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

Abstract

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 structure-activity relationship (SAR), hybrid 1,2,3-triazolic compounds containing a methylenedioxy group, showed the best antileishmanial and antitrypanosomal activities.

Keywords:
1,4-diaryl-1,2,3-triazole derivative; tetrahydrofuran neolignan; click chemistry; antitrypanosomatid activity

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.^2,3

Recent studies have focused on combinations of these drugs, such as sodium stibogluconate-paromomycin⁴ or miltefosine-paromomycin⁵ 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.^4,5

Chagas 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.⁶

Treatment 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.⁷ Recent 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.^8,9

In 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.^10,11 However, 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

Among natural products, our research group has focused on the tetrahydrofuran neolignans, a class that exhibits a great number of biological activities including antibacterial, antifungal, antitumor, and anti-inflammatory.^14-23 The neolignans veraguensin 1, grandisin 2, and machilin G 3 (Figure 1) have shown antitrypanosomal and antileishmanial activities.^14-23

Figure 1
Structures of veraguensin 1, grandisin 2 and machilin G 3.

Studies on the antitrypanosomal activity of compounds 1-3 found maximum inhibitory concentration (IC₅₀) values of 2.3, 3.7 and 2.2 µM, respectively.^21-23 Veraguensin 1 and machilin G 3 showed potential antileishmanial activity, with an IC₅₀ value of 18 µg mL^-1 (48.8 and 50.54 µM, respectively) against L. donovani.¹⁶ The 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.²¹

Bioactive compounds obtained via click chemistry strategy have provided chemical libraries of compounds with antitrypanosomal, anticancer, and antituberculosis activities.²⁴ This 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).

Figure 2
Design of 1,4-diaryl-1,2,3-triazole analogues of neolignans 1-3.

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.²⁶ This led us to consider the need to test the biological activity of possible positional isomers.

Results and Discussion

Chemistry

For the synthesis of 1,4-diaryl-1,2,3-triazoles 4-19 it was necessary to synthesize two building blocks: terminal acetylenes and aromatic azides, with appropriate substitution patterns (Scheme 1).²⁷

Scheme 1
Synthesis of aryl acetylenes 25a-d. Reagents and reaction conditions: (a) NBS, TsOH, SiO₂, CH₂Cl₂, 21b = 87%, 21c = 79%; (b) 2-methyl-3-butyn-2-ol, PdCl₂(Ph₃)₂, CuI, Et₃N, reflux, 24 h, 22a = 85%, 22b = 75%, 22c = 76%; (c) KOH, toluene, reflux, 24 h, 25a = 85%, 25b = 83%, 25c = 79%, (d) CCBr₄, CH₂Cl₂, 0 ºC, 5 h, 24 = 74%; (e) tetrahydrofuran (THF), n-BuLi, −25 ºC to room temperature, 1 h, 25d = 82%.

The synthesis of starting materials began by preparing aryl bromides 21b-c via a bromination reaction of 20b-c in the presence of N-bromosuccinimide (NBS), p-TsOH, CH₂Cl₂ and SiO₂.²⁸ Subsequently, a cross-coupling Sonogashira reaction between bromobenzenes 21a-c and 2-methyl-3-butyn-2-ol in the presence of PdCl₂(PPh₃)₂/CuI, Et₃N provided the acetylene alcohols 22a-c with 75-85% yields, after 24 h reaction time.^29,30 Retro-Favorski reaction of 22a-c with KOH under reflux in toluene generated the terminal acetylenes 25a-c with 79 to 85% yield.^29,30 Ethynyl-1,2,3-trimethoxybenzene 25d was synthesized by the Corey-Fuchs method (Scheme 1).^25,31

Next, the aromatic azides 27a-d were prepared by the reaction of aromatic amines 26a-d with t-BuONO/TMSN₃ using the protocol reported by Moses and co-workers³³ (Scheme 2).

Scheme 2
Synthesis of aryl azides 27a-d. Reagents and reaction conditions: (a) t-BuONO, CH₃CN, 15 min, 0 ºC, then TMSN₃, room temperature, 12 h; 27a = 73%, 27b = 78%, 27c = 75%, 27d = 87%.

The 1,3-dipolar cycloaddition occurred when terminal acetylenes 25a-d reacted with aryl azides 27a-d using CuSO₄.H₂O, sodium ascorbate and CH₂Cl₂/H₂O 1:1 as solvents, providing the compounds 4-19 in 78 to 92% yield (Table 1).³³

Table 1
Synthesis of 1,4-diaryl-1,2,3-triazoles 4-19

Synthetic compounds were characterized by nuclear magnetic resonance (NMR) ¹H and ¹³C, 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).

Table 2
In vitro antitrypanosomatid activity of 1,4-diaryl-1,2,3-triazoles 4-19

Compound	NIH/3T3 IC50a / µM	L. amazonensis IC50b/µM	SIc	L. infantum IC50b / µM	SIc	T. cruzi IC50b / µM	SIc
4	> 888.7	123.3	> 7.21	> 200	NCd	> 200	NCd
5	> 732.4	98.5	> 7.44	62.1	> 11.8	132.0	> 5.6
6	> 622.8	> 200	NCd	> 200	NCd	> 200	NCd
7	> 808.3	92.7	> 8.7	> 200	NCd	> 200	NCd
8	> 802.9	31.7	> 25.3	> 200	NCd	> 200	NCd
9	> 802.9	150	> 5.3	> 200	NCd	53.9	> 14.9
10	> 732.4	> 200	NCd	> 200	NCd	> 200	NCd
11	> 732.4	125.3	> 5.8	> 200	NCd	> 200	NCd
12	> 846.6	108.2	> 7.8	> 200	NCd	> 200	NCd
13	> 846.6	40.9	> 20.7	94.5	> 8.9	28.6	> 29.60
14	103.1	193.6	0.53	92.6	1.1	89.1	1.2
15	> 673.1	122.4	> 5.50	> 200	NCd	> 200	NCd
16	> 768.5	96.7	> 7.9	64.6	> 11.9	> 200	NCd
17	> 768.5	1.1	> 698.6	19.5	> 39.4	109.6	> 7.0
18	> 703.5	3.7	> 189.6	15.4	> 45.7	108.1	> 6.5
19	> 703.5	7.2	> 97.7	5.2	> 135.3	56.1	> 12.5
Pentamidine	78.7	8.9	8.8	−	−	−	−
Amphotericin B	5.7	−	−	0.7	8.2	−	−
Benznidazole	> 96.1	−	−	−	−	7.3	> 13.2
Doxorubicin	2.6	−	−	−	−	−	−

• ^a
  IC₅₀: half maximal inhibitory concentration on fibroblast cells;
• ^b
  IC₅₀: half maximal inhibitory concentration on L. amazonensis, L. infantum and T. cruzi;
• ^c
  SI: selectivity index: IC₅₀ on mammal cells per IC₅₀ on trypanosomatids;
• ^d
  NC: not calculated;
• e positive controls: pentamidine for L. amazonensis, amphotericin B for L. infantum, benznidazole for T. cruzi, and doxorubicin for fibroblast cells.

