Synthesis and Antitrypanosomastid Activity of 1 , 4-Diaryl-1 , 2 , 3-triazole Analogues of Neolignans Veraguensin , Grandisin and Machilin G

Sixteen 1,4-diaryl-1,2,3-triazole compounds derived from the natural products veraguensin, grandisin and machilin G were synthesized, with yields of 78-92%. Biological activity tests against Leishmania amazonensis promastigotes showed that three of these compounds were the most active, with maximum inhibitory concentration (IC50) values of 1.1, 3.71 and 7.23 μM. One compound was highly active against Leishmania infantum, with an IC50 value of 5.2 μM, and one derivative showed an IC50 value of 28.6 μM against Trypanosoma cruzi trypomastigotes. Regarding structureactivity relationship (SAR), hybrid 1,2,3-triazolic compounds containing a methylenedioxy group, showed the best antileishmanial and antitrypanosomal activities.


Introduction
Leishmaniases are a group of infectious diseases with diverse clinical manifestations, ranging from cutaneous to visceral forms.Visceral leishmaniasis is the most-often fatal protozoan disease, second only to malaria in the number of cases.The disease affects around two million people in endemic areas, with more than 350 million at risk. 1 The same chemotherapeutic drugs have been used for decades against leishmaniases. 1 Conventional therapeutic options include pentavalent antimony compounds such as, sodium stibogluconate (Pentostam®), N-methyl glucamine antimoniate (Glucantime®), amphotericin B, pentamidine, paromomycin, and miltefosine.All these drugs, however, have important limitations regarding toxicity, pronounced side effects or high cost. 2,3ecent studies have focused on combinations of these drugs, such as sodium stibogluconate-paromomycin 4 or miltefosine-paromomycin 5 in order to circumvent the resistance problems of different strains related to this disease.Despite the efforts of the Drugs for Neglected Diseases initiative (DNDi) in the clinical studies, only a few candidate drugs are under the development.Therefore, current and new strategies must be expanded in the search for antileishmanial compounds. 4,5hagas disease is also a neglected, potentially lethal disease caused by a protozoan parasite, Trypanosoma cruzi.This disorder is an important health problem in Latin America, where it affects about eight million people. 6reatment for Chagas disease includes two drugs, nifurtimox and benznidazole, which are active only in the acute phase of infection.Benznidazole is currently the first-choice drug in most Latin American countries.Unfortunately, narrow therapeutic windows, side effects, and variable susceptibility among T. cruzi strains result in low clinical efficacy for this nitroderivative. 7Recent studies have shown the potential antitrypanosomal activity of nitroheterocyclic fexinidazole.This candidate drug is now in phase II of a clinical trial against Chagas disease in a DNDi initiative for Latin America. 8,9n view of the serious health problems related to these two trypanosomatid diseases, it is imperative that new bioactive compounds be developed.
Natural products, mainly secondary metabolites, are an important source of bioactive compounds, due to their wide chemical diversity. 10,11However, natural bioactive compounds may possess unsuitable pharmacological characteristics, such as poor oral absorption, high lipophilicity, or cytotoxicity, which limit their use.These various properties can be modulated or improved with the development of synthetic derivatives. 10,12,13][23] Veraguensin 1 and machilin G 3 showed potential antileishmanial activity, with an IC 50 value of 18 µg mL -1 (48.8 and 50.54 µM, respectively) against L. donovani. 16he synthesis of derivatives could improve these biological effects.In fact, neolignan derivatives with greater hydrosolubility have been synthetized in order to reduce their lipophilicity, which limits in vivo studies. 21ioactive compounds obtained via click chemistry strategy have provided chemical libraries of compounds with antitrypanosomal, anticancer, and antituberculosis activities. 24This methodology could also be used to obtain derivatives of neolignans 1-3, containing the 1,2,3-triazole core, with potential biological activity against neglected diseases such as Chagas and Leishmaniases.
In this context, this study addressed the synthesis of sixteen 1,4-diaryl-1,2,3-triazole derivatives with substitution patterns based on neolignans 1-3, intending to clarify whether substitution of tetrahydrofuran by 1,2,3-triazole core (bioisosterism strategy), could providing compounds with improved antitrypanosomatid activity.Furthermore, click chemistry is a good strategy for synthesis of hybrids analogues of neolignans 1-3, what would permit to obtain preliminary information about structure-activity relationship (SAR) of the compounds 4-19 (Figure 2).
Although many studies have described the anticancer activity of triazole compounds with methoxy substitution patterns, 25 to date all possible position isomers of 1,4-diaryl-1,2,3-triazole derivatives of neolignans 1-3 have not yet synthesized and tested against Leishmania sp. and T. Cruzi.For instance, heterocyclic 1,2,3-triazole positional isomers derived from Combrestatin A4 showed different anticancer activities. 26This led us to consider the need to test the biological activity of possible positional isomers.
The  1). 33 Synthetic compounds were characterized by nuclear magnetic resonance (NMR) 1 H and 13 C, and the unknown compounds 7, 13, 17 and 19 were also analyzed by infrared (IR) and high resolution mass spectrometry.

