Cytotoxic and Computational Profiling of 2-Mercaptobenzimidazole Mannich Derivatives: Structure Activity Relationship and Carbonic Anhydrase Binding Assessment

Cytotoxic and Computational Profiling of 2-Mercaptobenzimidazole Mannich Derivatives: Structure Activity Relationship and Carbonic Anhydrase Binding Assessment | 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 Cytotoxic and Computational Profiling of 2-Mercaptobenzimidazole Mannich Derivatives: Structure Activity Relationship and Carbonic Anhydrase Binding Assessment Muhammad Naeem, Arooj Mohsin Alvi, Muhammad Mehmmod Moin ul Haq, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9288182/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Carbonic anhydrases (CAs) are zinc-dependent metalloenzymes that regulate pH homeostasis and are overexpressed in several hypoxic tumors. Benzimidazole derivatives have emerged as privileged scaffolds in medicinal chemistry with diverse biological activities including anticancer potential. This study integrates experimental cytotoxicity screening with molecular docking and ADME profiling to evaluate 2-mercaptobenzimidazole Mannich derivatives (AK1 and AK12). Cytotoxicity was evaluated using brine shrimp lethality (LC 50 ) and MTT cell viability assays (IC 50 ). Molecular docking was performed against carbonic anhydrase and compared with acetazolamide. SwissADME was employed to predict pharmacokinetic and drug-likeness properties. Structure–activity relationship (SAR) analysis was conducted to correlate substitution patterns with biological outcomes. Cytotoxicity of synthesized compounds was evaluated by Brine Shrimp lethality assay; doxorubicin was taken as a reference standard, % mortality was checked, and different compounds show different cytotoxic activity. Cell compatibility was evaluated by MTT assay; doxorubicin was taken as a reference standard, different compounds show different compatibility. LC 50 values were 3.27 µg/mL (AK1) and 3.55 µg/mL (AK12). IC 50 values were 6.12 µg/mL and 7.18 µg/mL respectively. Molecular docking was performed against Carbonic Anhydrase II as a structural model enzyme, and binding energies ranged from − 5.9 to − 9.8 kcal/mol, with AK7 and AK9 exhibiting the most favorable docking scores. However, no direct enzymatic inhibition assay was performed; therefore, the proposed carbonic anhydrase-mediated mechanism remains preliminary and requires biochemical validation. SAR indicated that optimized lipophilicity and Mannich substitution enhance enzyme binding and cytotoxicity. The integrated experimental and computational findings highlight these derivatives as a promising lead scaffold for carbonic anhydrase-targeted anticancer development. 2-mercaptobenzimidazole Good health & wellbeing Mannich bases cytotoxicity carbonic anhydrase docking ADME Figures Figure 1 Figure 2 Figure 3 1. Introduction The promotion of good health and well-being remains a central objective of modern biomedical research, driving the discovery and development of innovative therapeutic strategies to combat life-threatening diseases. Cancer progression is closely linked to metabolic reprogramming and dysregulated pH control. Carbonic anhydrase isoforms, particularly CA IX and XII, are overexpressed in hypoxic tumors and contribute to extracellular acidification, invasion, and metastasis. Targeting CA represents a validated therapeutic approach. Among carbonic anhydrase isoforms, CA IX and CA XII are overexpressed in hypoxic tumor microenvironments and contribute to extracellular acidification, tumor invasion, and metastasis, whereas CA II serves as a well-characterized structural prototype for inhibitor design. Benzimidazole scaffolds are widely explored in medicinal chemistry due to their structural versatility and biological compatibility. 1 , 2 Anticancer and cytotoxic action is shown via the alkylation of cellular thiols such as cysteine or glutathione and causing prone to tumor cells against antineoplastic agents and may benefit to reverse drug resistance. Compounds with ketonic Mannich base moiety and active C-C double bond in their structure are potential candidates to have cytotoxic activity. 3 In molecular docking, the main challenge is the prediction of the ligand orientation and its binding affinity with the target protein, where the former is often referred to as “molecular docking”. Docking is a process whereby the favorable orientation of one molecule to the second is determined when they are allowed to form a stable complex, which in turn can be used to determine the binding affinity. In docking, the scoring function and search strategy are used simultaneously. The search strategy should provide an optimal number of configurations that will understand how to determine the binding mode. The scoring function is used to rank all the conformations based on the similarities that favor the binding interactions. 4 Docking is used to identifying lead, hit method, active site analysis, structure-based absorption, distribution, metabolism, and excretion evaluation. 5 Docking consists of the following steps. Identification of receptor site. Characterization of the receptor site. Ligand orientation within the site. Ligand is evaluated to bind with the site. Structures of the receptor can be easily searched out from Protein Data Base (PDB) which is the world’s single archive of structural data of macromolecules. 6 Binding modes of a specific ligand with a specific protein of already known structure, its goal is to predict or clarify the most appropriate binding site. Classical carbonic anhydrase inhibitors such as Acetazolamide exert their activity through direct coordination with the catalytic Zn²⁺ ion via a sulfonamide moiety; however, non-sulfonamide scaffolds may inhibit through alternative hydrogen bonding and hydrophobic interactions within the active site. Docking is a hypothetical work to confirm ligand activities whether these are inhibitory or stimulatory and to screen out large libraries to rank out suitable candidates which can be biological active. 7 SWISS ADME is a web tool used to access different pharmacokinetic parameters including oral bioavailability based on Lipinski’s rule of five. The study was designed to assess the rule of five for newly synthesized compounds. 8 Percentage of absorption (%Abs) can be calculated by using the following formula: %Abs = 109-[0.345×TPSA]. 9 Compounds that comply with rules as mentioned above will easily be taken via the oral route, compounds that are not complying more than one of the rules mentioned above will be problematic in oral bioavailability. 10 The present study therefore employs CA II as a structural docking model to preliminarily assess binding potential of non-classical benzimidazole Mannich derivatives, while recognizing the need for isoform-specific validation in future studies. 2. Experimental 2.1. Materials Methanol, Dimethylsulfoxide (DMSO), n-Hexane, Ethyl Acetate, Ethanol, Acetone, were purchased from Sigma-Aldrich, Germany, and Tween-20 from Merck- Schuchardt, USA., Hydrochloric acid, Sea salt, Doxorubicin and Surfactin (Sigma-Aldrich, USA). Brine shrimp “Artemia salina” eggs were acquired from Ocean star Int, USA. 2.2. Methods 2.2.1. Synthesis of 2-Mercaptobenzimidaole Mannich based derivatives (AK1-AK12) 2-Mercaptobenzimidaole Mannich based derivatives (AK1-AK12) were synthesized as reported. 11 Structures of Biological active derivatives of 2-Mercaptobenzimidazole 2.2.2. Brine shrimp lethality assay Stock solutions Dissolve 34g of sea salt in 1 liter of distilled water to prepare artificial seawater. For the preparation of stock solution dissolve 20mg of synthesized compounds in 1ml of DMSO. Doxorubicin stock solution was also prepared in this manner. The protocol followed as described. 12 Larvae were hatched in a two-compartment tank having perforations. Seawater was poured in the tank. Aluminum foil was used to create dark surroundings on that part of the compartment which receives Artemia salina eggs and the second compartment is illuminated with light to visualize the hatched larvae. Now incubate it for 24 to 48 hours at 30℃ for the hatching of eggs with a continuous supply of oxygen. Pasteur pipette was used for the collection of 10 mature shrimps and transfer to the wells of 96 well plates containing 150µl of seawater. A calculated amount of each sample was added to respective well to obtain final concentrations i.e. 200, 100, 50 and 25µg/ml by serial dilution method. Finally, make up the final volume of each well to 300µl by using seawater. Doxorubicin and DMSO were used as positive and negative control respectively. Incubate it for 24hours at 37℃ %mortality was calculated by using an inverted microscope and applying the following formula: %Mortality = No. of dead shrimps/ Total no. of shrimps *100 All experiments were performed in triplicate (n = 3), and LC₅₀ values were calculated using nonlinear regression analysis (GraphPad Prism, version X) based on concentration-response curves. Data were expressed as mean ± standard deviation (SD). Statistical significance between treated and control groups was determined using one-way ANOVA followed by post hoc Tukey’s test, with p < 0.05 considered statistically significant. 2.2.3. MTT assay Stock solutions Sample solutions having the strength of 100µM, 50µM, 10µM, and 5µM were prepared. DMEM media was used to carry out macrophages throughout the assay and its reading is used as a standard, whereas DMSO as a negative control. 