Eco-friendly polyethylene glycol (PEG-400): PEG mediated synthesis, in-vitro, molecular dynamics, DFT, docking, ADMET and MM-GBSA studies of novel sulfonamides as potential antioxidant agents C.R. Santhosh a, Sampath Chinnam a,* , Hrushikesh S. Chaudhari b, Kondapalli Venkata Gowri Chandra Sekhar b, Gbolahan O. Oduselu c, Vishal Ahuja d, Kumarappan Chidambaram e , Nagaraju Kerru f, G.M. Madhu g,** a Department of Chemistry, M.S. Ramaiah Institute of Technology (Affiliated to Visvesvaraya Technological University, Belgaum), Bengaluru, Karnataka 560054, India b Department of Chemistry, Birla Institute of Technology and Science, Pilani, Hyderabad Campus, Jawahar Nagar, Kapra Mandal, Hyderabad, Telangana 500078, India c West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), P.O Box LG 54, University of Ghana, Accra, Ghana d Department of Biotechnology, University Centre for Research and Control, Chandigarh University, Mohali, Punjab 140413, India e Department of Pharmacology, College of Pharmacy, King Khalid University, Abha 61421, Saudi Arabia f Department of Chemistry, GITAM School of Sciences, GITAM University, Bengaluru, Karnataka 561203, India g Department of Chemical Engineering, M.S. Ramaiah Institute of Technology (Affiliated to Visvesvaraya Technological University, Belgaum), Bengaluru, Karnataka 560054, India A R T I C L E I N F O Keywords: Sulfonamides Antioxidant activity Molecular dynamics Docking DFT ADMET MM-GBSA A B S T R A C T Novel thiomorpholine bearing sulfonamide analogs 3(a-f) were synthesized by a facile one-pot, single-step process in good to excellent yields (87–95 %). Polyethylene glycol (PEG-400) was used as an eco-friendly, recyclable, and non-flammable solvent. In-vitro antioxidant studies were done using H2O2 and DPPH radical scavenging assays, while the docking studies were carried out using the Maestro software. In the DPPH assay, the activity of compound 3c with an IC50 value of 30.83±0.33 µg/mL was comparable to the standard, ascorbic acid (13.54±0.33 µg/mL). Interestingly, this compound demonstrated the highest scavenging antioxidant activity at all concentrations, with an IC50 value of 32.78±0.33 µg/mL in the H2O2 method. Compound 3b, with salt bridge interactions and hydrogen bonds, exhibited a docking score of -2.01 kcal/mol (PDB: 1H4O). ADME results indicated that all the synthesized compounds obeyed Lipinski’s rule, indicating the drug-like properties. The RMSD curve of complex 3b was observed to be closely matching with benzoic acid, the co-crystallized ligand, indicating alignment and stability with the reference ligand. The optimized molecules with lower energy gaps of 2.17, 4.23, and 1.21 eV indicating that the synthesized compounds shown potent biological activity. This pro tocol offers excellent yields, green solvent, catalyst-free, shorter reaction times, inexpensive, and no harsh re action conditions. These research findings reveal that these sulfonamide analogs could be potent antioxidant pharmacophores for future research in antioxidant drug discovery. 1. Introduction Antioxidants are important health-protecting factors as they fight against reactive free radicals [1]. Free radicals such as hydroxyl, su peroxide, and nitric oxide are oxygen-centered radicals, called reactive oxygen species (ROS). They are formed from the side reactions of various biochemical redox reactions inside the body, and they can be noxious when present in excess [2,3]. Free radicals in large quantities can impair lipids, enzymes, DNA, proteins, and tissues. Consequently, this may result in several illnesses, including aging, diabetes, cancer, cardiovascular disease, autoimmune diseases, and neurological disor ders [4,5]. Antioxidant molecules interact with free radicals and stop the * Corresponding author at: Department of Chemistry, M.S. Ramaiah Institute of Technology (Affiliated to Visvesvaraya Technological University, Belgaum), Bengaluru, Karnataka 560054, India. ** Corresponding author at: Department of Chemical Engineering, M.S. Ramaiah Institute of Technology (Affiliated to Visvesvaraya Technological University, Belgaum), Bengaluru, Karnataka 560054, India. E-mail addresses: sampath@msrit.edu (S. Chinnam), gmmadhu@msrit.edu (G.M. Madhu). Contents lists available at ScienceDirect Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstr https://doi.org/10.1016/j.molstruc.2025.143201 Received 29 March 2025; Received in revised form 19 June 2025; Accepted 2 July 2025 Journal of Molecular Structure 1346 (2025) 143201 Available online 3 July 2025 0022-2860/© 2025 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies. https://orcid.org/0000-0002-5334-9076 https://orcid.org/0000-0002-5334-9076 https://orcid.org/0000-0002-7981-4562 https://orcid.org/0000-0002-7981-4562 mailto:sampath@msrit.edu mailto:gmmadhu@msrit.edu www.sciencedirect.com/science/journal/00222860 https://www.elsevier.com/locate/molstr https://doi.org/10.1016/j.molstruc.2025.143201 https://doi.org/10.1016/j.molstruc.2025.143201 