Capsaicin-like analogue induced selective apoptosis in A2058 melanoma cells: Design, synthesis and molecular modeling

Article abstract of DOI:10.1016/j.bmc.2019.05.020

The use of molecules inspired by natural scaffolds has proven to be a very promising and efficient method of drug discovery. In this work, capsaicin, a natural product from Capsicum peppers with antitumor properties, was used as a prototype to obtain urea

Full text of DOI:10.1016/j.bmc.2019.05.020

Accepted Manuscript
Capsaicin-like analogue induced selective apoptosis in A2058 melanoma cells:
Design, synthesis and molecular modeling
Gustavo José Vasco Pereira, Maurício Temotheo Tavares, Ricardo Alexandre
Azevedo, Barbara Behr Martins, Micael Rodrigues Cunha, Rajesh Bhardwaj,
Yara Cury, Vanessa Olzon Zambelli, Euzébio Guimarães Barbosa, Matthias A.
Hediger, Roberto Parise-Filho
PII:
S0968-0896(19)30087-2
https://doi.org/10.1016/j.bmc.2019.05.020
BMC 14907
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 January 2019
13 April 2019
12 May 2019
Please cite this article as: Pereira, G.J.V., Tavares, M.T., Azevedo, R.A., Martins, B.B., Cunha, M.R., Bhardwaj,
R., Cury, Y., Zambelli, V.O., Barbosa, E.G., Hediger, M.A., Parise-Filho, R., Capsaicin-like analogue induced
selective apoptosis in A2058 melanoma cells: Design, synthesis and molecular modeling, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.020
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Capsaicin-like analogue induced selective apoptosis in A2058
melanoma cells: Design, synthesis and molecular modeling
Gustavo José Vasco Pereira,a† Maurício Temotheo Tavares,a† Ricardo Alexandre
Azevedo,b Barbara Behr Martins,c Micael Rodrigues Cunha,a,d Rajesh Bhardwaj,e
Yara Cury,c Vanessa Olzon Zambelli,c Euzébio Guimarães Barbosa,f Matthias A.
Hediger,e Roberto Parise-Filhoa*
aLaboratory of Design and Synthesis of Bioactive Substances (LAPESSB). Department of Pharmacy.
Faculty of Pharmaceutical Sciences. University of São Paulo. São Paulo. Brazil.
bExperimental Oncology Unit (UNONEX). Department of Microbiology, Immunology and Parasitology.
Federal University of São Paulo. São Paulo. Brazil
cLaboratory of Pain and Signaling. Butantan Institute. São Paulo. Brazil
dDepartment of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern.
eInstitute of Biochemistry and Molecular Medicine, National Center for Competence in Research, NCCR
TransCure, University of Bern, Bühlstrasse 28, 3012 Bern.
fDepartment of Pharmacy. Federal University of Rio Grande do Norte. Natal. Brazil
†These authors contributed equally for this work.
*Corresponding Author: R.P. E-mail: roberto.parise@usp.br
Graphical Abstract
Abstract
The use of molecules inspired by natural scaffolds has proven to be a very promising
and efficient method of drug discovery. In this work, capsaicin, a natural product from
Capsicum peppers with antitumor properties, was used as a prototype to obtain urea and
thiourea analogues. Among the most promising compounds, the thiourea compound 6g

exhibited significant cytotoxic activity against human melanoma A2058 cells that was
twice as high as that of capsaicin. Compound 6g induced significant and dose-
dependent G0/G1 cell cycle arrest in A2058 cells triggering cell death by apoptosis. Our
results suggest that 6g modulates the RAF/MEK/ERK pathway, inducing important
morphological changes, such as formation of apoptotic bodies and increased levels of
cleaved caspase-3. Compared to capsaicin, 6g had no significant TRPV1/6 agonist
effect either irritant effects on mice. Molecular modeling studies corroborate the
biological findings and suggest that 6g, besides being a more reactive molecule towards
its target, may also present a better pharmacokinetic profile than capsaicin. Inverse
virtual screening strategy found MEK1 as a possible biological target for 6g. Consistent
with these findings, our observations suggested that 6g could be developed as a
potential anticancer agent.
Keywords: peppers; anticancer agents; drug design; natural product; capsaicin; urea;
thiourea; cancer; chemotherapy; apoptosis.

Introduction
The term "cancer" is used to describe a set of changes that shares the common
characteristic of uncontrolled cell growth.1 In 2012, 14.1 million cases of cancer were
estimated around the world, where the expectation for 2035 could reach 24 million
cases.2 Thus, cancer is the second largest cause of death worldwide.2 Although there are
strategic therapies for the treatment of cancer, low selectivity to tumorigenic cells, high
toxicity, resistance and high cost are persistent issues in this field.3,4 In view of this,
natural products have played an important role for the design of new drugs, offering
active substances to various diseases.5 Capsaicin (Fig. 1A) is abundantly found in
Capsicum peppers and it is a secondary metabolite derived from the condensation of
vanillylamine with (E)-8-methylnon-6-enoic acid. A large number of independent
studies demonstrates the ability of capsaicin to prevent both mutagenic and carcinogenic
processes in vitro and in vivo.11–15 Moreover, several biological properties including
agonist effects over TRPV1 receptors are reported.6–10
According to Walpole and coworkers the structure of capsaicin can be divided
in three main regions.16–18 The A-region consists in the vanillyl moiety;16 the B-region
presents the secondary amide linker that connects both extremes of the molecule;17 and
C-region comprises an unsaturated hydrophobic tail derived from the fatty acid.18
Several structure-activity relationship (SAR) studies were carried out regarding the
design of TRPV1 agonists and antagonists, through systematic changes over the A-, B-,
and C-region of capsaicin.19–21 Noteworthy, unsaturation of C-region has no significant
influence in the potency of capsaicin-like compounds, which could be observed for –
but not only – dihydrocapsaicin who promotes equipotent cellular Ca2+ influx (via
TRPV1), and consequently similar pungency as capsaicin.21,22
Regarding the antitumor mechanism of capsaicin, this remains to be
elucidated,23–26 although several studies suggest that this effect may be the result of the
activation of two signaling pathways: i) the intrinsic or mitochondrial pathway, with late
activation of caspase 1 and 3; and ii) the extrinsic or receptor-dependent pathway,
through capsaicin action on TRPV-like channels.27 Our group has developed several
analogues of capsaicin that presented selective cytotoxicity for several breast, skin, and
lung tumorigenic cell lines.28–31 Thus, we have concentrated our recent efforts on the
production of new series of capsaicin analogues (Fig. 1B). The series were designed by
keeping the same vanillyl moiety of capsaicin in A-region and also by its ring closure,
generating the 1,3-benzodioxole bicyclic system (Fig. 1B) which is commonly found in

antineoplastic agents such as podophyllotoxin, etoposide and derivatives. The amide
linker in B-region suffered bioisosteric replacements, generating the urea and thiourea
series. In addition, the lipophilic tail of capsaicin in the C-region has been replaced by
alkyl and aryl substituents, conserving the hydrophobic character of this region for the
compounds. Herein, we describe the design and synthesis of two series of ureidic and
thioureidic analogues quite similar to capsaicin. The biological activities and anticancer
mechanism of these compounds were investigated. In silico studies were also performed
to provide information about SAR.
Fig. 1. (A) Chemical structure of capsaicin. (B) General structure of the designed analogues of this work.
Results and discussion
Chemistry
The method applied for the synthesis of the capsaicinoid derivatives was the
nucleophilic addition of primary amines to the carbonyl or thiocarbonyl, thus generating
ureidic and thioureidic derivatives. The final products were synthesized in a single step
by the reaction of piperonylamine (1) or vanillylamine (2) with isocyanates (3a-3g) or
isothiocyanates (4a-4g) as shown in Scheme 1. Twenty-seven analogues were
1
13
synthesized and characterized by H and C NMR, melting range, and the purity was
determined by HPLC. All compounds showed a solid appearance, with yields ranging
from 22 to 90%.

