A 2-sulfonylpyrimidine-4-amide compound and its uses

Abstract

This invention provides a 2-sulfonylpyrimidine-4-amide compound and its uses, belonging to the field of antitumor drug technology. The compound of this invention exhibits excellent antitumor activity targeting the colchicine site of tubulin, demonstrating antiproliferative activity against tumor cells such as HeLa, HepG2, H1299, and MCF-7 at the micromolar level, and significantly inhibiting colony formation in a dose-dependent manner. Furthermore, the compound inhibits tubulin polymerization and disrupts the microtubule network of H1299 cells in vitro, inducing cell cycle arrest in the G2 / M phase and apoptosis. More importantly, the compound can inhibit angiogenesis in HUVECs in vitro. The compound of this invention has great potential in the preparation of microtubule destabilizing agents targeting the colchicine site.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a 2-sulfonylpyrimidine-4-amide compound and its uses. Background Technology

[0002] Microtubules are a crucial component of the cytoskeleton, participating in various physiological processes, including maintaining cell structure, cell division, spindle formation, and intracellular transport. Disruption of microtubule polymerization homeostasis can lead to cell cycle arrest in the G2-M phase, the formation of abnormal mitotic spindles, and ultimately, apoptosis. Due to their vital role in mitosis, microtubules have become a highly attractive target for anticancer drug discovery. In recent decades, various natural and synthetic compounds with vincristine, colchicine, and paclitaxel as their primary binding sites have been extensively explored. Compared to paclitaxel and vincristine sites, drugs targeting colchicine sites can overcome P-gp multidrug resistance and inhibit angiogenesis. Currently, numerous colchicine binding site inhibitors (CBSIs) containing multiple cytoskeleton components have been discovered; however, due to their relatively narrow therapeutic indications, various toxicities, and multi-organ dysfunction side effects, none have yet been approved by the FDA for cancer treatment. Therefore, much more work is needed to explore novel microtubule destabilizers that interact with colchicine binding sites to overcome the above limitations and provide more potential active lead candidates for the development of anticancer drugs. Summary of the Invention

[0003] The purpose of this invention is to provide a 2-sulfonylpyrimidine-4-amide compound and its use in preparing an antitumor drug targeting the colchicine site.

[0004] This invention provides a 2-sulfonylpyrimidine-4-amide compound, characterized in that the structure of the 2-sulfonylpyrimidine-4-amide compound is shown in Formula I:

[0005]

[0006] in:

[0007] R 1 Selected from those that have not been replaced or have been R 5 The following groups are substituted: C 1-6 Alkyl, phenyl, heteroaryl, fused cycloalkyl, heterofused cycloalkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic; R 5 Selected from hydroxyl, amino, amide, guanidinyl, thiol, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6Alkylthio, phenyl;

[0008] R 2 Selected from hydrogen, hydroxyl, amino, amide, guanidinyl, mercapto, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl;

[0009] R 3 Hydrogen, hydroxyl, amino, amide, guanidinyl, mercapto, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl;

[0010] R 4 Selected from those that have not been replaced or have been R 5 The following groups are substituted: C 1-6 Alkyl, phenyl, heteroaryl, fused cycloalkyl, heterofused cycloalkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic; R 5 Selected from hydroxyl, amino, amide, guanidinyl, thiol, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl.

[0011] Furthermore, the structure of the 2-sulfonylpyrimidine-4-amide compound is shown in Formula II:

[0012]

[0013] in:

[0014] R 1 Selected from those that have not been replaced or have been R 5 The following groups are substituted: C 1-6 Alkyl, phenyl, heteroaryl, fused cycloalkyl, heterofused cycloalkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic; R 5 Selected from hydroxyl, amino, amide, guanidinyl, thiol, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl;

[0015] R 4Selected from those that have not been replaced or have been R 5 The following groups are substituted: C 1-6 Alkyl, phenyl, heteroaryl, fused cycloalkyl, heterofused cycloalkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic; R 5 Selected from hydroxyl, amino, amide, guanidinyl, thiol, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl.

[0016] Furthermore, the structure of the 2-sulfonylpyrimidine-4-amide compound is shown in Formula II:

[0017]

[0018] in:

[0019] R 4 Selected from those that have not been replaced or have been R 5 The following groups are substituted: C 1-6 Alkyl, phenyl, heteroaryl, fused cycloalkyl, heterofused cycloalkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic; R 5 Selected from hydroxyl, amino, amide, guanidinyl, thiol, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl;

[0020] m can be 0, 1, 2, 3 or 4;

[0021] R 6 Selected from hydrogen, hydroxyl, amino, amide, guanidinyl, mercapto, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl.

