Introduction

Cervical cancer, a malignant tumor originating in the cervix, represents the most prevalent gynecological malignancy within the female reproductive system. According to 2022 global cancer statistics, cervical cancer ranks fourth worldwide in both incidence and mortality among female malignancies1. Current therapeutic strategies for cervical cancer encompass surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, with chemotherapy serving as the cornerstone of treatment2. The efficacy of cisplatin-based first-line chemotherapy is suboptimal, with response rates of only 13% and 36% for cisplatin alone or in combination3,4. Cisplatin also has disadvantages such as ototoxicity, gastrointestinal toxicity, hepatotoxicity, nephrotoxicity, neurotoxicity, and drug resistance5,6. Therefore, these clinical challenges underscore the urgent need for novel therapeutic agents to enhance therapeutic outcomes in cervical cancer management.

The primary etiological factor for cervical cancer is the persistent infection with high-risk human papillomavirus (HPV). There is a significant correlation between HPV infection and the upregulation of the epidermal growth factor receptor (EGFR) signaling cascade7. Marlies Schrevel et al. analyzed 32 cases of cervical cancer and found that 29 of them had overexpression of EGFR in the cervical cancer tissues8. Therefore, EGFR inhibitors are potentially active against cervical cancer. Erlotinib, the first-generation oral EGFR tyrosine kinase inhibitor (TKI), has been approved by the FDA for use in non-small cell lung cancer patients with EGFR-sensitive mutations9. Studies have shown that EGFR-TKIs are also effective against cervical cancer. Yaping Lv et al. found that erlotinib inhibited the enrichment of cancer stem cells in paclitaxel-resistant cervical cancer cells and prevented the recurrence of cervical cancer10. Angelica Nogueira-Rodrigues et al. conducted a phase II clinical trial in patients with locally advanced cervical cancer and found that combining Erlotinib with cisplatin-based chemoradiotherapy (CRT) significantly enhanced treatment efficacy and improved patient tolerance11.

The 1,2,3-triazole moiety is an important nitrogen-containing heterocyclic group that can be used as a bioisostere for various structural modifications in medicinal chemistry12. The 1,2,3-triazole group is also frequently employed in the development of small molecule drugs with anti-cervical cancer activity13,14,15,16. Satheeshvarma Vanaparthi et al. synthesized a series of 1,2,3-triazole-tethered chalcone acetamide derivatives and evaluated their antitumor activity against human cervical cancer (HeLa cells), human alveolar adenocarcinoma (A549 cells), and human breast adenocarcinoma (MCF-7 cells) cells. Among them, compound 7c exhibited the best anti-proliferative activity against HeLa cells, with a half-maximal inhibitory concentration (IC50) value of 7.41 ± 0.8 µM13. Peng Deng et al. synthesized a series of erlotinib derivatives linked with 1,2,3-triazole, among which compound 4m had the strongest activity against HeLa cells (IC50 = 3.79 µM)14. In another study, Siva Kumar Gandham et al. synthesized a series of new tetrazolopyrrolidine-1,2,3-triazole analogues. Compounds 7a and 7i showed significant antiproliferative activity against HeLa cervical cancer cells, with IC50 values of 0.32 ± 1.00 µM and 1.80 ± 0.22 µM, respectively15. Murali Krishna Vanga et al. synthesized a series of hybrids of Meldrum’s acid, 7-azaindole, and 1,2,3-triazole. After evaluating the antiproliferative activity of the derivatives in five types of tumor cells, it was found that compound 6b exhibited significant anticancer activity against HeLa cells, with an IC50 value of 4.44 ± 0.32 µM16.

Due to the harmfulness of cervical cancer and its current treatment status, as well as the antitumor potential of erlotinib and the 1,2,3-triazole moiety, we used erlotinib as the core structure and employed click chemistry, a highly modular molecular assembly reaction17, to synthesize a series of erlotinib-1,2,3-triazole derivatives. The anti-cervical cancer activity of these compounds was screened using the Methylthiazolyldiphenyl-tetrazolium bromide assay (MTT assay), and the most potent compound was selected for further evaluation of its anti-cervical cancer activity, along with an exploration of its underlying mechanisms.

