New insights into highly potent tyrosinase inhibitors based on 3-heteroarylcoumarins: Anti-melanogenesis and antioxidant activities, and computational molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2017.01.037

Melanogenesis is a physiological pathway for the formation of melanin. Tyrosinase catalyzes the first step of this process and down-regulation of its activity is responsible for the inhibition of melanogenesis. The search for molecules capable of controll

Full text of DOI:10.1016/j.bmc.2017.01.037

Accepted Manuscript
New insights into highly potent tyrosinase inhibitors based on 3-heteroarylcou-
marins: Anti-melanogenesis and antioxidant activities, and computational mo-
lecular modeling studies
Francesca Pintus, Maria J. Matos, Santiago Vilar, George Hripcsak, Carla
Varela, Eugenio Uriarte, Lourdes Santana, Fernanda Borges, Rosaria Medda,
Amalia Di Petrillo, Benedetta Era, Antonella Fais
PII:
S0968-0896(16)30959-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.037
BMC 13516
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 October 2016
13 January 2017
21 January 2017
Please cite this article as: Pintus, F., Matos, M.J., Vilar, S., Hripcsak, G., Varela, C., Uriarte, E., Santana, L., Borges,
F., Medda, R., Di Petrillo, A., Era, B., Fais, A., New insights into highly potent tyrosinase inhibitors based on 3-
heteroarylcoumarins: Anti-melanogenesis and antioxidant activities, and computational molecular modeling
studies, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.037
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Bioorganic & Medicinal Chemistry
New insights into highly potent tyrosinase inhibitors based on 3-
heteroarylcoumarins: anti-melanogenesis and antioxidant activities, and
computational molecular modeling studies
Francesca Pintusa, Maria J. Matosb, c, , Santiago Vilarc, d, George Hripcsakd, Carla Varelae, Eugenio
Uriarteb, Lourdes Santanab, Fernanda Borgesc, Rosaria Meddaa, Amalia Di Petrilloa, Benedetta Eraa and
Antonella Faisa
a Department of Life Science and Environment, University of Cagliari, 09042 Monserrato, Cagliari, Italy
b Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain
c CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, 4169-007 Porto, Portugal
d Department of Biomedical Informatics, Columbia University Medical Center, New York, NY 10032, USA
e CNC-IBILI, Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal
Dr Maria Joao Matos
Department of Organic Chemistry
Faculty of Pharmacy
University of Santiago de Compostela,
15782 Santiago de Compostela
Spain
Phone: 0034881814936
Fax: not available
E-mail: mariacmatos@gmail.com

Corresponding author. Tel: +34 881 814936; fax: +34 981 544912; e-mail: mariacmatos@gmail.com

ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Melanogenesis is a physiological pathway for the formation of melanin. Tyrosinase catalyzes the
first step of this process and down-regulation of its activity is responsible for the inhibition of
melanogenesis. The search for molecules capable of controlling hyperpigmentation is a trend
topic in health and cosmetics. A series of heteroarylcoumarins have been synthesized and
evaluated. Compounds 4 and 8 exhibited higher tyrosinase inhibitory activities (IC50 = 0.15 and
0.38 M, respectively), than the reference compound, kojic acid (IC50 = 17.9 M). Compound 4
acts as competitive, while compound 8 as uncompetitive inhibitor of mushroom tyrosinase.
Furthermore, compounds 2 and 8 inhibited tyrosinase activity and melanin production in
B16F10 cells. In addition, compounds 2-4 and 8 proved to have an interesting antioxidant
profile in both ABTS and DPPH radicals scavenging assays. Docking experiments were carried
out in order to study the interactions between these heteroarylcoumarins and mushroom
tyrosinase.
Available online
Keywords:
3-heteroarylcoumarins
B16F10 melanoma cells
melanogenesis
tyrosinase inhibitors
2009 Elsevier Ltd. All rights reserved.
studies.12 However, only a few natural inhibitors have
practically been adapted as cosmetics due to other
1. Introduction
parameters, such as cytotoxicity, solubility, absorption,
effective cutaneous, etc..13 Many researchers are involved in
the development of novel synthetic inhibitors: many natural
inhibitors were modified to semi-synthetic analogues, and
several synthetic tyrosinase inhibitors with the novel
structural scaffold have been designed.14
Melanogenesis is a well-known physiological process
resulting in melanin production from melanocytes. Melanin
is a biopolymer that plays a key role in the protection of the
skin against damaging effects of ultraviolet radiation and it
is the main determinant of skin color. Tyrosinase (EC
1.14.18.1) is known as a key enzyme responsible for the
limiting step in melanogenesis. It catalyzes the
Coumarins (2H-1-benzopyran-2-one) are a large family of
compounds, of natural and synthetic origin, which presents
different pharmacological activities. Their structural variety
is responsible for the important place that they occupy in the
natural product and synthetic organic chemistry realm.
Recent studies have paid special attention to their
antioxidative,15 anti-inflammatory,16 cardioprotective,17
anticancer,18 antimicrobial,19 and enzymatic inhibition20
properties. In our previous work, we have described 3-
arylcoumarin and 3-heteroarylcoumarins derivatives as
hydroxylation
of
tyrosine
to
form
3,4-
dihydroxyphenylalanine (L-DOPA) and the oxidation of L-
DOPA to form o-dopaquinone.1 The next steps lead to the
synthesis of melanin pigments (pheomelanin and eumelanin)
through several oxidation and reduction reactions.2 Since
tyrosinase plays a critical role in melanin production, its
inhibitors have become increasingly important in medicinal
chemistry and in the cosmetic industry when related to
hyperpigmentation disorders.3 In fact, excess production or
abnormal distribution of melanin leads to hyperpigmentation
dysfunctions such as melasma, post-inflammatory
hyperpigmentation and solar lentigines, which become
prominent with aging.4 Because of the visible nature of these
dermatological diseases, affected patients have considerable
psychological consequences.5 Skin diseases have a strong
impact on the physical appearance and emotional state of the
patient and could result in a reduced quality of life.
Therefore, tyrosinase inhibitors are important to ensure a
decrease in the melanin content and to develop new
depigmenting drugs useful in pharmacological and cosmetic
fields.6
tyrosinase inhibitors.21-23 In particular, compounds
2 and 3,
included in this manuscript as antecedents and references for
the SAR study, acted as both good antioxidant and
tyrosinase inhibitors. Following these studies, in the current
work a series of heteroarylcoumarins have been synthesized
and evaluated for their inhibitory activities on mushroom
tyrosinase. The antimelanogenesis effect of active
compounds was also evaluate in cellular system.
2. Results and discussion
2.1. Chemistry
Compounds 1-12 were efficiently synthesized according to
Moreover, melanin synthesis is also due to non-enzymatic
oxidation. Therefore, the development of inhibitors with
antioxidant activity has received much attention.7 A large
number of tyrosinase inhibitors of natural or synthetic origin
have been identified up to now.8 A lot of plant extracts with
anti-tyrosinase properties have been described and a broad
spectrum of compounds have been isolated, which are
mainly polyphenols, flavonoids, aldehydes and their
derivatives.9,10 Besides higher plants, some compounds from
fungal sources have also been identified.11 Kojic acid is a
fungal metabolite produced by several species of fungi and it
is commonly used as positive control in skin-whitening
the synthetic strategy outlined in Scheme 1. The synthesis of
these compounds was carried out by two different
methodologies. The first one occurred in two different steps:
the first step was a Perkin–Oglialoro condensation of
different commercially available hydroxybenzaldehydes and
thiophenylacetic acids, using potassium acetate (CH3CO2K)
in acetic anhydride (Ac2O), under reflux, for 16 h, to obtain
the acetoxy-3-thiophenylcoumarins. Acetylation of the
hydroxy groups and pyrone ring closure occurs
simultaneously. The second step was the hydrolysis of the
obtained acetoxy derivatives in the presence of aqueous HCl

