Pharmaceutical composition based on 1,3,4-oxadiazole compounds and its use in the preparation of medicines for the treatment of systemic infections.

Abstract

The present invention falls within the context of pharmaceutical chemistry and relates to pharmaceutical compositions and their use in combating infections caused by fungi. More specifically, the present invention relates to two pharmaceutical compositions targeting the thioredoxin reductase enzyme of fungi, which is useful in the treatment of human infections caused by microorganisms of the genus Candida spp., Cryptococcus spp., and Paracoccidioides spp. The pharmaceutical compositions comprise the 1,3,4-oxydiazole compounds: 4-(n-benzyl-n-methylsulfamoyl)-n-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(n-cyclohexyl-n-ethylsulfamoyl)-n-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide.The present invention further describes the use of the compositions of the compounds 4-(n-benzyl-n-methylsulfamoyl)-n-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(n-cyclohexyl-n-ethylsulfamoyl)-n-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide in the formulation of a medicament, additive, agent or formulation for the treatment of systemic fungal infections, such as candidiasis, cryptococcosis and paracoccidioidomycosis.

Description

1 / 34 Pharmaceutical composition based on 1,3,4-oxadiazole compounds and its use in the preparation of medicines for the treatment of systemic infections.

[001] FIELD OF THE INVENTION

[002] The present invention falls within the context of pharmaceutical chemistry and generally refers to pharmaceutical compositions and the use of chemical compounds in combating systemic infections caused by fungi, targeting the inhibition of the thioredoxin reductase enzyme.

[003] More specifically, the present invention relates to pharmaceutical compositions comprising the 1,3,4-oxadiazole compounds: 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide or 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)1,3,4-oxadiazol-2-yl)benzamide, which are useful in the treatment of infections caused by fungi of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp. in humans.

[004] In addition, the present invention describes the use of the compositions and compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide in the formulation of a medicament, additive, agent or formulation also useful in the treatment of candidiasis, cryptococcosis and paracoccidioidomycosis.

[005] DESCRIPTION OF THE STATE OF THE ART

[006] Fungi are distributed throughout nature, living in a wide variety of places, such as: soil, swamps, tree trunks, dust, etc. They are classified as zoophilic (preferring animals), geophilic (soil-dwelling), and anthropophilic (human-dwelling). They comprise a vast field of study, from the industrial to the scientific sector, encompassing various branches of activity with both beneficial and harmful results. Among the negative aspects of fungi are fungal infections that affect humans, which are Petition 870180036380, dated 03 / 05 / 2018, page 19 / 74 2 / 34 are classified as ranging from superficial (e.g., tinea) to disseminated (e.g., systemic candidiasis, paracoccidioidomycosis, and cryptococcosis), which can be fatal.

[007] Hospital-acquired infections have been growing significantly in recent years, as a result of advances in medicine and the consequent increase in the lifespan of hospitalized patients. However, this has led to an increase in the number of patients who are immunologically vulnerable to fungi and bacteria, raising the frequency of infections caused by opportunistic pathogens.

[008] Fungi of the genus Candida are the main cause of fungal infections in hospital settings and are a significant cause of bloodstream infections. Among the most invasive species, which are responsible for severe cases of candidiasis, are C. albicans, C. glabrata, C. tropicalis, and C. krusei. This disease mainly affects transplant patients, those with immunodeficiency, diabetics, burn victims, etc.

[009] Cryptococcosis, a disease caused by fungi of the genus Cryptococcus spp., is also a serious infection, especially for patients in advanced stages of human immunodeficiency virus (HIV) infection, with meningitis being the most common clinical manifestation. The pathology of cryptococcosis is mainly related to the inhalation of the fungus from contaminated pigeon droppings, primarily affecting the lungs, followed by tropism for the cerebral epithelium.

[010] Among endemic infections in Latin America, Paracoccidioidomycosis (PCM) is the most prevalent, caused by fungi of the genus Paracoccidioides spp. Infected patients are generally rural workers who have significant deficiencies in access to and support from the health service network, leading to late diagnosis and, consequently, a high potential for disability with a significant number of premature deaths. Because it is a neglected disease, there are no precise records of its incidence, nor a specific treatment. All organs are susceptible to the disease, which generally presents as Petition 870180036380, dated 03 / 05 / 2018, page 20 / 74 3 / 34 pulmonary or extra-pulmonary lesions involving the oral mucosa, nose, and larynx.

[011] Antifungal treatment offers fewer therapeutic options compared to bacterial chemotherapy. Limitations to current therapeutic options include, among others: limited spectrum of activity, lack of efficiency due to increasing resistance, poor safety profile, multiple interactions with other drugs, inadequate pharmacokinetic profile, and excessive cost. The treatment of invasive fungal infections (IFIs) and opportunistic infections remains unsatisfactory with the use of most existing antifungal agents, such as itraconazole and amphotericin B, and furthermore, opportunistic fungi have increasingly developed resistance even with the use of new antifungal agents such as voriconazole.

[012] The search for new antifungal alternatives based on structure and mechanism of action has proven to be an efficient and less expensive strategy. Advances in computational techniques and hardware have enabled in silico methods to accelerate the identification and optimization of these potential new drugs. The virtual screening (VS) strategy of active compounds deposited in chemical libraries has been of great interest in improving the processes of developing new drugs, as it reduces the time and initial investment, allowing the rapid screening of large libraries of small molecules to identify those with the best hits (bioactive molecules), which can be purchased and / or synthesized, and evaluated experimentally.These in silico methodologies have contributed to the drug development process by preferentially targeting enzymes that are only part of pathogen metabolic pathways, which gives them the property of selective toxicity. Thioredoxin reductase (TRR1) is a flavoenzyme located in the cytoplasm and plays a critical role in maintaining the redox state of the cytoplasm. This enzyme is part of a complex known as the thioredoxin system, in which thioredoxin (Trx) is reduced by the flavoenzyme, thioredoxin reductase, using NADPH and protecting cells against stress. Petition 870180036380, dated 03 / 05 / 2018, page 21 / 74 4 / 34 oxidative. Two classes of thioredoxin reductase have been characterized. The low molecular weight isoform (~35 kDa) is present in prokaryotes, plants, some parasites, and fungi.

[013] Recently, the inventors of the present patent application, through molecular modeling and virtual scanning, selected eleven compounds that interact with the TRR1 of the fungus Candida albicans and exhibit antifungal activity against fungi that cause invasive and systemic mycoses. These compounds were selected using two conformations of TRR1 as templates: oxidized flavin and reduced flavin, the latter being the active form of this flavoenzyme. This new strategy allowed the obtaining of two compounds with in vitro antifungal activity against the three important pathogenic fungi (Candida, Cryptococcus and Paracoccidioides), showing promise in the development of new antifungal drugs.Through the following experiments, it was possible to verify the in vitro and in vivo antifungal activity against fungi of the genera Candida, Cryptococcus, and Paracoccidioides of the potential 1,3,4-oxydiazole compounds: 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)1,3,4-oxadiazol-2-yl)benzamide.

[014] In this context, the present invention presents a viable alternative for the treatment of infections caused by fungi of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp. by means of pharmaceutical compositions that target the inhibition of the thioredoxin reductase enzyme, and which, while presenting response rates similar to reference drugs, exhibit low cytotoxicity rates and efficiency in in vivo treatment.

[015] SUMMARY OF THE INVENTION

[016] The present invention provides pharmaceutical compositions for the treatment of infections caused by microorganisms of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp. by means of two pharmaceutical compositions that target the inhibition of the enzyme Petition 870180036380, dated 03 / 05 / 2018, page 22 / 74 5 / 34 thioredoxin reductase, comprising as active ingredient the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide or the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide or a salt or a hydrate or a solvate thereof.

[017] Another protected object of the present invention relates to the use of the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide or 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)1,3,4-oxadiazol-2-yl)benzamide, or a salt or a hydrate or a solvate thereof, useful in the preparation of pharmaceutical compositions for the treatment of infections caused by microorganisms of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp.

