Chemistry

In Fig. 1: Scheme 1, the final product, which is 4-(2-amino-1,3-thiazol-5-yl) benzohydrazide derivatives, were synthesized through a series of six steps. The process began by diazotizing para-aminobenzoic acid using NaNO2 and HCl, resulting in the formation of diazonium salt. A mixture of 2-aminothiazole, and CuCl2 was added to the diazonium salt and heated under reflux conditions to yield 4-(2-amino-1,3-thiazol-5-yl) benzoic acid, which was then treated with DCM and SOCl2 to convert the COOH group to COCl, achieved by refluxing for 2 h. Subsequently, the chlorine group in 4-(2-amino-1,3-thiazol-5-yl) benzoyl chloride was replaced with an NH-NH group in the presence of dry benzene and NH2-NH2, resulting in 4-(2-amino-1,3-thiazol-5-yl) benzohydrazide after refluxing for 7 h. Finally, various aromatic aldehydes were reacted with the resulting molecule, ethanol was employed in order to achieve the final products, denoted as (DT01–DT10).

Fig. 1

Scheme 1: Synthetic procedure of 2-aminothiazole derivatives (DT01–DT10)

Molecular docking of ligands with MurA dead-end complex with fosfomycin

Ten compounds were computationally designed and optimized with the MurA dead-end complex with fosfomycin (PDB ID: 3KR6) targeting the active binding site to observe the binding energy involved in forming complex and molecular interactions. The docking results are summarized in Table 1. From the ten compounds, DT03, DT05, and DT06 exhibited the highest binding energies when interacting with the above-mentioned protein, with values of − 10.0, − 9.5, and − 9.6 kcal/mol, respectively, which is considerably higher when compared with the co-crystallized ligand. Fosfomycin has a docking score of − 4.8 kcal/mol. The interactions can be observed in Figs. 2, 3 and 4. DT03 had the highest binding energy of − 10.0 kcal/mol when forming a complex attributed to hydrogen bonds with ALA:298 and VAL:327, as well as various interactions with active site amino acids, such as ARG:120, ALA165, and PRO:121.

Table 1 Docking results of compounds targeting MurA dead-end complex with fosfomycin (PDB ID: 3KR6)Fig. 2

3D and 2D interactions of protein-fosfomycin complex

Fig. 3

3D and 2D interactions of protein-DT03 complex

Fig. 4

3D and 2D interactions of protein-DT06 complex

In silico drug-likeness and toxicity profile

The highest TPSA (154.67) was observed in DT06, followed by DT03. DT06 was also found to have a molecular weight of 367.38 Daltons having two hydrogen bond donors and 5 acceptors. Some of the compounds were predicted to be very mild toxic belonging to toxicity class 4 and 5. However, no serious toxicity was observed in the synthesized derivatives.

Characterization4-(2-amino-1,3-thiazol-5-yl) benzoic acid (4)

Reddish powder, %Yield (4.59 g, 78.35%), MP-310.53 °C, IRύ: 3435(NH2), 3390, 2950, 2828, 1578, 1512, 1450, 1311, 1244, 647 cm−1. 1H NMR (DMSO-d6): 8.16 (d, 2H, Ar–H), 6.78 (s, 1H, NH), 12.73 (s, 1H, OH). MS m/z (%) 221.25 (M++1, 8), 218(M+, 30), 221(40), 207(21), 128(14), 104(43), 65(24), 53(19). Anal. Calcd. For C10H8N2O2S (220.17) Calcd: C,54.53; H,3.60; N,12.70; O,14.53; S,14.56. Found: C, 54.03; H,3.45; N, 12.20; O,14.19; S,14.37%.

4-(2-amino-1,3-thiazol-5-yl) benzoyl chloride (5)

Brownish powder, %Yield (4.26 g, 73.93%), MP-317.23 °C, IRύ: 3429(NH2), 3213, 2953, 2838, 1580, 1502, 1459, 1301, 1254, 1151 cm−1. 1H NMR (DMSO-d6) 8.26 (d, 4H, Ar–H), 6.68 (s, 1H, NH), 7.09 (s, 1H, CH). MS m/z (%) 237.57(M+ +1, 8), 228(M+, 30), 231(40), 217(32), 158(24), 124(43), 95(24), 73(19). Anal. Calcd. For C10H7ClN2OS (238.69) Calcd: C, 50.32; H,2.96; Cl, 14.85; N, 11.74; O,6.70; S,13.43. Found: C, 49.92; H, 2.36; Cl, 14.65; N, 11.24; O,6.56; S,13.09%.

