Design, synthesis and trypanocidal evaluation of novel 1,2,4-triazoles-3- thiones derived from natural piperine

Article abstract of DOI:10.3390/molecules18066366

The work reported herein describes the synthesis and the assessment of the trypanocidal activity of thirteen new 1,2,4-triazole-3-thiones obtained from natural piperine, the main constituent of the dry fruits of Piper nigrum. It is part of a research program aiming to use abundant and easily available natural products as starting materials for the design and synthesis of new molecules potentially useful as antiparasitic drugs. The variously substituted triazole derivatives were synthesized from the natural amide in four steps with the use of microwave irradiation on overall yields ranging from 32% to 51%. The cyclohexyl substituted derivative showed the best trypanocidal profile on proliferative forms of Trypanosoma cruzi (Y strain), with IC50s = 18.3 and 8.87 μM against epimastigotes and amastigotes, respectively.

Full text of DOI:10.3390/molecules18066366

Molecules 2013, 18, 6366-6382; doi:10.3390/molecules18066366
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis and Trypanocidal Evaluation of Novel
1,2,4-Triazoles-3-thiones Derived from Natural Piperine
Tatiany Nunes Franklim 1, Leonardo Freire-de-Lima 2, Julliana de Nazareth Sá Diniz 2,
José Osvaldo Previato 2, Rosane Nora Castro 1, Lucia Mendonça-Previato 2 and
Marco Edilson Freire de Lima 1,*
1
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro,
BR 465, Km 07, CEP: 23.890-000, Seropédica, RJ, Brazil;
E-Mails: tnfranklim@gmail.com (T.N.F.); nora@ufrrj.br (R.N.C.)
2
Cidade Universitária, Ilha do Fundão, Instituto de Biofísica Carlos Chagas Filho,
Universidade Federal do Rio de Janeiro, CEP: 21.941-902, Rio de Janeiro, RJ, Brazil;
E-Mails: leolima@biof.ufrj.br (L.F.-L.); jullianawinners@gmail.com (J.N.S.D.);
previato@biof.ufrj.br (J.O.P.); luciamp@biof.ufrj.br (L.M.-P.)
* Author to whom correspondence should be addressed; E-Mail: marco@ufrrj.br;
Tel./Fax: +55-21-2682-1872.
Received: 29 March 2013; in revised form: 16 May 2013 / Accepted: 21 May 2013 /
Published: 29 May 2013
Abstract: The work reported herein describes the synthesis and the assessment of the
trypanocidal activity of thirteen new 1,2,4-triazole-3-thiones obtained from natural
piperine, the main constituent of the dry fruits of Piper nigrum. It is part of a research
program aiming to use abundant and easily available natural products as starting materials
for the design and synthesis of new molecules potentially useful as antiparasitic drugs. The
variously substituted triazole derivatives were synthesized from the natural amide in four
steps with the use of microwave irradiation on overall yields ranging from 32% to 51%.
The cyclohexyl substituted derivative showed the best trypanocidal profile on proliferative
forms of Trypanosoma cruzi (Y strain), with IC50s = 18.3 and 8.87 μM against
epimastigotes and amastigotes, respectively.
Keywords: Trypanosoma cruzi; Piper nigrum; antiparasitic drugs; bioisosterism;
molecular hybridization

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1. Introduction
Among the most important natural molecules that have had strong impact on human health during
the last century we can mention quinine, extracted from Cinchona officinales (Rubiaceae) and
penicillin, obtained from Pennicillium chrysogenum (Trichocomaceae) through fermentation
processes [1]. Another naturally occurring molecule of great importance is taxol, first isolated from
Taxus brevifolia (Taxaceae). Taxol is an antitumor drug which has application in the treatment of
various cancers such as ovarian, lung, melanoma and breast cancer [1]. Thus it may be seen that the
natural products occupy a prominent position in drug development, mainly in the chemotherapy of
infectious diseases and cancer [1–4].
When planning to use natural products as drugs or even as raw materials for drug synthesis, there is
a limitation due to the fact that special metabolites of plant origin are biosynthesized in small
quantities and, normally, isolated in a very laborious way [5]. Another significant limitation to the use
of natural products in drug development is the fact that most of them are not obtained from sustainable
sources [5]. Natural piperine (1, Figure 1) is an exception, due to its abundance and ease of isolation.
Although found in different species of Piper, piperine is more abundant in the fruits of Piper nigrum,
from where it can be extracted in yields of about 3%–7% [6,7]. P. nigrum sees widespread use in folk
medicine in India and in Brazil, its main use being as a seasoning. Due to its great economic
importance in different countries, including Brazil, nowadays large areas are cultivated with Piper
nigrum [7,8]. The chemical composition of black pepper has motivated scientific studies for almost
two hundred years since the first report describing the isolation of piperine from P. nigrum was
published in 1819 by Hans Christian Oersted [8]. Various biological activities have been attributed to
piperine, including insecticidal properties and inhibition of liver metabolism, stimulation of
melanocyte proliferation, and antitumoral activity [7–10]. Piperine has been described as having toxic
activity against different species of protozoa from the Trypanosomatidae family such as Leishmania
donovani [11], Leishmania amazonensis [12], and Trypanosoma cruzi [13,14].
Figure 1. Chemical structures of piperine (1), nifurtimox (2) and benznidazole (3).
O
O
N
1
O
O
CH3
N
N
N
O2N
N
N
H
O
SO2
2
3
NO2
Despite the fact that neglected diseases, including tuberculosis, are responsible for 11.4% of the
cause of all disabling clinical conditions in the World, only 21 (1.3%) of the 1,556 new drugs
registered between 1,975 and 2,004 were developed specifically for the treatment of these infections [15].
These illnesses are known as neglected diseases due to the lack of investment from the pharmaceutical
industry in the development of new drugs to treat the patients and also to the shortage of public
policies aimed at their prevention [16]. The literature reports some studies concerning the development

