Methylthiazole derivatives with anti-hepatocellular carcinoma activity, their preparation methods and applications

Abstract

This invention relates to a method for preparing and applying a methylthiazole derivative with anti-hepatocellular carcinoma activity, belonging to the field of pharmaceutical synthesis technology. Based on the structure of 2-[3-acrylonitrile-4-(2-isobutoxy)-phenyl]-4-methylthiazole-5-carboxylic acid, a terminal alkyne is introduced into its structure. Then, through a click reaction with azide compounds, a variety of novel compounds are obtained. Activity tests in antitumor, anti-inflammatory, and antioxidant properties demonstrate that the obtained compounds possess significant anticancer activity, providing substantial application value for the development of anticancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a method for preparing and applying a methylthiazole derivative with anti-hepatocellular carcinoma activity. Background Technology

[0002] The thiazole ring is a five-membered aromatic heterocyclic structure containing nitrogen and sulfur, whose chemical properties and biological activities make it crucial in drug development. Thiazole ring structures are commonly found in small molecule drugs with diverse biological activities. For example, thiazole ring derivatives can possess a variety of pharmacological activities, including antibacterial, antifungal, antiviral, anti-inflammatory, antitumor, antidiabetic, and antihypertensive effects. This makes the thiazole ring an important structural unit in drug development. The thiazole ring can interact with various target proteins, acting as a key pharmacophore. Its aromaticity and heterocyclic nature allow for various interactions at the binding site, such as hydrogen bonds, hydrophobic interactions, and π-π interactions, thereby increasing the binding affinity and selectivity of drug molecules. The chemical structure of the thiazole ring allows for modifications at multiple positions, such as introducing different substituents onto different carbon atoms or nitrogen and sulfur atoms. This flexibility allows researchers to improve the pharmacokinetic properties of drugs, reduce toxicity, and enhance efficacy through structural optimization. Many marketed drugs contain a thiazole ring structure. For example, 2-[3-cyano-4-(2-isobutoxy)-phenyl]-4-methylthiazol-5-carboxylic acid is a non-purine analogue inhibitor of xanthine oxidase in both oxidized and reduced forms. It is a novel oral anti-hyperuric acid drug whose therapeutic effect is achieved by promoting uric acid excretion, thereby lowering serum uric acid levels. In the treatment of gout, it has advantages such as high efficacy, high selectivity, fewer side effects, and higher safety, and has gradually replaced allopurinol and benzbromarone, becoming the preferred drug for the current clinical treatment of gout and hyperuricemia.

[0003] 1,2,3-Triazole is an important building block for drug molecules. Its planar rigid structure allows it to insert into the DNA of tumor cells, interacting with the DNA and causing damage. This type of structure exhibits isosteric behavior similar to amides, esters, and carboxylic acids, enabling it to coordinate with metal ions. It can act on metal-containing proteases, inhibiting their activity and thus affecting tumor cell growth. Simultaneously, the 1,2,3-triazole ring can link different pharmacodynamic molecules together to form a new molecule; these derivatives often possess low toxicity and high activity. These properties of triazole structures make them widely used in modifying natural products, active azole compounds, or quinone compounds, and they are extensively applied in innovative drug development. Our team collaborated with Jinan Aisi Pharmaceutical Technology Co., Ltd. on the synthesis and activity testing of methylthiazole derivatives modified with 1,2,3-triazole. Jinan Aisi Pharmaceutical Technology Co., Ltd. was responsible for the compound synthesis, while our team was responsible for the activity testing. Summary of the Invention

[0004] To research and develop innovative drugs, the present invention aims to provide a method for preparing and applying a methylthiazole derivative with anti-hepatocellular carcinoma activity. The methylthiazole derivative is based on the structure of 2-[3-acrylonitrile-4-(2-isobutoxy)-phenyl]-4-methylthiazole-5-carboxylic acid, with a terminal alkyne introduced into its structure. Various novel compounds are obtained through reaction with azide compounds, exhibiting good antitumor, anti-inflammatory, and antioxidant activities.