Table 1. Antioxidant activity of a selected series of compounds (
and 11).
1-6
solution and MeOH, under reflux, for 3 h, to achieve the
hydroxy substituted 3-thiophenylcoumarins ( and 11).
8
-
1–6
8–
Structures of the synthesized compounds were established
on the basis of their spectral data.24,25 The second
methodology is a one-step traditional Perkin reaction
starting from the commercially available ortho-
EC50 (µM)a
Compound
ABTS
DPPH
hydroxybenzaldehyde
and
the
corresponding
thiophenylacetic acid, using N,N’-dicyclohexylcarbodiimide
o
(DCC) as dehydrating agent, DMSO as solvent, at 110 C,
7.08 ± 0.035
20.77 ± 0.42b
8.40 ± 0.20 b
8.51 ± 1.70
> 200
47.11 ± 10.09
18.65 ± 0.85b
9.42 ± 0.31b
15.83 ± 0.55
> 200
1
2
for 24 h, that allows obtaining compounds
7 and 12.
As described in previous works,23 to different approaches
were carried out for the synthesis of the studied derivatives.
Methoxy derivatives were prepared by the traditional Perkin
reaction, while hydroxyl derivatives were easily obtained by
Perkin-Oglialoro condensation. In this last case, we avoid
the traditional demethylation using hydriodic acid.22
3
4
5
A
CHO
CH3CO2K, Ac2O
HO
H3COCO
6
> 200
> 200
reflux, 16 h
OH
O
O
COOH
HCl, MeOH
reflux, 3 h
8
9
11.69 ± 3.83
> 200
14.13 ± 0.44
> 200
R5
R6
R7
10
> 200
> 200
O
O
R8
11
> 200
> 200
S
S
S
1: R5 = R7 = OH, R6 = R8 = H
2: R5 = R7 = OH, R6 = R8 = H
3: R5 = R7 = OH, R6 = R8 = H
4: R5 = R7 = OH, R6 = R8 = H
5: R7 = OH, R5 = R6 = R8 = H
=
=
=
=
=
6: R6 = OH, R5 = R7 = R8 = H
8: R5 = R7 = OH, R6 = R8 = H
9: R7 = OH, R5 = R6 = R8 = H
10: R8 = OH, R5 = R6 = R7 = H
11: R7 = R8 = OH, R5 = R6 = H
=
=
=
Br
Troloxc
5.28 ± 0.15
28.27 ± 0.45
Br
Br
S
a Data represent the mean (± standard deviation, SD) of three
independent experiments.
S
S
S
S
=
Br
Br
b Data previously reported in reference 23.
c Positive control.
=
B
S
S
R
MeO
CHO
MeO
DCC, DMSO
110 oC, 24 h
R
Based on the experimental results, compounds
not exert antioxidant activities in both assays. Regarding
ABTS·assay, compounds and showed the best EC50
5, 6, 9-11 did
OH
O
O
COOH
7: R = H
12: R = Br
1
-4
8
values in the same range (EC50 = 7.08, 20.77, 8.4, 8.51 and
11.69 μM, respectively) as Trolox (EC50 = 5.28 μM). The
same results were obtained for the DPPH·assay, except for
Scheme 1. Synthetic route for the obtention of the derivatives
represented in this study. A) Perkin–Oglialoro reaction and chemical
structure of the compounds and 11; B) Perkin reaction and
chemical structure of compounds and 12
1
-
6
8-
compound
the other studied compounds. The radical-scavenging
activity of compounds and proved to be even better
1, that showed a higher value (47.11 μM) than
7
.
2-4
8
2.2. Antioxidant activity. The free radical scavenging
activity of hydroxylated compounds was carried out using
ABTS26 and DPPH27 methods and Trolox was used as the
positive control for comparing the antioxidant capacity of
the studied molecules. Antioxidant capacity of compounds
without hydroxyl groups has been also determined but they
did not exert any antioxidant activity (data not shown). Half
maximal effective concentration (EC50) values for both
assays are reported in Table 1.
than the positive control (EC50 Trolox = 28.27 μM), with
EC50 values ranging between 9.42 and 18.65 μM. This effect
can be explained due to the presence of two hydroxy groups
on their structures. Comparing the EC50 values of
compounds
8 and 11, which differ only in the position of the
hydroxy groups in the coumarin scaffold (5,7 vs 7,8,
respectively), the position 5,7 seems to be important for the
antioxidant activity of this scaffold. This could be in
accordance with a previous study showing that resorcinol
has a higher antioxidant activity than catechol.28

