Synthesis and antimetastatic activity evaluation of cinnamic acid derivatives containing 1,2,3-triazolic portions

Article abstract of DOI:10.1016/j.tiv.2018.07.015

It is herein described the preparation and evaluation of antimetastatic activity of twenty-six cinnamic acid derivatives containing 1,2,3-triazolic portions. The compounds were prepared using as the key step the Copper(I)-catalyzed azide (A)-alkyne (A) cycloaddition (C) (CuAAC reaction), also known as click reaction, between alkynylated cinnamic acid derivatives and different benzyl azides. The reactions were carried in CH₂Cl₂/H₂O (1:1 v/v) at room temperature, and the triazole derivatives were obtained in yields ranging from 73%–99%. Reaction times varied from 5 to 40 min. The identity of the synthesized compounds was confirmed by IR and NMR (¹H and ¹³C) spectroscopic techniques. They were then submitted to in vitro bioassays to investigate how they act over metastatic behavior of murine melanoma. The most potent compound, namely 3-(1-benzyl-1H-1,2,3-triazol-4-yl)propyl cinnamate (9a), showed significant antimetastatic and antiproliferative activities against B16-F10 cells. In addition, gelatin zymography and molecular docking analyses pointed to the fact that this compound has potential to interact with matrix metalloproteinase 9 (MMP-9) and MMP-2, which are directly involved in melanoma progression. Therefore, these findings suggest that cinnamic acid derivatives containing 1,2,3-triazolic portions may have potential for development of novel candidates for controlling malignant metastatic melanoma.

Full text of DOI:10.1016/j.tiv.2018.07.015

Accepted Manuscript
Synthesis and antimetastatic activity evaluation of cinnamic acid
derivatives containing 1,2,3-triazolic portions
Graziela Domingues de Almeida Lima, Michelle Peixoto
Rodrigues, Tiago Antônio de Oliveira Mendes, Gabriela Alves
Moreira, Raoni Pais Siqueira, Adalberto Manoel da Silva, Boniek
Gontijo Vaz, Juliana Lopes Rangel Fietto, Gustavo Costa Bressan,
Mariana Machado-Neves, Róbson Ricardo Teixeira
PII:
DOI:
Reference:
S0887-2333(18)30387-4
doi:10.1016/j.tiv.2018.07.015
TIV 4335
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
2 April 2018
25 June 2018
20 July 2018
Please cite this article as: Graziela Domingues de Almeida Lima, Michelle Peixoto
Rodrigues, Tiago Antônio de Oliveira Mendes, Gabriela Alves Moreira, Raoni Pais
Siqueira, Adalberto Manoel da Silva, Boniek Gontijo Vaz, Juliana Lopes Rangel Fietto,
Gustavo Costa Bressan, Mariana Machado-Neves, Róbson Ricardo Teixeira , Synthesis
and antimetastatic activity evaluation of cinnamic acid derivatives containing
1,2,3-triazolic portions. Tiv (2018), doi:10.1016/j.tiv.2018.07.015
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ACCEPTED MANUSCRIPT
Synthesis and antimetastatic activity evaluation of cinnamic acid derivatives
containing 1,2,3-triazolic portions
Graziela Domingues de Almeida Limaa§, Michelle Peixoto Rodriguesb§, Tiago Antônio
de Oliveira Mendesc, Gabriela Alves Moreirac, Raoni Pais Siqueirac, Adalberto Manoel
da Silvad, Boniek Gontijo Vaze, Juliana Lopes Rangel Fiettoc, Gustavo Costa Bressanc*,
Mariana Machado-Nevesa*, Róbson Ricardo Teixeirab*
aDepartamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas
Gerais, Brazil
bDepartamento de Química, Universidade Federal de Viçosa, Viçosa, Minas Gerais,
Brazil
cDepartamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa,
Viçosa, Minas Gerais, Brazil
dInstituto Federal de Educação, Ciência e Tecnologia Catarinense, Araquari, Santa
Catarina, Brazil
eInstituto de Química, Universidade Federal de Goiás, Goiânia, Brazil
§These authors contributed equally to this work
*Corresponding authors
E-mail
addresses:
gustavo.bressan@ufv.br
(Gustavo
Costa
Bressan);
mariana.mneves@ufv.br (Mariana Machado-Neves); robsonr.teixeira@ufv.br (Róbson
Ricardo Teixeira).

