A sulfone hydrazone compound, a preparation method thereof and an anti-tumor application thereof

Abstract

The application relates to the technical field of pharmaceutical chemistry, and discloses a sulfuryl hydrazone compound, a preparation method thereof and anti-tumor application. The compound is synthesized from isatin derivatives and sulfuryl hydrazine through two-step nucleophilic substitution-elimination reaction, has a chemical structure as shown in formula (I), and is characterized by 1H NMR, 13C NMR and HRMS. The sulfuryl hydrazone compound has significant inhibitory activity on A549 (lung cancer), HepG2 (liver cancer) and Hela (cervical cancer) three human cancer cell strains, the value of some compounds is as low as 0.03 micromole / L, the compound can induce cell apoptosis by mediating ROS generation in tumor cells, inhibiting the NF-kappa B signal pathway and activating Caspase-3 activity, and can effectively inhibit cancer cell migration and colony formation. The compound has a mild synthesis process, simple operation and wide substrate applicability, can be used as a potential anti-tumor active ingredient, can be used for preparing drugs for resisting lung cancer, liver cancer, cervical cancer and other tumors, and can provide new candidate compounds and technical support for cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a sulfonylhydrazone compound, its preparation method, and its antitumor applications. Background Technology

[0002] Cancer is a major disease that seriously threatens human health worldwide, and chemotherapy is one of the main methods of clinical cancer treatment. However, existing chemotherapy drugs generally suffer from poor selectivity, strong side effects, and easy development of drug resistance, making it urgent to develop new anti-tumor drugs with high efficacy, low toxicity, and clear mechanisms of action.

[0003] Sulfonylhydrazones are a special class of Schiff bases containing a substructure (-SO₂NHN=C-), attracting widespread attention due to their unique chemical structure and diverse biological activities. Studies have shown that sulfonylhydrazones possess a variety of physiological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral, and antitumor effects. Furthermore, they can form complexes with metal ions with unique optical, electrical, and magnetic properties, demonstrating broad application prospects in medicine, pesticides, and materials science. In addition, the raw materials for the synthesis of sulfonylhydrazones are readily available, and the processes are flexible. Optimization of biological activities can be achieved through modification of the substrate structure, providing a favorable molecular framework for the development of novel antitumor drugs.

[0004] Currently, the synthesis methods for sulfonylhydrazones suffer from problems such as harsh reaction conditions, narrow substrate applicability, and unstable yields, and their antitumor mechanisms of action are not yet fully understood. Therefore, developing a mild and efficient synthetic process for sulfonylhydrazones and systematically exploring their antitumor activity and mechanisms of action is of great significance for the development of novel antitumor drugs. Summary of the Invention

[0005] 1. A sulfonylhydrazone compound The sulfonylhydrazone compounds provided by this invention have the chemical structure shown in formula (I):

[0006] In formula (I), R1 is selected from H, methyl, fluorine, chlorine, bromine, iodine, and methoxy; R2 is selected from p-tolyl, p-methoxyphenyl, ethyl, p-fluorophenyl, m-fluorophenyl, p-nitrophenyl, p-bromophenyl, m-bromophenyl, phenyl, 3-fluoro-4-bromophenyl, and m-trifluoromethylphenyl.

[0007] 2. Preparation methods of sulfonylhydrazone compounds The sulfonylhydrazone compounds of the present invention are prepared by the following two-step reaction: (1) Synthesis of sulfonylhydrazine: Under ice bath conditions, sulfonyl chloride derivative (10 mmol) is dissolved in 30 mL of tetrahydrofuran, and hydrazine hydrate (15 mmol) is slowly added dropwise with stirring. The reaction is maintained at 0 °C for 0.5 h, and the reaction progress is monitored by TLC. After the reaction is completed, the solvent is removed by rotary evaporation, and the residue is purified by column chromatography (eluent: petroleum ether / ethyl acetate = 10:1) to obtain a white solid sulfonylhydrazine derivative with a yield of 40%-81%. (2) Synthesis of sulfonylhydrazone: Indigo derivative (10 mmol) and sulfonylhydrazine derivative (12 mmol) are added to a reaction flask, dissolved in 15 mL of ethanol, and then 1 mmol of acetic acid is added as a catalyst. The mixture is heated to reflux and reacted for 12 h. The reaction is monitored by TLC until complete. After cooling to room temperature, the mixture was filtered, and the filter cake was washed three times with ethanol and dried to obtain an orange-yellow solid sulfonylhydrazone compound with a yield of 67%-86%.