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of new molecules derived from natural products for the treatment of neglected diseases [17]. Our
research group has been working during the last years on the discovery of new chemical entities that
may be applicable to the treatment of leishmaniasis and Chagas’ disease, using piperine as raw
material [12–14,18].
Chagas’ disease is an important parasitic infection, endemic in Latin America, which has the
hemoflagelated protozoa Trypanosoma cruzi as its etiological agent [19,20]. This infection is normally
transmitted by feces and urine of triatomine insects while blood feeding on a vertebrate host. The
parasite has a complex life cycle with evolutive forms present in vertebrate and invertebrate hosts. The
trypomastigote form when inside the insect differentiates into the epimastigote form and, after
replication, reaches the posterior insect’s intestinal tract, where it differentiates into the infective
metacyclic trypomastigote form. When inside of vertebrate cells, the metacyclic trypomastigote form
undergoes differentiation into amastigotes, which after several replicative cycles, cause the cell
breakage, being released into the blood stream of the vertebrate where it differentiates into
trypomastigotes, which are responsible for the dissemination of the infection [19]. According to the
World Health Organization (WHO), some 10 million people are infected with the T. cruzi, mainly in
Latin America, resulting in an estimated annual death toll of 10,000 [19,20]. Only two drugs are
available for the treatment of Chagas disease: benznidazole (2) and nifurtimox (3) (Figure 1) [19,20].
These compounds are responsible for undesirable side effects, including anorexia, vomiting, peripheral
polyneuropathy, and allergic dermopathy. Another matter of great importance is that the efficacy of
these drugs in the chronic phase of infection is still controversial [21]. Thus, considerable efforts have
being directed to developing new chemotherapeutical agents for treating chagasic patients, especially
using natural products as starting materials or even as a source of inspiration [10,22].
This work describes the synthesis and the evaluation of the trypanocidal activity of thirteen new
1,2,4-triazole-3-thiones 9a–9m derived from natural piperine (1). The 1,2,4-triazole-3-thiones are five-
membered heterocyclic rings present in the structure of numerous compounds with useful and wide-
ranging biological activities, including antidepressant [23], antifungal [24], anti-inflammatory [25],
antiviral [26] and antitumor effects [27].
2. Results and Discussion
2.1. Chemistry
When planning new drugs as chemotherapeutic agents for infectious diseases it is necessary to have
in mind the possible biochemical targets as well the differences between the parasite`s and host`s cells.
Ergosterol is the major sterol component of fungi and also of the T. cruzi cell membranes. This lipid
plays an important role in the regulation of membrane fluidity and integrity [28]. Azoles are a known
class of inhibitors of the cytochrome P-450 dependent sterol 14α-demethylase (CYP51) that promotes
the 14α-demethylation of lanosterol into ergosterol. The blockage of this biosynthetic pathway leads to
the accumulation of toxic pre-ergosterol lipids in the parasite membrane compromising the cell
viability [28]. Posaconazole (4, Figure 2) is an example of a broad spectrum anti-fungal azole that has
been investigated with respect to its trypanocidal activity. The azole 4 is currently entering phase II clinical
trials [29]. Another example of antifungal with the triazole moiety is the prothioconazole (5), a

Molecules 2013, 18
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1,2,4-triazole-3-thione and known inhibitor of 14α-demethylase, commercially available for the
treatment of plant-pathogenic fungal infections [24]. The nitrogen-containing heterocyclics present in
CYP51 inhibitors are important structural features for their coordination to the heme iron and
stabilization of the complex enzyme-inhibitor [30]. Having this information in mind the molecular
hybridization of piperine and the 1,2,4-triazole nucleus was planned [31], furnishing a series of new
compounds where the N4-substituents the triazole ring were modified changing their electronic and
lipophilic profiles in order to allow the investigation of the SAR (Figure 2). The molecular
modification approach used on the designing the new derivatives was also based in the bioisosterism
strategy [32], since there are different examples in the literature of the bioisosteric replacement of
amides by five membered nitrogen-containing rings as triazole, thiadiazole and oxadiazole (Figure 2) [33].
Figure 2. Strategies on the designing of new piperine-triazole hybrid.
O
N
N
HO
N
N
N
O
N
N
O
N
H
Posaconazole (4)
Bioisosterism
F
F
H
O
N
O
O
N
N
N
S
Cl
Cl
Piperine (1)
OH
Prothioconazole (5)
Molecular
hybridization
H
N
N
S
O
O
N
R
New hybrid chemical entity
Firstly, the isolation of suitable amounts of natural amide was carried out according to a previously
described methodology [6]. As shown in Scheme 1, piperic acid (6) was obtained in excellent yield by
basic hydrolysis of piperine (1). Originally this reaction was carried out in 24 h reflux [13], but after
improvements on the experimental conditions it could be carried out in 1 h and with 80% yield in a
microwave reactor in an open-vessel mode. Hydrazide 7 was prepared from the acid 6 after its reaction
with oxalyl chloride [13,18], followed by the treatment of the acyl chloride intermediate with
hydrazine monohydrate. The hydrazide 7 was the common intermediate for the preparation of all azole

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derivatives by the reaction with different thioisocyanates, furnishing the carbothioamides 8a–8m in
good yields. These intermediates underwent basic promoted ring closure, generating the targeted
triazoles (9a–9m) in moderate to good yields (Scheme 1). The literature describes that these reactions
normally require conventional heating for several hours [34]. After some improvements in the
experimental conditions, both reactions (preparation of carbothioamides and their ring closure to the
corresponding triazoles) were conducted in a microwave reactor using water or ethanol as solvents,
and resulting at reduced reaction times (30 min). Nowadays the principles of green chemistry are being
observed in our laboratory, due to their growing importance, mostly for ecological and health reasons.
Thus, chemical transformations have been conducted with the least expense of energy, decreased
reaction times and, whenever possible, avoiding both the use of toxic solvents and harmful waste
production [35].
Scheme 1. Preparation of new 1,2,4-triazole-2-thiones 9a–9m from piperine (1).
O
O
O
O
O
O
b, c
OH
N
a
6
1
O
S
O
O
O
NHNH
NHR
d
NHNH2
O
O
8a - 8m
7
8a, 9a: R= Methyl
8b, 9b: R= Ethyl
H
8c, 9c: R= Isopropyl
8d, 9d: R= n-Buthyl
8e, 9e: R= n-Hexyl
8f, 9f: R= Cyclohexyl
8g, 9g: R= Phenyl
8h, 9h: R= Benzyl
2
1
N
N
4
S
O
O
3
5
e
N
R
9a - 9m
8i, 9i: R= 3,4,5-Trimethoxyphenyl
8j, 9j: R= tert-Butyl
8k, 9k: R= 4-(Methylthio)phenyl
8l, 9l: R= 3-Methoxyphenyl
8m, 9m: R= 4-(Trifluoromethyl)phenyl
Reaction conditions: (a) KOH, ethanol, mw, 1 h (80%); (b) (COCl)2, 30 min, r.t.;
(c) NH2NH2.H2O, CH2Cl2, 0 °C to r.t. (71%); (d) suitable isothiocyanate, ethanol, mw, 30 min
(65%–91%); (e) NaOH aq., mw, 30 min; then HCl aq. to pH 3.0 (62%–90%).
The physical and spectroscopical data for the compounds 7, 8a–8m and 9a–9m are described herein
(Experimental section), while the spectral characterization of the isolated natural amide 1 and piperic
1
acid (6) has been described previously [36,37]. The H-NMR analysis of the newly prepared triazoles
9a–9m revealed characteristic absorptions assigned to the heterocyclic ring N-H protons at about δ 13
ppm that are quite different from absorptions assigned to the three different N-H protons present in the
carbothioamides 8a–8m, which showed signals between δ 7.65 to δ 10.24 ppm. The 13C-NMR spectra
showed signals at about δ 168 ppm and δ 149 ppm, assigned to the two carbon atoms of heterocyclic
ring (C3 and C4, respectively). The data observed in the high resolution mass spectra were consistent
with the calculated values for the expected molecular formulas of the final products.

