Formamide derivatives, their preparation methods and applications

Abstract

This invention belongs to the field of medicinal chemistry, specifically relating to 2-[2-methyl-6-(quinoline-3-yl)nicotinyl]-N-phenylhydrazine-1-formamide derivatives, their preparation methods, and applications. This invention is the first to synthesize 2-[2-methyl-6-(quinoline-3-yl)nicotinyl]-N-phenylhydrazine-1-formamide derivatives. Furthermore, the preparation method for these derivatives features low raw material costs, high yield, and a simple and easily controllable reaction process, making it suitable for industrial production. Moreover, these derivatives exhibit high NR4A1 protein-level inhibitory activity and can be used to prepare anti-hepatocellular carcinoma drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivatives, their preparation methods, and applications. Background Technology

[0002] The pathological type of primary liver cancer is hepatocellular carcinoma (HCC), accounting for 85% to 90%, with a 5-year survival rate of only 12.1%.

[0003] Currently, there are multiple treatment options for hepatocellular carcinoma (HCC). Early surgical treatment can cure HCC, but due to its insidious onset, rapid progression, and early metastasis, approximately 80% of HCC patients have already lost the opportunity for surgery at the time of diagnosis. Liver transplantation can cure advanced HCC, but it has limitations due to the scarcity of donors, high costs, and long waiting times. Radiotherapy, chemotherapy, and interventional treatments have drawbacks such as poor efficacy, numerous adverse reactions, high recurrence rates, and easy drug resistance, failing to meet the clinical needs of patients with advanced HCC. With the advent of the multi-target kinase inhibitor sorafenib, HCC treatment has entered the era of targeted therapy, but its tumor-suppressing effect is poor (ORR is only 2-3%, and OS is only extended by 28 months compared to placebo) and it has many adverse drug reactions. Therefore, understanding the development mechanism of HCC, finding therapeutic targets dependent on new signaling pathways, and designing and synthesizing novel anti-hepatocellular carcinoma drugs are currently the focus of research and development in the field of HCC treatment.

[0004] NR4A1 belongs to the steroid / thyroid hormone receptor superfamily. Its endogenous ligand has not yet been discovered, hence it is called an orphan nuclear receptor. As a product of the thyroid hormone receptor gene, NR4A1 can be induced in a cell-specific manner by various stimuli such as serum, growth factors, radiotherapy, chemotherapy, or pro-apoptotic drugs, thereby regulating cell survival, autophagy, and death. Numerous studies have shown that NR4A1, as a key factor, can determine the fate of cell survival or death. Furthermore, NR4A1 can shuttle between the nucleus and cytoplasm; when located in the cytoplasm, it often plays a role in promoting cell death, generally inducing apoptosis through the mitochondrial-Bcl2-BAX axis.

[0005] Studies have shown that NR4A1 levels decrease during the progression of liver cancer, and low NR4A1 levels are associated with poor prognosis. However, most current small molecule compounds targeting NR4A1 are derived from natural products, which have relatively low chemical activity in inhibiting tumor cells. Therefore, designing and synthesizing small molecule compounds targeting NR4A1, aiming to stabilize NR4A1 protein levels, and further exploring its mechanism of inducing liver cancer cell death, has significant guiding significance and clinical value for drug development and clinical treatment of liver cancer. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative, its preparation method, and its applications. This invention is the first to synthesize a 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative. Furthermore, the preparation method for this 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative features low raw material costs, high yield, and a simple and easily controllable reaction process, making it suitable for industrial production. Moreover, this type of derivative exhibits high NR4A1 protein-level inhibitory activity and can be used to prepare anti-liver cancer drugs.

[0007] The first object of the present invention is to provide a 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative, wherein the structural formula of the 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative is as follows:

[0008]

[0009] R is selected from H, halogen, C1-C5 alkyl or C1-C5 alkoxy.

[0010] Preferably, R is selected from H, -F, -Cl, -Br, C1-C3 alkyl or C1-C3 alkoxy.

[0011] Preferably, R is selected from H, -F, -Cl, -CH3 or -OCH3.

[0012] Preferably, the 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative is:

[0013] N-(4-chlorophenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide, N-(3,4-dichlorophenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide, N-(o-tolyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide, N-(4-methoxyphenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide or N-(3-fluorophenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide.