compound
(thiophene vs bromothiophene). The same is true between
compound and compound . Therefore, it seems that the
influence for the structure/activity relationship of the
aromatic side chain is as important as that of the hydroxyl
groups.
5 and compound 9 is aromatic side chain
2.3 Inhibition of mushroom tyrosinase activity. The
inhibition of tyrosinase displayed by the studied compounds
is shown in Table 2, and the inhibitory activity was
compared to that of the kojic acid (positive control).
1
2
Table 2. Half maximal inhibitory concentration (IC50) values of 3-aryl
and 3-heteroarylcoumarins against mushroom tyrosinase activity.
25
100
IC50 μMa
20
Compound
15
1b
408.37 ± 19.66
1.05 ± 0.056
0.19 ± 0.016
0.15 ± 0.028
> 1000
10
80
5
2b
0
60
-0.25
0
0.25 0.5 0.75
1
3b
Compound 4 (µM)
4
40
20
0
5
6
> 1000
7
> 1000
8
0.38 ± 0.084
31.8 ± 10.89
> 1000
9
0
1
2
3
[DOPA]-1 (µM-1)
10
11
259.03 ± 1.62
> 1000
12
Figure 1. Lineweaver–Burk plots for the inhibition of compound
4 on
Kojic acidb
17.9 ± 0.98
mushroom tyrosinase. The concentrations of inhibitor were 0 (○), 0.25
a Data represent the mean (± standard deviation), SD of three
independent experiments.
(●), 0.5 (■), and 1 (▲) μM. The secondary plot of slope (Km/Vmax
)
versus concentration of compound
constant (Ki) is showed as inset.
4
, to determine the inhibition
b Data previously reported in reference 23.
The experimental results showed that the studied compounds
present different profiles of inhibition of mushroom
tyrosinase activity. Among the new tested compounds,
16
12
30
compounds
4 and 8 appeared to be strong inhibitors, with
8
IC50 values of 0.15 and 0.38 μM, respectively. In fact, these
compounds proved to be approximately 120 and 50 times
more active than the standard inhibitor (IC50 = 17.90 μM).
4
0
0
0.2 0.4 0.6 0.8
1
20
10
0
Compound 8 (µM)
The inhibitory mechanisms of compounds
4 and 8 on
mushroom tyrosinase were determined from Lineaweaver-
Burk plots. The plots of the initial rates of tyrosinase activity
in the presence of increasing concentrations of compound
4
yielded a family of straight lines with different slopes that
crossed the Y-axis at similar points, indicating that
compound
4 is a competitive inhibitor. The inhibition
-4
-2
0
2
4
constant (Ki = 0.24 μM) for the inhibitor binding with the
free enzyme (E) was obtained from the secondary plot
(Figure 1).
As shown in Table 1 and 2, antioxidant activity and
tyrosinase inhibitory activity vary depending on the
hydroxyl group and the side chain. For example, analogues
[DOPA]-1 (µM-1)
Figure 2. Lineweaver-Burk plots for inhibition of compound
8
on
mushroom tyrosinase. The inhibitor concentrations were 0 (○), 0.25 (●),
0.5 (■) and 1 (▲) μM, respectively. Inset: plot of 1/Vmax versus
concentrations of compound
(KIS).
8 to determine the inhibition constant
without or with just one hydroxyl group (compounds
5, 6
and 12) showed almost no activity, while analogues with
9-
two hydroxyl groups showed very strong activity. It seems
that hydroxyl groups play an important role in the strong
binding to the receptor. However, as shown in Table 2,
Figure 2 shows the double-reciprocal plots of the enzyme
inhibition by compound . Results show that the plots 1/V
versus 1/[S] gave a family of parallel straight line with the
same slopes. As the inhibitor concentration increased, the
values of both Km and Vmax are reduced, but the ratios of
8
compound
compound
5
9
shows more than 30-fold less activity than
. The only structural difference between

Km/Vmax are unchanged. The slopes are independent of the
concentration of compound , which indicates that it is an
2.4 Antimelanogenic effects in B16F10 melanoma cells
2.4 Antimelanogenic effects in B16F10 melanoma cells
8
uncompetitive inhibitor of the enzyme. Results revealed that
the inhibitor binds at the site distinct from the substrate and
combines with enzyme-substrate complex (ES) but not with
enzyme free (E). The equilibrium constant for binding with
ES complex, KIS, is obtained from a plot of the vertical
intercept (1/Vmax) versus the inhibitor concentration. The
2.4.1 Effect on cell viability. Among all the tested
compounds, we selected the best tyrosinase inhibitors to
evaluate their depigmenting activity in B16F10 cells. The
compounds that showed the highest inhibition effect on
mushroom tyrosinase were compounds 2, 3, 4 and 8 (Table
value of KIS of compound
(Figure 2).
8
was determined to be 0.128 μM
2). First, we evaluated the potential cytotoxicity of these
compounds on melanoma cells by measuring cell viability.
Cells were treated with different concentration of each
compound (0–100 μM) for 48 h and were examined using
MTT test.29 Compounds
2
and
on cell viability in a previous work and showed no
cytotoxicity.23 Results also indicate that compounds
and
3 were tested for their effect
4
8
exhibited no considerable cytotoxic effect in B16F10
melanoma cells at the concentration in which tyrosinase
activity is inhibited (Figure 3). Cell viability was slightly
decreased at concentrations above 50 μM. Thus, the
following experiments were performed using up to this
concentration.
Figure 3. Effect of compounds
4 (○) and 8 (●) on B16F10 melanoma
cell viability. After 48 h incubation with the tested compounds, cell
viability was determined by MTT assay.
2.4.2 Effect on cellular tyrosinase activity and melanin
production. We examined the inhibitory effect of the
selected compounds on cellular tyrosinase activity and
melanin content of B16F10 melanoma cells treated with 100
nM
Compounds
α-Melanocyte-stimulating hormone
and did not exert any effect on both
(α-MSH).30
3
4
tyrosinase activity and melanin production in cellular system
(data not shown). On the other hand, cellular tyrosinase
activity, as well as levels of melanin content, was reduced in
a dose-dependent manner in presence of compounds
Figure 4A shows tyrosinase activity of cells treated with 100
nM α-MSH and compounds or at different concentration
(10, 25 and 50 μM). After treatment with compound
tyrosinase activity decreased to 94.3, 78.5 and 27.7 % of the
control. Compound showed a higher inhibitory effect with
18.1 % inhibition at 10 μM, 51.8 % at 25 M and 86.8 % at
2 or 8.
2
8
8,
2