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2.2. Biological Assays
All compounds were evaluated in vitro against the proliferative epimastigote form of Y strain
T. cruzi having benznidazole (Bdz) as a control drug. The compounds 9c, 9d, 9e, 9f and 9m which
showed IC50 values below 30 μM on epimastigotes were further selected for cell viability assays on
peritoneal macrophages (Mø) from Balb/c mice. The Mø were treated with the selected compounds
described above at the concentrations of 0.5, 1.0, 2.5 and 5 μg mL−1. The number of viable cells was
determined by a trypan blue dye exclusion assay after 72 h of treatment. In all the concentrations tested
the five derivatives showed no significant toxicity on host cells, presenting cellular viability values
ranging from 85% to 95%. The results obtained on the trypanocidal evaluation of the new triazoles are
summarized in Table 1.
Table 1. Toxic effects of new triazoles (9a–9m) on proliferative forms of T. cruzi.
IC50 (µM)
Triazoles
Epimastigotes
44.2 ± 7.30
Amastigotes
9a
nt
9b
50.16 ± 10.79
26.98 ± 6.98
13.22 ± 3.64
15.38 ± 4.47
18.30 ± 5.21
38.96 ± 8.88
39.39 ± 6.88
35.30 ± 6.26
50.75 ± 10.63
40.75 ± 10.50
48.02 ± 6.06
18.46 ± 3.95
2.20 ± 0.16
nt
9c
11.11 ± 1.26
9d
≥15.17 **
9e
≥13.98 **
9f
8.87 ± 2.39
9g
nt
9h
nt
9i
nt
9j
nt
nt
9k
9l
9m
nt
9.59 ± 2.87
2.50 ± 0.21
Benznidazole *
Notes:* Reference drug;** Maximum concentration allowed by trypan blue exclusion assay; nt = Not tested.
The N-alkyl-triazoles 9a and 9b had no important activity against the epimastigote form of T. cruzi,
with IC50 values of 44.2 and 50.16 μM, respectively. With increasing length of the alkyl chain, the
N-alkyl derivatives (9c–9f) exhibited a higher activity activity on epimastigotes, suggesting that
lipophilicity is an important parameter for the trypanocidal activity of this family of triazoles
interfering, for example, with their ability to cross cell membranes. In the N-benzyl and N-aryl series,
the triazoles 9g–9l showed no significant activity. Only 9m, which has a para-trifluoromethylaryl
substituent at N-4 of the heterocyclic ring, showed an important toxic effect against epimastigotes. In
the evaluation against amastigotes of T. cruzi the compounds 9c, 9f and 9m showed similar
trypanocidal effects, with IC50 values of 11.11, 8.87 and 9.59 μM, respectively. The triazoles 9d and
9e showed higher IC50 values on amastigotes. Among the thirteen new compounds prepared, the
triazole 9f (N-cyclohexyl derivative, Scheme 1) had the best trypanocidal profile, presenting
IC50 = 18.3 μM and 8.87 μM against epimastigotes and amastigotes of Trypanosoma cruzi (Y strain),
respectively.

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3. Experimental
3.1. General
1
Melting points were determined on a Büchi B-510 apparatus and are uncorrected. The H-NMR
(500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker Ultrashield Plus Spectrometer
1
13
(BrukerBioSpin GmbH, Rheinstetten, Germany) operating at 500 MHz for H and 125 MHz for C.
1H and 13C-NMR shifts (δ) are reported in parts per million (ppm) with respect to DMSO-d6 (2.50 ppm
1
13
for H and 39.7 ppm for C). Chemical shifts (δ) were reported in ppm and coupling constants (J) in
Hertz [Hz]. Signal multiplicity was assigned as singlet (s), doublet (d), doublet of doublets (dd), triplet
(t), quartet (q), multiplet (m) and broad signal (bs). The low-resolution mass spectra were carried out
on a Shimadzu GCMS-QP2010 Plus (Shimadzu Inc., Kyoto, Japan). Analytical conditions: Column:
VF-5MS, 30 m × 0.25 mm × 0.25 μm (Varian Inc., Santa Clara, CA, USA); Column temperature: 200 °C
for one minute and then increasing to 290 °C at a rate of 10 °C/min and holding for 40 minutes;
Injector temperature: 270 °C. The high-resolution mass spectra were recorded with Q-TOF Micromass
equipment (Waters, Milford, MA, USA). All the reactions involving microwave instrumentation used
the Discover SP system (CEM Inc., Matthews, NC, USA) and were performed in open vessel mode.
Analytical thin-layer chromatography (TLC) was performed on precoated silica gel plates (0.25 mm
layer thickness) in an appropriate solvent and the spots were visualized under UV light (254 nm and
356 nm). The natural piperine (1) was extracted from powdered dry fruits of P. nigrum purchased from
different sources on the market and presented physical and spectroscopic data consistent with that
originally described [6,36,37]. The isolation method employed in this study was an adaptation of that
previously reported [6].
3.1.1. Microwave-assisted Hydrolysis of Natural Piperine: Preparation of 1-[(2E,4E)-5-(1,3-
Benzodioxol-5-yl)penta-2,4-dienoyl]piperidine (6)
A suspension of piperine (1, 2.20 g, 7.72 mmol) in an 20% (w/v) ethanolic solution of NaOH
(22 mL) was placeded in a 50 mL round bottom flask equipped with a magnetic stirring bar and a
reflux apparatus. This suspension was then subjected to microwave irradiation (100 W) for 1 h in open
vessel mode. The evolution of the reaction was monitored by TLC. At the end of the reaction the
solvent was removed under reduced pressure and hot water was added to solubilize the carboxylate.
The warm mixture was filtered and the resulting solution was acidified with concentrated HCl (37%
w/v) until pH 3.0, whereupon the free acid precipitated as a yellow solid. After filtration the solid was
washed twice with ice water. The acid 6 (1.37 g; 82%) was thus obtained as a yellow solid after
1
13
recrystallization from ethanol and was characterized by its melting point, H- and C-NMR. All data
were consistent with those previously reported in the literature [37].
3.1.2. Synthesis of (2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienohydrazide (7)
Acid 6 (500 mg, 2.29 mmol) and oxalyl chloride (1.5 mL, 17.60 mmol) were added to a 10 mL
round bottom flask equipped with a magnetic stirring bar, rubber septum and kept under a dry N2
atmosphere. The resulting solution was subjected to stirring at room temperature for about 0.5 h. The