[0005] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a methylthiazole derivative with anti-hepatocellular carcinoma activity, the structure of which is shown in Formula I or Formula II below;

[0007] Equation I is: Equation II is: In Equations I and II, n is 0 or 1; R 1 R 2 R 3 R 4 and R 5 It can be one or more of the following: H, halogen, trifluoromethyl, ethyl, phenyl, hydroxyl, cyano, nitro, aromatic ring, etc.

[0008] Secondly, the present invention requests a method for preparing the above-mentioned methylthiazole derivative, which is based on the structure of 2-[3-acrylonitrile-4-(2-isobutoxy)-phenyl]-4-methylthiazole-5-carboxylic acid, to which a terminal alkyne is introduced, and then reacted with an azide compound by a click reaction; the product obtained varies depending on the type of azide compound.

[0009] Thirdly, the application of the above-mentioned methylthiazole derivatives in the preparation of anti-liver cancer drugs.

[0010] Beneficial effects: This invention is based on the structure of 2-[3-cyano-4-(2-isobutoxy)-phenyl]-4-methylthiazol-5-carboxylic acid, to which a terminal alkyne is introduced. Then, through click reaction with azide compounds, a variety of novel compounds are obtained. The antitumor, anti-inflammatory and antioxidant activities are tested. The results show that the obtained compounds have significant anticancer activity, providing great application value for the development of anticancer drugs. Attached Figure Description

[0011] Figure 1 This is the 1H NMR spectrum of compound 4.4 synthesized in Example 36.

[0012] Figure 2 This is the 1H NMR spectrum of compound 4.16 synthesized in Example 48. Detailed Implementation

[0013] This invention is based on the structure of 2-[3-cyano-4-(2-isobutoxy)-phenyl]-4-methylthiazol-5-carboxylic acid. Terminal alkynes were introduced into the structure, and then more than ten novel compounds were obtained by click reaction with azide compounds. The compounds were tested for antitumor, anti-inflammatory and antioxidant activities. Our team was responsible for the design and activity testing of the target compounds, while Jinan Aisi Pharmaceutical Technology Co., Ltd. was responsible for the specific synthesis research.

[0014] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter 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.

[0015] Unless otherwise specified, all reagents used in the following examples are commercially available reagents, and all operating methods employed are conventional techniques.

[0016] Example 1

[0017]

[0018] 31.6 g of 2-(3-acrylonitrile-4-isobutoxyphenyl)-4-methylthiazol-5-carboxylic acid, 11.7 g of 3-ethynylaniline, 38 g of HATU, and 26 g of DIPEA were added sequentially to a clean 500 mL round-bottom flask equipped with a stir bar. Then, 800 mL of N,N-dimethylformamide was added to the system, and the mixture was stirred at room temperature for 1 h. 600 mL of water was added to the reaction system. During stirring, a large amount of solid precipitated out. The solid was filtered and dried to obtain 35.1 g of 2-(3-acrylonitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazol-5-carboxamide.

[0019] Example 2

[0020]

[0021] 31.6 g of 2-(3-acrylonitrile-4-isobutoxyphenyl)-4-methylthiazol-5-carboxylic acid, 11.7 g of 4-ethynylaniline, 38 g of HATU, and 26 g of DIPEA were added sequentially to a clean 500 mL round-bottom flask equipped with a stirrer. Then, 800 mL of N,N-dimethylformamide was added to the system, and the mixture was stirred at room temperature for 1 h. 600 mL of water was added to the reaction system. During stirring, a large amount of solid precipitated out. The solid was filtered and dried to obtain 37.4 g of 2-(3-acrylonitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazol-5-carboxamide.

