Novel beta-lactam substituted benzenesulfonamides: in vitro enzyme inhibition, cytotoxic activity and in silico interactions

Abstract In this study, a library of twelve beta-lactam-substituted benzenesulfonamides (5a–l) was synthesized using the tail-approach method. The compounds were characterized using IR, 1H NMR, 13C NMR and elemental analysis techniques. These newly synthesized compounds were tested for their ability to inhibit the activity of two carbonic anhydrases (hCA) isoforms, I and II, and acetylcholinesterase (AChE) in vitro. The results showed that the synthesized compounds were potent inhibitors of hCA I, with KIs in the low nanomolar range (66.60–278.40 nM) than the reference drug acetazolamide (AAZ), which had a KI of 439.17 nM. The hCA II was potently inhibited by compounds 5a, 5d–g and 5l, with KIs of 69.56, 39.64, 79.63, 74.76, 78.93 and 74.94 nM, respectively (AAZ, KI of 98.28 nM). Notably, compound 5a selectively inhibited hCA II with a selectivity of > 4-fold over hCA I. In terms of inhibition of AChE, the synthesized compounds had KIs ranging from 30.95 to 154.50 nM, compared to the reference drug tacrine, which had a KI of 159.61 nM. Compounds 5f, 5h and 5l were also evaluated for their ability to inhibit the MCF-7 cancer cell line proliferation and were found to have promising anticancer activity, more potent than 5-fluorouracil and cisplatin. Molecular docking studies suggested that the sulfonamide moiety of these compounds fits snugly into the active sites of hCAs and interacts with the Zn2+ ion. Furthermore, molecular dynamics simulations were performed for 200 ns to assess the stability and dynamics of each enzyme-ligand complex. The acceptability of the compounds based on Lipinski’s and Jorgensen’s rules was also estimated from the ADME/T results. These results indicate that the synthesized molecules have the potential to be developed into effective and safe inhibitors of hCAs and AChE and could be lead agents. Communicated by Ramaswamy H. Sarma