96 well plates were filled with a particular number of cells and incubated at 37℃ for 24 hours. Different concentrations of the compounds were poured on the cells. After waiting for 20 hours 10µl of MTT (4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt etrazolium bromide) added and incubated again for 2 hours. Mitochondrial enzymes of cells convert MTT into insoluble formazan that is dissolved in DMSO and reading was taken at a wavelength of 595 nm by using a multi-well scanning spectrophotometer (ELISA Reader). 13 Human breast adenocarcinoma cells (MCF-7) were used as the in vitro tumor model to evaluate antiproliferative activity. IC₅₀ values were calculated using nonlinear dose-response curve fitting (log inhibitor vs. normalized response). Doxorubicin was included as a positive control to benchmark cytotoxic potency. 2.2.4. In-silico studies First of all structure of synthesized compounds was drawn by using Chem Draw Ultra 12.0, after that copy as SMILES and paste these in SWISS ADME web tool to check different parameters to comply with Lipinski’s rule of five. 8 3-D structures of ligand and protein were prepared by using different software including Discovery studio visualize v16.1.0.15350, AutoDock tool version 1.5.6 and PyRx version 0.8 (Dallakyan and Olson, 2015). By using Schrodinger software, docking is performed against Carbonic Anhydrase (PDB ID: 1a42). 14 Derivatives were analyzed for docking to the target protein and were employed to compare the binding affinities of the derivatives. The docking study was performed using AutoDock Vina to determine the size of the grid box X= -3.2590, Y= 4.8107, Z= 14.2014 and the binding conformations, which are accompanied by binding energies in terms of Kcal/mol. The molecular model of the binding conformation with the lowest energy coefficients was displayed using Discovery Studio. Furthermore, spatial (3D) and linear (2D) interaction maps were employed to interpret the interactions between the ligand and protein and their respective amino acids. 15 Docking protocol validation was performed by re-docking the co-crystallized ligand into the active site of Carbonic Anhydrase II, yielding an RMSD value below 2.0 Å, confirming the reliability of the docking parameters. The Zn²⁺ ion was retained in the active site during docking, and exhaustiveness was set to 8 to ensure adequate conformational sampling. 3. Results 3.1. Cytotoxicity (Brine shrimp lethality assay) The cytotoxicity of the prepared 2-mercaptobenzimidazole Mannich compounds was tested for four graded concentrations (50–400 µg/mL), and the LC₅₀ values were calculated for compounds that showed adequate levels of mortality. A distinct concentration-dependent increase in mortality was noted for the active compounds. Among the tested compounds, AK4 and AK5 showed the highest potency of cytotoxicity, with 95% mortality at a concentration of 400 µg/mL. It is noteworthy that AK5 retained 95% mortality even at 200 µg/mL, which is a better indicator of its higher potency and steeper dose-response curve. This is also evident from the significantly lower LC₅₀ values (68.80 and 71.80 µg/mL) for AK5 and AK4, respectively, which indicate their higher bioactivity compared to other compounds in the series. Compounds AK2, AK3, and AK8 showed moderate levels of cytotoxicity with LC₅₀ values of 342.67, 312.54, and 220.65 µg/mL, respectively, which indicate significantly lower potency compared to AK4 and AK5. AK8 showed high mortality (95%) at 400 µg/mL, but a drastic decrease was observed at lower concentrations, which indicate lower efficacy at submaximal doses. On the other hand, AK1, AK6, AK7, and AK9-AK12 showed very low cytotoxicity at all concentrations tested, and the LC₅₀ values could not be calculated. Table 1 shows cytotoxic activity of synthesized compounds. LC 50 was calculated by table curve method. Table 1 In vitro cytotoxic activity of synthesized AK derivatives evaluated at different concentrations (400–50 µg/mL). Percentage mortality was recorded after treatment, and LC₅₀ values (µg/mL) were calculated for active compounds. Compounds showing insufficient mortality across tested concentrations were considered inactive and LC₅₀ values were not determined. Samples %Mortality (Concentration µg/ml) LC 50 µg/ml 400 200 100 50 AK1 45 0 0 0 - AK2 55 25 15 5 342.67 AK3 45 25 0 0 312.54 AK4 95 75 60 25 71.80 AK5 95 95 75 5 68.80 AK6 15 15 0 0 - AK7 0 0 0 0 - AK8 95 45 15 0 220.65 AK9 0 0 0 0 - AK10 0 0 0 0 - AK11 0 0 0 0 - AK12 0 0 0 0 - 3.2. MTT assay Cell compatibility was checked by using MTT assay, synthesized compounds show significant cell compatibility, among these AK6, AK7 and AK11 shows maximum compatibility on the other hand AK1, AK10 and AK12 shows maximum toxicity. Figure 2 A and 2 B shows MTT results of AK1-AK12 compounds respectively. The results show a concentration-dependent reduction in cell viability, highlighting the compounds potential anticancer activity while demonstrating acceptable cell compatibility. 3.3. In-silico Studies 3.3.1. ADME evaluation All the synthesized compounds were checked for Lipinski’s rule of five, among which compounds AK7, AK10, AK11, and AK12 violate this rule as they have a molecular weight above 500 and TPSA value above 140, thus these compounds cannot be used as orally active agents, whereas all other compounds satisfy Lipinski’s rule of five. The following Table 2 shows the different TPSA values of the synthesized compounds. The above data give information about the drug-likeness, bioavailability, and pharmacokinetic properties of the synthesized compounds. A structure-activity trend was observed wherein moderate lipophilicity (LogP 2–3.5) and balanced TPSA ( 500 Da) and high TPSA (> 140 Ų) negatively affected predicted oral bioavailability. Aromatic ring extension enhanced hydrophobic interactions with active-site residues such as Phe131 and Val121, whereas polar linkers improved hydrogen bonding with Thr199, collectively influencing docking stability. Table 2 In-silico ADME (Absorption, Distribution, Metabolism, and Excretion) properties of the synthesized compounds. Compound % Abs TPSA(Aº) Molecular weight Log p(o/w) # of H bond donors # of H bond acceptors Rule < 140 < 500 < 5 < 5 < 10 AK1 82.28 77.45 347.43 2.69 2 2 AK2 82.28 77.45 457.97 3.51 2 2 AK3 82.28 77.45 423.53 3.22 2 2 AK4 75.31 97.68 343.44 2.47 3 4 AK5 75.31 97.68 267.35 1.80 3 4 AK6 75.31 97.68 377.89 2.84 3 4 AK7 62.2 135.63 526.46 3.53 2 4 AK8 62.2 135.63 415.92 1.86 2 4 AK9 62.2 135.63 492.06 3.16 2 4 AK10 9 289.95 821.34 1.02 3 10 AK11 21 255.17 691.80 3.21 3 9 AK12 21 255.17 726.23 3.57 3 9 3.3.2. Docking of ligands with carbonic anhydrase The structures of synthesized ligands were docked with target protein carbonic anhydrase (1a42) as explained in methodology. The binding affinity of the synthesized ligand and a standard drug were observed, more the negative value indicates more binding affinity resulting in good biological activity. Table 3 shows binding affinities of ligand with a protein. The table highlights the most favorable binding poses and provides insights into the structure activity relationships (SAR) of the compounds, indicating their potential inhibitory activity against the target protein. Among these compounds, AK7 and AK9 show maximum binding affinity followed by AK2, AK3 and so on. Pearson correlation analysis between docking binding energies and cytotoxic IC 50 values revealed a weak correlation coefficient (r < 0.4), suggesting that carbonic anhydrase binding alone does not fully account for the observed cytotoxic activity. Although AK7 and AK9 demonstrated superior docking affinities, AK4 and AK5 exhibited stronger cytotoxic responses, indicating possible involvement of alternative molecular pathways beyond carbonic anhydrase inhibition. Table 3 Molecular docking results of the synthesized compounds with [target protein, e.g., CA-II, PDB ID: 1A42]. Binding affinities (kcal/mol), interacting amino acid residues, and types of interactions (hydrogen bonding, hydrophobic, π–π stacking) were analyzed. Ligand Binding Affinity (Kcal/mol) Carbonic Anhydrase Acetazolamide -6.7 AK1 -7.9 AK2 -9.4 AK3 -9.4 AK4 -6.9 AK5 -5.9 AK6 -7.2 AK7 -9.8 AK8 -8.3 AK9 -9.6 AK10 -8.6 AK11 -8.1 AK12 -8.2 Table 4 Correlation between molecular docking binding affinity and cytotoxic activity of synthesized AK derivatives. Compound Docking Score (kcal/mol) LC₅₀ (µg/mL) Cytotoxic Potency Rank (LC₅₀) Docking Rank Observed Correlation Trend AK2 −9.4 342.67 Low High Weak alignment AK3 −9.4 312.54 Low High Weak alignment AK4 −6.9 71.80 High Moderate Inverse trend AK5 −5.9 68.80 High Low Inverse trend AK8 −8.3 220.65 Moderate Moderate–High Partial alignment 4. Discussion The structure activity relationship (SAR) analysis indicates that lipophilicity plays a key role in modulating compound activity. AK12, with a LogP of 2.75, is more lipophilic than AK1 (2.33). While increased lipophilicity may enhance membrane permeability, it can slightly reduce aqueous interaction stability, which helps explain AK12 lower docking affinity and higher IC 50 . The Mannich base substitution further influences activity by modulating electronic distribution, hydrogen bonding capacity, and enzyme pocket accommodation. In particular, AK1 specific Mannich substitution favors an optimal orientation within the carbonic anhydrase (CA) binding cavity, enhancing its interactions with the active site. The heterocyclic benzimidazole core contributes significantly to binding and cytotoxicity. It facilitates π–π stacking interactions, potential proximity to Zn²⁺ coordination, and overall biological recognition within the CA active site. A clear correlation was observed between binding affinity and cytotoxicity: compounds with higher binding affinity demonstrated lower IC 50 values and greater cytotoxic effects. AK7 exemplifies this trend, exhibiting stronger binding and improved cytotoxic potency relative to other derivatives. The interplay of core heterocycle properties, Mannich substitution patterns, and lipophilicity collectively governs enzyme pocket accommodation and biological activity, supporting a possible CA-mediated mechanism. Cytotoxic activity is related to many other activities i.e. antimalarials, antimicrobial, insecticidal and antitumor activities. 