Scheme 1. Synthesis of piperonyl (5a-g; 6a-g) and vanillyl (7a-g; 8a-f) analogues. Reagents and
conditions: i) CH2Cl2, cat. Et3N, atm. N2, r.t., 3 h; ii) CH2Cl2/MeOH (8:2), cat. Et3N, atm. N2, r.t., 4 h.
Cell viability assay and morphological evaluation
The cytotoxicity of all compounds (5a-g, 6a-g, 7a-g and 8a-f) were evaluated by
the MTT assay against a panel of tumorigenic cell lines including B16F10 (murine
melanoma), A2058 and SK-MEL-25 (human melanoma), MCF-7 (human breast
adenocarcinoma), HeLa (cervical carcinoma) and U-87 (glioblastoma). Furthermore, we
also tested the compounds against non-tumorigenic human fibroblasts (T75) cells to
verify the selectivity index (SI) of the compounds. Our results showed that compounds
(6g, 7b-d) showed superior cytotoxic activity compared to the prototype against human
melanoma tumor cells A2058 and SK-MEL-25, as well as human glioblastoma U-87,
with an effectiveness ranging from 55.17 μM to 98.7 μM (Table 1). On the other hand,
they were not able to replicate the same effect over MCF-7 breast adenocarcinoma,
human cervical adenocarcinoma HeLa (data not shown) and murine B16F10 melanoma
cells. Particularly, compound 6g showed pronounced activity with IC50 values of 55, 67,
and 87 μM against A2058, SK-MEL-25, and U-87 cells, respectively, which represent
one- to two-fold improvement in potency compared to capsaicin. Noteworthy,
compound 6g presented the highest SI for A2058 (SI: 1.8, Table 1).

Table 1. Biological data for compounds 6g, 7b-d.
IC50 (µM) ± SD€
Compounds
SK-MEL-25
A2058
>100
U-87
B16F10
T75
SI¥
>100
>100
>100
>100
-
67.19 ± 8.7
98.7 ± 7.35
55.17 ± 9.35 86.95 ± 7.44
98.20 ± 8.34 98.54 ± 8.55
>100
>100
>100
>100
>1.8
>1.1
90.5 ± 6.97
91.8 ± 7.45
84.45 ± 7.67 92.48 ± 7.98
>100
>100
>100 >1.18
>100 >1.08
92.54 ± 7.89
> 100
€Data are expressed as means ± SD obtained from three independent experiments.
¥SI: selectivity index, determined by the ratio between IC50 A2058 e T75
As a tendency, the compounds were more effective in A2058 and SK-MEL-25
human melanoma cells, both having a mutation in the B-RAF protein (B-RAFV600E
mutation).32,33 Preliminarily, we suggest that this mutation can be considered a possible
target of compound 6g, thereby triggering cytotoxic activity. This hypothesis became
more evident when compared to the effects found in B16F10 (wild-type B-RAF)
strains,34–36 which only showed an antiproliferative effect with IC50 higher than the
highest concentration used in the initial screenings. This situation is particularly
interesting since 50% of melanomas of all clinical types have B-RAF mutations.37
Among the most active compounds, 6g which has the 1,3-benzodioxole moiety
at the A-region and bearing a n-alkyl side chain bound to the thiourea linker exhibited
improved activity against A2058 cells relative to corresponding urea compounds 7b-d
bearing the bulky para-substituted benzyl group. Regarding urea analogues, those with
electron-donating groups, (EDG) such as compounds 7c and 7d, appear to result in
more active compounds, which may be due the increase in the electronic density of the
aromatic ring. Compound 7b corroborates this hypothesis, whereas its electron-
withdrawing nitro group (EWG, NO2), proved to be the least active analogue.
With these results, compound 6g was chosen for further investigations regarding
mechanistic studies of the cytotoxic effect. Our results confirm that 6g affects the
overall morphology of A2058 cells, disrupting the formation of structures that resemble
the in vivo architecture of those cells. The morphology of A2058 melanoma cells treated
with compound 6g is presented in Fig. 2A. As observed in comparison to the control,

the changes induced by 6g suggest cell death by apoptosis, since there is loss of cell
adhesion, significant withdrawal of the cytoplasm, loss of refringence and marked
formation of apoptotic bodies.38,39 Compared to the effects of 6g on B16F10 melanoma
cells (Fig. 2B), it is possible to observe that the cells are not dead, but there are, clearly,
fewer cells-per-well, suggesting cell cycle arrest.
Fig. 2. (A) Photomicrographs show the morphological aspects of melanoma control cultures of A2058
cells (top), and treated with 6g (bottom). (B) Morphological aspects of control vultures of B16F10
melanoma cells (top), and treated with 6g (bottom). Cultures were incubated for 24 h at 37 °C. Yellow
dashed circles indicate the appearance of apoptotic bodies. Grids (in red) indicate enlargement of the area.
Cell Cycle Analysis and Cell Death Studies
Analysis of the morphology of human A2058 and murine B16F10 melanoma
cells provided evidence that there are distinct biological effects, particularly related to
cell cycle arrest and possible death by apoptosis. To investigate effects on the cell cycle,
cells were treated with compound 6g at concentrations of 12.5, 25.0 and 50.0 μM and
then labeled with propidium iodide. The distribution of cell cycle phases was measured
by flow cytometry. The data showed that compound 6g at 50 μM was able to increase
the G0/G1 phase, suggesting an arrest at this stage of the cycle in murine B16F10
melanoma cells (Fig. 3A). As noted, this behavior has been shown to be dose-
dependent. In addition, there is no variation in the subG0/G1 population compared to the
control (vehicle), indicating that there is no cell death.40–42 On the other hand, A2058
human melanoma cells treated with compound 6g have shown a significant shift of the
cell population in subG0/G1, suggesting that those cells might be in the process of cell
death (Fig. 3B). Noteworthy, compound 6g induced a significant and dose-dependent
G0/G1 cell cycle arrest in A2058 cells.43

After obtaining evidence of the type of cell death involved in the effect triggered
by compound 6g, the next step was to characterize the cell death phenomenon by
analyzing the levels of anti-apoptotic mediator (Bcl-xl) and effector caspase 3 in its
active isoform (Casp3)44–46 by western blotting. It is important to note that capsaicin
derivatives as dihydrocapsaicin may act as autophagy inducers47 even though we did not
observed any evidences of autophagic bodies in our preliminary studies.
As observed in Fig. 3C, our results clearly show the up-regulation effect of the
active isoform of the pro-apoptotic protein and the downregulation of the antiapoptotic
protein. After 1 h, there is a considerable reduction in B-RAF (ratio 1 to 0.45) indicating
that the pathway is being modulated by compound 6g. These data are corroborated by
the abrupt reduction of C-RAF from 3 hours (1 to 0.49) of treatment.48 Also in Fig. 3C,
the 6-hour analysis reveals a reduction in the concentration of Bcl-xl antiapoptotic
protein, as well as reduction of total caspase 3 and, consequently, increase of the active
form Casp3. The involvement and increase of cleaved Casp3 corroborates our cellular
morphology data and altogether indicate that cell death is by apoptosis, in a possible
activation of the apoptotic RAF/MEK/ERK pathway.49,50
Fig. 3. (A) Distribution of the percentage of cell cycle phases in B16F10 cells treated with compound 6g.
(B) Distribution of cell cycle phases of A2058 cells treated with compound 6g. (C) Western blotting of
the A2058 cells treated with compound 6g, demonstrating the increase levels of caspase 3 over time.
Protein expression was quantified with ImageQuant LAS4000 software and normalized to β-actin
expression.