4-(2-amino-1,3-thiazol-5-yl) benzohydrazide (6)

Pale yellowish powder, %Yield (4.93 g, 79.74%), MP-343.23 °C. IRύ: 3440(NH2), 3213, 2953, 2838, 1580, 1502, 1459, 1301, 1214, 1151 cm−1. 1H NMR (DMSO-d6) 4.49 (s, 2H, NH2), 7.09 (s, 1H, CH), 8.18 (q, 4H, Ar–H), 9.64 (s, 1N, NH). MS m/z (%) 235.38(M+ +1, 8), 233(M+, 30), 231(40), 213(30), 151(29), 119(47), 96(34), 71(29). Anal. Calcd. For C10H10N4OS (234.28) Calcd: C,51.27; H,4.30; N,23.92; O,6.83; S,13.68. Found: C,50.897; H,4.09; N,23.22; O,5.93; S,13.18%.

4-(2-amino-1,3-thiazol-5-yl)-N′-[(Z)-(4-bromophenyl) methylidene] benzohydrazide (DT01)

%Yield (4.57 g, 79.13%). MP-361 °C. IRύ: 3437(NH2), 3032, 2960, 2753, 1725, 1680, 1628, 1436, 1367, 1231, 550 cm−1. 1H NMR (DMSO-d6) 6.35 (s, 2H, NH2), 7.09 (s, IH, CH), 7.74 (q, 4H, Ar–H), 7.89 (q, 4H, Ar–H), 8.71 (s, 1H, NH). 13C NMR (DMSO-d6) 122.2, 125.4, 127.6, 128.5, 130.2, 131.7, 132.7, 132.8, 137.1, 137.7, 146.8, 163.2, 168.10. MS m/z (%) 401.98(M+ +1, 8), 379(M+, 30), 293(41), 213(33), 154(28), 116(46), 97(33), 74(27). Anal. Calcd. For-C17H13N4BrOS (401.28). Calcd: C,50.88; H,3.27; Br,19.91; N,13.96; O,3.99; S,7.99. Found:C,50.38; H,3.07; Br,19.61; N,13.76; O,3.69; S,7.49%.

4-(2-amino-1,3-thiazol-5-yl)-N′-[(Z)-(3,4-dimethoxyphenyl) methylidene] benzohydrazide (DT02)

%Yield (4.98 g, 80.13%). MP-378.53 °C. IRύ: 3437(NH2), 3038, 2934, 2870, 2773, 1755, 1690, 1638, 1436, 1367, 1231, 1191 cm−1. 1H NMR (DMSO-d6) 3.94 (d, 3H, CH3), 6.90 (s, 2H, NH2), 7.24 (d, 2H, Ar–H), 7.53 (m, 4H, Ar–H), 7.98 (m, 4H, Ar–H), 8.40 (s, 1H, NH). 13C NMR (DMSO-d6) 56.1, 109.2, 111.7, 122.5, 122.3, 127.6, 130.2,130.6,137.1, 137.8, 149.9, 152.1, 163.2, 168.9. MS m/z (%) 383.02 (M+ +1, 15), 376(M+, 34), 361(23), 235(24), 169(63), 148(46), 119(48), 87(29), 81(79), 85(31), 70(37). Anal. Calcd. For-C19H18N4O3S (382.44). Calcd: C,59.67; H,4.74; N,14.65; O,12.55; S,8.38. Found:C,59.37; H,4.44; N,14.45; O,12.35; S,8.28%.

4-(2-amino-1,3-thiazol-5-yl)-N′-[(Z)-(3,4-dihydroxyphenyl) methylidene] benzohydrazide (DT03)

%Yield (4.23 g,78.53%), MP-371.21 °C, IRύ:3437(NH2), 3038, 2934, 2870, 2773, 1755, 1690, 1638, 1436, 1367, 1231, 1191 cm−1. 1H NMR (DMSO-d6) 6.65 (d, 2H, CH), 6.66 (s, 2H, NH2), 7.26 (s, 1H, CH), 7.45 (d, 2H, Ar–CH), 7.90–7.93 (m, 4H, Ar–CH), 8.04 (s, 1H, OH), 8.05 (s, 1H, NH). 13C NMR (DMSO-d6) 116.3,117.4, 122.2, 123.2, 127.6,130.2, 131.3,132.8,137.1, 137.7, 146.1,146.8,149.6,163.2,168.8. MS m/z (%) 355 (M+ +1, 5), 323(7), 261(25), 177(91), 132(28), 110(16), 92(69), 75(24). Anal. Calcd. For-C17H14N4O3S (354.38) Calcd: C,57.62; H,3.98; N,15.81; O,13.54; S,9.05. Found: C,57.42; H,3.78; N,15.61; O,13.44; S,9.01%.

