Design, synthesis, and biological evaluation of benzenesulfonyl chloride-substituted berberine derivatives as potential PGAM1 inhibitors

2.1. Chemicals and instrumentation used

Most of the chemical reagents and solvents used in this study were purchased from commercial suppliers and were subjected to further purification and drying prior to use to ensure experimental reliability. All organic solvents were dried over 4 Å molecular sieves and stored under appropriate conditions to prevent moisture contamination. Unless otherwise specified, analytical-grade solvents and reagents were employed throughout the experiments without additional treatment. Proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVANCE 400 and AVANCE 500 spectrometers operating at 400 MHz and 500 MHz, respectively. The acquired data were processed and analyzed using MestReNova software. NMR measurements were performed in deuterated solvents, including DMSO-d₆, CDCl₃, or D₂O, as appropriate for compound solubility, with tetramethylsilane (TMS) serving as the internal standard. Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are given in hertz (Hz). Electrospray ionization mass spectrometry (ESI-MS) was performed on Agilent 7250 and JEOL JMS-T100LP AccuTOF instruments to confirm molecular weights and structural integrity of the synthesized compounds. Thin-layer chromatography (TLC) was carried out on GF254 silica gel plates (Qingdao Marine Chemical Co., China) to monitor reaction progress, and flash column chromatography was conducted using 60 G silica gel from the same supplier to purify products. All commercially available solvents used in chromatography and extraction steps were employed as received without further purification. These standardized analytical and purification methods ensured the accuracy and reproducibility of the experimental results. All the spectras are shown in the Supplementary material.

2.2. General procedure for synthesizing the target compounds

2.3. PGAM1 inhibition

PGAM1 used in this study was purchased from Jiangsu Sumec Biotechnology Co., Ltd. (catalog number: MK530295A) and was stored and handled according to the manufacturer’s instructions. Prior to the experiment, all reagents, samples, and standards were equilibrated to room temperature to ensure uniformity and stability of the reactions. During the assay, appropriate volumes of reagents, samples, or standards were added to the designated wells of the microplate, followed by incubation at 37°C for 30 min to allow sufficient reaction. After incubation, the plate was washed five times with the provided wash buffer to remove unbound or nonspecific materials. Subsequently, an appropriate volume of enzyme-conjugated reagent was added to each well, and the plate was incubated again at 37°C for 30 min to ensure complete binding of the enzyme to the target molecules. After the second incubation, the plate was washed an additional five times to minimize background and improve detection specificity. Following the washing steps, 50 μL of chromogenic solution A and 50 μL of chromogenic solution B were sequentially added to each well. The plate was gently agitated to ensure thorough mixing of the reagents. The plate was then incubated at 37°C in the dark for 10 min to facilitate color development. Finally, 50 μL of stop solution was added to each well to terminate the reaction, which caused the color of the solution to change from blue to yellow. The optical density at 450 nm (OD₄₅₀) was measured within 15 min of adding the stop solution using a microplate reader, and the data were recorded for subsequent analysis.

2.4. Cell viability assays

The human cell lines A549, H460, ECA109, HepG2, SW620, and the normal human liver cell line LO2 used in this study were all obtained from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China) and were authenticated and maintained under recommended conditions. In this experiment, the A549, H460, ECA109, and LO2 cell lines were cultured in RPMI-1640 medium, while the HepG2 and SW620 cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) medium. Both media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin to support cell growth and prevent bacterial contamination. All cells were maintained in a humidified incubator at 37°C with 5% CO₂ atmosphere. To evaluate cell viability, approximately 4,000 cells from each cell line (A549, H460, ECA109, HepG2, SW620, and LO2) were seeded into individual wells of 96-well plates and allowed to adhere for 12-24 h. Once attachment was confirmed, cells were treated with the designated inhibitors for 48 h under standard culture conditions. Following drug exposure, 0.5 mg/mL of Methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution was added to each well, and the cells were incubated for an additional 4 h to allow for formazan crystal formation. The resulting crystals were then solubilized, and the optical density (OD) at 492 nm was measured using a microplate reader to quantify cell viability. All experiments were performed in triplicate to ensure reproducibility, and the data are presented as mean ± standard deviation (SD) of three independent experiments.

