was adjusted to neutral or acidic with hydrochloric acid solution until a pale yellow solid precipitated. The solid was filtered and the filter cake was dried naturally to give 1.36 g of pale yellow solid (compound E1), with a yield of approximately 71.14-84.98%.

[0069] (5) Dissolve 0.32 g (1 mmol) of compound E1 in 5 mL of dichloromethane, add 0.29 g (1.5 mmol) of EDCI and 0.03 g (0.2 mmol) of DMAP, and react at room temperature for 1 h. Then add (1 mmol) of different types of amines (see Table 2), and react overnight at room temperature (25 °C). Detect the reaction endpoint by TLC, extract with ethyl acetate, and dry with water and Na2SO4. Separate and purify by silica gel column chromatography (eluent: dichloromethane:methanol = 40:1), recrystallize from ethyl acetate, and a white solid precipitates. Dry under vacuum to obtain the target product series (I), with a yield of approximately 65-85%.

[0070] The specific structures of the corresponding compounds obtained by using different types of amines in step (5) are shown in Table 2.

[0071] Table 2

[0072]

[0073] The characterization data of compounds 9-16 are as follows:

[0074] Compound 9: 1 H NMR (600 MHz, Chloroform- d ) d 8.30 (s, 1H), 7.63 (d, J = 8.0Hz, 2H), 7.45 (s, 1H), 7.41-7.34 (m, 5H), 7.29 (d, J= 8.0 Hz, 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 CNMR (150 MHz, Chloroform - d ) d 173.23, 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 calcd forC 24 H 24 N4O3, [M + Na] + 439.1740, found: 439.1741。

[0075] Compound 10: 1 H NMR (600 MHz, Chloroform - d ) d 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). 13CNMR (150 MHz, Chloroform- d ) d 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。

[0076] Compound 11: 1 H NMR (600 MHz, Chloroform- d ) d 8.30 (d, J J = 1.2 Hz, 1H), 7.65 - 7.60 (m, 2H), 7.46 (d, J 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). 13 C NMR (150 MHz, Chloroform- d ) d 169.13, 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 24H 25 N3O3, [M+Na] + 426.1788, found:426.1788.

[0077] Compound 12: 1 H NMR (600 MHz, Chloroform- d ) d 8.32 (s, 1H), 7.73 – 7.66 (m,4H), 7.50 – 7.45 (m, 3H), 7.32 (d, J = 8.1 Hz, 2H), 5.21 (s, 2H), 4.91 (d, J =7.6 Hz, 1H), 4.26 – 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 (150 MHz, Chloroform- d ) d 172.08, 155.17, 149.11,138.72, 134.55, 134.02, 129.61, 128.55, 126.20, 126.17, 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 / z calcd for C 25 H 24 F3N3O3, [M+Na] + 494.1661, found: 494.1662.

[0078] Compound 13: 1 H NMR (600 MHz, Chloroform- d ) d8.33 – 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 C NMR (150 MHz, Chloroform- d ) d 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 H 24 F3N3O3, [M+Na] + 494.1661, found: 494.1662。

[0079] Compound 14: 1 H NMR (600 MHz, DMSO- d 6) d 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 C NMR (150 MHz, DMSO-d 6) d 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 H 19 F3N4O3, [M+Na] + 467.1301, found: 467.1301。

[0080] Compound 15: 1 H NMR (600 MHz, Chloroform- d ) d 8.29 (d, J J = 1.2 Hz, 1H), 7.66– 7.61 (m, 2H), 7.44 (d, J J = 1.3 Hz, 1H), 7.38 – 7.34 (m, 2H), 7.30 (dd, J J = 8.5, 2.9 Hz, 4H), 6.95 (s, 1H), 5.37 (s, 1H), 5.11 (s, 2H), 4.61 (dd, J J = 8.1, 2.1Hz, 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 ) d 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 C24 H 23 ClN4O3, [M+Na] + 473.1350, found: 473.1351.

[0081] Compound 16: 1 H NMR (600 MHz, Methanol- d 4) d 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). 13 C NMR (150 MHz, Methanol- d 4) d 174.15, 157.18, 148.81, 137.01, 131.57,131.52, 130.65, 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.

