Novel sulfonylurea derivatives as a potential agent: In-silico study, chemical synthesis, characterization and their evaluation

Sulfonylureas are a class of compounds that contain a central S-aryl sulfonylurea structure with a p-substituent on the phenyl ring and various groups terminating the urea N′ end group. They are primarily used for the treatment of type 2 diabetes mellitus, a pancreatic disorder, due to their ability to stimulate insulin release from pancreatic β-cells. 1-²

Structure of Sulphonylurea

Staphylococcus aureus is a major cause of hospital and community-acquired infections worldwide. The increasing drug resistance to antibiotics, such as vancomycin, has made the treatment of S. aureus infections more challenging. There is a significant need to discover alternative antibacterial agents to combat the rising antibiotic resistance. Interestingly, sulfonylurea herbicides, such as chlorsulfuron, have been reported to display inhibitory activities against human pathogens like Candida albicans and Mycobacterium tuberculosis due to their ability to inhibit acetohydroxy acid synthase (AHAS), an enzyme that exists only in plants and microbes. However, there are no published reports on the use of sulfonylurea derivatives as inhibitors of methicillin-resistant S. aureus (MRSA). In summary, molecular docking is a powerful tool in drug discovery that has been used to identify potential inhibitors of various targets. The sulfonylurea class of compounds, known for their antidiabetic properties, may also possess antibacterial activities and could be explored as potential leads for the development of novel anti-MRSA agents. ^3-10.  Molecular docking is a computational technique that has become a central tool in structure-based drug discovery since its introduction in 1982. It involves two main steps: 1) predicting the possible conformations, or poses, that a ligand can adopt within the binding pocket of a target protein using sampling algorithms, and 2) evaluating the binding affinity of these poses using scoring functions that estimate the interaction energy.^11-12

MATERIALS AND METHODS

The compounds diphenyl carbonate, liquid tetrahydrofuran, hexane, various amines and various solvents were commercially available (Sahyadri scientific and DODAL enterprises). reaction courses were monitored by TLC on silica gel precoated F254 merck plates. Developed plates were examined with iodine chamber.IR spectra were recorded on an FT-IR(PerkinElmer) melting points were recorded on Digital Melting Point Apparatus and are uncorrected.⁹ 1H NMR spectra were recorded on a BRUKER AVANCE II 500mz NMR spectrometer and were recorded in DMSO-d6 solvent. Chemical shifts are reported as δ ppm units (TMS). The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Mass spectra were taken with HRMS Model: 6540ba Qtof - Infinity 1290.

Step- I: Synthesis of Carbamates: Finely powdered diphenyl carbonates (15mol) were suspended in a mixture of THF (4ml) and water (36ml). The resulting suspension was stirred at room temperature followed by drop wise addition of different amine (15mmol). The reaction continued at room temperature and was monitored by TLC. After completion of reaction as indicated by TLC, the reaction mixture washes with ethyl acetate, and the organic layer was washed with cold aqueous NaOH solution (10%) and then brine. Organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure and purified by using hexane: ethyl acetate mixture as eluent to afford the desired carbamates (1-8).^10-12

Step-II: Synthesis of Sulphonylureas: Sulfonamide (2mmol) and prepared carbamate (2.2mmol) in step 1 were dissolved in acetonitrile (15ml), then DBU (3mmole) was added, and the reaction mixture was refluxed. The reaction was monitored by TLC. After completion of the reaction, as indicated by TLC, solvent was evaporated under reduced pressure. The residue was then dissolved in ethyl acetate and extracted with 0.1 N HCl. Organic layer was washed with brine, dried over Na2SO4, and concentrated again under reduced pressure. The crude product was purified by crystallization using hexane-ethyl acetate mixture to afford the desired Sulphonylurea derivatives (PG1-PG8).

Spectral data of synthesized compounds (PG1-PG8):

PG1: 4-methyl-N-(methylcarbamoyl) benzene-1-sulfonamide:- Yield - 80%; m.p, 126-128°C; Rf value: 0.8 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1 :1322(S=O Stretch), 3236(N-H Stretch), 1595(C=O Stretch), 3236(aliphatic CH), 3236(Ar C-H Stretch).1322(N-H Sr amine).H¹NMR : In H¹NMR spectrum of compound PG1: 4-methyl-N-(methylcarbamoyl)benzene-1-sulfonamide,there are well reserved resonance peak at (400 MHz,DMSO-D6),δ(ppm) = 2.4(-CH3, 3H, s), δ(ppm) 3.3 (CH3, 3H,s), δ(ppm) 7.2( Ar 2H, s) and δ (ppm)=7.8(-NH, 1H,s ) HRMS: Found 228.

