Synthesis, activity, and molecular modeling studies of 1,2,3-triazole derivatives from natural phenylpropanoids as new trypanocidal agents

Article abstract of DOI:10.1111/cbdd.13628

The search for compounds with new structural scaffolds is an important tool to the discovery of new drugs against Chagas disease. We report herein the synthesis of 1,2,3-triazoles obtained from eugenol and di-hydroeugenol and their in vitro and in vivo tr

Full text of DOI:10.1111/cbdd.13628

Received: 22 May 2019
Revised: 12 August 2019
Accepted: 21 September 2019
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DOI: 10.1111/cbdd.13628
R E S E A R C H A R T I C L E
Synthesis, activity, and molecular modeling studies of 1,2,3‐
triazole derivatives from natural phenylpropanoids as new
trypanocidal agents
Thiago Belarmino de Souza1
Camila Coelho Rodrigues3
Ivo Santana Caldas2
Favero Reisdorfer Paula3
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Diogo Teixeira Carvalho4
Danielle Ferreira Dias5
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1Escola de Farmácia, Universidade Federal
de Ouro Preto, Ouro Preto, Brazil
Abstract
2Instituto de Ciências
The search for compounds with new structural scaffolds is an important tool to the
discovery of new drugs against Chagas disease. We report herein the synthesis of
1,2,3‐triazoles obtained from eugenol and di‐hydroeugenol and their in vitro and in
vivo trypanocidal activity. These derivatives were obtained by a three‐step objective
Biomédicas, Universidade Federal de
Alfenas, Alfenas, Brazil
3Universidade Federal do Pampa,
Uruguaiana, Brazil
route and were suitably characterized by H and 13C nuclear magnetic resonance
1
4Faculdade de Ciências
spectroscopy and high‐resolution mass spectrometry. Two compounds (9 and 10)
showed activity against epimastigote forms of Trypanosoma cruzi (Y strain) in the
range 42.8–88.4 μM and were weakly toxic to cardiomyoblast cells (H9c2 cells). The
triazole 10 was the most active derivative and could reduce more than 50% of para-
sitemia after a 100‐mg/kg oral treatment of mice infected with T. cruzi. Molecular
docking studies suggested this compound could act as a trypanocidal agent by in-
hibiting cruzain, an essential enzyme for T. cruzi metabolism, usually inhibited by
triazole compounds.
Farmacêuticas, Universidade Federal de
Alfenas, Alfenas, Brazil
5Instituto de Química, Universidade Federal
de Alfenas, Alfenas, Brazil
Correspondence
Thiago Belarmino de Souza, Escola
de Farmácia, Universidade Federal de
Ouro Preto, Campus Morro do Cruzeiro,
35400‐000, Ouro Preto, MG, Brazil.
Email: thiagobs83@yahoo.com.br
K E Y W O R D S
Funding information
1,2,3‐triazoles, cruzain inhibitors, di‐hydroeugenol, eugenol, trypanocidal activity
Fundação de Amparo à Pesquisa do Estado
de Minas Gerais, Grant/Award Number:
APQ‐01268‐16 and CDS‐APQ‐03701‐17;
Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior, Grant/Award
Number: PNPD
2009), anti‐parasitic (Machado et al., 2011), and antioxidant
(Gülçin, 2011) actions.
1
INTRODUCTION
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Eugenol (4‐allyl‐2‐methoxyphenol) is a natural phenylpro-
panoid present in essential oils of some plants as India's
clove (Syzygium aromaticum) and cinnamon‐brava (Croton
zenhtneri). Several authors have reported different biological
activities for eugenol, analogues, and derivatives, such as the
antimicrobial (Dai et al., 2013; Yadav, Chae, Im, Chung, &
Song, 2015), antitumoral (Manikandan, Senthil, Priyadarsini,
Vinothini, & Nagini, 2010), anti‐inflammatory (Daniel et al.,
In view of this great interest, our research group has been
working on the chemical manipulation of this allylphenol
and its analogues, as isoeugenol and di‐hydroeugenol, in
order to identify new active derivatives from them. Recently,
we have discovered some derivatives of eugenol with anti-
bacterial (Cazelli et al., 2017; Souza et al., 2015) and anti-
fungal (Abrão et al., 2015; Souza et al., 2016, 2014) actions
(Figure 1).