[0008] The reaction route of the above preparation method is as follows: (reaction conditions: (i) tetrahydrofuran, ice bath, 0.5 h; (ii) ethanol, acetic acid, reflux, 12 h) 3. Applications of sulfonylhydrazones The sulfonylhydrazone compounds of this invention exhibit significant inhibitory activity against three human cancer cell lines: A549 (lung cancer cells), HepG2 (liver cancer cells), and HeLa (cervical cancer cells), with some compounds showing extremely strong targeted inhibitory effects. Among them, 5-bromoindigo ethylsulfonylhydrazone (A36) shows significant inhibitory activity against IC50 in A549 cells. 50 The value was 0.045 μmol / L, indicating that 5-methoxyindoindividu-p-fluorobenzenesulfonylhydrazone (A32) had an IC50 value of 0.045 μmol / L against HeLa cells. 50 The value was 0.034 μmol / L, indicating that 5-methoxyindoindividu-p-fluorobenzenesulfonylhydrazone (A32) had an IC50 value of 0.034 μmol / L against HepG2 cells. 50 The value was 0.349 μmol / L.

[0009] Mechanistic studies have shown that these compounds mediate the generation of reactive oxygen species (ROS) in tumor cells, increasing intracellular ROS levels, thereby inhibiting the NF-κB signaling pathway, activating Caspase-3 activity, and ultimately inducing tumor cell apoptosis. Simultaneously, scratch assays and colony formation assays confirmed that these compounds effectively inhibit cancer cell migration and proliferation, providing solid experimental evidence for their application as anti-tumor drugs. Attached Figure Description

[0010] Figure 1 Synthetic route of sulfonylhydrazones Figure 2Optimization results of reaction 1 (synthesis of sulfonyl hydrazide) Figure 3 Optimization results of reaction 2 (sulfonylhydrazone synthesis) Figure 4. Inhibitory effects of representative compounds on tumor cell migration ( Figure 4-1 Results of A549 cell scratch assay; Figure 4-2 Results of HepG2 cell scratch assay; Figure 4-3 (Results of HeLa cell scratch assay) Figure 5. Inhibitory effect of representative compounds on tumor cell colony formation ( Figure 5-1 A549 cell colony staining diagram; Figure 5-2 HepG2 cell colony staining diagram; Figure 5-3 (HeLa cell colony staining diagram) Figure 6. Fluorescence detection results of ROS generation in tumor cells induced by sulfonylhydrazone compounds ( Figure 6-1 A549 cells; Figure 6-2 HepG2 cells; Figure 6-3 HeLa cells) Figure 7 Effects of sulfonylhydrazones on Caspase-3 activity in tumor cells (A: A549 cells; B: HepG2 cells; C: HeLa cells) Detailed Implementation

[0011] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0012] Combination Figures 1 to 7 This invention provides a sulfonylhydrazone compound having the chemical structure shown in formula (I):

[0013] In formula (I), R1 is selected from H, methyl, fluorine, chlorine, bromine, iodine, and methoxy; R2 is selected from p-tolyl, p-methoxyphenyl, ethyl, p-fluorophenyl, m-fluorophenyl, p-nitrophenyl, p-bromophenyl, m-bromophenyl, phenyl, 3-fluoro-4-bromophenyl, and m-trifluoromethylphenyl.