[018] BRIEF DESCRIPTION OF THE FIGURES

[019] Figure 1 shows the Structural Formula of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide.

[020] Figure 2 shows the Structural Formula of the compound 4-(Ncyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide.

[021] Figure 3 shows the results of MFC (microscopic concentration assessment in Sabouraud Dextrose Agar (SDA) culture medium at 35°C, 24 hours) for the genus Candida spp. It was observed that the MFC of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide for the standard strains of the evaluated Candida species were all >256 pg / mL. The significant reduction in isolate growth shown in the MFC suggests that the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide likely has fungistatic behavior for the evaluated Candida species.

[022] Figure 4 shows the CFM (microscopic concentration of MICs in Sabouraud Dextrose Agar (SDA) culture medium at 35°C, 24 hours) results for the genus Candida spp. It was observed that the CFM of 4-(N-cyclohexyl-N-eth-sulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2yl)benzamide for the standard strains of the evaluated Candida species was >256 pg / mL for ATCC of C. krusei, C. glabrata and C. tropicalis, and 128 pg / mL Petition 870180036380, dated 03 / 05 / 2018, page 23 / 74 6 / 34 for ATCC of C. albicans and C. parapsilosis. The significant reduction in isolate growth observed in the CFM suggests that the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2yl)benzamide likely has fungistatic behavior against the Candida species evaluated.

[023] Figure 5 shows the CFM (Cultivation of MIC concentrations in Sabouraud Dextrose Agar (SDA) culture medium, at 35°C, 48 hours) results for the genus Cryptococcus spp. It was observed that the CFM of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide for the INCQS standard strain (C. neoformans) was 32 μg / mL and for the INCQS strain (C. gatti) it was 64 μg / mL. The significant reduction in isolate growth shown in the CFM suggests that the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide likely has fungicidal behavior against Cryptococcus spp.

[024] Figure 6 shows the CFM (Cultivation of MIC concentrations in Sabouraud Dextrose Agar (SDA) culture medium, at 35°C, 48 hours) results for the genus Cryptococcus spp. It was observed that the CFM of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide for the INCQS standard strain (C. neoformans) was 64 μg / mL and for the INCQS strain (C. gatti) it was 128 μg / mL. The significant reduction in isolate growth observed in the CFM suggests that the molecule 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2yl)benzamide likely possesses fungicidal behavior against Cryptococcus spp.

[025] Figure 7 shows the CFM (Cultivation of MIC concentrations in Heart Brain Infusion (BHI) agar culture medium, at 37°C) results for the genus Paracoccidioides spp. It was observed that the CFM of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2yl)benzamide was 16μg / mL for 16.67% (1 / 6), 32μg / mL for 16.67% (1 / 6), 64μg / mL for 16.67% (1 / 6), 128μg / mL for 33.33% (2 / 6) and 256μg / mL for 16.67% (1 / 6) of the clinical isolates of P. brasiliensis. And from 1μg / mL for 33.33% (1 / 3), 8μg / mL for 33.33% (1 / 3) and 64μg / mL for 33.33% (1 / 3) of the isolates Petition 870180036380, dated 03 / 05 / 2018, page 24 / 74 7 / 34 clinical cases of P. lutzii. The significant reduction in isolate growth shown in the CFM suggests that the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide likely has fungicidal behavior against Paracoccidioides spp.

[026] Figure 8 shows the CFM (microscopic concentration of MICs in heart and brain infusion (BHI) agar culture medium at 37°C) results for the genus Paracoccidioides spp. It was observed that the CFM of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2yl)benzamide was 8 μg / mL for 66.67% (4 / 6) and 16 μg / mL for 33.33% (2 / 6) of the clinical isolates of P. brasiliensis. And 8 μg / mL for 66.67% (2 / 3) and 16 μg / mL for 33.33% (1 / 3) of the clinical isolates of P. lutzii. The significant reduction in isolate growth observed in the CFM suggests that the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)1,3,4-oxadiazol-2-yl)benzamide likely possesses fungicidal behavior against Paracoccidioides spp.

[027] Figure 9 shows the results of the in vitro cytotoxicity assessment of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)1,3,4-oxadiazol-2-yl)benzamide. This experiment was performed on three cell lines using a colorimetric assay at 24 and 48 hours, evaluating concentrations from 0.5 to 256 pg / mL. There was a significant reduction (p<0.05) in cell viability only at the highest concentrations tested, 256 μg / mL (24 hours), 128 and 256 μg / mL (48 hours) for HeLa, and 256 μg / mL at 48 hours for HUVEC. The graph represents three independent experiments. The bars indicate the standard deviation.

[028] Figure 10 shows the results of the in vitro cytotoxicity assessment of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)1,3,4-oxadiazol-2-yl)benzamide. This experiment was performed on three cell lines using a colorimetric assay at 24 and 48 hours, evaluating concentrations from 0.5 to 256 pg / mL. There was a significant reduction (p<0.05) in cell viability only at the highest concentrations tested, for HeLa cell lines: at concentrations of 128 and 256 μg / mL (24 and 48 hours). Petition 870180036380, dated 03 / 05 / 2018, page 25 / 74 8 / 34 hours), for Vero cell line: at a concentration of 256 μg / mL (24 and 48 hours) and for HUVEC cell line: at concentrations of 128 and 256 μg / mL (24 and 48 hours). The graph is represented by three independent experiments. The bars indicate the standard deviation.

[029] Figure 11 shows the results of the in vitro cytotoxicity assessment of the diluent compounds, Dimethyl Sulfoxide (DMSO). This experiment was performed on three cell lines (HeLa, HUVEC, and Vero) using a colorimetric assay at 24 and 48 hours. The diluent (DMSO) did not show significant toxicity in any of the parameters evaluated. The graph is represented by three independent experiments. The bars indicate the standard deviation.

[030] Figures 12 and 13 show the results of the in vivo cytotoxicity assessment of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide. Male BALB / c mice were used, divided into four groups: no treatment, treatment with the compound diluent (PBS, DMSO and Pluronic F-127®), and single-dose intraperitoneal (50 mg / kg) and intravenous (Compound - 12.5 mg / kg) administration. Behavioral changes were observed within 30 minutes in all groups, with abdominal contortions and motor impairment being more evident in the intraperitoneal treatment and greater sedation in the intravenous treatment. There was no change in the animals' weight evolution, hematological profile, or macroscopic organ analysis. Intravenous administration showed a better profile for 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide.

[031] Figures 14 and 15 show the results of the in vivo cytotoxicity assessment of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)1,3,4-oxadiazol-2-yl)benzamide. Male BALB / c mice were used, divided into four groups: untreated, treated with the compound diluent (PBS, DMSO and Pluronic F-127®), and treated with a single intraperitoneal and intravenous dose of 50 mg / kg. Behavioral changes were observed within 30 minutes in all groups, being Petition 870180036380, dated 03 / 05 / 2018, page 26 / 74 9 / 34 more evident abdominal contortions and motor impairment were observed with intraperitoneal treatment, and greater sedation was achieved with intravenous administration. There was no change in the animals' weight gain, hematological profile, or macroscopic organ analysis.