[0022] Example 3

[0023]

[0024] 1 mmol of 2-(3-cyano-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazolyl-5-carboxamide, 1.2 mmol of 4-bromobenzyl azide, 2 mmol of sodium L-ascorbate, and 1 mmol of anhydrous CuSO4 were added to a 100 mL round-bottom flask equipped with a stir bar. Then, 50 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added to the flask, and the mixture was stirred at room temperature for 10 min until completely dissolved. The reaction was allowed to proceed overnight at room temperature with stirring for 2 h. After the reaction was complete as detected by TLC, the mixture was extracted three times with DCM, and the lower organic phase was collected. The mixture was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration. The crude product was obtained by vacuum distillation at 35°C to remove organic solvents such as DCM. The crude product was then separated and purified by column chromatography to obtain a white solid 3.1. 1H NMR (400MHz, DMSO-d6): 10.36 (s, 1H), 8.64 (s, 1H), 8.31 (d, J = 4.0Hz, 1H), 8.26-8.22 (m, 2H), 7.66-7.57 (m, 4H), 7. 45-7.40(m,2H),7.34(d,J=8.0Hz,2H),5.65(s,2H),4.03(d,J=4.0Hz,2H),2.67(s,2H),1.04(s,3H),1.03(s,3H).

[0025] Example 4

[0026]

[0027] The synthesis method of compound 3.2 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-acrylbenzyl azide. The crude product is purified by column chromatography to obtain solid 3.2.

[0028] Example 5

[0029]

[0030] The synthesis method of compound 3.3 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-bromobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.3.

[0031] Example 6

[0032]

[0033] The synthesis of compound 3.4 was similar to that of compound 3.1, except that 3-fluorobenzyl azide was used instead of 4-bromobenzyl azide. The crude product was purified by column chromatography to obtain solid 3.4.

[0034] Example 7

[0035]

[0036] The synthesis method of compound 3.5 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-chloro-6-fluorobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.5.

[0037] Example 8

[0038]

[0039] The synthesis method of compound 3.6 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-chloro-4-fluorobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.6.

[0040] Example 9

[0041]

[0042] The synthesis method of compound 3.7 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-chlorobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.7.

[0043] Example 10

[0044]

[0045] The synthesis method of compound 3.8 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-fluorophenyl azide. The crude product is purified by column chromatography to obtain solid 3.8.

[0046] Example 11

[0047]

[0048] The synthesis method of compound 3.9 is similar to that of compound 3.1, except that 3-chlorophenyl azide is used instead of 4-bromobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.9.

[0049] Example 12

[0050]

[0051] The synthesis method of compound 3.10 is similar to that of compound 4.1, except that 4-chlorophenyl azide is used instead of 4-bromobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.10.

[0052] Example 13

[0053]

[0054] The synthesis method of compound 3.11 is similar to that of compound 3.1, except that 4-fluorophenyl azide is used instead of 4-bromobenzyl azide. The crude product is purified by column chromatography to obtain solid 3.11.

[0055] Example 14

[0056]

[0057] The synthesis method of compound 3.12 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 2-trifluoromethylphenyl azide. The crude product is purified by column chromatography to obtain solid 3.12.

[0058] Example 15

[0059]

[0060] The synthesis of compound 3.13 was similar to that of compound 3.1, except that zidovudine was used instead of 4-bromobenzyl azide. The crude product was purified by column chromatography to obtain solid 3.13.

[0061] Example 16

[0062]

[0063] The synthesis of compound 3.14 was similar to that of compound 3.1, except that 4-bromobenzyl azide was replaced with 2-trifluoromethoxyphenyl azide. The crude product was purified by column chromatography to obtain solid 3.14.

[0064] Example 17

[0065]

[0066] The synthesis method of compound 3.15 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 3-trifluoromethylbenzyl azide. The crude product is purified by column chromatography to obtain solid 3.15.

[0067] Example 18

[0068]

[0069] The synthesis method of compound 3.16 is similar to that of compound 3.1, except that 4-bromobenzyl azide is replaced by 4-trifluoromethylbenzyl azide. The crude product is purified by column chromatography to obtain solid 3.16.

[0070] Example 19

[0071]

[0072] The synthesis of compound 3.17 was similar to that of compound 3.1, except that 4-fluorobenzyl azide was used instead of 4-bromobenzyl azide. The crude product was purified by column chromatography to obtain solid 3.17.