Introduction
Antibiotics are pharmaceuticals utilized to treat infections caused by bacteria in humans and animals.These agents exert their effects by either eradicating bacterial cells or inhibiting their growth and reproduction.Many antibiotics contain a beta-lactam ring in their chemical structure, characterized by a fused four-membered lactam ring, a free carboxylic acid group, and one or more substituted amino acid side chains (Osazee, 2016;Ram� ırez Granillo et al., 2015).Beta-lactams, a type of 2-azetidinone, are well-known heterocyclic compounds among organic and medicinal chemists.These compounds have a ring system that is a common structural feature of several broad-spectrum beta-lactam antibiotics, including penicillin, carbapenem, cephalosporin, monobactam, sulbactam, nocardicin and tazobactam, which are used as chemotherapeutic agents to treat microbial diseases (Mehta et al., 2010;Page, 1987;Singh, 2004aSingh, , 2004b)).
Anticancer agents are critical for the control and elimination of cancer.However, the rapid emergence of resistance to most anticancer agents and the low specificity of these agents present significant challenges to effective cancer therapy.In recent years, there has been a significant effort to devise new chemotherapeutic strategies for the treatment of various types of cancer.Beta-lactam derivatives, in particular, have gained attention as a versatile pharmacophore for drug discovery due to their broad spectrum of pharmacological activity and their ability to induce DNA damage and apoptosis in various cancer cell lines.As such, beta-lactam derivatives are a promising and efficient platform for the development of new anticancer agents (Malebari et al., 2020;Veinberg et al., 2004;Zhang & Jia, 2020).Additionally, these agents display a diverse range of biological activities such as anti-microbial (Kuskovsky et al., 2019), antibacterial (Carosso & Miller, 2015), antifungal (Wang et al., 2019), anti-hyperglycemic (Sahoo & Banik, 2020), anti-inflammatory (Saturnino et al., 2000), anti-HIV (Sperka et al., 2005), antimalarial (Rad et al., 2017), anti-tubercular (Kumar et al., 2012) and analgesic (Vigorita et al., 2001) activities.The biological activity of these compounds is often linked to the chemical reactivity of the lactam ring and the substituents, particularly at the nitrogen of the azetidinone ring (Brandi et al., 2008).
There are currently 15 different human carbonic anhydrase (hCA) isoenzymes that have been identified (Kakakhan et al., 2023;Leitans et al., 2015); they are found in many tissues and are involved in a wide range of metabolic processes (Alım et al., 2022;Ozer et al., 2022).With the highest turnover rate of all hCAs, hCA II is one of the most researched isoforms (k cat of 10 6 s À 1 ) (Singh et al., 2019).The His64 residue, located near the active site, plays a role in rapid proton transfer, contributing to the high turnover rate of the enzyme (Fisher et al., 2007).The hCA II is in charge of controlling the intraocular pressure that typically accompanies glaucoma because it participates in the main transport pathway of sodium into the eye (Carta et al., 2012).One of the main reasons to investigate more closely at hCA II inhibitors was for the treatment of glaucoma, which if left untreated can result in blindness (Scozzafava & Supuran, 2014).In addition, the physiologically dominant isoform of the hCAs family (Table 1), hCA II, is expressed in a variety of brain cells and tissues (Topal, 2019), including oligodendrocytes (Taslimi et al., 2019), astrocytes (Daryadel et al., 2018), myelin sheaths (Supuran, 2018), choroid plexus (Mishra et al., 2021) and myelinated tracts (Mishra et al., 2017).Particularly, hCA II is overexpressed in many tumor types, and this overexpression has frequently been linked to the aggressiveness of tumor cells (Parkkila et al., 2010), as in the case of colorectal cancer (Bekku et al., 2000) and synchronous distant metastases (Lankat-Buttgereit et al., 2004).
The hCAs that are catalytically active contain a Zn 2þ ion tetrahedrally coordinated by three histidine residues (His94, His96 and His119) (Aggarwal et al., 2013) and a water molecule at the base (Zhang et al., 1996).They also have a sizable active site split into two sides, one lined with hydrophobic residues and the other with polar, hydrophilic residues (Tawfik et al., 2022).Additionally, it is difficult to selectively inhibit one hCA isoform over others due to their catalytic regions' structural homology (Alterio et al., 2012) and amino acid sequence conservation (Mishra et al., 2020).The 'ring and tail' strategy is the most frequently utilized method to create hCA inhibitors (hCAIs) (Kumar et al., 2022).In this strategy, the 'ring' is made up of hetero/aromatic fragments that contain a zinc-binding group (ZBG) (Eldeeb et al., 2021), which is necessary for binding zinc (Figure 1).The ability of electron acceptor substituents to make the ZBG more acidic and hence raise its acidity, makes them valuable (Bonardi et al., 2020).A flexible piece attached to the aromatic ring called the 'tail' plays a role in the compound's increased binding to the hCAs active site or enhances its solubility in water (Elbadawi et al., 2021).Unquestionably, sulfonamides, which play a significant role as zinc binders, are among the most productive and extensively researched derivatives for developing numerous isoform-selective and potent hCAIs, as well as in the associated therapeutic setting (Elbadawi et al., 2022).
At this instance, as part of our ongoing work on developing more potent and selective hCAIs, a novel combination of biologically active but structurally dissimilar groups, such as beta-lactam and sulfonamide, has been designed using the ring and tail approach.The structural characteristics of the newly synthesized beta-lactam substituted benzenesulfonamide derivatives (5a-l) with this approach were determined using various spectroscopic techniques (Scheme 1).Afterward, the cytosolic hCA isoforms I, II and AChE were tested for the target derivatives' inhibitory effects.In addition, the synthesized compounds were tested for their antiproliferative effects against the human breast cancer (MCF-7) cell line in vitro.Molecular docking studies of the target derivatives in hCAs and AChE were also conducted to examine the predicted in silico binding interactions of the novel synthesized derivatives with the active sites of these enzymes.

General procedure for the preparation of the target compounds
The melting points of the compounds were determined using a Yanagimoto micro-melting point apparatus and were not corrected.Infrared spectra were obtained using a SHIMADZU Prestige-21 (200 VCE) spectrometer.Proton and carbon nuclear magnetic resonance spectra were acquired on a VARIAN Infinity Plus at 300 and 75 Hz, respectively,    and the chemical shifts were referenced to the internal deuterated solvent.Elemental analysis was conducted using a Leco CHNS-932 instrument.All chemicals used in the study were purchased from Merck, Alfa Aesar, and Sigma-Aldrich.

General procedure for preparation of 4sulfonylamide ester (2)
Sulfamoylbenzoic acid (10 mmol) was dissolved in 50 mL of ethanol and 1 mL of sulfuric acid and the mixture was refluxed for 24 h.The solvent was then removed by evaporation, and the resulting product was washed with cold water and dried.The product was characterized by NMR and used in the next step without further purification.

General procedure for preparation of 4sulfonylamidebenzohydrazide (3)
The reaction of 4-sulfonylamide ester (10 mmol) and hydrazine hydrate (25 mmol) in ethanol was carried out by refluxing the mixture at 80 � C for 24 h.Upon cooling to room temperature, the solid product was obtained by filtering the reaction mixture, washing with water, and drying.The product was characterized by NMR spectroscopy and used in the next step without further purification.