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ABSTRACT
It is herein described the preparation and evaluation of antimetastatic activity of twenty-
six cinnamic acid derivatives containing 1,2,3-triazolic portions. The compounds were
prepared using as the key step the Copper(I)-catalyzed azide (A)-alkyne (A)
cycloaddition (C) (CuAAC reaction), also known as click reaction, between alkynylated
cinnamic acid derivatives and different benzyl azides. The reactions were carried in
CH2Cl2/H2O (1:1 v/v) at room temperature, and the triazole derivatives were obtained in
yields ranging from 73%─99%. Reaction times varied from 5 to 40 min. The identity of
the synthesized compounds was confirmed by IR and NMR (1H and 13C) spectroscopic
techniques. They were then submitted to in vitro bioassays to investigate how they act
over metastatic behavior of murine melanoma. The most potent compound, namely 3-
(1-benzyl-1H-1,2,3-triazol-4-yl)propyl
cinnamate
(9a),
showed
significant
antimetastatic and antiproliferative activities against B16-F10 cells. In addition, gelatin
zymography and molecular docking analyses pointed to the fact that this compound has
potential to interact with matrix metalloproteinase 9 (MMP-9) and MMP-2, which are
directly involved in melanoma progression. Therefore, these findings suggest that
cinnamic acid derivatives containing 1,2,3-triazolic portions may have potential for
development of novel candidates for controlling malignant metastatic melanoma.
Keywords: cinnamic acid, 1,2,3-triazoles, antimetastatic, melanoma, click chemistry,
gelatinases, matrix metalloproteinase, virtual molecular docking

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1. Introduction
Malignant melanoma is an extremely aggressive and metastatic cancer
presenting the highest rate of mortality among skin cancers. In fact, less than 10% of
patients with metastatic melanoma survive up to 10 years after the diagnosis (Bathia et
al., 2009). Malignant melanoma can be cured by surgical intervention in its early stages,
but this approach is practically inefficient in its metastatic stage (Gray-Schopfer et al.,
2007; Elias et al., 2010). In this case, treatment is extremely difficult due to the
metastatic melanoma resistance to existing therapies. This resistance is related to the
complex cell signaling pathways modulating the proliferation and ability of tumor cells
to escape from apoptotic processes during metastasis (Gray-Schopfer et al., 2007;
Hanahan and Weinberg, 2011; Siegel et al., 2015; Agaësse et al., 2017). As the process
of tumor cells dissemination involves several events until the establishment of a
secondary tumor site, such as cellular detachment, migration, invasion, and adhesion, as
well as cell colonization and growth, these steps represent important therapeutic targets
(Kumar and Weaver, 2009; Guan, 2015). Hence, there is an urgent need to discover new
therapies for improving the current therapeutic strategies and, consequently, the survival
of cancer patients (Elias et al., 2010).
Natural products have been explored as a source of compounds for drug
discovery. Between 1981 and 2002, about 60% of the new drugs developed for cancer
treatments were derived from natural products (Gullo et al., 2006; Lam, 2007; Harvey,
2008; Schmidt et al., 2008; Dias et al., 2012). Compounds currently used in clinical
cancer chemotherapy, such as paclitaxel, etoposide, teniposide, and campthothecin are
examples of drugs based on the natural product pool (Ali et al., 2014).
The cinnamic acid (Fig. 1) consists of an aromatic acid found in several higher
plants. Typically, it occurs in nature in trans-geometry, and belongs to a class of plant
hormones regulating cell growth and differentiation known as auxins. This acid and its

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derivatives play an important role as intermediates in the production of other important
compounds, such as styrenes and stilbenes (Sharma et al., 2011). It has been
demonstrated that cinnamic acid derivatives display several activities including
antifungal, antibacterial, and anti-inflammatory (Chiriac et al., 2005).
The antimetastatic effect of cinnamic acid derivatives against tumor cell lines
has also been demonstrated (Yen et al., 2011; Tsai et al., 2013; Ling et al., 2015). For
instance, trans and cis forms of cinnamic acid exhibited anti-migratory activity against
human lung adenocarcinoma cells (Yen et al., 2011). A farnesyl thiosalicylic acid
(FTS)-diamine/cinnamic acid hybrid (1, Fig. 1) also showed significant anti-migratory
activity against colon carcinoma cells (Ling et al., 2015). Moreover, other derivatives of
cinnamic acid, such as caffeic acid (2), ferulic acid (3) and chlorogenic acid (4), were
capable of inhibiting the invasive behavior of human lung adenocarcinoma cells (Tsai et
al., 2013).
In this framework, we describe herein the synthesis of cinnamic acid derivatives
containing 1,2,3-triazolic portions. The Copper(I)-catalyzed azide (A)-alkyne (A)
cycloaddition (C) (CuAAC reaction), also known as click reaction, between cinnamates
having terminal triple bonds and different benzyl azides corresponded to the key step of
the synthetic route designed to obtain the twenty-six cinnamic acid derivatives
containing triazolic portions. Additionally, the antimetastatic activity of the derivatives
was screened against B16-F10 melanoma cell line. For that, the effects on cell
proliferation and colony formation were assessed using the most efficient compound
found in this study. Finally, gelatin zymography analyzes pointed to the fact that this
compound has the potential to reduce the matrix metalloproteinases (MMP) -9 and -2
activities, and the docking studies demonstrated that this compound have good score
and docking affinity with these enzymes.