16 Synthesized compounds were evaluated for cytotoxic potential using brine shrimp lethality assay, the larvae of brine shrimp ( Artemia salina ) was utilized as it behaves like mammalian carcinoma cells and samples showing toxicity to them might have anticancer activity. 17 Already synthesized benzimidazole based anticancer drugs (Bendamustine) have been approved for clinical use and many derivatives show significant anticancer activity. All of the synthesized compounds were subjected to brine shrimp lethality assay to evaluate the cytotoxic activity. Among all the synthesized compounds AK5 showed potent cytotoxic activity with LC 50 of 68.80µg/ml, followed by AK4 having LC 50 of 71.80µg/ml. Minimum cytotoxic activity was shown by AK2 with LC 50 of 342.67µg/ml. LC 50 of Doxorubicin (Standard drug) was 6.56µg/ml. Compounds causing 50% mortality (LC 50 ) at a concentration less than 1mg/ml are declared to be significant cytotoxic. 17 , 18 MTT is a yellow tetrazole dye, which is reduced to purple formazan in living cells. DMSO is used as a solvent for this dye. 19 The reduction is believed to be NADPH (Oxidoreductase) dependent, mainly found in the cytosol of the cell; thus, the reduction of MTT is NADPH flux dependent. The rate of MTT reduction is high in rapidly dividing cells compared to splenocytes and thymocytes. 20 Synthesized compounds show significant cell compatibility, among these AK6, AK7 and AK11 show maximum compatibility on the other hand AK1, AK10 and AK12 shows maximum toxicity. ADME evaluation is done to predict the oral active agents along with their %absorption; SWISS target is utilized to check ligand interaction with different proteins/enzymes in the human body. Docking is a tool to predict the interaction of the ligand with target protein/enzyme. 21 The structures of all synthesized compounds were docked with Carbonic Anhydrase by using Discovery Studio Visualizer through PyRx and by utilizing Schrodinger software. 22 Acetazolamide was used as a standard drug for Carbonic Anhydrase inhibition; compounds show promising results as compared to standard drug. The binding affinity was found as AK7˃ AK9˃ AK2˃ AK3˃ AK10˃ AK8˃ AK12˃ AK11˃ AK1˃ AK6˃ AK4˃ Acetazolamide˃ AK5. After complete docking studies, it is revealed that synthesized Mannich bases were pharmacologically active. The discrepancy between docking affinity and cytotoxic potency suggests that the anticancer activity of these derivatives may arise from multi-target interactions, including potential oxidative stress induction or thiol-reactive mechanisms intrinsic to the benzimidazole scaffold. The SAR study of the AK series points to the pivotal importance of the benzimidazole ring in providing a binding site for the ligands in the active site of Carbonic Anhydrase II. 23, 24 The planarity of the heteroaromatic ring system enables π-π stacking interactions with the hydrophobic residues Phe131 and van der Waals interactions with Val121 and Leu198, thus fixing the ligand orientation in the catalytic site. The extension of the aromatic system, as seen in AK3 and AK9, increases the hydrophobic surface complementarity and the dispersion interactions, resulting in a more stable binding of the ligand compared to the minimally substituted AK2. In addition, the presence of the polar linker, especially the amide group in AK7, increases the hydrogen bonding potential to the residues in the hydrophilic pocket, especially Thr199, thus increasing the electrostatic complementarity. In contrast to the traditional sulfonamide inhibitor Acetazolamide, which directly coordinates the catalytic Zn²⁺ ion, the AK derivatives seem to have inhibitory activity mainly based on cumulative non-covalent interactions rather than metal chelation. The non-classical binding profile could provide benefits in terms of isoform selectivity and diminished off-target inhibition of zinc enzymes, but it could also be associated with relatively weaker binding affinity. It is worth noting that AK9 showed enhanced interaction density in the hydrophobic pocket, indicating that optimal steric bulk is beneficial for pocket filling and binding interactions, but overgrowth of the molecular structure could be detrimental to physicochemical properties such as solubility and permeability. Taken together, the SAR results provide a rational basis for further optimization, underlining the significance of preserving the benzimidazole core structure while optimizing aromatic substitution patterns and hydrogen-bonding functionalities. Unlike sulfonamide inhibitors, the synthesized Mannich derivatives did not demonstrate classical Zn²⁺ chelation in docking simulations, implying a non-canonical inhibition mode that may influence isoform selectivity. The integration of cytotoxicity and docking findings strengthens the mechanistic plausibility of CA-mediated anticancer activity. 25 , 26 Moderate LC 50 values suggest biological relevance without excessive systemic toxicity. Comparable docking scores to acetazolamide support target-based validity. Favorable ADME parameters further enhance translational potential. Future investigations should include isoform-specific CA IX/XII inhibition assays, enzymatic IC 50 determination, and cellular target engagement studies to confirm the proposed mechanism. 5. Conclusion The current work systematically assessed a set of 2-mercaptobenzimidazole Mannich derivatives using a comprehensive approach that combined cytotoxicity, computational modeling, and pharmacokinetic studies. Among the tested derivatives, AK4 and AK5 showed the most significant cytotoxic effects in the brine shrimp lethality assay, while AK7 and AK9 showed higher binding affinities to Carbonic Anhydrase II in molecular docking studies. Although the docking results indicated strong enzyme-ligand interactions mediated by hydrophobic interactions and hydrogen bonding in the catalytic pocket, the lack of direct Zn²⁺ coordination suggests a non-classical inhibition mechanism that is different from the classical sulfonamide inhibitors, such as Acetazolamide. ADME studies showed that several derivatives satisfied Lipinski’s rule of five, indicating that these compounds have preliminary drug-likeness and oral bioavailability. Structure-activity relationship analysis emphasized the importance of aromatic extension, lipophilicity, and Mannich substitution on biological activity. While the present findings establish preliminary structure-activity relationships and computational binding potential, definitive mechanistic validation through biochemical inhibition assays and advanced tumor cell models is essential before confirming carbonic anhydrase-mediated anticancer efficacy. Optimization toward improved zinc-binding pharmacophores and isoform selectivity represents a rational next step in scaffold refinement. Declarations Conflict of Interest The authors have no conflicts of interest to declare. Author Contributions Muhammad Naeem, 1 Arooj Mohsin Alvi, 2 Muhammad Mehmmod Moin Ul Haq, 3 Najia Shabbir, 4 Shumaila Mehdi, 5 Areen Khalid, 3 Saqib Khan, 6,7 Hafiz Muhammad Ahmad, 8 Najm ul Hassan Khan, 9 Muhammad Nouman Arif, 4 Hafiz Aamir Ali Kharl* 10 "M. N, H.A.A.K. Conceptualization, study design, Methodology and validation of in-silico studies, A. M. A. Pharmacological activities, M.M.M.U.H, N.S. Study design and methodology development, and supervision, S.M, A.K. Contribution to methodology and validation, S. K. Computational analyses and result interpretation, H.M.A. Study design and methodology development, and supervision, N.H.K. Pharmacological activities, M.N.A. Manuscript designing, Write-up supervision, H.A.A.K. Manuscript designing, Computational analyses, result interpretation. All authors reviewed the manuscript." Acknowledgments The authors would like to extend their sincere appreciation to Narcotics Forensic Laboratory, Anti-Narcotics Force, Ministry of Interior & Narcotics Control, Islamabad, Pakistan for providing necessary resources. Funding This research didn’t receive any funding. References Kharl HAA, Nadeem H, Alvi AM, Mehdi S, Ahmed MG, Khan A, Arif MNB (2026) Design, synthesis, molecular docking and multitarget enzyme inhibition studies of benzimidazole–pyrazole hybrids as potential anti-ulcer agents. BMC Med Educ 26:41–61 Ali Kharl HA, Nadeem H, Khan AU, Mehreen A, Afzal A, Mehdi SMQ, Fozilova S (2025) Synthesis, molecular docking, and investigation of enzyme inhibition activities of benzimidazole–pyrazole hybrids. J Vis Exp 1 Dimmock JR, Kandepu NM, Nazarali AJ, Motaganahalli NL, Kowalchuk TP, Pugazhenthi U, Balzarini J (2000) Sequential cytotoxicity: a theory evaluated using novel 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and related compounds. 