[0014] A second object of the present invention is to provide a method for preparing the above-mentioned 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative, comprising the following steps:

[0015] Synthesis of intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one:

[0016] In a dry reaction flask, 3-acetylquinoline, N,N-dimethylformamide dimethyl acetal, and toluene were added sequentially. The mixture was heated to 80-90°C with stirring and the condensation reaction was carried out for 6-8 hours. Thin-layer chromatography (TLC) was used to confirm that the reaction was complete. Condensation reaction I was then stopped. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with toluene and dried at 80°C to obtain a white solid product (E)-3-(dimethylamino)-1-(quinoline-3-yl)propen-2-en-1-one crude product, wherein the molar ratio of 3-acetylquinoline to N,N-dimethylformamide dimethyl acetal was 1:2.5-3.

[0017] Synthesis of S2, intermediate ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid:

[0018] In a dry reaction flask, (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one, ethyl acetoacetate, ammonium acetate, and glacial acetic acid were added sequentially. The mixture was heated to 110-125℃ with stirring and the condensation reaction was carried out for 6-8 hours. Thin-layer chromatography (TLC) was used to confirm that the condensation reaction was complete, and the condensation reaction was stopped. After the reaction solution was cooled to room temperature, it was extracted three times with 15 ml of ethyl acetate and 5 ml of water. The extract was dried with anhydrous sodium sulfate, filtered, and rotary evaporated. Finally, the crude product was subjected to silica gel column chromatography (eluent: V(ethyl acetate) / V(petroleum ether) = 1 / 3) to obtain a white solid product, crude ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid ester 1, wherein the molar ratio of (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one to ethyl acetoacetate and ammonium acetate was 1:1:8.7-10.

[0019] Synthesis of S3, intermediate 2-methyl-6-(quinolin-3-yl)nicotinic acid hydrazide:

[0020] In a dry reaction flask, ethyl 2-methyl-6-(quinoline-3-yl)nicotinic acid, 80% hydrazine hydrate, and ethanol were added in one step. The mixture was heated to 70-80°C with stirring and reacted for 10-16 hours. The reaction was stopped after thin-layer chromatography (TLC) showed that the reaction was complete. The reaction solution was cooled to room temperature, filtered, the filter cake was washed with ethanol, and dried to obtain crude 2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazine, a yellow solid product. The molar ratio of ethyl 2-methyl-6-(quinoline-3-yl)nicotinic acid to hydrazine hydrate was 1:8-10.

[0021] Synthesis of S4, 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivatives:

[0022] In a dry reaction flask, substituted phenyl isocyanate, ethanol, and 2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazide were added sequentially. The mixture was heated to 70-80°C with stirring and the condensation reaction was carried out for 6-8 hours. Thin-layer chromatography (TLC) was used to confirm that the reaction was complete. The condensation reaction was then stopped. The reaction solution was cooled to room temperature, filtered, the filter cake was washed with ethanol, and dried to obtain a white solid product, crude 2-[2-methyl-6-(quinoline-3-yl)nicotinic acid]-N-phenylhydrazine-1-carboxamide derivative, wherein the molar ratio of substituted phenyl isocyanate to 2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazide was 1-1.1:1.

[0023] The synthetic route for the 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative is as follows:

[0024]

[0025] A third objective of this invention is to provide the use of the above-mentioned 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative in the preparation of NR4A1 inhibitors.

[0026] Preferably, the NR4A1 inhibitor comprises at least one of a pharmaceutically acceptable salt, hydrate, inclusion complex, solvate, isomer, or prodrug of a 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative and a 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative.

[0027] Preferably, the NR4A1 inhibitor is administered orally or via parenteral administration, wherein parenteral administration includes subcutaneous injection, intravenous injection, and intramuscular injection.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention provides a 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative, which has a urea group that can target NR4A1 and inhibit the protein level of NR4A1 to induce apoptosis. The 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative obtained by this invention has higher NR4A1 protein level inhibitory activity than the natural products of the prior art.