[032] Figure 16 shows the results of the antifungal activity of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2yl)benzamide and the commercial antifungal Fluconazole, in the experimental model of Systemic Candidiasis in mice. Figure 16A and 16B: Experimental Candidemia by Candida albicans. (A) Evaluation of the in vivo antifungal activity of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2yl)benzamide according to the target organ kidney. (B) Evaluation of the in vivo antifungal activity of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide according to the target organ spleen. In both (A) and (B), Colony Forming Units (Log10 CFU). Control: animals treated with diluent of the compound. Fluconazole: animals treated with Fluconazole 5 mg / kg. Compound: animals treated with the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 5 mg / kg. All treatments were performed for 5 days. The experiments were performed in triplicate, and the bars indicate the standard deviation. *p<0.05, statistically significant reduction when compared to the control. Figure 16C: Experimental Candidemia by Candida glabrata. Evaluation of the in vivo antifungal activity of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide according to the target organ kidney. Colony Forming Units (Log10 CFU). Control: animals treated with diluent of the compound. Fluconazole: animals treated with Fluconazole 5mg / kg. Compound: animals treated with the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 25mg / kg. All treatments were performed for 5 days. The experiments were performed in triplicate, and the bars indicate the standard deviation. *p<0.05, statistically significant reduction when compared to the control. Petition 870180036380, dated 03 / 05 / 2018, page 27 / 74 10 / 34

[033] Figure 17 shows the results of the antifungal activity of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2yl)benzamide and the commercial antifungal Fluconazole, in the experimental model of Systemic Candidiasis in mice. Figures 17A and 17B: Experimental Candidemia by Candida albicans. (A) Evaluation of the in vivo antifungal activity of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2yl)benzamide according to the target organ kidney. (B) Evaluation of the in vivo antifungal activity of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide according to the target organ spleen. In both (A) and (B), Colony Forming Units (Log10 CFU). Control: animals treated with diluent of the compound. Fluconazole: animals treated with Fluconazole 5 mg / kg. Compound: animals treated with the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, 5 mg / kg.All treatments were performed for 5 days. The experiments were performed in triplicate, and the bars indicate the standard deviation. *p<0.05, statistically significant reduction when compared to the control. Figure 17C: Experimental Candidemia by Candida krusei. Evaluation of the in vivo antifungal activity of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide according to the target organ kidney. Colony Forming Units (Log10 CFU). Control: animals treated with diluent of the compound. Fluconazole: animals treated with Fluconazole 5mg / kg. Compound: animals treated with the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, 5mg / kg. All treatments were performed for 5 days. The experiments were performed in triplicate, and the bars indicate the standard deviation. *p<0.05, statistically significant reduction when compared to the control.

[034] Figure 18 shows the results of the antifungal activity of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2yl)benzamide and the commercial antifungal Itraconazole, in the experimental model of Paracoccidioidomycosis in mice. Colony Forming Units (Log10 CFU) of the lung infected with Paracoccidioides brasiliensis. Control: Petition 870180036380, dated 03 / 05 / 2018, page 28 / 74 11 / 34 animals treated with the compound diluent. Itraconazole: animals treated with itraconazole 5 mg / kg. Compound: animals treated with the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 5 mg / kg. All treatments were performed for 14 days. The experiments were performed in triplicate, and the bars indicate the standard deviation. * p < 0.05, statistically significant reduction when compared to the control.

[035] Figure 19 shows the results of the antifungal activity of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and the commercial antifungal Amphotericin B, in the experimental model of Cryptococcosis in mice. Colony Forming Units (Log10 CFU) of the Brain and Spleen infected with Cryptococcus neoformans. Control: animals treated with diluent of the compound. Amphotericin B: animals treated with Amphotericin B 1 mg / kg. Compound: animals treated with the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, 5 mg / kg. All treatments were carried out for 5 days. The experiments were performed in triplicate, and the bars indicate the standard deviation. *p<0.05, statistically significant reduction when compared to the control.

[036] DETAILED DESCRIPTION OF THE INVENTION

[037] The present invention relates to two pharmaceutical compositions for the treatment of infections caused by microorganisms of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp., with the target of action being the inhibition of the enzyme thioredoxin reductase, having as active ingredient the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide or the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)1,3,4-oxadiazol-2-yl)benzamide, or a salt or a hydrate or a solvate thereof.

[038] The compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, represented by the structural formulas Figure 1 and 2, respectively, have important functional groups with biological properties in their chain. Petition 870180036380, dated 03 / 05 / 2018, page 29 / 74 12 / 34

[039] Additionally, the aforementioned pharmaceutical compositions of the invention comprise at least one pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient is understood to be any excipient that does not interfere with the efficacy of the active ingredient and is not toxic to the host in which it is administered.

[040] For human use, a pharmaceutical composition is generally administered together with a pharmaceutically acceptable excipient, selected according to the intended route of administration and in accordance with established pharmaceutical practice.

[041] In a preferred embodiment of the invention, the aforementioned protected pharmaceutical compositions are presented in pharmaceutical forms selected from powders, granules, tablets, dragees, capsules, solutions, syrups, elixirs, suspensions, emulsions, injectables, tinctures, extracts, ointments, creams, pastes, lotions, gels, aerosols, patches and other known forms of administration.

[042] Various excipients can be used in the pharmaceutical compositions of the present invention. Among the excipients that can be used for liquid pharmaceutical forms (solutions, suspensions, elixirs, lotions, among others), one can mention, among others, vehicles, viscosity-increasing agents, tonicity agents, suspending agents, acidifying agents, alkalizing agents, preservatives, buffers and sweetening agents.

[043] Among the excipients that can be used for solid pharmaceutical forms (powders, granules, tablets, capsules, among others), one can mention, among others, diluents, binders, disintegrants, lubricants, coating agents, absorbents and surfactants.

[044] Among the excipients that can be used for semi-solid pharmaceutical forms (creams, gels, ointments, lotions, among others), one can mention, among others, vehicles, thickeners, humectants, emollients, emulsifying agents, antioxidants, parabens, Petition 870180036380, dated 03 / 05 / 2018, page 30 / 74 13 / 34 preservatives, levigating or spraying agents, sequestering or chelating agents, adsorbents, buffers and sweetening agents.

[045] The present invention also relates to the use of the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, in one of their forms, selected from neutral, salt, hydrate or solvate, in the preparation of a medicament, additive, agent or formulation useful in the treatment of infections caused by microorganisms of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp., with the target of action being the inhibition of the thioredoxin reductase enzyme.

[046] These compounds may constitute an alternative to replace currently used active ingredients that have side effects that limit their use. The results of these tests demonstrated significant fungitoxic and specific potential against species of fungi of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp.

[047] EXAMPLES

[048] The examples below are presented in order to illustrate in more detail certain embodiments of the invention. It is important to note that the present invention is not limited to the examples cited, and may be used in all the applications described or in any other equivalent variations.

[049] It should be noted that the examples related to the activity of the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide are not restricted to one of their forms, whether neutral, salt, hydrate or solvate.

[050] EXAMPLE 1: IN SILICO EXPERIMENTATION OF COMPOUND SELECTION AND INTERACTION WITH THIOREDOXIN REDUCTASE ENZYME

[051] The compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl) Petition 870180036380, dated 03 / 05 / 2018, page 31 / 74 14 / 34 N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide were selected using in silico methods. In the absence of experimentally solved 3D structures, computational methods are used to predict three-dimensional protein models and provide information about their structures. Three templates were selected for homology modeling of the thioredoxin reductase (TRR1) protein: one from Saccharomyces cerevisiae in the oxidized conformation and two from Escherichia coli in the reduced conformation. In the second reduced conformation, the 3D structure of thioredoxin reductase is complexed with thioredoxin, NADP+ analogs, and AADP+. Thus, six TRR1 models were constructed: three for Candida albicans and three for Cryptococcus neoformans. Additionally, two TRR1 models from Paracoccidioides lutzii were obtained, based on the reduced and complexed reduced conformation templates. Thus, eight models were subjected to molecular dynamics (MD) simulations.The analysis of RMSd values ​​throughout the DM simulations showed stability. Secondary structures were preserved throughout the simulation, with the oxidized conformation of TRR1 always proving more stable than the reduced and complexed conformation of TRR1, regardless of the pathogen. Cluster analyses of the main conformational families were performed for each model, and the central frames of each conformational family were selected for molecular docking. From the Life Chemicals database of chemical compounds, 3,000 molecules with drug-like properties and chemical diversity were pre-selected. To this number were added 1080 molecules, which are analogous to 3 molecules from this database with previously evaluated antifungal activity. After selecting the best hits, eleven selected molecules were acquired and tested in vitro and in vivo for their antifungal activity.The compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide were two of the compounds that showed the best activities, therefore they are the target of this patent application.

