Novel Sulfonamide Derivatives as Anticancer Agents, VEGFR‑2 Inhibitors, and Apoptosis Triggers: Design, Synthesis, and Computational Studies

Abstract

Background: Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a pivotal role in angiogenesis and tumor progression. Targeting VEGFR-2 remains a validated strategy for anticancer drug development. Objective: This study aimed to design, synthesize, and evaluate a novel series of sulfonamide derivatives as potential VEGFR-2 inhibitors with anticancer activity. Methods: A series of sulfonamide derivatives were synthesized through multistep organic reactions. Their structures were confirmed by spectroscopic methods. In vitro antiproliferative activity was evaluated against HCT-116, HepG-2, and MCF-7 cancer cell lines using MTT assays. Selectivity was assessed via cytotoxicity against normal WI-38 fibroblasts. Compounds showing potent anticancer activity were further examined for VEGFR-2 and EGFR kinase inhibition, cell cycle progression, and apoptosis induction. Molecular docking and in silico ADMET/toxicity analyses supported the pharmacological evaluation. Results: Among the tested compounds, 3a, 6, and 15 exhibited potent cytotoxicity against all cancer cell lines, with compound 6 showing IC₅₀ values of 3.53, 3.33, and 4.31 μM against HCT-116, HepG-2, and MCF-7, respectively. These compounds showed minimal cytotoxicity against WI-38 cells (IC₅₀ > 69 μM), indicating favorable selectivity. Compound 15 exhibited the strongest VEGFR-2 inhibition (IC₅₀ = 0.0787 μM), while compound 3a was the most potent EGFR inhibitor (IC₅₀ = 0.17 μM). Flow cytometry revealed that compounds 3a, 6, and 15 induced G2/M and Pre-G1 phase arrest and significantly enhanced apoptosis. Docking studies demonstrated favorable binding interactions with VEGFR-2. ADMET predictions suggested acceptable drug-likeness and safety profiles. Conclusion: Compounds 3a, 6, and 15 represent promising VEGFR-2-targeting sulfonamides with potent, selective anticancer activity and favorable pharmacokinetic and safety profiles, warranting further development as lead candidates.

1. Introduction

Cancer, as a serious health trouble, comes in the second order as a life-threatening disease after cardiovascular diseases. According to WHO, there are 9.6 million deaths due to cancer in 2018. The efforts of scientists have introduced many anticancer drugs. However, many adverse effects characterize the discovered anticancer molecules. The drawbacks of anticancer drugs arise from their nonselectivity. The most famous drawbacks are hematological, gastrointestinal, and hair follicle toxicity. In addition, anticancer drugs have a bad effect on the nervous system, liver function, heart, urinary tract, and lung. Therefore, medicinal chemists still make high efforts to discover new anticancer agents with low toxicity and high selectivity. −

Protein tyrosine kinases (PTKs) are considered as vital macromolecules that participate in cell proliferation and differentiation. It was found that PTKs form about 60% of all oncoproteins and proto-oncoproteins (the player makers of tumor growth). Generally, PTKs have a central role in the growth and progression of cancer mass. Accordingly, much attention is paid to PTKs as a promising biological target in the field of anticancer agent drug discovery.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is a prominent member of the protein tyrosine kinase (PTK) family. It possesses a superior duty in the development of fresh blood vasculature (angiogenesis). In pathological conditions, VEGFR-2 is overexpressed in certain tissues to accelerate angiogenesis and facilitate tumor growth. , VEGFR-2 is the biological target of vascular endothelial growth factor (VEGF). Therefore, the blockage of VEGF/VEGFR-2 interaction can control the growth of tumor mass. Based on these facts, many FDA-approved anticancer agents play in the area of VEGFR-2 inhibition.

Until now, there have been many FDA-approved VEGFR-2 inhibitors, Figure . These inhibitors can be classified into chief categories in conformity with the main chemical nucleus. The first category is the pyridine derivatives such as sorafenib I and regorafenib II. , The second category is the indoline derivatives as sunitinib III and toceranib IV. The third category is the quinoline derivatives as lenvatinib V and cabozantinib VI. − Recently, many efforts have been done to modify such drugs to reach less toxic anticancer agents. ,

1.

Some FDA-approved VEGFR-2 inhibitors for the clinical treatment of cancer.

VEGFR-2 inhibitors have four pharmacophoric features to interact with the site of VEGFR-2. These features are a heteroaromatic moiety, a spacer group, a pharmacophore group, and a hydrophobic tail. − These features can bind the different pockets of the active site as the hinge region, gatekeeper region, DFG domain, and allosteric pocket, respectively, − Figure .

2.

Summary for the rationale of molecular design.

Sulfonamide derivatives are a family of compounds that exert a strong effect in tumor management. Some sulfonamide derivatives (such as pazopanib and its related compounds) have VEGFR-2 inhibitory activity. − So that targeting the sulfonamide derivatives may encourage the discovery of new promising anticancer agents. In continuation of our efforts to discover new VEGFR-2 inhibitors, − a new series of sulfonamide derivatives have been designed and synthesized to possess the essential pharmacophoric features of VEGFR-2 inhibitors, hoping to reach a promising anticancer agent.

1.1. The rationale of Molecular Design

Recently, our research group has designed and synthesized nicotinamide derivatives VII, VIII, and IX, Figure . These compounds showed promising antiproliferative and VEGFR-2 inhibitory activities. In addition, such compounds exhibited a promising apoptotic effect. So that, it was decided to modify these lead compounds to reach more effective anticancer agents targeting VEGFR-2 active site.

The VEGFR-2 active site comprises four key subregions. The hinge region is responsible for anchoring the core structure through hydrogen bonds, notably involving residues Cys917 and Leu838. The DFG motif domain plays a critical role in kinase activation and is often targeted via interactions with Glu883 and Asp1044. The gatekeeper region is generally hydrophobic and commonly involves residues such as Val914 and Phe1045. Lastly, the allosteric pocket contributes to inhibitor selectivity and stabilization, with key residues including Leu887, Ile890, and Lys866.

The modification tactic involved the change of the nicotinamide moiety of the lead compounds with N-sulfonylpiperidine moiety to occupy the hinge region of the active site. Such modification was based on the ability of the sulfonamide derivative to hinder cancer growth. In addition, the linker moiety (phenylethylidene) was retained without modification. Furthermore, the pharmacophore moiety was modified to be hydrazinecarbodithioate (compounds 3a,b), hydrazinecarbothioamide (compounds 4 and 5), (Z)-carbamohydrazonothioic acid (compounds 6 and 7), carbonohydrazonodithioic acid (compounds 8, 9, & 10), hydrazine (compound 12), and (Z)-2-hydrazono-2,3-dihydro-1,3,4-thiadiazole (compound 15) moieties. Finally, different aromatic and aliphatic moieties were utilized as a hydrophobic tail, Figure .

