New 1 H-benzimidazole-2-yl hydrazones with combined antiparasitic and antioxidant activity

Abstract

Parasitic infections, caused mainly by the species Trichinella spiralis (T. spiralis), are widespread around the world and lead to morbidity and mortality in the population. Meanwhile, some studies have showed that these parasites induce oxidative stress in the infected host. With the aim of developing a class of compounds combining anthelmintic with antioxidant properties, a series of new benzimidazolyl-2-hydrazones 5a-l, bearing hydroxyl- and methoxy-groups, were synthesized. The anthelmintic activity on encapsulated T. spiralis was studied in vitro thus indicating that all hydrazones were more active than the clinically used anthelmintic drugs albendazole and ivermectin. 5b and 5d killed the total parasitic larvae (100% effectiveness) after 24 hours incubation period at 37 °C in both concentrations (50 and 100 μg ml^-1). The antioxidant activity of the target compounds was elucidated in vitro against stable free radicals DPPH and ABTS as well as iron induced oxidative damage in model systems containing biologically relevant molecules lecithin and deoxyribose. The two 2,3- and 3,4-dihydroxy hydrazones 5b and 5d were the most effective radical scavengers in all studied systems. DFT calculations were applied to calculate the reaction enthalpies in polar and nonpolar medium and estimate the preferred mechanism of antioxidant activity. The relative radical scavenging ability of compounds 5a-l showed a good correlation to the experimentally observed trends. It was found that the studied compounds are capable to react with various free radicals - ˙OCH₃, ˙OOH and ˙OOCH₃, through several possible reaction pathways - HAT in nonpolar medium, SPLET in polar medium and RAF in both media.

Fig. 1. Designed prototype scaffold based on antioxidant and anthelmintic active pharmacophores.

Scheme 1. Synthesis of 1H-benzimidazole-2-yl hydrazone derivatives 5a-l.

Fig. 2. Anti-radical properties of compounds 5a-l in spectrophotometric model systems containing stable free radicals. Results have been presented as C-50 values calculated on the base of the concentration–RSA% relationship. (A) Data from the ABTS cation radical containing model systems; (B) data obtained using the DPPH assay. Melatonin (M) and quercetin (Q) have been used as reference compounds. Data are expressed as the mean ± SD. Groups were compared by one-way ANOVA with Bonferroni post hoc test. The results were considered statistically significant when p < 0.05. ** (p > 0.05) compared with Melatonin; ● (p > 0.05) compared with quercetin.

Fig. 3. Protection effects of compounds 5a-l on iron induced oxidative damage of biologically relevant molecules: (A) extent of molecular oxidative damage observed in lecithin containing model system (1 mg ml⁻¹) in the presence of compounds 5a-l (90 μM); (B) extent of molecular oxidative damage observed in deoxyribose containing model system (0.5 mmol l⁻¹) in the presence of compounds 5a-l (80 μM). Melatonin (M) and quercetin (Q) at the same concentration have been used as reference compounds in both systems. Data were compared by one-way ANOVA with Bonferroni post hoc test. Results are presented as “% of molecular damage” means ± SD. The results were considered statistically significant when p < 0.05. ** (p > 0.05) compared with melatonin; ● (p > 0.05) compared with quercetin.

Fig. 4. Optimized molecular structure and relative Gibbs energies (in kJ mol⁻¹) of the possible isomers of compound 5d in amino tautomeric form, obtained at B3LYP/6-311++G(d,p) level of theory in gas phase.

Fig. 5. Optimized molecular structure and relative Gibbs energies (in kJ mol⁻¹) of the possible isomers of compound 5b in imino tautomeric form, obtained at B3LYP/6-311++G(d,p) level of theory in gas phase.

Fig. 6. General view of the molecule of compound 5d (a) and benzimidazolyl-2-hydrazone of pyridine-2-carbaldehyde (b,) with selected bond lengths.

Scheme 2. Probable mechanisms of radical-scavenging activity.

Scheme 3. Possible sites for hydrogen atom transfer (in blue) and radical adduct formation (in red) for compound 5d along with the radicals formed by HAT and the respective bond dissociation enthalpies, calculated at B3LYP/6-311++G(d,p) level of theory in benzene.

Fig. 7. Visualisation of spin density of the three most stable radicals R4, R3 and R2 of 5d, calculated at B3LYP/6-311++G(d,p) level of theory in benzene.

Fig. 8. Reaction enthalpies for compounds 5a-l in gas phase (A) and water (B): dissociation enthalpy (BDE), ionization potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA), and electron transfer enthalpy (ETE), calculated at B3LYP/6-311++G** level of theory.

Fig. 9. Optimized structures of the TSs for the most stable radicals R2, R3 and R4 corresponding to the HAT mechanism. ΔΔG^‡ shows the difference in the activation energies between the TSs for every one of the studied radicals. Calculations are done on SP M06-2X/6-311++G** method in solvent water.

Fig. 10. Optimized structures of the most probable TSs corresponding to the RAF mechanism for every one of the studied radicals. Calculations are done in solvent water.