[052] EXAMPLE 2: FUNGAL SUSCEPTIBILITY TESTS Petition 870180036380, dated 03 / 05 / 2018, page 32 / 74 15 / 34 GENUS CANDIDA SPP. TO THE COMPOUNDS 4-(N-benzyl-N-methylsulfamoyl)N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-Methylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide AND AO ANTIFUNGAL FLUCONAZOLE

[053] To evaluate the susceptibility profile of clinical Candida spp. isolates to the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and fluconazole, the broth microdilution test was performed using the method proposed by CLSI (Clinical and Laboratory Standards Institute, 2010), according to protocol M27-A3 with modifications. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were determined for the pathogens used.

[054] The compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and fluconazole (Sigma-Aldrich) were tested. The drugs were diluted in RPMI medium [RPMI-1640 without bicarbonate, with L-glutamine, supplemented with 2% glucose and buffered (pH 7.0) with 0.165 mol / L morpholinopropanesulfonic acid (MOPS) (Sigma-Aldrich)]. Dimethyl sulfoxide (DMSO) and Pluronic F-127 (Sigma-Aldrich) were used as diluents for the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide. The two compounds were prepared by serial dilution at concentrations of 0.5 to 256 pg / mL, diluted in 1% DMSO and 0.2% Pluronic F-127, and fluconazole concentrations ranged from 0.125 to 64 pg / mL.The fungi were incubated in the absence and presence of 1% (vol / vol) DMSO and 0.2% (vol / vol) Pluronic F-127 to confirm that exposure to this solvent does not influence the growth of the tested isolates.

[055] In vitro susceptibility tests were performed on 79 Candida spp. isolates among standard and clinical strains (C. albicans (16), C. glabrata (16), C. parapsilosis (15), C. tropicalis (13) and C. krusei (19)), as Petition 870180036380, dated 03 / 05 / 2018, page 33 / 74 16 / 34 samples are part of the collection of the Medical Mycology Laboratory at the State University of Maringá. The fungi were maintained by subculture in semi-solid Sabouraud Agar (SDA) culture medium at 35°C and the fungi were used after 24 hours of growth.

[056] Yeast inocula were prepared in sterile saline buffer (0.85%) and RPMI, at a final concentration of 2 to 3 x 10³ cells / mL. Tests were performed in sterile microplates containing 96 wells. RPMI without inoculum (negative) was used as a control.

[057] The C. parapsilosis strain (ATCC 22019) was used as a reference yeast for all tests. The incubation time was 24 hours at 35°C. Data were presented as the minimum inhibitory concentration (MIC), which corresponds to the reading on an ELISA reader (405 nm), the concentration of the compound capable of inhibiting 50% of fungal growth. To determine the minimum fungicidal concentration (MFC), 5 μL from each well of the MIC plate were seeded onto SDA medium. The plates were incubated at 35°C for 24 hours. The lowest concentration of the compound at which no growth was observed was determined as the MFC concentration. Table 1 shows the results of the determination of the minimum inhibitory concentration (MIC) of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide acting directly on yeasts from different Candida spp. isolates (79 samples between clinical isolates and a standard strain). Samples Concentrations in pg / mL of 4-(N-benzyl-N-methylisutfamoH)-N-(5-(4-methoxybenzyl)- 256 128 64 1,3,4-oxadiazol-2-yl) benza mlda 2 1 0.5 32 16 8 4 C. albicans (16) 0 0 2 7 5 2 0 0 0 0 C. glabrata (16) 0 0 0 2 5 7 2 0 0 0 C. parapsilosis (15) 0 1 12 1 1 0 0 0 0 0 C. tropicalis (13) 0 0 2 2 6 2 1 0 0 0 C, Rru5e / (19) 0 0 1 15 3 0 0 0 0 0 Table 1. Minimum inhibitory concentration (MIC) of the compound 4-(N-benzyl-N-methylsulfamoii)-N(5-(4-methoxybenzii)-1,3,4-oxadiazol-2-ii)benzamide against clinical isolates and standard strain of Candida spp., distribution by species according to MIC results.

[058] Table 2 shows the results of the determination of the minimum inhibitory concentration (MIC) of the compound 4-(N-cyclohexyl-NPetition 870180036380, dated 03 / 05 / 2018, page 34 / 74 17 / 34 ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide acting directly on yeasts from different Candida spp. isolates (79 samples including clinical isolates and a standard strain). Samples Concentrations in mg / mL of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide 256 120 64 2 1 0.5 32 16 0 4 C. albicans (16) 0 0 2 12 2 0 0 0 0 0 C. glabrata (16) 0 0 0 0 12 4 0 0 0 0 C. parapsilosis (15) 0 4 5 5 0 1 0 0 0 0 C. tropicalis (13) 0 0 7 5 1 0 0 0 0 0 C. krusei (19) 0 0 6 13 0 0 0 0 0 0 Table 2. Minimum inhibitory concentration (MIC) of the compound 4-(N-cyclohexyl-N-ethylsulfamoii)-N(5-(furan-2-ii)-1,3,4-oxadiazol-2-ii)benzamide against clinical isolates and a standard strain of Candida spp., distribution by species according to the MIC results.

[059] The MIC of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide was 8 pg / mL for 12.5% ​​(2 / 16), 16 pg / mL for 31.25% (5 / 16), 32 pg / mL for 43.75% (7 / 16) and 64 pg / mL for 12.5% ​​(2 / 16) of the clinical isolates and standard strain of C. albicans. From 4 pg / mL for 12.5% ​​(2 / 16), 8 pg / mL for 43.75% (7 / 16), 16 pg / mL for 31.25% (5 / 16) and 32 pg / mL for 12.5% ​​(2 / 16) of the clinical isolates and standard strain of C. glabrata. From 16 pg / mL for 6.67% (1 / 15), 32 pg / mL for 6.67% (1 / 15), 64 pg / mL for 80% (12 / 15) and 128 pg / mL for 6.67% (1 / 15) of the clinical isolates and standard strain of C. parapsilosis. From 4 pg / mL to 7.70% (1 / 13), 8 pg / mL to 15.38% (2 / 13), 16 pg / mL to 46.16% (6 / 13), 32 pg / mL to 15.38% (2 / 13) and 64 pg / mL to 15.38% (2 / 13) of the clinical isolates and standard strain of C. tropicalis. And it was from 16 pg / mL to 15.79% (3 / 19), 32 pg / mL to 78.95% (15 / 19) and 64 pg / mL to 5.26% (1 / 19) of the clinical isolates and standard strain of C. krusei.

[060] The MIC of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano2-yl)-1,3,4-oxadiazol-2-yl)benzamide was 16 pg / mL for 12.5% ​​(2 / 16), 32 pg / mL for 75% (12 / 16) and 64 pg / mL for 12.5% ​​(2 / 16) of clinical isolates and standard strain of C. albicans. 8 pg / mL for 25% (4 / 16) and 16 pg / mL for 75% (12 / 16) of clinical isolates and standard strain of C. glabrata. 8 pg / mL for 6.67% (1 / 15), 32 pg / mL for 33.33% (5 / 15), 64 pg / mL for 33.33% (5 / 15) and 128 pg / mL for 26.67% (4 / 15) of clinical isolates and standard strain of C. Petition 870180036380, dated 03 / 05 / 2018, page 35 / 74 18 / 34 parapsilosis. From 16 μg / mL to 7.69% (1 / 13), 32 μρ / mL to 38.46% (5 / 13) and 64 μρ / mL to 53.85% (7 / 13) of the clinical isolates and standard strain of C. tropicalis. And it was from 32 μg / mL to 68.42% (13 / 19) and 64 μg / mL to 31.58% (6 / 19) of the clinical isolates and standard strain of C. krusei.

