Experimental

In silico studies

Molecular docking studies

Molecular docking studies were conducted using ADT software to assess the interactions between the designed compounds and the key enzymes targeted in mycobacteria (DprE1 and Mtb-DHFR) The crystal structures of these enzymes were obtained from the RCSB Protein Data Bank (http://www.rcsb.org) with the following protein data bank (PDB) codes: 4NCR for DprE1 and 1DF7 for Mtb-DHFR, [36,37]. The ADT program prepared the enzyme and ligand structures in protein data bank, partial charge Q and atom type T (PDBQT) format by adding polar hydrogens, removing water molecules and applying Kollman charges. Grid boxes (60×60×60 Å) were created to define the active sites, with specific coordinates for each enzyme: DprE1 (center_x = 17.29, center_y = -20.91, center_z = -1.71) and Mtb-DHFR (center_x = 3.32, center_y = 27.33, center_z = 11.77). To validate the docking process, the co-crystallized ligands were removed and the enzymes were re-docked. The docking results and receptor-ligand interactions were analysed and visualized using Discovery Studio 2021 Client [38].

Molecular dynamics simulations

The highest-ranked docking conformations of the three top-performing compounds, together with a reference ligand, were further evaluated through molecular dynamics (MD) simulations to gain deeper insight into ligand-target interactions. All simulations were carried out using the Desmond v7.6 package developed by D. E. Shaw Research [39]. For each ligand-protein complex, a 200 ns MD simulation was executed following a standardized workflow comprising system preparation, energy minimization, and production dynamics. Each system was embedded in an orthorhombic simulation box and solvated with explicit TIP3P water molecules, maintaining a minimum distance of 0.10 nm between the solute and the box edges. Appropriate amounts of Na⁺ and Cl⁻ ions were introduced to neutralize the system and achieve a physiological ionic strength of 0.15 M. Prior to the production run, energy minimization was performed under isothermal-isobaric ensemble (NTP) ensemble conditions at 300 K and 100 kPa to eliminate unfavourable contacts. The MD simulations were conducted for 200 ns, with trajectory snapshots saved at 10 ps intervals, yielding a total of 20,000 frames per system. Following completion of the simulations, trajectory analyses were performed using the Simulation Interaction Diagram module of Desmond. Stability and dynamic behaviour of the ligand-protein complexes were assessed by calculating key parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (_R_g) and protein-ligand interaction profiles throughout the simulation period [40].

Density function theory calculations

Density functional theory (DFT) calculations were carried out employing the hybrid B3LYP-D3 functional in conjunction with the 6-31G** basis set using the Jaguar v12.6 module of the Schrödinger Materials Suite [41]. The B3LYP functional is widely used owing to its reliable, balanced performance across a wide range of chemical systems. Inclusion of Grimme’s D3 dispersion correction [42] enhances the accuracy of modelling noncovalent interactions, conformational energetics, and reaction barriers. The polarized 6-31G** basis set provides a computationally efficient framework which, when combined with D3 (and optionally gCP), delivers qualitatively robust results suitable for preliminary geometry optimization and screening studies. Following geometry optimization, key electronic properties, including HOMO and LUMO energies, electrostatic potential, and related molecular parameters, were evaluated [43].

Chemistry

All chemicals used for the synthesis were procured from Spectrochem Private Limited, Sigma-Aldrich and Avra Synthesis Private Limited. Prior to use, reagents and solvents were purified according to standard laboratory procedures. The progress of reactions was monitored by thin-layer chromatography (TLC) on pre-coated silica gel GF254 plates, and the spots were visualized under UV light at 254 or 365 nm. Elution was carried out using different solvent systems, including hexane-ethyl acetate (7:3 and 6:4) and dichloromethane-methanol (9:1 volume ratio). Solvent removal during work-up was performed using a BUCHI R-300 rotary evaporator. Purification of the crude products was achieved by column chromatography using silica gel (100to 200 mesh). Melting points of the synthesized compounds were determined using a Veego VMP-D digital melting point apparatus and are reported without correction. Infrared spectra were recorded on a Bruker ALPHA-FT-IR spectrophotometer equipped with an ATR accessory, and absorption bands are expressed in cm-1. Molecular mass determination was carried out using a Waters Acquity QDA mass spectrometer. 1H and 13C NMR spectra were acquired on a Bruker 400 MHz NMR spectrometer using CDCl₃ or DMSO-d₆ as solvents, with tetramethylsilane (TMS) as the internal reference. Elemental composition and purity of the synthesized compounds were confirmed by elemental analysis using a Thermo Fisher FLASH 2000 organic elemental analyser, with the obtained values lying within ±0.4 % of the calculated carbon, hydrogen, and nitrogen contents.

