Novel 2-alkythio-4-chloro-N-[imino(heteroaryl)methyl]benzenesulfonamide Derivatives: Synthesis, Molecular Structure, Anticancer Activity and Metabolic Stability

A series of novel 2-alkythio-4-chloro-N-[imino-(heteroaryl)methyl]benzenesulfonamide derivatives, 8–24, were synthesized in the reaction of the N-(benzenesulfonyl)cyanamide potassium salts 1–7 with the appropriate mercaptoheterocycles. All the synthesized compounds were evaluated for their anticancer activity in HeLa, HCT-116 and MCF-7 cell lines. The most promising compounds, 11–13, molecular hybrids containing benzenesulfonamide and imidazole moieties, selectively showed a high cytotoxic effect in HeLa cancer cells (IC50: 6–7 μM) and exhibited about three times less cytotoxicity against the non-tumor cell line HaCaT cells (IC50: 18–20 μM). It was found that the anti-proliferative effects of 11, 12 and 13 were associated with their ability to induce apoptosis in HeLa cells. The compounds increased the early apoptotic population of cells, elevated the percentage of cells in the sub-G1 phase of the cell cycle and induced apoptosis through caspase activation in HeLa cells. For the most active compounds, susceptibility to undergo first-phase oxidation reactions in human liver microsomes was assessed. The results of the in vitro metabolic stability experiments indicated values of the factor t½ for 11–13 in the range of 9.1–20.3 min and suggested the hypothetical oxidation of these compounds to sulfenic and subsequently sulfinic acids as metabolites.


Introduction
Cancer is a major public health problem and a leading cause of death worldwide which caused nearly 10 million deaths in 2020 [1]. Basic and clinical research are still needed to increase our knowledge about cancer and accelerate progress in the fight against it. Breast, cervical and colorectal cancers are the most common female cancer types worldwide. In women, the incidence rates of breast cancer far exceed those of other cancers in both transitioned (55.9 per 100,000) and transitioning (29.7 per 100,000) countries, followed by those of colorectal cancer (20 per 100,000) in transitioned countries and of cervical cancer (18.8 per 100,000) in transitioning countries [2].
One of the most important strategies in the search for chemotherapeutics is the approach based on combining in one molecule building blocks, fragments of known drugs, leading structures or "hit" structures [3][4][5][6]. The conjugation of two pharmacophores into a
The structures of the final compounds 8-24 were confirmed by IR, 1 H NMR and 13 C NMR spectroscopy. The IR spectra showed the typical stretching vibration of the NH group at nearly 3300 cm −1 and the presence of two bands at approximately 1630 and 1540 cm −1 , corresponding to C=C and C=N stretching. Moreover, the sulfonyl group was identified by bands from S=O stretching (asymmetric and symmetric) at approximately 1370 and 1140 cm −1 . The appearance of NH signals at 10. 22-11.29  Compound 10 crystallized in the space group P2 1 /c with one molecule in the asymmetric unit. Details on data collection, structure solution and refinement are reported in Table S1 (Supplementary Materials). A molecular view is presented in Figure 2. In the solid state, the molecule is deprotonated in the sulfonamidic part, and the hydrogen atoms H2a and H2b are located on the amine nitrogen atom N2. They form hydrogen bonds with the neighbor O1 and S3 (for details, see Table S2 in Supplementary Materials). No distinct electron density peak was found in the vicinity of the sulfonamide nitrogen atom N1. Additionally, the CF 3 group was found disordered over two positions, with a site occupation factor of 0.58(2)/0.42 (2).  deprotonated in the sulfonamidic part, and the hydrogen atoms  H2a and H2b are located on the amine nitrogen atom N2. They form hydrogen bonds with  the neighbor O1 and S3 (for details, see Table S2 in Supplementary Materials). No distinct electron density peak was found in the vicinity of the sulfonamide nitrogen atom N1. Additionally, the CF3 group was found disordered over two positions, with a site occupation factor of 0.58(2)/0.42 (2).
We also performed an assay on the non-tumor cell line HaCaT (immortalized human keratinocytes) to assess if the effect of 11-13 was selective toward HeLa cells or resulted from a more general toxic activity. The test indicated that the compounds showed selectivity toward cancer cells. Values of IC 50 in the range of 18-20 µM for HaCaT cells indicated about three times less toxicity than that for HeLa cells. What is important, the cytotoxicity against non-cancerous cells of 11-13 was significantly lower than that of the reference drug cisplatin, which is a common drug used for cervical cancer treatment as a cell cycle non-specific drug in the clinic.
We also performed an assay on the non-tumor cell line HaCaT (immortalized human keratinocytes) to assess if the effect of 11-13 was selective toward HeLa cells or resulted from a more general toxic activity. The test indicated that the compounds showed selectivity toward cancer cells. Values of IC50 in the range of 18-20 µM for HaCaT cells indicated about three times less toxicity than that for HeLa cells. What is important, the cytotoxicity against non-cancerous cells of 11-13 was significantly lower than that of the reference drug cisplatin, which is a common drug used for cervical cancer treatment as a cell cycle non-specific drug in the clinic.

