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

Article abstract of DOI:10.5935/0103-5053.20160017

Sixteen 1,4-diaryl-1,2,3-triazole compounds derived from the natural products veraguensin, grandisin and machilin G were synthesized, with yields of 78-92percent. 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.

Full text of DOI:10.5935/0103-5053.20160017

http://dx.doi.org/10.5935/0103-5053.20160017
J. Braz. Chem. Soc., Vol. 27, No. 7, 1217-1228, 2016.
Printed in Brazil - ©2016 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole Analogues
of Neolignans Veraguensin, Grandisin and Machilin G
Tatiana B. Cassamale,a Eduarda C. Costa,a Diego B. Carvalho,a Nadla S. Cassemiro,a
Carolina C. Tomazela,b Maria C. S. Marques,b Mariáh Ojeda,c Maria F. C. Matos,c
Sérgio Albuquerque,d Carla C. P. Arrudab and Adriano C. M. Baroni*,a
aLaboratório de Síntese e Química Medicinal, LASQUIM, bLaboratório de Parasitologia Humana
and cLaboratório de Biologia Molecular e Culturas Celulares, Centro de Ciências Biológicas e da
Saúde, Universidade Federal do Mato Grosso do Sul, 79070-900 Campo Grande-MS, Brazil
dDepartamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências
Farmacêuticas de Ribeirão Preto, USP, 14040-930 Ribeirão Preto-SP, Brazil
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
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
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
In view of the serious health problems related to these
two trypanosomatid diseases, it is imperative that new
bioactive compounds be developed.
*e-mail: adriano.baroni@ufms.br

1218
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole
J. Braz. Chem. Soc.
Natural products, mainly secondary metabolites, are
core, with potential biological activity against neglected
diseases such as Chagas and Leishmaniases.
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
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
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 CombrestatinA4 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
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
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
Figure 1. Structures of veraguensin 1, grandisin 2 and machilin G 3.
Figure 2. Design of 1,4-diaryl-1,2,3-triazole analogues of neolignans 1-3.

Vol. 27, No. 7, 2016
Cassamale et al.
1219
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).
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
trypomastigotes forms of T. cruzi (Table 2).
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.
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
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
The antileishmanial activity of 4-19 was evaluated
against promastigote forms of L. amazonensis and
L. infantum, and the antitrypanosomal activity against
Scheme 1. Synthesisof 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%.
R1
R2
N3
R1
R2
NH2
a: R1 = H, R2 = OCH3, R 3= H
a
b: R1 = OCH3, R2 = OCH3, R 3= H
c: R1 and R2 = -OCH2O-, R3 = H
d: R1 = OCH3, R2 = OCH3, R3 = OCH3
R3
R3
27a-d
26a-d
Scheme 2. Synthesisof 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%.

1220
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole
J. Braz. Chem. Soc.
Table 1. Synthesis of 1,4-diaryl-1,2,3-triazoles 4-19
Compounda
Yield / %
79
Compounda
Yield / %
85
92
89
81
82
87
88
80
85
81
90
83
78
86
80
aReaction conditions: 25a-d (1.0 mmol), 27a-d (1.0 mmol), CuSO4.5H2O (0.15 mmol), sodium ascorbate (0.25 mmol), CH2Cl2 (3 mL), H2O (3 mL), room
temperature, 24 h.
moderate activity against T. cruzi trypomastigotes (IC50
values of 108.1 and 56.1 µM, respectively).
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 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).

Vol. 27, No. 7, 2016
Cassamale et al.
1221
Table 2. In vitro antitrypanosomatid activity of 1,4-diaryl-1,2,3-triazoles 4-19
NIH/3T3
IC50a / μM
L. amazonensis
IC50b /μM
L. infantum
IC50b / μM
T. cruzi
IC50b / μM
Compound
SIc
SIc
SIc
4
> 888.7
> 732.4
> 622.8
> 808.3
> 802.9
> 802.9
> 732.4
> 732.4
> 846.6
> 846.6
103.1
123.3
98.5
> 200
92.7
31.7
150
> 200
125.3
108.2
40.9
193.6
122.4
96.7
1.1
> 7.21
> 7.44
NCd
> 200
62.1
> 200
> 200
> 200
> 200
> 200
> 200
> 200
94.5
92.6
> 200
64.6
19.5
15.4
5.2
NCd
> 11.8
NCd
NCd
NCd
NCd
NCd
NCd
NCd
> 8.9
1.1
> 200
132.0
> 200
> 200
> 200
53.9
> 200
> 200
> 200
28.6
89.1
> 200
> 200
109.6
108.1
56.1
−
NCd
> 5.6
NCd
NCd
NCd
> 14.9
NCd
NCd
NCd
> 29.60
1.2
5
6
7
> 8.7
> 25.3
> 5.3
NCd
8
9
10
11
> 5.8
> 7.8
> 20.7
0.53
12
13
14
15
> 673.1
> 768.5
> 768.5
> 703.5
> 703.5
78.7
> 5.50
> 7.9
> 698.6
> 189.6
> 97.7
8.8
NCd
> 11.9
> 39.4
> 45.7
> 135.3
−
NCd
NCd
> 7.0
> 6.5
> 12.5
−
16
17
18
3.7
19
7.2
Pentamidine
Amphotericin B
Benznidazole
Doxorubicin
8.9
−
5.7
−
−
0.7
8.2
−
−
> 96.1
2.6
−
−
−
−
7.3
> 13.2
−
−
−
−
−
−
aIC50: half maximal inhibitory concentration on fibroblast cells; bIC50: half maximal inhibitory concentration on L. amazonensis, L. infantum and T. cruzi; cSI:
selectivity index: IC50 on mammal cells per IC50 on trypanosomatids; dNC: not calculated; epositive controls: pentamidine for L. amazonensis, amphotericin
B for L. infantum, benznidazole for T. cruzi, and doxorubicin for fibroblast cells.
compounds are potential candidates for further in vivo
studies (Table 2).
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.
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.
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
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,