[0030] 2. This invention provides a method for preparing 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivatives. The method features low raw material cost, high yield, and a simple and easily controllable reaction process, making it suitable for industrial production. The preparation principle is as follows: First, 3-acetylquinoline and N,N-dimethylformamide dimethyl acetal undergo a condensation reaction I under heating conditions to generate the intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one; subsequently, the intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one undergoes a condensation reaction II with ethyl acetoacetate under the catalysis of ammonium acetate and glacial acetic acid, and then... The mixture was purified to obtain the intermediate ethyl 2-methyl-6-(quinoline-3-yl)nicotinic acid. Then, ethyl 2-methyl-6-(quinoline-3-yl)nicotinic acid was converted to the intermediate 2-methyl-6-(quinoline-3-yl)nicotinamide via hydrazinolysis of hydrazine hydrate in ethanol. Finally, through condensation reaction III with substituted phenyl isocyanate in ethanol, the final product, 2-(2-methyl-6-(quinoline-3-yl)nicotinamide)-N-phenylhydrazine-1-carboxamide derivative, was obtained. The entire synthetic process ensured the efficient synthesis of the target product through precise control of reaction conditions and selective chemical transformations.

[0031] 3. The 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivative of the present invention has NR4A1 protein-level inhibitory activity and can be used to prepare anti-hepatocellular carcinoma drugs and to study the structure-activity relationship of such compounds. Attached Figure Description

[0032] Figure 1 The synthetic route diagrams for the 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivatives of Examples 1-5 are shown below.

[0033] Figure 2 The images show the initial screening results for the activity of 2-[2-methyl-6-(quinolin-3-yl)nicotinyl]-N-phenylhydrazine-1-carboxamide derivatives from Examples 1-5 against two liver cancer cell lines.

[0034] Figure 3 ICs M-4-6 in Example 1 and M-4-11 in Example 2 50 Value determination chart;

[0035] Figure 4 The diagram shows the apoptosis induced by M-4-6 in Example 1 and M-4-11 in Example 2 in liver cancer cells. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the data in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0038] In existing technologies, small molecule compounds targeting NR4A1 are mainly derived from natural products. Although these compounds possess biological activity and therapeutic efficacy, their ability to inhibit the chemical activity of tumor cells is often insufficient to achieve the desired clinical therapeutic effect. Furthermore, the inherent limitations of natural products, such as low yield, poor stability, and difficulty in controlling purity, significantly restrict their widespread application in drug development. More importantly, current small molecule compounds lack specificity for NR4A1, leading to side effects during treatment and even affecting the function of other non-target proteins, thus limiting their potential in liver cancer treatment.

[0039] To overcome the shortcomings of existing technologies, such as insufficient activity of small molecule compounds targeting NR4A1, limitations of natural products, and lack of specificity, this application utilizes computer-aided drug design technology to accurately predict and design small molecule compounds targeting specific binding sites of NR4A1, thereby significantly improving the chemical activity and specificity of anti-hepatocellular carcinoma. Simultaneously, by optimizing chemical synthesis methods, small molecule compounds with higher activity and stability can be produced, meeting the stringent requirements for compound quality and quantity in drug development. Ultimately, the small molecule compounds designed in this application can specifically target NR4A1, stabilizing its protein levels, and thus, by exploring its mechanism of inducing apoptosis in hepatocellular carcinoma cells, it is expected to reduce side effects and improve therapeutic efficacy.

[0040] Example 1

[0041] The preparation method of N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6) includes the following steps:

[0042] Preparation of intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one:

[0043] Take a clean 25 ml round-bottom flask, add 0.2 g (1.17 mmol) of 3-acetylquinoline, 600 μL (2.9 mmol) of N,N-dimethylformamide dimethyl acetal and 5 ml of toluene in sequence, stir and heat to 90 °C and reflux, keep in oil bath for 7 h, monitor the reaction with TLC until the reaction is complete, stop the reaction and cool to room temperature, filter under vacuum, dry at 80 °C to obtain 0.19 g of white solid product (E)-3-(dimethylamino)-1-(quinoline-3-yl)propen-2-en-1-one, yield 72.3%;