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Chem Biol Drug Des. 2019;00:1–6.
wileyonlinelibrary.com/journal/cbdd
© 2019 John Wiley & Sons Ltd
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de SOUZA et Al.
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Following our efforts in obtaining optimized deriva-
sodium azide (Carvalho et al., 2010). The reaction of alkyl
azides with different alkynes by a click reaction afforded
the triazoles 7–14 in good yields after purification by col-
umn chromatography, following the procedure described
by Souza et al. (2015). The click reaction is a 1,3‐dipo-
lar cycloaddition catalyzed by Cu(I) and has been largely
employed for the synthesis of biologically actives 1,2,3‐
triazoles (Deobald et al., 2011; Freitas et al., 2011). In
tives of eugenol, we have designed a new structural pat-
tern containing the eugenol or di‐hydroeugenol nucleus
attached to a 1,2,3‐triazole ring. This structural pattern
was designed considering the biological versatility of eu-
genol or di‐hydroeugenol combined with different reports
of di‐substituted 1,2,3‐triazole derivatives active against
T. cruzi (Andrade et al., 2015; Silva et al., 2008; Porta et
al., 2017). This heterocycle is isostere of 1,2,4‐triazole and
imidazole rings present in different drugs as fluconazole,
ketoconazole, benznidazole, and metronidazole. Then, we
hypothesized that the triazole core could help to improve
the antiparasitc properties of phenylpropanoids. Thus,
we synthesized these derivatives and evaluated them as
trypanocidal agents by in vitro and in vivo activity tests.
Further, we performed molecular docking studies to check
its ability to interact with cruzain as a possible target in
T. cruzi.
1
the H nuclear magnetic resonance spectra of compounds
7–14, it was possible to observe a signal corresponding to
the triazole proton near 8 ppm, besides the diastereotopic
methylene protons as a double of doublets between 3.5 and
4.5 ppm.
2.2
In vitro assays
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The cytotoxicity of the compounds was evaluated against
cardiac cells obtained from neonatal rat cardiomyoblasts
(H9c2 cells). Following, the selectivity indexes (SI) of the
synthesized compounds could be determined. All deriva-
tives and the two phenylpropanoids were evaluated against
epimastigote forms of Trypanosoma cruzi by the resazurin
microtiter assay (Table 1). The triazole 10, obtained from
di‐hydroeugenol, showed inhibition at 42.8 µM against
this form of the parasite, similarly to that presented by the
control drug benznidazole. Although this derivative had a
lower SI than benznidazole, it can be considered an inno-
vative structural core for optimization and design of new
tripanocidal agents. Moreover, the triazole 10 was twice
as active as the corresponding eugenol derivative 9 (IC50
2
RESULTS AND DISCUSSION
Chemistry
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2.1
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The 1,2,3‐triazole derivatives from eugenol and di‐hy-
droeugenol were synthesized in a short and objective syn-
thetic route as shown in Scheme 1.
Firstly, eugenol (1) and di‐hydroeugenol (2) were con-
verted to the epoxides 3 and 4, respectively, by reactions
with epychlorohydrine (Jin et al., 2004), which in sequence
afforded the alkylazides 5 and 6 after ring opening with
FIGURE 1 Eugenol (I) and derivatives with antibacterial (II and III) and antifungal (IV, V, and VI) activities

de SOUZA et Al.
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88.4 µM) pointing the importance of the n‐propyl side
chain for this activity. It is possible to note among these
1,2,3‐triazoles that the phenyl group (present in derivatives
9 and 10) was the best substituent at the triazole core, be-
cause derivatives with hydroxymethyl (derivatives 7 and
8), acetyl (derivatives 11 and 12) or cyclohexyl (deriva-
tives 13 and 14) groups showed lower or no trypanocidal
activity.
Spartan software (Shao et al., 2006), and the results from
the main interactions with cruzain are shown in Tables 2
and 3. According to these studies, it was possible to indi-
cate the individual poses relative to a selective binding to
the cruzain active site.