[0082] Example 3: Preparation of the series of compounds shown in formula (II)

[0083] Step (1) is the same as in Example 1.

[0084] (2) 0.66 g (5.00 mmol) of 4-chloro-6-hydroxypyrimidine (compound A2), 0.76 g (6.00 mmol) of benzyl chloride with 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 round-bottom 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 TCL. The solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate: petroleum ether = 1:6) to give 1.03 g of white powder (compound C2), with a yield of approximately 82.14-96.34%.

[0085] (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(diphenylphosphine)ferrocene]palladium dichloride were placed in a 50 mL round-bottom flask containing 20 mL of mixed solvent (ethanol:water = 1:1). Under argon protection, the mixture was heated to reflux at 85 °C for 2 h, and the reaction endpoint was detected by TCL. The ethanol in the mixed solvent was removed under reduced pressure, the reaction was quenched with water, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and dried over anhydrous Na2SO4. The mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate:petroleum ether = 1:4) to give 1.48 g of white powder (compound D2), with a yield of approximately 75.98-88.67%.

[0086] (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 round-bottom flask containing 20 mL of mixed solvent (methanol:water = 1:1). The mixture was heated under reflux at 50 °C for 3 h, and the reaction endpoint was detected by TCL. The methanol in the mixed solvent was removed under reduced pressure, and an appropriate amount of 1 mol·L⁻¹ was added. -1 The pH was adjusted to neutral or acidic with hydrochloric acid solution until a pale yellow solid precipitated. The solid was filtered and the filter cake was dried naturally to give 1.40 g of pale yellow solid (compound E2), with a yield of approximately 75.66-89.45%.

[0087] (5) Dissolve 0.32 g (1 mmol) of compound E2 in 5 ml of dichloromethane, add 0.29 g (1.5 mmol) of EDCI and 0.03 g (0.2 mmol) of DMAP, and react at room temperature for 1 h. Then add (1 mmol) of different types of amines (see Table 1), and react overnight at room temperature (25 °C). Detect the reaction endpoint by TLC, extract with ethyl acetate, and dry with water and Na2SO4. Separate and purify by silica gel column chromatography (eluent: dichloromethane:methanol = 60:1), recrystallize from ethyl acetate, and a white solid precipitates. Dry under vacuum to obtain the target product series (II), with a yield of approximately 65-86%.

[0088] The specific structures of the corresponding compounds obtained by using different types of amines in step (5) are shown in Table 3.

[0089] Table 3

[0090]

[0091] The characterization data of compounds 17-23 are as follows:

[0092] Compound 17: 1 H NMR (600 MHz, Methanol- d 4) d 8.42 (s, 1H), 7.95-7.90 (m,2H), 7.42-7.36 (m, 6H), 7.36-7.34 (m, 1H), 6.89 (d, J = 2.9 Hz, 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). 13 C NMR (150 MHz, Methanol- d 4) d 176.92, 172.11, 163.86, 162.90, 152.43,138.97, 136.80, 135.75, 131.08, 130.99, 130.25, 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.

[0093] Compound 18: 1 H NMR (600 MHz, Chloroform- d ) d 8.21 (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 ) d 171.74, 161.65, 160.64, 150.24, 137.22, 135.27, 134.74,129.55, 129.47, 129.10, 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.

[0094] Compound 19: 1 H NMR (600 MHz, Methanol- d 4) d8.57 (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 C NMR (150 MHz, Methanol - d 4) d 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。

[0095] Compound 20: 1 H NMR (600 MHz, DMSO - d 6) d 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 C NMR (150 MHz, DMSO - d 6) d169.76, 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。

[0096] Compound 21: 1H NMR (600 MHz, Chloroform-d) d 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- d 6) d 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。

[0097] Compound 22: 1 H NMR (600 MHz, DMSO- d 6) d 8.68 – 8.65 (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). 13 C NMR (150 MHz, DMSO- d 6) d 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 / zcalcd for C 21 H 19 ClFN3O3, [M+Na] + 438.0991, found:438.0991.

