Coumaric acid derivatives as tyrosinase inhibitors: Efficacy studies through in silico, in vitro and ex vivo approaches

Article abstract of DOI:10.1016/j.bioorg.2020.104108

p-Coumaric acid is a known inhibitor of tyrosinase, an enzyme involved in the initial steps of the melanin synthesis in human and other species. However, its low lipophilicity impairs its penetration through skin and efficacy as antimelanogenic agent indeed. Accordingly, this paper reports the assessment of several coumaric acid derivatives as tyrosinase inhibitors and antimelanogenic agents in in vitro, in silico and ex vivo assays. The compounds were designed with modifications in the aromatic and acid moieties of p-coumaric acid, being the coumarate esters the most promising derivatives. The compounds showed higher tyrosinase inhibitory activity (pIC₅₀ 3.7–4.2) than the parent acid, being compounds 1d, 1e and 1f the most potent inhibitors. Docking analysis showed that these esters are competitive inhibitors per se, and act independently of a redox mechanism as suggested by DPPH assays. Moreover, the esters showed efficacy in reducing the melanin deposition in human skin fragments at 0.1% concentration, especially compound 1e. In summary, there is an important equilibria between tyrosinase affinity and lipophilicity that must be considered to get effective antimelanogenic agents with adequate permeability in the skin.

Full text of DOI:10.1016/j.bioorg.2020.104108

BioorganicChemistry103(2020)104108
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Coumaric acid derivatives as tyrosinase inhibitors: Efficacy studies through
in silico, in vitro and ex vivo approaches
Marina Themoteo Varelaa,1, Márcio Ferrarinia,1, Vitória Gallo Mercaldia, Bianca da Silva Sufib,
⁎
Giovana Padovanib, Lucas Idacir Sbrugnera Nazatob, João Paulo S. Fernandesa,
a Department of Pharmaceutical Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences, Universidade Federal de São Paulo, Rua São Nicolau 210,
09913-030 Diadema-SP, Brazil
b Research and Development Department, Chemyunion Química Ltda, Av. Independência 1501, 18087-101 Sorocaba-SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
p-Coumaric acid is a known inhibitor of tyrosinase, an enzyme involved in the initial steps of the melanin
synthesis in human and other species. However, its low lipophilicity impairs its penetration through skin and
efficacy as antimelanogenic agent indeed. Accordingly, this paper reports the assessment of several coumaric
acid derivatives as tyrosinase inhibitors and antimelanogenic agents in in vitro, in silico and ex vivo assays. The
compounds were designed with modifications in the aromatic and acid moieties of p-coumaric acid, being the
coumarate esters the most promising derivatives. The compounds showed higher tyrosinase inhibitory activity
(pIC50 3.7–4.2) than the parent acid, being compounds 1d, 1e and 1f the most potent inhibitors. Docking
analysis showed that these esters are competitive inhibitors per se, and act independently of a redox mechanism
as suggested by DPPH assays. Moreover, the esters showed efficacy in reducing the melanin deposition in human
skin fragments at 0.1% concentration, especially compound 1e. In summary, there is an important equilibria
between tyrosinase affinity and lipophilicity that must be considered to get effective antimelanogenic agents
with adequate permeability in the skin.
Coumaric acid ester
Tyrosinase inhibitor
Antimelanogenic agent
Melanin synthesis inhibitor
Molecular docking
1. Introduction
the skin, such as acne [4].
Tyrosinase is an enzyme present in several organisms, including
Melanin biosynthesis is a natural process that occurs in several
living species, including plants, fungi and mammals. The limiting step
in melanogenesis is controlled by metalloprotein tyrosinase, a copper-
containing polyphenol oxidase that oxidizes L-tyrosine and L-DOPA into
DOPA-quinone (Fig. 1) [1]. The biological importance of melanin is
variable in each organism, but are mostly related to photoprotection
from UV light and pigmentation. In humans, melanin is responsible for
the different eye, hair and skin colors, as well as neural pigmentation,
associated with neuromelanin [2].
plant, fungal and animal species, but with marked differences among
them. For instance, human tyrosinase is an intracellular membrane-
bound glycoprotein [5], while fungal tyrosinase is a soluble enzyme.
Although the tyrosinases from different species differ considerably re-
garding several aspects of the protein structure, they share in common a
similar active site, comprised by two copper atoms coordinated by three
histidine residues, which catalyzes the oxidation reaction of phenols
[6,7].
Mushroom tyrosinase is a widely used model for identifying tyr-
osinase inhibitors for human applications, since it is an accessible,
commercially available enzyme due to its soluble characteristic [8,9].
Moreover, known inhibitors of mushroom tyrosinase usually exhibit
increased potency in murine and human tyrosinases, and thus by
identifying novel inhibitors in mushroom tyrosinase model, really ef-
fective antimelanogenic compounds may be expected [10,11].
There are many applications of melanin biosynthesis inhibitors in
both pharmacological and cosmetic aspects, especially to treat
Many dysfunctions and pathologies may come as a result of the
excess or lacking melanin pigments produced in the cells. Albinism is a
genetic disorder that is characterized by alteration of TYR gene, re-
sponsible for controlling the presence of tyrosinase in cells [3]. This
alteration causes cells to be unable to produce melanin, giving to the
individuals the characteristic pale skin and pink/red eyes and white
hair. Excessive production of melanin is usually caused by prolonged
and continuous sun exposure and also after inflammation processes in
⁎ Corresponding author at: Department of Pharmaceutical Sciences, Universidade Federal de São Paulo, Rua São Nicolau 210, 09913-030 Diadema-SP, Brazil.
E-mail address: joao.fernandes@unifesp.br (J.P.S. Fernandes).