chain reactions, thereby protecting the essential molecules [6]. Conse quently, research on developing novel and effective antioxidants with reduced side effects is always a prime focus [7,8]. The morpholine ring contains two different functionalities, such as ether and amine. In thiomorpholine, the oxygen atom is replaced by a sulfur atom [9]. Thiomorpholine scaffold is considered as a leading pharmacophore and has attracted significant attention owing to various applications in biologicals, including anti-fungal [10], anti-viral [11], antioxidant, anti-inflammatory [12], etc. Sulfonamides (sulfa drugs) are amide derivatives, containing the sulfamoyl (-SO2NH-) group. They are generally synthesized by a one-pot, two-component reaction by sulfo nylation of amines with sulfonyl halides using a base [13,14]. They are highly reactive molecules, mostly used as antibiotics and effective chemotherapeutic agents [15,16]. They were reported to have promi nent applications as antimicrobial [17], anti-tumor [18], anti-inflammatory [19], HIV protease inhibitors [20], etc. The syner gistic action of such biologically active compounds has encouraged re searchers to design, synthesize, and study their pharmacological properties [21,22]. In this context, several drugs containing sulfon amides have reached the markets and been effective in their activities Fig. 1. FDA-approved and marketed sulfonamide-containing drugs. Fig. 2. Our designed sulfonamide analogs containing thiomorpholine 3(a-f). C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 2 [23]. Common drugs containing sulfonamides are presented in Fig. 1. The use of eco-friendly, green solvents for organic synthesis is crucial in a way looking forward to green and sustainable chemistry. Consid erable efforts were being made in designing schemes based on solvent- free, water, ionic liquids, polyethylene glycol (PEG) etc., solvent sys tems [24–26]. PEG has attracted researchers for its non-toxic, non-flammable, inexpensive, and biodegradable properties because it acts as a green solvent. Moreover, it was reported to act as a catalyst for several organic transformations [27,28]. Computational techniques are an important part of drug discovery. As these methods can be employed to virtually screen the designed compounds, to predict their activities on the targeted proteins. Molec ular docking gives insights into the interaction types and binding af finities between proteins and ligand molecules [29]. The physicochemical parameters of the molecules, like absorption, lip ophilicity, toxicity, etc., can be predicted using ADME studies. Molecular dynamic simulation studies facilitate understanding protein-ligand complex stability, interaction fractions, etc. [30,31]. Likewise, quan tum chemical calculations further aid in deducing the molecular prop erties of the ligands. Therefore, these computational techniques are the Scheme 1. Synthesis of novel sulfonamide analogs 3(a-f). C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 3 key component of medicinal chemistry, supporting effective and safe drugs reaching the market [32,33]. We report a facile, catalyst-free synthesis and pharmacologically potent sulfonamide analogs containing thiomorpholine (Fig. 2) using PEG as a green solvent. In-vitro studies were performed by DPPH and H2O2 radical scavenging assays to understand their antioxidant prop erties. Further, in-silico ADME, docking, molecular dynamics, DFT, and MM-GBSA calculations were performed on the synthesized compounds to understand their molecular and drug-likeness properties. 2. Materials and methods 2.1 2.1.1. Experimental The required chemicals thiomorpholine (98 %), and various aro matic sulfonyl chlorides (95–98 %) were purchased from Merck, India, and used without purification. Mel-Temp apparatus with open capil laries was employed to get the melting points of the synthesized com pounds, which are expressed in ◦C (uncorrected). 1H and 13C NMR spectra’s were recorded in CDCl3 solvent on a Bruker 400 Avance spectrometer at 400 MHz. Chemical shifts (δ) were expressed in parts per million (ppm) and coupling constants (J) in Hertz (Hz). For HRMS, ambient temperatures and Bruker microTQF-Q II ESI high-resolution was used. 2.1.2. General synthesis procedure of novel sulfonamides 3(a-f) In a clean 50 mL round-bottom flask, thiomorpholine (0.001 mol) (1) was mixed with various aromatic sulfonyl chlorides (0.001 mol) 2(a-f) in the presence of PEG-400 as a green solvent under catalyst-free con ditions at 60 ◦C for 2–3 h. Reaction completion was monitored by thin- layer chromatography (TLC). Once the reaction was complete, the product was filtered, separated and removed water from PEG by rotary evaporator to get novel sulfonamide analogs containing thiomorpholine 3(a-f) (Scheme 1) in good to excellent yields (87–95 %). 2.1.3. 