Evaluation of TRPV1 activity
After this preliminary screening, our next goal was to test the effect of 6g on the
activity of human TRPV151 transiently expressed in HEK293T cells, in order to collect
data that point out toward a more detailed mechanistic explanation.52,53
Capsaicin was selected as standard TRPV1 activator and was used to develop
the protocol. For screening of compounds with antagonist activity, capsazepine was
selected as internal positive control. In our hands, capsaicin evoked an increase in Ca2+
transport through TRPV1, with an EC50 of 7.2 ± 4.1 nM,54 whereas the non-transfected
cells (HEK_nT) showed no transport (supplementary material - Fig. S1A and S1B).
Therefore, 100 nM capsaicin was sufficient to stimulate the channel to its maximum.
The antagonist of TRPV1, capsazepine, was found to inhibit the 100 nM capsaicin-
mediated activation of TRPV1, with an IC50 of 174.1 nM55,56 (supplementary material -
Fig. S1C).
Compound 6g profoundly evoked the influx of Ca2+ through TRPV1 in
HEK293T cells transiently transfected with TRPV1, but only marginally in
untransfected cells (Fig. S1D), suggesting that 6g acts as an agonist. However, the
activity was nearly 640-fold lower than that of capsaicin, with EC50 of 4.62 ± 0.08 µM
(Fig. 4A). To validate the agonist effects of 6g, we pretreated the cells with vehicle
(DMSO, 0.1% in buffer) or capsazepine (10 µM). Remarkably, both, the activity of
capsaicin (100 nM) and 6g (10 µM) were completely abolished by the pretreatment with
capsazepine (10 µM) (Fig. 4B).57 To evaluate, whether the human epithelial calcium
channel TRPV6 can be targeted by 6g, we tested the effect of 6g and also capsaicin on
the activity of the TRPV6 channel stably expressed in HEK293 cells.58 Fig. 4C indicates
that 6g at 50 µM, as well as capsaicin at 100 nM, showed no discernable effect on
TRPV6 activity, indicating that these compounds possess selectivity toward TRPV1,
whereas the previously published inhibitor cis-22a59 effectively blocked TRPV6 at 2.5
µM (Fig. 4C).

Fig. 4. (A) Dose-response curve of 6g in HEK_TRPV1 cells. Data shown is mean ± SEM
(n = 6/concentration). (B) Ca2+ traces of HEK_TRPV1 cells. I = first application (DMSO or
capsazepine – CPZ – 10 µM). II = second application (capsaicin – CPS – 100 nM or 6g, 10 µM). (C)
Evaluation of the capsaicin (CPS) and 6g in HEK-TRPV6 cells. Data shown is mean ± SEM (n =
3/concentration). Significance between the treated groups was determined by one-way ANOVA and
Tukey test. **p <0.01 (by comparison with vehicle).
Evaluation of nociceptive potential of 6g compared to capsaicin
TRPV1 plays a key role in the development of various pathological processes,
most prominently in the sensations of heat and inflammation, and mediates the
pungency and pain sensations associated with capsaicin.52,60–63 In order to verify
whether – in vivo – the compound 6g has the ability to activate TRPV1 channel in a
similar way as capsaicin, the flinching behavior was assessed.53 The test measured the
number of flinches during 5 minutes and indicates nociceptive intensity after
intraplantar application of 20 μL of vehicle (EtOH + saline), capsaicin and 6g – both at
0.26 M concentration (5.2 nmol/paw). Results are presented in Fig. 5.

8 0
6 0
4 0
2 0
0
*
#
V e h ic le
C a p s a ic in
6 g
Fig. 5. Evaluation of the capsaicin and 6g effect on the nociceptive behavior of mice. All animals
received the vehicle and tested compounds by intraplantar route (5.2 nmol/paw, 20 μL of a 0.26 M
solution). n = 8 animals. Significance between the treated groups was determined by one-way ANOVA
and Tukey test. * p <0.05 (by comparison with vehicle); # p <0.05 (by comparison with capsaicin).
As can be seen, capsaicin injection evoked nociceptive response when compared
to the vehicle. On the other hand, the compound 6g injection generated significantly
lower effect in treated animals, indicating that this compound is less effective to induce
nociceptive behavior as compared to capsaicin. These results suggest that 6g may not be
acting exclusively via TRPV1 receptor activation. Therefore, the more pronounced
antitumor effect compared to capsaicin is probably due to the action over other receptor
subtypes or, corroborating the data found in this work, the action may be due to
modulation of the RAF/MEK/ERK pathway. However, given that the potency of 6g to
activate TRPV1 is substantially lower than that of capsaicin, it seems unlikely that the
phenotype exhibited by 6g treated mice is related to activation of TRPV1.
Molecular modeling
The purpose of the theoretical approach was to find energetically more favorable
conformations of capsaicin and 6g, and thus to calculate molecular properties with
purpose of providing ideas about structure-activity relationships (SAR).64 The
calculation of the properties was performed in the Spartan'14 program, obtaining:
electrostatic potential map (MPE), energy of the HOMO and LUMO (EHOMO and
ELUMO) frontier orbitals, partition coefficient (ClogP), hydrogen bond acceptor site
(HBA), hydrogen bonding donor site (HBD), polar surface area (PSA), and dipole
moment (μ).

Table 2 shows the calculated values for the molecular properties of capsaicin
and compound 6g. According to the calculated values of ClogP, 3.66 for capsaicin and
3.92 for compound 6g, the molecules have similar hydrophobic character, although the
latter is slightly more lipophilic. In addition, PSA (48.21 Å 2 and 36.60 Å 2 for capsaicin
and 6g, respectively) showed that compound 6g presents the lower surface of all polar
atoms, corroborating its greater hydrophobicity. These data together indicate that the
more hydrophobic profile of 6g may result in higher cell membrane permeability,
suggesting the better activity of the compound compared to capsaicin.65 Regarding
HBD, HBA and MW properties, all have values that follow drug-like rules.66
Table 2. Calculated molecular properties for capsaicin and 6g.
Molecular Properties
ClogP
EHOMO (eV)
ELUMO (eV)
Gap = EHOMO - ELUMO
µ Debye (D)
PSA (Å 2)
capsaicin
3.66
-8.27
3.98
-12.25
3.96
48.21
4
6g
3.92
-8.51
3.70
-12.21
7.57
36.60
5
HBA
2
2
HBD
The energy values of the frontier molecular orbitals (EHOMO e ELUMO), as well as
the difference between them (Gap = EHOMO - ELUMO), were calculated from the lower
energy conformational models of capsaicin and compound 6g. The values are described
in Table 2 and the HOMO and LUMO maps are shown in Fig. 6A. Although the
calculated energy values are quite similar, 6g has a lower ELUMO (3.70 eV) than
capsaicin (ELUMO = 3.94 eV), characterizing it as being a more electron accepting
molecule. LUMO map distributions did not show significant differences between the
two investigated molecules.28
Regarding EHOMO energy values, 6g presents a slightly higher value when
compared to the prototype. In addition, the HOMO map distribution shows a greater
intensity of the HOMO orbital in the region where the bioisosteric replacement was
performed (replacing the amide function by thiourea). The gap values (see Table 2)
point out the benzodioxole derivative as a slightly more reactive molecule than
capsaicin.
The dipole moment vector () can be visualized as yellow arrows in Fig. 6B and
the values are listed in Table 2. The dipole moment vector is defined as the total
product of the amount of positive or negative charge and the distance between its
centroids. As seen in Fig. 6B, the vectors are in opposite sides of the two molecules.

Moreover, the difference of the values of dipole moment between the molecules is given
by the high electronic density of the thiourea moiety.65 Regarding the electrostatic
potential map (EPM, Fig. 6C), there is a higher electron density at the thiourea linker,
which may be an additional data to understand the reasons why 6g is more active than
the prototype.67
Fig.6. Electronic properties found for capsaicin and 6g. (A) Distribution of molecular orbital maps,
HOMO and LUMO (color range: −0.020 (red) to 0.020 (blue)). (B) Total dipole moment vector (yellow
arrows), and (C) Electrostatic potential maps (EPMs; color range: −200.0 (intense red) to 200.0 (intense
blue)). The molecules are presented as ball-wire models (carbon in gray, oxygen in red, nitrogen in blue,
and hydrogen in white).

