Avaliação do perfil antibacteriano e efeitos toxicológicos de fenilacrilamidas, fenilacrilatos, benzilidenemalononitrilas e hidrazina-1-carboticamidas

Abstract

The objective of this study was to evaluate the antibacterial activity and toxicological effects of synthetic molecules from the phenylacrylamide (EEs), phenylacrylate (ALs), benzylidenemalononitrile (APMs), and hydrazine-1-carbothioamide (GLs) classes, all synthesised through molecular modification strategies. According to the in silico analysis using Molinspiration©, all molecules exhibited excellent theoretical oral bioavailability. Based on the toxicological analysis via admetSAR©, the evaluated molecules are not theoretically carcinogenic, teratogenic, mutagenic, or Class III carcinogens, with LD50 values in rats ranging from 585.83 to 857.20 mg/kg. Regarding acute oral toxicity, EEs fall into Category III, ALs and APMs into Category II, and GLs

into Category I. In vitro analysis, while EEs, ALs, and APMs did not demonstrate anti- haemolytic protection, the disubstituted EE-04 (ortho-para) and phenylacrylate AL-09

(ortho-ortho) demonstrated a Minimum Lytic Concentration (MLC) of 400 μg/mL, which caused the rupture of more than 50% of the erythrocytes, and APMs showed no haemolytic activity. GLs presented an MLC of 200 μg/mL, with GL-02 and GL-04 reducing phenylhydrazine-induced oxidation—a known oxidative agent—by approximately 15%. AL-10 displayed a concentration-dependent profile, reducing oxidation by up to 20%, while APMs were the most effective, achieving a 30% reduction across all tested concentrations. The methaemoglobin levels of EEs, ALs, and GLs remained within normal ranges. In antibacterial activity assays, all molecules demonstrated significant efficacy against Viridans group Streptococcus strains. Notably, GL-08 exhibited bactericidal activity with an MBC < 8 μg/mL against Enterococcus faecalis ATCC 29212. In vitro combination assays with chloramphenicol against Staphylococcus aureus strains, EE-01 and APM-05 displayed synergism. Additionally, EEs and APMs modulated tetracycline activity, except against the methicillin-resistant S. aureus (MRSA 216) strain. The dichlorinated APM-04 isomer (ortho-ortho) stood out for its broad-spectrum antibacterial activity against Enterobacterales, Staphylococcus spp., and Streptococcus spp., as well as for modulating tetracycline activity in methicillin-sensitive S. aureus (MSSA) strains. Molecular docking analysis revealed a strong theoretical binding affinity to PBP4 (- 6.909 ± 0.14 kcal/mol) and α-HL (-6.183 ± 0.1 kcal/mol) compared with the antibiotic penicillin, and excellent theoretical binding affinity to the NorA efflux pump (-7.5 ± 0.14 kcal/mol) compared with ciprofloxacin. APM-04, 2,6-dichlorobenzylidenemalononitrile, possesses two proton-accepting groups, potentially enabling it to deprotonate NorA and situate itself between the β-sheets connecting chains A and B of PBP4. Furthermore, it is a candidate to inhibit the formation of the transmembrane channel created by α-haemolysin. APM-04 forms a hydrogen bond with PBP4, potentially inhibiting its activity. The analysed molecules may act as Michael acceptors due to their nucleophilic/electrophilic portions, which interact with proteins of opposing characteristics in bacterial cells. Combining licensed antibiotics with APM-04 could enhance efficacy, contributing to the treatment of infections caused by S. aureus.