Thiosemicarbazone compound containing naphthyl structure as well as preparation method and application of thiosemicarbazone compound

Abstract

The invention discloses a thiosemicarbazone compound containing a naphthyl structure as well as a preparation method and application of the thiosemicarbazone compound. The thiosemicarbazone compound has biological activity on human breast cancer cells. The anti-tumor activity of the compound is verified through in-vitro experiments, including an inhibition effect experiment of the compound on proliferation of human breast cancer cells, an influence experiment on oxidative stress level of the breast cancer cells, an influence experiment on mitochondrial membrane potential of the breast cancer cells and an experiment for inducing apoptosis of the breast cancer cells. The thiosemicarbazone compound containing the naphthyl structure has remarkable anti-tumor activity, can effectively inhibit proliferation of human breast cancer cells, can remarkably improve the oxidative stress level of the breast cancer cells in a concentration-dependent mode, and has a close correlation between the regulation effect on the mitochondrial membrane potential of the breast cancer cells and the treatment concentration; the compound can effectively induce apoptosis of breast cancer cells, has excellent anti-breast cancer activity, and provides important candidate compounds and theoretical basis for research and development of novel anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a naphthiourea compound containing a naphthyl structure, its preparation method, and its application in inhibiting the activity of breast cancer tumor cells. Background Technology

[0002] Breast cancer has become the most common malignant tumor among women worldwide, with its incidence showing significant regional differences and age correlation. According to the latest data from the International Agency for Research on Cancer (IARC) in 2025, more than 2.8 million new cases of breast cancer are diagnosed globally each year, accounting for 31.5% of new cancer cases in women, with a mortality rate of 15.2%, ranking first among cancer deaths in women. A 2025 report from the National Cancer Center of China shows that there are approximately 487,000 new cases of breast cancer and 132,000 deaths annually in my country, accounting for 17.4% of global cases. Furthermore, the incidence rate is increasing at a rate of 3.5% per year, showing a clear trend towards younger patients, with patients under 40 years old accounting for 21.3%. Therefore, developing effective drugs for the treatment of breast cancer has become a key research focus.

[0003] Thiourea is a widely used pharmaceutical intermediate. Its amino group condenses with the carbonyl group of an aldehyde or ketone to form thiourea. The hydrogen atom on the nitrogen atom in the molecule can be replaced by an alkyl or aryl group, thus forming a series of thiourea reagents with different properties. Furthermore, due to the presence of heteroatoms such as N and S in its structure, it possesses various biological activities, including antibacterial, antifungal, antitumor, antiparasitic, and cytotoxic activities. We successfully synthesized a thiourea compound (I) and characterized its structure using NMR and mass spectrometry. In vitro cytotoxicity experiments showed that this compound inhibited the proliferation of MCF-7 and JIMT-1 breast cancer cells by 17.97% and 26.77%, respectively. In addition, compound (I) significantly enhanced the antitumor efficacy, and its mechanism of action involves the accumulation of excess reactive oxygen species (ROS), dissipation of mitochondrial membrane potential (ΔΨm), and activation of intrinsic apoptosis pathways.

[0004] This invention relates to the application of a naphthiourea compound (I) containing a naphthyl structure in the pharmaceutical field. There are no prior art reports on the application of this compound or its pharmaceutical composition in the preparation or treatment of diseases such as tumors caused by various factors. Summary of the Invention

[0005] The purpose of this invention is to provide pharmaceutical uses for a thiourea compound (I) containing a naphthyl structure, specifically relating to its use in the preparation of antitumor drugs; The thiourea compound (I) containing a naphthyl structure used in this invention was chemically synthesized in our laboratory.

[0006] This invention investigated the inhibitory effect of a naphthiourea compound (I) containing a naphthyl group on the proliferation of human breast cancer cells, its influence on oxidative stress levels in breast cancer cells, its effect on mitochondrial membrane potential, and its induction of apoptosis in breast cancer cells. The results showed that compound (I) significantly inhibited breast cancer cell proliferation, significantly enhanced oxidative stress levels in a concentration-dependent manner, its effect on mitochondrial membrane potential in breast cancer cells was closely related to its treatment concentration, and it induced apoptosis in breast cancer cells, demonstrating antitumor activity.