Regarding EHOMO energy values, 6g presents a slightly higher value when
compared to the prototype. In addition, the HOMO map distribution shows a greater
intensity of the HOMO orbital in the region where the bioisosteric replacement was
performed (replacing the amide function by thiourea). The gap values (see Table 2)
point out the benzodioxole derivative as a slightly more reactive molecule than
capsaicin.
Inverse Virtual Screening and docking
The purpose of the inversed virtual screening was to find possible targets for the
compound 6g. In order to do so, a simplified version of 6g was created by removing the
long aliphatic chain attached to the thiourea moiety (6g_mod). The structure 6g_mod
was screened against all the PDB databank by ligand-based similarity search. Table 3
shows the entire cancer-related targets which the ligand has the highest similarities to
compound 6g_mod.
Table 3. Most promising targets for 6g as pointed out by the ligand-based inversed virtual screening.
Target (PDB ID)
Ligand ID
8MZ
Similarity
0.916
Proto-oncogene
serine/threonine-protein kinase
Pim-1 (5N4U)
Mitogen-activated protein
kinase kinase) MEK1 (5BX0)
4W5
0.910
Cyclin-dependent kinase 2
(2EXM)
ZIP
0.906
Phosphoinositide-Dependent
Kinase-1 (PDK1) (3QCQ)
3Q0
0.887
The mitogen/extracellular signal-regulated kinase (MEK1) from the MAPK
signaling cascade seems to be the best target among all. MEK1 regulates proliferation
and other cellular processes by the phosphorylation and activation of downstream ERK
proteins. These findings further corroborate the cell death study which the involvement
and increase of cleaved Casp3 trigger apoptosis in a possible activation of the apoptotic
RAF/MEK/ERK pathway. Fig. 7 shows a possible binding mode for 6g_mod and
MEK1 (PDB entry 5bx0).68 The interaction is quite similar to the bound ligand (BL)

indazole in the hydrophobic pocket. Another important feature is that the pointed-out
dipole moment vector created by the thiourea moiety points favorably to Gln153
forming a hydrogen bond like dipole-dipole interaction.
Fig. 7. Molecular docking pose of 6g_mod bound to MEK1 cancer target. BL and 6g_mod bind similarly
by the alignment of the fused rings differing only the side chain interactions.
Conclusion
In conclusion, we show that the capsaicin-like analogue 6g has a higher
antitumor activity than its prototype – capsaicin – by inducing significant and dose-
dependent G0/G1 cell cycle arrest in A2058 cells, which was associated with cellular
morphological changes, indicating cell death by apoptosis. In addition, it is suggested
that 6g acts by modulating the RAF/MEK/ERK pathway and exhibited insignificant
effects on TRPV-type vanilloid receptors. Molecular modeling studies suggest that 6g is
a more reactive molecule and may also present a better pharmacokinetic profile when
compared to capsaicin. The inverse virtual screening strategy identified MEK1 as a
promising target for compound 6g. In summary, our observations suggested that
compound 6g could be developed as a potential anticancer agent.
Experimental
Chemistry
Reagents and solvents were purchased from Sigma-Aldrich, and were used
without further purification. The following reagents were used to synthesize
capsaicinoid derivatives: piperonylamine, vanilylamine, phenyl isocyanate, 4-

nitrophenyl isocyanate, 4-chlorophenyl isocyanate, 4-methoxyphenyl isocyanate, 4-tolyl
isocyanate, butyl isocyanate, hexyl isocyanate, phenyl isothiocyanate, 4-nitrophenyl
isothiocyanate, 4-chlorophenyl isothiocyanate, 4-methoxyphenyl isothiocyanate, 4-tolyl
isothiocyanate, butyl isothiocyanate, hexyl isothiocyanate.
The melting points were measured by the Büchi M-565 apparatus. TLC was
performed on silica gel plates (60 F254 TLC, Merck Darmstadt, Germany) for
monitoring the reaction and checking for impurity traces. Plate readings were performed
under UV light at 254 nm. Flash chromatography was used in the purification of the
compounds. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded using
a Bruker Advanced DPX-300 spectrometer at 300 MHz and 75 MHz, respectively.
Chemical shifts were expressed as  units, using tetramethylsilane (TMS) or solvent
residual peak as internal standard. The spectral splitting patterns are described as
follows: s, singlet; bs, broad singlet; d,doublet; dd, double doublet; q, quartet; t, triplet;
m, multiplet peak. The chromatographic purity of the compounds was determined using
a high performance liquid chromatograph (Shimadzu®-PROMINENCE) coupled to a
C18 column (Shimadzu®-Shim-pack CLC-ODS 150 x 4.6 mm).
General procedure for the synthesis of compounds.
A) Synthesis of derivatives 5a-5g and 6a-6g:
Piperonylamine (1.1 mmol) and triethylamine (1 mmol) were diluted in 20 mL
dichloromethane (DCM) in a 100 mL double neck round bottom flask coupled with an
addition funnel of 50 mL. Isocyanate (1 mmol) or isothiocyanate (1 mmol) was
dissolved in DCM (5 mL) and added dropwise to the solution of piperonylamine. The
mixture was kept under stirring in nitrogen atmosphere for 3 hours. Then, the solvent
was evaporated under reduced pressure and the product was purified by flash
chromatography or recrystallization.
B) Synthesis of derivatives 7a-7g e 8a-8f:
Vanillylamine (1.1 mmol) and triethylamine (1 mmol) were diluted in 40 mL of
dichloromethane:methanol (DCM:MeOH - 8:2) in a 100 mL double neck round bottom
flask coupled with an addition funnel of 50 mL. Isocyanate (1 mmol) or isothiocyanate
(1 mmol) was dissolved in DCM (5 mL) and added dropwise to the solution of

vanillylamine. The mixture was kept under stirring in nitrogen atmosphere for 4 hours.
Then, the solvent was evaporated under reduced pressure and the product was purified
by recrystallization.
1-(benzo[d][1,3]dioxol-5-ylmetyl)-3-phenylurea (5a):
White solid (Yield: 50%), m.p. 162–163.5 °C. 1H NMR (DMSO-d6, 300 MHz):
8.50 (s, 1H), 7.41 (d, J = 7.7 Hz, 2H), 7.23 (t, J = 7.3 Hz, 2H), 6.91 (d, J = 7.3 Hz, 1H),
6.87 (s, 1H), 6.85 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.54 (t, J = 5.8 Hz, 1H), 5.98 (s,
13
2H), 4.21 (d, J = 5.9 Hz, 2H). C NMR (DMSO-d6, 75 MHz): 155.2, 147.2, 146.0,
140.4, 134.2, 128.6, 121.1, 120.3, 117.7, 108.0, 107.8, 100.7, 42.5. HPLC analyses:
99.3%.
1-(benzo[d][1,3]dioxol-5-ylmetil)-3-(4-nitrophenil)urea (5b):
1
Yellow solid (Yield: 40%), m.p.160,3–162,1 °C. H NMR (DMSO-d6, 300
MHz): 9.36 (s, 1H), 8.04 (bs, 1H), 7.96 (dd, J1= 9.0 Hz, J2= 3.1 Hz, 2H), 7.81 – 7.78
(m, 2H), 7.40 (s, 1H), 7.31 (d, J = 7.9 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 6.42 (s, 2H),
13
5.23 (d, J = 4.0 Hz, 2H). C NMR (DMSO-d6, 75 MHz): 154.4, 147.2, 147.1, 146.1,
140.5, 133.7, 125.1, 120.4, 116.9, 108.0, 107.9, 100.8, 42.6. HPLC analyses: 95.4%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-chlorophenyl)urea (5c):
1
White solid (Yield: 63%), m.p. 187.3–189.0 °C. H NMR (DMSO-d6, 300
MHz): 8.65 (s, 1H), 7.44 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 6.87 (s, 1H),
6.86 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.59 (t, J = 7.9 Hz, 1H), 5.48 (s, 2H),
13
4.21 (d, J = 4.2 Hz, 2H). C NMR (DMSO-d6, 75 MHz): 155.0, 147.2, 146.0, 139.4,
134.1, 128.4, 124.5, 120.3, 119.2, 108.0, 107.8, 100.8, 42.6. HPLC analyses: 98.8%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-methoxyphenyl)urea (5d):
1
White solid (Yield: 75%), m.p.197,4–199,5 °C. H NMR (DMSO-d6, 300
MHz): 8.27 (s, 1H), 7.28 (d, J = 8.4 Hz, 2H), 6.86 (s, 1H), 6.84–6.75 (m, 4H), 6.41 (t, J
13
= 5.9 Hz, 1H), 5.97 (s, 2H), 4.18 (d, J = 5.9 Hz, 2H), 3.69 (s, 3H). C NMR (DMSO-
d6, 75 MHz): 155.4, 154.0, 147.2, 145.9, 134.4, 133.5, 120.2, 119.5, 113.9, 108.0,
107.8, 100.7, 55.1, 42.6, 28.6. HPLC analyses: 97.6%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(p-tolyl)urea (5e):
1
White solid (Yield: 90%;), m.p. 151.0–152.5 °C. H NMR (DMSO-d6, 300
MHz): 8.25 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 7.35 (s, 1H),
7.28 (d, J = 7.9 Hz, 1H), 7.23 (d, J = 7.3 Hz, 1H), 6.51 (bs, 1H), 6.41 (s, 2H), 4.77 (d, J
= 5.8 Hz, 2H), 2.70 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 155.3, 147.2, 146.0,
137.8, 134.3, 129.9, 129.0, 120.3, 117.8, 108.0, 107.8, 100.7, 42.5, 20.2. HPLC
analyses: 99.4%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-butylurea (5f):
1
White solid (Yield: 75%), m.p. 197.0–198.5 °C. H NMR (DMSO-d6, 300
MHz): 6.83 (d, J = 7.9 Hz, 1H), 6.80 (s, 1H), 6.71 (d, J = 7.9 Hz, 1H), 6.19 (t, J = 5.7
Hz, 1H), 5.97 (s, 2H), 5.83 (t, J = 5.3 Hz, 1H), 4.10 (d, J = 6.0 Hz, 2H), 3.00 (q, J = 6.4
Hz, 2H), 1.38 – 1.23 (m, 4H), 0.87 (t, J = 7,1 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz):