Synthesis of (E)-4-(2-benzylidenehydrazinecarbonyl) benzenesulfonamide derivatives (4)
In the present study, the synthesis of benzaldehyde derivatives (2.5 mmol) through the reaction of 4-sulfonylamidebenzohydrazide (2.5 mmol) and DMF (10 mL) was carried out by refluxing the mixture at 80 � C for 12 h.Upon completion, the mixture was cooled and poured into ice-cold water before being filtered and crystallized from acetone.The resulting compounds were confirmed to have structural characteristics consistent with Schiff-base compounds based on 1 H and 13 C NMR spectra.

hCA and AChE inhibitory effect study
The human erythrocyte hCA I and II isoforms were isolated using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography.The esterase activity of the hCAs was measured using Verpoorte's method (Verpoorte et al., 1967;Yararli et al., 2023), which involves the assessment of the change in absorbance at 348 nm, in order to determine the inhibitory effects of the newly synthesized beta-lactam substituted benzenesulfonamides (5a-l).The activity of hCAs was measured at 25 � C using the same substrate, 4-nitrophenyl acetate (PubChem CID: 13244) as in our earlier tests (Askin et al., 2021;G€ ulec¸ et al., 2022;Lolak et al., 2022;Osmaniye et al., 2022;T€ urkes ¸ et al., 2019).The reaction mix (approx. 1 mL) consisted of substrate (3 mM), hCA, and Tris-sulfate buffer (50 mM, pH 7.4).The enzyme unit was calculated using the absorption coefficient (e ¼ 5.4 � 10 3 M À 1 cm À 1 ) (Karakılıc ¸ et al., 2022).Additionally, using Ellman's method (Ellman et al., 1961), spectrophotometric measurements at room temperature were conducted to determine the effects of these novel synthesized derivatives on AChE (Sigma C2888, Type V-S) activity (Yakan et al., 2023).As in our earlier research, the results were achieved using the substrate acetylthiocholine iodide (AChI, Sigma 01480, PubChem CID: 74629) (Demir et al., 2023;G€ uller et al., 2021).The reaction mixture contained 10 mM of AChI and 0.5 mM 5,5 0 -dithiobis(2-nitrobenzoic acid) in 1 mM Tris/HCl (pH 8.0) containing 5 mM EDTA.The assay for AChE activity was based on the reaction of DTNB (e ¼ 13.6 M À 1 cm À 1 ) at 412 nm (Atmaca et al., 2022;G€ ok et al., 2021).These compounds (5a-l), as well as the reference medications AAZ and THA, were initially dissolved in DMSO at a concentration of 1 mg/mL.DMSO was present in the final reaction mixture at a concentration of around 1%.Each sample underwent three measurements.V max , K m and K I values, and inhibition types for beta-lactam substituted benzenesulfonamide compounds 5a-l were calculated from the data acquired by Michaelis-Menten (Michaelis et al., 2011) and Lineweaver-Burk (Kim et al., 2007) plots as in our earlier research (Yapar et al., 2021).The analysis of data and creation of graphs were carried out using GraphPad Prism V9 (GraphPad Software, La Jolla California USA) for Mac.The inhibition constants were calculated using SigmaPlot V12 (Systat Software, San Jose, CA, USA) for Windows.Statistical comparisons between data sets were performed using the extra sum-of-squares F test and the Akaike information criterion approach, and results are presented as mean ± standard error of the mean (95% confidence intervals).Statistical significance was determined when the p-value was less than 0.05.

Cytotoxic activity study
The human breast cancer (MCF-7) cell line was thawed and cultured in a 75 cm 2 flask with DMEM and 10% FBS.After 48 h, the cells were transferred to a 96-well plate and incubated at 37 � C in a 5% CO 2 atmosphere.The cells were then exposed to various concentrations (1-100 mM) of compounds 5d, 5f-i and 5l for 24 h.The cytotoxic effects of these compounds were then evaluated using the MTT assay (Buza et al., 2023).Specifically, 10 mL of MTT solution (5 mg/mL) was added to each well, and the plate was incubated for an additional 3 h in a 5% CO 2 atmosphere.The absorbance of the formazan crystals formed as a result of the MTT assay was then measured at 570 nm using an Epoch Microplate Spectrophotometer.The cell viability rates were calculated by comparing the absorbance of the treated cells to that of the untreated control cells and expressed as a percentage.
The IC 50 values were determined using GraphPad Prism V9 (GraphPad Software, La Jolla, CA, USA) for Mac.

