Design and synthesis of chalcone derivatives as potent tyrosinase inhibitors and their structural activity relationship

Article abstract of DOI:10.1016/j.molstruc.2014.12.073

Browning of fruits and vegetables is a serious issue in the food industry, as it damages the organoleptic properties of the final products. Overproduction of melanin causes aesthetic problems such as melisma, freckles and lentigo. In this study, a series of chalcones (1-10) have been synthesized and examined for their tryrosinase inhibitory activity. The results showed that flavokawain B (1), flavokawain A (2) and compound 3 were found to be potential tyrosinase inhibitors, indicating IC₅₀ 14.20-14.38 μM values. This demonstrates that 4-substituted phenolic compound especially at ring A exhibited significant tyrosinase inhibition. Additionally, molecular docking results showed a strong binding affinity for compounds 1-3 through chelation between copper metal and ligands. The detailed molecular docking and SARs studies correlate well with the tyrosinase inhibition studies in vitro. The structures of these compounds were elucidated by the 1D and 2D NMR spectroscopy, mass spectrometry and single X-ray crystallographic techniques. These findings could lead to design and discover of new tyrosinase inhibitors to control the melanine overproduction and overcome the economic loss of food industry.

Full text of DOI:10.1016/j.molstruc.2014.12.073

Journal of Molecular Structure 1085 (2015) 97–103
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Design and synthesis of chalcone derivatives as potent tyrosinase
inhibitors and their structural activity relationship
b
a
c
d
Muhammad Nadeem Akhtar a, , Nurshafika M. Sakeh , Seema Zareen , Sana Gul , Kong Mun Lo ,
⇑
Zaheer Ul-Haq c, Syed Adnan Ali Shah e,f, , Syahida Ahmad b
⇑
a Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak 26300, Kuantan, Pahang, Malaysia
b Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
c Dr. Panjwani Center for Molecular Medicine & Drug Research, International Center for Chemical & Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
e Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
f Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
In this study, a series of chalcones (1–10) have been synthesized and examined for their tryrosinase
inhibitory activity. The results showed that flavokawain B (1), flavokawain A (2) and compound 3 were
found to be potential tyrosinase inhibitors, indicating IC50 14.20–14.38 lM values. This demonstrates
ꢀ
Chalcones derivatives were prepared
through the Claisen–Schmidt
condensation reaction.
that 4-substituted phenolic compound especially at ring A exhibited significant tyrosinase inhibition.
Additionally, molecular docking results showed a strong binding affinity for compounds 1–3 through che-
lation between copper metal and ligands.
ꢀ
Compounds were characterized by
detailed spectroscopic techniques and
single-crystal X-ray structural
analysis.
ꢀ
ꢀ
Flavokawain B (1), flavokawain A (2)
and compound 3 were found to be
potential tyrosinase inhibitors.
Detailed molecular docking and SARs
studies were correlated well with the
tyrosinase inhibition studies in vitro.
a r t i c l e i n f o
a b s t r a c t
Article history:
Browning of fruits and vegetables is a serious issue in the food industry, as it damages the organoleptic
properties of the final products. Overproduction of melanin causes aesthetic problems such as melisma,
freckles and lentigo. In this study, a series of chalcones (1–10) have been synthesized and examined for
their tryrosinase inhibitory activity. The results showed that flavokawain B (1), flavokawain A (2) and
Received 5 September 2014
Received in revised form 3 December 2014
Accepted 23 December 2014
Available online 31 December 2014
compound 3 were found to be potential tyrosinase inhibitors, indicating IC50 14.20–14.38 lM values. This
demonstrates that 4-substituted phenolic compound especially at ring A exhibited significant tyrosinase
inhibition. Additionally, molecular docking results showed a strong binding affinity for compounds 1–3
through chelation between copper metal and ligands. The detailed molecular docking and SARs studies
correlate well with the tyrosinase inhibition studies in vitro. The structures of these compounds were
elucidated by the 1D and 2D NMR spectroscopy, mass spectrometry and single X-ray crystallographic
Keywords:
Chalcones
1D and 2D NMR spectroscopy
Tyrosinase inhibitors
Flavokawain A
⇑
Corresponding authors. Tel.: +60 9 5492393; fax: +60 9 5492766 (M.N. Akhtar). Tel.: +60 3 32584616; fax: +60 3 32584602 (S.A.A. Shah).
