Tyrosine-like condensed derivatives as tyrosinase inhibitors

Article abstract of DOI:10.1111/j.2042-7158.2012.01467.x

Objectives We report the pharmacological evaluation of a new series of 3-aminocoumarins differently substituted with hydroxyl groups, which have been synthesized because they include in their structures the tyrosine fragment (tyrosine-like compounds), with the aim of discovering structural features necessary for tyrosinase inhibitory activity. Methods The synthesized compounds 4 and 7-9 were evaluated in vitro as mushroom tyrosinase inhibitors. Key findings Two of the described compounds showed lower IC50 (concentration giving 50% inhibition of tyrosinase activity) than umbelliferone, used as a reference compound. Conclusions Compound 7 (IC50 = 53 μm) was the best tyrosinase inhibitor of this small series, having an IC50 value 10-fold lower than umbelliferone. Compound 7 (3-amino-7-hydroxycoumarin) had amino and hydroxyl groups precisely mimicking the same positions that both groups occupy on the tyrosine molecule. 2012 The Authors. JPP

Full text of DOI:10.1111/j.2042-7158.2012.01467.x

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Research Paper
Journal of Pharmacy
And Pharmacology
Tyrosine-like condensed derivatives as tyrosinase inhibitors
a
a
a
a,b
c
Maria João Matos , Lourdes Santana , Eugenio Uriarte , Silvia Serra , Marcella Corda ,
c
c
c
Maria Benedetta Fadda , Benedetta Era and Antonella Fais
a
Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain and
b
c
Dipartimento Farmaco Chimico Tecnologico, Facoltà di Farmacia and Dipartimento di Scienze della Vita e dell’Ambiente, Università degli Studi di
Cagliari, Cagliari, Italy
Keywords
depigmenting agents; mixed-type inhibitor;
Abstract
tyrosinase inhibition; tyrosine-like compounds
Objectives We report the pharmacological evaluation of a new series of
-aminocoumarins differently substituted with hydroxyl groups, which have been
synthesized because they include in their structures the tyrosine fragment (tyrosine-
like compounds), with the aim of discovering structural features necessary for
tyrosinase inhibitory activity.
3
Correspondence
Maria João Matos, Department of Organic
Chemistry, University of Santiago de
Compostela, Fac Farmacia, Campus Vida,
Santiago de Compostela, 15782, Spain.
E-mail: mariajoao.correiapinto@rai.usc.es
Methods The synthesized compounds 4 and 7–9 were evaluated in vitro as mush-
room tyrosinase inhibitors.
Key findings Two of the described compounds showed lower IC50 (concentration
giving 50% inhibition of tyrosinase activity) than umbelliferone, used as a reference
compound.
Received October 11, 2011
Accepted January 5, 2012
Conclusions Compound 7 (IC50 = 53 mm) was the best tyrosinase inhibitor of this
small series, having an IC50 value 10-fold lower than umbelliferone. Compound 7
doi: 10.1111/j.2042-7158.2012.01467.x
(3-amino-7-hydroxycoumarin) had amino and hydroxyl groups precisely mimick-
ing the same positions that both groups occupy on the tyrosine molecule.
Introduction
Tyrosinase (EC 1.14.18.1) is a multifunctional dinuclear
copper centre metalloenzyme widely distributed in nature.
many tyrosinase inhibitors have been reported, including
vitamin C (ascorbic acid), kojic acid, umbelliferone, resvera-
[1]
[7–12]
This enzyme catalyses two distinct reactions of conversion
of the tyrosine: 3′-hydroxylation of l-tyrosine into l-3,4-
dihydroxyphenylalanine (L-DOPA) and oxidation of the
resultant L-DOPA into DOPA quinone, which further poly-
trol, hydroquinone and oxyresveratrol.
Because of the
structural similarity, umbelliferone was used as a reference
inhibitor (Figure 1).
Coumarins are a large family of compounds,of natural and
synthetic origin, which present different pharmacological
[2]
merizes spontaneously into melanin. Most melanin-
biosynthesis inhibitors are phenol or catechol analogues,
which are structurally similar to tyrosinase substrates:
[13]
actions. Chemically they are lactones of cinnamic acid.
Their structural variety is responsible for the important
place that they occupy in the natural product and synthetic
[3]
tyrosine or L-DOPA. Therefore, tyrosine-like molecules
could be an interesting scaffold to the tyrosinase inhibition
process. Tyrosinase is mainly involved in the formation of
pigments such as melanins and other polyphenolic com-
[14]
organic chemistry realm. Some important studies pay
special attention to their antioxidative, anti-cancer anti-
inflammatory, cardioprotective and enzymatic inhibitory
[
1]
[15–21]
pounds. Tyrosinase oxidizes phenols and diphenols using a
catalytic mechanism dependent on the presence of copper at
properties.
In recent studies, some coumarins proved
[22,23]
to be mushroom tyrosinase inhibitors.
In those studies,
[
1,2]
its active site.
This enzyme is responsible for unwanted
esculetin (6,7-dihydroxycoumarin) and umbelliferone (7-
hydroxycoumarin) exhibited some of the strongest inhibi-
tory activity of the tested series, with esculetin proving to
[4]
browning of fruits and vegetables. Therefore, it is involved
in the process of maintaining the appearance, flavour, texture
and nutritional value of many fresh-cut products. Tyrosi-
nase is also responsible for the colouring of skin,hair and eyes
in animals, including humans. In fact, tyrosinase inhibitors
have been used as depigmenting agents for the treatment or
prevention of hyperpigmentation disorders. In recent years,
[4]
[22]
be the strongest inhibitor of the series. Recently, and
in contrast to the initial findings, Sollai et al. showed that
esculetin is a tyrosinase substrate rather than an inhibitor,
whereas umbelliferone seems to be an inhibitor of
[5]
[6]
[24]
tyrosinase. These studies revealed that tyrosinase affinity
©
2012 The Authors. JPP © 2012
7
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Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 742–746