1 These authors contributed equally.
https://doi.org/10.1016/j.bioorg.2020.104108
Received 11 May 2020; Received in revised form 23 May 2020; Accepted 15 July 2020
Availableonline21July2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
Fig. 1. Oxidation of the amino acid L-tyrosine to L-DOPA and then to DOPA-quinone.
Fig. 2. Tested compounds.
dermatological hyperpigmentation. Natural products were historically
used as drugs and drug prototypes [12] and the literature brings that p-
coumaric acid is a natural inhibitor of tyrosinases [8,10,11]. p-Cou-
maric acid is a phenolic phenylpropanoid acid that have shown in-
hibitory activity on mushroom and human tyrosinases, as described in
recent literature, which presents evident structural similarities with the
tyrosinase substrate, the amino acid L-tyrosine [10,11]. Curiously, both
phenylalanine and tyrosine are also substrates to the biosynthetic
pathway of p-coumaric acid [13]. These considerable facts may explain
the reason why p-coumaric acid can bind to the tyrosinases and inhibit
their activities on melanin synthesis.
compound in vivo. To overcome this issue, its methyl ester was already
evaluated as tyrosinase inhibitor [8], and showed to be more potent
than the parent acid. However, limited information about other cou-
maric acid derivatives is reported in literature. Regarding this, the
objective of the present study was to assess the activity of a set of
coumaric acid analogues as antimelanogenic compounds, which con-
sidered a possible better penetration in the skin in the molecule’s de-
sign. For this purpose, the inhibitory activity of several derivatives on
mushroom tyrosinase through experimental and in silico assays (mole-
cular docking) were performed, and ex vivo studies on human skin
fragments for evaluating the efficacy as antimelanogenic agent were
also done.
Although p-coumaric acid has inhibitory activity on tyrosinase, its
poor penetration in the skin limited its application as antimelanogenic
2

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
2. Material and methods
J = 6.7 Hz, 2H), 1.84 – 1.61 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H).
i-Propyl p-coumarate (1e). Yellowish solid (m.p. 69–70 °C); 65%
yield. 1H NMR (CDCl3) δ 7.62 (d, J = 15.9 Hz, 1H), 7.50 – 7.34 (m,
2H), 6.91 – 6.83 (m, 2H), 6.28 (d, J = 15.9 Hz, 1H), 5.14 (dt, J = 12.4,
6.2 Hz, 1H), 1.32 (d, J = 6.2 Hz, 6H).
2.1. Chemicals and equipment
The chemicals were acquired from Sigma-Aldrich Co. in adequate
purity to use in the experiments. The p-coumaric (1a), cinnamic (2a), p-
methoxycinnamic (3a) and caffeic (4a) acids were used as starting
materials for preparing the derivatives described below as well in the
activity assays. Eugenol (5), isoeugenol (6) and 2-allylphenol (7) were
also tested as acquired from commercial source. The mushroom tyr-
osinase (> 1000 U) solution was prepared in accordance to the man-
ufacturer’s instructions. The compounds were characterized through 1H
NMR spectroscopy in a Bruker Advance 300 spectrometer, operating at
300 MHz frequency, using TMS as internal standard and the chemical
shifts (δ) are presented in ppm. The NMR data are in accordance to
previous literature reports [14–21,47]. The purity of the compounds
was chromatographically assessed through GC-MS in a Shimadzu
QP2010 equipment and considered adequate when > 95%. The tyr-
osinase and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assays
were performed in a 96-well plates Biotek Synergy HT plate reader, and
read in the described wavelength. The compounds selected for the ex
vivo testing were incorporated in a simple base comprised by Uniox C
(cetearyl alcohol and polysorbate 60) at 0.1% (w/v) concentration. The
base alone was also used as sham group.
n-Butyl p-coumarate (1f). Yellowish solid (m.p. 75–78 °C); 40%
yield. 1H NMR (CDCl3) δ 7.63 (d, J = 16.0 Hz, 1H), 7.41 (d, J = 8.5 Hz,
2H), 6.85 (d, J = 8.5 Hz, 2H), 6.30 (d, J = 16.0 Hz, 1H), 4.21 (t,
J = 6.6 Hz, 2H), 1.75 – 1.59 (m, 2H), 1.50 – 1.37 (m, 2H), 0.96 (t,
J = 7.3 Hz, 3H).
Methyl cinnamate (2b). Yellowish oil; 59% yield. 1H NMR (CDCl3) δ
7.69 (d, J = 16.0 Hz, 1H), 7.59 – 7.44 (m, 2H), 7.44 – 7.28 (m, 3H),
6.44 (d, J = 16.0 Hz, 1H), 3.79 (s, 3H).
Ethyl cinnamate (2c). Yellowish oil; 57% yield. 1H NMR (CDCl3) δ
7.69 (d, J = 16.0 Hz, 1H), 7.57 – 7.46 (m, 2H), 7.41 – 7.36 (m, 3H),
6.44 (d, J = 16.0 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.33 (t, J = 7.1 Hz,
3H).
i-Propyl cinnamate (2e). Yellowish oil; 48% yield. 1H NMR (CDCl3)
δ 7.67 (d, J = 16.0 Hz, 1H), 7.52 – 7.48 (m, 2H), 7.38 – 7.34 (m, 3H),
6.41 (d, J = 16.0 Hz, 1H), 5.14 (sept, J = 6.2 Hz, 1H), 1.31 (d,
J = 6.2 Hz, 6H).
n-Butyl cinnamate (2f). Yellowish oil; 41% yield. 1H NMR (CDCl3) δ
7.68 (d, J = 16.0 Hz, 1H), 7.52 (dd, J = 6.6, 2.9 Hz, 2H), 7.45 – 7.33
(m, 3H), 6.44 (d, J = 16.0 Hz, 1H), 4.21 (t, J = 6.7 Hz, 2H), 1.78 – 1.61
(m, 2H), 1.54 – 1.34 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H).
