Preparation method and application of urea triazole derivative with anti-hepatoma effect

Abstract

The invention discloses a preparation method and application of a urea triazole derivative which is simple and easy to operate, cheap and easily available in raw materials, high in reaction efficiency and good in inhibition effect on human liver cancer cells Huh-7, and belongs to the technical field of medicine synthesis. According to the key points of the technical scheme, the urea triazole derivative molecule has a structure, or R1 is hydrogen atom, fluorine, chlorine, bromine, iodine, nitryl, alkyl (such as methyl, ethyl or alkynyl) or alkoxy (such as methoxyl) and various substituent groups, R2 is phenyl derivative or benzyl derivative, and X is oxygen atom or sulfur atom. The invention designs and synthesizes a urea triazole derivative with a novel structure, and the compound has a good inhibition effect on human liver cancer cells Huh-7.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis technology, specifically relating to a method for preparing and applying a urea triazole derivative with anti-liver cancer activity. Background Technology

[0002] Given that most anticancer drugs become ineffective due to drug resistance, the development of new molecules with novel mechanisms of action to combat cancer is particularly urgent.

[0003] Molecular hybridization, which involves covalently binding two or more pharmacophores to form a single molecule, has become an effective strategy for designing highly active new drug entities. These combined pharmacophores can act on multiple therapeutic targets, potentially enhancing the ability of drugs to circumvent drug resistance. Furthermore, drug hybridization can minimize unwanted side effects and exhibit synergistic effects. Currently, molecular hybridization methods are widely used in the development of novel antitumor drugs to overcome the challenge of drug resistance.

[0004] 1,2,3-Triazole has long been a significant source of inspiration in medicinal chemistry, thanks to its efficient click chemistry synthesis and rich bioactivity. As a key nitrogen-containing heterocyclic compound, 1,2,3-triazole is a five-membered heterocycle consisting of three nitrogen atoms and two carbon atoms, with the molecular formula C2N3H3. This structure possesses unique planar rigidity, endowing it with a strong ability to intercalate into DNA. Simultaneously, its large dipole moment allows it to form various non-covalent interactions with a wide range of biological targets (including hydrophobic interactions, hydrogen bonds, van der Waals forces, and dipole-dipole bonds, etc.).

[0005] Furthermore, the structural properties of 1,2,3-triazole allow it to function as an electronically equivalent substitute for amides, esters, carboxylic acids, and olefin analogs, thus exhibiting a wide range of biological activities. Therefore, it is often used as an important molecular building block in the synthesis of compounds with antibacterial, antimalarial, antifungal, antiviral, antituberculosis, and anticancer activities. In the field of medicinal chemistry, 1,2,3-triazole has broad application prospects.

[0006] Many clinical drugs, after being modified and engineered with 1,2,3-triazole, not only exhibit enhanced original biological activities but also acquire new ones. As a common five-membered aromatic heterocyclic structure in drug molecule design, 1,2,3-triazole is weakly basic and exists as two isomers: 1,2,3-triazole and 1,2,4-triazole. The nitrogen atom in its structure can act as a hydrogen bond acceptor, while the NH atom can act as a hydrogen bond donor, interacting with biological targets.

[0007] Due to the difference in electronegativity between carbon and nitrogen, 1,2,3-triazole has a low logP value, a characteristic that gives it the potential to improve the water solubility of compounds. Furthermore, the relatively stable metabolism of triazole further increases its application value in drug design.

[0008] For example, the triazolylpyrazine compound SAR125844 (1-6-[6-(4-fluorophenyl)-[1,2,4]triazol[4,3-b]pyrazin-3-ylsulfonamide]benzothiazol-2-yl)-3-(2-morpholinone-4-ethyl)urea) designed by Schio's research group exhibits highly efficient and selective inhibition of wild-type (WT) MET kinase and various clinical mutants. This discovery suggests that this type of compound holds promise as a potential parenteral drug for treating MET-dependent cancers, providing new ideas and directions for drug development.