50

M. Similar effects were observed on melanin
6). Tyrosinase activity is very low in non-stimulated cells while
α-MSH treatment created a dark band compared to that of
untreated control. Upon incubation with compounds, activity of
tyrosinase decreased and lighter bands were observed. Results
confirmed the inhibitory effects of the compounds with
compound 2 exerting the strongest inhibition also compared with
the standard inhibitor.
production (Figure 4B).
Although compounds
effects on mushroom tyrosinase as compounds
didn't exhibit inhibitory effect on both tyrosinase activity
and melanin production in cellular systems. This can be due
to the complex cellular mechanisms, not reflected in the
preliminary tyrosinase inhibition screening assays.
3
and
4
possess similar inhibitory
and , they
2
8
120
A
100
80
60
40
20
0
-MSH (100 nM)
Kojic acid ( M)
-
-
-
-
+
-
+
50
-
+
-
+
-
+
-
+
-
+
-
+
-
10 25 50
-
-
-
-
Compound 2 ( M)
Compound 8 ( M)
10 25 50
-
-
-
-
-
120
B
Figure 5. Effects of compound
2 on B16F10 cells, by L-DOPA
100
80
60
40
20
0
staining. Tyrosinase activity was estimated by zymography (A) and the
relative intensity of bands was determined with ImageJ software (B).
-
+
-
+
+
-
+
-
+
+
-
+
-
+
-
-MSH (100 nM)
Kojic acid ( M)
-
-
50
Compound 2 ( M)
Compound 8 ( M)
10 25 50
-
-
-
-
-
-
-
-
-
10 25 50
-
-
-
Figure 4. Effects of compound 2 and 8 on cellular tyrosinase activity (A) and
melanin content (B) in α-MSH stimulated B16F10 cells.
Also in this case, both compounds reduced melanin content with
compound 2 being more active regarding to compound 8.
Moreover, all these data showed that compound 2 and 8 resulted
to be more effective as antimelanogenic agents than kojic acid,
the standard tyrosinase inhibitor, which did not show any
inhibition at the highest sample concentration (50 μM) as
compared with the control (Figures 4 A and B).
The inhibitory effect on melanogenesis of compound 2 and 8 was
confirmed through the results of tyrosinase zymography. DOPA
staining assay was carried out with lysates of α-MSH-stimulated
B16F10 cells treated with or without compounds (Figures 5 and
Figure 6. Effects of compound 8 on B16F10 cells, by L-DOPA staining.
Tyrosinase activity was estimated by zymography (A) and the relative
intensity of bands was determined with ImageJ software (B).