Results

Synthesis of erlotinib-1,2,3-triazole derivatives

With erlotinib as the core structure, 1,2,3-triazole moieties with various substituents were linked to the erlotinib scaffold via click reactions, yielding the compounds 3a-3h and 5a-5h. 4-chloro-6,7-di(ethoxymethoxy)quinazoline (compound 1) was reacted with 3-aminophenylacetylene or propargylamine to generate terminal alkyne compounds 2 or 4. Subsequently, compound 2 was reacted with azide compounds containing various substituents to synthesize 8 new target compounds, namely 3a-3h. Using the same method, compound 4 was reacted with azide compounds to synthesize 8 new compounds 5a-5h. All compounds were purified by column chromatography and extensively characterized by spectroscopic techniques, including 1H Nuclear Magnetic Resonance (NMR), 13C NMR, and high-resolution mass spectrometry (HRMS). Detailed information for the 1H NMR, 13C NMR, and HRMS spectra could be found in the supplementary files.

Scheme 1
scheme 1

Synthesis of 1,2,3-triazole hybrids of erlotinib derivatives.

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Anti-cervical cancer activity screening of erlotinib-1,2,3-triazole derivatives

MTT assays were performed to measure the cell viability of cervical cancer HeLa cells treated with candidate compounds. After culturing for 24 h, the cells were treated with the compounds at a final concentration of 10 µM or with vehicle control (normal control group) for 48 h. Subsequently, 10 µL of MTT solution (5 mg/mL) was added to each well and incubated for 3 h. The absorbance was measured at 490 nm to calculate cell viability for each group. The cell viability of the normal control group was normalized to 100%, and the inhibitory effects of the structural derivatives on HeLa cells are shown in Table 1. After treatment with the positive control doxorubicin (10 µM), the cell viability was 11.95 ± 1.20%, while treatment with erlotinib (10 µM) resulted in a cell viability of 53.02 ± 0.51%. Among the 10 µM of candidate compounds, the 3a-3h series exhibited better anticancer activity compared to the 5a-5h series, with compound 3h showing the lowest cell viability at 10.44 ± 0.80%. Furthermore, the IC50 values of the structural derivatives were determined. Based on the effects of different concentrations (0, 1, 5, 10, 20, 40 µM) on HeLa cell viability, the IC50 values for the compounds were calculated, as shown in Table 1. The IC50 value of compound 3h was 1.35 ± 0.74 µM, while that of erlotinib was 25.91 ± 1.35 µM. Furthermore, the effects of the compounds on the cell viability of NIH3T3 cells (mouse embryonic fibroblasts) were assessed to evaluate their cytotoxic effects on normal cells (Table S1). As shown in Table S1, there was no obvious toxicity of compound 3h (10 µM) on NIH3T3 cells, with a cell viability of 90.41 ± 1.36%. Therefore, compound 3h was identified as the most promising active compound for the treatment of cervical cancer, and subsequent experiments will focus on compound 3h.

Table 1 Inhibitory effect of erlotinib derivatives on Hela cells.
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Inhibitory effect of compound 3h on HeLa cell proliferation

To investigate the effect of compound 3h on the colony formation of HeLa cells, the colony formation assay was conducted. The number and size of cell colonies were observed and quantitatively analyzed after crystal violet staining. As shown in Fig. 1A-B, the CON group exhibited a high number of colonies with large cell clusters, whereas the colony formation ability of cells treated with compound 3h was inhibited to varying degrees. With increasing treatment concentration of compound 3h, a significant reduction was shown in the number of colonies and colony size, with the colony formation rate reduced to 52.15 ± 10.45% of the control group (P < 0.001) after being treated with compound 3h (10 µM). These results indicate that compound 3h could significantly inhibit the colony formation of HeLa cells.

