Design, DFT, molecular Docking and ADMET evaluation of novel imine-sulphonamide analogues as carbonic anhydrase II inhibitors

Design, DFT, molecular Docking and ADMET evaluation of novel imine-sulphonamide analogues as carbonic anhydrase II inhibitors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design, DFT, molecular Docking and ADMET evaluation of novel imine-sulphonamide analogues as carbonic anhydrase II inhibitors Yasmin Momin, Ashwini Patil, Shailaja Desai, Amruta Pawar, Kiran Shinde, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8421792/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Carbonic anhydrases (CAs) are zinc containing metalloenzymes distributed in human tissues, helps to regulate ion and pH cellular homeostasis. Statement of Problem : Discovery of carbonic anhydrase II (CA II) inhibitor is essential to minimize off-target effects and related complications including oxidative stress, cancer, glaucoma, and obesity. However, current therapeutic efficacy and safety profiles are still below ideal, which leads to more potent and specific CA II inhibitors discovery. In this work, new non-heterocyclic imine compounds with sulfonamide groups that were specially designed to inhibit CA II are designed. Methods: Based on structure-activity relationship (SAR) insights, twelve novel non-heterocyclic imine derivatives were designed by incorporating sulfonamide moieties and aromatic rings to enhance CA II binding affinity and inhibitory activity. Molegro Virtual Docker, SwissADME were utilized computational analysis. Furthermore, to evaluate molecular stability and reactivity, frontier molecular orbitals and chemical reactivity descriptors were analyzed using Density Functional Theory (DFT) computations. Result: Docking scores demonstrated significant binding affinities for compounds C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4 (scores ranging from − 63.58 to -73.23). ADMET analysis confirmed favorable drug-likeness, with compounds C-2, C-4, E-2, E-4, P-2, and P-4 exhibiting good oral bioavailability and minimal toxicity.DFT study supported the compounds' possible reactivity and binding efficiency by revealing favorable HOMO-LUMO energy gaps and high electrophilicity indices. Conclusion: The designed imine sulphonamido derivatives showed promising potential as safe CA II inhibitors in silico. These findings permit further in vivo validation and toxicity studies. This work contributes in the advancement of targeted therapies for CA-related disorders. Drug Discovery, Design, & Development Carbonic anhydrase II molecular docking DFT ADMET imine-sulfonamide cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Carbonic anhydrases (CAs) are zinc metallo-enzymes widely distributed in human tissues, cells, and body fluids for regulation of ion and pH cellular homeostasis [ 1 ]. In mammals, at least 15 distinct isoforms of α-carbonic anhydrase (EC 4.2.1.1) have been identified[ 1 ], all of which are zinc-containing enzymes with diverse physiological functions. The isozymes CA I, CA II, CA III, CA VII, and CA XIII are cytosolic; CA IV, CA IX, CA XII, CA XIV, and CA XV are membrane-bound; CA VA and CA VB are mitochondrial, and CA VI is released in milk and saliva [ 1 ]. Catalytic action of carbonic anhydrase is illustrated in Fig. 1 . Numerous physiological processes, including breathing, calcification, bone resorption, and renal functions, can be influenced by CAs. Numerous health conditions may result from functional disruption of distinct CA isoform. Inhibition of CA has vital roles in glaucoma, cancer, convulsions, bacterial infection, fungal infection, osteoporosis, renal tubular acidosis, cerebral calcification [ 2 , 3 ]and obesity. Since targeted carbonic anhydrase (CA) inhibitors shows diverse therapeutic potential, new compounds with improved isoform specificity and selectivity have been developed in an effort to reduce the detrimental effects of non-selective CA inhibition. Conventional drugs like acetazolamide and methazolamide, non-selective CA inhibitors lacks selectivity leads to off-target effects, as they hinders physiological balances, pH regulations and maintenance of ion in the kidney, brain, and blood. Improved selectivity of CAs isoform is also one of the most emerging aspects in cancer treatment due to adaptive mechanisms of microorganisms in tumor growth. In order to design novel CAs inhibitors, excellent CA isoform selectivity plays crucial role in medicinal chemistry and opens newer avenues to medicinal chemists. Amongst the various isoform, carbonic anhydrase II (CA II) is most catalytically active human isoform which is strongly inhibited by sulphonamide. Sulfonamide moiety is identified as good CA II inhibitors [ 4 ]. Moreover imine analogues have multiple pharmacological activities as anti-leishmanial, antioxidant, antimicrobial and carbonic anhydrase inhibition [ 5 ],[ 6 ]. Durgun et al., demonstrated sulfonamide-benzyl amine derivatives as CA I, II, IX, and XII inhibitors [ 7 ]. Molecular docking process provides optimum information regarding binding interactions of compounds to receptor of interest in living organisms[ 8 ].In part to developments in docking and computational techniques, it is now possible to predict the interactions of compounds with receptor in terms of docking score, and signal transduction which measured in both pathological and physiological states. Various reported literatures revealed the significance of designing, synthesizing and evaluating new CA inhibitors in glaucoma and cancer [ 9 ]. Zimmermann-Franco DC, Esteves B, et al., reported in vitro and in vivo anti-inflammatory pharmacological activities of imine derivatives that supports significance of imine derivatives in CA inhibition [ 10 ]​.By taking into an account the significant role of imine compounds and importance of sulfonamide moiety which has ability to significantly inhibit various forms of CA enzyme [ 11 ],we attempted to design 12 novel imine derivatives reasonable parallels with currently available Cox-2 inhibitors like Celecoxib, Etoricoxib, and Parecoxib and CA inhibitors. But prolonged use of existed carbonic anhydrase drugs caused liver and kidney damage and having low efficacy. The invention of non-heterocyclic imine derivatives associated with distinctive pharmacological characteristics of imine scaffolds with the CA-inhibiting potential of sulfonamides helps to overcome these difficulties by using computational chemistry such as DFT calculations, ADMET profiling, and molecular docking. Non-selectivity, specificity and poor pharmacokinetic properties of existing CA inhibitors disrupts various physiological roles of other CAs isoform in brain, kidney, tissue and blood and affects their therapeutic effectiveness in cancer or other associated diseases. Some carbonic anhydrase inhibitors (CAIs) exhibit systemic toxicity or organ-specific adverse effects, particularly at higher doses. These effects highlight the need for safer and more tolerable therapeutic options. The objective of the study is to design and evaluate novel non-heterocyclic imine derivatives that cater the CA-inhibiting potential with sulfonamides and who tailored CA II, minimizing off-target effects and associated adverse reactions. With respect to these objectives we attempted to designed non-heterocyclic imine derivatives which have ability to reduce undesirable symptoms and off-target effects. The addition of drug-likeness analysis results implies that these compounds possess favorable pharmacokinetic characteristics, potentially eliminating solubility and bioavailability limitations. The probability of systemic or organ-specific toxicity is decreased by computational screening for toxicity. These advances make the developed compounds excellent candidates of further experimental validation, with future possibilities in treating disorders that demand specific CA II inhibition. 