The first step was to access the Protein Data Bank and retrieve the Helicobacter pylori crystal structure (PDB: 4YGF). We eliminated water molecules over 5 Å from the binding site to focus on the ligand interaction region. To mimic the conditions in the body, the current study prepared the proteins at a pH of 7. This procedure involved optimizing hydrogen atoms and assigning bond ordering. Following this, the current study optimized the protein structure using the OPLS4 force field, a well-known force field for biomolecular simulations in molecular mechanics. Additionally, the ligand was prepared at a pH of 7 ± 2 using the OPLS4 force field. This was done to ensure that the ligand molecule’s shape and net charge matched physiological conditions. The current study also considered the addition of the metal binding state of the ligands. We used a grid-based approach to accurately identify the precise region of interest for the docking simulations, where the binding site is located. This process involved creating a grid with the co-crystallized ligand, which provided crucial information about the appropriate binding pocket for the new ligand. We placed the native ligand at the center of the grid and constructed a rectangular solid with measurements of 20 Å around it. For the docking simulation, we used Glide, an advanced docking software program developed by Schrödinger (version 2023). We used typical precision docking with flexible sampling of the ligand to thoroughly investigate the interactions between the ligand and protein inside the specific binding pocket. By employing this method, we evaluated candidate binding positions and anticipated the binding configurations that would yield the highest energy stability between the ligand and the protein target (Lu et al. 2021; Yang et al. 2021).

Chemicals and solvents used in the synthesis were obtained from various companies without further purification before use. The Stuart SMP30 was utilized to measure the melting point of intermediate and final compounds. Thin-layer chromatography (TLC) was utilized to confirm the reactions’ progress and assess the compounds’ purity by employing different mobile phases. The FT-IR spectrophotometer is manufactured by the Japanese company Shimadzu. A Bruker model Ultra Shield (300, and 400 MHz) spectrophotometer was utilized to acquire ¹H-NMR spectra, with DMSO-d6 serving as the solvent.

The process for producing intermediate and final compounds is described in Scheme 1. The reaction of acetanilide with chlorosulfonic acid produces N-acetylsulfonylchloride as an intermediate (1). The fusion of different carboxylic acids with thiocarbohydrazide results in the formation of a 1, 2, 4-Triazole-3-thiol ring (2a–c) with good yield and purity. The combination of intermediate (1) with a 1, 2, 4-Triazole-3-thiol ring in the presence of pyridine as a catalyst creates compounds (3a–c).

Scheme 1. 

General synthetic pathway of target compounds.

The minimum inhibitory concentration (MIC), along with the agar diffusion method, was used to assess the antimicrobial activity of the target derivatives against several microorganisms, including Gram-positive bacteria (S. aureus, S. pneumoniae, and B. subtilis), Gram-negative bacteria (P. aeruginosa, E. coli, N. gonorrhoeae, and H. pylori), and fungal species (C. albicans). Diluted solutions were created from a stock solution (10 mg/ml) of each derivative, at concentrations ranging from 10–1000 mcg/ml. The solutions were prepared on a microtiter plate. The diluent employed was Mueller-Hinton broth. Each well was inoculated with 20 μl of a bacterial suspension with an equivalent concentration to McFarland standard no. 0.5 (1.5×108CFU/ml), except for the negative control wells. After that, the microtiter plates were put in an incubation chamber and kept at a temperature of 37 °C for 18 to 20 hours. After the incubation period, a volume of 20 µl of resazurin dye was added to each well. The samples were incubated for 2 hours to observe any color changes. The sub-MIC concentrations in the resazurin broth assay have been determined by visually observing the lowest concentrations in the broth micro dilutions at which the color transformed from blue to pink. Sulfadiazine, sulfamethoxazole, and fluconazole served as standard antibiotics (Franconi and Lupetti 2023).

The well diffusion assay was carried out using a bacterial suspension of approximately 1.5×108 CFU/ml obtained from the McFarland turbidity standard (number 0.5). The procedure involved applying the substance to the surface of MHA plates using a swab and allowing the excess fluids to dry in a sterile hood. Four wells were created in each agar plate containing the microorganisms under examination, and 80 μL of the test chemical was added to each well. The plates were then placed in an incubator at 30 °C for 72 hours for fungal species and at 37 °C for 24 hours for bacterial species. The zone of inhibition (ZI) width around each well was measured in millimeters to assess the antimicrobial activity (Subramaniam et al. 2020).

All the experiments were performed and reported in triplicate. The average mean values were reported along with standard deviation values. The T-test was used to assess the data’s significance and compare the mean (α < 0.05). The software used for statistical analysis is (R Studio 4.5 and the figures by Origin Lab 2021 software).