1222
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole
J. Braz. Chem. Soc.
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.
4-(4-Methoxyphenyl)-2-methyl-3-butyn-2-ol (22a)38
1
Yield: 85%; yellow crystal; mp 53 ºC; H NMR
(300 MHz, CDCl3) d 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) d 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
1
Yield: 75%; yellow crystal; mp 48-50 ºC; H NMR
General procedure for the preparation of aryl bromides
(300 MHz, CDCl3) d 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) d 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.
(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-(1,3-Benzodioxol-5-yl)-2-methyl-3-butyn-2-ol (22c)40
1
Yield: 76%; yellow crystal; mp 44 ºC; H NMR
(300 MHz, CDCl3) d 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) d 31.50, 61.57, 81.94, 92.14,
101.24, 108.34, 111.64, 115.97, 126.21, 147.32, 147.79.
4-Bromo-1,2-dimethoxybenzene (21b)35
General procedure for the preparation of terminal acetylenes
Yield: 87%; colorless oil; 1H NMR (300 MHz, CDCl3)
d 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) d 55.97,
112.56, 112.66, 114.80, 123.34, 148.36, 149.78.
(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.
5-Bromo-1,3-benzodioxole (21c)36,37
Yield: 79%; colorless oil; 1H NMR (300 MHz, CDCl3)
d 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) d 101.59, 109.54, 112.29,
113.06, 124.30, 147.01, 148.61.
General procedure for the preparation of acetylene alcohols
(22a-c)
1-Ethynyl-4-methoxybenzene(25a)40
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.
Yield: 85%; colorless oil; 1H NMR (300 MHz, CDCl3)
d 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) d 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) d 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,

Vol. 27, No. 7, 2016
Cassamale et al.
1223
Ar-H), 7.05 (dd, 1H, J 8.2, 1.8 Hz, Ar-H); 13C NMR
(75 MHz, CDCl3) d 55.83, 75.63, 83.72, 110.84, 114.12,
114.61, 125.43, 148.50, 149.79.
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.
5-Ethynyl-1,3-benzodioxole (25c)42
1
Yield: 79%; yellow oil; H NMR (300 MHz, CDCl3)
d 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) d 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
1-Azide-4-methoxybenzene (27a)26
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) d 3.83 (s, 9H,
3OCH3), 6.77 (s, 2H, Ar-H), 7.38 (s, 1H, Ar-H); 13C NMR
(75 MHz, CDCl3) d 56.16, 60.84, 88.77, 105.79, 130.54,
136.59, 138.36, 152.99.
Yield: 73%; yellow solid; 1H NMR (300 MHz, CDCl3)
d 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) d 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)
d 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) d 55.92, 56.17, 103.15, 110.40,
112.01, 132.72, 146.48, 149.95.
1,3-Benzodioxol-5-yl-azide(27c)25
1
Yield: 75%; brown oil; H NMR (300 MHz, CDCl3)
d 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) d 100.66; 101.59; 108.75;
111.58; 133.74; 145.01; 148.64.
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) d 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) d 55.99, 60.11, 79.70, 83.71, 109.11,
116.80, 138.64, 152.87.
5-Azide-1,2,3-trimethoxybenzene(27d)26
Yield: 87%; yellow solid; 1H NMR (300 MHz, CDCl3)
d 3.78 (s, 3H, OCH3), 3.82 (s, 6H, 2OCH3), 6.21 (s, 2H,
Ar-H); 13C NMR (75 MHz, CDCl3) d 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