The empirical scoring function of iGemdock is character-
ized by the sum of Van der Waals, H‐bonding, and electro-
static energies. At this point, the best poses for ligands 9 and
10 showed affinity energies of −102.25 and −102.05 kcal/
mol, respectively. Calculated values for both molecules were
similar to each other but the compound 9 showed lower Van
der Waal and higher H‐bonding values in comparison to
compound 10. No electrostatic interactions were observed
for these compounds and the amino acid residues in cruzain
active site.
The data of docking experiments indicate that the main
interactions between the triazoles 9 and 10 and cruzain ac-
tive site involve the residues GLY 23, CYS 25, TRP 26, SER
64, GLY 65, GLY 66, LEU 67, MET 68, LEU 160, HIS
162, and GLY 163. Additionally, the active interactions be-
tween the compounds 9 and 10 and cruzain active site are
shown in Figure 2. After applying the postscreening analy-
sis GLY 65 was detected as the main residue evolved in this
2.3
Docking studies
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Some azoles are known as compounds that show anti‐tryp-
anosomal activity and this action may come from cruzain
inhibition (Brak et al., 2010). The compounds synthesized
in this work present a di‐substituted 1,2,3‐triazole ring,
so they could act as such possibly by inhibiting this en-
zyme, which is crucial in trypanosomatides survival. In
the current study, a crystallographic complex formed by
this enzyme and a di‐substituted 1,2,3‐triazole inhibitor
(1,2,3‐triazole‐tetrafluorophenoxymethyl ketone) was se-
lected as the model (Brak et al., 2010). Docking studies
were carried using the conformers of minimal energy from
compounds 9 and 10 with chemical structures generated by
SCHEME 1 Synthesis of 1,2,3‐triazole derivatives from eugenol and di‐hydroeugenol

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de SOUZA et Al.
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ligand–receptor binding (with Z‐score −1.09 and WPharma
1.00). Some of these residues (CYS 25, GLY 65, GLY 66,
and LEU 67) are the same observed in the docking study per-
formed by Brak et al. (2010) with tetrafluorophenoxymethyl
ketone and the active site of cruzain (figure available in
“Supporting Information”).
According to the results generated by these docking stud-
ies, 1,2,3‐triazoles 9 and 10 interact in the same region at
the cruzain active site as observed in the model generated
with the 1,2,3‐triazole inhibitor (Brak et al., 2010). The main
intermolecular interactions are those observed between GLY
23 and the triazole ring/N3 (2.74 Å), CYS 25 thiol and the
N‐2 from the same ring (2.20 Å). Other important H‐bonding
interaction observed in this model occurs between the sec-
ondary alcohol and the GLY 163 nitrogen (2.5 Å). These re-
sults show a good predicted binding pose between derivatives
9 and 10 and suggest they may be candidates to structural
optimization to follow development as trypanocidal agents
acting probably as cruzain inhibitors, a special target used in
anti‐trypanosomatide drug design.
TABLE 1 In vitro activity (IC50) against epimastigote forms of
T. cruzi, cytotoxicity (CC50) against H9c2 cells and SI for eugenol (1),
di‐hydroeugenol (2) and derivatives 7–14
T. cruzi
IC50 (µM)
H9c2 cells
CC50 (µM)
Compound
Selectivity Index
1
2
7
8
383.3
658.6
>1,000
>1,000
144.4
319.4
141.4
155.3
>500
>500
363.6
375.0
655.1
>3.2
>1.8
—
1.1
1.6
3.6
—
a
—
293.9
88.4
42.8
—
9
10
11
12
13
14
Bzn
2.4
In vivo assay
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The two most potent derivatives against T. cruzi epimastigote
forms (compounds 9 and 10) were evaluated in mice infected
with T. cruzi (Y strain) trypomastigotes. These compounds
were given orally for seven consecutive days as suspensions
at 100 mg/kg weight, and benznidazole was used as the con-
trol drug (Table 4).
—
—
108.2
178.9
35.8
3.3
2.1
18.3
Abbreviation: —a: no significant activity; Bzn: Benznidazole; SI: CC50/IC50
.