E-mail addresses: nadeemupm@gmail.com (M.N. Akhtar), syedadnan@salam.uitm.edu.my, benzene301@yahoo.com (S.A.A. Shah).
http://dx.doi.org/10.1016/j.molstruc.2014.12.073
0022-2860/Ó 2014 Elsevier B.V. All rights reserved.

98
M.N. Akhtar et al. / Journal of Molecular Structure 1085 (2015) 97–103
Flavokawain B
Molecular docking studies
techniques. These findings could lead to design and discover of new tyrosinase inhibitors to control the
melanine overproduction and overcome the economic loss of food industry.
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
Single-crystal X-ray crystallography
Skin health and appearance is a major concerned issue for peo-
ple globally. As early as last century, pharmaceuticals and tradi-
tional remedies have become incrementally popular for this
purpose. Skin disorders are often related to deleterious effect of
ultraviolet radiations. The failure of melanogenesis regulation
may causes overproduction of melanin and resulting hyperpig-
mentation trauma such as post-inflammatory melanoderma, mel-
asma and solar lentigines [1,2]. Tyrosinase (EC 1.14.18.1) is a
copper-containing enzyme, which plays a key role in melanin bio-
The molecular structure of the of (E)-1-(20,40-dihydroxy-phe-
nyl)-3-(2,3-dimethoxyphenyl)-propenone (9) was determined by
single X-ray crystal diffraction. The data were collected at 100 K
by using a Bruker APEXII with a CCD area-detector X-ray diffrac-
tometer. The structure was solved by direct method with SHELXS97
program and refined on F2 by full-matrix least-squares methods
with anisotropic non-hydrogen atoms. The compound crystallizes
in the orthorhombic Pna2(1)/c space group with the crystallo-
graphic detail presented in Table 1. The 1,3-diaryl-2-propen-1-
one molecule (excluding the hydroxyl and methoxy substituents)
is a planar molecule with r.m.s deviation of 0.018 Å (Fig. 1). The
molecule adopts a trans configuration at the C@C bond with a dis-
tance of 1.3372(19) Å. One of the hydroxyl group is intramolecu-
larly hydrogen bonded with the carbonyl substituent (O5AH5ꢁ ꢁ ꢁO3
2.5059(15) Å), while the other hydroxyl group is intermolecular
hydrogen bonded to the methoxy oxygen of an adjacent molecule
(O4AH4ꢁ ꢁ ꢁO1 = 2.7849(14) Å, symmetry code: ꢂ0.5 + x, 0.5 ꢂ y,
ꢂ1 + z) giving rise to a polymeric chain structure. The crystal
synthesis. Tyrosinase catalyzes the aerobic hydroxylation of
sine to -DOPA and the subsequent oxidation of -dopa to
-dopaquinone, which lead to the accumulation of melanin and
L-tyro-
L
L
L
hyperpigmentation [3]. In addition, unfavorable enzymatic brown-
ing of plant-derived foods by tyrosinase causes a decrease in nutri-
tional values and economic loss in food industry. Tyrosinase
inhibitors are used for the treatment of melasma, postinflammato-
ry hyperpigmentation and Addison’s disease [4].
