Exploring the Potential of New Benzamide-Acetamide Pharmacophore Containing Sulfonamide as Urease Inhibitors: Structure–Activity Relationship, Kinetics Mechanism, and In Silico Studies

Abstract

The search for novel drug scaffolds that can improve effectiveness and safety through drug conjugates is a promising approach. Consequently, drug conjugates constitute a dynamic field of study and advancement within medicinal chemistry. This research demonstrates the conjugation of diclofenac and mefenamic acid with sulfa drugs and their screening for urease inhibition. These conjugates’ structural confirmation was performed using elemental analysis and spectroscopic methods, including IR, ¹H NMR, and ¹³C NMR. Diclofenac conjugated with sulfanilamide (4), sulfacetamide (10), and mefenamic acid conjugated with sulfanilamide (12), and sulfamethoxazole (17) was found potent and demonstrated urease inhibition competitively, with IC₅₀ (μM) values 3.59 ± 0.07, 5.49 ± 0.34, 7.92 ± 0.27, and 8.35 ± 0.26, respectively. Diclofenac conjugated with sulfathiazole (6), sulfamerazine (8), and sulfaguanidine (11), while mefenamic acid conjugated with sulfisoxazole (13), sulfathiazole (14), and sulfadiazine (15) exhibited a mixed mode of urease inhibition. The IC₅₀ (μM) values were 16.19 ± 0.21, 9.50 ± 0.28, 4.35 ± 0.23, 15.86 ± 0.25, 14.80 ± 0.27, and 7.92 ± 0.27, respectively. Furthermore, molecular docking studies were employed to predict the binding pose of competitive inhibitors at the urease active site. These conjugates generated stable complexes with the urease protein observed through molecular dynamics (MD) simulations, where no conformational changes occurred throughout the simulations. These results highlight the potential for approved therapeutic molecule conjugates to give rise to new categories of pharmacological agents for urease inhibition. The structural similarity of sulfonamides with urea allows them to compete with urea for binding to the active site of the urease enzyme. Sulfonamides and nonsteroidal anti-inflammatory drugs (NSAIDs) can interact hydrophobically with the active site of the urease enzyme, which may disturb its structure and catalytic activity. Therefore, these conjugates may be helpful in the development of novel pharmacological agents for the treatment of a variety of illnesses in which the urease enzyme is involved.

1. Introduction

Enzyme inhibitors exist both naturally and are purposefully synthesized as pharmaceuticals. Most naturally occurring toxins function as organic enzyme inhibitors. Synthetic enzyme inhibitors are employed as medications for treating various medical conditions.¹⁻³ Inhibitors are molecules capable of interfering with enzymatic activity by attaching to the enzyme’s active site either temporarily or permanently. They obstruct the enzyme’s active sites, thereby halting the enzymatic biological reaction.⁴ Urease, also referred to as urea amidohydrolase, is an enzyme that contains the metal (Ni).⁵ Of the various ureases employed in enzyme inhibition studies, the one derived from jack bean was the initial specimen to undergo comprehensive characterization and crystallization. The active site-bearing unit contains approximately 840 amino acids, and the catalytic process relies on the presence of Ni²⁺ within the active site.⁶ Developing efficient and safe urease inhibitors has received significant attention from the pharmacological research community primarily because ureases play a crucial role in various disease conditions. The ongoing production of ammonia contributes to heightened gastric mucosa permeability, resulting in states such as ulcers, inflammation, lymphoma, and adenocarcinoma.^7,8 To remain alive in the stomach’s extremely acidic environment, the pathogenic bacteria Helicobacter pylori uses the enzyme “urease” to produce excessive amounts of ammonia through the hydrolysis of urea in the gastric mucosa, and about 50% of the world’s population is affected by this bacterial infection.⁹ Focusing on inhibiting urease activity can potentially eliminate the H. pylori bacterium at the initial stages of the disease because it relies on urease for its survival in the acidic stomach environment. Our primary area of research revolves around designing new urease inhibitors, as urease plays a critical role in bacterial infections, and the existing options for urease inhibitors are currently quite limited.^10,11

Sulfonamides, also known as sulfa drugs, feature the –SO₂NH– functional group, obtained by replacing the hydroxyl group of the sulfonic acid group (RSO₃H) through a reaction with an amino group. Sulfonamides exhibit diverse pharmacological effects because of the presence of a sulfonyl functional group. This diversity places them in a distinctive position within the pharmaceutical industry and the field of medicinal chemistry.^12,13 Sulfonamides display an extensive range of pharmacological effects, encompassing antibacterial, antifungal, diuretic, antihypertensive, antithyroid, anticonvulsant, hypoglycemic, antidiabetic, protease inhibitory, antimigraine, anticarbonic anhydrase, anti-inflammatory, antiurease, and herbicidal properties.¹⁴⁻¹⁹ There are four types of urease inhibitors, including chelators of the Ni atom at the active site, hydroxamic acid, phosphoramidates, and thiolate compounds.²⁰ Finding efficient and secure urease inhibitors has recently proven difficult for many scientists working in the pharmaceutical sector. To prepare safe and secure urease inhibitors, a variety of sulfa drugs and NSAID derivatives have been thoroughly studied in the past decade (Figure 1). This research identified a few drug-based conjugates that would be the best pharmacological agents for the inhibition of the urease enzyme.^11,21,22 The nonsteroidal anti-inflammatory drugs (NSAIDs) and their derivatives, such as acetylsalicylic acid, ibuprofen, diclofenac, mefenamic acid, flurbiprofen, naproxen, and piroxicam, were investigated for their ability to exhibit a range of biological activities, including inhibiting enzymes like α-glucosidase, β-glucuronidase, α-amylase, and urease.^4,11,23−27

Figure 1.

NSAIDs and sulfa-drug-derived compounds as urease inhibitors.

Our objective was to design and synthesize the conjugates that combine mefenamic acid and diclofenac with sulfa drugs, employing the multitarget approach that is gaining popularity among pharmaceutical chemists worldwide. The synthesis of derivatives plays a crucial role, primarily focused on altering the effects of current medications, with a specific emphasis on minimizing adverse effects while enhancing their efficacy. Existing literature has established that over 60% of drugs are derived from previously known molecules. Therefore, in light of this, novel combinations were synthesized by linking NSAIDs (mefenamic acid and diclofenac) with sulfa drugs (including sulfaguanidine, sulfacetamide, sulfamethoxazole, sulfanilamide, sulfamerazine, sulfisoxazole, sulfadiazine, and sulfathiazole) through an amide coupling reaction conducted in situ. We subjected the recently created combinations to urease inhibition screening. Preserving the essential pharmacophore in our intended compounds is a fundamental aspect of our design approach. This approach entails identifying and developing new therapeutic applications for existing drugs that rationalize the repurposing of drugs. This strategy may be less expensive and time-consuming than creating entirely new therapeutic agents from scratch. The present study was rationalized on the structural similarity of sulfonamides with urea. This structural mimic allows them to compete with urea to bind the urease enzyme’s active site. Also, the structure and catalytic activity of the urease enzyme can be disturbed by the hydrophobic interaction of sulfonamides with the active site of the urease enzyme. Additionally, we conducted molecular docking and dynamic simulation analyses to assess the performance of the most effective inhibitors as ligands versus the urease enzyme. An investigation of the molecular dynamics (MD) trajectories produced over a 300 ns simulation helped to further validate the stability of these highly potent inhibitors in their interaction with the enzyme.

