Phenylsulfonylethyl‐Substituted Benzimidazolium Salts: Synthesis, Characterization, Crystal Structure, Molecular Docking, and Inhibitory Properties Against Various Enzymes

ABSTRACT

N‐heterocyclic carbenes (NHCs) are chemical structures called heterocyclic structures that contain one or more nitrogen atoms. N‐heterocyclic compounds are the basic building blocks of many drugs and biologically active molecules. One of these compounds is benzimidazolium salts. Herein, we present the synthesis of a series of phenylsulfonylethyl‐substituted benzimidazolium salts. These compounds were characterized using NMR (¹H‐ and ¹³C‐) and FTIR spectroscopic techniques. Compound 1b exhibits a U‐shaped benzimidazolium cation paired with a chloride anion. A hierarchical supramolecular architecture arises from C–H⋯Cl chains, C–H⋯π layers, and π⋯π stacking, demonstrating cooperative non‐covalent stabilization of the crystal structure. The synthesized benzimidazolium derivatives exhibited notable inhibitory effect against human carbonic anhydrases (hCA I, hCA II)and acetylcholinesterase (AChE), with several compounds showing markedly lower K_i values than the clinical standards. Molecular docking of the NHCs with different receptors showed excellent binding capability with the active site of the target receptors. Moreover, the physicochemical and ADME analysis of the compounds were performed with SwissADME software. Overall examined compounds were found with very good oral bioavailability determined by Lipinski and Verber's rule. The compounds showed Pgp susceptibility with reasonable blood brain barrier permeability.

Herein, we present the synthesis of a series of phenylsulfonylethyl‐substituted benzimidazolium salts. These compounds were characterized using NMR and FTIR spectroscopic techniques. Also, crystal structures of the one compound was recorded by using crystal X‐ray diffraction assay. The synthesized benzimidazolium salts exhibited notable inhibitory activity against hCA I, hCA II and AChE, with several compounds showing markedly lower Ki values than the clinical standards. Moreover, the physicochemical and ADME analysis of the compounds were performed with SwissADME software. Overall examined compounds were found with very good oral bioavailability determined by Lipinski and Verber's rule.

1. Introduction

Benzimidazolium salts belongs to a class of bicyclic organic compounds that are formed by the fusion of a benzene ring to an imidazole ring [1, 2]. These salts have been attracting a great deal of attention in many fields due to their unique properties and applications. For example, benzimidazolium salts usually have a positive charge localized at the nitrogen atom of the imidazole ring [3]. This affects the stability and electronic properties of benzimidazolium salts. Additionally, the solubilities of these salts vary depending on the anion present in their structure and substituents on the aromatic ring. Many are useful for synthesis and reaction conditions because they are soluble in polar organic solvents such as water, methanol, and ethanol [4]. Benzimidazolium compounds exhibit ionic properties that can affect their melting points, thermal stability, and reactivity in ionic exchange reactions. Furthermore, some of these salts can exhibit interesting electrochemical behavior, being conductive due to having a positive charge [5]. They can also participate in ionic interactions, making them suitable for batteries and sensors. Benzimidazolium salts are part of a class of important ligands known as N‐heterocyclic carbene (NHC) precursors. Organometallic complexes obtained because of the coordination of these ligands to metal centers attract attention in organo‐catalysis and transition metal catalysis [6]. Some benzimidazolium salts exhibit a wide range of biological activities, including antibacterial [7], antifungal [8], anticancer [9] anti‐inflammatory, and analgesic [10] properties. This has made them of great interest to scientists in the fields of medicinal chemistry and the development of new drugs. All these properties make benzimidazolium salts versatile compounds with applications ranging from organic synthesis to material science and drug discovery.

Carbonic anhydrase (CAs, EC 4.2.1.1) enzymes are ubiquitous metalloproteins that are known to play a vital role in prokaryotes and eukaryotes alike, as it facilitates the straightforward biological reaction of carbon dioxide hydration, producing a proton and a bicarbonate anion [11, 12]. Consequently, they serve as one of the principal controllers of pH homeostasis within the cell. These enzymes also play a role in many important biological processes, such as electrolyte secretion, respiration, carbon dioxide and bicarbonate ion transfer, urea synthesis, bone resorption, lipogenesis, gluconeogenesis, etc [13, 14].

This family of enzymes has been recognized for several decades as a potentially important class of biologically relevant targets for possible pharmacological intervention [15]. A common alteration in the expression level of the human CAs has been correlated with human diseases: certain central‐nervous system syndromes, such as idiopathic intracranial hypertension, epilepsy and neuropathic pain (hCAs I, II, and VII), glaucoma (hCAs I, II, IV, XII), osteoporosis (hCA IV and XIV), cancer (hCAs IX and XII), and edema (hCA II, IV, XII, and XIV) [16, 17]. Sulfonamides are one of the most significant and well‐known inhibitors of CA, and many members have been in clinical use for many decades [18, 19]. On structural basis and even for their subcellular targeting, many of the CA isoforms are quite similar to each other, while the major pitfall of CA inhibitors (CAIs) is represented by their targeted inhibition of several CA isoforms without selectivity and undesired side effects [20, 21].

Designing selective medicines with distinct inhibitory characteristics for various isoforms remains a problem. Expansion of our knowledge in this area is important since CAs are involved in a variety of physiological and pathological processes, and the usefulness of potent and selective inhibitors has been demonstrated in disease models, so new compounds based on our pharmacophore models could ultimately help to advance strategies for selective targeting in diseases where dysregulated CA activity has been implicated, such as cancer, glaucoma and other disseases [22, 23].

Alzheimer's disease (AD) is a persistent neurodegenerative condition that results in the demise of brain cells, leading to deficits in behavior, cognition, and memory [24]. There have been various theories to explain Tau, damage to cholinergic neurons, comprising amyloid‐β (Aβ), the etiology of AD, inflammation, oxidative stress, and so on [25, 26]. The current primary therapy strategies for AD are founded on the cholinergic hypotesis. This concept pertains to the degeneration of cholinergic neurons and the expedited breakdown of the neurotransmitter ACh (acetylcholine) by cholinesterases [27]. The human brain has two cholinesterases: butyrylcholinesterase (BChE) and AChE, both of which facilitate the breakdown of ACh. Thereby, the inhibition of these enzymes could serve as a critical determinant in AD treatment through the blockade of ACh hydrolysis [28, 29]. Researchers are now developing novel cholinesterase inhibitors for more effective AD treatment.