[0009] To screen compounds with significant inhibitory activity against Huh-7 human hepatocellular carcinoma cells, this study designed and synthesized a series of novel urea triazole derivatives. Specifically, we reacted phenyl isocyanates with different substituents with aminoalkynyl compounds to successfully introduce terminal alkynyl groups, constructing the target urea compound skeleton. Subsequently, using a click chemistry strategy, 1,2,3-triazole groups were introduced into the molecular structure to obtain compounds with anti-Huh-7 human hepatocellular carcinoma cell activity.

[0010] To evaluate the antitumor activity of the target compound, we conducted in vitro proliferation inhibition experiments on Huh-7 human hepatocellular carcinoma cells using the CCK-8 assay. The results showed that the synthesized compound exerts a multiple antitumor mechanism by inducing DNA damage and autophagy in Huh-7 cells, ultimately inhibiting cell proliferation and inducing apoptosis. These findings provide important theoretical and experimental basis for the development of novel anticancer drugs. Summary of the Invention

[0011] The technical problem solved by this invention is to provide a method for preparing and applying a ureatriazole derivative that is simple to operate, uses inexpensive and readily available raw materials, has high reaction efficiency, and exhibits good inhibitory effects on human liver cancer cells (Huh-7). The molecular structure of this ureatriazole derivative is characterized by: or R1 is a hydrogen atom, fluorine, chlorine, bromine, iodine, nitro, alkyl (such as methyl, ethyl or alkynyl) or alkoxy (such as methoxy) substituent groups, R2 is a phenyl derivative or benzyl derivative, and X is an oxygen atom or sulfur atom.

[0012] The present invention relates to a method for preparing and applying a urea triazole derivative with anti-hepatocellular carcinoma activity, characterized in that: (1) Using benzoic acid derivatives and diphenyl azidophosphate (DPPA) as raw materials, a Curtius rearrangement reaction was carried out. After the reaction solution was concentrated and dissolved, it was washed, dried, and concentrated in sequence to obtain phenyl isocyanate. A certain amount of benzoic acid derivative was dissolved in chloroform, and a certain amount of diphenyl azidophosphate (DPPA) and triethylamine were added in sequence at room temperature. After the addition was completed, the system temperature was gradually raised to 60-80℃. The system was reacted at this temperature for several hours, and the reaction was monitored by TLC. After the reaction was completed, the system was quenched with water, extracted with dichloromethane, dried with anhydrous sodium sulfate, and concentrated to obtain phenyl isocyanate. The molar ratio of benzoic acid derivative, DPPA, and triethylamine was 1:1~1.2:1~1.2. The reaction temperature was 60~80℃.

[0013] (2) Using aminoyne (m-aminophenylacetylene, p-aminophenylacetylene and propargylamine) and phenyl isocyanate (and other substituent phenyl isocyanates) as raw materials, a nucleophilic addition reaction is carried out. The reaction solution is concentrated and purified to obtain urea compounds containing terminal alkynes. The molar ratio of aminoyne to phenyl isocyanate (and other substituent phenyl isocyanates) is 1:1-1.2. The reaction temperature is 0~40℃.

[0014] (3) A certain amount of alkynyl urea compound and benzyl azide are dissolved in a mixed solution of tert-butanol / water / tetrahydrofuran. The system is heated and reacted for several hours. The reaction is monitored by TLC. After the reaction is completed, the urea triazole product is obtained by column chromatography. The reaction temperature is 60~100℃, the reaction time is 6h-18h, and the molar ratio of alkynyl urea to benzyl azide is 1:1.1~1.6.

[0015] The urea triazole derivatives described in this invention have the following technical advantages: (1) This invention designs and synthesizes novel urea triazole derivatives based on the phenyl isocyanate structure; (2) The urea triazole derivatives utilize their rigid planar structure to achieve DNA interleaving, leading to DNA damage and ultimately affecting the death of related cells. These findings provide important theoretical basis and experimental foundation for the development of novel anti-liver cancer drugs. Attached Figure Description