2. Experimental Section

2.1. Chemistry

2.1.1. General

The newly developed conjugates were prepared using high-purity sulfa drugs sourced from Sigma-Aldrich and obtained from Falcon Scientific, Lahore-Pakistan. NSAIDs were generously provided by Novamed Pharmaceuticals, Lahore-Pakistan. The structural characteristics of the conjugates were determined through spectral investigations, employing techniques such as Fourier transform infrared (FTIR) spectroscopy, ¹H NMR-400 MHz, and ¹³C NMR-100 MHz (Bruker). Elemental analysis for carbon (C), hydrogen (H), nitrogen (N), and sulfur (S) was performed using Thermo Scientific HT+ elemental analyzer from the U.K. Purity assessment of the synthesized conjugates under UV light was performed using precoated silica thin-layer chromatography (TLC) plates from Merck, Germany, whereas the melting point was determined using the Gallenkamp apparatus.

2.1.2. Synthesis Protocol for New Conjugates

A solvent mixture containing methanol (MeOH) and acetonitrile (ACN) in 50:50 ratio was employed to couple the NSAIDs (diclofenac and mefenamic acid) with sulfa drugs including sulfaguanidine, sulfacetamide, sulfamethoxazole, sulfanilamide, sulfamerazine, sulfisoxazole, sulfadiazine, and sulfathiazole. 1 mmol each of diclofenac and coupling agent N,N′-dicyclohexylcarbodiimide (DCC) was charged to a 100 mL round-bottom flask, and the reaction was initiated by 4-dimethylaminopyridine (DMAP) as a catalyst, and the reaction was carried out for 30 min at 80 °C. Following the addition of 1 mmol of the respective sulfa drug, the reaction mixture was refluxed for 42 h to precipitate the dicyclohexylurea (DCU) (dicyclohexylurea), confirming the amide bond formation. The eluent composed of n-hexane:EtOAc:DCM:MeOH in a ratio of 50:24:15:10 was employed to check the progress of the reaction by applying thin-layer chromatography (TLC). DCU precipitates were filtered out, and the filtrate contained the product, which was obtained in a solid form by evaporating the solvent using a rotary evaporator. The raw product was purified by using flash chromatography with ACN/MeOH (25:1) as the eluent. The mefenamic acid-sulfa drug conjugates were synthesized by opting for the same procedure.

2.1.3. Analytical Details of Diclofenac-Sulfa Drug Conjugates

2.1.3.1. 2-((2,3-Dimethylphenyl)amino)-N-(4-sulfamoylphenyl)benzamide (4)

White crystalline solid; yield: 71.5%; mp: 173–175 °C; R_f: 0.76 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3463 (–NH, sulfonyl), 3011 (=C–H, aromatic), 2921 (–NH, amide), 1712 (–C = O), 1591 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δH 3.75 (s, 2H, CH₂), 5.82 (s, 2H, NH₂), 6.22 (d, 1H, J = 8.0 Hz, ArH), 6.56 (d, 1H, J = 8.0 Hz, ArH), 6.91 (app td, 1H, J = 8.0, 4.0 Hz, ArH), 7.09–7.02 (m, 4H, ArH), 7.46–7.41 (m, 3H, ArH), 7.52 (app dd, 1H, J = 8.0, 4.0 Hz, ArH), 7.78 (brs, 1H, NH), 10.5 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 167.7 (C = O), 140.7 (=C-NH–), 139.9, 136.9, 135.2, 130.3, 129.5, 129.1, 128.0, 126.9, 126.2, 121.2, 119.7, 116.2, 32.2 (–CH₂–). Anal. calculated for C₂₀H₁₇Cl₂N₃O₃S (450.33 g/mol): C, 53.34; H, 3.81; N, 9.33; O, 10.66; S, 7.12%, Found; C, 53.55; H, 3.62; N, 9.25; O, 10.88; S, 7.07%.

2.1.3.2. 2-(2-((2,6-Dichlorophenyl)amino)phenyl)-N-(4-(N-(3,4-dimethylisoxazol-5-yl)sulfamoyl)phenyl)acetamide (5)

White crystalline solid; yield: 67.4%; mp 202–205 °C; R_f: 0.77 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3465 (–NH, sulfonyl), 3016 (=C–H, aromatic), 2926 (–NH, amide), 2836 (O–CH₃), 1710 (–C = O), 1628 (–C = N–, imine), 1594 (–CH = CH–), 1361 (–NH-S = O, asymmetric), 1138 (–NH-S = O, symmetric), 1024 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.90 (s, 6H, 2CH₃), 3.75 (s, 2H, CH₂), 5.60 (s, 1H, NH), 6.33 (d, 1H, J = 8.0 Hz, ArH), 6.67 (app td, 1H, J = 1.2, 8.0 Hz, ArH), 7.19–7.05 (m, 7H, ArH), 7.52 (d, 2H, J = 8.0 Hz, ArH), 7.78 (brs, 1H, NH), 8.53 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 169.3 (–C = O), 159.0 (–O–C=), 153.7 (–N = C-NH–), 143.5, 140.6, 139.2, 137.7, 135.1, 131.4, 129.7, 127.9, 125.6, 121.4, 116.9, 100.1, 32.2 (–CH₂–), 13.7 (–CH₃). Anal. calculated for C₂₅H₂₂Cl₂N₄O₄S (545.43 g/mol): C, 54.05; H, 4.07; N, 10.27; O, 11.73; S, 5.88% Found; C, 54.25; H, 3.91; N, 10.25; O, 11.61; S, 6.01%.

2.1.3.3. 2-(2-((2,6-Dichlorophenyl)amino)phenyl)-N-(4-(N-(thiazol-2-yl)sulfamoyl)phenyl)acetamide (6)

White crystalline solid; yield: 67.4%; mp: 202–205 °C; R_f: 0.77 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3011 (=C–H, aromatic), 2919 (–NH, amide), 1708 (–C = O), 1622 (–C = N–, amide), 1594 (–CH = CH–), 1361 (–NH-S = O, asymmetric), 1138 (–NH-S = O, symmetric), 1021 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δH 3.75 (s, 2H, CH₂), 5.58 (s, 1H, NH), 6.59 (d, 1H, J = 8.0 Hz, CH), 6.87 (d, 1H, J = 8.0 Hz, CH), 7.00 (d, 1H, J = 4.0 Hz, ArH), 7.11–7.05 (m, 6H, ArH), 7.18 (d, 2H, J = 8.0 Hz, ArH), 7.40 (d, 1H, J = 8.0 Hz, ArH), 7.52 (d, 1H, J = 8.0 Hz, ArH), 7.79 (brs, 1H, NH), 8.53 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 169.3 (–N = C-NH–), 166.5 (–C = O), 143.5, 138.5, 137.8, 135.9, 131.4, 129.7, 128.1, 127.9, 125.6, 121.4, 119.0, 116.9, 112.7 (=C–S–), 107.4, 32.2 (–CH₂–). Anal. calculated for C₂₃H₁₈Cl₂N₄O₃S₂ (533.44 g/mol): C, 51.79; H, 3.40; N, 10.50; O, 9.00; S, 12.02% Found; C, 51.55; H, 3.62; N, 10.25; O, 9.13; S, 12.07%.