To further evaluate the anti-proliferative effect of compound 3h against HeLa cells, a live/dead cell staining assay was used. HeLa cells were treated with compound 3h at concentrations of 5 µM and 10 µM for 48 h, followed by live and dead cells stained with calcein-AM and Propidium Iodide (PI), respectively. Then, the cells were imaged and counted. With increasing concentration of compound 3h, the number of live cells gradually decreased, while the number of dead cells increased (Fig. 1C-E).

Fig. 1
figure 1

Inhibitory effect of compound 3h on the proliferation of HeLa cells. A-B. The effect of the compound 3h (5 µM and 10 µM) on colony formation and quantitative analysis of Hela cells. C-E. Fluorescence images and quantitative analysis of Hela cells stained with PI and calcein-AM after compound 3h treatment (5 µM and 10 µM) for 48 h (n = 3, *P<0.05, **P<0.01, ***P<0.001 compared with the CON group).

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Compound 3h inhibits the migration of HeLa cells

To assess the impact of compound 3h on the migration of HeLa cells, a wound healing assay was employed. As shown in Fig. 2, the migration rate of cells in the CON group was 36.03 ± 3.44% after 48 h. However, with increasing concentrations of compound 3h, the migration rate of HeLa cells significantly decreased. Specifically, after treatment with 10 µM of compound 3h, the migration rate of HeLa cells was reduced to 20.75 ± 2.58%, which was significantly lower than that of the normal control group (P < 0.001). These results indicate that compound 3h has a pronounced inhibitory effect on the migration of HeLa cells.

Fig. 2
figure 2

The inhibitory effects of compound 3h on the migration of HeLa cells. (A) Wound healing assay. Images were captured by microscope at 0 h, 24 h, and 48 h after treatment with compound 3h. (B) Quantitative analysis of migration distance. (n = 5, ***P < 0.001 compared with the CON group)

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Cell cycle arrest induced by compound 3h in HeLa cells

To explore the effect of compound 3h on the cell cycle of HeLa cells, flow cytometry was used to analyze the distribution of cells in different phases of the cell cycle. After treatment with compound 3h, the proportion of cells in the G1 phase gradually decreased, while the proportion of cells in the G2/M phase significantly accumulated, increasing from 11.88 ± 1.01% (CON group) to 19.21 ± 1.94% (10 µM group) (Fig. 3A-D), indicating that compound 3h induced G2/M phase arrest in HeLa cells.

Fig. 3
figure 3

Effect of compound 3h on the cell cycle of HeLa cells. A-C. Cell cycle distribution detected by flow cytometry after 24 h of treatment with compound 3h; D. Quantitative analysis results. (n = 3, *P < 0.05 compared with the CON group)

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Compound 3h induced apoptosis in HeLa cells

To further investigate the role of compound 3h in inducing apoptosis in HeLa cells, Annexin V FITC/PI double staining was performed after HeLa cells were treated with different concentrations of compound 3h for 48 h and the proportion of apoptotic cells was detected by flow cytometry. As shown in Fig. 4A-B, after 48 h of treatment with 10 µM of compound 3h, the total apoptosis rate of HeLa cells was 37.63 ± 9.92%. Compared with the normal control group, the apoptosis rate gradually increased with the higher concentration of compound 3h (P < 0.05). Subsequently, 4’,6-diamidino-2-phenylindole (DAPI) staining was used to observe the apoptosis of cells under a fluorescence microscope. After 48 h of treatment with compound 3h (5 and 10 µM), cell shrinkage, intensified nuclear staining, and even apoptotic features such as nuclear condensation and fragmentation were observed (Fig. 4C), suggesting that compound 3h significantly promoted apoptosis in HeLa cells.