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2. Materials and methods
2.1. Reagents and synthetic procedures
Solvents were purchased from Vetec (Rio de Janeiro, Brazil). Commercially
available pent-4-yn-1-ol, prop-2-yn-1-ol, benzyl alcohols, triethyl amine, dimethyl
sulfoxide
(DMSO),
N,N’-dicyclohexylcarbodiimide
(DCC),
4-N,N’-
dimethylaminopyridine (DMAP), sodium ascorbate, sodium azide and copper(II) sulfate
pentahydrate were purchased from Sigma Aldrich (St. Louis, MO, USA) and used
without further purification. Details concerning the preparation of the compounds
investigated herein can be found in the supplementary material.
2.2. Biological evaluation
2.2.1. Cell culture and culture conditions
Metastatic melanoma cells (B16-F10) were kindly provided by Dr. Anésia
Aparecida dos Santos (Department of General Biology, Universidade Federal de Viçosa
(UFV), Viçosa, Minas Gerais, Brazil). Embryonic fibroblasts cell line (NIH3T3) was
kindly provided by Dr. Leandro Licursi de Oliveira (Department of General Biology,
UFV, Viçosa, Minas Gerais, Brazil). The African green monkey kidney cell line (Vero)
was kindly provided by Dr. Juliana Lopes Rangel Fietto (Department of Biochemistry
and Molecular Biology, UFV, Viçosa, Minas Gerais, Brazil). Cell lines were grown in
RPMI-1640 medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS;
LGC Biotecnologia, São Paulo, Brazil), 100 μg mL-1 of streptomycin, and 100 units mL-
1 of penicillin (Sigma) at pH 7.2 and 37 ºC under 5% CO2 atmosphere.
2.2.2. Cell viability assay
Cell viability was determined with the MTT ((3-(4,5 dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide; Sigma). B16-F10 cells were seeded onto 96-well plates at
a concentration of 1 x 104 cells/well. Each well contained 100 μL of complete RPMI

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medium and 100 μL of each compound solution at different concentrations (0-200 μmol
L-1). The compounds 8a─8m and 9a─9m were diluted in RPMI medium with 10% FBS
plus 0.4% v/v DMSO (Sigma). After 48 h of culture, MTT (5 mg mL-1; Sigma) was
added to each well, and incubated for 4 h at 37 ºC. The MTT solution was then
removed, and DMSO at 100 µL/well was added to solubilize the formazan. Absorbance
was measured at 540 nm in a microplate reader (SpectraMax M5, Molecular Devices,
California, USA). Results were normalized considering the cultures treated with 0.4%
DMSO (control), and the half-maximal inhibitory concentration (IC50) was calculated
according to Siqueira et al. (2015).
2.2.3. Cell migration assay
The wound-healing assay is a method widely used to study directional cell
migration in vitro, which mimics cell migration during wound healing in vivo
(Rodrigues et al., 2005). Thus, it was conducted to evaluate the ability of cinnamic acid
derivatives to inhibit cell migration, based on a previous published methodology (Liang
et al., 2007; Anasamy et al., 2017) with modifications. B16-F10 cells were seeded onto
24-well plate at a concentration of 2 x 105 cells/well, and allowed to reach full
confluence after incubation overnight at 37 °C under 5% CO2 atmosphere. Monolayers
were then wounded using a sterile 200 µL pipette tip. Cells were washed twice with
phosphate buffered saline (PBS) to remove detached cells, and then treated with
compounds 8a─8m and 9a─9m at the concentration of 100 μmol L-1. The compounds
with inhibitory effect on cell migration were tested at the concentration of 50 μmol L-1.
DMSO (0.4% v/v) was used as control. Photos of the wound were taken using an
inverted microscope (EVOS fl, Life Technologies, Canada). Wound closure rates were
then calculated quantitatively as the difference between wound width at 0 and 24 h.
Results were expressed as percentage of cell migration.