PG2: N-(ethylcarbamoyl)benzenesulfonamide:- Yield - 83%; m.p, 124-126°C; Rf value: 0.7 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1:1322(S=O Stretch), 3235(N-H Stretch), 1572(C=O Stretch), 2879(aliphatic CH), 2879 (Ar C-H Stretch).1151(N-H Sr. Amine) H¹NMR: (400MHz,DMSO-D6), δ(ppm) 38.85(3H,s), δ(ppm) 3.49(3H,s), δ(ppm) 57.65(2H) .HRMS: Found 153

PG3: N- (propyl carbamoyl) benzenesulfonamide: - Yield - 68%; m.p, 170-173°C; Rf value: 0.9 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1 :1365(S=O Stretch), 3025(N-H Stretch), 1736(C=O Stretch), 3025(aliphatic CH), 3025 (Ar C-H Stretch). 464(N-H Sr. amine)

PG4: N-(hexylcarbamoyl)-4-methylbenzene-1-sulfonamide: - Yield - 84%; m.p, 94-96°C; Rf value: 0.9 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1 :1304(S=O Stretch), 3262(N-H Stretch), 1737(C=O Stretch), 3262(aliphatic CH), 3262 (Ar C-H Stretch).3262(N-H Sr. amine)

PG5: N-(benzylcarbamoyl) benzenesulfonamide: - Yield - 78%; m.p, 170-172°C; Rf value: 0.7 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1: 1322(S=O Stretch), 3237(N-H Stretch), 1737(C=O Stretch), 3123(aliphatic CH), 3123 (Ar C-H Stretch).3324(N-H Sr. amine)

PG6: N-(dimethylcarbamoyl)benzenesulfonamide:- Yield -80%; m.p, 168-170°C; Rf value: 0.8 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1 :1366(S=O Stretch), 3238(N-H Stretch), 1746(C=O Stretch), 3238(aliphatic CH), 3238(Ar C-H Stretch).3238(N-H Sr. amine)

PG7: N-(dimethylcarbamoyl)-4-methylbenzene-1-sulfonamide:- Yield - 68%; m.p, 150-152°C; Rf value: 0.9 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1 :1365(S=O Stretch), 2970(N-H Stretch), 1737(C=O Stretch), 2970(aliphatic CH), 2970(Ar C-H Stretch).3238(N-H Sr. amine)

PG8: N-(4-methylbenzene-1-sulfonyl) piperidine-1-carboxamide: - Yield - 66%; m.p, 170-172°C; Rf value: 0.7 (mobile phase: ethyl acetate: hexane: 7:3) IR cm^-1: 1306(S=O Stretch),2799 (N-H Stretch), 1737(C=O Stretch), 2970(aliphatic CH), 2970 (Ar C-H Stretch).3238(N-H Sr. amine)

2 Antimicrobial studies: -

The antimicrobial activity of compounds was tested against two bacteria; Bacillus subtitles, Escherichia coli.¹² The antibacterial activity of compounds was monitored by observing their Minimum inhibitory concentration (MIC, μg mL-1) as previously mentioned by broth dilution methods with penicillin as control drugs. The antibacterial potential of synthesized compounds was evaluated using Kirby-Bauer disk diffusion method. Briefly, each sterile disk (Himedia Pvt. Ltd. Mumbai) loaded with synthesized compounds 50 µL (1 mg/ mL) and dried.  Each disk was then placed on the surface of the sterile solidified Nutrient agar medium which was spreaded with 24 hrs old inoculums E. coli and B. subtilis and Penicillin (1mg/ mL) were used as standard ^13,14.  The plates were kept in refrigerator for diffusion for 1 hr and then transferred to the incubator at 37ºC for 24-48 hrs. After incubation, the zones around the discs were measured by the zone scale (Himedia Pvt. Ltd. Mumbai). Result was recorded by measuring the zone of inhibition in millimeter (mm) using zone recorded.