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evolution of the reaction was accompanied by TLC (indirectly by the reaction of an aliquot with
methanol leading to the spontaneous formation of the corresponding methyl ester). After the removal
of excess oxalyl chloride the resulting residue was dissolved in dry dichloromethane (5 mL). The
resulting solution was added dropwise to an ice-cooled mixture of hydrazine monohydrate (1.0 mL,
20.64 mmol) and dichloromethane (7 mL) placed in a 25 mL round bottom flask equipped with stirring
bar, rubber septum and kept under a dry N2 atmosphere. The reaction medium was allowed to reach
room temperature (about thirty minutes) and then the solvent was removed under reduced pressure.
The product precipitated with addition of ice water to the residue previously obtained and then was
filtered, furnishing 375 mg (71% yield) of the hydrazide 7 as a yellow amorphous solid. m.p. =
237–239 °C. 1H-NMR δ (ppm): 9.30 (s, 1H, -NH-), 7.27 (d, 1H, J = 1.40 Hz), 7.19 (dd, 1H, J = 15.01 and
10.40 Hz), 7.00 (dd, 1H, J = 8.00 and 1.40 Hz), 6.86–6.97 (m, 3H), 6.05 (d, 1H, J = 15.10 Hz), 6.06
13
(s, 2H, -O-CH2-O-), 4.45 (s, 2H, -NH2); C-NMR δ (ppm): 165.23, 148.43, 148.19, 139.38, 138.26,
131.27, 125.82, 123.13, 123.05, 108.93, 106.10, 101.75.
3.1.3. General Procedure for the Synthesis of Carbothioamides 8a to 8m
The suitable alkylisothiocyanates (0.517 mmol, 1.2 equiv.) were added to a suspension of hydrazide
7 (100 mg, 0.431 mmol) in ethanol (5 mL). The mixture was placed in a 10 mL round bottom flask
equipped with a magnetic stirring bar and a reflux apparatus and then subjected to microwave
irradiation (100 W) for 0.5 h in open vessel mode. The evolution of the reaction was monitored by
TLC. After elimination of ethanol under reduced pressure and addition of water to the vessel the
products were collected and recrystallized from ethanol. All compounds were characterized by 1H- and
13C-NMR. The yields, melting points and spectral data observed for each carbothioamide 8a–8m
prepared are as follows:
2-[(2E,4E)-5-(1,3-Bbenzodioxol-5-yl)penta-2,4-dienoyl]-N-methylhydrazinecarbothioamide
(8a).
Brown amorphous solid, yield 67%, m.p. = 195–196 °C (ethanol). 1H-NMR δ (ppm): 9.89 (s, 1H, NH),
9.27 (s, 1H, NH), 7.98 (s, 1H, NH), 7.32 (s, 1H), 7.29 (dd, 1H, J = 14.98 and 10.56 Hz), 6.93–7.04 (m,
4H), 6.09 (d, 1H, J = 15.00 Hz), 6.07 (s, 2H, -O-CH2-O-), 2.87 (d, 3H, J = 15.00 Hz, -NH-CH3);
13C-NMR δ (ppm): 165.65, 148.46, 141.37, 139.53, 131.20, 125.53, 123.51, 122.40, 108.96, 106.18,
101.81, 31.40.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-ethylhydrazinecarbothioamide (8b). Brown
1
amorphous solid, yield 90%, m.p. = 192–193 °C. H-NMR δ (ppm): 9.86 (s, 1H, NH), 9.18 (s, 1H,
NH), 8.00 (t, 1H, J = 5.20 Hz, NH), 7.29 (d, 1H, J = 1.40 Hz), 7.25 (dd, 1H, J = 15.18 and 9.91 Hz),
7.00 (dd, 1H, J = 8.28 and 1.51 Hz), 6.91–6.99 (m, 3H), 6.08 (d, 1H, J = 15.30 Hz), 6.05 (s, 2H,
-O-CH2-O-), 3.39-3.49 (m, 2H, -NH-CH2-CH3), 1.05 (t, 3H, J = 7.00 Hz, -NH-CH2-CH3); 13C-NMR δ
(ppm): 181.65, 165.61, 148.42, 141.35, 139.46, 131.16, 125.48, 123.38, 122.36, 108.94, 106.20,
101.76, 38.96, 14.87.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-isopropylhydrazinecarbothioamide
(8c).
1
Pale yellow amorphous solid, yield 66%, m.p. = 193–194 °C. H-NMR δ (ppm): 9.82 (s, 1H, NH),
9.16 (s, 1H, NH), 7.67 (d, 1H, J = 7.80 Hz, NH), 7.31 (s, 1H), 7.26 (dd, 1H, J = 14.98 and 10.25 Hz),