TABLE 2 Docking results and Van der Waals (VDW), H‐bond, and electrostatic interactions (in kcal/mol) for derivatives 9 and 10 in
Trypanosoma cruzain active site (PDB code 3IUT), using IGemdock 2.1 software
Compounds
Affinity energy (Kcal/mol) VDW (Kcal/mol)
H‐bond (Kcal/mol)
Electrostatic (Kcal/mol)
9
10
−102.25
−102.05
−82.88
−87.37
−19.37
−14.68
0.0
0.0
FIGURE 2 Binding poses of triazoles 9 (blue) and 10 (purple) in the Trypanosoma cruzi cruzain active site (3IUT) calculated by Igemdock
2.1 and visualized using Chimera software (v. 1.10.1)

de SOUZA et Al.
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TABLE 3 Central Van der Waals and H‐bond interactions (in kcal/mol) of compounds 9 and 10 with the Trypanosoma cruzi cruzain active
site (PDB code 3IUT) applying the Residues Consensus Analysis using Igemdock 2.1 software
Compounds
interactionsa
GLY 23 CYS 25 TRP 26 SER 64 GLY 65 GLY 66 LEU 67 MET 68 LEU 160 HIS 162 GLY 163
9
−5.9
−4.0
−5.1
−6.3
−7.3
−5.4
−4.6
−5.5
−4.7
−5.4
0.0
Van der Waalsa
H‐Bonda
−5.8
−6.1
−6.5
−4.2
−3.5
−7.6
0.0
−6.3
0.0
−7.8
0.0
−7.1
0.0
−7.2
0.0
−6.7
0.0
−2.5
0.0
−6.3
−3.5
0.0
10
Van der Waalsa
H‐Bonda
−2.6
−5.1
−3.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
−3.5
aTotal interaction energy in kcal/mol.
TABLE 4 In vivo evaluation of compounds 9 and 10 in the
ACKNOWLEDGMENTS
reduction of parasitaemia
The authors acknowledge the funding agencies FAPEMIG
(APQ‐01268‐16; CDS‐APQ‐03701‐17: Programa pesquisa
para o SUS/CNPq/FAPEMIG) and CAPES/PNPD for the
financial support.
Compound
Number of parasites/0.1 ml blood
UA
Bzn
9
1,425.000
18.333
1,101.000
625.000
10
CONFLICT OF INTEREST
Note: The values represent the means of parasitemia obtained in peripheral
blood samples from mice infected with 5,000 of T. cruzi (Y strain) trypomastig-
otes in the maximum peak of parasitemia.
The authors declare no conflict of interest.
Abbreviation: UA, untreated animals.
ETHICAL APPROVAL
All experimental protocols were conducted in accordance
with the guidelines issued by the Brazilian College of Animal
Experimentation (COBEA) and approved by the Ethics
Committee in Animal Research at UNIFAL‐MG (number
59/2017).
Untreated infected animals showed maximal parasite-
mia (1.425 × 106 parasites/ml blood) as expected. The di‐
hydroeugenol derivative 10 reduced more than 50% of the
parasitemia when compared to the untreated animals. The
compound 9 showed no significant ability to reduce the in-
fection. Although compound 10 was not as good as benzni-
dazole in reducing parasitaemia, it is important to note that
it has an innovative structural core. As such it can be seen
as an alternative in acting against benznidazole‐resistant
parasites.
DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as all data have
been made available in the “Supplementary Material” section.
ORCID
3
CONCLUSION
|
Thiago Belarmino de Souza
org/0000-0003-3627-6407
Diogo Teixeira Carvalho
org/0000-0002-6362-2822
Danielle Ferreira Dias
org/0000-0001-9129-4734
https://orcid.
https://orcid.
Eight new 1,2,3‐triazoles were synthesized from eugenol and
di‐hydroeugenol and were assayed as trypanocidal agents.
Some important structure–activity relationships could be
noted for best trypanocidal activity and higher selectivity
index, as a preference for a propyl instead of an allyl side
chain in the phenylpropanoid residue and for a phenyl group
as the substituent attached to the triazole ring. Docking stud-
ies showed that the triazoles 9 and 10 interact with the active
site of cruzain similarly to a well‐known 1,2,3‐triazole inhib-
itor of this enzyme. In vivo studies showed that the triazole
10 could reduce parasitaemia in infected mice and, as such,
can be seen as a good prototype for the development of new
anti‐trypanosomal agents.
https://orcid.