4-(2,4-difluorophenylsulfonyl)thiomorpholine (3a) White solid, Yield: 89 %, mp 156–158 ◦C. 1H NMR (400 MHz, CDCl3) δ 8.13 (dd, J = 8.1, 1.4 Hz, 1H, Ar-H), 7.75 (td, J = 7.9, 1.4 Hz, 1H, Ar- H), 7.68 (dd, J = 7.8, 1.4 Hz, 1H, Ar-H), 3.07 (t, J = 6.9 Hz, 2H, CH2), 2.03 (t, J = 7.0 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 135.27, 131.27, 130.64, 130.21, 129.05, 128.82, 128.07, 124.97, 53.21, 27.90; HRMS of [C10H11NO2S2F2 + H]+ (m/z) 280.1294; Calcd: 280.1290. 2.1.4. 4-(2-nitrophenylsulfonyl)thiomorpholine (3b) Yellow solid, Yield: 95 %, mp 178–180 ◦C. 1H NMR (400 MHz, CDCl3) δ 8.63 (t, J = 2.1 Hz, 1H, Ar-H), 8.34 (dd, J = 6.2, 0.9 Hz, 1H, Ar- H), 8.21 (dd, J = 2.2, 0.9 Hz, 1H, Ar-H), 7.79 (t, J = 8.2 Hz, 1H, Ar-H), 2.97 (t, J = 8.1 Hz, 2H, CH2), 1.89 (t, J = 7.9 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 135.20, 131.83, 130.02, 128.07, 125.14, 122.56, 51.89, 26.98; HRMS of [C10H12N2O4S2 + H]+ (m/z) 289.1337; Calcd: 289.1341. 2.1.5. 4-(4-chloro-2-fluorophenylsulfonyl)thiomorpholine (3c) White solid, Yield: 90 %, mp 178–180 ◦C. 1H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 2.1 Hz, 1H, Ar-H), 7.59 (dd, J = 8.3, 2.1 Hz, 1H, Ar-H), 7.42 (d, J = 8.3 Hz, 1H, Ar-H), 2.97 (t, J = 8.0 Hz, 2H, CH2), 1.89 (t, J = 8.0 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 132.50, 130.58, 130.12, 130.02, 127.99, 124.90, 121.28, 121.10, 52.76, 28.18; HRMS of [C10H11NO2S2FCl + H]+ (m/z) 295.1445; Calcd: 295.1447. 2.1.6. 4-(4-nitrophenylsulfonyl)thiomorpholine (3d) Yellow solid, Yield: 91 %, mp 187–189 ◦C. 1H NMR (400 MHz, CDCl3) δ 8.90 (d, J = 7.5 Hz, 2H, Ar-H), 8.20 (d, J = 8.0 Hz, 2H, Ar-H), 2.92 (t, J = 8.0 Hz, 2H, CH2), 2.07 (t, J = 8.0 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 130.51, 128.91, 128.00, 126.28, 51.92, 26.18; HRMS of [C10H12N2O4S2 + H]+(m/z) 289.9911; Calcd: 289.9906. 2.1.7. 4-(4-iodophenylsulfonyl)thiomorpholine (3e) White solid, Yield: 89 %, mp 190–192 ◦C. 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 8.5 Hz, 2H, Ar-H), 7.86 (d, J = 8.5 Hz, 2H, Ar-H), 2.93 (t, J Table 1 Performance of different solvents and temperatures on the synthesis of sulfon amide 3a. S.No. Medium Temp. / ◦C Time in hours % of yieldsa 1 Solvent-free rt 8.0 9 2 Acetonitrile rt 8.0 26 3 DMF rt 6.0 18 4 THF rt 6.0 31 5 EtOH rt 6.0 49 6 PEG 400 rt 6.0 61 7 PEG 400 40 4.0 73 8 PEG 400 50 4.0 77 9 PEG 400 60 3.0 89 10 PEG 400 70 3.0 89 Reaction conditions: Thiomorpholine (1.1 mmol), 2,4-difluro-sulfonyl chloride (2a, 1.1 mmol), and solvent (5 mL); a Isolated yields. Fig. 3. Scavenging activity of synthesized compounds 3(a-f) on DPPH radicals at varied concentrations in comparison with the standard drug ascorbic acid. Fig. 4. Percentage scavenging activity of H2O2 radicals by the synthesized compounds 3(a-f) at various concentrations compared with the standard drug ascorbic acid. C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 4 Fig. 5. Structure-activity relationship (SAR) studies of novel sulfonamide analogs 3(a-f). Table 2 Predicted ADMET properties of the synthesized compounds 3(a-f). S. No. Compound MW Log P HBD HBA TPSA RB nAH MR 1 3a 279.33 1.69 0 5 71.06 2 6 66.16 2 3b 288.34 1.35 0 5 116.88 3 6 75.07 3 3c 295.78 2.22 0 4 71.06 2 6 71.21 4 3d 288.34 1.32 0 5 116.88 3 6 75.07 5 3e 369.24 2.14 0 3 71.06 2 6 78.96 6 3f 257.37 1.86 0 3 71.06 2 6 71.21 Fig. 6. Geometry optimized compounds 3b, 3c, and 3d (B3LYP/6–311G* level). Fig. 7. Molecular electrostatic potential analysis of compounds 3b, 3c, and 3d by color range (in a.u.) (Iso-surface ±0.002 a.u). C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 5 = 7.8 Hz, 2H, CH2), 2.07 (t, J = 7.9 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 129.51, 123.36, 122.09, 121.30, 52.91, 29.95; HRMS of [C10H12NO2S2I + H]+ (m/z) 369.1486; Calcd: 369.1487. 2.1.8. 4-tosylsulfonylthiomorpholine (3f) White solid, Yield: 87 %, mp 190–192 ◦C. 1H NMR (400 MHz, CDCl3) δ 7.38 (d, J = 8.3 Hz, 2H, Ar-H), 6.89 (d, J = 8.3 Hz, 2H, Ar-H), 2.89 (t, J = 8.1 Hz, 2H, CH2), 2.31 (s, 3H, CH3), 2.08 (t, J = 8.0 Hz, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 124.67, 123.09, 121.89, 120.51, 52.79, 28.38, 18.15; HRMS of [C10H15NO2S2 + H]+ (m/z) 258.0689; Calcd: 258.0698. 2.2. Biological studies 2.2.1. In-vitro antioxidant studies The antioxidant activity of compounds 3(a-f) was assessed using the DPPH free radical scavenging assay [34]. To compare the antioxidant activity of these compounds, ascorbic acid was treated as a positive control. A 0.004 % DPPH solution was obtained by mixing 4 mg of it in 100 mL of methanol. A series of solutions with different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/mL) of the synthesized compounds were obtained by diluting them with methanol. Each of the test solutions (1 mL) was mixed with 4 mL of DPPH: methanol solution. Samples were incubated for 30 min in the dark. Using a UV–visible spectrophotometer, the sample’s absorbance was measured at 517 nm. The scavenging activities of the samples were computed by the following equation. % Inhibition of DPPH = ( Abscontrol − Abssample ) Abscontrol × 100 Where ‘Abscontrol’ indicates the DPPH solution absorbance and ‘Abssample’ defines the sample and the DPPH solution’s absorbance. Synthesized compounds 3(a-f) were also examined for the hydrogen peroxide radical scavenging activity [35]. A hydrogen peroxide solution of 40 mM was obtained in phosphate buffer (pH=7.4–7.5). Test Fig. 8. HOMO-LUMO band gap calculations 3b, 3c, and 3d of optimized ligands. Fig. 9. Validation of docking protocol by re-docking benzoic acid (co-crystallized ligand). C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 6 compounds in 3.4 mL of phosphate buffer were added to the H2O2 so lution (0.6 mL, 40 mM). Dark incubation (30 min) was carried out for the samples. Samples and phosphate buffer’s absorbance values were then measured without hydrogen peroxide at 230 nm by a UV spectro photometer. As a standard, ascorbic acid was employed. The scavenging activity of the hydrogen peroxide radical was computed using the given formula. % Inhibition of H2O2 = ( Abscontrol − Abssample ) Abscontrol × 100 Where Abscontrol indicates standard H2O2 absorbance; Abssample de scribes the absorbance values in the presence of the sample and standard H2O2. In both methods, IC50 values were obtained from a plot between the concentration of test compounds and the percentage of scavenging activity. Fig. 10. Docking poses of 3b, 3c, 3d, BA, ascorbic acid, and quercetin with targeted protein. C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 7 2.3. In-silico ADMET SwissADME and pkCSM tools were used to predict the ADME prop erties of the synthesized compounds 3(a-f). ChemDraw Ultra 22.0.0 software was employed to draw the 2D structures of the compounds. And they were converted into SMILES notations, and further used for analysis [36,37]. 2.4. MM-GBSA calculations Gaussian 09 and GaussView 6.0 were used to analyze the com pounds’ molecular structures. The molecular properties of the ligands were evaluated by Quantum chemical calculations. Optimization of the ground-state geometric parameters was achieved by the DFT method at the B3LYP/6–311G* level. GaussView 6.0 was employed to visualize the MEP and FMOs [38,39]. 2.5. Molecular docking studies Docking investigations were performed on the three molecules that demonstrated the highest antioxidant activity. Using Maestro 2022–1 version 4.2.667, Glide XP docking was employed to evaluate the compatibility and interactions of thiomorpholine-based sulfonamides with the PrxV receptor (PDB: 1H4O). Molecules were docked using Glide XP at the PrxV receptor’s active sites [40–43]. 2.6. Molecular dynamics (MD) studies Desmond v5.9 (Schrödinger 2019–3) was used to perform MD sim ulations to understand the effects of solvent on the interactions of the protein-ligand complex. Simulation data were recorded every 10 ps, and the NPT ensemble was run for 100 ns. Interaction diagrams were analyzed to study trajectories and frame variations [44–46]. 3. Results and discussion 3.1. Chemistry A novel series of thiomorpholine-containing sulfonamide analogs 3 (a-f) were synthesized in good to excellent yields (87–95 %) by the re action of thiomorpholine (1) with various aromatic sulfonyl chlorides 2 (a-f) using PEG-400 as a green solvent under catalyst-free conditions. Reaction conditions were optimized for variables like different me diums, time, and temperature to get maximum yields. Initially, a pilot reaction was conducted with an equimolar mixture of thiomorpholine (1) and 2,4-difluorosulfonyl chloride (2a) in 5 mL of solvent (Table 1). Subsequently, a detailed examination focused on the influence of varying solvents and the impact of temperature was done. Initially, the reaction was carried out at room temperature in reflux condition for 8 h without any medium, and no product was observed (Table 1, entry 1); it indicates the essential role of a medium for the successful transformation to the desired product. The reaction was then explored using different aprotic solvents (Acetonitrile, DMF, and THF). When comparing the aprotic medium, a low-yielding product was ob tained even after extended reaction times for 8 h (Table 1, entries 2–4). Further, a moderate yield of product was observed by using protic sol vent, ethanol (Table 1, entry 5). Surprisingly, PEG-400 yielded markedly superior results (61 %), establishing PEG-400 as the most effective medium and as an activator for the one-pot benchmark reaction (Table 1, entry 6). Further investigations into the optimal temperature conditions for the model reaction were conducted. The reaction rate accelerated noticeably with a temperature increase from room temperature to 70 ◦C, which also significantly enhanced the yield of the expected product. It was particularly encouraging to note that at a moderate temperature range of 60 ◦C, the reaction proceeded smoothly and yielded almost complete conversion of the reactants. This temperature range facilitated the production of the desired product (3a) with an impressive 89 % yield for 3 h (Table 1, entries 7–10). Subsequent temperature elevation showed no further