Triazole compounds 4-7 showed low activity against T. cruzi (132 to > 200 µM). The triazole 5 derivative from veraguensin exhibited low activity against all trypanosomatids tested (98.5, 62.1 and 132 µM, respectively), and the triazole 6 derivative from grandisin was the least active against all of them (> 200 µM). Compound 7, containing two methylenedioxy groups, also showed low activity against all species tested.

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.

For instance, isomers 8 and 9 exhibited IC₅₀ values of 31.7 and 150 µM, respectively, against L. amazonensis, and IC₅₀ values of > 200 and 53.9 µM against T. cruzi. None of these isomers was active against L. infantum (> 200 µM).

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 IC₅₀ 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).

Positional isomer 16 was less active than 17, with IC₅₀ values of 96.7 and 1.1 µM, respectively for L. amazonensis, 64.6 and 19.5 µM for L. infantum, and > 200 and 109.6 µM for T. cruzi.

Positional isomers 18 and 19 excelled among all the synthetic compounds, showing high activity against L. amazonensis (IC₅₀ values of 3.71 and 7.23 µM) and L. infantum (15.4 and 5.2 µM). Both also exhibited moderate activity against T. cruzi trypomastigotes (IC₅₀ values of 108.1 and 56.1 µM, respectively).

Compounds 17, 18 and 19 were more active against L. amazonenis than was pentamidine (IC₅₀ 8.9 µM). Compound 17, an analogue of machilin G 3, was 8 times more active than pentamidine, while 18 and 19 were 2.5 and 1.2 times more active than pentamidine, respectively (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.

The replacement of tetrahydrofuran by the 1,2,3-triazole core increases the antileishmania activity, since an IC₅₀ of 18 µg mL^-1 (48.8 µM) of machilin G 3 against promastigotes of L. donovani was reported.¹⁶ Last, the different biological activities of 1,2,3-triazole positional isomers synthesized in this study are notable.

Conclusions

In summary, this article describes the synthesis and the antitrypanosomatid activities of 16 1,4-diaryl-1,2,3-triazole compounds 4-19. Compound 13 was the most active agaisnt T. cruzi. The 1,2,3-triazole compound 17, an analogue of machilin G, and the hybrid compounds 18 and 19, analogues of grandisin 2 and machilin G 3, showed very good activity against L. amazonensis and L. infantum.

Experimental

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 ¹H and ¹³C NMR spectra were recorded in CDCl₃ solutions using a Brucker 75 or 300 MHz spectrometer, as noted. Chemical shifts (δ) 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 NaHCO₃ solution and the product was extracted with ethyl acetate (3 × 150 mL). The organic phase was dried over MgSO₄ and the solvent removed under reduced pressure. The products were purified by distillation at low pressure (3 mmHg).

4-Bromo-1,2-dimethoxybenzene (21b)³⁵

Yield: 87%; colorless oil; ¹H NMR (300 MHz, CDCl₃) δ 3.84 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 6.73 (d, 1H, J 8.3 Hz, Ph-H), 6.98 (d, 1H, J 1.6 Hz, Ph-H), 7.03 (dd, 1H, J 8.3, 1.6 Hz, Ph-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.97, 112.56, 112.66, 114.80, 123.34, 148.36, 149.78.

5-Bromo-1,3-benzodioxole (21c)^36,37

Yield: 79%; colorless oil; ¹H NMR (300 MHz, CDCl₃) δ 5.92 (s, 2H, CH₂), 6.64 (d, 1H, J 8.2 Hz, Ph-H), 6.91 (dd, 1H, J 8.2, 1.9 Hz, Ph-H), 6.92 (d, 1H, J 1.9 Hz, Ph-H); ¹³C NMR (75 MHz, CDCl₃) δ 101.59, 109.54, 112.29, 113.06, 124.30, 147.01, 148.61.

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), PdCl₂(PPh₃)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 MgSO₄, and the solvent removed under reduced pressure. The products were purified by column chromatography on silica gel using hexane/ethyl acetate as eluent.

4-(4-Methoxyphenyl)-2-methyl-3-butyn-2-ol (22a)³⁸

Yield: 85%; yellow crystal; mp 53 ºC; ¹H NMR (300 MHz, CDCl₃) δ 1.59 (s, 6H, 2CH₃), 2.07 (s, 1H, OH), 3.78 (s, 3H, OCH₃), 6.81 (d, 2H, J 8.8 Hz, Ar-H), 7.33 (d, 2H, J 8.8 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 31.56, 55.25, 65.65, 81.99, 92.38, 113.86, 114.81, 133.07, 159.53.

4-(3,4-Dimethoxyphenyl)-2-methyl-3-butyn-2-ol (22b)³⁹

Yield: 75%; yellow crystal; mp 48-50 ºC; ¹H NMR (300 MHz, CDCl₃) δ 1.59 (s, 6H, 2CH₃), 2.11 (sl, 1H, OH), 3.84 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 6.75 (d, 1H, J 8.3 Hz, Ar-H), 6.89 (d, 1H, J 1.8 Hz, Ar-H), 6,99 (dd, 1H, J 8.2, 1.8 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 31.03, 31.55, 55.86, 55.87, 65.65, 82.13, 92.27, 110.92, 114.39, 114.89, 124.87, 148.54, 149.41.

4-(1,3-Benzodioxol-5-yl)-2-methyl-3-butyn-2-ol (22c)⁴⁰

Yield: 76%; yellow crystal; mp 44 ºC; ¹H NMR (300 MHz, CDCl₃) δ 1.57 (s, 6H, 2CH₃), 2.21 (sl, 1H, OH), 5.93 (s, 2H, CH₂), 6.70 (d, 1H, J 8.0 Hz, Ar-H), 6.83 (d, 1H, J 1.6 Hz, Ar-H), 6.90 (dd, 1H, J 8.1, 1.6 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 31.50, 61.57, 81.94, 92.14, 101.24, 108.34, 111.64, 115.97, 126.21, 147.32, 147.79.

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 NH₄Cl (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 MgSO₄, the solvent was removed under reduced pressure and the residue purified by column chromatography on silica gel using hexane/ethyl acetate as eluent.

1-Ethynyl-4-methoxybenzene(25a)⁴⁰

Yield: 85%; colorless oil; ¹H NMR (300 MHz, CDCl₃) δ 2.98 (s, 1H, CH), 3.79 (s, 3H, OCH₃), 6.83 (d, 2H, J 8.9 Hz, Ar-H), 7.41 (d, 2H, J 8.9 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.26, 75.74, 83.63, 113.90, 114.12, 133.56, 159.90.

