Design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.07.029

The synthesis, molecular modeling and biological evaluation of substituted deoxybenzoins and optimized dihydrostilbenes are reported. Preliminary structure-activity relationship data were elucidated and lead compounds suitable for further optimization wer

Full text of DOI:10.1016/j.bmcl.2012.07.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5523–5526
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and molecular simulation studies of dihydrostilbene
derivatives as potent tyrosinase inhibitors
Argyro Vontzalidou a, Grigoris Zoidis a,b, Eliza Chaita a, Maria Makropoulou a, Nektarios Aligiannis a,
George Lambrinidis b, Emmanuel Mikros b, Alexios-Leandros Skaltsounis a,
⇑
a Faculty of Pharmacy, Department of Pharmacognosy and Natural Products Chemistry, University of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece
b Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Panepistimiopolis Zografou, GR-15771, Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, molecular modeling and biological evaluation of substituted deoxybenzoins and optimized
dihydrostilbenes are reported. Preliminary structure–activity relationship data were elucidated and lead
compounds suitable for further optimization were discovered. Dihydrostilbene 7 is a particularly potent
Received 16 May 2012
Revised 3 July 2012
Accepted 6 July 2012
Available online 14 July 2012
inhibitor (IC50 = 8.44 lM, more potent than kojic acid).
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Deoxybenzoins
Dihydrostilbenes
Tyrosinase inhibitors
Kojic acid
Gallic acid
Molecular simulations
Tyrosinase (EC 1.14.18.1) is a multifunctional copper-contain-
ing enzyme, playing a key role in melanin biosynthesis by catalyz-
ing the oxidation of phenol to o-quinone observed in the early
stage of various browning phenomena in nature,1 such as melani-
sation in animals and browning in plants. Tyrosinase inhibitors can
therefore be clinically useful for the treatment of some dermato-
logical disorders associated with melanin hyperpigmentation. In
addition, these inhibitors are also known to be useful in cosmetics
for whitening and depigmentation after sunburn.2,3
against L-DOPA oxidation catalyzed by mushroom tyrosinase. The
recently published crystal structures of the fungal tyrosinase in
complex with the inhibitor Tropolone (PDB entry 2Y9X)14 and of
the bacterial tyrosinase (PDB entry 3NQ1)15 in complex with the
inhibitor kojic acid encouraged us to perform virtual screening cal-
culations (data not shown) using our in house virtual library of
Natural, Synthetic and semi-Synthetic compounds (ꢀ1500 com-
pounds). Top ranked molecules exhibited scaffold variety including
deoxybenzoins, flavonoids, stilbenoids and some polyphenols. At
this early stage we emphasized to in silico against in vitro screen-
ing. So among the top ranked molecules only 10 structures were
selected and tested for inhibition of tyrosinase activity, evaluated
Moreover, the presence of the oxidation products of L-tyrosine
has recently been linked to the demise of neurons in several
neurodegenerative disorders such as Parkinson’s and Huntington’s
diseases.4,5 Thus, the study of tyrosinase inhibition is clearly impor-
tant for the development of therapeutic agents, and also for that of
antioxidants and cosmeceuticals.6
In the past decade, a number of polyphenol tyrosinase inhibi-
tors from both natural and synthetic sources, including flavonoids,
stilbenes and terpenoids, have been intensively investigated.7–11
Reports on the relation between the structure of such polyphenol
derivatives and their enzyme inhibition have emphasized the con-
tribution of the 2,4-substituted resorcinol structure.12a To our
knowledge, however, only a few biphenyl inhibitors have been re-
ported to date.12,13 Most of the in vitro evaluations are made
against L-DOPA oxidation catalyzed by mushroom tyrosinase. Gal-
lic and kojic acid (Fig. 1) were used as the reference standards. Gal-
lic acid (3,4,5-trihydroxybenzoate) has been isolated and identified
as a tyrosinase inhibitor from many plants, and its inhibitory
mechanism together with those of its ester derivatives has been
well studied by Kubo et al.16
The deviation of inhibition percentage at concentration 100
lg/mL was from 16% to 92% for all compounds. Since our research
effort is focused on drug optimization, we selected deoxybenzoin
1 exhibiting 33% inhibition at 100 lg/mL, for further investigation
and optimization. Deoxybenzoin 117 was considered as new hit
for the discovery of novel tyrosinase inhibitors. To assess the role
of the substitution on the B ring in the activity, we tested deoxy-
benzoins with various functional groups such as hydroxy, fluoride
⇑
Corresponding author. Tel.: +30 2107274598; fax: +30 2107274594.
E-mail address: skaltsounis@pharm.uoa.gr (A.-L. Skaltsounis).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.029

