Design, synthesis, trypanocidal activity, and studies on human albumin interaction of novel s-alkyl-1,2,4-triazoles

Article abstract of DOI:10.21577/0103-5053.20190033

Chagas disease is a neglected tropical disease caused by the hemoflagellated parasite Trypanosoma cruzi (Kinetoplastida). The only available drug to treat chagasic patients in Brazil, the nitroheterocycle benznidazole, is effective solely during the acute phase of the infection. There is accordingly a need to develop new therapeutic tools for the treatment of Chagas disease. This work reports the synthesis, trypanocidal evaluation and human serum albumin (HSA) interactions of a novel series of 1,2,4-triazoles. The new derivatives were synthesized via microwave irradiation in good yields. Most compounds showed toxic effects against T. cruzi with low toxicity to host cells. Three S-alkylated-triazoles showed the best activity profile against amastigotes, with half maximal inhibitory concentration (IC₅₀) values of 3.95 ± 1.41, 4.15 ± 0.92 and 3.61 ± 0.65 μmol L^-1, respectively. The interaction between HSA and 3-[(1E,3E)-4-(1,3-benzodioxol-5-yl)buta-1,3-dien-1-yl]-5-(butylthio)-4-cyclohexyl-4,5-dihydro-1H-1,2,4-triazole was investigated using multiple spectroscopic techniques and molecular docking, revealing that serum albumin is a potential endogenous carrier to this compound in the human bloodstream.

Full text of DOI:10.21577/0103-5053.20190033

http://dx.doi.org/10.21577/0103-5053.20190033
J. Braz. Chem. Soc., Vol. 30, No. 7, 1378-1394, 2019
Printed in Brazil - ©2019 Sociedade Brasileira de Química
Article
Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin
Interaction of Novel S-Alkyl-1,2,4-triazoles
Tatiany N. Franklim,a,b Leonardo Freire-de-Lima,b Otávio A. Chaves,a
Isabel F. LaRocque-de-Freitas,b Joana D. da Silva-Trindade,a José C. Netto-Ferreira,a,c
Célio G. Freire-de-Lima,b Debora Decoté-Ricardo,d José O. Previato,b
Lucia Mendonça-Previatob and Marco E. F. de Lima *,a
aInstituto de Química, Departamento de Química Orgânica, Universidade Federal Rural do Rio de Janeiro,
BR 465, km 07, 23890-000 Seropédica-RJ, Brazil
bInstituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Cidade Universitária, Ilha do Fundão, 21941-902 Rio de Janeiro-RJ, Brazil
cDivisão de Metrologia Química, Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO),
25250-020 Duque de Caxias-RJ, Brazil
dDepartamento de Microbiologia e Imunologia Veterinária, Instituto de Veterinária,
Universidade Federal Rural do Rio de Janeiro, BR 465, km 07, 23890-000 Seropédica-RJ, Brazil
Chagas disease is a neglected tropical disease caused by the hemoflagellated parasite
Trypanosoma cruzi (Kinetoplastida). The only available drug to treat chagasic patients in Brazil,
the nitroheterocycle benznidazole, is effective solely during the acute phase of the infection. There
is accordingly a need to develop new therapeutic tools for the treatment of Chagas disease. This
work reports the synthesis, trypanocidal evaluation and human serum albumin (HSA) interactions
of a novel series of 1,2,4-triazoles. The new derivatives were synthesized via microwave irradiation
in good yields. Most compounds showed toxic effects against T. cruzi with low toxicity to host
cells. Three S-alkylated-triazoles showed the best activity profile against amastigotes, with half
maximal inhibitory concentration (IC50) values of 3.95 1.41, 4.15 0.92 and 3.61 0.65 μmol L-1,
respectively. The interaction between HSA and 3-[(1E,3E)-4-(1,3-benzodioxol-5-yl)buta-1,3-dien-
1-yl]-5-(butylthio)-4-cyclohexyl-4,5-dihydro-1H-1,2,4-triazole was investigated using multiple
spectroscopic techniques and molecular docking, revealing that serum albumin is a potential
endogenous carrier to this compound in the human bloodstream.
Keywords: Trypanosoma cruzi, piperine, molecular hybridization, 1,2,4-triazole-3-thioether,
human serum albumin
Introduction
most neglected infections. Approximately 8 million people
in 21 countries in Latin America are infected with T. cruzi.
Of these patients, 20-30% will progress to the chronic phase
of the infection, developing severe and irreversible cardiac,
digestive, and neurological disorders.1 Furthermore, CD is
the parasitic disease with the highest socioeconomic impact,
being responsible for the loss of approximately US$ 1.2 billion
a year in productivity in Latin America.1,2 This infection is
spreading to the United States and Europe, because of the
conjunction of the increasing population exodus that occurs
from poor countries towards these regions and the current
climate changes that facilitate the adaptation of vector insects
to different regions.3,4
Chagas disease (CD), also known as American
trypanosomiasis, is a chronic parasitic illness that, since its
first description in 1909, had no effective chemotherapeutic
treatment.1 CD is caused by the hemoflagellate
protozoan Trypanosoma cruzi (T. cruzi; Kinetoplastida,
Trypanosomatidae) and is considered to be one of the human
diseases most related to underdevelopment and poverty.
These socioeconomic characteristics make critical the
situation of millions of chagasic patients that face one of the
*e-mail: marco@ufrrj.br

Vol. 30, No. 7, 2019
Franklim et al.
1379
The availability of drugs applicable to the treatment
of CD is extremely limited in the acute phase. Only the
nitroheterocycles benznidazole and nifurtimox are used
as anti-chagasic drugs. These drugs have no efficacy in
the chronic phase of the disease showing serious and
deleterious side effects.5,6 Different molecular targets
have been investigated, with the aim of developing new
alternatives for the treatment of CD.5-8 The structural
diversity of natural products points out them as an attractive
alternative for the development of new and more effective
anti-chagasic drugs.9 Several classes of natural products and
their derivatives have shown promising anti T. cruzi activity.
Among them we can highlight quinones,10,11 terpenes,12
alkaloids,13 and diarylheptanoids.14
In planning the use of natural products as drugs or as raw
materials for drug preparation, it is important to consider
that most special metabolites of plant origin are ordinarily
biosynthesized in small quantities; are difficult to isolate;
and in most cases are not obtained from sustainable sources.
Despite the great contribution of natural products in drug
discovery, these limitations remain as a bottleneck for the
use of products isolated from natural sources straight as
precursors of new drugs. The natural amide piperine (1,
Figure 1) is an exception among the special metabolites
of plant origin. This natural amide, present in different
species of the genus Piper, is most abundant in the fruits
of Piper nigrum.15
have demonstrated the toxic effects of piperine derivatives
and analogues against different evolutive forms of
L. amazonensis and T. cruzi.20-24
Azole derivatives were evaluated for CD experimental
treatment and exhibited highly promising results. These
azoles are commercially available drugs or molecules
that were tested against other type of infections.25-28 The
triazole ring has been applied in the development of new
anticonvulsant, antidepressant, anti-inflammatory, analgesic,
anticancer and anti-infective drugs and as a scaffold for
the synthesis of other bioactive molecules.29,30 Recent
studies have reported that 3-nitrotriazole-based piperazides
derivatives showed activity against Trypanosoma brucei
rhodesiense.31 Furthermore, a series of 1,2,3-triazole
benznidazole analogs were synthesized and many of them
exhibited activity comparable to that of the standard drug
against trypomastigotes, with selective toxicity.2
Recently, we described the synthesis and trypanocidal
evaluation of a new series of N4-substituted 1,2,4-triazole-
3-thiones (e.g., 2) prepared from natural piperine (Figure 1).24
This heterocyclic core is an important framework for the
development of bioactive molecules in several therapeutic
fields.32 The new derivatives synthesized were planned
through molecular hybridization33 as the main strategy for
molecular modification. In the molecular planning of the
new 1,2,4-triazole-3-thiones, the antifungal prothioconazole
(Figure 1) was used as a starting point. Prothioconazole is a
known inhibitor of the enzyme 14-α-demethylase (CYP51),
which plays an important role in T. cruzi cell membrane
composition and structure due to its involvement in the
biosynthesis of ergosterol.34 The design of the new derivatives
was based on the conjugation of the known trypanocidal
effects of piperine to the ability of molecules possessing
a triazole moiety to block the activity of CYP51, since
nitrogen containing heterocycles play a pharmacophoric
role in the structure of CYP51 inhibitors, by anchoring
Piperine was isolated for the first time about
200 years ago. Since then, it has attracted great interest
due to its biological properties.16-19 The structural
features of piperine (1) allow the planning of different
synthetic manipulations. In addition, its occurrence in
significant amounts in the fruits of Piper nigrum renders
it a very promising starting material for the design of
new bioactive molecules. Studies from our group aimed
at tracing a structure-activity relationship (SAR) profile
Figure 1. Chemical structure of the N4-cyclohexyl-1,2,4-triazole-3-thione (2) prepared based on both piperine (1) and prothioconazole structures (3).