2.5 Molecular docking simulations We studied the most
active compounds using molecular docking simulations to
determine the key residues important for the ligand-protein
interaction. For this purpose, we downloaded the crystal
structures 2Y9W and 2Y9X (Crystal structure of Agaricus
bisporus mushroom tyrosinase: identity of the tetramer
subunits and interaction with tropolone) of the Agaricus
bisporus tyrosinase from the RCSB Protein Data Bank
the series were also docked to the protein crystal structures
using Glide module.31 Compound
showed a binding mode
4
in the 2Y9W where the coumarin ring is directed towards
the bottom of the cavity whereas the 3-heteroaryl substituent
is placed close to the Arg268 towards the protein surface
(Figure 7B). The hydroxy groups placed at positions 5 and 7
of the coumarin skeleton play an important role for the
anchoring with the enzyme. Hydroxyl at position 5
established a hydrogen bond with the residue Met280. The
compound also established a hydrogen bond with the amide
moiety of the residue Asn260 using the 7-hydroxy group
(Figure 7C). Moreover, residues His263 and Phe264
established π-π stacking interactions with the coumarin
(RCSB
Protein
Data
Bank
-
RCSB
PDB.
http://www.rcsb.org/).30 The initial docking was performed
31
with Glide using the 2Y9W structure in which there is no
ligand bound to the protein. The crystallized structure
presents a water molecule or hydroxyl ion between the
copper ions that was maintained for the docking
calculations. Docking simulations were also performed
using the 2Y9X-crystallized structure to evaluate the
stability of our results. In this case, the protein is bound to
the ligand tropolone and no water is present in the pocket.
We validated the docking protocol measuring the root mean
square deviation (RMSD) between the co-crystallized heavy
atoms coordinates of the inhibitor tropolone and the
theoretical pose determined in the calculations.
scaffold. The docking pose for compound
4 showed a XP
energy score of -4.67 kcal/mol, similar to the score obtained
for the crystallized tropolone (-4.05 kcal/mol). Additionally,
we calculated the residue energy contribution for ligand
binding. The main residues favorable for the ligand-protein
recognition are shown in Figure 7D. The residues that play
an important role in stabilizing the complex are: Asn260,
Met280, Phe264 and Val283. Compounds that do not
present hydroxy substituents at the mentioned positions
showed lower tyrosinase activity with the limitation to
establish the described H-bonds with both residues. Similar
results for compound
2Y9X crystallized enzyme in the docking. The proposed
binding mode for compound is in accordance with our
4 were obtained using the alternative
4
experimental assays in which the compound did not interact
with the copper but showed competitive inhibition.
Our experimental results showed a different type of
inhibition for compound
tyrosinase in an uncompetitive mode. Although docking
results for compound yielded some poses in the active site
8. This compound inhibited the
8
of the enzyme, our results also showed some alternative
binding modes more in accordance with the uncompetitive
inhibition. Compound
8 was found to bind the protein in a
shallower area than the tyrosinase active site. The described
binding mode would allow the substrate to interact with the
protein, providing a useful uncompetitive model. However,
our docking does not provide significant information to
differentiate between non-competitive and uncompetitive
inhibition. Figure 8 shows the pose determined by molecular
docking for compound
8. The compound established
hydrogen bonds with residues Asn81, His85, and Glu322.
Hydroxy group at position 5 and the carbonyl group of the
benzopyrone played an important role for the anchoring with
the enzyme. Moreover, although hydroxy group at position 7
did not yield a hydrogen bond in our simulation, this
substituent is also important in ligand binding through
Coulomb and van der Waals interactions. The compound
established Coulomb interactions mainly with residues
Glu322, Asn81, Glu256, His85 and His244. On the other
hand, the residues that contribute to the binding through van
der Waals interactions are: Glu322, His85, Asn81, His244
and Val283. A XP energy score of -3.40 kcal/mol was
Figure 7. a) Co-crystallized ligand tropolone (green CPK) in the
tyrosinase crystal structure (PDB: 2Y9X). b) Hypothetical binding
mode extracted from docking for compound
c) Detailed pose for compound determined by docking. Hydrogen
bonds are represented in yellow dashed lines. d) Residue interaction
scores (kcal/mol) when compound is interacting with the tyrosinase
4 in the 2Y9W tyrosinase.
4
4
(sum of Coulomb, van der Waals and H-bond energies).
It is worth noting that the 2Y9X crystal structure is a
tetramer with four different tropolone inhibitors. Each
inhibitor binds a protein subunit in a slight different mode.
The lower RMSD between theoretical and experimental
conformation was 1.24 and 2.30 Å for 2Y9W and 2Y9X
respectively (see Figure 7A with the protein co-crystallized
with the inhibitor tropolone). The most active compounds in
obtained for the docking pose of compound 8.
Besides the hydroxyl substitutions in the coumarin nucleus,
the 3-heteroaryl fragment based on thiophene or
bromothiophene, could be an important factor for the

3. Conclusion
activity. There is divergence in the activity between some
compounds with the only structural difference based on the
In the current study, a series of heteroarylcoumarins have
been synthesized and studied for their inhibitory activities
on mushroom tyrosinase and murine melanoma B16F10
cells, as well as for their antioxidant capacity. Among these
bromine substituent of the thiophene fragment. Compound
showed more than 30-fold higher activity than compound
9
5
and similar results were observed between compounds
2
and
1. Assuming a competitive mode similar to the described for
coumarin derivatives, compounds
tyrosinase inhibitory activity than kojic acid. The kinetic
studies of tyrosinase inhibition revealed that compound
acts as a competitive inhibitor, while compound proved to
be an uncompetitive inhibitor of mushroom tyrosinase.
Furthermore, compounds and inhibited cellular
4 and 8 exhibited higher
compound
4
, molecular docking studies showed that the
could be located close to
the residues Arg268 and Leu275. The main differences in
the residue contribution interactions for compound and
were observed in both residues. The residue contributions of
Arg268 and Leu275 in the interaction with compound are -
2.86 and -1.39 kcal/mol respectively (van der Waals +
Coulomb energies). However, in the case of compound
bromine substituent in compound
9
4
8
9
5
2
8
9
tyrosinase activity and melanin production in B16F10 cells.
Docking experiments were carried out in order to study the
key interactions between these coumarin derivatives and the
mushroom tyrosinase enzyme. The remarkable results found
for these compounds encourage us to continue our research
based on the coumarin scaffold. The analyzed data so far is
very important to define which compound will be the best
candidate for further in vivo studies. In summary, this study
is a noteworthy contribution in the area and allows the
scientific community a new approach in the optimization of
new molecules as depigmenting agents.
5
with no bromine substituent, the contributions decrease to -
0.35 and -0.20 kcal/mol for Arg268 and Leu275. This could
be a reason to explain the different activity shown by some
bromine derivatives. A different inhibition mode could be
another hypothesis to explain the different activity shown by
the thiophene and bromothiophene derivatives. In fact,
compound
4 and 8, with the only structural difference of a
bromine substituent, interact with the protein in
a
competitive and uncompetitive mode respectively.
Additional data would be necessary to establish the type of
interaction, competitive or uncompetitive, shown by
4. Experimental Section
compounds
9 and 2 and to study deeply the causes of a
4.1 Chemistry
different nature in the inhibition.
The starting materials and reagents were obtained from
commercial suppliers (Sigma-Aldrich) and were used
without further purification. Melting points (Mp) are
uncorrected and were determined using a Reichert Kofler
Thermopan or in capillary tubes in a Buchi 510 apparatus.
1H NMR (300 MHz) and 13C NMR (75.4 MHz) spectra were
recorded using a Bruker AMX spectrometer using CDCl3 or
DMSO-d6 as solvent. Chemical shifts (δ) are expressed in
parts per million (ppm) using TMS as an internal standard.
Coupling constants J are expressed in Hertz (Hz). Spin
multiplicities are given as s (singlet), d (doublet), dd
(doublet of doublets) and m (multiplet). Mass spectrometry
was carried out using
a Hewlett-Packard 5988 A
spectrometer. Elemental analyses were performed by using a
Perkin-Elmer 240B microanalyzer and the results are within
± 0.4% of calculated values in all cases. The analytical
results document ≥98% purity for all compounds. Flash
chromatography (FC) was performed on silica gel (Merck
60, 230–400 mesh); analytical TLC was performed on
precoated silica gel plates (Merck 60 F254). Organic
solutions were dried over anhydrous sodium sulfate.
Concentration and evaporation of the solvent after reaction
or extraction were carried out on a rotary evaporator (Buchi
Rotavapor) operating under reduced pressure.
4.2 General procedure for the synthesis of acetoxy-3-
arylcoumarins. Compounds were synthesized under
anhydrous conditions, using a material previously dried at
60 °C for at least 12 h and at 300 °C for a few minutes
immediately before use. A solution containing anhydrous
CH3CO2K (2.94 mmol), the corresponding arylacetic acid
(1.67 mmol) and the corresponding hydroxysalicylaldehyde
Figure 8. a) Hypothetical binding mode extracted from docking for
compound
8. The described binding mode is in agreement with
uncompetitive inhibition. Crystallized ligand tropolone is also shown to
point out the active site of the enzyme. b) More detailed view of the
binding of compound
8 along with important residues in ligand
recognition. Hydrogen bonds with residues Asn81, His85 and Glu322
are represented in yellow color.