Phenylsulfonyl ethyl‐substituted organic compounds, characterized by the presence of a sulfonyl group (–SO₂–) attached to the benzene ring, exhibit several distinctive properties due to the influence of the functional group on chemical reactivity, polarity, and intermolecular interactions [30]. The sulfonyl group is highly polar due to the electronegative sulfur and oxygen atoms. This polarity can increase the solubility of phenylsulfonyl compounds in polar solvents such as water and alcohols compared to non‐polar compounds. Such groups increase the hydrophilicity of the compound, making it useful in applications where increased solubility in aqueous media is desired (e.g., pharmaceuticals). Many of the phenylsulfonyl compounds have good thermal stability, which is useful in a wide range of industrial applications [31]. The sulfonyl group can act as a leaving group in nucleophilic substitution reactions. This makes the phenylsulfonyl compounds reactive towards nucleophiles and leads to the formation of sulfonamides or other substituted derivatives. Additionally, phenylsulfonyl compounds may exhibit acidity due to the ability of the sulfonyl group to stabilize the negative charge (as in sulfonic acids). This property could be of use in acid‐base reactions. Furthermore, phenylsulfonyl groups are commonly found in biologically active compounds, including pharmaceuticals [31]. For example, sulfonamide antibiotics use the properties of the sulfonyl group to inhibit bacterial enzymes [32]. The properties of phenyl sulfonyl ethyl‐substituted organic compounds are greatly influenced by the sulfonyl group, which affects their solubility, reactivity, and biological activity. Their versatility makes them important in both industrial and pharmaceutical chemistry. So, in this work, a series of phenylsulfonylethyl‐substituted benzimidazolium salts was designed and synthesized. All synthesized compounds were characterized using NMR and FTIR spectroscopic techniques. Furthermore, the structure of a compound was also characterized using X‐ray diffraction (XRD).

The primary aim of this study was to evaluate the inhibitory potential of newly synthesized phenylsulfonyl ethyl‐substituted benzimidazolium salts against key metabolic enzymes, particularly hCA I and II as well as AChE. By investigating how structural modifications influence enzyme binding and inhibitory potency, this work seeks to identify promising lead molecules for the development of selective and potent CA and AChE inhibitors. Molecular docking analysis of the respective NHCs were conducted with Autodoc Vina to determine the capability of the ligand to bind with the active pocket of the selected proteins. Physicochemical and ADME analysis were performed to investigate the oral bioavailability, cytotoxicity, and permeability of the NHCs.

2. Experimental

2.1. Material and Methods

All phenylsulfonyl ethyl‐substituted benzimidazolium salts (1a–f) have been prepared by standard Schlenk's techniques under an inert argon atmosphere. The solvents were used without any purification. All reagents were purchased from well‐known suppliers such as Abcr, Isolab, Tekkim, and Sigma‐Aldrich Chemical Co. The melting points were measured using an Electrothermal‐9200 device in glass capillaries under atmospheric air. The FT‐IR spectra were recorded in the range 400–4000 cm^{− 1} on Perkin Elmer Spectrum 100 FTIR spectrometer. Carbon and Proton NMR spectra were recorded using a Bruker Avance III 400 MHz NMR spectrometer operating at 100 MHz (¹³C‐), 400 MHz (¹H‐) in CDCl₃ and DMSO‐d₆.

2.2. Synthesis

2.2.1. 1‐Benzyl‐3‐(2‐(phenylsulfonyl)ethyl)benzimidazolium Chloride, 1a

To synthesize compound 1a; the 1‐benzylbenzimidazole (624 mg, 3 mmol) and ((2‐chloroethyl)sulfonyl)benzene (614 mg, 3 mmol) were mixed by equivalent. The mixture was added to a Schlenk under an inert atmosphere. The mixture was stirred at 80°C for 24 h in acetonitrile (25 mL). Excess of the acetonitrile was then removed under vacuum. Diethyl ether was added to the remaining mixture and stirred at room temperature. The crude product was then recrystallized at room temperature using a mixture of ethanol and diethyl ether (1:2) [33]. Yield: 82% (1.02 g); m.p.: 180°C–182°C; FTIR for: ν _sym(S=O): 1147 cm^{− 1}; ν _asym(S=O): 1299 cm^{− 1}; ν _(CN): 1557 cm^{− 1}. ¹H NMR (400 MHz, CDCl₃), δ; 5.67 (s, 2H, N‐C H ₂‐C₆H₅); 4.14 and 5.21 (s, 4H, N‐C H ₂C H ₂(SO₂)C₆H₅); 7.27–7.92 (m, 14H, Ar‐ H ); 11.80 (s, 1H, ‐C H‐ ). ¹³C NMR (100 MHz, CDCl₃), δ; 40.8 and 51.8 (N C H₂ C H₂(SO₂)C₆H₅); 54.6 (N C H₂C₆H₅); 111.5, 113.8, 127.2, 127.4, 128.0, 128.1, 129.3, 129.5, 130.9, 131.8, 132.2, 134.4, and 138.4 (Ar‐ C ); 144.3 (‐ C H‐).

Compounds 1b–f were also synthesized with a similar method. For the amount of starting material used for the synthesis of compounds 1b–f see details of the individual compounds.

2.2.2. 1‐(2‐Methylbenzyl)‐3‐(2‐(phenylsulfonyl)ethyl)benzimidazolium Chloride, 1b

1‐(2‐Methylbenzyl)benzimidazole (666 mg, 3 mmol). Yield: 80% (1.03 g). m.p.: 227°C–228°C; FTIR for: ν _sym(S=O): 1143 cm^{− 1}; ν_asym(S=O): 1320 cm^{− 1}; ν_(CN): 1562 cm^{− 1}. ¹H NMR (400 MHz, CDCl₃), δ; 2.35 (s, 3H, N‐CH₂‐C₆H₄‐2‐C H ₃); 4.09 and 5.23 (s, 4H, N‐C H ₂C H ₂(SO₂)C₆H₅); 5.66 (s, 2H, N‐C H ₂‐C₆H₄‐2‐CH₃); 7.05‐7.95 (m, 13H, Ar‐ H ); 11.58 (s, 1H, ‐C H‐ ). ¹³C NMR (100 MHz, CDCl₃), δ; 19.6 (N‐CH₂‐C₆H₄‐2‐ C H₃); 41.1 and 50.4 (N C H₂ C H₂(SO₂)C₆H₅); 55.1 (N C H₂C₆H₄‐2‐CH₃); 113.6, 114.0, 127.1, 127.4, 127.5, 128.1, 128.4, 129.6, 129.7, 130.0, 131.0, 131.4, 132.0, 134.5, 136.5, and 138.5 (Ar‐ C ); 143.5 (‐ C H‐).