MariaJoão Matos et al.
Tyrosine-like tyrosinase inhibitors
O
3-nitrocoumarins 1–3 in good yields (75–95%). They were
synthesized in a dry Schlenk tube, with acetic anhydride
as solvent, in the presence of sodium hydride, at room tem-
perature, for 3 h. The reaction mixture was purified by
flash chromatography, using hexane/AcOEt (9 : 1) as eluent.
The 3-aminocoumarins 4–6 were prepared from previously
synthesized 3-nitrocoumarins, in EtOH, with Pd/C as cata-
OH
NH2
HO
HO
O
O
Tyrosine
Umbelliferone
lyst, under H
purified by crystallization in AcOEt to give the desired
-aminocoumarins, in a yield of 95%. The hydroxy deriva-
tives 7 and 8 were obtained from the methoxy derivatives
compounds 5 and 6, respectively) by a hydrolysis reaction
2
atmosphere. The obtained products were
3
HO
NH2
O
(
with hydriodic acid, in the presence of acetic acid and acetic
anhydride, at 110°C, for 5 h. The residue was purified by
crystallization of acetonitrile, and the hydroxy derivatives
O
Tyrosine-like compounds
[31]
were obtained in a yield of 60%. Compound 9
was
Figure 1 Chemical structures of tyrosine (tyrosinase substrate),
tyrosine-like condensed molecules and umbelliferone (tyrosinase
inhibitor).
obtained via reduction reaction, under the same conditions
as described above, of the commercially available 4-hydroxy-
3-nitrocoumarin, in a yield of 93%.
The biological assays were carried out using the following
protocol. Pre-incubation with the enzyme was carried out:
/15 m phosphoric acid buffer solution (pH 6.8, 1.8 ml),
an aqueous solution of mushroom tyrosinase (1000 U/ml;
Sigma Chemical Co., Milan, Italy, 0.1 ml) and dimethyl
sulfoxide (DMSO) (0.1 ml) with or without the sample.
The mixture was incubated at 25°C for 10 min. Then, 1.5 mm
L-DOPA solution (1 ml) was added and the reaction was
monitored at 475 nm for 5 min. The percent of tyrosinase
activity inhibition was calculated as: inhibition (%) =
can be efficiently modulated by appropriate substitutions in
the coumarin moiety. The introduction of different numbers
of hydroxyl groups in different positions of the coumarin is
1
[22,23]
one of the most important modifications.
Also, until
recently, polyphenols were the largest scaffold in tyrosinase
[
25,26]
inhibition.
Tyrosinase inhibitors could have broad applications.As the
ideal drug candidate has not been attained, an intensive
search for new tyrosinase inhibitors is still needed. This effort
has considerably increased in the recent years. In this context,
and in an attempt to develop novel tyrosinase inhibitors, we
had previously synthesized and described 3-arylcoumarin
derivatives in which both the coumarin and the resveratrol
(
A - B)/A ¥ 100, where A represents the difference in the
absorbance of control sample between 0.5 and 1.0 min, and
B represents the difference in absorbance of the test sample
between 0.5 and 1.0 min. The mushroom tyrosinase acti-
vity was determined by spectrophotometric assays (Varian
Cary 50). Umbelliferone was used as a reference tyrosinase
inhibitor.
[23,27]
templates were present.
Based on this work, and with the
aim of finding new structure–activity features, we propose
the study of a series of tyrosine-like condensed molecules
(
Figure 1). The similarity of these compounds to the tyrosi-
nase substrate is the novelty of this study. The proposed com-
pounds are structurally related to the amino acid tyrosine,the
natural substrate of this enzyme. Tyrosinase inhibitors based
on the aminocoumarin scaffold have not been previously
studied.
Statistical methods
All experiments were carried out three times. Continuous
variables were expressed as mean Ϯ SD.The IC50 value (con-
centration giving 50% inhibition of tyrosinase activity) was
determined by interpolation of dose–response curves and all
data were statistically evaluated using Student’s t-test or
Mann–Whitney test (Statistica 6; Statsoft Tulsa, Tulsa, OK,
USA). To assess the slopes of curves in Figure 3 the Kruskal–