4-Ethynyl-1,2-dimethoxybenzene(25b)⁴¹

Yield: 83%; white solid; mp 73 ºC; ¹H NMR (300 MHz, CDCl₃) δ 2.95 (s, 1H, CH), 3.82 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 6.74 (d, 1H, J 8.3 Hz, Ar-H), 6.93 (d, 1H, J 1.8 Hz, Ar-H), 7.05 (dd, 1H, J 8.2, 1.8 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.83, 75.63, 83.72, 110.84, 114.12, 114.61, 125.43, 148.50, 149.79.

5-Ethynyl-1,3-benzodioxole (25c)⁴²

Yield: 79%; yellow oil; ¹H NMR (300 MHz, CDCl₃) δ 2.95 (s, 1H, CH), 5.96 (s, 2H, CH₂), 6.73 (d, 1H, J 8.0 Hz, Ar-H), 6.91 (d, 1H, J 1.4 Hz, Ar-H), 7.00 (dd, 1H, J 7.9, 1.4 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 75.50, 83.52, 101.33, 108.40, 112.02, 115.28, 126.87, 147.36, 148.27.

Synthesis of 5-(2,2-dibromovinyl)-1,2,3-trimethoxybenzene (24)⁴³

To a solution of CBr₄ (100 mmol, 1.0 equiv) in dry CH₂Cl₂ (100 mL) under nitrogen atmosphere, at 0 °C, was added dropwise a solution of PPh₃ (200 mmol) in dry CH₂Cl₂ (100 mL). After 1 h was added 3,4,5-trimethoxybenzaldehyde 23 (50 mmol) in dry CH₂Cl₂ (50 mL). The reaction was stirred by 3 h. Then, the reaction was extracted with ethyl acetate (4 × 100 mL), organic phase was dried over anhydrous MgSO₄ and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel using hexane/ethyl acetate 9:1 as eluent to give 24 as a yellow crystal in 74% yield; mp 59 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.83 (s, 9H, 3OCH₃), 6.77 (s, 2H, Ar-H), 7.38 (s, 1H, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.16, 60.84, 88.77, 105.79, 130.54, 136.59, 138.36, 152.99.

General procedure for the preparation of 5-ethynyl-1,2,3-trimethoxybenzene (25d)⁴³

To a solution of 5-(2,2-dibromovinyl)-1,2,3-trimethoxybenzene 24 (14.08 g, 40 mmol, 1.0 equiv) in tetrahydrofuran (THF) (60 mL) at -25 °C, under nitrogen atmosphere, and vigorous stirred, was added dropwise n-BuLi (78.63 mL, 92 mmol, 2.3 equiv). After complete addition of n-BuLi, the reaction solution was kept at room temperature for one hour. Then, it was added 100 mL of saturated solution of NH₄Cl and the product was extracted with ethyl acetate (3 × 100 mL). The organic phase was dried over anhydrous MgSO₄ and the solvent was removed under reduced pressure. The residue purified by column chromatography on silica gel using hexane/ethyl acetate as eluent to give 25d as white solid; 83% yield; mp 68-69 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.01 (s, 1H, CH), 3.83 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 6.71 (s, 2H, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.99, 60.11, 79.70, 83.71, 109.11, 116.80, 138.64, 152.87.

General procedure for the preparation of azides (27a-d)³²

To a solution of anilines 26a-d (20 mmol, 1.0 equiv) in acetonitrile (76 mL) under nitrogen atmosphere, at 0 °C, was added t-BuONO (43 mmol, 2.15 equiv) and TMSN₃ (32.6 mmol, 1.63 equiv) dropwise. After 15 minutes, the reaction was kept at ambient temperature for 12 hours. Then, 40 mL of water was added and reaction solution was extracted with ethyl acetate (3 × 100 mL). The organic phase was dried over anhydrous MgSO₄ and the solvent was removed under reduced pressure. The residue purified by column chromatography on silica gel using hexane/ethyl acetate 8:2 as eluent.

1-Azide-4-methoxybenzene (27a)²⁶

Yield: 73%; yellow solid; ¹H NMR (300 MHz, CDCl₃) δ 3.78 (s, 3H, OCH₃), 6.87 (d, 2H, J 9.0 Hz, Ar-H), 6.94 (d, 2H, J 8.9 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.49, 115.05, 119.93, 132.26, 156.93.

4-Azide-1,2-dimethoxybenzene (27b)²⁵

Yield: 78%; orange solid; ¹H NMR (300 MHz, CDCl₃) δ 3.84 (s, 6H, 2OCH₃), 6.49 (d, 1H, J 2.5 Hz, Ar-H), 6.58 (dd, 1H, J 8.4, 2.4 Hz, Ar-H), 6.81 (d, 1H, J 8.6 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.92, 56.17, 103.15, 110.40, 112.01, 132.72, 146.48, 149.95.

1,3-Benzodioxol-5-yl-azide(27c)²⁵

Yield: 75%; brown oil; ¹H NMR (300 MHz, CDCl₃) δ 5.95 (s, 2H, CH₂), 6.47 (dd, 1H, J 8.2, 2.3 Hz, Ar-H); 6.51 (d, 1H, J 2.1 Hz, Ar-H); 6.75 (d, 1H, J 8.3 Hz, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 100.66; 101.59; 108.75; 111.58; 133.74; 145.01; 148.64.

5-Azide-1,2,3-trimethoxybenzene(27d)²⁶

Yield: 87%; yellow solid; ¹H NMR (300 MHz, CDCl₃) δ 3.78 (s, 3H, OCH₃), 3.82 (s, 6H, 2OCH₃), 6.21 (s, 2H, Ar-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.17, 61.00, 96.40, 135.35, 135.64, 154.07.

General procedure for the preparation of triazole derivatives (4-19)

To a solution of terminal acetylenes 25a-d (2 mmol, 1.0 equiv) and azides 27a-d (2 mmol, 1.0 equiv) in dichloromethane (2 mL) and water (2 mL), were added CuSO₄.5H₂O pentahydrate (0.128 mmol, 0.064 equiv) and sodium ascorbate (0.352 mmol, 0.176 equiv). The reaction mixture was stirred for 24 h. Then was added a saturated solution of NH₄Cl (30 mL) and the product was extracted with dichloromethane (3 × 20 mL). The organic phase was dried over anhydrous MgSO₄, and the solvent was evaporated under reduced pressure. The product was purified by recrystallization from ethyl acetate.

1,4-Bis(4-ethoxyphenyl)-1H-1,2,3-triazole (4)²⁵

Yield: 79%; yellow solid; mp 205-206 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.84 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 6.97 (d, 2H, J 8.6 Hz, Ar-H), 7.02 (d, 2H, J 9.0 Hz, Ar-H), 7.65 (d, 2H, J 9.0 Hz, Ar-H), 7.81 (d, 2H, J 8.6 Hz, Ar-H), 8.01 (s, 1H, CH, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.33, 55.63, 114.31, 114.77, 117.02, 122.14, 123.07, 127.13, 130.62, 148.09, 159.74, 159.78; HRMS (ESI+) m/z calcd. for C₁₆H₁₅N₃O₂ [M + H]⁺: 282.1242; found: 282.1242; *Tr: triazole hydrogen.