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Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
to the porphyrin iron present at the active site of the
enzyme.35 Among the triazole derivatives prepared, the
N4-cyclohexyl-1,2,4-triazole (2, Figure 1) was the one with
the best trypanocidal profile with half maximal inhibitory
concentration (IC50) values of 18.30 and 8.87 μmol L-1
against the epimastigote and amastigote forms of T. cruzi,
respectively. Additionally, these compounds exhibited low
toxic effects against host cells (murine macrophages) with
85-95% cell viability at the highest tested drug concentration
(median lethal dose (DL50) > 14.08 μmol L-1).24
of two subdomains (A and B); subdomain IIA presents
the Trp-214 residue, which is responsible for the intrinsic
fluorescent properties of HSA. This intrinsic fluorescence
is extensively used in ligand binding studies.39-41
Experimental
General: organic synthesis
Melting points were determined on a Büchi B-510
(Flawil, Switzerland) apparatus and were not corrected.
The 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker Ultrashield Plus Spectrometer
(BrukerBioSpin GmbH, Rheinstetten, Germany) operating
at 500 MHz for 1H and 125 MHz for 13C. 1H and 13C NMR
shifts (d) are reported in parts per million (ppm) with
respect to dimethyl sulfoxide (DMSO-d6) and acetone
Herein a novel series of eight 1,2,4-triazole derivatives
was planned with the N4-cyclohexyl-1,2,4-triazole moiety
(2, Figure 1)24 as a starting point; our goal was to optimize
the antiparasitic profile of this substance. The trypanocidal
activities of the novel derivatives prepared were assessed
against the different evolutive forms of T. cruzi as well as their
toxic effects against murine macrophages. The novel triazoles
were prepared (Figure 2) with changes in both the substitution
at the sulfur atom of the thioether function (4a-4d),36
and the butadienyl-benzodioxole moiety (5-8). The new
derivatives and analogues were designed to obtain additional
information on the SAR for this family of compounds.
Additionally, it was evaluated the interaction between
one of the prepared triazoles with the best activity profile
and human serum albumin (HSA), which is the most
abundant human plasma protein. HSA has attracted great
interest from the pharmaceutical industry because it can
bind to a remarkable variety of endogenous and exogenous
molecules, impacting their delivery and efficacy, and
altering mainly their pharmacokinetic properties.37 Thus,
binding affinity of any substance to HSA is one of the
major factors that determine its pharmacokinetics (e.g., the
time course of drug absorption, distribution, metabolism,
and excretion).38 From a structural point of view, HSA is a
non-glycosylated polypeptide (585 amino acid residues),
with a heart-shaped structure composed of three structurally
similar α-helical domains (I-III). Each domain consists
1
(DMSO-d6, 2.50 ppm for H and 39.7 ppm for 13C;
1
acetone-d6, 2.05 ppm for H and 206.7 ppm for 13C).
Coupling constants (J) were reported 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
(LRMS) 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 1 min and then increasing to 290 °C at a rate of
10 °C min-1 and holding for 40 min; injector temperature:
270 °C. High-resolution mass analysis was taken in the
positive ion mode under electrospray inoziation (ESI)
methods on a Bruker 9.4 T Apex-Qh (FT-ICR) (Bruker
DaltonikGmbH Life Sciences, Bremen, Germany). 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 pre-coated silica
Figure 2. New series of 1,2,4-triazoles (4a-4d and 5) derived from natural piperine and lower chain analogues (6-8).