Wallis test followed by Dunn’s post-hoc test (Statistica 6;
Statsoft Tulsa) was used. The criterion for statistical signifi-
cance was generally taken as P < 0.05.
Materials and Methods
We synthesized (Figure 2) and evaluated a small series of
3-aminocoumarins.We decided to explore the importance of
the position of a hydroxyl group under the 3-aminocoumarin
moiety, based on the idea of mimicking the molecular struc-
ture of the tyrosinase substrate.
[28–30]
The coumarin derivatives 4 and 7–9
were efficiently
Results
synthesized according to the protocol outlined in Figure 2.
Starting from different substituted commercially available
salicylaldehydes and ethyl nitroacetate, we afforded
The tyrosinase inhibitory activity of compounds 4 and 7–9
was evaluated in vitro by measuring the enzymatic activity of
©
2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 742–746
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Tyrosine-like tyrosinase inhibitors
MariaJoão Matos et al.
O
NO2
O
O
H
a
O2N
O
R1
OH
R1
O
R2
R2
1
2
3
R1 = R2 = H
R1 = OMe, R2 = H
R1 = H, R2 = OMe
b
NH2
O
NH2
c
R1
O
R1
O
O
R2
R2
7
8
R1 = OH, R2 = H
R1 = H, R2 = OH
4 R1 = R2 = H
5 R1 = OMe, R2 = H
6
R1 = H, R2 = OMe
OH
O
OH
NO2
O
NH2
O
b
O
9
Figure 2 Protocols for synthesis of coumarin derivatives. Reagents and conditions: (a) acetic anhydride, NaH, r.t., 3 h; (b) H
AcOH, Ac O, 110°C, 5 h.
2
, EtOH, Pd/C, r.t., 5 h; (c) HI,
2
mushroom tyrosinase enzyme extracted from the mushroom
Agaricus bisporus. Then, the IC50 values for the inhibitory
effects of the new compounds were calculated (Table 1).
In the presence of compound 7, kinetic studies on mush-
room tyrosinase, using a Lineweaver–Burk double reciprocal
plot (Figure 3), showed that compound 7 was a mixed-type
inhibitor. Increasing the concentration of 7 resulted in a
family of lines with different slope and intercept, which inter-
sected in the second quadrant. This behaviour showed that
compound 7 can bind not only with free enzyme but also
with the enzyme–substrate complex, with different equilib-
rium constants. The inhibition constants for the inhibitor
moiety was studied. In this way a small series of compounds
that are both tyrosine (tyrosinase substrate) and umbellifer-
one (tyrosinase inhibitor) analogues was obtained.
It is known that umbelliferone (7-hydroxycumarin) has a
tyrosinase inhibitor effect (IC50 = 0.42 mm), despite having
no amino group in its position 3. The 3-aminocoumarin
(compound 4) was synthesized and evaluated and did not
show tyrosinase inhibitory activity. Then, other coumarins
were prepared, maintaining the amino group in the
3-position, also incorporating a hydroxyl group in different
positions of the coumarin skeleton.
Different synthetic methodologies were carried out to
obtain compounds 7 and 8, which have the hydroxyl group in
positions 7 and 8, respectively, of the coumarin nucleus
(benzene ring) and compound 9, with the hydroxyl group in
position 4 of the coumarin (pyrone ring). As shown in
Table 1, compound 7 was the most active compound of this
series, with an IC50 value in the micromolar range (53 mm).
This compound was more than 10 times more active than
umbelliferone, the reference compound. Compound 7 had
the amino and hydroxyl groups that precisely mimicked the
same positions that both groups occupy on the tyrosine mol-
ecule. Compound 8 was inactive against tyrosinase whereas
binding with free enzyme, K
substrate complex, KIS (0.78 mm), were obtained from the
linear secondary plots of 1/Vmax and K /Vmax versus the con-
I
(2.14 mm), and with enzyme–
m
centration of compound 7, respectively.
Discussion
In this communication, the possible tyrosinase inhibitory
effect of coumarins that incorporate a portion of tyrosine on
their skeletons was described. The introduction of a hydroxyl
substituent into different positions of the 3-aminocoumarin
©
2012 The Authors. JPP © 2012
7
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Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 742–746