2.1.3.4. 2-(2-((2,6-Dichlorophenyl)amino)phenyl)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)acetamid (7)

White crystalline solid; yield: 68.1%; mp: 172–174 °C; R_f: 0.84 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2921 (–NH, amide), 1708 (–C = O), 1621 (–CH = N–, imine), 1588 (–CH = CH–), 1359 (–NH-S = O, asymmetric), 1139 (–NH-S = O, symmetric), 1023 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δH 3.75 (s, 2H, CH₂), 5.79 (brs, 1H, NH), 6.33 (d, 1H, J = 8.0 Hz, ArH), 6.59–6.53 (m, 2H, ArH), 6.86 (t, 1H, J = 8.0 Hz, ArH), 7.15–7.05 (m, 4H, ArH), 7.18 (d, 2H, J = 8.0 Hz, ArH), 7.52 (d, 2H, J = 8.0 Hz, ArH), 8.09 (brs, 1H, NH), 8.36 (brs, 1H, NH), 8.48 (d, 2H, J = 8.0 Hz, ArH). ¹³C NMR (100 MHz, DMSO-d₆): δC 169.2 (–N = C-NH–), 160.2 (–C = O), 153.7 (–C = N–), 149.8 (=C–N = C–), 143.5, 139.9, 137.8, 136.2, 133.2, 131.3, 129.7, 127.9, 125.6, 121.4, 116.9, 32.2 (–CH₂–). Anal. calculated for C₂₄H₁₉Cl₂N₅O₃S (528.41 g/mol): C, 54.55; H, 3.62; N, 13.25; O, 9.08; S, 6.07% Found; C, 54.75; H, 3.69; N, 13.45; O, 9.28; S, 6.19%.

2.1.3.5. 2-(2-((2,6-Dichlorophenyl)amino)phenyl)-N-(4-(N-(4-methylpyrimidin-2-yl)sulfamoyl)phenyl)acetamide (8)

White crystalline solid; yield: 70.2%; mp 168–173 °C; R_f: 0.79 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3463 (–NH, sulfonyl), 3021 (=C–H, aromatic), 2924 (–NH, amide), 2836 (O–CH₃), 1708 (–C = O), 1618 (–C = N–, imine), 1591 (–CH = CH–), 1362 (–NH-S = O, asymmetric), 1142 (–NH-S = O, symmetric), 1031 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δH 1.91 (s, 3H, CH₃), 3.75 (s, 2H, CH₂), 5.57 (brs, 1H, NH), 6.33 (d, 1H, J = 8.0 Hz, ArH), 6.87 (t, 1H, J = 8.0 Hz, ArH), 7.11–7.05 (m, 6H, ArH), 7.17 (d, 2H, J = 8.0 Hz, ArH), 7.52 (d, 2H, J = 8.0 Hz, ArH), 7.79 (brs, 1H, NH), 8.23 (brs, 1H, NH), 8.52 (d, 1H, J = 8.0 Hz, ArH). ¹³C NMR (100 MHz, DMSO-d₆): δC 174.0 (–N = C-NH–), 169.3 (–C = O), 153.7 (–C = N–), 143.5 (=C–N = C–), 140.4, 139.2, 137.7, 136.9, 133.0, 131.4, 129.7, 127.9, 125.6, 125.5, 121.4, 121.4, 116.9, 32.2 (–CH₂–), 25.8 (–CH₃). Anal. calculated for C₂₅H₂₁Cl₂N₅O₃S (542.43 g/mol): C, 55.36; H, 3.90; N, 12.91; O, 8.85; S, 5.91% Found; C, 55.45; H, 3.95; N, 12.78; O, 9.08; S, 6.07%.

2.1.3.6. 2-(2-((2,6-Dichlorophenyl)amino)phenyl)-N-(4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl)acetamide (9)

White crystalline solid; yield: 67.9%; mp: 165–167 °C; R_f: 0.81 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3459 (–NH, sulfonyl), 3022 (=C–H, aromatic), 2923 (–NH, amide), 1712 (–C = O), 1617 (–C = N–, imine), 1590 (–CH = CH–), 1361 (–NH-S = O, asymmetric), 1138 (–NH-S = O, symmetric), 1031 (–S = O), 550–870 (C–Cl), 1710 (C = O); ¹H NMR (400 MHz, DMSO-d₆): δH 1.90 (s, 3H, CH₃), 3.75 (s, 2H, CH₂), 5.58 (brs, 1H, NH), 5.76 (s, 1H, CH), 6.33 (d, 1H, J = 8.0 Hz, ArH), 6.87 (t, 1H, J = 8.0 Hz, ArH), 7.11–7.05 (m, 6H, ArH), 7.18 (d, 2H, J = 8.0 Hz, ArH), 7.52 (d, 1H, J = 8.0 Hz, ArH), 7.78 (brs, 1H, NH), 8.52 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 172.4 (–N = C-NH–), 169.3 (–C = O), 153.7 (C = N), 143.5, 141.5, 140.0, 137.7, 134.8, 131.4, 129.7, 127.9, 125.6, 123.2, 121.4, 120.0, 116.9, 93.6, 32.2 (–CH₂–), 20.0 (2 × –CH₃). Anal. calculated for C₂₄H₂₀Cl₂N₄O₄S (531.40 g/mol): C, 54.25; H, 3.79; N, 10.54; O, 12.04; S, 6.03% Found; C, 54.55; H, 3.62; N, 10.25; O, 12.18; S, 6.07%.

2.1.3.7. N-(4-(N-Acetylsulfamoyl)phenyl)-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetamide (10)

White crystalline solid; yield: 66.7%; mp: 235–237 °C; R_f: 0.81 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3458 (–NH, sulfonyl), 3021 (=C–H, aromatic), 2921 (–NH, amide), 1710 (–C = O), 1594 (–CH = CH–), 1361 (–NH-S = O, asymmetric), 1142 (–NH-S = O, symmetric), 1031 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δH 1.70 (s, 3H, CH₃), 3.75 (s, 2H, CH₂), 5.37 (brs, 1H, NH), 6.22 (d, 1H, J = 8.0 Hz, ArH), 6.45 (d, 1H, J = 8.0 Hz, ArH), 6.72 (app td, 1H, J = 8.0, 4.0 Hz, ArH), 6.91 (app td, 1H, J = 8.0, 4.0 Hz, ArH), 7.08–7.03 (m, 3H, ArH), 7.38 (d, 2H, J = 8.0 Hz, ArH), 7.45 (d, 1H, J = 8.0 Hz, ArH), 7.52 (app dd, 1H, J = 8.0, 4.0 Hz, ArH), 8.42 (brs, 1H, NH), 10.48 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 173.2 (–HN-C = O), 167.0 (–C = O), 144.5, 141.2, 138.8, 137.0, 132.8, 129.5, 128.6, 125.9, 124.2, 120.1, 119.2, 115.8, 112.3, 126.9, 34.8 (–CH₂–), 25.8 (–CH₃). Anal. Calculated for C₂₃H₂₆Cl₂N₄O₃S (492.37 g/mol): C, 53.67; H, 3.89; N, 8.53; O, 13.00; S, 6.51% Found; C, 53.55; H, 3.62; N, 8.25; O, 13.23; S, 6.66%.

2.1.3.8. N-(4-(N-Carbamimidoylsulfamoyl)phenyl)-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetamide (11)

White crystalline solid; yield: 63.8%; mp 105–107 °C; R_f: 0.73 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3019 (=C–H, aromatic), 2921 (–NH, amide), 1710 (–C = O), 1619 (–CH = N–, imine), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O), 550–870 (C–Cl). ¹H NMR (400 MHz, DMSO-d₆): δH 3.75 (s, 2H, CH₂), 5.65 (s, 1H, NH), 5.73 (s, 1H, NH), 6.55 (d, 1H, J = 8.0 Hz, ArH), 6.74 (brs, 2H, NH₂), 7.20–7.01 (m, 6H, ArH), 7.39 (d, 2H, J = 8.0 Hz, ArH), 7.46 (d, 1H, J = 8.0 Hz, ArH), 7.52 (app dd, 1H, J = 8.0, 4.0 Hz, ArH), 8.43 (brs, 1H, NH), 10.05 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 171.0 (–C = NH), 158.4 (–C = O), 143.7, 142.1, 138.5, 134.9, 131.4, 130.9, 129.4, 127.8, 126.1, 124.3, 121.3, 120.3, 112.8, 33.8 (–CH₂–). Anal. Calculated for C₂₁H₁₉Cl₂N₅O₃S (492.75 g/mol): C, 51.23; H, 3.89; N, 14.22; O, 9.75; S, 6.51% Found; C, 51.55; H, 3.62; N, 14.35; O, 9.81; S, 6.61%.