Molecules 2013, 18
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6.93–7.03 (m, 4H), 6.10 (d, 1H, J = 15.00 Hz), 6.07 (s, 2H, -O-CH2-O-), 4.34–4.46 (m, 1H, -NH-CH-
(CH3)2), 1.12 (d, 6H, J = 6.60 Hz, -NH-CH-(CH3)2); 13C-NMR δ (ppm): 165.49, 148.46, 141.20,
139.47, 131.22, 125.53, 123.48, 122.56, 108.96, 106.17, 101.81, 46.24, 22.39.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-buthylhydrazinecarbothioamide
(8d).
Yellow amorphous solid, yield 87%, m.p. = 173–175 °C. 1H-NMR δ (ppm): 9.87 (s, 1H, NH), 9.19 (s,
1H, NH), 8.00 (s, 1H, NH), 7.33 (s, 1H), 7.27 (dd, 1H, J = 14.82 and 10.72 Hz), 6.94–6.98 (m, 2H),
7.02–7.30 (m, 2H), 6.10 (d, 1H, J = 15.00 Hz), 6.08 (s, 2H,-O-CH2-O-), 3.48-3.44 (m, 2H, -NH-CH2-
CH2-CH2-CH3), 1.49 (m, 2H, -NH-CH2-CH2-CH2-CH3), 1.28 (m, 2H, -NH-CH2-CH2-CH2-CH3), 0.90
(t, 3H, J = 6.90 Hz, NH-CH2-CH2-CH2-CH3); 13C-NMR δ (ppm): 165.57, 148.46, 148.40, 141.27,
139.49, 131.22, 125.53, 123.49, 122.49, 108.96, 106.17, 101.81, 43.82, 31.39, 19.92, 14.31.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-hexylhydrazinecarbothioamide (8e). Yellow
1
amorphous solid, yield 90%, m.p. = 165–167 °C. H-NMR δ (ppm): 9.86 (s, 1H, NH), 9.18 (s, 1H,
NH), 7.99 (s, 1H, NH), 7.32 (s, 1H), 7.26 (dd, 1H, J =14.98 and 10.56 Hz), 6.93–7.02 (m, 2H),
7.03–7.24 (m, 2H), 6.11 (d, 1H, J = 15.00 Hz), 6.07 (s, 2H, -O-CH2-O-), 3.41–3.42 (m, 2H, -NH-CH2-
CH2-CH2-CH2-CH2-CH3), 1.48-1.66 (m, 2H, -NH-CH2-CH2-CH2-CH2-CH2-CH3), 1.27–1.37 (m, 6H,
-NH-CH2-CH2-CH2-CH2-CH2-CH3), 0.88 (t, 3H, J = 5.00 Hz, -NH-CH2-CH2-CH2-CH2-CH2-CH3);
13C-NMR δ (ppm): 165.65, 164.17, 148.48, 141.35, 139.56, 131.30, 125.62, 123.57, 122.56, 109.04,
106.25, 101.88, 44.21, 31.62, 29.26, 26.48, 22.65, 14.50.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-cyclohexylhydrazinecarbothioamide
(8f).
Yellow amorphous solid, yield 91%, m.p. = 192–194 °C. 1H-NMR δ (ppm): 9.84 (s, 1H, NH), 9.18 (s,
1H, NH), 7.65 (d, 1H, NH, J = 2.52 Hz), 7.32 (s, 1H), 7.25 (dd, 1H, J = 14.98 and 10.56 Hz), 7.03 (d,
1H, J = 8.20 Hz), 6.93-7.04 (m, 3H), 6.10 (d, 1H, J = 15.13 Hz), 6.07 (s, 2H, -O-CH2-O-), 4.07–4.19
(bs, 1H, -NH-CH-(CH2-CH2)2-CH2), 1.06–1.89 (m,10H, -NH-CH-(CH2-CH2)2-CH2); 13C-NMR δ
(ppm): 162.92, 148.39, 141.20, 139.46, 131.22, 125.54, 123.47, 122.55, 108.96, 106.17, 101.81, 53.37,
32.35, 25.67, 25.40.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-phenylhydrazinecarbothioamide
(8g).
1
Yellow amorphous solid, yield 87%, m.p. = 149–150 °C. H-NMR δ (ppm): 10.11 (s, 1H, NH), 9.74
(s, 1H, NH), 9.68 (s, 1H, NH), 7.44–7.48 (bs, 2H), 7.34–7.36 (m, 2H), 7.33 (s, 1H), 7.27–7.33 (m, 1H),
7.16–7.18 (m, 1H), 6.84-7.04 (m, 4H), 6.15 (d, 1H, J = 13.87 Hz), 6.07 (s, 2H, -O-CH2-O-); 13C-NMR
δ (ppm): 148.46, 141.32, 139.70, 139.54, 131.22, 130.43, 128.47, 126.47, 125.56, 123.51, 122.53,
108.97, 106.18, 101.81.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-benzylhydrazinecarbothioamide
(8h).
Yellow amorphous solid, yield 84%, m.p. = 196–197 °C. 1H-NMR δ (ppm): 9.98 (s, 1H, NH), 9.41 (s,
1H, NH), 8.59 (bs, 1H, NH), 7.32 (s, 1H), 7.29–7.32 (m, 5H), 7.24–7.27 (m, 1H), 6.94–7.04 (m, 4H),
13
6.11 (d, 1H, J = 15.00 Hz), 6.07 (s, 2H, -O-CH2-O-), 4.74 (d, 2H, J = 5.30 Hz); C-NMR δ (ppm):
165.71, 148.41, 141.37, 139.94, 139.56, 131.21, 128.51, 127.60, 127.08, 125.53, 123.51, 122.45,
108.97, 106.18, 101.82, 41.17.

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2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-(3,4,5-trimethoxyphenyl) hydrazinecarbo-
thioamide (8i). Pale yellow amorphous solid, yield 77%, m.p. = 189–191 °C. 1H-NMR δ (ppm): 10.08
(s, 1H, NH), 9.67 (s, 1H, NH), 9.60 (s, 1H, NH), 7.32 (s, 1H), 7.27–7.32 (m, 1H), 7.03 (dd, 1H,
J = 7.88 and 1.26 Hz), 6.86–7.06 (m, 3H), 6.91 (s, 2H), 6.16 (d, 1H, J = 11.03 Hz), 6.07 (s, 2H,
13
-O-CH2-O-), 3.76 (s, 6H), 3.66 (s, 3H); C-NMR δ (ppm): 153.79, 148.46, 148.42, 141.42, 139.59,
135.40, 135.07, 131.21, 125.55, 123.51, 122.43, 108.97, 106.19, 104.42, 101.81, 60.56, 56.31.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-tert-butylhydrazinecarbothioamide
(8j).
1
Yellow amorphous solid, yield 80%, m.p. = 135–136 °C. H-NMR δ (ppm): 10.18 (s, 1H, NH), 9.95
(s, 1H, NH), 9.15 (s, 1H, NH), 7.30 (s, 1H), 7.24–7.30 (m, 1H), 7.02 (dd, 1H, J = 8.16 and 1.38 Hz),
6.92–6.99 (m, 3H), 6.13 (d, 1H, J = 14.81 Hz), 6.07 (s, 2H, -O-CH2-O-), 1.46 (s, 9H, -C-(CH3)3);
13C-NMR δ (ppm): 178.73, 169.55, 148.44, 141.54, 139.58, 131.17, 125.50, 123.48, 121.91, 108.94,
106.14, 101.81, 53.19, 29.09.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-[4-(methylthio)phenyl] hydrazinecarbothio-
1
amide (8k). Yellow amorphous solid, yield 84%, m.p.= 175–177 °C. H-NMR δ (ppm): 10.24 (s, 1H,
NH), 9.80 (s, 2H, NH), 7.55-7.64 (m, 1H), 7.55 (bs, 1H), 7.36-7.45 (m, 4H),7.06–7.18 (m, 4H), 6.27
(d, 1H, J = 14.10 Hz), 6.20 (s, 2H, -O-CH2-O-), 2.65 (s, 3H, -S-CH3); 13C-NMR δ (ppm): 148.48,
141.36, 139.57, 136.96, 131.24, 126.30, 125.57, 123.52, 122.53, 121.67, 108.99, 106.22, 101.83, 15.63.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-(3-methoxyphenyl)
hydrazinecarbothio-
1
amide (8l). Dark green amorphous solid, yield 65%, m.p.= 174–175 °C. H-NMR δ (ppm): 10.09 (s,
1H, NH), 9.70 (s, 2H, NH), 7.32 (s, 1H), 7.22–7.38 (m, 3H), 6.93–7.08 (m, 5H), 6.74 (d, 1H,
13
J = 8.03 Hz), 6.13 (d, 1H, J = 15.30 Hz), 6.07 (s, 2H, -O-CH2-O-), 3.75 (s, 3H); C-NMR δ (ppm):
181.15, 172.77, 164.55, 148.45, 141.33, 140.79, 139.54, 131.19, 131.13, 125.53, 123.50, 122.45,
118.68, 114.96, 111.62, 108.94, 106.15, 101.79, 55.54.
2-[(2E,4E)-5-(1,3-Benzodioxol-5-yl)penta-2,4-dienoyl]-N-[4-(trifluoromethyl)phenyl] hydrazinecarbo-
1
thioamide (8m). Pale yellow amorphous solid, yield 83%, m.p. = 177–178 °C. H-NMR δ (ppm):
10.18 (s, 1H, NH), 10.15 (s, 1H, NH), 9.93 (s, 1H, NH), 7.69–7.80 (m, 4H), 7.33 (s, 1H), 7.27–7.34
(m, 1H), 6.93–7.08 (m, 4H), 6.16 (d, 1H, J = 14.80 Hz), 6.07 (s, 2H, -O-CH2-O-); 13C-NMR δ (ppm):
181.27, 164.55, 148.45, 143.54, 141.54, 139.69, 131.16, 130.10, 125.49, 124.81 (q, CF3,
JC-F = 270 Hz), 123.53, 122.28, 121.60, 117.13, 108.94, 106.16, 101.80.
3.1.4. General Procedure to the Synthesis of Triazoles 9a to 9m
In a 10 mL bottom rounded flask equipped with a magnetic stirring bar and a reflux apparatus were
placed the corresponding hydrazine carbothioamide 8a–8m (1.37 mmol) and an aqueous solution of
NaOH (2 mL, 1 equiv.). This mixture was then subjected to microwave irradiation (100 W) for 0.5 h in
open vessel mode. The evolution of the reaction was monitored by TLC. Then the mixture was
acidified with a 10% aqueous solution of HCl (w/v) until pH 3, filtered and the solids were washed
twice with ice water (5 mL). After recrystallization from ethanol all the triazoles were characterized by
1
NMR and low- and high-resolution mass spectra. All compounds were characterized by H- and