In silico study
In this study, we used the Small-Molecule Drug Discovery Suite 2023-1 for Mac (Schr€ odinger, LLC, NY, USA) to perform molecular docking analysis.The hCA I (PDB ID 1AZM, A chain, 2.00 Å) (Chakravarty & Kannan, 1994), hCA II (PDB ID 3HS4, A chain, 1.10 Å) (Sippel et al., 2009), and AChE (PDB ID 7E3I, A chain, 2.85 Å) (Cheung et al., 2012) protein structures were obtained from the RCSB Protein Data Bank (https:// www.rcsb.org)and prepared for docking using the Protein Preparation Wizard (Madhavi Sastry et al., 2013; Schr€ odinger release 2023-1: Protein preparation wizard, 2023).The betalactam substituted benzenesulfonamides (5a-l) were drawn using ChemDraw V21.0 (PerkinElmer, Inc., Waltham, MA, USA) for Mac (Sever et al., 2021) and optimized in the OPLS4 force field with Epik (Schr€ odinger release 2023-1: Epik, 2023; Shelley et al., 2007) using the LigPrep module (Schr€ odinger release 2023-1: LigPrep, 2023) at pH 7.4 ± 0.5.The active site residues of the proteins were identified using the SiteMap tool (Halgren, 2009) and defined in the Receptor Grid Generation module to create the receptor grid in the Maestro panel (Schr€ odinger release 2023-1: Receptor grid generation, 2023; Yararli et al., 2023).The SiteMap tool (Schr€ odinger release 2023-1: SiteMap, 2023) determines one or more regions on or near the protein surface, called sites, and particularly calculates SiteScore, which has proven effective at identifying known binding sites in co-crystallized complexes.Here, the site of binding of the compounds was determined as Site 1, which had the best SiteScore (0.864, 0.790 and 1.087 for 1AZM, 3HS4 and 7E3I, respectively) among five suitable active sites (Table 2).The Glide application with the enhanced precision (XP) approach (Ece, 2020;Friesner et al., 2004;Halgren et al., 2004; Schr€ odinger release Contact, the contact property measures how strongly the average site point interacts with the surrounding receptor via van der Waals nonbonded interactions, when the site point is given nominal van der Waals parameters.The contact score has been calibrated so that the average score for a tight-binding site is 1.0.
f Hydrophobic and hydrophilic character, and balance, these properties, labeled phob and phil, measure the site's relative hydrophobic and hydrophilic character.
g Donor/acceptor character, this property, labeled don/acc, indicates the degree to which a well-structured ligand might be expected to donate, rather than accept, hydrogen bonds, as inferred from the sizes and intensities of donor and acceptor SiteMap regions.
2023-1: Glide, 2023) was used to dock the ligands to hCAs and AChE with default settings (T€ urkes ¸ et al., 2021).The relative binding affinity of the protein-ligand complexes was predicted using the MM-GBSA approach (Barreiro et al., 2007; Schr€ odinger release 2023-1: Prime, 2023) in the VSGB energy model and OPLS4 force field.Molecular dynamics (MD) simulations were performed using Desmond (Bowers et al., 2006) package implemented in Schr€ odinger software (Schr€ odinger release 2023-1: Desmond, 2023).The input protein and ligand complexes were obtained from docking studies.The system was solvated with the transferable intermolecular potential with three points (TIP3P) model in an orthorhombic simulation box.Sodium counterions were placed to neutralize the systems.Moreover, the physiological conditions were mimicked by adding 0.15 M NaCl.Normal pressure (1.01325 bar) and temperature (300 K) (NPT) ensemble were used in MD simulations.All equilibrated systems were first relaxed and then subjected to an MD run for 200 ns with a recording interval of 200 ps that generated approximately 1000 frames.Simulation Interaction Diagram (SID) tool to analyze MD results involving the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and protein-ligand contacts.