[0044] Spectral data: 1 H NMR (600MHz, DMSO-d6): 9.38(d,J=1.8Hz,1H),8.89(s,1H),8.12(d,J=7.7Hz,1H),8.07(d,J=8.4 Hz,1H),7.88-7.78(m,2H),7.69-7.63(m,1H),6.05(d,J=12.1Hz,1H),3.18(s,3H),2.98(s,3H); 13 C NMR (150MHz, DMSO-d6): 184.3, 155.1, 149.9, 148.8, 135.5, 132.7, 131.2, 129.7, 129.1, 127.5, 127.4, 91.6, 45.1, 37.8;

[0045] Preparation of intermediate 2-methyl-6-(quinolin-3-yl)nicotinic acid ethyl ester (S2):

[0046] In a clean 10 ml round-bottom flask, 0.1 g (0.44 mol) of (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one was added, followed by 118 μL (0.93 mmol) of ethyl acetoacetate, 0.57 g (0.74 mol) of ammonium acetate, and 5 ml of glacial acetic acid. The mixture was stirred and heated to 125 °C and refluxed for 8 h. The reaction was monitored by TLC until the reaction was complete, at which point stirring was stopped. The mixture was cooled to room temperature and extracted three times with 15 ml of ethyl acetate and 5 ml of water. The extract was dried over anhydrous sodium sulfate, filtered, rotary evaporated, and precipitated. The crude product was then subjected to silica gel column chromatography (eluent: V(ethyl acetate) / V(petroleum ether) = 1 / 3) to give 0.16 g of ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid, with a yield of 65%.

[0047] Spectral data: 1H NMR (600MHz, DMSO-d6): 9.65(d,J=2.2Hz,1H),9.08(d,J=1.8Hz,1H),8.31(d,J=8.0Hz,1H),8.17(d,J=8.0Hz,1H),8.13(d,J=7.7H z,1H),8.08(d,J=8.4Hz,1H),7.87-7.79(m,1H),7.68(t,J=7.1Hz,1H),4.34(q,J=7.2Hz,2H),2.84(s,3H),1.36(t,J=7.1Hz,3H): 13 C NMR(150MHz,DMSO-d6):172.5,166.3,159.4,156.0,149.5,148.4,139.9,134.7,131.0,1 30.5,129.5,129.2,127.7,127.7,124.6,118.7,61.5,21.5,14.5; HRMS-ESⅠ(m / z):calcd for C 18 H 17 N₂O₂[M+H] + : 293.1212,found,293.1210.

[0048] Preparation of S3 and intermediate 2-methyl-6-(quinolin-3-yl)nicotinamide:

[0049] In a clean 25 ml round-bottom flask, 0.16 g (0.54 mmol) of 2-methyl-6-(quinoline-3-yl)nicotinic acid ethyl ester, 406 μL of 80% hydrazine hydrate, and 5 ml of ethanol were added. The mixture was stirred and heated to 80 °C under reflux. The reaction was maintained in an oil bath for 16 h. The reaction was monitored by TLC until it was completed. After cooling to room temperature, a yellow precipitate was formed. The precipitate was vacuum filtered and dried to obtain 0.09 g of the yellow solid product 2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazine, with a yield of 60.2%.

[0050] Spectral data: 1 HNMR (600MHz, DMSO-d6): 9.71 (s, 1H), 9.64 (d, J = 2.2Hz, 1H), 9.04 (d, J = 1.8Hz, 1H), 8.13 (d, J = 7.7Hz, 1H), 8.09 (t,J=8.6Hz,2H),7.88(d,J=8.0Hz,1H),7.84-7.80(m,1H),7.68(t,J=7.3Hz,1H),4.60(brs,2H),2.67(s,3H); 13C NMR (150MHz, DMSO-d6): 167.5,156.6,154.1,149.5,148.2,137.1,134.1,131.1 ,130.8,130.3,129.4,129.2,127.8,127.7,118.3,23.5; HRMS-ESⅠ(m / z)::calcd for C 17 H 16 N3[M+H] + :279.1168,found,279.1170.

[0051] Preparation of S4, N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6):

[0052] In a 25 ml flask, 0.1 g (0.36 mmol) of 2-methyl-6-(quinolin-3-yl)nicotinamide and 5 ml of anhydrous ethanol were added sequentially. After dissolution, 0.055 g (0.39 mmol) of 4-chloroisocyanurate was added. The mixture was stirred and heated to 80 °C under reflux for 8 h. The reaction was stopped by TLC, filtered, and washed with ethanol to give 0.11 g of a white solid product, with a yield of 65%.