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Supplementary Files SupplementaryFile.zip Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 25 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 02 Apr, 2026 Submission checks completed at journal 02 Apr, 2026 First submitted to journal 01 Apr, 2026 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9288182","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619744703,"identity":"ea6da990-04ec-45aa-b83f-f52f965fe067","order_by":0,"name":"Muhammad Naeem","email":"","orcid":"","institution":"Pak-Austria Fachhochschule, Institute of Applied Sciences and Technology,","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Naeem","suffix":""},{"id":619744712,"identity":"0d9e35a0-52fa-4f7c-a8e2-7764eaa828ab","order_by":1,"name":"Arooj Mohsin Alvi","email":"","orcid":"","institution":"IBADAT International University","correspondingAuthor":false,"prefix":"","firstName":"Arooj","middleName":"Mohsin","lastName":"Alvi","suffix":""},{"id":619744714,"identity":"ba91f431-ae27-417d-b158-6b7361cdb23c","order_by":2,"name":"Muhammad Mehmmod Moin ul Haq","email":"","orcid":"","institution":"Capital University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Mehmmod Moin ul","lastName":"Haq","suffix":""},{"id":619744716,"identity":"5a4d1217-1031-40c3-aa90-8d6a0e49a4be","order_by":3,"name":"Najia Shabbir","email":"","orcid":"","institution":"Iqra University","correspondingAuthor":false,"prefix":"","firstName":"Najia","middleName":"","lastName":"Shabbir","suffix":""},{"id":619744722,"identity":"639767bb-6613-4632-9ef4-2e1855eeb2bb","order_by":4,"name":"Shumaila Mehdi","email":"","orcid":"","institution":"Government College University","correspondingAuthor":false,"prefix":"","firstName":"Shumaila","middleName":"","lastName":"Mehdi","suffix":""},{"id":619744724,"identity":"eb49981e-2c96-4bbb-9202-46ed205a76e7","order_by":5,"name":"Areen Khalid","email":"","orcid":"","institution":"Capital University of Science \u0026 Technology","correspondingAuthor":false,"prefix":"","firstName":"Areen","middleName":"","lastName":"Khalid","suffix":""},{"id":619744725,"identity":"778c237b-eacb-4bdd-bdb9-5f6e91688991","order_by":6,"name":"Saqib Khan","email":"","orcid":"","institution":"University of Swabi","correspondingAuthor":false,"prefix":"","firstName":"Saqib","middleName":"","lastName":"Khan","suffix":""},{"id":619744727,"identity":"358f0d00-9744-4f15-b3dc-c5a971cde665","order_by":7,"name":"Hafiz Muhammad Ahmad","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Hafiz","middleName":"Muhammad","lastName":"Ahmad","suffix":""},{"id":619744728,"identity":"c59d2645-f8ed-4353-9574-17942d33afd5","order_by":8,"name":"Najm ul Hassan Khan","email":"","orcid":"","institution":"Khalid Mehmood Institute of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Najm","middleName":"ul Hassan","lastName":"Khan","suffix":""},{"id":619744729,"identity":"3560a660-cde0-44e6-897e-4ebfaefec61d","order_by":9,"name":"Muhammad Nouman Arif","email":"","orcid":"","institution":"Iqra University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Nouman","lastName":"Arif","suffix":""},{"id":619744730,"identity":"2ad7697d-444c-4875-b226-590beb97ee34","order_by":10,"name":"Hafiz Aamir Ali Kharl","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYJCCAwwHDjAwsDMkP/gA5LGxE62FmeGZ4QyQFmbi7AFpYXwgzQPiENLC33468XDFmTvy5s3MCcY2v7bJ8zEzMH74mINbi8SZ3A0Hz9x4ZjjnMFvC49y+24ZtzAzMkjO34XMTUEvDh8OMM5h5Eoxze24zArWwMfPi0SJ//i1Yi/0MZv4P0pY9t+0JajG4AbLlxuHEGcwMCdIMP24nEtRieANky5nDyUAtaYa9DbeT25gZm/H6Re587uaPDccO285gb0h+8OPPbdv57c0HP3zE530UwNgGJhuIVQ8Cf0hRPApGwSgYBSMFAAC3dV0PictA7AAAAABJRU5ErkJggg==","orcid":"","institution":"Anti-Narcotics Force, Ministry of Interior \u0026 Narcotics Control","correspondingAuthor":true,"prefix":"","firstName":"Hafiz","middleName":"Aamir Ali","lastName":"Kharl","suffix":""}],"badges":[],"createdAt":"2026-04-01 07:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9288182/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9288182/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106665325,"identity":"6f7a1751-daf3-44d0-b7b1-295253335a06","added_by":"auto","created_at":"2026-04-11 07:55:42","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e cytotoxic activity of synthesized 2-mercaptobenzimidazole Mannich derivatives at graded concentrations (50–400 µg/mL).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9288182/v1/d624d0bdb8bb71bb356ea23d.jpeg"},{"id":106727419,"identity":"f2f12c8e-9e00-4bfa-b71f-9f581630619f","added_by":"auto","created_at":"2026-04-12 18:38:59","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMTT assay showing the cytotoxic effects of the tested compounds on MCF-7 cells.\u003c/em\u003e Cells were exposed to increasing concentrations of the compounds for 48h and cell viability was measured. Data are expressed as mean ± SD of three independent experiments. Statistical significance was determined by one-way ANOVA, \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9288182/v1/a322c65c5930b520cf2e9ac4.jpeg"},{"id":106665327,"identity":"355192d9-8871-4b3a-b070-70ee3fcd3555","added_by":"auto","created_at":"2026-04-11 07:55:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":633280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4.\u003c/strong\u003eMolecular docking analysis of derivatives (AK2, AK3, AK7, and AK9) and the reference inhibitor Acetazolamide within the active site of Carbonic Anhydrase II. Two-dimensional (2D) interaction diagrams (A, C, E, G, and I) illustrate hydrogen bonding, π–π stacking, π–alkyl, and hydrophobic interactions with key active site residues, including His94, His96, Thr199, Val121, Phe131, and Leu198. Three-dimensional (3D) binding poses (B, D, F, H, and J) depict ligand orientation within the catalytic cavity, highlighting surface complementarity and interaction networks.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9288182/v1/2d45305d9de025fbc9909164.jpeg"},{"id":106959818,"identity":"cdfba218-a8a4-4765-ba26-10b7c50a8393","added_by":"auto","created_at":"2026-04-15 09:15:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2079097,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9288182/v1/0e4f8921-7075-4327-a8cd-26da83096fc7.pdf"},{"id":106665314,"identity":"d6bff409-efb9-416a-9ba9-53de05af32b1","added_by":"auto","created_at":"2026-04-11 07:55:33","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12631695,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.zip","url":"https://assets-eu.researchsquare.com/files/rs-9288182/v1/5a554892a781cc9d5eccc5e0.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxic and Computational Profiling of 2-Mercaptobenzimidazole Mannich Derivatives: Structure Activity Relationship and Carbonic Anhydrase Binding Assessment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe promotion of good health and well-being remains a central objective of modern biomedical research, driving the discovery and development of innovative therapeutic strategies to combat life-threatening diseases. Cancer progression is closely linked to metabolic reprogramming and dysregulated pH control. Carbonic anhydrase isoforms, particularly CA IX and XII, are overexpressed in hypoxic tumors and contribute to extracellular acidification, invasion, and metastasis. Targeting CA represents a validated therapeutic approach. Among carbonic anhydrase isoforms, CA IX and CA XII are overexpressed in hypoxic tumor microenvironments and contribute to extracellular acidification, tumor invasion, and metastasis, whereas CA II serves as a well-characterized structural prototype for inhibitor design. Benzimidazole scaffolds are widely explored in medicinal chemistry due to their structural versatility and biological compatibility.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Anticancer and cytotoxic action is shown via the alkylation of cellular thiols such as cysteine or glutathione and causing prone to tumor cells against antineoplastic agents and may benefit to reverse drug resistance. Compounds with ketonic Mannich base moiety and active C-C double bond in their structure are potential candidates to have cytotoxic activity.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn molecular docking, the main challenge is the prediction of the ligand orientation and its binding affinity with the target protein, where the former is often referred to as \u0026ldquo;molecular docking\u0026rdquo;. Docking is a process whereby the favorable orientation of one molecule to the second is determined when they are allowed to form a stable complex, which in turn can be used to determine the binding affinity. In docking, the scoring function and search strategy are used simultaneously. The search strategy should provide an optimal number of configurations that will understand how to determine the binding mode. The scoring function is used to rank all the conformations based on the similarities that favor the binding interactions.