2. MATERIALS AND METHODs 2.1 Designing of compounds The structure activity relationship (SAR) for carbonic anhydrase II (CA II) inhibitors is represented in Fig. 2 (a), which indicates that there are three essential structural components, namely the zinc-binding group (ZBG), the heterocyclic/aromatic core, as well as the substitution components. The zinc-binding group is one of the most vital pharmacophore components, which helps to retain the inhibitors in the active site of the carbonic anhydrase II enzyme by acting on the catalytic zinc metal centered in the active site. This is specifically because of the presence of zinc-coordinated residues, namely His94, His96, and His119, which helps to displace the bound zinc water molecules, thus inhibiting the catalytic activity of the enzyme. In Fig. 2 (b), a classical CA II inhibitor, acetazolamide, demonstrates the SAR which has used the sulfonamide group as the major ZBG, coordinating with zinc efficiently. The heterocyclic structure in acetazolamide contributes to the overall rigidity of the structure, which is essential for positioning the ZBG efficiently at the active site. Moreover, the heterocyclic or aromatic moiety, as depicted in Fig. 2 (a), also has a notable role in the stabilization of the IC/E complex via hydrophobic interactions, along with π-π stacking, with the amino acid residues that line the catalytic active site pocket. It has been observed that incorporation of aromatic moieties has increased the potency of CAs as inhibitors. Based on structure activity relationship of CA II (Fig. 2 ), a set of novel molecules has been designed; Acetazolamide is a clinically approved CA inhibitor and is the main reference compound for CA II inhibition. And, Acetazolamide provides the validated Carbonic Anhydrase (CA)–binding pharmacophore. Celecoxib, Etoricoxib, and Parecoxib provide drug-like aryl-sulfonamide scaffolds with validated pharmacokinetic behaviour [ 7 , 12 ]. The zinc center is concerned with the polarization of the water molecule, resulting in the release of a hydroxide ion which further binds with carbon dioxide to produce carbonic acid[ 13 , 14 ]. The incorporation of an aromatic part in a structure increases the potential of CA enzyme inhibition 15 . An effort has also been made to design molecules with these substitutes by considering the presence of these substitutes in a reference compound (Table 1 ). 2.2 Molecular Docking analysis The two-dimensional (2D) structure of Compound C-1 to C-4, Compound E-1 to E-4, and Compound P-1, to P-4 were drawn in chem-sketch and cleaned into 3D. All 3D structures were converted to aromatic form using Marvin sketch software. X-ray crystallography structure of carbonic anhydrase II (PDB: 2AW1) was compiled from the protein data bank ( www.rcsb.org ) by filtering various proteins for resolution below 2A 0 and method of isolation of x ray diffraction. The water and ligand were extracted of (PDB: 2AW1) in Molegro Virtual Docker 6.0[ 16 ]. The energy of receptor was minimized with Hamiltonian Merk Molecular Universal Force Field (MMUFF) with RMS gradient of 0.001 Kcal/mol/A2[ 17 ]. This optimized protein structure was saved from further analysis. Molecular Docking analysis of designed compounds was performed in Molegro Virtual Docker software [ 18 , 19 ]. Before going for docking study, validation of docking result was checked by redocking of 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide to protein receptor 2AW1 in Molegro Virtual Docker software. 2.3 Density Functional theory analysis (DFTs) Analysis of best docked poses was conducted for frontier molecular orbitals energies by using Density Functional theory method. B3LYP/def2-SVP level theory is used in Orca 4.2.1 [ 20 ] for molecular system energies and shape optimization [ 21 ]. The programs Orca and Avogadro were used to create the input and output files [ 22 , 23 ]. Using previously published Koopmans' theory equations, the chemical reactivity descriptors were computed [ 24 ]. 2.4 Evaluation of drug-likeness and ADMET properties of designed compounds and standard drug These designed compounds were then used to predict in silico pharmacokinetics (ADME) using the SwissADME [ 19 , 25 ], Osiris Property Explorer, and PreADMET tools. 2D Structure of designed 12 compounds was drawn in Swiss ADME and the PreADMET software. Physicochemical descriptors are computed by Swiss ADME. While PreADMET [ 18 ] offers information on toxicity and ADME, pharmacokinetics, and the drug-like qualities of compounds. Their drug-likeness was assessed using the physicochemical properties of substances necessary for pharmacokinetics (absorption, distribution, metabolism, and excretion) and pharmacodynamics (biological effects). Hydrogen bond donors and acceptors, molecular weight (MW), topological polar surface area (TPSA), and partition coefficient (log P) all affect drug similarity. Of these, Lipinski's "Rule of Five" says that a molecule is having a molecular weight under 500 Daltons is more likely to have adequate oral bioavailability, a log P value under five, fewer than five hydrogen bond donors, and fewer than ten hydrogen bond acceptors, are equally important for a compound to act as a drug. Furthermore, compounds having ten or fewer rotatable bonds and a TPSA of 140 Ų or less are more likely to have adequate oral bioavailability in rats, according to Veber's rule. When evaluating the oral drug-likeness of possible therapeutic compounds in the drug discovery stages, these guidelines act as essential filters. 3. RESULT 3.1 Protein and ligand preparation Targeted protein structure was retrieved from RCSB website ( www.rcsb.org ). The protein was prepared by extracting water molecules and ligand in molegro virtual docker 6.0. Designed compounds were also prepared by assigning missing bonds, hydrogen to the compounds. Revalidation of docking was performed in molegro virtual docker 6.0. To confirm the accuracy of the docking algorithm, the results of the docking study was validated. The first steps were to re-docked with the same co-crystallized ligand PDB: 2AW1 after removing it from the same binding cavity [ 26 ] and superimposed root mean square deviation (RMSD) value was calculated which was found less than 2Å [ 27 ]. Co-crystallized and re-docked ligands' superimposed structures with the RMSD report produced by BIOVIA Discovery Studio were added in supplementary file. The re-docking of the co-crystallized ligand is the widely applied method to estimate the accuracy of a used docking protocol [ 28 ]. 3.2 Molecular Docking analysis In this study, the interactions of the designed compounds with the CA II enzyme were evaluated by In Silico to examine their selectivity profiles. We screened our designed molecules against CA II receptor to identify superior docking scores with respect to reference ligand of PDB 2AW1 (4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide. Docking results were reported with molegro virtual docker (MVD) in terms of plant score, and MolDock score[ 29 ]. Among 12 designed compounds, nine compounds (C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2 and P-4) had shown better binding affinity similar to co-crystal protein ligand 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide. Ligand 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide shown binding affinity of -70.41 plants score to protein receptor CA II (PDB: 2AW1, Table 2 ). 2D interactions and 3D interactions were reported for best docked compounds in Figs. 3 and 4 . 3.3 Density Functional Theory Analysis (DFTs) This study was focused on investigation of various molecular properties of best docked compounds on the basis of frontier molecular orbitals (FMOs) energies. Reactivity of compounds was calculated on the basis of highest occupied molecular orbital (HOMO) energy and lowest unoccupied molecular orbital (LUMO) energy in best docked compounds [ 30 ]. The tendency to donate electrons during reaction is largely determined by its HOMO, whereas LUMO is responsible for electron-accepting tendency of molecule. Small difference gap between HOMO and LUMO indicated that the molecule was more polarizable and chemically reactive. A large gap between the HOMO and LUMO gaps indicated a less reactive and more stable molecule [ 31 ]. Table 4 represents Frontier molecular orbitals of best docked compounds in the study. Other molecular properties such as chemical potential (µ), ionization potential (IE), electron affinity (EA), electronegativity(χ), chemical hardness (η), and electrophilicity (ω), were calculated based on Koopmans’s theory (Table 3 ). 3.4 Evaluation of drug-likeness and ADMET properties of designed compounds and standard drug All designed compounds and standard drugs (Acetazolamide, Celecoxib, Etoricoxib and Parecoxib) were studied for drug likeness by SwissADME online platform and presented satisfactory observations. Amongst 12 compounds, 9 compounds and standard drugs (Acetazolamide, Celecoxib, Etoricoxib and Parecoxib) were obeyed Lipinski rule of 5 with molecular weight below 500. Log P values were less than 5 (0.44 to 2.68). Total number of Hydrogen bond acceptor moieties was less than 10 & Hydrogen Bond Donor moieties were less than 5. The physicochemical properties of drug play an important role in pharmacokinetics and pharmacodynamics profile. The physicochemical properties and pharmacokinetic profile of 12 compounds was computed using SwissADME, OSIRIS Property Explore, and PreADMET. Compound C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4 show high Human Intestinal Absorption and hence good water solubility and effective bioavailability[ 32 ], good binding affinity to receptor, unlikely to cross the blood-brain barrier that decreases the chances of central nervous system related side effects. Further favorable metabolic profiles of these compounds reduce the risk of drug-drug interactions and poor transdermal absorption. The synthesized imine-sulphonamide compounds were evaluated for drug-likeness using Veber's Rule and Lipinski's Rule of Five, both of which forecast oral bioavailability. The majority of drugs showed good pharmacokinetic behaviour by following these guidelines. Notably, compounds C-2, C-4, E-2, E-4, and P-2 showed no violations and satisfied all requirements, including log P (< 5), suitable hydrogen bond donors and acceptors, molecular weight (< 500 g/mol), and topological polar surface area (TPSA < 140 Ų). These compounds also showed good oral absorption, with a tolerable number of rotatable bonds (ROTB < 10). Compounds C-3, E-3, and P-3, on the other hand, violated Lipinski's and Veber's requirements, mainly because of their high TPSA and an abundance of hydrogen bond acceptors, which may have a detrimental effect on membrane permeability and absorption. Toxicity analysis of these 12 designed compounds was performed using OSIRIS Property Explorer and pkCSM. The results indicated that compounds C-2, C-4, E-2, and E-4 were found to be safe and produced no toxicity in terms of mutagenicity, tumorigenicity, irritation, and effect on the reproductive system. Celecoxib, Etoricoxib, Parecoxib and Acetazolamide drugs were showing hepatotoxicity. 