[0007] The present invention has the following advantages: The antitumor activity experiments of this invention have demonstrated that the compound can significantly inhibit the proliferation of breast cancer cells, induce apoptosis of breast cancer cells, and affect changes in mitochondrial membrane potential, thus exhibiting significant antitumor activity. Attached Figure Description

[0008] Figure 1 The chemical structure of thiourea compound (I) is shown. Figure 2 The inhibitory activity of compound (I) against breast cancer cells; Figure 3 This study investigated the effects of compound (I) on the oxidative stress levels of breast cancer cells MCF-7(a) and JIMT-1(b). Figure 4 Experiments on the effect of compound (I) on the mitochondrial membrane potential of breast cancer cells MCF-7(e) and JIMT-1(f); Figure 5 Experiments to induce apoptosis in breast cancer cells MCF-7(e) and JIMT-1(f) by compound (I). Detailed Implementation Plan

[0009] The present invention will now be analyzed in more detail with reference to the accompanying drawings and embodiments. Example 1

[0010] Experimental study on the inhibitory effect of thiourea compound (I) on the proliferation of MCF-7, JIMT-1, and MDA-MB-231 human breast cancer cells.

[0011] 1. Experimental reagents Thioaminourea (5 mM) and 2-hydroxy-1-naphthaldehyde (10 mM) were dissolved in ethanol (20 mL) in a round-bottom flask and refluxed at 80°C for 4 hours. After removing the solvent by vacuum distillation, the crude product was recrystallized from methanol to obtain thiourea compound (I). 1HNMR (400MHz, DMSO) δ12.10(s,2H),7.94(d,J=12.0Hz,2H),7.89(d,J=8.0Hz,2H),7.62(t,J=4.0Hz,2H),7.41(t,J=8.0Hz,2H),7.24(d,J=8.0Hz,2H). 13 CNMR(101MHz,DMSO)δ174.3,133.1,132.1,129.2,128.3,124.0.MS(ESI):m / z415.19[M+H] + 2. Cell lines MCF-7, JIMT-1, and MDA-MB-231 breast cancer cell lines were purchased from Wuhan Pronosei Biotechnology Co., Ltd. Cells were cultured in DMEM medium containing 10% bovine serum peptide at 37°C, 5% CO2, and under fully saturated humidity conditions in air. The medium was changed after 48 hours. When the cells reached saturation, they were passaged using 0.25% trypsin. Subculture was performed every 2-3 days. Cells in the logarithmic growth phase were used in the experiments.

[0012] 3. Cell viability detection The effect of thiourea compound (I) on the proliferation activity of MCF-7, JIMT-1, and MDA-MB-231 cells was detected using the tetrazolium salt WST-8 (CCK-8) assay. MCF-7, JIMT-1, and MDA-MB-231 cells in the logarithmic growth phase were harvested, trypsinized, and resuspended. At initial cell seeding, 1×10⁶ cells were added to the culture medium. 4 Cells were seeded in each well of a 96-well plate (n = 5) and subsequently exposed to different concentrations of the compound (0, 1, 5, and 50 µM). After 24 hours of incubation, the culture medium was aspirated, and the cell monolayer was washed twice with 100 μL of serum-free medium. Next, CCK-8 reagent (5 mg / mL) was diluted with culture medium to a final concentration of 0.5 mg / mL, added to each well, and incubated at 37°C for another 4 hours. Absorbance was measured at 450 nm using a Spectramax M2 microplate reader (Molecular Devices, USA).

[0013] The results showed that after 24 hours of culture, the half-maximal inhibitory concentrations (IC50) for MCF-7, JIMT-1, and MDA-MB-231 cells were 1.20±0.23 μM, 2.17±0.49 μM, and 47.87±29.13 μM, respectively. Cell proliferation inhibition activity is shown below. Figure 2 . Example 2

[0014] Experiment on the effect of thiourea compound (I) on oxidative stress levels in breast cancer cells MCF-7 and JIMT-1.

[0015] 1. Experimental reagents Same as Example 1.

[0016] 2. Cell lines MCF-7 and JIMT-1 breast cancer cell lines were purchased from Wuhan Pronosei Biotechnology Co., Ltd. They were cultured in DMEM medium containing 10% bovine serum peptide at 37°C, 5% CO2, and under fully saturated humidity conditions in air. The medium was changed after 48 hours. When the cells reached saturation, they were passaged using 0.25% trypsin. Subculture was performed every 2-3 days. Cells in the logarithmic growth phase were used in the experiment.