Table 1: Antimicrobial activity of the compounds (PG1-PG8)

3 In-silico molecular docking studies-

3.1 Target Protein Retrieval and Preparation: On the basis of literature survey, we found that 4YVP is major target to study antidiabetic study. Therefore, the fasta sequence of 4YVP for 4YVP species and activity taxon was retrived from National centre for biotechnology information server and searched similar biological sequence available on Protein Data Bank using basic local alignment search tool (BLAST) where we sorted top 5 to 10 selected sequence for its better query coverage, percentage identity and E- value. Three-dimensional X-ray crystallographic structure of 4YVP was obtained from PDB databank.²⁰

3.2 Grid Generation: AutoDock Tools, Chimera and Maestro were used for receptor grid identification. The prepared 4YVP protein was displayed in the workspace. The volume of grid was calculated using dimensions of pocket. The enclosing box was made small so that it will be consistent with the shape and character of the proteins active site and with ligands that were expected to be docked.

3.3 Ligands Preparation: Ligand molecules were designed in Marvin Sketch v21.13 and saved in 3D MOL2 format. All the three compounds were processed, and optimized by UCSF Chimera v1.15 using AMI-BCC semi-emperical force field other parameters defaults like steepest descent steps: 1000; Conjugate gradient steps 100, etc.

3.4 Molecular Docking of Target Protein with Ligands: After obtaining the ligands and proteins their structures were converted to pdbqt format, using in-house bash script made using Auto dock Tools 1.5.6 for ligand and ADFR suit for proteins, in which all the rotatable bonds of ligands were allowed to rotate freely, and the receptor were considered rigid. For docking studies, we used the Auto dock Vina 1.2.3 with 0.375 A⁰ of space in between the grid points. the grid box was centered on the active site if the enzyme with high resolution, allowing the program to search for additional places of probable interactions between the ligands and the receptor. Other configurations were considered default. The XYZ center coordinates(X*Y*Z) and size of the grid box is 20*20*20 A⁰. Other parameters like CPU were set for 23 exhaustiveness was 32 number of modes were 9 and energy range was set for 3 redockings were performed with the same configuration of the previously performed dockings.

3.5 Visualization: Results obtained after Auto dock Vina processing were subjected to make a complex using Biovia Discovery Studio Visualizer, 2D and 3D images for complexes were made using Maestro 12.3 and Ligplot 1.2. interactions and binding energies of test compounds were compared with standard inhibitor.

RESULTS AND DISCUSSION

Sulfonamide (2mmol) and prepared carbamate (2.2mmol) in step 1 were dissolved in acetonitrile (15ml), then DBU (3mmole) was added, and the reaction mixture was refluxed. The reaction was monitored by TLC. After completion of the reaction, as indicated by TLC, solvent was evaporated under reduced pressure. The residue was then dissolved in ethyl acetate and extracted with 0.1 N HCl. Organic layer was washed with brine, dried over Na2SO4, and concentrated again under reduced pressure. The crude product was purified by crystallization using hexane-ethyl acetate mixture to afford the desired Sulphonylurea derivatives (PG1-PG8). Newly synthesized compounds (1a-8a) were characterized by IR, NMR, UV and Thin layer chromatography. Formation of all the newly synthesized compounds (PG1-PG8) were confirmed by recording their IR, NMR, UV and thin layer chromatography. All compounds were characterized after recrystallization from appropriate solvents. Similarly, the spectral values for all the compounds are given in the experimental part and the characterization is mentioned above. The synthesized compounds were then subjected to antibacterial activity Eight compounds were subjected for antibacterial activity, out of which compound PG2 and PG5 shows good activity against E. Coli whereas the compound PG2 and PG4 shows good activity against B. subtilis as compared to standard one. All the compounds were subjected to molecular docking studies to determine the mode of binding and energy changes. The crystal structure of AKR1C1(PDB: 4YVP) was used for this study and ligands were prepared for docking by method described earlier.^17,18 The Alpha ketoreductase is co-crystallized with Glibenclamide. which we have used as reference ligand for molecular docking studies. It was observed that compound PG7(-10.266), PG6 (-10.062), PG16 (-10.037), PG24 (-9.94), PG15 (-9.507) showed highest interaction with the enzyme in their molecular docking studies.

Fig 01: Molecular docking studies 2D and 3D images of compound PG7 and PG6