2. Result and Discussion

2.1. Organic Synthesis

Four Schemes – were followed to clarify the synthetic methods of target compounds. The inaugural ketone derivative 1. , was heated with methyl/benzyl hydrazinecarbodithioate 2c,d − [prepared from the reaction of aqueous hydrazine (80%) and an appropriate inorganic sulfur compound, such as carbon disulfide in isopropyl alcohol (IPA), in the presence of aqueous caustic potash (KOH), producing a nonisolable intermediate known as potassium hydrazinecarbodithioate 2a,b. These intermediates were then treated with monoiodomethane (CH₃I) and/or (Chloromethyl)­benzene (C₆H₅CH₂Cl) through methylation and/or benzoylation processes, leading to the yielding of the required chemicals] in ethanol to afford the dithioesters 3a,b The reactivity of compound 3a toward some secondary amines was studied. Thus, compound 3a was allowed to react with dimethylamine and/or diethyl amine under refluxing ethanol to produce the corresponding thiosemicarbazone derivatives 4 and 5. This reaction is characterized by the evolution of methyl mercaptan (tested by sodium nitroprusside moistened filter paper, which converts into a pink color). Using n-butanol as the refluxing solvent in the reaction of compound 3a with dimethyl amine resulted in compound 6. The formation of compound 6 is assumed to proceed via adding dimethyl amine to the thiocarbonyl group of dithioester 3a followed by loss of hydrogen sulfide (tested by lead acetate paper), Scheme . Similarly, the treatment of compound 3a with piperidine in boiling n-butanol afforded the corresponding compound 7.

1. General Synthetic Method of the Thiosemicarbazone-Sulfonamide Conjugates 4–7 .

^a Reagents and conditions: (i). Iso propyl alcohol, potassium hydroxide, H₂O, stirring, ice-bath, 2 h; (ii) monoiodomethane, and/or (Chloromethyl)­benzene, stirring, ice-bath, 2 h; (iii) Ethyl alcohol, reflux, 3 h; (iv) Dimethyl amine, Ethyl alcohol, reflux, overnight; (v) diethyl amine, (vi) Dimethyl amine, n-Butyl alcohol, reflux, overnight; (vii) Piperidine, n-Butyl alcohol, reflux, overnight;

4. Synthesis of the 1,3,4-Thiadiazol-Sulfonamide Derivative 15 .

^a Reagents and conditions: (i) Ethyl alcohol, reflux, 6 h.

Moreover, experimental evidence has indicated that the methylation of 3a using dimethyl sulfate in an ethanolic aqueous solution of sodium hydroxide under stirring at 25 °C led to the formation of dimethyl thioester derivative 8 with a favorable yield. When compounds 3a and 3b were subjected to the reaction with an alkyl halide, such as (Chloromethyl)­benzene, in boiling anhydrous ethyl alcohol in the presence of a catalytic amount of TEA, achieved bis-alkylthio derivatives 9 and 9,10 were obtained in a good yield, as depicted in Scheme .

2. General Synthetic Procedure of the S-Dimethyl/Benzyl Thiosemicarbazone-Sulfonamide Conjugates 8–10 .

^a Reagents and conditions: (i) Dimethyl sulfate, sodium hydroxide, stirring, r.t, 3 h; (ii) (Chloromethyl)­benzene, Ethyl alcohol, triethyl amine, reflux, 3–4 h.

Prompted by the results, the dithioester derivative 3a was exploited as a precursor for preparing a new sulfonamide derivative. Hence, when the dithioester derivative 3a was treated with 99% Hydrazinium hydroxide in refluxing ethyl alcohol, the hydrazine derivative 12 was afforded. The last was formed by the elimination of methyl hydrazinecarbodithioate of the proposed intermediate 11. On the other hand, the thiocarbohydrazide derivative 13 was not formed.

The confirmation of the proposed structure for compound 12 was achieved by the synthesis of this compound via an alternative reaction route. Hence, the reaction between precursor 1 and hydrazine hydrate in absolute ethanol under reflux led to the formation of a compound with identical characteristics (melting point, mixed melting point, and spectral analysis), indicating that it is compound 12, Scheme .

3. Synthesis Procedure for the Hydrazone-Sulfonamide Derivative 12 .

^a Reagents and conditions: (i) 99% NH₂NH₂.H₂O, Ethyl alcohol, reflux, 4 h.

Lastly, the reaction of dithioester derivative 3a with hydrazonoyl halide derivative − ((Z)-2-oxo-N-2-diphenylacetohydrazonoyl bromide) 14, in boiling ethyl alcohol in the existence of triethylamine as a catalyst, furnished ((5Z)-5-((1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl) ethylidene) hydrazineylidene)-4-phenyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)­(phenyl)­methanone 15, Scheme .

In conformity with antecedent results, the mechanism illustrated in Figure is the most presumable route for the formation of 15 in the reaction of 3a with hydrazonoyl bromide 14. The reaction anticipates the first formation of the hydrazonic thioanhydride 14a which tolerates intramolecular ring closure as soon as it is formed to yield the intermediate 14c or be subjected to 1,3-dipolar cycloaddition of nitrilimine 14b [originated in situ from 14 with triethylamine] to the thiocarbonyl (CS) of 3a. Mercapto methane was eliminated from compound 14c to produce compound 15.

3.

Proposed reaction mechanism of formation of 1,3,4-thiadiazole derivative 15.

The structural characterization of the newly synthesized compound was determined through a comprehensive analysis of various analytical and spectroscopic findings, particularly FTIR, ¹H NMR, ¹³C NMR, mass spectrometry, and elemental analysis.

2.2. Biological Results

2.2.1. In Vitro Antiproliferative Activities

2.2.1.1. In Vitro Antiproliferative Activities Against Tumor Cells

Overexpression of VEGF have been noticed in several types of cancer including colorectal cancer, breast cancer and hepatocellular carcinoma. − Accordingly, the antiproliferative effects of the synthesized compounds were assessed using the MTT assay against a panel of human cancer cell lines, including colorectal carcinoma (HCT-116), hepatocellular carcinoma (HepG-2), and breast cancer (MCF-7). Doxorubicin and vinblastine were employed as reference anticancer drugs. The IC₅₀ values of the tested derivatives are shown in Table .

1. In Vitro Anti-Proliferative Effects of the Synthesized Candidates against HCT-116, HepG-2, and MCF-7 Cell Lines.

	IC 50 (μM)
compounds	HCT-116	HepG-2	MCF-7	WI-38
3a	5.58 ± 0.12	4.82 ± 0.22	11.15 ± 0.87	86.26 ± 0.25
3b	117.34 ± 5.15	91.34 ± 3.71	121.99 ± 4.22
4	11.76 ± 0.82	10.09 ± 0.81	19.29 ± 0.77
5	15.12 ± 0.75	10.67 ± 0.63	18.41 ± 0.85
6	3.53 ± 0.22	3.33 ± 0.03	4.31 ± 0.36	86.52 ± 0.35
7	38.71 ± 1.10	33.94 ± 0.15	46.29 ± 2.09
8	12.59 ± 0.55	10.59 ± 0.71	18.77 ± 0.86
9	51.76 ± 1.15	44.93 ± 1.15	78.06 ± 2.36
10	31.75 ± 1.90	24.36 ± 1.11	32.11 ± 0.54
12	87.34 ± 6.20	59.65 ± 3.30	96.14 ± 5.23
15	3.66 ± 0.25	3.31 ± 0.67	4.29 ± 0.03	69.68 ± 0.25
vinblastine	3.21 ± 0.21	7.35 ± 0.52	5.83 ± 0.61
doxorubicin	6.74 ± 0.40	7.52 ± 0.31	8.19 ± 0.62
sorafenib	9.30 ± 0.20	7.40 ± 0.25	6.72 ± 0.16	35.71 ± 0.013

^aThe results are the meaning of three experiments.

HCT-116 (colorectal carcinoma), HepG2 (hepatocellular carcinoma), and MCF-7 (breast adenocarcinoma) were chosen for their clinical relevance and molecular diversity. Importantly, these cell lines are reported to exhibit elevated VEGFR-2 expression, which aligns with the proposed mechanism of action of our sulfonamide derivatives as VEGFR-2 inhibitors. −

The results demonstrated a diverse range of activities among the synthesized compounds. The most potent effects were observed with compounds 3a, 6, and 15, with compounds 6 and 15 exhibiting comparable activity. Specifically, compound 6 showed promising antiproliferative effects against HCT-116, HepG-2, and MCF-7, with IC₅₀ values of 3.53, 3.33, and 4.31 μM, respectively. Likewise, compound 15 displayed strong cytotoxicity against the same cell lines, with IC₅₀ values of 3.66, 3.31, and 4.29 μM, respectively.

