The aim of the study was a synthesis and investigation of the dose-dependent anti-inflammatory effect of new thiourea derivatives of naproxen with selected aromatic amines and esters of aromatic amino acids. The results of the in vivo study indicate that derivatives of m-anisidine (4) and N-methyl tryptophan methyl ester (7) showed the most potent anti-inflammatory activity four hours after injection of carrageenan, with the percentage of inhibition of 54.01% and 54.12%, respectively. In vitro assays of COX-2 inhibition demonstrated that none of the tested compounds achieved 50% inhibition at concentrations lower than 100 µM. On the other hand, the aromatic amine derivatives (1–5) accomplished significant inhibition of 5-LOX, and the lowest IC₅₀ value was observed for compound 4 (0.30 μM). High anti-edematous activity of compound 4 in the rat paw edema model, together with potent inhibition of 5-LOX, highlight this compound as a promising anti-inflammatory agent.

The objective of this study was to synthesize seven novel thiourea derivatives of naproxen (8–14), examine the anti-inflammatory activity of the newly synthesized compounds, investigate the cytotoxic potential of both sets of synthesized compounds (1–7 and 8–14), and select the most promising anti-inflammatory and antitumor drug candidates. The results of the in vivo anti-inflammatory study clearly showed that compounds 8 and 9 were capable of decreasing paw edema, as evident from a high percentage of inhibition (44.83% and 49.29%, respectively). In addition, the results of in vitro enzyme inhibition assays demonstrated that neither of the newly synthesized compounds reached 50% inhibition of 5-LOX at concentrations lower than 100 µM. In terms of antitumor potential, derivatives 3 and 8 exhibited strong cytotoxic effects on the HeLa cell line, suggesting the involvement of the extrinsic pathway of apoptosis. According to the overall results obtained for both sets of synthesized molecules, derivatives 4 and 8 can be underlined as molecules with the strongest anti-inflammatory activity, while derivatives 3 and 8 are the most promising cytotoxic agents.

Administration of current non-steroidal anti-inflammatory drugs is often associated with serious adverse effects. Therefore, there is a constant need to develop new molecules with anti-inflammatory activity. On the other hand, thiourea derivatives of non-steroidal anti-inflammatory drugs demonstrated significant anti-inflammatory activity in numerous studies. To clarify anti-inflammatory mechanism of action, in silico study was performed on four thiourea derivatives of naproxen, which were selected from the initial group of compounds synthesized by our research group. Tested compounds contain p-fluoroaniline (16), p-methoxyaniline (17), p-ethoxyaniline (18) and aniline (19) in the side chains. Selected 3D structures of enzymes COX-2 (3NT1) and 5-LOX (6NCF) were taken from PDB database. MAKE Receptor 3.2.0.2 software (OpenEye Scientific Software, Inc, Santa Fe, NM, United States) was used for preparation of enzymes’ active sites, while ligands were prepared in OMEGA 2.5.1.4. FRED 3.2.0.2 software (OpenEye Scientific Software, Inc, Santa Fe, NM, United States) was employed for the analysis of binding poses into enzymes’ active sites. All tested compounds showed key binding interactions with both enzymes. The highest number of key binding interactions was observed during molecular fitting of derivative 19 into the active site of COX-2 and derivatives 16 and 18 into the 5-LOX. Derivative 17 had the lowest value of free binding energy for both target enzymes (−14.90 kcal/mol for COX-2 and −9.57 kcal/mol for 5-LOX). Therefore, all analyzed compounds represent potential dual inhibitors of mentioned enzymes.

Masking the carboxyl group of naproxen with other functional groups may be a promising strategy to decrease its gastrointestinal toxicity. Thiourea moiety has been described as an important pharmacophore in a variety of pharmacologically active compounds, including anti-inflammatory, antiviral, anticancer, hypoglycemic and antimicrobial agents. Our research group has previously designed twenty novel thiourea derivatives of naproxen, containing amino acids (glycine, L-alanine, β-alanine, L-valine and L-phenylalanine – compounds 1,2,3,4 and 5, respectively), their methyl (6–10) and ethyl esters (11–15), as well as aromatic amines (16–20). Pharmacokinetic properties and druglikeness of these compounds were predicted using SwissADME web tool (http://www.swissadme.ch/). Predicted pharmacokinetic properties include potential for gastrointestinal absorption, blood-brain barrier permeability, skin permeability, transport mediated by P-glycoproteins and enzyme inhibitory potential. Druglikeness was evaluated using Lipinski’s, Ghose’s, Veber’s, Egan’s and Muegge’s rules, as well as on the basis of bioavailability score. All tested compounds had high-predicted gastrointestinal absorption and low blood-brain barrier permeability. Also, derivatives 2, 4, 7, 9, 10, 12, 14, 15 and 18 were predicted to be substrates for P-glycoprotein. Derivatives with aromatic amines (16–20) showed inhibitory potential against all tested CYP isoforms. Derivative 19 had the highest, while derivative 13 demonstrated the lowest predicted skin permeability. Finally, derivatives 1–12, except 5 and 10, have druglike structures, since they obey to all imposed rules.

Cyclooxygenase enzymes (COX-1 and COX-2) are bifunctional homodimeric enzymes which catalyze the biological oxidation of arachidonic acid and biosynthesis of the proinflammatory prostaglandins, such as PGD₂, PGE₂, PGF_2α and PGI₂. COX-1 isoform is a constitutive form distributed in most tissues where it performs mostly physiological functions of the gastrointestinal organs, the kidneys, and platelets. The enzyme COX-2 is an inducible form, expressed during inflammation and with only minor physiological functions like stimulating prostacyclin (PGI₂) production and thus preventing platelet aggregation. Both of these enzymes (COX-1 and COX-2) convert arachidonic acid into prostaglandin, which leads to pain and inflammation^1,2. The design of selective cyclooxygenase inhibitors has been of great interest over the past few years due to side effects associated with existing nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit both COX-1 and COX-2. This study aims to investigate the molecular docking of S-alkyl derivatives of thiosalicylic acid (S-benzyl, S-methyl, S-ethyl, S-propyl and S-butyl) towards COX-1 and COX-2 enzymes. Molecular docking studies were performed in order to explain the possible interactions between the inhibitors and both COX isoforms binding pockets. The docking studies were analysed with AutoDock Vina program. The crystal structures of the enzymes (COX-1 and COX-2), complexed with co-crystallized ligands, were obtained from Protein Data Bank (PDB ID: 1HT5 and 4FM5). COX-1 and COX-2 enzymes have very similar amino acid sequences. The ligand binding analysis in the active site of COX-1 showed that S-benzyl, S-propyl and S-butyl derivatives of thiosalicylic acid possess the best interactions. S-butyl derivative of thiosalicylic acid shows unique binding mode and forms five key binding interactions (Ala527, Leu352, Ser530, Val349 and Tyr355) with active site of COX-1. S-benzyl, S-ethyl, S-propyl and S-butyl derivatives of thiosalicylic acid possess some of the key binding interactions with the active site of COX-2. The docking results indicated that S-butyl derivative of thiosalicylic acid shows the key binding interactions (Leu352, Ser530, Val349, Tyr355, Ala527 and Gly526) with selected target. Binding energies of all tested compounds were similar to the corresponding co-crystallized ligands for both targets (COX-1 and COX-2). References: Fitzpatrick FA. Cyclooxygenase enzymes: regulation and function. Curr Pharm Des. 2004; 10(6): 577-88. Rao P, Knaus EE. Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs): cyclooxygenase (COX) inhibition and beyond. J Pharm Pharm Sci. 2008; 11(2): 81-110.