4. DISCUSSION 4.1 Molecular Docking analysis According to the molecular docking studies the designed imine-sulphonamide compounds showed a substantial binding affinity towards the carbonic anhydrase II enzyme, with a binding affinity comparable to the reference ligand. Significant hydrogen bonding and steric interactions were demonstrated by compounds C-2, P-2, E-2, and P-1 with important residues of CA II (PDB: 2AW1), such as ASN 232, PHE231, and Lys 170. According to the docking score, these substances are likely to exhibit inhibitory activity since they fit well into the active pocket of enzyme. The proposed compounds are structurally optimized for CA II inhibition, as evidenced by the steady binding energies and consistent interaction pattern. These results demonstrated the potency of designed compounds as promising candidates for additional biological analysis. 4.2 Density Functional Theory Analysis (DFTs) The electrical characteristics and reactivity of the imine-sulphonamide derivatives were assessed using Density Functional Theory (DFT) analysis, which provided information on their stability and potential for binding as CA II inhibitors, but not directly correlates inhibition of CA enzyme. Thus, DFT analysis is supportive chemical descriptor method over the pharmacological activity. A crucial indicator of chemical reactivity is the HOMO-LUMO energy gap (ΔE); a narrower gap denotes greater reactivity and decreased kinetic stability. P-1 had the highest electrophilicity index (ω = 18.22 eV) and the shortest ΔE (1.44 eV) of any chemical, indicating that it is highly reactive and capable of effectively receiving electrons at the active site. In addition, it has a low hardness (η = 0.7206 eV) and a high chemical potential (µ = -5.1256 eV), suggesting that it tends to interact favorably with nucleophilic residues in the CA II binding pocket. Promising candidates were compounds E-1 (ΔE = 1.62 eV, ω = 14.43 eV) and E-2 (ΔE = 1.78 eV, ω = 13.04 eV), which also shown substantial reactivity. Conversely, However, the biggest HOMO-LUMO gaps were seen in C-2 (ΔE = 3.37 eV, ω = 4.25 eV) and P-2 (ΔE = 3.36 eV, ω = 5.02 eV), which showed greater electronic stability but lower reactivity. This may have limited their capacity to interact with the enzyme quickly, but it may have improved their long-term structural integrity. P-1 (χ = 5.13 eV) had the highest electronegativity (χ), another measure of a molecule's propensity to attract electrons, followed closely by C-1 (χ = 4.85 eV) and E-1 (χ = 4.84 eV), confirming their functions as electron acceptors in enzyme binding. To further balance reactivity and selectivity, the majority of compounds fell within an ideal range for chemical hardness (η) and softness (σ). Remarkably, C-1 (ω = 12.57 eV) and P-4 (ω = 9.99 eV) also displayed encouraging results, supporting their potential to suppress CA II. All things considered, DFT descriptors such as HOMO-LUMO gaps and electrophilicity indices indicate a variation in reactivity among compounds, although these do not correlate with biological inhibition capabilities and are only used in this study to support docking and ADMET prioritization. Compound P-1 shown highest electrophilicity (ω = 18.22 eV), suggesting more reactive towards nucleophilic attack, followed by compound C-1, E-1, E-2,C-4, E-4, P-4,C-2. The order of chemical reactivity of best docked compounds depends upon the HOMO–LUMO energy gap in descending manner was as follows: P-1 > E-1 > E-2 > C-1 > P-4 > C-4 > E-4 > P-2 > C-2. 4.3 Evaluation of drug-likeness and ADMET properties of designed compounds and standard drug The potential of compounds C-2, C-4, E-2, and E-4 as safe and efficient inhibitors of Carbonic Anhydrase II (CA II) is well supported by the pharmacokinetic and toxicity studies. Strong oral bioavailability and favorable drug-likeness, which are essential for successful therapeutic development, are indicated by their adherence to both Lipinski's and Veber's principles. The systemic action with low central side effects is suggested by the strong HIA and weak BBB permeability, making it perfect for treating peripheral CA II-associated illnesses. The observed high HIA in combination with low BBB permeability has important therapeutic implications. This pharmacokinetic behavior, indicating a predominately peripheral mode of action minimizes central nervous system exposure and thereby reduces the risk of CNS-related adverse effects. The chosen compounds had a better safety profile than conventional medications, which had a higher lipophilicity and potential hepatotoxicity. These compounds may lessen systemic adverse effects and drug-drug interactions in clinical applications due to their good metabolic compatibility and lack of toxicity hazards. It's interesting to note that common medications like Celecoxib, Etoricoxib, and Parecoxib meet Lipinski's requirements as well, but their larger log P values may have an impact on solubility. All things considered, C-2, C-4, E-2, E-4, and P-2 stood out as the best options with encouraging drug-likeness and bioavailability profiles, indicating the need for additional experimental research. From the drug likeness and ADMET study, the compounds C-2, C-4, E-2, and E-4 found safe with good pharmacokinetic properties. This study presents a promising opportunity for targeted drug discovery of CA II inhibitors with reduced systemic side effects and better pharmacokinetics. Taking all into consideration, integrated docking, DFT, and ADMET analyses therefore supported the prioritization of the C-2, C-4, E-2, and E-4 for further studies. These results therefore provided a rationale for follow-up in vitro and in vivo studies in an effort to validate their inhibiting action on CA II, pharmacokinetic behavior, and safety. 5. LIMITATIONS OF THE STUDY Notwithstanding the favorable results, the study is limited by its support with In silico methods only. Molecular docking and ADMET predictions, while robust, require experimental validation to confirm biological activity and pharmacokinetic behavior In vivo. Another limitation is the absence of molecular dynamics simulations, which could provide insights into the stability of compound-receptor interactions under physiological conditions. Additionally, necessitating structural modifications in potent compounds found toxic to improve their safety profile. 6. CONCLUSION This study attempted to design novel imine analogues based upon structural resemblance with Celecoxib, Etoricoxib and Parecoxib and Acetazolamide that act as CA II inhibitors. Incorporation of sulfonamide moiety with imine scaffold shows increased affinity to CA II protein via docking and DFT analysis. The docking results highlight the ability of the designed compounds to effectively mimic the interactions of standard inhibitors while offering improved docking scores, supporting their potential to inhibit CA II (PDB: 2AW1) via hydrophobic interaction, and hydrogen bond interaction. Amongst 12 novel imine analogues, compounds C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4 exhibited better binding affinity as that of standard drugs that confirms the affinity to carbonic anhydrase II enzyme. Importantly, Density Functional Theory (DFT) analysis of frontier molecular orbitals (HOMO-LUMO) supported the observed binding potential by confirming favorable electronic characteristics and high electrophilicity, especially for compound P-1. These results provide deeper insight into molecular stability, reactivity, and drug-likeness. ADME analysis and drug likeness analysis ensures that the designed compounds not only have favorable binding characteristics but also possess desirable pharmacokinetic properties, making them viable for oral administration. Toxicity analysis of C-2, C-4, E-2, E-4, P-2 and P-4 predicted favorable toxicity profile in silico . The observed low toxicity profiles for these compounds further emphasize their therapeutic potential in diseases. Current investigation demonstrate that the designed imine derivatives have the potential to bridge existing gaps in CA inhibitor therapies by offering improved selectivity and reduced toxicity. It was concluded that these novel imine derivatives were shown noteworthy potential as CA II inhibitors; safer and nontoxic. Further synthesis of potent imine compounds needs to be conducted and consequently, to confirm the results of these studies, more research utilizing a variety of experimental models, such as in-vivo testing and toxicity studies, is required. Those compounds presented best binding energy and having more toxicity profile need to be modified further to be better and potent drug candidates. 