improvement in the product yield, affirming that 60 ◦C is the most efficient temperature for this transformation. Spectral techniques such as 1H, 13C NMR and HR-MS confirmed the formation of the designed compounds. In 1H NMR, peaks at δ 7.38–8.90 ppm were assigned for the aromatic hydrogen atom of compounds 3(a- f). Doublets were shown at δ 6.89–8.34 ppm corresponding to aromatic protons for 3(a-f) compounds. Further, doublets appeared at δ 7.42–8.21 ppm, confirming protons in the aromatic ring of 3(a-c) compounds. Triplets were seen at δ 2.89–3.07 ppm, which was attributed to the –CH2 group of 3(a-f) compounds. Peaks at δ 1.89–2.08 ppm were assigned for the –CH2 group of 3(a-f) compounds. In the 13C NMR spectrum, all the signals corresponding to aromatic carbon were observed in the expected δ range from 120.0–135.0 ppm. Also, carbon peaks of the -CH2- group were observed in the ranges of δ 25.0–55.0 ppm. Additionally, the m/z peaks in the HRMS spectra confirmed the formation of all the compounds. 3.2. Biological assay 3.2.1. In-vitro analysis Scavenging activities of the synthesized compounds 3(a-f), on DPPH and H2O2 free radicals were evaluated at serial concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg/mL. Activity results were compared with the ascorbic acid standard. Scavenging results indicated that the majority of the compounds exhibit good to promising antioxi dant activities. The order of DPPH free radical scavenging activity of these compounds was found to be 3c>3b>3d>3e>3f>3a based on in hibition percentage values. Results disclosed that compounds 3b, 3c, and 3d had higher scavenging activity at all concentrations in compar ison to other compounds. In particular, compound 3c had a maximum inhibition percentage of 82.8 ± 0.43 % at 100 µg/mL, these results were comparable with those of the standard at that concentration. We also calculated corresponding IC50 values for these compounds and found them to be 35.61±0.45, 30.83±0.33, and 33.52±0.19 µg/mL for 3b, 3c, and 3d compounds, respectively. Generally, higher scavenging activity trends are associated with lower IC50 values, indicating greater potency to inhibit free radicals. From this study, it was observed that these compounds exhibit lower IC50 values. This implies that for 50 % inhi bition of respective free radicals, smaller concentrations of compounds were required, indicating their potency. Table S1 and Fig. 3 outline the percentage of scavenging activity of the synthesized compounds 3(a-f) at various concentrations. In H2O2 radical scavenging studies, we found the order of percentage Table 3 Docking scores, interaction types with amino acids of the co-crystal ligand (BA) and potent compounds. Compound Amino acid involved Type of bond Distance (Å) Dock score (kcal/mol) BA (Co- crystallized) GLY 46 CYS 47 ARG 127 H-Bond H-Bond Salt bridge 1.98 2.29 3.42 − 5.27 3b GLY 46 CYS 47 ARG 127 ARG 127 H-Bond H-Bond H-Bond Salt bridge 2.16 1.83 1.86 3.34 − 2.01 3c – – – − 1.99 3d PHE120 ARG 127 Pi-Pi stacking Salt bridge 5.35 2.09 − 2.07 Ascorbic acid GLY 46 CYS 47 THR 44 ARG 127 H-Bond H-Bond H-Bond Salt bridge 2.10 1.93 1.76 3.04 − 6.67 Quercetin GLY 46 H-Bond 2.31 − 5.58 C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 8 inhibition among the synthesized compounds to be 3c>3b>3d>3e>3f>3a. Specifically, compound 3c presented the highest inhibition percentage at almost all the concentrations in com parison to other compounds. The percentage inhibition of compound 3c at 100 µg/mL was 84.8 ± 0.73 %, comparable with the standard at that concentration (92.42±0.43 %). However, compounds 3b and 3d also exhibited significant activity, as the percentage of inhibition was com parable with that of the standard. As discussed earlier, these compounds demonstrated lower IC50 values (3c: 32.78±0.33 µg/mL, 3b: 36.05 ±0.61 µg/mL, and 3d: 34.78±0.43 µg/mL) indicating their potency in inhibition of free radicals. Table S2 and Fig. 4 outline the H2O2 radical scavenging activities of compounds 3(a-f) at varied concentrations. Structure-activity relationship (SAR) studies The structure-activity relationship (SAR) studies of the synthesized novel sulfonamide analogs 3(a-f) are presented in Fig. 5. From Fig. 5, it is evident that the presence of the sulfonyl group is very crucial for the activity. The sulfur atom present in the ring contributes to activity, and overall thiomorpholine scaffold shows good activity. Activity has enhanced significantly when various substitutions of R groups contain ing benzyl groups and different substitutions of functional groups at various positions on the benzyl group are clubbed to the sulfonyl group. The order of increasing activity of all the synthesized novel sulfonamide analogs with R groups follows as: 3c:4‑chloro-2-fluorobenzene>3b:o- nitrobenzene>3d:p-nitrobenzene>3e:p-nitrobenzene>3f:o-methyl benzene>3a:2,4-difluorobenzene. Fig. 11. (A) RMSD plot of simulated compounds. (B) RMSF plot of 3b, 3c, and 3d, along with BA. (C) Rg plot of