[0017] 3. Experimental Methods MCF-7 and JIMT-1 cells in the logarithmic growth phase were harvested, digested with trypsin, and resuspended. At initial cell seeding, MCF-7 or JIMT-1 cells were seeded at a density of 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density in 6-well plates and treated with different concentrations (0, 0.25, 0.5, 0.75, 1 µM) of compound (I) for 24 hours. After treatment, cells were collected, and reactive oxygen species (ROS) were detected using a DCF-DA assay kit (Beyotime Biotechnology, Shanghai). Flow cytometry data were analyzed using FlowJo 7.6 software (BD Biosciences, USA).

[0018] The results showed that quantitative analysis using the DCFH-DA assay revealed that compound (I) significantly enhanced oxidative stress levels in a concentration-dependent manner. Figure 3 ab). When compound (I) was used in combination with the apoptosis inhibitor z-VAD-FMK, the ROS accumulation in the co-treated cells continued to increase and was not significantly reduced. Example 3

[0019] Experiment on the effect of thiourea compound (I) on mitochondrial membrane potential of breast cancer cells.

[0020] 1. Experimental reagents Same as Example 1.

[0021] 2. Cell lines Same as Example 2.

[0022] 3. Experimental Methods MCF-7 and JIMT-1 cells in the logarithmic growth phase were harvested, digested with trypsin, and resuspended. At initial cell seeding, MCF-7 or JIMT-1 cells were seeded at a density of 2.5 × 10⁶ cells per well. 5Cells were seeded at a density of [insert density here] in 6-well plates and treated with different concentrations (0, 0.25, 0.5, 0.75, 1 µM) of compound (I) for 24 hours. After treatment, cells were collected, and mitochondrial membrane potential was measured using the JC-1 assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). Flow cytometry data were analyzed using FlowJo 7.6 software (BD Biosciences, Inc., USA). Changes in mitochondrial membrane potential are shown below. Figure 4 .

[0023] The results showed that the mitochondrial membrane potential of the control group cells remained stable, and JC-1 aggregated in the mitochondrial matrix in polymer form, exhibiting red fluorescence. However, when the concentration of compound (I) was gradually increased from 0.25 μM to 1 μM, after 24 hours of treatment, the proportion of JC-1 monomers in the cells significantly increased, and the intensity of green fluorescence also gradually increased. Figure 4 In MCF-7 cells, quantitative analysis showed that compared with the control group, the red / green fluorescence intensity ratios of the 0.25 μM, 0.5 μM, 0.75 μM, and 1 μM treatment groups decreased to 11.77 ± 1.42, 11.23 ± 1.18, 10.5 ± 1.23, and 8.17 ± 1.42, respectively (P < 0.05). Figure 4 The concentration-dependent decrease in mitochondrial membrane potential (ef) was observed. Furthermore, compared to the group treated with 0.5 μM compound (I) alone, the addition of z-VAD-FMK significantly reversed the downward trend in mitochondrial membrane potential. These results indicate that the effect of compound (I) on mitochondrial membrane potential is closely related to its treatment concentration. Example 4

[0024] An experiment on the effect of thiourea compound (I) on apoptosis in breast cancer cells.

[0025] 1. Experimental reagents Same as Example 1.

[0026] 2. Cell lines Same as Example 2.

[0027] 3. Experimental Methods MCF-7 and JIMT-1 cells in the logarithmic growth phase were harvested, digested with trypsin, and resuspended. At initial cell seeding, MCF-7 or JIMT-1 cells were seeded at a density of 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density in 6-well plates and treated with 0–1 µM of compound (I). After 24 hours of treatment, cells were collected, stained with 7-AAD and Annexin V (Beyotime Biotechnology, China), and analyzed by flow cytometry. Final data were processed using FlowJo 7.6 analysis software (BD Biosciences, USA).

[0028] The results showed that compound (I) significantly induced breast cancer cell death. Induction of breast cancer cell apoptosis is shown in [see figure]. Figure 5 ef.

Claims

1. A thiourea compound containing a naphthyl structure, its preparation method, and its antitumor application, wherein the compound (I) has the following chemical structure: 。 2. The antitumor use according to claim 1, characterized in that, The compound (I) is used to prevent or treat breast cancer, wherein the tumor cell line corresponding to the breast cancer includes one or more of MCF-7, JIMT-1, and MDA-MB-231.

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