Methyl thiazole derivative with anti-hepatoma activity as well as preparation method and application thereof

Abstract

The invention relates to a preparation method and application of a methylthiazole derivative with anti-hepatoma activity, and belongs to the technical field of synthesis of medicines. On the basis of a 2-[3-cyano-4-(2-isobutoxy)-phenyl]-4-methylthiazole-5-formic acid structure, terminal alkyne is introduced to the structure, then a click reaction and an azide compound react to obtain a plurality of compounds with novel structures, and activity tests in anti-tumor, anti-inflammatory and anti-oxidation aspects show that the compound has a broad application prospect. The obtained compound has obvious anti-cancer activity and provides huge application value for research and development of anti-cancer drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and specifically relates to a preparation method and application of a methylthiazole derivative with anti-liver cancer activity. Background Art

[0002] The thiazole ring is a five-membered aromatic heterocyclic structure containing nitrogen and sulfur. Its chemical properties and biological activities make it play an important role in drug development. The thiazole ring structure is commonly found in small molecule drugs with various biological activities. For example, thiazole ring derivatives can have a variety of pharmacological activities such as antibacterial, antifungal, antiviral, anti-inflammatory, antitumor, antidiabetic and antihypertensive. This makes the thiazole ring an important structural unit in drug development. The thiazole ring can interact with a variety of target proteins and play a key pharmacophore role. Its aromatic and heterocyclic properties enable it to form a variety of interactions in 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 modification of multiple positions, such as the introduction of different substituents on different carbon atoms or nitrogen and sulfur atoms of the thiazole ring. This flexibility of modification enables researchers to improve the pharmacokinetic properties of drugs, reduce toxicity and improve efficacy through structural optimization. Many drugs on the market contain thiazole ring structures, such as 2-[3-cyano-4-(2-isobutyloxy)-phenyl]-4-methylthiazole-5-carboxylic acid, which is a non-purine analog inhibitor of xanthine oxidase in both oxidized and reduced forms. It is a new oral anti-hyperuricemia drug, and its therapeutic effect is achieved by promoting uric acid excretion, thereby reducing serum uric acid. In the treatment of gout, it has the advantages of being potent, highly selective, with fewer side effects and higher safety. It has gradually replaced allopurinol and benzbromarone and has become the preferred drug for the current clinical treatment of gout and hyperuricemia.

[0003] 1,2,3-triazole is an important drug molecule building block. Its planar rigid structure can be inserted into the DNA of tumor cells, interact with DNA and cause damage to tumor cell DNA. This type of structure has electronic isosteric effects similar to amides, esters, carboxylic acids, etc., which enables it to coordinate with metal ions, and can act on proteases containing metal ions, inhibiting their activity and thus affecting the growth of tumor cells; at the same time, the 1,2,3-triazole ring can link different pharmacodynamic molecules to form a new molecule, and this derivative often has low toxicity and high efficiency. These characteristics of triazole structures make it widely used in modifying natural products, active azole compounds or quinone compounds, and are widely used in the development of innovative drugs. Our team cooperated with Jinan Aisi Pharmaceutical Technology Co., Ltd. to synthesize and test the activity of methylthiazole derivatives modified with 1,2,3-triazole. Jinan Aisi Pharmaceutical Technology Co., Ltd. is responsible for the synthesis of compounds, and our team is responsible for activity testing. Summary of the invention

[0004] In order to research and develop innovative drugs, the purpose of the present invention is to provide a preparation method and application of methylthiazole derivatives with anti-liver cancer activity. The methylthiazole derivatives are based on the structure of 2-[3-nitrile-4-(2-isobutyloxy)-phenyl]-4-methylthiazole-5-carboxylic acid, and terminal alkynes are introduced into the structure, and then various novel structural compounds are obtained by reacting with azide compounds, which have good anti-tumor, anti-inflammatory and antioxidant activities.