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2.2.4. Cell proliferation assay
B16-F10 cells were seeded onto 6-well plates at a concentration of 1 x 105
cells/well in the presence of 100 μmol L-1compound 9a. Additionally, untreated cells
were used as control using DMSO (0.4% v/v). The effect of the compound on cell
growth was evaluated by trypan blue (Invitrogen, São Paulo, Brazil) dye exclusion.
After 24, 48, and 72 h, cells were loaded on a hemocytometer to obtain the viable cell
count (Pietraszek et al., 2013).
2.2.5. Cell invasion assay
This assay was carried out using transwell inserts with pore diameter of 8 µm
(Millicell, Ireland) based on protocols previously described (Chen, 2005; Hulkower and
Herber, 2011) with modifications. Briefly, 60 μL of matrigel (BD Biosciences) diluted
in serum free medium (1:12) was added to the upper chambers, and incubated for 1 h at
37 °C to gel formation. Thereafter, B16-F10 cells at 1 × 105 cells/well were diluted in
serum free medium and treated with compound 9a at different concentrations (12.50, 25
and 50 μmol L-1) for 1 h. Cells were then seeded onto the upper surface, whereas a
medium containing 10% FBS was added in the lower surface. After 24 h of incubation,
filter inserts were removed from the wells. The cells on the upper surface of the filter
were wiped with a cotton swab. The cells that invaded the lower surface were fixed with
methanol for 10 min, stained with 0.5% toluidine blue solution (Sigma), and then
counted under a microscope using ten randomly chosen fields/ group. DMSO-vehicle
treatment at 0.4% (v/v) was used as control. Results were expressed as percentage of
cell migration.
2.2.6. Cell-matrix adhesion assay
After treatment with 12.50, 25 and 50 μmol L-1 of compound 9a for 30 min, the
cells (5 × 104 cells/well) were seeded onto 96-well matrigel-coated plates, and incubated

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overnight at 37 °C under 5% CO2 atmosphere. Non-adherent cells were removed by
gentle washing with PBS. The adherent cells on the plate were measured by MTT assay.
2.2.7. Clonogenic assay
For clonogenic assay, B16-F10 melanoma cells (1000 cells per well) were
seeded onto a 6-well plate and incubated overnight at 37 °C under 5% CO2 atmosphere.
Subsequently, exhausted medium was changed with fresh medium containing 12.50, 25
and 50 μmol L-1 of compound 9a. Culture treatment was carried out for 24 h and the
cells were cultured for 14 days with growth medium replaced at every two days. On the
15th d, colonies were fixed with methanol, stained with 0.5% toluidine blue solution,
and finally counted (Mao et al., 2016). Results were expressed as percentage of
untreated control cultures.
2.2.8. Cytotoxicity on non-tumor cell lines
The cytotoxicity of compound 9a was tested on two non-tumor cell lines. Cell
NIH3T3 (1x104 cells/well) and Vero (3x104 cells/well) were seeded onto 96-well plates,
and cell viability was determined by MTT assay previously described (Pietraszek et al.,
2013).
2.2.9. Hemolysis assay
This assay was used to determine the compatibility of erythrocytes to compound
9a. Blood was collected from a five healthy C57BL/6J male mice, centrifuged at 700 g
for 10 min, washed three times with 0.9% saline solution (m/v), and resuspended in the
same buffer to prepare 2% red blood cell suspension (Xu et al., 2017). Subsequently,
different concentrations (0─100 µmol L-1) of the compound 9a were incubated with the
suspension at 37 °C. The red blood cells were also incubated with saline solution (0.9%
m/v) and triton (1% v/v) as negative and positive control, respectively. After 2 h, the
samples were centrifuged for 10 min at 700 g and it was determined the release of

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hemoglobin by photometric analysis at 540 nm. The described procedures above are in
agreement with the ethical principles of animal research adopted by the Animal Ethics
Committees (CEUA protocol number 27/2015).
2.2.10. Predicting ADMET properties of 9a
In order to predict ADMET (Absorption, distribution, metabolism, excretion,
toxicity) properties of compound 9a, it was utilized the pkCSM approach which uses
graph-based signatures to develop predictive models of central ADMET properties for
drug development (Pires et al., 2015). Herein, it was evaluated the in silico ADMET
characteristics of compound 9a using the freely accessible web server
http://biosig.unimelb.edu.au/pkcsm/prediction
and
the
SMILES
related to
O=C(OCCCC1=CN(CC2=CC=CC=C2)N=N1)\C=C\C1=CC=CC=C1
compound 9a.
2.2.11. Gelatin zymography
B16-F10 cells were incubated with 50, 100 and 150 μmol L-1 compound 9a for
24 h. The media were then collected and centrifuged at 1,500 rpm for 6 min at 4 °C to
remove cells debris. The cell-free supernatant was mixed with a 4X non reducing
sample buffer and electrophoresis was performed using precast gel (10%
polyacrylamide and 0.1% gelatin as a protease substrate). Following electrophoresis, the
gel was washed twice in 2.5% Triton X-100 for 1 h to remove sodium dodecyl sulfate
(SDS), subsequently washed in an activation buffer containing CaCl2 and incubated in
this buffer at 37 °C for 18 h. The gel was, then, stained with Coomassie blue R-250
(0.125% Coomassie blue R-250, 50% methanol, and 10% acetic acid) followed by
distaining (methanol/acetic acid/water, 40/10/50, v/v) (Lee et al., 2015). The protein
content was determined according to the method described by Bradford using BSA as a
standard (Bradford, 1976).

