New Cinnamamide and Derivatives as New Larvicides against <i>Aedes aegypti</i> Vector Larvae: Facile Synthesis, <i>In Silico</i> Study, <i>In Vitro</i> Noncytotoxicity, and Nontoxicity against Zebrafish

Every year, millions of people are infected by arboviruses, such as Dengue, a neglected disease that mainly affects regions with lower socioeconomic development. One of the main strategies to combat these diseases is population control of mosquito vectors, such as <i>Aedes aegypti</i>. Chemical control is an effective approach to reducing the population of these vectors; however, challenges such as toxicity and increasing resistance in populations make it necessary to search for new bioactive and selective substances continuously. In this sense, this study presents the synthesis and evaluation of the larvicidal activity of cinnamamides derived from cinnamic acid against the larvae of the <i>A. aegypti</i> mosquito. Given the increase in resistance to traditional larvicides, this research aims to offer safer and more effective alternatives for vector control. In this study, we synthesized 21 cinnamamides featuring scaffolds with and without electron-withdrawing substituents as well as variations in the amide side chain with aliphatic and aromatic groups (the latter containing a -CH₂- spacer). The compounds were obtained via Steglich coupling or acid chloride formation followed by amidation and characterized by ¹H NMR, ¹³C DEPTQ, and HRMS (for novel molecules). Larvicidal assays against fourth-instar <i>A. aegypti</i> revealed two active compounds: (<i>E</i>)-<i>N</i>-allyl-3-(4-chlorophenyl)-acrylamide (<b>AF03</b>, LC₅₀ = 41.6 μg/mL) and (<i>E</i>)-<i>N</i>-allyl-3-(4-(trifluoromethoxy)-phenyl)-acrylamide (<b>AF18</b>, LC₅₀ = 45.4 μg/mL). Cytotoxicity tests indicated that both molecules were nontoxic to A549 cells (5-100 μg/mL). <b>AF03</b> was further evaluated in chicken erythrocytes, showing no effect on oxygen transport, and in zebrafish, where it caused mild irritation but no mortality. Enzymatic assays showed that <b>AF03</b> increased acetylcholinesterase (AChE) activity in the zebrafish brain, slightly elevated catalase (CAT) activity in the liver and heart (similar to solvent effects), and increased superoxide dismutase (SOD) activity in the brain but not in the heart. Morphological analysis suggested potential involvement of receptor, transport, or digestive proteins as biological targets. Molecular docking identified sterol carrier protein-2 (AeSCP-2, PDB: 2ksi) and kynurenine aminotransferase (AeKAT, PDB: 1yiy) as promising targets, with AeSCP-2 selected for molecular dynamics simulations. MD results confirmed a stable and specific interaction between <b>AF03</b> and AeSCP-2, supported by the global structural stability of the protein and the low RMSD values of the ligand. This study suggests that cinnamamides may lead to the development of new, effective larvicidal agents with a low environmental impact.