Biological activity
The antileishmanial activity of 4-19 was evaluated against promastigote forms of L. amazonensis and L. infantum, and the antitrypanosomal activity against trypomastigotes forms of T. cruzi (Table 2).
Hybrid derivatives 8-19 showed different activities against L. amazonensis, L. infantum and T. cruzi (Table 2).Of these, the positional isomers also showed different biological activities.
Isomers 10 and 11 showed low or no activity against all parasite species (Table 2).Isomer 12 was less active than 13 against all species, with IC 50 values of 108.2 and 40.9 µM, respectively against L. amazonensis, > 200 and 94.5 µM against L. infantum, and > 200 and 28.6 µM against T. cruzi.Isomers 14 and 15 showed low activity (Table 2).
An important criterion in the search for compounds with antiprotozoal activity is their toxicity to mammalian host cells.Compounds 17, 18 and 19 showed low cytotoxicity, with high selectivity indexes (SI), 34 tens to hundreds of times higher than those of the recommended drugs for leishmaniasis, such as pentamidine and amphotericin B (SI 8.8 and 8.2, respectively) and then benznidazole (SI 13.2) for T. cruzi, indicating that these compounds are potential candidates for further in vivo studies (Table 2).
Regarding SAR, hybrid 1,2,3-triazole compounds 13 and 17 and positional isomers 18 and 19, containing the methylenedioxy group present in machilin G 3, were the most active against the trypanosomatids, indicating that this group is responsible for the high antileishmanial activity and moderate antitrypanosomal activity of these compounds.

Conclusions
In summary, this article describes the synthesis and the antitrypanosomatid activities of 16

General remarks
All solvents were distilled before use according to the standard procedure.All reactions were performed under an atmosphere of dry nitrogen and monitored by thin-layer chromatography (TLC) using prepared plates (Silica Gel 60 F254 on aluminum).The chromatograms were examined under both 254 and 360 nm UV light or with the developing agent ethanolic vanillin and heat.Flash column chromatography was performed on silica gel 60 (particle size 200-400 mesh ASTM, purchased from Aldrich) and eluted with hexane or hexane/ethyl acetate in different ratios.Melting points were determined using Fisatom 430D equipment.Infrared (IR) spectra were recorded on Nicolet iS5 spectrometer from Thermo Scientific.The 1 H and 13 C NMR spectra were recorded in CDCl 3 solutions Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole J. Braz.Chem.Soc.1222 using a Brucker 75 or 300 MHz spectrometer, as noted.Chemical shifts (d) are expressed as parts per million (ppm) downfield from tetramethylsilane as the internal standard.High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) measurements were carried out on a quadrupole time-of-flight instrument (UltrOTOF-Q, BrukerDaltonics, Billerica, MA).Compound 21a was purchased from Sigma-Aldrich.

General procedure for the preparation of aryl bromides (21b-c)
To a solution of compounds 20b and 20c (75 mmol) in dichloromethane (210 mL) containing TsOH (10 mmol), silica gel G 60 230-400 mesh (37 g) in nitrogen atmosphere at 0 °C, was added NBS (75 mmol) slowly.The reaction was stirred at room temperature by 3 hours.The work up was performed with 300 mL of saturated NaHCO 3 solution and the product was extracted with ethyl acetate (3 × 150 mL).The organic phase was dried over MgSO 4 and the solvent removed under reduced pressure.The products were purified by distillation at low pressure (3 mmHg).

General procedure for the preparation of acetylene alcohols (22a-c)
To a solution of the bromines 21a-c (3.0 mmol) in triethylamine (15 mL), PdCl 2 (PPh 3 ) 2 (0.075 mmol), CuI (0.15 mmol) in nitrogen atmosphere was added 2-methyl-3-butyn-2-ol (11.0 mmol).The mixture was stirred under refluxed for 20 hours.Then, the excess triethylamine was removed by distillation, and the reaction was extracted with ethyl acetate, dried over MgSO 4 , and the solvent removed under reduced pressure.The products were purified by column chromatography on silica gel using hexane/ethyl acetate as eluent.

General procedure for the preparation of terminal acetylenes (25a-c)
To a solution of compounds 22a, 22b and 22c (47 mmol, 1.0 equiv) in toluene (353 mL), were added KOH (141 mmol, 3.0 equiv).The reaction was stirred under reflux in nitrogen atmosphere by 18 hours.Toluene was evaporated under reduced pressure, the residue diluted with ethyl acetate (150 mL) and then, it was added a saturated solution of NH 4 Cl (100 mL).The products were extracted with ethyl acetate (3 × 100 mL) and washed with water (3 × 100 mL).After organic phase was dried over anhydrous MgSO 4 , the solvent was removed under reduced pressure and the residue purified by column chromatography on silica gel using hexane/ethyl acetate as eluent.