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. 2023 Nov 9;16(11):1584.
doi: 10.3390/ph16111584.

Analgesic Activity of 5-Acetamido-2-Hydroxy Benzoic Acid Derivatives and an In-Vivo and In-Silico Analysis of Their Target Interactions

Cleydson B R Santos  1   2 , Cleison C Lobato  1   2 , Sirlene S B Ota  2 , Rai C Silva  1   2 , Renata C V S Bittencourt  1 , Jofre J S Freitas  3 , Elenilze F B Ferreira  4 , Marília B Ferreira  1   3 , Renata C Silva  3 , Anderson B De Lima  3 , Joaquín M Campos  5   6 , Rosivaldo S Borges  2 , José A H M Bittencourt  1
Affiliations

Analgesic Activity of 5-Acetamido-2-Hydroxy Benzoic Acid Derivatives and an In-Vivo and In-Silico Analysis of Their Target Interactions

Cleydson B R Santos et al. Pharmaceuticals (Basel). .

Abstract

The design, synthesis, and evaluation of novel non-steroidal anti-inflammatory drugs (NSAIDs) with better activity and lower side effects are big challenges today. In this work, two 5-acetamido-2-hydroxy benzoic acid derivatives were proposed, increasing the alkyl position (methyl) in an acetamide moiety, and synthesized, and their structural elucidation was performed using 1H NMR and 13C NMR. The changes in methyl in larger groups such as phenyl and benzyl aim to increase their selectivity over cyclooxygenase 2 (COX-2). These 5-acetamido-2-hydroxy benzoic acid derivatives were prepared using classic methods of acylation reactions with anhydride or acyl chloride. Pharmacokinetics and toxicological properties were predicted using computational tools, and their binding affinity (kcal/mol) with COX-2 receptors (Mus musculus and Homo sapiens) was analyzed using docking studies (PDB ID 4PH9, 5KIR, 1PXX and 5F1A). An in-silico study showed that 5-acetamido-2-hydroxy benzoic acid derivates have a better bioavailability and binding affinity with the COX-2 receptor, and in-vivo anti-nociceptive activity was investigated by means of a writhing test induced by acetic acid and a hot plate. PS3, at doses of 20 and 50 mg/kg, reduced painful activity by 74% and 75%, respectively, when compared to the control group (20 mg/kg). Regarding the anti-nociceptive activity, the benzyl showed reductions in painful activity when compared to acetaminophen and 5-acetamido-2-hydroxy benzoic acid. However, the proposed derivatives are potentially more active than 5-acetamido-2-hydroxy benzoic acid and they support the design of novel and safer derivative candidates. Consequently, more studies need to be conducted to evaluate the different pharmacological actions, the toxicity of possible metabolites that can be generated, and their potential use in inflammation and pain therapy.

Keywords: 5-acetamido-2-hydroxy benzoic acid; ADME; analgesic; molecular docking; toxicity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of 5-acetamido-2-hydroxy benzoic acid (PS1).
Figure 2
Figure 2
The 5-acetamido-2-hydroxy benzoic acid (PS1) and derivatives (PS2 and PS3).
Figure 3
Figure 3
Synthetic methodology for the preparation of 5-acetamido-2-hydroxy benzoic acid and derivatives.
Figure 4
Figure 4
Superpositions of crystallographic ligands poses (in red) with the calculated poses (in green): (A) COX-2 (Mus musculus, PDB ID 4PH9–RMSD 0.33 Å), (B) COX-2 (Homo sapiens, PDB ID 5KIR–RMSD 0.90 Å), (C) COX-2 (Mus musculus, PDB ID 1PXX–RMSD 1.34 Å) and (D) COX-2 (Homo sapiens, PDB ID 5F1A–RMSD 0.88 Å) with respective crystallographic ligands.
Figure 5
Figure 5
Binding affinity values of the ligands with COX-2 (Mus musculus) PDB ID 4PH9, by molecular docking.
Figure 6
Figure 6
Binding affinity values of the ligands with COX-2 (Homo sapiens) PDB ID 5KIR, obtained by molecular docking.
Figure 7
Figure 7
Binding affinity values of the ligands with COX-2 (Mus musculus) PDB ID 1PXX, obtained by molecular docking.
Figure 8
Figure 8
Binding affinity values of the ligands with COX-2 (Homo sapiens) PDB ID 5F1A, obtained by molecular docking.
Figure 9
Figure 9
Interactions and distances (Å) predicted between the active site of COX-2 and compound PS1 (A), PS2 (B) and PS3 (C).
Figure 10
Figure 10
Effect of PS1, PS2, PS3 and Acetaminophen (Acet) at a dose of 20 mg/kg and morphine at a dose of 10 mg/kg on the nociceptive heat stimulus (55 ± 0.1 °C) induced in mice. Each point represents mean ± e.p.m. of five animals. * p < 0.05, when compared to the control group; ANOVA, Dunn’s method.
Figure 11
Figure 11
(A) Effect of PS1 and Acetaminophen (Acet) on the nociceptive stimulus induced by intraperitoneal injection of 0.6% acetic acid in mice. (B) Effect of PS3 and Acet on the nociceptive stimulus induced by the intraperitoneal injection of 0.6% acetic acid in mice. (C) Comparison of compounds (PS1, PS2 and PS3 at a dose of 50 mg/kg) with Acet at a dose of 20 mg/kg on the nociceptive stimulus induced by the intraperitoneal injection of 0.6% acetic acid in mice. Each column represents mean ± e.p.m. of five animals. * p < 0.05, when compared to the control group; ANOVA, Student-Newman–Keuls test.
Figure 12
Figure 12
Structure and numeration of all derivatives studied here (PS1, PS2 and PS3).
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