Methyl p-methoxycinnamate (3b). White solid (m.p. 85–86 °C); 77%
yield. 1H NMR (CDCl3) δ 7.65 (d, J = 16.0 Hz, 1H), 7.47 (d, J = 8.7 Hz,
2H), 6.90 (d, J = 8.7 Hz, 2H), 6.31 (d, J = 16.0 Hz, 1H), 3.83 (s, 3H),
3.79 (s, 3H).
2.2. Design of the compounds
The compounds were designed to explore the importance of each
moiety in the p-coumaric acid’s structure allied to an increased lipo-
philicity, which could help to achieve a better penetration in the skin
than the prototype. Regarding this, ester, amide and ketone derivatives
were prepared and tested in order to investigate whether these com-
pounds are behaving as prodrugs or if they possess any activity per se.
Different lengths of the alkyl chain were tested to explore the lipophi-
licity and potential hydrophobic interactions on the active site.
Furthermore, the role of the phenol hydroxy group was also assessed
through several phenolic compounds (such as 5–7), since phenol group
has radical scavenging activity that may chemically interfere with the
oxidation process of tyrosinase over the substrate. Additionally, the
phenolic pattern of the aromatic moiety seems to affect the inhibitory
activity [9,22]. The set of compounds proposed are shown in Fig. 2.
Ethyl p-methoxycinnamate (3c). Yellowish solid (m.p. 49–50 °C);
44% yield. 1H NMR (CDCl3) δ 7.64 (d, J = 16.0 Hz, 1H), 7.47 (d,
J = 8.7 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.30 (d, J = 16.0 Hz, 1H),
4.25 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H).
Methyl caffeate (4b). White solid (m.p. 151–153 °C); 57% yield. 1H
NMR (CDCl3) δ 7.59 (d, J = 15.9 Hz, 1H), 7.08 (d, J = 1.9 Hz, 1H),
7.01 (dd, J = 8.2, 1.9 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.27 (d,
J = 15.9 Hz, 1H), 5.84 (br.s, 2H), 3.80 (s, 3H).
2.4. Synthesis of the amide 1g
The compound 1 g was prepared following previous method de-
scribed elsewhere [23]. In a flask 1.1 mmol of p-coumaric acid (1a),
1.1 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)
and 1.1 mmol of hydroxybenzotriazole (HOBt) hydrate were dissolved
in 15 mL of dicloromethane. The mixture was stirred for 1 h, when
1 mmol of 1-butylamine was added. The reaction was carried out
overnight and then washed with 2x10 mL 1 M HCl, 2x10 mL of satu-
rated NaHCO3 solution and 10 mL of water. The organic layer was
separated, dried with anhydrous Na2SO4 and evaporated to give the
pure product 1 g.
2.3. Synthesis of the esters
The ester derivatives (b-f) were synthesized following classic
Fischer esterification [18] using 1 mmol of the corresponding acid,
0.1 mL of concentrated sulfuric acid and the corresponding alcohol as
solvent. The reaction proceeded for 3 h or until no more acid was ob-
served in TLC. The reaction mixture was neutralized with aqueous
NaHCO3 saturated solution, and the solvent excess was evaporated
under reduced pressure. The residue was taken up in 10 mL ethyl
acetate and washed with 3x10 mL of saturated NaHCO3 solution and
10 mL of water. The organic layer was dried with anhydrous Na2SO4.
The desired compounds were further purified in a silica gel column
chromatography, using hexane:ethyl acetate as eluent.
N-Butyl-4-coumaramide (1g). White solid (m.p. 209–212 °C); 56%
yield. 1H- NMR (DMSO‑d6) δ 9.82 (s, 1H), 7.93 (t, J = 5.2 Hz, 1H), 7.38
(d, J = 8.5 Hz, 2H), 7.30 (d, J = 15.6 Hz, 1H), 6.78 (d, J = 8.5 Hz,
2H), 6.40 (d, J = 15.6 Hz, 1H), 3.15 (q, J = 6.0 Hz, 2H), 1.48 – 1.36
(m, 2H), 1.35 – 1.24 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). 13C- NMR
(DMSO‑d6) δ 165.71, 159.21, 138.89, 129.58, 126.50, 119.30, 116.18,
38.74, 31.80, 20.10, 14.15.
Methyl p-coumarate (1b). White solid (m.p. 134–136 °C); 57% yield.
1H NMR (CDCl3) δ 7.65 (d, J = 16.0 Hz, 1H), 7.42 (d, J = 8.6 Hz, 2H),
6.86 (d, J = 8.6 Hz, 2H), 6.30 (d, J = 16.0 Hz, 1H), 6.11 (s, 1H), 3.81
(s, 3H).
2.5. Synthesis of the ketones 1 h and 1i
Ethyl p-coumarate (1c). Yellowish solid (m.p. 70–71 °C); 53% yield.
1H NMR (CDCl3) δ 7.56 (d, J = 16.0 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H),
6.99 (s, 1H), 6.80 (d, J = 8.6 Hz, 2H), 6.21 (d, J = 16.0 Hz, 1H), 4.19
(q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
The ketones were prepared following the previous report from our
group [17,47]. Briefly, 1 mmol of 4-hydroxybenzaldehyde, and 1 mL of
the corresponding ketone were dissolved in ethanol, and then 1 mL of
40% sodium hydroxide solution was added. The reaction proceeded for
24 h and after neutralization, ethanol was evaporated. The pure pro-
duct was obtained by recrystallization from water.
n-Propyl p-coumarate (1d). Yellowish solid (m.p. 73–75 °C); 72%
yield. 1H NMR (CDCl3) δ 7.64 (d, J = 16.0 Hz, 1H), 7.41 (d, J = 8.6 Hz,
2H), 6.88 (d, J = 8.6 Hz, 2H), 6.30 (d, J = 16.0 Hz, 1H), 4.17 (t,
3

M.T. Varela, et al.