7. FUTURE PROSPECTS Future research must emphasize on synthesis and biological assay for best docked and nontoxic compounds to validate the predicted efficacy. Advanced simulations, such as free energy calculations, can further enhance the understanding of binding interactions. Expanding the compound library by suitable structural modifications found through In silico analysis can lead to develop structurally diverse analogues and discover new opportunities for optimizing CA II inhibition. 8. SUMMARY The goal of this project is to rationally design twelve new imine-based sulphonamide derivatives that inhibit Carbonic Anhydrase II (CA II), a crucial enzyme linked to a number of illnesses, including obesity, glaucoma, and cancer. Structure-activity relationships (SAR) and structural resemblance to well-known medications like acetazolamide, Celecoxib, Etoricoxib, and Parecoxib were used in the design of the compounds.The compounds were assessed for toxicity, pharmacokinetic characteristics, reactivity, and binding affinity using Molecular Docking, Density Functional Theory (DFT), and ADMET analysis. CA II was significantly bound by nine compounds (C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4). Compound P-1 showed the highest electrophilicity, indicating considerable reactivity, according to DFT analysis. For a number of compounds, good oral bioavailability was confirmed by drug-likeness and pharmacokinetic evaluations, and toxicity studies revealed that C-2, C-4, E-2, and E-4 were non-toxic and had favorable ADMET profiles. This investigation will support to validate further therapeutic potential of these designed compounds in vitro and in vivo. Abbreviations HOMO Highest occupied molecular orbital LUMO Lowest unoccupied molecular orbital DFT Density functional theory PDB Protein data bank FMO Frontier molecular orbital CA II Carbonic anhydrase II. Declarations Competing Interests “The authors have no relevant financial or non-financial interests to disclose.” Funding “The authors declare that no funds, grants, or other support were received during the Preparation of this manuscript.” ETHICS “No human or animal subjects were involved in this study.” INFORMED CONSENT “Not applicable. This study does not involve human participants or animals. ” ACKNOWLEDGEMENT The authors are deeply thankful to the management, and principal of Annasaheb Dange College of B.Pharmacy, Ashta for providing state of the art research facilities. References Supuran CT (2007) Carbonic anhydrases as drug targets–an overview. Curr Top Med Chem 7:825–833 Roth DE, Venta PJ, Tashian RE, Sly WS (1992) Molecular basis of human carbonic anhydrase II deficiency. 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J Biomol Struct Dyn 40:585–611. https://doi.org/10.1080/07391102.2020.1815584 Azam F (2021) Elucidation of teicoplanin interactions with drug targets related to COVID-19. https://doi.org/10.3390/antibiotics10070856 . Antibiotics 10: Abdusalam AAA, Murugaiyah V (2020) Identification of Potential Inhibitors of 3CL Protease of SARS-CoV-2 From ZINC Database by Molecular Docking-Based Virtual Screening. Front Mol Biosci 7:1–11. https://doi.org/10.3389/fmolb.2020.603037 Saxena DP (2021) Intoxication of Cypermethrin on Binding Site of Human Oxyhaemoglobin. Sch Acad J Biosci 9:145–148. https://doi.org/10.36347/sajb.2021.v09i05.005 Adindu EA, Godfrey OC, Agwupuye EI et al (2023) Structural analysis, reactivity descriptors (HOMO-LUMO, ELF, NBO), effect of polar (DMSO, EtOH, H2O) solvation, and libido-enhancing potential of resveratrol by molecular docking. Chem Phys Impact 7:100296. https://doi.org/10.1016/j.chphi.2023.100296 Hadigheh Rezvan V (2024) Molecular structure, HOMO–LUMO, and NLO studies of some quinoxaline 1,4-dioxide derivatives: Computational (HF and DFT) analysis. Results Chem 7:101437. https://doi.org/10.1016/j.rechem.2024.101437 B ZH a BQC, A MC (2021) Novel carbohydrate-based sulfonamide derivatives as selective carbonic anhydrase II inhibitors: Synthesis, biological and molecular docking analysis. Bioorg Med Chem Lett 51:128291. https://doi.org/10.1016/j.bmcl.2021.128291 Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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08:13:51","extension":"xml","order_by":104,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121335,"visible":true,"origin":"","legend":"","description":"","filename":"rs84217920structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/dc7ba1c5a687d925932224c6.xml"},{"id":99308564,"identity":"526691f5-19a4-4d05-bf41-f45b345325c9","added_by":"auto","created_at":"2025-12-31 16:08:47","extension":"html","order_by":105,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133699,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/1831b4fef513d076ec626ad1.html"},{"id":98858160,"identity":"1ac7c4bf-34ab-4099-a5a1-f6a23ced0356","added_by":"auto","created_at":"2025-12-23 08:13:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":303556,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the catalytic mechanism of carbonic anhydrase (CA), illustrating the role of the zinc ion and water molecule in the reversible hydration of carbon dioxide to bicarbonate.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/e8ae729a92287519e1e3c40b.png"},{"id":98858161,"identity":"968a5783-2cd0-443a-9b44-9500ac0bc16e","added_by":"auto","created_at":"2025-12-23 08:13:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":144880,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Structure–Activity Relationship (SAR) features relevant to CA II inhibitors, highlighting key interactions with active site residues (His94, His96, His119).(b) Chemical structure of the reference CA inhibitor acetazolamide, showing the zinc-binding sulfonamide moiety.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/cbc853ac8cb7ec21231ba930.png"},{"id":98858172,"identity":"6786c9a7-fc8c-47d7-a621-ff8380544de2","added_by":"auto","created_at":"2025-12-23 08:13:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":335912,"visible":true,"origin":"","legend":"\u003cp\u003e2D interaction diagrams of the best-docked imine sulphonamide compounds (C-1, C-2, C-4, E-1, P-1, P-2, P-4) and reference compound with the active site residues of CA II (PDB: 2AW1), showing hydrogen bonds and steric interactions.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/051c14142abaefca90f2a2f4.png"},{"id":98858164,"identity":"24d112f8-a624-4279-a986-68abdd76cad4","added_by":"auto","created_at":"2025-12-23 08:13:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":204215,"visible":true,"origin":"","legend":"\u003cp\u003eHydrophobic interactions of best docked compounds and ligand Ligand 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulphonamide with target protein CA II (PDB: 2AW1).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/c928894a44b91fcb0c5a9ed3.png"},{"id":99322693,"identity":"cb4483e6-b705-40f7-b685-76b09c20404c","added_by":"auto","created_at":"2025-12-31 16:44:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1545014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/dfac5612-7c21-4d48-8fff-3431839eb1da.pdf"},{"id":98858224,"identity":"d1249bed-ec67-4a14-b800-306466cbd01f","added_by":"auto","created_at":"2025-12-23 08:13:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3035799,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8421792/v1/311eec424c664663be8a77f0.