1,4-Bis(3,4-dimethoxyphenyl)-1H-1,2,3-triazole (5)⁴⁴

Yield: 92%; yellow solid; mp 156-157 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.90 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 6.91 (d, 1H, J 8.2 Hz, Ar-H), 6.94 (d, 1H, J 8.4 Hz, Ar-H), 7.19 (dd, 1H, J 8.6, 2.5 Hz, Ar-H), 7.34 (dd, 1H, J 8.3, 1.9 Hz, Ar-H), 7.37 (d, 1H, J 2.4 Hz, Ar-H); 7.51 (d, 1H, J 1.7 Hz, Ar-H), 8.05 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.94, 56.01, 56.18, 56.22, 105.02, 109.08, 111.20, 111.40, 112.42; 117.27; 118.27; 123.30, 130.70, 148.14, 149.26, 149.36, 149.39, 149.78; HRMS (ESI+) m/z calcd. for C₁₈H₁₉N₃O₄ [M + H]⁺: 342.1454; found: 342.1458; *Tr: triazole hydrogen.

1,4-Bis(3,4,5-trimethoxyfenyl)-1H-1,2,3-triazole (6)²⁵

Yield: 89%; yellow solid; mp 172-174 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.88 (s, 6H, 2OCH₃), 3.94 (s, 12H, 4OCH₃), 6.97 (s, 2H, Ar-H), 7.12 (s, 2H, Ar-H); 8.07 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.31, 56.50, 60.99, 61.09, 98.60, 103.10, 117.71, 125.76, 132.88, 138.42, 148.27, 153.73, 153.96; HRMS (ESI+) m/z calcd. for C₂₀H₂₃N₃O₆ [M + H]⁺: 402.1665; found: 402.1677; *Tr: triazole hydrogen.

1,4-Bis(1,3-benzodioxol-5-yl)-1H-1,2,3-triazole (7)

Yield: 87%; light-yellow solid; mp 214-215 ºC; IR (KBr): ν / cm^-1 3104, 2989, 2888, 2796, 1500, 1477, 1359, 1257, 1216, 1182, 1108, 1037, 935, 817, 808, 607; ¹H NMR (300 MHz, DMSO-d₆) δ 6.07 (s, 2H, CH₂), 6.16 (s, 2H, CH₂), 7.02 (d, J 8.6 Hz, 1H, Ar-H), 7.12 (d, J 8.5 Hz, 1H, Ar-H), 7.38 (dd, 1H, J 8.4, 2.2 Hz, Ar-H), 7.43 (m, 2H, Ar-H), 7.49 (d, 1H, J 2.2 Hz, Ar-H), 9.05 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, DMSO-d₆) δ 101.67, 102.27, 102.61, 106.11, 109.12, 109.26, 114.11,119.44, 119.57, 124.80, 131.55, 147.42, 147.66, 147.87, 148.28, 148.66; HRMS (ESI+) m/z calcd. for C₁₆H₁₁N₃O₄ [M + H]⁺: 310.0828; found: 310.0833; *Tr: triazole hydrogen.

1-(3,4-Dimethoxyphenyl)-4-(4-methoxyphenyl)-1H-1,2,3-triazole (8)²⁵

Yield: 80%; white solid; mp 163-165 ºC; ¹H NMR (300 MHz, DMSO-d₆) δ 3.84 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 6.96 (m, 3H, Ar-H), 7.18 (dd, 1H, J 8.4, 2.3 Hz, Ar-H), 7.38 (d, 1H, J 8.6 Hz, Ar-H), 7.12 (d, 1H, J 2.3 Hz, Ar-H), 7.81 (d, 2H, J 8.7 Hz, Ar-H), 8.03 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, DMSO-d₆) δ 55.35, 56.20, 56.25; 87.58, 104.96, 111.13, 112.36, 114.30, 117.09, 122.98, 127.15, 130.73, 148.13, 149.32; 149.32, 149.75, 159.76; HRMS (ESI+) m/z calcd. for C₁₇H₁₇N₃O₃ [M + H]⁺: 312.1348, found: 312.1350; *Tr: triazole hydrogen.

4-(3,4-Dimethoxyphenyl)-1-(4-methoxyphenyl)-1H-1,2,3-triazole (9)^25,45

Yield: 85%; white solid; mp 141-143 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.86 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.97(s, 3H, OCH₃), 6.92 (d, 1H, J 8.3 Hz, Ar-H), 7.02 (m, 2H, Ar-H), 7.34 (dd, 1H, J 8.3, 1.5 Hz, Ar-H), 7.53 (d, 1H, J 1.5 Hz, Ar-H), 7.66 (m, 2H, Ar-H), 8.03 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.64, 55.98, 56.04, 109.09, 111.42, 114.79, 117.23, 118.26, 122.18, 123.40; 130.60; 149.25; 149.39; 159.83; HRMS (ESI+) m/z calcd. for C₁₇H₁₇N₃O₃ [M + H]⁺: 312.1348; found: 312.1347; *Tr: triazole hydrogen.

4-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole(10)²⁵

Yield: 83%; yellow solid; mp 180-181 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.83 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 3.92 (s, 6H, 2OCH₃), 6.97 (m, 4H, Ar-H), 7.81 (d, 2H, J 8.8 Hz, Ar-H), 8.04 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.35, 56.45, 61.06, 98.48, 114.32, 117.08, 122.87, 127.16, 133.00, 138.28, 153.92, 159.83; HRMS (ESI+) m/z calcd. for C₁₈H₁₉N₃O₄ [M + H]⁺: 342.1454; found: 342.1443; *Tr: triazole hydrogen.

1-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole(11)²⁵

Yield: 86%; white solid; mp 166-167 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.87 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 3.94 (s, 6H, 2OCH₃), 7.02 (d, 2H, J 9.0 Hz, Ar-H), 7.12 (s, 2H, Ar-H), 7.67 (d, 2H, J 8.9 Hz), 8.09 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.64, 56.27, 60.96, 103.02, 114.79, 117.68, 122.19, 125.99, 130.50, 138.29, 153.70, 159.89; HRMS (ESI+) m/z calcd. for C₁₈H₁₉N₃O₄ [M + H]⁺: 342.1454; found: 342.1451; *Tr: triazole hydrogen.