2.1.4. Analytical Details of Mefenamic Acid-Sulfa Drug Conjugates

2.1.4.1. 2-((2,3-Dimethylphenyl)amino)-N-(4-sulfamoylphenyl)benzamide (12)

White crystalline solid; yield: 70.8%; mp 202–210 °C; R_f: 0.82 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3462 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2921 (–NH, amide), 1710 (–C = O), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δH 2.28 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 5.81 (brs, 2H, NH₂), 6.60 (d, 1H, J = 8.0 Hz, ArH), 7.08–7.00 (m, 4H, ArH), 7.20–7.11 (m, 3H, ArH), 7.46 (d, 2H, J = 8.0 Hz, ArH), 7.89 (dd, 1H, J = 8.0, 4.0 Hz, ArH), 8.11 (brs, 1H, NH), 9.19 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 168.8 (–C = O), 154.6 (=C-NH–), 152.4, 149.1, 140.5, 138.6, 136.0, 135.0, 132.0, 131.7, 130.5, 127.9, 126.6, 123.0, 116.9, 112.9, 20.7 (–CH₃), 14.1 (–CH₃). Anal. Calculated for C₂₁H₂₁N₃O₃S (395.48 g/mol): C, 63.78; H, 5.35; N, 10.63; O, 12.14; S, 8.11% Found; C, 63.69; H, 5.48; N, 10.87; O, 12.41; S, 8.31%

2.1.4.2. N-(4-(N-(3,4-Dimethylisoxazol-5-yl)sulfamoyl)phenyl)-2-((2,3-dimethylphenyl)amino)benzamide (13)

White crystalline solid; yield: 72.5%; mp: 241–248 °C; R_f: 0.83 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3460 (–NH, sulfonyl), 3021 (=C–H, aromatic), 2921 (–NH, amide), 1712 (–C = O), 1633 (–CH = N–, imine), 1594 (–CH = CH–), 1364 (–NH-S = O, asymmetric), 1138 (–NH-S = O, symmetric), 1029 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δH 2.08 (s, 3H, CH₃), 2.16 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 6.58 (d, 1H, J = 8.0 Hz, ArH), 6.75–6.67 (m, 3H, ArH), 7.07–6.99 (m, 3H, ArH), 7.20–7.11 (m, 1H, ArH), 7.37 (d, 2H, J = 8.0 Hz, ArH), 7.89 (dd, 1H, J = 8.0, 2.0 Hz, ArH), 7.95 (brs, 1H, NH), 8.20 (brs, 1H, NH), 9.18 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 168.8 (–C = O), 161.4 (=C-NH–), 153.4 (–C = N–), 149.1, 140.2, 138.4, 135.0, 132.0, 131.7, 129.0, 126.6, 126.3, 123.0, 118.1, 116.9, 113.8, 102.7, 31.2 (–CH₃), 14.1 (–CH₃), 10.8 (–CH₃), 6.4 (–CH₃). Anal. Calculated for C₂₆H₂₆N₄O₄S (390.58 g/mol): C, 63.66; H, 5.34; N, 11.42; O, 13.05; S, 6.54% Found; C, 63.91; H, 5.58; N, 11.67; O, 13.21; S, 6.31%.

2.1.4.3. 2-((2,3-Dimethylphenyl)amino)-N-(4-(N-(thiazol-2-yl)sulfamoyl)phenyl)benzamide (14)

White crystalline solid; yield: 78.1%; mp 183–186 °C; R_f: 0.84 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2921 (–NH, amide), 2838 (O–CH₃), 1710 (–C = O), 1631 (–C = N-, imine), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δH 2.10 (s, 3H, CH₃), 2.29 (s, 3H, CH₃), 6.56 (d, 1H, J = 8.0 Hz, ArH), 6.75 (d, 1H, J = 8.0 Hz, CH), 6.96 (d, 1H, J = 8.0 Hz, CH), 7.15–7.04 (m, 6H, ArH), 7.19 (d, 1H, J = 4.0 Hz, ArH), 7.44 (d, 2H, J = 8.0 Hz, ArH), 7.92 (dd, 1H, J = 8.0, 2.0 Hz, ArH), 7.96 (brs, 1H, NH), 8.17 (brs, 1H, NH), 9.19 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 171.3 (N = C-NH–), 168.8 (–C = O), 149.1 (=C-NH–), 146.7, 139.6, 138.6, 135.0, 133.4, 132.3, 131.7, 130.8, 128.6, 127.2, 126.3, 125.6, 123.0, 121.3, 116.9, 110.9, 20.7 (–CH₃), 14.1 (–CH₃). Anal. calculated for C₂₄H₂₂N₄O₃S₂ (478.59 g/mol): C, 60.23; H, 4.63; N, 11.71; O, 10.03; S, 13.40% Found; C, 60.23; H, 4.78; N, 11.87; O, 10.15; S, 3.34%.

2.1.4.4. 2-((2,3-Dimethylphenyl)amino)-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)benzamide (15)

White crystalline solid; yield: 74.3%; mp: 163–165 °C; R_f: 0.82 0.84 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2921 (–NH, amide), 1710 (–C = O), 1632 (–CH = N–, imine), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δH 2.09 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 6.56 (d, 1H, J = 8.0 Hz, ArH), 6.75 (d, 1H, J = 8.0 Hz, CH), 7.07–6.98 (m, 4H, ArH), 7.16 (d, 1H, J = 4.0 Hz, ArH), 7.32 (d, 2H, J = 8.0 Hz, ArH), 7.61 (d, 1H, J = 8.0 Hz, ArH), 7.89 (dd, 2H, J = 8.0, 1.6 Hz, ArH), 7.96 (s, 1H, NH), 8.11 (brs, 1H, NH), 8.47 (d, 2H, J = 4.0 Hz, ArH), 9.19 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 168.8 (N = C-NH–), 158.7 (–C = O), 154.1 (–C = N–), 149.1, 138.6, 138.4, 135.0, 132.0, 131.7, 130.2, 127.2, 126.6, 123.0, 116.9, 113.8, 112.6, 110.9, 20.7 (–CH₃), 14.1 (–CH₃). Anal. Calculated for C₂₅H₂₃N₅O₃S (473.55 g/mol): C, 63.41; H, 4.90; N, 14.79; O, 10.14; S, 6.77%, Found; C, 63.59; H, 4.94; N, 14.87; O, 10.25; S, 6.91%.

2.1.4.5. 2-((2,3-Dimethylphenyl)amino)-N-(4-(N-(4-methylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (16)

White crystalline solid; yield: 75.6%; mp 159–161 °C; R_f: 0.81 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3462 (–NH, sulfonyl), 3021 (=C–H, aromatic), 2923 (–NH, amide), 1710 (–C = O), 1628 (–C = N–, imine), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δ_H 2.09 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 2.31 (s, 3H, CH₃), 6.56 (d, 2H, J = 8.0 Hz, ArH), 6.89 (d, 2H, J = 8.0 Hz, ArH), 7.15–6.99 (m. 3H, ArH), 7.36–7.29 (m. 2H, ArH), 7.63 (d, 1H, J = 8.0 Hz, ArH), 7.89 (dd, 2H, J = 8.0, 2.0 Hz, ArH), 7.96 (brs, 1H, NH), 8.10 (brs, 1H, NH), 8.31 (d, 1H, J = 4.0 Hz, ArH), 9.18 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 168.4 (N = C-NH–), 158.1 (–C = O), 153.4 (–C = N–), 149.5, 144.4, 140.5, 138.4, 135.0, 131.7, 130.5, 127.2, 126.6, 125.5, 123.0, 121.3, 119.2, 116.9, 115.2, 113.8, 112.5, 110.9, 23.8 (–CH₃), 20.7 (–CH₃), 14.1 (–CH₃). Anal. calculated for C₂₆H₂₅N₅O₃S (487.58 g/mol): C, 64.05; H, 5.17; N, 14.36; O, 9.84; S, 6.58% Found; C, 64.71; H, 5.28; N, 14.37; O, 9.95; S, 6.35%.