2.5. Apoptosis detection assay

H460 cells were seeded at a density of approximately 1.5 × 10⁵ cells per well in six-well culture plates and allowed to adhere for 12 h under standard culture conditions (37°C, 5% CO₂, humidified atmosphere). After cell attachment, the medium was replaced with fresh medium containing various concentrations of the test compounds. Specifically, cells were treated with compound 18 at final concentrations of 2.5 μM, 5 μM, and 10 μM, or with compound 46 at 5 μM, 10 μM, and 20 μM, and incubated for an additional 24 h. Following treatment, cells were harvested by trypsinization without ethylenediaminetetraacetic acid (EDTA) to minimize damage to cell surface markers. The cells were collected, washed twice with ice-cold phosphate-buffered saline (PBS), and centrifuged to remove the supernatant. The resulting cell pellets were gently resuspended in 500 μL of 1× binding buffer provided in the apoptosis detection kit. Subsequently, 5 μL of Annexin V–Fluorescein Isothiocyanate (FITC) and 10 μL of propidium iodide (PI) staining solution were added to each suspension, and the cells were incubated in the dark at room temperature for 5 min to allow for staining. After incubation, apoptosis was analyzed immediately using a PrismTech flow cytometer (Chengdu, China) according to the manufacturer’s instructions. Data acquisition and analysis were performed to quantify the proportions of viable, early apoptotic, late apoptotic, and necrotic cells in each treatment group. All assays were performed in triplicate to ensure reproducibility.

2.6. Cell cycle assay

H460 cells were seeded in six-well plates at a density of approximately 1.5 × 10⁵ cells per well and incubated for 12 h under standard culture conditions (37°C, 5% CO₂, humidified atmosphere) to allow for cell attachment. Once adherent, the cells were treated with compound 18 at final concentrations of 1.5 μM, 5 μM, and 10 μM, or with compound 46 at 5 μM, 10 μM, and 20 μM, and incubated for an additional 24 h. At the end of the treatment period, the cells were harvested by trypsinization and collected into centrifuge tubes. The cell suspensions were washed twice with ice-cold phosphate-buffered saline (PBS) to remove residual medium and detached debris, followed by centrifugation to discard the supernatant. The resulting cell pellets were gently resuspended in 1 mL of ice-cold 70% (v/v) ethanol and fixed at 4°C for at least 4 h to preserve cellular morphology and nuclear integrity for subsequent analysis. After fixation, the cells were centrifuged again to remove the ethanol, and the pellets were resuspended in 0.5 mL of staining buffer. Subsequently, 25 μL of propidium iodide (PI, 20× stock solution) and 10 μL of RNase A (50× stock solution) were added to each sample to stain DNA and eliminate RNA interference, respectively. The samples were incubated at room temperature in the dark for 30 min to ensure complete staining. Finally, cell cycle distribution was analyzed by flow cytometry using a PrismTech cytometer (Chengdu, China) according to the manufacturer’s instructions. Data were acquired and processed to quantify the proportions of cells in different cell cycle phases (G₀/G₁, S, and G₂/M), and all experiments were performed in triplicate to ensure reproducibility and statistical validity.

2.7. ROS burst assay

H460 cells were seeded into six-well plates at a density of approximately 1.5 × 10⁵ cells per well and incubated for 12 h under standard culture conditions (37°C, 5% CO₂, humidified atmosphere) to allow proper cell attachment. Once the cells were adherent, they were treated with compound 18 at final concentrations of 2.5 μM, 5 μM, and 10 μM, or with compound 46 at concentrations of 5 μM, 10 μM, and 20 μM, and incubated for an additional 24 h. Following the treatment period, the culture medium was carefully aspirated, and the cells were incubated with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C for 30 min in the dark. DCFH-DA is a cell-permeable fluorescent probe that, upon oxidation by intracellular reactive oxygen species (ROS), is converted to the highly fluorescent compound dichlorofluorescein (DCF), enabling quantification of ROS levels. After staining, the cells were washed three times with PBS to remove excess probe and minimize background fluorescence. The cells were then resuspended in 500 μL of PBS, and the fluorescence intensity of DCF in each sample was immediately measured using a PrismTech flow cytometer (Chengdu, China) according to the manufacturer’s instructions. The data were acquired and analyzed to assess ROS production in a concentration-dependent manner, and all experiments were performed in triplicate to ensure reproducibility and reliability of the results.