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eDesign, DFT, molecular Docking and ADMET evaluation of novel imine-sulphonamide analogues as carbonic anhydrase II inhibitors\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eCarbonic anhydrases (CAs) are zinc metallo-enzymes widely distributed in human tissues, cells, and body fluids for regulation of ion and pH cellular homeostasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In mammals, at least 15 distinct isoforms of α-carbonic anhydrase (EC 4.2.1.1) have been identified[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], all of which are zinc-containing enzymes with diverse physiological functions. The isozymes CA I, CA II, CA III, CA VII, and CA XIII are cytosolic; CA IV, CA IX, CA XII, CA XIV, and CA XV are membrane-bound; CA VA and CA VB are mitochondrial, and CA VI is released in milk and saliva [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Catalytic action of carbonic anhydrase is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Numerous physiological processes, including breathing, calcification, bone resorption, and renal functions, can be influenced by CAs. Numerous health conditions may result from functional disruption of distinct CA isoform. Inhibition of CA has vital roles in glaucoma, cancer, convulsions, bacterial infection, fungal infection, osteoporosis, renal tubular acidosis, cerebral calcification [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]and obesity. Since targeted carbonic anhydrase (CA) inhibitors shows diverse therapeutic potential, new compounds with improved isoform specificity and selectivity have been developed in an effort to reduce the detrimental effects of non-selective CA inhibition. Conventional drugs like acetazolamide and methazolamide, non-selective CA inhibitors lacks selectivity leads to off-target effects, as they hinders physiological balances, pH regulations and maintenance of ion in the kidney, brain, and blood. Improved selectivity of CAs isoform is also one of the most emerging aspects in cancer treatment due to adaptive mechanisms of microorganisms in tumor growth. In order to design novel CAs inhibitors, excellent CA isoform selectivity plays crucial role in medicinal chemistry and opens newer avenues to medicinal chemists.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmongst the various isoform, carbonic anhydrase II (CA II) is most catalytically active human isoform which is strongly inhibited by sulphonamide. Sulfonamide moiety is identified as good CA II inhibitors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover imine analogues have multiple pharmacological activities as anti-leishmanial, antioxidant, antimicrobial and carbonic anhydrase inhibition [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e],[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Durgun et al., demonstrated sulfonamide-benzyl amine derivatives as CA I, II, IX, and XII inhibitors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMolecular docking process provides optimum information regarding binding interactions of compounds to receptor of interest in living organisms[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].In part to developments in docking and computational techniques, it is now possible to predict the interactions of compounds with receptor in terms of docking score, and signal transduction which measured in both pathological and physiological states. Various reported literatures revealed the significance of designing, synthesizing and evaluating new CA inhibitors in glaucoma and cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eZimmermann-Franco DC, Esteves B, et al., reported \u003cem\u003ein vitro and in vivo\u003c/em\u003e anti-inflammatory pharmacological activities of imine derivatives that supports significance of imine derivatives in CA inhibition [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]​.By taking into an account the significant role of imine compounds and importance of sulfonamide moiety which has ability to significantly inhibit various forms of CA enzyme [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e],we attempted to design 12 novel imine derivatives reasonable parallels with currently available Cox-2 inhibitors like Celecoxib, Etoricoxib, and Parecoxib and CA inhibitors. But prolonged use of existed carbonic anhydrase drugs caused liver and kidney damage and having low efficacy. The invention of non-heterocyclic imine derivatives associated with distinctive pharmacological characteristics of imine scaffolds with the CA-inhibiting potential of sulfonamides helps to overcome these difficulties by using computational chemistry such as DFT calculations, ADMET profiling, and molecular docking.\u003c/p\u003e \u003cp\u003eNon-selectivity, specificity and poor pharmacokinetic properties of existing CA inhibitors disrupts various physiological roles of other CAs isoform in brain, kidney, tissue and blood and affects their therapeutic effectiveness in cancer or other associated diseases. Some carbonic anhydrase inhibitors (CAIs) exhibit systemic toxicity or organ-specific adverse effects, particularly at higher doses. These effects highlight the need for safer and more tolerable therapeutic options. The objective of the study is to design and evaluate novel non-heterocyclic imine derivatives that cater the CA-inhibiting potential with sulfonamides and who tailored CA II, minimizing off-target effects and associated adverse reactions. With respect to these objectives we attempted to designed non-heterocyclic imine derivatives which have ability to reduce undesirable symptoms and off-target effects. The addition of drug-likeness analysis results implies that these compounds possess favorable pharmacokinetic characteristics, potentially eliminating solubility and bioavailability limitations. The probability of systemic or organ-specific toxicity is decreased by computational screening for toxicity. These advances make the developed compounds excellent candidates of further experimental validation, with future possibilities in treating disorders that demand specific CA II inhibition.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODs","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Designing of compounds\u003c/h2\u003e \u003cp\u003eThe structure activity relationship (SAR) for carbonic anhydrase II (CA II) inhibitors is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a), which indicates that there are three essential structural components, namely the zinc-binding group (ZBG), the heterocyclic/aromatic core, as well as the substitution components. The zinc-binding group is one of the most vital pharmacophore components, which helps to retain the inhibitors in the active site of the carbonic anhydrase II enzyme by acting on the catalytic zinc metal centered in the active site. This is specifically because of the presence of zinc-coordinated residues, namely His94, His96, and His119, which helps to displace the bound zinc water molecules, thus inhibiting the catalytic activity of the enzyme. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (b), a classical CA II inhibitor, acetazolamide, demonstrates the SAR which has used the sulfonamide group as the major ZBG, coordinating with zinc efficiently. The heterocyclic structure in acetazolamide contributes to the overall rigidity of the structure, which is essential for positioning the ZBG efficiently at the active site.\u003c/p\u003e\u003cp\u003eMoreover, the heterocyclic or aromatic moiety, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (a), also has a notable role in the stabilization of the IC/E complex via hydrophobic interactions, along with π-π stacking, with the amino acid residues that line the catalytic active site pocket.\u003c/p\u003e \u003cp\u003eIt has been observed that incorporation of aromatic moieties has increased the potency of CAs as inhibitors. Based on structure activity relationship of CA II (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), a set of novel molecules has been designed; Acetazolamide is a clinically approved CA inhibitor and is the main reference compound for CA II inhibition. And, Acetazolamide provides the validated Carbonic Anhydrase (CA)\u0026ndash;binding pharmacophore. Celecoxib, Etoricoxib, and Parecoxib provide drug-like aryl-sulfonamide scaffolds with validated pharmacokinetic behaviour [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The zinc center is concerned with the polarization of the water molecule, resulting in the release of a hydroxide ion which further binds with carbon dioxide to produce carbonic acid[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The incorporation of an aromatic part in a structure increases the potential of CA enzyme inhibition \u003csup\u003e15\u003c/sup\u003e. An effort has also been made to design molecules with these substitutes by considering the presence of these substitutes in a reference compound (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Molecular Docking analysis\u003c/h2\u003e \u003cp\u003eThe two-dimensional (2D) structure of Compound C-1 to C-4, Compound E-1 to E-4, and Compound P-1, to P-4 were drawn in chem-sketch and cleaned into 3D. All 3D structures were converted to aromatic form using Marvin sketch software. X-ray crystallography structure of carbonic anhydrase II (PDB: 2AW1) was compiled from the protein data bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rcsb.org\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by filtering various proteins for resolution below 2A\u003csup\u003e0\u003c/sup\u003e and method of isolation of x ray diffraction. The water and ligand were extracted of (PDB: 2AW1) in Molegro Virtual Docker 6.0[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The energy of receptor was minimized with Hamiltonian Merk Molecular Universal Force Field (MMUFF) with RMS gradient of 0.001 Kcal/mol/A2[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This optimized protein structure