2.1.4.6. 2-((2,3-Dimethylphenyl)amino)-N-(4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl)benzamide (17)

White crystalline solid; yield: 73.5%; mp: 177–180 °C; R_f: 0.74 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2921 (–NH, amide), 1710 (–C = O), 1634 (–C = N–, imine), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1028 (–S = O). ¹H NMR (400 MHz, DMSO-d₆) δ_H 2.08 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 6.09 (s, 1H, CH), 6.58 (d, 1H, J = 8.0 Hz, ArH), 6.75–6.65 (m, 3H, ArH), 7.14–6.99 (m. 3H, ArH), 7.36–7.30 (m, 1H, ArH), 7.47 (d, 2H, J = 8.0 Hz, ArH), 7.89 (dd, 1H, J = 8.0, 2.0 Hz, ArH), 7.95 (brs, 1H, NH), 8.13 (brs, 1H, NH), 9.18 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO) δ_C 170.1 (–O-C = C–), 168.8 (–C = O), 158.7 (–C = N–), 153.6 (=C-NH–), 149.1, 138.6, 138.4, 135.0, 132.0, 131.7, 129.2, 127.2, 126.6, 125.0, 123.0, 116.9, 113.8, 110.9, 95.8, 30.9, 20.7 (–CH₃), 14.1 (–CH₃). Anal. calculated for C₂₅H₂₄N₄O₄S (476.55 g/mol): C, 63.01; H, 5.08; N, 11.76; O, 13.43; S, 6.73%, Found; C, 62.99; H, 5.28; N, 11.87; O, 13.45; S, 6.66%.

2.1.4.7. N-(4-(N-Acetylsulfamoyl)phenyl)-2-((2,3-dimethylphenyl)amino)benzamide (18)

White crystalline solid; yield: 73.5%; mp 177–180 °C; R_f: 0.74 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3461 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2921 (–NH, amide), 1710 (–C = O), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δH 2.09 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 2.29 (s, 3H, CH₃), 6.47 (d, 1H, J = 8.0 Hz, ArH), 6.74–6.66 (m, 4H, ArH), 7.08–6.98 (m, 1H, ArH), 7.20–7.10 (m, 1H, ArH), 7.35–7.30 (m, 1H, ArH), 7.40 (d, 2H, J = 8.0 Hz, ArH), 7.89 (dd, 1H, J = 8.0, 1.6 Hz, ArH), 9.18 (brs, 1H, NH), 9.52 (brs, 1H, NH), 11.54 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 168.8 (–NH-C = O), 150.7 (–C = O), 149.1 (=C-NH–), 138.6, 138.4, 135.0, 133.5, 132.0, 131.7, 128.5, 127.2, 126.6, 123.0, 116.9, 113.8, 112.3, 110.9, 27.2 (–CH₃), 20.7 (–CH₃), 14.1 (–CH₃). Anal. calculated for C₂₃H₂₃N₃O₄S (437.52 g/mol): C, 63.14; H, 5.30; N, 9.60; O, 14.63; S, 7.33%, Found; 63.23; H, 5.38; N, 9.67; O, 14.75; S, 7.32%.

2.1.4.8. N-(4-(N-Carbamimidoylsulfamoyl)phenyl)-2-((2,3-dimethylphenyl)amino)benzamide (19)

White crystalline solid; yield: 79.2%; mp 202–205 °C; R_f: 0.85 (EtOAc:MeOH:n-hexane:DCM); IR (ATR, υ cm^–1): 3462 (–NH, sulfonyl), 3018 (=C–H, aromatic), 2923 (–NH, amide), 1710 (–C = O), 1628 (–CH = N–, imine), 1594 (–CH = CH–), 1366 (–NH-S = O, asymmetric), 1141 (–NH-S = O, symmetric), 1027 (–S = O). ¹H NMR (400 MHz, DMSO-d₆): δH 2.10 (s, 3H, CH₃), 2.30 (s, 3H, CH₃), 5.69 (s, 1H, NH), 6.55 (d, 1H, J = 8.0 Hz, ArH), 6.59 (brs, 2H, NH₂), 6.75–6.65 (m, 4H, ArH), 7.08–6.99 (m, 1H, ArH), 7.20–7.11 (m, 1H, ArH), 7.36–7.30 (m, 1H, ArH), 7.39 (d, 2H, J = 8.0 Hz, ArH), 7.89 (dd, 1H, J = 8.0, 1.6 Hz, ArH), 7.96 (brs, 1H, NH), 8.12 (brs, 1H, NH), 9.17 (brs, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆): δC 168.8 (–C = O), 158.3 (–C = NH), 151.8 (=C-NH–), 149.1, 140.5, 138.6, 135.0, 132.0, 131.7, 131.3, 127.7, 126.6, 123.0, 116.9, 113.8, 112.8, 20.7 (–CH₃), 14.1 (–CH₃). Anal. Calculated for C₂₂H₂₃N₅O₃S (437.52 g/mol): C, 60.40; H, 5.30; N, 16.01; O, 10.97; S, 7.33% Found; C, 60.56; H, 5.48; N, 16.13; O, 10.95; S, 7.21%.

2.2. Antiurease Assay

With a few minor adjustments, we conducted the urease inhibition experiment following our prior studies.^7,8,10 In brief, 250 μM stock solutions of each conjugate and thiourea (standard inhibitor of urease) were prepared in DMSO and serial dilution was made up to 0.49 μM. Each Falcon tube contained 20 μL of the respective inhibitor, 100 μL of buffer (K₂HPO₄, 50 mM) with a pH range of 6.8–7.0, and 20 μL of jack bean urease. The contents of each tube were thoroughly mixed and incubated for 30 min at 37 °C. Subsequently, 400 μL of urea (20 mM) was added to each tube as a substrate and incubated for an additional 10 min at the same temperature. Next, 400 μL of phenol reagent and 750 μL of alkali reagent containing 0.1% active chlorine were added to each tube, and the mixture was left for 50 min at 37 °C. Following this, the absorbance of each tube’s mixture was measured at 595 nm using a spectrophotometer (Labdex, LX210DS, U.K.), and the percentage of urease inhibition was calculated using the following formula:

The results in mean ± standard error of the mean (SEM) were presented, with T representing the absorbance of wells containing the inhibitor and C representing the absorbance of blank wells. IC₅₀ values for each inhibitor were determined using a regression equation that showed 50% inhibition. To investigate the binding mechanism of each inhibitor, a range of doses (0–20 μM) were tested. Various concentrations of urea (0.5–4.0 mM) were used as substrates to determine whether the inhibitors acted uncompetitive, mixed (noncompetitive), or competitively. Lineweaver–Burk plots were created using GraphPad PRISM 8.0, and these plots were utilized to calculate K_m (app), V_max (app), and K_i (inhibition constant) values.