2.2.3. 1‐(3‐Methylbenzyl)‐3‐(2‐(phenylsulfonyl)ethyl)benzimidazolium Chloride, 1c

1‐(3‐Methylbenzyl)benzimidazole (666 mg, 3 mmol). Yield: 84% (1.08 mg); m.p.: 185°C–187°C; FTIR for: ν _sym(S=O): 1141 cm^{− 1}; ν _asym(S=O): 1300 cm^{− 1}; ν_(CN): 1565 cm^{− 1}. ¹H NMR (400 MHz, CDCl₃), δ; 2.23 (s, 3H, N‐CH₂‐C₆H₄‐3‐C H ₃); 5.62 (s, 2H, N‐C H ₂‐C₆H₄‐2‐CH₃); 4.14 and 5.22 (s, 4H, N‐C H ₂C H ₂(SO₂)C₆H₅) 7.07–7.93 (m, 13H, Ar‐ H ); 11.70 (s, 1H, ‐C H‐ ). ¹³C NMR (100 MHz, CDCl₃), δ; 21.3 (N‐CH₂‐C₆H₄‐3‐ C H₃); 40.8 and 51.8 (N‐ C H₂ C H₂(SO₂)C₆H₅); 54.6 (N‐ C H₂C₆H₄‐3‐CH₃); 113.6, 113.8, 125.1, 127.2, 127.4, 128.0, 128.6, 129.3, 129.4, 130.1, 131.8, 132.1, 134.3, 138.5, and 139.4 (Ar‐ C ); 144.2 (‐ C H‐).

2.2.4. 1‐(4‐Methylbenzyl)‐3‐(2‐(phenylsulfonyl)ethyl)benzimidazolium Chloride, 1d

1‐(4‐methylbenzyl)benzimidazole (666 mg, 3 mmol). Yield: 86% (1.10 g); m.p.: 190°C‐191°C; FTIR for: ν_sym(S=O): 1146 cm^‐1; ν_asym(S=O): 1290 cm^‐1; ν_(CN): 1560 cm^‐1. ¹H NMR (400 MHz, CDCl₃), δ; 2.21 (s, 3H, N‐CH₂‐C₆H₄‐4‐C H ₃); 5.61 (s, 2H, N‐C H ₂‐C₆H₄‐4‐CH₃); 4.14 and 5.20 (s, 4H, N‐C H ₂C H ₂(SO₂)C₆H₅); 7.07‐7.93 (m, 13H, Ar‐ H ); 11.67 (s, 1H, ‐C H‐ ). ¹³C NMR (100 MHz, CDCl₃), δ; 21.2 (N‐CH₂‐C₆H₄‐4‐ C H₃); 40.9 and 51.6 (N‐ C H₂ C H₂(SO₂)C₆H₅); 54.4 (N‐ C H₂C₆H₄‐4‐CH₃); 113.6, 113.8, 127.1, 127.3, 128.0, 128.3, 138.4, and 139.2 (Ar‐ C ); 144.1 (‐ C H‐).

2.2.5. 1‐(2,4,6‐Trimethylbenzyl)‐3‐(2‐(phenylsulfonyl)ethyl)benzimidazolium Chloride, 1e

1‐(2,4,6‐Trimethylbenzyl)benzimidazole (750 mg, 1 mmol). Yield: 81% (1.106 mg); m.p.: 197°C–198°C; FTIR for: ν _sym(S=O): 1137 cm^{− 1}; ν_asym(S=O): 1243 cm^{− 1}; ν_(CN): 1588 cm^{− 1}. ¹H NMR (400 MHz, CDCl₃), δ; 2.26 and 2.27 (s, 9H, N‐CH₂‐C₆H₂‐2,4,6‐(C H ₃)₃); 4.00 and 5.27 (s, 4H, ‐NC H ₂C H ₂(SO₂)C₆H₅₎; 5.58 (s, 2H, N‐C H ₂‐C₆H₂‐2,4,6‐(CH₃)₃); 6.92 (s, 2H, N‐CH₂‐C₆ H ₂‐2,4,6‐(CH₃)₃); 7.31 (d, 1H, J = 8.4 Hz, Ar‐ H ); 7.48 (m, 3H, Ar‐ H ); 7.61 (m, 2H, Ar‐ H ); 7.79 (d, 2H, J= 7.7 Hz, Ar‐ H ); 8.06 (d, 1H, J = 8.4 Hz, Ar‐ H ); 10.78 (s, 1H, ‐C H‐ ). ¹³C NMR (100 MHz, CDCl₃), δ; 20.1 and 21.2 (N‐CH₂‐C₆H₂‐2,4,6‐( C H₃)₃); 40.9 and 47.0 (N C H₂ C H₂(SO₂)C₆H₅); 54.9 (N‐ C H₂‐C₆H₂‐2,4,6‐(CH₃)₃); 113.1, 114.2, 124.2, 127.4, 127.6, 128.0, 130.3, 134.5, 138.2, 138.5, and 140.3 (Ar‐ C ); 143.1 (‐ C H‐).

2.2.6. 1‐Ethyl‐3‐(2‐(phenylsulfonyl)ethyl)benzimidazolium chloride, 1f

1‐Ethylbenzimidazole (396 mg, 3 mmol). Yield: 85% (859 mg); m.p.: 181°C–183°C; FTIR for: ν _sym(S=O): 1141 cm^{− 1}; ν _asym(S=O): 1303 cm^{− 1}; ν_(CN): 1565 cm^{− 1}. ¹H NMR (400 MHz, DMSO‐d₆), δ; 1.51 (t, 3H, J = 7.3 Hz, N‐CH₂C H ₃); 4.49 (m, 2H, N‐C H ₂CH₃); 4.29 and 4.92 (m, 4H, N‐C H ₂C H ₂(SO₂)C₆H₅); 7.50‐8.05 (m, 9H, Ar‐ H ); 10.16 (s, 1H, ‐C H‐ ). ¹³C NMR (100 MHz, DMSO‐d₆), δ; 14.7 (N‐CH₂ C H₃); 41.3 (N‐ C H₂CH₃); 42.5 and 53.0 (N C H₂ C H₂(SO₂)C₆H₅); 114.0, 114.3, 127.1, 127.8, 127.9, 129.8, 130.8, 131.0, 131.2, 134.6, and 138.9 (Ar‐ C ); 143.2 (‐ C H‐).