The synthesis of new sulfonamide derivatives (3a–c) was achieved through the straightforward substitution of an aryl sulfonyl chloride with 1,2,4-triazole-3-thiol, as illustrated in Scheme 1. The fusion of liquid carboxylic acid with thiocarbohydrazide under microwave irradiation for an appropriate time and temperature yields compounds (2a–c). Microwave irradiation facilitates the nucleophilic attack of the amine group of thiocarbohydrazide on the carbonyl group of the carboxylic acid, forming a tetrahedral intermediate. The intramolecular cyclization proceeds through the formation of an intermediate known as (1-acylthiocarbohydrazide) (Kurzer and Wilkinson 1970), as shown in Scheme 2. The compounds (3a–c) are formed by stirring compounds (2a–c) with compound (1) in the presence of pyridine as a catalyst and THF as a solvent. The substitution reaction at RSO₂X is similar to the nucleophilic attack at RCOX. Although sulfonyl halides are less reactive than carboxylic acid halides, many reactions share fundamental similarities. The reaction proceeded through the S_N-2 type mechanism (Smith 2007; Dakhel and Mohammed 2017). Compounds (2a–c) are characterized by the presence of two bands around 3270–3150 cm^-1, representing the asymmetrical and symmetrical stretching vibration of primary amine, respectively. Also, the bands around 1615 cm^-1 for (C=N) stretching, and around 1200 cm^-1 for (C=S) stretching identified the synthesis of these compounds. A single band around 3370 cm^-1, corresponding to the vibrations of sulfonamide groups’ (N-H) bonds, confirms the successful synthesis of the desirable compounds (3a–c). The successful chemical synthesis of the intermediate and target compounds was confirmed via ¹H-NMR analysis. All the synthesized compounds (2a–c), show a singlet peak of around 13.5 ppm for (SH), and a singlet peak of around 5.5 ppm for (NH₂). Compound (2c), shows two additional peaks, the quartet peak around 2.5 ppm for (CH₂) and the triplet peak around 1 ppm for (CH₃). Compound (2b), shows an additional peak of around 2 ppm for (CH₃) that directly binds to the triazole ring. Compound (2a), has characterized a peak of around 8.5 ppm for the (C-H) of triazole ring. There are clear singlet peaks in all of the spectra at 10 ppm for the (N-H) of the triazole ring, and around 9 ppm for (N-H) of sulfonamide groups. In addition, there is a singlet peak at around 8 ppm for the (N-H) of the amide bonds.

Scheme 2. 

Mechanism of synthesis of compound (2a–c).

This section will focus on the binding interactions and affinities of various compounds with carbonic anhydrase, particularly the critical interactions within the enzyme’s active site and their impact on binding efficiency. A relatively high binding affinity of acetazolamide is indicated by its docking score of -6.2 kcal/mol. Several significant interactions support this strong binding. These include hydrogen bonds between the amide group and Asn 108, the sulfonamide group and Thr 191, and the sulfonamide group and the zinc ion Zn301. The ligand is stabilized within the enzyme’s active site as a result of these interactions, which also enhance the inhibitory effect of the enzyme. The highest docking score, -7.05, as compared to acetazolamide was achieved for compound (3a). This high affinity arises from extensive interactions, including multiple bonds with Zn301 via the sulfur substituent of the triazole, two π-π stacking interactions with His 110 and His 84, and a hydrogen bond with Trp 23 through its amide group. These strong interactions make clear the superior binding efficiency of this compound. The poorer binding affinity of compound (3c), with a score of -1.2, is evident from its limited interactions, including only a coordination bond between the triazole sulfur substituent and Zn301 and a hydrogen bond with Trp 23, with a lack of other major interactions contributing to its low binding score. A strong binding affinity with a score of -6.75 was displayed by the compound (3b). This compound formed extensive coordination bonds, a total of four, with Zn301 and hydrogen bonds between its sulfonamide group and Asn 108, its amide group, and Thr 191, as shown in Table 1.

Docking validation was conducted by redocking the reference ligand and comparing the redocked conformation with the original conformation of the co-crystallized (Wadi et al. 2023), as shown in Fig. 1.

Figure 1. 

The docking validation was performed by redocking the co-crystallized ligand.

The red molecule represents the reference ligand from the crystal structure, while the blue molecule represents the redocked ligand. The minimal differences in conformation between the red and blue ligands indicate a successful docking protocol validation. The RMS value is 3.5 Å.

