New Sulfonate-Semicarbazone Hybrid Molecules: Synthesis, Theoretical Evaluations, Molecular Simulations, and Butyrylcholinesterase Inhibition Activity

Abstract

In the present work, 18 novel semicarbazone-sulfonate hybrids were synthesized to evaluate their potential as butyrylcholinesterase (BChE) inhibitors. Among all compounds, 4-[(E)-(2-carbamoylhydrazinylidene)­methyl]­phenyl 2-(trifluoromethoxy)­benzene-1-sulfonate (12), 4-[(E)-(2-carbamoylhydrazinylidene)­methyl]­phenyl naphthalene-1-sulfonate (17), and 4-[(E)-(2-carbamoylhydrazinylidene)­methyl]­phenyl naphthalene-2-sulfonate (18) exhibited the most potent BChE inhibition, with IC₅₀ values of 61.88, 77.02, and 93.67 μM, respectively, outperforming the reference drug pyridostigmine bromide (IC₅₀: 130.04 μM). As shown by molecular docking studies, numerous interactions, including hydrogen bonds, π–π stacking, π–sulfur contacts, and halogen bonds, supported the high binding of compounds’ affinities to the BChE active site. Also, this study employed molecular dynamics (MD) simulations to assess the inhibitory potential of compounds 12, 17, and 18 against BChE. All ligands retained structural integrity during simulations. Compound 17 exhibited the highest conformational stability (minimal RMSD values) and formed robust interactions with critical binding site residues. Additionally, compound 18 demonstrated superior hydrogen bonding capacity, while compound 17 achieved the strongest binding affinity. Furthermore, in silico ADME predictions for the most active molecules showed good pharmacokinetic profiles and drug-likeness. Consequently, the results suggested that semicarbazone-sulfonate hybrid compounds 12, 17, and 18 were promising potential multifunctional agents targeting cholinergic dysfunction in Alzheimer’s disease (AD).

1. Introduction

The chronic neurodegenerative disease known as Alzheimer’s disease (AD) causes the brain’s neurons and synaptic connections to deteriorate gradually, which eventually impairs memory, reduces cognitive function, and changes behavior. It is also the primary cause of dementia, a general term for conditions characterized by severe cognitive decline that impairs daily functioning and self-sufficiency. − Globally, AD affects more than 50 million individuals, a figure that is projected to nearly triple, reaching up to 150 million cases by 2050. AD advances through distinct stages, beginning with preclinical changes before progressing to mild, moderate, and eventually severe forms, with symptoms intensifying at each stage. Although its precise cause is still not fully understood, its development is thought to result from a complex interplay of genetic predisposition, lifestyle choices, and environmental influences, with advancing age being a predominant risk factor. , On a pathological level, AD is marked by the accumulation of amyloid-β plaques, the formation of neurofibrillary tangles, and the deterioration of neuronal connections, all of which contribute to cognitive decline. While a definitive cure remains unavailable, current interventions, including cholinesterase inhibitors and memantine, lifestyle modifications, and comprehensive supportive care, can help alleviate symptoms and improve patients’ quality of life. ,, The loss of cholinergic neurons leads to a significant decline in acetylcholine (ACh), a neurotransmitter vital for cognition. In AD, the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) play crucial roles in both its progression and treatment. AChE inhibitors such as donepezil, rivastigmine, and galantamine help increase ACh levels, thereby improving cognitive function in early to moderate AD. As the disease advances, BChE activity rises, further contributing to ACh depletion. − Thus, inhibiting BChE to enhance the level of ACh has gained importance as an alternative therapeutic target for managing symptoms and slowing AD progression. Also, BChE has been reported to slow the formation of Aβ fibrils by acting as a molecular chaperone, stabilizing soluble Aβ groups. From this point of view, it is thought that compounds that inhibit BChE by binding to both the catalytic active site (CAS) and the PAS region can prevent Aβ-associated toxicity while simultaneously reducing ACh hydrolysis. Only rivastigmine is the most selective for BChE and is particularly suitable for moderate to severe AD, among FDA-approved anticholinesterase drugs. However, in addition to side effects such as nausea, vomiting, diarrhea, stomach pain, anorexia, and weight loss, 17–35% of patients experienced delusions, hallucinations, agitation and aggression, disinhibition, irritability and lability, and abnormal motor behavior. Consequently, the exploration of potential BChE inhibitor candidates for the management of AD has become imperative.

In light of this knowledge, semicarbazones are one of the scaffolds that could be used as drug candidates due to their structural modifiability and diverse biological activities, including anticonvulsant, anticancer, antimicrobial, antidiabetic, and anticholinesterase properties. − They are also imine derivatives that can be easily and simply obtained by the condensation reaction between a ketone or aldehyde and a semicarbazide. There is evidence that semicarbazones can interact more readily with cholinesterases by mimicking the structure of the substrate, thanks to the presence of the imine group, which facilitates hydrogen bonding and electrostatic interactions with critical residues in the active sites of cholinesterases, and the carbonyl group, which polarizes the ligand and makes it more electrophilic. , Although there are some studies proving that semicarbazone derivatives are effective on AChE and BChE, the therapeutic potential of semicarbazones in the treatment of AD has not been fully appreciated. − Tripathi et al. reported that among semicarbazone derivatives derived from 2-amino-5-nitrothiazole, a compound bearing a chloro group in the para position was the lead compound with an IC₅₀: 0.024 μM against BChE. Also, in another study, a series of carbazole-based semicarbazones and hydrazones were designed, synthesized, and assessed for their anticholinesterase inhibitory activity. Neto et al. synthesized a thiosemicarbazone and a semicarbazone containing an acridine group and evaluated their cholinesterase inhibitory potential. They also reported that the compounds were weaker than tacrine against BChE but less toxic in silico and noted that semicarbazones likely have a more rigid or specific structure, allowing for stronger and more stable interactions with ChEs. However, these limited studies suggest that semicarbazones may have significant potential in the development of more selective and effective drug candidates for the treatment of AD. Therefore, a new series of semicarbazone derivatives was designed in this study to contribute to a more comprehensive investigation of semicarbazones, as they may have significant potential as effective BChE inhibitor candidates for anti-Alzheimer’s agents. On the other hand, due to their high lipophilicity, sulfonates are molecules with excellent binding capacity to the active sites of target enzymes, owing to their favorable physicochemical properties. Moreover, arylsulfonate derivatives are one of the most remarkable pharmacophore groups recently and are promising, especially in terms of their inhibitory activity against butyrylcholinesterase. ,

In this study, to identify new hybrid scaffolds and continue our studies on cholinesterase inhibitors, we designed a series of hybrid compounds consisting of 18 semicarbazone-sulfonate hybrid molecules to investigate the effect of substituents present on the aryl ring attached to the sulfonate structure and ring size on BChE inhibitory activity. These new hybrid compounds were designed and synthesized by a molecular hybridization approach (1–18), which is a drug design method based on combining pharmacophore groups to discover new drug candidates. Furthermore, molecular docking studies were conducted to evaluate the inhibition mechanisms and determine the stability of the ligand–protein complexes for the most active compounds. The physicochemical properties of the compounds and their binding affinities to the target protein were investigated by using molecular docking, molecular dynamics simulations, and quantum chemical calculations. Finally, in silico ADME (Absorption, Distribution, Metabolism, and Excretion) profiling was performed for all compounds to explore their drug-likeness.

2. Results and Discussion

2.1. Chemistry

In this study, 18 novel (E)-4-[(2-carbamoylhydrazinylidene)­methyl]­phenyl-substituted benzenesulfonate derivatives (1–18) were designed and synthesized for the first time, as illustrated in Scheme . p-Hydroxybenzaldehyde was reacted with sulfonyl chloride derivatives in the presence of triethylamine to afford arylsulfonyloxybenzaldehyde intermediates (A1–A18). Subsequent reactions of compounds A1–A18 with semicarbazide yielded the corresponding semicarbazones (1–18) in excellent yields (93–98%). The substituents of the synthesized compounds are listed in Scheme . The chemical structures of the new derivatives were confirmed by spectroscopic techniques, including FT-IR, ¹H NMR, and ¹³C NMR analyses.

