Antiparasitic Behavior of Trifluoromethylated Pyrazole 2-Amino-1,3,4-thiadiazole Hybrids and Their Analogues: Synthesis and Structure-Activity Relationship

A series of trifluoromethylated pyrazole thiosemicarbazone, trifluromethylated pyrazole isothiosemicarbazone, and trifluoromethylated pyrazole 2-amino-1,3,4-thiadiazole hybrids were synthesized and evaluated in vitro against the promastigote form of Leishmania amazonensis and the epimastigote form of Trypanosoma cruzi, the pathogens causing the neglected tropical diseases leishmaniasis and Chagas disease, respectively. The results show the potential of these compounds regarding their antiparasitic properties. Studies on the structure-activity relationship demonstrated that compounds containing a bulky group at the para position of the phenyl ring attached to the 5-position of the pyrazole core had better antiparasitic effects. Among the substituents attached at the 3-position of the pyrazole ring, the insertion of the 2-amino-1,3,4-thiadiazole nucleus led to the most potent compounds compared to the thiosemicarbazone derivative.


INTRODUCTION
Every year, more than 700,000 deaths occur due to infectious diseases caused by parasites (Habercom, 2016). Among them, neglected tropical diseases (NTD) are responsible for about 530,000 deaths (WHO). Leishmaniasis is a NTD caused by Leishmania species and transmitted by the phlebotomine sandfly vector that can be found throughout the world, but mostly in developing countries, putting at risk more than 350 million lives (Habercom, 2016;Gradoni et al., 2017).
The drugs that are used nowadays for the treatment of leishmaniasis include pentavalent antimonials as first-line drugs, such as meglumine antimoniate. In case of resistance, other drugs are used like amphotericin B, paromomycin, miltefosine, and pentamidine (Alvar et al., 2012;Sangshetti et al., 2015;Hurrell et al., 2016). However, all these drugs present high toxicity and several collateral effects, beyond resistance of the protozoan to the drugs (Sangshetti et al., 2015).
Furthermore, Chagas disease (CD), also known as American trypanosomiasis, is another NTD that also occurs in developing countries, caused by the vector-borne flagellate protozoan parasite Trypanosoma cruzi. Chagas disease has infected over 20 million people in Central and South America and is responsible for around 20,000 deaths per year. Current treatments for Chagas disease are based on two old drugs, benznidazole (BZN) and nifurtimox (NFX) (Goupil and McKerrow, 2014;Chain et al., 2019). They are more effective in the acute phase of the disease, show several side effects, and require prolonged treatment. Therefore, there is remarkable urgency for the development of effective, inexpensive, and safe drugs for the treatment of NTD such as leishmaniasis and Chagas disease.
Additionally, the presence of fluorine atoms in organic compounds affect the chemical, biological, and physical properties of the molecules. Therefore, a trifluoromethyl group (-CF 3 ) can improve the pharmacological activities of compounds bearing this moiety (Petrov, 2009).
With the purpose of developing potent antiprotozoal compounds, that are less toxic and more selective, in this study, we adopted the molecular hybridization strategy for molecules design and synthesized a new series of trifluoromethylated pyrazoles and thiosemicarbazone/Smethylated thiosemicarbazone/2-amino 1,3,4-thiadiazoles hybrids ( Figure 1F), and evaluated their antiprotozoal activity against the promastigote form of Leishmania amazonensis and the epimastigote form of Trypanosoma cruzi.

RESULTS AND DISCUSSION
A one-pot synthesis of thiosemicarbazone derivatives 2a-f starting from trifluoromethylated b-enamino diketone (TBED) 1a-f was developed based on a methodology previously reported by our research group (Pianoski et al., 2020) (Figure 2). This reaction exhibited high regioselectivity, good scope, and efficiency, providing six new thiosemicarbazone derivatives (2a-f) in moderate to excellent yields (63-94%). The electronic effects of aryl group in the TBED displayed an important role, since aryl groups with electron withdrawing group had the best yields, while neutral and electron donors had the worst.
Next, we synthesized a series of S-methylated thiosemi carbazone derivatives 3a-f from the methylation reaction of the sulfur atom of their respective thiosemicarbazones 2a-f. For the synthesis of these derivatives, we performed some tests where the optimal condition was found when the reaction was carried out in DMSO, at room temperature, for 5 min, using 3.0 equiv. of iodomethane and 1.2 equivalents of Na 2 CO 3 . Satisfactorily, the methodology was simple and effective, and the products 3a-f were obtained with moderate to good yields (61-85%) (Figure 3).
The syntheses of the 1,3,4-thiadiazole-pyrazole hybrids 4a-f were conducted from the oxidative cyclisation of their respective thiosemicarbazones 2a-f. Various methods have been reported for the synthesis of this core on literature (Hu et al., 2014;Haider et al., 2015), so we used two different oxidant agents and conditions for the cyclisation (Table 1). Initially, the compound 2a was subject to the oxidative cyclisation reaction using 1.2 equiv. of molecular iodine, 3.0 equivalents of Na 2 CO 3 , in THF, at room temperature. However, most of the starting material was recovered, even after prolonged reaction time (Entry 1, Table 1). On the other hand, when the reaction was carried out at reflux, the desired product 4a was obtained with a yield of 44% (Entry 2, Table 1). The yield of the product 4a was slightly improved when employed 1,4-dioxane reflux (Entry 3, Table 1). We also test ferric chloride as an oxidant. Nevertheless, a longer time was necessary for the total conversion to the desired product (Entries 4-6, Table 1). Thus, the best condition found for the oxidative cyclisation was using molecular iodine as oxidant with 3.0 equivalents of Na 2 CO 3 under 1,4-dioxane reflux for 4 h (Entry 3, Table 1).
The efficacy of the methodology was demonstrated when applied for the other thiosemicarbazones 2b-f containing R groups with distinct electronic properties. In general, the properties of these groups did not affect the reaction, and the 2-amino-1,3,4-thiadiazole pyrazole hybrids 4a-f were obtained with yields from 50 to 56% (Figure 4).
Finally, all the structures of the synthesized compounds were elucidated based on 1 H and 13 C supplemented with 2D NMR measurements.