[061] For Fluconazole, most of the isolates tested were inhibited at a concentration of 0.125 μg / mL for C. albicans, 8 μρ / mL for C. glabrata, 0.5 μg / mL for C. parapsilosis and C. tropicalis, and 16 μg / mL for C. krusei. Based on the MIC, it was possible to determine the minimum fungicidal concentration (MFC) (Figures 3 and 4), revealing that the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide exhibit a fungistatic profile (which does not kill the fungus, but inhibits its growth) for Candida spp. This profile is interesting since fluconazole, one of the most widely used conventional antifungals for treating candidiasis, is also a fungistatic agent.Based on these results, it was possible to compare the in vitro antifungal efficacy between the tested compound and the reference drug fluconazole, verifying that, despite the reference drug presenting the lowest MIC values, it has a known resistance profile for the treatment of C. glabrata and C. krusei. Therefore, the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide show relevant antifungal efficacy against Candida spp., and may be promising for the development of a new antifungal drug.

[062] EXAMPLE 3: SUSCEPTIBILITY TESTS OF THE FUNGUS OF THE GENUS CRYPTOCOCCCUS SPP. TO THE COMPOUNDS 4-(N-benzyl-Nmethylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(Ncyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide AND TO ANTIFUNGAL AMPHOTERICIN B.

[063] To evaluate the susceptibility profile of clinical isolates of Cryptococcus spp. to the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 4-(N-cyclohexyl-N-ethylsulfamoyl)-N Petition 870180036380, dated 03 / 05 / 2018, page 36 / 74 For the pathogens 19 / 34 (5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and amphotericin B, a broth microdilution test was performed using the method proposed by CLSI (Clinical and Laboratory Standards Institute, 2010), according to protocol M27-A3 with modifications. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined for the pathogens used.

[064] The compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and amphotericin B (Sigma-Aldrich) were tested. The drugs were diluted in RPMI medium [RPMI-1640 without bicarbonate, with L-glutamine, supplemented with 2% glucose and buffered (pH 7.0) with 0.165 mol / L morpholinopropanesulfonic acid (MOPS) (Sigma-Aldrich)]. Dimethyl sulfoxide (DMSO) and Pluronic F-127 (Sigma-Aldrich) were used as diluents for the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide. The two compounds were prepared by serial dilution at concentrations from 1 to 512 pg / mL, diluted in 1% DMSO and 0.2% Pluronic F-127, and amphotericin B concentrations ranged from 0.03 to 16 pg / mL.The fungi were incubated in the absence and presence of 1% (vol / vol) DMSO and 0.2% (vol / vol) Pluronic F-127 to confirm that exposure to this solvent does not influence the growth of the tested isolates.

[065] In vitro susceptibility tests were performed on 16 isolates of the fungus Cryptococcus spp. between standard and clinical strains (13 clinical isolates of the species C. neoformans and three clinical isolates of the species C. gatti). The samples are part of the collection of the Medical Mycology Laboratory of the State University of Maringá. The fungi were maintained by subculture in semi-solid Sabouraud Agar (SDA) culture medium at 25°C and the fungi were used after 48 hours of growth.

[066] Yeast inocula were prepared in sterile saline buffer (0.85%) and RPMI, at a final concentration of 2 to 3 x 10³ cells / mL. The Petition 870180036380, dated 03 / 05 / 2018, page 37 / 74 20 / 34 tests were performed in sterile microplates containing 96 wells. RPMI without inoculum (negative) was used as a control.

[067] The C. parapsilosis strain (ATCC 22019) was used as a reference yeast for all tests. The incubation time was 24 hours at 35°C. Data were presented as the minimum inhibitory concentration (MIC), which corresponds to the reading on an ELISA reader (405 nm), the concentration of the compound capable of inhibiting 100% of fungal growth. To determine the minimum fungicidal concentration (MFC), 5 µL from each well of the MIC plate were seeded onto SDA medium. The plates were incubated at 35°C for 48 hours. The lowest concentration of the compound at which no growth was observed was determined as the MFC concentration. Table 3 shows the results of the determination of the minimum inhibitory concentration (MIC) of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide acting directly on yeasts from different isolates of Cryptococcus spp. (16 samples between clinical isolates and a standard strain). Samples Concentrations in pg / mL of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-metaxybenzyl)-1,3,4-oxadiazol-2-I-I)benzamide 512 256 128 4 2 1 64 32 16 8 C. neoformans (13) 1 0 0 0 11 1 0 0 0 0 C. gatti(3) 0 0 0 0 3 0 0 0 0 0 Table 3. Minimum Inhibitory Concentration (MIC) of the compound 4-(N-benzyl-N-methylsulfamoii)-N(5-(4-methoxybenzii)-1,3,4-oxadiazol-2-ii)benzamide against clinical isolates and standard strain of Cryptococcus spp., distribution by species according to MIC results.

[068] Table 4 shows the results of the determination of the minimum inhibitory concentration (MIC) of the compound 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide acting directly on yeasts from different Cryptococcus spp. isolates (16 samples between clinical isolates and standard strain). Samples Concentrations in pg / mL of 4-(N-cyclohexyl-N-ethylsulfamyl)-N-(5-(fibro-2-yl)-1,314-512256oxadiazol-2-yl)benzamide 2 1 128 64 32 16 8 4 C. neofarmans (13) 1 0 10 2 0 0 0 0 0 0 C. gatti (3) 0 0 3 0 0 0 0 0 0 0 Table 4. Minimum Inhibitory Concentration (MIC) of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-Petition 870180036380, dated 03 / 05 / 2018, page 38 / 74 21 / 34 (5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide against clinical isolates and standard strain of Cryptococcus spp., species distribution according to MIC results.

[069] The MIC of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)1,3,4-oxadiazol-2-yl)benzamide was 16μg / mL for 7.69% (1 / 13), 32μg / mL for 84.62% (11 / 13) and 512pg / mL for 7.69% (1 / 13) of the clinical isolates of C. neoformans and standard strain. And it was 32μg / mL for 100% (3 / 3) of the clinical isolates of C. gatti and standard strain.

[070] The MIC of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide was 64 μg / mL for 15.39% (2 / 13), 128 μg / mL for 76.92% (10 / 13) and 512 μg / mL for 7.69% (1 / 13) of the clinical isolates of C. neoformans and standard strain. And it was 128 μg / mL for 100% (3 / 3) of the clinical isolates of C. gatti and standard strain.

[071] For amphotericin B, most of the isolates tested were inhibited at a concentration of 0.25 μg / mL.

[072] From the MIC it was possible to determine the minimum fungicidal concentration (MFC) (Figures 5 and 6), for the standard strain of C. neoformans it was 32 μg / mL and for C. gatti it was 64 pg / mL, revealing that the two compounds, 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, have a fungicidal profile for Cryptococcus spp.Based on these results, it was possible to compare the in vitro antifungal efficiency between the tested compound and the reference drug amphotericin B. It was found that, although the reference drug presented the lowest MIC values ​​and also had a fungicidal profile, its use is limited due to numerous side effects and its intravenous administration, which can only be performed in a hospital setting. Therefore, it was concluded that the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide exhibit relevant fungicidal antifungal efficiency against Cryptococcus spp. and may be promising in the development of a new antifungal drug.

[073] EXAMPLE 4: FUNGAL SUSCEPTIBILITY TESTS Petition 870180036380, dated 03 / 05 / 2018, page 39 / 74 22 / 34 GENUS PARACOCCIDIOIDES SPP. TO THE COMPOUND 4-(N-benzyl-Nmethylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(Ncyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide AND TO THE ANTIFUNGAL ITRACONAZOLE.

[074] To evaluate the susceptibility profile of clinical isolates of Paracoccidioides spp. to the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and to itraconazole, the broth microdilution test was performed using the method proposed by CLSI (Clinical and Laboratory Standards Institute, 2010), according to protocol M27-A3 with modifications. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were determined for the pathogens used.