Cytotoxic Activity
The cytotoxic activity of 11-13 was determined in a time-dependent manner with the MTT assay ( Figure 3).  HeLa cells were treated with 11, 12 and 13 in the concentration range of 0-20 µM. After 24 h of treatment, the IC 50 values were not reached by the compounds 11 and 12, whereas for compound 13, the IC 50 value was reached at the concentration of 18 µM. After 48 h, the IC 50 values for compounds 11, 12 and 13 were 6, 7 and 6 µM, respectively. Further treatment with the compounds did not increase their cytotoxic activity.

Apoptosis Induction
In order to determine whether the anti-proliferative effects of 11, 12 and 13 were associated with their ability to induce apoptosis in HeLa cells, the induction of phosphatidylserine externalization by compounds 11, 12 and 13 was examined by flow cytometric analysis. The cells were treated with 2.5, 5 and 10 µM concentrations of 11, 12 and 13 for 24 and 48 h and stained with Annexin V-PE and 7-AAD. The results shown in Figure 4 indicated that compounds 11-13 induced apoptosis in a concentration-and time-dependent manner. After 24 h of treatment, a significant increase in the early apoptotic population of cells was visible starting from the concentration of 5 µM.

Apoptosis Induction
In order to determine whether the anti-proliferative effects of 11, 12 and 13 were associated with their ability to induce apoptosis in HeLa cells, the induction of phosphatidylserine externalization by compounds 11, 12 and 13 was examined by flow cytometric analysis. The cells were treated with 2.5, 5 and 10 µM concentrations of 11, 12 and 13 for 24 and 48 h and stained with Annexin V-PE and 7-AAD. The results shown in Figure 4 indicated that compounds 11-13 induced apoptosis in a concentration-and time-dependent manner. After 24 h of treatment, a significant increase in the early apoptotic population of cells was visible starting from the concentration of 5 µM.  These results provide valuable insights into the mechanism of action of compoun 11, 12 and 13 as potential anti-proliferative agents targeting HeLa cells. The concentrati and time-dependent induction of apoptosis highlighted the effectiveness of these co pounds in promoting programmed cell death in the tested cell line.

Caspase Activation
Apoptosis induction was further determined by examining the effects of 11, 12 a 13 on caspase activation in HeLa cells. Caspase activity induction was determined w the use of the fluorescently labeled caspase inhibitor-FAM-VAD-FMK (a carboxyfluor cein derivative of valylalanylaspartic acid fluoromethyl ketone). The caspase inhib binds to active caspases inhibiting their enzymatic activity, thus allowing caspase activ These results provide valuable insights into the mechanism of action of compounds 11, 12 and 13 as potential anti-proliferative agents targeting HeLa cells. The concentration-and time-dependent induction of apoptosis highlighted the effectiveness of these compounds in promoting programmed cell death in the tested cell line.