[0016] Figure 1 The image shows the hydrogen nuclear magnetic resonance spectrum of compound 3 obtained in Example 3. Figure 2 The image shows the 1H NMR spectrum of compound N-1 obtained in Example 6. Figure 3 The image shows the 1H NMR spectrum of compound N-2 obtained in Example 7. Figure 4 The image shows the fluorescence staining of Huh-7 human hepatocellular carcinoma cells after treatment with different concentrations of the N-series target compounds (N3, N10, N11, N19) for 24 hours. Figure 5 Flow cytometry analysis of Huh-7 human hepatocellular carcinoma cells after treatment with different concentrations of N-series target compounds (N3, N10, N11, N19) for 72 hours. Figure 6 Microscopic images showing DNA damage 24 hours after treatment of Huh-7 human hepatocellular carcinoma cells with N-series target compounds (N3, N10, N11, N19) by immunofluorescence staining. Figure 7 Microscopic images showing the autophagy levels of Huh-7 human hepatocellular carcinoma cells 24 hours after treatment with N-series target compounds (N3, N10, N11, N19) using specific fluorescent staining. Figure 8 Electrophoretic images showing the expression of key proteins in Huh-7 human hepatocellular carcinoma cells after 72 hours of treatment with N-series target compounds (N3, N10, N11, N19) using Western blotting. Detailed Implementation

[0017] 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.

[0018] Example 1:

[0019] p-Chlorobenzoic acid (1.0 g), diphenyl azidophosphate (DPPA) (1.76 g), 1.0 mL triethylamine (TEA), and 20 mL chloroform were sequentially added to a round-bottom flask and stirred at 60 °C for 6 h. The reaction was monitored by TLC [V(dichloroform):V(methanol) = 15:1] until complete and the mixture was concentrated under reduced pressure. Dichloroform (30 × 3 mL) was added for extraction, and the mixture was stirred for 15 min. The mixture was washed with saturated sodium bicarbonate (20 mL x 3), dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 0.9 g of p-chlorophenyl isocyanate.

[0020] Example 2:

[0021] In a microwave chemical reactor (MCR-3), 1.0 g of p-chlorobenzoic acid, 1.76 g of DPPA, and 1.0 mL of TEA obtained in the previous step were dissolved in 25 mL of dichloroethane. The power was turned on, the temperature sensor was inserted, and the microwave reaction power was set to 300-400 watts, the temperature to 60 °C, and the time to 25 min. The reaction was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure, extracted with 30 × 3 mL of dichloromethane, stirred for 15 min, washed with 20 mL × 3 saturated sodium bicarbonate, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain 0.91 g of p-chlorophenyl isocyanate.

[0022] Example 3: ; The p-chlorophenyl isocyanate (0.5 g) obtained in the previous step was dissolved in anhydrous dichloromethane (20 mL), and m-aminophenylacetylene (0.38 g) was added at room temperature. The system was reacted at room temperature, and the reaction was monitored by TLC until the starting material disappeared and the product was formed. After the system was dried by rotary evaporation, ethyl acetate / petroleum ether (1:10) mixed solvent (20 mL) was added and stirred to diffuse. After filtration, urea compound 3 (0.8 g) was obtained. 1 HNMR (400 MHz, DMSO- d 6) δ 8.87 (s, 1H), 8.82 (s, 1H), 7.66 (s, 1H), 7.49 (d, J = 8.9 Hz, 2H), 7.40 (d, J = 1.2 Hz, 1H), 7.36 – 7.27 (m, 3H), 7.09 (dt, J =7.6, 1.3 Hz, 1H), 4.16 (s, 1H). Example 4:

[0023] In a reaction flask, p-chlorophenyl isocyanate (0.5 g) was dissolved in anhydrous dichloromethane (10 mL) to prepare solution A; then, a mixture of m-aminophenylacetylene (0.38 g) and anhydrous dichloromethane (26 g) was dissolved to prepare solution B (approximately 20 mL in volume); solutions A and B were simultaneously introduced into a microchannel reactor at flow rates of 1 mL / min and 2 mL / min, respectively, with the reactor temperature set at 10–20 °C. After the reaction was completed, the reaction liquid discharged from the reactor was collected and poured into methyl tert-butyl ether. A large amount of solid product appeared, which was recrystallized from isopropanol to obtain urea compound 3 (0.85 g).