Vol. 30, No. 7, 2019
Franklim et al.
1381
gel plates (0.25 mm layer thickness) in an appropriate
solvent and the spots were visualized under UV light (254
or 356 nm). Natural piperine used in this work was isolated
from dry fruits of Piper nigrum as previously described.24
1H NMR (500 MHz, acetone-d6) d 7.45 (dd, 1H, J 15.13 and
10.72 Hz), 7.01-7.14 (m, 3H), 6.81-6.87 (m, 3H), 6.04 (s, 2H,
–O–CH2–O–), 4.36 (bs, 1H, –N‑CH‑(CH2‑CH2)2‑CH2),
3.80 (m, 1H), 1.39 (d, J5 Hz, 6H, –S–CH–(CH2)2), 1.39-1.50
(m, 10H, –N‑CH‑(CH2‑CH2)2‑CH2) (Figure S4, SI section);
13C NMR (125 MHz, acetone-d6) d 152.89, 148.55, 148.38,
147.96, 135.71, 135.46, 131.58, 126.58, 122.20, 115.12,
108.36, 105.29, 101.43, 55.97, 39.48, 31.48, 25.75, 24.73,
22.75 (Figure S5, SI section); LRMS m/z, 397; HRMS (ES)
m/z, calcd. for C20H23N3O2S [M + H]+: 398.19022; found:
398.18967 (Figure S6, SI section).
General procedure for the synthesis of new 1-cyclohexyl-
2-thioalkyl-1,2,4-triazole derivatives (4a-4d)
Triazole-thione 2 (100 mg, 0.28 mmol) was obtained
according to previous work procedures.24 Compound 2 was
dissolved in 5 mL of ethanol in a 10 mL round bottom flask
equipped with a magnetic stirring bar and a reflux apparatus.
To this solution KOH (33.6 mg, 0.60 mmol) dissolved in
ethanol (2 mL) was added dropwise. The reaction mixture
was then subjected to microwave irradiation (MW: 100 W)
for 5 min in open vessel mode. Thereafter, the appropriate
stoichiometric amount of the corresponding alkyl halide
(methyl, isopropyl, n-butyl and benzyl iodides) was added.
The mixture was subjected to MW for 1 h and the evolution
of the reaction was monitored by TLC. After elimination
of solvent under reduced pressure, the solid formed was
suspended in 5% KOH aqueous solution and extracted three
times with ethyl acetate. The organic solvent was evaporated
under reduced pressure, giving the desired product in
appropriate degree of purity after recrystallization from
ethanol.All compounds were characterized by NMR as well
as by low- and high-resolution mass spectra (HRMS). The
yields, melting points and spectral data observed for each
compound (4a-4d) are described below:
3-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-
5-(butylthio)-4-cyclohexyl-4,5-dihydro-1H-1,2,4-triazole (4c)
Yellow amorphous solid, yield 65%, m.p. 138-140 °C;
1H NMR (500 MHz, acetone-d6) d 7.43 (dd, 1H, J 14.98 and
10.88 Hz), 7.00-7.13 (m, 3H), 6.78-6.87 (m, 3H), 6.05 (s, 2H,
–O–CH2–O–), 4.29 (bs, 1H, ‑N‑CH‑(CH2‑CH2)2‑CH2),
3.24 (t, 2H, J 8.20 Hz, S–CH2), 1.31-1.94 (m, 14H), 0.95
(t, 3H, J 7.41 Hz, ‑S‑CH‑(CH2)2‑CH3) (Figure S7, SI
section); 13C NMR (125 MHz, acetone-d6) d 153.01, 148.99,
148.39, 147.95, 135.51, 135.14, 131.61, 126.61, 122.15,
115.15, 108.34, 105.28, 101.43, 55.83, 33.03, 31.51,
31.38, 25.74, 24.76, 21.53, 13.00 (Figure S8, SI section);
LRMS m/z, 411; HRMS (ES) m/z, calcd. for C23H29N3O2S
[M + H]+: 412.20587, found: 412.20532 (Figure S9, SI
section).
3-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-
1-yl]-5-(benzylthio)-4-cyclohexyl-4,5-dihydro-
1H-1,2,4-triazole(4d)
3-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-
4-cyclohexyl-5-(methylthio)-4,5-dihydro-1H-1,2,4-triazole
(4a)
Yellow amorphous solid, yield 62%, m.p. 140-
1
142 °C; H NMR (500 MHz, acetone-d6) d 7.28-7.41
Brown amorphous solid, yield 73%, m.p. 123-125 °C;
1H NMR (500 MHz, acetone-d6) d 7.42 (dd, 1H,
J 14.81 and 11.04 Hz), 6.99-7.12 (m, 3H), 6.76-6.86
(m, 3H), 6.03 (s, 2H, –O–CH2–O–), 4.24 (bs, 1H,
–N‑CH‑(CH2‑CH2)2‑CH2), 2.70 (s, 3H, –S–CH3),
1.47-1.98 (m, 10H, –N–CH‑(CH2‑CH2)2–CH2) (Figure S1,
Supplementary Information (SI) section); 13C NMR
(125 MHz, acetone-d6) d 153.28, 149.85, 148.38, 147.95,
135.61, 135.39, 131.61, 126.60, 122.12, 114.92, 108.33,
105.37, 101.40, 55.88, 31.29, 25.69, 24.77, 15.05
(Figure S2, SI section); LRMS m/z, 369; HRMS (ES)
m/z, calcd. for C20H23N3O2S [M + H]+: 370.15892, found:
370.15837 (Figure S3, SI section).
(m, 6H), 6.84-7.13 (m, 5H), 6.76 (d, 1H, J 15.45 Hz),
6.05 (s, 2H, –O–CH2–O–), 4.44 (s, 2H), 4.20 (bs,
1H, ‑N‑CH‑(CH2‑CH2)2‑CH2), 1.22-1.85 (m, 10H)
(Figure S10, SI section); 13C NMR (125 MHz, acetone-d6)
d 153.04, 148.51, 148.40, 147.97, 137.68, 135.65, 135.31,
131.58, 129.04, 128.53, 127.46, 126.56, 122.18, 115.12,
108.34, 105.28, 101.43, 55.94, 38.36, 31.31, 25.72, 24.69
(Figure S11, SI section); LRMS m/z, 445; HRMS (ES)
m/z, calcd. for C26H27N3O2S [M + H]+: 446.19022, found:
446.18967 (Figure S12, SI section).
General procedure for the synthesis of hydrazides 11, 14,
16 and 19
3-[(1E,3E)-4-(1,3-Benzodioxol-5-yl)buta-1,3-dien-1-yl]-
4-cyclohexyl-5-(isopropylthio)-4,5-dihydro-1H-1,2,4-triazole
(4b)
The corresponding carboxylic acid (1.0 mmol) and
oxalyl chloride (10 mmol) were added to a 10 mL round
bottom flask equipped with a magnetic stirring bar. A
rubber septum was used and the reaction was kept under
Yellow amorphous solid, yield 65%, m.p. 134-136 °C;

1382
Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
a dry N2 atmosphere. The resulting solution was subjected
to stirring at room temperature for about 0.5 h and the
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 oxalyl chloride
excess the residue was dissolved in dry dichloromethane
(3.0 mL). The resulting solution was added dropwise
to an ice-cooled mixture of hydrazine monohydrate
(15 mmol) and dichloromethane (5 mL) placed in a 25 mL
round bottom flask equipped with a stirring bar. A rubber
septum was used and the reaction was kept under a dry
N2 atmosphere. After reaching room temperature (about
30 min), the solvent was removed under reduced pressure.
The product precipitated after the addition of ice water
to the previously obtained residue and was then filtered,
leading to the formation of the desired hydrazides as a
solid material (72-91% yield).All the hydrazides obtained,
e.g., 11, 14, 16 and 19, were characterized by LRMS, 1H
and 13C NMR.
General procedure for the synthesis of the new 1,2,4-triazole-
3-thiones 5, 6, 7 and 8
Cyclohexylisothiocyanate (0.6 mmol) was added to
a suspension of the corresponding hydrazide (0.5 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. Then the reaction was subjected to
microwave irradiation (MW: 100W) for 0.5 h in open vessel
mode. The evolution of the reaction was monitored by TLC.
After total consumption of the starting hydrazide, all the
volatile compounds were removed under reduced pressure.
It was added an aqueous solution of NaOH (1 mmol in
2 mL of water) to the solid residue. This mixture was
then subjected again to additional 0.5 h of MW (100 W)
in open vessel mode. The evolution of the reaction was
monitored by TLC until its completion. Then, the mixture
was acidified until pH 3 with a 10% m/v HCl aqueous
solution. The solid obtained was filtered and washed twice
with ice water (5 mL). After recrystallization from ethanol
the corresponding cyclohexyltriazoles 5, 6, 7, and 8 were
obtained as yellow amorphous solids and characterized by
1H and 13C NMR and HRMS.
5-(1,3-Benzodioxol-5-yl)pentanohydrazide (11)
1
Yellow amorphous solid, yield 72%; H NMR
(500 MHz, acetone-d6) d 6.74-6.76 (m, 2H), 6.67-6.69 (m,
1H), 5.95 (s, 2H, –O–CH2–O–), 2.56-2.59 (t, 2H), 2.06-
2.07 (t, 2H), 1.63 (s, 4H); 13C NMR (125 MHz, acetone-d6)
d 165.17, 147.62, 136.39, 121.06, 108.67, 107.84, 100.73,
35.06, 33.21, 31.99, 31.33; MS m/z: 236.
5-[4-(1,3-Benzodioxol-5-yl)butyl]-4-cyclohexyl-2,4-dihydro-
3H-1,2,4-triazole-3-thione (5)
Yellow amorphous solid, yield 72%, m.p.
1
152-154 °C; H NMR (500 MHz, acetone-d6) d 6.88-
6.75 (m, 3H), 5.95 (s, 2H, –O–CH2–O–), 4.52 (bs,
1H, ‑NH‑CH‑(CH2‑CH2)2‑CH2), 2,10-2.84 (m, 6H),
1.15-2.00 (m, 12H) (Figure S13, SI section); 13C NMR
(125 MHz, acetone-d6) d 174.25, 152.28, 147.63, 145.70,
136.04, 121.13, 108.67, 107.89, 100.79, 56.22, 34.98,
34.82, 31.12, 30.78, 25.81, 24.88 (Figure S14, SI section);
LRMS m/z, 359; HRMS (ES) m/z, calcd. for C19H25N3O2S
[M + H]+: 360.17457, found 360.17402 (Figure S15, SI
section).
(2E)-3-(1,3-Benzodioxol-5-yl)acrylohydrazide (14)
1
Yellow amorphous solid, yield 80%; H NMR
(500 MHz, acetone-d6) d 9.2-9.6 (d, 1H), 7.6 (d, 1H),
7.4-7.5 (d, 1H), 7.1-7.2 (m, 1H), 6.9 (d, 1H), 6.1 (s, 2H,
–O–CH2–O–); 13C NMR (125 MHz, acetone-d6) d 165.21,
148.85, 148.38, 141.5, 129.9, 123.9, 115.9, 108.4, 106.4,
101.7; MS m/z: 206.
3-(1,3-Benzodioxol-5-yl)propanohydrazide (16)
1
Yellow amorphous solid, yield 77%; H NMR
5-[(E)-2-(1,3-Benzodioxol-5-yl)vinyl]-4-cyclohexyl-
2,4-dihydro-3H-1,2,4-triazole-3-thione (6)
(500 MHz, DMSO-d6) d 6.7-6.84 (m, 3H), 5.97 (s, 2H,
‑O‑CH2‑O‑), 2.77 (t, J 7.7 Hz, 2H), 2.41 (t, J 7.7 Hz,
2H); C NMR (125 MHz, DMSO-d6) d 170.80, 147.58,
145.86, 135.23, 121.55, 109.27, 108.54, 101.08, 35.64,
30.84; MS m/z: 208.
Yellow amorphous solid, yield 70%, m.p. 203-205 °C;
1H NMR (500 MHz, acetone-d6) d 7.45 (d, 1H,
J 15.76 Hz), 7.36 (s, 1H), 7.15 (d, 1H, J 7.88 Hz), 7.08
(d, 1H, J 16.08 Hz), 6.91 (d, 1H, J 8.20 Hz), 6.09 (s, 2H,
‑O‑CH2‑O‑), 4.89 (bs, 1H, –NH–CH–(CH2–CH2)2‑CH2),
1.30-1.93 (m, 10H, –N–CH–(CH2–CH2)2–CH2)
(Figure S16, SI section); 13C NMR (125 MHz, acetone-d6)
d 167.87, 149.86, 148.95, 148.51, 136.47, 129.98,
123.60, 109.84, 108.33, 105.90, 101.66, 55.72, 30.10,
25.88, 24.74 (Figure S17, SI section); LRMS m/z, 329;
HRMS (ESI-positive mode) m/z, calcd. for C17H19N3O2S
13
(2E)-3-(3-Methoxyphenyl)acrylohydrazide (19)
Yellow amorphous solid, yield 91%; 1H NMR (500 MHz,
acetone-d6) d 7.56-7.68 (m, 2H), 6.87-7.36 (m, 4H), 3.84 (s,
3H, –O–CH3); 13C NMR (125 MHz, acetone-d6) d 166.01,
160.16, 141.7, 140.31, 136.89, 129.88, 120.22, 115.41,
113.10, 54.71; MS m/z: 192.