[0005] In order to achieve the above object, the specific scheme adopted by the present invention is:

[0006] In the first aspect, the present invention provides a methylthiazole derivative having anti-liver cancer activity, the structure of which is shown in the following formula I or formula II; The formula I is: The formula II is: In Formula I and Formula II, n is 0 or 1; R 1 , R 2 , R 3 , R 4 and R 5 It is one or more of H, halogen, trifluoromethyl, ethyl, phenyl, hydroxyl, cyano, nitro, aromatic ring, etc.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned methylthiazole derivatives, which is based on the structure of 2-[3-nitrile-4-(2-isobutyloxy)-phenyl]-4-methylthiazole-5-carboxylic acid, introduces a terminal alkyne into its structure, and then reacts with an azide compound through a click reaction to obtain the derivatives; different products are obtained depending on the type of azide compound.

[0008] In a third aspect, the present invention provides an application of the above-mentioned methylthiazole derivatives in the preparation of anti-liver cancer drugs.

[0009] Beneficial effects: The present invention is based on the structure of 2-[3-nitrile-4-(2-isobutyloxy)-phenyl]-4-methylthiazole-5-carboxylic acid, introduces a terminal alkyne into its structure, and then reacts with azide compounds through a click reaction to obtain a variety of novel structural compounds, and conducts anti-tumor, anti-inflammatory and antioxidant activity tests. The results show that the obtained compounds have obvious anti-cancer activity, which provides great application value for the research and development of anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the H-NMR spectrum of compound 4.4 synthesized in Example 36.

[0011] Figure 2 This is the H-NMR spectrum of compound 4.16 synthesized in Example 48. DETAILED DESCRIPTION

[0012] This invention was developed by our team in collaboration with Jinan Aisi Pharmaceutical Technology Co., Ltd. Based on the structure of 2-[3-nitrile-4-(2-isobutyloxy)-phenyl]-4-methylthiazole-5-carboxylic acid, a terminal alkyne was introduced into its structure, and then more than ten compounds with novel structures were obtained by reacting with azide compounds through a click reaction, and anti-tumor, anti-inflammatory and antioxidant activity tests were carried out. Our team was responsible for the design and activity testing of the target compounds, and Jinan Aisi Pharmaceutical Technology Co., Ltd. was responsible for the specific synthesis research.

[0013] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0014] In the following examples, unless otherwise specified, the reagents used are conventional commercially available reagents, and the operating methods adopted are conventional technical means.

[0015] Example 1

[0016] Into a 500 mL clean round-bottom flask equipped with a stirring bar, 31.6 g of 2-(3-nitrile-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic acid, 11.7 g of 3-ethynylaniline, 38 g of HATU and 26 g of DIPEA were added in sequence, and then 800 mL of N,N-dimethylformamide was added to the system. The mixture was stirred at room temperature for 1 h. 600 mL of water was added to the reaction system. A large amount of solid was precipitated during the stirring process. The solid was filtered and dried to obtain 35.1 g of 2-(3-nitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide.

[0017] Example 2

[0018] Into a 500 mL clean round-bottom flask equipped with a stirring bar, 31.6 g of 2-(3-nitrile-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic acid, 11.7 g of 4-ethynylaniline, 38 g of HATU and 26 g of DIPEA were added in sequence, and then 800 mL of N,N-dimethylformamide was added to the system. The mixture was stirred at room temperature for 1 h. 600 mL of water was added to the reaction system. A large amount of solid was precipitated during the stirring process. The solid was filtered and dried to obtain 37.4 g of 2-(3-nitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide.

[0019] Example 3 Take 2-(3-nitrile-4-isobutyloxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide (1mmol), 4-bromobenzyl azide (1.2mmol), L-sodium ascorbate (2mmol) and anhydrous CuSO4 (1mmol) and add them to a 100mL round-bottom flask equipped with a stirrer, then add 50mL of a mixed solvent of water: tert-butyl alcohol: THF = 1:1:1 (v / v / v) to the flask, stir at room temperature for 10min to dissolve it completely. Keep room temperature and stir overnight for 2h. After the reaction is completed by TLC detection, extract with DCM 3 times and collect the lower organic phase. Then extract with saturated brine 3 times, collect the lower organic phase, add anhydrous sodium sulfate to it, stir well and let stand for 30min, then filter to remove anhydrous sodium sulfate. Organic solvents such as DCM were distilled off under reduced pressure at 35°C to obtain a crude product, which was then separated and purified by column chromatography to obtain a white solid 3.1. 1 H NMR (400 MHz, DMSO-d 6):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).