Drug design strategy and chemistry
The ring and tail strategy has emerged as an effective approach due to the shared similarities in the active sites of hCAs (Bozdag et al., 2014).This method targets specific hydrophobic or hydrophilic residues in the outer region of the isoform active site by introducing various aryl or heterocyclic scaffolds to the aromatic sulfonamide ring of hCAIs (Nocentini & Supuran, 2019).The target derivatives (5a-l) in this study have a benzenesulfonamide-based ZBG attached to a beta-lactam scaffold.The beta-lactam moiety increases the flexibility and hydrophilicity of the designed compounds through advantageous interactions with specific residues in the hydrophilic region of the active site, enabling selective inhibition of hCA II.The hydrophobic tail is constructed using the phenyl moiety, which is connected to the hydrophobic or hydrophilic rims of the active site through the scaffold, ensuring proper orientation.
Ethyl 4-(aminosulfonyl)benzoate was synthesized from sulfamoylbenzoic acid in ethanol with a catalytic amount of sulfuric acid, with the reaction being carried out under reflux for 24 h.The ester group of ethyl 4-(aminosulfonyl)benzoate was then converted to the 4-sulfonylamidebenzohydrazide compound using hydrazine hydrate in ethanol at 80 � C for 24 h.The aldehyde derivatives and 4-sulfonylamidebenzohydrazide were dissolved in DMF and stirred at 80 � C for 24 h to form the synthesized imine derivatives.These derivatives were then dissolved in a mixture of dry dioxane-THF in the presence of a TEA base in an ice bath, and chloroacetylchloride was slowly added.The mixture was refluxed for 12 h to yield the prepared compounds (5a-l) shown in Scheme 1.
These compounds were characterized by 1 H NMR, 13 C NMR, IR, and elemental analysis.The 1 H NMR spectra of the compounds showed NH 2 and ¼ CH proton peaks on the aromatic ring at around 7.50-7.60and 8.00-8.30ppm, respectively.The amide NH peak resonances were observed between 7.20 and 7.40 ppm (Arya et al., 2013).Hydrogen atoms on the lactam ring appeared as a doublet at around 4.75 ppm.The lactam ring and amide carbonyl carbon signals were seen at around 165 ppm and 155 ppm, respectively, in the 13 C NMR spectra.The infrared spectra of compounds 5al displayed absorptions around 3250 cm À 1 and 1670 cm À 1 corresponding to N-H and C ¼ O stretchings, respectively.Two peaks were assigned to SO 2 stretching, appearing at around 1300 cm À 1 and 1100 cm À 1 as asymmetric and symmetric stretches, respectively (Kılıcaslan et al., 2016).All spectra data support the structure of the synthesized compounds.