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A. Vontzalidou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5523–5526
COOH
3
3
O
4
2
4
HO
OH
O
2
HO
HO
HO
OH
R1
R1
OH
7'
7'
1
1
R2
R3
2'
2'
1'
1'
R2
R3
5
5
3'
4'
6
3'
HO
Kojic acid
6
7
HO
Gallic acid
O
6'
6'
6-10
4'
1-5
5'
5'
1
2
3
1
2
3
1
2
3
6
7
8
. R =R =H, R =OCH3
. R =R =H, R =OCH3
1
2
3
1
2
3
. R =R =H, R =OH
. R =R =H, R =OH
1
2
3
1
2
3
. R =R =H, R =F
. R =R =H, R =F
4. R1=Cl, R2=R3=H
5. R1=R2=H, R3=Cl
9. R1=Cl, R2=R3=H
10.R1=R2=R3=H
Figure 1. Structures of gallic acid, kojic acid, prevalent tyrosinase inhibitor, and synthesized compounds.
Figure 2. Schematic representation of the two possible binding modes of molecule 1 as derived from virtual screening, depicting potential interaction with Asn260.
R1
3
6
3
6
R2
R3
4
2
4
HO
OH
HO
OH
O
2
HO
OH
HO
R1
R1
a
b
1
7'
1'
R2
R3
1
2'
2'
+
1'
R2
R3
O
5
5
3'
4'
3'
4'
7
6'
6'
5'
5'
Scheme 1. Reagents and conditions: (a) BF3ꢁOEt2, 95 °C, 3–5 h, 45–90%; (b) H2, Pd/C 10%, EtOH/AcOEt (1/1), 55 psi, 5 h, 43–90%.
and chloride. Moreover, preliminary docking calculations showed
two possible binding modes for deoxybenzoin 1. In the first mode,
(having the lowest docking score) a hydrogen bond (HB) is formed
between OH group in position 2 of the phenyl ring and the side
chain of Asn260 (Fig. 2a) which is placed near the entrance of the
binding pocket. In the second mode, the internal HB formed be-
tween OH-2 and the carbonyl group could reduce its binding affin-
ity and thus the biological potency (Fig. 2b). The relative docking
score for the two possible binding modes was 1 kcal/mol. Therefore,
we considered to design new molecules eliminating the carbonyl
group by hydrogenation, in order to identify its role in tyrosinase
inhibition.
Based on the above initial results we present herein the synthesis,
tyrosinase inhibitoryactivity and structure-activity analysis of some
dihydrostilbenes 6–10 (Fig. 1). The intermediates deoxybenzoins 1–
5 (Scheme 1) were also in vitro re-evaluated as tyrosinase inhibitors
and compared to dihydrostilbenes 6–10. As shown in Scheme 1, the
synthesis of substituted deoxybenzoins and corresponding dihydro-
stilbenes started from resorcinol and phenylacetic acids. Resorcinol
reacted with variously substituted phenylacetic acids, giving excel-
lent to moderate yields of deoxybenzoins catalyzed by BF3ꢁOEt2. The
latter compounds, upon catalytic hydrogenation in the presence of
Pd/C 10% in ethanol/ethyl acetate (1/1) at 55 psi, were converted
to the corresponding dihydrostilbenes. It is noteworthy, that during
the hydrogenation of deoxybezoin 5 cleavage of the chloride at the
para position took place, whereas in the case of deoxybenzoin 4
reductive dechlorination of the ortho moiety did not occur.
All new compounds were fully characterized by spectroscopic
methods (1H, 13C NMR, COSY, HMQC and HMBC), and high resolu-
tion mass spectrometry (APCI-HRMS).
The newly synthesized dihydrostilbenes were potently active
tyrosinase inhibitors, with IC50 values ranging from 8.44 to
49.58
l
M. Compound 7 was the most potent with activity in low
M, Table 1), while dihydrostilbenes
micromolar range (IC50 = 8.44
l
6, 9 and 10 were also significantly active. Eliminating the carbonyl
group from the middle chain of deoxybenzoins (1–5) progressively
increased tyrosinase inhibitory activity (Table 1). This marked en-
hance of activity demonstrates the negative effect of carbonyl
group and identifies the major role of the free to interact OH group
in position 2. Moreover, the role of the substitution on the B ring of
dihydrostilbens, such as hydroxy, O-methyl, fluoride, chloride or
unsubstituted, in the activity was also investigated. Specifically,
the hydroxy group at 40 position (ring B) is indispensable for signif-
icant tyrosinase inhibition in this class of compounds.
Preliminary conformational analysis suggested that an intramo-
lecular hydrogen bond formed, between the carbonyl oxygen and