[0073] Example 20

[0074]

[0075] 1 mmol of 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazolyl-5-carboxamide, 1.2 mmol of 2-cyanobenzyl azide, 2 mmol of sodium L-ascorbate, and 1 mmol of anhydrous CuSO4 were added to a 100 mL round-bottom flask equipped with a stir bar. Then, 50 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min until completely dissolved. The reaction was allowed to proceed overnight at room temperature with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with DCM, and the lower organic phase was collected. The mixture was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to the organic phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration. The crude product was obtained by vacuum distillation at 35°C to remove organic solvents such as DCM. The crude product was then separated and purified by column chromatography to obtain a white solid 4.1 (yield 77%). 1 H NMR(400MHZ,DMSO-d6):10.37(s,1H),8.62(s,1H),8.30(d,J=4.0Hz,1H),8.2 3(dd,J1=4.0Hz,J2=4.0Hz,1H),7.95(d,J=8.0Hz,1H),7.86(t,J=8.0Hz,2H), 7.80-7.74(m,3H),7.59(t,J=12.0Hz,1H),7.43(dd,J1=8.0Hz,J2=12.0Hz,2H ),5.87(s,2H),4.02(d,J=8.0Hz,2H),2.66(s,3H),1.04(s,3H),1.02(s,3H).

[0076] Example 21

[0077]

[0078] The synthesis of compound 4.2 was similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide was replaced with 4-bromobenzyl azide. The crude product was purified by column chromatography to obtain solid 4.2 (yield 81%). 1 H NMR (400MHZ, DMSO-d6): 10.36 (s, 1H), 8.60 (s, 1H), 8.31 (s, 1H), 8.23 ​​(d, J = 12.0Hz, 1H), 7.86 (d, J = 12.0Hz, 2H), 7.78 (d, J = 4 .0Hz,2H),7.43-7.35(m,6H),5.66(s,2H),4.03(d,J=4.0Hz,2H),2.66(s,3H),2.12-2.06(m,1H),1.05(s,3H),1.03(s,3H).

[0079] Example 22

[0080]

[0081] The synthesis method of compound 4.3 is similar to that of compound 4.1, except that 2-chloro-5-fluorobenzyl azide is used instead of 2-cyanobenzyl azide. The crude product is purified by column chromatography to obtain solid 4.3 (yield 69%). 1 H NMR(400MHZ,DMSO-d6):10.36(s,1H),8.58(s,1H),8.30(d,J=4.0Hz,1H),8 .23(dd,J1=4.0Hz,J2=4.0Hz,1H),7.86(d,J=8.0Hz,2H),7.77(d,J=8.0Hz,2 H),7.60(dd,J1=8.0Hz,J2=4.0Hz,1H),7.41-7.23(m,3H),5.75(s,2H),4.02 (d,J=4.0Hz,2H),2.65(s,3H),2.15-2.05(m,1H),1.04(s,3H),1.02(s,3H).

[0082] Example 23

[0083]

[0084] The synthesis of compound 4.4 was similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide was replaced with 3-fluorobenzyl azide. The crude product was purified by column chromatography to obtain solid 4.4 (yield 72%). 1 H NMR (400MHZ, DMSO-d6): 10.36 (s, 1H), 8.62 (s, 1H), 8.30 (d, J = 4.0Hz, 1H), 8.23 ​​(dd, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 7.86 (d, J = 8.0Hz, 2H), 7.77 (d, J = 1 2.0Hz,2H),7.48-7.39(m,2H),7.24-7.18(m,3H),5.69(s,2H),4.02(d, J=8.0Hz,2H),2.65(s,3H),2.15-2.05(m,1H),1.04(s,3H),1.02(s,3H).

[0085] 1H NMR spectrum as follows Figure 1 As shown.

