Newly formulated conjugates were synthesized by utilizing high-purity sulfa drugs originating in Sigma Aldrich, United States, and were purchased from Falcon Scientific, Lahore, Pakistan. NSAIDs were kindly gifted by Novamed Pharmaceuticals, Lahore-Pakistan. Conjugate structures were elucidated through spectral investigations using techniques including FTIR, ¹HNMR-500 MHz, and ¹³CNMR-125 MHz (Bruker, United States). Thermo Scientific, UK’s HT + elemental analyzer was used for the analysis of the elements (C, H, N, and S). While pre-coated silica TLC plates (Merck, Germany) were used to check the purity of the synthesized conjugates under UV light whereas the Gallenkamp apparatus was used to find out the melting point and are uncorrected.

In a 100 mL flask, naproxen (1 mmol) was dissolved in a solvent containing the mixture of methanol and acetonitrile (30 mL) in a 50:50 ratio. Then 1 mmol of N, N′-Dicyclohexylcarbodiimide (DCC) was added to the solution and the reaction proceeded with the addition of 4-dimethylaminopyridine (DMAP) as a catalyst. The reaction of naproxen and DCC is continued for 30 min at 80°C. Then for amide bond formation, a respective sulfa drug such as sulfanilamide, sulfisoxazole, sulfathiazole, sulfadiazine, sulfamerazine, sulfamethoxazole, sulfacetamide, and sulfaguanidine (1 mmol) was added in the reaction mixture. The refluxing of the reaction mixture was continued for 42 h to complete the reaction, after amide bond formation, dicyclohexylurea (DCU) become precipitated. The TLC was run to monitor the progress of the reaction using ethyl acetate: methanol: n-hexane: DCM (24: 10: 50: 15) as eluent. From the reaction mixture, insoluble DCU was filtered, and the filtrate was separated. The solid product was obtained by evaporating the solvent in rotary evaporated and further purification was done by flash chromatography using acetonitrile/MeOH (25:1) as eluent.

With a few minor modifications, the urease inhibition experiment was carried out as described in our past research (Ahmed et al., 2017; Ahmed et al., 2020; Imran et al., 2020). Briefly, DMSO was used to dissolve the synthesized conjugates (inhibitors, 250–0.49 µM) and reference urease inhibitor (thiourea). Each falcon tube contains the respective inhibitor (20 µL), a buffer of pH = 6.8–7.0 (K₂HPO₄, 100 μL, 50 mM), and jack bean urease (20 µL), each tube was mixed well, and the mixture was incubated at 37°C for 30 min. Each tube received 400 µL of urea (20 mM) as substrate, which was then incubated for 10 min at the same temperature. Afterward, each tube received 400 µL of phenol reagent and 750 µL of alkali reagent containing 0.1% active chlorine and was left at 37°C for 50 min. Following the use of a spectrophotometer (Labdex, LX210DS, United Kingdom) to measure the absorbance of the mixture in each tube at 595 nm, the percentage of urease inhibition was calculated using the equation below. % Urease   inhibition = 1 − T / C × 100 (1)

Where T and C are the absorbances of each well-containing inhibitor and blank respectively, each assay was performed in triplicate, and results are presented as mean ± SEM. Using a regression equation where 50% inhibition was seen, the IC₅₀ values of each inhibitor were determined. Each inhibitor’s binding mechanism was tested at various doses (0–20 µM) for kinetics investigations. Urea was used as substrate in different concentrations (0.5–4.0 mM) to determine the mode of inhibition of inhibitors whether these acted as uncompetitive, mixed (non-competitive), or competitive. Lineweaver Burk plots were drawn using GraphPad PRISM 7.0 to determine the values of K_{m (app)}, V_{max (app)}, and K_i (inhibition constant).

As previously mentioned in our study, carrageenan-induced acute inflammation in mice was used to measure the anti-inflammatory effect (Ahmed et al., 2018b). Six groups of mice were formed and each group comprised six mice, the mice fasted 16 h before of induction of inflammation by injecting the phlogistic agent (carrageenan). The right hind paw of each mouse was measured before and after the 100 µL injection of the carrageenan (1% in 0.9% saline). The test substances, standard medication (indomethacin), and control (0.9% saline) were administered intraperitoneally at 10 mg/kg body weight. For 3 h, and after the 1-h interval, the thickness (mm) of the right hind paw of each mouse was measured. The difference in thickness of paw edema of control and test substance was used to determine the percentage inhibition of inflammation by the formula given below. Percentage inhibition of inflammation = C t − C o control C t − C o treated C t − C o control × 100 (2)

Where C_t and C_o are the right hind paw thickness after and before carrageenan injection.