To further explore the mechanism by which compound 3h induces apoptosis in HeLa cells, Western blot analysis was used to examine the expression of apoptosis-related proteins B-cell lymphoma-2 (Bcl-2)/ BCL2-associated X protein (Bax) and the activation of apoptotic signaling pathways (Caspase-3, Caspase-9, and poly(ADP-ribose) polymerase 1 (PARP-1)) after treatment with compound 3h for 48 h. As shown in Fig. 4D-J, compared with the CON group, with increasing concentrations of compound 3h, the expression of the anti-apoptotic protein Bcl-2 gradually decreased, while the expression of the pro-apoptotic protein Bax gradually increased. Also, the ratio of Bax to Bcl-2 also significantly increased after treatment with 10 µM of compound 3h (Fig. 4D). Further detection showed that the expression levels of mitochondrial apoptosis pathway proteins Caspase-3, Caspase-9, and PARP-1 decreased with increasing concentrations of compound 3h, while the expression of Cleaved PARP-1 increased (Fig. 4D). The above results indicate that compound 3h significantly induces apoptosis in HeLa cells, and the activation of the mitochondrial apoptosis pathway is involved in the apoptosis induced by compound 3h.

Fig. 4
figure 4

Compound 3h induced apoptosis of HeLa cells. A-B. Detection of apoptosis levels in HeLa cells and quantitative analysis by flow cytometry; C. Detection of apoptotic morphological changes by DAPI staining. D-J. Detection and quantitative analysis of the expression of key proteins in the mitochondrial apoptosis signaling pathway by Western blot. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the CON group)

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Compound 3h promotes ROS generation in HeLa cells

Reactive Oxygen Species (ROS) is a key factor in inducing DNA damage and apoptosis18. Therefore, the generation of ROS in HeLa cells under the action of compound 3h was detected by flow cytometry. As shown in Fig. 5, the fluorescence intensity reflects the content of ROS. Compound 3h dose-dependently increased the intracellular ROS level, with the fluorescence intensity in cells treated with 10 µM of compound 3h being 277.0 ± 16.10% of that of the control group (P < 0.001), indicating that compound 3h significantly induced accumulation of ROS in HeLa cells.

Fig. 5
figure 5

Compound 3h increased the intracellular ROS levels in HeLa cells. A: Detection of ROS levels in HeLa cells by flow cytometry; B. Quantitative analysis of mean fluorescence intensity. (n = 3, **P < 0.01, ***P < 0.001 compared with the CON group)

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Compound 3h triggered DNA damage in HeLa cells

γ-H2AX (H2A histone family member X) is a highly specific and sensitive molecular marker of DNA damage19. To investigate whether the anti-cancer effect of compound 3h on HeLa cells is related to the induction of DNA damage, immunofluorescence staining and Western blot were performed to examine the effect of compound 3h on the expression of γ-H2AX. The results of immunofluorescence staining showed that after treatment with compound 3h, the fluorescence intensity of γ-H2AX in the nuclei of HeLa cells increased in a concentration-dependent manner (Fig. 6A-B). Western blot analysis confirmed the above findings, showing that compound 3h significantly upregulated γ-H2AX expression in the HeLa cell nucleus (Fig. 6C). Taken together, compound 3h induced DNA damage in HeLa cells.

Fig. 6
figure 6

Compound 3h induces DNA damage in HeLa cells. A-B. Detection of γ-H2AX expression in HeLa cells induced by compound 3h using immunofluorescence staining; C. Detection of γ-H2AX expression in nuclear proteins of HeLa cells by Western blot. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the CON group)

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Compound 3h modulated the MAPK signaling pathway

Mitogen-activated protein kinase (MAPK) pathways, including extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 are involved in various cellular activities related to cancer development, such as proliferation, differentiation, apoptosis, inflammation, and immunity20. However, different kinase subtypes exert distinct biological functions. The sustained activation of ERK and JNK promotes cell proliferation while suppressing the expression of anti-apoptotic proteins21,22. In contrast, p38 is generally recognized for its tumor-suppressive role. The tumor-inhibitory function of p38α is primarily mediated through negative regulation of cell cycle progression and induction of apoptosis23. Therefore, we further evaluated the effect of compound 3h on the MAPK signaling pathway. The phosphorylated and total protein levels of ERK1/2, JNK, and p38 in HeLa cells were detected by Western blot. Compound 3h dose-dependently inhibited the levels of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated JNK (p-JNK), while upregulating the phosphorylation of p38. Meanwhile, compound 3h had no effect on the total protein levels of ERK1/2, JNK, and p38 (Fig. 7). The results indicated that compound 3h regulated MAPK signaling pathways.