1. Synthetic Pathway of Semicarbazone-Sulfonate Hybrid Molecules.

In the FT-IR spectra of compounds (1–18), it was determined that the CN stretching band, which is one of the characteristic absorptions of the semicarbazone moiety, appeared in the range of 1567–1596 cm^–1 and the N–H stretching vibrations were observed in the range of 3545–3145 cm^–1. Also, the asymmetric and symmetric stretching bands of SO₂ were determined at 1350–1384 and 1133–1199 cm^–1, respectively. In the ¹H NMR spectra, evidence for the formation of semicarbazones was provided by the appearance of N–H proton signals between δ 10.36 and 10.30 ppm as a singlet, together with the – NH₂ protons appearing as a doublet (2H) between δ 6.56 and 6.50 ppm. In addition, the −CH proton of the azomethine (CN) group was observed as a singlet at δ 7.80–7.71 ppm. In the ¹³C NMR spectra, the azomethine (CN) carbon and the carbonyl (CO) carbon, which are among the most significant signals of hydrazone-type compounds, resonated in the range of δ 166.92–149.05 ppm. Signals of aromatic carbons were also clearly observed in their expected regions. All spectroscopic data obtained for the characterization of semicarbazone-sulfonate derivatives (1–18) were consistent with the previously reported values. −

2.2. DFT

2.2.1. Frontier Molecular Orbitals

In quantum chemistry, frontier molecular orbitals (FMOs)namely, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO)along with their energy difference (ΔE = E _HOMO – E _LUMO), are fundamental indicators of a molecule’s electronic behavior. These parameters provide important information about the kinetic stability, biological potential, and reactivity of a molecule. The FMO profiles and associated HOMO–LUMO energy gaps of compounds 12, 17, and 18 are shown in Figure .

1.

HOMO and LUMO plots for 12, 17, and 18 at the B3LYP/6–311++G­(d,p) level.

The evaluation of HOMO and LUMO energy levels in compounds facilitates the determination of several global reactivity parameters, including the ionization energy (I), electron affinity (A), chemical hardness (η), chemical softness (σ), electronegativity (χ), chemical potential (μ), and electrophilicity index (ω). These descriptors of quantum chemistry are essential for forecasting the behavior of molecules in various chemical environments, providing information on their stability, reactivity, and possible interactions with biological targets.

2.2.2. Global Reactivity Parameters

The global reactivity descriptors are presented in Table , which provides important information about the electrical properties of compounds 12, 17, and 18. A chemical with a lower E _LUMO value indicates a better ability to accept electrons, while a compound with a higher E _HOMO value generally has a greater tendency to donate electrons. Among the compounds, 18 stood out with its remarkable capacity to donate electrons, with the highest E _HOMO level of −6.368 eV. On the other hand, compound 12 had the lowest E _LUMO value of −2.047 eV in the gas phase, indicating a strong capacity to accept electrons.

1. Global Reactivity Parameters (in eV) for 12, 17, and 18 .

compound	E HOMO	E LUMO	ΔE	IP	EA	μ	η	σ	χ	ω
12	–6.362	–2.047	4.315	6.362	2.047	–4.204	2.158	0.463	4.204	4.096
17	–6.351	–2.252	4.099	6.351	2.252	–4.302	2.049	0.488	4.302	4.515
18	–6.368	–2.239	4.129	6.368	2.239	–4.303	2.064	0.484	4.303	4.485

In conclusion, the analysis of global reactivity descriptors revealed notable differences in the electronic properties of the studied compounds. While compound 12, which had the lowest electronegativity and electrophilicity index, mostly functions as an electron donor, compound 18, which had the highest electronegativity, was a notable strong electron acceptor. On the other hand, compound 17 exhibited the highest electrophilicity, indicating its enhanced electrophilic character. Notably, increased biological activity was typically linked to reduced electrophilicity. These findings suggested that the electron-donating capacity and reduced electrophilicity could be advantageous features for designing biologically active molecules.

2.2.3. Molecular Electrostatic Potential (MEP) Maps

The molecular electrostatic potential (MEP) maps of compounds 12, 17, and 18 are given in Figure . These maps provided visual insights into the charge distribution over the molecular surfaces, highlighting regions prone to electrophilic and nucleophilic interactions. The color gradient ranged from red (electron-rich, negative potential) to blue (electron-deficient, positive potential). Those with a high electron density were shown on these maps as red, and those with a low electron density were shown as blue. , In compound 12, intense red regions were observed around the carbonyl and sulfonyl oxygen atoms, indicating a high electron density and potential nucleophilic attack sites. The blue areas near hydrogen atoms suggested regions susceptible to electrophilic attack. Compounds 17 and 18 showed a more balanced distribution with prominent red regions at the sulfonyl moiety and medium blue regions near the amine groups, indicating the potential for both electrophilic and nucleophilic interactions. Overall, the MEP profiles indicated that differences in electrostatic potential distribution could influence the binding behavior and biological activity of these molecules.

2.

MEP maps of compounds at the B3LYP/cc-pVDZ level.

2.3. In Vitro Cholinesterase Inhibition Studies

As a result of in vitro inhibition studies of the BChE enzyme, IC₅₀ values of semicarbazone-sulfonate hybrid compounds were found in the range of 61.88–346.57 μM (Table ). The IC₅₀ value of pyridostigmine bromide used as a reference inhibitor was 130.04 μM, and compounds 11, 12, 13, 14, 16, 17, and 18 remained below this value and showed a stronger inhibitory effect.

2. Inhibitory Effects of Semicarbazone-Sulfonate Derivatives on BChE.

Comp.	BChE IC50 (μM)	Comp.	BChE IC50 (μM)
1	139.466	10	138.629
2	161.197	11	113.631
3	141.459	12	61.888
4	176.823	13	115.525
5	173.287	14	129.560
6	177.730	15	346.574
7	136.178	16	111.798
8	172.424	17	77.016
9	147.165	18	93.669
Pyridostigmine bromide	130.04

The butyrylcholinesterase (BChE) inhibitory activities of the compounds in the series show marked differences depending on the electronic, steric, and hydrophobic properties of the R groups on the aromatic ring in the structure. The IC₅₀ values range from 61.88 to 346.57 μM.

The highest inhibitory activity was observed in compound 12 (R = OCF₃, IC₅₀ = 61.88 μM). This result may be due to the strong electron-withdrawing and polar trifluoromethoxy (−OCF₃) group, providing a balanced combination of hydrophobic and polar interactions in the active site of BChE. Similarly, the relatively high activities of compounds 17 (R = naphthyl, IC₅₀ = 77.02 μM) and 18 (R = 2-naphthyl, IC₅₀ = 93.67 μM) may be attributed to the potential of their broad aromatic surfaces to form π–π stacking interactions within the enzyme’s hydrophobic pocket. This is consistent with the large hydrophobic active site of BChE.

In contrast, compound 15 (R = NO₂, IC₅₀ = 346.57 μM) exhibits the lowest inhibitory activity. The strong electron-withdrawing character and steric bulk of the nitro group likely impede access to the active site or a suitable binding conformation. Furthermore, the nonhydrophobic nature of the NO₂ group may result in weak interactions with the apolar residues in the enzyme’s active site.

Halogen derivatives (e.g., 1–4; F, Cl, Br, and I) generally exhibited moderate activity (IC₅₀ ≈ 130–175 μM). Differences in size and electronegativity among the halogens did not cause significant variation in activity, suggesting that the interaction is primarily based on hydrophobic contacts. Similarly, compounds containing CF₃ (e.g., 8–11, 13) showed moderate activity; although these groups are electron-withdrawing, their high steric volume may have limited their complete integration into the active site.

Overall, it can be concluded that electron-withdrawing substituents that do not create excessive steric hindrance (e.g., −OCF₃) or groups providing a large aromatic surface (e.g., naphthyl derivatives) are more advantageous for BChE inhibition. This result is consistent with the literature information that BChE has a larger active site compared to that of AChE and can tolerate large aromatic systems.

Overall, a strong correlation between the BChE inhibition activity and structural characteristics was found. These findings implied that compounds’ structural characteristics, particularly those of 12, 17, and 18, can be assessed as pharmacophores and optimized by further molecular modeling.

2.4. Enzyme Kinetic Studies

Enzyme kinetic studies were performed on butyrylcholinesterase (BChE) using the most potent inhibitors, compounds 12, 17, and 18, to elucidate their inhibition mechanisms. The enzymatic activity of BChE was evaluated at various concentrations of the substrate butyrylthiocholine iodide (BTChI) in the presence of fixed concentrations of each inhibitor. The resulting data were analyzed by Lineweaver–Burk double reciprocal plots to determine kinetic parameters such as K _m, V _max, and the inhibition type (Figure ).

3.

IC₅₀ graph, Lineweaver–Burke plot of the inhibition kinetics of BChE for 12 (a), 17 (b), and 18 (c).

The Lineweaver–Burk plots demonstrated that for all tested compounds, the lines intersected at a common point on the y-axis while exhibiting progressively steeper slopes as the inhibitor concentration increased. This characteristic pattern indicates a competitive inhibition mechanism, where the inhibitors compete with the substrate for the enzyme’s active site. Consequently, the Km values increased with increasing inhibitor concentrations, reflecting a reduction in substrate affinity, while Vmax remained constant, confirming that the inhibition can be overcome by higher substrate levels.