was saved from further analysis. Molecular Docking analysis of designed compounds was performed in Molegro Virtual Docker software [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Before going for docking study, validation of docking result was checked by redocking of 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide to protein receptor 2AW1 in Molegro Virtual Docker software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Density Functional theory analysis (DFTs)\u003c/h2\u003e \u003cp\u003eAnalysis of best docked poses was conducted for frontier molecular orbitals energies by using Density Functional theory method. B3LYP/def2-SVP level theory is used in Orca 4.2.1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] for molecular system energies and shape optimization [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The programs Orca and Avogadro were used to create the input and output files [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Using previously published Koopmans' theory equations, the chemical reactivity descriptors were computed [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Evaluation of drug-likeness and ADMET properties of designed compounds and standard drug\u003c/h2\u003e \u003cp\u003eThese designed compounds were then used to predict \u003cem\u003ein silico\u003c/em\u003e pharmacokinetics (ADME) using the SwissADME [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], Osiris Property Explorer, and PreADMET tools. 2D Structure of designed 12 compounds was drawn in Swiss ADME and the PreADMET software. Physicochemical descriptors are computed by Swiss ADME. While PreADMET [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] offers information on toxicity and ADME, pharmacokinetics, and the drug-like qualities of compounds.\u003c/p\u003e \u003cp\u003eTheir drug-likeness was assessed using the physicochemical properties of substances necessary for pharmacokinetics (absorption, distribution, metabolism, and excretion) and pharmacodynamics (biological effects). Hydrogen bond donors and acceptors, molecular weight (MW), topological polar surface area (TPSA), and partition coefficient (log P) all affect drug similarity. Of these, Lipinski's \"Rule of Five\" says that a molecule is having a molecular weight under 500 Daltons is more likely to have adequate oral bioavailability, a log P value under five, fewer than five hydrogen bond donors, and fewer than ten hydrogen bond acceptors, are equally important for a compound to act as a drug. Furthermore, compounds having ten or fewer rotatable bonds and a TPSA of 140 \u0026Aring;\u0026sup2; or less are more likely to have adequate oral bioavailability in rats, according to Veber's rule. When evaluating the oral drug-likeness of possible therapeutic compounds in the drug discovery stages, these guidelines act as essential filters.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULT","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Protein and ligand preparation\u003c/h2\u003e\n \u003cp\u003eTargeted protein structure was retrieved from RCSB website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rcsb.org\u003c/span\u003e\u003c/span\u003e). The protein was prepared by extracting water molecules and ligand in molegro virtual docker 6.0. Designed compounds were also prepared by assigning missing bonds, hydrogen to the compounds. Revalidation of docking was performed in molegro virtual docker 6.0. To confirm the accuracy of the docking algorithm, the results of the docking study was validated. The first steps were to re-docked with the same co-crystallized ligand PDB: 2AW1 after removing it from the same binding cavity [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] and superimposed root mean square deviation (RMSD) value was calculated which was found less than 2\u0026Aring; [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. Co-crystallized and re-docked ligands\u0026apos; superimposed structures with the RMSD report produced by BIOVIA Discovery Studio were added in supplementary file. The re-docking of the co-crystallized ligand is the widely applied method to estimate the accuracy of a used docking protocol [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Molecular Docking analysis\u003c/h2\u003e\n \u003cp\u003eIn this study, the interactions of the designed compounds with the CA II enzyme were evaluated by \u003cem\u003eIn Silico\u003c/em\u003e to examine their selectivity profiles. We screened our designed molecules against CA II receptor to identify superior docking scores with respect to reference ligand of PDB 2AW1 (4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide. Docking results were reported with molegro virtual docker (MVD) in terms of plant score, and MolDock score[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Among 12 designed compounds, nine compounds (C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2 and P-4) had shown better binding affinity similar to co-crystal protein ligand 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide. Ligand 4-(5-methyl-3-phenylisoxazol-4-yl) benzene sulfonamide shown binding affinity of -70.41 plants score to protein receptor CA II (PDB: 2AW1, Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). 2D interactions and 3D interactions were reported for best docked compounds in Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Density Functional Theory Analysis (DFTs)\u003c/h2\u003e\n \u003cp\u003eThis study was focused on investigation of various molecular properties of best docked compounds on the basis of frontier molecular orbitals (FMOs) energies. Reactivity of compounds was calculated on the basis of highest occupied molecular orbital (HOMO) energy and lowest unoccupied molecular orbital (LUMO) energy in best docked compounds [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. The tendency to donate electrons during reaction is largely determined by its HOMO, whereas LUMO is responsible for electron-accepting tendency of molecule. Small difference gap between HOMO and LUMO indicated that the molecule was more polarizable and chemically reactive. A large gap between the HOMO and LUMO gaps indicated a less reactive and more stable molecule [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e represents Frontier molecular orbitals of best docked compounds in the study. Other molecular properties such as chemical potential (\u0026micro;), ionization potential (IE), electron affinity (EA), electronegativity(\u0026chi;), chemical hardness (\u0026eta;), and electrophilicity (\u0026omega;), were calculated based on Koopmans\u0026rsquo;s theory (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Evaluation of drug-likeness and ADMET properties of designed compounds and standard drug\u003c/h2\u003e\n \u003cp\u003eAll designed compounds and standard drugs (Acetazolamide, Celecoxib, Etoricoxib and Parecoxib) were studied for drug likeness by SwissADME online platform and presented satisfactory observations. Amongst 12 compounds, 9 compounds and standard drugs (Acetazolamide, Celecoxib, Etoricoxib and Parecoxib) were obeyed Lipinski rule of 5 with molecular weight below 500. Log P values were less than 5 (0.44 to 2.68). Total number of Hydrogen bond acceptor moieties was less than 10 \u0026amp; Hydrogen Bond Donor moieties were less than 5.\u003c/p\u003e\n \u003cp\u003eThe physicochemical properties of drug play an important role in pharmacokinetics and pharmacodynamics profile. The physicochemical properties and pharmacokinetic profile of 12 compounds was computed using SwissADME, OSIRIS Property Explore, and PreADMET. Compound C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4 show high Human Intestinal Absorption and hence good water solubility and effective bioavailability[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e], good binding affinity to receptor, unlikely to cross the blood-brain barrier that decreases the chances of central nervous system related side effects. Further favorable metabolic profiles of these compounds reduce the risk of drug-drug interactions and poor transdermal absorption.\u003c/p\u003e\n \u003cp\u003eThe synthesized imine-sulphonamide compounds were evaluated for drug-likeness using Veber\u0026apos;s Rule and Lipinski\u0026apos;s Rule of Five, both of which forecast oral bioavailability. The majority of drugs showed good pharmacokinetic behaviour by following these guidelines. Notably, compounds C-2, C-4, E-2, E-4, and P-2 showed no violations and satisfied all requirements, including log P (\u0026lt;\u0026thinsp;5), suitable hydrogen bond donors and acceptors, molecular weight (\u0026lt;\u0026thinsp;500 g/mol), and topological polar surface area (TPSA\u0026thinsp;\u0026lt;\u0026thinsp;140 \u0026Aring;\u0026sup2;). These compounds also showed good oral absorption, with a tolerable number of rotatable bonds (ROTB\u0026thinsp;\u0026lt;\u0026thinsp;10). Compounds C-3, E-3, and P-3, on the other hand, violated Lipinski\u0026apos;s and Veber\u0026apos;s requirements, mainly because of their high TPSA and an abundance of hydrogen bond acceptors, which may have a detrimental effect on membrane permeability and absorption. Toxicity analysis of these 12 designed compounds was performed using OSIRIS Property Explorer and pkCSM. The results indicated that compounds C-2, C-4, E-2, and E-4 were found to be safe and produced no toxicity in terms of mutagenicity, tumorigenicity, irritation, and effect on the reproductive system. Celecoxib, Etoricoxib, Parecoxib and Acetazolamide drugs were showing hepatotoxicity.