Fig. 7
figure 7

Compound 3h modulated the MAPK signaling pathway. A. detection of the expression of p-ERK/ERK, p-JNK/JNK, and p-p38/p38 in HeLa cells after treatment with compound 3h by Western blot assay; B-G. Quantitative analysis of blots. (n = 3, *P < 0.05, **P < 0.01 compared with the CON group)

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Identification of the binding modes of compound 3h with ERK2, JNK-1, and p38α

To further investigate whether compound 3h exerts its regulatory effects through direct targeting of MAPKs, molecular docking studies were performed using the crystal structures of ERK2, JNK-1, and p38α proteins. First, the binding free energies of compounds 3h with ERK2, JNK-1, and p38α were calculated using the Prime MM-GBSA approach, a reliable and widely used method for estimating protein-ligand interactions. The results demonstrated strong binding affinities, with calculated values of -50.17 kcal/mol (ERK2), -104.88 kcal/mol (JNK-1), and − 66.5 kcal/mol (p38α). For all three complexes, van der Waals (vdW) interactions and hydrophobic effects (Lipophilic) were identified as the dominant stabilizing contributions (Table 2).

Table 2 The relative binding-free energies (kcal/mol) obtained by prime MM-GBSA.
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Moreover, the 2D and 3D molecular interaction models (Fig. 8) suggest that compound 3h exhibits robust binding affinity to all three proteins, primarily driven by significant hydrophobic interactions within the binding pockets. At the same time, compound 3h forms two hydrogen bonds with residues Tyr34 and Lys52 in ERK2. In JNK-1, compound 3h establishes two hydrogen bonds with residues Thr178 and Gln253, along with a π-π stacking interaction with Tyr259. Additionally, in p38α, compound 3h engages in a hydrogen bond with residue Arg67 and a π-π stacking interaction with residue Phe169. These extensive interactions contribute to the stable binding of compound 3h within the active sites of the respective proteins.

Fig. 8
figure 8

Molecular interaction models of compound 3h with ERK2, JNK-1, and p38α proteins. A-C. The three-dimensional binding modes of compound 3h with ERK2, JNK-1, and p38α, respectively. Compound 3h is represented as yellow sticks, while key interacting residues are depicted as sticks, colored according to their respective protein structures. Hydrogen bonds are indicated by red dashed lines, and π-π stacking interactions are highlighted with cyan dashed lines. D-F. Two-dimensional interaction diagrams illustrating the detailed binding interactions of compound 3h with ERK2, JNK-1, and p38α proteins, respectively.

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Conclusions

In this study, a total of 15 novel erlotinib-1,2,3-triazole derivatives were synthesized through click chemistry reactions. After screening for anti-proliferative activity against HeLa cells, compound 3h was found to have the most potent antitumor activity, with an IC50 value of 1.35 ± 0.74 µM. Further investigations revealed that compound 3h significantly inhibited colony formation, reduced the number of live cells, suppressed cell migration, and caused cell cycle arrest in the G2/M phase in HeLa cells. Mechanistic studies demonstrated that compound 3h was found to induce apoptosis through the mitochondrial pathway by increasing the Bax/Bcl-2 ratio, decreasing Caspase-9, Caspase-3, and full-length PARP levels, and increasing cleaved PARP levels. Additionally, compound 3h markedly elevated intracellular ROS levels, induced DNA damage, and regulated the phosphorylation levels of ERK, JNK, and p38 by binding to MAPKs. These findings collectively suggested that compound 3h possesses significant anti-cervical cancer activity by modulating the MAPK signaling pathway to induce ROS-mediated DNA damage, thereby promoting mitochondrial apoptosis in HeLa cervical cancer cells.