1224
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole
J. Braz. Chem. Soc.
phase was dried over anhydrous MgSO4, and the solvent
was evaporated under reduced pressure. The product was
purified by recrystallization from ethyl acetate.
(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
1,4-Bis(4-ethoxyphenyl)-1H-1,2,3-triazole (4)25
1
1
Yield: 79%; yellow solid; mp 205-206 ºC; H NMR
Yield: 80%; white solid; mp 163-165 ºC; H NMR
(300 MHz, CDCl3) d 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) d 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.
(300 MHz, DMSO-d6) d 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) d 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.
1,4-Bis(3,4-dimethoxyphenyl)-1H-1,2,3-triazole (5)44
4-(3,4-Dimethoxyphenyl)-1-(4-methoxyphenyl)-1H-1,2,3-
triazole (9)25,45
1
Yield: 92%; yellow solid; mp 156-157 ºC; H NMR
1
(300 MHz, CDCl3) d 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)
d 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.
Yield: 85%; white solid; mp 141-143 ºC; H NMR
(300 MHz, CDCl3) d 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) d 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
1,4-Bis(3,4,5-trimethoxyfenyl)-1H-1,2,3-triazole (6)25
1
1
Yield: 89%; yellow solid; mp 172-174 ºC; H NMR
Yield: 83%; yellow solid; mp 180-181 ºC; H NMR
(300 MHz, CDCl3) d 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) d 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.
(300 MHz, CDCl3) d 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) d 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-Bis(1,3-benzodioxol-5-yl)-1H-1,2,3-triazole (7)
1-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-1,2,3-
triazole(11)25
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) d 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) d 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
1
Yield: 86%; white solid; mp 166-167 ºC; H NMR
(300 MHz, CDCl3) d 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) d 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.

Vol. 27, No. 7, 2016
Cassamale et al.
1225
1-(1,3-Benzodioxol-5-yl)-4-(4-methoxyphenyl)-1H-1,2,3-
triazole(12)25
1-(1,3-Benzodioxol-5-yl)- 4-(3,4-dimethoxyphenyl)-1H-
1,2,3-triazole (16)25
1
1
Yield: 85%; white solid; mp 186-187 ºC; H NMR
Yield: 81%; brown solid; mp 152-153 ºC; H NMR
(300 MHz, CDCl3) d 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) d 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.
(300 MHz, CDCl3) d 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) d 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-(4-methoxyphenyl)-1H-1,2,3-
triazole (13)
4-(1,3-Benzodioxol-5-yl)-1-(3,4-dimethoxyphenyl)-1H-1,2,3-
triazole (17)
Yield: 81%; white solid; mp 175-176 ºC; IR (KBr)
ν / cm-1 3106, 3012-2794, 1608, 1519, 1481, 1255, 1230,
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) d 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) d 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
1214, 1108, 1037, 935, 838, 811; H NMR (300 MHz,
CDCl3) d 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) d 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
1
Yield: 82%; white solid; mp 141-142 ºC; H NMR
1-(1,3-Benzodioxol-5-yl)-4-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole (18)25
(300 MHz, CDCl3) d 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) d 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.
Yield: 78%; a yellow solid; mp 126-127 ºC; 1H NMR
(300 MHz, CDCl3) d 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) d 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.
1-(3,4-Dimetyhoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole (15)25
1
Yield: 88%; yellow solid; mp 136-137 ºC; H NMR
4-(1,3-Benzodioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)-1H-
1,2,3-triazole (19)
(300 MHz, CDCl3) d 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) d 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.
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) d 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) d 56.43, 61.03, 98.45, 101.24, 106.43,

1226
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole
J. Braz. Chem. Soc.
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.
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.
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).
For both kinds of assays the percentages of parasite lysis
were determined from the following formula:
% lysis = 100 − {[(X − CP) / (PC − NC)]} × 100
(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.
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.
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
OriginVersion 6.0 e Microsoft Office Excel 2007). Selectivity
index (SI) was calculated according to de Medeiros et al.49
The percentage of activity was calculated by the
following formula:
Supplementary Information
% Activity = 100 − [(N − Y) / (N − P)] × 100
(1)
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
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.
Acknowledgments
This study was supported by grants from FUNDECT-MS

Vol. 27, No. 7, 2016
Cassamale et al.
1227
(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.
fexichagas-po.html accessed in December 2015.
9. Bahia, M. T.; deAndrade, 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.
References
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.
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.
12. Wermuth, C. G.; The Practice of Medicinal Chemistry, 3rd 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.
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.
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.
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.
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.
5. http://www.dndi.org/images/stories/pdf_portfolios/DNDi_
Portfolio_2013_POR.pdf, Drugs for Neglected Diseases
initiative, accessed in December 2015.
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
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-

1228
Synthesis and Antitrypanosomastid Activity of 1,4-Diaryl-1,2,3-triazole
J. Braz. Chem. Soc.
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.
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.
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.
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.
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.
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.
30. Dabdoub, M. J.; Dabdoub, V. B.; Guerrero Jr., P. G.; Hurtado,
G. R.; Tetrahedron Lett. 2012, 53, 5302.
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.
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.
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.
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.
Submitted: November 20, 2015
Published online: January 25, 2016
35. Pan, J.; Wang, X.; Zhang,Y.; Buchwald, S. L.; Org. Lett. 2011,
13, 4974.
FAPESP has sponsored the publication of this article.