[075] The compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide and itraconazole (Sigma-Aldrich) were tested. The drugs were diluted in RPMI medium [RPMI-1640 without bicarbonate, with L-glutamine, supplemented with 2% glucose and buffered (pH 7.0) with 0.165 mol / L morpholinopropanesulfonic acid (MOPS) (Sigma-Aldrich)]. Dimethyl sulfoxide (DMSO) and Pluronic F-127 (Sigma-Aldrich) were used as diluents for the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide. The two compounds were prepared by serial dilution at concentrations from 1 to 512 pg / mL, diluted in 1% DMSO and 0.2% Pluronic F-127, and itraconazole concentrations ranged from 0.03 to 16 pg / mL.The fungi were incubated in the absence and presence of 1% (vol / vol) DMSO and 0.2% (vol / vol) Pluronic F-127 to confirm that exposure to this solvent does not influence the growth of the tested isolates.

[076] In vitro susceptibility tests were performed on 6 isolates of P. brasiliensis (Airton, João, Orlando, Luiz, Apdo and PB18) and three Petition 870180036380, dated 03 / 05 / 2018, page 40 / 74 23 / 34 isolates of P. lutzii (Pb01,8334 and ROSC), the samples are part of the Medical Mycology Laboratory collection of the State University of Maringá. The fungi were maintained by weekly subculture in Fava Neto semi-solid culture medium containing 4% glucose at 35°C and the fungi were used between the fifth and seventh day of growth in culture medium.

[077] Yeast inocula were prepared in sterile saline buffer (0.85%), at a final concentration of 0.5 to 2.5 x 10⁵ cells / mL. Tests were performed in sterile microplates containing 96 wells. RPMI without inoculum (negative) was used as a control.

[078] The C. parapsilosis strain (ATCC 22019) was used as a reference yeast for all tests. The incubation time was 5 days at 37°C. Data were presented as the minimum inhibitory concentration (MIC), which corresponds to the concentration of the compound capable of inhibiting 80% of growth. To determine the minimum fungicidal concentration (MFC), 5 µL from each well of the MIC plate, which showed growth inhibition, were plated onto BHI agar medium. The plates were incubated at 37°C for 7 days. The lowest concentration of the compound at which no growth was observed was determined as the MFC concentration. Table 5 shows the results of the determination of the minimum inhibitory concentration (MIC) of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide acting directly on yeasts from different isolates of Paracoccidioides spp. (9 samples). Samples Concentrations in pg / mL of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzH)-1,3,4-oxadiazol-2-yl)benzamide 512 256 2 1 126 64 32 16 8 4 P. brasiliensis (6) 0 1 2 1 1 1 0 0 0 0 P. lutzír (3) 0 0 0 1 0 0 1 0 0 1 Table 5. Minimum Inhibitory Concentration of the compound 4-(N-benzyl-N-methylsulfamoii)-N-(5-(4-methoxybenzii)-1,3,4-oxadiazol-2-ii)benzamide against clinical isolates of Paracoccidioides spp., distribution by species according to MIC results.

[079] Table 6 shows the results of the determination of the minimum inhibitory concentration (MIC) of the compound 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide acting Petition 870180036380, dated 03 / 05 / 2018, page 41 / 74 24 / 34 directly onto yeasts from different isolates of Paracoccidioides spp. (9 samples). Samples Concentrations in pg / mL of 4-(N-cyclohexí!-N-ethylsu!fsmoiI}-N-(5-(furan-2-ii)-1,3,4- 512 256 oxadiazol-2-yl)benzamide 2 1 128 64 32 16 8 4 P. brasiliensis (6) 0 0 0 0 0 2 4 0 0 0 P. 0 0 0 0 0 1 2 0 0 0 Table 6. Minimum Inhibitory Concentration of the compound 4-(N-cyclohexyl-N-ethylsulfamoii)-N-(5(furan-2-ii)-1,3,4-oxadiazol-2-ii)benzamide against clinical isolates of Paracoccidioides spp., distribution by species according to MIC results.

[080] The MIC of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)1,3,4-oxadiazol-2-yl)benzamide was 16 pg / mL for 16.67% (1 / 6), 32 pg / mL for 16.67% (1 / 6), 64 pg / mL for 16.67% (1 / 6), 128 pg / mL for 33.33% (2 / 6) and 256 pg / mL for 16.67% (1 / 6) of the clinical isolates of P. brasiliensis. And it was 1 pg / mL for 33.33% (1 / 3), 8 pg / mL for 33.33% (1 / 3) and 64 pg / mL for 33.33% (1 / 3) of the clinical isolates of P. lutzii.

[081] The MIC of 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide was 8 pg / mL for 66.67% (4 / 6) and 16 pg / mL for 33.33% (2 / 6) of the clinical isolates of P. brasiliensis. And it was 8 pg / mL for 66.67% (2 / 3) and 16 pg / mL for 33.33% (1 / 3) of the clinical isolates of P. lutzii.

[082] For itraconazole, all isolates tested were inhibited at a concentration of 1 pg / mL. From the MIC, it was possible to determine the minimum fungicidal concentration (MFC) (Figures 7 and 8), revealing that the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-H)-1,3,4-oxadiazol-2-yl)benzamide have a fungicidal profile. This is an extremely relevant characteristic, since among the drugs available for the treatment of Paracoccidioidomycosis, only amphotericin B has a fungicidal profile, but its use is limited due to numerous side effects and its intravenous administration, which can only be performed in a hospital setting. Itraconazole, one of the most commonly used treatments for PCM, is a fungistatic agent that does not kill the fungus, but only inhibits its growth. Petition 870180036380, dated 03 / 05 / 2018, page 42 / 74 25 / 34 Based on these results, it was possible to compare the antifungal efficiency between the tested compounds and the reference drug itraconazole, verifying that, despite the reference drug presenting the lowest MIC values, the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)1,3,4-oxadiazol-2-yl)benzamide show great antifungal efficiency due to their fungicidal profile.

[083] EXAMPLE 5: CYTOTOXICITY TESTS OF COMPOUNDS 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide IN THREE HUMAN CELL LINES

[084] The cytotoxicity of the compounds 4-(N-benzyl-N-methylsulfamoyl)-N(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide was performed in three cell lines, HeLa, Vero and HUVEC, using the CellTiter 96 assay (Promega, Madison, WI, USA), based on the reduction of MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium). Cells were seeded in 96-well microdilution plates at a density of 2 χ¹⁰⁴ cells / well in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v / v) fetal bovine serum (FBS) (fetal bovine serum, Gibco BRL, Invitrogen, Paisley, Scotland). Cells were cultured in a humid environment with 5% CO₂ and 95% O₂ at 37°C.After 24 hours of incubation, 200 μL of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide or the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide were added to row 1 of the microdilution plates and diluted 1:1 serially to obtain final concentrations ranging from 0.5 to 256 pg / mL. Cells cultured with the compound were incubated for 24 hours and 48 hours in a humid environment with 5% CO2 and 95% O2 at 37°C. The cells were washed twice with PBS and 100 µl of MTS was added at a 1:100 dilution. Petition 870180036380, dated 03 / 05 / 2018, page 43 / 74 26 / 34 cells were incubated in 5% CO2 and 95% O2 at 37°C, and after 4 hours the supernatant was transferred to a new plate, which was then transferred to an ELISA microdilution plate reader at 492nm. Cytotoxicity was expressed as the percentage of cell viability compared to untreated control cells.

[085] Figures 9 and 10 show cell viability (%) as a function of the concentration of the compounds, which showed antifungal activity in vitro. The in silico methods used sought compounds with selective activity against the fungus, without causing damage to mammalian cells. Thus, before the animal assay to evaluate the antifungal activity of the compounds, the in vitro cytotoxicity assay was performed.

[086] Figure 11 shows cell viability (%) as a function of the concentration of the diluent (DMSO) used for the compounds.