[0086] Example 24

[0087]

[0088] The synthesis of compound 4.5 was similar to that of compound 4.1, except that 2-bromobenzyl azide was used instead of 2-nitrile benzyl azide. The crude product was purified by column chromatography to obtain solid 4.5 (yield 70%). 1 H NMR (400MHZ, DMSO-d6): 10.36 (s, 1H), 8.56 (s, 1H), 8.29 (d, J = 4.0Hz, 1H), 8.23 ​​(dd, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 7.86 (d, J = 8.0Hz, 2H), 7.78-7.71 (m,3H),7.46-7.32(m,3H),7.24(d,J=8.0Hz,1H),5.74(s,2H),4.02(d, J=4.0Hz,2H),2.65(s,3H),2.13-2.07(m,1H),1.04(s,3H),1.02(s,3H).

[0089] Example 25

[0090]

[0091] The synthesis of compound 4.6 was similar to that of compound 4.1, except that zidovudine was used instead of 2-acrylbenzyl azide. The crude product was purified by column chromatography to obtain solid 4.6 (yield 76%). 1 H NMR(400MHZ,DMSO-d6):11.38(s,1H),10.38(s,1H),8.75(s,1H),8.31(d,J=4.0Hz,1H),8 .24(dd,J1=4.0Hz,J2=4.0Hz,1H),7.87-7.79(m,5H),7.41(d,J=8.0Hz,1H),6.47(t,J=4. 0Hz,1H),5.44-5.30(m,2H),4.32-4.28(m,1H),4.02(d,J=4.0Hz,2H),3.78-3.66(m,2H), 2.85-2.71(m,2H),2.66(s,3H),2.16-2.06(m,1H),1.83(s,3H),1.04(s,3H),1.02(s,3H).

[0092] Example 26

[0093]

[0094] The synthesis of compound 4.7 was similar to that of compound 4.1, except that 2-chlorophenyl azide was used instead of 2-cyanobenzyl azide. The crude product was purified by column chromatography to obtain solid 4.7 (yield 63%). 1H NMR (400MHZ, CDCl3): 8.18-8.08(m,2H),7.95(d,J=8.0Hz,1H),7.74-7.68(m,1H),7.65-7.58(m,2H),7.53-7.49(m,2H),7. 06-7.03(m,1H),3.93(d,J=8.0Hz,2H),3.10(s,1H),2.81(s,2H),2.27-2.18(m,1H),1.65(s,3H),1.12(s,3H),1.10(s,3H).

[0095] Example 27

[0096]

[0097] The synthesis of compound 4.8 was similar to that of compound 4.1, except that 2-fluorophenyl azide was used instead of 2-cyanobenzyl azide. The crude product was purified by column chromatography to obtain solid 4.8 (yield 66%). 1 H NMR (400MHZ, DMSO-d6): 10.41 (s, 1H), 9.05 (d, J = 4.0Hz, 1H), 8.31 (d, J = 4.0Hz, 1H), 8.23 ​​(dd, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 7.98-7.82 (m, 4H), 7.73-7.6 0(m,2H),7.49(t,J=8.0Hz,1H),7.41(d,J=8.0Hz,1H),4.02(d,J=8.0Hz,2 H),3.31(s,1H),2.67(s,3H),2.16-2.06(m,1H),1.04(s,3H),1.03(s,3H).

[0098] Example 28

[0099]

[0100] The synthesis of compound 4.9 was similar to that of compound 4.1, except that 3-chlorophenyl azide was used instead of 2-acrylonitrile benzyl azide. The crude product was purified by column chromatography to obtain solid 4.9 (yield 72%). 1H NMR(400MHZ,DMSO-d6):10.45(s,1H),9.38(s,1H),8.34(d,J=4.0Hz,1H),8.27(dd,J 1=4.0Hz,J2=4.0Hz,1H),8.12(s,1H),8.01(d,J=8.0Hz,1H),7.96(d,J=8.0Hz,2H),7 .86(d,J=8.0Hz,2H),7.71(t,J=8.0Hz,1H),7.63(d,J=8.0Hz,1H),7.44(d,J=8.0Hz, 1H), 4.06 (d, J = 4.0Hz, 2H), 2.69 (s, 3H), 2.17-2.10 (m, 1H), 1.07 (s, 3H), 1.05 (s, 3H).