The calculated inhibition constants (K _i) further supported these findings, with values of 11.87 ± 3.31 μM for compound 12, 9.08 ± 3.51 μM for compound 17, and 5.49 ± 0.33 μM for compound 18. Among these, compound 18 exhibited the strongest binding affinity toward the enzyme, suggesting a more effective competition with the substrate at the catalytic site. The relatively low K _i values observed for all three compounds indicate a high inhibitory potency against BChE.

Collectively, these results reveal that compounds 12, 17, and 18 act as reversible competitive inhibitors of BChE by occupying the active site and preventing substrate binding. This behavior aligns with the structural features predicted from molecular docking studies, reinforcing the conclusion that the interaction occurs primarily within the catalytic pocket. Therefore, these compounds may serve as promising scaffolds for the further design and development of potent and selective BChE inhibitors with potential therapeutic applications in neurodegenerative disorders such as Alzheimer’s disease.

2.5. Molecular Docking

Molecular docking analyses were performed to evaluate the binding affinity and interaction modes of the compounds with the target enzyme. In the analyses conducted with the Molegro Virtual Docker 6.0 program, compound 18 showed the highest binding score (MolDock score: −138.27), followed by compounds 12 (−128.81) and 17 (−111.27). These results indicated that compound 18 bound most strongly to the enzyme’s active site. It was thought that compound 12 established multifaceted interactions with the enzyme’s active site, attributed to the aromatic ring and halogen group in its structure, and also the conventional hydrogen bonds were observed, especially with the amino acid residues Trp82, Gln67, Asn83, Ser198, Gly116, and Gly117. In addition, alkyl interactions with Trp231 and Leu286, π-alkyl interaction with Trp231, π-sulfur interactions with Phe329 and His438, and halogen bond with Leu286 also made important contributions (Table ).

3. Interactions between Compounds 12, 17, and 18 with the BChE Active Site.

types	category	12	17	18
hydrogen bond		Trp82, Gln67, Asn83, Ser198, Gly116, Gly117	Ile69, Gln71, Ser72	Asn83, Gln67, Asn68, Gly121, Trp82
hydrophobic	Pi–pi stacked		Tyr332, His438	Trp82
	Amide–pi stacked	Gly16
	Alkyl	Trp231, Leu286
	Pi–alkyl	Trp231	Ala328
halogen		Gly117, Leu286
π-sulfur		Phe329, His438	His438	His438
π-anion				Asp70
MolDock score		–128.81	–111.27	–138.27

These interactions supported the stable placement of the compounds in the active site. The bond between the halogen group in compound 12 and Leu286 revealed the binding stability (Figure a,b). Compound 17, in which hydrogen bonds were limited to Ile69, Gln71, and Ser72, showed fewer interactions than the other compounds. The π–π interactions were observed with Tyr332 and His438, while π–alkyl interaction occurred with Ala328 and π–sulfur interaction occurred with His438. Although the aromatic groups in the molecular structure entering into π–sulfur interaction with His438 slightly increased the binding affinity, the total interaction diversity and number were lower (Figure c,d). Compound 18 exhibited the most extensive profile in terms of both hydrogen bonds and hydrophobic interactions and achieved the lowest MolDock score. The hydrogen bonds with Gln67, Asn68, Asn83, Gly121, and Trp82 ensured that the compound fits tightly into the active site. In addition, the π–π interaction with Trp82, the π–sulfur interaction with His438, the π–anion interaction with Asp70, and the van der Waals interactions with Tyr332 were important interactions that increase the stability of the complex. In particular, the interactions between the large conjugated π-system in compound 18 and aromatic amino acids such as Trp82 and His438 strengthened the binding energy (Figure e,f). In conclusion, compound 18 exhibited a strong binding affinity with the enzyme active site due to the establishment of numerous and diverse bond interactions.

4.

BChE binding modes of compounds 12 (a, b), 17 (c, d), and 18 (e, f) in both 2D and 3D configurations.

2.6. Molecular Dynamics Simulations

To assess the binding stability of bioactive compounds, molecular dynamics (MD) simulations were carried out for five systems: apo-4DBS, 4DBS-Tacrine, and 4DBS complexes with compounds 12, 17, and 18 (Figure ), each simulated for 100 ns. The trajectories were analyzed using statistical metrics, including root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), hydrogen bond interactions (with occupancy percentages), and MMGBSA binding energy calculations throughout the simulation time frame.

5.

Graphical representation of protein and protein–ligand complexes: (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18, where protein is shown in cartoon representation, and the ligand is shown in CPK representation with transparent surface.

2.6.1. RMSD Analysis

The Cα-RMSD profiles showing the structural stability of the 4DBS protein upon binding with various ligands, including the native ligand Tacrine, which was docked at its known binding site, are presented in Figure . RMSD values stabilized across all systems after ∼20 ns, indicating convergence and equilibration of the simulations. The apo form exhibited the lowest mean RMSD (0.15 nm, Std: 0.01), confirming its intrinsic structural stability. Tacrine and compound 18 demonstrated low mean RMSD values (0.16 nm), comparable to the apoprotein, suggesting that these ligands maintain the native-like conformation of 4DBS with minimal perturbation. In contrast, compounds 12 and 17 showed slightly higher average RMSDs (0.18 and 0.19 nm, respectively) yet remained well below 0.3 nm, indicating acceptable structural integrity. Overall, these results implied that ligand binding, particularly with Tacrine and compound 18, did not significantly disrupt the protein backbone, reinforcing the suitability of these compounds for further analysis.

6.

Graphical representation of the plots showing protein RMSD (nm) versus time (100 ns); (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18.

The ligand RMSD trajectories over 100 ns (Figure ) provided valuable insight into the conformational stability and binding persistence of the compounds within the 4DBS active site. Notably, 4DBS-Tacrine, the native ligand docked into its crystallographic position, exhibited a relatively low mean RMSD of 0.19 nm with a moderate standard deviation of 0.05, suggesting a stable binding pose and validating the docking protocol. Similarly, compounds 12 and 17 maintained low RMSD values (0.17 and 0.18 nm, respectively) with minimal deviations (Std: 0.04), indicating consistent and well-retained interactions throughout the simulation. In contrast, compound 18 showed a significantly higher mean RMSD of 0.29 nm and the largest fluctuation range (Max: 0.38 nm; Std: 0.09), implying less stable binding and larger conformational shifts. These results suggest that compounds 12 and 17 are promising candidates with stability profiles comparable to those of the native ligand, while 18 may require further optimization for enhanced binding persistence.

7.

Graphical representation of the plots showing ligand RMSD (nm) versus time (100 ns) for (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18.

2.6.2. RMSF Analysis

The Cα-RMSF analysis (Figure ) delineates residue-specific flexibility variations in the 4DBS protein across ligand-free (apo) and ligand-bound states during simulations. The apo form displayed the lowest overall fluctuations (Mean: 0.08 nm, Max: 0.35 nm), reflecting intrinsic structural rigidity. While global flexibility trends remained conserved across ligand-complexed systems, localized differences emerged. Notably, the native ligand Tacrine induced minimal perturbations (Mean: 0.09 nm, Std: 0.05), closely resembling the apo state. In contrast, compound 12 exhibited pronounced flexibility at the C-terminal region (∼residue 530), reaching a maximum of 0.79 nm, suggesting destabilization in the peripheral loop regions. Crucially, the core active site (residues 120–180) retained low RMSF values (<0.15 nm) in all systems, confirming that ligand bindingwhether natural (Tacrine) or synthetic (17/18)preserves the catalytic pocket’s stability. These results indicate that Tacrine, compound 17, and compound 18 maintain native-like conformational dynamics, whereas compound 12 introduces localized flexibility outside the functional site, potentially influencing noncatalytic regions.

8.

Graphical representation of the plots showing the protein RMSF (nm) versus residue index number of protein for (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18.

2.6.3. H-Bond Interaction

Figure illustrates the number of hydrogen bonds formed between the 4DBS protein and each ligand throughout the simulation, providing insights into binding consistency and stability. Compound 18 formed the highest average number of hydrogen bonds (1.93) with a maximum of 7, indicating frequent and stable interactions with the protein’s binding site. Compound 17 and the native ligand Tacrine followed with average hydrogen bond counts of 1.03 and 0.92, respectively, and maximum values of 6, suggesting moderately stable interactions, consistent with their favorable binding poses. Compound 12 showed the lowest hydrogen bonding performance (Mean: 0.62, Max: 4), reflecting fewer or less persistent contacts within the binding pocket. The relatively stable hydrogen bonding behavior of Tacrine, docked into its crystallographic binding site, further validates the simulation setup. Overall, the data highlight compound 18 as the ligand with the strongest hydrogen bonding profile, supporting its potential for high-affinity binding to 4DBS.