Caspase Activation
Apoptosis induction was further determined by examining the effects of 11, 12 and 13 on caspase activation in HeLa cells. Caspase activity induction was determined with the use of the fluorescently labeled caspase inhibitor-FAM-VAD-FMK (a carboxyfluorescein derivative of valylalanylaspartic acid fluoromethyl ketone). The caspase inhibitor binds to active caspases inhibiting their enzymatic activity, thus allowing caspase activity quantification through determining the fluorescent intensity of the bound inhibitor. The results shown in Figure 6 indicated that compounds 11, 12 and 13 induced caspase activity in HeLa cells in a dose-dependent manner. Increased caspase activation was shown by the increased fluorescence of the caspase inhibitor in the cell population, as indicated in Figure 6. The results showed that compounds 11, 12 and 13 induced apoptosis through caspase activation in HeLa cells. quantification through determining the fluorescent intensity of the bound inhibitor. The results shown in Figure 6 indicated that compounds 11, 12 and 13 induced caspase activity in HeLa cells in a dose-dependent manner. Increased caspase activation was shown by the increased fluorescence of the caspase inhibitor in the cell population, as indicated in Figure 6. The results showed that compounds 11, 12 and 13 induced apoptosis through caspase activation in HeLa cells. By targeting caspase activation, compounds 11, 12 and 13 initiate the cascade of events leading to programmed cell death. This observation supports the hypothesis that the anti-proliferative effects of these compounds in HeLa cells are mediated through the induction of apoptosis. The evaluation of caspase activity added valuable insight into the mechanism of action of compounds 11, 12 and 13, highlighting their potential as apoptotic inducers in cancer therapy.  By targeting caspase activation, compounds 11, 12 and 13 initiate the cascade of events leading to programmed cell death. This observation supports the hypothesis that the antiproliferative effects of these compounds in HeLa cells are mediated through the induction of apoptosis. The evaluation of caspase activity added valuable insight into the mechanism of action of compounds 11, 12 and 13, highlighting their potential as apoptotic inducers in cancer therapy.  Taken together, these results indicated that compounds 11, 12 and 13 exerted their anti-proliferative effects in HeLa cells by inducing DNA fragmentation and potentially triggering apoptosis through the activation of CAD. The cell cycle analysis provided valuable insights into the mechanisms of action of these compounds and their potential as anti-cancer agents.

In Vitro Metabolic Stability Assay
The three most potent compounds (11, 12 13) were tested in an in vitro metabolic stability assay. Human liver microsomes along with NADPH were used to assess their susceptibility to undergo first-phase oxidation reactions. The progress of biotransformation was followed by liquid chromatography-mass spectrometry. The results derived from triplicate incubations, expressed as in vitro metabolic half-life (t½), are shown in Figure 8. In order to support the in vitro results, we performed in silico calculations using the Human Liver Microsome-based model for CYP-mediated oxidations provided by the Xenosite online tool [45].
In relation to their stability, the compounds could be ordered as 12 > 13 > 11, with decreasing stability. All substituents in the R 1 position can undergo oxidation, and this occurs for substituents in several positions for 4-chlorophenyl (11) and 1-naphthyl (13). Interestingly, in opposition to the Xenosite's results, the derivative bearing a piperonyl moiety (12) exhibited the best stability in vitro among the studied set of compounds. A detailed survey of possible reasons for this property suggested that another part of the studied molecules can be more important for metabolic stability than the R 1 substituents, thus diminishing their influence.
The most probable hypothesis includes oxidation of the sulfur atom in the thione functionality, resulting in the formation of sulfenic and, subsequently, sulfinic acid. This kind of biotransformation was reported several times in the literature, including in a detailed study of several thioureas and thiones by Henderson and others [46] and also in a paper by Yamazaki et al. for methimazole [47]. Taken together, these results indicated that compounds 11, 12 and 13 exerted their anti-proliferative effects in HeLa cells by inducing DNA fragmentation and potentially triggering apoptosis through the activation of CAD. The cell cycle analysis provided valuable insights into the mechanisms of action of these compounds and their potential as anti-cancer agents.

In Vitro Metabolic Stability Assay
The three most potent compounds (11, 12 13) were tested in an in vitro metabolic stability assay. Human liver microsomes along with NADPH were used to assess their susceptibility to undergo first-phase oxidation reactions. The progress of biotransformation was followed by liquid chromatography-mass spectrometry. The results derived from triplicate incubations, expressed as in vitro metabolic half-life (t 1 2 ), are shown in Figure 8. In order to support the in vitro results, we performed in silico calculations using the Human Liver Microsome-based model for CYP-mediated oxidations provided by the Xenosite online tool [45].