A. Vontzalidou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5523–5526
5525
Table 1
Tyrosinase inhibitory activities18 of deoxybenzoins 1–5, dihydrostilbenes 6–10, gallic acid and kojic acid. (For experimental details see Supplementary data).
a
Compound
R1
R2
R3
% Inhibition (100
lg/mL)
IC50 (lM)
3
4
2
HO
OH
R1
A
6
1
1'
R2
R3
2'
5
3'
B
5'
O
6'
4'
1
2
3
4
5
H
H
H
Cl
H
H
H
H
H
H
OCH3
OH
F
H
Cl
33.30 0.56
16.32 0.41
65.80 1.24
5.70 0.25
9.70 0.96
—
—
—
—
—
3
4
2
HO
OH
R1
A
6
1'
R2
R3
1
2'
5
3'
B
5'
6'
4'
6
7
8
9
H
H
H
Cl
H
H
H
H
H
OCH3
OH
F
H
H
94.20 0.03
95.10 0.09
88.00 0.24
92.30 0.17
89.70 0.05
31.64 6.38
8.44 0.88
49.58 9.95
33.81 7.80
30.56 2.81
10
H
Gallic acid
Kojic acid
31.70 0.79
96.9 0.24
—
9.96 1.23
a
The IC50 values represent means SEM of three different experiments.
showed that compound 7 is bound at the entrance of the receptor
similar to Tropolone (Fig. 3) forming three HB, and more specifi-
cally OH-2 with the amide carbonyl of Asn260, OH-4 with the
backbone carbonyl group of Val283 and OH-40 with His244 imidaz-
ole nitrogen.
The interaction of OH-2 with Asn260 emphasizes the impor-
tance of this residue located close to the entrance of the receptor
and could explain the loss of activity for analogues 1–5 compared
to analogues 6–10 validating our initial hypothesis. Furthermore,
the HB of OH-40 with His244 imidazole nitrogen illustrates the
strong inhibition of mushroom tyrosinase (IC50 = 8.44
lM), higher
than kojic acid (IC50 = 9.96 M), that compound 7 exhibited com-
l
pared to analogues 6 and 8–10.
Synopsis: The aim of this work was to develop lead compounds
by investigating the structural and stereoelectronic requirements
for optimal tyrosinase inhibition. Virtual screening calculations
proved valuable tool for drug design. All dihydrostilbenes 6–10
demonstrated higher inhibitory activity than gallic acid, while
dihydrostilbene 7 was more potent than kojic acid. Two interesting
points arise from this analysis: (i) the presence of the carbonyl
group in analogues 1–5 causes a dramatic reduction in their po-
tency. One possible explanation for this, based on conformational
analysis, could be an intramolecular hydrogen bond formed, be-
tween the carbonyl oxygen and the OH-2 (ring A) in compounds
1–5 that hampers the latter to interact with the receptor and (ii)
the presence of OH-40 (ring B) is critical for the antityrosinase
activity, probably due to the HB of OH-40 with His244 imidazole
nitrogen. New synthetic efforts will be made to obtain structural
analogues by introducing a variety of substituents at the two aro-
matic rings, as well as by functionalizing the carbonyl group. The
future experiments should acquire more SAR information to help
refine the requirements for optimal activity.
Figure 3. Superposition of the crystal structure of fungal tyrosinase in complex
with tropolone (red) and global minimum structure of fungal tyrosinase in complex
with analogue 7 after docking calculations. For clarity reasons only few crucial
aminoacids are visible. Hydrogen bonds are shown with yellow dashed lines.
the OH-2 in compounds 1–5, can hamper the latter to interact with
the receptor.
As mentioned, the crystal structure of the bacterial and the fun-
gal tyrosinase are solved in complex with known antagonists, kojic
acid and Tropolone respectively. In both crystal structures the li-
gands do not interact with the bi-nuclear copper-binding site,
but mainly with residues’ side chains in the active-site entrance.
Our hypothesis, that the OH-2 (analogues 6–10) could form stabi-
lizing interactions with one or more aminoacids near that entrance
was examined by molecular docking calculations. Starting with the
crystal structure of the fungal tyrosinase in complex with Tropo-
lone, the inhibitor was replaced manually by compound 7, which
showed the strongest inhibition. 500 steps of flexible docking cal-
culations using LowMode Search were run, as implemented on
Macromodel 9.0 (Schrödinger Inc). Global minimum structure
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
07.029.

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