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1-(4-Hydroxyphenyl)but-1-en-3-one (1h). Yellowish solid (m.p.
109–112 °C); Yield 62%. 1H NMR (CDCl3) δ 7.55 – 7.36 (m, 3H), 6.87
(d, J = 8.7 Hz, 2H), 6.61 (d, J = 16.1 Hz, 1H), 5.55 (br. s, 1H), 2.37 (s,
3H).
evaluated using the Fontana Masson methodology using human skin
explants. These fragments were obtained from patients between 35 and
65 years old submitted to ophthalmic plastic surgery (Surgical Center of
the Eye Bank of the Sorocaba Ophthalmological Hospital - BOS-HOS,
Sorocaba-SP, Brazil), and kept in 0.9% saline buffer until the time of the
experiment. The collection and use of these human skin explants derive
from voluntary donation of the patient with their consent and approved
by the Ethics Committee of the Hospital (HOS-BOS), and it is under the
consubstantiate report number 3.065.484 registered in CONEP
(National Committee of Research Ethics).
1-(4-Hydroxyphenyl)hex-1-en-3-one (1i). Yellowish solid (m.p.
116–121 °C); Yield 68%. 1H NMR (CDCl3) δ 7.58 – 7.40 (m, 3H), 6.86
(d, J = 8.62 Hz, 2H), 6.63 (d, J = 16.1 Hz, 1H), 5.26 (br. s, 1H), 2.63
(t, J = 7.3 Hz, 2H), 1.81–1.61 (m, 2H), 0.98 (m, 3H).
2.6. Tyrosinase inhibition assay
The samples were then immersed in 70% ethanol for 60 s for de-
contamination and rinsed twice in fresh saline buffer. The samples were
cut on ~0.5 cm2 fragments and transferred to a cell culture dish con-
taining culture medium (Dulbecco’s Modified Eagle Medium, DMEM,
supplemented with 10% fetal bovine serum) leaving the skin to the top
side without contact with the medium, for maintenance and nutrition of
the tissue. The skin side of these fragments were immediately treated
with the formulation of the compounds (base alone for sham group, no
formulation for control group) and kept in contact with them for 72 h.
During this period, the culture dish was maintained in a 95% humid
atmosphere at 37 °C and 5% CO2. The compounds were applied to the
skin fragments at a rate of 12 mg/cm2.
The compounds were assessed for their tyrosinase inhibition activity
on the monophenolase activity (when using L-tyrosine as substrate),
following the previously described method elsewhere [9,15]. To these
assays, the compounds were freshly prepared by dissolving them in
phosphate buffer (50 mL KH2PO4 0.2 M + 11.5 mL NaOH 0.2 M, pH
6.5) with 0.1% DMSO to give a 1 mM stock solution. In a 96-well plate,
100 µL of each compound were transferred from the stock solution and
serially diluted (50%) with phosphate buffer. After this, 50 µL of
mushroom tyrosinase (~100 U/mL) were added and the solutions were
incubated for 10 min at 25 °C. After this, 50 µL of 1.5 mM L-tyrosine
solution were added to reach a final volume of 200 µL/well. The plate
was immediately read at 490 nm wavelength and every 3 min during
30 min. Compound 1a, a known tyrosinase inhibitor, was used as
standard and phosphate buffer as negative control. The assay was
performed in triplicate (n = 3), and the IC50 values were calculated by
non-linear regression in the GraphPad Prism 6.0 software.
After the treatment, the fragments were fixed in 4% paraformalde-
hyde (pH 7.4) for 24 h and transferred to 30% saccharose solution for
cryopreservation and inserted in tissue freezing medium. The samples
were sectioned in 10 μm thickness slices in a cryostat (CN1850, Leica
Biosystems) and directly collected onto silanized glass slides, where
were stained by Fontana Masson technique [26].
2.7. Antiradical activity
2.10. Measument of the melanin content
The antiradical activity of the compounds was assessed using the
DPPH assay following the methodology described by Gaspar et al. [24].
Briefly, 100 µL of DPPH methanolic solution (250 µM) were added to a
96-well plate and afterward 100 µL of the stock solutions of the com-
pounds (2 mM in methanol). The optical density (OD) from the plates
were read for every 3 min until 30 min at 25 °C at 515 nm wavelenght.
Methanol was used as negative control. The radical scavenging activity
(%) was calculated by using the formulae 100 × [(control OD – sample
OD) / control OD].
Melanin content was measured by optical semi-quantitative method
in the ImageJ software [27], adapting the guidelines proposed by Miot
et al. [28]. Automatic segmentation of the pixels corresponding to the
melanin of the other skin structures was used. In parallel, the selection
of the black and white (B&W) color threshold, hue, saturation and
brightness (HSB) color space and dark background were adjusted.
Afterwards, the selection of the area of interest for the quantifica-
tion of melanin pigments was done from the viable epidermis using the
freehand selection tool in the software. The pixels were semi-quantified
using the measure function from the software. The average melanin
content was normalized by the value of the selected area, and thus
eventual variations in the thickness of the epidermis, according to the
analyzed field, do not result in deviations from the evaluation.