Both compounds 6 and 15 demonstrated activity comparable to or greater than vinblastine, which exhibited IC₅₀ values of 3.21, 7.35, and 5.83 μM against HCT-116, HepG-2, and MCF-7, respectively. Also, these compounds showed higher activities than sorafenib (a standard VEGFR-2 inhibitor) which showed IC₅₀ values of 9.30 ± 0.20, 7.40 ± 0.25, and 6.72 ± 0.16 μM against HCT-116,, HepG2, and MCF-7, respectively.

Additionally, these compounds outperformed doxorubicin, which showed IC₅₀ values of 6.74, 7.52, and 8.19 μM against the same cell lines. Compound 3a also exhibited notable activity, with IC₅₀ values of 5.58, 4.82, and 11.15 μM against HCT-116, HepG-2, and MCF-7, respectively. Notably, compound 3a demonstrated higher potency than vinblastine against HepG-2 and surpassed doxorubicin in activity against both HCT-116 and HepG-2. This compound showed higher activities than Sorafenib against HCT-116 and HepG2.

Additionally, compounds 7, 10, and 14 exhibited moderate antiproliferative activity, with IC₅₀ values ranging from 11.16 to 19.48 μM. In contrast, compounds 3b, 5, 6, 11, 12, and 13 showed weak cytotoxicity, with IC₅₀ values between 24.62 and 121.99 μM.

2.2.1.2. Cytotoxicity against Normal (WI-38) Cells

The cytotoxicity data against the normal human lung fibroblast WI-38 cell line provides insight into the selectivity and safety profile of the tested compounds. A high IC₅₀ value against WI-38 suggests lower toxicity to normal cells, which is a desirable trait for potential anticancer agents (Table ).

Among the tested compounds, 3a and 6 showed potent anticancer activity (e.g., IC₅₀ values of 3.53–5.58 μM on cancer cell lines), yet both exhibited high IC₅₀ values against WI-38 (86.26 ± 0.25 and 86.52 ± 0.35 μM, respectively). This large therapeutic window implies a high degree of selectivity toward cancer cells, making them promising candidates for further development.

Compound 15 also showed potent cytotoxicity against cancer cell lines (IC₅₀ = 3.3–4.3 μM), with a moderately high IC₅₀ of 69.68 ± 0.25 μM against WI-38. Although its selectivity is somewhat lower than that of 3a and 6, it still suggests a favorable safety profile.

By contrast, sorafenib, a reference drug, displayed a WI-38 IC₅₀ of 35.71 ± 0.013 μM, indicating higher toxicity to normal cells compared to 3a, 6, and 15. Overall, the data suggest that compounds 3a and 6 combine potent anticancer effects with low cytotoxicity to normal cells.

2.2.2. Kinase Profiling

2.2.2.1. In Vitro VEGFR-2 Inhibitory Activity

The most potent antiproliferative compounds, 3a, 6, and 15, were evaluated for their in vitro VEGFR-2 inhibitory activity, with sorafenib serving as the reference compound. The IC₅₀ values summarizing their inhibitory effects on VEGFR-2 are presented in Table .

2. VEGFR-2 and EGFR Inhibitory Activities of the Tested Compounds.

comp.	VEGFR-2(IC 50  μM)	EGFR(IC 50  μM)
3a	0.2007 ± 0.007	0.17 ± 0.001
6	1.5073 ± 0.055	0.25 ± 0.005
15	0.0787 ± 0.003	1.31 ± 0.04
sorafenib	0.0416 ± 0.002	0.2002 ± 0.001

^aThe results are the meaning of three experiments.

Notably, the results of the VEGFR-2 inhibition assay aligned with the cytotoxicity data, confirming that the most effective antiproliferative compound, 15, also exhibited significant VEGFR-2 inhibitory activity. It achieved an IC₅₀ value of 0.0787 μM, which was comparable to that of sorafenib (0.0416 μM). Additionally, compounds 3a and 6 displayed moderate VEGFR-2 inhibition, with IC₅₀ values of 0.2007 and 1.5073 μM, respectively.

2.2.2.2. In Vitro EGFR Inhibitory Activity

The inhibitory activities of compounds 3a, 6, and 15 against EGFR were assessed and compared with the reference drug sorafenib. The IC₅₀ values (μM) indicate the concentration at which each compound inhibits 50% of EGFR activity. Lower IC₅₀ values correspond to higher inhibitory potency.

Compound 3a exhibited the most potent EGFR inhibition among the tested compounds, with an IC₅₀ of 0.17 ± 0.001 μM, slightly more potent than sorafenib (0.2002 ± 0.001 μM). This suggests that 3a may serve as a promising lead candidate for EGFR-targeted anticancer therapy, potentially offering greater efficacy than the standard comparator.

Compound 6 also demonstrated strong inhibitory activity with an IC₅₀ of 0.25 ± 0.005 μM, close to that of sorafenib, indicating its potential as an effective EGFR inhibitor as well. Although slightly less potent than 3a, its comparable activity suggests structural features contributing to effective binding at the EGFR active site.

In contrast, compound 15 showed considerably weaker inhibition, with an IC₅₀ of 1.31 ± 0.04 μM, indicating a lower binding affinity and suggesting that its structural features are less favorable for EGFR interaction.

2.2.3. Structure–Activity Relationship

The structure–activity relationship (SAR) analysis of the synthesized compounds provides insights into how structural modifications influence their antiproliferative activity against the tested cancer cell lines (HCT-116, HepG-2, and MCF-7). The detailed SAR analysis based on the IC₅₀ values is provided in Table . All compounds share a common sulfonamide-piperidine scaffold, which appears to be essential for maintaining baseline activity. Modifications to this core structure (e.g., removal or replacement) would likely result in a loss of activity. Compounds 6 and 15 were the most potent against HCT-116, with IC₅₀ values (3.53 and 3.66 μM, respectively) comparable to Vinblastine (3.21 μM) and Doxorubicin (6.74 μM). Similar trends were observed, with 6 and 15 showing the highest potency (IC₅₀: 3.33 and 3.31 μM, respectively) against HepG-2 as compared to Vinblastine (7.35 μM) and Doxorubicin (7.52 μM), and 6 and 15 remained the most active, their IC₅₀ values were slightly higher (4.31 and 4.29 μM, respectively) against MCF-7, as compared to the standard drugs Vinblastine (5.83 μM) and Doxorubicin (8.19 μM), while compounds 3b, 9, 10, and 12 showed significantly lower activity, with IC₅₀ values exceeding 30 μM in most cases. This indicates that the substitution at the hydrazine/carbohydrazonothioate moiety influenced activity. In addition, the presence of a methyl group at the hydrazine and the thiadiazole moieties appears to enhance activity, likely by improving lipophilicity and binding affinity of compound, 6,15. Furthermore, compounds containing benzyl groups (e.g., 3b, 9, 10) showed markedly reduced activity compared to methyl-substituted analogs. This suggests that the bulkier benzyl group may hinder interactions with the target protein or reduce cellular uptake. Finally, the other compounds had moderate and weak-to-fair cytotoxic activities.