[0022] Furthermore, the structure of the 2-sulfonylpyrimidine-4-amide compound is shown in Formula III:

[0023]

[0024] in:

[0025] R 1 Selected from those that have not been replaced or have been R 5 The following groups are substituted: C 1-6Alkyl, phenyl, heteroaryl, fused cycloalkyl, heterofused cycloalkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclic; R 5 Selected from hydroxyl, amino, amide, guanidinyl, thiol, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl;

[0026] m can be 0, 1, 2, 3 or 4;

[0027] R 7 Selected from hydrogen, hydroxyl, amino, amide, guanidinyl, mercapto, halogen, C 1-6 Alkyl, halogen-substituted C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 alkylamine group, C 1-6 Alkylthio, phenyl.

[0028] Furthermore, the 2-sulfonylpyrimidine-4-amide compounds are selected from:

[0029]

[0030] The present invention also provides a pharmaceutical composition comprising the above-described 2-sulfonylpyrimidine-4-amide compounds and pharmaceutically acceptable excipients.

[0031] The present invention also provides the use of the above-mentioned 2-sulfonylpyrimidine-4-amide compounds in the preparation of antitumor drugs.

[0032] In some implementations, the tumor is selected from lung cancer, cervical cancer, breast cancer, and liver cancer.

[0033] The present invention also provides the use of the above-mentioned 2-sulfonylpyrimidine-4-amide compounds in combination with other antitumor drugs or sensitizers in the preparation of antitumor drugs.

[0034] The present invention also provides that the target of the above-mentioned 2-sulfonylpyrimidine-4-amide compounds is the colchicine site of tubulin.

[0035] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0036] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～bAlkyl groups refer to any alkyl group containing one to two carbon atoms. For example, C 1～6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0037] "Heteroaryl" refers to a heteroaryl group containing at least one heteroatom, which includes, but is not limited to, oxygen, sulfur, and nitrogen. For example...

[0038] "Fused cycloalkyl" refers to a polycyclic cycloalkyl group in which two rings share two adjacent carbon atoms.

[0039] "Heterocyclic alkyl" refers to a polycyclic heterocyclic group in which two rings share two adjacent carbon atoms or heteroatoms.

[0040] "3- to 6-membered saturated cycloalkyl" refers to saturated cycloalkyl groups with 3 to 6 carbon atoms in the ring.

[0041] "3- to 6-membered saturated heterocyclic groups" refers to saturated heterocyclic groups with 3 to 6 ring atoms, where the ring atoms contain at least one heteroatom.

[0042] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0043] This invention provides a novel 2-sulfonylpyrimidine-4-amide compound that targets the colchicine site of tubulin and exhibits excellent antitumor activity at the micromolar level, and significantly inhibits colony formation in a dose-dependent manner.

[0044] The compound of this invention inhibits tubulin polymerization and disrupts the microtubule network of H1299 cells in vitro, inducing cell cycle arrest in the G2 / M phase and apoptosis in H1299 cells.

[0045] The compounds of this invention can inhibit angiogenesis in HUVECs in vitro.

[0046] The compounds of this invention have great potential in the preparation of microtubule-destabilized antitumor drugs that target colchicine sites.

[0047] Obviously, based on the above description of the present invention, and according to common technical knowledge and effective means in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Attached Figure Description

[0048] Figure 1The effects of the preferred compounds of this invention on cancer cell proliferation and colony formation. (A) Inhibitory effect of a 40 μM concentration of the compound on the growth of MCF-7 cells. (BC) Antiproliferative activity (IC50) of colchicine (B) and the preferred compound of this invention, hit22 (C), against HeLa, HepG2, H1299, and MCF-7 cancer cells. 50 (D) Effect of different doses of the preferred compound hit22 of the present invention on H1299 cell colony formation. (E) Statistical test of the effect of the preferred compound hit22 of the present invention on H1299 cell colony formation. The data shown represent the mean ± standard deviation (sd) of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, compared with the control group.

[0049] Figure 2 The preferred compound of this invention, hit22, is known for its ability to inhibit microtubule polymerization in vitro.