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2.2.12. Computational simulation of the interaction between compound 9a and MMP-9
and -2
Molecular docking analyses of the synthesized compounds 8a−8m and 9a9m
were simulated to determine the relative affinity with MMP-9 and MMP-2 enzymes
using AutoDock Vina release 1.1.2 (Trott and Olson, 2010). The crystal structure of
MMP-9 (PDB ID: 1L6J) and MMP-2 (PDB ID: 1QIB) was extracted from Protein Data
Bank. The activity site of enzymes was identified by selection of amino acids within 12
Å radius from zinc ions that participate of the catalytic mechanism. The 3D structures of
all compounds were drawn, optimized and protonate using Marvinsketch software
(Chemaxon®). Binding energy was estimated using Auto Docking Tools implemented
in PyRx plataform. Docked structures were visualized and edited using PyMOL
software.
2.3. Statistical Analysis
All numeric data were done in three independent replicates. The values were
expressed as mean ± standard error mean (S.E.M). Statistical analyses were performed
using Microsoft Excel (Microsoft Office Software), and GraphPad Prism (GraphPad
Software Inc.). Statistical analyses were submitted to one-way ANOVA followed by
Dunnett's test.
3. Results and discussion
3.1. Synthesis
The synthesis of cinnamic acid derivatives containing triazolic portions required
the preparation of esters 5 and 6, as well as a series of azides 7 (Fig. 2). Thus, the
Steglich esterification (Sova et al., 2006) between trans-cinnamic acid and different
alcohols afforded compounds 5 and 6 in good yields (68% and 83%, respectively) after
purification by silica gel column chromatography. Azides 7 were prepared from the

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corresponding benzylic alcohols (Fig. 2) using a methodology previously described
(Borgati et al., 2013).
The click reaction between esters 5 and 6 and azides 7 (Fig. 3) afforded the
desired cinnamic acid derivatives 8a─8m and 9a─9m in yields ranging from 73% to
99%. Further details concerning the structures of the synthesized compounds and
characterization of them can be found in the supplementary material.
It should be mentioned that the synthesis of these compounds was planned in
order to evaluate the influence of different benzyl groups (attached to the triazole ring),
as well as the size of the aliphatic carbon chain (connecting the cinnamic acid portion
and the triazole functionality) on biological responses.
3.2. Cinnamic acid derivatives reduce metastatic potential of B16-F10 cells
Migration is an important step in cancer metastasis, and cancer cells have a
strong migration behavior (Shtivelman et al., 2006; Astin et al., 2010). In order to
examine the effect of the synthesized compounds 8a─8m and 9a─9m on B16-F10 cell
migration, we performed wound-healing assays in the presence of compounds to
evaluate the cell motility. In this assay, we used the concentration of 100 μmol L-1
considering that all compounds had low cytotoxicity based on IC50 (data not shown).
Nineteen of the twenty-six derivatives synthesized significantly suppressed B16-F10
cell migration at 100 μmol L-1 (8a, 8b, 8f, 8h, 8i, 8j, 8l, 8m, 9a, 9b, 9c, 9d, 9e, 9g, 9h,
9i, 9k, 9l and 9m) compared to vehicle-treated cells (0.4% DMSO) (Fig. 4A and B).
Moreover, compounds 8a, 8b, 8l, 8m, 9a, 9c, 9e, 9h, 9l and 9m were the most active in
suppressing B16-F10 cell migration in at least 50% compared to control. Thereafter, it
was evaluated the anti-migratory efficacy of these ten most effective derivatives at 50
μmol L-1 (Fig. 4C). In this case, only compound 9a significantly suppressed the cell