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13C- NMR. The yields, melting points and spectral data observed for each carbothioamide 9a–9m
prepared are as follows:
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-methyl-2,4-dihydro-3H-1,2,-triazole-3-
1
thione (9a). Brown amorphous solid, yield 71%, m.p. = 220–222 °C. H-NMR δ (ppm): 13.78 (s, 1H,
NH), 7.28 (dd, 1H, J = 15.40 and 11.00 Hz), 7.23 (s, 1H), 6.90–7.07 (m, 4H), 6.54 (d, 1H, J = 15.40 Hz),
13
6.07 (s, 2H, -O-CH2-O-), 3.39 (s, 3H, -N-CH3); C-NMR δ (ppm): 167.11, 150.15, 148.45, 148.24,
137.55, 137.02, 131.31, 126.50, 123.00, 113.95, 109.03, 105.91, 101.77, 30.40; MS (m/z): 287; HRMS
(ES)-m/z: [M+H]+, found 288.0807. C15H15N3O2S requires 288.0806.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-ethyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9b). Brown amorphous solid, yield 62%, m.p. = 208–209 °C. 1H-NMR δ (ppm): 13.76 (s, 1H,
NH), 7.28 (dd, 1H, J = 15.30 and 10.70 Hz), 7.20 (s, 1H), 6.88–7.07 (m, 4H), 6.57 (d, 1H, J = 15.30
Hz), 6.05 (s, 2H, -O-CH2-O-), 4.08 (q, 2H, J = 7.03 Hz, -N-CH2-CH3), 1.19 (t, 3H, J = 7.03 Hz,
13
-NH-CH2-CH3); C-NMR δ (ppm): 166.46, 149.55, 148.44, 148.23, 137.56, 137.21, 131.32, 126.50,
122.95, 113.50, 109.02, 105.90, 101.75, 38.33, 14.22; MS (m/z): 301; HRMS (ES)-m/z: [M+H]+,
found 302.0963. C15H15N3O2S requires 302.0963.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-isopropyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9c). Pale yellow amorphous solid, yield 90%, m.p. = 210–211 °C. 1H-NMR δ (ppm): 13.78 (s,
1H, NH), 7.15-7.27 (m, 2H), 7.20 (s, 1H), 6.87–7.00 (m, 3H), 6.75 (d, 1H, J = 15.00 Hz), 6.07 (s, 2H,
-O-CH2-O-), 5.09-5.14 (m, 1H, -NH-CH-(CH3)2), 1.47 (d, 6H, J = 5.00 Hz, -NH-CH-(CH3)2);
13C-NMR δ (ppm): 166.46, 149.57, 148.46, 148.22, 137.23, 131.44, 126.77, 122.96, 114.55, 109.06,
105.84, 101.81, 46.61, 20.78; MS (m/z): 315; HRMS (ES)-m/z: [M+H]+, found 316.1120.
C16H17N3O2S requires 316.1119.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-butyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9d). Yellow amorphous solid, yield 71%, m.p. = 173–174 °C. 1H-NMR δ (ppm): 13.78 (s, 1H,
NH), 7.29 (dd, 1H, J = 15.10 and 11.00 Hz), 7.22 (s, 1H), 6.89–7.09 (m, 4H), 6.58 (d, 1H, J = 15.10 Hz),
6.07 (s, 2H, -O-CH2-O-), 4.04 (t, 2H, J = 6.78 Hz, -NH-CH2-CH2-CH2-CH3), 1.60–1.63 (m, 2H,
-NH-CH2-CH2-CH2-CH3), 1.31-1.36 (m, -NH-CH2-CH2-CH2-CH3), 0.93 (t, 3H, J = 7.20 Hz,
-NH-CH2-CH2-CH2-CH3); 13C-NMR δ (ppm): 166.74, 149.79, 148.48, 148.27, 137.58, 137.17, 131.55,
125.53, 126.55, 123.06, 113.56, 109.06, 105.89, 101.81, 42.92, 30.87, 19.78, 14.12; MS (m/z): 329;
HRMS (ES)-m/z: [M+H]+, found 330.1276. C17H19N3O2S requires 330.1276.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9e). Dark green amorphous solid, yield 79%, m.p. = 124–125 °C. 1H-NMR δ (ppm): 13.77 (s,
1H, NH), 7.27–7.32 (m, 1H), 7.22 (s, 1H), 6.89–7.06 (m, 4H), 6.57 (d, 1H, J = 15.45 Hz), 6.07 (s, 2H,
-O-CH2-O-), 4.03 (t, 2H, J = 7.57 Hz, -NH-CH2-CH2-CH2-CH2-CH2-CH3), 1.62–1.64 (m, 2H,
-NH-CH2-CH2-CH2-CH2-CH2-CH3), 1.27–1.30 (m, 6H, -NH-CH2-CH2-CH2-CH2-CH2-CH3), 0.84–0.90
(m, 3H, -NH-CH2-CH2-CH2-CH2-CH2-CH3); 13C-NMR δ (ppm): 166.73, 149.79, 148.48, 137.58,
137.19, 131.35, 126.54, 123.06, 113.56, 109.07, 105.89, 101.81, 43.09, 31.26, 28.66, 26.06, 22.47,
14.36; MS (m/z): 357; HRMS (ES)-m/z: [M+H]+, found 358.1589. C19H23N3O2S requires 358.1589.