Synthesis
The melting points were uncorrected and measured using a Thermogalen (Leica, Vienna, Austia) apparatus. The IR spectra were measured on a Thermo Mattson Satellite FTIR spectrometer (Thermo Mattson, Madison, WI, USA) in KBr pellets; the absorption range was 400-4000 cm −1 . The 1 H NMR and 13 C NMR spectra were recorded on a Varian Gemini 200 apparatus or a Varian Unity Plus 500 apparatus (Varian, Palo Alto, CA, USA), In relation to their stability, the compounds could be ordered as 12 > 13 > 11, with decreasing stability. All substituents in the R 1 position can undergo oxidation, and this occurs for substituents in several positions for 4-chlorophenyl (11) and 1-naphthyl (13).
Interestingly, in opposition to the Xenosite's results, the derivative bearing a piperonyl moiety (12) exhibited the best stability in vitro among the studied set of compounds. A detailed survey of possible reasons for this property suggested that another part of the studied molecules can be more important for metabolic stability than the R 1 substituents, thus diminishing their influence.
The most probable hypothesis includes oxidation of the sulfur atom in the thione functionality, resulting in the formation of sulfenic and, subsequently, sulfinic acid. This kind of biotransformation was reported several times in the literature, including in a detailed study of several thioureas and thiones by Henderson and others [46] and also in a paper by Yamazaki et al. for methimazole [47].

Synthesis
The melting points were uncorrected and measured using a Thermogalen (Leica, Vienna, Austia) apparatus. The IR spectra were measured on a Thermo Mattson Satellite FTIR spectrometer (Thermo Mattson, Madison, WI, USA) in KBr pellets; the absorption range was 400-4000 cm −1 . The 1 H NMR and 13 C NMR spectra were recorded on a Varian Gemini 200 apparatus or a Varian Unity Plus 500 apparatus (Varian, Palo Alto, CA, USA), as well as on a Bruker Ascend 600 spectrometer (Bruker, Billerica, MS, USA). The chemical shifts are expressed at δ values relative to Me 4 Si (TMS) as an internal standard. The apparent resonance multiplicity is described as: s (singlet), br s (broad singlet), d (doublet), t (triplet) and m (multiplet). Elemental analyses were performed on a PerkinElmer 2400 Series II CHN Elemental Analyzer (Perkin Elmer, Shelton, CT, USA), and the results indicated by the symbols of the elements were within ±0.4% of the theoretical values. Thin-layer chromatography (TLC) was performed on Merck Kieselgel 60 F254 plates (Merck, Darmstadt, Germany) and visualized by UV spectroscopy. An HPLC-UV analysis was performed on anAgilent 1260 liquid chromatograph equipped with a VWD detector (Agilent, Santa Clara, CA, USA). A Poroshell EC-C18 column (150 × 3 mm, 2.7 um) (Agilent, Santa Clara, CA, USA) was used at the flow rate of 0.2 mL/min. The injection volume was 5 µL. Gradient elution was applied as follows: a linear increase of acetonitrile in water from 5% to 100% over 30 min. Detection was performed at 254 nm.
General procedure for the synthesis of 2-alkythio-4-chloro-N-[imino(heteroaryl)methyl] benzenesulfonamide (8-24) Method A. A mixture of monopotassium salt (1.5 mmol), p-toluenesulfonic acid monohydrate (PTSA) (1.5 mmol) and an appropriate thiol (1.5 mmol) in dry toluene (25 mL) was stirred at reflux for 14-28 h. After cooling to room temperature, an insoluble side product was filtered out. The organic layer was washed with water (2 × 10 mL), then dried with MgSO 4 and concentrated in vacuum. The residue was dissolved in a hot solvent (acetonitrile for compounds 8 and 12, benzene for 14, ethanol for 15) and left to crystallize at room temperature. The precipitate was collected by filtration and dried.
Method B. A mixture of monopotassium salt (1.5 mmol), PTSA (1.5 mmol) and an appropriate thiol (1.5 mmol) in dry p-dioxane (8 mL) was stirred at 105 • C for 4-5 h. After cooling to room temperature, the mixture was concentrated in vacuum to dryness, and the residue was treated with water (20 mL) and stirred using an ultrasonic bath for 5 min. The precipitate was filtered off, dried and crystallized from ethanol (compounds 9 and 16) or ethanol/acetonitrile mixture (v/v = 4:1) (compound 13).
Method C. A mixture of monopotassium salt (1.5 mmol), PTSA (1.5 mmol) and an appropriate thiol (1.5 mmol) in dry toluene (25 mL) was stirred at reflux for 14 h. After cooling to room temperature, the solid was filtered off and dried. The products were purified by crystallization from acetonitrile (compound 19).