9.

Pictorial representation of the number of h-bond contacts formed by (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18.

Hydrogen bond occupancy analysis across ligand-bound 4DBS complexes (Figure ) highlights distinct interaction patterns with critical residues, elucidating their binding mechanisms and stability. The native ligand Tacrine, bound to its crystallographic site, demonstrated moderate yet stable hydrogen bonding, primarily with Thr120, Tyr332, and Tyr440, with peak occupancy of ≤2%, reflecting dynamic but stable interactions characteristic of natural ligand–protein associations. Compound 17 displayed the most robust interactions, forming highly stable hydrogen bonds with Thr120 (29% occupancy) and Tyr440 (38% occupancy), underscoring its targeted binding to conserved active-site residues. Compound 18 exhibited notable interactions, including strong engagement with Tyr114 (22% occupancy) and moderate bonding with Thr120, Trp112, and Gly116, suggesting a wider interaction network within the active site. While compound 12 showed weaker overall hydrogen bonding, it maintained modest interactions with Gly439, Thr120, and Tyr440, aligning with its lower overall interaction capacity observed in time-dependent analyses. These results emphasize Thr120 and Tyr440 as pivotal anchoring points for ligand binding, with compound 17 demonstrating superior stability through focused, high-occupancy hydrogen bonds in the catalytic pocket.

10.

Histogram representation of % occupancies of the H-bond protein–ligand contacts of (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18.

2.6.4. MMGBSA Calculations

Figure illustrates the binding energy profiles, offering a comparative analysis of the ligand interactions with the 4DBS protein during simulations. Compound 17 emerged as the strongest binder, displaying the lowest average binding energy (−54.74 kJ/mol) and a peak interaction strength of −99.90 kJ/mol, signifying robust, consistent engagement with the protein. Compounds 18 (−53.28 kJ/mol) and 12 (−48.98 kJ/mol) followed, both showing stable binding but with marginally greater variability. The crystallographically docked native ligand Tacrine had a moderate average energy of −47.57 kJ/mol, aligning with its experimentally confirmed affinity but underperforming relative to those of the leading compounds. The ligand-free apo system recorded a similar mean energy (−46.24 kJ/mol), implying that nonspecific interactions prevail without a bound ligand. These results position compound 17 as the most thermodynamically stable binder, surpassing even the native ligand in predicted efficacy, and underscore its viability as a prospective inhibitory agent.

11.

MMGBSA ΔG binding energy calculations for (A) 4DBS-Tacrine, (B) 4DBS Apoprotein, (C) 4DBS-Compound 12, (D) 4DBS-Compound 17, and (E) 4DBS-Compound 18.

2.7. ADMET

A molecule must reach its target location in the body in a high enough concentration and stay in a physiologically active state for an extended period of time in order to elicit the desired biological reaction. Due to limited access to physical samples, predicting ADMET properties with computer-aided models at an early stage in the drug development process offers an important alternative. In this study, the ADMET properties of compounds 12, 17, and 18 were calculated using online tools such as SwissADME, ProTox-II, and PKCS,M and these results are given in Table .

4. ADMET Profile of Compounds 12, 17, and 18 .

	value
properties	12	17	18
molecular weight (g/mol)	403.33	369.39	369.39
rotatable bonds	8	6	6
consensus log P o/w	2.55	2.59	2.58
topological polar surface area (TPSA, Å)	128.46	119.23	119.23
water solubility (log mol/L)	–4.618	–4.219	–4.237
intestinal absorption (human) (%)	81.26	86.121	89.338
BBB permeant	No	No	No
predicted LD50 (mg/kg)	10750	10750	10750
predicted toxicity class	6	6	6
	Prediction	Prediction	Prediction
hepatotoxicity	inactive	inactive	inactive
immunotoxicity	inactive	inactive	inactive
cytotoxicity	inactive	inactive	inactive
carcinogenicity	inactive	active	active
mutagenicity	inactive	inactive	inactive
Caco-2 permeability (log Papp in 10–6 cm/s)	0.865	0.889	0.979
AMES toxicity	no	yes	yes
P-gp substrate	yes	yes	yes
Lipinski	yes; 0 violation	yes; 0 violation	yes; 0 violation

Accordingly, compounds 12, 17, and 18 meet Lipinski’s “drug-likeness” rules without violating them, which is a positive indicator in terms of oral bioavailability. Their molecular weights are in the range of 369.39–403.33 g/mol, and the rotatable bond numbers vary between 6 and 8, indicating that they can establish a balance between structural flexibility and target site interaction potential. The efficacy of drugs targeting the central nervous system is predicated on their ability to traverse the blood–brain barrier (BBB). However, no compounds have the capacity to penetrate this physiological barrier. Their water solubility values were similar, and although they showed low solubility, they were within acceptable limits. Their intestinal absorption rates were high (81.26 to 89.34%), indicating that the compounds could be effectively absorbed from the gastrointestinal tract. It was observed that compounds 12, 17, and 18 had good oral absorption rates in the human body. The Ames test is employed to evaluate the mutagenic potential of a drug or compound by determining its ability to cause DNA damage, thereby serving as an important indicator of genetic toxicity. According to the toxicity parameters, compound 12 was negative for AMES, indicating that the mutagenic potential was low. On the other hand, compounds 17 and 18 gave positive AMES results, which was important for their potential genotoxic effects. For optimal polarity-related properties, the topological polar surface area (TPSA) should generally be in the range of 20–130 Ų. Compounds 12, 17, and 18 exhibited TPSA values within this acceptable range, indicating favorable polarity for potential bioavailability. Caco-2 value is a remarkable parameter for human colorectal cancer cell lines. This cell line is used as a model that mimics the intestinal epithelial barrier. The Caco-2 permeability values of compounds 12, 17, and 18 varied between 0.865 and 0.979. LD50, in toxicology, is the amount of a drug that causes the death of 50% of the total population when given at once. As can be seen from Table , the estimated LD₅₀ value of these compounds was determined as 10750 mg/kg. A high value indicates that the acute toxicity of the compound is low and therefore may have a positive potential in terms of safety profile. According to Protox-II estimation, all three molecules are classified as toxicity class 6 and are not expected to exhibit harmful effects if ingested (ref: 10.1093/nar/gky318). Additionally, the ProTox-II web server predicts five toxicological parameters: hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity. According to the results, all three compounds were considered inactive in terms of hepatotoxic, immunotoxic, cytotoxic, and mutagenic effects (Table ). In contrast, compounds 17 and 18 were predicted to be carcinogenic, while compound 12 was predicted to be noncarcinogenic. These findings indicate that the investigated compounds generally have a favorable toxicological profile with low acute toxicity potential.

3. Experimental Section

3.1. Reagents and Instruments

All starting materials and reagents were obtained from Sigma-Aldrich and Merck in analytical-grade purity and used without any purification. The progress of the reactions and the purity of the products were monitored by thin-layer chromatography (TLC) on silica gel 60 F ₂₅₄ plates (20 cm × 20 cm, 0.25 mm thickness, Merck). Hexane:ethyl acetate (2:1, v/v) was employed as the mobile phase, and chromatographic development was carried out at room temperature in a solvent-saturated chamber. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum-100 FT-IR spectrometer equipped with a Universal ATR Sampling Accessory in the range of 4000–400 cm^–1, using approximately 2 mg of each solid sample. The ¹H and ¹³C NMR spectra of the compounds (1–18) were obtained with Bruker AVANCE III 400 and 600 MHz spectrometers. Samples (≈20 mg) were dissolved in DMSO-d ₆ , and the chemical shifts (δ) were reported in parts per million (ppm) relative to tetramethylsilane (TMS) as the internal reference. Mass spectra (MS) were obtained using a liquid chromatography–tandem mass spectrometer (LC–MS/MS, Shimadzu 8045) operated in electrospray ionization (ESI) mode. To determine the purity of the compounds, an Agilent 1260 Infinity HPLC instrument was used with a C18 column (3 μm, 4.6 mm × 50 mm) under gradient elution conditions using an acetonitrile/water (80:20) mobile phase. Analysis by integrating the areas of the main peaks detected at 254 nm revealed purities of ≥ 90% for all tested compounds (Figures S76–S93).