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5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-cyclohexyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9f). Yellow amorphous solid, yield 91%, m.p. = 192–194 °C. 1H-NMR δ (ppm): 13.76 (s, 1H,
NH), 7.19 (d, 1H, J = 1.89 Hz), 7.19–7.24 (m, 1H), 7.11–7.17 (m, 1H), 6.99 (dd, 1H, J = 10.00 and
5.00 Hz), 6.93 (d, 1H, J = 8.20 Hz), 6.85 (d, 1H, J = 15.13 Hz), 6.75 (d, 1H, J = 14.50 Hz), 6.05 (s,
2H, -O-CH2-O-), 4.55–4.75 (bs, 1H, -NH-CH-(CH2-CH2)2-CH2), 1.33–2.08 (m, 10H, -NH-CH-(CH2-
CH2)2-CH2); 13C-NMR δ (ppm): 166.63, 149.63, 148.47, 148.21, 141.20, 137.22, 131.45, 126.80,
122.97, 114.63, 109.06, 105.82, 101.81, 55.45, 32.35, 26.13, 24.87; MS (m/z): 355; HRMS (ES)-m/z:
[M+H]+, found 356.1433. C19H21N3O2S requires 356.1432.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione
1
(9g). Yellow amorphous solid, yield 89%, m.p. = 244–245 °C. H-NMR δ (ppm): 14.00 (s, 1H, NH),
7.58–7.65 (m, 3H), 7.44 (dd, 2H, J = 8.04 and 1.42 Hz), 7.20 (d, 1H, J = 1.58 Hz), 7.15 (dd, 1H,
J = 15.00 and 10.00 Hz), 6.94–7.00 (m, 2H), 6.89 (d, 1H, J = 7.88 Hz), 6.78 (d, 1H, J = 15.45 Hz),
6.04 (s, 2H, -O-CH2-O-) 5.94 (d, 1H, J = 15.45 Hz);13C-NMR δ (ppm): 168.03, 149.83, 148.25,
137.79, 137.37, 134.02, 131.31, 130.10, 129.06, 126.54, 123.29, 113.40, 108.93, 105.91, 101.75; MS
(m/z): 349; HRMS (ES)-m/z: [M+H]+, found 350.0963. C19H15N3O2S requires 350.0963.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-benzyl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (9h). Yellow amorphous solid, yield 76%, m.p. = 187–188 °C. 1H-NMR δ (ppm): 13.97 (s, 1H,
NH), 7.37–7.40 (m, 2H), 7.28–7.32 (m, 3H), 7.21 (d, 1H, J = 1.26 Hz), 7.21–7.26 (m, 1H), 6.93 (d,
1H, J = 15.45 Hz), 6.93–6.99 (m, 2H), 6.85 (d, 1H, J = 15.45 Hz), 6.42 (d, 1H, J = 15.45 Hz), 6.06 (s,
2H, -O-CH2-O-), 5.35 (s, 2H); 13C-NMRδ (ppm): 167.49, 150.06, 148.30, 137.94, 137.38, 136.42,
131.24, 129.24, 128.21, 127.37, 126.35, 123.20, 113.40, 109.01, 105.95, 101.80, 45.92; MS (m/z): 363;
HRMS (ES)-m/z: [M+H]+, found 364.1120. C20H17N3O2S requires 364.1119.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-(3,4,5-trimethoxyphenyl)-2,4-dihydro-3H-
1
1,2,4-triazole-3-thione (9i). Pale yellow amorphous solid, yield 81%, m.p. = 242–243 °C. H-NMR δ
(ppm): 13.94 (s, 1H, NH), 7.21 (d, 1H, J = 1.58 Hz), 7.17–7.22 (m, 1H), 7.03 (dd, 1H, J = 15.00 and
10.00 Hz), 6.96 (dd, 1H, J = 10.00 and 5.00 Hz), 6.90 (d, 1H, J = 7.88 Hz), 6.81 (d, 1H, J = 15.00 Hz),
6.80 (s, 2H), 6.03 (d, 1H, J = 15.00 Hz), 6.04 (s, 2H, -O-CH2-O-), 3.80 (s, 6H), 3.78 (s, 3H); 13C-NMR
δ (ppm): 168.01, 153.73, 150.06, 148.43, 148.22, 138.41, 137.58, 137.16, 131.39, 129.53, 126.74,
123.23, 113.65, 108.94, 106.85, 105.89, 101.75, 60.59, 56.77; MS (m/z): 439; HRMS (ES)-m/z:
[M+H]+, found 440.1280. C22H21N3O5S requires 440.1280.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-tert-butyl-2,4-dihydro-3H-1,2,4-triazole-3-
1
thione (9j). Yellow amorphous solid, yield 50%, m.p. = 183–184 °C. H-NMR δ (ppm): 7.63 (s, 1H,
NH), 7.22 (s, 1H), 6.84 (d, 1H, J = 15.06 Hz), 6.89–7.04 (m, 4H), 6.48 (d, 1H, J = 15.31 Hz), 6.06 (s,
2H, -O-CH2-O-), 1.36 (s, 9H, -C-(CH3)3); 13C-NMR δ (ppm): 161.97, 157.91, 148.42, 148.12, 136.76,
135.29, 131.41, 126.51, 122.87, 113.17, 109.00, 105.87, 101.76, 51.50, 28.79; MS (m/z): 329; HRMS
(ES)-m/z: [M+H]+, found 330.1276. C17H20N3O2S requires 330.1276.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-[4-(methylthio)phenyl]-2,4-dihydro-3H-
1,2,4-triazole-3-thione (9k). Pale yellow amorphous solid, yield 75%, m.p. = 209–211 °C. 1H-NMR δ