The interaction of acetazolamide, and compounds (3a–c) with the target proteins (PBD: 4YGF) are shown in Fig. 2.

Figure 2. 

The (2D, and 3D) interaction of acetazolamide, and compounds (3a–c) with the target protein, Where A for (Acetazolamide), B, C, and D for compounds (3a, 3b, and 3c) respectively.

The antibacterial activity of the targeted compounds was tested against various strains of bacteria as compared to the standard antibacterial drugs (Sulfamethoxazole and sulfadiazine). Additionally, the antifungal activity against (C. albicans) was evaluated as compared to the standard antifungal drug (fluconazole) as displayed in Table 2.

The MIC results of the target compounds can represented in a column chart, as shown in Fig. 3.

Figure 3. 

This figure illustrates the more potent compound with a small MIC value.

The zone of inhibition was measured in millimeters using the agar diffusion method based on MIC results and is illustrated in Table 3.

Based on the data presented in (Table 3), the mean ± SD for S. aureus is 29.8 ± 2.55. The T-test indicates a strong correlation between the zone of Inhibition (3a–c, Sulfadiazine, Sulfamethoxazole, Fluconazole, and DMSO) and S. aureus, with a p-value less than 0.001. Similarly, for S. pneumonia, the mean ± SD is 31.6 ± 2.07. The T-test shows a strong correlation with the same set of inhibitors, with a p-value less than 0.00001.B. subtilis has a mean ± SD of 23.4 ± 3.13. The T-test indicates a strong correlation with the Zone of Inhibition (3a–c, Sulfadiazine, Sulfamethoxazole, Fluconazole, and DMSO), with a p-value less than 0.005. For E. coli, the mean ± SD is 17 ± 6.29, and the T-test also shows a strong correlation with the same inhibitors, with a p-value less than 0.01. P. aeruginosa exhibits a mean ± SD of 25 ± 3.2, and the T-test reveals a strong correlation with the zone of Inhibition (3a–c, Sulfadiazine, Sulfamethoxazole, Fluconazole, and DMSO), with a p-value less than 0.001. N. gonorrhoeae has a mean ± SD of 11.5 ± 7.23, with the T-test showing no correlation with the inhibitors, resulting in a p-value of 0.05. H. pylori, with a mean ± SD of 33.2 ± 3.96, shows a strong correlation with the zone of Inhibition (3a–c, Sulfadiazine, Sulfamethoxazole, Fluconazole, and DMSO), with a p-value less than 0.05. Lastly, C. albicans, with a mean ± SD of 31.25 ± 2.36, demonstrates a strong correlation with the same set of inhibitors, indicated by a p-value less than 0.05. The data indicate varying levels of susceptibility to the inhibitors across different microorganisms. S. aureus and S. pneumonia: Both show strong correlations with the inhibitors, with very low p-values (<0.001 and <0.00001, respectively), suggesting the high effectiveness of the inhibitors against these bacteria. The close mean values and low standard deviations indicate consistent results within each group. B. subtilis: Exhibits a strong correlation with the inhibitors, with a p-value less than 0.005, indicating that the inhibitors are quite effective. However, the slightly higher standard deviation compared to S. aureus and S. pneumonia suggests more variability in response. E. coli: Shows a significant correlation with the inhibitors, but the p-value is slightly higher (<0.01), and the standard deviation is notably larger, indicating more variability in its susceptibility. P. aeruginosa: Also exhibits a strong correlation, with a p-value less than 0.001, suggesting high susceptibility to the inhibitors, though variability is still present as indicated by the standard deviation. N. gonorrhoeae: Displays the lowest mean Zone of Inhibition and the highest standard deviation, with a p-value of 0.05, suggesting no significant correlation with the inhibitors. This indicates that N. gonorrhoeae is likely resistant to the inhibitors tested. H. pylori and C. albicans show strong correlations with the inhibitors, with p-values less than 0.05, indicating effectiveness. H. pylori, with the highest mean Zone of Inhibition, shows a relatively lower standard deviation, suggesting consistent susceptibility. C. albicans also shows consistent results but with a slightly higher variability than H. pylori. In summary, while the inhibitors are broadly effective against most tested microorganisms, N. gonorrhoeae appears to be an outlier with resistance to the inhibitors. These findings highlight the need for tailored antimicrobial strategies for different pathogens, particularly those demonstrating resistance. The boxplot of the zone of the inhibition is shown in Fig. 4.

Figure 4. 

The boxplot of the zone of inhibition (3a–c, Sulfadiazine, Sulfamethoxazole, Fluconazole, and DMSO) and the isolations.