Based on the data provided in Tables and , here is a structure–activity relationship (SAR) analysis for the synthesized compounds:

• 1.Core structure:

• All compounds contain a 4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl ethylidene hydrazine core.

• 2.Antiproliferative activity:

• Compounds 6 and 15 showed the highest potency across all three cancer cell lines (HCT-116, HepG-2, and MCF-7), with IC50 values comparable to or better than the reference drugs vinblastine and doxorubicin.

• Compound 3a also demonstrated good activity, especially against HCT-116 and HepG-2 cell lines.

• 3.Substituent effects:

• Methyl carbodithioate derivatives 3a showed significantly better activity compared to benzyl carbodithioate derivatives 3b, suggesting smaller alkyl groups are preferred.

• N,N-dimethyl substitution 4 resulted in better activity than N,N-diethyl substitution 5, further supporting the preference for smaller alkyl groups.

• The presence of a phenyl ring, as in compound 15, appears to enhance activity.

• 4.VEGFR-2 Inhibition:

• Compound 15, containing a thiadiazole ring and two phenyl groups, showed the highest VEGFR-2 inhibitory activity among the tested compounds, approaching the potency of sorafenib.

• Compound 3a also demonstrated good VEGFR-2 inhibition, while compound 6 was less potent but still active.

• 5.Structure–activity trends:

• The presence of a thiadiazole ring 15 or a carbodithioate group 3a appears to enhance both antiproliferative and VEGFR-2 inhibitory activities.

• Bulkier substituents (e.g., benzyl groups in 3b, 9, 10) generally resulted in reduced antiproliferative activity.

• The unsubstituted hydrazine 12 showed poor activity, highlighting the importance of additional functional groups for potency.

In conclusion, compounds 6, 15, and 3a emerge as the most promising candidates, demonstrating potent antiproliferative activity across multiple cancer cell lines and significant VEGFR-2 inhibition. The SAR suggests that smaller alkyl substituents, thiadiazole rings, and carbodithioate groups contribute positively to the compounds’ biological activities.

2.2.4. Cell Cycle Analysis against HepG2 Cells

The flow cytometric analyses for cell cycle progression and apoptosis were conducted as single measurements for each of the most active compounds. While these data provide informative indications of the compounds’ effects on cell cycle arrest and apoptosis induction, they should be considered as preliminary mechanistic evidence.

Subsequently, the effects of compounds 3a, 6, and 15 on the cell cycle progression of HepG2 cells were evaluated. The cells were treated with concentrations of 4.82, 3.33, and 3.31 μM, respectively. As shown in Table and Figure , treatment with these compounds resulted in an increased cell population in the G2/M and Pre-G1 phases, while a reduction was observed in the G0-G1 and S phases.

3. Effect of Compounds 3a, 6, and 15 on Cell Cycle Progression in HepG2 Cells.

sample	%G0-G1	%S	%G2/M	%Pre-G1
compound3a/HepG2	32.59	32.56	34.85	11.82
compound6/HepG2	35.29	33.42	31.29	22.04
compound15/HepG2	26.89	34.62	38.49	22.8
doxorubicin/HepG2	48.26	30.28	21.46	31.28
Cont.HepG2	46.72	40.55	12.73	1.92

4.

Cell cycle analysis of the synthesized compounds in the HepG2 cell line.

In the G2/M phase, the percentage of HepG2 cells increased from 12.73% in the control group to 34.85, 31.29, and 38.49% following treatment with compounds 3a, 6, and 15, respectively. Similarly, for the Pre-G1 phase, the cell population rose from 1.92% in untreated HepG2 cells to 11.82, 22.04, and 22.80%, respectively. These findings were in line with doxorubicin’s effects, which elevated the cell population to 21.46% in the G2/M phase and 31.28% in the Pre-G1 phase.

In conclusion, compounds 3a, 6, and 15 effectively induced cell cycle arrest in HepG2 cells at both the G2/M and Pre-G1 phases.

2.2.5. Apoptosis Analysis against HepG2 Cells

The Annexin V and PI double staining experiment was used to assess the most promising candidates’ induction of apoptosis in tumor cells (3a, 6, and 15). The positive control in this study was doxorubicin.

The apoptotic effects of compounds 3a, 6, and 15 on HepG2 cells are presented in Table and illustrated in Figure . The overall apoptosis rates induced by these compounds were significant, with values of 10.24, 18.92, and 20.28% for compounds 3a, 6, and 15, respectively. Among them, compound 15 exhibited the most pronounced apoptotic effect, markedly higher than the negative control, which showed only 1.40% apoptosis.

4. Apoptotic Effect of Compounds 3a, 6, and 15 on HepG2 Cells.

	apoptosis
sample	total	early	late	necrosis
compound3a/HepG2	11.82	3.59	6.65	1.58
compound6/HepG2	22.04	3.94	14.98	3.12
compound15/HepG2	22.8	6.38	13.9	2.52
doxorubicin/HepG2	31.28	7.43	21.13	2.72
Cont. HepG2	1.92	1.17	0.23	0.52

5.

Apoptosis of the synthesized compounds in the HepG2 cell line.

2.3. Computational Studies

2.3.1. Molecular Docking

Identifying a possible mechanism of action for the synthesized derivatives was a key aspect of our study. This was achieved using MOE 2019 to perform molecular docking with the crystal structure of VEGFR-2 (PDB ID: 2OH4). The reliability of the docking procedure was confirmed by redocking the native ligand into the active site. The results indicated a strong overlap between the native and docked ligands, as illustrated in Figure .

6.

Overlay of the redocked (blue) and native (pink) ligands in the VEGFR-2 active site (RMSD = 1.25 Å) is shown by the wire mesh network.

The binding energies of the synthesized compounds and sorafenib are summarized in Table . Sorafenib used as the reference molecule, exhibited a binding energy of −20.88 kcal/mol. Within the DFG domain, its urea group established three hydrogen bonds with Glu883 and Asp1044. The central phenyl moiety contributed to hydrophobic interactions with Val914, Val846, and Val897. Additionally, the N-methylpicolinamide core formed four hydrophobic interactions with Leu1033, Leu838, Val846, and Ala864 in the hinge region, along with two hydrogen bonds with Cys917. In the allosteric pocket, the 1-chloro-2-(trifluoromethyl)­benzene moiety created five hydrophobic interactions with Leu1017, Ile890, Leu887, and Ile886, as well as one electrostatic interaction with Asp1044, Figure .

5. Docking Energies of the Synthesized Compounds against VEGFR-2.

comp.	ΔG [kcal/mol]	comp.	ΔG [kcal/mol]
3a	–19.26	8	–20.67
3b	–20.62	9	–24.00
4	–18.95	10	–25.55
5	–21.65	12	–16.37
6	–20.78	15	–24.27
7	–21.88	sorafenib	–20.88

7.

3D and 2D interactions of sorafenib with VEGFR-2.

Compound 3a exhibited a binding energy of −19.26 kcal/mol. Within the DFG domain, its hydrazinecarbodithioate moiety established two hydrogen bonds with Glu883 and Asp1044. The central phenyl core contributed to hydrophobic interactions with Val914, Val897, Cys1043, Leu887, and Val897. Additionally, the N-sulfonylpiperidine moiety formed five hydrophobic interactions with Leu1033, Leu838, Phe916, and Cys917 in the hinge region. In the allosteric pocket, the methyl group created a hydrophobic interaction with His1024, Figure .

8.

3D and 2D interactions of compound 3a with VEGFR-2.