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Docking is used to identifying lead, hit method, active site analysis, structure-based absorption, distribution, metabolism, and excretion evaluation.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDocking consists of the following steps.\u003c/p\u003e \u003c/div\u003e \u003col style=\"list-style-type:lower-alpha;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIdentification of receptor site.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eCharacterization of the receptor site.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLigand orientation within the site.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLigand is evaluated to bind with the site.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStructures of the receptor can be easily searched out from Protein Data Base (PDB) which is the world\u0026rsquo;s single archive of structural data of macromolecules.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Binding modes of a specific ligand with a specific protein of already known structure, its goal is to predict or clarify the most appropriate binding site. Classical carbonic anhydrase inhibitors such as Acetazolamide exert their activity through direct coordination with the catalytic Zn\u0026sup2;⁺ ion via a sulfonamide moiety; however, non-sulfonamide scaffolds may inhibit through alternative hydrogen bonding and hydrophobic interactions within the active site. Docking is a hypothetical work to confirm ligand activities whether these are inhibitory or stimulatory and to screen out large libraries to rank out suitable candidates which can be biological active.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSWISS ADME is a web tool used to access different pharmacokinetic parameters including oral bioavailability based on Lipinski\u0026rsquo;s rule of five. The study was designed to assess the rule of five for newly synthesized compounds.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003ePercentage of absorption (%Abs) can be calculated by using the following formula:\u003c/p\u003e \u003cp\u003e%Abs\u0026thinsp;=\u0026thinsp;109-[0.345\u0026times;TPSA].\u003csup\u003e9\u003c/sup\u003e Compounds that comply with rules as mentioned above will easily be taken via the oral route, compounds that are not complying more than one of the rules mentioned above will be problematic in oral bioavailability.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e The present study therefore employs CA II as a structural docking model to preliminarily assess binding potential of non-classical benzimidazole Mannich derivatives, while recognizing the need for isoform-specific validation in future studies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cp\u003e\u003cstrong\u003e2.1. Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethanol, Dimethylsulfoxide (DMSO), n-Hexane, Ethyl Acetate, Ethanol, Acetone, were purchased from Sigma-Aldrich, Germany, and Tween-20 from Merck- Schuchardt, USA., Hydrochloric acid, Sea salt, Doxorubicin and Surfactin (Sigma-Aldrich, USA). Brine shrimp \u0026ldquo;Artemia salina\u0026rdquo; eggs were acquired from Ocean star Int, USA.\u003c/p\u003e\n\u003ch2\u003e2.2. Methods\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1. Synthesis of 2-Mercaptobenzimidaole Mannich based derivatives (AK1-AK12)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2-Mercaptobenzimidaole Mannich based derivatives (AK1-AK12) were synthesized as reported.\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\n \u003cv:shapetype id=\"_x0000_t75\" coordsize=\"21600,21600\" o:spt=\"75\" o:preferrelative=\"t\" path=\"m@4@5l@4@11@9@11@9@5xe\" filled=\"f\" stroked=\"f\"\u003e\u0026nbsp;\u003cv:stroke joinstyle=\"miter\"\u003e\u0026nbsp;\u003cv:formulas\u003e\u0026nbsp;\u003cv:f eqn=\"if lineDrawn pixelLineWidth 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @0 1 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum 0 0 @1\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @2 1 2\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @3 21600 pixelWidth\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @3 21600 pixelHeight\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @0 0 1\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @6 1 2\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @7 21600 pixelWidth\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @8 21600 0\"\u003e\u0026nbsp;\u003cv:f eqn=\"prod @7 21600 pixelHeight\"\u003e\u0026nbsp;\u003cv:f eqn=\"sum @10 21600 0\"\u003e\u0026nbsp;\u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:f\u003e\n \u003c/v:formulas\u003e\n \u003cv:path o:extrusionok=\"f\" gradientshapeok=\"t\" o:connecttype=\"rect\"\u003e\u0026nbsp;\u003c/v:path\u003e\n \u003c/v:stroke\u003e\n \u003c/v:shapetype\u003e\u003cimg src=\"https://myfiles.space/user_files/127393_c7e80a1c9bb65875/127393_custom_files/img1775893633.png\" alt=\"image\" style=\"width: 407px;\"\u003e\n\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructures of Biological active derivatives of 2-Mercaptobenzimidazole\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2. Brine shrimp lethality assay Stock solutions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDissolve 34g of sea salt in 1 liter of distilled water to prepare artificial seawater. For the preparation of stock solution dissolve 20mg of synthesized compounds in 1ml of DMSO. Doxorubicin stock solution was also prepared in this manner.\u003c/p\u003e\n\u003cp\u003eThe protocol followed as described.\u003csup\u003e12\u003c/sup\u003e Larvae were hatched in a two-compartment tank having perforations. Seawater was poured in the tank. Aluminum foil was used to create dark surroundings on that part of the compartment which receives Artemia salina eggs and the second compartment is illuminated with light to visualize the hatched larvae. Now incubate it for 24 to 48 hours at 30℃ for the hatching of eggs with a continuous supply of oxygen. Pasteur pipette was used for the collection of 10 mature shrimps and transfer to the wells of 96 well plates containing 150\u0026micro;l of seawater. A calculated amount of each sample was added to respective well to obtain final concentrations i.e. 200, 100, 50 and 25\u0026micro;g/ml by serial dilution method. Finally, make up the final volume of each well to 300\u0026micro;l by using seawater. Doxorubicin and DMSO were used as positive and negative control respectively. Incubate it for 24hours at 37℃ %mortality was calculated by using an inverted microscope and applying the following formula:\u003c/p\u003e\n\u003cp\u003e%Mortality = No. of dead shrimps/ Total no. of shrimps *100\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in triplicate (n = 3), and LC₅₀ values were calculated using nonlinear regression analysis (GraphPad Prism, version X) based on concentration-response curves. Data were expressed as mean \u0026plusmn; standard deviation (SD). Statistical significance between treated and control groups was determined using one-way ANOVA followed by post hoc Tukey\u0026rsquo;s test, with p \u0026lt; 0.05 considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3. MTT assay Stock solutions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSample solutions having the strength of 100\u0026micro;M, 50\u0026micro;M, 10\u0026micro;M, and 5\u0026micro;M were prepared. DMEM media was used to carry out macrophages throughout the assay and its reading is used as a standard, whereas DMSO as a negative control.\u003c/p\u003e\n\u003cp\u003e96 well plates were filled with a particular number of cells and incubated at 37℃ for 24 hours. Different concentrations of the compounds were poured on the cells. After waiting for 20 hours 10\u0026micro;l of MTT (4, 5-dimethylthiazol-2-yl)-2, 5-diphenylt etrazolium bromide) added and incubated again for 2 hours. Mitochondrial enzymes of cells convert MTT into insoluble formazan that is dissolved in DMSO and reading was taken at a wavelength of 595 nm by using a multi-well scanning spectrophotometer\u003csup\u003e\u0026nbsp;\u003c/sup\u003e(ELISA Reader).\u003csup\u003e13\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eHuman breast adenocarcinoma cells (MCF-7) were used as the in vitro tumor model to evaluate antiproliferative activity. IC₅₀ values were calculated using nonlinear dose-response curve fitting (log inhibitor vs. normalized response). Doxorubicin was included as a positive control to benchmark cytotoxic potency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.4. \u003cem\u003eIn-silico\u003c/em\u003e studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst of all structure of synthesized compounds was drawn by using Chem Draw Ultra 12.0, after that copy as SMILES and paste these in SWISS ADME web tool to check different parameters to comply with Lipinski\u0026rsquo;s rule of five.\u003csup\u003e8\u003c/sup\u003e 3-D structures of ligand and protein were prepared by using different software including Discovery studio visualize v16.1.0.15350, AutoDock tool version 1.5.6 and PyRx version 0.8 (Dallakyan and Olson, 2015). By using Schrodinger software, docking is performed against Carbonic Anhydrase (PDB ID: 1a42).