[0101] Example 29

[0102]

[0103] The synthesis of compound 4.10 was similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide was replaced with 4-chlorophenyl azide. The crude product was purified by column chromatography to obtain solid 4.10 (yield 74%). 1 H NMR (400MHZ, DMSO-d6): 10.41(s,1H),9.30(s,1H),8.31(s,1H),8.25(dd,J1=4.0Hz,J2=4.0Hz,1H),8.01(d,J=8.0Hz,1H),7.94(d,J=8.0Hz,1H),7 .84(d,J=8.0Hz,2H),7.73(d,J=8.0Hz,2H),7.42(d,J=8.0Hz,1H),4.03(d ,J=8.0Hz,2H),2.67(s,3H),2.15-2.08(m,1H),1.05(s,3H),1.03(s,3H).

[0104] Example 30

[0105]

[0106] The synthesis method of compound 4.11 is similar to that of compound 4.1, except that 2-trifluoromethoxyphenyl azide is used instead of 2-acrylonitrile benzyl azide. The crude product is purified by column chromatography to obtain solid 4.11 (yield 55%). 1H NMR(400MHZ,DMSO-d6):10.42(s,1H),9.03(s,1H),8.31(s,1H),8.25(dd,J1=4.0Hz,J2=4.0Hz,1H),7.97-7.90(m,3H),7.83(d,J=8.0Hz,1H),7 .77(d,J=4.0Hz,2H),7.73-7.68(m,1H),7.42(d,J=8.0Hz,1H),4.03(d, J=8.0Hz,2H),2.67(s,3H),2.16-2.11(m,1H),1.04(s,3H),1.03(s,3H).

[0107] Example 31

[0108]

[0109] The synthesis method of compound 4.12 is similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide is replaced by 4-fluorophenyl azide. The crude product was purified by column chromatography to obtain solid 4.12 (yield 90%). 1 H NMR (400MHZ, DMSO-d6): 10.41(s,1H),9.25(s,1H),8.31(s,1H),8.25(dd,J1=4.0Hz,J2=4.0Hz,1H),8.02-7.99(m,2H),7.94(d,J=8.0Hz,1H),7.8 3(d,J=8.0Hz,2H),7.51(t,J=8.0Hz,2H),7.42(d,J=12.0Hz,1H),4.03(d ,J=8.0Hz,2H),2.67(s,3H),2.14-2.08(m,1H),1.04(s,3H),1.03(s,3H).

[0110] Example 32

[0111]

[0112] The synthesis of compound 4.13 was similar to that of compound 4.1, except that 2-trifluoromethoxybenzyl azide was used instead of 2-nitrile benzyl azide. The crude product was purified by column chromatography to obtain solid 4.13 (yield 77%). 1H NMR (400MHz, CDCl3): 8.20(d,J=4.0Hz,1H),8.12(dd,J1=4.0Hz,J2=4.0Hz,1H),7.86(d,J=8.0Hz,2H),7.78(d,J=8.0Hz,1H),7.74(s,1H),7.68(d,J =8.0Hz,2H),7.59-7.48(m,3H),7.05(d,J=8.0Hz,1H),5.82(s,2H),3.94( d,J=4.0Hz,2H),2.83(s,3H),2.28-2.19(m,1H),1.13(s,3H),1.11(s,3H).

[0113] Example 33

[0114]

[0115] The synthesis method of compound 4.14 is similar to that of compound 4.1, except that 2-chloro-6-fluorobenzyl azide is used instead of 2-cyanobenzyl azide. The crude product is purified by column chromatography to obtain solid 4.14. 1 H NMR (400MHZ, DMSO-d6): 10.35 (s, 1H), 8.56 (s, 1H), 8.30 (d, J = 4.0Hz, 1H), 8.23 ​​(dd, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 7.86 (d, J = 8.0Hz, 2H), 7 .76(d,J=8.0Hz,2H),7.56-7.35(m,4H),5.76(s,2H),4.03(d,J=8.0Hz,2H),2.65(s,3H),2.14-2.07(m,1H),1.04(s,3H),1.02(s,3H).