(1.67 mmol), in Ac2O (1.2 mL), was refluxed for 16 h. The
reaction mixture was cooled, neutralized with 10% aqueous
NaHCO3, and extracted with EtOAc (3 x 30 mL). The
organic layers were combined, washed with water, dried
over anhydrous Na2SO4 and the solvent was evaporated
under reduced pressure. The product was purified by
recrystallization in EtOH and dried in vacuum to afford the
desired compound.
d6) δ: 6.79 (d, 1H, J=2.1, H-8), 6.87 (dd, 1H, J=8.5, J=2.1,
H-6), 7.59 (d, 1H, J=8.5, H-5), 7.72 (d, 1H, J=1.2, H-5’),
7.77 (d, 1H, J=1.2, H-3’), 8.62 (s, 1H, H-4), 10.78 (s, 1H,
OH). 13C NMR (75.5 MHz, DMSO-d6) δ: 99.8, 102.1, 109.3,
111.7, 114.2, 114.8, 125.3, 126.9, 130.3, 137.7, 138.3,
154.5, 161.9. MS m/z (%): 321.9 ([M+1]+, 97 %). Anal.
Elem. Calc. for C13H7BrO3S: C, 48.32; H, 2.18. Found: C,
48.34; H, 2.20.
4.2.1 General procedure for the synthesis of hydroxy-3-
4.2.8 3-(4-Bromothiophen-2-yl)-8-hydroxycoumarin (10).
o
1
arylcoumarins (1–6 and 8–11). Compounds
1–
6
and
8–
11
Yield 88%. Mp: 246-247 C. H NMR (250 MHz, DMSO-
d6) δ: 7.11-7.16 (m, 3H, H-5, H-6, H-7), 7.79 (s, 1H, H-5’),
7.88 (s, 1H, H-3’), 8.66 (s, 1H, H-4), 10.37 (s, 1H, OH). 13C
NMR (75.5 MHz, DMSO-d6) δ: 103.3, 118.5, 118.7, 118.9,
120.0, 125.0, 126.4, 127.9, 136.9, 137.7, 139.1, 140.9,
144.4. MS m/z (%): 324 ([M+1]+, 100). Anal. Elem. Calcd.
for C13H7BrO3S: C, 48.32; H, 2.18. Found: C, 48.31; H,
2.16.
were obtained by hydrolysis of their acetoxylated
counterparts, respectively. The appropriate acetoxylated
coumarin, mixed with 2N aqueous HCl and MeOH, was
refluxed for 3 h. The resulting reaction mixture was cooled
in an ice-bath and the reaction product and the obtained
solid was filtered, washed with cold distilled water, and
dried under vacuum, to afford the desired compounds (
and 11).
1–6
8–
4.2.9 3-(4-Bromothiophen-2-yl)-7,8-dihydroxycoumarin
(11) Described in reference 24.
4.2.2 Compounds 1-3. Described in reference 23.
4.2.3 5,7-Dihydroxy-3-(thiophen-2-yl)coumarin (4). Yield
93%. H NMR (250 MHz, DMSO-d6) δ: 6.25 (d, 1H, J=2.1,
4.3 General procedure for the synthesis of hydroxy-3-
arylcoumarins (7 and 12). A solution of 2-hydroxy-5-
methoxybenzaldehyde (7.34 mmol) and the corresponding
thiophenylacetic acid (9.18 mmol) in DMSO (15 mL) was
prepared. DCC (11.46 mmol) was added and the mixture
was heated in an oil bath at 110 o C for 24 h. Ice (100 mL)
and acetic acid (10 mL) were added to the reaction mixture.
After being kept at room temperature for 2 h, the mixture
was extracted with diethyl ether (3 x 25 mL). The organic
layer was extracted with sodium bicarbonate solution (50
mL, 5%) and then water (20 mL). The solvent was
evaporated under vacuum and the dry residue was purified
by FC (hexane/ethyl acetate, 9:1).
1
H-8), 6.31 (d, 1H, J=2.1, H-6), 7.12 (dd, 1H, J=5.1, J=3.8,
H-4’), 7.56 (dd, 1H, J=5.1, J=1.0, H-3’), 7.72 (dd, 1H,
J=1.0, J=3.8, H-5’), 8.33 (s, 1H, H-4), 10.52 (s, 1H, OH),
10.88 (s, 1H, OH). 13C NMR (75.5 MHz, DMSO-d6) δ: 94.0,
102.4, 107.1, 113.9, 125.2, 127.1, 127.6, 132.3, 136.6,
149.5, 155.2, 156.4, 162.4. MS m/z 260.9 ([M+1]+, 99 %).
Anal. Elem. Calc. for C13H8O4S: C, 59.99; H, 3.10. Found:
C, 59.97; H, 3.08.
4.2.4
7-Hydroxy-3-(thiophen-2-yl)coumarin
(5)
Described in reference 32.
4.2.5 6-Hydroxy-3-(thiophen-2-yl)coumarin (6). Yield
87%. H NMR (250 MHz, DMSO-d6) δ: 7.03 (dd, 1H,
4.3.1
6-Methoxy-3-(thiophen-2-yl)coumarin
(7)
1
Described in reference 34.
J=8.7, J=2.0, H-7), 7.10 (d, 1H, J=2.0, H-5), 7.17-7.22 (m,
1H, H-4’), 7.30 (d, 1H, J=8.7, H-8), 7.68 (d, 1H, J=5.0, H-
5’), 7.87 (d, 1H, J=3.7, H-3’), 8.52 (s, 1H, H-4), 9.82 (s, 1H,
OH). 13C NMR (75.5 MHz, DMSO-d6) δ: 112.3, 116.9,
119.8, 119.9, 120.0, 126.7, 127.4, 129.0, 129.0, 135.5,
136.3, 154.2, 154.2. MS m/z (%): 245.0 ([M+1]+, 81 %).
Anal. Elem. Calc. for C13H8O3S: C, 63.92; H, 3.30. Found:
C, 63.93; H, 3.32.
4.3.2 3-(4-Bromothiophen-2-yl)-6-methoxycoumarin (12).
o
1
Yield 73%. Mp: 188-189 C. H NMR (250 MHz, DMSO-
d6) δ: 3.82 (s, 3H, CH3), 7.20-7.27 (m, 3H, H-5, H-7, H-5’),
7.41 (d, 1H, J=1.2, H-3’), 7.80-7.84 (m, 2H, H-4, H-8), 8.66
(s, 1H, OH). 13C NMR (75.5 MHz, DMSO-d6) δ: 56.1,
110.8, 111.0, 117.9, 119.0, 123.2, 123.9, 125.7, 130.1,
138.6, 145.1, 147.2, 156.9, 163.1. MS m/z (%): 338
([M+1]+, 100). Anal. Elem. Calcd. for C14H9BrO3S: C,
49.87; H, 2.69. Found: C, 49.89; H, 2.71.
4.2.6 3-(4-Bromothiophen-2-yl)-5,7-dihydroxycoumarin
1
(8). Yield 85%. H NMR (250 MHz, DMSO-d6) δ: 6.26 (d,
1H, J=2.0, H-8), 6.31 (d, 1H, J=2.0, H-6), 7.67 (d, 1H,
J=1.3, H-5’), 7.76 (d, 1H, J=1.3, H-3’), 8.44 (s, 1H, H-4),
10.61 (s, 1H, -OH), 10.94 (s, 1H, -OH). 13C NMR (75.5
MHz, DMSO-d6) δ: 111.1, 114.3, 124.2, 133.2, 136.3,
144.0, 148.0, 159.8, 166.5, 173.5, 174.1, 184.6. MS m/z (%):
337.9 ([M+1]+, 98 %). Anal. Elem. Calc. for C13H7BrO4S: C,
46.04; H, 2.08. Found: C, 46.01; H, 2.09.
4.4 Biological protocols
Mushroom tyrosinase, L-3,4-dihydroxyphenylalanine (L-
DOPA), α-melanocyte stimulating hormone (α-MSH) and
other chemical reagents were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, USA), unless specified otherwise.
4.4.1 ABTS radical cation assay (ABTS·+). The total free
radical-scavenging capacity of compounds was determined
4.2.7 3-(4-Bromothiophen-2-yl)-7-hydroxycoumarin (9).
Yield 90%. Mp: 244-245 C. H NMR (250 MHz, DMSO-
by
sulfonic
ABTS·+
[2,2’-azinobis-(3-ethylbenzothiazoline-6-
acid)] method using 6-hydroxy-2,5,7,8-
o
1