1-(1,3-Benzodioxol-5-yl)-4-(4-methoxyphenyl)-1H-1,2,3-triazole(12)²⁵

Yield: 85%; white solid; mp 186-187 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.84 (s, 3H, OCH₃), 6.06 (s, 2H, CH₂), 6.89 (d, 1H, J 8.3 Hz, Ar-H), 6.97 (d, 1H, J 8.6 Hz, Ar-H), 7.16 (dd, 1H, J 8.3, 2.0 Hz, Ar-H), 7.28 (d, 1H, J 2.0 Hz, Ar-H), 7.80 (d, 2H, J 8.6 Hz, Ar-H), 7.98 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.33, 102.09, 102.74, 108.49, 113.15, 114.12, 114.30, 117.11, 122.90, 127.12, 131.63, 147.94, 148.61, 159.79; HRMS (ESI+) m/z calcd. for C₁₆H₁₃N₃O₃ [M + H]⁺: 296.1035; found: 296.1028; *Tr: triazole hydrogen.

4-(1,3-Benzodioxol-5-yl)-1-(4-methoxyphenyl)-1H-1,2,3-triazole (13)

Yield: 81%; white solid; mp 175-176 ºC; IR (KBr) ν / cm^-1 3106, 3012-2794, 1608, 1519, 1481, 1255, 1230, 1214, 1108, 1037, 935, 838, 811; ¹H NMR (300 MHz, CDCl₃) δ 3.86 (s, 3H, OCH₃), 6.00 (s, 2H, CH₂), 6.87 (d, 1H, J 7.6 Hz, Ar-H), 7.02 (d, 2H, J 8.5 Hz, Ar-H), 7.37 (m, 2H, Ar-H), 7.65 (d, 2H, J 8.5 Hz, Ar-H), 7.99 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.64, 101.22, 106.48, 108.72, 114.80, 117.23, 119.56, 122.15, 124.57, 130.57, 147.74, 148.18, 159.84; HRMS (ESI+) m/z calcd. for C₁₆H₁₃N₃O₃ [M + H]⁺: 296.1035; found: 296.1036; *Tr: triazole hydrogen.

4-(3,4-Dimethoxiphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (14)²⁵

Yield: 82%; white solid; mp 141-142 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.88 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.93 (s, 6H, 2 OCH₃), 3.97 (s, 3H, OCH₃), 6.92 (d, 1H, J 8.3 Hz, Ar-H), 6.97 (s, 2H, Ar-H), 7.35 (dd, 1H, J 8.3, 1.6 Hz, Ar-H), 7.52 (d, 1H, J 1.6 Hz, Ar-H), 8.06 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.98, 56.05, 56.48, 61.06, 98.60, 109.11, 111.43, 117.24, 118.33, 123.20, 132.99, 138.40, 149.37, 149.42, 153.95; HRMS (ESI+) m/z calcd. for C₁₉H₂₁N₃O₅ [M + H]⁺:372.1559; found: 372.1561; *Tr: triazole hydrogen.

1-(3,4-Dimetyhoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (15)²⁵

Yield: 88%; yellow solid; mp 136-137 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.88 (s, 3H, OCH₃), 3.94 (s, 9H, 3OCH₃), 3.97 (s, 3H, OCH₃), 6.95 (d, 1H, J 8.6 Hz, Ar-H), 7.18 (m, 3H, Ar-H), 7.38 (d, 1H, J 1.9 Hz, Ar-H), 8.11 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.21, 56.27, 56.28, 60.96, 103.03, 105.06, 111.23, 112.55, 126.02, 130.73, 138.34, 149.49, 149.84, 153.73; HRMS (ESI+) m/z calcd. C₁₉H₂₁N₃O₅ [M + H]⁺: 372.1559; found: 372,1571; *Tr: triazole hydrogen.

1-(1,3-Benzodioxol-5-yl)- 4-(3,4-dimethoxyphenyl)-1H-1,2,3-triazole (16)²⁵

Yield: 81%; brown solid; mp 152-153 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.90 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 6.06 (s, 2H, CH₂), 6.89 (d, 1H, J 8.3 Hz, Ar-H), 6.91 (d, 1H, J 8.3 Hz, Ar-H), 7.16 (dd, 1H, J 8.3, 2.2 Hz, Ar-H), 7.27 (d, 1H, J 2.1 Hz, Ar-H), 7.32 (dd, 1H, J 8.3, 1.8 Hz, Ar-H), 7.51 (d, 1H, J 1.8 Hz, Ar-H), 8.00 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 55.95, 56.01, 102.10, 102.70, 108.48; 109.02, 111.37, 114.14, 117.31, 118.25; 123.22, 131.60, 147.99, 148.13, 148.33, 149.27, 149.36; HRMS (ESI+) m/z calcd. C₁₇H₁₅N₃O₄ [M + H]⁺: 326.1141; found: 326.1136; *Tr: triazole hydrogen.

4-(1,3-Benzodioxol-5-yl)-1-(3,4-dimethoxyphenyl)-1H-1,2,3-triazole (17)

Yield: 90%; light-brown solid; mp 151-152 ºC; IR (KBr) ν / cm^-1 3133-3099, 3014-2834, 1602, 1515, 1481, 1440, 1378, 1346, 1267, 1234, 1214, 1137, 1035, 931, 885, 813, 769; ¹H NMR (300 MHz, CDCl₃) δ 3.93 (s, 3H, OCH₃), 3.96 (s, 3H, OCH₃), 6.00 (s, 2H, CH₂), 6.87 (d, 1H, J 7.9 Hz, Ar-H), 6.95 (d, 1H, J 8.6 Hz, Ar-H), 7.18 (dd, 1H, J 8.6, 2.4 Hz, Ar-H), 7.37 (m, 3H, Ar-H), 8.00 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.20, 56.24, 101.23, 104.97, 106.46, 108.72, 111.16, 112.36, 117.26, 119.56, 124.47, 130.67, 147.75, 148.17, 149.38, 149.78; HRMS (ESI+) m/z calcd. C₁₇H₁₅N₃O₄ [M + H]⁺: 326.1141; found: 326.1134; *Tr: triazole hydrogen.

1-(1,3-Benzodioxol-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (18)²⁵

Yield: 78%; a yellow solid; mp 126-127 ºC; ¹H NMR (300 MHz, CDCl₃) δ 3.87 (s, 3H, OCH₃), 3.93 (s, 6H, OCH₃), 6.07 (s, 2H, CH₂), 6.90 (d, 1H, J 8.3 Hz, Ar-H), 7.11 (sl, 2H, Ar-H), 7.18 (dd, 1H, J 8.4, 2.1 Hz, Ar-H), 7.28 (d, 1H, J 2.0 Hz, Ar-H), 8.05 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.23, 60.94, 102.12, 102.69, 102.97, 108.47, 114.14, 117.77, 125.82, 131.49, 138.28, 148.04, 148.63, 153.67; HRMS (ESI+) m/z calcd. for C₁₈H₁₇N₃O₅ [M + H]⁺: 356.1246; found: 356.1253; *Tr: triazole hydrogen.