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migration in approximately 86% (p ≤ 0.0001) in comparison with the control (Fig. 4D).
Therefore, compound 9a (Fig. 4E) was chosen for further experiments.
In addition to migration, B16-F10 cells may close the scratch by cell
proliferation. Based on that, we also evaluated the effect of compound 9a on the
proliferative activity. Our findings revealed that compound 9a significantly impaired
B16-F10 cell proliferation at 100 μmol L-1 in a time-dependent manner (Fig. 4F).
Taking these findings together, we may suggest that the compound 9a was able to
suppress cell migration and proliferation.
Invasion and cell adhesion are also key steps in melanoma metastasis. Thus, the
development of compounds with anti-invasive and anti-adhesive properties is of
potential interest (Shtivelman et al., 2006). Herein, we also evaluated the effect of
compound 9a on the invasion and adhesion of metastatic B16-F10 cells. B16-F10 cell
invasion decreased after 24 h of treatment with compound 9a at 12.5 (52.3%), 25
(64.2%) and 50 (76%) μmol L-1 compared to vehicle-treated cells (0.4% DMSO) (Fig.
5A and B). Similarly, cell adhesion was significantly decreased at 25 (8.6%) and 50
(32.2%) μmol L-1 (Fig. 5C). This remarkable activity of compound 9a over the
metastatic behavior of melanoma is in agreement with other studies that previously
reported antimetastatic activity for cinnamic acid derivatives against human lung
adenocarcinoma and colon carcinoma cells (Yen et al., 2011; Tsai et al., 2013; Ling et
al., 2015).
Finally, the colony formation assay was performed (Franken et al., 2006) to
investigate whether cinnamic acid derivative 9a could influence the colony-formation of
B16-F10 cells. The colony-forming activity of B16-F10 cells was suppressed after
treatment with compound 9a at 12.5 (25.5%), 25 (20.6%) and 50 (93.1%) μmol L-1 (Fig.
6), with a reduction in the number of colonies when compared to vehicle-treated cells
(0.4% DMSO).

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Altogether, we may suggest that the compound 9a was efficient in reducing
metastatic potential of B16-F10 cells in vitro. The endpoints tested herein, including
cell migration, proliferation, invasion, adhesion, and colonization, have been described
as pivotal steps in the metastasis process in vivo (Kumar e Weaver, 2009; Guan, 2015).
For instance, mouse models have been widely used for unraveling the complex
interactions involved in the metastatic cascade, as well as for testing potential therapies
against tumor cell migration (Gómez-Cuadrado et al. 2017). In this context, our findings
could be considered a baseline for the study of the efficiency of cinnamic acid
derivatives in animal model.
3.3. Effect of compound 9a in non-tumor cell and erythrocyte viability
Herein, we evaluated the cytotoxicity of compound 9a over non-tumoral cell
lineages, as well as primary erythrocytes, since it has proved to be a promising
antimetastatic agent. Vero and fibroblast (NIH3T3) cells were slightly sensitive to the
compound 9a at 100 μmol L-1 (Fig. 7A). Moreover, there was no indicative of
hemolysis for treatments with the derivative at different concentrations (Fig. 7B).
The slightly sensitivity of fibroblasts to the compound 9a may also indicates that
it is not a skin sensitizer (Tomankova et al., 2011). The in silico ADMET analysis, in
addition, revealed that derivative 9a has no ability to induce allergic contact dermatitis,
and relatively low skin permeability (Table S2).
Moreover, this analysis showed that the most active derivative does not violate
the Lipinsky´s rules of five (Lipinsk, 2000), and therefore this compound is drug like.
The prediction of absorption indicated that the compound 9a has a high percentage of
intestinal absorption, especially if orally administered. In the kidney, the compound 9a
is not an organic cation transporter 2 substrate, and it is potentially filtrated and partially
reabsorbed in renal tubules. This result is important for determining dosing rates to

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achieve steady-state concentrations. With respect to its toxicity, the compound 9a is not
a mutagenic compound and, therefore, may not act as a carcinogen. Although it is not a
skin sensitizer, the compound 9a may be associated with disrupted normal hepatic
function (Table S2). The latter, in turn, can be verified in studies using animal models.
Collectively, these findings indicate that compound 9a seems to be non-toxic agent,
enabling its application in more advanced in vivo studies.
3.4 Gelatin zymography and molecular docking help to understand the activity suppress
of MMP -2 and -9 by compound 9a
It is well documented that MMP-9 and -2 are directly involved in melanoma
progression (van den Oord et al., 1997; Hofmann et al., 2000; Kondratiev et al., 2008;
Zhang et al., 2017). Both MMP enzymes are capable of degrading components from
basement membranes. For that reason, they are probably related to the metastasis steps
in melanoma (Hofmann et al., 2000; Gu et al., 2016). In this context, MMPs are
important targets for the development of new drugs with antimetastatic activity.
Cinnamic acid and its derivatives have demonstrated to be inhibitors of the MMPs
activities (Yen et al., 2011; Tsai et al., 2013; Ling et al., 2015; Gopi et al., 2016). In the
current study, B16-F10 cells were treated with compound 9a for 24 h and analyzed by
gelatin zymography in order to clarify whether the MMP-9 and -2 are related to the
antimetastatic behavior of this compound. Our results showed that MMP-9 activity was
suppressed after treatment with compound 9a at 100 (64.26%) and 150 (63.85%) μmol
L-1 compared to vehicle-treated cells (0.4% DMSO; Fig. 8). Similarly, MMP-2 activity
was suppressed by compound 9a at 100 (48.75%) and 150 (43.90%) μmol L-1 (Fig. 8).
These findings are in accordance to previous reports revealing the cinnamic acid and its
derivatives ability to inhibit the activities of MMP-9 and -2 in tumor cells, as well as