REFERENCES
Abrão, P. H. O., Pizi, R. B., Souza, T. B., Silva, N. C., Fregnan, A. M.,
Silva, F. N., … Carvalho, D. T. (2015). Synthesis and biological
evaluation of new eugenol Mannich bases as promising antifungal
agents. Chemical Biology & Drug Design, 86, 459–465. https://doi.
org/10.1111/cbdd.12504

6
de SOUZA et Al.
|
Andrade, P., Galo, O. A., Carvalho, M. R., Lopes, C. D., Carneiro, Z.
A., Sesti‐Costa, R., … Carvalho, I. (2015). 1,2,3‐Triazole‐based
analogue of benznidazole displays remarkable activity against
Trypanosoma cruzi. Bioorganic and Medicinal Chemistry, 23,
6815–6826. https://doi.org/10.1016/j.bmc.2015.10.008
Machado, M., Dinis, A. M., Salgueiro, L., Custódio, J. B. A., Cavaleiro,
C., & Sousa, M. C. (2011). Anti‐Giardia activity of Syzygium aro-
maticum essential oil and eugenol: Effects on growth, viability,
adherence and ultrastructure. Experimental Parasitology, 127, 732–
739. https://doi.org/10.1016/j.exppara.2011.01.011
Brak, K., Kerr, I. D., Barrett, K. T., Fuchi, N., Debnath, M., Ang, K., …
Ellman, J. A. (2010). Nonpeptidic tetrafluorophenoxymethyl ketone
cruzain inhibitors as promising new leads for Chagas disease che-
motherapy. Journal of Medicinal Chemistry, 53, 1763–1773. https
://doi.org/10.1021/jm901633v
Carvalho, I., Andrade, P., Campo, V. L., Guedes, P. M. M., Sesti‐Costa,
R., Silva, J. S., … Field, R. A. (2010). ‘Click chemistry’ synthesis of
a library of 1,2,3‐triazole‐substituted galactose derivatives and their
evaluation against Trypanosoma cruzi and its cell surface trans‐sial-
idase. Bioorganic & Medicinal Chemistry, 18, 2412–2427. https://
doi.org/10.1016/j.bmc.2010.02.053
Cazelli, D. S. P., Barroso, M. E. S., Pizi, R. B., Orlandi, M., Souza, T.
B., Carvalho, D. T., … Endringer, D. C. (2017). The relationship be-
tween the antimicrobial activity of eugenol and the LPETG peptide
structure and associated analysis for docking purposes. Chemical
Papers, 71, 1877–1886. https://doi.org/10.1007/s11696-017-0181-0
da Silva, E. N., Menna‐Barreto, R. F. S., Pinto, M. D. C. F. R., Silva,
R. S. F., Teixeira, D. V., de Souza, M. C. B. V., … Pinto, A. V.
(2008). Naphthoquinoidal [1,2,3]‐triazole, a new structural moi-
ety active against Trypanosoma cruzi. European Journal of
Medicinal Chemistry, 43, 1774–1780. https://doi.org/10.1016/j.
ejmech.2007.10.015
Dai, J. P., Zhao, X. F., Zeng, J., Wan, Q. Y., Yang, J. C., Li, W. Z., …
Li, K. S. (2013). Drug screening for autophagy inhibitors based on
the dissociation of Beclin1‐Bcl2 complex using BiFC technique and
mechanism of eugenol on anti‐influenza a virus activity. PLoS ONE,
8, 1–16. https://doi.org/10.1371/journal.pone.0061026
Daniel, A. N., Sartoretto, S. M., Schmidt, G., Caparroz‐Assef, S. M.,
Bersani‐Amado, C. A., & Cuman, R. K. N. (2009). Anti‐inflamma-
tory and antinociceptive activities of eugenol essential oil in exper-
imental animal models. Brazilian Journal of Pharmacognosy, 19,
212–217. https://doi.org/10.1590/S0102-695X2009000200006
Deobald, A. M., Camargo, L. R. S., Alves, D., Zukerman‐Schpector, J.,
Corrêa, A. G., & Paixão, M. W. (2011). Click chemistry: An effi-
cient synthesis of heterocycles substituted with steroids, saponins,
and digitalis analogues. Synthesis, 24, 4003–4010.