2.8. Docking studies
To simulate the in silico protein–ligand interaction the compounds
were built in the software MarvinSketch 30.3 (ChemAxon Inc.) in a .mol
format. The starting geometry was optimized subsequently through
MM + molecular mechanics forcefield and semi-empirical PM3 method
using the GaussView 5.0 software (Gaussian Inc.). GOLD software
(CCDC, version 2020.1) was used for the docking simulations. The
protein used was a mushroom tyrosinase (Agaricus bisporus) retrieved
from the RCSB Protein Data Bank (PDB ID: 2Y9X) and the energy was
minimized using the online server YASARA [25]. The binding site was
defined as the atoms within 18 Å from the copper atoms in the active
site. GoldScore and ChemPLP were used as scoring functions to predict
the most likely binding pose of these compounds. The “allow early
termination” default setting was deactivated and results with a maximal
difference of 0.75 Å were clustered. The rest of the parameters were left
on the default settings. Then, genetic algorithm was used to perform
100 docking runs for each compound. The highest scoring poses were
considered for the evaluation of protein–ligand interactions, using a
cut-off value of 4 Å for the interactions. Molecular interactions and
visualization were done by using Discovery Studio Visualizer 20.1.0
(Dassault SystèmesBiovia Corp.).
2.11. ClogP estimation
The calculated n-octanol/water partition coefficient value was es-
timated using the calculator plugin from the Marvin software version
20.3.0 (ChemAxon Ltd.). The software uses a consensus method that
weights the PhysProp data with the calculated logP (ClogP) using the
Viswanadhan’s method [29]. The electrolyte concentration for Na+, K+
and Cl- used in the estimation was from default configuration (0.1 M).
2.12. Statistical analysis
The statistical analysis was carried out by analysis of variance
(ANOVA). The Bonferroni’s test was used for comparing the tested
groups with the control group. The considered significance level was
95% (p < 0.05).
3. Results and discussion
2.9. Ex vivo efficacy assessment
3.1. Tyrosinase inhibition
The efficacy of the selected compounds 1a, 1b, 1c, 1e and 1f was
Melanin overproduction may be caused by many different reasons,
4

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
leading to the darkening of the skin as a whole or in specific sites where
the damage was made. This may lead to hyperpigmentation on the skin
and melasma. Tyrosinase inhibitors are very useful in the treatment of
such hyperpigmentations of the skin, especially if they show to be ef-
fective through topical administration. Therefore, promising com-
pounds must have a good permeability to penetrate the skin and reach
the melanocytes in the deepest epidermis layers.
than the prototype (1a). This is an interesting finding because the
compounds not only could facilitate the permeation into the skin and
produce higher efficacy as tyrosinase inhibitors, but also promote long
lasting activity, since they will inevitably be hydrolyzed generating the
acid 1a, which is also an inhibitor. On the other hand, the esters from
acids 2a, 3a and 4a showed no relevant activity up to 1 mM, suggesting
a unique ability from the coumaric esters to exhibit inhibitory activity
and the essential role of the hydroxy group on this effect.
Several tyrosinase inhibitors are already available for such pur-
poses. Hydroquinone is chemically the 1,4-dihydroxybenzene and is
widely used as a skin-whitening agent alone, or usually in combination
with retinoids [4]. Arbutin, a glycosylated hydroquinone derivative, is
also a tyrosinase inhibitor, but it is less associated to skin irritation and
cytotoxicity related to hydroquinone [30]. Kojic acid is another natural
product produced by fungi, such as Penicillium sp. and Aspergillus sp.
This compound acts by chelating the copper ions present in the active
site of tyrosinases and thus, inhibiting its activity [31,32]. The main
problems related to these compounds are the safety, skin permeation
issues (due to high hydrophilicity) and unwanted side effects.
It is noteworthy that the p-coumaric acid derivatives (1) possess a
comparable or higher inhibitory effect on tyrosinase activity when
compared to the prototype 1a, and thus some SAR rules may be derived
from this data. The methyl (1b) and ethyl (1c) derivatives have very
similar IC50 values (~95 µM), which are 3-fold higher than the parent
acid (296 µM). When increasing the length of the alkyl chain, the in-
hibitory activity improved considerably. The n-butyl ester compound 1f
is the most potent in the set, showing an IC50 value of 69 µM. This
suggests that hydrophobic interactions in the binding site of the enzyme
may occur, improving the affinity of such compounds. In counterpart,
branching this chain seems negative contribution to the affinity. For
instance, the n-propylic derivative 1d (IC50 124 µM) showed to be 2-
fold more potent than the isopropylic ester 1e (IC50 221 µM). Similar
findings were already observed with other phenolic acids such as or-
sellinates (2,4-dihydroxy-6-methyl benzoates), which had growing in-
hibitory activity when alkyl chains are elongated up to 8-carbon length
[33].
The tested compounds showed inhibitory activity in micromolar
range, which is compatible or even superior than other reports from
literature [10,11,33]. The results presented in Table 1 indicate that the
hydroxy group is very important to the activity, since its change by
hydrogen (2) or methoxyl (3) led to considerable drop in the enzyme
inhibition, leading to poorly active compounds. Although compound 4a
showed to be the less active acid, it was noted a quick darkening of the
medium during the assay, suggesting that it may serve as a substrate on
the diphenolase step (second step in Fig. 1) instead of a competitive
inhibitor. Literature describes that 4a is a covalent inhibitor of tyr-
osinase, since the enzyme can oxidize it to the corresponding o-caf-
feoquinone which is dark and a very reactive compound [34], ex-
plaining why it is also considered a suicide tyrosinase inhibitor.
However, covalent inhibitors are frequently associated with severe side
effects, such as allergies, tissue damage and carcinogenesis [35].
By definition, prodrugs should not possess biological activity; they
should only serve as transport molecule of the active compound to the
target tissue [36]. Accordingly, if the esters were to be prodrugs they
should not have any inhibitory activity over the enzyme. However, the
results from Table 1 show that the analogues 1b-f were active inhibitors
of the enzyme per se in the monophenolase step, with higher potencies
Considering that the p-coumarate esters are active without the acid
moiety, and the activity obtained with 1f, the amide 1g was also tested,
considering the higher stability of this group. Additionally, non-hy-
drolysable analogues such as the ketones 1h and 1i were also evaluated.