2.8. Mitochondrial member potential assay

In the experiment, H460 cells were seeded into six-well plates at a density of approximately 1.5 × 10⁵ cells per well and cultured for 12 h under standard conditions (37°C, 5% CO₂, humidified atmosphere) to allow proper adhesion. After cell attachment, the culture medium was replaced with fresh medium containing either compound 18 at final concentrations of 2.5, 5, or 10 μM, or compound 46 at concentrations of 5, 10, or 20 μM. The cells were incubated with the respective compounds for 24 h. Following treatment, the culture medium was carefully aspirated, and the cells were incubated with 1 mmol/L of the JC-1 fluorescent probe at 37°C for 30 min in the dark to stain mitochondria. JC-1 is a cationic dye that selectively accumulates in mitochondria; in healthy cells with intact mitochondrial membrane potential (MMP), JC-1 aggregates and emits red fluorescence, whereas in cells with depolarized mitochondria, JC-1 remains in its monomeric form and emits green fluorescence, enabling detection of mitochondrial dysfunction. After staining, the cells were washed three times with PBS to remove excess dye and reduce background signal. The cells were then resuspended in 500 μL of PBS, and the fluorescence intensity of JC-1 was measured immediately by flow cytometry using a PrismTech instrument (Chengdu, China). Data were collected and analyzed to quantify the proportion of cells with mitochondrial membrane depolarization, and all experiments were performed in triplicate to ensure reproducibility and statistical reliability of the results.

2.9. Western blotting assay

In the experiment, H460 cells were seeded into six-well plates at a density of approximately 2.0 × 10⁵ cells per well and treated with compound 18 at final concentrations of 2.5, 5, or 10 μM, or with compound 46 at concentrations of 5, 10, or 20 μM. After 24 h of incubation at 37°C in a humidified atmosphere with 5% CO₂, total cellular proteins were extracted using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. The lysates were collected and stored at –20°C until further analysis. The protein concentrations were determined using the bicinchoninic acid (BCA) assay according to the manufacturer’s protocol. For western blot analysis, equal amounts of protein (30 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes using a semi-dry transfer apparatus. The membranes were then blocked at room temperature for 4 h in Tris-Buffered Saline with Tween-20 (TBST) buffer (Tris-buffered saline containing 0.1% Tween-20) supplemented with 5% non-fat dry milk to prevent nonspecific binding. After blocking, the membranes were incubated overnight at 4°C with the appropriate primary antibodies, gently shaking to ensure even binding. Following incubation with primary antibodies, the membranes were washed three times with TBST (10 min per wash) to remove unbound antibodies. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C for 1.5 h. After secondary antibody incubation, the membranes were washed again three times with TBST (10 min each) to minimize background signal. Finally, the immunoreactive protein bands were visualized using the Pierce enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific), and images were captured with the Tanon 5200 chemiluminescence imaging system. Band intensities were quantified using appropriate software, and representative results from at least three independent experiments are presented. All gray values are detailed in Tables S1 and S2.

2.10. Experimental animals, diets, and housing conditions

Sixty SPF-grade KM mice, half male and half female, with a body weight ranging from 18 to 22 grams, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. The mice were housed in the SPF-grade Animal Laboratory of Shaanxi University of Chinese Medicine, and the feeding conditions and animal welfare complied with relevant requirements. This experiment was approved by the Laboratory Animal Ethics Committee of Shaanxi University of Chinese Medicine, with the ethical approval number: SUCMDL20250926002.

2.11. Statistical analysis

Data are presented as means ± standard deviation (SD) from three independent experiments. Student’s t-test was employed to assess statistical significance between cells treated with various target compounds and control groups. All statistical analyses and graph plotting were performed using GraphPad Prism version 8.0.2 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS Statistics 27 (IBM Corp., Armonk, NY, USA). Statistical significance was defined as p < 0.05.

3.1. Synthetic chemistry

The process of preparing BBR derivatives is illustrated in Scheme 1. Initially, BBR (compound 1; 1.0 g) is converted into its de-methylated form, compound 2, by removing the 9-O methyl group at 190°C under negative pressure. In a separate step, berberrubine (100 mg; 1.0 mmol) is dissolved in acetonitrile (5 mL). Once completely dissolved, triethylamine (175 μL) is added, and the solution is heated to 70°C. Subsequently, 4-trifluoromethyl benzenesulfonyl chloride (90.51 mg; 1.2 mmol) is introduced, and the mixture is refluxed at 70°C for 5-6 h, with the reaction monitored by TLC. The resulting mixture was subjected to three extractions using 50 mL of CH₂Cl₂ and 50 mL of H₂O (v/v ratio) to remove acetonitrile and water-soluble impurities. Anhydrous sodium sulfate was subsequently added to the mixture for drying purposes. Following vacuum distillation, the residue is purified by silica gel column chromatography using an eluent of CH₂Cl₂:CH₃OH in a ratio of 10:1 to 6:1 (v/v), yielding a dark brown solid with a final yield of 82.6%. Additional compounds (4-31) were synthesized using this method.