2.3. Crystal Structure Determination and Refinement

Single‐crystal X‐ray diffraction data for compound 1b were collected at 294(2) K on a Rigaku‐Oxford Xcalibur diffractometer equipped with an Eos‐CCD detector. Graphite‐monochromated MoK_α radiation (λ = 0.71073 Å, standard wavelength) was used for the measurements. Data collection parameters and refinement details are provided in Table 1. Data reduction and analytical absorption corrections were carried out using the CrysAlisPro software package [34]. The crystal structure of 1b was solved via the Intrinsic Phasing method implemented in ShelXT [35], using Olex2 [36] as the graphical interface. Refinement was performed with ShelXL [37] through full‐matrix least‐squares minimization. All non‐hydrogen atoms were refined anisotropically. Hydrogen atoms were positioned geometrically, optimized using a riding model, and assigned isotropic displacement parameters U _iso(H) set to 1.2U _eq of the parent atom (1.5U _eq for methyl groups). Bond lengths for aromatic C–H, methylene C–H, and methyl C–H were constrained to standard distances of 0.93, 0.97, and 0.96 Å, respectively.

Table 1.

Crystallographic data and structure refinement summary for compound 1b.

	1b
CCDC number	2441321
Chemical formula	Cl·C23H23N2O2S
Formula weight	426.94
Temperature (K)	150
Crystal system	Monoclinic
Space group	P21 /c
Crystal size (mm3)	0.36 × 0.19 × 0.05
Crystal shape/color	Plate/colorless
a, b, c (Å)	15.352 (3), 13.1922 (13), 10.866 (2)
α, β, γ (°)	90, 102.30 (2), 90
Volume (Å3)	102.30 (2)
Formula Z	4 (Z′ = 1)
T min, T max	0.969, 0.995
F(000)	896
Calc. density (g cm−3)	1.319
Abs. Coeff. μ (mm−1)	0.296
θ range (°)	3.736–20.707
Index ranges	−18 ≤ h ≤ 13
	−13 ≤ k ≤ 15
	−7 ≤ l ≤ 12
Unique reflections	3780
Parameters/restraints	263/0
R int	0.0717
Goodness‐of‐fit on F 2	0.998
R indices [I > 2σ(I)]	R 1 = 0.0670, wR 2 = 0.1247
Δρ min, Δρ max (eÅ−3)	−0.283, 0.349
(Δ/σ)max	0.062

2.4. Biochemical Assays

2.4.1. hCAs and AChE Inhibitory Effect Study

The esterase activities of the hCA I and hCA II were tested using Verpoorte's method [38] of detecting the change in absorbance at 348 nm to determine the inhibitory effects of new benzimidazolium salts bearing phenylsulfonyl ethyl substituent according to previous studies [39, 40]. The assay was performed in 50 mM Tris–SO₄ buffer (pH 7.4) at 25°C. The final substrate concentration was 3 mM 4‐nitrophenyl acetate (4‐NPA). The enzyme concentration was adjusted to 0.20 EU/mL for hCA I and 0.27 EU/mL for hCA II. The hydrolysis of 4‐NPA was monitored by measuring the absorbance increase at 348 nm due to the formation of 4‐nitrophenolate [41, 42, 43]. In vitro effects on AChE activity of the new synthesized benzimidazolium salts bearing phenylsulfonyl ethyl substituent were evaluated by the method of Ellman's method [44]. The assay mixture contained 0.1 M Tris buffer (pH 8.0), 1 mM 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DTNB), 0.075 U/mL electric eel AChE (Electrophorus electricus, Type VI‐S, Sigma‐Aldrich), and the test compounds. After 15 min of pre‐incubation at room temperature, the reaction was initiated by adding 1 mM acetylthiocholine iodide (AChI). The formation of the yellow 5‐thio‐2‐nitrobenzoate anion, resulting from the reaction between DTNB and thiocholine, was monitored spectrophotometrically at 412 nm for 5 min. These compounds 1a–f, acetazolamide (AZA) and tacrine (TAC) were dissolved in DMSO at an initial concentration of 1 mg/mL. The final DMSO concentration did not exceed 1% (v/v). Parallel solvent controls containing 1% (v/v) DMSO were included in each experimental set, and no statistically significant difference was observed compared to buffer‐only controls. To investigate the in vitro inhibitory mechanisms of the compounds, kinetic assays were performed using varying substrate and inhibitor concentrations. The observed data were analyzed by constructing Lineweaver–Burk plots [45] and IC₅₀ curves. The inhibition type was determined based on the pattern of Lineweaver–Burk plots obtained at different inhibitor concentrations. The inhibition constants (Kᵢ) were calculated from secondary plots derived from the slopes or intercepts of the Lineweaver–Burk plots, depending on the inhibition model. All kinetic parameters were obtained by nonlinear regression analysis using appropriate enzyme kinetics models. All measurements were performed in triplicate, and results are reported as means ± standard deviations.

2.5. Computational Details

In silico molecular docking was conducted for predicting the binding affinities of compounds 1a–f with various target proteins using AutoDock vina. The structures of compounds were drawn in ChemDraw 22.0.0, and energy was minimized using Avogadro 1.2.0. The protein structures were retrieved from the RCSB database. The downloaded protein structures were prepared for docking by removing water, adding hydrogen atoms, and isolating active pockets using AutoDock tools 1.5.7. Additionally, the SwissADME was utilized to evaluate the physicochemical and ADME (Absorption, Distribution, Metabolism, and Excretion) properties of the compounds 1a–f.

3. Results and Discussion

3.1. Synthesis

For the synthesis of benzimidazolium salts bearing a phenylsulfonyl group 1a–f, 1 equivalent of 1‐alkylbenzimidazole and respective sulfonyl halides were mixed in 25 mL of acetonitrile at 80°C (Scheme 1) [33]. Yields ranging from 80% to 86% were obtained for all compounds as white solids. All benzimidazolium salts are readily soluble in polar organic solvents such as water, ethyl alcohol, dimethylformamide, and dimethyl sulfoxide. However, it is partially soluble in halogenated organic solvents such as chloroform and dichloromethane, but insoluble in organic solvents such as hexane, pentane, toluene, and diethyl ether.

Scheme 1.

The benzimidazolium salts bearing phenylsulfonyl ethyl group 1a–f.