However, these inhibitors suffer from toxicity and lack of effi-
cacy. Even, the most popular drug hydroquinone is used as a tyros-
inase inhibitor [5] causes DNA damage [6] and carcinogenic effect
[7]. The use of hydroquinone strong depigmentation with adverse
effect reported in long term of usage [8,9]. Therefore, the use of
hydroquinone in cosmetic products was banned in the European
Union and is under scrutiny by the United States Food and Drug
Administration (FDA).
packing of the compound is formed via
p-p stacking interaction
[distance between ring centroids, Cg(1)ACg(2)i :3.8495(9) Å, sym-
metry operator i: x, y, 1 + z and dihedral angle between planes:
3.53(7)°]. The full crystallographic data of compound 9 has been
deposited at the Cambridge Crystallographic Data Center (CCDC)
as supplementary publication (number is CCDC938805). Union
Road, Cambridge CB2 1EZ, UK [Fax: þ44 1223 336033. email:
deposit@ccdc.cam.ac.uk or at www.ccdc.cam.ac.uk].
Tyrosinase inhibition has been indicating as an important target
for inhibition of melanin production [5,10]. Most of the enzyme
inhibitors, such as kojic acid, tropolone and arbutin are structurally
Chemicals
similar to L-DOPA and tyrosine, which act competitively for tyros-
inase [11–13]. Naturally derived compounds have a major contri-
bution for the development effective inhibitors for treatment
with low side effects. Chalcones form a group of polyphenols,
which are widely distributed in edible plants and possess diverse
pharmacological activities including anti-inflammatory, antipy-
retic, analgesic, bactericidal, insecticidal, anti-fungal, anticancer
and antioxidant [14,15]. Flavokawain B (1) and flavokawain A (2)
were isolated from Piper methysticum [16] exhibited a strong cyto-
toxicity againist various cancer cell lines [17,18]. The main purpose
of this study is to discover efficient tyrosinase inhibitors. This may
contribute in the development of effective inhibitors to control the
melanin overproduction and improve the quality of fruits, vegeta-
bles and plant-derived foods.
The following reagents were purchased commercially: mush-
room tyrosinase (EC 1.14.18.1), dimethyl sulfoxide (DMSO),
L-3,4-
dihydroxyphenylalanine (
L
-DOPA), Dulbecco’s Modified Eagle0s
Medium (DMEM), Kojic acid from Sigma Chemical Co. (St. Louis,
MO, USA).
Tyrosinase enzymatic assay
Tyrosinase inhibition assays were performed according to a
modified method described by Kubo [26]. Mushroom tyrosinase
(EC 1.14.18.1, Sigma Product T3824 with an activity of 3320 U/mg)
was used in this bioassay protocol [26]. The solutions compounds
1–10 were prepared (200 lg/mL) in DMSO and mixed with 0.5 mL
Table 1
Material and methods
Percentage Inhibitions and IC50 values of compounds (1–10) and Kojic acid (11).
General
Compound
Percentage of inhibition (%)
IC50 (lM)
1
2
3
4
5
6
7
8
90.61 0.42
95.20 0.47
97.36 0.36
84.85 0.17
63.68 0.56
75.23 0.31
82.31 1.72
29.51 2.80
87.20 0.69
20.29 0.97
99.21 0.16
14.38 0.12
14.26 0.08
14.20 0.03
15.61 0.31
20.59 0.33
15.64 0.01
15.01 0.42
NT
Melting points were determined on a melting point apparatus,
XSP-12 500X and were uncorrected. UV spectra were recorded on
UV–visible spectrophotometer CARY 100 in MeOH. IR spectra were
recorded on a Perkin–Elmer RXI Fourier Transform Infrared spec-
trometer (FTIR) as KBr disks. Mass spectra were measured on a
Finnigan MAT SSQ 710 spectrometer by electron impact at 70 eV.
NMR spectra were recorded in CDCl3 or acetone-d6 using a Varian
500 MHz NMR spectrometer. Column chromatography was per-
formed on silica gel (60 Merck 9385 (230–400 mesh, ASTM).