[0116] Example 34

[0117]

[0118] The synthesis method of compound 4.15 is similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide is replaced with 3-trifluoromethoxybenzyl azide. The crude product is purified by column chromatography to obtain solid 4.15. 1H NMR (400MHZ, DMSO-d6): 10.36 (s, 1H), 8.65 (s, 1H), 8.30 (d, J = 4.0Hz, 1H), 8.24 (dd, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 7.85 (d, J = 8.0Hz, 2H), 7.79-7.73 (m ,4H),7.66(d,J=4.0Hz,2H),7.41(d,J=8.0Hz,1H),5.78(s,2H),4.03(d, J=4.0Hz,2H),2.66(s,3H),2.12-2.07(m,1H),1.04(s,3H),1.02(s,3H).

[0119] Example 35

[0120]

[0121] The synthesis of compound 4.16 was similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide was replaced with 4-fluorobenzyl azide. The crude product was purified by column chromatography to obtain solid 4.16. 1 H NMR (400MHZ, DMSO-d6): 10.36 (s, 1H), 8.58 (s, 1H), 8.30 (d, J = 4.0Hz, 1H), 8.24 (dd, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 7.84 (d, J = 8.0Hz, 2H), 7.77 (d, J = 8. 0Hz,2H),7.46-7.40(m,3H),7.24(t,J=4.0Hz,2H),5.65(s,2H),4.02(d, J=8.0Hz,2H),2.66(s,3H),2.14-2.07(m,1H),1.04(s,3H),1.02(s,3H).

[0122] 1H NMR spectrum as follows Figure 2 As shown.

[0123] Example 36

[0124]

[0125] The synthesis method of compound 4.17 is similar to that of compound 4.1, except that 2-acrylonitrile benzyl azide is replaced by 4-trifluoromethylbenzyl azide. The crude product is purified by column chromatography to obtain solid 4.17. 1H NMR (400MHZ, DMSO-d6): 10.38(s,1H),8.64(s,1H),8.30(d,J=4.0Hz,1H),8.24(dd,J1=4.0Hz,J2=4.0Hz,1H),7.86(d,J=12.0Hz,2H),7.80-7.77(m ,4H),7.56(d,J=8.0Hz,2H),7.41(t,J=12.0Hz,1H),5.82(s,2H),4.02(d ,J=8.0Hz,2H),2.65(s,3H),2.14-2.05(m,1H),1.04(s,3H),1.02(s,3H).

[0126] Example 37

[0127] HepG2 cells were seeded into 96-well plates, with approximately 2000-5000 cells per well. Cells were treated with a 40 μM compound, with four auxiliary wells for each concentration group. After culturing for 72 hours, CCK8 reagent was added to each well at a ratio of 10 μL of CCK8 reaction solution per 100 μL. Cells were incubated for another hour, and the absorbance at 450 nm was measured using a microplate reader. The experiment was performed in triplicate. Cell viability at different concentrations was determined based on the absorbance values, and the cell survival rate at different concentrations was calculated.

[0128]

[0129]

[0130] Example 38

[0131] HUH-7, A549, PC-9, H460, H1299, and LO2 cells were seeded in 96-well plates, approximately 2000 cells per well. Cells were treated with a 20 μM concentration of the compound, with four auxiliary wells for each concentration group. After culturing in a cell culture incubator for 72 hours, CCK8 reagent was added to each well at a ratio of 10 μL of CCK8 reaction solution per 100 μL. Cells were incubated for another 30 minutes, and the absorbance at 450 nm was measured using a microplate reader. The experiment was performed in triplicate. Cell viability at different concentrations was obtained based on the absorbance values, and the cell inhibition rate at different concentrations was calculated.

[0132]

[0133]

[0134] Example 39

[0135] A549: F4.16 treatment led to increased cell death, and the number of dead cells increased with increasing concentration; F4.4 treatment did not significantly change cell apoptosis, while F4.16 treatment significantly increased cell apoptosis; F4.4 and F4.16 significantly increased autophagy; F4.4 had no significant effect on cell DNA damage, while F4.16 significantly increased cell DNA damage.