MD-simulated compounds. (D) Protein-ligand interaction fractions of the 3b complex throughout MDS. C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 9 3.3. In silico predicted ADMET properties All the synthesized compounds 3(a-f), were evaluated for their drug- likeness and ADMET properties. This includes count of aromatic heavy atoms (nAH), molecular weight (M.W.), hydrogen bond acceptors (HBA), number of rotatable bonds (R.B.), topological polar surface area (TPSA), molecular refractivity (M.R.), LogP, hydrogen bond donors (HBD), and octanol/water partition coefficient (iLOGP). ADME analysis validated that all the compounds follow Lipinski’s Rule of Five with zero violations. This demonstrates the suitability for further studies and drug- like properties of these compounds, as shown in Table 2. The predicted in-silico ADME properties disclosed the important physicochemical characteristics of these compounds and are presented in supplementary data (Tables S3 and S4). 3.4. Geometry optimization of ligands Quantum chemistry techniques were used to analyze the molecular geometries of the compounds. Tuning of ligands was done to produce lower-energy and stable molecular structures, which is common in computational research, as shown in Fig. 6. In detail, the labelled opti mized structure of compound 3b is shown in the supplementary file (Figure S19). 3.5. Molecular electrostatic potential (MEPs) diagrams The ground state distributions of charge across the complete energy surface of molecules are explained by MEPs. They are essential for interpreting a molecule’s electronic structure. A study on structure- property relationships by indicating the polarity of molecules from coloration is done by MEP analysis. Red, yellow, blue, and green, respectively, stand for negative, slightly negative, positive, and neutral Fig. 12. The secondary structural and torsional profile of the PrxV-3b complex. Table 4 Ligand properties of the simulated ligands. Compound RMSD Å Rg Å Intra HB Mol SA sq. Å SASA sq. Å PSA sq. Å 3b 1.3976 3.0220 0 233.931 249.309 123.446 3c 1.1806 3.2431 0 232.017 306.930 68.600 3d 0.8499 3.3970 0 237.931 287.405 161.613 Table 5 Pre- and post-simulation binding free energies of BA (co-crystal ligand), 3b, 3c, and 3d. S. No. Compound Pre-MDS-binding free energy (kcal/mol) Post MDS-binding free energy (kcal/mol) 1 BA − 22.94 − 6.32 2 3b − 29.12 − 15.61 3 3c − 30.33 − 30.56 4 3d − 39.19 − 16.26 Fig. 13. MM-GBSA binding energy plot over run-time for all the complexes. C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 10 regions in an MEP plot. MEPs represent the distributions of charge across the complete energy surface of the molecules. The molecule is more likely to be attacked by electrophiles in regions with high negative MEP (red or yellow), whereas nucleophiles are more likely to attack regions with high positive MEP (blue). It helps in used to identify potential sites for hydrogen bond interactions. MEPs are useful in determining behaviour of enzyme-substrate complex. Fig. 7 depicts the MEP of the optimized ligands. 3.6. Frontier molecular orbitals (FMOs) Understanding of the Lowest Unoccupied Molecular Orbital (LUMO) and Highest Occupied Molecular Orbital (HOMO) are given by FMOs. Calculations of HOMO and LUMO were done using the molecular structure (optimized) of the compounds. The presence of electron-withdrawing substituents (Halogens like F, Cl and NO2) resulted in charge transfer towards the aromatic ring from HOMO (donor fragment) to LUMO (acceptor fragment) in all the Fig. 14. Trajectory images of BA-PrxV during the 100 ns MDS. Fig. 15. Trajectory images of 3b complex during 100 ns MDS. C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 11 optimized structures. In case of molecules 3b and 3d, HOMO is found at one end of molecule and LUMO is found at other end, showing charge transfer through the molecules from one end to the other. Whereas, molecule 3c showed localized HOMO-LUMO (both present in same re gion). Optimised molecules 3b, 3c, and 3d exhibited the highest bio logical activity as indicated by the lower energy gaps of 2.17, 4.23, and 1.21 eV (Fig. 8) [47]. 3.7. Molecular docking studies Peroxiredoxins (Prxs) are a class of Oxidoreductase enzymes. With a remarkably high catalytic efficiency, they engage in redox signalling and antioxidant defence, which makes them essential for cellular peroxide reduction. Some Prxs in eukaryotes are involved in the regulation of hydrogen peroxide signaling pathways in addition to their antioxidant action. Peroxiredoxins (Prxs) are vital peroxidases that use a conserved cysteine (Cys) residue to reduce peroxide substrates involved in redox signalling and antioxidant defence [48–50]. When compared with docking results of designed compounds 3b, 3c and 3d with PDBs for PrxV receptor, 1H4O (1.95 Å) and 1HD2 (1.50 Å) with the same cocrystal ligand benzoic acid (BA), we found that a similar kind of interactions between the ligands and the PrxV receptors. Dock ing