9
10
14.23 0.41
NT
12.01 0.11
Kojic acid (11)

M.N. Akhtar et al. / Journal of Molecular Structure 1085 (2015) 97–103
99
Fig. 1. The structures of compounds (1–11).
of
L
-DOPA solution (1.25 mM). About 0.9 mL of sodium acetate buf-
docking was performed. So far there is no crystal bound structure
available for tyrosinase target enzyme, hence experimentally
resolved X-ray structure of apo-tyrosinase enzyme (PDBcode
1WX2 with resolution 1.82 Å) was retrieved from Protein Data Bank
(PDB) [27]. Data set containing kojic acid (11) as a standard and
nine chalcone derivatives were used for docking studies. All the
compounds were docked in tyrosinase enzyme by using GOLD soft-
ware [28]. First, all compounds were drawn and then each com-
pound was converted into mol2 format by using babel program.
Moreover, all compounds were filtered using OE Filter program
from OpenEyes to adjust protonation state and followed by minimi-
zation with MMFF94 force field with gradient of 0.05 kcal/(molÅ)
by Molecular Operating Environment [29–31]. Before docking
protein structure of tyrosinase enzyme (PDB code 1WX2) was sub-
jected to partial charge calculation and then followed by energy
minimization with MMFF94 force field gradientof0.05 kcal/molÅ
in MOE. Initially, by GOLD software, minimized structure of tyrosi-
nase protein (PDB code 1WX2) was prepared by correcting proton-
ation state (hydrogen atoms were added) and all water molecules
were deleted. Copper ions play an important role for compound
placement in active site [32]. After preparation of tyrosinase protein
structure, active site containing Cu2+ ions metals were marked for
docking as active site, where the compounds can dock. Docking
was performed by keeping default parameters in which Lamarkican
genetic algorithm was integrated. It provided full ligand flexibility
in docking. One scoring function i.e. Gold score, was used as fitness
score to quantify ligand binding interaction.
fer solution (0.05 M, pH 6.8) was added and preincubated for
10 min at 25 °C. Then 0.05 mL of an aqueous solution of mushroom
tyrosinase was added in the last mixture. The activity was
expressed as the sample concentration that gave a 50% inhibition
in the enzyme activity (IC50). For the measurement of diphenolase
inhibitory activity, 0.1 mL of a sample solution and 1.5 mL of 1.5 M,
L-DOPA solution (0.1 M phosphate buffer, pH 6.8) were mixed with
0.4 mL H2O, and preincubated at 25 °C for 5 min. Then, 0.5 mL of
tyrosinase solution (125 U) was added and the reaction was mon-
itored by using a UV microplate reader (SpectraMax, Plus 384,
Molecular Devices, Inc., USA). The amount of dopachrome pro-
duced was monitored at 475 nm using UV microplate reader. For
the measurement of monophenolase inhibitory activity, 0.1 mL of
a sample solution and 1 mL of 2 M L-tyrosine solution were mixed
with 0.9 mL of 0.1 M phosphate buffer (pH 6.8) and pre-incubated
at 25 °C for 5 min. Then, 0.5 mL of tyrosinase solution (250 U) was
added and the reaction was monitored at 475 nm for 20 min for
detection of dopachrome formed. Kojic acid was used as positive
control. Triplicate absorbance readings of control and test samples
were averaged. The inhibition percentage of each sample was then
calculated by using the following formula.
Tyrosinase inhibition ð%Þ ¼ ðAcontrolÞ ꢂ ðAsampleÞ=ðAcontrolÞ:
Statistical analysis
Statistical analysis were performed by using one-way analysis
of variance (ANOVA), followed by Dunnett test post hoc for
multi-group comparison test using GraphPadPrsim software ver-
sion 5.0. Statistical significance of differences between groups
was accepted at P < 0.05.
General procedure for the synthesis of chalcones 1–10
To a stirred solution of appropriate acetophenone (1 equiv),
substituted benzaldehyde (1 equiv) in ethanol, KOH (20% w/v
aqueous solution) were added. The mixture was stirred at room
temperature for 48–72 h. The reaction mixture was cooled to
0 °C (ice-water bath) and acidified with HCl (10% v/v aqueous solu-
tion). In most cases, yellow precipitate were formed, filtered and
washed with 10% aqueous HCl solution. The mixture was extracted
Docking protocol
In order to gain more insight at molecular level about binding
mode of tyrosinase enzyme, computational studies involving

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M.N. Akhtar et al. / Journal of Molecular Structure 1085 (2015) 97–103
with ethyl acetate, the extracts were dried over Na2SO4 anhydrous
and the solvent was evaporated to obtain final product as in solid
or crystalline form hot MeOH. The crude products were purified
with column chromatography by elution with ethyl acetate and
hexane.