[0024] Example 5:

[0025] Dissolve the p-chlorophenyl isocyanate (0.5 g) obtained in the previous step in anhydrous dichloromethane (20 mL), add m-aminophenylacetylene (0.38 g) at room temperature, place the system in a HY-3 multifunctional shaker, shake for 0.5 h, evaporate the system to dryness, add 20 mL of n-hexane, shake again for 10 min, centrifuge for 10 min, remove the supernatant to obtain product 3 (0.81 g).

[0026] Example 6: ; Synthesis of the target product 1-(3-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (N-1): In a reaction flask, intermediate 3 (90 mg), 4-chlorobenzyl azide (67 mg), 10 mL tert-butanol / 10 mL water / 10 mL tetrahydrofuran, copper sulfate pentahydrate (9 mg), and sodium ascorbate (13 mg) were added sequentially. The reaction was carried out at 85 °C for 12 h. After the reactants were completely converted into the product, 20 mL of dichloromethane was added. The reaction solution was filtered to obtain a yellowish liquid. The organic phase was then separated, and the aqueous phase was extracted twice with 15 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain 120 mg of the product. 1 H NMR (400 MHz, DMSO- d 6) δ 8.86 (s, 1H), 8.62 (s, 1H), 8.04 (s, 1H), 7.53 – 7.22 (m, 12H), 5.66 (s, 2H).

[0027] Example 7: ; Synthesis of the target product 1-(3-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (N-2): In a parallel reactor, intermediate 3 (200 mg), 3-chlorobenzyl azide (149 mg), 10 mL of tert-butanol / 10 mL of water / 10 mL of tetrahydrofuran, 20 mg of copper sulfate pentahydrate, and 30 mg of sodium ascorbate were added sequentially. The reaction was carried out at 85 °C for 10 h. After the reactants were completely converted into the product, excess tetrahydrofuran and tert-butanol were evaporated from the system. 50 mL of deionized water was added, and the mixture was sonicated in an ultrasonic instrument for 20 min. After standing for 1 h, the solid was generated. After centrifugation for 10 min, the supernatant was removed, and the product was recrystallized from a small amount of isopropanol and filtered to obtain 252 mg of the product. 1 H NMR (400 MHz, DMSO- d 6) δ 8.84 (s, 1H), 8.66 (s, 1H), 8.05 (s, 1H), 7.52-7.51 (m, 1H), 7.50 – 7.49 (m,1H), 7.48 – 7.46 (m, 1H), 7.46 – 7.40 (m, 3H), 7.38 – 7.30 (m, 6H), 5.67 (s, 2H).

[0028] Example 8: ; Synthesis of the target product 1-(3-(1-(4-bromochlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (N-3): In a microwave chemical reactor (MCR-3), intermediate 3 (200 mg), 4-bromobenzyl azide (188 mg), copper sulfate pentahydrate (20 mg), and sodium ascorbate (30 mg) were added sequentially to a solution of tert-butanol (10 mL / water / tetrahydrofuran). The power was turned on, a temperature sensor was inserted, and the microwave reaction power was set to 300-400 watts, the temperature to 80 °C, and the time to 45 min. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated under reduced pressure. Excess tetrahydrofuran and tert-butanol were evaporated from the system. 50 mL of deionized water was added, and the mixture was sonicated for 20 min. After standing for 1 h to allow solid formation, the mixture was centrifuged for 10 min, the supernatant was removed, and the product was recrystallized from a small amount of isopropanol and filtered to obtain 281 mg of product. 1 H NMR (400 MHz, DMSO- d 6) δ 8.84 (s, 1H), 8.61 (s, 1H), 8.04 (d, J = 1.9 Hz, 1H), 7.60 (d, J =8.4 Hz, 2H), 7.51 (d, J = 8.9 Hz, 2H), 7.47 – 7.41 (m, 2H), 7.38 – 7.27 (m, 6H), 5.64 (s, 2H).