results with PDB: 1H4O were comparatively better in terms of docking score as well as binding interactions with active site residues than with PDB: 1HD2. Thereby, we preferred to proceed with PDB 1H4O for our current study. Re-docking benzoic acid (co-crystallized ligand) to its native position confirmed the docking protocol with the RMSD value of 0.0633 Å (Fig. 9). Further, by keeping all parameters the same, docking was done on all the synthesized compounds into the binding pockets of the tar geted protein. Co-crystallized ligand (BA) along with other antioxidant standard drugs like ascorbic acid, and quercetin, highly active compounds were analysed by the molecular docking technique to understand their binding affinities. Fig. 10 depicts the docking postures, and Table 3 outlines the docking score and interaction types with the PrxV receptor (PDB ID: 1H4O). In comparison with the co-crystal ligand benzoic acid (BA), the docking score of all the active compounds is low, around − 2 kcal/mol. Compound 3b has a docking score of − 2.012 kcal/mol, but it has pro duced similar binding interactions as the co-crystal ligand (BA) with the active site residues GLY 46, CYS 47, and ARG 127 of PrxV (PDB 1H4O). Furthermore, docking scores of the additional reference compounds, ascorbic acid and quercetin, were higher when compared with the most active compound 3b Therefore, despite having low binding scores in comparison with co-crystal ligand benzoic acid (BA) and one of the reference compounds, ascorbic acid, the binding interaction given by compound 3b is similar to that of these two compounds (BA and ascorbic acid). 3.8. Molecular dynamics simulations (MDS) Simulations studies of the bound molecule (3b) with PDB 1H4O for 100 ns were performed to examine complex stability. PrxV receptor’s backbone RMSD value at 2.95 ns, increased to 1.582 Å, and then by 43.75 ns, the value reduced to 1.055 Å. The 3b complex, in the RMSD curve, exhibited a close match with BA (co-crystallized ligand) (Fig. 11A), indicating alignment and stability with the reference ligand. Mean RMSD value for receptor atoms was 2.541 Å and 1.880 Å for the PrxV receptor. Significant differences in the PrxV receptor’s protein chain were found by root mean square fluctuation (RMSF) analysis (Fig. 11B), especially in the N- and C-terminal regions when compared to the other regions. These fluctuations indicate the conformational changes of the receptor on binding to BA, the reference ligand. Compound BA, which demonstrated considerable docking scores, underwent 100 ns MD simulations to analyze the stability of the complex. Hydrogen-bonding and hydrophobic interactions were dis played by stable regions during simulation. BA demonstrated a stable radius of gyration (Rg) with an average value of 2.11 Å (Fig. 11C) and presented a high SASA during MDS. Interactions with stability were seen at residues ARG 124, ALA 42, ASN 76, PHE 43, VAL 80, PHE 120, and ARG 127 in the simulation study of compound 3b ALA 42, ARG 124, ASN 76, and ARG 127 formed hydrogen bonds. Also, hydrophobic interactions were exhibited by PHE 120. Hydrogen bonding interactions with the highest frequencies were exhibited by compound 3b During the study, H-bond networks were observed for 27 % at ARG 127, and hydrophobic interactions for 7 % of the time with PHE 120. For stabilizing the PrxV-3b complex, no water- mediated H-bond networks contributed during simulation (Fig. 11D) [51]. The selection of a target for docking studies plays a crucial role in supporting the biological results obtained. Our target peroxiredoxins (Prxs) have remarkably high catalytic efficiency in redox signaling and antioxidant defence via a conserved cysteine (Cys) residue. The required interactions in the active site of PrxV by co-crystal ligand BA which are essential for anti-oxidant activity, were also observed in case of com pound 3b with active site residue Cys 47 in molecular docking and MD simulation, thereby supporting the potent anti-oxidant activity shown in biological studies. The secondary structural and torsional profiles of the PrxV-3b complex are presented in Fig. 12. Ligand properties of the simulated compounds 3b, 3c and 3d are outlined in Table 4. Figures S20–S27 present the interaction graphs and secondary structural properties of the compounds 3c, 3d and BA during MDS. 3.9. Estimation of binding-free energy To compute binding free energies, MM-GBSA studies were performed on complexes from docking and dynamic simulations. Stronger protein- ligand interactions are indicated by lower binding energy values, explaining improved binding affinity. Specifically, the BA (co-crystal lized ligand) displayed maximum binding energy. The total binding energies (ΔG) after docking was − 22.94 kcal/mol for BA, for 3b it was − 29.12 kcal/mol, − 30.33 kcal/mol for compound 3c, and for com pound 3d it was − 39.19 kcal/mol. Binding free energies for 3b, 3c, 3d, and BA pre-MD simulations and post-MD simulations are outlined in Table 