155.3, 147.2, 146.0, 137.8, 134.3, 129.9, 129.0, 120.3, 117.8, 108.0, 107.8, 100.7, 42.5,
20.2. HPLC analyses: 97.6%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-hexylurea (5g):
1
White solid (Yield: 42%), m.p. 130.0–131.0 °C. H NMR (DMSO-d6, 300
MHz): 6.83 (d, J = 7.9 Hz, 1H), 6.80 (s, 1H), 6.71 (d, J = 7.8 Hz, 1H), 6.17 (t, J = 5.8
Hz, 1H), 5.96 (s, 2H), 5.84 (t, J = 5.5 Hz, 1H), 4.09 (d, J = 6.0 Hz, 2H), 2.99 (q, J = 6.7
Hz, 2H), 1.36 – 1.25 (m, 8H), 0.86 (t, J = 6.5 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz):
158.2, 147.9, 146.8, 133.3, 120.6, 108.2, 108.1, 101.0, 44.4, 40.6, 31.5, 30.1, 26.5, 22.5,
14.0. HPLC analyses: 96.7%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-phenylthiourea (6a):
1
White solid (Yield: 40%), m.p. 160,3–162,1 °C. H NMR (CDCl3, 300 MHz):
7.72 (s, 1H), 7.41 (t, J = 7.7 Hz, 2H), 7.28 – 7.21 (m, 1H), 7.19 (d, J = 7.6 Hz, 2H),
6.81 (s, 1H), 6.74 (s, 2H), 6.17 (bs, 1H), 5.94 (s, 2H), 4.77 (d, J = 5.4 Hz, 2H). 13C
NMR (CDCl3, 75 MHz): 180.2, 147.2, 146.3, 141.9, 131.9, 124.4, 121,0, 120.6, 108.3,
108.1, 100.9, 54.8, 47.0. HPLC analyses: 99.9%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-nitrophenyl)thiourea (6b):
1
Yellow solid (Yield: 50%), m.p. 142.1–143.9°C. H NMR (DMSO-d6, 300
MHz): 10.16 (s, 1H), 8.58 (bs, 1H), 8.19 (d, J= 9.2 Hz, 2H), 7.86 (d, J= 9.2 Hz, 2H),
6.96 (s, 1H), 6.90 (d, J = 7.9 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.00 (s, 2H), 4.66 (d, J
= 4.0 Hz, 2H). 13C NMR (DMSO-d6, 75 MHz): 180.7, 147.2, 146.2, 138.3, 132.6,
128.4, 128.0, 124.8, 120.8, 108.1, 108.0, 100.8, 46.9. HPLC analyses: 99.5%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-chlorophenyl)thiourea (6c):
1
White solid (Yield: 65%), m.p. 151.0–152.5 °C. H NMR (CDCl3, 300 MHz):
7.73 (s, 1H), 7.37 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.6 Hz, 2H), 6.81 (s, 1H), 6.74 (s,
13
2H), 6.09 (bs, 1H), 5.94 (s, 2H), 4.74 (d, J = 5.4 Hz, 2H). C NMR (DMSO-d6, 75
MHz): 180.6, 147.1, 146.1, 139.1, 132.7, 128.6, 124.4, 123.4, 120.8, 108.1, 108.0,
100.8, 46.9. HPLC analyses: 99.8%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-methoxyphenyl)thiourea (6d):
1
White solid (Yield: 90%), m.p. 161.1–162.5 °C. H NMR (DMSO-d6, 300
MHz): 9.33 (s, 1H), 7.82 (s, 1H), 7.27 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 4.2 Hz, 2H),
6.88 (d, J = 7.3 Hz, 2H), 6.85 (d, J = 5.9 Hz, 1H), 6.79 (s, 1H), 5.97 (s, 2H), 4.64 (d, J
= 4.5 Hz, 2H), 3.74 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 183.3, 158.8, 148.6,
147.6, 134.1, 127.9, 121.8, 115.3, 109.1, 108.7, 101.9, 55.7, 48.7. HPLC analyses:
99.0%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(p-tolyl)thiourea (6e):
1
White solid (Yield: 48%), m.p. 135.0–137.0 °C. H NMR (DMSO-d6, 300
MHz): 9.45 (s, 1H), 7.95 (bs, 1H), 7.23 (d, J = 8.3 Hz, 2H), 7.13 (d, J = 8.3 Hz, 2H),
6.93 (s, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 5.99 (s, 2H), 4.62 (d, J
= 5.7 Hz, 2H), 2.28 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 180.7, 147.1, 146.1,