hCA and AChE inhibitory effect of the target compounds
The inhibitory effects of newly synthesized beta-lactam substituted benzenesulfonamide derivatives (5a-l) were examined against hCA I, II isoforms, and AChE.Acetazolamide (AAZ, PubChem CID: 1986) and tacrine (THA, PubChem CID: 1935) were included as standard inhibitors in the experiments.The inhibitory effect of these pharmacophores on the hCA II isoform was found to be significantly impacted by the diversity of substituents.The structure-activity relationship (SAR) was determined using enzyme inhibition constants (K I ) and coefficients of determination (R 2 ) and is presented in Table 3.The cytosolic isoform hCA I was potently inhibited by herein reported novel synthesized beta-lactam substituted benzenesulfonamide derivatives with K I s in the low nanomolar range of 66.60-278.40nM, indicating that all synthesized molecules (5a-l) are higher selective and more potent inhibitors than reference drug AAZ (K I of 439.17 nM).The most active derivatives in this series enclose 4-fluoro 5h, 4-nitro 5 l, and 4-bromo 5d groups, which have K I s of 66.60, 68.63 and 85.53 nM, respectively.The 3-bromo 5e, 4-cyano 5k, and unsubstituted 5a derivatives (K I s of 202.00, 260.50 and 278.40 nM, respectively) recorded the least inhibiting activity in the series.Exploring the inhibitory activity of novel synthesized beta-lactam substituted benzenesulfonamides (5a-l), all molecules showed potent inhibitory action towards the physiologically dominant hCA II isoform with K I s ranging from 39.64 to 250.10 nM, which is even better than the standard drug AAZ (K I of 98.28 nM).In particular, 4-bromo derivative 5d exhibited the best hCA II inhibitory activity with K I of 39.64 nM, whereas 5b,c and 5h-k exerted the least activity with three-digit K I s ranging between 106.80 and 250.10 nM.All the synthesized beta-lactam substituted benzenesulfonamide derivatives (5a-l) strongly inhibited the AChE, with two to three-digit K I s of 30.95-154.50 nM compared to standard drug THA (K I of 159.61 nM).The 4-nitro substituted 5 l derivative showed the most robust inhibitory effect with K I of 30.95 nM.Also, unsubstituted 5a (K I of 34.02 nM) displayed a similar activity to both the 3-bromo 5e and 4-chloro 5f derivatives (K I s of 34.46 nM and 35.58 nM, respectively).Also, 4-metoxy 5c and 4-cyano 5k derivatives had the weakest AChE inhibitory activity with three-digit nanomolar K I s of 102.00 nM and 154.50 nM, respectively.
Regarding SAR, the presence of the electron-withdrawing groups, 3-to 4-chlorophenyl (5g and 5f), 3-to 4-bromophenyl (5e and 5d), 4-fluorophenyl (5h), 2-chloro-4-fluorophenyl (5j), 2-chloro-6-fluorophenyl (5i), 4-cyanophenyl (5k), and 4-nitrophenyl (5 l) derivatives or the electron-donating groups, 4-methyphenyl (5b) and 4-methoxyphenyl (5c) resulted in an increase of affinity against the cytosolic isoform hCA I when compared to the unsubstituted phenyl (5a, K I of 278.40 nM).In particular, derivatives 5h, 5l, and 5d, having 4-fluorophenyl, 4-nitrophenyl, and 4-bromophenyl moiety with K I s of 66.60, 68.63 and 85.53 nM, respectively, were found to be 4.18, 4.06 and 3.26 times more effective than its 5a.Substituting phenyl ring with 4-bromide (5d, K I of 39.64 nM) resulted in the highest activity against hCA II isoform, whereas, the presence of electron-donating groups such as methyl and methoxy significantly led to decreased activity versus this isoform.On the other hand, replacing at the 4-position bromide moiety (5d) with a chloride or fluoride group decreased the inhibitory activity against hCA II by 1.89 and 3.21 times, as seen in the 5f and 5h, respectively (K I s of 74.76 nM and 127.30nM).Substituting phenyl ring with halogens such as 3-bromo 5e with K I of 34.46 nM and 4-chloride 5f with K I of 35.58 nM resulted in a little reduction of activity against AChE.But, the decline of the K I constant (i.e.increased inhibitory activity towards AChE) was observed when the substituting phenyl ring with 4-nitro was as in 5l (K I of 30.95 nM).
The hCAs have a high degree of similarity in their primary sequences, with over 30% sequence identity.This makes it  difficult to design isoform-selective hCAIs, as many of the residues that are conserved across the isoforms are located in the active site.However, the novel beta-lactam substituted benzenesulfonamide derivatives (5a-l) developed in this study exhibit notable selectivity for the target isoform hCA II over the off-target isoform hCA I.The selectivity index (S I ), calculated as the ratio of the K I for hCA I to the K I for hCA II, was used to measure enzyme selectivity (see Table 3).The S I s for the beta-lactam substituted benzenesulfonamides (5a-l) ranged from 4.00 to 0.44, with the unsubstituted derivative 5a showing the highest selectivity towards hCA II (S I of 4.00).
The presence of electron-donating groups such as 4-methyl (5b) or 4-methoxy (5c) significantly reduced the selectivity (S I s of 0.67 and 0.44, respectively).On the other hand, the presence of strong electron-withdrawing groups such as 4-chloro (5d), 3-chloro (5e), 3-bromo (5f) and 4-bromo (5g) had a negative impact on selectivity, with S I s of 2.16, 2.54, 1.28 and 1.51, respectively.Replacing these halogen groups at the 4 position with a fluoro group in 5h also decreased the selectivity (S I s of 1.27 and 1.14, respectively).

Cytotoxic activity of the target compounds
Based on in vitro enzymatic assays, selected beta-lactam substituted benzenesulfonamides (5d, 5f-5i and 5l) were screened for their cytotoxic activity against the human breast