3.2. General Procedure for the Synthesis of 4-Formylphenyl-Substituted Sulfonates (A1–A18)

A mixture of 4-hydroxybenzaldehyde (1 mmol) and triethylamine (TEA, 2 mmol) was dissolved in dichloromethane (DCM, 10 mL) and stirred vigorously at room temperature for 45 min. Subsequently, substituted benzenesulfonyl chloride (1 mmol) in DCM (10 mL) was added dropwise, and the reaction mixture was refluxed for approximately 5 h. The progress of the reaction was monitored by thin-layer chromatography (TLC). After being completed, the mixture was cooled to room temperature and extracted twice with 2 M HCl. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by recrystallization from ethanol to afford the compound in pure form.. − The ¹H NMR spectra of the intermediate 4-formylphenyl-substituted sulfonates (A8, A12, and A13), reported here for the first time, are included in the Supporting Information (Figures S1–S3). The other 4-formylphenyl-substituted sulfonates were previously reported. −

3.2.1. 4-Formylphenyl 2-(Trifluoromethyl)­benzenesulfonate (A8)

¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 9.99 (s, 1H, HCO), 8.25 (t, 2H, J ₁ = 8.0, J ₂ = 8.4 Hz, ArH), 8.15 (s, 1H, ArH), 7.97 (d, 3H, J = 8.8 Hz, ArH), 7.35 (d, 2H, J = 8.8 Hz, ArH).

3.2.2. 4-Formylphenyl 2-(Trifluoromethoxy)­benzenesulfonate (A12)

¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 9.98 (s, 1H, HCO), 7.97 (d, J = 7.4 Hz, 4H, ArH), 7.79 (d, J = 7.4 Hz, 1H, ArH), 7.63 (t, J = 7.3, 7.3 Hz, 1H, ArH), 7.32 (d, J = 7.8 Hz, 2H, ArH).

3.2.3. 4-Formylphenyl 3-(Trifluoromethoxy)­benzenesulfonate (A13)

¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 9.99 (s, 1H, HCO), 7.98 (s, 1H, ArH), 7.96 (d, 2H, J = 8.7 Hz, ArH), 7.84–7.91 (m, 3H, ArH), 7.33 (d, 2H, J = 8.5 Hz, ArH).

3.3. General Procedure for the Synthesis of Semicarbazone-Sulfonate Derivatives (1–18)

Semicarbazide hydrochloride (1 mmol) and sodium acetate (2 mmol) were added to a solution of 4-formylphenyl-substituted sulfonates (1 mmol, A1–A18) in methanol (10 mL) with continuous stirring. The reaction mixture was stirred until the formation of a precipitate, which was subsequently filtered, washed with cold methanol, dried, and recrystallized from ethanol to afford the corresponding semicarbazone-sulfonate derivatives. The purity and completion of the reaction were confirmed by thin-layer chromatography (TLC, single spot). All spectral data employed for the structural characterization of the compounds are provided in the Supporting Information (Figures S4–S75).

3.3.1. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl Benzenesulfonate (1)

White solid, yield: 81%, mp: 227–228 °C. IR (ν, cm^–1): 3480, 3435 (N–H); 3070 (Ar–CH); 2988, 2923 (aliphatic CH); 1695 (CO); 1585 (CN stretching band); 1498, 1472 (Ar–CC); 1370, 1199 (SO₂); 688 (Ar–CH). ¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 10.32 (s, 1H, NH), 7.86 (d, J = 7.6 Hz, 2H, ArH), 7.82 (t, J = 7.6 Hz, 1H, ArH), 7.78 (s, 1H, NCH), 7.72 (d, J = 8.7 Hz, 2H, ArH), 7.67 (t, J = 7.8 Hz, 2H, ArH), 7.00 (d, J = 8.6 Hz, 2H, ArH), 6.53 (s, 2H, NH ₂). ¹³C NMR (151 MHz, DMSO-d ₆) δ ppm: 157.12 (CO), 149.52 (CN), 138.00, 135.58, 134.64, 131.82, 130.30, 128.73, 128.45, 122.71 (ArC). ESI-MS (m/z): 318 [M – H]⁺. Elemental analysis, C₁₄H₁₃N₃O₄S (319,34 g/mol). Found, %: C, 52.77; H, 4.41; N, 13.40; S, 10.23. Calculated, %: C, 52.66; H, 4.10; N, 13.16; S, 10.04. HPLC: purity 90%.

3.3.2. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-Fluorobenzene-1-sulfonate (2)

White solid, yield: 86%, mp: 205–206 °C. IR (ν, cm^–1): 3491, 3405 (N–H); 3051 (Ar–CH); 2969, 2923 (aliphatic CH); 1694 (CO); 1591 (CN stretching band); 1574, 1495 (Ar–CC); 1371, 1150 (SO₂);1091 (Ar–C–F); 688 (Ar–CH). ¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 10.35 (s, 1H, NH), 7.94 (dd, J = 8.8, 5.0 Hz, 2H, ArH), 7.79 (s, 1H, NCH), 7.74 (d, J = 8.7 Hz, 2H, ArH), 7.51 (t, J = 8.7 Hz, 2H, ArH), 7.02 (d, J = 8.7 Hz, 2H, ArH), 6.56 (s, 2H, NH ₂). ¹³C NMR (151 MHz, DMSO-d ₆) δ ppm: 166.92 (CO), 165.23, 157.17, 149.43 (CN), 138.05, 134.70, 132.18, 128.51, 122.78, 117.73 (ArC). ESI-MS (m/z): 336 [M – H]⁺. Elemental analysis, C₁₄H₁₂FN₃O₄S (337,33 g/mol). Found, %: C, 50.09; H, 3.65; N, 12.60; S, 10.00. Calculated, %: C, 49.85; H, 3.59; N, 12.46; S, 9.50. HPLC: purity 98%.

3.3.3. 4-[(E)-(2-carbamoylhydrazinylidene)­methyl]­phenyl 4-Chlorobenzene-1-sulfonate (3)

White solid, yield: 84%, mp: 213–215 °C. IR (ν, cm^–1): 3492, 3294 (N–H); 3095 (Ar–CH); 2968, 2925 (aliphatic CH); 1692 (CO); 1575 (CN stretching band); 1497, 1481 (Ar–CC); 1373, 1152 (SO₂); 1087 (Ar–C–Cl); 680 (Ar–CH). ¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 10.35 (s, 1H, NH), 7.87 (d, J = 7.3 Hz, 2H, ArH), 7.80 (s, 1H, NCH), 7.75 (d, J = 8.5 Hz, 4H, ArH), 7.04 (d, J = 7.5 Hz, 2H, ArH), 6.55 (s, 2H, NH ₂). ¹³C NMR (151 MHz, DMSO-d ₆) δ ppm: 157.14 (CO), 149.38 (CN), 140.64, 138.00, 134.77, 133.39, 130.71, 130.49, 128.54, 122.77 (ArC). ESI-MS (m/z): 353 [M – H]⁺. Elemental analysis, C₁₄H₁₂ClN₃O₄S (353,78 g/mol). Found, %: C, 47.38; H, 3.25; N, 11.65; S, 9.18. Calculated, %: C, 47.53; H, 3.42; N, 11.88; S, 9.06. HPLC: purity 93%.

3.3.4. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-Bromobenzene-1-sulfonate (4)

White solid, yield: 89%, mp: 219–220 °C. IR (ν, cm^–1): 3491, 3288 (N–H); 3025 (Ar–CH); 2972, 2902 (aliphatic CH); 1692 (CO); 1574 (CN stretching band); 1531, 1449 (Ar–CC); 1070 (Ar–C–Br); 1372, 1151 (SO₂); 670 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.33 (s, 1H, NH), 7.90 (t, J = 8.5 Hz, 2H, ArH), 7.80–7.74 (m, 5H, ArH), 7.04 (t, J = 9.0 Hz, 2H, ArH), 6.53 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.13 (C O), 149.35 (CN), 137.92, 134.78, 133.78, 133.44, 130.69, 129.84, 128.55, 122.78 (ArC). ESI-MS (m/z): 398 [M+2]⁺. Elemental analysis, C₁₄H₁₂BrN₃O₄S (396,97 g/mol). Found, %: C, 42.30; H, 3.11; N, 10.72; S, 8.33. Calculated, %: C, 42.23; H, 3.04; N, 10.55; S, 8.05. HPLC: purity 99%.

3.3.5. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-Iodobenzene-1-sulfonate (5)

White solid, yield: 91%, mp: 106–107 °C. IR (ν, cm^–1): 3485, 3145 (N–H); 3063 (Ar–CH); 2919 (aliphatic CH); 1745 (CO); 1567 (CN stretching band); 1504, 1446 (Ar–CC); 1088 (Ar–C–I); 1361, 1198 (SO₂); 669 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.33 (s, 1H, NH), 8.06 (d, J = 8.6 Hz, 2H, ArH), 7.78 (s, 1H, NCH), 7.74 (d, J = 8.7 Hz, 2H, ArH), 7.59 (d, J = 8.6 Hz, 2H, ArH), 7.02 (d, J = 8.7 Hz, 2H, ArH), 6.54 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.12 (CO), 149.37 (CN), 139.22, 137.92, 134.74, 134.13, 130.16, 128.54, 122.75, 104.78 (ArC). ESI-MS (m/z): 445 [M – H]⁺. Elemental analysis, C₁₄H₁₂IN₃O₄S (445,23 g/mol). Found, %: C, 37.91; H, 2.80; N, 9.49; S, 7.28. Calculated, %: C, 37.77; H, 2.72; N, 9.44; S, 7.20. HPLC: purity 98%.