[0050] Figure 3 The effect of the preferred compound hit22 of this invention on microtubule network damage in H1299 cells. (A) Confocal microscopy images illustrating the effect of the preferred compound hit22 of this invention on the microtubule network of H1299 cells. (B) Mean fluorescence intensity of microtubules treated with the preferred compound hit22 of this invention. Laser scanning confocal microscopy, 40×, Bar, 7.5 μm was used. Images represent typical cells from at least three independent experiments. Data shown represent the mean ± standard deviation (sd) of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, compared with the control group.

[0051] Figure 4 The effect of the preferred compound hit22 of this invention on the migration of H1299 cells. (A) Scratch assay of H1299 cells treated with different doses of the preferred compound hit22 of this invention, and photographs. (B) Statistical test of the effect of the preferred compound hit22 of this invention on the migration of H1299 cells. The data shown represent the mean ± standard deviation (sd) of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared with the control group. Detailed Implementation

[0052] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0053] The following embodiments better illustrate the content of the present invention. However, the present invention is not limited to the following embodiments.

[0054] Example 1

[0055] Preparation of compound (3)

[0056]

[0057] Compound (1) (2.89 g, 0.02 mol) was dispersed evenly in 100 mL of ethanol, and then thiourea (1.52 g, 0.02 mol) was added. The mixture was then heated to 96 °C and stirred for 10 h. After the reaction was completed as detected by TLC, the reaction solution was concentrated under reduced pressure to obtain 4.38 g of crude solid compound (3), with a yield of 99%.

[0058] Example 2

[0059] Preparation of compound (5)

[0060]

[0061] Compound (3) (4.42 g, 0.02 mol) was dispersed evenly in 100 mL of water in an ice bath, and then chlorogenic acid (5.07 g, 0.03 mol) was added, followed by the slow addition of triethylamine (3.03 g, 0.03 mol), and the mixture was stirred overnight. After the reaction was completed as detected by TLC, the pH of the reaction solution was adjusted to 5, and then extracted with ethyl acetate and concentrated under reduced pressure to obtain 4.07 g of crude compound (5), with a yield of 68%.

[0062] Example 3

[0063] Preparation of compound (7)

[0064]

[0065] Compound (5) (5.98 g, 0.02 mol) and compound (6) (4.83 g, 0.03 mol) were dispersed evenly in 100 mL of dichloromethane. Then, HATU (7.6 g, 0.02 mol) and DIPEA (2.58 g, 0.02 mol) were added sequentially to the above reaction system, and the mixture was stirred at room temperature for 12 h. After removing the solvent by vacuum distillation, the mixture was recrystallized in ethyl acetate to give 8.4 g of compound (7), with a yield of 95%.

[0066] 1 H NMR(400MHz,Chloroform-d)δ10.18(s,1H),8.71(s,1H),8.49(d,J=8.3Hz,1H),7.69–7.5 6(m,2H),7.47–7.36(m,1H),7.30–7.23(m,2H),7.06(dt,J=14.6,8.2Hz,2H),4.48(s,2H).

[0067] 13 C NMR (150MHz, Chloroform-d) δ 169.28, 161.89, 161.17, 160.24, 159.26, 150.72, 134.50, 133.04, 130.97, 130.94, 129.60, 129.54, 126.32, 126.29, 126.25, 126.21, 125.78, 124.89, 124.29, 124.27, 123.23, 123.20, 123.10, 120.21, 120.01, 115.69, 115.55, 77.21, 77.00, 76.79, 29.09, 29.06. Example 4

[0068] Preparation of compound (hit-22)

[0069]

[0070] Compound (7) (8.84 g, 0.02 mol) was dispersed evenly in 100 mL of dichloromethane. Meta-chloroperoxybenzoic acid (8.65 g, 0.05 mol) was added in portions, and the mixture was stirred for 3 h. After the reaction was complete as detected by TLC, the reaction solution was quenched with saturated sodium thiosulfate solution and saturated sodium bicarbonate solution. The reaction solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Recrystallization in ethyl acetate solution yielded 7.94 g of compound (hit-22), with a yield of 84%.

[0071] 1 H NMR(400MHz,Chloroform-d)δ10.12(s,1H),9.09(s,1H),8.43(d,J=8.3Hz,1H),7.74–7.61(m,2H),7.49(td,J=7.5,1.8 Hz,1H),7.34(ddt,J=7.9,5.4,2.5Hz,2H),7.17(td,J=7.6,1.2Hz,1H),7.01(ddd,J=9.7,8.3,1.3Hz,1H),4.87(s,2H).

