Design, synthesis, biological and computational screening of novel pyridine-based thiadiazole derivatives as prospective anti-inflammatory agents

Abstract

In this study, a novel series of pyridine-based thiadiazole derivatives (NTD1-NTD5) were synthesized as prospective anti-inflammatory agents by combining substituted carboxylic acid derivatives of 5-substituted-2-amino-1,3,4-thiadiazole with nicotinoyl isothiocyanate in the presence of acetone. The newly synthesized compounds were characterized by FTIR, ¹H NMR, ¹³C NMR, and mass spectrometry. First, the compounds underwent rigorous in vivo testing for acute toxicity and anti-inflammatory activity and the results revealed that three compounds-NTD1, NTD2, and NTD3, displayed no acute toxicity and significant anti-inflammatory activity, surpassing the efficacy of the standard drug, diclofenac. Notably, NTD3, which featured benzoic acid substitution, emerged as the most potent anti-inflammatory agent among the screened compounds. To further validate these findings, an in silico docking study was carried out against COX-2 bound to diclofenac (PDB ID: 1pxx). The computational analysis demonstrated that NTD2, and NTD3, exhibited substantial binding affinity, with the lowest binding energies (-8.5 and -8.4, kcal/mol) compared to diclofenac (-8.4 kcal/mol). This alignment between in vivo and in silico data supported the robust anti-inflammatory potential of these derivatives. Moreover, molecular dynamics simulations were conducted, extending over 100 ns, to examine the dynamic interactions between the ligands and the target protein. The results solidified NTD3's position as a leading candidate, showing potent inhibitory activity through strong and sustained interactions, including stable hydrogen bond formations. This was further confirmed by RMSD values of 2-2.5 Å and 2-3Ǻ, reinforcing NTD3's potential as a useful anti-inflammatory agent. The drug likeness analysis of NTD3 through SwissADME indicated that most of the predicted parameters including Lipinski rule were within acceptable limits. While these findings are promising, further research is necessary to elucidate the precise relationships between the chemical structures and their activity, as well as to understand the mechanisms underlying their pharmacological effects. This study lays the foundation for the development of novel anti-inflammatory therapeutics, potentially offering improved efficacy and safety profiles.

Keywords: Acute toxicity; Anti-inflammatory activity; Cyclooxygenase-2; In silico studies; Pyridine derivatives; Thiadiazole derivatives.

Fig. 1

Structures of pyridine and 1,3,4-thiadiazole containing drug molecules.

Fig. 2

Structures of selected pyridine and thiadiazole derivatives with potential anti-inflammatory activity.

Fig. 3

General structure of the designed compounds.

Scheme 1

Synthesis of nicotinoyl chloride and nicotinoyl isothiocyanate a) thionyl chloride (SOCl₂), stirring for 15–20 min; b) Ammonium thiocyanate (NH₄SCN), Acetone, Stirring for 1 h, rt.

Scheme 2

Synthesis of 5-substituted-2-amino-1,3,4-thiadiazole derivatives a) Ethanol, conc. H₂SO₄, reflux for 1.5 h.

Scheme 3

Synthesis ofpyridine linked 1,3,4-thiadiazole derivatives (NTD1-NTD5) a) Reflux in the presence of acetone.

Fig. 4

Effect of selected derivatives on paw edema in rats: *p < 0.05, **p < 0.01, ***p < 0.001 Assessed using two-way ANOVA followed by Tukey's multiple comparison test.

Fig. 5

Anti-inflammatory activity in Wistar rats. A: Paw edema before treatment; B: Paw edema after treatment.

Fig. 6

Interactions of selected derivatives (NTD2 and NTD3) with active pocket residues of COX-2.

Fig. 7

Root mean square deviation (RMSD) graph of (a) COX-2 protein and co-crystal ligand (Diclofenac) (b) COX-2 protein and NTD3.

Fig. 8

Root mean square fluctuation (RMSF) graph of (a) COX-2 protein and co-crystal ligand diclofenac, and (b) COX-2 protein and NTD3.

Fig. 9

Plot (stacked bar charts) of ligand-protein interactions during MD simulation of (a) protein-internal ligand (diclofenac) complex and (b) protein-NTD3 ligand complex. [green: H-bonds, lavender: hydrophobic, pink: ionic, and blue: water bridges]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

Specific interactions of the protein with the (a) internal ligand: diclofenac, and (b) NTD3 throughout the trajectory. (Dark color indicates a more specific contact with the ligand). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

2D interactions of amino acid residues of protein with (a) internal ligand: diclofenac, and (b) NTD3.

Fig. 12

Representation of the ligand properties of (a) internal ligand: diclofenac, and (b) NTD3.

Fig. 13

Structure activity relationship features of pyridine-1,3,4-thiadiazole derivatives.