General method for acid-amine coupling (11 to 19)

To the solution of substituted benzoic acids (2-10) (1.0 g) in DMF (10 mL), EDC.HCl (1.1 equiv) and HOBt (1.1 equiv) were added, and the reaction mixture was stirred at a temperature between 5 and 10 °C for a time of 20 min. The 1-Boc-piperazine (1.0 equiv) was added to the above solution, followed by triethylamine (3 equiv). Stirring was continued at RT for 8-10 h and the progress of the reaction was monitored by TLC using (50 % ethyl acetate in hexane). After consumption of the starting materials, the reaction mixture was poured into ice-cold water to obtain solid products (11-19).

Tert-butyl 4-benzoylpiperazine-1-carboxylate (11): Unsing benzoic acid (2) (1.0 g, 8.1 mM) to obtain the desired product (12) as a white solid (1.65 g, 70 %), m.p. 187-189 °C, TLC (_R_f): 0.7 (50 % ethyl acetate in hexane), IR: 3004, 2977, 2928 2867, 1686, 1620, 1425, 1241, 1005 cm-1.

Tert-butyl 4-(4-chlorobenzoyl)piperazine-1-carboxylate (12): Unsing 4-chlorobenzoic acid (3) (1.0 g, 6.38 mM) to obtain the desired product (13) as a white solid (1.86 g, 90 %), melting point (m.p.) 183-186 °C, TLC (_R_f): 0.7 (50 % ethyl acetate in hexane), IR: 2967, 2930, 2846, 1681, 1621, 1439, 1242, 1158, 1116, 1077 cm-1.

Tert-butyl 4-(4-fluorobenzoyl)piperazine-1-carboxylate (13): Unsing 4-fluorobenzoic acid (4) (1.0 g, 7.13 mM) to obtain the desired product as a white solid (2.02 g, 92 %), m.p. 185-187 °C, TLC (_R_f): 0.67 (50 % ethyl acetate in hexane), IR: 3003, 2978, 2927, 1688, 1621, 1427, 1286, 1243, 1068 cm-1.

Tert-butyl 4-(4-bromobenzoyl)piperazine-1-carboxylate (14): Unsing and 4-bromobenzoic acid (5) (1.0 g, 4.97 mM) to obtain the desired product as a white solid (1.62 g, 89 %), m.p. 180-183 °C, TLC (_R_f): 0.77 (50 % ethyl acetate in hexane), IR: 3002, 2977, 2928, 1683, 1620, 1426, 1285, 1243, 1069, 1005 cm-1.

Tert-butyl 4-(4-nitrobenzoyl)piperazine-1-carboxylate (15): Unsing 4-nitrobenzoic acid (6) (1.0 g, 5.98 mM) to obtain the desired product as a yellow solid (1.84 g, 92 %), m.p. 176-179 °C, TLC (_R_f): 0.82 (50 % ethyl acetate in hexane), IR: 3002, 2979, 2927, 1688, 1624, 1427, 1285, 1244, 1064, 1006 cm-1.

Tert-butyl 4-(4-methoxybenzoyl)piperazine-1-carboxylate (16): Unsing 4-methoxybenzoic acid (7) (1.0 g, 6.57 mM) to obtain the desired product as a white solid (1.93 g, 92 %), m.p. 189-192 °C, TLC (_R_f): 0.75 (50 % ethyl acetate in hexane), IR: 3002, 2979, 2927, 1685, 1624, 1422, 1285, 1246, 1067, 1004 cm-1.