3.3.6. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-Methylbenzene-1-sulfonate (6)

White solid, yield: 94%, mp: 203–205 °C. IR (ν, cm^–1): 3492, 3297 (N–H); 3090 (Ar–CH); 2972, 2925 (aliphatic CH); 1690 (CO); 1596 (CN stretching band); 1574, 1497 (Ar–CC); 1365, 1152 (SO₂); 686 (Ar–CH). ¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 10.34 (s, 1H, NH), 7.79 (s, 1H, NCH), 7.74 (d, J = 7.8 Hz, 4H, ArH), 7.47 (d, J = 8.0 Hz, 2H, ArH), 7.00 (d, J = 8.5 Hz, 2H, ArH), 6.55 (s, 2H, NH ₂), 2.42 (s, 3H, CH ₃). ¹³C NMR (151 MHz, DMSO-d ₆) δ ppm: 157.16 (CO), 149.60 (CN), 146.35, 138.10, 134.51, 131.74, 130.70, 128.75, 128.44, 122.72 (ArC), 21.63 (CH₃). ESI-MS (m/z): 332 [M – H]⁺. Elemental analysis, C₁₅H₁₅N₃O₄S (333,36 g/mol). Found, %: C, 54.17; H, 4.63; N, 12.81; S, 9.70. Calculated, %: C, 54.04; H, 4.54; N, 12.61; S, 9.62. HPLC: purity 98%.

3.3.7. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-Methoxybenzene-1-sulfonate (7)

White solid, yield: 87%, mp: 173–175 °C. IR (ν, cm^–1): 3485, 3145 (N–H); 3063 (Ar–CH); 2972 (aliphatic CH); 1693 (CO); 1596 (CN stretching band); 1574, 1445 (Ar–CC); 1350, 1154 (SO₂); 1028 (Ar–C–OCH₃); 686 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.31 (s, 1H, NH), 7.75 (dd, J = 16.1, 8.7 Hz, 6H, ArH), 7.16 (d, J = 9.0 Hz, 2H, ArH), 6.98 (d, J = 8.7 Hz, 2H, ArH), 6.52 (s, 2H, NH ₂), 3.86 (s, 3H, CH ₃). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 164.52 (CO), 157.12, 149.62 (CN), 138.01, 134.46, 131.18, 128.42, 125.75, 122.79, 115.42 (ArC), 56.41 (OCH3). ESI-MS (m/z): 348 [M – H]⁺. Elemental analysis, C₁₅H₁₅N₃O₅S (349,36 g/mol). Found, %: C, 51.70; H, 4.56; N, 12.31; S, 9.05. Calculated, %: C, 51.57; H, 4.33; N, 12.03; S, 9.18. HPLC: purity 99%.

3.3.8. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 2-(Trifluoromethyl)­benzene-1-sulfonate (8)

White solid, yield: 83%, mp: 204–206 °C. IR (ν, cm^–1): 3447, 3329 (N–H); 3080 (Ar–CH); 2988 (aliphatic CH); 1694 (CO); 1570 (CN stretching band); 1499, 1441 (Ar–CC); 1381, 1172 (SO₂); 1146, 1122 (Ar–C–CF₃); 688 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.34 (s, 1H, NH), 8.21 (d, J = 7.4 Hz, 1H, ArH), 8.05 (t, J = 7.2 Hz, 2H, ArH), 7.92 (t, J = 7.5 Hz, 1H, ArH), 7.79 – 7.74 (m, 3H, ArH), 7.03 (d, J = 8.6 Hz, 2H, ArH), 6.54 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.09 (CO), 149.19 (CN), 142.58, 137.85, 136.35, 134.89, 134.21, 133.52, 132.66, 129.82, 128.63, 127.72, 122.52 (ArC). ESI-MS (m/z): 386 [M – H]⁺. Elemental analysis, C₁₅H₁₂F₃N₃O₄S (387,33 g/mol). Found, %: C, 46.63; H, 14.85; N, 10.68; S, 8.33. Calculated, %: C, 46.51; H, 14.71; N, 10.85; S, 8.28. HPLC: purity 99%.

3.3.9. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl-3-(trifluoromethyl)­benzene-1-sulfonate (9)

Off-white solid, yield: 87%, mp: 38–40 °C. IR (ν, cm^–1): 3512, 3483 (N–H); 3063 (Ar–CH); 2972 (aliphatic CH); 1694 (CO); 1569 (CN stretching band); 1496, 1473 (Ar–CC); 1382, 1165 (SO₂); 692 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.33 (s, 1H, NH), 7.87 (dd, J = 25.7, 11.2 Hz, 4H, ArH), 7.79 – 7.73 (m, 3H, ArH), 7.05 (d, J = 5.9 Hz, 2H, ArH), 6.54 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 170.28 (CO), 157.10, 149.26 (CN), 149.25, 148.83, 137.84, 136.35, 134.87, 132.91, 128.54, 128.11, 122.69, 121.40 (ArC). ESI-MS (m/z): 386 [M – H]⁺. Elemental analysis, C₁₅H₁₂F₃N₃O₄S (387,33 g/mol). Found, %: C, 46.46; H, 14.76; N, 10.89; S, 8.35. Calculated, %: C, 46.51; H, 14.71; N, 10.85; S, 8.28. HPLC: purity 98%.

3.3.10. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl-4-(trifluoromethyl)­benzene-1-sulfonate (10)

White solid, yield: 90%, mp: 38–40 °C. IR (ν, cm^–1): 3482, 3301 (N–H); 3072 (Ar–CH); 2988 (aliphatic CH); 1697 (CO); 1587 (CN stretching band); 1496, 1435 (Ar–CC); 1380, 1152 (SO₂); 1124, 1085 (Ar–C–CF₃); 629 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.35 (s, 1H, NH), 8.09 (dd, J = 12.8, 6.6 Hz, 4H, ArH), 7.80–7.75 (m, 3H, ArH), 7.06 (d, J = 10.9 Hz, 2H, ArH), 6.55 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 192.38, 157.12 (CO), 153.10, 149.26 (CN), 138.37, 135.61, 132.06, 129.86, 127.60, 123.39, 122.72 (ArC). Elemental analysis, C₁₅H₁₂F₃N₃O₄S (387,33 g/mol). Found, %: C, 46.39; H, 14.70; N, 10.88; S, 8.37. ESI-MS (m/z): 386 [M – H]⁺. Calculated, %: C, 46.51; H, 14.71; N, 10.85; S, 8.28. HPLC: purity 98%.

3.3.11. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 3,5-Bis­(trifluoromethyl)­benzene-1-sulfonate (11)

White solid, yield: 88%, mp: 65–66 °C. IR (ν, cm^–1): 3545, 3486, 3414 (N–H); 3087 (Ar–CH); 2915 (aliphatic CH); 1691 (CO); 1572 (CN stretching band); 1494, 1432 (Ar–CC); 1384, 1171 (SO₂); 1138, 1109 (Ar–C–CF₃); 697 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.36 (s, 1H, NH), 8.72 (s, 1H, NCH), 8.46 (s, 2H, ArH), 7.79 (d, J = 9.1 Hz, 3H, ArH), 7.17 (d, J = 8.6 Hz, 2H, ArH), 6.56 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.12 (CO), 149.05 (CN), 137.81, 137.17, 135.14, 132.52, 132.18, 129.55, 128.61, 122.87, 121.34 (ArC). ESI-MS (m/z): 454 [M – H]⁺. Elemental analysis, C₁₆H₁₁F₆N₃O₄S (455,33 g/mol). Found, %: C, 42.10; H, 2.39; N, 9.17; S, 7.10. Calculated, %: C, 42.21; H, 2.44; N, 9.23; S, 7.04. HPLC: purity 99%.