The inhibition of COX-2 was performed by using a kit procured from Cayman Chemical Company, United States (Item No. 760151). For the initial activity assay, 150 µL buffer, 10 µL heme, and 10 µL COX-2 enzyme were mixed in a well whereas 110 µL buffer, 10 µL heme, 10 µL COX-2 enzyme, and 40 µL test inhibitor (10 µM) were mixed for inhibitory assay using 96 wells plate. The well plate was placed in a shaker for 5 min at 25°C, then each well received the colorimetric substrate (20 µL), and arachidonic acid (20 µL) was added to initiate the reaction. Then the well plate was incubated at 25°C for 5 min, following the use of a microplate reader (Labtech, LT-4500, United Kingdom) to measure the absorbance of the mixture in each well at 590 nm, the percentage of COX-2 inhibition was calculated using the equation below (Ahmed et al., 2018b), each assay was performed in triplicate. % COX − 2   inhibition = 1 − T C × 100 (3)

Where T and C are the absorbances of each well-containing inhibitor and blank.

We evaluated the conjugates that had been successfully synthesized for their in vitro anti-urease action. In urease inhibition investigations, thiourea has served as a reference and exhibited the IC₅₀ value of 22.61 µM. Table 1 presented the enzyme (urease) inhibition data, all of the conjugates are effective against it.

SAR studies (Scheme 2) were carried out purely based on the central core containing naproxen moiety coupled with various substituted sulfonamides (sulfa drugs) through an amide linker. The effective structural feature of the most active inhibitor comprised of five membered heterocyclics such as thiazole, and isoxazole substituted sulfonamide. Naproxen coupled to thiazole substituted sulfonamides (sulfathiazole) demonstrated the five time more activity (5, IC₅₀ = 5.82 ± 0.28 µM) than the methylisoxazole (8, IC₅₀ = 29.64 ± 0.27 µM, sulfamethoxazole) and dimethylisoxazole (4, IC₅₀ = 25.63 ± 0.24 µM, sulfafurazole/sulfisoxazole) substitutes sulfa drugs. It is evident that the dimethylisoxazole is more active than the mono-methylisoxazole which is due to electron donating effect of methyl group. In contrast, the pyrimidine (6, IC₅₀ = 4.08 ± 0.10) and methylpyrimidin (7, IC₅₀ = 16.57 ± 0.14) had shown more activity than isoxazole substituted sulfonamides (4 and 8). Furthermore, guanidine (10, IC₅₀ = 5.06 ± 0.29), and amino (3, IC₅₀ = 6.69 ± 0.11) groups on sulfonamide side showed the excellent urease inhibition activities as compared to five and six membered heterocyclic substituents (Table 1). Whereas acetyl (9, IC₅₀ = 20.32 ± 0.12) substitution on sulfonamide side demonstrated less urease inhibition than the guanidine and amino substituent but more active than methylisoxazole and dimethylisoxazole on sulfonamide side.

Naproxen conjugated (Figure 2) with sulfanilamide (3), sulfathiazole (5), and sulfaguanidine (10) was found potent and showed a competitive mode of urease inhibition, with IC₅₀ (µM) values 6.69 ± 0.11, 5.82 ± 0.28, 5.06 ± 0.29, and urease inhibition was 89.4%, 88.9%, and 89.1% respectively (Table 1).

The competitive mode of inhibition of conjugates (3, 5, and 10) was demonstrated by kinetic studies. The kinetics studies were performed by using five different concentrations (0.0, 5.0, 10.0, 15.0, and 20.0 µM) of each conjugate while using four different conditions of urea (0.5, 1.0, 2.0, and 4.0) as substrate.

Lineweaver-Burk plot is a powerful tool for analyzing enzyme kinetics and determining the mode of inhibition of an enzyme by a particular conjugate. The inhibitor molecule (conjugate) binds to the enzyme’s active site in competitive inhibition and prevents the substrate from attaching. In a Lineweaver-Burk plot, competitive inhibition is characterized by a change in the slope of the line (K_m, also called the Michaelis constant), while the intercept remains the same (V_max, the maximum rate). The increase in the K_m value of the urease enzyme while the value of V_max remains constant at 20 µM of inhibitor concentration demonstrated that the conjugates (3, 5, and 10) inhibit the enzyme in a competitive way (Figure 3).

The inhibition constant (K_i) value of each conjugate was also calculated by plotting the slope of each line vs. different concentrations of each conjugate, also called secondary Lineweaver Burk secondary plots. The K_i value of conjugates (3, 5, and 10) was found 2.40, 5.05, and 3.56 µM respectively (Table 1). The plots of enzymatic kinetics of competitive inhibitors are presented below in Figure 3.