Tert-butyl 4-(2-chlorobenzoyl)piperazine-1-carboxylate (17): Unsing 2-chlorobenzoic acid (8) (1.0 g, 6.38 mM) water to obtain the desired product as a white solid (1.84 g, 89 %), m.p. 179-182 °C, TLC (_R_f): 0.69 (50 % ethyl acetate in hexane), IR: 2967, 2930, 1681, 1621, 1483, 1287, 1242, 1053, 1007 cm-1.

Tert-butyl 4-(4-methylbenzoyl)piperazine-1-carboxylate (18): Unsing 4-methylbenzoic acid (9) (1.0 g, 7.34 mM) to obtain the desired product as a white solid (1.98 g, 89 %), m.p. 185-188 °C, TLC (_R_f): 0.83 (50 % ethyl acetate in hexane), IR: 3002, 2979, 2929, 1687, 1625, 1456, 1423, 1285, 1245, 1120, 1006 cm-1.

Tert-butyl 4-(2,4-dichlorobenzoyl)piperazine-1-carboxylate (19): Unsing 2,4-dicholobenzoic acid (10) (1.0 g, 5.20 mM) to obtain the desired product as a white solid (1.81 g, 87 %), m.p. 185-187 °C, TLC (Rf): 0.82 (50 % ethyl acetate in hexane), IR: 2967, 2930, 1681, 1621, 1439, 1408, 1287, 1242, 1116, 1007 cm-1.

General method for Boc-deprotection to obtain 20 to 28 (Method B)

To the solution of corresponding products (11 to 19) in DCM (7.5 mL), dioxane HCl (7.5 mL) was added and stirred at 25 °C for 3 h. The reaction was monitored by TLC after completion; the reaction mixture was then removed under reduced pressure to obtain the solid product (20 to 28).

Phenyl(piperazin-1-yl)methanone (20): tert-butyl 4-benzoylpiperazine-1-carboxylate (10) (1.5 g, 5.16 mM) offered the compound (20) (0.93 g, 95 %), which was further processed for the next step of the reaction. TLC (_R_f): 0.30 (10 % methanol in dichloromethane).

(4-chlorophenyl)(piperazin-1-yl)methanone (21): tert-butyl 4-(4-chlorobenzoyl)piperazine-1-carboxylate (11) (1.5 g, 4.61 mM) offered the compound (21) (1.03 g, 98 %), which was further processed for next step of the reaction. TLC (_R_f): 0.28 (10 % methanol in dichloromethane).

(4-fluorophenyl)(piperazin-1-yl)methanone (22): tert-butyl 4-(4-fluorobenzoyl)piperazine-1-carboxylate (12) (1.5 g, 4.86 mM) offered the compound (22) (0.98 g, 97 %), which was further processed for next step of the reaction. TLC (_R_f): 0.26 (10 % methanol in dichloromethane).

4-bromophenyl)(piperazin-1-yl)methanone (23): tert-butyl 4-(4-bromobenzoyl)piperazine-1-carboxylate (13) (1.5 g, 4.06 mM) offered the compound (23) (1.03 g, 95 %) was further processed for next step of the reaction. TLC (_R_f): 0.29 (10 % methanol in dichloromethane).

(4-nitrophenyl)(piperazin-1-yl)methanone (24): tert-butyl 4-(4-nitrobenzoyl)piperazine-1-carboxylate (14) offered the compound (24) (0.99 g, 95 %) was further processed for the next step of the reaction. TLC (_R_f): 0.31 (10 % methanol in dichloromethane).

(4-methoxyphenyl)(piperazin-1-yl)methanone (25): tert-butyl 4-(4-methoxybenzoyl)piperazine-1-carboxylate (15) (1.5 g, 4.68 mM) offered the compound (25) (1.0 g, 97 %) was further processed for next step of the reaction. TLC (_R_f): 0.32 (10 % Methanol in dichloromethane).