Vol. 30, No. 7, 2019
Franklim et al.
1383
[M + H]+: 330.12762, found 330.12707 (Figure S18, SI
section).
was monitored by cell counting in a Neubauer chamber.42
Tissue culture-derived trypomastigotes (TCT) 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 Roswell Park
Memorial Institute (RPMI) 1640 medium containing 10%
(v/v) FBS under an atmosphere of 5% CO2 at 37 °C. The
TCT forms were collected from the culture supernatants,
washed with medium, and then used to infect murine
peritoneal macrophages (MP-Mf) (see below).
5-[2-(1,3-Benzodioxol-5-yl)ethyl]-4-cyclohexyl-2,4-dihydro-
3H-1,2,4-triazole-3-thione (7)
Yellow amorphous solid, yield 75%, m.p. 160-162 °C;
1H NMR (500 MHz, acetone-d6) d 6.74-6.81 (m, 2H),
6.56 (s, 1H), 5.96 (s, 2H, –O–CH2–O–), 4.14 (bs, 1H,
–NH–CH–(CH2–CH2)2–CH2), 2.84 (t, 2H, J 7.6 Hz),
2.59 (t, 2H, J 7.6 Hz), 1.16-1.35 (m, 10H, –N–CH–(CH2–
CH2)2–CH2) (Figure S19, SI section); 13C NMR (125 MHz,
acetone-d6) d 173.00, 145.94, 144.32, 134.95, 121.13,
108.72, 107.94, 100.84, 52.14, 35.35, 32.64, 30.37, 25.45,
24.81 (Figure S20, SI section); LRMS m/z, 331; HRMS
(ESI-negative mode) m/z, calcd. for C17H20N3O2S [M – H]–:
330.12762, found 330.12850 (Figure S21, SI section).
Anti-epimastigote effect
The toxic effect of the drugs on epimastigote forms was
evaluated as previously described.20-22,24 The drugs were
stored as 10 mg mL-1 stock solution in DMSO and used
in serial dilution (1:2) in BHI-FBS medium. Drug-free
control medium contained comparable final concentration
of DMSO (0.25%). Epimastigotes (2.0 × 105 cells) were
incubated in BHI-FBS medium with or without the drug
to a final volume of 2.0 mL in 24-well plates. After 7 days
of treatment, the toxic effect of the drugs was quantified
by the direct count of the live epimastigotes in a Neubauer
chamber.
4-Cyclohexyl-5-[(E)-2-(3-methoxyphenyl)vinyl]-2,4-dihydro-
3H-1,2,4-triazole-3-thione (8)
White amorphous solid, yield 81%, m.p. 202-204 °C;
1H NMR (500 MHz, DMSO-d6) d 7.28-7.40 (m, 4H), 7.15
(d, 1H, J 15.76 Hz), 6.95 (d, 1H, J 7.25 Hz), 4.72 (bs,
1H, –NH–CH–(CH2–CH2)2–CH2), 3.81 (s, 3H, –O–CH3),
1.13-1.86 (m, 10H) (Figure S22, SI section); 13C NMR
(125 MHz, DMSO-d6) d 166.96, 160.06, 149.59, 137.28,
130.33, 120.24, 114.99, 113.53, 55.65, 55.33, 30.51, 26.08,
25.05 (Figure S23, SI section); LRMS m/z, 315; HRMS
(ESI-positive mode) m/z, calcd. for C17H21N3OS [M + H]+:
316.14835, found 316.14781 (Figure S24, SI section).
Cytotoxicity to macrophages
The assessment of the toxic effects of each compound
was carried out as previously described.20-22,24 MP-Mf
adhered to 24-well plates were treated with the compounds
at the indicated concentrations (1-20 μg mL-1) for 72 h at
37 °C in 5% CO2. The cells were then washed and incubated
with the vital dye trypan blue in a final concentration
0.3%. The number of viable cells was scored by the count
of 200 cells where plasma membrane permeability was
evaluated.24
Ethical statement
All animal procedures were approved by the animal-
care ethics committee of the Centro de Ciências da Saúde,
UFRJ (License No. 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).
Anti-amastigote effect
Assays of anti-amastigote activity were performed in
MP-Mf. Cells were seeded at a density of 2.0 × 105 cells
per well in 24-well microplates with rounded coverslips
on the bottom. After infection in a parasite-to-cell ratio of
10:1 for 24 h at 37 °C, extracellular parasites were removed
by six washes with phosphate buffer solution (PBS),
pH 7.2, and fresh medium containing or not increasing
concentrations (1.0-20 μg mL-1) of each compound was
added. Seventy-two hours later, the coverslips were fixed
with methanol (Merck, Darmstadt, Germany), stained with
Giemsa (Sigma, St. Louis, MO, USA), and the number of
amastigotes per 100 cells was calculated by use of light
microscopy. Anti-amastigote activity was determined by
the counting of 200 cells in triplicate, where the percentage
Biological assays
Parasites
Trypanosoma cruzi (Y-strain) was provided by the
Trypanosomatid Collection of the Oswaldo Cruz Institute,
Fiocruz, Rio de Janeiro, Brazil. Parasites were grown at
28 °C for 7 days in brain-heart infusion medium (BHI,
BD Bacto, Sparks, Maryland, USA) supplemented with
10 μg L-1 hemin (Sigma, MO, USA), 20 μg L-1 folic acid
(Sigma, St. Louis, MO, USA), and 10% (v/v) fetal bovine
serum (FBS; Gibco, Dublin, Ireland). Parasite growth