Materials and methods

General synthetic procedure for compounds 3a-3h (5a-5h)

4-chloro-6,7-di(ethoxymethoxy)quinazoline (Compound 1, 0.1 mmol) was dissolved in 25 ml of isopropanol, followed by the addition of 3-aminophenylacetylene (0.11 mmol). The reaction mixture was refluxed for 3 h, during which a large amount of solid was formed. The progress of the reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the mixture was filtered and dried to obtain N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (compound 2). Compound 2 (0.1 mmol) was then added to a 1:1:1 mixture of water, t-butanol, and tetrahydrofuran (10 ml), and the corresponding azide compound was added, along with anhydrous copper sulfate (0.1 mmol) and sodium ascorbate (0.2 mmol). The reaction mixture was placed in a constant-temperature device and refluxed with stirring to ensure the reaction proceeded fully. The completion of the reaction was monitored by TLC. After the reaction, the mixture was extracted with saturated NaCl solution and ethyl acetate. The organic phase was concentrated via rotary evaporation, and the crude product was purified by column chromatography to yield the target compounds 3a-3h. The methods are applicable to the preparation of compounds 5a-5h.

A Bruker 400 spectrometer (Bruker, Billerica, MA, USA) was used to capture NMR spectra of compounds. Both 1H NMR and 13C NMR spectra were recorded in solutions of DMSO-d6 solution. Chemical shifts (δ) were presented in parts per million (ppm), referenced internally to tetramethylsilane. Coupling constants were denoted in hertz, with the following abbreviations used: s (singlet), d (doublet), t (triplet), and m (multiplet).

Cell culture

Hela cells and NIH3T3 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Sijiqing Biological Engineering Materials, Hangzhou, China), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Cell cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

MTT assay

HeLa cells in the logarithmic growth phase were seeded in a 96-well plate at a density of 4 × 104 cells/mL and cultured in the incubator for 24 h. The cells were divided into three groups: the negative control group, the positive control group, and the drug treatment group. The negative control group was treated with complete medium containing solvent control, the positive control group was treated with doxorubicin, and the drug treatment group was treated with the corresponding structural derivatives. After 48 h of incubation, 10 µL of 5 mg/mL MTT solution (Sangon Biotech, Shanghai, China) was added to each well, mixed, and then cultured for an additional 3 h. The culture medium was removed, and 100 µL of DMSO solution was added to each well to dissolve the formazan crystals. The absorbance (OD) was measured at 490 nm using a microplate reader.

Live/dead cell staining assay

Hela cells were seeded in 24-well plates at a density of 4 × 104 cells/mL and incubated for 24 h. The cells were treated with compound 3h at final concentrations of 0, 5, and 10 µM for 48 h. After the medium was removed, cells were washed with PBS. Then, 200 µL of Calcein-AM/PI staining solution (final concentrations of 4 µM Calcein-AM and 6 µM PI) was added to each well to incubate at 37℃ for 15 min. Images were captured under a fluorescence microscope, and the percentages of live and dead cells were calculated using ImageJ software.

Colony formation assay

HeLa cells were seeded in 6-well plates at a density of 200 cells/mL and cultured for 24 h. The cells were then treated with complete medium containing different concentrations of compound 3h (0, 5, and 10 µM) for 14 days. After washing with PBS, the cells were fixed with 4% paraformaldehyde overnight. Following three washes with PBS, the cells were stained with crystal violet for 10 min at room temperature. After washing three times with PBS, images were taken for each group.

