Synthesis and Characterization of New Hexahydroquinoline Derivatives, Evaluation of Their Cytotoxicity, Intracellular ROS Production and Inhibitory Effects on Inflammatory Mediators

Inflammation is a response to injury and infection in an organism. It can be categorized as acute or chronic. Chronic inflammation is the underlying cause of many diseases such as Alzheimer disease, diabetes, rheumatoid arthritis, atherosclerosis, and cardiovascular diseases. Recent studies have proven the antiinflammatory properties of 1,4-dihydropyridines (1,4-DHPs) and their derivatives, which have many biological activities including the blocking of calcium channels. In this study, 15 compounds that are condensed derivatives of 1,4-DHPs, with the general structure of hexahydroquinoline-3-carboxylate, were synthesized. These compounds, expected to show inhibitory activity against inflammatory mediators, were obtained by the reaction of 4-(difluoromethoxy)benzaldehyde, substituted/nonsubstituted 1,3-cyclohexanedione derivatives, and appropriate alkyl acetoacetate compounds in the presence of ammonium acetate as a nitrogen source according to the Hantzsch synthesis method. The structures of the synthesized compounds were elucidated by IR, ¹H NMR, ¹³C NMR, and HRMS methods. The cytotoxic properties of the compounds were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method in the 3T3 cell line. Among the 15 compounds, the three compounds with the lowest levels of cytotoxic effects were selected for further experiments. Inflammation was induced by lipoxygenase and the effects of the selected compounds on the levels of reactive oxygen species, cytokines, and complement C3 and C9 regulatory proteins were investigated. It was found that the three selected compounds decreased the levels of transforming growth factor-beta 1 (TGF-β1). Among these compounds, compound <b>3e</b> provided the most significant decrease in this cytokine. Moreover, <b>3e</b> increased both C3 and C9 levels. Molecular modeling studies also showed that <b>3e</b> had better affinity for TGF-β1. When the binding modes of these compounds in the active site of TGF-β1 were analyzed, it was found that compound <b>3e</b> had hydrophobic interactions with amino acids Leu142, Tyr84, and Ile13; halogen bond interactions with Asp92; and hydrogen bond interactions with Ser89, Gly88, and Gly14 in the active binding site. Further in vitro and in vivo studies are needed to show the possible mechanism of action of compound <b>3e</b>.