(d, J = 15.6 Hz, 1H, Ha), 7.76–7.90 (m, 4H, C6H4), 8.05 (d,
J = 15.6 Hz, 1H, Hb), MS m/z: 275 (M + 1)+. Anal. for C15H11ClO3.
(E)-1-(20,40-Dihydroxy-phenyl)-3-(2,3-dimethoxyphenyl)-propenone
(9)
Yield 82.4 %. Yellow flat crystals: mp 184–186 °C. IR (KBr): 1652
(E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-phenylprop-2-en-1-one
(1)
(C@O), 2987 (Ar CAH str.), 1622, 1378, 1590 (C@C ring) cmꢂ1 1H
.
NMR (CDCl3, 500 MHz): d 12.4 (s, 1H, OH), 8.15 (d,1H, J = 15.5 Hz,
Hb), 7.95 (br,d,1H, J = 9.0 Hz, H-60), 7.81 (d,1H, J = 15.5 Hz, H
a),
Yield 82.4%. Yellow needles crystals: m.p. 102–104 °C. IR (KBr):
3060 (Ar CAH), 1642 (C@O), 1589, 1588, 1478, (C@C ring), 1202
6.41 (d, J = 2.5, 1H, H-30), 7.12 (t, J = 8.0 Hz, each H- 5), 7.44 (d,
J = 8.0 Hz, H-6), 7.15 (dd, J = 8.0 each, H-4), 6.30 (d, J = 2.5, 1H,
H-50), 3.88 (s, 3H, OMe, C-2), 3.87 (s, 3H, OMe, C-3). EI-MS m/z
284.23, (Molcular formula C17H16O4).
(CAO) cmꢂ1 1H NMR (CDCl3, 500 MHz): d 14.3 (s, 1H, OH), 7.92
.
(d,1H, J = 15.0 Hz, Hb), 7.80 (d,1H, J = 15.0 Hz, Ha), 7.64 (brd, 2H,
H-2, 6), 7.48 (m, 3H, H-3, 4, 5), 6.98 (br,s, 1H, H-5’), 6.21 (br,s,
1H, H-3’), 3.93 (s, 3H, OMe, C-60), 3.85 (s, 3H, OMe, C-40). 13C
NMR (CDCl3, 125 MHz): d193.2 (C@O), 168.9 (C20), 166.7 (C40),
162.5 (C60), 142.51 (Cb), 141.0 (C4), 132.9 (C1), 130.2 (C2, C6),
(2E)-3-[4-(Dimethylamino)phenyl]-1-(20,40,60-trimethoxy-phenyl)-2-
propen-1-one (8)
128.1 (C3,C5), 126.6 (C
55.53 (OCH3), 55.7 (OCH3). EI-MS m/z 284.23, (Molcular formula
17H16O4).
a
), 108.14 (C10), 93.6 (C30), 90.94 (C50),
Yield 73%. Yellowsolid, mp = 149–152 °C. IR (KBr): 3103, 2937,
2839, 1648, 1622, 1529, 1463, 1452, 1413, 1184, 968, 945, 815
C
(Ar) cmꢂ1 1H NMR (CDCl3): d 7.41 (d, 2H, H2, H6), 7.27 (d, 1H,
.