[0053] Spectral data: 1 HNMR (600MHz, DMSO-d6): 10.28(s,1H),9.67(d,J=2.2Hz,1H),9.12(brs,1H),9.08(d,J=1.5Hz,1H),8.43(s,1H),8.16(dd,J=8.1,15.0Hz,2H),8 .10(d,J=8.4Hz,1H),8.05(d,J=5.9Hz,1H),7.85-7.79(m,1H),7.69(t, J=7.5Hz,1H),7.56(d,J=8.8Hz,2H),7.34(d,J=8.8Hz,2H),2.75(s,3H); 13 C NMR (150MHz, DMSO-d6): 168.3,157.0,155.8,154.6,149.5,148.3,139.2,137.4,134.3,131.0 ,130.8,129.4,129.2,129.0,127.9,127.7,126.0,120.5,118.2,23.6; HRMS-ESⅠ(m / z):calcd forC 23 H 18 ClN5O2,[M+H] +:432.1149,found,432.1221.

[0054] Example 2

[0055] The preparation method of N-(3,4-dichlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-11) is the same as steps S1-S3 in Example 1, except that:

[0056] In a 25 ml round-bottom flask, add 0.1 g (0.36 mmol) of 2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazide and 5 ml of anhydrous ethanol. After the 2-methyl-6-(quinoline-3-yl)pyridine-3-carboxyhydrazide has completely dissolved, add 0.0761 g (0.39 mmol) of 3,4-dichloroisocyanurate, stir, and heat to 80 °C under reflux. React for 8 h. TLC analysis shows that the reaction has ended. Stop the reaction, filter, and wash with ethanol to obtain 0.11 g of the white solid product N-(3,4-dichlorophenyl)-2-(2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazide-1-carboxamide, yield 66.4%.

[0057] Spectral data: 1 H NMR (600MHz, DMSO-d6): 10.32(s,1H),9.67(s,1H),9.31(brs,1H),9.08(s,1H),8.61(brs,1H),8.18(d,J=8.0Hz,1H),8.15(d,J=8.0Hz,1H),8. 10(d,J=8.4Hz,1H),8.06(brs,1H),7.93(brs,1H),7.83(t,J=7.5Hz,1H),7.68(t,J=7.3Hz,1H),7.54-7.51(m,1H),7.47(brs,1H),2.75(s,3H); 13 C NMR (150MHz, DMSO-d6): 168.3,157.0,154.6,149.5,148.2,140.4,137.4,134.3,131.4,131.0,13 0.9,129.4,129.3,129.2,127.8,127.7,124.2,123.7,118.2,100.0,23.6; HRMS-ESⅠ(m / z):calcd for C 23 H 17 Cl2N5O2,[M+H] + :466.0759,found,466.0826.

[0058] Example 3

[0059] The preparation method of N-(o-tolyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-13) is the same as steps S1-S3 in Example 1, except that:

[0060] In a 25 mL round-bottom flask, add 0.1 g (0.36 mmol) of 2-methyl-6-(quinoline-3-yl)nicotinamide and 5 mL of anhydrous ethanol. After the 2-methyl-6-(quinoline-3-yl)pyridine-3-carboxylamide is completely dissolved, add 0.052 g (0.39 mmol) of o-methyl isocyanate, stir, and reflux at 80 °C for 8 h. The reaction is stopped when TLC shows that the reaction is complete, the mixture is filtered, and washed with ethanol to give 0.10 g of a white solid product, yield 72.2%.

[0061] Spectral data: 1 H NMR (600MHz, DMSO-d6): 10.05(d,J=1.5Hz,1H),9.65(d,J=2.2Hz,1H),9.07(d,J=1.8H z,1H),8.14(d,J=7.7Hz,2H),8.09(d,J=8.4Hz,1H),7.99(d,J=8.0Hz,1H),7.86(s,1H) ,7.85-7.79(m,1H),7.68(t,J=7.3Hz,1H),6.43(d,J=7.3Hz,1H),3.99-3.86(m,1H),2. 72(s,3H),1.92-1.76(m,2H),1.68-1.58(m,2H),1.56-1.46(m,2H),1.44-1.35(m,2H); 13 C NMR (150MHz, DMSO-d6): 168.1,158.1,156.9,154.4,149.5,148.2,137.3,134.2,131.0,130 .8,129.6,129.4,129.2,127.8,127.7,118.2,51.6,33.1,23.7,23.5; HRMS-ESⅠ(m / z):calcd for C 22 H 23 N5O2,[M+H] + :390.1852,found,390.1931.