[0020] Example 4

[0021] The synthesis method of compound 3.2 is similar to that of compound 3.1, except that 2-nitrobenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.2.

[0022] Example 5

[0023] The synthesis method of compound 3.3 is similar to that of compound 3.1, except that 2-bromobenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.3.

[0024] Example 6

[0025] The synthesis method of compound 3.4 is similar to that of compound 3.1, except that 3-fluorobenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.4.

[0026] Example 7

[0027] The synthesis method of compound 3.5 is similar to that of compound 3.1, except that 2-chloro-6-fluorobenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.5.

[0028] Example 8

[0029] The synthesis method of compound 3.6 is similar to that of compound 3.1, except that 2-chloro-4-fluorobenzyl azide is used to replace 4-bromobenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 3.6.

[0030] Example 9

[0031] The synthesis method of compound 3.7 is similar to that of compound 3.1, except that 2-chlorobenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.7.

[0032] Example 10

[0033] The synthesis method of compound 3.8 is similar to that of compound 3.1, except that 2-fluorophenyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.8.

[0034] Embodiment 11

[0035] The synthesis method of compound 3.9 is similar to that of compound 3.1, except that 3-chlorophenyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.9.

[0036] Example 12

[0037] The synthesis method of compound 3.10 is similar to that of compound 4.1, except that 4-chlorophenyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.10.

[0038] Embodiment 13

[0039] The synthesis method of compound 3.11 is similar to that of compound 3.1, except that 4-fluorophenyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.11.

[0040] Embodiment 14

[0041] The synthesis method of compound 3.12 is similar to that of compound 3.1, except that 2-trifluoromethylphenyl azide is used to replace 4-bromobenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 3.12.

[0042] Embodiment 15

[0043] The synthesis method of compound 3.13 is similar to that of compound 3.1, except that zidovudine is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.13.

[0044] Example 16

[0045] The synthesis method of compound 3.14 is similar to that of compound 3.1, except that 2-trifluoromethoxyphenyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.14.

[0046] Embodiment 17

[0047] The synthesis method of compound 3.15 is similar to that of compound 3.1, except that 3-trifluoromethylbenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.15.

[0048] Embodiment 18

[0049] The synthesis method of compound 3.16 is similar to that of compound 3.1, except that 4-trifluoromethylbenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.16.

[0050] Embodiment 19

[0051] The synthesis method of compound 3.17 is similar to that of compound 3.1, except that 4-fluorobenzyl azide is used to replace 4-bromobenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 3.17.

[0052] Embodiment 20

[0053] Take 2-(3-cyano-4-isobutyloxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide (1mmol), 2-nitrile benzyl azide (1.2mmol), L-sodium ascorbate (2mmol) and anhydrous CuSO4 (1mmol) and add them to a 100mL round-bottom flask equipped with a stirrer, then add 50mL of a mixed solvent of water: tert-butyl alcohol: THF = 1:1:1 (v / v / v) to the flask, stir at room temperature for 10min to dissolve it completely. Keep room temperature, stir overnight to react, and after the reaction is completed by TLC detection, extract with DCM 3 times, and collect the lower organic phase. Then extract with saturated brine 3 times, collect the lower organic phase, add anhydrous sodium sulfate to it, stir well and let stand for 30min, then filter to remove anhydrous sodium sulfate. DCM and other organic solvents were removed by distillation under reduced pressure at 35°C to obtain a crude product, which was then separated and purified by column chromatography to obtain a white solid 4.1 (yield 77%); 1 H NMR (400 MHZ, DMSO-d 6):10.37(s,1H),8.62(s,1H),8.30(d,J=4.0Hz,1H),8.23(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).

[0054] Embodiment 21

[0055] The synthesis method of compound 4.2 is similar to that of compound 4.1, except that 4-bromobenzyl azide is used to replace 2-nitrilebenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.2 (yield 81%). 1 H NMR (400 MHZ, DMSO-d 6 ):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).

[0056] Embodiment 22

[0057] The synthesis method of compound 4.3 is similar to that of compound 4.1, except that 2-chloro-5-fluorobenzyl azide is used to replace 2-nitrilebenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.3 (yield 69%). 1 H NMR (400 MHZ, DMSO-d 6):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,2H),7.60(d d, 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).

