Avaliação da estabilidade da cumarina 8-metoxi-3-(4- nitrobenzoil)-6- propil-2H-croman-2-ona e estudos de identificação e do potencial biológico de seus produtos de degradação

Abstract

Neglected diseases are a group of tropical illnesses that mainly affect the most vulnerable populations. Studies have shown the potential of the nitrobenzoylcoumarin 8-methoxy-3-(4- nitrobenzoyl)-6-propyl-2H-chromen-2-one against Chagas disease and leishmaniasis. Meanwhile, stability studies are required by regulatory agencies during the initial stages of drug candidate development, as molecule instability can lead to the formation of degradation products (DPs) harmful to human health. Thus, the objective of this work was to synthesize the mentioned nitrobenzoylcoumarin, evaluate its stability through forced degradation studies, identify its DPs, verify if the trypanocidal potential will be maintained in these by-products and assess the cytotoxic and mutagenic potential of both the coumarin and the identified DPs. The synthesis of nitrobenzoylcoumarin was carried out using the Duff method followed by a Knovenaegel reaction between formyl-dihydroeugenol and ethyl 4-nitrobenzoylacetate. The cytotoxic and mutagenic potentials were evaluated, respectively, using the Thiazolyl Blue Tetrazolium Bromide colorimetric assay and the micronucleus test. The evaluation of intrinsic stability was conducted through forced degradation studies following the conditions recommended by ICH Q3A (R2), ICH Q1B guidelines and ANVISA resolution No. 53/2015. Additionally, the nitrobenzoylcoumarin was subjected to accelerated stability conditions, incubated at 75% ± 5% of relative humidity and 40°C ± 2°C. An analytical method employing high-performance liquid chromatography and ultraviolet detection was developed and validated for the quantification of nitrobenzoylcoumarin and its DPs. The structural elucidation of these by-products was performed by mass spectrometry and their mutagenic potentials were predicted by in silico assays employing QSAR (Quantitative Structure-Activity Relationships) techniques. Biological studies demonstrated dose-dependent cytotoxicity of nitrobenzoylcoumarin with a mean inhibitory concentration of 238.98 μM ± 23.11 μM, similar to values reported for the drugs benznidazole and amphotericin B, that are used, respectively, in the treatment of Chagas disease and leishmaniasis. Genotoxicity studies revealed an absence of genotoxic effects at low concentrations for nitrobenzoylcoumarin. In the evaluation of intrinsic stability, it was found that nitrobenzoylcoumarin undergoes degradation when exposed to alkaline hydrolysis, metal ion solutions and oxidative conditions. Accelerated stability studies demonstrated that nitrobenzoylcoumarin did not undergo degradation after six months of incubation under the recommended temperature and humidity conditions for the assay. In

silico mutagenicity assessments indicated the absence of structural alerts in the related by- products concerning the researched endpoint or the absence of discrepant structural alerts

between the parent molecule and its by-products. This suggests that the compounds may be considered safe. The results demonstrated the potential of nitrobenzoylcoumarin as a promising prototype of new drug candidate to be used in the treatment of Chagas disease and leishmaniasis. Additional in vitro studies are being conducted to confirm the biological safety of the PDs.