[0136] Huh-7: F4.4 treatment led to increased cell death; F4.4 significantly increased apoptosis, while F4.16 had little effect on apoptosis; F4.4 and F4.16 significantly increased autophagy; F4.4 and F4.16 significantly increased cellular DNA damage.

[0137] PC-9: F4.16 treatment led to increased cell death, with the number of dead cells increasing with increasing concentration; it had little effect on apoptosis; F4.4 and F4.16 significantly increased autophagy; F4.4 and F4.16 significantly increased cellular DNA damage.

[0138] Example 40

[0139] BV2 cells were used at a rate of 2 × 10 4 Each sample was seeded per well in a 96-well plate and incubated at 37°C in a 5% CO2 incubator. After 24 hours of incubation, the corresponding concentration of the test compound was added to the drug-treated group and incubated for 2 hours. Subsequently, LPS was added to both the drug-treated group and the LPS model group at a final concentration of 100 ng / ml. After 24 hours of incubation, 50 μL of supernatant was mixed with 50 μL of Greiss buffer from each well and reacted at room temperature for 15 minutes. The OD values ​​of each group were measured at 540 nm using a Biotek microplate reader.

[0140] Table 3. Results of the compounds in improving LPS-induced inflammation in BV2 cells

[0141]

[0142]

[0143] In the NO generation rate detection, the NO generation rate of the LPS model group was set to 100% (n=3). The data in the table are expressed as mean ± SEM. * P<0.05, ** P<0.01 relative to the LPS group.

[0144] Example 41

[0145] PC3 cells were seeded into 96-well plates, with approximately 3000 cells per well. Cells were treated with a 10 μM compound, with four auxiliary wells for each concentration group. After incubation for 72 hours, the cells were analyzed using the MTT assay. The experiment was performed in triplicate. Cell viability at different concentrations was obtained based on absorbance values, and the cell inhibition rate at each concentration was calculated.

[0146]

[0147]

[0148] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A methylthiazole derivative with anti-hepatocellular carcinoma activity, characterized in that: The structure of the methylthiazole derivative is shown in the following formula: 。 2. The method for preparing the methylthiazole derivative according to claim 1, characterized in that: Includes the following steps: S1. Using 2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazol-5-carboxylic acid as a raw material, prepare 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazol-5-carboxamide; S2. Using 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide as a raw material, a methylthiazole derivative is prepared by a click reaction with 3-fluorobenzyl azide.

3. The preparation method according to claim 2, characterized in that: In step S1, The preparation method of 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazol-5-carboxamide is as follows: 2-(3-cyano-4-isobutoxyphenyl)-4-methylthiazol-5-carboxylic acid, 4-ethynylaniline, HATU, and DIPEA are added sequentially to a round-bottom flask. Then, N,N-dimethylformamide is added to the system, and the mixture is stirred at room temperature. Water is added to the reaction system and stirring is continued. During the stirring process, a solid precipitates out. The solid is then filtered and dried to obtain 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazol-5-carboxamide.

4. The preparation method according to claim 2, characterized in that: In step S2, the preparation method of the methylthiazole derivative is as follows: 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide, 3-fluorobenzyl azide, sodium L-ascorbate, and anhydrous CuSO4 are placed in a round-bottom flask, followed by the addition of a mixed solvent of water, tert-butanol, and THF. The mixture is stirred at room temperature until completely dissolved. The reaction is carried out overnight at room temperature with stirring. After the reaction is completed as detected by TLC, the mixture is extracted with DCM and the lower organic phase is collected. The mixture is then back-extracted with saturated brine and the lower organic phase is collected. Anhydrous sodium sulfate is added, and the mixture is stirred thoroughly and allowed to stand. The anhydrous sodium sulfate is then removed by filtration. The crude product is obtained by vacuum distillation at 35°C. The crude product is then separated and purified by column chromatography to obtain a white solid, which is the methylthiazole derivative.

5. The use of the methylthiazole derivative according to claim 1 in the preparation of anti-hepatocellular carcinoma drugs.

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