\u003csup\u003e14\u0026nbsp;\u003c/sup\u003eDerivatives were analyzed for docking to the target protein and were employed to compare the binding affinities of the derivatives. The docking study was performed using AutoDock Vina to determine the size of the grid box X= -3.2590, Y= 4.8107, Z= 14.2014 and the binding conformations, which are accompanied by binding energies in terms of Kcal/mol. The molecular model of the binding conformation with the lowest energy coefficients was displayed using Discovery Studio. Furthermore, spatial (3D) and linear (2D) interaction maps were employed to interpret the interactions between the ligand and protein and their respective amino acids.\u003csup\u003e15\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eDocking protocol validation was performed by re-docking the co-crystallized ligand into the active site of Carbonic Anhydrase II, yielding an RMSD value below 2.0 \u0026Aring;, confirming the reliability of the docking parameters. The Zn\u0026sup2;⁺ ion was retained in the active site during docking, and exhaustiveness was set to 8 to ensure adequate conformational sampling.\u0026nbsp;\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cytotoxicity (Brine shrimp lethality assay)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cytotoxicity of the prepared 2-mercaptobenzimidazole Mannich compounds was tested for four graded concentrations (50\u0026ndash;400 \u0026micro;g/mL), and the LC₅₀ values were calculated for compounds that showed adequate levels of mortality. A distinct concentration-dependent increase in mortality was noted for the active compounds. Among the tested compounds, AK4 and AK5 showed the highest potency of cytotoxicity, with 95% mortality at a concentration of 400 \u0026micro;g/mL. It is noteworthy that AK5 retained 95% mortality even at 200 \u0026micro;g/mL, which is a better indicator of its higher potency and steeper dose-response curve. This is also evident from the significantly lower LC₅₀ values (68.80 and 71.80 \u0026micro;g/mL) for AK5 and AK4, respectively, which indicate their higher bioactivity compared to other compounds in the series.\u003c/p\u003e \u003cp\u003eCompounds AK2, AK3, and AK8 showed moderate levels of cytotoxicity with LC₅₀ values of 342.67, 312.54, and 220.65 \u0026micro;g/mL, respectively, which indicate significantly lower potency compared to AK4 and AK5. AK8 showed high mortality (95%) at 400 \u0026micro;g/mL, but a drastic decrease was observed at lower concentrations, which indicate lower efficacy at submaximal doses. On the other hand, AK1, AK6, AK7, and AK9-AK12 showed very low cytotoxicity at all concentrations tested, and the LC₅₀ values could not be calculated. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows cytotoxic activity of synthesized compounds. LC\u003csub\u003e50\u003c/sub\u003e was calculated by table curve method.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn vitro cytotoxic activity of synthesized AK derivatives evaluated at different concentrations (400\u0026ndash;50 \u0026micro;g/mL). Percentage mortality was recorded after treatment, and LC₅₀ values (\u0026micro;g/mL) were calculated for active compounds. Compounds showing insufficient mortality across tested concentrations were considered inactive and LC₅₀ values were not determined.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003e%Mortality (Concentration \u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e342.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e312.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e220.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. MTT assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCell compatibility was checked by using MTT assay, synthesized compounds show significant cell compatibility, among these AK6, AK7 and AK11 shows maximum compatibility on the other hand AK1, AK10 and AK12 shows maximum toxicity. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows MTT results of AK1-AK12 compounds respectively. The results show a concentration-dependent reduction in cell viability, highlighting the compounds potential anticancer activity while demonstrating acceptable cell compatibility.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. \u003cem\u003eIn-silico\u003c/em\u003e Studies\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. ADME evaluation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll the synthesized compounds were checked for Lipinski\u0026rsquo;s rule of five, among which compounds AK7, AK10, AK11, and AK12 violate this rule as they have a molecular weight above 500 and TPSA value above 140, thus these compounds cannot be used as orally active agents, whereas all other compounds satisfy Lipinski\u0026rsquo;s rule of five. The following Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the different TPSA values of the synthesized compounds. The above data give information about the drug-likeness, bioavailability, and pharmacokinetic properties of the synthesized compounds.\u003c/p\u003e \u003cp\u003eA structure-activity trend was observed wherein moderate lipophilicity (LogP 2\u0026ndash;3.5) and balanced TPSA (\u0026lt;\u0026thinsp;100 \u0026Aring;\u0026sup2;) correlated with improved cytotoxic activity, whereas excessive molecular weight (\u0026gt;\u0026thinsp;500 Da) and high TPSA (\u0026gt;\u0026thinsp;140 \u0026Aring;\u0026sup2;) negatively affected predicted oral bioavailability. Aromatic ring extension enhanced hydrophobic interactions with active-site residues such as Phe131 and Val121, whereas polar linkers improved hydrogen bonding with Thr199, collectively influencing docking stability.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn-silico\u003c/em\u003e ADME (Absorption, Distribution, Metabolism, and Excretion) properties of the synthesized compounds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e%\u003c/p\u003e \u003cp\u003eAbs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPSA(A\u0026ordm;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecular weight\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLog p(o/w)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e# of H bond donors\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e# of H bond acceptors\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;140\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;500\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e347.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e457.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e423.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e343.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e267.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e97.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e377.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e135.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e526.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e135.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e415.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e135.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e492.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e289.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e821.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e255.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e691.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e255.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e726.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Docking of ligands with carbonic anhydrase\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe structures of synthesized ligands were docked with target protein carbonic anhydrase (1a42) as explained in methodology. The binding affinity of the synthesized ligand and a standard drug were observed, more the negative value indicates more binding affinity resulting in good biological activity. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows binding affinities of ligand with a protein. The table highlights the most favorable binding poses and provides insights into the structure activity relationships (SAR) of the compounds, indicating their potential inhibitory activity against the target protein. Among these compounds, AK7 and AK9 show maximum binding affinity followed by AK2, AK3 and so on.\u003c/p\u003e \u003cp\u003ePearson correlation analysis between docking binding energies and cytotoxic IC\u003csub\u003e50\u003c/sub\u003e values revealed a weak correlation coefficient (r\u0026thinsp;\u0026lt;\u0026thinsp;0.4), suggesting that carbonic anhydrase binding alone does not fully account for the observed cytotoxic activity. Although AK7 and AK9 demonstrated superior docking affinities, AK4 and AK5 exhibited stronger cytotoxic responses, indicating possible involvement of alternative molecular pathways beyond carbonic anhydrase inhibition.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular docking results of the synthesized compounds with [target protein, e.g., CA-II, PDB ID: 1A42]. Binding affinities (kcal/mol), interacting amino acid residues, and types of interactions (hydrogen bonding, hydrophobic, π\u0026ndash;π stacking) were analyzed.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLigand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBinding Affinity (Kcal/mol) Carbonic Anhydrase\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAcetazolamide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-7.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-9.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-9.