[0098] Compound 23: 1 H NMR (600 MHz, DMSO- d 6) d 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 C NMR (150 MHz, DMSO- d 6) d 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 forC 24 H 23 Cl2N3O3, [M+Na] + 494.1008, found: 494.1009.

[0099] Example 4: Activity study of the series of compounds of the present invention against SphK2

[0100] This embodiment investigated the activity of all the above compounds on SphK2.

[0101] (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 detection kit

[0102] (2) Experimental method:

[0103] All of the above samples were measured at 10 m M, 100 mThe inhibition rate of M on SphK2 at different concentrations (n=3, three replicates) is shown in Table 4.

[0104] Table 4. Compounds of the present invention in 10 m M, 100 m Inhibition rate of M at different concentrations on SphK2

[0105]

[0106] Note: a Standard deviation b Inactive

[0107] Compounds 7, 8, and 12, which showed good inhibition rates, were selected. Different concentration gradients were set for each compound with different inhibition rates, using three replicates. Nonlinear curve fitting analysis was performed on the inhibition rate at each concentration, and the half-maximal inhibitory concentration (IC50) was calculated. 50 value.

[0108] The 384-well plate contains four systems: a blank control group, a negative control group, a positive control group, and a sample group. The blank control group consists of 3 wells. m L intact buffer, 2 µ L substrate SPH+ATP; Negative group: 1 m L intact buffer, 2 µ L substrate SPH+ATP, 2 µ LSphK2 protein kinase; Sample group: 1 m L different concentrations of compounds, 2 µ L substrate SPH+ATP, 2 µ L SphK2 protein kinase; Positive control group: 1 m L different concentrations of ABC294640, 2 µ L substrate SPH+ATP, 2 µ L SphK2 protein kinase; then incubated at room temperature (25°C) for 60 min, followed by the addition of ADP-Glo TM Kinase assay reagents. Fluorescence intensity (F-value) was measured using a Bio-Stack microplate reader at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The inhibition rate of different concentrations of the compound against SphK2 enzyme was calculated. The correction values ​​for the negative control group and the treated group were obtained by subtracting the blank control group. Inhibition rate = (Corrected value of negative control group - Corrected value of treated group) / Corrected value of negative control group x 100%. Compounds with relatively high inhibition rates were selected, and six different concentration gradients were set up with three replicates. Nonlinear curve fitting analysis was performed on the inhibition rate at each concentration, and the half-maximal inhibitory concentration (IC50) was calculated. 50 Value. Half-maximal inhibitory concentration (IC50) 50 The lower the value, the stronger the inhibitory effect of the compound on SphK2. The experimental results are shown in Table 5.

[0109] Table 5. Inhibitory activity of some compounds of the present invention against SphK2.

[0110]

[0111] Compounds 7, 8, and 12 showed significant inhibitory effects on SphK2, and their effects were significantly better than those of the positive control drug ABC294640.

[0112] Example 5: Study on the effects of the series of compounds of the present invention on cancer cell proliferation

[0113] (1) Experimental materials: target compound and positive control ABC294640, trypsin, washing solution PBS, fetal bovine serum (Gemini), human hepatocellular carcinoma (HepG2), murine hepatocellular carcinoma (Hepa1-6), human colon cancer (HCT116), murine colon cancer (MC-38), cervical cancer (MCF-7), gastric cancer (HGC-27) cells (Shanghai Cell Bank of Chinese Academy of Sciences), microplate reader (thermoscientific) and 96-well plates, DMEM medium and 1640 medium (Solarbio, in which the final concentrations of penicillin and streptomycin were 100 U / mL and 100 U / mL, respectively).

[0114] (2) Experimental method:

[0115] Cells grown to the logarithmic growth phase were prepared into a uniformly dispersed single-cell suspension, with 90 cells per well. µ L single-cell suspension approximately (2-5) x 10 3 Cells were seeded into 96-well plates and incubated in a 5% CO2, 37°C incubator for 48 hours. After cell attachment, 10 μL of each of the six different concentrations of the sample was added. m For each concentration, set up 3-5 replicates. After incubation for 48 hours, add 10 μL to each well. m After incubation with CCK8 for 1-2 hours, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Each experiment was conducted independently in triplicate. Inhibition rate = 1 - [(OD experimental group - OD blank group) / (OD negative control - OD blank group)] × 100%. Graphpad 7.0 software was used for analysis; linear regression was performed between the logarithm of sample concentration and cell inhibition rate to calculate the half-maximal inhibitory concentration (IC50) of the compound for each cell type. 50 Value (mean ± standard deviation).