3.2. NMR Study

The ¹H NMR spectra of benzimidazolium salts bearing a phenylsulfonyl group (1a–f) were examined. The characteristic ‐C H‐ protons in these salts were observed at 11.80, 11.58, 11.70, 11.67, 10.78, and 10.16 ppm, respectively (Figures S20–S25) [46, 47, 48, 49, 50]. However, for benzimidazolium salts, the ethyl group protons in the phenylsulfonyl ethyl group were observed as single peaks at 4.00–4.29 and 4.92–5.27 ppm in compounds 1a–e. In contrast, they were observed as multiple peaks in compound 1f. In compounds 1b–d, methyl proton peaks at the ortho, meta, and para positions on the benzene ring were observed as singlets at 2.35, 2.23, and 2.21 ppm, respectively. However, for compound 1e, proton peaks at the ortho and para positions on the benzene ring were observed as singlets at 2.26 and 2.27 ppm. In compound 1f, ethyl proton peaks attached to the nitrogen atom in the benzimidazole ring were observed as a triplet at 1.51 ppm and a multiplet at 4.49 ppm. Aromatic proton peaks in the structures of all salts were observed mostly as multiplets at approximately 7.00 to 8.00 ppm.

The characteristic ‐ CH‐ carbon peaks in the ¹³C NMR spectra of compounds 1a–f were observed at 144.3, 143.5, 144.2, 144.1, 143.1, and 143.2 ppm, respectively (Figures S20–S25) [46, 47, 48, 49, 50]. In all benzimidazolium salts 1a–f, the carbon peaks of the ethyl group in the phenylsulfonyl ethyl group were observed to be around 41 and 52 ppm. For compounds 1b–d, methyl carbon peaks at the ortho, meta, and para positions on the benzene ring were observed at 19.6, 21.3, and 21.2 ppm, respectively. However, for compound 1e, carbon peaks at the ortho and para positions on the benzene ring were observed at 20.1 and 21.2 ppm, respectively. For compound 1f, ethyl carbon peaks attached to the nitrogen atom in the benzimidazole ring were observed at 14.7 and 41.3 ppm. Aromatic carbon peaks in the structures of all salts were observed to be between 112 and 140 ppm.

3.3. FTIR Study

The characteristic C–N stretching of benzimidazolium salts (1a–f) bearing phenylsulfonyl ethyl groups were observed at 1557, 1562, 1565, 1560, 1588, and 1565 cm^{− 1}, respectively (Figures S26–S31). The sulfonyl group (S=O) stretching in all benzimidazole salts was observed at 1147–1299, 1143–1320, 1141–1300, 1146–1290, 1137–1243, and 1142–1303 cm^{− 1}, respectively (Figures S26–S31). However, aromatic and aliphatic C–H stretching were observed at around 2900 and 3000 cm^{− 1}, respectively. The data obtained from the FTIR spectra of benzimidazolium salts bearing a phenylsulfonyl group are in agreement with the existing studies [46, 47, 48, 49, 50].

3.4. Structure Description of Compound 1b

Single‐crystal X‐ray diffraction analysis confirms that compound 1b crystallizes in the monoclinic crystal system with the P2₁/c space group. The asymmetric unit consists of a chloride anion and a benzimidazole cation substituted with (phenylsulfonyl)ethyl and 2‐methylbenzyl groups (Figure 1). Selected bond distances and angles are provided in the figure caption. The cationic units adopt a U‐shaped conformation in the solid state. Both the sulfonyl and methylbenzyl substituents are oriented toward the same face of the benzimidazole ring, as evidenced by torsion angles of −114.5(4)° (C1–N1–C8–C9) and 104.8(5)° (C1–N2–C16–C17). The benzimidazole core is nearly planar, exhibiting a mean deviation of 0.019 Å from ideal planarity. The pendant C10/C15 and C17/C22 benzyl rings form dihedral angles of 24.2(2)° and 70.4(2)°, respectively, with the central benzimidazole plane. Bond length analysis reveals partial electron delocalization within the imidazole ring: C–N bond distances range from 1.326(5) to 1.398(5) Å, intermediate between typical single (1.48 Å) and double (1.28 Å) bonds [51]. This behavior aligns with the well‐documented partial double‐bond character observed in NHCs. Notably, no anomalous bond lengths or angles are observed in the benzimidazole ring. The structural features of 1b are consistent with those of related derivatives reported in the literature [52, 53, 54, 55].

Figure 1.

The asymmetric unit of compound 1b showing the atom‐labeling scheme. Displacement ellipsoids are drawn at the 40% probability level. Selected bond lengths (Å): N1–C1 1.328(5); N1–C7 1.398(5); N1–C8 1.468(5); S1–O1 1.436(3); S1–O2 1.432(3); S1–C9 1.772(4); S1–C10 1.765(4); N2–C1 1.326(5); N2–C2 1.400(5); N2–C16 1.481(5). Selected bond angles (°): C1–N1–C8 125.2(4); N1–C8–C9 114.1(4); C9–S1–C10 105.5(2); O1–S1–O2 119.72(19); C1–N2–C16 125.5(4); N2–C16–C17 112.2(3).

3.5. Crystal Packing and Supramolecular Architecture

In the crystal structure, the cationic units are interconnected via chloride anions, propagating along the c‐axis. The chloride ions act as hydrogen bond acceptors, forming three C–H⋯Cl interactions: C1–H1⋯Cl1 [H⋯Cl = 2.62 Å, C⋯Cl = 3.268(4) Å, ∠C–H⋯Cl = 129°], C9–H9B⋯Cl1ⁱ [H⋯Cl = 2.65 Å, C⋯Cl = 3.415(4) Å, ∠C–H⋯Cl = 136°], and C16–H16⋯Cl1ⁱⁱ [H⋯Cl = 2.66 Å, C⋯Cl = 3.627(5) Å, ∠C–H⋯Cl = 174°]. These interactions generate 1D zigzag chains of edge‐fused $R_4^2\left(13\right)$ rings with a C(7) motif (Figure 2). Symmetry codes: (i) x, 1/2 − y, −1/2 + z, ii: x, 1/2 − y, 1/2 + z.

Figure 2.

A partial view of the compound 1b along the a‐axis of C–H⋯Cl hydrogen bonded molecules. The bond path, which generates the graphset motif shown as ball and stick drawing style, the other atoms shown as stick drawing style. For clarity, hydrogen atoms not involved in bonding are omitted.

The chains are further stabilized by a C21–H⋯π interaction between the methylbenzyl group (C21) and the sulfone phenyl ring (centroid Cg1: C10–C15) of a symmetry‐related molecule [C21⋯Cg1ⁱⁱⁱ = 3.705(7) Å; symmetry code: (iii) 1 − x, 1 − y, 1 − z]. This interaction bridges adjacent chains, resulting in a 2D layered architecture parallel to the bc‐plane.

Finally, π⋯π stacking between benzimidazole rings [centroid Cg2: C2–C7; Cg2⋯Cg2^iv = 3.705(7) Å; symmetry code: (iv) −x, 1 − y, 1 − z] consolidates the layers into a 3D supramolecular network (Figure S19).