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Molecular Docking analysis\u003c/h2\u003e \u003cp\u003eAccording to the molecular docking studies the designed imine-sulphonamide compounds showed a substantial binding affinity towards the carbonic anhydrase II enzyme, with a binding affinity comparable to the reference ligand. Significant hydrogen bonding and steric interactions were demonstrated by compounds C-2, P-2, E-2, and P-1 with important residues of CA II (PDB: 2AW1), such as ASN 232, PHE231, and Lys 170. According to the docking score, these substances are likely to exhibit inhibitory activity since they fit well into the active pocket of enzyme. The proposed compounds are structurally optimized for CA II inhibition, as evidenced by the steady binding energies and consistent interaction pattern. These results demonstrated the potency of designed compounds as promising candidates for additional biological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Density Functional Theory Analysis (DFTs)\u003c/h2\u003e \u003cp\u003eThe electrical characteristics and reactivity of the imine-sulphonamide derivatives were assessed using Density Functional Theory (DFT) analysis, which provided information on their stability and potential for binding as CA II inhibitors, but not directly correlates inhibition of CA enzyme. Thus, DFT analysis is supportive chemical descriptor method over the pharmacological activity. A crucial indicator of chemical reactivity is the HOMO-LUMO energy gap (ΔE); a narrower gap denotes greater reactivity and decreased kinetic stability. P-1 had the highest electrophilicity index (ω\u0026thinsp;=\u0026thinsp;18.22 eV) and the shortest ΔE (1.44 eV) of any chemical, indicating that it is highly reactive and capable of effectively receiving electrons at the active site. In addition, it has a low hardness (η\u0026thinsp;=\u0026thinsp;0.7206 eV) and a high chemical potential (\u0026micro; = -5.1256 eV), suggesting that it tends to interact favorably with nucleophilic residues in the CA II binding pocket. Promising candidates were compounds E-1 (ΔE\u0026thinsp;=\u0026thinsp;1.62 eV, ω\u0026thinsp;=\u0026thinsp;14.43 eV) and E-2 (ΔE\u0026thinsp;=\u0026thinsp;1.78 eV, ω\u0026thinsp;=\u0026thinsp;13.04 eV), which also shown substantial reactivity. Conversely, However, the biggest HOMO-LUMO gaps were seen in C-2 (ΔE\u0026thinsp;=\u0026thinsp;3.37 eV, ω\u0026thinsp;=\u0026thinsp;4.25 eV) and P-2 (ΔE\u0026thinsp;=\u0026thinsp;3.36 eV, ω\u0026thinsp;=\u0026thinsp;5.02 eV), which showed greater electronic stability but lower reactivity. This may have limited their capacity to interact with the enzyme quickly, but it may have improved their long-term structural integrity. P-1 (χ\u0026thinsp;=\u0026thinsp;5.13 eV) had the highest electronegativity (χ), another measure of a molecule's propensity to attract electrons, followed closely by C-1 (χ\u0026thinsp;=\u0026thinsp;4.85 eV) and E-1 (χ\u0026thinsp;=\u0026thinsp;4.84 eV), confirming their functions as electron acceptors in enzyme binding. To further balance reactivity and selectivity, the majority of compounds fell within an ideal range for chemical hardness (η) and softness (σ). Remarkably, C-1 (ω\u0026thinsp;=\u0026thinsp;12.57 eV) and P-4 (ω\u0026thinsp;=\u0026thinsp;9.99 eV) also displayed encouraging results, supporting their potential to suppress CA II.\u003c/p\u003e \u003cp\u003eAll things considered, DFT descriptors such as HOMO-LUMO gaps and electrophilicity indices indicate a variation in reactivity among compounds, although these do not correlate with biological inhibition capabilities and are only used in this study to support docking and ADMET prioritization. Compound P-1 shown highest electrophilicity (ω\u0026thinsp;=\u0026thinsp;18.22 eV), suggesting more reactive towards nucleophilic attack, followed by compound C-1, E-1, E-2,C-4, E-4, P-4,C-2. The order of chemical reactivity of best docked compounds depends upon the HOMO\u0026ndash;LUMO energy gap in descending manner was as follows: P-1\u0026thinsp;\u0026gt;\u0026thinsp;E-1\u0026thinsp;\u0026gt;\u0026thinsp;E-2\u0026thinsp;\u0026gt;\u0026thinsp;C-1\u0026thinsp;\u0026gt;\u0026thinsp;P-4\u0026thinsp;\u0026gt;\u0026thinsp;C-4\u0026thinsp;\u0026gt;\u0026thinsp;E-4\u0026thinsp;\u0026gt;\u0026thinsp;P-2\u0026thinsp;\u0026gt;\u0026thinsp;C-2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Evaluation of drug-likeness and ADMET properties of designed compounds and standard drug\u003c/h2\u003e \u003cp\u003eThe potential of compounds C-2, C-4, E-2, and E-4 as safe and efficient inhibitors of Carbonic Anhydrase II (CA II) is well supported by the pharmacokinetic and toxicity studies. Strong oral bioavailability and favorable drug-likeness, which are essential for successful therapeutic development, are indicated by their adherence to both Lipinski's and Veber's principles. The systemic action with low central side effects is suggested by the strong HIA and weak BBB permeability, making it perfect for treating peripheral CA II-associated illnesses. The observed high HIA in combination with low BBB permeability has important therapeutic implications. This pharmacokinetic behavior, indicating a predominately peripheral mode of action minimizes central nervous system exposure and thereby reduces the risk of CNS-related adverse effects. The chosen compounds had a better safety profile than conventional medications, which had a higher lipophilicity and potential hepatotoxicity. These compounds may lessen systemic adverse effects and drug-drug interactions in clinical applications due to their good metabolic compatibility and lack of toxicity hazards.\u003c/p\u003e \u003cp\u003eIt's interesting to note that common medications like Celecoxib, Etoricoxib, and Parecoxib meet Lipinski's requirements as well, but their larger log P values may have an impact on solubility. All things considered, C-2, C-4, E-2, E-4, and P-2 stood out as the best options with encouraging drug-likeness and bioavailability profiles, indicating the need for additional experimental research. From the drug likeness and ADMET study, the compounds C-2, C-4, E-2, and E-4 found safe with good pharmacokinetic properties. This study presents a promising opportunity for targeted drug discovery of CA II inhibitors with reduced systemic side effects and better pharmacokinetics.\u003c/p\u003e \u003cp\u003eTaking all into consideration, integrated docking, DFT, and ADMET analyses therefore supported the prioritization of the C-2, C-4, E-2, and E-4 for further studies. These results therefore provided a rationale for follow-up \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies in an effort to validate their inhibiting action on CA II, pharmacokinetic behavior, and safety.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. LIMITATIONS OF THE STUDY","content":"\u003cp\u003eNotwithstanding the favorable results, the study is limited by its support with \u003cem\u003eIn silico\u003c/em\u003e methods only. Molecular docking and ADMET predictions, while robust, require experimental validation to confirm biological activity and pharmacokinetic behavior \u003cem\u003eIn vivo.\u003c/em\u003e Another limitation is the absence of molecular dynamics simulations, which could provide insights into the stability of compound-receptor interactions under physiological conditions. Additionally, necessitating structural modifications in potent compounds found toxic to improve their safety profile.