(2-chlorophenyl)(piperazin-1-yl)methanone (26): tert-butyl 4-(2-chlorobenzoyl)piperazine-1-carboxylate (16) (1.5 g, 4.61 mM) offered the compound (26) (0.97 g, 94 %), which was further processed for next step of the reaction. TLC (_R_f): 0.31 (10 % methanol in dichloromethane).

Piperazin-1-yl(p-tolyl)methanone (27): tert-butyl 4-(4-methylbenzoyl)piperazine-1-carboxylate (17) (1.5 g, 4.92 mM) offered the compound (27) (0.96 g, 96 %) was further processed for the next step of the reaction. TLC (_R_f): 0.27 (10 % methanol in dichloromethane).

(2,4-dichlorophenyl)(piperazin-1-yl)methanone (28): tert-butyl 4-(2,4-dichlorobenzoyl)piperazine-1-carboxylate (19) (1.5 g, 4.82 mM) offered the compound (28) (0.97 g, 95 %) was further processed for the next step of the reaction. TLC (_R_f): 0.23 (10 % methanol in dichloromethane).

General method for chloro-amine coupling (29 to 37) (Method C)

To the solution of corresponding products (20 to 28) (1.0 g) in DCM (10 mL), triethylamine (1.5 equiv) was added. and the reaction mixture was stirred at ice-cold conditions at 0 °C. Chloroacetyl chloride (1 equiv) was added dropwise, and the reaction was allowed to proceed at room temperature for 4 to 5 h. The progress of the reaction was monitored by TLC using (60 % ethyl acetate in hexane). After the consumption of starting materials, the resulting residue was washed with water and extracted in DCM. The organic layer was removed under reduced pressure to obtain the desired product (29 to 37).

1-(4-benzoylpiperazin-1-yl)-2-chloroethan-1-one (29): Using phenyl(piperazin-1-yl)methanone (20) (0.85 g, 4.46 mM) to obtained (29) as - solid (0.95 g, 80 %), m.p. 149-151 °C, TLC (_R_f): 0.37 (70 % ethyl acetate in hexane), IR: 2924, 2863, 1629, 1509, 1426, 1223, 1005, 847 cm-1.

2-chloro-1-(4-(4-chlorobenzoyl)piperazin-1-yl)ethan-1-one (30): Using (4-chlorophenyl)(piperazin-1-yl)methanone (21) (0.85 g, 3.78 mM) to obtained (30) as - solid (0.96 g, 85 %), m.p. 165-168 °C, TLC (_R_f): 0.37 (70 % ethyl acetate in hexane), IR: 2924, 2866, 1743, 1632, 1605, 1430, 1246, 1194, 1004 744 cm-1.

_2-chloro-1-(4-(4-fluorobenzoyl)piperazin-1-yl)ethan-1-_one (31): Using (4-fluorophenyl)(piperazin-1-yl)methanone (22) (0.85 g, 3.78 mM) to obtained (31) (0.99 g, 86 %), m.p. 149-151 °C, TLC (_R_f): 0.49 (70 % ethyl acetate in hexane), IR: 2924, 2865, 1740, 1626, 1427, 1268, 1006, 788 cm-1.

1-(4-(4-bromobenzoyl)piperazin-1-yl)-2-chloroethan-1-one (32): Using (4-bromophenyl)(piperazin-1-yl)methanone (23) (0.85 g, 3.15 mM) to obtained (32) (0.96 g, 88 %), m.p. 149-151°C, TLC (_R_f): 0.39 (70 % ethyl acetate in hexane), IR: 2965, 2925, 1630, 1427, 1266, 1148, 1003, 834 cm-1.

2-chloro-1-(4-(4-methoxybenzoyl)piperazin-1-yl)ethan-1-one (33): Using (4-methoxyphenyl)(piperazin-1-yl)methanone (24) (0.85 g, 3.85 mM) to obtained (33) (0.98 g, 86 %), m.p. 153-157 °C, TLC (_R_f): 0.31 (70 % ethyl acetate in hexane), IR: 2928, 2859, 1626, 1512, 1425, 1248, 1173, 1001, 841 cm-1.