[0029] Example 9: ; Synthesis of the target product 1-(3-(1-(3-bromochlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (N-4): Intermediate 3 (20 mg), 3-bromobenzyl azide (19 mg), copper sulfate pentahydrate (2 mg), sodium ascorbate (3 mg), and tert-butanol (5 mL / water / tetrahydrofuran 5 mL) were transferred to a 25 mL round-bottom high-temperature flask and irradiated with sonic waves for 10 min. The flask was then irradiated with a 100 W UV LED lamp under stirring, and the reaction was monitored by TLC. After the product was formed and the starting material disappeared, a small amount of solvent was evaporated from the system, 15 mL of double-distilled water was added, the mixture was sonicated for 10 min, and allowed to stand for 0.5 h until a solid was formed. After centrifugation for 10 min, the supernatant was removed, and the mixture was recrystallized with a small amount of acetone and filtered to obtain 30 mg of the product. 1 H NMR (400 MHz, DMSO- d 6) δ 8.88(s, 1H), 8.87 (s, 1H), 8.66 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.63 – 7.60(m, 1H), 7.58 – 7.54 (m, 1H), 7.51 (d, J = 8.9 Hz, 2H), 7.44 (dt, J = 5.1 Hz,1H), 7.38 – 7.31 (m, 6H), 5.67 (s, 2H). The preparation methods for target compounds N5-N20 are similar to those for N1-N4, the difference being the different substituents of the active groups with azide structures. Their structural data are as follows:

[0030] The target compound 1-(3-(1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (N-5): yield: 89%.

[0031] The target compound 1-(3-(1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-fluorophenyl)urea (N-6): yield: 96%.

[0032] The target compound 1-(3-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-fluorophenyl)urea (N-7): yield: 93%.

[0033] The target compound 1-(3-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-fluorophenyl)urea (N-8): yield: 79%.

[0034] The target compound 1-(3-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-fluorophenyl)urea (N-9): yield: 86%.

[0035] The target compound 1-(3-(1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-fluorophenyl)urea (N-10): yield: 80%.

[0036] The target compound 1-(3-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (N-11): yield: 85%.

[0037] The target compound 1-(3-(1-(3-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (N-12) had a yield of 87%.

[0038] The target compound 1-(3-(1-(2-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (N-13): yield: 90%.

[0039] The target compound 1-(3-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (N-14): yield: 88%.

[0040] The target compound 1-(3-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (N-15): yield: 84%.

[0041] The target compound 1-(3-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (N-16): yield: 78%.

[0042] The target compound 1-(3-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (N-17): yield: 75%.

[0043] The target compound 1-(3-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (N-18): yield: 92%.

[0044] Target compound 1-(3-(1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (N-19): yield: 86%.

[0045] The target compound 1-(3-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (N-20): yield: 91%.

[0046] The preparation methods for target compounds NN1~NN20 are similar to those for N1~N20, and their structural data are as follows:

[0047] The target compound 1-(4-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (NN-1): yield: 90%.

[0048] The target compound 1-(4-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (NN-2) had a yield of 94%.

[0049] The target compound 1-(4-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-chlorophenyl)urea (NN-3) had a yield of 91%.

[0050] The target compound 1-(4-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (NN-4): yield: 76%.

[0051] The target compound 1-(4-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(2-bromophenyl)urea (NN-5): yield: 85%.

[0052] The target compound 1-(4-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (NN-6): yield: 66%.

[0053] The target compound 1-(4-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (NN-7) had a yield of 86%.

[0054] The target compound 1-(4-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (NN-8): yield: 88%.

[0055] The target compound 1-(4-(1-(2-methylchlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)ureaNN-9): yield: 94%.

[0056] The target compound 1-(4-(1-(4-methylchlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-3-(4-methoxyphenyl)urea (NN-10) had a yield of 89%.

[0057] The target compound 1-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-chlorophenyl)urea (NN-11) had a yield of 88%.

[0058] Target compound 1-(1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-chlorophenyl)urea (NN-12): Yield: 87%.

[0059] The target compound 1-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-chlorophenyl)urea (NN-13) had a yield of 79%.

[0060] The target compound 1-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(2-bromophenyl)urea (NN-14): yield: 85%.

[0061] The target compound 1-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(2-bromophenyl)urea (NN-15): yield: 87%.

[0062] The target compound 1-(1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)urea (NN-16): yield: 90%.

[0063] The target compound 1-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)urea (NN-17) had a yield of 88%.