[0058] Embodiment 23

[0059] The synthesis method of compound 4.4 is similar to that of compound 4.1, except that 3-fluorobenzyl azide is used to replace 2-nitrilebenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.4 (yield 72%). 1 H NMR (400 MHZ, DMSO-d 6 ):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=12.0Hz,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).

[0060] H NMR spectrum Figure 1 shown.

[0061] Embodiment 24

[0062] The synthesis method of compound 4.5 is similar to that of compound 4.1, except that 2-bromobenzyl azide is used to replace 2-nitrilebenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.5 (yield 70%). 1 H NMR (400 MHZ, DMSO-d 6):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).

[0063] Embodiment 25

[0064] The synthesis method of compound 4.6 is similar to that of compound 4.1, except that zidovudine is used to replace 2-nitrile benzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 4.6 (yield 76%). 1 H NMR (400 MHZ, DMSO-d 6 ):11.38(s,1H),10.38(s,1H),8.75(s,1H),8.31(d,J=4.0Hz,1H),8.24(dd,J1=4.0 Hz,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).

[0065] Embodiment 26

[0066] The synthesis method of compound 4.7 is similar to that of compound 4.1, except that 2-chlorophenyl azide is used to replace 2-nitrile benzyl azide. The crude product is separated and 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).

[0067] Embodiment 27

[0068] The synthesis method of compound 4.8 is similar to that of compound 4.1, except that 2-fluorophenyl azide is used to replace 2-nitrile benzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.8 (yield 66%). 1 H NMR (400 MHZ, DMSO-d 6 ):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),7.73-7.60(m,2H),7 .49(t,J=8.0Hz,1H),7.41(d,J=8.0Hz,1H),4.02(d,J=8.0Hz,2H),3.31(s,1H),2.67(s,3H),2.16-2.06(m,1H),1.04(s,3H),1.03(s,3H).

[0069] Embodiment 28

[0070] The synthesis method of compound 4.9 is similar to that of compound 4.1, except that 3-chlorophenyl azide is used to replace 2-nitrile benzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.9 (yield 72%). 1 H NMR (400 MHZ, DMSO-d 6):10.45(s,1H),9.38(s,1H),8.34(d,J=4.0Hz,1H),8.27(dd,J1=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).

[0071] Embodiment 29

[0072] The synthesis method of compound 4.10 is similar to that of compound 4.1, except that 4-chlorophenyl azide is used to replace 2-nitrile benzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.10 (yield 74%). 1 H NMR (400 MHZ, DMSO-d 6 ):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).

[0073] Embodiment 30

[0074] The synthesis method of compound 4.11 is similar to that of compound 4.1, except that 2-trifluoromethoxyphenyl azide is used to replace 2-nitrilebenzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.11 (yield 55%). 1 H NMR (400 MHZ, DMSO-d 6):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).

[0075] Embodiment 31

[0076] The synthesis method of compound 4.12 is similar to that of compound 4.1, except that 4-fluorophenyl azide is used to replace 2-nitrile benzyl azide. The crude product is separated and purified by column chromatography to obtain solid 4.12 (yield 90%). 1 H NMR (400 MHZ, DMSO-d 6 ):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),7.83(d,J=8.0 Hz,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).

[0077] Embodiment 32

[0078] The synthesis method of compound 4.13 is similar to that of compound 4.1, except that 2-trifluoromethoxybenzyl azide is used to replace 2-nitrilebenzyl azide. The crude product is separated and 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).

[0079] Embodiment 33

[0080] The synthesis method of compound 4.14 is similar to that of compound 4.1, except that 2-chloro-6-fluorobenzyl azide is used to replace 2-nitrilebenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 4.14; 1 H NMR (400 MHZ, DMSO-d 6 ):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).

[0081] Embodiment 34

[0082] The synthesis method of compound 4.15 is similar to that of compound 4.1, using 3-trifluoromethoxybenzyl azide to replace 2-nitrilebenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 4.15; 1 H NMR (400 MHZ, DMSO-d 6):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).