Compound 15 exhibited a binding energy of −24.27 kcal/mol. Its 4-methylpiperidine moiety established a hydrophobic interaction with Leu838 and a hydrogen bond with Cys917 by fitting into the hinge region. The central phenyl core occupied the linker region, forming hydrophobic contacts with Cys1043, Val846, Leu1033, and Ala864. Within the DFG motif area, the (Z)-2-hydrazono-2,3-dihydro-1,3,4-thiadiazole moiety of compound 15 engaged in one hydrogen bond and two electrostatic interactions with Glu883, Asp1044, and Phe1045. Additionally, the terminal phenyl moiety occupied the allosteric pocket, forming four hydrophobic interactions with Ala864, Val846, Val914, and Lys866, Figure .

9.

3D and 2D interactions of compound 15 with VEGFR-2.

2.3.2. In Silico ADMET Analysis

Computational ADMET descriptor predictions were performed using Discovery Studio software, with sorafenib as the reference compound. The summarized results of the ADMET analysis are presented in Table .

6. ADMET Parameters of the Synthesized Compounds.

comp.	BBB level	solubility level	absorption level	CYP2D6 prediction	PPB prediction
3a	2	2	0	false	true
3b	1	2	0	false	true
4	2	3	0	false	true
5	2	3	0	false	true
6	2	3	0	false	true
7	1	2	0	false	true
8	1	2	0	false	true
9	1	2	0	false	true
10	4	1	3	false	true
12	3	3	0	false	true
15	4	1	2	false	true
sorafenib	4	1	0	false	true

^aBBB Penetration: 0 = Very High, 1 = High, 2 = Medium, 3 = Low, 4 = Very Low.

^bSolubility Level: 1 = Very Low, 2 = Low, 3 = Good, 4 = Optimal.

^cAbsorption Level: 0 = Good, 1 = Moderate, 2 = Poor, 3 = Very Poor.

^dCYP2D6 (Cytochrome P2D6): TRUE = Inhibitor, FALSE = Non-Inhibitor.

^ePBB (Plasma Protein Binding): FALSE = Less than 90%, TRUE = More than 90%.

The predictions indicated that compounds 3b, 7, 8, and 9 exhibited high blood-brain barrier (BBB) penetration, while compounds 3a, 4, 5, and 6 were predicted to have medium BBB penetration. The remaining compounds were categorized as having low to very low BBB penetration.

Regarding aqueous solubility, all synthesized compounds, except for compounds 10 and 15, were predicted to have low to good solubility levels. In terms of absorption, most tested compounds demonstrated good absorption, except for compounds 10 and 15, which showed poor to very poor absorption levels.

Additionally, all tested compounds were predicted to be noninhibitors of CYP2D6. Lastly, the analysis suggested that the tested compounds exhibited strong plasma protein binding, exceeding 90%.

2.3.3. In Silico Toxicity Analysis

Computational toxicity assessments were conducted using Discovery Studio software, with the predicted values for each compound summarized in Table . For the FDA rodent carcinogenicity (Rat-Male) and Ames prediction models, all tested compounds were predicted to be noncarcinogenic and nonmutagenic, respectively. The carcinogenic potency (TD₅₀) values of the tested compounds were generally higher than that of sorafenib, except for compounds 10 and 15.

7. Predicted Toxicity Profiles of the Synthesized Compounds.

comp.	carcinogenic potency TD50 (mouse)	FDA rodent carcinogenicity (rat-male)	ames prediction	rat oral LD50	rat chronic LOAEL	skin irritancy	ocular irritancy
3a	83.097	non-carcinogen	non-mutagen	0.066	0.049	none	moderate
3b	30.745	non-carcinogen	non-mutagen	0.068	0.056	none	none
4	19.872	non-carcinogen	non-mutagen	0.042	0.056	none	moderate
5	23.233	non-carcinogen	non-mutagen	0.106	0.046	none	moderate
6	22.847	non-carcinogen	non-mutagen	0.039	0.062	none	mild
7	38.469	non-carcinogen	non-mutagen	0.044	0.072	none	moderate
8	91.614	non-carcinogen	non-mutagen	0.048	0.063	none	moderate
9	33.699	non-carcinogen	non-mutagen	0.045	0.073	mild	none
10	12.671	non-carcinogen	non-mutagen	0.100	0.060	mild	none
12	55.102	non-carcinogen	non-mutagen	0.194	0.099	none	moderate
15	8.390	non-carcinogen	non-mutagen	0.195	0.030	none	mild
sorafenib	19.236	non-carcinogen	non-mutagen	0.823	0.005	none	mild

^aUnit: mg/kg body weight/day.

^bUnit: g/kg body weight.

The predicted rat oral LD₅₀ values for the tested compounds ranged from 0.039 to 0.194 mg/kg body weight per day, which are lower than that of sorafenib (LD₅₀ = 0.823 mg/kg body weight per day). Conversely, the tested compounds exhibited rat chronic LOAEL values between 0.030 and 0.099 g/kg body weight, exceeding sorafenib’s value of 0.005 g/kg body weight. Regarding skin irritation potential, all tested compounds, except for compounds 9 and 10, were predicted to be nonirritants. However, all compounds were predicted to cause eye irritation, with the exception of compounds 3b, 9, and 10.

3. Conclusions

A novel series of 11 sulfonamide derivatives was designed, synthesized, and evaluated as potential VEGFR-2 inhibitors with anticancer properties. Among them, compounds 3a, 6, and 15 exhibited the most potent antiproliferative activity against HCT-116, HepG-2, and MCF-7 cancer cell lines, with minimal cytotoxicity against normal WI-38 fibroblasts, indicating good selectivity. Compound 15 showed the strongest VEGFR-2 inhibition (IC₅₀ = 0.0787 μM), while compound 3a demonstrated superior EGFR inhibition (IC₅₀ = 0.17 μM). These compounds also induced G2/M and Pre-G1 phase cell cycle arrest and significantly increased apoptosis in HepG-2 cells. Structure–activity relationship (SAR) analysis revealed that smaller alkyl groups (e.g., methyl in 3a and 6) enhanced antiproliferative potency compared to bulkier benzyl groups (e.g., 3b, 9, 10). The presence of a thiadiazole moiety in compound 15 and carbodithioate in compound 3a improved both VEGFR-2 inhibition and cytotoxicity. Additionally, piperidine-containing hydrophobic tails supported better target engagement. Molecular docking confirmed favorable binding at the VEGFR-2 active site, and in silico ADMET/toxicity studies supported drug-likeness and safety. Collectively, compounds 3a, 6, and 15 represent promising leads for further development as targeted anticancer agents.

4. Material and Method

4.1. Chemistry

The starting materials and solvents were bought from mercantile purveyors and were utilized without further purification. Additional clarification about chemicals and equipment exploited in chemical preparation and analyses were added in the Supporting data. As described in previous articles, ,, compound 1 was prepared following the same procedure.

4.1.2. Synthesis of Intermediates 2c, d

The preparation of the intermediates adhered to the methodology detailed in prior publications.. −

4.1.3. Synthesis of Compounds 3a and b

Intermediates 3a and b were synthesized from derivative 1 and methyl/benzylhydrazine carbodithioate 2a, b according to the prescribed procedure. In brief, Compound 1 (0.01 mmol) was placed in anhydrous ethanol (20 mL) at a temperature of reflux. Reagents 2a, b (0.01 mmol), were introduced into the mixture, and the resulting mixture underwent warming for a duration of 3 h. After that, the precipitate formed was filtered while hot, laundered with hot ethanol, and desiccated with direct air to obtain the desired products 3a and 3b.