3.6. Results of CA Inhibition Study

CAIs have been extensively studied in their potential therapeutic use against many disseases conditions like glaucoma, seizure disorders, and cancer [56, 57]. Yet, development of selective and potent inhibitors that specifically target individual CA isoforms represents a major hurdle partly owing to the high structural similarity shared between isoforms and the overlap in subcellular localization of these enzymes [58]. One such physiologically relevant isoform is hCA I, which has been implicated in multiple pathologies such as retinal diseases and a variety of neurologic conditions [59]. Here, we investigated the inhibitory effectiveness of six newly synthesized compounds 1a–f on hCA I and compared activity with clinically applied standard inhibitor, AZA (Table 2). The K _i derived for these compounds offered pivotal information regarding the structure‐activity relationship (SAR), as comparison on modification of particular moieties would attribute significant influences on the inhibition activity.

Table 2.

Inhibition data of AChE and hCA I, II isoforms with the new a series benzimidazolium salts bearing phenylsulfonylethyl group.

Compounds	IC50 (nM)						K i (nM)		AChE
	hCA I	r 2	hCA II	r 2	AChE	r 2	hCA I	hCA II
1a	94.12	0.9711	12.44	0.9844	95.32	0.9744	97.46 ± 21.68	19.44 ± 4.80	101.84 ± 6.54
1b	63.56	0.9832	145.66	0.9747	49.50	0.9805	61.06 ± 5.39	151.51 ± 7.04	58.92 ± 6.67
1c	45.56	0.9888	198.65	0.9812	20.55	0.9766	51.21 ± 9.32	205.67 ± 15.68	26.78 ± 4.88
1d	83.45	0.9744	61.88	0.9721	19.22	0.9721	87.69 ± 10.13	74.08 ± 3.38	25.00 ± 1.69
1e	14.54	0.9807	160.76	0.9799	53.31	0.9832	17.56 ± 0.42	167.15 ± 22.24	55.27 ± 11.32
1f	134.67	0.9813	55.78	0.9743	110.21	0.9813	131.41 ± 8.84	66.44 ± 3.67	124.81 ± 22.78
AZA	173.50	0.9788	165.78	0.9766	—	—	167.21 ± 4.21	159.56 ± 4.78	—
TAC	—	—	—	—	92.56	0.9821	—	—	115.21 ± 8.98

The K _i values for the tested compounds against hCA I indicated a distinct trend toward increasing potency by making specific structural modifications. Notably, the control compound AZA showed a K _i value of 167.21 ± 4.21 nM, used as a reference. The most potent compound among the newly synthesized compounds, compound 1e, exhibited the strongest inhibitory potency (K _i: 17.56 ± 0.42 nM), and approximately 9.5‐fold stronger inhibition than AZA. As the inhibitory activity had significantly improved, the structural changes in compound 1e appear to interact well in the hCA I active site, possibly leading to an increased binding affinity and stability. Compounds 1d and 1f exhibited K _i values of 87.69 ± 10.13 and 131.41 ± 8.84 nM, respectively, both compounds demonstrate improved inhibition when compared to AZA (Table 2). The structural difference between these compounds likely accounts for the 1.5‐fold increase in potency observed in compound 1d. Compound 1a exhibited a K _i value of 97.46 ± 21.68 nM, indicating a slight reduction in potency compared to compound 1d but still demonstrating improved inhibition over AZA (1.72‐fold stronger). This observed decrease in potency relative to compound 1d indicated that the specific electronic and steric properties of its substituents may mediate enzyme affinity. An even subtle steric hindrance introduced by its functional group might alter the ideal binding conformation in the active site of the enzyme.

Compound 1b displayed 2.74 times more potent inhibition (K _i: 61.06 ± 5.39 nM) than AZA. The increase in action was potentially due to the substituents on compound 1b creating enhanced van der Waals interactions, π‐π stacking or hydrogen bonding in the active site, thus allowing for a longer residence time of the inhibitor in the enzyme. This finding emphasized the need to carefully evaluate the placement of substituents to enhance the interactions between enzymes and ligands. The inhibition constant of compound 1c (K _i: 51.21 ± 9.32 nM) showed an even greater improvement compared to AZA (3.27‐fold stronger inhibition) (Table 2). This trend demonstrated that the changes observed in compound 1c afford the most favorable electronic effects relative to steric accessibility at the binding site.

Inhibition potency summary of hCA II inhibitors 1a–f, a well‐known standard inhibitor, AZA, has a K _i: 159.56 ± 4.78 nM. The inhibition data showed a broad range of active compounds with K _i values between 19.44 ± 4.80 to 205.67 ± 15.68 nM. Compound 1a inhibited with the highest potency (8.21 times superior to AZA) while compound 1c with 1.29 times lower potency than AZA. These results suggested that slight differences in the chemical structure have a pronounced effect on inhibition.

Compounds 1d and 1f, with K _i values of 74.08 ± 3.38 and 66.44 ± 3.67 nM, respectively (Table 2), had rather impressive inhibition potency improvements over AZA, with compound 1f being a 2.40 and compound 1d a 2.15‐fold inhibition effect. The increase in inhibition ability suggested that these compounds contained functional groups that facilitate the interaction with the hCA II active site via an improved coordination to the catalytic Zn²⁺ ions. The relatively similar K_i values for compounds 1f and 1d indicated that the introduced substituents contribute to a stabilization of the enzyme‐inhibitor complex but do not drastically alter binding affinity. Compound 1a was the most potent compound aganist hCA II isoform. This impressive improvement likely indicated that the substituents at position compound 1a facilitate a much more favorable binding orientation in the hCA II active site. In contrast to the above compounds, compounds 1b (K _i: 151.51 ± 7.04 nM) and 1c (K _i: 205.67 ± 15.68 nM) exhibited weaker inhibition effects when compared to AZA. These findings suggested that the modifications present in compounds 1b and 1c either introduce steric hindrance or reduce critical electrostatic interactions within the active site, ultimately diminishing binding efficiency. Compound 1e (K _i: 167.15 ± 22.24 nM) exhibited an inhibitory potency nearly identical to AZA, indicating that while its structural modifications do not significantly enhance inhibition, they also do not introduce considerable steric or electronic hindrance.

3.7. Results of AChE Inhibition Study

AChE is an essential enzyme that hydrolyzes ACh at cholinergic synapses, an important step in neurotransmission itself [42, 60]. AChE dysregulation has been implicated in a number of neurodegenerative disorders, most notably AD, but also Parkinson's disease and myasthenia gravis [61]. Thus, the design and synthesis of potent and selective AChE inhibitors (AChEIs) have become a central theme of new drug discovery in medicinal chemistry to try and produce effective therapeutics.