[0072] 13C NMR(150MHz,Chloroform-d)δ162.59,162.03,161.46,160.38,157.90,152.11,133.86,133.08,133.02,133.00,131.56,131.50 ,126.50,126.47,125.59,124.85,124.83,124.75,123.60,115.86,115.71,113.77,113.68,77.21,77.00,76.79,51.78,51.76.

[0073] Example 5

[0074] Preparation of compound (9)

[0075]

[0076] Compound (5) (0.598 g, 0.002 mol) and compound (8) (0.279 g, 0.003 mol) were dispersed evenly in 10 mL of dichloromethane. Then, HATU (0.76 g, 0.002 mol) and DIPEA (0.258 g, 0.002 mol) were added sequentially to the above reaction system, and the mixture was stirred at room temperature for 12 h. After removing the solvent by vacuum distillation, 0.718 g of crude compound (9) was obtained, with a yield of 96%.

[0077] Example 6

[0078] Preparation of compound (hit-15)

[0079]

[0080] Compound (9) (0.748 g, 0.002 mol) was dispersed evenly in 10 mL of dichloromethane. Meta-chloroperoxybenzoic acid (0.865 g, 0.005 mol) was added in portions, and the mixture was stirred for 3 h. After the reaction was complete as detected by TLC, the reaction solution was quenched with saturated sodium thiosulfate solution and saturated sodium bicarbonate solution. The reaction solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Recrystallization in ethyl acetate solution yielded 0.69 g of compound (hit-15), with a yield of 85%.

[0081] 1 H NMR(400MHz,Chloroform-d)δ10.12(s,1H),9.09(s,1H),7.80–7.74(m,2H),7.41 –7.24(m,3H),7.20–7.12(m,2H),7.02(ddd,J=10.1,7.7,1.4Hz,1H),4.87(s,2H).

[0082] 13 C NMR(150MHz,Chloroform-d)δ162.59,162.03,161.08,160.91,158.36,153.90,136.96,131.45,129.84,129. 80,129.45,129.39,128.49,126.10,126.07,123.54,120.52,120.40,120.25,116.42,116.25,51.78,51.76.

[0083] Example 7

[0084] Preparation of compound (11)

[0085]

[0086] Compound (5) (0.598 g, 0.002 mol) and compound (10) (0.429 g, 0.003 mol) were dispersed evenly in 10 mL of dichloromethane. Then, HATU (0.76 g, 0.002 mol) and DIPEA (0.258 g, 0.002 mol) were added sequentially to the above reaction system, and the mixture was stirred at room temperature for 12 h. After removing the solvent by vacuum distillation, 0.772 g of crude compound (11) was obtained, with a yield of 91%.

[0087] Example 8

[0088] Preparation of compound (hit-16)

[0089]

[0090] Compound (11) (0.848 g, 0.002 mol) was dispersed evenly in 10 mL of dichloromethane. Meta-chloroperoxybenzoic acid (0.865 g, 0.005 mol) was added in portions, and the mixture was stirred for 3 h. After the reaction was complete as detected by TLC, the reaction solution was quenched with saturated sodium thiosulfate solution and saturated sodium bicarbonate solution. The reaction solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Recrystallization in ethyl acetate solution yielded 0.748 g of compound (hit-16), with a yield of 82%.

[0091] 1H NMR(400MHz,Chloroform-d)δ10.14(s,1H),9.40(s,1H),7.82(dd,J=8.0,1.6Hz ,1H),7.79(ddd,J=7.9,6.7,1.1Hz,1H),7.70(dt,J=8.1,1.5Hz,1H),7.52(ddd,J =8.1,6.7,1.6Hz,1H),7.41–7.33(m,2H),7.32–7.24(m,1H),7.15(dddt,J=8.8,3 .6,1.9,0.9Hz,1H),7.08–6.98(m,2H),6.73(dt,J=7.7,1.4Hz,1H),4.97(s,2H).

[0092] 13 C NMR(150MHz,Chloroform-d)δ165.04,162.87,161.08,160.91,158.36,153.90,134.43,132.41,131.45,129.84,129.80,129.47,129. 45,129.39,128.03,127.55,127.01,126.91,126.10,126.07,126.01,122.83,121.65,120.52,120.40,116.42,116.25,54.97,54.92.