4.1.3.1. Methyl (E)-2-(1-(4-((4-Methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­hydrazine-1-carbodithioate, 3a

Pale-yellow solid (80%, dioxane), M. p.; 180–182 °C; IR (KBr disc): 3140 cm^–1 (−NH), 3030 cm^–1 (=C–H aromatic), 2928 (−CH aliphatic), 1339 cm^–1 (-S = O, asymmetric), and 1163 (-S = O, symmetric); ¹H NMR (dimethyl sulfoxde-d₆, 400 MHz): δ _H (ppm) = 0.85­(d, 3H, CH₃-piperidinyl moiety), 1.11,1.63 (br, 4H, 2CH₂–piperidinyl moiety), 1.30 (br, 1H, CH-piperidinyl moiety), 2.22, 3.60 (t, 4H, CH₂–N-CH₂–piperidinyl moiety), 2.43 (s, 3H, CH₃C = N–), 2.49 (s, 3H, SCH₃) 7.77, 8.06 (dd, J = 8 Hz, 4H, AB–Ar-H), 12.59 (s, 1H, NH); ¹³C NMR (dimethyl sulfoxde-d₆, 101 MHz) δ _C (ppm)= 15.14, 17.56, 21.77, 29.70, 29.70, 33.33, 46.52, 46.52,127.73, 127.73, 128.10, 128.10, 136.83, 141.88, 150.19 and 201.26; MS (m/z): 385 [%]: [M⁺, (10%)], Anal. Calcd for C₁₆H₂₃N₃O₂S₃ (385.56): C, 49.84; H, 6.01; N, 10.90; O; Found: C, 49.76; H, 5.52; N, 9.3%.

4.1.3.2. Benzyl (E)-2-(1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­hydrazine-1-carbodithioate, 3b

Pale-yellow solid (88%, dioxane), M.p.; 194–196 °C; IR (KBr, disc): 3147 cm^–1 (−NH), 3029 cm^–1 (−CH aromatic), 2924 cm^–1 (−CH aliphatic) 1337 cm^–1(–S = O, asymmetric), and 1172 cm^–1 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxde-d_6, 400 MHz): δ _H (ppm)= 0.80­(d, 3H, CH₃-piperidinyl moiety), 1.08,1.61­(br, 4H, 2CH₂–piperidinyl moiety), 1.22 (br, 1H, CH-piperidinyl moiety), 2.15, 3.56 (t, 4H, CH₂–N-CH₂–piperidinyl moiety), 2.38 (s, 3H, CH₃C = N–), 4.45­(s,2H, -SCH₂) 7.22,7.38 (m, 5H, phenyl group), 7.71, 8.01­(dd, 4H, J = 8 Hz, AB–Ar-H), 12.65 (s, 1H, NH); ¹³C NMR (dimethyl sulfoxde-d₆, 101 MHz) δ_C (ppm) = 15.25, 21.73, 29.67, 29.7, 33.3, 46.50, 46.50, 56.50, 127.74–138.98, 141.5, 150.03 and 183.35; MS (m/z): 461 [%]:[M⁺, (26%)], Anal. Calcd for C₂₂H₂₇N₃O₂S₃ (461.66): C, 57.24; H, 5.90; N, 9.10; O; Found: C, 57.16; H, 5.80; N, 9.02%.

4.1.3.3. General Procedure for the Synthesis of Compounds 4, 5, and 6

To an ethanolic or n-butanolic solution of 3a (0.01 mol), dimethyl amine or diethyl amine (0.01 mol) was added sequentially, and the reaction contents were refluxed for no more mercapto methane (CH₃SH↑) or hydrogen sulfide (H₂S↑) gas evolved, about 18 h approximately. The reaction mixture was concentrated and allowed to cool; the crude product was filtered, washed with light petroleum (Bp 60–80 °C), and crystallized from methanol. The crystals were filtered, washed with methanol, and dried to yield compounds 4, 5, and 6, respectively.

4.1.3.4. (E)-N,N-Dimethyl-2-(1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene) Hydrazine-1-carbothioamide (4)

White solid (88%), M.p.; 208–210 °C; IR (KBr, disc): 3343 cm^–1 (−NH), 3063 cm^–1 CH aromatic, 2949 cm^–1 (−CH aliphatic), 1338 cm^–1 (–S = O, asymmetric), and 1161 cm^–1 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxde-d_6, 400 MHz) δ_H (ppm) = 0.85 (d, J = 6.3 Hz, 3H, CH₃-piperidinyl moiety),1.15 (t, 2H, CH₂–piperidinyl moiety), 1.30 (br, 1H, CH-piperidinyl moiety), 1.65 (t, J = 11.6 Hz, 2H, CH₂–piperidinyl moiety), 2.21 (t, J = 11.3 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 2.35 (s, 3H, CH₃ C = N–), 3.06 (s, 6H, N­(CH₃)₂), 3.62 (t, J = 11.4 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 7.71 (d, J = 8.4 Hz, 2H, AB–Ar-H), 8.18 (d, J = 8.3 Hz, 2H, AB–Ar-H), 10.41 (s, 1H, NH, commutable by D₂O), ¹³C NMR (Chloroform-d, 101 MHz) δ_C (ppm) = 13.51,21.43, 30.11, 31.43, 33.32, 46.42, 126.74, 127.87,128.80, 128.80,137.02, 141.42, 144.61, 178.95; MS (m/z):382 [%]:[M⁺, (12.25%)], Anal. Calcd for C₁₇H₂₆N₄O₂S₂ (382.54): C,53.38; H, 6.85; N, 14.65; Found: C, 53.30; H, 6.78; N, 14.57%.

4.1.3.5. (E)-N,N-Diethyl-2-(1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­hydrazine-1-carbothioamide (5)

Yellow solid (76%), M.p.; 219–221 °C; IR (KBr, disc): 3290 cm^–1 (−NH), 3020 cm^–1 (−CH aromatic), 2926 cm^–1 (−CH aliphatic), 1336 cm^–1 (–S = O, asymmetric), and 1156 cm^–1 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxide-d_6, 400 MHz) δ_H (ppm) = 0.85 (d, J = 6.4 Hz, 3H, CH₃-piperidinyl moiety), 1.21 (t, J = 7.0 Hz, 8H, CH₂–piperidinyl moiety and 2CH₂CH₃), 1.65 (d, J = 12.6 Hz, 2H, CH₂), 1.30 (s, 1H, CH-piperidinyl moiety), 2.23 (t, J = 11.4 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 2.35 (s, 3H, CH₃ C = N–), 3.63 (d, J = 12.1 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 3.76 (q, J = 7.0 Hz, 4H, 2CH₂CH₃), 7.77 (d, J = 8.1 Hz, 2H AB–Ar-H), 8.00 (d, J = 8.4 Hz, 1H, AB–Ar-H), 9.52 (s, 1H, NH); ¹³C NMR (Chloroform-d,101 MHz) δ_C (ppm) = 12.61,16.56, 21.46, 30.14, 30.14, 33.36, 46.43, 46.43, 46.55, 126.37, 126.64, 127.80, 127.83, 136.45, 141.61, 147.44, 172.31; MS (m/z): 410 [%]:[M⁺, (29%)], Anal. Calcd for C₁₉H₃₀N₄O₂S₂ (410.60): C, 55.58; H, 7.36; N, 13.65; Found: C, 55.51; H, 7.28; N, 13.57%.