Method D.
A mixture of monopotassium salt (1.5 mmol), PTSA (1.5 mmol) and an appropriate thiol (1.5 mmol) in dry p-dioxane (8 mL) was stirred at 105 • C for 1.5-7 h. After cooling to room temperature, an insoluble side product was filtered out, then the filtrate was concentrated in vacuum to dryness, and the residue was treated with water (20 mL) and stirred using an ultrasonic bath for 5 min. The precipitate was filtered off, dried and crystalized from ethanol (compounds 20-24) or purified by gravity liquid chromatography using silica gel with pore size 60 Å, 220-440 mesh particle size and 35-75 µm particle size (compounds 10, 11, 17, 18).
Data collection and data reduction were controlled by the X-Area 1.75 program (STOE, 2015). An absorption correction was performed on the integrated reflections by a combination of frame scaling, reflection scaling and a spherical absorption correction. Outliers were rejected according to the Blessing's method. The structures were solved by direct methods and refined anisotropically using the program packages OLEX2 and SHELX-2015. The positions of the C-H hydrogen atoms were calculated geometrically and taken into account with isotropic temperature factors. The amine hydrogen atoms H2a and H2b were found in the Fourier residual electron density map and were refined with the N-H distance restrained to 0.88(2) Å.
The crystal data, data collection and structure refinement details are summarized in Tables S1-S2.
Crystallographic data for the structure of 10 reported in this paper were deposited in the Cambridge Crystallographic Data Centre as a supplementary publication, No. CCDC 1832901. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: (þ44) 1223-336-033; Email: deposit@ccdc.cam.ac.uk).

Cell Culture and Cell Viability Assay
All chemicals, if not stated otherwise, were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MCF-7, HeLa and HaCaT cell lines were purchased from Cell Lines Services (Eppelheim, Germany), and the HCT-116 cell line was purchased from ATCC (ATCC-No: CCL-247). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL of penicillin and 100 µg/mL of streptomycin. The cultures were maintained in a humidified atmosphere with 5% CO 2 at 37 • C, in an incubator (Heraeus, HeraCell).
Cytotoxicity assay: Cell viability was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) assay. The cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and treated for 24, 48 and 72 h with the examined compounds in the concentration range 1-100 µM. Following treatment, MTT (0.5 mg/mL) was added to the medium, and the cells were further incubated for 2 h at 37 • C. the cells were lysed with DMSO, and the absorbance of the formazan solution was measured at 550 nm with a plate reader (Victor, 1420 multilabel counter). The experiment was performed in triplicate. The values are expressed as the mean ± SD of at least three independent experiments.
Detection of apoptosis by Annexin V-PE and 7-AAD staining: Apoptosis induction was detected with an Annexin V-PE Apoptosis Detection Kit I (BD Biosciences, Belgium) according to the manufacturer's instructions. The cells were treated with 11-13 (2.5, 5 and 10 µM) for 24 and 48 h. Following treatment, the cells were collected and stained with Annexin V-phycoerythrin (PE) and 7-amino-actinomycin (7-AAD) in Annexin-binding buffer for 15 min at RT in the dark. The acquisition was performed on a FACSCalibur cytometer (BD), and the data were analyzed with Flowing software (version 2.5).
Caspase Activity Determination: Caspase activity was determined with the FLICA Apoptosis Detection Kit (Immunochemistry Technologies) according to the manufacturer's instructions. The cells were treated with comp. 11, 12 and 13 (5, 7 and 10 µM) for 24 h, after which the cells were collected and suspended in a buffer containing the caspase inhibitor, i.e., a carboxyfluorescein-labeled fluoromethyl ketone peptide. The cells were subsequently incubated for 1 h at 37 • C under 5% CO 2 and, next, they were washed with a washing buffer. The fluorescence intensity of fluorescein was determined by flow cytometry (BD FACSCalibur), and caspase activity was determined as the amount of fluorescence emitted from the caspase inhibitors bound to the caspases. The data were analyzed with Flowing software (version 2.5).
Cell Cycle Distribution Analysis: The effects of 11, 12 and 13 on the cell cycle distribution in HeLa cells were determined by flow cytometry analysis. The cells were treated with 11, 12 and 13 (5, 7 and 10 µM) for 48 h, after which they were fixed in cold 70% ethanol for 24 h. The fixed cells were treated with 100 µg/mL of RNAse (Invitrogen, Germany) and stained with 10 µg/mL of PI (Invitrogen, Germany) for 30 min at RT. The acquisition was performed on a FACSCalibur cytometer (BD), and the data were analyzed with Flowing software (version 2.5).
Statistical Analysis: Values are expressed as means ± SD of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad software). Differences between control and treated samples were analyzed by one-way ANOVA with Tukey's post hoc tests. A p-value < 0.05 was considered statistically significant in each experiment.