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Scheme 1.

Synthesis of compounds 2-59. Reagents and conditions:(a) negative pressure, rt, 190°C, 0.5 h. (b) methyl alcohol, NaBH₄, rt, 0°C, 12 h. (c) acetonitrile, triethylamine, rt, 70°C, 5-6 h. (d) acetonitrile, triethylamine, rt, 70°C, 5-6 h.

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To synthesize tetrahydroberberrubine (compound 3), berberrubine is initially reduced using NaBH₄. Tetrahydroberberrubine (100 mg; 1.0 mmol) is dissolved in 5 mL of acetonitrile. Once fully dissolved, triethylamine (175 μL) is added, and the solution is heated to 70°C. Following this, 4-trifluoromethyl benzenesulfonyl chloride (90.98 mg; 1.2 mmol) is introduced, and the mixture is refluxed at 70°C for 5-6 h, with the reaction monitored via TLC. The mixture is then extracted three times with 50 mL of CH₂Cl₂ and 50 mL of H₂O (v/v) to eliminate acetonitrile and water-soluble impurities. Anhydrous sodium sulfate was added to the mixture for drying. After vacuum distillation, the residue is purified through silica gel column chromatography using an eluent of CH₂Cl₂:CH₃OH in a ratio of 50:1 (v/v), resulting in a reddish-brown solid with a yield of 50.2%. Additional compounds (32-59) were synthesized following this procedure.

3.2. PGAM1 inhibition activity of target compounds

The inhibitory activity of the target compounds against PGAM1 was evaluated using BBR, berberrubine, and PGMI-004A as reference controls. As shown in Table 3, most synthesized compounds exhibited moderate to strong inhibition of PGAM1, with half-maximal inhibitory concentration (IC₅₀) values all below 10 μM, indicating their promising potential as enzyme inhibitors. Notably, compound 18 displayed an IC₅₀ of 0.081 μM, compound 22 exhibited an IC₅₀ of 0.076 μM, and compound 35 showed an IC₅₀ of 0.087 μM, all of which were significantly more potent than the parent compound BBR and its oxidized derivative berberrubine. More importantly, the inhibitory potency of these three compounds approached that of the positive control PGMI-004A (IC₅₀ = 0.052 μM), suggesting that structural modifications of the BBR core substantially enhanced its binding affinity toward PGAM1.

3.3. The effect of different compounds on inhibiting the proliferation of cancer cells

The antiproliferative activities of the synthesized compounds were evaluated using the MTT assay against five human solid tumor cell lines: human NSCLC cell line (A549), human large cell lung cancer cell line (H460), human esophageal cancer cell line (ECA109), human liver cancer cell line (HepG2), and human colon cancer cell line (SW620), as well as one normal human liver cell line (LO2) over 48 h. BBR, berberrubine, PGMI-004A, and tetrahydroberberrubine were used as control drugs. As shown in Tables 3 and 4, most compounds exhibited certain antiproliferative effects against these tumor cell lines. Overall, the BBR derivatives (Table 3) demonstrated better activity compared to the tetrahydroberberrubine derivatives (Table 4). This can be attributed to the quaternary ammonium ion (N⁺), which has good solubility and biocompatibility, allowing it to better permeate cell membranes and exert antitumor effects [31].

Compounds 18, 19, 23, 24, and 26 in Table 3 exhibited significant antiproliferative effects against A549 and H460 cell lines. In A549 cells, the IC₅₀ values were 5.41 µM, 6.51 µM, 8.56 µM, 5.12 µM, and 6.56 µM, respectively, showing better antiproliferative activity compared to the control drugs. In H460 cells, the IC₅₀ values were 4.50 µM, 5.78 µM, 9.01 µM, 5.63 µM, and 5.03 µM, respectively, also outperforming the three control drugs. Meanwhile, compounds 46 and 47 in Table 4 demonstrated notable effects on the H460 cell line, with IC₅₀ values of 10.82 µM and 12.01 µM, respectively, which also exceeded the efficacy of the control drugs. Further observation revealed that among the compounds with better antiproliferative activity, compounds 18 and 46 share the same substituent group (-R), as do compounds 19 and 47. The latter compounds (46 and 47) were derived from the former (18 and 19) through reduction reactions. Moreover, compounds 18, 19, 46, and 47 not only exhibited strong antiproliferative effects on the H460 cancer cell line but also significantly improved their selectivity index (SI), which is the ratio of the IC₅₀ value in normal liver cells (LO2) to that in H460 cells. These compounds showed reduced cytotoxicity towards LO2 cells, indicating that they selectively kill cancer cells rather than normal cells. The concentration-response curves illustrating the inhibitory effects of BBR, compound 18, and compound 46 on the proliferation of H460 cells have been shown in Figure 2. Finally, compounds 18 and 46 with the same substituents and the highest activity were selected to further verify their biological activities.