[087] EXAMPLE 6: IN VIVO CYTOTOXICITY TESTS OF THE COMPOUNDS 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2yl)-1,3,4-oxadiazol-2-yl)benzamide

[088] The in vivo cytotoxicity of the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide was performed with male BALB / c mice divided into four groups: no treatment, treatment with the compound diluent (PBS, DMSO and Pluronic F-127 ® F-127) and treated with a single dose via intraperitoneal (50 mg / kg) and intravenous (12.5 mg / kg) routes for the compound 4-(N-benzyl-Nmethylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide, and intraperitoneally and intravenously (50mg / Kg) for the compound 4-(N-cyclohexyl-Methylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide.

[089] Prior to treatment, an initial hematological evaluation was performed on all animals, and after the administration of each treatment, they were observed individually at 0, 15, 30, 60, 120, 240, and 480 minutes. During this period, the parameters were evaluated. Petition 870180036380, dated 03 / 05 / 2018, page 44 / 74 27 / 34 behavioral observations included: motor activity, abdominal contortion, seizures, salivation, sedation, and piloerection. Additionally, the animals were observed for a maximum period of 14 days for weight and survival assessment. On this day, the animals were anesthetized and subsequently sacrificed; blood was collected for analysis of biochemical parameters (aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and glucose) using enzymatic colorimetric methods; and final hematological analysis. Following this, the liver and kidneys of each animal were macromorphologically examined and weighed.

[090] Behavioral changes were observed up to 30 minutes in all groups, with abdominal contortions and motor impairment being more evident in the intraperitoneal treatment and greater sedation via intravenous administration. After this period, no changes were observed in the analyzed parameters. As can be seen in Figures 12A and 14A, there was a change in liver weight in animals that received the vehicle and the compounds 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide via intraperitoneal administration (p <0.05). For this same route of administration, the same compounds were also able to alter kidney weight in animals that received a single dose of the same compounds (Figures 12B and 14B). There were no significant changes in the heart (Figures 12C and 14C).However, intravenous administration showed a better profile for 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide (Figures 12D, 12E, and 12F), showing no significant changes in any of the organs evaluated. For 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, intravenous administration also showed a better profile, significantly altering liver weight only in the group that received the compound dose (Figures 14D, 14E, and 14F). As can be seen in Figures 13A and 13B, the values ​​of the liver enzymes AST and ALT, respectively, showed an increased profile when compared to the normal group and the group that received the vehicle. However, via the intravenous route, only the AST enzyme is released. Petition 870180036380, dated 03 / 05 / 2018, page 45 / 74 28 / 34 showed altered levels for the groups that received the vehicle and 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide (Figure 13E). Regarding the biochemical parameters for 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, via the intraperitoneal and intravenous routes, only the AST enzyme (Figures 15A and 15E) showed altered levels for the groups that received the compound. And for the intravenous route, serum glucose levels were altered (Figure 15H).

[091] It is important to emphasize that there was no change in the weight evolution of the animals, in the hematological profile and in the macroscopic analysis of the organs for both compounds.

[092] EXAMPLE 7: EVALUATION OF THE IN VIVO ANTIFUNGAL ACTIVITY OF THE COMPOUNDS 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide NO Experimental Model of Systemic Candidiasis in Mice

[093] For these in vivo assays, six-week-old female Balb / c mice were used, bred under specific conditions free of pathogens at the animal origin facilities of the State University of Maringá, Brazil. All procedures were performed in accordance with the regulations of the Institutional Ethics Committee for Animal Experimentation, State University of Maringá, Brazil (CEUA / UEM No. 9810191015). The animals were treated in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (http: / / oacu.od.nih.gov / ARAC / index.htm; accessed November 21, 2015).

[094] For each Candida species evaluated, Balb / c mice were separated into three groups of 5 mice: compound treatment group, fluconazole treatment group, and control group (treated with PBS). For the preparation of fungal cell inocula, the standard strains ATCC90028 of C. albicans and ATCC90030 of Petition 870180036380, dated 03 / 05 / 2018, page 46 / 74 29 / 34 C. glabrata and ATCC6258 of C. krusei, grown in culture medium for 24 hours, fungal viability was verified with the vital stain Trypan Blue (generally more than 90%) and colony-forming units were counted in a Neubauer chamber. A saline suspension of 5x10⁵ (Candida albicans), 1x10⁷ (Candida krusei) or 5x10⁷ (Candida glabrata) was inoculated into the lateral tail vein and treatment was initiated 3 hours after infection. The compound was administered at 5 mg / kg for C. albicans and C. krusei species and at 25 mg / kg for C. glabrata, fluconazole at 5 mg / kg, twice daily for 5 days, intraperitoneally. The animals in each group were euthanized 5 days after treatment, part of the organs were sent for histopathological analysis and the other part for maceration. The organs (kidney and spleen) of each mouse were weighed and macerated using a mortar and pestle in 1 ml of PBS, and 20 µl (pure, diluted 1:10 and 1:100) of this macerate was plated on SDA.The number of viable yeast cells in the organs was estimated by counting colony-forming units (CFU) grown for 24 hours at 35°C. The results were presented as the log10 of the mean value ± standard deviation. The experiment was performed in triplicate.

[095] As shown in Figures 16A and 16B (4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide) and Figures 17A and 17B (4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide), both compounds showed efficacy in controlling infection caused by C. albicans in animals. Female Balb / c mice infected with ATCC90028 and treated with the compounds at a concentration of 5 mg / kg intraperitoneally for five days significantly reduced the number of remaining C. albicans colony-forming units (CFU) in the kidneys and spleen of the animals compared to the control group of infected animals treated only with the vehicle.

[096] As shown in Figure 16C, the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide also showed efficiency in controlling infection caused by C. glabrata in animals. It was found that female Balb / c mice infected with ATCC90030 and Petition 870180036380, dated 03 / 05 / 2018, page 47 / 74 30 / 34 treated with 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide at a concentration of 25 mg / kg via intraperitoneal injection for five days significantly reduced the number of remaining C. glabrata colony-forming units (CFU) in the kidneys of the animals compared to the control group of infected animals treated only with the vehicle.

[097] As shown in Figure 17C, the compound 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide also showed efficiency in controlling infection caused by C. krusei in animals. It was found that female Balb / c mice infected with ATCC6258 and treated with 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide at a concentration of 5 mg / kg via intraperitoneal injection for five days significantly reduced the number of remaining C. krusei colony-forming units (CFU) in the kidneys of the animals compared to the control group of infected animals treated only with the vehicle.

[098] Thus, due to the low cytotoxicity of the two compounds evaluated and their ability to inhibit the growth of fungi of the genus Candida (fungistatic action), the same activity found in Fluconazole, which is one of the most widely used conventional antifungals in the treatment of mycoses and which has been showing therapeutic failure due to the appearance of resistant strains, the compounds are quite promising for the development of new drugs to treat systemic mycoses, such as candidiasis. In this context, the present invention presents a viable alternative for the treatment of infections caused by fungi of the genus Candida spp.

[099] EXAMPLE 8: EVALUATION OF THE IN VIVO ANTIFUNGAL ACTIVITY OF THE COMPOUND 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)1,3,4-oxadiazol-2-yl)benzamide E IN THE EXPERIMENTAL MODEL OF PARACOCCIDIOIDOMYCOSIS IN MICE

[100] For these in vivo assays, six-week-old male Balb / c mice were used, bred under specific pathogen-free conditions at the facilities of Petition 870180036380, dated 03 / 05 / 2018, page 48 / 74 31 / 34 animal origin from the State University of Maringá, Brazil. All procedures were performed in accordance with the regulations of the Institutional Ethics Committee for Animal Experimentation, State University of Maringá, Brazil (approval no. 053 / 2014 CEP, 2014 / 02 / 10). The animals were treated in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (http: / / oacu.od.nih.gov / ARAC / index.htm; accessed November 21, 2015).

