Molecular docking, microwave-assisted synthesis, characterization, and preliminary evaluation of the anti-microbial activity of new sulfonamide derivatives of the 1, 2, 4-triazole-3-thiol ring system

A new sulfonamide derivative containing a 1,2,4-Triazole-3-thiol ring was synthesized and characterized using FT-IR and 1 H-NMR. The microwave-assisted chemical synthesis of the 1,2,4-Triazole-3-thiol ring system resulted in good yield and purity. The synthetic compounds were then subjected to in vitro evaluation for antimicrobial study. All synthetic compounds show high activity against Gram-positive bacteria ( S. aureus , S. pneumonia , and B. subtilis ) and high activity against Gram-negative bacteria ( P. aeruginosa , and H. pylori ), with less activity against ( N. gonorrhea , and E. coli ). Also, it shows high activity against fungi ( C. albicans ). In this study, we utilized computational methods to design new derivatives that target the carbonic anhydrase enzyme of H. pylori (PDB: 4YGF). All the target compounds interact with the enzyme’s active site, resulting in a disturbance of the acid-base balance affecting the virulence and pathogenicity


Introduction
The main cause of drug resistance in the healthcare setting is the excessive and incorrect consumption of different antimicrobial agents.Infections caused by resistant bacteria are treated by administering different antibiotics to which they may still be sensitive.However, bacteria may acquire resistance to these new classes of antibiotics, becoming multi-resistant organisms.Therefore, it is crucial to discover new types of antibiotics that can overcome the resistance in these microorganisms (Campestre et al. 2021).Many strategies have been taken to find novel antibacterial compounds capable of fighting resistant organisms (Alani et al. 2024).Sulfonamides are an important class in the area of medicinal chemistry due to their wide variety of pharmacological activities, including antimicrobial (Lihumis et al. 2023), anti-inflammatory (Jiao et al. 2024), anti-cancer (Elsayad et al. 2024), anti-glaucoma (Angeli et al. 2024), and anti-diabetic properties (Naim et al. 2018).Sulfonamides work as antimicrobial agents through two main mechanisms.The primary mechanism involves targeting and inhibiting the bacterial dihydropteroate synthase (DHPS).This inhibition leads to a defect in the synthesis of tetrahydrofolic acid (THF), disrupting the supply of thymidine, which results in the arrest of DNA biosynthesis and ultimately affects bacterial growth (Zessel et al. 2014).Several DHPS, mutations are responsible for the sulfonamide resistance; there is another mechanism involved in the inhibition of bacterial carbonic anhydrase by targeting the zinc-binding domain that leads to disturbances in the acid/base balance, which reduces the pathogenicity and virulence of various bacteria, including H. pylori, V. cholera, E. coli, P. aeruginosa, and S. enterica (Supuran and Capasso 2020).The chemical synthesis of the heterocyclic ring is an integral part of medicinal chemistry, with different pharmacological activities including anti-inflammatory (Paprocka et al. 2023), antiviral (Farghaly et al. 2024), anti-cancer (Abbas et al. 2023), and antimicrobial (Karnaš et al. 2024).The triazole ring system can target various mammalian and pathogenic proteins by creating a network of bonds at the proteins' active sites, easily forming various weak interactions, such as H-bonds, dipole-dipole interactions, and π-stacking (Rahman et al. 2023).This capability to inhibit various proteins makes the triazole ring system an intriguing option for the chemical design of new therapeutic agents.The incorporation of a triazole ring into various drug candidates has been of interest in recent years for designing new classes or improving existing ones.Among them, the sulfonamide-containing triazole ring system is noteworthy (Naaz et al. 2018).

Molecular docking
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).

Materials
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 1 H-NMR spectra, with DMSO-d6 serving as the solvent.

General procedure
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).

Synthesis of 4-acetamidobenzenesulfonylchloride, compound (1)
In a dry conical flask containing (2.7 g, 20 mmol) of acetanilide, immerse the flask in an ice bath.Chloro-sulfonic acid (8 ml, 120 mmol) was added from the dropping funnel all at once and immediately connected to the conical flask, which contained some amount of water to trap the liberated gas.The solution is rapidly stirred, keeping the temperature below 20 °C.The flask was heated in a water bath at 70-80 °C for 20 min, to complete the reaction after the acetanilide had mostly dissolved and the initial exothermic reaction had subsided.Once the flask had cooled to room temperature, it was transferred into a 250 ml beaker holding 100 g of crushed ice, and stirred by glass road to prevent the formation of large lumps.The precipitate was collected by vacuum filtration and washed with cold distilled water till the filtrate was neutral to PH paper (Ovung and Bhattacharyya 2021).After the product was dried, washed with toluene, and recrystallized from chloroform to give a white to cream-beige solid, yield 75%, melting point 143-145 °C.

Synthesis of 1, 2, 4-Triazole-3-thiol, compound (2a-c)
A mixture consisting of (10 mmol) of thiocarbohydrazide and (15 mmol) of the liquid carboxylic acids was microwave-irradiated for the appropriate duration of time at (145 °C).The reaction's progress was tracked using thin-layer chromatography (TLC).The reactions were typically completed within a time frame of 30 to 60 minutes.Following the completion of the reaction, chilled distilled water was added, and the solid that had been formed was separated by filtration and subsequently purified through recrystallization with ethanol (Safonov et al. 2021).

In vitro anti-microbial study
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).

Statistical analysis
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).

Chemistry
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 RSO 2 X 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 1 H-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 (NH 2 ).Compound (2c), shows two additional peaks, the quartet peak around 2.5 ppm for (CH 2 ) and the triplet peak around 1 ppm for (CH 3 ).Compound (2b), shows an additional peak of around 2 ppm for (CH 3 ) 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.

Docking study
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.
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.

Anti-microbial study
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.
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, Scheme 2. Mechanism of synthesis of compound (2a-c).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.

Conclusion
The synthesis of a new series of sulfonamide derivatives was accomplished with successful results.Their chemical structures were determined using FT-IR spectroscopy and 1 H-NMR spectroscopy.The presence of a new band around 3370 cm -1 in FT-IR, and a singlet peak around 9 ppm in 1 H-NMR indicate the synthesis of target compounds.Compounds (3b, and 3c) can target the zinc-binding domain of the carbonic anhydrase enzyme of H. pylori (PDB: 4YGF) via the sulfur atom in the triazole ring system.In contrast, compound (3a) interacts via sulfur atom, and the sulfonamide group reflected the higher binding affinity.All final compounds exhibit significant efficacy against Gram-positive bacteria, including (S. aureus, S. pneumoniae, and B. subtilis).Most of the synthesized compounds show high activity against G-negative bacteria, including (P. aeruginosa, and H. pylori), with lower activity against (N.gonorrhoeae and E. coli).All the synthesized target compounds exhibit a high activity against C. albicans.The MIC results show that compound (3a) can inhibit a variety of bacterial strains at low concentrations when compared to another synthetic compound.According to both the MIC and zone on inhibitions results compound (3a) has slightly more potent activity against C. albicans than the standard antifungal drug fluconazole.

Figure 1 .
Figure 1.The docking validation was performed by redocking the co-crystallized ligand.

Figure 3 .Figure 4 .
Figure 3.This figure illustrates the more potent compound with a small MIC value.

Table 2 .
The minimum inhibitory concentration in (mcg/ml) for new compounds with references.

Table 3 .
Correlation between the zone of inhibition and the isolations.