2-chloro-1-(4-(4-nitrobenzoyl)piperazin-1-yl)ethan-1-one (34): Using (4-nitrophenyl)(piperazin-1-yl)methanone (25) (0.85 g, 3.61 mM) to obtained (34) (0.97 g, 87 %), m.p. 144-147 °C, TLC (_R_f): 0.43 (70 % ethyl acetate in hexane), IR: 2996, 2922, 2361, 1634, 1521, 1433, 1349, 1152, 1004, 848 cm-1.

2-chloro-1-(4-(2-chlorobenzoyl)piperazin-1-yl)ethan-1-one (35): Using (2-chlorophenyl)(piperazin-1-yl)methanone (26) (0.85 g, 3.78 mM) to obtained (35) (0.94 g, 83 %), m.p. 150-153 °C, TLC (_R_f): 0.38 (70 % ethyl acetate in hexane), IR: 2924, 1742, 1633, 1481, 1431, 1284, 1194, 1051, 1004, 740 cm-1.

2-chloro-1-(4-(4-methylbenzoyl)piperazin-1-yl)ethan-1-one (36): Using piperazin-1-yl(p-tolyl)methanone (27) (0.85 g, 4.16 mM) to obtained (36) (1.0 g, 86 %), m.p. 175-178 °C, TLC (_R_f): 0.3 (70 % ethyl acetate in hexane), IR: 2921, 2864, 1632, 1430, 1274, 1147, 1005, 831 cm-1.

3-chloro-1-(4-(2,4-dichlorobenzoyl)piperazin-1-yl)propan-1-one (37): Using (2,4-dichlorophenyl) (piperazin- -1-yl) methanone (28) (0.85 g, 4.16 mM) to obtained (37) (0.93 g, 88 %), m.p. 185-187 °C, TLC (_R_f): 0.39 (70 % ethyl acetate in hexane), IR: 2924, 2865, 1742, 1632, 1430, 1284, 1030 cm-1.

General procedure for the synthesis of 5,6-diphenyl-1,2,4-triazine-3-thiol (38)

The compound (38) was prepared according to the reported procedure [44,45]. Briefly, a solution of benzil (1.0 g, 1 mM) in glacial acetic acid (10 mL) was stirred at 100 °C for 1 h. After that thiosemicarbazide (0.86 g, 9.6 mM) was added, the reaction was refluxed for 2-3 h, the progress of the reaction was monitored by TLC using (20 % ethyl acetate in hexane) After the consumption of starting materials, the reaction mixture cooled down and filter out to get orange precipitate and washed with cold acetic acid and water and recrystallized it form ethanol to get desired product (34). Yield: 85 %; m.p. 234 to 235 °C (m.p. 234 to 236 °C [44])

General procedure for the synthesis of the target compounds (FP1 to FP8) (Method D)

To the solution of compound (38) (1 equiv) in DMF (10 V), K2CO3 (1 equiv) was added. The reaction mixture was stirred at room temperature for 1 h. After that, the corresponding product (29 to 37) (1.2 equiv) and potassium iodide (0.6 equiv) were added to the above solution. The progress of the reaction was monitored by TLC using (60 % ethyl acetate in hexane). After consumption of the starting materials, the reaction mixture was poured into ice-cold water to obtain solid products (FP1-FP9), which were further purified by column chromatography using 100-200 mesh silica gel as the stationary phase and Ethyl acetate:hexane as the mobile phase to afford the desired pure products (FP1 to FP9).

