The use of computational chemistry, associated with bioinformatics, has been playing an innovative role in directing studies and drug planning, through the use of computer simulations (Oprea 2005). In this context, the use of in silico models stands out, which have evolved with remarkable progress in several areas, such as: correlation, prediction, simplification, automation, among other expressive aspects (Cruz et al. 2019).
The evolution in the development of a new drug is without a doubt a very complex process that needs time and financial resources. Based on this, computer-aided studies aim to create new approaches that boost research and provide subsidies for further testing. The virtual screening for identification and optimization of other testing methodologies has been one of the advantages of these studies, from it it is possible to predict which pharmacological activities can be performed by a certain molecule, besides quantifying by a probability score of activity and inactivity (Yu et al. 2016; Surabhi et al. 2018).
In the PASS tests performed with the substances with the purpose of having an initial screening regarding which pharmacological activity to follow with the studies, it was possible to observe that the four chalcones showed similar results for the 15 activities with higher probability of occurrence. The fact that the four substances belong to the same class could explain the occurrence of similar results among them, taking into account that the test works from the decomposition of the molecular structure in 2D and 3D descriptors, which are already expected to be common among them.
Based on a literature search, seeking to elect the pharmacological activity that was most related to the chemical class of chalcones, it was observed that the antileishmania activity has been one of the main ones to be researched and that, concomitantly, these substances showed a promising action with regard to potency and efficacy against the different existing species (Mello et al. 2018; N'guessan et al. 2021; Osman et al. 2022).
A number of studies have reported a leishmanicidal action of compounds of this class, such as: fisetin, a polyphenolic flavonoid, which has potent action against Leishmania spp. in vitro tests (Adinehbeigi et al. 2017); purified dimeric flavonoids from Arrabidaea brachypoda with in vitro against promastigotes and amastigotes forms of L. amazonensis (Rocha et al. 2018); flavonoids isolated from Polygonum salicifolium performed leishmanicidal activity in vitro against L. mexicana (Zheoat et al. 2021); rusflavone, a biflavonoid isolated from the pollen of Attalea funifera, has been shown to act against the promastigote and amastigote forms of L. amazonensis, through a mechanism that involves the production of ROS, mitochondrial dysfunction, and membrane disruption in the parasites (Gomes et al. 2021).
Departing specifically for chalcones, scientific research can also be found indicating a broad leishmanicidal activity against different species of the genus Leishmania (Alonso et al. 2020). The studies of Nardella et al. (2018) indicated that the most active compounds against Leishmania spp. belong to the chalcone, biflavone and aurone classes, regardless of the assay performed, whether in vitro or in vivo. The phytochemical evaluation of the chalcones, 2',4'-dimethoxy-6'-hydroxychalcone and 2',5'-dimethoxy-4',6'-dihydroxichalcone, showed a promising antileishmania activity against L. mexicana, with absence of any toxicity in tests with a human cell line (Zheoat et al. 2021). The leishmanicidal activity of 31 synthetic chalcones was analysed using promastigotes and amastigotes of L. donovani, L. tropica, L. major and L. infantum in vitro tests, the results indicated that 16 of these compounds were active against the strains, showing high selectivity and low toxicity against mammalian cells (Ortalli et al. 2018). In view of this, it was decided to continue the studies in this area.
Cytotoxicity assays are among the main in vitro tests used at the beginning of studies with natural products, from these it is possible to predict the toxicity of substances in several cell types, providing a fundamental means for the evaluation, safety screening and classification of compounds. The monitoring of cell response within these assays provides reliable results and can serve as a basis for measuring other parameters such as cell viability and SI (Tolosa et al. 2014; Costa et al. 2020).
The results obtained by the cytotoxicity test performed on murine macrophages demonstrated a low cytotoxicity of the four chalcones when compared with gentian violet (CC50 = 0.6 ± 0.01 µM), with CC50 values above 50 µM. Encouraging results, as it is essential that new antileishmanial drug candidates have reduced cytotoxicity in order to overcome the disadvantages of drugs currently used in therapy.
Similar results were found in a study investigating the effects of twenty brominated chalcones against four cancer cell lines, where these substances exhibited lower cytotoxicity for non-malignant gastric epithelial cells than for diseased ones, demonstrating selectivity (Zhang et al. 2016). Researchers investigated the in vitro cytotoxicity of ten chalcones against the HeLa cell line through the MTT viability assay and the calculation of the SI, the results pointed out a low cytotoxicity of these substances in this cell type (Sinha et al. 2019).