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their capability of adhesion, migration, and invasion (Chiriac et al., 2005; Ali et al.,
2014).
In order to shed light on the way that compound 9a interacts with the
aforementioned metalloproteinases, this compound, the cinnamic acid (Yen et al.,
2011), and their derivatives that activity on MMP-9 and -2 have been previously
described (Tsai et al., 2013) were anchored with human MMP-9 (PDB-ID: 1L6J) and -2
(PDB-ID: 1QIB). Those MMPs are directly involved in the progression of melanoma
and indicates a poor prognosis (Hofmann et al., 2000; Gu et al., 2016). The anchorage
investigation showed that compound 9a displayed the best predicted binding affinity
values for MMP-9 (-8.2 kcal / mol) and -2 (-8.6 kcal / mol) (Table 1).
The binding region of compound 9a with MMP-9 (Fig. 9A) and -2 (Fig. 9C) is
predicted to match to the active site, as well as observed for the cinnamic acid and their
derivatives. As shown in Fig. 9B, the compound 9a has potential to interact with
catalytic aspartate residues Asp182 and Asp185 of MMP-9 with distance of 3.5 Å and
3.6 Å, respectively. The Fig. 9D showed that compound 9a in the docking model
interacts with catalytic glutamate residues, Glu150, Glu184 and Glu202 of MMP-2 with
distance of 9.6 Å, 9.1 Å and 2.2 Å, respectively.
Our results revealed that compound 9a is effective in inhibiting the metastatic
behavior of B16-F10 melanoma cells. Furthermore, the prediction of molecular binding
indicated that the inhibitory ability of this compound is mainly related to its interaction
with MMP-9 and MMP-2. Similarly, previous studies have shown that cinnamic acid
derivatives are potent inhibitors of MMPs (Zhang et al., 2006; Yen et al., 2011; Melo
and Qsar, 2012; Tsai et al., 2013; Ling et al., 2015; Gopi et al., 2016). The reason for
this inhibitory activity has been ascribed to the presence of heteroatoms (especially the
hydroxyl group) in these compounds. The heteroatoms are capable of establishing
hydrogen bonds with the proteins. In addition, the π electrons present in this compound

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facilitate interactions with hydrophobic regions of the MMP-9 and -2 (Zhang et al.,
2006; Yen et al., 2011; Melo and Qsar, 2012; Tsai et al., 2013; Ling et al., 2015; Gopi et
al., 2016).
4. Conclusions
In the present study, twenty-six cinnamic acid derivatives containing 1,2,3-
triazolic portions were synthesized and submitted to in vitro bioassays to investigate
their activity over the metastatic behavior of murine melanoma. The most potent
compound 9a, a 1,2,3-triazolic-cinnamic acid derivative containing a benzyl group
attached to the triazolic portion, presented significant antimetastatic activity against
B16-F10 melanoma cells. The molecular docking simulation contributes to
understanding our in vitro experimental results, demonstrating that compound 9a has
binding affinity with MMP-9 and -2. Therefore, our findings suggest that cinnamic acid
derivatives containing a 1,2,3-triazolic portion may represent a scaffold to be explored
towards the development of novel candidates for controlling malignant metastatic
melanoma.
Acknowledgements
This work was supported by the Programa Nacional de Cooperação Acadêmica
(PROCAD) of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The
funders had no involvement in the study design, in the collection, analysis or
interpretation of data, in the writing of the report, or in the decision to submit the article
for publication. The authors also thank CNPq, FAPEMIG and CAPES for the
fellowships to students and researchers involved in this work.

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Figure captions:
Fig. 1. Structures of cinnamic acid and some derivatives.
Fig. 2. Synthesis of esters 5 and 6, and azides 7. Reagents and conditions: (i) DCC,
DMAP (10 mol%), CH2Cl2, r.t; (ii) Et3N, CH3SO2-Cl, CH2Cl2, -50 ºC; (iii) NaN3, DMSO,
r.t.
.
Fig. 3. Reagents and conditions: (iv) sodium ascorbate (40 mol%), CuSO4 5H2O (20
mol%), CH2Cl2/H2O (1:1 v/v), r.t.
Fig. 4. Effect of cinnamic acid derivatives containing 1,2,3-triazolic portions on
wound healing and proliferation of B16-F10 melanoma cells in vitro. (A and B)
B16-F10 cells were wounded with a pipette tip and then treated with 100 μmol L-1 of
each synthesized compound for 24 h. (C) B16-F10 cells were wounded and treated for