136.4, 133.7, 132.9, 129.1, 123.0, 120.7, 108.1, 107.9, 100.8, 46.9, 20.4. HPLC
analyses: 96.4%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-butylthiourea (6f):
Yellow solid (Yield: 75%), m.p. 71.2–73.0 °C. 1H NMR (DMSO-d6, 300 MHz):
7.68 (bs, 1H), 7.41 (bs, 1H), 6.85 (d, J = 10.1 Hz, 2H), 6.76 (d, J = 7.9 Hz, 1H), 5.97 (s,
2H), 4.54 (d, J = 6.0 Hz, 2H), 1.50 – 1.40 (m, 2H), 1.34 – 1.21 (m, 4H), 0.87 (t, J
= 7.3 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz): 177.4, 147.1, 146.0, 133.2, 120.5,
107.9, 100.7, 46.6, 43.2, 30.8, 19.5, 13.6. HPLC analyses: 99.1%.
1-(benzo[d][1,3]dioxol-5-ylmethyl)-3-hexylthiourea (6g):
1
White solid (Yield: 52%), m.p. 116.0–117.0 °C. H NMR (DMSO-d6, 300
MHz): 7.69 (bs, 1H), 7.42 (bs, 1H), 6.87 (s, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.76 (d, J =
9.3 Hz, 1H), 5.98 (s, 2H), 4.54 (d, J = 4.2 Hz, 2H), 1.45 (q, J = 6.6 Hz, 2H), 1.33 – 1.25
(m, 8H), 0.87 (t, J = 6.7 Hz, 3H). 13C NMR (DMSO-d6, 75 MHz): 158.2, 147.9, 146.8,
133.3, 120.6, 108.2, 108.1, 101.0, 44.4, 40.6, 31.5, 30.1, 26.5, 22.5, 14.0. HPLC
analyses: 99.8%.
1-(4-hydroxy-3-methoxybenzyl)-3-phenylurea (7a):
1
White solid (Yield: 42%), m.p. 181.7–183.2 °C. H NMR (DMSO-d6, 300
MHz): 8.79 (s, 1H), 8.47 (s, 1H), 7.40 (d, J = 7.9 Hz, 2H), 7.21 (t, J = 7.8 Hz, 2H), 6.90
(t, J = 8.1 Hz, 2H), 6.75 (s, 1H), 6.72 (s, 1H), 6.46 (t, J = 4.8 Hz, 1H), 4.19 (d, J = 5.1
Hz, 2H), 3.75 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 155.1, 147.5, 145.5, 140.5,
130.9, 128.6, 121.0, 119.7, 117.6, 115.3, 111.8, 55.6, 42.7. HPLC analyses: 99.6%.
1-(4-hydroxy-3-methoxybenzyl)-3-(4-nitrophenyl)urea (7b):
1
Yellow solid (Yield: 48%), m.p. 187.0–183.2°C. H NMR (DMSO-d6, 300
MHz): 8.79 (bs, 1H), 8.32 (s, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 8.1 Hz, 2H),
6.87 (s, 1H), 6.75 – 6.68 (m, 2H), 6.38 (t, J = 5.4 Hz, 1H), 4.17 (d, J = 5.4 Hz, 2H),
3.75 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 155.2, 147.5, 145.4, 137.9, 131.0, 129.7,
129.0, 119.7, 117.8, 115.3, 111.8, 55.6, 42.7. HPLC analyses: 95.0%.
1-(4-chlorophenyl)-3-(4-hydroxy-3-methoxybenzyl)urea (7c):
1
White solid (Yield: 59%), m.p. 198.4–199.9 °C. H NMR (DMSO-d6, 300
MHz): 8.80 (bs, 1H), 8.61 (s, 1H), 7.44 (d, J = 7.4 Hz, 2H), 7.25 (d, J = 7.4 Hz, 2H),
6.87 (s, 1H), 6.73 (d, J = 9.0 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 6.50 (t, J = 5.0 Hz, 1H),
13
4.18 (d, J = 4.8 Hz, 2H), 3.75 (s, 3H). C NMR (DMSO-d6, 75 MHz): 154.9, 147.5,
145.5, 139.5, 130.8, 128.4, 124.5, 119.8, 119.1, 115.3, 113.6, 55.7, 42.7. HPLC
analyses: 99.8%.
1-(4-hydroxy-3-methoxybenzyl)-3-(4-methoxyphenyl)urea (7d):
1
White solid (Yield: 72%), m.p. 154.9–156.8 °C. H NMR (DMSO-d6, 300
MHz): 8.81 (s, 1H), 8.25 (s, 1H), 7.31 (d, J = 8.9 Hz, 2H), 6.88 (s, 1H), 6.82 (d, J = 8.9
Hz, 2H), 6.74 (d, J = 8.2 Hz, 1H), 6.71 (d, J = 9.0 Hz, 1H), 6.34 (t, J = 5.6 Hz, 1H),
4.18 (d, J = 5.7 Hz, 2H), 3.76 (s, 3H), 3.70 (s, 3H). 13C NMR (DMSO-d6, 75 MHz):

155.4, 154.0, 147.4, 145.4, 133.6, 131.1, 119.7, 119.5, 115.3, 113.9, 111.8, 55.6, 55.1,
42.7. HPLC analyses: 99.4%.
1-(4-hydroxy-3-methoxybenzyl)-3-(p-tolyl)urea (7e):
1
White solid (Yield: 63%), m.p. 198.4–199.5 °C. H NMR (DMSO-d6, 300
MHz): 8.80 (bs, 1H), 8.32 (s, 1H), 7.27 (d, J = 7.6 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H),
6.87 (s, 1H), 6.73 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 9.0 Hz, 1H), 6.38 (t, J = 5.1 Hz, 1H),
4.17 (d, J = 5.2 Hz, 2H), 3.17 (s, 3H), 2.12 (s, 3H). 13C NMR (DMSO-d6, 75 MHz):
154.9, 147.5, 145.5, 139.5, 130.8, 128.4, 124.5, 119.8, 119.1, 115.3, 113.6, 55.7, 42.7.
HPLC analyses: 98.6%.
1-butyl-3-(4-hydroxy-3-methoxybenzyl)urea (7f):
1
White solid (Yield: 20%), m.p. 96.7–98,5 °C. H NMR (DMSO-d6, 300 MHz):
8.76 (bs, 1H), 6.81 (s, 1H), 7.60 (d, J = 7.9 Hz, 1H), 6.63 (d, J = 7.8 Hz, 1H), 6.11 (bs,
1H), 5.84 (bs, 1H), 4.08 (s, 2H), 3.73 (s, 3H), 1.39 – 1.16 (m, 6H), 0.86 (t, J = 7.1 Hz,
13
3H). C NMR (DMSO-d6, 75 MHz): 158.1, 147.4, 145.2, 131.7, 119.5, 115.2, 111.6,
55.8, 55.5, 42.8, 32.2, 19.5, 13.7. HPLC analyses: 98.3%.
1-hexyl-3-(4-hydroxy-3-methoxybenzyl)urea (7g):
Yellow solid (Yield: 42%), m.p. 83.4–84.3 °C. 1H NMR (DMSO-d6, 300 MHz):
6.79 (d, J = 8.0 Hz, 1H), 6.79 (s, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.02 (s, 1H), 5.26 (t,
J = 5.3 Hz, 1H), 4.98 (t, J = 5.0 Hz, 1H), 4.17 (d, J = 5.5 Hz, 2H), 3.79 (s, 3H), 3.08 (q,
13
J = 6.7 Hz, 2H), 1.43 – 1.36 (m, 8H), 0.89 (t, 3H). C NMR (DMSO-d6, 75 MHz):
158.8, 146.8, 144.9, 131.2, 120.2, 114.4, 110.3, 55.8, 44.3, 40.5, 31.5, 30.2, 26.5, 22.5,
14.0. HPLC analyses: 98.8%.
1-(4-hydroxy-3-methoxybenzyl)-3-phenylthiourea (8a):
1
White solid (Yield: 37%), m.p. 131.4–133.3 °C. H NMR (DMSO-d6, 300
MHz): 9.50 (s, 1H), 8.84 (s, 1H), 7.98 (s, 1H), 7.46 (d, J = 7.4 Hz, 2H), 7.34 (d, J = 7.3
Hz, 2H), 7.12 (t, J = 7.1 Hz, 1H), 6.96 (s, 1H), 6.76 (d, J = 6.7 Hz, 2H), 4.61 (d, J = 4.6
Hz, 2H), 3.76 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 180.5, 147.5, 145.7, 139.3,
129.5, 128.5, 124.1, 123.2, 120.3, 115.3, 112.3, 55.6, 47.3. HPLC analyses: 99.8%.
1-(4-hydroxy-3-methoxybenzyl)-3-(4-nitrophenyl)thiourea (8b):
1
Yellow solid (Yield: 39%), m.p. 163.7–164.9°C. H NMR (DMSO-d6, 300
MHz): 10.13 (s, 1H), 8.90 (bs, 1H), 8.50 (s, 1H), 8.17 (d, J = 9.2 Hz, 2H), 7.87 (d, J =
9.2 Hz, 2H), 6.97 (s, 1H), 6.79 (d, J = 8.2 Hz, 2H), 6.75 (d, J = 8.0 Hz, 1H), 4.61 (d, J
= 5.1 Hz, 2H), 3.77 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 179.9, 147.5, 146.4,
145.9, 141.8, 128.6, 124.4, 120.5, 115.3, 112.4, 55.7, 47.3. HPLC analyses: 99.1%.
1-(4-chlorophenyl)-3-(4-hydroxy-3-methoxybenzyl)thiourea (8c):
1
White solid (Yield: 51%), m.p. 154.0–154.7 °C. H NMR (DMSO-d6, 300
MHz): 9.56 (s, 1H), 8.85 (bs, 1H), 8.08 (s, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.6
Hz, 2H), 6.95 (s, 1H), 6.77 (d, J = 9.3 Hz, 1H), 6.74 (d, J = 8.2 Hz, 1H), 4.60 (d, J = 5.1
Hz, 2H), 3.76 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 180.5, 147.5, 145.7, 138.4,
129.3, 128.3, 127.8, 124.6, 120.3, 115.3, 112.3, 55.6, 47.3. HPLC analyses: 98.5%.