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Figure 2.

The concentration-response curves of the inhibitory effects of BBR(0-50 μM), compound 18 (0-50 μM), and compound 46 (0-50 μM) on the proliferation of H460 cells.

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The substituent group (-R) for compounds 18 and 46 is 1,3,5-triisopropyl-2-methylbenzene, while the substituent group (-R) for compounds 19 and 47 is 1-(tert-butyl)-4-methylbenzene. Their common feature is the presence of a methyl (-CH₃) substituent. Firstly, the methyl group acts as an electron-donating group, which increases the electron density of the benzene ring through resonance and inductive effects, thereby enhancing the interaction between the compound and target sites such as enzymes, receptors, or DNA. Secondly, the spatial effect of the methyl substituent alters the 3D structure of the compound, making it more suitable for fitting into target sites [32]. Thirdly, the methyl group increases the hydrophobicity of the compound, which can affect its membrane permeability and distribution within the body, thus enhancing its antitumor activity [33]. Lastly, the methyl substituent can improve the metabolic stability of the compound, prolonging its half-life in the body, which may also positively impact its antitumor activity [34].

3.4. Preliminary study on the structure-activity relationship of BBR derivatives

We have preliminarily explored the relationship between the structure and anti-cancer activity of BBR derivatives (Figure 3a). Because compounds 9, 18, and 25 exhibit good enzyme activity against PGAM1, we selected these three compounds as representatives to study the SAR of BBR derivatives. The study found that introducing a halogen atom at the R1 position can enhance the anti-cancer activity, and if it is disubstituted with R5, the activity will be further improved. Introducing an ether bond or a nitro group at the R3 position can enhance the activity, and introducing a methyl group is more effective in enhancing the anti-cancer activity. Moreover, introducing isopropyl groups at the R1, R3, and R5 positions simultaneously can effectively enhance the anti-cancer activity (Figure 3b). Similarly to the above, we took compounds 35, 40, and 46 as representatives to study the SAR of deionized BBR derivatives. For the deionized BBR derivatives, introducing a halogen atom at the R4 position can enhance the activity, and the effect is better than that at the R1, R2, R3, and R5 positions. Introducing an ether bond at the R2 position does not enhance the anti-cancer activity, but introducing an ether bond or a nitro group at the R3 position can enhance the activity. In addition, introducing isopropyl groups at the R1, R3, and R5 positions can effectively enhance the anti-cancer activity (Figure 3c). Compounds 18 and 46 contain a benzene ring structure substituted with six methyl groups. As an electron-donating group, the methyl group (-CH₃) increases the electron density on the aromatic ring through resonance and inductive effects, thereby enhancing the interaction between the compound and the target protein [35]. The methyl substituents change the 3D structure of the compound, making it more suitable for binding to the active site of the target protein. This steric effect may enable the compound to more easily form a stable complex with the target protein, thus enhancing its inhibitory effect. Moreover, the methyl substituents increase the hydrophobicity of the compound, which indicates that the substituents at this position are in the hydrophobic pocket [36]. Therefore, our subsequent research is mainly based on Compounds 18 and 46.

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Figure 3.

SAR of BBR derivatives. (a) The SAR of the derivatives of BBR and their deionized derivatives. (b) Compounds 9, 18, and 25, and their activities against some types of cancer cells. (c) Compounds 35, 40, and 46 and their activities against certain types of cancer cells.