2.3. Molecular Docking and Dynamics Simulation Studies

The competitive conjugates were docked to the urease protein to analyze their molecular interactions. The crystal structure of the first jack bean urease with a resolution of 1.52 Å (PDB ID: 4H9M) was retrieved and prepared for molecular docking by using the protein preparation wizard of Maestro.²⁸ During the preparation of the receptor, hydrogen atoms were added, structural integrity was ensured, and zero bond orders for metals were created. Additionally, unnecessary ligand atoms and water molecules were removed. The tautomeric states were adjusted to pH 7.4. The hydrogen atoms were optimized to optimize the geometry of the receptor, and then the protein was minimized using the OPLS_2005 force field.²⁹ Finally, a three-dimensional (3D)-grid box was generated at specific residues to conduct site-specific docking. After the preparation of the receptor, the competitive inhibitors were prepared using the LigPrep tool, and the conformers with the lowest energy were obtained for the docking analysis. The standard precision mode of the glide tool was used to conduct the molecular docking, and the best binding modes of conjugates against the urease protein were subjected to explore the structural stability and protein dynamics by running 300 ns long simulation using VMD³⁰ and NAMD³¹ tools. The parameter and coordinate files were generated using the modules of Ambertools 21.³² The ligand topology and parameter files were generated by antechamber, while the missing hydrogen atoms were added through Leap Program.³³ After generating the parameter files, a periodic box of 10 Å containing the TIP3P water model³⁴ was added to the complexes, and then the system was neutralized by adding Na⁺ ions. The system was minimized for 10000 steps to avoid energy clashes by applying ff14SB force field³⁵ for protein and GAFF for ligands. After minimization, the solvation was equilibrated for 10000 steps, followed by temperature equilibrations at 200, 250, and 300 K. The prepared systems were then subjected to the production run to compute 300 ns simulation and store the trajectories at every 20 ps interval. The post-simulation analysis was performed by using the BIO3D package of R.³⁶

3. Results and Discussion

3.1. Chemistry

The synthesis of the target conjugates (Scheme 1) involved combining diclofenac and mefenamic acid with a range of sulfa drugs, including sulfaguanidine, sulfacetamide, sulfamethoxazole, sulfanilamide, sulfamerazine, sulfisoxazole, sulfadiazine, and sulfathiazole. To facilitate the coupling of the amino group of sulfa drugs with the hydroxyl group of diclofenac/mefenamic acid, we utilized DCC as a coupling agent, with DMAP as a catalyst. Numerous studies have highlighted that one of the most commonly used reactions in medicinal chemistry is amide coupling, which enables the synthesis of a wide variety of compounds by combining two readily available synthons: a carboxylic acid and an amine. In recent years, sulfonamide linkers have increased prominence in medicinal chemistry.³⁷ The synthesis procedures can be found in the Experimental Section, while several spectroscopic methods, such as ¹H NMR, ¹³C NMR, IR, and elemental analysis, were used to characterize the newly synthesized conjugates. Further information is provided in Section 2.1.2.

Scheme 1. Synthesis Procedure for Diclofenac and Mefenamic Acid-Sulfa Drug Conjugates.

In the IR spectroscopic analysis, the absorption band for all of the conjugates appeared in the 3463–3472 cm^–1 range, which corresponds to the –NH group within the sulfonamides. The –NH functional group of the acetamide moiety demonstrated the absorption band in the 2923–2929 cm^–1 range for all of the conjugates. The –NH-S = O group displayed absorption bands in unsymmetrical (1338–1373 cm^–1) and symmetrical (1137–1145 cm^–1) regions. The appearance of the absorption band in the 1024–1032 cm^–1 range corresponds to sulfoxide in –NH-S = O for all of the conjugates. In the proton ¹H NMR spectra of the conjugates, the peaks within the chemical shift range of δ 9.17–10.5 ppm indicate the presence of the –NH proton from the sulfonamide group, confirming the existence of the –SO₂NH– group. The singlet at δ 2.09–2.30 ppm signifies the presence of the methyl group (–CH₃) protons. In the ¹³C NMR spectra, the 167.7–173.2 ppm peaks correspond to the –C = O carbon in all of the conjugates. In Section 2.1.2, the details of chemical shifts and integrals corresponding to aliphatic and aromatic protons and carbon can be found, and spectra can be seen in Supporting Information.

3.2. Pharmacological Activities

3.2.1. Urease Inhibition

We assessed the synthesized conjugates for their antiurease activity. In our investigations of urease inhibition, we used thiourea as a reference compound, which showed an IC₅₀ value of 22.61 μM. The enzyme inhibition data, presented in Table 1, demonstrates that all of the conjugates effectively inhibit the enzyme (urease). A summary of the results for all 16 conjugates can be seen in Supporting Information.

Table 1. IC₅₀ and Kinetics Parameters of NSAIDs-Sulfa Drug Conjugates (4–19)^a.

conjugate	IC50 (μM); mean ± SEM (% inhibition)	Vmax (app) (μM/min)b	Km (app) (mM)b	Ki (μM)c	mode of inhibition
4	3.59 ± 0.07 (89.7)	2.94	6.41	7.45	competitive
5	23.13 ± 0.49 (73.4)
6	16.19 ± 0.21 (71.3)	0.755	1.19	16.29	mixed
7	30.49 ± 0.28 (83.6)
8	9.50 ± 0.28 (85.9)	2.27	2.01	10.80	mixed
9	33.34 ± 0.21 (77.3)
10	5.49 ± 0.34 (84.5)	3.46	11.76	2.73	competitive
11	4.35 ± 0.23(78.7)	1.11	2.46	1.09	mixed
12	18.69 ± 0.21 (91.6)	8.62	7.14	3.06	competitive
13	15.86 ± 0.25 (94.1)	3.03	0.99	4.82	mixed
14	14.80 ± 0.27 (82.9)	2.28	1.01	1.65	mixed
15	7.92 ± 0.27 (84.9)	6.36	2.63	1.04	mixed
16	94.99 ± 0.22 (68.3)
17	8.35 ± 0.26 (86.4)	8.54	7.40	0.46	competitive
18	36.08 ± 0.28 (64.1)
19	31.32 ± 0.29 (79.2)
thiouread	22.61 ± 0.23 (92.3)	18.61	2.18	18.18	competitive

^aIntercept, the maximum rate, at an inhibitor concentration of 20 μM.

^bMichaelis constant (slope of the line) at an inhibitor concentration of 20 μM.

^cInhibition constant.

^dStandard urease inhibitor.

3.2.2. Enzyme Kinetic Studies

Diclofenac combined with sulfanilamide (4), sulfacetamide (10), and mefenamic acid combined with sulfanilamide (12) and sulfamethoxazole (17) exhibited significant potency. These conjugates demonstrated a competitive mode of inhibition against urease, with IC₅₀ (μM) values of 3.59 ± 0.07, 5.49 ± 0.34, 7.92 ± 0.27, and 8.35 ± 0.26, respectively (Figure 2). The respective urease inhibition percentages were 89.7, 84.5, 91.6, and 84.6%.

Figure 2.

Competitive-type urease inhibitors.

The competitive inhibition mode of the conjugates depicted in Figure 2 (4, 10, 12, and 17) was confirmed through kinetic investigations. In competitive inhibition, the enzyme cannot be bound to the substrate and the inhibitor simultaneously. This typically happens because the substrate and inhibitor fight for access to an enzyme’s active site due to the inhibitor’s affinity for the same region, where the substrate also binds. This type of inhibition may be overcome by increasing the substrate concentration. However, the value of maximal velocity (V_max) remains constant, whereas the value of K_m increases, calculated from the primary Lineweaver–Burk plot. The enzyme’s active site is attached to the inhibitor; consequently, adhering to the substrate is prevented in the competitive mode of inhibition of the enzyme. As depicted in Figure 3, at different concentrations (0.0, 5.0, 10.0, 15.0, and 20.0 μM, details in the Supporting Information) of each conjugate (4, 10, 12, and 17), the value of V_max remains constant, whereas the value of K_m increases, which demonstrated the competitive type of urease inhibition. The concentration of substrate was also varied from 0.5 to 2.0 mM. The K_i values were 7.45, 2.73, 3.06, and 0.46 μM for conjugates (4, 10, 12, and 17), respectively (as shown in Table 1), calculated from secondary Lineweaver–Burk plots. The enzymatic kinetic plots illustrating mixed inhibition are presented in Figure 3.