1-(4-hydroxy-3-methoxybenzyl)-3-(4-methoxyphenyl)thiourea (8d):
1
White solid (Yield: 56%), m.p. 140.0–141.3 °C. H NMR (DMSO-d6, 300
MHz): 9.30 (s, 1H), 8.82 (s, 1H), 7.77 (bs, 1H), 7.25 (d, J = 8.8 Hz, 2H), 6.93 (s, 1H),
6.89 (d, J = 8.8 Hz, 2H), 4.58 (d, J = 5.1 Hz, 2H), 3.75 (s, 3H), 3.73 (s, 3H). 13C NMR
(DMSO-d6, 75 MHz): 180.9, 156.5, 147.4, 145.6, 131.8, 129.7, 125.9.120.1, 115.2,
113.9, 112.1, 55.6, 55.2, 47.2. HPLC analyses: 98.8%.
1-(4-hydroxy-3-methoxybenzyl)-3-(p-tolyl)thiourea (8e):
1
White solid (Yield: 48%), m.p. 131.4–133.3 °C. H NMR (DMSO-d6, 300
MHz): 7.89 (s, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.07 (d, J = 8.2 Hz, 2H), 6.87 (s, 1H),
6.83 (d, J = 8.0 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.14 (bs, 1H), 5.63 (s, 1H), 4.76 (d, J
= 5.3 Hz, 2H), 3.86 (s, 3H), 2.33 (s, 3H). 13C NMR (DMSO-d6, 75 MHz): 180.8, 146.7,
145.3, 137.6, 133.2, 130.8, 129.2, 125.5, 120.8, 114.5, 110.7, 56.0, 49.4, 21.0. HPLC
analyses: 99.3%.
1-hexyl-3-(4-hydroxy-3-methoxybenzyl)thiourea (8f):
1
White solid (Yield: 22%), m.p. 81.1–82.0 °C. H NMR (CDCl3, 300 MHz):
6.89 – 6.87 (m, 2H), 6.80 (d, J = 8.0 Hz, 1H), 6.01 (bs, 1H), 5.80 (bs, 1H), 5.65 (s, 1H),
4.55 (s, 2H), 3.88 (s, 3H), 3.35 (d, J = 4.1 Hz, 2H), 1.60 – 1.48 (m, 8H), 0.86 (d,
J = 6.1 Hz, 3H). 13C NMR (CDCl3, 75 MHz): 181.7, 147.0, 145.6, 128.7, 120.7, 114.6,
110.4, 56.0, 48.6, 44.4, 31.4, 28.8, 26.5, 22.5, 13.9. HPLC analyses: 97.3%.
Biological Assays
Cytotoxic activity
All compounds were submitted to cytotoxic activity assays in tumorigenic and
non-tumorigenic cell lines. The tumorigenic cell lines were murine melanoma
(B16F10), human melanoma (SK-MEL-25 and A2058), human glioblastoma (U-87),
human mammary adenocarcinoma (MCF-7), human cervical adenocarcinoma (HeLa),
and human fibroblast (T75) as non-tumorigenic cell line. The MTT assay was used to
evaluate cell viability and proliferation. Cells in the logarithmic growth phase were
plated at a density of 104 cells/mL in 96-well plates. 24 hours later, cells were treated
with capsaicin and compounds 5a-5g, 6a-6g, 7a-7g and 8a-8f at the concentrations of
3.0 to 100 µM for further 24 h. Then, 5 μL of MTT (5 mg/mL) was then added to each
well and incubated again under same conditions for 3 h. Subsequently, 100 μL of 30%
sodium dodecyl sulfate (SDS) was added and plates were stored overnight. Then, the
absorbance was quantified at 570 nm by spectrophotometry (Spectramax-M2, Software
Molecular Pro 5.4, Sunnyvale, CA, EUA).69 Cell viability was expressed as percentage
values compared to the negative control.

Aa: sample absorbance; Ab: negative control absorbance; Ac: positive control
absorbance.
The data were plotted on Prism 5 software version 5.03 (GraphPad Software,
Inc, 2009), where the IC50 was calculated according to the tutorial provided by Prism.
Cell cycle analysis
B16F10 murine melanoma and A2058 human melanoma cells were plated in 12-
well plates at the concentration of 105 cells/well. For synchronization, the cells were
plated with RPMI medium without addition of fetal bovine serum (FBS). After 12 h of
incubation, the FBS-free medium was removed and the cells were treated with the
highest activity analog 6g at concentrations relative to the IC50 value, diluted in RPMI
medium supplemented with 10% FBS and incubated in an oven containing 5% CO2 at
37 °C. After 24 h of incubation, cells were washed with PBS and centrifuged for 10 min
at 1200 RPM. The pellet was resuspended in 300 μL of HSF (0.1% Triton X-100, 0.1%
sodium citrate of 50 μg/mL propidium iodide), 10 μg/mL RNAse (Sigma-Aldrich) and
incubated at 37 °C for 1 h in the absence of light. Data were acquired by flow cytometry
(FACSCalibur). The Dean Jett-Fox algorithm of the FlowJo software (TreeStar Inc,
Ashland, OR, USA) was used to determine the percentage of cells in the home phase of
the cycle.
Analysis of cell signaling induced by the compound 6g
For analysis of cell cycle and cell migration mediators, Western blotting assay
was standardized from the cell lysates obtained from the culture of A2058 melanoma
cells treated with the 6g. Proteins from each sample were separated by gel (10% SDS-
PAGE) electrophoresis using buffer 2.5 mM Tris-HCl (pH 8.0), 19 mM glycine and 0.3
mM SDS. The samples were then transferred to nitrocellulose membranes (Protran,
Schleicher and Schuell, USA) in buffer containing 1.2 mM Tris-HCl (pH 8.0), 9.6 mM
glycine and 20% methanol.