Antiproliferative and Cytotoxic Activities
All the novel trifluoromethylated pyrazoles derivatives 2a-f, 3af, and 4a-f were evaluated against the promastigote form of L. amazonensis and the epimastigote form of T. cruzi. The results are reported as the concentration causing a 50% inhibition in cell growth (IC 50 ). Additionally, the toxicity of compounds was evaluated against two different cell lines: epithelial cell LLCMK 2 and macrophages J774A1. The antiproliferative and cytotoxic activities are shown in Table 2, as well as the calculated selectivity index (SI). In general, 18 compounds showed antiproliferative activity against L. amazonensis and T. cruzi.
As shown in Table 2, the series of the compounds 2a-f exhibited IC 50 values in the range 18.9 to 61.7 µM against L. amazonensis. The unsubstituted phenyl ring led to the compound with the worst activity (2e, IC 50 = 61.7 µM). Among the halogens, the para-bromo phenyl ring exhibited the best activity (2d, IC 50 = 18.9 µM), and was also the most active compound among the series 2a-f. The electronwithdrawing nitro group (2a, IC 50 = 26.7 µM) and the electron-donating methoxy group (2f, IC 50 = 22.4 µM) at the para position were also tolerated, presumably for steric reasons. In general, the compounds containing the bulkiest groups at the para position exhibited the best results against L. amazonensis. The selective index against macrophages ranged from 8 to 9.
For the series of compounds 3a-f, again bulky groups such as bromo, methoxy, and nitro at the para position led to the most active compounds with IC 50 values of 13.9, 14.2, and 18.3 µM, respectively. Furthermore, the methylation of the sulfur atom    (3a-f) offered an improvement on activity, leading to compounds about 1.5-fold more active compared to 2a-f. The most active compound, 3d, exhibited a selectivity index of 10.55 against macrophages. Among the series of the 2-amino-1,3,4-thiadiazole pyrazole hybrids 4a-f, it was also found that the presence of bulky groups such as bromo, methoxy, and nitro at the para position led to the most active compounds with IC 50 values of 14.7 µM (4d), 17.1 µM (4f), and 19.6 µM (4a), respectively. These compounds showed SI in the range 9.14 to 14.56. Moreover, the molecular hybridization of 2-amino-1,3,4-thiadiazole and pyrazole moieties offered the most potent compounds (4a-f) compared to their thiosemicarbazone analogues (2a-f), demonstrating the importance of the heterocyclic ring.
All compounds of the series 2a-f, 3a-f, and 4a-f were less potent against T. cruzi compared to L. amazonensis. The most active compounds for L. amazonensis (2d, 3d, and 4d) were about 2fold less potent for T. cruzi. Again, the compounds with bulky groups at the para position had the best IC 50 values in each series.
Aiming to evaluate the influence of the thiosemicarbazone group attached at the 3-position of the pyrazole core (2a), we choose to assess the antileishmanial and antitrypanosomal activities of the 4-formyl pyrazole derivative 5a. The insertion of the formyl group at the 4-position of the pyrazole ring led to a 3-fold and 2-fold decrease in activity against L. amazonensis and T. cruzi, respectively. Compound 5a also exhibited a worse SI compared to 2a.
It is worth mentioning that comparing the values IC 50 of the series 2a-f with 3a-f and 4a-f it is observed a low variance between the results obtained. In general, the compounds of the series 3a-f were 1.4-fold most active compared to 2a-f, and the compounds 4a-f were 1.2-fold most active than 2a-f. Comparing the works of Batista (Batista et al., 2019) and Vandresen (Vandresen et al., 2017), the transformation of the thiosemicarbazone group into 2-amino-1,3,4-thiadiazole derivative led to compounds in the range 1.4 to 6-fold less actives.

CONCLUSIONS
In summary, we synthesized 18 new trifluormethylated pyrazole hybrids by a simple and efficient methodology. Screening of the compounds against Leishmania amazonensis and Trypanosoma cruzi demonstrated the importance of the substitution pattern of the trifluoromethylated N-aryl pyrazole system. The presence of a bulky group such as bromo, methoxy, or nitro at the para position of the aryl ring attached at the 5-position of pyrazole was essential for antiparasitic activity. Also, the nature of the substituent attached at the 3-position of pyrazole influenced the activity, as the thiosemicarbazone derivative led to active compounds, whereas the formyl derivative was inactive. Furthermore, the transformation of the thiosemicarbazone into S-methyl and 2-amino-1,3,4-thiadiazole derivatives increased the activity against both protozoans. However, the compounds were more active against L. amazonensis. From the results obtained in this study, the S-methyl thiosemicarbazones 3a, 3d, and 3f, and 2-amino-1,3,4-thiadiazole pyrazole hybrids 4a, 4d, and 4f could be considered as lead structures for the optimization of antileishmanial and antitrypanosomal properties.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/ Supplementary Material.

AUTHOR CONTRIBUTIONS
JC and KP contributed to the synthesis, analysis of the results, and writing of the paper. MS contributed to synthesis and formal analysis. DL-B and HV contributed to pharmacological analyses. SM contributed to the HRMS analysis. CN contributed to the supervision of pharmacological analyses. FR contributed to designing the work, synthesis supervision, project administration, funding acquisition, and in the review and editing of the writing. All authors contributed to the article and approved the submitted version.