[101] Groups of 6 Balb / c mice were used to evaluate the in vivo antifungal activity of 4-(N-benzyl-N-methylsulfamoyl)-N(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide. For the preparation of the fungal cell inoculum, Pb18 grown in culture medium for 5 days was used, fungal viability was verified with the vital stain Trypan Blue (generally more than 90%) and colony-forming units were counted in a Neubauer chamber. The three groups of mice (6-8 weeks old) were infected intratracheally (IT) with 1 χ 106 viable yeast cells in 50 µl of phosphate-buffered saline (PBS). After 24 hours of infection, treatment was initiated with 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide (5 mg / kg), itraconazole (5 mg / kg) or vehicle (PBS, 1% DMSO and 0.2% Pluronic F-127).The animals in each group were euthanized fifteen days after treatment. Part of the organs (lung and liver) were sent for histopathological analysis, and the other part for maceration. The organs of each mouse were weighed and macerated using a mortar and pestle in 1 ml of PBS, and 300 µl of this macerate was plated on supplemented BHI. The number of viable yeasts in the organs was estimated by counting the colony-forming units (CFU) grown for up to 15 days at 35°C. The results were presented as the log10 of the mean value ± standard deviation. The experiment was performed in triplicate.

[102] As shown in Figure 18, the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide showed efficiency in controlling infection caused by P. brasiliensis in animals. It was found that male Balb / c mice infected with Pb18 and treated Petition 870180036380, dated 03 / 05 / 2018, page 49 / 74 Intraperitoneal administration of 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide for fifteen days significantly reduced the number of remaining colony-forming units (CFU) of P. brasiliensis in the lungs of animals compared to the control group of infected animals treated only with the vehicle. The results of treatment with 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide were similar to the group of animals treated with itraconazole (p<0.05).Thus, due to the low cytotoxicity of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide and its ability to kill fungi (fungicidal action), the compound is quite promising for the development of new drugs to treat systemic mycoses compared to itraconazole, which is the drug of choice for non-hospitalized patients and which, however, has a fungistatic profile and not a fungicidal one, as is the case with the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide. In this context, the present invention presents a viable alternative for the treatment of infections caused by fungi of the genus Paracoccidioides spp.

[103] EXAMPLE 9: EVALUATION OF THE IN VIVO ANTIFUNGAL ACTIVITY OF THE COMPOUND 4-(N-cyclohexH-N-ethylsulfamoyl)-N-(5-(furan-2-H)1,3,4-oxadiazol-2-yl)benzamide IN THE EXPERIMENTAL MODEL OF CRYPTOCOCCOSIS IN MICE

[104] For these in vivo assays, six-week-old female Balb / c mice were used, bred under specific conditions free of pathogens at the animal origin facilities of the State University of Maringá, Brazil. All procedures were carried out in accordance with the regulations of the Institutional Ethics Committee for Animal Experimentation, State University of Maringá, Brazil (CEUA / UEM No. 9810191015). The animals were treated in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (http: / / oacu.od.nih.gov / ARAC / index.htm; accessed November 21, 2015). Petition 870180036380, dated 03 / 05 / 2018, pages 50 / 74 33 / 34

[105] Balb / c mice were separated into three groups of 5 mice: a treatment group with 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, a treatment group with amphotericin B, and a control group (treated with PBS). For the preparation of the fungal cell inoculum, the INCQS standard strain of C. neoformans was used, grown in culture medium for 48 hours, fungal viability was verified with the vital dye Trypan Blue (generally more than 90%), and colony-forming units were counted in a Neubauer chamber. A saline suspension of 2x10⁵ cells / mL was inoculated into the lateral tail vein, and treatment was initiated 3 hours after infection. The compound was administered at 5 mg / kg and amphotericin B at 1 mg / kg, twice daily for 5 days, intravenously. The animals in each group were euthanized 5 days after treatment; some organs were sent for histopathological analysis and others for maceration.The organs (brain and spleen) of each mouse were weighed and macerated using a mortar and pestle in 1 ml of PBS, and 20 µl (pure, diluted 1:10 and 1:100) of this macerate was plated on SDA. The number of viable yeasts in the organs was estimated by counting the colony-forming units (CFU) grown for 48 hours at 25°C. The results were presented as the log10 of the mean value ± standard deviation. The experiment was performed in triplicate.

[106] As shown in Figure 19, the compound 4-(N-cyclohexyl-Netylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide was effective in controlling infection caused by C. neoformans in animals. Female Balb / c mice infected with the INCQS standard strain of C. neoformans and treated with 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)benzamide at a concentration of 5 mg / kg intravenously for five days significantly reduced the number of remaining C. neoformans colony-forming units (CFU) in the brain and spleen of the animals compared to the control group of infected animals treated only with the vehicle. Thus, due to the low cytotoxicity of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furan-2-yl) Petition 870180036380, dated 03 / 05 / 2018, pp. 51 / 74 34 / 34 Given its 1,3,4-oxadiazol-2-yl)benzamide composition and fungicidal action, the compound is quite promising for the development of new drugs to treat systemic mycoses, unlike amphotericin B, which is the drug of choice for hospitalized patients but presents hepatotoxicity and other side effects. In this context, the present invention presents a viable alternative treatment for infections caused by fungi of the genus Cryptococcus spp. Petition 870180036380, dated 03 / 05 / 2018, pages 52 / 74

Claims

1 / 2 CLAIMS 1. PHARMACEUTICAL COMPOSITION, characterized by comprising as an active ingredient the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide with action in inhibiting the thioredoxin reductase enzyme of fungi.

2. PHARMACEUTICAL COMPOSITION, characterized by comprising as its active ingredient the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)1,3,4-oxadiazol-2-yl)benzamide, with action in inhibiting the thioredoxin reductase enzyme of fungi.

3. PHARMACEUTICAL COMPOSITION, according to Claim 1, characterized by comprising as an active ingredient the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide in one of its forms, these being neutral, salt, hydrate or solvate.

4. PHARMACEUTICAL COMPOSITION, according to Claim 2, characterized by comprising as its active ingredient the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide, in one of its forms, these being neutral, salt, hydrate or solvate.

5. PHARMACEUTICAL COMPOSITION, according to Claim 3, characterized by comprising at least one pharmaceutically acceptable excipient.

6. PHARMACEUTICAL COMPOSITION, according to Claim 4, characterized by comprising at least one pharmaceutically acceptable excipient.

7. PHARMACEUTICAL COMPOSITION, according to Claim 1, or 3 or 5, characterized by comprising one of the presentation forms selected from powders, granules, tablets, dragees, capsules, elixirs, solutions, suspensions, emulsions, injectable syrups, tinctures, extracts, lotions, creams, pastes, gels, aerosols, patches and other forms of administration known or to be known.

8. PHARMACEUTICAL COMPOSITION, according to Claim 2, or 4 or 6, characterized by comprising one of the presentation forms selected from powders, granules, tablets, dragees, capsules, elixirs, solutions, suspensions, emulsions, injectable syrups, tinctures, extracts, lotions, creams, pastes, gels, aerosols, patches and other known or future forms of administration.

9. USE OF THE PHARMACEUTICAL COMPOSITION, according to Claims 1, 3, 5 and 7 and of the compound 4-(N-benzyl-N-methylsulfamoyl)-N-(5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)benzamide in one of its forms, characterized by being in the preparation of a medicament, additive, agent or formulation to be used in the treatment of infections caused by microorganisms of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp.

10. USE OF THE PHARMACEUTICAL COMPOSITION, according to Claims 2, 4, 6 and 8, and of the compound 4-(N-cyclohexyl-N-ethylsulfamoyl)-N-(5-(furano-2-yl)-1,3,4-oxadiazol-2-yl)benzamide in one of its forms, characterized by being in the preparation of a medicament, additive, agent or formulation to be used in the treatment of infections caused by microorganisms of the genus Candida spp., Cryptococcus spp. and Paracoccidioides spp. Petition 870180036380, dated 03 / 05 / 2018, pp. 54 / 74

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