3.3.12. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 2-(Trifluoromethoxy)­benzene-1-sulfonate (12)

White solid, yield: 92%, mp: 172–174 °C. IR (ν, cm^–1): 3460, 3293 (N–H); 3065 (Ar–CH); 2988 (aliphatic CH); 1689 (CO); 1596 (CN stretching band); 1498, 1479 (Ar–CC); 1382, 1194 (SO₂); 1155, 1130 (Ar–C–OCF₃); 694 (Ar–CH). ¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 10.35 (s, 1H, NH), 7.98 (t, J = 8.0 Hz, 1H, ArH), 7.94 (d, J = 7.9 Hz, 1H, ArH), 7.79 (s, 2H, ArH), 7.76 (d, J = 8.6 Hz, 2H, ArH), 7.62 (t, J = 7.7 Hz, 1H, ArH), 7.04 (d, J = 8.5 Hz, 2H, ArH), 6.54 (s, 2H, NH ₂). ¹³C NMR (151 MHz, DMSO-d ₆) δ ppm: 157.12 (CO), 149.22 (CN), 145.98, 138.27, 137.89, 134.90, 132.49, 128.64, 128.56, 127.16, 122.30, 122.06, 121.14 (ArC). ESI-MS (m/z): 402 [M – H]⁺. Elemental analysis, C₁₅H₁₂F₃N₃O₅S (403,33 g/mol). Found, %: C, 44.60; H, 3.05; N, 10.48; S, 8.00. Calculated, %: C, 44.67; H, 3.00; N, 10.42; S, 7.95. HPLC: purity 99%.

3.3.13. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 3-(Trifluoromethoxy)­benzene-1-sulfonate (13)

White solid, yield: 90%, mp: 137–139 °C. IR (ν, cm^–1): 3477, 3381 (N–H); 3082 (Ar–CH); 2987 (aliphatic CH); 1788 (CO); 1587 (CN stretching band); 1497, 1474 (Ar–CC); 1378, 1173 (SO₂); 1148, 1092 (Ar–C–OCF₃); 692 (Ar–CH). ¹H NMR (600 MHz, DMSO-d ₆) δ ppm: 10.35 (s, 1H, NH), 7.95–7.92 (m, 1H, ArH), 7.88 (s, 1H, ArH), 7.83 (d, J = 7.1 Hz, 2H, ArH), 7.79 (s, 1H, N = CH), 7.76 (d, J = 8.6 Hz, 2H, ArH), 7.05 (d, J = 8.6 Hz, 2H, ArH), 6.55 (s, 2H, NH ₂). ¹³C NMR (151 MHz, DMSO-d ₆) δ ppm: 157.13 (CO), 149.30 (CN), 148.82, 137.93, 136.42, 134.88, 132.90, 131.89, 128.54, 128.40, 128.08, 122.67, 121.35 (ArC). ESI-MS (m/z): 402 [M – H]⁺. Elemental analysis, C₁₅H₁₂F₃N₃O₅S (403,33 g/mol). Found, %: C, 44.70; H, 2.95; N, 10.45; S, 7.90. Calculated, %: C, 44.67; H, 3.00; N, 10.42; S, 7.95. HPLC: purity 93%.

3.3.14. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-(Trifluoromethoxy)­benzene-1-sulfonate (14)

White solid, yield: 90%, mp: 170–172 °C. IR (ν, cm^–1): 3486, 3149 (N–H); 3069 (Ar–CH); 2972 (aliphatic CH); 1748 (CO); 1576 (CN stretching band); 1491, 1439 (Ar–CC); 1363, 1198 (SO₂); 1153, 1092 (Ar–C–OCF₃); 702 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.34 (s, 1H, NH), 8.02 (d, J = 8.9 Hz, 2H, ArH), 7.78 (s, 1H, NCH), 7.75 (d, J = 8.7 Hz, 2H, ArH), 7.66 (d, J = 8.4 Hz, 2H, ArH), 7.04 (d, J = 8.6 Hz, 2H, ArH), 6.54 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.13 (CO), 153.03, 149.33 (CN), 137.90, 134.82, 133.29, 131.67, 128.55, 123.41, 122.74, 122.14 (ArC). ESI-MS (m/z): 402 [M – H]⁺. Elemental analysis, C₁₅H₁₂F₃N₃O₅S (403,33 g/mol). Found, %: C, 44.71; H, 3.07; N, 10.50; S, 8.01. Calculated, %: C, 44.67; H, 3.00; N, 10.42; S, 7.95. HPLC: purity 98%.

3.3.15. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl 4-Nitrobenzene-1-sulfonate (15)

White solid, yield: 95%, mp: 251–253 °C. IR (ν, cm^–1): 3489 (N–H); 3075 (Ar–CH); 2988 (aliphatic CH); 1694 (CO); 1576 (CN stretching band); 1530, 1446 (Ar–CC); 1351, 1149 (SO₂); 679 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.34 (s, 1H, NH), 8.45 (d, J = 9.0 Hz, 2H, ArH), 8.16 (d, J = 9.0 Hz, 2H, ArH), 7.79 (s, 1H, NCH), 7.75 (d, 2H, ArH), 7.07 (d, J = 8.8 Hz, 2H, ArH), 6.53 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.18 (CO), 151.54 (CN), 149.12, 139.79, 137.83, 134.98, 130.58, 128.64, 125.49, 122.76 (ArC). Elemental analysis, C₁₄H₁₂N₄O₆S (364,33 g/mol). Found, %: C, 46.21; H, 3.40; N, 15.40; S, 8.85. Calculated, %: C, 46.15; H, 3.32; N, 15.38; S, 8.80. HPLC: purity 99%.

3.3.16. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl [1,1′-Biphenyl]-4-sulfonate (16)

Off-white solid, yield: 89%, mp: 144–146 °C. IR (ν, cm^–1): 3488 (N–H); 3091 (Ar–CH); 2923 (aliphatic CH); 1690 (CO); 1573 (CN stretching band); 1497, 1456 (Ar–CC); 1373, 1150 (SO₂); 671 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.32 (s, 1H, NH), 7.97 (d, J = 8.6 Hz, 2H, ArH), 7.92 (d, J = 7.2 Hz, 2H, ArH), 7.78 (s, 1H, N = CH), 7.74 (d, J = 7.3 Hz, 2H, ArH), 7.57 – 7.46 (m, 5H, ArH), 7.05 (d, J = 7.1 Hz, 2H, ArH), 6.53 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.16 (CO), 149.53 (CN), 146.69, 138.19, 138.01, 134.63, 133.27, 129.70, 129.54, 129.44, 128.50, 128.26, 127.69, 122.78 (ArC) ESI-MS (m/z): 396 [M + H]⁺. Elemental analysis, C₂₀H₁₇N₃O₄S (395,43 g/mol). Found, %: C, 61.00; H, 4.36; N, 10.58; S, 8.15. Calculated, %: C, 60.75; H, 4.33; N, 10.63; S, 8.11. HPLC: purity 94%.

3.3.17. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl Naphthalene-1-sulfonate (17)

Brown solid, yield: 94%, mp: 88–90 °C. IR (ν, cm^–1): 3486, 3454, 3320 (N–H); 3100 (Ar–CH); 2968 (aliphatic CH); 1708 (CO); 1576 (CN stretching band); 1497, 1444 (Ar–CC); 1359, 1133 (SO₂); 675 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.30 (s, 1H, NH), 8.67 (d, J = 8.8 Hz, 1H, ArH), 8.43 (d, J = 8.4 Hz, 1H, ArH), 8.23 (d, J = 8.2 Hz, 1H, ArH), 8.10 (d, J = 7.3 Hz, 1H, ArH), 7.94 (t, J = 7.8 Hz, 1H, ArH), 7.81 (t, J = 7.5 Hz, 1H, ArH), 7.71 (s, 1H, N CH), 7.64 (dd, J = 8.2, 3.2 Hz, 3H, ArH), 6.83 (d, J = 8.7 Hz, 2H, ArH), 6.50 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.09 (CO), 149.48 (CN), 137.80, 136.98, 134.63, 134.20, 132.03, 129.98, 128.45, 128.16, 128.03, 125.11, 124.53, 122.20 (ArC). ESI-MS (m/z): 368 [M – H]⁺. Elemental analysis, C₁₈H₁₅N₃O₄S (369,40 g/mol). Found, %: C, 58.60; H, 4.11; N, 11.42; S, 8.71. Calculated, %: C, 58.53; H, 4.09; N, 11.38; S, 8.68. HPLC: purity 98%.

