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)

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.

本研究以分离自亚马逊古巴香脂油（Copaifera multijuga Hayne）的3-羟基古巴酸与抗炎药物布洛芬为原料，合成得到一种新型抗炎杂合体——3-布洛芬基古巴酸（3-IbuCA）。经全面结构表征后，通过体外、体内及计算机辅助分子对接实验验证该杂合体的抗炎活性。本研究开展了细胞毒性与肿瘤坏死因子-α（TNF-α）抑制活性的体外实验，以及胸膜炎模型、蛋白表达检测与胃黏膜细胞毒性的体内实验。采用诱导契合对接技术，探究该杂合体与环氧合酶-1（COX-1）及环氧合酶-2（COX-2）的相互作用模式。针对COX-1与COX-2的分子对接结果显示，3-IbuCA的结合自由能（ΔG）分别为-2.2与-7.8 kcal·mol⁻¹；而对照药物莫非唑酮与吲哚美辛的结合自由能分别为-8.5与-10.1 kcal·mol⁻¹，表明3-IbuCA对COX-2具有选择性抑制作用。在浓度高达2 μM时，该杂合体对人巨噬细胞无毒性，且可抑制脂多糖（LPS）刺激的巨噬细胞中TNF-α的生成。胸膜炎实验结果显示，3-IbuCA可降低总白细胞与单核细胞数量，同时伴随磷酸化核因子κB抑制剂α（p-IKBα）蛋白表达水平的下调。与单独使用布洛芬相比，该杂合体引发的胃黏膜损伤程度更低。综上，分子对接结果结合体外与体内实验均表明，该新型抗炎杂合体具备开发为新型抗炎药物的潜力。