4-(2-amino-1,3-thiazol-5-yl)-N′-[(Z)-(2-methoxyphenyl) methylidene] benzohydrazide (DT04)

%Yield (4.07 g, 76.86%), MP-352.53 °C, IRύ: 3432 (NH2), 3038, 2954, 2890, 2820, 2376, 1765, 1670, 1658, 1446, 1397, 1291 cm−1. 1H NMR (DMSO-d6) 3.93 (s, 3H, OCH3), 6.64 (s, 2H, NH2), 7.36–7.42 (m, 4H, Ar–CH), 8.37 (s, 1H, NH). 13C NMR (DMSO-d6) 55.8, 111.2, 116.9, 121.1, 122.2, 127.6, 132.0, 130.2, 131.7, 132.8, 137.1, 137.7, 146.0, 157.6, 163.2, 168.9. MS m/z (%) 352.93(M+,8), 337(9), 271(35), 197(81), 137(36), 117(26), 72(53). Anal. Calcd. For: C18H16N4O2S (352.41) Calcd: C,61.35; H,4.58; N,15.90; O,9.08; S,9.10. Found: C,61.15; H,4.39; N,15.10; O,8.9; S,9.01%.

4-(2-amino-1,3-thiazol-5-yl)-N′-{(Z)-4-(dimethylamino) phenyl methylidenebenzohydrazide (DT05)

%Yield (3.96 g, 71.35%), MP-367.43 °C, IRύ: 3437(NH2), 3103, 2971, 2859, 2820, 2510, 1795, 1684, 1617, 1596, 1437, 1316, 1253 cm−1. 1H NMR (DMSO-d6) 3.03 (s, 3H, CH3), 6.72 (s, 2H, NH2), 7.25 (d, 2H, Ar–CH), 7.69–7.71 (m, 4H, Ar–CH). 13C NMR (DMSO-d6) 41.3, 111.9, 122.2, 123.2, 127.6, 128.3, 130.2, 132.8, 137.1, 137.7, 146.8, 153.4, 163.2, 168.9. MS m/z (%) 366.21(M++1,24), 352(59), 283(31), 194(67), 139(76), 121(34), 67(43). Anal. Calcd. For: C19H19N5OS (365.46) Calcd: C,62.45; H,5.24; N,19.16; O,4.38; S,8.77. Found: C,62.13; H,5.09; N,19.01; O,4.17; S,8.36%.

4-(2-amino-1,3-thiazol-5-yl)-N′-[(Z)-(2-nitrophenyl) methylidene] benzohydrazide (DT06)

%Yield (3.73 g, 70.03%), MP-482 °C. IRύ: 3437(NH2), 3210, 2860, 2490, 1927, 1637, 1594, 1489, 1356, 1292, 1253 cm−1. 1H NMR (DMSO-d6) 7.26 (s, 2H, NH2), 7.74 (d, 1H, Ar–CH), 8.09–8.11 (m, 4H, Ar–CH), 8.30 (s, 1H, CH), 9.11 (s, 1H, NH).13C NMR (DMSO-d6) 122.3, 124.0, 127.6, 128.4, 130.1, 130.2, 131.8, 132.8, 137.1, 134.9, 137.6, 143.3, 147.9, 163.3, 168.7. MS m/z (%) 368.27 (M++1,32), 354(64), 278(29), 193(76), 147(53), 117(76), 78(43). Anal. Calcd. For C17H13N5O3S (367.38) Calcd: C,55.58; H,3.57; N,19.06; O,13.06; S,8.73. Found: C,55.26; H,3.19; N,18.96; O,13.01; S,8.57%.

4-(2-aminothiazol-5-yl)-N′-[2-(dimethylamino) benzylidene] benzohydrazide (DT07)

%Yield (3.94 g, 70.64%). MP-364.24 °C. IRύ: 3433(NH2), 3209, 2862, 2492, 1929, 1641, 1589, 1481, 1359, 1297, 1249 cm−1. 1H NMR (DMSO-d6) 7.98–7.96 (m, 1H), 7.93–7.91 (m, 1H), 7.83 (s, 1H), 7.67–7.65 (m, 1H), 7.52–7.50 (m, 1H), 6.19 (s, 2H). 13C NMR (DMSO-d6) 126.32, 128.29, 129.07, 129.10, 129.13, 132.31, 132.86, 133.93, 136.87, 139.87, 147.52, 162.59, 168.31. MS m/z (%) 356.83 (M+ +1,17), 341(15), 276(27), 197(61), 141(26), 113(71), 71(49). Anal. Calcd. For C17H13ClN4OS (356.83) Calcd: C,57.54; H,3.84; N,18.21; O,12.84; S,7.27; Cl,14.47. Found: C,57.36; H,3.81; O,12.79; S,7.22; Cl,14.41%.