Figure 3.

Conjugates (4, 10, 12, and 17) exhibited a competitive mode of inhibition demonstrated by kinetic studies employing Lineweaver–Burk plots. (a) K_m and V_max values calculation by primary plot and (b) K_i value calculation by secondary plot.

Diclofenac conjugated with sulfathiazole (6), sulfamerazine (8), and sulfaguanidine (11), while mefenamic acid conjugated with sulfisoxazole (13), sulfathiazole (14), and sulfadiazine (15) exhibited the mixed mode of urease inhibition (Figure 4). The IC₅₀ (μM) values were 16.19 ± 0.21, 9.50 ± 0.28, 4.35 ± 0.23, 15.86 ± 0.25, 14.80 ± 0.27, and 7.92 ± 0.27, and urease inhibition was 71.3, 85.9, 78.7, 94.1, 82.9, and 84.9% of conjugates (6, 8, 11, 13, 14, and 15), respectively.

Figure 4.

Mixed-type urease inhibitors.

When mixed-type inhibition occurs, the inhibitor molecule binds to the complex of the substrate and enzyme to stop the enzymatic reaction. Unlike competitive inhibition, this sort of inhibition is not overcome by increasing the substrate concentration. Changes in the line’s intercept (V_max) and slope (K_m) in a primary Lineweaver–Burk plot are indicators of inhibition in mixed ways. The conjugates (6, 8, 11, 13, 14, and 15) were put through a kinetics mechanism utilizing different concentrations (details in the Supporting Information) of each conjugate (0.0–20.0 μM) and substrate (0.5–4.0 mM, urea). Although mixed-type inhibitors have the potential to bind in the active site, this inhibition typically happens due to an inhibitor’s allosteric action. This type of inhibitor will lower V_max but raise K_m. As depicted in Figures 5 and 6, at different concentrations (0.0–20.0 μM) of each conjugate (6, 8, 11, 13, 14, and 15), the value of V_max decreases, whereas the value of K_m increases, which demonstrated the mixed type of urease inhibition. The K_i values were found to be 16.29, 10.80, 1.09, 4.82, 1.65, and 1.04 μM for conjugates (6, 8, 11, 13, 14, and 15), respectively (as shown in Table 1), which were calculated from secondary Lineweaver–Burk plots. The enzymatic kinetic plots illustrating mixed inhibition are presented in Figures 5 and 6.

Figure 5.

Conjugates (4, 8, and 11) exhibited mixed mode of inhibition demonstrated by kinetic studies employing Lineweaver–Burk plots. (a) K_m and V_max values calculation by primary plot and (b) K_i value calculation by secondary plot.

Figure 6.

Conjugates (13, 14, and 15) exhibited mixed mode of inhibition demonstrated by kinetic studies employing Lineweaver–Burk plots. (a) K_m and V_max values calculation by primary plot and (b) K_i value calculation by secondary plot.

While the remaining conjugates (5, 7, 9, and 19) likewise demonstrated effective urease inhibition in the 73.4–83.6% range, the conjugates (16 and 18) exhibited moderate urease inhibition.

The introduction of the additional ring on the sulfonamide moiety in the conjugates (6, 8, 11, 13, 14, and 15) did not closely cooperate with the active site to support their mixed mode of urease inhibition. Therefore, it could be summarized from the differences in structures of conjugates with the substitutions on heteroring or without the additional ring (4, 10, 12, and 17) are electronegative, and also show polarizability, may improve the interaction with the active site of urease. The –NH₂ and –C=O groups bind well to the enzyme’s active site through hydrogen-bond interactions, which might be responsible for these conjugates’ competitive mode of inhibition. Additionally, some of the coordination sites in these conjugates (4, 10, 12, and 17) proved their competitive mode of inhibition due to the presence of –NH₂ and –C = O functional groups, which may enhance the coordination properties of the urease active site containing Ni (II).

3.2.3. Structure–Activity Relationship (SAR)

SAR investigations were primarily focused on the central core structure (depicted in Figure 7), which includes the acetamide and substituted sulfonamide scaffold. To provide a logical basis for our findings, we divided the target compounds into two groups: one group involved diclofenac and the other group involved mefenamic acid, each coupled with various substituted sulfonamides (sulfa drugs)

Figure 7.

Central core for the depiction of SAR studies.

As demonstrated from kinetics studies, conjugates 4 and 12 showed a competitive mode of inhibition of urease. Still, conjugate 4 is more potent than conjugate 12, which might be due to the attachment of the chloro group on the ortho-position (Figure 8). An electron-withdrawing group like chloro can change a molecule’s electrical characteristics and impact how it interacts with urease. These groups may bind with specific metal ions or amino acid residues in the enzyme’s active site, which would change the enzyme’s activity.

Figure 8.

SAR studies of the competitive mode of inhibition of urease.

Numerous noncovalent interactions, including hydrogen bonds (hydrogen-bond acceptor N and O), pi–pi stacks (unsaturated five-membered rings), and hydrophilic contacts, are made feasible by the structural characteristics of isoxazole. The acetamide moiety also has a broad spectrum of biological targets, and acetaminophen is one of the most widely used therapeutic agents globally. So, conjugate 10 showed better inhibition of urease than conjugate 17. In conjugate 10, the acetamide moiety and electron-withdrawing chloro group on the ortho-position of the terminal phenyl ring made it more potent than conjugate 17, where the terminal phenyl ring contains an electron-donating methyl group (Figure 8). Therefore, the substituent’s ability to withdraw electrons can promote more significant contact with the urease enzyme, increasing its inhibition. It is essential to remember that the precise effects of electron-withdrawing groups on urease inhibition can change depending on the chemical structure in which they are used.

The reported studies about the antiurease activities of the different substituents on aromatic rings support our findings. It has already been reported that isoxazole substituted on the sulfonamide side and electron-withdrawing groups such as fluoro-substituted biphenyl groups on the acetamide side showed better urease inhibition activities as compared to methyl substituent phenyl groups on the acetamide side. Furthermore, guanidine and amino groups on the sulfonamide side showed excellent urease inhibition activities compared to five- and six-membered heterocyclic substituents.^26,27 Overall, the electron-withdrawing and -donating groups attached to phenyl groups play a pivotal role in the inhibition activities of urease enzymes. However, this effect largely depends on the overall structure of the molecule under investigation.^38,39

3.3. Molecular Docking and Dynamics Simulation Studies

The possible binding mechanisms of the competitive inhibitors with the urease were predicted through molecular docking studies, and their molecular interactions were analyzed. Analysis revealed that conjugate 4 has two hydrophobic interactions, one pi–sulfur interaction with His492, and three hydrogen bonds with Ala440, His519, and Ala636. Also, conjugate 10 showed van der Waals interactions with Cys592, His492, and Ala440, formed hydrogen bonds with His519 and Arg609, and a pi–sulfur interaction with Asp494. Conjugate 12 made two hydrogen bonds, two van der Waals interactions, and three pi–alkyl bonds. Lastly, conjugate 17 made a hydrogen bond with His593, three van der Waals interactions, and seven pi–alkyl bonds. Figures 9 and 10 display the protein binding pocket’s molecular interactions and conjugate-binding mechanisms.

Figure 9.