In this study, the K _i values of six newly synthesized compounds 1a–f were evaluated against AChE and compared with the reference AChE inhibitor TAC (K _i: 115.21 ± 8.98 nM), which has been widely used for the treatment of neurodegenerative diseases in clinical applications. The data showed significant differences in inhibition potency, demonstrating how structural changes can influence enzyme‐inhibitor interfaces. The K _i values of the synthesized compounds ranged from 25.00 ± 1.69 nM (compound 1d) to 124.81 ± 22.78 nM (compound 1f), indicating substantial variability in inhibition potency. The most potent inhibitors (1d and 1c) demonstrated a 4.61‐ and 4.3‐fold increase in potency compared to TAC, respectively, highlighting the critical role of group modifications in optimizing AChE inhibition (Table 2).

Compound 1b (K _i: 58.92 ± 6.67 nM, 1.96‐fold stronger than TAC) and 1a (K _i: 55.27 ± 11.32 nM, 2.08‐fold stronger than TAC) demonstrated improved inhibition to TAC, but not as potent as compounds 1c and 1d. These results imply that the functional groups in compounds 1a and 1b support better interaction between the drug and the enzyme, but due to their structure introduce some steric or electronic restrictions that cannot allow full optimization.

In contrast, with a K _i: 124.81 ± 22.78 nM, compound 1f inhibited less than TAC, implying that its functional derivatives may not enhance affinity towards AChE. Notably, though compound 1a showed only modest improvement in inhibition (1.13‐fold compared to TAC), indicating that this compound, while benefitting from some favorable interactions, also has less overall potency than other inhibitors in the series. The placement of bulky substituents may create unfavorable steric interactions, hindering the inhibitor from fully occupying the enzyme's catalytic gorge.

Although the synthesized compounds demonstrated potent inhibitory effect against hCA I, hCA II and AChE as in vitro, these findings should be interpreted with caution. Enzyme inhibition data alone do not directly translate into therapeutic efficacy, as in vivo activity is influenced by complex pharmacokinetic and pharmacodynamic factors. Therefore, the present results should be considered as preliminary biochemical evidence, suggesting that these compounds may serve as promising lead structures rather than confirmed therapeutic agents. Further in vivo studies and detailed pharmacological evaluations are required to validate their therapeutic potential.

3.8. Molecular Docking Simulation Analysis

In silico molecular docking simulations of compounds (1a–f) were performed to evaluate the binding interactions and affinities of compounds (1a–f) and standards (tacrine and acetazolamide) with three target proteins, hCA I (PDB ID: 1CZM) [62], hCA II (PDB ID: 1XCV) [63], and AChE (PDB ID: 1B41) [64]. The binding affinities of compounds (1a–f) and standards with target proteins are summarized in Table 3.

Table 3.

Binding affinity of compounds (1a–f) and standards acetazolamide (AZA) and tacrin (TAC) with hCA I (PDB ID: 1CZM), hCA II (PDB ID: 1XCV), and AChE (PDB ID: 1B41).

Compounds	PDB ID: 1CZM	PDB ID: 1XCV	PDB ID: 1B41
	Binding affinity (Kj/mol)	Binding affinity (Kj/mol)	Binding affinity (Kj/mol)
1a	−8.6	−7.7	−8.7
1b	−8.7	−5.2	−9.4
1c	−8.0	−7.1	−9.2
1d	−7.2	−7.4	−8.8
1e	−8.9	−6.5	−9.3
1f	−6.8	−6.5	−8.2
AZA	−5.2	−4.3	—
TAC	—	—	−8.6

The docking results showed that all compounds (1a–f) have shown strong interactions with target proteins and possess various types of key interactions with target protein involving hydrogen bonding, carbon‐hydrogen, pi–pi, pi–alkyl, and pi–sigma interactions.

Furthermore, it was found that all compounds have higher binding affinity values (range −6.8 to −8.9 Kj/mol) with target protein hCA I (PDB ID: 1CZM) as compared to standard compound acetazolamide (−5.2 Kj/mol). Among the studied compounds, compound 1e showed the highest binding affinity (−8.9 Kj/mol) with target protein hCA I (PDB ID: 1CZM) and showed various important interactions including hydrogen bonding with GLN 92 and HIS 67, ARG 296, pi‐sigma with LEU 198 and pi‐alkyl interactions with HIS 94 and ALA 121. The interaction diagrams of 1e with target protein hCA I (PDB ID: 1CZM) are illustrated in Figure 3. The interaction diagrams of compounds 1a–d, f, and AZA are provided in supplementary information Figures S1–S6.

Figure 3.

Interaction diagrams of compound 1e with target protein (PDB ID:1CZM) (a) surface view of binding pocket. (b) Detailed view of 1e within the binding site (c) Close‐up of key interactions. (d) 2 d interaction diagram.

Docking results of compounds (1a–f) with target protein hCA II (PDB ID: 1XCV also showed that all compounds (1a–f) have higher binding affinity value (range −5.2 to −7.7 Kj/mol) with target protein hCA II (PDB ID: 1XCV), as compared to standard compound acetazolamide (−4.3 Kj/mol). Compound 1a showed the highest binding affinity (−7.7 Kj/mol) with target protein and involved in various important interactions including electrostatic pi‐cation interaction with HIS 98 and pi‐anion interactions with GLU 21, pi‐pi interactions with PHE 128 and ARG 33. The interaction diagrams of 1a with target protein (PDB ID: 1XCV) are illustrated in Figure 4. The interaction diagrams of compounds 1b–f and AZA are provided in supplementary information Figures S7–S12.

Figure 4.

Interaction diagrams of compound 1a with target protein (PDB ID: 1XCV) (a) Surface view of binding pocket. (b) Detailed view of 1a within the binding site (c) Close‐up of key interactions. (d) 2d interaction diagram.

In case of docking of compounds (1a–f) with target protein AChE (PDB ID: 1B41) revealed that compounds 1a–e have higher binding affinity value (range −8.6 to −9.4 Kj/mol) with target protein AChE (PDB ID: 1B41) except compound 1f as compared to standard compound tacrin (−8.6 Kj/mol). Compound 1b showed the highest binding affinity (−9.4 Kj/mol) with target protein and involved in various important interactions including hydrogen bonding with ARG 296, carbon‐hydrogen interaction with VAL 294, electrostatic (pi‐cation) interaction with TRP 286 and pi‐pi interaction with TYR72 and HIS 287. The interaction diagrams of 1b with target protein (PDB ID: 1B41) are illustrated in Figure 5. The interaction diagrams of compounds 1a, c–f and TAC are provided in supplementary information Figures S13–S18.