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(ppm): 13.98 (s, 1H, NH), 7.47 (d, 2H, J = 10.00 Hz), 7.37 (d, 2H, J = 10.00 Hz), 7.20 (d, 1H, J = 1.00
Hz), 7.16–7.22 (m, 1H), 7.00 (dd, 1H, J = 15.00 and 10.00 Hz), 6.95 (dd, 1H, J = 10.00 and 5.00 Hz),
6.90 (d, 1H, J = 8.03 Hz), 6.81 (d, 1H, J = 15.31 Hz), 6.04 (s, 2H, -O-CH2-O-), 5.95 (d, 1H, J = 15.56
Hz), 2.52 (s, 3H, -S-CH3); 13C-NMR δ (ppm): 148.48, 141.36, 139.57, 136.96, 131.24, 126.30, 125.57,
123.52, 122.53, 121.67, 108.99, 106.22, 101.83, 15.63; MS (m/z): 396; HRMS (ES)-m/z: [M+H]+,
found 396.0840. C20H18N3O2S2 requires 396.0840.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-
1
triazole-3-thione (9l). Yellow amorphous solid, yield 80%, m.p. = 248–249 °C. H-NMR δ (ppm):
13.97 (s, 1H, NH), 7.53 (t, 1H, J = 8.16 Hz), 7.20 (d, 1H, J = 1.51 Hz), 7.13–7.18 (m, 2H), 7.04–7.05
(m, 1H), 6.95–6.99 (m, 3H), 6.90 (d, 1H, J = 8.03 Hz), 6.80 (d, 1H, J = 15.56 Hz), 5.97 (d, 1H,
J = 15.31 Hz), 6.04 (s, 2H, -O-CH2-O), 3.83 (s, 3H); 13C-NMR δ (ppm): 167.62, 160.36, 149.81,
148.40, 148.22, 137.71, 137.27, 135.00, 131.32, 130.87, 126.57, 123.23, 121.07, 115.74, 114.84,
113.42, 108.91, 105.88, 101.73, 56.01; MS(m/z): 379; HRMS (ES)-m/z: [M+H]+, found 380.1069.
C20H18N3O3S requires 380.1068.
5-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-4-[4-(trifluoromethyl)phenyl]-2,4-dihydro-
1
3H-1,2,4-triazole-3-thione (9m). Brown amorphous solid, yield 70%, m.p. = 232–233 °C. H-NMR δ
(ppm): 14.09 (s, 1H, NH), 8.03 (d, 2H, J = 8.53Hz), 7.75 (d, 2H, J = 8.28 Hz), 7.20 (d, 1H, J = 1.51
Hz), 7.13–7.18 (m, 1H), 6.95–7.00 (m, 2H), 6.90 (d, 1H, J = 8.03Hz), 6.83 (m, 1H), 5.96 (d, 1H,
13
J = 15.31 Hz), 6.04 (s, 2H, -O-CH2-O-); C-NMR δ (ppm): 167.78, 149.61, 148.41, 148.27,137.87,
137.70, 137.55, 131.29, 130.29, 130.30, 127.04 (q, JC-F = 270.00 Hz)127.19, 126.51, 123.26, 113.14,
108.93, 105.82, 101.75; MS(m/z): 417; HRMS (ES)-m/z: [M+H]+, found 418.0837. C20H15F3N3O2S
requires 418.0837.
3.2. Biological Assays
3.2.1. Parasites
Trypanosoma cruzi (Y strain) was obtained from the Fundação Oswaldo Cruz (Rio de Janeiro,
Brazil) culture collection, and maintained in the Laboratory of Glycobiology (UFRJ, Rio de Janeiro, Brazil)
facilities. Epimastigote forms were maintained by weekly transfers at 28 °C in brain-heart-infusion
(BHI) medium (Acumedia, Lansing, MI, USA), supplemented with 10 mg of hemin (Sigma, St. Louis,
MO, USA), 20 mg of folic acid L−1 (Sigma) and 10% heat-inactivated fetal calf serum (FCS) (BHI-FCS
medium, Gibco, Grand Island, NY, USA). Tissue culture-derived trypomastigotes were obtained after
infection of confluent monolayers of Vero cells (kidney epithelial cells extracted from African green
monkeys) with blood trypomastigotes (Y strain) to establish the intracellular cycle and maintained in RPMI
1640 medium containing 10% FCS under an atmosphere of 5% CO2 at 37 °C. The tissue culture-derived
trypomastigotes were used to infect Mø in vitro to evaluate the toxic effects of the drugs [14].
3.2.2. Anti-Epimastigote Effect
For evaluation of the anti-epimastigote effects, parasites (1 × 105 cells per mL) were subcultured in
the absence or presence of different concentrations (0.5 to 25 μg.mL−1) of the drugs 9a–9m, from a

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5 mg mL−1 stock solution in DMSO (Sigma). The parasites were incubated in BHI-FCS medium with
or without drugs in a final volume of 1.0 mL in 24-well plates (TPP, Trasadingen, Switzerland), and
after 7 days, the toxic effects of the drugs were quantified by the direct count of the live epimastigotes
in a Neubauer chamber [14]. Drug-free medium contained comparable final concentration of DMSO
(0.05%) has been used as control.
3.2.3. Cytotoxicity to Macrophages
The evaluation of the toxic effects of the compounds was carried out as previously described [18].
Murine peritoneal Mø were seeded (1 × 106 cells per well) in 24-well plates with 1 mL of RPMI
medium containing 10% FCS. The cells were allowed to attach for 24 h at 37 °C and then exposed for
72 h to the five compounds (9c, 9d, 9e, 9f and 9m) which showed highest activity against epimastigote
forms (maximum final concentration of DMSO was 0.05%). The five drugs were assayed in increasing
concentrations (0.5, 1.0, 2.5, 5.0 µg mL−1). Afterwards, the cells were washed with PBS, and RPMI
was added to the culture before the addition of vital dye trypan blue in a final concentration of 0.01%.
The toxic effects of the drugs were monitored by the count of 200 cells in a Neubauer chamber where
plasma membrane permeability was evaluated [13,18].
3.2.4. Anti-Amastigote Effect
Resident peritoneal Mø from non-infected BALB/c mice were harvested and cultured as described
above. Adhered Mø were infected with T. cruzi trypomastigotes at a 10:1 parasite/macrophage ratio
and incubated at 37 °C under 5% CO2 for 1 h. Subsequently, non-infective cells were removed by
extensive washing with PBS, and the infected macrophage cultures were treated with increasing
concentrations (0.5, 1.0, 2.5, 5.0 µg mL−1) of the compounds 9c, 9d, 9e, 9f and 9m which showed
anti-epimastigote activity and non-toxic effects on Mø at the same concentrations assayed. After,
monolayers were washed with PBS at 37 °C, fixed in methanol, and stained with Giemsa (Sigma). The
amastigote survival was determined by the counting of 200 cells in triplicate, the percentage of
infected cells was analyzed, as well as the number of amastigotes per macrophage and the endocytic
index of the infected cells [13,18].
3.2.5. Statistical Analysis
The 50% inhibitory concentrations (IC50) values shown in the Table 1 represent the mean of
experiments carried out in triplicate. The IC50 of all compounds were determined by linear regression
analysis using the program IGOR Pro 2.03 (WaveMetrics, Inc, Lake Oswego, OR, USA).
3.2.6. Ethics Statement
All animal procedures were approved by the animal-care ethics committee of the Centro de
Ciências da Saúde/UFRJ (License #DAHEICB 055) and were performed under the guidelines from
SBCAL (Brazilian Society of Science in Laboratory Animals) and strictly followed the Brazilian law
for Procedures for the Scientific Use of Animals (11.794/2008).

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4. Conclusions
The results shown herein highlight the potential use of natural piperine as a precursor for new
molecules suitable for the treatment of Chagas’s disease. Through the use of bioisosterism and
molecular hybridization as molecular modification strategies we were able to design and synthesize a
new series of 1,2,4-triazole-2-thiones, derivatives of the natural amide piperine, which showed activity
against proliferative forms of T. cruzi. The cyclohexyl triazole derivative 9f showed the best activity
profile, presenting IC50 = 18.3 and 8.87 μM, against epimastigotes and amastigotes of Trypanosoma
cruzi, respectively, and low toxic effects on host cells (murine macrophages).
Acknowledgments
We thank CAPES for scholarship to T.N. Franklim. The authors are also grateful for financial
supports from FAPERJ, CNPq, CAPES and PROCAD-NF. The authors wish to acknowledge Dr.
Victor M. Rumjanek (UFRRJ) for reading the manuscripts.
Conflict of Interest
The authors declare no conflict of interest.
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