4-(2-aminothiazol-5-yl)-N′-benzylidene benzohydrazide (DT08)

%Yield (4.12 g, 72.89%). MP-316.28 °C. IRύ: 3437(NH2), 3215, 2861, 2497, 1922, 1637, 1585, 1489, 1351, 1295, 1251 cm−1. 1H NMR (DMSO-d6) 7.98–7.96 (m, 2H), 7.93–7.91 (m, 2H), 7.81 (s, 1H), 7.61 (ddd, 2H), 7.42–7.40 (m, 3H), 6.19 (s, 2H). 13C NMR (DMSO-d6) 126.32, 127.70, 128.29, 128.88, 129.07, 130.59 132.86, 133.93, 134.23, 139.54, 147.43 162.59, 168.31. MS m/z (%) 322.38 (M+ +1,41), 308(19), 269(34), 196(46), 137(34), 109(29), 69(41). Anal. Calcd. For C17H14N4OS (322.38) Calcd: C,56.81; H,3.76; N,18.34; O,12.75; S,7.31. Found: C,56.75; H,3.72, N,18.29; O,12.69; S,7.29%.

4-(2-aminothiazol-5-yl)-N′-(2,3,4-trimethoxy) benzylidene benzohydrazide (DT09)

%Yield (3.94 g, 75.76%). MP-356.14 °C. IRύ: 3438 (NH2), 3203, 2854, 2490, 1929, 1631, 1598, 1482, 1351, 1296, 1258 cm−1. 1H NMR (DMSO-d6) 8.30 (s, 1H), 7.98–7.91 (m, 4H), 7.25 (d, 1H), 6.87 (d, 1H), 6.19 (s, 2H), 3.87–3.85 (m, 9H). 13C NMR (DMSO-d6) 168.31, 162.59, 156.48, 151.26, 146.91, 142.15, 139.54, 133.93, 132.95, 129.07, 128.29, 126.32, 121.89, 121.53, 107.48, 61.53, 61.01, 56.56. MS m/z (%) 412.46 (M+ +1,37), 401(19), 376(21), 327(32), 275(49), 207(18), 176(25), 139(46), 102(42), 72(41). Anal. Calcd. For. C20H20N4O4S (412.46) Calcd: C,54.54; H,3.54; N,17.37; O,12.76; S,7.25. Found: C,54.52; H,3.51, N,17.33; O,12.72; S,7.21%.

4-(2-aminothiazol-5-yl)-N′-[2-(dimethylamino) benzylidene] benzohydrazide (DT10)

%Yield (3.89 g, 79.61%). MP-388.16 °C. IRύ: 3432(NH2), 3214, 2851, 2493, 1932, 1636, 1591, 1491, 1359, 1299, 1257 cm−1. 1H NMR (DMSO-d6) 7.98–7.91 (m, 4H), 7.55 (s, 1H), 7.51–7.49 (m, 1H), 7.17–7.14 (m, 2H), 6.73–6.73 (m, 1H), 6.19 (s, 2H), 2.94 (s, 6H). 13C NMR (DMSO-d6) 168.31, 162.59, 150.33, 144.05, 139.54, 133.93, 132.90, 131.56, 129.07, 128.29, 127.44, 126.32, 126.18, 123.02, 115.55, 44.89. MS m/z (%) 365.45 (M++1,57) 341(12), 309(37), 267(23), 194(49), 135(31), 107(27), 71(43). Anal. Calcd. For. C19H19N5OS (365.45) Calcd: C,55.53; H,3.64; N,15.37; O,12.67; S,7.34. Found: C,55.51; H,3.61; N,15.32; O,12.62; S,7.28%.

Anti-microbial activity and MIC

The anti-microbial activity of the synthesized derivatives was tested by disc diffusion method against different strains. From the results, it was observed that almost all the prepared molecules showed varying degrees of inhibition against the tested microorganisms as depicted in Figs. 5 and 6.