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24 h with 50 μmol L-1 of the most active compounds as determined in (A and B). (D)
Photos of the wound were taken at 0 and 24 h after treatment with 50 μmol L-1 of
compound 9a under 100 x magnitude microscope. (E) Chemical structure of compound
9a. (F) Trypan blue dye exclusion assay of compound 9a on the proliferation of B16-
F10 cells. Mean ± S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 ou #p < 0.0001 versus
control (0.4% DMSO) by Dunnett's test.
Fig. 5. Effect of compound 9a on invasion and adhesion of B16-F10 melanoma cells
in vitro. (A) Photomicrography represents the cells invasion through matrigel coating
(100 x magnification). (B) The bar graphs represent invasive cell number that was
treated with 12.5, 25 and 50 μmol L-1 of compound 9a for 24 h. (C) The bar graph
represents the adhered cell number that was treated with 12.5, 25, and 50 μmol L-1 of
compound 9a for 12 h. Mean ± S.E.M. *p < 0.05, **p < 0.01 or ***p < 0.001versus
control (0.4% DMSO) by Dunnett's test.
Fig. 6. Effect of compound 9a on colony formation of B16-F10 melanoma cells in
vitro. Photomicrography showing the formation of B16-F10 colonies after treatment
with 12.5, 25 and 50 μmol L-1 of compound 9a for 14 days. After that, the colonies were
fixed with methanol, stained with toluidine blue solution and counted. Bar graphs
represent the colony number that were formed after 14 d of treatment with compound 9a
at 12.5, 25 and 50 μmol L-1. Mean ± S.E.M. *p < 0.05, **p < 0.01 or ***p < 0.001
versus control (0.4% DMSO) by Dunnett's test.
Fig. 7. Cytotoxic effects of compound 9a on non-neoplastic cell lines and
erythrocytes viability. (A) Vero (African green monkey kidney cell) and NIH3T3
(Embryonic fibroblasts) cells were exposed to 100 μmol L-1 compound 9a for 48 h. (B)

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Graphical of hemolysis assay. Mean ± S.E.M.**p < 0.01 versus control (0.4% DMSO),
and #p < 0.0001 versus control (Triton) by Dunnett's test.
Fig. 8. Representative gelatin zymogram showing the effects of compound 9a on
matrix metalloproteinase (MMP)-9 and -2 activities in treated B16-F10 melanoma
cells. Cells were incubated with 0 (Control), 50, 100 and 150 μmol L-1 of compound 9a
for 24 h. The density of each band was quantified by densitometric analysis. Fetal
bovine serum was used as weight standard (WE). M: medium. Mean ± S.E.M. *p < 0.01
versus control (0.4% DMSO) by Dunnett's test.
Fig. 9. Molecular interactions of compound 9a, cinnamic acid, and its derivatives
with the active site (orange) of matrix metalloproteinases (MMP)-9 and -2. (A)
Docking of compound 9a (blue), cinnamic acid (green), ferulic acid (red), chorogenic
acid (magenta), caffeic acid (yellow) with MMP-9. (B) Docking view showing
compound 9a bond interaction of ligands with residues in the active site of MMP-9.
Important residues Asp182 and Asp185 (green) of MMP-9 interaction between
compound 9a (blue). (C) Docking of compound 9a (blue), cinnamic acid (green),
ferulic acid (red), chorogenic acid (magenta), caffeic acid (yellow) with MMP-2. (D)
Docking view showing the compound 9a bond interaction of ligands with residues in
the active site of MMP-2. Important residues Glu 150, Glu184 and Glu202 (green) of
MMP2 interaction between compound 9a (blue). Zinc ion indicated in yellow (B, D).

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Table 1. Predicted binding affinities of compound 9a, cinnamic acid,
ferulic acid, chlorogenic acid, and caffeic acid to matrix
metalloproteinase (MMP)-9 and -2 by Autodock Vina program.
MMP-9 (PDB–ID:1L6J) MMP-2 (PDB–ID:1QIB)
Compounds
Bond energy (kcal/mol)
Bond energy (kcal/mol)
9a
-8.2
-6.7
-6.2
-6.8
-5.8
-8.6
-6.5
-6.9
-6.5
-7.6
Cinnamic acid
Ferulic acid
Chorogenic acid
Caffeic acid

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Highlights
 Twenty six 1,2,3-triazole cinnamic acid derivatives were synthesized
 The antimetastic activity of derivatives on B16F10 cell line was assessed
 3-(1-benzyl-1H-1,2,3-triazol-4-yl)propyl cinnamate (9a) was the most active one
 The antimetastatic effect of 9a seems to be linked with inhibition of MMP-9 and
-2
 Docking studies revealed interactions of 9a with the active site of MMP-9 and -2

Figure 1

Figure 2

Figure 3