The results obtained with these compounds denote that the ester moiety
is the best functional group to the interaction with the enzyme, since all
presented lower affinity than the ester derivatives. The amide 1g
showed improved potency than the parent acid 1a, with an IC50 value
of 209 µM (1.4-fold higher). Regarding the ketones 1h and 1i the role of
lipophilicity was different than in the esters, since the more lipophilic
compound 1i (IC50 508 µM) showed decreased affinity than 1 h (IC50
386 µM). In summary, these results strongly suggest that both phenol
and carboxylate moieties may perform favorable interactions with the
enzyme, but also that the carboxylic acid is not essential to the inter-
action with tyrosinase, as suggested by the natural substrate, L-tyrosine.
In order to evaluate the role of the phenol group in the inhibitory
effect on tyrosinase, simplified derivatives containing the phenolic
moiety but lacking the carbonyl/carboxyl groups (5–7) were tested.
These compounds showed inhibitory activity on tyrosinase with dif-
ferent degrees. The lower activity shown by eugenol (5) when com-
pared to its isomer 6 suggests an important electronic effect given by
the extended conjugation of the phenol group with the double bond,
which is in line with previous reports [33]. In fact, non-conjugated
analogues of p-coumaric acid were already tested, showing very low
inhibitory activity on tyrosinase [9]. However, the increased potency of
compound 7 in comparison to 5 and 6, and also to 1a denotes that the
presence of the hydroxyl ortho-positioned in the aromatic ring is fa-
vourable to the interaction with the enzyme [16]. It has been demon-
strated that both ortho- and para-phenolic compounds tend to have a
better inhibitory activity than the meta-analogues, but the potency also
depends on the remaining structural characteristics of the compounds
[22].
Table 1
Percentage of monophenolase activity at 1 mM (L-tyrosine as substrate) and
IC50 of the tested compounds.
Compound
R
R’
% control (1 mM
SEM) pIC50
(
SEM)
1a
1b
1c
1d
1e
1f
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
H
OH
32
2.6
1.3
2.4
0.6
2.7
3.52
4.02
4.01
3.90
3.66
4.16
3.67
3.41
3.29
3.06
0.03
0.03
0.03
0.02
0.02
0.02
0.04
0.03
0.03
0.04
OMe
OEt
11
15
OnPr
OiPr
OnBu
10
27
< 10
1 g
1 h
1i
NHnBu 22
4.3
1.7
1.1
1.1
5.5
7.4
0.8
2.8
3.7
Me
27
16
52
68
75
87
85
56
> 90
70
75
77
74
nPr
2a
2b
2c
2e
2f
OH
H
OMe
OEt
OiPr
OnBu
OH
< 3.00
< 3.00
< 3.00
< 3.00
3.07
H
H
H
3a
3b
3c
4a
4b
5
4-OMe
4-OMe
4-OMe
3,4-diOH
3,4-diOH
3-OMe, 4-
OH
0.06
OMe
OEt
OH
< 3.00
< 3.00
< 3.00
< 3.00
< 3.00
3.2. Antiradical activity
1.5
3.6
3.2
2.6
It is widely known that phenols present radical scavenging activity
that may also contribute totally or in part to the antimelanogenic effect
of tyrosinase inhibitors. To investigate if the inhibitory activity ob-
served for the compounds was due to an interaction with tyrosinase’s
active site or due to a redox effect, DPPH assay was performed with the
OMe
–
6
7
3-OMe, 4-
OH
–
–
52
19
2.9
3.7
3.11
3.71
0.06
0.04
2-OH
compounds [37]. DPPH is
a stable radical compound used to
5

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
Fig. 3. Percentage of the maximum radical scavenging activity of the compounds in the DPPH assay. C- is the negative control (methanol). Black bars
represent
SEM. *p < 0.05 relative to the C-.
Table 2
Atoms or amino acid residues involved in the interactions with the ligands and respective measured distances, and the docking score for each studied compound.
Atoms / Residues
Type of interaction
Distance (Å)
tyrosine
1a
1b
1c
1d
1e
1f
Cu2+
Coordinate bond
Hydrogen bond
Hydrogen bond
Hydrogen bond
π-π stacking
π-alkyl
2.64
2.84
2.06
–
2.46
2.96
2.07
–
2.47
2.70
1.95
–
2.27
–
2.47
2.70
2.05
–
2.48
2.73
1.97
–
2.51
2.73
2.14
–
His84
His60
–
His295
His262
Val282
Ala285
Gly280
Gly280
Ser281
Val247
Phe263
Met256
CHEMPLP Score
2.50
4.06
2.99
5.21
–
3.61
4.14
4.54
2.44
2.63
–
3.90
4.00
4.77
–
4.06
3.00
5.20
–
4.12
2.95
5.31
–
4.03
3.02
5.19
–
4.19
2.79
5.38
–
π-alkyl
Hydrogen bond
Hydrogen bond
Hydrogen bond
Hydrophobic
Hydrophobic
Hydrophobic
–
–
–
–
–
–
2.54
–
–
–
–
–
–
–
5.07
4.79
–
5.06
4.77
–
5.00
4.78
–
4.30
5.46
–
5.32
5.12
4.91
62.82
–
–
–
–
64.48
58.75
55.13
58.34
60.86
59.50
Fig. 4. Ligand interactions between L-tyrosine (A) and 1a (B) and the amino acids in the active site of mushroom tyrosinase. Dashed lines represents the observed
interactions. Green: hydrogen bond; purple: π-alkyl interactions; pink: π-π stacking; grey: coordinate bond. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
monitoring reactions involving radicals, which can be correlated to the
antioxidant potential of tested compounds. DPPH presents a vibrant
purple color that can be quantified by spectrophotometric method. In
the presence of a radical scavenger, this compound is reduced to the
corresponding hydrazine, which is a colorless or pale yellow compound.
