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

## TL;DR

This study develops new cinnamamide compounds that effectively kill Aedes aegypti larvae without harming human cells or zebrafish, offering a safer alternative to traditional larvicides.

## Contribution

The study introduces new cinnamamide derivatives with larvicidal activity and low toxicity, supported by in silico and in vitro evaluations.

## Key findings

- Two cinnamamides, AF03 and AF18, showed larvicidal activity against Aedes aegypti larvae with LC50 values of 41.6 and 45.4 μg/mL, respectively.
- AF03 was noncytotoxic to A549 cells and caused no mortality in zebrafish, indicating low environmental and human toxicity.
- Molecular docking and dynamics simulations identified sterol carrier protein-2 as a stable target for AF03, suggesting a potential mechanism of action.

## Abstract

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 Aedes aegypti. 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 A. aegypti 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 A. aegypti revealed two active compounds: (E)-N-allyl-3-(4-chlorophenyl)­acrylamide (AF03, LC50 = 41.6 μg/mL) and (E)-N-allyl-3-(4-(trifluoromethoxy)­phenyl)­acrylamide
(AF18, LC50 = 45.4 μg/mL). Cytotoxicity
tests indicated that both molecules were nontoxic to A549 cells (5–100
μg/mL). AF03 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 AF03 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 AF03 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.