In Vitro Metabolic Stability Assay
Stock solutions of the studied compounds were prepared at a concentration of 10 mM in DMSO. Working solutions were prepared by dilution of the stock solutions with reaction buffer or acetonitrile; the final concentration of the organic solvent did not exceed 1%. The incubation mixture contained the studied derivative at a 10 µM concentration, 1 mM NADPH (Sigma-Aldrich) and 0.5 mg/mL of human liver microsomes (HLM, Sigma-Aldrich) in potassium phosphate buffer (0.1 M, pH 7.4). The incubation was carried out in a thermostat at 37 • C and started by the addition of the compound of interest. Then, 20 µL samples were taken after 5, 15, 30, 45 and 60 min. The enzymatic reaction was terminated by the addition of an equal volume of ice-cold acetonitrile containing 10 µM sulfamethoxazole as an internal standard. Control incubations were performed without NADPH as a negative control reflecting NADPH-independent processes, such as chemical degradation and precipitation. After collection, the samples were immediately centrifuged (10 min, 10,000 rpm), and the resulting supernatant was directly analyzed or kept at −80 • C until LC-MS analysis. The natural logarithm of a compound over the IS peak area ratio was plotted vs. the incubation time. The metabolic half-time (t 1 2 ) was calculated from the slope of the linear regression, as demonstrated [51].
The LC-MS analysis was performed on an Agilent 1260 system coupled to SingleQuad 6120 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Poroshell EC-C18 (2.1 mm × 150 mm, 2.7 µm, Agilent Technologies, Santa Clara, CA, USA) was used in reversed-phase mode with gradient elution starting with 90% of phase A (0.1% formic acid in water) and 10% of phase B (0.1% dormic acid in acetonitrile). The amount of phase B was linearly increased to 100% in 15 min and equilibrated. The total analysis time was 21 min at 25 • C, the flow rate was 0.5 mL/min, and the injection volume was 20 µL. The mass spectrometer was equipped with an electrospray ion source and operated in positive ionization mode. The mass analyzer was set for each individual compound to detectthe [M+H] + protonated molecule. The MSD parameters of the ESI source were as follows: nebulizer pressure 40 psig (N 2 ), drying gas 10 mL/min (N 2 ), drying gas temperature 300 • C, capillary voltage 3.0 kV, fragmentor voltage 150 V.

Conclusions
In conclusion, our study focused on the design and synthesis of novel benzenesulfonamide derivatives containing imidazole rings as potential anticancer agents. The cytotoxic evaluation revealed that compounds 11-13 exhibited remarkable selectivity and efficacy against HeLa cervical cancer cells, with IC 50 values of 6-7 µM. These compounds showed significantly lower cytotoxicity towards the non-tumor cell line HaCaT (IC 50 : 18-20 µM). The enhanced anticancer activity of compounds 11-13 was further supported by their ability to induce apoptosis, as demonstrated by increased phosphatidylserine externalization, caspase activation, and DNA fragmentation. Additionally, an in vitro metabolic stability assay suggested potential oxidation pathways for these compounds. These findings highlight the potential of benzenesulfonamide-imidazole hybrids as promising candidates for the development of new and effective anticancer agents. However, further research is necessary to optimize their design, enhance their efficacy and ensure their safety.