[0014] The compound is one of the following: (1) indigo p-toluenesulfonylhydrazone; (2) 5-methylindigo p-methoxybenzenesulfonylhydrazone; (3) 5-fluoroindigo p-fluorobenzenesulfonylhydrazone; (4) 5-chloroindigo p-nitrobenzenesulfonylhydrazone; (5) 5-bromoindigo ethylsulfonylhydrazone; (6) 5-methoxyindigo m-trifluoromethylbenzenesulfonylhydrazone; (7) indigo m-bromobenzenesulfonylhydrazone; (8) 5-iodoindigo p-bromobenzenesulfonylhydrazone.

[0015] The preparation method of sulfonylhydrazone compounds includes the following steps: (1) Synthesis of sulfonyl hydrazine: under ice bath conditions, sulfonyl chloride derivative is dissolved in tetrahydrofuran, and hydrazine hydrate is added dropwise with stirring. After reacting for 0.5 h, the sulfonyl hydrazine derivative is obtained by concentration and column chromatography; (2) Synthesis of sulfonylhydrazone: indigo derivative and sulfonyl hydrazine derivative obtained in step (1) are dissolved in ethanol, acetic acid is added as a catalyst, the mixture is refluxed for 12 h, filtered, and washed with ethanol to obtain sulfonylhydrazone compounds.

[0016] In step (1), the molar ratio of sulfonyl chloride derivative to hydrazine hydrate is 1:1.5, the reaction solvent is tetrahydrofuran, and the reaction temperature is 0℃; in step (2), the molar ratio of indigo derivative to sulfonyl hydrazine derivative is 1:1.2, the amount of acetic acid catalyst added is 1 mmol, the reaction solvent is ethanol, and the reaction temperature is reflux temperature.

[0017] The application of the sulfonylhydrazone compounds in the preparation of antitumor drugs. The tumors include lung cancer, liver cancer, and cervical cancer.

[0018] The sulfonylhydrazone compounds exert their antitumor effects by mediating ROS generation in tumor cells, inhibiting the NF-κB signaling pathway, and activating Caspase-3 activity.

[0019] An antitumor drug comprising a sulfonylhydrazone compound as the active ingredient, and pharmaceutically acceptable excipients.

[0020]

[0021] Example 1: Synthesis of p-Toluenesulfonylhydrazine Under ice bath conditions, 10 mmol of p-toluenesulfonyl chloride was added to a reaction flask containing 30 mL of tetrahydrofuran. After stirring to dissolve, 15 mmol of hydrazine hydrate was slowly added dropwise, and the reaction was maintained at 0 °C for 0.5 h. After the reaction was complete as monitored by TLC, the solvent was removed by rotary evaporation, and the residue was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 10:1) to give a white solid p-toluenesulfonyl hydrazine in 77% yield. The structure of the product was confirmed by ¹H NMR.

[0022] Example 2: Synthesis of indigo-p-toluenesulfonylhydrazone Indigo (10 mmol), p-toluenesulfonylhydrazine (12 mmol), and acetic acid (1 mmol) were added to a reaction flask containing 15 mL of ethanol, and the mixture was heated to reflux for 12 h. After cooling to room temperature, the mixture was filtered, and the filter cake was washed three times with ethanol and dried to obtain an orange-yellow solid of indigo p-toluenesulfonylhydrazone, with a yield of 74%. The structure of the product was confirmed by ¹H NMR, ¹³C NMR, and HRMS.

[0023] Example 3: Synthesis of 5-bromoindigo ethylsulfonylhydrazone 5-Bromoindigo (10 mmol), ethylsulfonyl hydrazine (12 mmol), and acetic acid (1 mmol) were added to a reaction flask containing 15 mL of ethanol, and the mixture was heated to reflux for 12 h. After cooling to room temperature, the mixture was filtered, and the filter cake was washed three times with ethanol and dried to obtain an orange-yellow solid of 5-bromoindigodigo ethylsulfonyl hydrazone, with a yield of 79%. The structure of the product was confirmed by ¹H NMR, ¹³C NMR, and HRMS. This compound showed activity against IC50 in A549 cells. 50 The concentration was 0.045 μmol / L, which significantly inhibited A549 cell migration and colony formation.