[0093] Example 9

[0094] Preparation of compound (13)

[0095]

[0096] Compound (1) (2.53 g, 0.02 mol) was dispersed evenly in 100 mL of ethanol, and then thiourea (1.52 g, 0.02 mol) was added. The mixture was then heated to 96 °C and stirred for 10 h. After the reaction was completed as detected by TLC, the reaction solution was concentrated under reduced pressure to obtain 4.02 g of crude solid compound (13), with a yield of 99%.

[0097] Example 10

[0098] Preparation of compound (14)

[0099]

[0100] In an ice bath, compound (13) (4.06 g, 0.02 mol) was dispersed evenly in 100 mL of water, then chlorogenic acid (5.07 g, 0.03 mol) was added, followed by the slow addition of triethylamine (3.03 g, 0.03 mol), and the mixture was stirred overnight. After the reaction was completed as detected by TLC, the pH of the reaction solution was adjusted to 5, and then extracted with ethyl acetate and concentrated under reduced pressure to obtain 3.65 g of crude compound (14), with a yield of 65%.

[0101] Example 11

[0102] Preparation of compound (15)

[0103]

[0104] Compound (14) (5.62 g, 0.02 mol) and compound (6) (4.83 g, 0.03 mol) were dispersed evenly in 100 mL of dichloromethane. Then, HATU (7.6 g, 0.02 mol) and DIPEA (2.58 g, 0.02 mol) were added sequentially to the above reaction system, and the mixture was stirred at room temperature for 12 h. After removing the solvent by vacuum distillation, 7.97 g of crude compound (15) was obtained, with a yield of 94%.

[0105] Example 12

[0106] Preparation of compound (hit-1)

[0107]

[0108] Compound (15) (8.48 g, 0.02 mol) was dispersed evenly in 100 mL of dichloromethane. Then, m-chloroperoxybenzoic acid (8.65 g, 0.05 mol) was added in portions, and the mixture was stirred for 3 h. After the reaction was complete as detected by TLC, the reaction solution was quenched with saturated sodium thiosulfate solution and saturated sodium bicarbonate solution. The reaction solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The solution was then recrystallized in ethyl acetate solution to give 8.03 g of compound (hit-1), with a yield of 88%.

[0109] 1 H NMR(400MHz,Chloroform-d)δ10.02(s,1H),9.30(s,1H),7.71(dd,J=10.2,1.9Hz,1H),7.6 2(dd,J=6.8,1.7Hz,1H),7.53–7.42(m,2H),7.38(dtd,J=6.5,4.2,2.1Hz,5H),4.77(s,2H).

[0110] 13C NMR(150MHz,Chloroform-d)δ165.89,161.08,158.36,153.90,136.23,136.18, 136.13,136.08,131.58,131.45,131.06,129.96,129.48,128.19,127.83,126. 81,126.78,126.76,126.73,126.04,126.01,125.98,125.96,125.64,123.45,121.99,121.73,121.47,121.25,121.20,119.93,119.91,119.88,119.86,58.81.

[0111] Example 13

[0112] Preparation of compound (17)

[0113]

[0114] Compound (1) (3.13 g, 0.02 mol) was dispersed evenly in 100 mL of ethanol in an ice bath, and then thiourea (1.52 g, 0.02 mol) was added. The mixture was then heated to 96 °C and stirred for 10 h. After the reaction was completed as detected by TLC, the reaction solution was concentrated under reduced pressure to obtain 4.61 g of crude solid compound (17), with a yield of 99%.

[0115] Example 14

[0116] Preparation of compound (18)

[0117]

[0118] Compound (17) (4.66 g, 0.02 mol) was dispersed evenly in 100 mL of water in an ice bath, and then chlorogenic acid (5.07 g, 0.03 mol) was added, followed by the slow addition of triethylamine (3.03 g, 0.03 mol), and the mixture was stirred overnight. After the reaction was completed as detected by TLC, the pH of the reaction solution was adjusted to 5, and then extracted with ethyl acetate and concentrated under reduced pressure to obtain 3.73 g of crude compound (18), with a yield of 60%.