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3.5. Apoptosis of H460 induced by compounds 18 and 46

Cell apoptosis, also known as programmed cell death, exerts a pivotal role in governing the development and growth of both normal and neoplastic cells [37]. Inducing apoptosis is considered one of the key mechanisms by which anticancer agents exert their therapeutic effects. In this study, the effects of compounds 18 and 46 on apoptosis induction in H460 lung cancer cells were evaluated using Annexin V-FITC/propidium iodide (PI) double staining followed by flow cytometry. BBR and the positive control PGMI-004A were included for comparison. As shown in Figure 4(a), treatment of H460 cells with increasing concentrations of compound 18 for 24 h resulted in a dose-dependent increase in the proportion of apoptotic cells. At a concentration of 10 μM, compound 18 increased the apoptosis rate (sum of Q1 and Q4 quadrants) by 46.81%, which was markedly higher than that observed in the untreated control group (13.62% and 6.41% in Q1 and Q4, respectively). Similarly, as illustrated in Figure 4(b), treatment with different concentrations of compound 46 for 24 h also promoted apoptosis in a concentration-dependent manner. When the concentration of compound 46 reached 20 μM, the apoptosis rate increased significantly to 37.67%, compared to the control group (22.92% and 11.36% in Q1 and Q4, respectively). These results demonstrate that both compounds 18 and 46 effectively induce apoptosis in H460 cells, and the extent of this effect correlates positively with the drug concentration, suggesting a clear dose-response relationship. The observed apoptotic induction is consistent with the proposed mechanism of action of these PGAM1 inhibitors and further supports their potential as promising anticancer agents.

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Figure 4.

Effects of compounds on cell apoptosis. (a) Compound 18 and positive controls PGMI-004A and BBR-treated H460 cells for 24 h. (b) Compound 46 and positive controls PGMI-004A and BBR-treated H460 cells for 24 h. ***P < 0.001,**P < 0.01, ns: not significant (P > 0.05),determined by Student’s t-test. At least three independent experiments were performed for each experimental condition.

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3.6. Effects of compounds 18 and 46 on the cell cycle

One of the characteristics of tumor cells is the disruption of cell cycle regulation, which results in uncontrolled cell growth [38]. To investigate the effects of compounds 18 and 46 on the cell cycle distribution of H460 lung cancer cells, flow cytometry analysis was performed after treatment with varying concentrations of the compounds. As shown in Figure 5, both compounds 18 and 46 significantly induced cell cycle arrest at the G2/M phase in a dose-dependent manner. Specifically, treatment with 10 μM of compound 18 resulted in a marked increase in the proportion of cells in the G2/M phase, reaching 44.34%, whereas the positive control PGMI-004A and the parent compound BBR induced only 11.28% and 19.23% of cells in G2/M phase, respectively, under the same conditions. Similarly, treatment with 20 μM of compound 46 led to a substantial accumulation of cells in the G2/M phase, with a proportion of 41.28%, which was significantly higher than that observed for PGMI-004A (12.46%) and comparable to or slightly higher than BBR (35.75%). These results demonstrate that both compounds 18 and 46 are capable of markedly perturbing cell cycle progression by inducing G2/M phase arrest in H460 cells. The observed G2/M blockade is consistent with their antiproliferative activity and suggests that these compounds interfere with critical mitotic checkpoints, thereby preventing proper cell division and contributing to their anticancer mechanism of action. Taken together, these findings highlight the ability of the designed BBR derivatives to target cell cycle regulation pathways more effectively than the parent compound and underscore their potential as promising antitumor agents.

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Figure 5.

Effects of compounds on the cell cycle. (a) Compound 18 and positive controls PGMI-004A and BBR-treated H460 cells for 24 h. (b) Compound 46 and positive controls PGMI-004A and BBR-treated H460 cells for 24 h. At least three independent experiments were performed for each experimental condition.

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3.7. Compounds 18 and 46 caused an ROS burst in H460 cells

ROS are a class of highly reactive molecules containing oxygen free radicals that play a crucial role in regulating various cellular processes, including proliferation, differentiation, and apoptosis. Elevated intracellular ROS levels have been shown to suppress tumor growth by persistently enhancing cell cycle inhibition and promoting programmed cell death. Therefore, triggering excessive ROS production has emerged as a viable strategy for inducing apoptosis in tumor cells. In this study, the intracellular ROS levels in H460 lung cancer cells treated with compounds 18 (5a) and 46 (5b) were assessed using the fluorescent probe DCFH-DA, which specifically detects ROS by emitting green fluorescence upon oxidation. As shown in Figure 6, treatment with increasing concentrations of compounds 18 and 46 for 24 h resulted in a significant and dose-dependent elevation of ROS levels in H460 cells, suggesting that both compounds exert their antiproliferative effects, at least in part, by inducing oxidative stress. Notably, the fluorescence intensity of ROS in treated cells was markedly higher compared to the untreated control, indicating robust ROS generation upon drug exposure. These findings imply that compounds 18 and 46 disrupt the cellular redox balance and enhance oxidative stress, thereby contributing to cell cycle arrest and apoptosis in lung cancer cells [39]. This ROS-mediated mechanism further supports the potential of these BBR-based derivatives as effective anticancer agents targeting redox homeostasis.