[0083] Embodiment 35

[0084] The synthesis method of compound 4.16 is similar to that of compound 4.1, except that 4-fluorobenzyl azide is used to replace 2-nitrilebenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 4.16; 1 H NMR (400 MHZ, DMSO-d 6 ):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).

[0085] H NMR spectrum Figure 2 shown.

[0086] Embodiment 36

[0087] The synthesis method of compound 4.17 is similar to that of compound 4.1, except that 4-trifluoromethylbenzyl azide is used to replace 2-nitrilebenzyl azide, and the crude product is separated and purified by column chromatography to obtain solid 4.17; 1 H NMR (400 MHZ, DMSO-d 6):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).

[0088] Embodiment 37

[0089] HepG2 cells were plated in 96-well plates, with about 2000-5000 cells per well. Compounds at a concentration of 40 μM were added to treat the cells, and 4 sub-wells were set 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 ul cck8 reaction solution per 100 ul. The cells were placed in the incubator and continued to incubate for 1 hour, and the absorbance of the cells at 450 nm was measured with an enzyme marker. The experiment was repeated three times. The activity of the cells at different concentrations was obtained based on the absorbance, and then the cell survival rate at different concentrations was calculated.

[0090] Embodiment 38

[0091] HUH-7, A549, PC-9, H460, H1299 and LO2 cells were plated in 96-well plates, with about 2000 cells per well. Compounds were added at a concentration of 20 μM to treat the cells, and 4 wells were set 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 cck8 reaction solution per 100 μl. The cells were placed in the incubator and continued to incubate for 30 minutes, and the absorbance of the cells at 450 nm was measured with an enzyme marker. The experiment was repeated three times. The activity of the cells at different concentrations was obtained based on the absorbance, and the cell inhibition rate at different concentrations was calculated.

[0092] Embodiment 39

[0093] A549: F4.16 treatment led to increased cell death, and the number of dead cells increased with increasing concentration; there was no obvious change in cell apoptosis after F4.4 treatment, but F4.16 treatment significantly increased cell apoptosis; F4.4 and F4.16 significantly increased cell autophagy; F4.4 had no significant effect on cell DNA damage, but F4.16 significantly increased cell DNA damage.

[0094] Huh-7: F4.4 treatment leads to increased cell death; F4.4 can significantly increase cell apoptosis, while F4.16 has little effect on cell apoptosis; F4.4 and F4.16 can significantly increase cell autophagy; F4.4 and F4.16 significantly increase cell DNA damage.

[0095] PC-9: F4.16 treatment leads to increased cell death, and the number of dead cells increases with increasing concentration; it has little effect on cell apoptosis; F4.4 and F4.16 can significantly increase cell autophagy; F4.4 and F4.16 significantly increase cell DNA damage.

[0096] Embodiment 40

[0097] BV2 cells were collected at 2×10 4 Each well was inoculated in a 96-well culture plate and placed in a 5% CO 2 After 24 hours of culture, the test compound of the corresponding concentration was added to the drug group and incubated for 2 hours, and then LPS with a final concentration of 100 ng / ml was added to the drug group and LPS model group. After 24 hours of culture in the incubator, 50 μL of supernatant and 50 μL of Greiss buffer were taken from each well and reacted at room temperature for 15 minutes. The OD value of each group at a wavelength of 540 nm was detected by an enzyme marker (Biotek).

[0098] Table 3. Improvement of compounds on LPS-induced BV2 cell inflammation In the NO generation rate detection, the NO generation rate of the LPS model group was set as 100% (n=3). The data in the table are expressed as mean±SEM. * P<0.05, ** P<0.01 compared with the LPS group.

[0099] Embodiment 41

[0100] PC3 cells were plated in 96-well plates, with about 3,000 cells per well. Compounds at a concentration of 10 μM were added to treat the cells, and 4 replicate wells were set up for each concentration group. After culturing in a cell culture incubator for 72 hours, the MTT method was used for detection. The experiment was repeated three times. The activity of cells at different concentrations was obtained based on the absorbance value, and the cell inhibition rate at different concentrations was calculated.