Competitive inhibitors’ (4, 10, 12, and 17) molecular interactions with urease. In this illustration, various bonds and interactions are visually depicted by colored spheres. Hydrogen, sigma, pi–sulfur, and pi–alkyl bonds are presented by green, purple, orange, and magenta spheres, respectively.

Figure 10.

Binding modes of competitive inhibitors.

The stability of the protein–ligand complexes was evaluated through a series of molecular dynamics simulation analyses. The structural stability of these complexes was explicitly assessed by computing the root-mean-square deviation (RMSD) of the backbone atoms in the urease complexes when bound with the conjugates (Figure 11).⁴⁰ Up until 175 ns, the conjugate 4 complex’s RMSD remained within the range of 2.5–3 Å, steadily increasing to 3.5 Å at 200 ns. With a few tiny variances noted at 225 ns, the RMSD values stayed in this range until the simulation ended. The RMSD of the conjugate 10 complex likewise followed the same pattern, with values remaining in the 2.5–3 Å range until 225 ns before progressively rising to 3.5 Å at the end of the simulation. The RMSD values of conjugate 12 complex showed significant deviations in the first 50 ns of the simulation, where the values increased to ∼3.5 Å at 50 ns, but then the RMSD maintained a range of ca. 2.5–3 Å until the end of the simulation. The protein showed minor deviations during the simulation according to the examination of the RMSD for the conjugate 17 complex. It consistently maintained a range of ca. 2–3 Å, with only minor variations of around 1 Å observed between the 125 and 160 ns interval. These minor fluctuations in the trajectories serve as indicators of the overall stability of the urease complexes.

Figure 11.

Plots of the urease backbone atoms determined throughout the 300 ns simulation in root-mean-square deviation.

An analysis of the root-mean-square fluctuation (RMSF) was conducted to assess the flexibility of protein residues.⁴¹ Higher RMSF values are indicative of flexible loops, while lower values suggest the presence of stable α helices and β sheets. The RMSF analysis exhibited consistent patterns across all complexes with elevated fluctuations in the initial residues owing to N-terminal loops. Notably, amino acid residues within the ranges of 50–60, 90–100, 110–125, 260–270, 420–530, 590–610, and 630–640 displayed significant fluctuations, signifying the existence of flexible loops in these regions. The conjugate 10 complex showed substantial variations in residues 650–660 compared to the other complexes, whereas the rest of the protein remained rather stiff throughout the simulation (Figure 12).

Figure 12.

RMSF plots of the urease protein residues.

We used a radius of gyration (R_g) assay to gauge how compact the urease proteins are when they are in contact with conjugates.⁴² Lower R_g values indicate structural stability, whereas higher R_g values suggest structural distortions during simulation. Higher R_g values suggest structural deformation during simulation, whereas lower R_g values indicate structural stability. The R_g plots of the complexes showed that their R_g values were consistently between 30.06 and 31 Å. These stable R_g values demonstrated that when attached to conjugates, the protein structures remained compact and stable throughout the simulation (Figure 13).

Figure 13.

Radius of gyration calculation to analyze the compactness of urease protein structure during simulation.

The QikProp tool was used to calculate the conjugates’ physicochemical and ADMET parameters. Most conjugates fell within the octanol/water partition coefficient’s permissible range. The anticipated cell permeability and brain/blood partition coefficient yielded similar results. The compounds did, however, show that their expected IC₅₀ values for blocking HERG K⁺ channels were slightly greater than expected. The following parameters have cutoff values for ADMET: “QPlogHERG” (−5), “QPlogPo/w” (−2.0 to 6.5), “QPlogBB” (−3.0 to 1.2), “QPPCaco” (<25 poor, >500 great), and “QPlogKhsa” (−1.5 to 1.5). In Table 2, the conjugates’ physicochemical and ADMET characteristics are listed.

Table 2. Physicochemical and ADMET Properties of the Conjugates^a.

conjugates	MW	HBD	HBA	QPlogPo/w	QPlogHERG	QPPCaco	QPlogBB	QPlogKhsa
4	450.339	4	7	3.263	–6.881	249.261	–1.414	0.131
6	533.446	3	9	4.508	–7.142	542.052	–0.976	0.374
8	542.439	3	9	4.423	–7.53	424.696	–1.339	0.446
10	492.376	3	8	3.812	–6.619	196.067	–1.511	0.399
11	492.379	4	5	3.9	–6.539	48.44	–2.254	0.611
12	395.475	3	6	3.216	–6.736	206.315	–1.676	0.344
13	490.576	2	8	4.795	–7.643	344.386	–1.72	0.828
14	478.583	2	8	4.555	–7.769	456.18	–1.394	0.596
15	473.548	2	8	4.156	–7.827	305.81	–1.686	0.52
17	476.549	2	8	4.358	–7.577	279.917	–1.78	0.664

^aMW = molecular weight, HBD = estimated no. of hydrogen-bond donor, HBA = estimated no. of hydrogen-bond acceptor, QPlogPo/w = predicted octanol/water partition coefficient, QPlogHERG = predicted IC₅₀ value for blockage of HERG K⁺ channels, QPPCaco = predicted Caco2 cell permeability in nm/s, QPlogBB = predicted brain/blood partition coefficient, QPlogKhsa = prediction of binding to human serum albumin.

Conclusions

In this study, 16 conjugates were designed, successfully synthesized, and characterized. These conjugates were synthesized by coupling the diclofenac and mefenamic acid (NSAIDs) and sulfa drugs that feature biologically significant acetamide-benzamide and sulfonamide structures. These drug conjugates were synthesized in high yield (ranging from 63.8 to 79.2%) by coupling the amino group of various substituted sulfa drugs and the hydroxyl group of NSAIDs by employing DCC as coupling agents. Their properties were thoroughly examined by using spectroscopic techniques. Among these conjugates, the most effective inhibitor against urease was conjugate 4 (diclofenac-sulfanilamide), with a K_i value of 7.45 μM. The most potent inhibitor’s structural feature (89.7% inhibition) is an electron-withdrawing group like chloro, which can change a molecule’s electrical characteristics and impact how it interacts with urease. The substituent’s ability to withdraw electrons can promote more significant contact with the urease enzyme, increasing its inhibition. In a nutshell, the findings of these SAR experiments demonstrate the significant influence that changes the aromatic ring. It is imperative to thoroughly investigate the potential of acetamide-benzamide integrating sulfonamide as a urease inhibitor, and its structure needs to be improved for maximum effectiveness.

Additionally, the analysis of binding stability was conducted by aligning various snapshots from the MD trajectories, revealing that the ligands maintained close interactions with the protein throughout the simulation. According to ADMET predictions, the synthesized drug conjugates had few adverse side effects, low toxicity profiles, and drug-like characteristics. Last but not least, the in silico analysis of these conjugates (4–19) within the urease active sites explained the observed biological outcomes.

Data Availability Statement

This research is part of Ph.D. dissertation of Saghir Ahmad, and data is in the repository of Higher Education Commission (HEC) of Pakistan.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c07275.

• ¹H NMR and ¹³C NMR spectral data and summary of antiurease data (PDF)

Author Contributions

^◆ M.A.Q. and M.A. contributed equally to this work as second authors. Conceptualization, methodology, formal analysis: S.A. Validation, resources, project administration, supervision: M.A.Q. Conceptualization, writing—original draft, writing—review & editing: M.A. Formal analysis, investigation: M.A. Formal analysis, investigation, resources: M.I. Software, data curation, visualization: N.Y. Writing—review & editing, funding acquisition: T.A.W. Validation, funding acquisition: S.Z. Formal analysis, validation: I.A. Methodology, software, visualization: M.M.

The authors declare no competing financial interest.

Notes

Informed Consent Statement This study was not performed on humans.