[0024] Example 4: Antitumor activity test of the compound 1. MTT assay for cell viability: A549, HepG2, and HeLa cells were seeded into 96-well plates at 5 × 10³ cells per well and cultured at 37°C with 5% CO2 for 24 h. Then, different concentrations of sulfonylhydrazone compounds were added, and the cells were cultured for another 48 h. MTT solution (5 mg / mL) was added, and after incubation for 4 h, the supernatant was discarded, and formazan crystals were dissolved in DMSO. The absorbance at 490 nm was measured using a microplate reader, and the cell inhibition rate and IC50 were calculated. 50 Values ​​(see Table 2 for results). The results show that sulfonylhydrazone compounds with different substituents have varying degrees of inhibitory activity against the three types of tumor cells, and some compounds exhibit extremely strong targeting.

[0025] 2. Scratch assay for cell migration: A549, HepG2, and HeLa cells were seeded in 6-well plates and cultured to 90% confluence. The cells were then scratched with the tip of a 200 μL pipette, washed three times with PBS, and cultured with medium containing different concentrations of the compound. Images of the scratched areas were captured at 0 h and 48 h. Results showed that after 48 h of treatment with 5-bromoindigo ethylsulfonylhydrazone (20 μmol / L), the scratch healing rate of A549 cells was significantly lower than that of the control group (DMSO group) (see results below). Figure 4-1 A26 and A32 significantly inhibited the migration of HepG2 cells (see results). Figure 4-2 A17, A21, and A32 significantly inhibited HeLa cell migration (see results). Figure 4-3 ).

[0026] 3. Colony formation assay to detect cell proliferation: A549, HepG2, and HeLa cells were seeded in 6-well plates at a density of 1.25 × 10⁶ cells per well. 4 Cells were cultured for 24 h, and then different concentrations of the compound were added. The cells were cultured for 6 days (the medium was changed every 3 days). The medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, stained with crystal violet for 15 min, and the number of colonies was counted after washing. The results showed that 5-bromoindigo ethylsulfonylhydrazone effectively inhibited A549 cell colony formation (see results below). Figure 5-1 A26 and A32 significantly inhibited HepG2 cell colony formation, with A26 showing a better inhibitory effect than A32 (see results). Figure 5-2 A17, A21, and A32 significantly reduced the number of HeLa cell colonies, with A17 showing the best inhibitory effect (see results). Figure 5-3 ).

[0027] 4. ROS detection: Cells were seeded in 12-well plates and cultured for 24 h. Afterward, the compound was added for another 24 h, followed by the addition of the DCFH-DA probe (1:1000 dilution). The cells were incubated at 37°C for 20 min, washed three times with serum-free medium, and observed under a fluorescence microscope (results are shown in [link to results]). Figure 6-1 , Figure 6-2 , Figure 6-3 The results showed that the intracellular fluorescence intensity in the compound-treated group was significantly higher than that in the control group, indicating that it could induce ROS generation in tumor cells; after the addition of the ROS inhibitor NAC, the fluorescence intensity was significantly reduced, confirming that ROS generation is an important pathway for the compound to exert its anti-tumor effect.

[0028] 5. Caspase-3 Activity Assay: Cells were seeded in 6-well plates and cultured for 24 h. Afterward, the compound was added and treated for another 24 h. Cells were then collected, lysis buffer was added, and the cells were incubated on ice for 15 min. After centrifugation, the supernatant was collected, and the absorbance was measured at 405 nm using a microplate reader according to the instructions of the Caspase-3 assay kit (results are shown in Figure 7). The results showed that the Caspase-3 activity in the compound-treated group was significantly higher than that in the control group, indicating that it could activate the Caspase-3 signaling pathway and induce apoptosis. The addition of NAC decreased Caspase-3 activity, further confirming that the compound exerts its antitumor effect through ROS-mediated NF-κB signaling pathway inhibition.