4.1.3.6. Methyl (Z)-N,N-Dimethyl-N′-((E)-1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene) Carbamohydrazono Thioate (6)

Yellow solid (70%), M.p.: 238–240 °C; IR (KBr, disc): 3063 cm^–1 (−CH aromatic), 2922 (−CH aliphatic), 1337 (–S = O, asymmetric), and 1165 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxide-d_6, 400 MHz) δ_H (ppm) = 0.85 (d, J = 6.3 Hz, 3H, CH₃-piperidinyl moiety), 1.14 (t, 2H, CH₂–piperidinyl moiety), 1.29 (s, 1H, CH-piperidinyl moiety), 1.64 (d, J = 12.8 Hz, 2H, CH₂–piperidinyl moiety), 2.21 (t, J = 11.9 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 2.31 (s, 3H, CH₃ C = N–), 2.67 (s, 3H, SCH₃), 2.97 (s, 6H, N­(CH₃)₂), 3.61 (d, J = 11.8 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 7.72 (d, J = 8.3 Hz, 2H, AB–Ar-H), 7.94 (d, J = 8.4 Hz, 2H, AB–Ar-H); MS (m/z):396 [%]:[M⁺, (42.48%)], Anal. Calcd for C₁₈H₂₈N₄O₂S₂ (396.57): C,54.52; H, 7.12; N, 14.13; Found: C, 54.44; H, 7.04; N, 14.06%.

4.1.3.7. Synthesis of Methyl­(Z)-N-((E)-1-(4-((4-methylpiperidin-1-yl) sulfonyl) phenyl) ethylidene) piperidine-1-carbohydrazonothioate (7)

A solution of dithioester 3a (0.01 mol) in 30 mL of n-butanol was combined with heterocyclic aliphatic amine, such as piperidine (0.01 mol). The mixture was stirred and refluxed for 18 h, ensuring that no more hydrogen sulfide (H₂S↑) gas was released. Following the evaporation of the solvent through distillation under reduced pressure, the residual material was gathered and recrystallized using an amalgamation of ethanol and benzene, resulting in the formation of compound 7.

Pale-yellow solid (77%), M. p.; 258–260 °C; IR (KBr, disc): 3058 cm^–1 (−CH aromatic), 2928 cm^–1 (−CH aliphatic), 1339 cm^–1 (–S = O, asymmetric), and 1167 cm^–1 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxide-d_6, 400 MHz) δ_H (ppm)= 0.85 (d, J = 6.4 Hz, 3H, CH₃-piperidinyl moiety), 1.15 (t, 2H, CH₂–piperidinyl moiety), 1.29 (s, 1H, CH-piperidinyl moiety), 1.61–1.51 (m, 6H, piperidine-H_{3′,4′,5′}), 1.65 (t, J = 11.9 Hz, 2H, CH₂–piperidinyl moiety), 2.21 (t, J = 12.0 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 2.31 (s, 3H, CH₃C = N–), 2.66 (s, 3H, SCH₃), 3.18 (d, J = 5.0 Hz, 4H, piperidine-H_2′,6′), 3.61 (t, J = 11.5 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 7.72 (d, J = 8.5 Hz, 2H, AB–Ar-H), 7.94 (d, J = 8.5 Hz, 2H, AB–Ar-H); MS (m/z):436 [%]: [M⁺, (4.49%)], Anal. Calcd for C₂₁H₃₂N₄O₂S₂ (436.63): C, 57.77; H, 7.39; N, 12.83; Found: C, 56.69; H, 7.30; N, 12.75%.

4.1.3.8. Synthesis of Dimethyl­(E)-(1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­carbono-hydrazonodithioate (8)

For 3 h, a vigorously agitated suspension solution of dithioester 3a (0.01 mol) in 10% NaOH (25 mL) was supplemented with portions of dimethyl sulfate (0.15 mol). Compound 8 was prepared by filtering the resulting solid, washing it with distilled water (3 × 10 mL) to remove the catalyst, drying it, and recrystallizing it from ethanol.

Pale-yellow solid (69%), M. p; 164–166 °C; IR (KBr, disc): 3065 cm^–1 (−CH-aromatic), 2951 cm^–1 (−CH-aliphatic) and 1333 cm^–1 (–S = O, asymmetric), and 1165 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxde-d_6, 400 MHz) δ_H (ppm) = 0.86 (d, J = 6.3 Hz, 2H, CH₃-piperidinyl moiety);1.16 (t, 2H, CH₂–piperidinyl moiety), 1.31 (s, 1H, CH-piperidinyl moiety), 1.66 (t, J = 11.5 Hz, 2H, CH₂–piperidinyl moiety), 2.24 (t, 2H, CH₂–N-CH₂–piperidinyl moiety), 2.43 (s, 3H, CH₃C = N–), 2.58 (s, 6H, SCH₃), 3.63 (t, J = 8.8 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 7.79 (d, J = 8.1 Hz, 2H, AB–Ar-H), 8.09 (d, J = 8.4 Hz, 2H, AB–Ar-H), MS (m/z): 399 [%]: [M⁺, (100%)], Anal. Calcd for C₁₇H₂₅N₃O₂S₃ (399.59): C, 51.10; H, 6.31; N, 10.52; Found: C, 51.02; H, 6.23; N, 10.43%.

4.1.3.9. General Preparation Procedure for Synthesis of the Target Compounds 9,10

Triethylamine was added catalytically in a volume of 0.5 mL to a combination of 3a, b, and chloromethyl)­benzene in ethyl alcohol (25 mL). For three to 4 h, the reaction mixture was heated under reflux. Compounds 9 and 10 were obtained by filtering the result after the reaction mixture had cooled, washing it with ethanol, and then recrystallizing it from either ethanol or an ethanol and benzene combination.

4.1.3.10. Benzyl Methyl­((E)-1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­carbonohydra-zonodithioate (9)

Pale-yellow solid (90%), M. p; 136- 138 °C; IR (KBr, disc): 3061 cm^–1 (−CH-aromatic), 2927 cm^–1 (CH-aliphatic) and 1336 cm^–1 (–S = O, asymmetric), and 1171 cm^–1 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxide-d_6, 400 MHz) δ_H (ppm) = 0.83 (d, 3H,CH₃-piperidinyl moiety), 1.15 (t, 2H,CH₂–piperidinyl moiety), 1.29 (s, 1H,CH-piperidinyl moiety), 1.64 (t, 2H,CH₂–piperidinyl moiety), 2.23 (t, 2H,CH₂–N-CH₂–piperidinyl moiety), 2.41 (s, 3H,CH₃C = N–), 2.60 (s, 3H,SCH₃), 3.61 (t, J = 10.9 Hz, 2H,CH₂–N-CH₂–piperidinyl moiety), 4.47 (s, 2H, SCH₂Ph), 7.51–7.25 (m, 5H, Ph-H), 7.79 (d, J = 8.7 Hz, 2H, AB–Ar-H), 8.10 (d, J = 8.5 Hz, 2H, AB–Ar-H), ¹³C NMR (dimethyl sulfoxide-d_6, 101 MHz) δ_C (ppm) = 13.90, 15.09, 21.77, 29.67, 29.70, 33.33, 36.28, 46.51, 46.51, 127.82, 127.85, 128.02, 128.02, 128.97, 128.97, 129.09, 129.09, 129.59, 136.78, 137.11, 142.15, 160.05, 165.81; MS (m/z):= 475[%]:[M+, (95.7%)], Anal. Calcd for C₂₃H₂₉N₃O₂S₃ (475.68): C, 58.07; H, 6.15; N, 8.83; Found: C, 57.99; H, 6.08; N, 8.75%.