[0101] 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 above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A methylthiazole derivative having anti-hepatocarcinoma activity, characterized in that: The structure of the methylthiazole derivative is shown in Formula I or Formula II below; The formula I is: ; The formula II is: ; In Formula I and Formula II, n is 0 or 1; R 1 , R 2 , R 3 , R 4 and R 5 It is one or more of H, halogen, trifluoromethyl, ethyl, phenyl, hydroxyl, cyano, nitro, aromatic ring, etc.

2. The method for preparing a methylthiazole derivative according to claim 1, characterized in that: The following steps are involved: S1, using 2-(3-nitrile-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic acid as a raw material, preparing 2-(3-nitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide and 2-(3-nitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide respectively; S2. Using 2-(3-nitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide or 2-(3-nitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide as raw materials, a methylthiazole derivative with a structure shown in Formula I or Formula II is prepared by click reaction with an azide compound.

3. The preparation method according to claim 2, characterized in that: In step S1, The preparation method of 2-(3-nitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide is as follows: 2-(3-nitrile-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic acid, 3-ethynylaniline, HATU and DIPEA are added to a round-bottom flask in sequence, and 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 the stirring is continued. Solid is precipitated during the stirring process, and the solid is filtered and dried to obtain 2-(3-nitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide. The preparation method of 2-(3-nitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide comprises: sequentially adding 2-(3-nitrile-4-isobutoxyphenyl)-4-methylthiazole-5-carboxylic acid, 4-ethynylaniline, HATU, and DIPEA into a round-bottom flask, then adding N,N-dimethylformamide into the system, stirring at room temperature, adding water into the reaction system and continuing to stir, solid precipitates during the stirring process, and filtering and drying to obtain 2-(3-nitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-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: take 2-(3-nitrile-4-isobutoxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide or 2-(3-cyano-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide, azide, sodium L-ascorbate and anhydrous CuSO4 in a round-bottom flask, then add a mixed solvent of water, tert-butyl alcohol and THF, and stir at room temperature to dissolve it completely; keep room temperature and stir overnight to react; after the reaction is completed by TLC detection, extract with DCM and collect the lower organic phase; then back-extract with saturated brine, collect the lower organic phase, add anhydrous sodium sulfate, stir well and let stand, then filter to remove the anhydrous sodium sulfate; distill under reduced pressure at 35°C to obtain a crude product, and then separate and purify the obtained crude product by column chromatography to finally obtain a white solid, which is a methylthiazole derivative.

5. The preparation method according to claim 4, characterized in that: In the process of preparing a methylthiazole derivative of the structural formula I using 2-(3-nitrile-4-isobutyloxyphenyl)-N-(3-ethynylphenyl)-4-methylthiazole-5-carboxamide as a raw material, the azide compound used is any one of 2-bromobenzyl azide, 2-nitrile benzyl azide, 4-bromobenzyl azide, 3-fluorobenzyl azide, 2-chloro-6-fluorobenzyl azide, 2-chloro-4-fluorobenzyl azide, 2-chlorobenzyl azide, 2-fluorophenyl azide, 3-chlorophenyl azide, 4-chlorophenyl azide, 4-fluorophenyl azide, 2-trifluoromethylphenyl azide, zidovudine, 2-trifluoromethoxyphenyl azide, 3-trifluoromethylbenzyl azide, 4-trifluoromethylbenzyl azide and 4-fluorobenzyl azide; In the process of preparing a methylthiazole derivative of structural formula II using 2-(3-nitrile-4-isobutoxyphenyl)-N-(4-ethynylphenyl)-4-methylthiazole-5-carboxamide as a raw material, the azide compound used is 2-nitrile benzyl azide, 4-bromobenzyl azide, 2-chloro-5-fluorobenzyl azide, 3-fluorobenzyl azide, 2-bromobenzyl azide, zidovudine, 2-chlorophenyl azide, 2-fluorophenyl azide, 3-chlorophenyl azide, 4-chlorophenyl azide, 2-trifluoromethoxyphenyl azide, 4-fluorophenyl azide, 2-trifluoromethoxybenzyl azide, 2-chloro-6-fluorobenzyl azide, 3-trifluoromethoxybenzyl azide, 4-fluorobenzyl azide, and 4-trifluoromethylbenzyl azide.

6. Use of the methylthiazole derivative according to claim 1 in the preparation of anti-liver cancer drugs.

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