Figure 5.

Interaction diagrams of compound 1b with target protein (PDB ID: 1B41). (a) Surface view of binding pocket. (b) Detailed view of 1b within the binding site. (c) Close‐up of key interactions. (d) 2d interaction diagram.

3.9. Physicochemical/Drug Likeness Properties Analysis

The drug likeness properties of synthesized compounds (1a–f) were assessed by SwissADME. The physicochemical parameters of compounds (1a–f) are given in Table 4.

Table 4.

Physicochemical property profile parameters of compound 1a–f.

ID	MW ≤ 500	HBD ≤ 5	HBA ≤ 10	clog P ≤ 5	TPSA < 140 Ų	RB ≤ 10	Lipinski	Veber
1a	412.93	0	2	1.91	51.33	6	Yes	Yes
1b	426.96	0	2	2.57	51.33	6	Yes	Yes
1c	426.96	0	2	2.92	51.33	6	Yes	Yes
1d	426.96	0	2	2.84	51.33	6	Yes	Yes
1e	455.01	0	2	3.19	51.33	6	Yes	Yes
1f	336.84	0	2	1.12	51.33	4	Yes	Yes

Abbreviations: HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; MW, molecular weight; RB, rotatable bonds.

All the compounds (1a–f) exhibited molecular weight (MW) below 500 Da, ensuring good permeability and absorption. The hydrogen bonds donor (HBA) was zero for all compounds, while the hydrogen bond acceptor (HBA) count as 2, indicating a balance hydrogen bonding capacity. The calculated clogP value is within the acceptable range of lipophilicity. Moreover, the TPSA values are also below the 140 A² threshold, suggesting efficient cellular permeability. The number of rotatable bonds range from 4 to 6, ensuring molecule flexibility without compromising the bioavailability. The physicochemical parameters including Lipinski and Veber's rule were also determined to predict the oral bioavailability. All the compounds follow the set of guidelines of Lipinski and Veber's rule which shows that all the compounds (1a–f) have the favorable oral bioavailability profile. The bioavailability radar of compounds are shown in Figure 6.

Figure 6.

The bioavailability radar chart of compounds (1a–f).

3.10. ADME Analysis

The Absorption, Distribution, Metabolism and Excretion (ADME) parameters of synthesized compounds (1a–f) were also evaluated by SwissADME to get insight into their pharmacokinetics behavior. The ADME parameters of compounds are given in Table 5.

Table 5.

ADME parameters of compounds 1a–f.

ID	BS	BBB Permeant	P‐gp substrate	Log K p cm/s	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 Inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor
1a	0.55	Yes	Yes	−5.39	No	No	No	No	No
1b	0.55	Yes	Yes	−5.21	No	No	No	No	No
1c	0.55	Yes	Yes	−5.21	No	Yes	Yes	No	No
1d	0.55	Yes	Yes	−5.21	No	Yes	Yes	No	No
1e	0.55	Yes	Yes	−4.87	No	No	No	No	No
1f	0.55	No	Yes	−6.05	No	No	No	No	No

Abbreviations: BS, bioavailability score; Log K _p, (Skin permeation) cm/s; BBB permeant, blood brain barrier permeant.

All the compounds have moderate bioavailability score (0.55) indicating reasonable oral absorption. Compounds 1a–e except 1f were predicted to permeate the blood brain barrier suggesting them as potential candidate for neurodegenerative disorders. However, all compounds are found to be P‐glycoprotein (P‐gp) substrates, show susceptibility to efflux mechanisms that could reduce intracellular bioavailability. The Skin permeation (log K _p) values ranged from −4.87 to −6.05 cm/s, indicating less skin transdermal permeability. Furthermore, except compound 1c and 1d, all compounds showed no inhibition of major CYP isoforms, indicating a safer metabolic profile with no potential effect on drug metabolism.

4. Conclusions

In conclusion, a new series of benzimidazolium salts with phenylsulfonyl ethyl‐substituted was synthesized. All compounds were characterized using ¹H NMR, ¹³C NMR, and FTIR spectroscopic techniques. The single crystal of compound 1b was elucidated using the XRD method. This compound exhibits a U‐shaped benzimidazolium cation paired with a chloride anion. A hierarchical supramolecular architecture arises from C–H⋯Cl chains, C–H⋯π layers, and π⋯π stacking, demonstrating cooperative non‐covalent stabilization of the crystal structure. Physicochemical and ADME analysis of the compounds found them very good Pgp substrates with excellent bioavailability scores. This research summarizes the crucial importance of structural changes in the structures as hCAs and AChE inhibitors. Compounds 1a were the most potent inhibitors of hCA II tested resulting in 8.2‐fold increase in potency over AZA, whereas compounds 1b and 1c had rather lower activity. For hCA I, compound 1a had the highest inhibition with an 8.2‐fold increase in strong potency to AZA; compound 1c had lower inhibition than AZA with 1.3‐fold lower potency. Compounds 1c and 1d showed remarkable inhibitory ability (4.6‐ and 4.3‐fold for TAC, respectively). By contrast, inhibitors 1a and 1f displayed weaker inhibition, suggesting that steric hindrance and unsatisfactory electrostatic interactions can reduce binding efficiency. The results indicated that the location of the functional groups could play a key role in improving binding of enzymes.

Accession Code

The supplementary crystallographic data for the compound has been deposited at the Cambridge Crystallographic Data Centre (CCDC) and is available upon request from www.ccdc.cam.ac.uk/data_request/cif, quoting the deposition number CCDC 2441321.

Author Contributions

Yetkin Gök: investigation, writing – original draft, writing – review and editing, supervision. Ali Kazancı: investigation, methodology, resources. Yeliz Demir: investigation, writing – original draft, writing – review and editing, methodology, data curation. Zaroon Sajid: investigation, writing – original draft, conceptualization, methodology, writing – review and editing, software. Merve İzmirli: conceptualization, writing – original draft, software, data curation, investigation. Muhammad Haroon: conceptualization, investigation, software, data curation, writing – original draft, writing – review and editing, formal analysis. Muhittin Aygün: investigation, writing – review and editing, supervision, methodology. Aydın Aktaş: conceptualization, investigation, writing – original draft, methodology, visualization. İlhami Gülçin: conceptualization, investigation, writing – review and editing, supervision, methodology, validation.

Supporting information

Supporting File

Data Availability Statement

The data that supports the findings of this study are available in the supporting material of this article.