Wound healing assay

HeLa cells were seeded in 6-well plates at a density of 3 × 105 cells/mL and cultured for 24 h. A scratch was made in the cell monolayer, and the cells were washed three times with D-Hanks solution to remove debris and non-adherent cells. The cells were then treated with culture medium containing 1% FBS and different concentrations of compound 3h (0, 5, and 10 µM). Images were taken at the same location under a microscope at 0, 24, and 48 h after treatment and the wound area was analyzed quantitatively using ImageJ software through the following procedure24: All images underwent spatial calibration followed by grayscale conversion. Edge enhancement was achieved through iterative Sobel filtering, with adaptive thresholding applied for binarization. After secondary edge refinement, leading margins were demarcated using automated wand selection (Process-Find Edges), and planimetric quantification of denuded areas was performed. Wound closure percentage was computed as: Cell migration rate = [(A0 - At)/A0] × 100%, where A0 and At represent initial (t = 0) and terminal (t = 24–48 h) wound areas, respectively.

Cell cycle analysis

HeLa cells were seeded in 12-well plates at a density of 1.5 × 105 cells/mL and cultured for 24 h. The cells were then treated with compound 3h at final concentrations of 0, 5, and 10 µM for 48 h. Cells were digested with 0.25% trypsin, collected by centrifugation at 500 g for 5 minutes, and stained with DNA Staining solution and Permeabilization solution according to the manufacturer’s instructions (Multi Sciences (Lianke) Biotech Co., Ltd, Hangzhou, China). After incubation for 20 min at room temperature in the dark, cell cycle distribution was analyzed by BD Accuri™ C6 Plus flow cytometry (BD Biosciences, Franklin Lake, NJ, USA).

Apoptosis assay

HeLa cells were seeded in 12-well plates at a density of 1.5 × 105 cells/mL and cultured for 24 h. The cells were treated with different concentrations of the compound (0, 5, 10 µM) for 48 h. The cells were digested with 0.25% trypsin (without EDTA), and collected by centrifugation at 500 g for 5 minutes. Then, the cells were stained with Annexin V-FITC and PI staining solution according to the manufacturer’s instructions (Vazyme Biotech Co., Ltd., Nanjing, China). After incubation for 20 min at room temperature in the dark, cell apoptosis was analyzed by BD Accuri™ C6 Plus flow cytometry (BD Biosciences, Franklin Lake, NJ, USA).

DAPI staining

HeLa cells were seeded in 24-well plates at a density of 4 × 104 cells/mL and cultured for 24 h. The cells were then treated with compound 3h at final concentrations of 0, 5, and 10 µM for 48 h. After washed with PBS, the cells were fixed with 4% paraformaldehyde for 15 min, and stained with DAPI solution for 5 min at room temperature in the dark. After discarding the staining solution and washing once with PBS, images were captured under a fluorescence microscope.

Western blot analysis

HeLa cells were seeded in 12-well plates at a density of 1.5 × 104 cells/mL and cultured for 24 h. The cells were treated with culture medium containing varying concentrations of compound 3h (0, 5, and 10 µM) for 48 h. After treatment, the cells were washed with PBS, and total cellular proteins were extracted using RIPA lysis buffer. Alternatively, nuclear proteins were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific, Rockford, IL, USA). Protein concentration was determined by the BCA method, and 5 × Loading buffer was added to the protein samples, which were then denatured by heating at 98 °C for 10 min. The denatured proteins were separated by SDS-PAGE and transferred to nitrocellulose (NC) membranes. After transfer, the membranes were blocked with 5% skim milk at room temperature for 1 h. Primary antibodies, diluted according to manufacturer protocols, were applied and incubated overnight at 4°C. After incubation, the membrane was washed three times with 1 × TBST for 10 min each ), followed by incubation with secondary antibodies at room temperature for 1 h. After three additional TBST washes, the membrane was then treated with ECL chemiluminescent substrate and imaged using a Gel Imaging System (Shanghai Tanon Life Science Co., Ltd., Shanghai, China). GAPDH, TBP, and β-actin were used as internal references.