In the analysis of antileishmanial activity axenic culture by calculating the IC50, it was observed that chalcones 1, 2 and 3 did not show promising inhibitory potency, unlike chalcone 4, which was the most potent in inhibiting the growth of L. braziliensis species, interesting result when added to the fact that this substance has low cytotoxicity when compared to AB, being a promising candidate for future tests
A number of studies have reported chalcones with potent antileishmanial activity against L. braziliensis species (Mello et al. 2014; Escrivani et al., 2021; Osman et al. 2022). Two synthetic chalcones were evaluated in vitro against L. braziliensis promastigotes for inhibitory activity and cytotoxicity against macrophages. The results of IC50 and CC50 were, respectively, 1.38 ± 1.09; 6.36 ± 2.04 µM, and 13.49 ± 3.13; 199.43 ± 4.11 µM, indicating that both chalcones showed an effect on L. braziliensis promastigotes, with low toxicity to mammalian cells (Mello et al. 2014).
Similar results were found in a study involving three methoxychalcones, which showed significant antileishmanial activity against L. braziliensis promastigotes in vitro tests (IC50 = 2.7 µM; 3.9 µM; and 4.6 µM), with a more potent action than the control drug pentamidine (IC50 = 6.0 µM) (Bello et al. 2011).
Given the results, it was hypothesized that the addition of bromine to molecule 4 optimized its antileishmanial effects in relation to the other molecules. Reports in the literature already relate brominated synthetic substances with a superior bioactive potential, although the mechanisms that explain these results are not yet fully elucidated, one can cite several possibilities, such as: elevation of lipophilicity and permeability through biological membranes, increased half-life or the ability to form intermolecular bonds (attractive interactions) between the electrophilic region of the molecule containing bromine atoms and nucleophilic active sites of biomolecules. Thus, bromination can lead to increased potency of therapeutic agents and research in the area of chemoinformatics can provide important contributions to the elucidation of molecular interactions (Jitareanu et al. 2018). Thus, we decided to continue our studies with chalcone 4, now entitled FERAI, with tests investigating its action on L. braziliensis amastigotes and evaluating the compound's SI.
The L. braziliensis species is related to a possible metastasis and mucosal form of leishmaniasis, hence the importance of developing more effective and less toxic drugs for the treatment of this disease (Brasil 2017; Vasconcelos et al. 2018). When the pharmacological activity of FERAI was evaluated against amastigotes of L. braziliensis, an IC50 value of 10.13 ± 1.7 µM was found. AB, a drug commercially available for the treatment of leishmaniasis, showed an IC50 value of 0.7 ± 0.004 µM. Although FERAI presented a higher inhibitory concentration, this result still showed promise and added to its low toxicity compared to AB, this compound shows that it can be a candidate for future drug for the treatment of leishmaniasis. In addition, macrophages were infected with amastigotes of L. braziliensis and treated with different concentrations of FERAI resulted in a reduction both in the number of infected macrophages and in the number of amastigotes per macrophage, confirming a significant action of this substance on the intracellular forms of L. braziliensis.
Seeking new therapeutic targets (Miranda-Sapla et al. 2019), he carried out an in vitro study to investigate the action of trans-chalcone (TC) against amastigotes of L. amazonensis. The exciting results that CT treatment of macrophages infected with L. amazonensis attempted to reduce the percentage of infected cells, as well as the number of amastigotes per macrophage, also generated a selectivity index (SI) of 53.8 for the parasite.
Investigating a possible mechanism of action of FERAI, ROS levels in L. braziliensis promastigotes were evaluated. The results indicated a possible action of this compound in the increase of ROS levels, concentration of 10 µM (56.33%), 20 µM (61.76%) and 30 µM (67.13%) in comparison with the untreated control. Similar results were found by Santiago-Silva et al., (2022), who evaluated the production of ROS by chalcone ((E)-1-(4,8-dimethoxynaphthalen-1-yl)-3-(4-nitrophenyl) prop-2-en-1-one), 4f. 4f induced several morphological and ultrastructural changes in free promastigotes, loss of plasma membrane integrity and increase in ROS.