\u003c/p\u003e"},{"header":"6. CONCLUSION","content":"\u003cp\u003eThis study attempted to design novel imine analogues based upon structural resemblance with Celecoxib, Etoricoxib and Parecoxib and Acetazolamide that act as CA II inhibitors. Incorporation of sulfonamide moiety with imine scaffold shows increased affinity to CA II protein via docking and DFT analysis. The docking results highlight the ability of the designed compounds to effectively mimic the interactions of standard inhibitors while offering improved docking scores, supporting their potential to inhibit CA II (PDB: 2AW1) via hydrophobic interaction, and hydrogen bond interaction. Amongst 12 novel imine analogues, compounds C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4 exhibited better binding affinity as that of standard drugs that confirms the affinity to carbonic anhydrase II enzyme. Importantly, Density Functional Theory (DFT) analysis of frontier molecular orbitals (HOMO-LUMO) supported the observed binding potential by confirming favorable electronic characteristics and high electrophilicity, especially for compound P-1. These results provide deeper insight into molecular stability, reactivity, and drug-likeness. ADME analysis and drug likeness analysis ensures that the designed compounds not only have favorable binding characteristics but also possess desirable pharmacokinetic properties, making them viable for oral administration. Toxicity analysis of C-2, C-4, E-2, E-4, P-2 and P-4 predicted favorable toxicity profile \u003cem\u003ein silico\u003c/em\u003e. The observed low toxicity profiles for these compounds further emphasize their therapeutic potential in diseases. Current investigation demonstrate that the designed imine derivatives have the potential to bridge existing gaps in CA inhibitor therapies by offering improved selectivity and reduced toxicity. It was concluded that these novel imine derivatives were shown noteworthy potential as CA II inhibitors; safer and nontoxic. Further synthesis of potent imine compounds needs to be conducted and consequently, to confirm the results of these studies, more research utilizing a variety of experimental models, such as \u003cem\u003ein-vivo\u003c/em\u003e testing and toxicity studies, is required. Those compounds presented best binding energy and having more toxicity profile need to be modified further to be better and potent drug candidates.\u003c/p\u003e"},{"header":"7. FUTURE PROSPECTS","content":"\u003cp\u003eFuture research must emphasize on synthesis and biological assay for best docked and nontoxic compounds to validate the predicted efficacy. Advanced simulations, such as free energy calculations, can further enhance the understanding of binding interactions. Expanding the compound library by suitable structural modifications found through \u003cem\u003eIn silico\u003c/em\u003e analysis can lead to develop structurally diverse analogues and discover new opportunities for optimizing CA II inhibition.\u003c/p\u003e"},{"header":"8. SUMMARY","content":"\u003cp\u003eThe goal of this project is to rationally design twelve new imine-based sulphonamide derivatives that inhibit Carbonic Anhydrase II (CA II), a crucial enzyme linked to a number of illnesses, including obesity, glaucoma, and cancer. Structure-activity relationships (SAR) and structural resemblance to well-known medications like acetazolamide, Celecoxib, Etoricoxib, and Parecoxib were used in the design of the compounds.The compounds were assessed for toxicity, pharmacokinetic characteristics, reactivity, and binding affinity using Molecular Docking, Density Functional Theory (DFT), and ADMET analysis. CA II was significantly bound by nine compounds (C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4). Compound P-1 showed the highest electrophilicity, indicating considerable reactivity, according to DFT analysis. For a number of compounds, good oral bioavailability was confirmed by drug-likeness and pharmacokinetic evaluations, and toxicity studies revealed that C-2, C-4, E-2, and E-4 were non-toxic and had favorable ADMET profiles. This investigation will support to validate further therapeutic potential of these designed compounds in vitro and in vivo.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHOMO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHighest occupied molecular orbital\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLUMO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLowest unoccupied molecular orbital\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDFT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDensity functional theory\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein data bank\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFMO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFrontier molecular orbital\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCA II\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbonic anhydrase II.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;The authors have no relevant financial or non-financial interests to disclose.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;The authors declare that no funds, grants, or other support were received during the Preparation of this manuscript.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;No human or animal subjects were involved in this study.\u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eINFORMED CONSENT\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026ldquo;Not applicable. This study does not involve human participants or animals. \u0026rdquo;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are deeply thankful to the management, and principal of Annasaheb Dange College of B.Pharmacy, Ashta for providing state of the art research facilities.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSupuran CT (2007) Carbonic anhydrases as drug targets\u0026ndash;an overview. Curr Top Med Chem 7:825\u0026ndash;833\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoth DE, Venta PJ, Tashian RE, Sly WS (1992) Molecular basis of human carbonic anhydrase II deficiency. 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Results Chem 7:101437. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.rechem.2024.101437\u003c/span\u003e\u003cspan address=\"10.1016/j.rechem.2024.101437\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB ZH a BQC, A MC (2021) Novel carbohydrate-based sulfonamide derivatives as selective carbonic anhydrase II inhibitors: Synthesis, biological and molecular docking analysis. Bioorg Med Chem Lett 51:128291. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bmcl.2021.128291\u003c/span\u003e\u003cspan address=\"10.1016/j.bmcl.2021.128291\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Annasaheb Dange College of B.Pharmacy Ashta","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carbonic anhydrase II, molecular docking, DFT, ADMET, imine-sulfonamide, cancer","lastPublishedDoi":"10.21203/rs.3.rs-8421792/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8421792/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCarbonic anhydrases (CAs) are zinc containing metalloenzymes distributed in human tissues, helps to regulate ion and pH cellular homeostasis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement of Problem\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDiscovery of carbonic anhydrase II (CA II) inhibitor is essential to minimize off-target effects and related complications including oxidative stress, cancer, glaucoma, and obesity. However, current therapeutic efficacy and safety profiles are still below ideal, which leads to more potent and specific CA II inhibitors discovery. In this work, new non-heterocyclic imine compounds with sulfonamide groups that were specially designed to inhibit CA II are designed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on structure-activity relationship (SAR) insights, twelve novel non-heterocyclic imine derivatives were designed by incorporating sulfonamide moieties and aromatic rings to enhance CA II binding affinity and inhibitory activity. Molegro Virtual Docker, SwissADME were utilized computational analysis. Furthermore, to evaluate molecular stability and reactivity, frontier molecular orbitals and chemical reactivity descriptors were analyzed using Density Functional Theory (DFT) computations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDocking scores demonstrated significant binding affinities for compounds C-1, C-2, C-4, E-1, E-2, E-4, P-1, P-2, and P-4 (scores ranging from − 63.58 to -73.23). ADMET analysis confirmed favorable drug-likeness, with compounds C-2, C-4, E-2, E-4, P-2, and P-4 exhibiting good oral bioavailability and minimal toxicity.DFT study supported the compounds' possible reactivity and binding efficiency by revealing favorable HOMO-LUMO energy gaps and high electrophilicity indices.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe designed imine sulphonamido derivatives showed promising potential as safe CA II inhibitors \u003cem\u003ein silico.\u003c/em\u003e These findings permit further \u003cem\u003ein vivo\u003c/em\u003e validation and toxicity studies. This work contributes in the advancement of targeted therapies for CA-related disorders.\u003c/p\u003e","manuscriptTitle":"Design, DFT, molecular Docking and ADMET evaluation of novel imine-sulphonamide analogues as carbonic anhydrase II inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-23 08:13:43","doi":"10.21203/rs.3.rs-8421792/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b5337672-84a0-4d00-9da9-a73e8f89dc96","owner":[],"postedDate":"December 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60045301,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2025-12-23T08:13:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-23 08:13:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8421792","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8421792","identity":"rs-8421792","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}