4.1.3.11. Dibenzyl (E)-(1-(4-((4-methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­carbonohydrazono-dithioate (10)

Pale-yellow solid (87%), M. p; 155–157 °C; IR (KBr, disc): 3060 cm^–1 (−CH-aromatic), 2918 cm^–1 (−CH-aliphatic), 1338 cm^–1 (–S = O, asymmetric), and 1169 cm^–1 (–S = O, symmetric); MS (m/z): = 551 [%]: [M-1, (91%)], Anal. Calcd for C₂₉H₃₃N₃O₂S₃ (551.78): C, 63.13; H, 6.03; N, 7.62;′ Found: C, 63.06; H, 5.94; N, 7.55%.

4.1.3.12. Synthesis of (E)-1-((4-(1-hydrazineylideneethyl)­phenyl)­sulfonyl)-4-methylpiperidine (12)

A mixture of intermediate 3a (0.01 mol) and 99% Hydrazinium hydroxide (0.01 mol) in absolute ethyl alcohol (30 mL) was subjected to boiling for a duration of 4 h. Upon cooling, the resultant solid precipitate was isolated via filtration. The solid was subjected to an ethanol wash, followed by recrystallization from ethanol, yielding compound 12.

Pale-yellow solid (93%), M. p.; 138–140 °C; IR (KBr, disc): 3410:3325 cm^–1 (NH₂), 3064 cm^–1 (−CH-aromatic), 2954 cm^–1 (−CH-aliphatic), 1323 cm^–1 (–S = O, asymmetric), and 1161 (–S = O, symmetric); ¹H NMR (dimethyl sulfoxide-d_6, 400 MHz) δ_H (ppm) = 0.85 (d, J = 6.4 Hz, 3H, CH₃-piperidinyl moiety), 1.14 (t, 2H, CH₂–piperidinyl moiety), 1.29 (br, 1H, CH-piperidinyl moiety), 1.64 (t, J = 12.5 Hz, 2H, CH₂–piperidinyl moiety), 2.05 (s, 3H, CH₃C = N–), 2.18 (t, J = 10.8 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 3.59 (t, J = 11.6 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 6.81 (s, 2H, NH₂ commutable by D₂O), 7.64 (d, J = 8.5 Hz, 2H, AB–Ar-H), 7.84 (d, J = 8.5 Hz, 2H, AB–Ar-H), MS (m/z)= 295[%]:[M⁺, (27.61%)], Anal. Calcd for C₁₄H₂₁N₃O₂S (295.40): C, 56.92; H, 7.17; N, 14.23; O; Found: C, 56.85; H, 7.09; N, 14.16%.

4.1.3.13. Synthesis of ((Z)-5-(((E)-1-(4-((4-Methylpiperidin-1-yl)­sulfonyl)­phenyl)­ethylidene)­hydraziney- lidene)-4-phenyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)­(phenyl)­methanone (15)

Alkyl carbodithioate 3a (0.01 mol) and hydrazonoyl bromide 14 (0.01 mol) were dissolved in 25 mL of ethyl alcohol, followed by the dropwise addition of triethylamine (1 mL). The reaction mixture was subjected to 6 h of reflux and then cooled to ambient temperature. Filtration yielded a solid product, which was purified by washing with ethanol, drying, and recrystallization from an ethanol-benzene solution, resulting in thiadiazole derivative 15.

Orange solid (86%), M. p.; 200–202 °C; IR (KBr, disc): 3063 cm^–1 (−CH-aromatic), 2919 cm^–1 (−CH-aliphatic), 1650 cm^–1 (C = O) and 1354 cm^–1 (–S = O, asymmetric), 1160 cm^–1 (–S = O, symmetric).

¹H NMR (dimethyl sulfoxide-d_6, 400 MHz) δ_H (ppm)= 0.86 (d, J = 6.1 Hz, 3H, CH₃-piperidinyl moiety), 1.17 (t, 2H, CH₂–piperidinyl moiety), 1.32 (s, 1H, CH-piperidinyl moiety),1.64 (t, 2H, CH₂–piperidinyl moiety), 2.25 (t, 2H, CH₂–N-CH₂–piperidinyl moiety), 2.47 (s, 3H, CH₃C = N–), 3.64 (t, J = 8.7 Hz, 2H, CH₂–N-CH₂–piperidinyl moiety), 7.44 (t, J = 7.2 Hz, 1H, Ph-H), 7.65–7.56 (m, 4H, Ph-H), 7.78 (dd, J = 23.9, 8.0 Hz, 3H, Ph-H), 8.05 (d, J = 7.8 Hz, 2H, AB–Ar-H), 8.12 (d, J = 7.9 Hz, 2H, AB–Ar-H), 8.25 (d, J = 7.6 Hz, 2H, AB–Ar-H), ¹³C NMR (Chloroform-d,101 MHz) δ_C (ppm)= 15.53, 21.48, 30.15, 30.15, 33.38, 46.46, 46.46, 122.23, 127.02, 127.02, 127.19,127.19, 127.70,127.70, 128.61, 128.61, 129.04, 129.04, 130.57, 130.57, 133.94, 134.54, 136.86, 139.32, 141.90, 152.13, 158.95, 166.20, 182.68; MS (m/z):=559 [%]:[M⁺, (61.16%)], Anal. Calcd for C₂₉H₂₉N₅O₃S₂ (559.70): C, 62.23; H, 5.22; N, 12.51; O; Found: C, 62.15; H, 5.15; N, 12.12.43%.

4.2. Biological Examinations

4.2.1. In Vitro Antiproliferative Activity

HCT-116, HepG-2, and MCF-7 cell lines were subjected to the synthesized compounds to assess the In vitro antiproliferative effect using MTT assay (Supporting data). −

4.2.2. In Vitro VEGFR-2 Inhibition

The synthesized compounds were tested for their VEGFR-2 inhibitory activities using a VEGFR-2 ELISA kit (Supporting data).

4.2.3. Flow Cytometry Assay

cell cycle arresting and apoptosis potential of compounds 3a, 4, 8, and 9 on HCT-116, HepG-2, and MCF-7 cells was evaluated using flow cytometry (Supporting data).

4.3. In Silico Studies

4.3.1. Docking Studies

MOE2019 software was used to study the interaction of the synthesized compounds against VEGFR-2 [PDB ID: 2OH4, resolution: 2.05 Å] (Supporting data). ,

4.3.2. ADMET and Toxicity Studies

Discovery Studio 4.0 software was used to study the ADMET and toxicity profiles of the synthesized compounds (Supporting data). −

4.3.3. Quantum Chemical Calculations

Quantum chemical calculations, based on DFT are performed using the DMol module of BIOVIA’s Materials Studio 7.0 software to determine the various molecular parameters, and global and local properties, of compounds 15, 3a, and 6.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03503.

• 1, Chemistry; 2, Biological testing; 2.2. In vitro antiproliferative activity; 2.3. In vitro VEGFR-2 inhibition; 2.4. Cell cycle analyses: cell cycle analysis against HCT-116 cells (Table and Figure ); cell cycle analysis against MCF-7 cells (Table and Figure ); apoptosis analysis against HCT-116 cells (Table and Figure ); apoptosis analysis against MCF-7 cells (Table and Figure ); 3. In silico studies: IC₅₀ graphs for in vitro VEGFR-2 inhibitory activity (Figure ); dose–response curves for the viability and concentration and the IC₅₀ calculation (Figure ); 4. Spectral Data Includes (¹H NMR, ¹³C NMR, FT-IR, and Mass Spectrum for the synthesized compounds (3a-15)) (Figures S7–S46); this additional material provides further confirmation and details relevant to the experimental section of the manuscript (PDF)

The authors extend their appreciation to Taif University, Saudi Arabia for supporting this work through the project number (TU-DSPP-2024-293).

The authors declare no competing financial interest.