The efficacy of the blots was verified by staining the membranes with Ponceau S
(Sigma) dye, followed by blocking in solution containing 5% milk powder (Nestlé,
Brazil) diluted in PBS. After the blockade, (3x) 10 min washes were performed with
TTBS (20 mM Tris-HCl (pH 7.6), 150 mM NaCl and 0.1% Tween 20 (pH 7.6) for 2 h.
After washing, membranes were incubated for 24 h at 4 °C under constant stirring and
with the following primary antibodies: Anti-Cyclin D1 mouse, anti-Cyclin D3 mouse,
anti-Cyclin A mouse, antiNFк-B rabbit, anti-fosfo Akt (Thr308) rabbit, anti-β-Catenin
rabbit, anti-c-Raf, antifosfo c-Ras, anti-fosfo p38 (Thr180/Tyr182) rabbit, anti-fosfo
MAPK rabbit, anti-fosfo GSK3β (Ser9) rabbit; anti-N-Cadherin rabbit, anti-Src rabbit,
anti-fosfo Src (Tyr416) rabbit, anti-FAK rabbit, anti-fosfo FAK (Tyr925) rabbit and
anti-fosfo FAK (Tyr397) rabbit. All primary antibodies were purchased from Cell
Signaling Technology (Beverly, MA). After incubation with the primary antibodies,
membranes were washed with TTBS for 30 min and incubated for 1 h at room
temperature under stirring with the secondary antibody conjugated with IgG horseradish
peroxidase, purchased from Sigma-Aldrich (St. Louis, MO). Then, membranes were
washed with TTBS and the Luminata™ chemiluminescent substrate was added and the
membranes were developed and analyzed in ImageQuant LAS4000 (GE)
Photodocumentator system. All bands were normalized in comparison with β-actin.
FLIPR assays – TRPV1/6 activity
Cell culture reagents and Lipofectamine 2000 were purchased from Invitrogen
Life Technologies Europe B.V. The calcium indicator, Calcium-5 was bought from
Molecular Devices LLC. All other chemicals were purchased from Sigma-Aldrich.
HEK293T cells (ATCC) were cultured at 37 °C in DMEM cell culture media
supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 1%
penicillin/streptomycin. The attached cells were trypsinized and plated at 27,000
cells/well density onto Corning® 96-well black polystyrene clear bottom microplates
(CLS3603 Sigma-Aldrich) coated with 100 μg/mL poly-D-lysine (P6407 Sigma-
Aldrich) using 100 µL phenol-red free DMEM with 10% FBS and 2 mM glutamine
without antibiotics. Transfection was performed 24 h later using 200 ng of TRPV1-
pcDNA 3.1 and 0.6 µL Lipofectamine 2000 reagent/well.
TRPV1 activity was measured using HEK293T cells transiently overexpressing
TRPV1 as previously reported.57 24 h after the transfection, the medium was replaced

with 90 µL of loading buffer (modified Krebs buffer containing 117 mM NaCl, 4.8 mM
KCl, 1 mM MgCl2, 5 mM D-glucose, 10 mM HEPES, 1.8 mM CaCl2 and calcium-5
fluorescence dye (50 µL/mL loading buffer). Cells were incubated in the loading buffer
at 37 °C for 1 h in dark. Fluorescence Ca2+ measurements were carried out using
FLIPRTETRA high-throughput, fluorescence microplate reader. Cells were excited using
a 470–495 nm LED module, and the emitted fluorescence signal was filtered with a
515–575 nm emission filter (manufacturer’s guidelines). Stable baselines were
established for 50 seconds before 10 µL of a 10X compound prepared in 1.8 mM CaCl2-
containing Krebs buffer was robotically administered to the cells. Cells were incubated
and fluorescence was monitored in the presence of compound for an additional 5
minutes before administration of 100 µL of 2X capsaicin or 6g prepared in 1.8 mM
CaCl2-containing Krebs buffer (final concentration of capsaicin in the assay plate was
100 nM). The activity of TRPV1 was measured by quantifying the area under the curve
(AUC) of the fluorescence intensity, following administration of the agonists.
HEK293 cells stably over-expressing hTRPV6 (HEK-TRPV6, previously
established58) were seeded at a density of 70,000 cells per well onto Corning® 96-well
black polystyrene clear bottom microplates coated with 100 μg/mL poly-D-lysine using
100 µL phenol-red free DMEM with 10% FBS and 2 mM glutamine without antibiotics.
TRPV6 activity was measured using the HEK-TRPV6 cell line as previously
reported.59,70 16 h later the medium was replaced with 50 µL of nominally calcium-free
(NCF) loading buffer (modified Krebs buffer containing 117 mM NaCl, 4.8 mM KCl, 1
mM MgCl2, 5 mM D-glucose, 10 mM HEPES, and calcium-5 fluorescence dye (50 µL
per mL loading buffer). Cells were incubated in the NCF-loading buffer at 37 °C for 1 h
in dark. Fluorescence Cd2+ measurements were carried out using FLIPRTETRA high-
throughput, fluorescence microplate reader. Cells were excited using a 470–495 nm
LED module, and the emitted fluorescence signal was filtered with a 515–575 nm
emission filter (manufacturer’s guidelines). Stable calcium-free baselines were
established for 50 seconds before 50 µL of 3X compound either cis-22a or 6g was
robotically administered to the cells. Cells were incubated and fluorescence was
monitored in the presence of compound for an additional 5 minutes before
administration of 50 µL of a 4X CdCl2 substrate (final concentration of CdCl2 in the
assay plate was 50 µM). The activity of hTPRV6 was measured by quantifying the AUC
of the fluorescence intensity following administration of the substrate.

Experiments were done with 3 to 6 repeats per group on at least two different
days. Relative IC50 values were determined using GraphPad® Prism (GraphPad®
Software, v. 5.0, San Diego, CA, US). Inhibition curves were obtained by non-linear
regression using the built-in log (inhibitor) versus normalized response function.
Assessment of the nociceptive behavior induced by compound 6g
Male Swiss mice, weighting between 18 to 20 g, were housed in a room with
controlled temperature (22 ± 2 °C) and lighting (lights on from 6 am to 6 pm), with free
access to water and food. All experiments were conducted between 9 am and 5 pm and
were in accordance with the ethical guidelines of the International Association for the
Study of Pain.71 Mice were individually positioned in a fully transparent box with an
upper opening for air intake. Animal remained for 20 min for its adaptation prior to the
experiment. Capsaicin, compound 6g (5.2 nmol/paw, 20 μL) and the corresponding
vehicle (EtOH + saline), under the same experimental conditions, were administered by
the intraplantar route. Immediately after injection, the animal returned to the transparent
box positioned in front of a mirror to facilitate viewing and measuring the time (in
seconds) of flinches or licks of the treated paw for 5 min.53 The animal assays were
approved by the Ethics Committee for the use of animals of the Butantan Institute,
under the protocol number: 1904030518.
Statistical analysis
Statistical analysis was performed using analysis of variance72 associated with
the Tukey test73 for comparison of more than two means. The significance level was p <
0.05.
Molecular modeling approach
The three-dimensional (3D) molecular models of capsaicin and its derivative
were built up, in their neutral forms, using ChemBio Draw® Ultra 12.0 and the
electronic, steric, topological and lipophilicity properties were calculated by Spartan'14®

software. Energy minimization was performed by semi-empirical method PM6,
followed by Hartree-Fock 3-21G method, thus obtaining the descriptors: electrostatic
potential maps (EPM), energy of the frontier orbitals HOMO and LUMO, calculated
partition coefficient (ClogP), hydrogen bond acceptor (HBA), hydrogen bond donor
(HBD), polar surface area (PSA), and dipole moment (μ).
Reverse virtual screening approach
In order to find possible targets for the most promising compound 6g, a virtual
screening strategy was employed. Compound 6g was simplified (6g_mod) by removing
its aliphatic chain attached to the thiourea linker and then compared to all ligands bound
to proteins available on PDB. This task was accomplished using the software
PharmACOphore74 and ShaEP.75 The first software changed 6g_mod conformation to
align it to all the co-crystallographic ligands available on PDB. The latter was used to
quantitatively express the shape and electrostatic similarity between aligned 6g_mod
and the PDB ligands. 6g was simplified to reduce biasing the alignment to the aliphatic
portion. This approach helped to find possible targets for 6g, since similar compounds
tent to have similar biological activities. The top best cancer-related targets were filtered
out and analyzed via molecular docking with AutoDock Vina.76
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements
The authors are grateful to Faculty of Pharmacy of University of São Paulo,
which allowed the development of this work and to 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
São Paulo (FAPESP, grant numbers: 2013/19311-6 and 2017/00689-0) for financial
support. R.B. and M.A.H. acknowledge the SNF Sinergia grant number:
CRSII5_180326 for the funding to support this work. M.R.C. acknowledge the Swiss
Excellence Scholarship for Foreign Students and Scholars (ESKAS – 2017.0670). The
authors are also grateful to Guilherme Galdino for technical assistance in biological

assays. The human TRPV1 plasmid was a kind gift from Dr. Yoshiro Suzuki,
Department of Physiology, Iwate Medical University, Japan and Dr. Makoto Tominaga,
Division of Cellular Signaling, Okazaki Institute for Integrative Bioscience, Japan.
Supplementary material
1H and 13C NMR spectra, HPLC, and supplementary figures are available online
at https://doi.org/10.1016/
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