3.3.18. 4-[(E)-(2-Carbamoylhydrazinylidene)­methyl]­phenyl Naphthalene-2-sulfonate (18)

Yellow solid, yield: 90%, mp: 81–83 °C. IR (ν, cm^–1): 3484 (N–H); 3100 (Ar–CH); 2968 (aliphatic CH); 1688­(CO); 1573 (CN stretching band); 1497, 1457 (Ar–CC); 1372, 1152 (SO₂); 659 (Ar–CH). ¹H NMR (400 MHz, DMSO-d ₆) δ ppm: 10.30 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.23 (d, J = 8.8 Hz, 2H, ArH), 8.13 (d, J = 8.4 Hz, 1H, ArH), 7.90 (d, J = 8.8 Hz, 1H, ArH), 7.82–7.77 (m, 1H, ArH), 7.75 (s, 1H, N = CH), 7.73–7.68 (m, 3H, ArH), 7.02 (d, J = 8.7 Hz, 2H, ArH), 6.51 (s, 2H, NH ₂). ¹³C NMR (101 MHz, DMSO-d ₆) δ ppm: 157.12 (CO), 149.56 (CN), 137.93, 135.55, 134.60, 131.89, 131.63, 130.85, 130.53, 130.13, 128.61, 128.47, 123.01, 122.75 (ArC). ESI-MS (m/z): 368 [M – H]⁺. Elemental analysis, C₁₈H₁₅N₃O₄S (369,40 g/mol). Found, %: C, 58.60; H, 4.06; N, 11.41; S, 8.70. Calculated, %: C, 58.53; H, 4.09; N, 11.38; S, 8.68. HPLC: purity 99%.

3.4. DFT Studies

Quantum chemical investigations were performed using density functional theory (DFT) as implemented in the Gaussian 09 software suite. Compounds with good enzyme activity (12, 17, and 18) were optimized, and vibrational frequency analyses were carried out to verify that these structures represent true local minima on the potential energy surface. The B3LYP functional combined with the 6–311++G­(d,p) basis set was employed for all calculations. − To better understand the electronic distribution and reactive tendencies of the molecules, frontier molecular orbitals (FMOs) and molecular electrostatic potential (MEP) maps were generated. The energies of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) served as the basis for calculating various global reactivity descriptors (GRPs) (eq ). These parameters include

$$\begin{array}{ll}I=-E_{\mathrm{HOMO}}& \left(1\right)\\ A=-E_{\mathrm{LUMO}}& \left(2\right)\\ \chi =-\left[\frac{E_{\mathrm{LUMO}}+E_{\mathrm{HOMO}}}{2}\right]& \left(3\right)\\ \chi =\frac{(I-A)}{2}& \left(4\right)\\ \mu =-\chi & \left(5\right)\\ \sigma =\frac{1}{\eta }& \left(6\right)\\ \omega ={\mu }^2/2\eta & \left(7\right)\end{array}$$ 1

3.5. In Vitro Colinesterase Inhibition Studies

The semicarbazone derivatives were evaluated for their inhibitory activities against human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using standard in vitro assays. The Ellman’s technique that was tailored to our lab’s needs was used to measure the activity of the cholinesterase enzyme. The test chemicals were first dissolved in DMSO at a concentration of 1 mg/mL to create stock solutions, which were then diluted 10 times with distilled water for use in the inhibition tests. Each compound’s inhibitory effects on the enzymes were evaluated at five different concentrations. Fifty microliters of distilled water, 50 μL of 0.1 M Tris–HCl buffer (pH 8.0), 15 μL of 0.015 M acetylcholine iodide or butyrylcholine iodide (as substrates), 30 μL of 0.06 M DTNB (5,5′-dithiobis­(2-nitrobenzoic acid)), and 10 μL of enzyme solution made up the reaction mixture for the AChE and BChE tests. The absorbance of the blank solutions was measured before the substrate was added. The substrate was then added to each well to start the enzymatic reaction. Enzyme activity was kinetically assessed at 408 nm by using a microplate reader. Each inhibition measurement was repeated three times, and average values were taken when measuring the inhibitory potentials of the molecules. The IC₅₀ values, indicating each compound’s inhibitory strength against cholinesterase, were determined by plotting percent activity versus inhibitor concentration.

3.6. Enzyme Kinetics

To determine the inhibition type of BChE, kinetic studies were performed with the most active compounds (12, 17, and 18). Different concentrations of butyrylthiocholine iodide were used in the presence of three concentrations of each compound. Lineweaver–Burk plots were constructed by plotting the reciprocal of reaction velocity (1/V) against the reciprocal of substrate concentration (1/[S]). Based on these plots, the inhibition types were determined. The inhibition constant (K _i) was calculated using the following equation (eq ).

$$\mathrm{slope}=K_{\mathrm{m}}/V_{\max }\times (1+[I]/K_{\mathrm{i}})$$ 2

3.7. Molecular Docking

Molecular docking studies were conducted to elucidate the interactions between the synthesized compounds and the enzyme butyrylcholinesterase (BChE) at the atomic level. The docking simulations were carried out using Molegro Virtual Docker (MVD) 6.0. The crystal structure of BChE was obtained from the Protein Data Bank (4BDS). The MVD software automatically performed protein and ligand preparation, removal of water molecules, and automatic identification of the enzyme’s active site. The binding region targeted for docking corresponds to the site where a known reference ligand interacts. Each compound underwent 15 independent docking runs, and the best binding poses were selected based on MolDock scoring. Interaction details and 2D binding visualizations were further examined using BIOVIA Discovery Studio.

3.8. Molecular Dynamic Simulation and Enzyme Preparation

Molecular dynamics (MD) simulations were conducted using GROMACS 2024, leveraging the VSmartMD tool for automated execution and visualization. Initial.pdb files of protein–ligand complexes were prepared, and simulations were performed with the CHARMM27 force field. Protein topologies were derived via pdb 2gmx, while ligand parameters were assigned using the SwissParam server.

The systems were solvated in a TIP3P water model within a cubic box, maintaining a 1 nm buffer between the protein and the box edges under periodic boundary conditions. Charge neutrality was achieved by adding Na⁺ ions, followed by energy minimization (50,000 steepest descent steps). Equilibration included NVT (100 ps, 300 K) and NPT (100 ps, 1 bar) phases, with temperature/pressure regulated via the Berendsen method (time constants: 0.1 ps for temperature and 2 ps for pressure). The Leapfrog integrator was used with separate coupling for protein, ligand, solvent, and ions.

Production runs spanned 100 ns under NPT conditions (1 ps pressure coupling), with LINCS-constrained bonds, a 1.2 nm cutoff for nonbonded interactions, and PME for long-range electrostatics. Trajectories were analyzed using VMD 1.9.2, HeroMDAnalysis, and VSmartMD.

3.9. ADMET

ADME studies evaluate a compound’s absorption, distribution, metabolism, and excretion within the body through the application of predictive mathematical models. Since pharmacodynamic and pharmacokinetic properties are critical in drug development, such assessments play a vital role in guiding early-stage drug design. In silico ADMET analysis offers insight into a molecule’s drug-likeness and suitability as a therapeutic agent, contributing to more cost-effective and time-efficient drug discovery. In this study, the ADMET profiles of all synthesized compounds were predicted using the SwissADME web tool, while toxicity-related parameters were assessed with the ProTox-II platform.

In this study, the ADMET profiles of all synthesized compounds were predicted using the SWISS ADME, ProTox-II, and PKCSM online sites.

4. Conclusion

In this study, a novel series of 18 (E)-4-((2-carbamoylhydrazinylidene)­methyl)­phenyl-substituted sulfonate derivatives (1–18) were successfully synthesized in high yields (93–98%) and structurally characterized by FT-IR, ¹H NMR, and ¹³C NMR spectroscopy. In vitro inhibitory studies against BChE revealed that several compounds, particularly 11, 12, 13, 14, 16, 17, and 18, exhibited stronger inhibition than the reference drug pyridostigmine bromide. Among them, compound 12 emerged as the most potent inhibitor (IC₅₀ = 61.88 μM). Molecular dynamics and binding energy analyses were conducted to evaluate the inhibitory effectiveness of “Compounds 12, 17, and 18” on the 4DBS protein, using “tacrine” as a reference ligand. The RMSD and RMSF findings indicated that all ligands preserved the structural integrity of the protein with “Compound 17” and “tacrine” showing the smallest conformational fluctuations. Crucial binding site residues such as “THR120” and “TYR440” remained consistently involved across all complexes, with “Compound 17” demonstrating the highest occupancy of hydrogen bonds at these positions. Additionally, “Compound 18” recorded the highest average number of hydrogen bond interactions. Binding free energy results identified “Compound 17” as having the most favorable interaction energy (−54.74 kJ/mol), exceeding that of tacrine. Overall, these findings highlight “compound 17” as the most promising BChE inhibitor candidate, with “compound 18” also demonstrating strong and stable binding characteristics, thereby supporting the potential of this newly synthesized semicarbazone series as lead scaffolds for further drug development.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09763.

• 1H NMR, ¹³C NMR, FT-IR, and Mass spectrum of compounds A8, A12–13, and 1–18 (PDF)

The authors declare no competing financial interest.