[0064] The target compound 1-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)urea (NN-18) had a yield of 84%.

[0065] The target compound 1-(1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)urea (NN-19): yield: 80%.

[0066] The target compound 1-(1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(4-methoxyphenyl)urea (NN-20): yield: 85%.

[0067] Example 10:

[0068] We prepared compounds N1-N20 at a concentration of 50 μM and applied them to different cancer cell lines (including human hepatocellular carcinoma cell line Huh-7, human breast cancer cell line MCF-7, and human lung cancer cell lines A549, H460, PC-9, and H1299) and normal hepatocyte cell line L02. After 72 hours of treatment, we found that the N-series compounds had the best inhibitory activity against human hepatocellular carcinoma cell line Huh-7, with cell viability below 20%, which was significantly better than the inhibitory activity against other cell lines. At the same time, the N-series compounds did not have any inhibitory or damaging effect on normal human hepatocytes, indicating the safety of compounds N1-N20 in normal cells (Table 1).

[0069] Table 1: Cellular activities of target compounds N1-N20 against different human cancer cells:

[0070] The half-maximal inhibitory concentration (IC50) of compounds N1-N20 against the human hepatocellular carcinoma cell line Huh-7 was [value missing]. 50 Detection revealed that most N-series compounds exhibited excellent inhibition efficiency, IC50... 50 The values ​​are mostly below 20 μM, with IC values ​​for N3, N10, N11, and N19 being particularly high. 50 The lowest values ​​were 10.23±1.32μM, 11.62±3.72μM, 12.25±1.31μM, and 10.80±0.14μM (Table 2).

[0071] Table 2: Half-maximal inhibitory rate (IC50) of the N1-N20 series target compounds against Huh-7 human hepatocellular carcinoma cells 50：

[0072] Example 11: We prepared compounds NN1-NN20 at a concentration of 50 μM and applied them to different cancer cell lines (including human hepatocellular carcinoma cell line Huh-7, human breast cancer cell line MCF-7, human lung cancer cell lines A549, H460, PC-9, and H1299) and normal hepatocyte cell line L02. After 72 hours of treatment, we found that the inhibitory efficiency of the NN series compounds against different human cancer cell lines was weaker than that of the N series compounds, and the cell viability was higher than 50% (Table 3).

[0073] Table 3: Cellular activities of target compounds NN1-NN20 against different human cancer cells:

[0074] In addition to NN9's IC50 on Huh-7 human liver cancer cells 50 Besides the value of 8.32 μM, other NN compounds showed IC50 values ​​against Huh-7 human hepatocellular carcinoma cells. 50 The values ​​are all greater than 50 μM (Table 4).

[0075] Table 4: Half-maximal inhibitory rate (IC50) of NN series target compounds against Huh-7 human hepatocellular carcinoma cells 50：

[0076] Example 12: We treated Huh-7 cells at different concentrations (5, 10, 20 μM) of four compounds (N3, N10, N11, and N19) with the lowest cell viability for 24 hours and examined their effects on cell proliferation. Treatment with N3, N10, N11, and N19 significantly increased the number of dead cells and decreased the number of viable cells, and this effect was concentration-dependent. Figure 4 ).

[0077] Example 13:

[0078] For the detection and analysis of apoptosis in Huh-7 cells, treatment with different concentrations (5, 10, 20 μM) of compounds N3, N10, N11, and N19 for 72 hours significantly increased the number of apoptotic cells, with the increase increasing with increasing concentration. Figure 5The percentage of apoptotic cells was 1.73% in untreated cells with compound N3, 2.48% at 5 μM, 4.48% at 10 μM, and 11.73% at 20 μM. In the control group, the percentages of apoptotic cells with compound N10 at concentrations of 5 μM, 10 μM, and 20 μM were 1.81%, 2.10%, 2.26%, and 4.29%, respectively. In the control group, the percentages of apoptotic cells with compound N11 at concentrations of 5 μM, 10 μM, and 20 μM were 2.17%, 3.19%, 7.04%, and 15.84%, respectively. In the control group, the percentages of apoptotic cells with compound N19 at concentrations of 5 μM, 1.92%, 3.46%, and 6.44%, respectively.