5. Protein residues contrib uting to the lowest binding energy include ASN 76, PHE 120, ALA 42, ARG 124, VAL 80, PHE 43, and ARG 127. In case of post MD MMGBSA analysis, co-crystal ligand BA showed the highest binding energy (− 6.32 kcal/mol) in all of them because of the maximum instability of the ligand in the active site. The 3b complex displayed an average ΔG binding energy of − 15.61 kcal/mol, while 3d exhibited − 16.26 kcal/mol. Minimum binding energy of − 30.56 kcal/ mol was shown by the 3c complex. Fig. 13 depicts the binding energy plot for all the complexes. Post-simulation MMGBSA analysis was performed on BA-1H4O complex to check the stability of the complex in terms of binding free energy throughout the course of simulation. In trajectory analysis (Fig. 14), it was observed that the co-crystal ligand came out of the binding pocket, because of missing binding interactions with active site key residues such as GLY 46, CYS 47, and ARG 127 over period of time somewhere around 76.35 ns to the end of the MD simulation, which led the MMGBSA value to shift drastically. Post-simulation binding free energy was calculated as the average of all the binding free energies of protein-ligand complex at particular in tervals throughout simulation. When the average was taken, there was a decrease in binding free energy of post-MD 3d-1H4O complex in com parison with the pre-MD 3d-1H4O complex. The MMGBSA value was decreased from − 39.19 kcal/mol to − 16.26 kcal/mol, making it less stable. The fluctuations were seen less in case of compound 3b over co- C.R. Santhosh et al. Journal of Molecular Structure 1346 (2025) 143201 12 crystal BA while analyzing MD simulation trajectory snapshots (Fig. 15) but when compared with the co-crystal ligand benzoic acid i.e., BA-PrxV complex, it was found to be more stable. Docking followed by dynamic results disclosed that effective binding interactions with receptor (PrxV) was exhibited by 3b compound. By presenting good stability during the simulations, the 3b compound can be the lead for binding at the active site of PrxV. 4. Conclusion A new series of novel thiomorpholine-bearing sulfonamides were synthesized in good to excellent yields (87–95 %). To the best of our knowledge, this study gives the synthesis of thiomorpholine-containing sulfonamides as potentially effective antioxidant drug candidates. In both H2O2 and DPPH radical scavenging methods, compounds 3b, 3c and 3d shown significant antioxidant activity compared with the stan dard drug, ascorbic acid. These compounds with the highest percentage of inhibition presented lower IC50 values, as it indicates that minimum amounts were required to achieve 50 % inhibition, and these values were comparable to those of a standard drug, ascorbic acid. In-silico ADME results validated the drug-like properties of all the compounds as they obeyed Lipinski’s rule. Computational analysis revealed the sta bility followed by strong binding affinity of the PrxV-3b complex. In- vitro results were validated by the binding studies of 3b complex at the active site of PrxV receptor. Future research should focus on optimizing the structural features of these sulfonamide analogs to enhance their binding affinities, biological activity, and drug-likeness, including sol ubility, stability, and bioavailability. Experimental validation through comprehensive in-vitro and in-vivo studies are essential to evaluate their antioxidant potential, pharmacokinetics, toxicity, and overall safety. CRediT authorship contribution statement C.R. Santhosh: Conceptualization, Data curation, Formal analysis, Resources, Writing – original draft, Writing – review & editing. Sam path Chinnam: Conceptualization, Supervision, Methodology, Re sources, Formal analysis, Writing – original draft, Writing – review & editing. Hrushikesh S. Chaudhari: Conceptualization, Data curation, Formal analysis, Resources, Writing – original draft, Writing – review & editing. Kondapalli Venkata Gowri Chandra Sekhar: Conceptualiza tion, Supervision, Methodology, Resources, Formal analysis, Writing – original draft, Writing – review & editing. Gbolahan O. Oduselu: Writing – review & editing. Vishal Ahuja: Conceptualization, Supervi sion, Methodology, Resources, Formal analysis, Writing – original draft, Writing – review & editing. Kumarappan Chidambaram: Writing – review & editing. Nagaraju Kerru: Conceptualization, Supervision, Methodology, Resources, Formal analysis, Writing – original draft, Writing – review & editing. G.M. Madhu: Writing – review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful to the M. S. Ramaiah Institute of Technol ogy, Bengaluru 560054, Karnataka, India under the SEED money proj ect: MSRIT/Admin/1575/23–24. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University, Saudi Arabia for supporting this work through a Large Group Research Project under grant number RGP.2/189/46. The author C. R. Santhosh is grateful to M.S. 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