[0119] Example 15

[0120] Preparation of compound (19)

[0121]

[0122] Compound (18) (6.22 g, 0.02 mol) and compound (6) (4.83 g, 0.03 mol) were dispersed evenly in 100 mL of dichloromethane. Then, HATU (7.6 g, 0.02 mol) and DIPEA (2.58 g, 0.02 mol) were added sequentially to the above reaction system, and the mixture was stirred at room temperature for 12 h. After removing the solvent by vacuum distillation, 8.17 g of crude compound (19) was obtained, with a yield of 90%.

[0123] Example 16

[0124] Preparation of compound (hit-8)

[0125]

[0126] Compound (19) (9.08 g, 0.02 mol) was dispersed evenly in 100 mL of dichloromethane. Meta-chloroperoxybenzoic acid (8.65 g, 0.05 mol) was added in portions, and the mixture was stirred for 3 h. After the reaction was complete as detected by TLC, the reaction solution was quenched with saturated sodium thiosulfate solution and saturated sodium bicarbonate solution. The reaction solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Recrystallization in ethyl acetate solution yielded 7.68 g of compound (hit-8), with a yield of 79%.

[0127] 1 H NMR(400MHz,Chloroform-d)δ9.65(s,1H),9.30(s,1H),7.71(dd,J=10.2,1.8Hz,1H),7.62(dd,J=6.8,1 .7Hz,1H),7.53–7.42(m,2H),7.14–7.08(m,2H),6.82–6.76(m,2H),4.70(d,J=0.9Hz,2H),3.82(s,3H).

[0128] 13 C NMR(150MHz,Chloroform-d)δ164.89,161.08,159.39,158.36,153.90,136.23,1 36.18,136.13,136.08,131.45,130.39,129.48,127.83,126.81,126.78,126.76, 126.73, 126.04, 126.01, 125.98, 125.96, 125.64, 125.18, 123.45, 121.99, 121.73, 121.47, 121.25, 121.20, 119.93, 119.91, 119.88, 119.86, 114.92, 58.71, 55.35.

[0129] The following experimental examples demonstrate the beneficial effects of the compounds of this invention.

[0130] Experimental Example 1: The compound of this invention exhibits inhibitory activity against tumor cell proliferation and colony formation.

[0131] Using colchicine as a positive control, an MTT assay was performed on four cancer cell lines (HeLa, MCF-7, HepG2, and H1299). First, the effects of 25 compounds on MCF-7 cells at a concentration of 40 μM for 72 hours were assessed. Figure 1 As shown in (A), eight compounds exhibited inhibition rates exceeding 50% at a concentration of 40 μM. Among them, the preferred compound of this invention, hit22, showed the strongest antiproliferative activity against MCF-7 cells, with an inhibition rate of 94.87%. The antiproliferative activity of the preferred compound hit22 against HeLa, H1299, MCF-7, and HepG2 cells was then determined. Figure 1 As shown in Figure (B), among these four cell types, HeLa and MCF-7 cancer cells were sensitive to colchicine, while HepG2 and H1299 cells showed relatively low sensitivity. The IC50 values ​​for the antiproliferative activity of colchicine against HeLa, HepG2, H1299, and MCF-7 cell lines were [not specified in the original text]. 50 The values ​​were 0.017, 0.071, 0.135, and 0.023 μM, respectively. For example... Figure 1 As shown in (C), in the treatment group with the preferred compound hit22 of the present invention, H1299 and MCF-7 cancer cells were sensitive to the preferred compound hit22 of the present invention, while HeLa and HepG2 showed relatively low sensitivity to the preferred compound hit22 of the present invention. The IC50 values ​​of the preferred compound hit22 of the present invention for HeLa, HepG2, H1299, and MCF-7 are... 50 The values ​​were 32.28, 16.41, 3.93, and 7.65 μM, respectively. Clearly, among these four cancer cell lines, the preferred compound hit22 of this invention exhibited the strongest anti-proliferative activity against H1299 cancer cells. Furthermore, the inhibitory effect of the preferred compound hit22 of this invention on H1299 cell colony formation was investigated. H1299 cells were exposed to different concentrations of the preferred compound hit22 of this invention (1, 3, 6, and 9 μM) for 72 h, then washed and incubated in conventional culture medium for 7 days. Figure 1 As shown in (D), the number of colonies decreased significantly with increasing concentration of the preferred compound hit22. In particular, when the concentration reached 9 μM, the preferred compound hit22 almost completely inhibited colony formation in H1299 cells. Figure 1As shown in (E), the preferred compound of the present invention, hit22, inhibits the proliferation of H1299 cell colonies in a dose-dependent manner. In summary, these preliminary observations demonstrate that the preferred compound of the present invention, hit22, exhibits good anti-proliferative activity in vitro, as evidenced by its ability to inhibit H1299 cell proliferation and colony formation.