[0029]

[0030]

[0031]

[0032]

[0033]

[0034] Invention Effects This invention provides a series of novel sulfonylhydrazone compounds with mild, simple synthetic processes, broad substrate applicability, and high product yields and purity. These compounds exhibit significant inhibitory activity against various tumor cell types, with a clearly defined mechanism of action. They exert their antitumor effects by inducing ROS generation, inhibiting the NF-κB signaling pathway, and activating Caspase-3 activity, while also effectively inhibiting cancer cell migration and proliferation. This invention provides new candidate compounds and technical solutions for the development of novel antitumor drugs, possessing significant pharmaceutical value and application prospects.

Claims

1. A sulfonylhydrazone compound, characterized in that, It has the chemical structure shown in formula (I): In formula (I), R1 is selected from H, methyl, fluorine, chlorine, bromine, iodine, and methoxy; R2 is selected from p-tolyl, p-methoxyphenyl, ethyl, p-fluorophenyl, m-fluorophenyl, p-nitrophenyl, p-bromophenyl, m-bromophenyl, phenyl, 3-fluoro-4-bromophenyl, and m-trifluoromethylphenyl.

2. The sulfonylhydrazone compound according to claim 1, characterized in that, The compound is one of the following: (1) indigo p-toluenesulfonylhydrazone; (2) 5-methylindigo p-methoxybenzenesulfonylhydrazone; (3) 5-fluoroindigo p-fluorobenzenesulfonylhydrazone; (4) 5-chloroindigo p-nitrobenzenesulfonylhydrazone; (5) 5-bromoindigo ethylsulfonylhydrazone; (6) 5-methoxyindigo m-trifluoromethylbenzenesulfonylhydrazone; (7) indigo m-bromobenzenesulfonylhydrazone; (8) 5-iodoindigo p-bromobenzenesulfonylhydrazone.

3. The method for preparing the sulfonylhydrazone compound according to claim 1 or 2, characterized in that, Includes the following steps: (1) Synthesis of sulfonyl hydrazine: Under ice bath conditions, sulfonyl chloride derivatives were dissolved in tetrahydrofuran, and hydrazine hydrate was added dropwise with stirring. After reacting for 0.5 h, the sulfonyl hydrazine derivatives were obtained by concentration and column chromatography. (2) Synthesis of sulfonylhydrazone: Indigo derivatives and sulfonyl hydrazine derivatives obtained in step (1) were dissolved in ethanol, acetic acid was added as a catalyst, and the reaction was refluxed for 12 h. After filtration and washing with ethanol, sulfonylhydrazone compounds were obtained.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of sulfonyl chloride derivative to hydrazine hydrate is 1:1.5, the reaction solvent is tetrahydrofuran, and the reaction temperature is 0℃; in step (2), the molar ratio of indigo derivative to sulfonyl hydrazine derivative is 1:1.2, the amount of acetic acid catalyst added is 1 mmol, the reaction solvent is ethanol, and the reaction temperature is reflux temperature.

5. The use of the sulfonylhydrazone compound of claim 1 or 2 in the preparation of antitumor drugs.

6. The application according to claim 5, characterized in that, The tumors include lung cancer, liver cancer, and cervical cancer.

7. The application according to claim 5, characterized in that, The sulfonylhydrazone compounds exert their antitumor effects by mediating ROS generation in tumor cells, inhibiting the NF-κB signaling pathway, and activating Caspase-3 activity.

8. An antitumor drug, characterized in that, It contains the sulfonylhydrazone compound as described in claim 1 or 2 as the active ingredient, and pharmaceutically acceptable excipients.

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