4-(1,3-Benzodioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (19)

Yield: 80%; light-brown solid; mp 182-183 ºC; IR (KBr) ν / cm^-1 2996-2796, 1604, 1511, 1482, 1452, 1419, 1309, 1238, 1128, 1037, 997, 827, 779; ¹H NMR (300 MHz, CDCl₃) δ 3.87 (s, 3H, OCH₃), 3.92 (s, 6H, 2OCH₃), 5.99 (s, 2H, CH₂), 6.86 (d, 1H, J 8.0 Hz, Ar-H), 6.96 (s, 2H, Ar-H), 7.36 (m, 2H, Ar-H), 8.02 (s, 1H, *Tr-H); ¹³C NMR (75 MHz, CDCl₃) δ 56.43, 61.03, 98.45, 101.24, 106.43, 108.70, 117.27, 119.57, 124.32, 132.89, 138.32, 147.81, 148.18, 153.91; HRMS (ESI+) m/z calcd. for C₁₈H₁₇N₃O₅ [M + H]⁺: 356.1246; found: 356.1244; *Tr: triazole hydrogen.

Antileishmanial assays

Leishmania amazonensis

Synthetic compounds were tested in triplicates in 96-well microplates, with final concentrations from 0.78 to 50 µg mL^-1. L. amazonensis promastigotes (IFLA/BR/1967/PH8 strain, 1 × 10⁶ parasites mL^-1) in exponential phase cultivated in Schneider's Insect Medium were added to the plates and then incubated at 26 °C for 72 h. 20 µL (5 mg mL^-1) of thiazolyl blue tetrazolium bromide-MTT (Sigma-Aldrich®) were added in each well and incubated at 37 ºC, 5% CO₂ for 2 h as described by Marques et al.⁴⁶ Pentamidine (Sigma-Aldrich) was used as positive control. Dimethyl sulfoxide (DMSO, Vetec) in Schneider's Insect Medium was used as negative control at the concentration used to solubilize the highest concentration of the test samples and did not interfered on cell viability (1%). A non-linear dose-response regression curve was used to calculate the half maximum inhibitory concentration (IC₅₀).

Leishmania infantum

The compounds were applied in triplicate under promastigotes forms of Leishmania infantum (0.5, 2.0, 8.0 and 32.0 µM) previously adjusted to 1 × 10⁶ cells mL^-1 in a 96 well plate. The plate was incubated in a humidified atmosphere at 22 °C for 72 hours. After this period the viability of the promastigotes was performed by XTT tetrazol salt, which was added in 50 mL of a XTT solution containing PMS (phenazine methosulfate), in the proportion of 1 mg mL^-1 of XTT to 0.001 mg mL^-1 of PMS. After, the plate was incubated in humid atmosphere at 37 °C and 5% CO₂ for 4 hours, protected from light. At the end, the reaction results were obtained in spectrophotometer (Biotek) at 450 nm. As positive control only culture medium was used, and parasites with 1.5% DMSO as used as negative control.

The percentage of activity was calculated by the following formula:

(1)

where: Y = optical density reading of cells and wells with different concentrations of the compounds; N = optical density reading of parasites in wells with 1.5% DMSO; P = optical density reading of the wells with only culture medium.

Trypanocidal assays

The assays against trypomastigote forms of T. cruzi were carried out in 96 well microplates. The trypomastigotes were obtained from supernatant of cellular culture. The concentration of parasites was adjusted to 10⁶ forms mL^-1 in RPMI medium, supplemented by antibiotics and fetal bovine serum. The compounds were added at concentrations of 0.5, 2.0, 8.0 and 32.0 µM. All the compounds were evaluated in triplicate. The materials were incubated for 24 hours at 4 °C. After this period, to each well was added 10 µL solution of FluoReporter lacZ/Galactosidase quantitative Kit (Life Technologies), and the plates were incubated again for 30 minutes. The colorimetric reaction was quantified by fluorescence microplate reader (BIOTEK) at 386 nm excitation and 448 nm emissions.

For both kinds of assays the percentages of parasite lysis were determined from the following formula:

(2)

where: X = optical density value of the samples; CP = optical density value of the positive controls; CN = optical density value of the negative controls.

Culture medium was used as positive control (CP) and medium with DMSO 0.6% as negative control.

Cytotoxicity assay

Fibroblasts (NIH/3T3) obtained from Rio de Janeiro Cell Bank (Brazil), were seeded in 96 well plates (1 × 10⁴ cells mL^-1) and incubated with synthetic compounds at 37 °C, 5% CO₂ for 48 h at the concentrations of 0.25 to 250 µg mL^-1 to calculate the IC₅₀. Amphotericin B (Sigma-Aldrich) was used as the reference drug at the concentrations of 0.025 to 25 µg mL^-1. Cell growth was estimated by the sulforhodamine B colorimetric method (SRB).⁴⁷ DMSO (Vetec) was used as negative control at the concentration used to solubilize the highest concentration of the test samples and did not interfered on cell viability. The percentage of growth was calculated as described by Monks et al.⁴⁸ IC₅₀ was determined by non-linear regression analysis (Microcal Origin Version 6.0 e Microsoft Office Excel 2007). Selectivity index (SI) was calculated according to de Medeiros et al.⁴⁹

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

https://minio.scielo.br/documentstore/1678-4790/zNJLR9qRbH7jkhxqZnTwM5S/7dd59073a6021f45830f96dd67841edd43dbb83e.pdf

Acknowledgments

This study was supported by grants from FUNDECT-MS (process No. 23/200.137/2010), PROPP-UFMS, CNPq (process No. 563977/2010-8) and CAPES. Special thanks to the Laboratory of Natural Products and Mass Spectrometry (LaPNEM) of the Federal University of Mato Grosso do Sul for the HPLC-DAD-MS/MS analysis.