Fig. 5

IZD of the synthesized derivatives at concentration 300 µg

Fig. 6

MBC and MFC of the synthesized derivatives

Cytotoxicity studies (MTT assay)

In vitro cytotoxicity assessments using the MTT assay were conducted to evaluate the viability of bacterial cell lines across a range of concentrations of hybrid analogues. The MTT assay relies on metabolically active cells with functioning mitochondria to transform MTT into a blue formazan product. Notably, the synthesized compound’s cytotoxicity was found to be lower compared to standard levofloxacin and doxorubicin, as indicated in Fig. 7.

Fig. 7

MIC, cytotoxicity, IC50 and selectivity index of the synthesized compounds

Molecular dynamic simulation

The stability of the docked protein–ligand complex was assessed by measuring the root-mean-square deviation (RMSD) of each trajectory frame during the molecular dynamics simulation (MDS), as shown in Fig. 8. The RMSD values were calculated for a period of 1 ns (1000 ps) and are plotted. The RMSD analysis is based on the comparison of the ligand and protein’s positions against their initial docking coordinates to evaluate the structural stability over time. The RMSD values for the protein are represented by the black curve, while the ligand’s RMSD is depicted by the green curve.

Fig. 8

The RMSD plot reveals an initial increase in the protein’s RMSD due to ligand binding, observed within the first 200 ps of the simulation as the complex undergoes conformational adjustments. After this period, the protein reaches a stable conformation, and fluctuations observed are minor and fall within the expected range for a flexible biomolecular system indicating that the protein–ligand complex has reached an equilibrium state. Similarly, the ligand exhibits initial fluctuations, stabilizing around 100 ps with minimal deviation thereafter, confirming its tight binding to the protein. While the protein continues to show slight fluctuations post-stabilization, the ligand remains largely unchanged, suggesting a strong and stable interaction between the two components throughout the simulation.

The root-mean-square fluctuation (RMSF) was analysed to evaluate residue-specific flexibility during the molecular dynamics simulation (MDS), as shown in Fig. 9. The RMSF plot highlights fluctuations along the protein chain, providing insights into its structural stability. A significant peak at (1036.88, 0.481 nm) corresponds to an alanine (ALA) residue located within the active site. This suggests dynamic movement in the binding region, which may influence ligand interactions. Another notable fluctuation is observed at (52.95, 0.454 nm), corresponding to a phenylalanine (PHE) residue located away from the binding site. This flexibility is unlikely to impact the stability of the protein–ligand complex. In contrast, the core residues, particularly those in alpha-helices and beta-strands, exhibit RMSF values below 0.20 nm, indicating a stable and rigid structure. As expected, terminal regions show higher flexibility, occasionally exceeding 0.30 nm.

Fig. 9

The total free energy analysis provides insights into the thermodynamic stability and binding efficiency of the protein and its complex with the ligand. Key energetic components such as van der Waals interactions, electrostatic energy, polar solvation energy, and solvent-accessible surface area (SASA) energy were calculated to evaluate the energetics of the protein both with and without the ligand. For the protein with ligand, the van der Waals energy for the protein–ligand complex was calculated to be − 187.513 ± 11.260 kJ/mol, indicating significant non-covalent interactions between the protein and ligand. These interactions play a key role in stabilizing the complex. The electrostatic energy contribution was − 71.231 ± 9.099 kJ/mol, highlighting the importance of charge-based interactions between the protein and the ligand. A positive polar solvation energy of 138.928 ± 13.305 kJ/mol was observed, reflecting the energy required for desolvation of polar residues during the binding process. This value is counteracted by other stabilizing contributions. The solvent-accessible surface area (SASA) energy was − 17.709 ± 0.587 kJ/mol, which suggests a reduction in solvent exposure upon ligand binding, further contributing to the stabilization of the complex. The total binding energy of the protein–ligand complex was calculated to be − 137.525 ± 8.199 kJ/mol, which reflects the overall stabilization resulting from the combination of favourable van der Waals and electrostatic interactions, as well as SASA effects. For the protein without the ligand, several energy components could not be computed, as indicated by NaN (not a number) values for van der Waals energy, electrostatic energy, and binding energy. The polar solvation energy was 206.627 ± 71.925 kJ/mol, significantly higher than the corresponding value for the protein–ligand complex. This indicates that the protein alone has greater solvent exposure, likely due to the absence of ligand shielding. The SASA energy for the protein was − 0.713 ± 1.408 kJ/mol, suggesting minimal changes in solvent-accessible surface area compared to the protein–ligand complex.