INTRODUCTION: Leishmaniasis is considered a Neglected Tropical Disease (NCD), which occurs with a higher incidence in emerging and underdeveloped countries, being considered an endemic disease in Brazil 1,2. This disease is caused by a protozoan of the genus Leishmania spp., which belongs to the Trypanosomatidae family. Its transmission in the Americas occurs through the bite of the female sandfly mosquito Lutzomya longipalpais 3. Based on the above, infected individuals present three main clinical manifestations, including Visceral Leishmaniasis (VL), known as Kala-azar, which is the most serious form of the disease, causing mild splenomegaly followed by hepatomegaly and lymphadenopathy; Cutaneous Leishmaniasis (CL), being the most common, presenting ulcers on the skin and Mucocutaneous Leishmaniasis (CML), in which partial or total destruction of the mucous membranes occurs, due to lymphatic metastasis2,4. Therefore, despite the large number of cases of infection and great epidemiological importance, treatment is long, has a high cost and a limited route of administration. The main first-choice drugs for treatment are pentavalent antimonials, such as sodium stibogluconate (Pentostam®) and N-methyl glucamine antimoniate (Glucantime®). Second-line treatment uses amphotericin B (Amphotericin B®) and pentamidine 5,6. However, high doses cause serious side effects due to toxicity. Considering the need to develop research on leishmaniasis, the present work aimed to synthesize new leishmanicidal agents based on the thienopyrimidine heterocycle, designed by FBDD, in addition to investigating their activity, given the pharmacological potential of this class. OBJECTIVES: From this perspective, an FBDD planning technique was carried out, making it possible to filter and select potential chemical skeletons, in order to rationally plan molecules that may be promising against leishmaniasis. Through in silico studies, 5 top hit compounds were synthesized. The synthesized compounds were structurally characterized using hydrogen and carbon thirteen Nuclear Magnetic Resonance ( 1H and 13C NMR), high performance liquid chromatography (HPLC) and melting point techniques. Soon after, cell viability analysis was carried out on macrophages from the J774.A1 line. METHODS: Using a proposed synthetic route, containing four steps, a series of thienopyrimidines were synthesized. The initial step is the Gewald synthesis 7,8, which takes place in an ice bath, resulting in the first intermediate, heterocycle-2-aminothiophene. Subsequently, the synthesis of the second intermediate, tetrahydrobenzothiophene thiourea 9, occurs. Then, cyclization takes place to insert four bromoacetophenone substituents, including 2-bromo-4-methoxyacetophenone; 2-bromo-4-methylacetophenone; 2-bromo-2,4-dichloroacetophenone and 2-bromo-4-chloroacetophenone. Following the same synthetic route, due to the similarity between the thienopyrimidine compound, two new hydroquinazoline derivative molecules were inserted, making synthesis viable. Soon after confirmation of the new prototypes, an in vitro cytotoxicity test was carried out on macrophages of the J774.A1 lineage, being tested in triplicates with 200µl of the synthesized substances, diluted in RPMI medium in six concentrations of 100, 30, 10, 3, 1 and 0.3 µg/mL. In this way, cell viability is determined by MTT reduction assay and reading the absorbances on a spectrophotometer at 550nm. The cell viability of macrophages treated with the compounds was compared to the death pattern obtained in control cultures with dimethyl sulfoxide (DMSO). RESULTS AND DISCUSSIONS: 12 compounds were synthesized with satisfactory yield (between 44.49% - 78.36%), in addition to a purity level greater than 86% analyzed by CLEA. All derivatives obtained had their structures confirmed by 1H and 13C NMR. Next, in vitro cytotoxicity was evaluated in macrophages of the J774.A1 lineage. The analyzes of the data obtained were carried out using analysis of variance (ANOVA), using GraphPad Prism 8. Thus, the first series tested were the compounds obtained with the thienopyrimidine group, with only the compound VM22 (maximum effect of 55 .42 ± 3.59 % and IC50 of 44.69 µg/mL), presented a greater potency and lower cytotoxicity, when compared to the standard drug, amphotericin B (maximum effect of 72.17 ± 1.73 % and IC50 86 .26 µg/mL). For compounds from the hydroquinazoline series, VM30 (maximum effect 70.18 ± 3.34% and IC50 of 63.50 µg/m) showed better results. CONCLUSION: The search for chemical fragments in a virtual and experimental way demonstrated a selection of the best potentially promising fragments against leishmaniasis. Furthermore, it was possible to synthesize previously planned derivatives effectively. Given the results presented, some compounds with satisfactory results in in vitro tests were identified, such as VM20 and VM30. Therefore, the results reported here may assist in research aimed at developing new prototypes to combat the disease caused by the Leishmania protozoan, which are safe, effective and low-cost, especially for the population with greater socioeconomic vulnerability.
References
[1]
[2]Organización Panamericana de la Salud Organización Mundial de la Salud. [s.l: s.n.]. Disponível em: <https://iris.paho.org/bitstream/handle/10665.2/50524/9789275320631_spa.pdf?seque%0Ance=1&isAllowed=y.>.
[3]LOKESH KUMAR TRIPATHI; NAILWAL, T.K. Leishmaniasis: na overview of evolutions, classification, distribution, and historical aspects of parasite and its vector. Elsevier eBooks, p. 1-25, 1 jan. 2021
[4]Da Rocha FF, Campos MG. Leishmaniose tegumentar americana. Vol. 52. 2006. https://doi.org/10.1590/S0104-42302006000600015
[5]BRAZIL. Guia De Vigilância Em Saúde Lv. 2016
[6]Gabriel Á, Valério-Bolas A, Palma-Marques J, Mourata-Gonçalves P, Ruas P, Dias-Guerreiro T, et al. Cutaneous Leishmaniasis: The Complexity of Host’s Effective Immune Response against a Polymorphic Parasitic Disease. J Immunol Res 2019; 2019:1-16. https://doi.org/10.1155/2019/2603730
[7]Gewald, K.; Schinke, E. Heterocyclen Aus CH?aciden Nitrilen, X. Notiz Zur Reaktion von Aceton Mit Cyanessigester Und Schwefel. Chem. Ber., 1966, 99, 2712–2715.
[8]Abaee, MS; Hadizadeh, A.; Mojtahedi, MM; Halvagar, MR Explorando o escopo da reação de Gewald: expansão para um processo de quatro componentes. Tetraedro Lett., 2017, 58, 1408–1412.
[9] Silva-Júnior EF, Silva EPS, França PHB, Silva JPN, Barreto EO, Silva EB, et al. Desing, synthesis, molecular docking and biological evaluation of thiophen-2-iminothiazolidine derivatives for use against Trypanossoma cruzi. Bioorganic Med Chem 2016:24:4228-40. https://doi.org/10.1016/j.bmc.2016.07.013.
Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
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