References

• ¹
  World Health Organization (WHO); Control of the Leishmaniases; Geneva; WHO Tech. Rep. Ser. 2010, 949, 1.
• ²
  Croft, S. L.; Coombs, G. H.; Trends Parasitol. 2003, 19, 502; Oliveira, L. F. O.; Schubach, A. O.; Martins, M. M.; Passos, S. L.; Oliveira, R. O.; Marzochi, M. C.; Andrade, C. A.; Acta Trop. 2011, 118, 87; Sundar, S.; Chaterjee, M.; Indian J. Med. Res. 2006, 123, 345; Rizk, Y. S.; Fischer, A.; Cunha, M. C.; Rodrigues, P. O.; Marques, M. C. S.; Matos, M. F. C.; Kadri, M. C. T.; Carollo, C. A.; Arruda, C. C. P.; Mem. Inst. Oswaldo Cruz 2014, 109, 1050.
• ³
  Musa, A. M.; Younis, B.; Fadlalla, A.; Royce, C.; Balasegaram, M.; Wasunna, M.; Hailu, A.; Edwards, T.; Omollo, R.; Mudawi, M.; Kokwaro, G.; El-Hassan, A.; Khalil, E.; PLoS Negl. Trop. Dis. 2010, 4, e855; Ben Salah, A.; Ben Messaoud, N.; Guedri, E.; Zaatour, A.; Bem, A. N.; Bettaieb, J.; Gharbi, A.; Belhadj, H. N.; Boukthir, A.; Chlif, S.; Abdelhamid, K.; El Ahmadi, Z.; Louzir, H.; Mokni, M.; Morizot, G.; Buffet, P.; Smith, P. L.; Kopydlowski, K. M.; Kreishman-Deitrick, M.; Smith, K. S.; Nielsen, C. J.; Ullman, D. R.; Norwood, J. A.; Thorne, G. D.; McCarthy, W. F.; Adams, R. C.; Rice, R. M.; Tang, D.; Berman, J.; Ransom, J.; Magill, A. J.; Grogl, M.; N. Engl. J. Med. 2013, 7, 524; Silva-Jardim, I.; Thiemann, O. H.; Anibal, F. F.; J. Braz. Chem. Soc. 2014, 25, 1810.
• ⁴
  Musa, A.; Khalil, E.; Hailu, A.; Olobo, J.; Balasegaram, M.; Omollo, R.; Edwards, T.; Rashid, J.; Mbui, J.; Musa, B.; Abuzaid, A. A.; Ahmed, O.; Fadlalla, A.; El-Hassan, A.; Mueller, M.; Mucee, G.; Njoroge, S.; Manduku, V.; Mutuma, G.; Apadet, L.; Lodenyo, H.; Mutea, D.; Kirigi, G.; Yifru, S.; Mengistu, G.; Hurissa, Z.; Hailu, W.; Weldegebreal, T.; Tafes, H.; Mekonnen, Y.; Makonnen, E.; Ndegwa, S.; Sagaki, P.; Kimutai, R.; Kesusu, J.; Owiti, R.; Ellis, S.; Wasunna, M.; PLoS Negl. Trop. Dis. 2012, 6, e1674.
• ⁵
  http://www.dndi.org/images/stories/pdf_portfolios/DNDi_Portfolio_2013_POR.pdf, Drugs for Neglected Diseases initiative, accessed in December 2015.
  » http://www.dndi.org/images/stories/pdf_portfolios/DNDi_Portfolio_2013_POR.pdf
• ⁶
  World Health Organization (WHO); Research Priorities for Chagas Disease, Human African Trypanosomiasis and Leishmaniasis; Geneva; WHO Tech. Rep. Ser. 2012, 975, 1.
• ⁷
  Ferreira, E. I.; Rev. Virtual Quim. 2012, 4, 225; Zingales, B.; Miles, M. A.; Moraes, C. B.; Luquetti, A.; Guhl, F.; Schijman, A. G.; Ribeiro, I.; Mem. Inst. Oswaldo Cruz 2014, 109, 828.
• ⁸
  http://www.dndi.org/media-centre/press-releases/354-media-centre/press-releases/langues-press-releases/1913-pr-fexichagas-po.html accessed in December 2015.
  » http://www.dndi.org/media-centre/press-releases/354-media-centre/press-releases/langues-press-releases/1913-pr-fexichagas-po.html
• ⁹
  Bahia, M. T.; de Andrade, I. M.; Martins, T. A.; do Nascimento, Á. F.; Diniz, L. F.; Caldas, I. S.; Talvani, A.; Trunz, B. B.; Torreele, E.; Ribeiro, I.; PLoS Negl. Trop. Dis. 2012, 6, e1870.
• ¹⁰
  Barreiro, E. J.; Fraga, C. A. M.; Química Medicinal: As Bases Moleculares da Ação de Fármacos, 3ª ed.; Artmed: Porto Alegre, Brasil, 2015.
• ¹¹
  Calderon, L. D.; Silva-Jardim, I.; Zuliani, J. P.; Silva, A. D. E.; Ciancaglini, P.; da Silva, L. H. P.; Stabeli, R. G.; J. Braz. Chem. Soc. 2009, 20, 1011.
• ¹²
  Wermuth, C. G.; The Practice of Medicinal Chemistry, 3^rd ed.; Academic Press: San Diego, 2008.
• ¹³
  Jing, Y. R.; Zhou, W.; Li, W. L.; Zhao, L. X.; Wang, Y. F.; Bioorg. Med. Chem. 2014, 22, 194.
• ¹⁴
  Verza, M.; Arakawa, N. S.; Lopes, N. P.; Kato, M. J.; Pupo, M. T.; Said, S.; Carvalho, I.; J. Braz. Chem. Soc. 2009, 20, 195.
• ¹⁵
  Lopes, N. P.; Chicaro, P.; Albuquerque, S.; Yoshida, M.; Kato, M. J.; Planta Med. 1998, 64, 667.
• ¹⁶
  Silva Filho, A. A.; Costa, E. S.; Cunha, W. R.; Silva, M. L.; Nanayakkara, D.; Bastos, J. K.; Phytother. Res. 2008, 22, 1307.
• ¹⁷
  Oliveira, R. B.; Vaz, A. B. M.; Alves, R. O.; Liarte, D. B.; Donicci, C. L.; Romanha, A. J.; Zani, C. L.; Mem. Inst. Oswaldo Cruz 2006, 101, 169.
• ¹⁸
  de Oliveira, R. B.; Zani, C. L.; Ferreira, R. S.; Leite, R. S.; Alves, T. M. A.; da Silva, T. H. A.; Romanha, A. J.; Quim. Nova 2008, 31, 261.
• ¹⁹
  Carvalho, A. V.; Galdino, P. M.; Nascimento, M. V.; Kato, M. J.; Valadares, M. C.; Cunha, L. C.; Costa, E. A.; Phytother. Res. 2010, 24, 113.
• ²⁰
  Jean-Moreno, V.; Rojas, R.; Goyeneche, D.; Coombs, G. H.; Walker, J.; Exp. Parasitol. 2006, 112, 21.
• ²¹
  Bernardes, L. S. C.; Kato, M. J.; Albuquerque, S.; Carvalho, I.; Bioorg. Med. Chem. 2006, 14, 7075.
• ²²