1-(4-Benzoylpiperazin-1-yl)-2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (FP1): Using 1-(4-benzoylpiperazin-1-yl)-2-chloroethan-1-one (26) the desired compound (FP1) was obtained as a white solid (0.72 g, 60 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: hexane as mobile phase, m.p. 173-176 °C. TLC (_R_f): 0.43 (70 % ethyl acetate in hexane); IR: 2916, 1620, 1429, 1287,1258, 1184, 696 cm-1; 1H NMR: δ 7.49-7.45 (t, J = 9.3 Hz, 11H, ArH), 7.42 (s, 2H, ArH), 7.40-7.36 (d, J = 8.0 Hz, 2H, ArH), 4.45 (s, 2H, CH2), 3.64 (m, 8H, CH2); C28H25N5O2S requires: C, 67.86; H, 5.08; N, 14.13; found requires: C, 67.76; H, 5.03; N, 14.08; mass (m/z): 496.2 (M+H)+.

1-(4-(4-Chlorobenzoyl)piperazin-1-yl)-2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (FP2): Using 2-chloro-1-(4-(4-chlorobenzoyl)piperazin-1-yl)ethan-1-one (27) the desired compound (FP2) was obtained as a light orange solid (0.81 g, 63 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p. 177-79 °C; TLC (R_f): 0.47 (70 % ethyl acetate in hexane); IR: 2918, 2859, 1629, 1426, 1263, 1180, 1088, 754 cm-1; 1H NMR: δ 7.55-7.52 (d, J = 8.5 Hz, 2H, Ar_H), 7.46-7.36 (m, 12H, ArH), 4.45 (s, 2H, CH2), 3.71-3.57 (m, 8H, CH2); C28H24ClN5O2S Rrequires: C, 63.45; H, 4.56; N, 13.21; found requires: C, 63.35; H, 4.47; N, 13.18; mass (m/z): 531.2 (M+H)+, 532.4 (M+2); HR-MS (m/z): [M+H]+ calculated 530.1373; found 530.1389.

2-((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(4-fluorobenzoyl)piperazin-1-yl)ethan-1-one (FP3): Using 2-chloro-1-(4-(4-fluorobenzoyl)piperazin-1-yl)ethan-1-one (28) the desired compound (FP 3) was obtained as a light orange solid (0.80 g, 64 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p. 172-75 °C; TLC (Rf): 0.52 (70 % ethyl acetate in hexane); IR: 2916, 2858, 1629, 1424, 1333, 1180, 1000, 755, 695 cm-1; 1H NMR: δ 7.70-7.67 (m, 2H, ArH), 7.49-7.36 (m, 12H, ArH), 4.45 (s, 2H, CH2), 3.70-3.57 (m, 8H, CH2); C28H24FN5O2S Requires: C, 65.48; H, 4.71; N, 13.64; found requires: C, 65.38; H, 4.68; N, 13.59; Mass (m/z): 514.3 (M+H)+; HR-MS (m/z): [M+H]+ calculated 514.1668; found 514.1684.

1-(4-(4-Bromobenzoyl)piperazin-1-yl)-2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (FP4): Using 1_-(4-(4-bromobenzoyl)piperazin-1-yl)-2-chloroethan-1-one_ (29) the desired compound (FP4) was obtained as a light yellow solid (0.97 g, 69 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p. 175-78 °C; TLC (Rf): 0.46 (70 % ethyl acetate in hexane); IR: 2920, 2859, 1633, 1427, 1334, 1182, 1000, 757, 696 cm_-_1; 1H NMR: δ 7.54-7.29 (m, 14H, ArH), 4.44 (s, 2H, CH2), 3.69-3.34 (m, 8H, CH2); C28H24BrN5O2S Requires: C, 58.54; H, 4.21; N, 12.19; found requires: C, 58.50; H, 4.18; N, 12.22; mass (m/z): 576.0 (M+2H)+.