MariaJoão Matos et al.
Tyrosine-like tyrosinase inhibitors
4
3
2
1
0
.00
.00
.00
.00
.00
35
30
0
0.02
0.04 0.06
I] (mM)
0.08
25
[
2
1
1
0
5
0
5
0
3
2
1
0
.00
.00
.00
.00
0
0.02
0.04 0.06
I] (mM)
0.08
[
–
4
–2
0
2
4
6
8
10
12
1
/[S] mM–1
Figure 3 Lineweaver–Burk plots for inhibition of compound 7 on mushroom tyrosinase for catalysis of L-DOPA. Inhibitor concentrations were 0 (᭜),
0
m
.010 mM (᭹), 0.025 mM (x), 0.050 mM (᭡), 0.070 mM ( ). The insets are the secondary plots of 1/Vmax and K /Vmax versus concentration of compound
7
, respectively.
Table 1 Inhibitory effect of compounds 4 and 7–9 and umbelliferone
on mushroom tyrosinase activity
presence of a hydroxyl group in the seven position of the
-aminocoumarin, the same position as in tyrosine and
3
Compounds
IC50 (mM) (L-DOPA 0.5 mM)
umbelliferone, improves the inhibitory activity with respect
to the other synthesized derivatives and the reference com-
pound. So, the introduction of hydroxyl groups improves the
pharmacological potential of these 3-aminocoumarins, con-
firming that this lead could be effectively optimized in a can-
didate for the treatment of some hyperpigmentation skin
diseases. These findings have encouraged us to continue the
effort towards the optimization of the pharmacological
profile of these coumarins.
4
>5.0
0.05 Ϯ 0.01
7
8
>5.0
9
0.25 Ϯ 0.003
a
Umbelliferone
0.42
a
[23]
Obtained from Fais et al. . These results are average results of three
experiments.
compound 9 was active against this enzyme (IC50 =
Declarations
0
.26 mm).Compound 9 presented only slightly higher tyrosi-
Conflict of interest
nase inhibitory activity than umbelliferone.
Therefore, it can be inferred that the tyrosinase inhibitory
activity depends on the position of the hydroxyl group in the
coumarin moiety. Also, the presence of the hydroxyl group at
the same position that of tyrosine and umbelliferone is
important to the inhibitory activity.
The Author(s) declare(s) that they have no conflicts of inter-
est to disclose.
Funding
This work was partially supported by the Ministerio de
Sanidad y Consumo [grant number FIS PS09/00501], Xunta
da Galicia [grant number INCITE09E2R203035ES and
PGIDIT09CSA030203PR], RAS–LR7/2007 [grant number
CRP2_133] and Università degli Studi di Cagliari. This work
was also partially supported by Fundação de Ciência e Tecno-
logia [grant number SFRH/BD/61262/2009].
Conclusions
This study showed that some of the synthesized tyrosine-like
condensed derivatives have inhibitory activity against mush-
room tyrosinase. The two active compounds present tyrosi-
nase inhibitory activity in the micromolar range. The
©
2012 The Authors. JPP © 2012
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 742–746
745

Tyrosine-like tyrosinase inhibitors
MariaJoão Matos et al.
hydro- quinone and glycolic acid. Der- 20. Matos MJ et al. Synthesis and study
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