The primary antibodies used were as follows: anti-Bcl-2 (ABclonal, Wuhan, China), anti-Bax (Wanleibio, Shenyang, China), anti-Caspase 9/p35/p10 (Proteintech, Wuhan, China), anti-Caspase 3/p17/p19 (Proteintech, Wuhan, China), anti-PARP-1 (Proteintech, Wuhan, China), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA, USA), anti-p44/42 MAPK (ERK1/2) (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling Technology, Beverly, MA, USA), anti-SAPK/JNK (Cell Signaling Technology, Beverly, MA, USA), anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology, Beverly, MA, USA), anti-p38 MAPK (Cell Signaling Technology, Beverly, MA, USA), anti-TBP (Proteintech, Wuhan, China), anti-GAPDH (Proteintech, Wuhan, China), and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA).

Measurement of intracellular ROS

HeLa cells were seeded in 12-well plates at a density of 1.5 × 105 cells/mL and cultured for 24 h. The cells were treated with culture medium containing different concentrations of compound 3h (0, 5, and 10 µM) for 48 h. The cells were digested with 0.25% trypsin and centrifuged at 500 g for 5 minutes to collect the cells. DCFH-DA staining solution was prepared in serum-free culture medium and added to the cells. After incubation at 37 ℃ for 30 min, the cells were washed with PBS, and then analyzed by flow cytometry. Flow cytometry was then used to detect the fluorescence intensity of each group.

Immunofluorescence staining

HeLa cells were seeded in 24-well plates containing glass slides at a density of 2 × 104 cells/well and cultured for 24 h. The cells were treated with culture medium containing different concentrations of compound 3h (0, 5, and 10 µM) for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min followed by three washes with PBS. The cells were permeabilized with methanol at -20℃ for 10 min, washed three times with PBS, and then blocked with blocking buffer at room temperature for 60 min. The primary antibody was added at the recommended dilution and incubated overnight at 4℃. Then, the cells were washed three times with 1× TBST for 5 min each. The fluorescently labeled secondary antibody was applied and incubated at room temperature in the dark for 1 h. DAPI staining solution was added and incubated for 10 min. After washing with 1× TBST, an appropriate amount of anti-fade reagent was added, and the slides were mounted. The cells were observed using a confocal laser scanning microscopy (Nikon, Tokyo, Japan).

System preparation

The crystallographic data of the MAP kinase ERK2 (PDB:5UMO25), c-Jun N-terminal kinase 1 (Jnk-1) (PDB: 3O2M 26), and human p38α MAP kinase (PDB:6Y4T 27) were obtained from the PDB database (https://www.rcsb.org) at a resolution of 2.3 Å, 2.7 Å, and 1.98 Å, respectively. Protein structures were prepared for molecular docking using the Protein Preparation Wizard module in Maestro, following a standardized computational protocol 27. Compound 3h was energy-minimized and optimized using the LigPrep tool within Maestro 28. The docking region was defined by a grid box generated automatically using the Receptor Grid Generation tool, based on the ligand coordinates in the crystal structures of Jnk-1 and p38α 29. For ERK2, the binding site was predicted using the SiteMap tool in Maestro and further validated by alignment with previously reported binding sites25,30. Finally, the generated grid files were employed to perform molecular docking of compound 3h into ERK2, Jnk-1, and p38α to explore potential binding modes.

Prime MM-GBSA calculation

The binding free energy (△Gbind) of each ligand was calculated using the Prime MM-GBSA method based on the docking complex, as described by the following Equation.31.

△Gbind= △EMM + △Gsolv + △GSA.

Where △EMM measures the difference in the minimized energies between the protein-ligand complex and the sum of the energies of the protein and ligand. △GSA represents the difference between in surface area energies of the complex and the sum of the surface area energies for the protein and ligand. △Gsolv shows the distinguish in Generalized Born Surface Area (GBSA) solvation energy of the protein-ligand complex and the sum of the solvation energies for protein and ligand.

Statistical analysis

All data were analyzed using GraphPad Prism version 9.5. Each experiment was performed in triplicate or more, and the results are presented as the mean ± standard error of the mean (Mean ± SEM). For comparisons involving multiple groups, one-way analysis of variance (One-Way ANOVA) was used, whereas for comparisons between two groups, a t-test was employed. A P-value of less than 0.05 was considered to indicate statistical significance.