[0062] Example 4

[0063] The preparation method of N-(4-methoxyphenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-16) is the same as steps S1-S3 in Example 1, except that:

[0064] In a 25 ml round-bottom flask, add 0.1 g (0.36 mmol) of 2-methyl-6-(quinolin-3-yl)nicotinamide and 5 ml of anhydrous ethanol. After dissolving, add 0.064 g (0.39 mmol) of 4-methoxyphenyl isocyanate, stir, and heat to 80 °C under reflux. React for 8 h. TLC analysis shows that the reaction has ended. Stop the reaction, filter, and wash with ethanol to give 0.104 g of white solid product, yield 67.7%.

[0065] Spectral data: 1 HNMR (600MHz, DMSO-d6): 10.24(s,1H),9.66(d,J=2.2Hz,1H),9.08(d,J=1.5Hz,1H),8.77(s,1H),8.24(s,1H),8.18-8.13(m,2H),8.10(d,J=8.1 Hz,1H),8.05(d,J=7.3Hz,1H),7.85-7.80(m,1H),7.68(t,J=7.3Hz,1H) ,7.41(d,J=8.8Hz,2H),6.88(d,J=9.2Hz,2H),3.72(s,3H),2.75(s,3H); 13 C NMR(150MHz,DMSO-d6):168.3,157.0,156.1,155.0,154.5,149.5,148.2,137.4,134.3,133.1,131. 0,130.9,129.5,129.4,129.2,127.8,127.7,120.8,118.2,114.3,55.6,23.6; HRMS-ESⅠ(m / z):calcd forC 24 H 21 N5O3,[M+H] + :428.1664,found,427.1668.

[0066] Example 5

[0067] The preparation method of product N-(3-fluorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-18) is the same as steps S1-S3 in Example 1, except that:

[0068] In a 25 ml round-bottom flask, add 0.1 g (0.36 mmol) of 2-methyl-6-(quinolin-3-yl)nicotinamide and 5 ml of anhydrous ethanol. After dissolving, add 0.053 g (0.39 mmol) of 3-fluoroisocyanate, stir, and heat to 80 °C under reflux. React for 10 h. TLC analysis shows that the reaction has ended. Stop the reaction, filter, and wash with ethanol to give 0.103 g of white solid product, yield 69.0%.

[0069] Spectral data: 1 HNMR (600MHz, DMSO-d6): 10.31 (s, 1H), 9.67 (d, J=2.2Hz, 1H), 9.22 (brs, 1H), 9.09 (d, J= 1.5Hz,1H),8.48(brs,1H),8.18(d,J=7.7Hz,1H),8.15(d,J=8.1Hz,1H),8.10(d,J=8.1Hz ,1H),8.06(d,J=4.0Hz,1H),7.85-7.81(m,1H),7.69(t,J=7.3Hz,1H),7.53(d,J=11.7Hz ,1H),7.35-7.28(m,1H),7.25(d,J=7.0Hz,1H),6.80(dt,J=1.8,8.4Hz,1H),2.75(s,3H); 13 C NMR (150MHz, DMSO-d6): 168.3,163.6,162.0,157.0,155.7,154.6,149.5,148.2,142.0,137.4,134.3,13 1.0,130.9,130.7,129.4,129.2,127.8,118.3,114.7,108.7(d,J=21.0Hz),23.6; HRMS-ESⅠ(m / z):calcd for C 23 H 18 FN5O2,[M+H] + :416.1445,found,416.1513.

[0070] Example 6

[0071] The preparation method of N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6) is the same as steps S2-S4 in Example 1, except that:

[0072] Preparation of intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one:

[0073] Take a clean 25 ml round-bottom flask, add 0.17 g (1 mmol) of 3-acetylquinoline, 620.7 μL (3 mmol) of N,N-dimethylformamide dimethyl acetal and 5 ml of toluene in sequence, stir and heat to 80 °C and reflux, keep the reaction in an oil bath for 8 h, monitor the reaction with TLC until the reaction is complete, stop the reaction and cool to room temperature, perform vacuum filtration, dry at 80 °C to obtain a white solid product (E)-3-(dimethylamino)-1-(quinoline-3-yl)propen-2-en-1-one.