[0079] Example 14:

[0080] DNA damage plays a crucial role in cell proliferation, and increased DNA damage can lead to cell death. Staining of Huh-7 cells with different concentrations (5, 10, 20 μM) of compounds N3, N10, N11, and N19 showed that treatment with these compounds for 24 hours significantly increased DNA damage within Huh-7 cells, with the degree of DNA damage increasing with increasing compound concentration. Figure 6 ).

[0081] Example 15:

[0082] Autophagy is an important pathway leading to cell death. To investigate the effects of compounds N3, N10, N11, and N19 on autophagy, we treated Huh-7 cells with different concentrations (5, 10, 20 μM) of these compounds. After 24 hours, fluorescence staining was used to detect changes in intracellular autophagosomes. The results showed that compounds N3, N10, N11, and N19 significantly increased intracellular autophagosomes, greatly promoting the occurrence of autophagy. Figure 7 ).

[0083] Example 16:

[0084] Analysis of the expression of key proteins involved in the proliferation and death processes of Huh-7 cells by compounds N3, N10, N11, and N19 revealed that treatment of cells with 10 μM of these compounds for 72 hours significantly promoted the expression of autophagy and the key DNA damage proteins LC3 and H2AX. Figure 8 This inhibits cell proliferation and promotes cell death, thereby exerting the antitumor activity of N-series compounds.

[0085] 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 method for preparing and applying a ureatriazole derivative that is simple to operate, uses inexpensive and readily available raw materials, has high reaction efficiency, and exhibits good inhibitory effects on human hepatocellular carcinoma cells Huh-7, characterized in that... The structure of this ureatriazole derivative is as follows: , or R1 is a hydrogen atom, fluorine, chlorine, bromine, iodine, nitro, alkyl (such as methyl, ethyl or alkynyl or alkoxy) or various substituent groups (such as methoxy), R2 is a phenyl derivative or a benzyl derivative, and X is an oxygen atom or a sulfur atom.

2. The method for preparing a urea triazole derivative with anti-hepatocellular carcinoma activity according to claim 1, characterized in that... The specific process is as follows: using benzoic acid derivatives and diphenyl azidophosphate (DPPA) as raw materials, a Curtius rearrangement reaction is carried out. After the reaction solution is concentrated and dissolved, it is washed, dried, and concentrated sequentially to obtain phenyl isocyanate. The molar ratio of the benzoic acid derivative, DPPA, and triethylamine is 1:1~1.2:1~1.2; the reaction temperature is 60~80℃.

3. The method for preparing a ureatriazole derivative with anti-hepatocellular carcinoma activity according to claim 1, characterized in that... The specific process is as follows: a certain amount of phenyl isocyanate is added... Using aminoyne (m-aminophenylacetylene, p-aminophenylacetylene, and propargylamine) as raw materials, a nucleophilic addition reaction is carried out, and the reaction solution is concentrated and purified to obtain urea compounds containing terminal alkynes. , or The molar ratio of aminoacetylene to phenyl isocyanate (and other substituted phenyl isocyanates) is 1:1-1.2, and the reaction temperature is 0~40℃.

4. The method for preparing a ureatriazole derivative with anti-hepatocellular carcinoma activity according to claim 1, characterized in that... The specific process is as follows: A certain amount of alkyne urea compounds, azide compounds, sodium ascorbate, and CuSO4 are added to a mixed solution of water, tert-butanol, and tetrahydrofuran. The mixture is heated to reflux and reacted for a period of time. After stirring, brine is added, and the mixture is extracted multiple times with dichloromethane. The organic phases are combined, dried over anhydrous sodium sulfate, concentrated under vacuum to remove the organic solvent, and then separated by silica gel column chromatography to obtain the target compound. , or The reaction temperature is 60~100℃, the reaction time is 6h-18h, and the molar ratio of alkynyl urea to benzyl azide is 1:1.1~1.

6.

5. The rigid planar structure of the ureatriazole derivatives as described in claim 1 can achieve DNA interleaving, leading to DNA damage, thereby inhibiting cell proliferation and ultimately inducing apoptosis.

Images