While the rest of the conjugates also showed good inhibition for the urease in the range between 84.3% and 94.1% and IC₅₀ values ranged between 4.08 ± 0.10 and 29.64 ± 0.27 µM. Naproxen conjugated with sulfadiazine (6), sulfamerazine (7), and sulfacetamide (9) exhibited a mixed mode of urease inhibition (Figure 4).

When there is a mixed type of inhibition, the inhibitor molecule binds to the enzyme-substrate complex, preventing the reaction from occurring. By boosting the concentration of the substrate, this form of inhibition cannot be overcome. In a Lineweaver-Burk plot, mixed-type inhibition is characterized by a change in both the slope (K_m) and the intercept of the line (V_max).

The mixed type of inhibition of conjugates (6, 7, and 9) were also demonstrated after kinetics studies by using five different concentration (0.0, 5.0, 10.0, 15.0, and 20.0 µM) of each conjugate while using four different conditions of urea (0.5, 1.0, 2.0, and 4.0) as substrate. The increase in K_m value of the urease enzyme while the value of V_max decreases at 20 µM of inhibitor concentration demonstrated that the conjugates (6, 7, and 9) inhibit the enzyme inhibition in a mixed way (Figure 5). The K_i values calculated from secondary Lineweaver Burk secondary plots and were obtained 5.58, 9.98, and 2.61 µM (Table 1) for conjugates (6, 7 and 9) respectively. The plots of enzymatic kinetics of mixed inhibitors are presented below in Figure 3.

Naproxen conjugated containing isoxazole and thiazole moieties (4, 5, and 8) were evaluated for their potential anti-inflammatory action whereas indomethacin was used as a reference NSAID (Table 2). Heterocyclic scaffolds have a wide variety of structural variations and have been shown to simultaneously target several inflammatory pathways. Numerous mediators and signaling channels interact intricately during inflammation. Contrary to medications that predominantly target a specific enzyme or pathway, heterocyclics have the potential to affect various elements of the inflammatory cascade, offering a wider spectrum of anti-inflammatory efficacy. In this context, the conjugates (4, 5, and 8) contain the five-membered heterocyclics, and their selection as anti-inflammatory agents was hypothesized as celecoxib contains the five-membered heterocyclic. The inhibitory activities of these conjugates were also assessed in vitro against both COX-2 and celecoxib was used as the standard inhibitor of COX-2. COX-2 enzyme screening kit was used to evaluate the inhibition of enzyme and the % inhibition of COX-2 enzyme by each conjugate under study is presented in Table 2.

Using carrageenan-induced paw edema, the anti-inflammatory efficacy of synthetic conjugates was evaluated in the current study. When carrageenan is injected into the paw of an animal, it induces a local inflammatory response that results in swelling (edema). This response involves the release of pro-inflammatory mediators such as prostaglandins, leukotrienes, and cytokines. The biphasic nature of carrageenan-induced edema makes it a useful model for studying the mechanisms of acute inflammation and evaluating the anti-inflammatory activities of drugs and natural compounds. Broad-spectrum anti-inflammatory compounds exhibit action against both the early and late stages of carrageenan-induced edema (Rathod et al., 2023).

Histamine, serotonin, and bradykinin are released during the early stages of carrageenan-induced edema, which starts within the first few hours of carrageenan administration. Usually lasting 1–2 h, this phase is characterized by increased vascular permeability, vasodilation, and neutrophil infiltration. The second phase of carrageenan-induced edema occurs approximately 2–3 h after carrageenan injection and is characterized by the release of prostaglandins and other mediators of inflammation. This phase is more sustained than the first phase, typically lasting for several hours, and is associated with increased vascular permeability, leukocyte infiltration, and tissue damage (de Siqueira Patriota et al., 2022). Table 2, presented the anti-inflammatory data which revealed that the conjugates significantly inhibited the induced edema in the late phase.

Naproxen-sulfamethoxazole conjugate (8), among the tested conjugates, demonstrated better anti-inflammatory action by inhibiting 82.8% of induced edema, and the inhibition result is comparable to the indomethacin (86.8%) which was used as reference anti-inflammatory drug. For the COX-2 inhibition studies, the results demonstrated that conjugate 8 also exhibited 75.4% inhibition which is comparable with the reference drug (celecoxib, 77.1% inhibition). Naproxen conjugated with sulfamethoxazole exhibited better COX-2 inhibition than thiazole-conjugated moiety.