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Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
of infected cells was analyzed, as well as the number of
where, Fcor and Fobs are the corrected and observed
fluorescence intensity values, respectively, whileAex andAem
represent the absorbance value at the excitation (λ = 280 nm:
ε = 23,905 cm-1 M-1, in PBS) and emission wavelengths
(λ = 340 nm: ε = 29,744 cm-1 M-1, in PBS).
amastigotes per MP-Mf.24
Statistical analysis
The 50% inhibitory concentrations (IC50) values
shown in Table 1 represent the mean of three experiments
carried out in triplicate. The IC50 of all compounds were
determined by linear regression analysis using the program
IGOR Pro 2.03.43
The UV-Vis absorption spectrum for 4c (1.32 × 10-5 M,
in PBS) was measured in the 200-600 nm range at room
temperature. The steady state fluorescence measurements
were carried out in the 290-450 nm range (λexc = 280 nm)
at 296, 303, 310, and 317 K and using 3.0 mL of an HSA
solution (1.00 × 10-5 M, in PBS). The addition of 4c to the
HSA solution was done manually by using a micro syringe,
achieving final concentrations of 0.17; 0.33; 0.50; 0.66;
0.83; 0.99; 1.15; and 1.32 × 10-5 M.
Table 1. Trypanocidal activity of new cyclohexyl-1,2,4-triazole-
3-thioethers 4a-4d and cyclohexyl-1,2,4-triazole-3-thiones 2, 5-8 against
replicative forms of T. cruzi (Y strain)
In order to obtain quantitative information on the binding
ability of the interaction between HSA and 4c, Stern-Volmer
(equation 2), double logarithmic (equation 3), modified
Stern-Volmer (equation 4), van’t Hoff (equation 5) and
Gibbs’ free energy (equation 6) analyses were applied:45,46
IC50 / (μmol L-1)
Triazole
Epimastigote
18.30 5.21
7.52 1.65
3.52 0.91
3.18 0.78
3.36 0.55
12.46 3.01
11.83 2.82
7.93 1.14
> 79
Amastigote
8.87 1.16
9.51 2.33
3.95 1.41
4.15 0.92
3.61 0.65
12.81 1.88
13.35 3.82
9.41 3.26
ntb
2a
4a
4b
4c
4d
(2)
(3)
(4)
5
6
7
8
Benznidazolec
2.20 0.16
2.50 0.21
aLead compound; bnot tested;creference drug.
Procedure for the studies of the interaction between HSA
and 4c: spectroscopic measurements
(5)
(6)
Commercially available HSA, PBS buffer (pH = 7.4),
warfarin, ibuprofen and digitoxin were obtained from
Sigma-Aldrich Chemical Company (St. Louis, MO,
USA). Water used in all experiments was Millipore grade.
Acetonitrile (spectroscopic grade) was obtained from Tedia
(Fairfield, OH, USA).
UV-Vis and steady state fluorescence spectra were
measured on a Jasco J-815 fluorimeter (Easton, MD, USA)
using a quartz cell (1.00 cm optical path) and employing
a thermostatic cuvette holder Jasco PFD-425S15F.
The circular dichroism spectra were measured in a
spectropolarimeter Jasco J-815 (Easton, MD, USA),
employing the same thermostatic cuvette holder as
described above.All spectra were recorded with appropriate
background corrections. Due the fact that 4c shows
absorption higher than 0.10 a.u. at 280 and 340 nm
(Figure S25, SI section), the correction for the inner filter
effect was made, according to the equation 1:44
where F0 and F are the steady state fluorescence
intensities of HSA in the absence and in the presence
of 4c, respectively. [Q], KSV and kq are the ligand
concentration, Stern-Volmer quenching constant and
bimolecular quenching rate constant, respectively. τ0 is the
experimental fluorescence lifetime of HSA in the absence
of 4c (5.46 0.08) × 10-9 s. Kb and n are the binding
constant and the number of binding sites, respectively. Ka
and ƒ are the modified Stern-Volmer binding and fraction
of the initial fluorescence intensity corresponding to the
fluorophore that is accessible by the quencher (ƒ ca. 1.00),
respectively. ΔH°, ΔS° and ΔG° are the enthalpy, entropy
and Gibbs’ free energy change, respectively. T and R
are the temperature (296, 303, 310 and 317 K) and gas
constant (8.3145 J mol-1 K-1), respectively.
The circular dichroism analysis was carried out using
3.0 mL of an HSA solution (1.00 × 10-6 M in PBS) in
(1)

Vol. 30, No. 7, 2019
Franklim et al.
1385
the absence and presence of 4c (1.32 × 10-5 M) in the
200-250 nm range, at 310 K. The intensity of the signal
from the circular dichroism spectra was expressed as
mean residue ellipticity (MRE), defined according to
equation 7:47
Theoretical calculations
clog P calculations
The chemical structures of the samples under study
(2, 4a-4d) were built and energy-minimized by density
functional theory (DFT) calculations (B3LYP potential)
with basis set 6-31G*, available in the Spartan’14
program.48
(7)
where θ, n, l and Cp are the observed circular dichroism
(in milli-degrees), number of amino acid residues (585 for
HSA),39 optical pathlength of the cell (1.00 cm) and molar
concentration of HSA (1.00 × 10-6 M), respectively.
To obtain quantitative information on the percentage of
changes in the α-helix content upon 4c binding, the results
of mean residue ellipticity (MRE) at 208 and 222 nm were
analyzed according to equations 8 and 9:47
Molecular docking studies on HSA:4c
The 4c structure was built and energy-minimized
by DFT calculations, with B3LYP potential and basis
set 6-31G*, available in the Spartan’14 program.48 The
crystallographic structure of HSA was obtained in the
Protein Data Bank (1N5U).39 The molecular docking
studies were performed with GOLD 5.2 program (CCDC,
Cambridge Crystallographic Data Centre).49
The hydrogen atoms were added to the albumin
structure according to the data inferred by GOLD 5.2
program49 on the ionization and tautomeric states. Docking
interaction cavity in the protein was established with a 10Å
radius from the Trp-214 residue. The number of genetic
operations (crossover, migration, mutation) in each docking
run used in the search procedure was set to 100,000. The
scoring function used was ‘ChemPLP’, which is the default
function of the GOLD 5.2 program.49 For more details, see
previous publications.44,47
(8)
(9)
Time-resolved fluorescence measurements were
performed on a model FL920 CD fluorimeter from
Edinburgh Instruments (Edinburgh, UK), equipped with an
electrically pumped laser (EPL, λexc = 280 10 nm; pulse
of 850 ps with energy of 1.8 μW per pulse; monitoring
emission at 340 nm). The time-resolved fluorescence
decay of a 3.0 mL solution of HSA (1.00 × 10-5 M, in
PBS) was measured in the absence and presence of 4c
(1.32 × 10-5 M).
Synchronous fluorescence spectroscopy (SFS) and
3D fluorescence were performed in a model Xe900
fluorimeter from Edinburgh Instruments (Edinburgh, UK).
Synchronous fluorescence spectra of a 3.0 mL solution
of HSA (1.00 × 10-5 M, in PBS) were measured without
and in the presence of 4c, in the 240-320 nm range by
setting Δλ = 60 and 15 nm for tryptophan and tyrosine
residues, respectively. The ligand concentration was the
same used in the steady state fluorescence studies at
room temperature. 3D fluorescence spectra of HSA were
recorded in the absence and presence of 4c, using an
excitation wavelength range of 200-340 nm and emission
wavelength range of 210-460 nm, at room temperature.
3D spectra were recorded for 3.0 mL of HSA solution
(1.00 × 10-5 M, at pH = 7.4) and for HSA:4c in the
maximum concentration of quencher used in the steady
state fluorescence measurements (1.32 × 10-5 M).
Results and Discussion
Chemistry
The synthesis of the novel triazoles derivatives
4a-4d from natural piperine 1 was based on molecular
hybridization strategy.33 The synthetic approach for the
preparation of triazole derivatives has been described by
Franklim et al.24 This approach yielded the N-cyclohexyl-
triazole derivative 2 in five steps and a 47% overall yield
from natural amide (Figure 1). Among the 13 triazoles
synthesized by Franklim et al.,24 derivative 2 exhibited the
best activity profile against T. cruzi with low toxic effects
against mammalian cells. For the preparation of the new
S-alkylated triazoles 4a-4d, the N4-cyclohexyl triazole 2
was reacted with different alkyl iodides (methyl, isopropyl,
n-butyl and benzyl) in refluxing basic ethanolic solution36
to afford the corresponding S-alkyl derivatives in yields
ranging from 62 to 73% (Scheme 1).
Synthesis of the saturated triazole 5 (Scheme 2, entry 1)
started with the catalytic reduction of the double bonds
present in the piperic acid 9.20,50 The resulting saturated
acid 10 generated the saturated hydrazide 11 by treating the