[0074] Example 7

[0075] The preparation method of N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6) is the same as steps S1 and S3-S4 in Example 1, except that:

[0076] Preparation of intermediate 2-methyl-6-(quinolin-3-yl)nicotinic acid ethyl ester (S2):

[0077] In a clean 10 ml round-bottom flask, add 0.227 g (1 mol) of (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one, followed by 126.88 μL (1 mmol) of ethyl acetoacetate, 7.7 g (10 mol) of ammonium acetate, and 5 ml of glacial acetic acid. Stir and heat to 110 °C, reflux for 6 h, and monitor the reaction by TLC until the reaction is complete. Then stop stirring, cool to room temperature, and extract three times with 15 ml of ethyl acetate and 5 ml of water. Dry the extract with anhydrous sodium sulfate, filter, rotary evaporate, and prepare slurry. Finally, the crude product is subjected to silica gel column chromatography (eluent: V(ethyl acetate) / V(petroleum ether) = 1 / 3) to give the white solid product ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid.

[0078] Example 8

[0079] The preparation method of N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6) is the same as steps S1-S2 and S4 in Example 1, except that:

[0080] Preparation of S3 and intermediate 2-methyl-6-(quinolin-3-yl)nicotinamide:

[0081] In a clean 25 ml round-bottom flask, 0.3 g (1 mmol) of ethyl 2-methyl-6-(quinoline-3-yl)nicotinic acid, 507.5 μL of 80% hydrazine hydrate, and 5 ml of ethanol were added. The mixture was stirred and heated to 80 °C under reflux. The reaction was maintained in an oil bath for 10 h. The reaction was monitored by TLC until it was completed and then stopped. After cooling to room temperature, a yellow precipitate was formed. The precipitate was then vacuum filtered and dried to obtain the yellow solid product 2-methyl-6-(quinoline-3-yl)nicotinic acid hydrazide.

[0082] Example 9

[0083] The preparation method of N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6) is the same as steps S1-S3 in Example 1, except that:

[0084] Preparation of S4, N-(4-chlorophenyl)-2-[2-methyl-6-(quinolin-3-yl)nicotinyl]hydrazide-1-carboxamide (M-4-6):

[0085] In a 25 ml flask, 0.3 g (1.1 mmol) of 2-methyl-6-(quinolin-3-yl)nicotinamide and 5 ml of anhydrous ethanol were added sequentially. After dissolution, 0.141 g (1 mmol) of 4-chloroisocyanurate was added. The mixture was stirred and heated to 70 °C under reflux for 6 h. The reaction was stopped by TLC, filtered, and washed with ethanol to obtain a white solid product.

[0086] The compounds from Examples 1-5 of this invention are used as examples to illustrate their application in inducing apoptosis in liver cancer cells. The specific application methods and results are shown below:

[0087] (1) In vitro toxicity test on tumor cells:

[0088] The compounds (M-4-6, M-4-11, M-4-13, M-4-16, and M-4-18) from Examples 1-5 were subjected to preliminary in vitro toxicity tests against tumor cells using the MTT assay. Three concentrations (5.6 μM, 16.7 μM, and 50 μM) were used, and the compounds were treated for 24 h. OD values ​​were measured to screen for concentration-dependent active compounds with good efficacy. Experimental results showed that product M-4-6 inhibited the growth of HepG2 and Huh7 cells in a dose-dependent manner; M-4-11 showed significant inhibition rates against both liver cancer cell lines; and products M-4-13, M-4-16, and M-4-18 also exhibited inhibitory effects on the growth of HepG2 and Huh7 cells. Figure 1 As shown.

[0089] (2) The effects of the compounds in Examples 1-5 on the IC50 of HepG2 and Huh7 cells 50 Value determination:

[0090] Using HepG2 and Huh7 cells, five concentrations of the compound (0.625 μM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM) were established, and treatment was performed for 24 h. Results showed that the IC50 values ​​of M-4-6 and M-4-11 for both cell lines were [not specified]. 50 The values ​​were as follows: Huh7 cells: 3.89±0.68μM, 0.55±0.13μM; HepG2 cells: 4.68±0.68μM, 2.22±0.23μM, as shown in Table 1.

