Synthesis, Characterization and in vitro, in vivo and in silico Anti-Inflammatory Studies of the Novel Hybrid Based on Ibuprofen and 3-Hydroxy-Copalic Acid Isolated from Copaiba Oil (Copaifera multijuga)

Fábio De Souza,Lirlândia Sousa,Larissa Brito,Kelson De Oliveira,Almir Wanderley,Fernanda Guilhon-Simplicio,Marcos Silva,Michelle Sugimoto,Ana Cristina Pinto,Emerson Lima,Valdir Da Veiga-Junior,Rodrigo Souza,Renyer Costa,Patrícia De Almeida

doi:10.21577/0103-5053.20190266

Abstract

A novel anti-inflammatory hybrid 3-ibuprofenyl-copalic acid (3-IbuCA) was synthesized from 3-hydroxy-copalic acid isolated from Amazonian copaiba oil (Copaifera multijuga Hayne), and the anti-inflammatory ibuprofen. After full characterization, several assays to verify its anti-inflammatory effects were performed in vitro, in vivo and in silico (molecular docking). Induced fit docking was performed to observe the interactions with the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). In vitro tests of cytotoxicity and tumor necrosis factor (TNF)-α inhibition, and in vivo tests of pleurisy, protein expression and gastrocytotoxicity were performed. Molecular docking studies with COX-1 and 2 showed binding free energies (ΔG) of -2.2 and -7.8 kcal mol-1, respectively, while for mofezolac and indomethacin, the binding free energies ΔG presented values of -8.5 and -10.1 kcal mol-1, which makes 3-IbuCA selective for COX-2 inhibition. This hybrid showed no toxicity against human macrophage at concentrations up to 2 µM, and inhibited TNF-α production in lipopolysaccharide (LPS)-stimulated macrophages. In the pleurisy assays, 3-IbuCA reduced the total leukocytes and mononuclear cells, which was followed by reduction of p-IKBα (phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) protein expression. Compared with ibuprofen alone, the hybrid caused less gastric damage. Thus, the docking, together with in vitro and in vivo studies suggest that this novel hybrid has potential as a new anti-inflammatory agent.

Highlights

Copaiba oil is an oilresin exuded from the trunk of trees of the Copaifera genus (Fabaceae), which is widely distributed throughout the Amazon region, as well as in Central and Eastern regions of Brazil
In the case of nonselective COX-2 inhibitors, such as ibuprofen, it is of the utmost importance to clarify the affinity relationships of COXs after chemical modifications by synthetic processes.[9]. In this sense, taking advantage of the biological activities of substances in copaiba oil such as 3-hydroxy-copalic acid (3-HCA), and the properties of ibuprofen, we propose the synthesis of a new substance, the hybrid 3-ibuprofenyl-copalic acid (3-IbuCA) as a potential new anti-inflammatory agent
In vivo model of pleurisy induced by LPS The anti-inflammatory activity of the semi-synthetic derivative of 3-hydroxy-copalic acid with ibuprofen in an LPS-induced pleurisy model was evaluated (Figure 7). 3-IbuCA caused a significant reduction in the total number of cells and mononuclear cells, which was concluded by an important anti-inflammatory action, being considered the gold-standard model in the evaluation of this biological activity with important prospects for new studies.[25,26]

Summary

Introduction

Copaiba oil is an oilresin exuded from the trunk of trees of the Copaifera genus (Fabaceae), which is widely distributed throughout the Amazon region, as well as in Central and Eastern regions of Brazil. In this sense, taking advantage of the biological activities of substances in copaiba oil such as 3-HCA, and the properties of ibuprofen, we propose the synthesis of a new substance, the hybrid 3-ibuprofenyl-copalic acid (3-IbuCA) as a potential new anti-inflammatory agent. The potential anti-inflammatory activity was evaluated by measuring the cytokine tumor necrosis factor-α (TNF-α) produced by LPS-stimulated THP1 macrophages, as previously described.[16] For this, the supernatant was collected, and TNF-α was measured using the cytokinespecific sandwich quantitative enzyme-linked immunesorbent assay (ELISA) according to the manufacturer’s instructions (TNF-α duo set Elisa kits, R&D Systems, Minneapolis, USA).

Results

Conclusion