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Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
Scheme 1. Preparation of new S-alkylated N4-cyclohexyl-1,2,4-triazole 4a-4d. Reaction conditions: (a) suitable alkyl iodide, KOH, ethanol,
reflux (62-73% yield).
former with oxalyl chloride. Next, there was a reaction with
hydrazine monohydrate (Scheme 2, entry 1). The reaction
of hydrazide 11 with cyclohexyl-isothiocyanate, followed
by subsequent treatment of the respective carbothioamide
intermediate with ethanolic KOH under reflux in an one-pot
reaction, led to good yields of triazole 5 (Scheme 1, entry 1).
Synthesis of the lower vinyl homolog 6 (Scheme 2,
entry 2), a derivative with a carbon-carbon double bond
between the triazole group and the phenyl ring, started
with a Knoevenagel reaction between 3,4-methylenedioxy
benzaldehyde 12 and malonic acid, which generated the
unsaturated acid 13. Preparation of triazole 6 from acid 13
passed through hydrazide 14 using the same methodology
applied in the synthesis of triazole 5 (Schemes 2, entry 1).24
To obtain the triazole 7 (Scheme 2, entry 3) the saturated
precursor 15 was prepared via the hydrogenation of the
double bond present in the carboxylic acid 13 (Scheme 2,
entry 3). Once the carboxylic acid 15 was prepared, the
same methodology applied for the synthesis of the triazoles
5 and 6 was used,24 which generated a good yield of the
triazole 7.
For the preparation of triazole 8 (Scheme 2, entry 4), the
Knoevenagel reaction between 3-methoxy benzaldehyde
17 and malonic acid yielded the commercially available
α,β-unsaturated acid 17 (Scheme 1, entry 4), which was
precursor of triazole 8 via the same reaction sequence
described in the previous examples.24
All of the new triazoles prepared from natural
piperine, e.g. 4a-4d and 5, as well as the new modified
triazole analogues 6, 7 and 8 were fully characterized
and their respective spectrometric data (1H and 13C NMR,
MS, and HRMS) and melting points are described in
the Experimental section. All spectra are shown in the
Supplementary Information section.
benznidazole as positive controls. The data are summarized
in Table 1.
The novel synthesized triazole derivatives 4a-4d, 5-8
exhibited toxic effects against epimastigotes with IC50
values at or below 12.46 3.01 μmol L-1, the exception
was compound 8 (IC50 ≥ 79 μmol L-1). Then, all of the
compounds were evaluated for their cytotoxicity using
trypan blue exclusion test in peritoneal macrophages from
Balb/c mice. After 72 h of treatment, all concentrations
tested of the triazole compounds (0.5; 1.0; 2.5 and
5.0 μg mL-1) showed no significant toxicity for the host
cells; cellular viability ranged from 90-95% at the highest
concentration assessed.
The S-alkyl-triazoles 4a-4d showed significant
activity against epimastigote forms of T. cruzi with IC50
values ranging from 3.18 0.78 to 7.52 1.65 μmol L-1.
The introduction of the S-alkyl chain into the triazole
ring contributed to the increased activity against
epimastigotes compared with their precursor: the triazole 2
(IC50 = 18.30 5.21 μmol L-1).24 These results highlight
the lipophilicity of the sample, which may be a relevant
parameter for the trypanocidal activity exhibited by this
S-alkylated triazole series. To a better understanding of
the relationship between lipophilicity and trypanocidal
activity, the clogP values for 4a-4d triazoles were calculated
(Table 2). The best trypanocidal profile was achieved by
derivatives 4b, 4c and 4d, whose calculated clogP values
were 6.60, 7.18 and 7.67, respectively. These results suggest
an optimal lipophilicity range to the trypanocidal activity
that may be related to the ability of these molecules to cross
cell membranes. Similar correlations with different series
of compounds have been observed by other authors such as
Silva et al.51 These authors studied the tripanocidal activity
of 1-N- and 3-N-alkyl-naphthoimidazoles.
Compounds 5-7, which had their butenodienyl-
benzodioxole moieties modified, exhibited toxic activity
against epimastigotes (Table 1). The lack of trypanocidal
effect exhibited by the methoxylated derivative 8 (Table 1)
highlights the relevance of methylenedioxy substitution
in the aromatic ring to the toxic activity against T. cruzi.20
Compound 5, which has a saturated spacer chain, showed
an improved toxic activity against epimastigotes when
compared to the unsaturated triazole 2. This finding
Biological assays
The eight novel triazole derivatives prepared herein
were evaluated in vitro against the replicative epi- and
amastigote forms of T. cruzi (Y strain). The cytotoxicity
evaluation was assessed on murine macrophages. First, the
anti-epimastigote activity of the new derivatives 4a-4d and
5-8 were evaluated using triazole 2 and the commercial drug

Vol. 30, No. 7, 2019
Franklim et al.
1387
Entry 1
Reaction conditions: (a) H2, Pd/C, ethyl acetate, r.t., 30 min (89% yield); (b) (COCl)2, 30 min, r.t., then: NH2NH2.H2O, CH2Cl2, 273 K to r.t. (72% yield);
(c) cyclohexyl isothiocyanate, ethanol, MW, 30 min; (d) NaOH aq., MW, 30 min, then HCl aq. until pH 3.0 (72%; 46% yield from acid 9).
Entry 2
Reaction conditions: (a) malonic acid, piperidine (catalytic), pyridine (reflux), then, HCl aq. 10% (75% yield); (b), (c) and (d): same reaction conditions
used in entry 1 (61% yield for the three steps).
Entry 3
Reaction conditions: (a) H2, Pd/C, ethyl acetate, r.t., 30 min (89% yield); (b), (c) and (d): same reaction conditions used in entry 1 (57% yield for the
three steps).
Entry 4
Reaction conditions: (a) malonic acid, piperidine (catal.), pyridine (reflux), then HCl 10% (70% yield); (b), (c) and (d): same reaction conditions used in
entry 1 (73% yield for the three steps).
Scheme 2. Preparation of novel modified N4-cyclohexyl-1,2,4-triazole derivatives 5-8.