[0116] Table 6 Comparison of experimental results of half-maximal inhibitory concentrations (IC50) of some compounds of the present invention against cancer cells.

[0117]

[0118] Experimental results showed that compound 7 had a significant inhibitory effect on all six cancer cell lines, and its effect was significantly better than that of the positive control drug ABC294640; compound 8 had a significant inhibitory effect on MC-38 murine colon cancer cells, which was better than that of the positive control ABC294640; compound 12 had a significant inhibitory effect on human liver cancer (HepG2), murine liver cancer (Hepa1-6), and cervical cancer (MCF-7) cells, which was better than that of the positive control ABC294640.

[0119] In summary, the above embodiments demonstrate that, according to the present invention, a series of compounds with higher enzyme inhibitory and anticancer activities than the positive control ABC294640 have been discovered, among which compound 7 exhibits the best enzyme inhibitory and anticancer cell proliferation effects, indicating that it can be further developed into a new generation of anticancer drugs.

[0120] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be easily made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Any partial changes to the formulation and process therein should be within the scope of protection 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): ， In the formula: X is C or N; R1 is any one of 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 may be the same or different; when n = 3, it is trisubstituted, in which case R1 may be the same or different. R2 is , Any one of them.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Selected from the following structure: 、 、 、 Compound 7, Compound 8, Compound 12 、 ， Compound 13 Compound 21.

3. 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.

4. A method for preparing the compound according to claim 1, characterized in that, The synthesis route is as follows: or ， In the formula, X, R1 and R2 are defined as in claim 1.

5. The preparation method according to claim 4, characterized in that, Includes the following steps: (1) Compound F and pinacol boronic acid ester were heated under reflux for 2 h in a 1,4-dioxane solution under N2 protection to generate compound G; the molar ratio of compound F to pinacol boronic acid ester was 1:1.

2. (2) Compound A and compound B are heated to reflux at 65°C in acetone solution for 3 hours to produce compound C; The molar ratio of compounds A to B is 1:1.5; (3) Compound C and compound G undergo Suzuki coupling under anaerobic conditions and react under reflux at 95°C for 5 hours to generate compound D; the molar ratio of compound C to G is 1:

1. (4) Compound D reacts with sodium hydroxide in a methanol:water = 4:1 system by heating and reflux at 70°C for 1 to 1.5 h to generate compound E. The molar ratio of compound D to sodium hydroxide is 1:

3. (5) Compound E reacts with an amine having an R2 structure in dichloromethane solvent overnight at room temperature (25°C) to produce the compound shown in formula (I); the molar ratio of compound E to the amine is 1:

1. or, (2') Compound A2 and compound B2 are heated under reflux in acetone solution at 65°C for 3 hours to produce compound C2; the molar ratio of compound A2 to B2 is 1:1.

5. (3') Compound C2 and compound G undergo Suzuki coupling under anaerobic conditions, and react under reflux at 95°C for 5 h to generate compound D2; the molar ratio of compound C2 to G is 1:1; (4') Compound D2 and sodium hydroxide reacted in a methanol:water = 4:1 system under reflux at 70°C for 1 to 1.5 h to generate compound E2. The molar ratio of compound D2 to sodium hydroxide was 1:

3. (5') Compound E2 was reacted with an amine having the R2 structure in dichloromethane solvent overnight at room temperature (25°C) to produce the compound shown in formula (II); the molar ratio of compound E2 to the amine was 1:

1.

6. Use of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug.

7. The application according to claim 6, characterized in that, The tumor is a human tissue cell gastric cancer, cervical cancer, human colon cancer, human liver cancer, murine liver cancer, or murine colon cancer.

8. A pharmaceutical composition, characterized in that, It comprises the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

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