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Figure 6.

Effects of compounds on ROS levels. (a) H460 cells treated with compound 18 for 24 h; (b) H460 cells treated with compound 46 for 24 h. Pink: low ROS cell population; Blue: high ROS cell population. Data are presented as mean ± standard deviation (n = 3). ***P < 0.001, ns: not significant (P > 0.05), determined by Student’s t-test. All experiments were independently repeated at least three times.

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3.8. Compounds 18 and 46 caused mitochondrial damage in H460 cells

Apoptosis, also referred to as programmed cell death, is primarily mediated through two canonical pathways: the intrinsic, mitochondria-dependent pathway and the extrinsic pathway triggered by external death signals [40]. Increasing evidence suggests that mitochondrial dysfunction plays a critical role in initiating and amplifying the apoptotic process [41]. To determine whether the apoptosis induced by compounds 18 and 46 is associated with the mitochondrial pathway, we assessed the MMP in H460 cells using the fluorescent probe JC-1. After treatment with different concentrations of compounds 18 and 46 for 24 h, the cells were stained with JC-1 and analyzed by flow cytometry (Figure 7). JC-1 aggregates in mitochondria with intact membrane potential and emits red fluorescence, whereas in depolarized mitochondria, it remains in the monomeric form, emitting green fluorescence. Thus, the increase in green fluorescence intensity serves as an indicator of MMP collapse, reflecting mitochondrial dysfunction. As shown in Figure 7, treatment with 2.5 μM, 5 μM, and 10 μM of compound 18 for 24 h resulted in a concentration-dependent increase in the proportion of cells exhibiting mitochondrial membrane depolarization, with rates of 11.54%, 23.17%, and 63.14%, respectively. In comparison, treatment with 10 μM of PGMI-004A and BBR resulted in MMP collapse rates of only 13.51% and 17.40%, respectively, indicating that compound 18 was more potent in disrupting mitochondrial integrity. Similarly, treatment with 5 μM, 10 μM, and 20 μM of compound 46 also led to a significant, dose-dependent increase in the proportion of cells with MMP loss, with rates of 10.81%, 33.13%, and 47.55%, respectively. By contrast, treatment with 20 μM of PGMI-004A and BBR yielded MMP collapse rates of 21.46% and 25.68%, respectively. These findings demonstrate that mitochondrial dysfunction induced by compounds 18 and 46 intensifies with increasing drug concentration and substantially exceeds that of the parent compound BBR and the positive control PGMI-004A. The pronounced depolarization of the MMP strongly suggests that both compounds trigger apoptosis in H460 cells primarily through the intrinsic, mitochondria-dependent pathway, highlighting their potential as effective anticancer agents targeting mitochondrial function and disrupting tumor cell proliferation.

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Figure 7.

Effects of compounds on mitochondrial function. (a) H460 cells treated with compound 18 for 24 h; (b) H460 cells treated with compound 46 for 24 h. Data are presented as mean ± standard deviation (n = 3). ***P < 0.001,**P < 0.01, *P < 0.05,determined by Student’s t-test. Each experiment was independently repeated at least three times.