In silico study
The binding patterns of the newly synthesized beta-lactam substituted benzenesulfonamides (5a-l) were examined using X-ray crystallographic structures of hCA I (PDB code 1AZM), hCA II (PDB code 3HS4), and AChE (PDB code 7E3I).
The native ligands AZM (5-acetamido-1,3,4-thiadiazole-2-sulfonamide) and THA (1,2,3,4-tetrahydro-9-acridinamine) were   respectively, with K I s of 66.6, 39.6 and 31.0 nM, respectively.These inhibitors demonstrated predicted binding scores of À 7.50 kcal/mol and MM-GBSA value of À 24.20 kcal/mol in hCA I, a docking score of À 7.11 kcal/mol and MM-GBSA value of À 5.95 kcal/mol in hCA II, and a docking score of À 8.20 kcal/mol and MM-GBSA value of À 49.87 kcal/mol in AChE.Compounds 5h and 5d displayed similar binding patterns in hCAs, which included the positioning of the sulfonamide moiety in the active site and interaction with the active site Zn 2þ ions as well as hydrogen bonding with the gatekeeper residues Thr199 (1.86 Å distance for hCA I and 2.60 Å distance for hCA II) and Thr200 (2.02 Å distance for hCA II).The beta-lactam ring engaged in a hydrogen bond with the polar residue Gln92 in hCA I (2.30Å distance) and a water molecule in hCA II (1.69 Å distance).In hCA II, a hydrogen bond interaction also occurred between the amide moiety of 5d and a water molecule (1.88 Å distance).Compound 5 l and AChE formed a hydrogen bond interaction between the oxygen atom of the benzenesulfonamide moiety and Phe295 residue (1.77 Å distance).Additionally, the hydrophobic residue Tyr337 (2.30 Å distance) formed a hydrogen bond with the carboxy moiety, while the nitro moiety exhibited a p-cation interaction with Trp86 (Figures 2-4).
The binding of compound 5a to the hCA II isoform exhibits notable selectivity, as indicated by the selectivity index of 4.00.This selectivity may be attributed to the interactions of 5a with the gatekeeper residue Thr200 and other residues, such as Gln92 and water molecules (distances of 2.15, 2.28, 1.60 and 1.69 Å), in the hCA II binding pocket.In contrast, in the hCA I binding pocket, 5a primarily interacts with Thr199 (distance of 2.06 Å) and a water molecule (distance of 1.90 Å) through hydrogen bonding.These specific interactions in the hCA II binding pocket may allow for a stronger binding of 5a to this isoform.It is worth noting that the hydrogen bonds and distances between 5a and hCA II are important for the observed selectivity.These unique binding characteristics may enable 5a to fit more securely in the hCA II binding cavity (Figure 5).
The docked conformers of the potent compounds 5h, 5d and 5l were subjected to MD simulations to assess the stability of the enzyme-ligand complexes under physiological conditions and also, to confirm the interaction profiles of the ligands with vital amino acid residues at the molecular level as a function of simulation time.In all three cases, the RMSD plots shown in Figure 6 indicate a stable binding interaction with slight deviations from the initial position that remained within an acceptable thermal average of 1-3 Å.
The RMSFs were used to depict the fluctuations of each residue present in enzymes over the simulation time (Figure 7).Although there seem to be a few high fluctuations, most of the amino acid fluctuations were less than 1.2 Å. Green vertical lines in Figure 7 represent ligand contacts.Low fluctuations were observed for most of the residues with which the ligands interact, which can be concluded that the interactions are quite stable.On the other hand, some fluctuations of the ligands were observed in each case.Inspection of the 200 ns trajectories revealed that the mainly aromatic ring bonded to the beta-lactam was flexible CNS, Central nervous system activity (-2 inactive and þ2 active); MW, molecular weight of the compound (130.00-725.00);Dipole, computed dipole moment of the compound (1.00-12.50);Volume, total solvent-accessible volume in cubic angstroms using a probe with a 1.4 A˚ Radius (500.00-2000.00);donorHB, estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution (0-6); accptHB, estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution (2-20); QPlogPoct, octanol/gas partition coefficient (8.00-35.00);QPlogPw, water/gas partition coefficient (4.00-45.00);QPlogPo/w, octanol/water partition coefficient (À 2.00 to 6.50); QPlogS, aqueous solubility (À 6.50 to 0.50); QPPCaco, apparent Caco-2 cell permeability in nm/sec (<25 poor, great > 500); QPlogBB, brain/blood partition coefficient (À 3.00 to 1.20); QPlogKp, skin permeability (À 8.00 to À during the course of the simulation resulting in minor conformational changes throughout the simulation.However, the ligands were still remaining in the binding pocket. To see how long the ligand-amino acid interactions were stable during the simulation time, ligand-protein contacts were also evaluated (Figure 7).Contacts that were observed for 50% of the course of the simulations were evaluated.In addition to water bridges, the interaction of compound 5h with Zn metal in hCAI remained for 100% of the time, while hydrogen bonds between Thr199 and Gln92 were also stable for 98% and 82% of the simulation, respectively.A p interaction was also observed with His94 residue.In the hCAII-5d system, the critical metallic interaction with Zn was also conserved for 100% of the simulation time, and also hydrogen bond with Gln92 was formed over 70% of the time.In the MD study of AChE complex, during at least 80% of the time, compound 5l interacted mainly with the peripheral anionic site (Thy41, Tyr124 and Trp286) and anionic site (Trp86) of AChE.
Additionally, the novel synthesized beta-lactam substituted benzenesulfonamides (5a-l) were evaluated for their drug-like properties using the QikProp module of the Schr€ odinger Suite 2022-3 for Mac.The selected ADME/T parameters were computed and listed in Table 5.These results showed that, based on their physicochemical characteristics, the derivatives 5a-l met the criteria for drug-like molecules as defined by Lipinski's (Lipinski et al., 1997) and Jorgensen's (Duffy & Jorgensen, 2000) rules.