[0132] Experimental Example 2: The preferred compound of this invention, hit22, blocks the microtubule network by targeting tubulin.

[0133] In this invention, immunofluorescence staining was used to detect the effect of the preferred compound hit22 on cellular microtubule structure. H1299 cells were treated with 0.5, 1.0, 3.0, and 6.0 μM concentrations of the preferred compound hit22 for 24 h. Colchicine was used as a positive control group. The results are as follows: Figure 3 As shown in (A), the untreated microtubule network exhibits normal arrangement and organization, while cells treated with different doses of the preferred compound of the present invention, hit22, show severe disruption of the microtubule network, similar to colchicine. With increasing concentration of the preferred compound hit22, the microtubule structure becomes increasingly disordered. The relative fluorescence intensity of the microtubules is shown in Figure (A). Figure 3 As shown in (B), the relative fluorescence intensity of microtubules treated with 10 nM colchicine was 80.04%. At concentrations of 0.5, 1.0, 3.0, and 6.0 μM, the relative fluorescence intensities of H1299 microtubules were 87.45%, 80.23%, 67.18%, and 54.39%, respectively. These data and the morphological changes in the microtubules indicate that the preferred compound of this invention, hit22, can stably disrupt the H1299 cell microtubule network in a dose-dependent manner.

[0134] Experimental Example 3: The preferred compound of this invention, hit22, inhibits the migration of H1299 cells.

[0135] Cell migration is closely related to cancer cell invasion and the formation of metastatic tumors, and microtubules are important components of cell migration. In this invention, the effect of the preferred compound hit22 on the migration potential of H1299 cells was evaluated using a wound healing assay. Figure 4 As shown in (A), different doses of the preferred compound hit22 of the present invention showed reduced scratch closure of H1299 compared to the control group. Figure 4As shown in Figure (B), H1299 cells treated with colchicine at 10 nM for 12 and 24 h showed migration rates of 21.53% and 35.72%, respectively. After treatment with the preferred compound hit22 of this invention at 0, 1, 3, and 6 μM for 12 h, the migration rates of H1299 cells were 33.24%, 32.13%, 28.85%, and 26.37%, respectively. After treatment with the preferred compound hit22 of this invention at 0, 1, 3, and 6 μM concentrations for 24 h, the migration rates of H1299 cells were 58.74%, 56.98%, 50.99%, and 37.69%, respectively. These results indicate that the preferred compound hit22 of this invention has a significant inhibitory effect on the migration of H1299 cells, and this effect is time- and concentration-dependent.

[0136] In summary, this invention provides a 2-sulfonylpyrimidine-4-amide compound that exhibits excellent antitumor activity targeting the colchicine site of tubulin. It demonstrates antiproliferative activity against tumor cells such as HeLa, HepG2, H1299, and MCF-7 at the micromolar level and significantly inhibits colony formation in a dose-dependent manner. Furthermore, the compound inhibits tubulin polymerization and disrupts the microtubule network of H1299 cells in vitro, inducing cell cycle arrest at the G2 / M phase and apoptosis. More importantly, the compound inhibits angiogenesis in HUVECs in vitro. This compound shows great potential in the preparation of microtubule destabilizing agents targeting the colchicine site.

Claims

1. A 2-sulfonylpyrimidine-4-amide compound, characterized in that, The structure of the 2-sulfonylpyrimidine-4-amide compound is as follows: or .

2. A pharmaceutical composition, characterized in that, It comprises the 2-sulfonylpyrimidine-4-amide compound of claim 1 and pharmaceutically acceptable excipients.

3. Use of the 2-sulfonylpyrimidine-4-amide compound of claim 1 in the preparation of antitumor drugs.

4. The use of the 2-sulfonylpyrimidine-4-amide compound of claim 1 in combination with other antitumor drugs or sensitizers in the preparation of antitumor drugs.

5. The use according to claim 3 or 4, characterized in that, The target site of the 2-sulfonylpyrimidine-4-amide compound is the colchicine site of tubulin.

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