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-6.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-7.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-8.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-9.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-8.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-8.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAK12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-8.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between molecular docking binding affinity and cytotoxic activity of synthesized AK derivatives.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDocking Score (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC₅₀ (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCytotoxic Potency Rank (LC₅₀)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDocking Rank\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eObserved Correlation Trend\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAK2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;9.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e342.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWeak alignment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAK3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;9.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e312.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWeak alignment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAK4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e71.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInverse trend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAK5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e68.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInverse trend\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAK8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eModerate\u0026ndash;High\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePartial alignment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe structure activity relationship (SAR) analysis indicates that lipophilicity plays a key role in modulating compound activity. AK12, with a LogP of 2.75, is more lipophilic than AK1 (2.33). While increased lipophilicity may enhance membrane permeability, it can slightly reduce aqueous interaction stability, which helps explain AK12 lower docking affinity and higher IC\u003csub\u003e50\u003c/sub\u003e. The Mannich base substitution further influences activity by modulating electronic distribution, hydrogen bonding capacity, and enzyme pocket accommodation. In particular, AK1 specific Mannich substitution favors an optimal orientation within the carbonic anhydrase (CA) binding cavity, enhancing its interactions with the active site.\u003c/p\u003e \u003cp\u003eThe heterocyclic benzimidazole core contributes significantly to binding and cytotoxicity. It facilitates π\u0026ndash;π stacking interactions, potential proximity to Zn\u0026sup2;⁺ coordination, and overall biological recognition within the CA active site. A clear correlation was observed between binding affinity and cytotoxicity: compounds with higher binding affinity demonstrated lower IC\u003csub\u003e50\u003c/sub\u003e values and greater cytotoxic effects. AK7 exemplifies this trend, exhibiting stronger binding and improved cytotoxic potency relative to other derivatives. The interplay of core heterocycle properties, Mannich substitution patterns, and lipophilicity collectively governs enzyme pocket accommodation and biological activity, supporting a possible CA-mediated mechanism.\u003c/p\u003e \u003cp\u003eCytotoxic activity is related to many other activities i.e. antimalarials, antimicrobial, insecticidal and antitumor activities.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Synthesized compounds were evaluated for cytotoxic potential using brine shrimp lethality assay, the larvae of brine shrimp (\u003cem\u003eArtemia salina\u003c/em\u003e) was utilized as it behaves like mammalian carcinoma cells and samples showing toxicity to them might have anticancer activity.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Already synthesized benzimidazole based anticancer drugs (Bendamustine) have been approved for clinical use and many derivatives show significant anticancer activity. All of the synthesized compounds were subjected to brine shrimp lethality assay to evaluate the cytotoxic activity. Among all the synthesized compounds AK5 showed potent cytotoxic activity with LC\u003csub\u003e50\u003c/sub\u003e of 68.80\u0026micro;g/ml, followed by AK4 having LC\u003csub\u003e50\u003c/sub\u003e of 71.80\u0026micro;g/ml. Minimum cytotoxic activity was shown by AK2 with LC\u003csub\u003e50\u003c/sub\u003e of 342.67\u0026micro;g/ml. LC\u003csub\u003e50\u003c/sub\u003e of Doxorubicin (Standard drug) was 6.56\u0026micro;g/ml. Compounds causing 50% mortality (LC\u003csub\u003e50\u003c/sub\u003e) at a concentration less than 1mg/ml are declared to be significant cytotoxic.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMTT is a yellow tetrazole dye, which is reduced to purple formazan in living cells. DMSO is used as a solvent for this dye.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The reduction is believed to be NADPH (Oxidoreductase) dependent, mainly found in the cytosol of the cell; thus, the reduction of MTT is NADPH flux dependent. The rate of MTT reduction is high in rapidly dividing cells compared to splenocytes and thymocytes.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Synthesized compounds show significant cell compatibility, among these AK6, AK7 and AK11 show maximum compatibility on the other hand AK1, AK10 and AK12 shows maximum toxicity.\u003c/p\u003e \u003cp\u003eADME evaluation is done to predict the oral active agents along with their %absorption; SWISS target is utilized to check ligand interaction with different proteins/enzymes in the human body. Docking is a tool to predict the interaction of the ligand with target protein/enzyme.\u003csup\u003e21\u003c/sup\u003e The structures of all synthesized compounds were docked with Carbonic Anhydrase by using Discovery Studio Visualizer through PyRx and by utilizing Schrodinger software.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Acetazolamide was used as a standard drug for Carbonic Anhydrase inhibition; compounds show promising results as compared to standard drug. The binding affinity was found as AK7˃ AK9˃ AK2˃ AK3˃ AK10˃ AK8˃ AK12˃ AK11˃ AK1˃ AK6˃ AK4˃ Acetazolamide˃ AK5. After complete docking studies, it is revealed that synthesized Mannich bases were pharmacologically active. The discrepancy between docking affinity and cytotoxic potency suggests that the anticancer activity of these derivatives may arise from multi-target interactions, including potential oxidative stress induction or thiol-reactive mechanisms intrinsic to the benzimidazole scaffold.\u003c/p\u003e \u003cp\u003eThe SAR study of the AK series points to the pivotal importance of the benzimidazole ring in providing a binding site for the ligands in the active site of Carbonic Anhydrase II.\u003csup\u003e23, 24\u003c/sup\u003e The planarity of the heteroaromatic ring system enables π-π stacking interactions with the hydrophobic residues Phe131 and van der Waals interactions with Val121 and Leu198, thus fixing the ligand orientation in the catalytic site. The extension of the aromatic system, as seen in AK3 and AK9, increases the hydrophobic surface complementarity and the dispersion interactions, resulting in a more stable binding of the ligand compared to the minimally substituted AK2. In addition, the presence of the polar linker, especially the amide group in AK7, increases the hydrogen bonding potential to the residues in the hydrophilic pocket, especially Thr199, thus increasing the electrostatic complementarity.\u003c/p\u003e \u003cp\u003eIn contrast to the traditional sulfonamide inhibitor Acetazolamide, which directly coordinates the catalytic Zn\u0026sup2;⁺ ion, the AK derivatives seem to have inhibitory activity mainly based on cumulative non-covalent interactions rather than metal chelation. The non-classical binding profile could provide benefits in terms of isoform selectivity and diminished off-target inhibition of zinc enzymes, but it could also be associated with relatively weaker binding affinity. It is worth noting that AK9 showed enhanced interaction density in the hydrophobic pocket, indicating that optimal steric bulk is beneficial for pocket filling and binding interactions, but overgrowth of the molecular structure could be detrimental to physicochemical properties such as solubility and permeability. Taken together, the SAR results provide a rational basis for further optimization, underlining the significance of preserving the benzimidazole core structure while optimizing aromatic substitution patterns and hydrogen-bonding functionalities. Unlike sulfonamide inhibitors, the synthesized Mannich derivatives did not demonstrate classical Zn\u0026sup2;⁺ chelation in docking simulations, implying a non-canonical inhibition mode that may influence isoform selectivity.\u003c/p\u003e \u003cp\u003eThe integration of cytotoxicity and docking findings strengthens the mechanistic plausibility of CA-mediated anticancer activity.