[0091] Table 1. Effects of the compounds in Examples 1-5 on IC50 in HepG2 and Huh7 cells. 50 value

[0092]

[0093]

[0094] (3) Determination of apoptosis induced by M-4-6 in Example 1 and M-4-11 in Example 2 in liver cancer cells:

[0095] Studies have shown that NR4A1 levels decrease to some extent during the development of liver cancer. HepG2 cells were further treated with 4 μM M-4-6 and 2 μM M-4-11, with samples collected at 0h, 2h, 4h, 8h, 12h, and 24h. The results showed that both active compounds affected NR4A1 protein levels. M-4-6 reduced NR4A1 protein levels in a short period but recovered them at 24h; M-4-11 downregulated NR4A1 protein levels and maintained them within a certain range. Furthermore, treatment with M-4-11 increased the cleavage of the apoptosis-related protein PARP, indicating that M-4-11 can induce hepatocyte death through apoptosis; treatment with M-4-6 did not significantly increase PARP cleavage, suggesting the possible existence of other death mechanisms, such as… Figure 3 As shown.

[0096] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0097] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A formamide derivative, characterized in that, The chemical structural formula of the formamide derivative is: The R is selected from H, -F, -Cl, -Br, C1-C3 alkyl or C1-C3 alkoxy.

2. The formamide derivative according to claim 1, characterized in that, The R is selected from H, -F, -Cl, -CH3 or -OCH3.

3. A formamide derivative, characterized in that, Formamide derivatives are: N-(4-chlorophenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide, N-(3,4-dichlorophenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide, N-(o-tolyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide, N-(4-methoxyphenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide or N-(3-fluorophenyl)-2-[2-methyl-6-(quinoline-3-yl)nicotinic acidyl]hydrazine-1-carboxamide.

4. A method for preparing the formamide derivative according to claim 1, characterized in that, Includes the following steps: Using toluene as a solvent, 3-acetylquinoline was condensed with N,N-dimethylformamide dimethyl acetal to obtain the intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one; Using acetic acid as a solvent, (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one and ethyl acetoacetate were subjected to a condensation reaction under ammonium acetate catalysis to obtain the intermediate ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid. Using ethanol as a solvent, ethyl 2-methyl-6-(quinoline-3-yl)nicotinic acid was subjected to hydrazinolysis with hydrazine hydrate to obtain the intermediate 2-methyl-6-(quinoline-3-yl)nicotinamide. Using ethanol as a solvent, 2-methyl-6-(quinoline-3-yl)nicotinamide hydrazide was condensed with substituted phenyl isocyanate to obtain a formamide derivative.

5. The method for preparing the formamide derivative according to claim 4, characterized in that, In preparing the intermediate (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one, the molar ratio of 3-acetylquinoline to N,N-dimethylformamide dimethyl acetal is 1:2.5-3; the reaction temperature is 80-90℃.

6. The method for preparing the formamide derivative according to claim 4, characterized in that, When preparing the intermediate ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid, the molar ratio of (E)-3-(dimethylamino)-1-(quinolin-3-yl)propen-2-en-1-one, ethyl acetoacetate, and ammonium acetate is 1:1:8.7-10; the reaction temperature is 110-125℃.

7. The method for preparing the formamide derivative according to claim 4, characterized in that, When preparing the intermediate 2-methyl-6-(quinolin-3-yl)nicotinamide, the molar ratio of ethyl 2-methyl-6-(quinolin-3-yl)nicotinic acid to hydrazine hydrate is 1:8-10; the reaction temperature is 70-80℃.

8. The method for preparing the formamide derivative according to claim 4, characterized in that, When preparing formamide derivatives, the molar ratio of the substituted phenyl isocyanate to 2-methyl-6-(quinolin-3-yl)nicotinamide is 1-1.1:1, and the reaction temperature is 70-80℃.

9. The use of a formamide derivative according to any one of claims 1-3 in the preparation of an NR4A1 inhibitor, characterized in that, The NR4A1 inhibitors include formamide derivatives and pharmaceutically acceptable salts of formamide derivatives.

Images