2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(4-nitrobenzoyl)piperazin-1-yl)ethan-1-one (FP5): Using that 2-chloro-1-(4-(4-nitrobenzoyl)piperazin-1-yl)ethan-1-one (30) the desired compound (FP5) was obtained as a light yellow solid (0.86 g, 65 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p. 176-79 °C; TLC (_R_f): 0.46 (70 % ethyl acetate in hexane); IR: 2956, 2923, 1632, 1514, 1427, 1336, 1177, 1004, 696 cm-1; 1H NMR: δ 8.33-8.31 (d, J = 8.4 Hz, 2H, ArH), 7.74-.72 (d, J = 8.5 Hz, 2H, ArH), 7.49-7.39 (m, 10H, ArH), 4.49-4.41 (d, J = 30.4 Hz, 2H, CH2), 3.76-3.51 (t, J = 49.2 Hz, 6H, CH2), 3.35-3.25 (d, J = 42.1 Hz, 2H, CH2); C28H24N6O4S requires: C, 62.21; H, 4.48; N, 15.22; found requires: C, 62.18; H, 4.40; N, 15.18; mass (m/z): 541.4 (M+H)+.

2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)ethan-1-one (FP6): Using 2-chloro-1-(4-(4-methoxybenzoyl)piperazin-1-yl)ethan-1-one (31) the desired compound (FP 6) was obtained as a white solid (0.87 g, 68 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p. 178-81°C; TLC (_R_f): 0.43 (70 % ethyl acetate in hexane); IR: 2955, 2922, 1630, 1425, 1335, 1174, 1002, 758, 696 cm-1; 1H NMR: δ 7.50-7.36 (m, 12H, ArH), 7.03-7.00 (m, 2H, ArH), 4.44 (s, 2H, CH2), 3.81 (s, 3H, OCH3), 3.68-3.48 (m 8H, C_H_2); C29H27N5O3S requires: C, 66.27; H, 5.18; N, 13.32; found requires: C, 66.20; H, 5.16; N, 13.28; mass (m/z): 526.2(M+H)+.

1-(4-(2-Chlorobenzoyl)piperazin-1-yl)-2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (FP7): Using 2_-chloro-1-(4-(2-chlorobenzoyl)piperazin-1-yl)ethan-1-one_ (32) the desired compound (FP 7) was obtained as a light orange solid (0.89 g, 69 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p. 174-77 °C; TLC (_R_f): 0.42 (70 % ethyl acetate in hexane); IR: 2922, 1631, 1482, 1336, 1177, 1002, 696 cm-1; 1H NMR: δ 8.34-8.27 (m, 2H, ArH), 7.96-7.90 (d, J = 16.9 Hz, 1H, ArH), 7.80-7.76 (t, J = 7.9 Hz, 1H, ArH), 7.55-7.39 (m, 11H, ArH), 4.49-4.40 (d, J = 25.8 Hz, 2H, CH2), 3.76-3.40 (m, 8H, CH2); C28H24ClN5O2S Requires: C, 63.45; H, 4.56; N, 13.21; Found Requires: C, 63.40; H, 4.50; N, 13.16; mass (m/z): 530.3 (M+).

2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(4-methylbenzoyl)piperazin-1-yl)ethan-1-one (FP8): Using 2-chloro-1-(4-(4-methylbenzoyl)piperazin-1-yl)ethan-1-one (33) the desired compound (FP8) was obtained as a white solid (0.87 g, 70 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: hexane as mobile phase, m.p.173-76 °C; TLC (_R_f): 0.43 (70 % ethyl acetate in hexane); IR: 2958, 2921, 1632, 1429, 1363, 1178, 1004, 766, 696 cm-1; 1H NMR: δ 7.49-7.26 (m, 14H, ArH), 4.44 (s, 2H, CH2), 3.68-3.36 (m, 8H, CH2), 2.35 (s, 3H, CH3); C29H27N5O2S requires: C, 68.35; H, 5.34; N, 13.74; found requires: C, 68.30; H, 5.30; N, 13.70; mass (m/z): 510.4 (M+H)+; HR-MS (m/z): [M+H]+ calculated 510.1919; found 510.1946.