tetramethylchromane-2-carboxylic
acid
(Trolox)
as
well plate (104 cells/well) and incubated with samples at
concentration ranging from 10 to 100 μM for 48 h. As
DMSO was used as solvent for compounds, all activities
were performed also in the presence of DMSO alone, as
solvent control. After incubation time, cells were labelled
with MTT solution for 3 h at 37 °C. The resulting violet
formazan precipitates were dissolved in isopropanol and the
absorbance of each well was determined at 590 nm using a
microplate reader with a 630 nm reference.
antioxidant standard.26 Briefly, the free radical ABTS·+ was
produced by reacting 7 mM ABTS with 2.45 mM potassium
persulfate (final concentration) in aqueous solution and kept
in the dark at room temperature for 24 h before use. Samples
of each compound (10 μL) were added to 1 ml of ABTS·+
and the absorbance at 734 nm was recorded after 1 min
incubation. Results were expressed as concentration of
sample necessary to give a 50% reduction in the original
absorbance (EC50 values).
4.4.6 α-MSH treatment. B16F10 cells were seeded at a
density of 105 cells/ml in 6-well plates containing 10% fetal
bovine serum and 1% penicillin/streptomycin at 37 °C in a
humidified atmosphere with 5% CO2. After 24 h, the
medium was substituted by fresh one supplemented with
100 nM α-MSH and different concentrations of compounds
(0–50 μM) and incubated for 48 h. Cells treated with 100
nM α-MSH and 100 μM kojic acid were used as positive
control and to compare the inhibitory strength of the
inhibitors. All experiments with these stimulated-cells were
performed by using the procedures above reported.
4.4.2 DPPH radical scavenging activity. The 2,2-diphenyl-
1-picrylhydrazyl (DPPH) scavenging activity of compounds
was analyzed according to the procedure previously
described.27 Each compound, in DMSO solution (20 µL),
was added to a mixture of 100 mM acetate buffer (pH 6.5,
630 µL) and 0.3 mM DPPH in ethanol (350 µL) in a cuvette,
and let at room temperature, in the dark, for 15 min. The
absorbance of the resulting solutions was measured at 515
nm. Scavenging capacity of each sample was compared to
that of DMSO (control) and trolox (positive control). Results
were expressed as concentration of compounds capable of
scavenging 50% of free radicals (EC50).
4.4.7 Measurements of cellular tyrosinase activity. Cells
were plated in 60π-dishes at a density of 105cells/mL,
incubated with 100 nM α-MSH and treated for 48 h with
compounds at different concentration (0–50 μM). Cells were
washed with PBS and lysed in 50 mM phosphate buffer (pH
6.8) containing 1% Triton X-100 and 0.1 mM phenylmethyl-
sulfonyl fluoride. Cellular lysates were clarified by
centrifuging samples at 12000 g for 20 min at 4 °C. The
protein content was determined by Bradford method using
BSA as standard. The cellular extract containing 3 μg of
total proteins, was incubated with L-DOPA (1.25 mM) in 25
mM phosphate buffer (pH 6.8) and the absorbance at 492
nm was read until the reaction has finished.
4.4.3 Measurement of mushroom tyrosinase activity.
Tyrosinase inhibition assay was carried out using the
following protocol. Briefly, in a 96-well plate, 80 μL of
phosphate buffer (0.5 M, pH 6.5), 60 μL of mushroom
tyrosinase (240 UmL-1), 20 μL of inhibitor dissolved in
DMSO at the different concentrations or DMSO (control),
were mixed. The assay mixture was then incubated at 37 °C
for 10 min. Finally, 40 μL of 2.5 mM L-DOPA in phosphate
buffer were added and the amount of the dopachrome
produced was determined spectrophotometrically at 492 nm,
using a microplate reader (Fluostar Optima BMGLabtech).
The IC50 was determined by interpolation of dose-response
curves. Kojic acid was used as a positive control. Each
measurement was made in triplicate. The mode of inhibition
of the enzyme was performed using the Lineweaver-Burk
plot. The assay was performed varying the concentration of
inhibitor and L-DOPA (0.3, 0.5, 0.7 and 1.0 mM). Kinetics
constants were determined by the second plots of the
apparent Km/Vmax and/or 1/Vmax versus the inhibitor
concentration.
4.4.8 Determination of melanin content. α-MSH-
stimulated cells were plated on 60π-dishes at a density of
105cells/mL. 3-Heteroarylcoumarins were added to the cell
culture at different concentration (0–50 μM). After
incubation for 48 h cells were harvested and an aliquot was
used for protein quantification while the remaining cells
were centrifuged and lysed with NaOH 1 M at 100 °C for 1
h. Melanin content was calculated by comparison of the
absorbance at 405 nm using a standard curve of synthetic
melanin.
4.4.4 Cell culture system. Murine melanoma B16F10 cells
(CRL-6475) were purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA). Cells
were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) containing 10% fetal bovine serum (FBS, Gibco,
NY, USA), and 1% penicillin/streptomycin at 37°C in a
humidified atmosphere containing 5% CO2.
4.4.9 Tyrosinase zymography (L-DOPA staining). The
DOPA-staining assay was performed as reported
previously.30 Cells were treated for 48 h with α-MSH alone
or α-MSH plus compounds (at 10, 25 and 50 μM) or DMSO
(as solvent control) or kojic acid at 50 μM (positive control).
After treatment, cells were harvested with lysis buffer, as
described in the section Measurements of cellular tyrosinase
activity. Protein extracts (5 μg) were mixed with 10 mM
Tris-HCl buffer, pH 7.0, containing 1% SDS, without
mercaptoethanol or heating, and resolved by 8% SDS-
polyacrylamide gel electrophoresis. After running, gel was
rinsed in 0.1 M phosphate buffer (pH 6.8) and equilibrated
4.4.5 Cell viability assay. Cell viability was detected by the
colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay.29 This is a
colorimetric assay for measuring the activity of
mitochondrial enzymes in living cells that convert MTT into
purple formazan crystals. Briefly, cells were seeded in a 96-