  Filho, A. A. S.; Albuquerque, S.; Silva, M. L. A.; Eberlin, M. N.; Tomazela, D. M.; Bastos, J. F.; J. Nat. Prod. 2004, 67, 42.
• ²³
  Schmidt, T. J.; Khalid, S. A.; Romanha, A. J.; Alves, T. M. A.; Biavatti, M. W.; Brun, R.; da Costa, F. B.; de Castro, S. L.; Ferreira, V. F.; de Lacerda, M. V. G.; Lago, J. H. G.; Leon, L. L.; Lopes, N. P.; Amorim, R. C. N.; Niehues, M.; Ogungbe, I. V.; Pohlit, A. M.; Scotti, M. T.; Setzer, W. N.; de Soeiro, M. N. C.; Steindel, M.; Tempone, A. G.; Curr. Med. Chem. 2012, 19, 2176.
• ²⁴
  da Cruz, E. H. G.; Hussene, C. M. B.; Dias, G. G.; Diogo, E. B. T.; de Melo, I. M. M.; Rodrigues, B. L.; da Silva, M. G.; Valença, W. O.; Camara, C. A.; de Oliveira, R. N.; de Paiva, Y. G.; Goulart, M. O. F.; Cavalcanti, B. C.; Pessoa, C.; da Silva Jr., E. N.; Bioorg. Med. Chem. 2014, 22, 1608; Guimarães, T. T.; Pinto, M. D. C. F. R.; Lanza, J. S.; Melo, M. N.; do Monte-Neto, R. L.; de Melo, I. M. M.; Diogo, E. B. T.; Ferreira, V. F.; Camara, C. A.; Valença, W. O.; de Oliveira, R. N.; Frézard, F.; da Silva Jr., E. N.; Eur. J. Med. Chem. 2013, 63, 523; da Silva Jr., E. N.; de Moura, M. A. B. F.; Pinto, A. V.; Pinto, M. C. F. R.; Souza, M. C. B. V.; Araújo, A. J.; Pessoa, C.; Costa-Lotufo, L. V.; Montenegro, R. C.; de Moraes, M. O.; Ferreira, V. F.; Goulart, M. O. F.; J. Braz. Chem. Soc. 2009, 20, 635; da Silva, G. B.; Guimarães, B. M.; Assis, S. P. O.; Lima, V. L. M.; de Oliveira, R. N.; J. Braz. Chem. Soc. 2013, 24, 914; Jardim, G. A. M.; Cruz, E. H. G.; Valença, W. O.; Resende, J. M.; Rodrigues, B. L.; Ramos, D. F.; Oliveira, R. N.; Silva, P. E. A.; da Silva Jr., E. N.; J. Braz. Chem. Soc. 2015, 26, 1013; Gonzaga, D. T. G.; da Rocha, D. R.; da Silva, F. C.; Ferreira, V. F.; Curr. Top. Med. Chem. 2013, 13, 2850.
• ²⁵
  Pagliai, F.; Pirali, T.; del Grosso, E.; di Brisco, R.; Tron, G. C.; Sorba, G.; Genazzani, A. A.; J. Med. Chem. 2006, 26, 467.
• ²⁶
  Odlo, K.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.; Hansen, T. V.; Bioorg. Med. Chem. 2010, 18, 6874.
• ²⁷
  Kolb, H. C.; Finn, M. G.; Sharpless, K. B.; Angew. Chem. Int. Ed. 2001, 40, 2004.
• ²⁸
  Konishi, H.; Aritomi, K.; Okano, T.; Kiji, J.; Bull. Chem. Soc. Jpn. 1989, 62, 521.
• ²⁹
  Vuligonda, V.; Thacher, S. M.; Chandraratna, R. A. S.; J. Med. Chem. 2001, 44, 2298.
• ³⁰
  Dabdoub, M. J.; Dabdoub, V. B.; Guerrero Jr., P. G.; Hurtado, G. R.; Tetrahedron Lett. 2012, 53, 5302.
• ³¹
  Gibtner, T.; Hampel, F.; Gisselbrecht, J.-P.; Hirsch, A.; Chem. Eur. J. 2002, 68, 408; Corey, E. J.; Fuchs, P. L.; Tetrahedron Lett. 1972, 13, 3769.
• ³²
  Barral, K.; Moorhouse, A. D.; Moses, J. E.; Org. Lett. 2007, 9, 1809.
• ³³
  Lee, B. Y.; Park, S. R.; Jeon, H. B.; Kim, K. S.; Tetrahedron Lett. 2006, 47, 5105.
• ³⁴
  Badisa, R. B.; Darling-Reed, S. F.; Joseph, P.; Cooperwood, J. S.; Latinwo, L. M.; Goodman, C. B.; Anticancer Res. 2009, 29, 2993.
• ³⁵
  Pan, J.; Wang, X.; Zhang, Y.; Buchwald, S. L.; Org. Lett. 2011, 13, 4974.
• ³⁶
  Porter, M. B.; Tetrahedron 1999, 55, 13927.
• ³⁷
  Liu, A. H.; He, L. N.; Hua, F.; Yang, Z. Z.; Huang, C. B.; Yu, B.; Li, B.; Adv. Synth. Catal. 2011, 353, 3187.
• ³⁸
  Cheng, J.; Sun, Y.; Wang, F.; Guo, M.; Xu, J. H.; Pan, Y.; Zhang, Z.; J. Org. Chem. 2004, 69, 5428.
• ³⁹
  Klyatskaya, S. V.; Tretyakov, E. V.; Vasilevsky, S. F.; Russ. Chem. Bull. 2001, 50, 868.
• ⁴⁰
  Xiong, Y. P.; Wu, M. Y.; Zhang, X. Y.; Ma, C. L.; Huang, L.; Zhao, L. J.; Tan, B.; Liu, X. Y.; Org. Lett. 2014, 16, 1000.
• ⁴¹
  Chang, H. K.; Liao, Y. C.; Liu, R. S.; J. Org. Chem. 2007, 72, 8139.
• ⁴²
  Fang, Z.; Song, Y.; Sarkar, T.; Hamel, E.; Fogler, W. E.; Agoston, G. E.; Fanwick, P. E.; Cushman, M.; J. Org. Chem. 2008, 73, 4241.
• ⁴³
  Burroughs, L.; Ritchie, J.; Ngwenya, M.; Khan, D.; Lewis, W.; Woodward, S.; Beilstein J. Org. Chem. 2015, 11, 273.
• ⁴⁴
  Massarotti, A.; Aprile, S.; Mercalli, V.; Delgrosso, E.; Grosa, G.; Sorba, G.; Tron, G. C.; ChemMedChem 2014, 9, 2497.
• ⁴⁵
  Chen, Y.; Zhuo, Z. J.; Cui, D. M.; Zhang, C.; J. Organomet. Chem. 2014, 749, 215.
• ⁴⁶
  Marques, M. C. S.; Hamerski, L.; Garcez, F. R.; Tieppo, C.; Vasconcelos, M.; Torres-Santos, E. C.; Chang, M.; Garcez, W. S.; J. Med. Plants Res. 2013, 7, 957.
• ⁴⁷
  Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; Mcmahon, J.; Vistica, D.; J Natl. Cancer Inst. 1990, 82, 1107.
• ⁴⁸
  Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Pau, K.; Vistica, D.; J. Natl. Cancer Ins. 1991, 83, 757.
• ⁴⁹
  de Medeiros, M. G. F.; da Silva, A. C.; Citó, A. M. G. L.; Borges, A. R.; de Lima, S. G.; Lopes, J. A. D.; Cham. Parasitol Int. 2011, 60, 237.

Publication Dates

• Publication in this collection
  July 2016

History

• Received
  20 Nov 2015