The difference in absorbance indicates the antiradical capacity of the
tested compound [38]. Fig. 3 presents the results from the DPPH assay
with the reported compounds.
As observed in the results, several compounds exhibited significant
radical scavenging activity, and some other had strong DPPH scaven-
ging activity such as the caffeates 4a and 4b (87.7 and 85.1%, re-
spectively), and the phenolic compounds 5 and 6 (81.7 and 84.7%,
respectively). This observation could be explained by the fact that the
presence of an additional hydroxyl group (in comparison to p-coumaric
acid) better stabilizes the phenoxyl radicals formed, prior to conversion
to the corresponding quinone [9,39], and our results is also in line with
6

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
Fig. 5. Docking poses showing the hydrogen bonds of 1a (A) and 1f (B) with His60, His84 (dashed green lines), coordinate bond with copper atom (dashed grey
lines), π-π stacking interactions (dashed pink lines) and π-alkyl interactions (purple dashed lines). (C) Hydrophobic potential surface of the binding site showing the
fitting of the alkyl chain of 1f (blue) into a hydrophobic pocket whereas the parent acid 1a (purple) interacts with Arg267 in a more hydrophilic site. The
hydrophobicity scale is shown, being the more hydrophobic site, the more red while the more hydrophilic site, the more blue. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
other literature reports [40].
eugenol and isoeugenol in DPPH assays [41,42].
Noteworthy, the other cinnamic acid derivatives, including p-cou-
maric acid, did not display any significant radical scavenging activity.
This is in line with previous reports from literature [9]. However, Ta-
kahashi and Miyazawa [9] showed that the 2,3-dihydrocinnamates
display stronger antiradical activity than the corresponding cinnamates.
This suggests that the extended conjugation of the phenol hydroxyl
reduces the radical scavenging effect. Moreover, the presence of elec-
tron-donating groups ortho or para to the phenol hydroxyl increases the
radical scavenging activity, which explains the stronger effect of
caffeates [9].
Interestingly, the position isomer 2-allylphenol 7 did not show
significant antiradical activity, suggesting that the position of the hy-
droxy group in the ring strongly affects the activity. This lower activity
may be due to the fact that compound 7 does not have the ortho
methoxy group, as in both eugenol and isoeugenol, which may make it
less capable of stabilizing the phenoxyl radical, or even due to the ab-
sence of an electron donating group in para position For instance, butyl-
hydroxytoluene (BHT) is a known antioxidant which presents two ortho
t-butyl groups and a para methyl group related to the hydroxyl [43].
Considering these aspects, it can be concluded that the observed
tyrosinase inhibitory activity for the compounds 1a-i is independent of
an antioxidant effect, similarly to other reports from literature for other
cinnamates [9].
The report from Takahashi and and Miyazawa [9] clearly shows
that the dihydrocinnamates are also less potent tyrosinase inhibitors
than the corresponding cinnamates, being practically inactive. Our re-
sults, together with Takahashi and Miyazawa’s [9] data, strongly sug-
gests that the mechanism of tyrosinase inhibition for the compounds
1a-1i are independent of a redox effect, since the more potent tyr-
osinase inhibition activity the less potent radical scavenging effect.
The results for the phenolic compounds 5–7 also shed light in this
discussion. Eugenol (5) and isoeugenol (6) showed very strong anti-
radical activity. This is in line with previous literature reports for
3.3. Docking studies
To better understand how the compounds may interact with the
catalytic site of mushroom tyrosinase, molecular docking studies were
carried out. These studies were done with the compounds selected as
more interesting. The highest scoring poses were chosen to investigate
7

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
Fig. 6. Overlap of the isopropyl ester 1e (blue) and n-propyl ester 1d (red) in the binding site surrounded by a hydrophobic surface. The hydrophobicity scale is
shown, being the more hydrophobic site, the more red while the more hydrophilic site, the more blue. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
the interactions between ligand and active site, and the summary of the
interactions are presented in Table 2.
interacts with the hydrophobic amino acids in the same pocket, how-
ever the residues Val247 and Phe263 create a steric hindrance in the
cavity, which hinders the adequate interaction of the isopropyl group as
suggested by the distance values for these interactions. This seems to be
the reason for the lower affinity of the isopropyl analogue 1e in com-
parison to the non-branched analogue 1d.
Compound 1a interacts with the catalytic site in a similar manner as
does L-tyrosine (Fig. 4), with the oxygen of the phenol ring coordinating
to the copper ions and performing hydrogen bonds with the histidine
residues His60 and His84. The protonated amino group from L-tyrosine
seems to interact by hydrogen bonding with Gly281, and an additional
hydrogen bond between the carbonyl group and Asn260 was also
identified. Additionally, the negative charged carboxylate group of 1a
showed a salt bridge with the protonated guanidine group from Arg267
(Fig. 4B) and both ligands exhibit π-π stacking interactions with the
histidines from the active site. These observed interactions are in line
with other literature results from molecular docking [14,15,34,44].
The data from Table 2 clearly shows that the esters 1b-1f perform
additional hydrophobic interactions with the hydrophobic pocket
comprised by the amino acids Val247, Phe263 and Met256 in the en-
zyme. These interactions thermodynamically contribute to better in-
teraction with the target (as described by the CHEMPLP score), ex-
plaining why these compounds showed improved affinity to the enzyme
than the acid prototype. Moreover, the esters seems to perform closer
interactions with the Val282 and His84 residues. The remaining inter-
actions seems very similar in the prototype and derivatives.