Conclusion
In the present study, we designed, synthesized, and evaluated the inhibitory effects of twelve beta-lactam-substituted benzenesulfonamides (5a-l) on the hCA I, II isoforms, and AChE.These new inhibitors contained benzenesulfonamide as a ZBG motif, linked to a phenyl tail through a beta-lactam linker.The SAR analysis showed that substitution of the phenyl tail with unsubstituted phenyl (5a, K I of 69.6 nM), bromo (5d and 5e, K I s of 39.6 nM and 79.6 nM, respectively), chloro (5f and 5g, K I s of 74.8 nM and 78.9 nM, respectively), and nitro (5 l, K I of 75.0 nM) groups conferred potent hCA II inhibitory activity.Notably, the unsubstituted sulfanilamide derivative 5a selectively inhibited hCA II isoform with a selectivity of > 4-fold over hCA I. On the other hand, all derivatives (5a-l) incorporating both electron-donating and electron-withdrawing groups exhibited effective hCA I and AChE inhibitory activity.In addition, selected beta-lactam substituted benzenesulfonamides were screened for their cytotoxic activity against the MCF-7 cell line using the MTT assay protocol.Compounds 5f and 5l showed promising anticancer activities, with IC 50 values of 38.3 mM and 32.0 mM, respectively, after 24 h of treatment, which were more potent than 5-fluorouracil and cisplatin against the MCF-7 cell line.Moreover, molecular docking was used to investigate the binding of the sulfonamide moiety of the beta-lactam substituted benzenesulfonamides to the active sites of hCAs and AChE.The results suggested that these compounds fit well into the active sites of these enzymes and interact with the Zn 2þ ions present hCAs.The MD simulations revealed significant stability of each enzyme-ligand complex during 200 ns and confirmed favorable interactions with the crucial amino acid residues in the binding pocket.Also, it is determined that these compounds under investigation displayed druglike traits based on physicochemical properties, and all the derivatives (5a-l) comply with Lipinski's and Jorgensen's rules.These results suggest that the reported derivatives have the potential to be exploited in the design of novel, next-generation inhibitors, based on the insights of the inhibition data in terms of SARs.

Disclosure statement
No potential conflict of interest was reported by the authors.

Figure 5 .
Figure 5.A comparison of the 3D and 2D binding modes of the hCA I isoform (PDB ID 1AZM) complexed with compound 5a (N-(3-chloro-2-oxo-4-phenylazetydin-1-yl)-4-sulfamoylbenzamide) and the hCA II isoform (PDB ID 3HS4) complexed with compound 5a is shown.The left image displays the hCA I isoform complex, while the right image displays the hCA II isoform complex.The interacting amino acids are shown in the figures, which include both 3D (top) and 2D (bottom) images that are synchronized for clarity.

Figure 6 .
Figure 6.Protein RMSD plots as a function of simulation time.

.
Organ or tissue distribution and subcellular localization of the hCAs.

Table 2 .
Active binding sites of 1AZM, 3HS4, and 7E3I.To accept the effective binding of the compounds, site analysis by the SiteMap tool has a significant role in molecular docking studies.For this purpose, the essential binding site was selected according to the site score from among the five active sites.Exposure and enclosure, these two properties provide different measures of how open the site is to solvent.
b Druggability score.c Site volume.d e

Table 4 .
Antiproliferative activities of the selected beta-lactam substituted benzenesulfonamides against human breast cancer (MCF-7) cell line with the IC 50 values and inhibition percentages.
a Non-determined.

Table 3 .
The inhibitory effects of the novel beta-lactam substituted benzenesulfonamides 5a-l on cytosolic human CA isoforms hCA I, II, and AChE.
a Human carbonic anhydrase I. b Human carbonic anhydrase II.c Acetylcholinesterase. d Selectivity index of inhibitors for cytosolic hCA II over target isoform hCA I, calculated as the ratio of K I off-target hCA/K I target hCA.A potent, selective inhibitor is characterized by a high-value ratio.e Acetazolamide.f Tacrine.

Table 5 .
ADME/T related parameters a of novel synthesized beta-lactam substituted benzenesulfonamides 5a-l and the reference inhibitors acetazolamide and tacrine, the clinically used drugs.Compounds ID CNS MW Dipole Volume donorHB accptHB QPlogPoct QPlogPw QPlogPo/w QPlogS QPPCaco QPlogBB QPlogKp Metab QPlogKhsa HOA PSA Rule of Five Rule of Three PAINS