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3.9. Western blot

To elucidate the mechanisms underlying apoptosis induction by compounds 18 and 46, we performed Western blot analysis on relevant proteins. The experimental results (Figure 8) demonstrated that after treating H460 cells with compounds 18 and 46 for 24 h, the expression of P53 was upregulated, while the expression of PGAM1 was downregulated, and the expression of α-smooth muscle actin (Acta2) was upregulated. PGAM1, a key enzyme in the glycolytic pathway, plays a pivotal role in maintaining the glycolytic flux and energy homeostasis of cells. Its inhibited expression is likely to disrupt the normal metabolic flux of glycolysis, leading to the accumulation of upstream metabolites such as 2-phosphoglycerate and the reduction of downstream 3-phosphoglycerate. This metabolic imbalance can trigger a series of metabolism-related stress responses, including the activation of AMPK-dependent energy-sensing pathways, which in turn affects multiple cellular physiological processes. Acta2, widely recognized as a classic marker for stromal cell activation and matrix remodeling, its altered expression strongly indicates dynamic changes in the tumor microenvironment [42]. In specific tumor contexts, the upregulation of Acta2 in cancer cells has been functionally linked to enhanced migratory and invasive capacities, possibly through the reorganization of the cytoskeleton and the modulation of cell-matrix adhesion molecules. P53, a well-characterized tumor suppressor gene, is tightly regulated at both transcriptional and post-translational levels. It is typically upregulated in response to various cellular stresses, including metabolic perturbations and DNA damage, and subsequently initiates a cascade of events leading to cell cycle arrest and apoptosis [43]. Compounds 18 and 46 appear to trigger stress responses by inhibiting PGAM1, thereby activating the P53 pathway and modulating Acta2 expression. This multi-target regulatory pattern suggests that these compounds may exert dual effects of effective tumor growth inhibition and apoptosis induction. Furthermore, the upregulation of BAX, Caspase-3, and Caspase-9 proteins, coupled with the downregulation of BCL2, provides compelling evidence that these compounds induce programmed cell death through the activation of intrinsic apoptotic pathways [44]. Specifically, the increased BAX/BCL2 ratio promotes mitochondrial outer membrane permeabilization, leading to the release of cytochrome c and the subsequent activation of the caspase cascade, ultimately resulting in apoptotic cell death. Additionally, the reduced expression levels of CDK2, CDK4, and Cyclin A2 indicate that these compounds may interfere with cell cycle regulation, particularly during the transition from the G1/S phase to the G2/M phase[45]. These cell cycle-related proteins are critical for the progression of cell division, and their downregulation can lead to cell cycle arrest, thereby inhibiting cell proliferation. Such changes in protein expression collectively suggest that the compounds may suppress cell proliferation by targeting cell cycle-associated pathways. Moreover, these findings strongly support that PGAM1 holds great promise as a candidate target for cancer-targeted therapy. They also indicate that while inhibiting PGAM1 activity, these compounds may regulate multiple key cellular stress response pathways, forming a complex regulatory network that contributes to their anti-tumor effects. This comprehensive understanding of the molecular mechanisms lays a solid foundation for further in vivo studies and potential clinical applications of these compounds.

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Figure 8.

(a) H460 cells were incubated with compound 18 (0, 2.5, 5, 10µM) for 24 h, respectively. (b) H460 cells were incubated with compound 46 (0, 5, 10, 20µM) for 24 h, respectively. ***P < 0.001,**P< 0.01, *P<0.05,determined by Student’s t-test. At least three separate experiments were done in each case.

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3.10. Acute toxicity induced by compounds 18 in mice

To investigate the acute toxic effect of Compound 18 (a compound with relatively prominent activity) on mice, the animals in the experiment received a single intraperitoneal injection of Compound 18 at different doses (0, 30, 60, 120, and 240 mg/kg) and were continuously monitored for 14 days. On the first day after administration, eight mice died in the highest dose group (240 mg/kg) and two died in the 120 mg/kg group, while no mouse deaths were observed in the other groups. On the second day, all mice in the highest dose group died, six mice died in the 120 mg/kg group, and two mice died in the 60 mg/kg group. On the third day, four mice died in the 60 mg/kg group. On the fourth day, one mouse died in the 60 mg/kg group. No additional mouse deaths occurred during the remaining observation period. Ultimately, the mortality rate in the 240 mg/kg dose group was 100%, the mortality rate in the 120 mg/kg dose group was 66.7%, and the mortality rate in the 60 mg/kg dose group was 58.3%. Based on these data, an optimal “concentration-mortality” effect curve was established, and the LD₅₀ of Compound 18 was determined to be 72.2 mg/kg (Figure 9a), while the LD₅₀ of BBR for acute toxicity in mice was 57.6 mg/kg [46]. This indicates that Compound 18 has better safety.

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Figure 9.

(a) Dose-mortality relationship curve of compound 18 (b) Body weight changes over time (days) in mice treated with different doses.

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Acute toxicity tests showed that some mice treated with compound 18 at doses of 60, 120, and 240 mg/kg exhibited reduced activity when in a moribund state. To evaluate the effect of compound 18 on the body weight of mice, all mice were weighed. During the 14-day monitoring period, all tested mice showed weight loss, and some of them died within 14 days (Table S3), while the body weight of the surviving mice gradually increased (Figure 9b). These results indicate that compound 18 induces acute toxicity in mice and affects their body weight.