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Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
Table 2. Calculated clogP values for the triazole-thione 2 and its
S-alkylated derivatives 4a-4d
antiparasitic drug was able to decrease the fluorescence
emission of HSA (λem = 340 nm), indicating that it may be
located near to the Trp-214 residue.40 Furthermore, the blue
shift observed at the wavelength of the maximum emission
of HSA upon ligand binding (from 340 to 316 nm) is a clear
evidence of an increase in the hydrophobicity around the
Trp-214 residue.53
Triazole
clogP
5.08
5.94
6.60
7.18
7.67
2
4a
4b
4c
4d
suggests that the flexibility of this chain may be an
important structural feature for trypanocidal activity in
this series of compounds. This observation contrasts with
the results described by Otero et al.,52 where the double
bond saturation in a series of cinnamic acid esters resulted
in a total lack of leishmanicial activity compared to its
unsaturated precursor. Similarly, the saturated triazole 7
exhibited better activity than its unsaturated analogue 6
(Table 1). Compounds 6 and 7, containing two carbon
atoms in the spacer chain, exhibited a slight increase in
their trypanocidal activity when compared with their
higher homologues with four carbon atoms (i.e., 2 and 4c,
respectively), which highlights the fact that chain size may
have an impact on the toxic activity against the parasite.
All new derivatives exhibited significant toxic activity
against amastigotes of T. cruzi, particularly the S-alkylated
triazoles 4b, 4c and 4d, which showed trypanocidal activity
comparable to the standard drug benznidazole. These
triazoles had IC50 values of 3.95 1.41, 4.15 0.92 and
3.61 0.65 μmol L-1, respectively. Among the derivatives
that presented the highest toxic activity against T. cruzi, the
triazole 4c was selected as a model to study the interactions
with HSA, the main carrier protein in the human
bloodstream, which is responsible for the distribution of
several endogenous and exogenous molecules in the human
organism.38
Figure 3. Steady state fluorescence emission of HSA and its quenching
via successive additions of 4c at pH = 7.4 and 310 K. Inset: Stern-
Volmer plots for the interaction HSA:4c at 296, 303, 310 and 317 K.
[HSA] = 1.00 × 10-5 M and [4c] = 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15;
and 1.32 × 10-5 M.
Fluorescence quenching corresponds to a decrease
in fluorescence quantum yield induced by a variety of
molecular interactions with the quencher molecule, such
as excited-state reactions, molecular rearrangement, energy
transfer, ground-state complex formation, and collisional
quenching. However, in many instances the fluorophore can
be quenched both by collisions and by complex formation
with the same quencher.40
Table 3 lists the KSV and kq values at 296, 303, 310,
and 317 K. The kq values are in the order of 1012 M-1 s-1,
higher than the maximum diffusion rate constant for
water (kdiff ca. 7.40 × 109 M-1 s-1 at 298 K, according
to Smoluchowski-Stokes-Einstein theory at 298 K),55
suggesting that a ground-state association can occur
between HSA and 4c, which is characteristic of a static
mechanism.41 On the other hand, upward-trending curves
in the Stern-Volmer plot at high ligand concentration can
be observed (inset in the Figure 3). In addition, the KSV
values increase with increasing temperature (Table 3),
suggesting that the Trp-214 fluorescence can be quenched
both by collision and by complex formation with the same
quencher.40
Interaction between HSA and 4c
Binding characterization for HSA:4c
Steady state fluorescence spectroscopy can be used
to monitor perturbations on HSA upon ligand binding.
This interaction can lead to a decrease in the fluorescence
quantum yield of the Trp-214 residue, which can result in
changes in its position or orientation and consequently alter
its exposure to a solvent.53,54 Figure 3 depicts the steady
state fluorescence emission of HSA (1.00 × 10-5 M) and its
fluorescence quenching upon successive additions of 4c, at
310 K. It is worth noting that 4c exhibits some fluorescence
emission in the 400-450 nm range, which does not affect
the fluorescence behavior of the albumin. This potential
To further confirm the presence of both fluorescence
quenching mechanisms, static and dynamic, or the prevalence
of only a static mechanism on the HSA:4c interaction, time

Vol. 30, No. 7, 2019
Franklim et al.
1389
Table 3. Binding constant values and thermodynamic parameters (KSV, kq, n, Ka, ΔH°, ΔS° and ΔG°) for the interaction between HSA:4c at 296, 303, 310
and 317 K
T / K
296
303
310
317
KSV × 105 / M-1
4.46 0.22
4.86 0.31
5.13 0.33
5.64 0.36
kq × 1013 / (M-1 s-1)
n
Ka × 105 / M-1
1.66 0.26
1.73 0.26
1.83 0.26
1.90 0.26
ΔH° / (kJ mol-1)
5.13 0.29
5.13 0.29
5.13 0.29
5.13 0.29
ΔS° / (kJ mol-1 K-1)
0.117 0.001
0.117 0.001
0.117 0.001
0.117 0.001
ΔG° / (kJ mol-1)
–29.5
8.17
8.90
9.40
10.3
1.28 0.03
1.31 0.04
1.33 0.04
1.39 0.06
–30.3
–31.1
–32.0
T: temperature; KSV: Stern-Volmer quenching constant; kq: bimolecular quenching rate constant; n: number of binding sites; Ka: modified Stern-Volmer
constant; ΔH°: enthalpy; ΔS°: entropy; ΔG°: Gibbs’ free energy change.
resolved fluorescence measurements were carried out at
pH 7.4 for HSA in the absence and presence of the ligand
in a higher concentration than that used in the steady state
fluorescence quenching studies. Figure S26 and Table S1 (SI
section) depict the time-resolved fluorescence decay and its
parameters for HSA without and in the presence of 4c. Free
HSA showed two fluorescence lifetimes (τ1 = 1.56 0.09 ns
and τ2 = 5.46 0.08 ns), with the longest lifetime showing
the greatest contribution to the decay: 22.3% for τ1 and
77.7% for τ2. These results are in agreement with those
in the literature.54 The addition of 4c to the HSA solution
in PBS changes the albumin fluorescence lifetime inside
the experimental error, e.g. τ2 showed a slight decrease to
5.39 0.10 ns for HSA:4c at the ligand concentration of
1.32 × 10-5 M. The time-resolved fluorescence measurements
suggested that only a static process was functioning as the
main fluorescence quenching mechanism. This mechanism
involved the formation of a ground-state complex.56 The
natural product piperine (1) exhibited the same behavior
when associated with HSA.45,57
change (ΔS°) are important for the study of the interaction
forces.58 The thermodynamic parameters associated with
the interaction between HSA and 4c were calculated
according to the van’t Hoff (Figure S29, SI section) and
Gibbs’ free energy analysis.
The negative ΔG° values for the interaction HSA:4c
are consistent with the spontaneity of the binding process
in all temperatures under study (Table 3).56 The calculated
ΔH° and ΔS° values are positive, indicating that only
the entropy change value can contribute to ΔG° < 0,
suggesting that the association is entropically driven. In
other words, the unfavorable positive enthalpy change
(ΔH° = 5.13 0.29 kJ mol-1) is compensated by the
positive entropy change (ΔS° = 0.117 0.001 kJ mol-1 K-1),
suggesting that the binding of 4c to HSA may be governed
by desolvation and/or hydrophobic factors.59 In addition,
according to the Ross and Subramanian60 theory, hydrogen
bonding can also be considered as the main binding force
due to ΔH° > 0.
Based on double logarithmic analysis for HSA:4c, the
calculated number of binding sites (n) is approximately
1.00 at 296, 303, 310 and 317 K (Figure S27 and Table 3,
SI section), indicating the existence of just one main
binding site in the serum albumin structure for 4c.56
Since n ca. 1.00, the binding constant parameter can be
calculated according to the modified Stern-Volmer analysis
(Figure S28, SI section). Table 3 lists the Ka values for the
interaction HSA:4c; these values are on the order of 105 M-1,
the same order of magnitude noted for HSA:piperine.45
This findings indicates that 4c can interact strongly with
serum albumin.57,58 In addition, the slight increase in the Ka
values with increasing temperature suggests that at body
temperature (310 K), the antiparasitic triazole 4c presents
better binding ability than at lower temperatures.57
Investigation of HSA conformational changes
The conformational change of the HSA structure
induced by 4c binding was studied using circular dichroism
spectroscopy. Serum albumin exhibits two negative signals
at 208 (π-π* transition) and 222 nm (n-π* transition), which
are due to the amide forming groups of the peptide bonds.61
Figure S30 (SI section) depicts the circular dichroism
spectra for HSA in the absence and presence of 4c at
pH = 7.4 and 310 K.
The circular dichroism signal for HSA decreases with
the addition of 4c (Figure S30, SI section), indicating
changes in the protein structure upon ligand binding.41
However, the quantitative α-helical percentage content
for HSA with and without 4c did not change significantly,
varying from 63.4-55.9% at 208 nm and from 58.6-56.4%
at 222 nm. These results indicate that the presence of 4c
results in a moderate perturbation to the secondary structure
of the albumin.62,63
The non-covalent interaction forces between xenobiotics
and biomolecules may include hydrogen bonding,
electrostatic forces, van der Waals forces and hydrophobic
interactions. The sign and magnitude of the thermodynamic
parameters such as enthalpy change (ΔH°) and entropy
Synchronous fluorescence spectroscopy (SFS) is a
sensitive technique for analyzing micro-environmental