Freitas, L. B. O., Ruela, F. A., Pereira, G. R., Alves, R. B., Freitas,
R. P., & Santos, L. J. (2011). The "click" reaction in the synthesis
of 1,2,3‐triazoles: Chemical aspects and applications. Química
Nova, 34, 1791–1804. https://doi.org/10.1590/S0100-40422
011001000012
Manikandan, P., Senthil, R. M., Priyadarsini, R. V., Vinothini, G., &
Nagini, S. (2010). Eugenol induces apoptosis and inhibits invasion
and angiogenesis in a rat model of gastric carcinogenesis induced
byMNNG. Life Science, 86, 936–941. https://doi.org/10.1016/j.
lfs.2010.04.010
Porta, E. O. J., Jäger, S. N., Nocito, I., Lepesheva, G. I., Serra, E. C.,
Tekwani, B. L., & Labadie, G. R. (2017). Antitrypanosomal and an-
tileishmanial activity of prenyl‐1,2,3‐triazoles. MedChemComm, 8,
1015–1021. https://doi.org/10.1039/c7md00008a
Shao, Y., Molnar, L. F., Jung, Y., Kussmann, J., Ochsenfeld, C.,
Brown, S. T., … Head‐Gordon, M. (2006). Advances in methods
and algorithms in a modern quantum chemistry program package.
Physical Chemistry Chemical Physics, 8, 3172–3191. https://doi.
org/10.1039/B517914A
Souza, T. B., Brito, K. M. O., Silva, N. C., Rocha, R. P., Sousa, G. F.,
Duarte, L. P., … Dias, D. F. (2016). New eugenol glucoside‐based
derivative shows fungistatic and fungicidal activity against oppor-
tunistic Candida glabrata. Chemical Biology & Drug Design, 87,
83–90. https://doi.org/10.1111/cbdd.12625
Souza, T. B., Orlandi, M., Coelho, L. F. L., Malaquias, L. C. C., Dias,
A. L. T., Carvalho, R. R., … Carvalho, D. T. (2014). Synthesis and
in vitro evaluation of antifungal and cytotoxic activities of eugenol
glycosides. Medicinal Chemistry Research, 23, 496–502. https://doi.
org/10.1111/cbdd.12625
Souza, T. B., Raimundo, P. O. B., Andrade, S. F., Hipólito, T. M. M.,
Silva, N. C., Dias, A. L. T. D., … Dias, D. F. (2015). Synthesis
and antimicrobial activity of 6‐triazolo‐6‐deoxy eugenol gluco-
sides. Carbohydrate Research, 410, 1–8. https://doi.org/10.1016/j.
carres.2015.04.002
Yadav, M. K., Chae, S. W., Im, J. G., Chung, J. W., & Song, J. J.
(2015). Eugenol: A phyto‐compound effective against methicillin‐
resistant and methicillin‐sensitive Staphylococcus aureus clinical
strain biofilms. PLoS ONE, 10, 1–21. https://doi.org/10.1371/journ
al.pone.0119564
SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section at the end of the article.
Gülçin, I. (2011). Antioxidant activity of eugenol: A structure–activity
relationship study. Journal of Medicinal Food, 14, 975–985. https://
doi.org/10.1089/jmf.2010.0197
Jin, C., Nagasawa, H., Shimamura, M., Uto, Y., Inayama, S., Takeuchi,
Y., … Hori, H. (2004). Angiogenesis inhibitor TX‐1898: Synthesis
of the enantiomers of sterically diverse haloacetylcarbamoyl‐2‐
How to cite this article: de Souza TB, Caldas IS,
Paula FR, Rodrigues CC, Carvalho DT, Dias DF.
Synthesis, activity, and molecular modeling studies of
1,2,3‐triazole derivatives from natural
phenylpropanoids as new trypanocidal agents. Chem
Biol Drug Des. 2019;00:1–6. https://doi.org/10.1111/
cbdd.13628
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