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for 30 min twice. Then, it was transferred in a staining
solution containing 0.1 M phosphate buffer (pH 6.8) with 5
mM L-DOPA and incubated in the dark for 1 h at 37 °C.
Tyrosinase activity was visualized in the gel as dark
melanin-containing bands.
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4.5 Molecular docking simulations: ligand preparation.
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different tautomers and protonation states at pH of 7.0 ± 2.0
and optimized the ligands structures.
4.5.1
Molecular
docking
simulations:
protein
preparation. The X-ray crystallized structures of the
Agaricus bisporus tyrosinase (PDB codes: 2Y9W and
2Y9X)31 were downloaded from the Protein Data Bank. We
used the Protein Preparation Workflow31 to prepare the
proteins for the docking. Through this step we added and
optimized hydrogen bond networks, including cap termini,
optimized tautomers and protonation states of the residues,
etc.
4.5.2 Molecular docking. In the first step, we defined a grid
box centered in the tyrosinase active site and with an outer
box dimension of 20 Å. The van der Waals scaling factor in
the grid generation was 1.0. In the second step, we
performed molecular docking simulations with Glide XP
(extra precision) from the Schrödinger package.32 The top
ranked poses using XP scoring and Emodel were selected as
representative of the docking and for visual inference.
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Acknowledgments
This work was partially supported by University of Cagliari and
University of Santiago de Compostela. Authors would like to
thank Angeles Alvariño, Plan Galego de Investigación,
Innovación e Crecemento 2011–2015 (I2C), European Social
Fund (ESF) and Fundação para a Ciência e Tecnologia (FCT),
POPH and QREN for their grants.
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Molecular docking
R5
Anti-melanogenesis activity
Mushroom tyrosinase assay
Cell-based assay
R6
R7
O
O
R8
Antioxidant activity
Coumarin derivatives