1-(4-(2,4-dichlorobenzoyl)piperazin-1-yl)-2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (FP9): Using 2-chloro-1-(4-(2,4-dichlorobenzoyl)piperazin-1-yl)ethan-1-one (34) the desired compound (FP9) was obtained as a white solid (0.82 g, 69 %), which was further purified by column chromatography using 100-200 silica gel as stationary phase and ethyl acetate: Hexane as mobile phase, m.p.171-73 °C; TLC (_R_f): 0.41 (70 % ethyl acetate in hexane); IR: 2958, 2921, 1632, 1429, 1363, 1178, 1004, 766, 696 cm-1; 1H NMR: δ 7.78-7.75 (dd, J = 6.5, 2.0 Hz, 1H, ArH), 7.57-7.33 (m, 13H, ArH), 4.50-4.37 (m, 2H, CH2), 3.74 (s, 2H, CH2), 3.60 (m, 2H, CH2), 3.48-3.41 (m, 2H, CH2), 3.19-3.10 (m, 2H, CH2); C28H23N5O2S requires: C, 59.58; H, 4.11; N, 12.14; found requires: C, 59.48; H, 4.04; N, 12.11; mass (m/z): 565.3 (M+H)+

Biological Activity

Anti-tubercular activity

The synthesized analogues (FP1 to FP9) were screened in vitro for their anti-mycobacterial activity against the H37Rv strain using the Microplate Alamar Blue Assay method [46]. The reference standard drugs, such as isoniazid, ethambutol, and pyrazinamide, were also used for comparison. The steps involved in the Microplate Alamar Blue Assay method for screening the anti-mycobacterial activity of the synthesized analogues (FP1 to FP9) are as follows: 200 μL of sterile deionized water was added to all the outer perimeter wells of the sterile 96-well plate to prevent the evaporation of the medium in the wells during the incubation period. The 96-well plate was then supplemented with 100 μL of Middlebrook 7H9 broth, and the serial dilutions of the test drugs were directly prepared on the plate. The concentration range of the test drugs was maintained between 100 and 0.2 μg mL-1. The plates were covered, sealed with parafilm, and incubated for five days at 37 °C. After the incubation period, 25 μL of the freshly prepared mixture of Alamar Blue reagent and Tween 80 (10 wt.%) was added to the plate, followed by further incubation for 24 hours. The colour change in the wells, i.e. blue colour indicating the absence of growth, and pink colour indicating the presence of growth, was recorded, and the minimum inhibitory concentration was determined.

In vitro cytotoxicity assay

The cytotoxic potential of the test samples was studied on the RAW 264.7 cell line obtained from the National Centre for Cell Science (Pune). The MTT assay was used for the purpose of the experiment. Cells were seeded at a density of 104 cells per well in a 96-well plate and allowed to adhere for 24 h in DMEM medium supplemented with 10 % foetal bovine serum and 1 % penicillin-streptomycin solution. The cell cultures were maintained at 37° C in a humidified incubator with 5 % CO2 supplementation. After 24 h of cell seeding, the cells were treated with different concentrations of the test compounds. The stock solution of the test compounds was prepared in dimethyl sulfoxide and then diluted with incomplete medium to obtain the required concentrations. After 24 h of treatment of the cells with the test compounds, MTT solution (5 μg mL-1) was added to each well of the plate and incubated for a period of 2 h in a CO2 incubator. The wells containing untreated cells were considered the control, while the wells devoid of cells were considered the blank. After the experiment was completed, the medium was carefully discarded, and the resulting crystals were dissolved in 100 μL of DMSO. The absorbance was measured at 540 nm using an ELISA microplate reader (iMark Bio-Rad USA). The IC50 values for the compounds were obtained using GraphPad Prism software version 6 [47-48].

In silico drug-likeness and ADMET prediction studies

The pharmacokinetic profiles and drug-likeness of the studied compounds were also predicted using the SWISSADME tool. This tool was used to investigate key physicochemical parameters of the studied compounds, including molecular weight, the number of hydrogen-bond donors and acceptors, the n-octanol/water partition coefficient (log P), and the number of rotatable bonds. Other important parameters related to absorption, distribution, metabolism, and excretion were also studied using the same tool [49].