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Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
changes in a vicinity of a chromophore. This technique
involves simultaneous scanning of excitation and emission
monochromators of a fluorimeter, with a fixed wavelength
difference (Δλ) between them. In the case of HSA,
when Δλ = 15 and 60 nm the synchronous fluorescence
spectra exhibits the spectral character of the tyrosine and
tryptophan residues, respectively.64 These spectra are shown
in Figure 4 without and in the presence of successive
additions of 4c at room temperature (pH = 7.4). At
Δλ = 15 nm, there is no significant change in the maximum
fluorescence wavelength position upon increasing the
ligand concentration, indicating that 4c binding does not
exert any influence on the polarity around the tyrosine
residue.63 On the other hand, at Δλ = 60 nm, a slight blue
shift from 277 to 273 nm was observed, suggesting that the
presence of 4c can increase the hydrophobicity around the
Trp-214 residue.65,66 This result is fully consistent with the
steady state fluorescence results described above.
changes of the protein. Peak II (λex = 225 nm) is related
to the spectral behavior of Trp and Tyr residues caused
by a π→π* transition and reflects the polarity of the
microenvironment.68
The presence of 4c (1.32 × 10-5 M) in the HSA solution
led to a significant reduction in the fluorescence intensity
of the latter, showing 42.5 and 42.2% quenching for
peaks I and II, respectively. A decrease in the magnitude
of the Stokes shift, from 330 to 320 nm for peak I and
from 335 to 320 nm for peak II can also be observed.
These changes in the 3D fluorescence spectrum of
HSA when in the presence of 4c can fully confirm the
structural and microenvironment perturbations upon
drug binding.69 On the basis of comparing the 3D and
SFS results, there is strong evidence that the perturbation
on the microenvironment occurs mainly around the Trp
residue.
Recently, three-dimensional (3D) fluorescence
spectroscopy has become a significant analytical technique
for studying macromolecules, because it provides very
detailed information about the conformational changes in
proteins. This information may include a shift in excitation
or fluorescence emission wavelength, as well as the
appearance of a new peak or the disappearance of an already
existing peak.67 To further confirm the conformational and
microenvironmental changes observed for HSA from the
circular dichroism and SFS results described above, 3D
spectra were recorded for pure HSA and HSA:4c (Figure 5
and Table 4).
Peaks ‘a’ (λex = λem) and ‘b’ (2λex = λem) are referred to
Rayleigh and second order scattering peaks, respectively.58
Peak I (λex = 280 nm) mainly represents the structural
characteristics of the polypeptide backbone caused
by a π→π* transition of the amide group responsible
for the peptide bond and reflects the conformational
Competitive binding studies and theoretical analysis on
HSA:4c
The HSA structure possesses different binding sites
with distinct specificities, with domains I (Sudlow’s site I,
in subdomain IIA, warfarin binding site) and II (Sudlow’s
site II, in subdomain IIIA, ibuprofen binding site) being
considered the most important; however, the subdomain
IB (digitoxin binding site) is also an important site due
to its ability to interact with some ligands.39 To identify
the main binding site in the HSA structure responsible
for the interaction with 4c, competitive binding studies
were performed with the site markers warfarin, ibuprofen
and digitoxin (Figure S31, SI section). For the interaction
HSA:4c, without the presence of site markers, a Ka value
of (1.83 0.26) × 105 M-1 at 310 K was obtained (Table 3),
while Ka values of (1.52 0.26) × 105, (1.79 0.26) × 105
and (1.81 0.26) × 105 M-1 at 310 K were measured in the
presence of warfarin, ibuprofen and digitoxin, respectively.
Figure 4. Synchronous fluorescence spectra of HSA with and without successive additions of 4c at (a) Δλ = 15 nm and (b) Δλ = 60 nm. [HSA] = 1.00 × 10-5 M
and [4c] = 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15; and 1.32 × 10-5 M.

Vol. 30, No. 7, 2019
Franklim et al.
1391
Figure 5. 3D fluorescence spectral projections and the corresponding contour maps for HSA (A and B) and HSA:4c (C and D), at pH 7.4 and
r.t. [HSA] = 1.00 × 10-6 M and [4c] = 1.32 × 10-5 M.
Table 4. 3D fluorescence spectral characteristics for HSA and HSA:4c
at pH = 7.4 and r.t.
that the hydrogen atom from the guanidinium group of
Arg-221 residue is a donor for hydrogen bonding with
the sulfur group present in the ligand structure, within a
Peak position (λexc/λem) /
System
HSA
Peak
Intensity × 106 / a.u.
distance of 2.80Å. In addition, the peptide NH hydrogen of
theArg-484 residue is a donor group for hydrogen bonding
with one of the oxygen atoms present on the benzodioxole
ring of the ligand, within 2.40 Å. The molecular docking
nm/nm
220/220 → 360/360
230/460
a
b
I
2.55 → 2.05
1.56
280/330
1.27
II
a
225/335
2.89
220/220 → 360/360
230/460
2.80 → 2.20
1.93
b
I
HSA:4c
280/320
0.73
II
225/320
1.67
These results clearly indicate that warfarin alters Ka more
significantly (17.0%) than either ibuprofen (2.19%) or
digitoxin (1.10%). Therefore, one can conclude that
the main binding site for the association HSA:4c is the
Sudlow’s site I.62,70
To offer a molecular level explanation on the binding
ability of 4c toward HSA, theoretical studies using
molecular docking calculation were performed. Figure 6
shows the molecular docking results for the interaction
between HSA and 4c inside Sudlow’s site I, which suggest
Figure 6. Best score pose for HSA:4c in Sudlow’s site I (ChemPLP
function). Selected amino acids residues and 4c are represented in beige
and pink, respectively. Black dots represent the interaction via hydrogen
bonding. Hydrogen: white; oxygen: red; nitrogen: dark blue and sulfur:
yellow.

1392
Design, Synthesis, Trypanocidal Activity, and Studies on Human Albumin Interaction of Novel S-Alkyl-1,2,4-triazoles
J. Braz. Chem. Soc.
results also suggest the presence of hydrophobic interactions
between 4c and the Leu-197, Trp-214, Leu-237, Val-342,
Val-343, and Leu-480 residues. These theoretical results
are in full accordance with the intermolecular interactions
suggested by the experimental thermodynamic parameters
described above.59
Científico e Tecnológico (CNPq); Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior, Brasil
(CAPES), Finance Code 001; and Fundação de Amparo à
Pesquisa do Estado do Rio de Janeiro (FAPERJ). Authors
are grateful to Prof Dr Carlos M. R. Sant’Anna (Institute
of Chemistry, UFRRJ) for the molecular docking facilities
and Prof Dr Nanci Camara de Lucas Garden (Institute of
Chemistry, UFRJ) for the time-resolved, synchronous and
3D fluorescence facilities.
Conclusions
Eight new triazole derivatives, that is 4a-4d and
5-8, were synthesized from natural piperine using a
molecular hybridization strategy. All triazole derivatives
were fully characterized by spectrometric analysis.
Seven of the eight triazole derivatives (4a-4d, 5, 6 and 7)
displayed trypanocidal activities on proliferative forms of
T. cruzi, and low level of toxicity on host cells (murine
macrophages). Indeed, the S-alkylated triazole derivatives
4b, 4c and 4d exhibited trypanocidal activity comparable
to the standard drug benznidazole, with IC50 values
of 3.95 1.41; 4.15 0.92; and 3.61 0.65 μmol L-1,
respectively.
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Submitted: November 14, 2018
Published online: February 26, 2019
This is an open-access article distributed under the terms of the Creative Commons Attribution License.