Overall, these modeling results suggest that the unsubstituted car-
boxylic acid is not essential to the activity of 1a, and that the additional
hydrophobic interactions provided by the alkyl chain from the esters
play an important role in the affinity of such compounds. Although
there is still some space for longer alkyl chains in the binding pocket,
the excessive lipophilicity of such compounds limit their solubility in
water and in the enzymatic assay medium. Therefore, the butyl ester 1f
was considered the optimal derivative for tyrosinase inhibitor in vitro.
3.4. Ex vivo efficacy assessment
Considering the potential of these compounds to be used as an ac-
tive ingredient on formulations to hyperpigmentation treatment, the
evaluation of their efficacy in human skin is necessary. Therefore, the
prototype 1a and some selected esters (1b, 1c, 1e and 1f) were sub-
mitted to an ex vivo efficacy assay in order to evaluate the melano-
genesis inhibition potential of these analogues.
Docking analysis for the most active compound from the set, 1f,
showed similar interactions to the prototype 1a with the amino acids
His60 and His84 through hydrogen bonding, as well as the hydrophobic
and π-π stacking interactions of the aromatic ring with Val282, Ala 285
and His262, respectively (Fig. 5A and B), which is also in line for si-
milar compounds from literature [34]. However, due to the absence of
the negatively charged carboxylate group, no interaction was observed
with the Arg267 residue. On the other hand, the longer alkyl chain fits
nicely into a hydrophobic pocket close to the catalytic site (Fig. 5C),
comprised by the hydrophobic amino acids Met256 and Phe263, thus
possibly conferring this analogue a higher activity than the prototype
and also than the other less active esters. All these interactions provided
the higher CHEMPLP score among the esters, and also corroborates the
experimental data.
This assay allows the evaluation of total depigmentation capacity of
the tested compounds on human skin tissue [28]. Accordingly, the
assay design allows the indirect evaluation of the permeability through
the skin. The methodology uses ImageJ software to measure the mel-
anin content per area through pixel analysis, which is compared to
untreated control skin images. The higher pixel density indicates a
higher melanin production. For this, evaluation, the compounds were
applied at a test concentration of 0.1% (w/v).
The results are presented in Fig. 7, and clearly show that although
1a is a known tyrosinase inhibitor, its efficacy was not observed in the
performed assays. However, the esters 1c and 1e showed significant
efficacy on reducing the melanin content in the skin fragments. This
indicates that the analogues may present increased permeation through
skin, and confirms their potential as antimelanogenic compounds by
tyrosinase inhibition mechanism. Interestingly, compound 1e showed
the highest efficacy among the tested esters, although it is the less
Interestingly, isopropyl ester 1e showed about half of the potency
compared to the n-propyl analogue 1d, and the docking results are in
line with the experimental data (Fig. 6). The isopropyl group from 1e
8

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
Fig. 7. (A) Melanin content measured in the ex vivo assay with the compounds. *p < 0.05; ***p < 0.001 relative to the control. Sample micrographs obtained with
the treatments with sham (B), 1a (C), 1e (D) and 1f (E). The arrows indicate melanin deposits on the measured area. Note that compound 1e produced less pigmented
epidermis.
potent tyrosinase inhibitor. Moreover, the most potent compound from
the series (1f) showed no efficacy on reducing the melanin content in
the tested skin fragments. This suggests that the tyrosinase inhibitory
activity should not be directly correlated to the efficacy in the skin.
There is no consensus in literature on which lipophilicity range
(usually expressed by the calculated n-octanol/water partition coeffi-
cient value, ClogP) is adequate to good permeation through the skin,
but it is accepted that molecules with moderate lipophilicity (ClogP
1–3) can diffuse through the skin [45,46]. Excessive lipophilicity may
provide poor solubility and permeation issues accordingly, while low
lipophilicity is not favorable to permeation through the hydrophobic
layers of the epidemis and cell membranes. Moreover, low molecular
weight (< 500) and low melting point are also favorable to adequate
permeation [45]. Although p-coumaric acid 1a present ClogP (1.83)
value inside the expected range, it must be considered that it is pre-
dominantly deprotonated in the skin pH, and this ionized form presents
ClogP −1.70, which explains its poor permeation through the skin.
On the other hand, the compounds presented herein are more li-
pophilic than the parent acid, and may better penetrate in the skin.
Compound 1e presents the highest ClogP value (2.99) inside the ex-
pected range, with lower melting point and the best efficacy in the ex
vivo assays. Regarding this, possibly the most potent ester as tyrosinase
inhibitor 1f was ineffective due to excessive lipophilicity that may
impair its penetration in the skin. This suggests that the balance be-
tween potency and lipophilicity must be considered when designing
novel tyrosinase inhibitors
4. Conclusion
The results here presented showed that p-coumaric acid esters are
effective antimelanogenic compounds with remarkable inhibitors of
tyrosinase independent of radical scavenging activity. Docking studies
indicate that the alkyl chain from these esters contributes to the affinity
for the enzyme, but the lipophilicity balance with water solubility must
be considered to design novel and more potent agents. Overall, these
analogues should be considered for lead optimization to obtain effective
compounds for hyperpigmentation topical treatment.
9

M.T. Varela, et al.
BioorganicChemistry103(2020)104108
Declaration of Competing Interest
https://doi.org/10.1016/j.ijbiomac.2018.07.173.
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Acknowledgements
The authors are grateful to the São Paulo Research Foundation –
FAPESP for the financial support to JPSF working group (grant 2016/
25028-3) and for the scholarship to MTV (grant 2018/03918-2), to the
National Council for Scientific and Technological Development – CNPq
for the scholarships to VGM and the fellowship to JPSF (grant 306355/
2018-3) and to Coordination of Superior Level Staff Improvement –
CAPES (financial code 001) for the scholarship to MF. Special thanks to
Dr. Wagner Vidal Magalhães for the support to the Chemyunion team
and to the contributions to this work, and to Dr. Daniela Rando for the
tips in the molecular docking experiments.
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104108.
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