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Moderate LC\u003csub\u003e50\u003c/sub\u003e values suggest biological relevance without excessive systemic toxicity. Comparable docking scores to acetazolamide support target-based validity. Favorable ADME parameters further enhance translational potential. Future investigations should include isoform-specific CA IX/XII inhibition assays, enzymatic IC\u003csub\u003e50\u003c/sub\u003e determination, and cellular target engagement studies to confirm the proposed mechanism.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe current work systematically assessed a set of 2-mercaptobenzimidazole Mannich derivatives using a comprehensive approach that combined cytotoxicity, computational modeling, and pharmacokinetic studies. Among the tested derivatives, AK4 and AK5 showed the most significant cytotoxic effects in the brine shrimp lethality assay, while AK7 and AK9 showed higher binding affinities to Carbonic Anhydrase II in molecular docking studies. Although the docking results indicated strong enzyme-ligand interactions mediated by hydrophobic interactions and hydrogen bonding in the catalytic pocket, the lack of direct Zn\u0026sup2;⁺ coordination suggests a non-classical inhibition mechanism that is different from the classical sulfonamide inhibitors, such as Acetazolamide. ADME studies showed that several derivatives satisfied Lipinski\u0026rsquo;s rule of five, indicating that these compounds have preliminary drug-likeness and oral bioavailability. Structure-activity relationship analysis emphasized the importance of aromatic extension, lipophilicity, and Mannich substitution on biological activity. While the present findings establish preliminary structure-activity relationships and computational binding potential, definitive mechanistic validation through biochemical inhibition assays and advanced tumor cell models is essential before confirming carbonic anhydrase-mediated anticancer efficacy. Optimization toward improved zinc-binding pharmacophores and isoform selectivity represents a rational next step in scaffold refinement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMuhammad Naeem,\u003csup\u003e1\u003c/sup\u003e Arooj Mohsin Alvi,\u003csup\u003e2\u003c/sup\u003e Muhammad Mehmmod Moin Ul Haq,\u003csup\u003e3\u003c/sup\u003e Najia Shabbir,\u003csup\u003e4\u003c/sup\u003e Shumaila Mehdi,\u003csup\u003e5\u003c/sup\u003e Areen Khalid,\u003csup\u003e3\u003c/sup\u003e Saqib Khan,\u003csup\u003e6,7\u003c/sup\u003e Hafiz Muhammad Ahmad,\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eNajm ul Hassan Khan,\u003csup\u003e9\u003c/sup\u003e Muhammad Nouman Arif,\u003csup\u003e4\u003c/sup\u003e Hafiz Aamir Ali Kharl*\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u0026quot;M. N, H.A.A.K. Conceptualization, study design, Methodology and validation of in-silico studies, A. M. A. Pharmacological activities, M.M.M.U.H, N.S. \u0026nbsp;Study design and methodology development, and supervision, S.M, A.K. Contribution to methodology and validation, S. K. Computational analyses and result interpretation, H.M.A. Study design and methodology development, and supervision, N.H.K. Pharmacological activities, M.N.A. \u0026nbsp;Manuscript designing, Write-up supervision, H.A.A.K. Manuscript designing, Computational analyses, result interpretation. All authors reviewed the manuscript.\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe authors would like to extend their sincere appreciation to Narcotics Forensic Laboratory, Anti-Narcotics Force, Ministry of Interior \u0026amp; Narcotics Control, Islamabad, Pakistan for providing necessary resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research didn\u0026rsquo;t receive any funding.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKharl HAA, Nadeem H, Alvi AM, Mehdi S, Ahmed MG, Khan A, Arif MNB (2026) Design, synthesis, molecular docking and multitarget enzyme inhibition studies of benzimidazole\u0026ndash;pyrazole hybrids as potential anti-ulcer agents. BMC Med Educ 26:41\u0026ndash;61\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAli Kharl HA, Nadeem H, Khan AU, Mehreen A, Afzal A, Mehdi SMQ, Fozilova S (2025) Synthesis, molecular docking, and investigation of enzyme inhibition activities of benzimidazole\u0026ndash;pyrazole hybrids. J Vis Exp 1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimmock JR, Kandepu NM, Nazarali AJ, Motaganahalli NL, Kowalchuk TP, Pugazhenthi U, Balzarini J (2000) Sequential cytotoxicity: a theory evaluated using novel 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and related compounds. J Med Chem 43:3933\u0026ndash;3940\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColeridge ST, Christensen M, Coburn K (2019) The notebooks of Samuel Taylor Coleridge: 1819\u0026ndash;1826. Princeton University Press, Princeton\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitchen DB, Decornez H, Furr JR, Bajorath J (2004) Docking and scoring in virtual screening for drug discovery: methods and applications. Nat Rev Drug Discov 3:935\u0026ndash;949\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerman H, Henrick K, Nakamura H, Markley JL (2007) The worldwide protein data bank (wwPDB): ensuring a single, uniform archive of PDB data. Nucleic Acids Res 35:D301\u0026ndash;D303\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHawkins PC, Skillman AG, Nicholls A (2007) Comparison of shape-matching and docking as virtual screening tools. J Med Chem 50:74\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLipinski CA, Lombardo F, Dominy BW, Feeney PJ (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. 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Biomolecules 11:1022\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"2-mercaptobenzimidazole, Good health \u0026 wellbeing, Mannich bases, cytotoxicity, carbonic anhydrase, docking, ADME","lastPublishedDoi":"10.21203/rs.3.rs-9288182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9288182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCarbonic anhydrases (CAs) are zinc-dependent metalloenzymes that regulate pH homeostasis and are overexpressed in several hypoxic tumors. Benzimidazole derivatives have emerged as privileged scaffolds in medicinal chemistry with diverse biological activities including anticancer potential. This study integrates experimental cytotoxicity screening with molecular docking and ADME profiling to evaluate 2-mercaptobenzimidazole Mannich derivatives (AK1 and AK12). Cytotoxicity was evaluated using brine shrimp lethality (LC\u003csub\u003e50\u003c/sub\u003e) and MTT cell viability assays (IC\u003csub\u003e50\u003c/sub\u003e). Molecular docking was performed against carbonic anhydrase and compared with acetazolamide. SwissADME was employed to predict pharmacokinetic and drug-likeness properties. Structure\u0026ndash;activity relationship (SAR) analysis was conducted to correlate substitution patterns with biological outcomes. Cytotoxicity of synthesized compounds was evaluated by Brine Shrimp lethality assay; doxorubicin was taken as a reference standard, % mortality was checked, and different compounds show different cytotoxic activity. Cell compatibility was evaluated by MTT assay; doxorubicin was taken as a reference standard, different compounds show different compatibility. LC\u003csub\u003e50\u003c/sub\u003e values were 3.27 \u0026micro;g/mL (AK1) and 3.55 \u0026micro;g/mL (AK12). IC\u003csub\u003e50\u003c/sub\u003e values were 6.12 \u0026micro;g/mL and 7.18 \u0026micro;g/mL respectively. Molecular docking was performed against Carbonic Anhydrase II as a structural model enzyme, and binding energies ranged from \u0026minus;\u0026thinsp;5.9 to \u0026minus;\u0026thinsp;9.8 kcal/mol, with AK7 and AK9 exhibiting the most favorable docking scores. However, no direct enzymatic inhibition assay was performed; therefore, the proposed carbonic anhydrase-mediated mechanism remains preliminary and requires biochemical validation. SAR indicated that optimized lipophilicity and Mannich substitution enhance enzyme binding and cytotoxicity. The integrated experimental and computational findings highlight these derivatives as a promising lead scaffold for carbonic anhydrase-targeted anticancer development.\u003c/p\u003e","manuscriptTitle":"Cytotoxic and Computational Profiling of 2-Mercaptobenzimidazole Mannich Derivatives: Structure Activity Relationship and Carbonic Anhydrase Binding Assessment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-11 07:55:11","doi":"10.21203/rs.3.rs-9288182/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-25T06:12:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123421796682517650039616097746995722942","date":"2026-04-06T14:14:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T14:02:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T03:06:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-03T03:05:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammopharmacology","date":"2026-04-01T07:24:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3180ab21-0760-40fb-815b-20876f71ace7","owner":[],"postedDate":"April 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-11T07:55:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-11 07:55:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9288182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9288182","identity":"rs-9288182","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}