J = 15.0 Hz, Hb), 6.78 (d, 1H, J = 15.0 Hz, Ha), 6.65 (d, 2H, H3, H5),
(E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(4-methoxyphenylprop-
2-en-1-one (2)
6.16 (s, 2H, H30, H50), 3.86 (s, 3H, OCH3), 3.76 (s, 6H, OCH3), 3.01
13
(6H, NA(CH3)2), C NMR (CDCl3): d 194.95 (C@O), 162.19 (C40),
Yield 82.4%. Yellow needles crystals. mp 110–112 °C. IR (KBr):
1654 (C@O), 3200 (Ar CAH str.), 1590, 1589, 1476, (C@C), 1202
158.80 (C20, C60), 152.05 (C4), 146.20 (Cb), 130.47 (C2, C6),
124.67 (C1), 122.84 (Ca
), 112.49 (C10), 110.99 (C3, C5), 90.90
(CAO str.), 850 cmꢂ1 1H NMR (CDCl3, 500 MHz): d 13.6 (s, 1H,
.
(C30, C50), 56.15 (p0-OCH3), 55.70 (p0-OCH3), 41.09 (NA(CH3)2.
OH), 8.02 (d,1H, J = 15.5 Hz, Hb), 7.81 (d,1H, J = 15.5 Hz, Ha), 7.73
(brd, 2H, H-2, 6), 7.49 (m, 2H, H-3, 5), 6.87 (br,s, 1H, H-50), 6.02
(br,s, 1H, H-30), 3.93 (s, 3H, OMe, C-60), 3.85 (s, 3H, OMe, C-40),
3.82 (s, 3H, OMe, C-4). 13C NMR (CDCl3, 125 MHz): d 192.6
(C@O), 168.7 (C20), 166.2 (C40), 163.1 (C60), 160.9 (C4), 141.6 (Cb),
Table 2
Crystal data and structure refinement for compound 9.
Empirical formula
C17H16O5
130.1(C2, C6), 127.5 (C1), 125.4 (Ca
), 114.3 (C3, C5), 93.56 (C30),
Formula weight
300.30
91.4 (C50), 55.79 (OCH3), 55.53 (OCH3), 55.35 (OCH3). EI-MS m/z
Temperature
100(2) K
Wavelength
0.71073 A
298.33, (Molcular formula C18H18O5).
Crystal system, space group
Unit cell dimensions
Orthorhombic, Pna2(1)
a = 18.982(2) Å alpha = 90°
b = 9.9786(12) Å beta = 90°
c = 7.6015(9) Å gamma = 90°
1439.9(3) Å3
(E)-3-(4-Dimethylamino)phenyl)-1-(40-hydroxyphenyl)prop-2-en-1-
one (3)
Volume
Yield: 66%. mp: 184–186 °C. IR (KBr) cmꢂ1: 3384 (OH), 1658
(C@O str.), 3081 (Ar CAH), 1590, 1482, 1434 (C@C), 1222 (CAO
Z, Calculated density
Absorption coefficient
Crystal size
Theta range for data collection
Reflections collected/unique
Completeness to theta = 28.25
Max. and min. transmission
Refinement method
4, 1.385 Mg/m3
0.102 mmꢂ1
str.), 748 cmꢂ1 1H NMR (CDCl3, 500 MHz): d 8.13–7.86 (m, 4H,
.
0.20 ꢃ 0.10 ꢃ 0.08 mm
2.15–28.25°
C6H4ArAH), 7.82 (d, J = 15.6 Hz, 1H, Hb), 7.35 (d, J = 15.6 Hz, 1H,
16141/3549 [R(int) = 0.0383]
99.8 %
0.9919 and 0.9798
Full-matrix least-squares on F2
3549 / 1 / 199
Ha), 6.87–6.93 (m, 4H, C6H4), 2.99 (s, 6H, 4-N(CH3)2), MS m/z:
268 (M + 1)+. Anal. For C17H17NO2.
(E)-3-(4-Chlorophenyl)-1-(20,40-dihydroxyphenyl)prop-2-en-1-one
(5)
Data/restraints/parameters
Goodness-of-fit on F2
1.044
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
r
(I)]
R1 = 0.0326, wR2 = 0.0753
R1 = 0.0360, wR2 = 0.0774
0.236 and ꢂ0.183 e. Åꢂ3
Yield: 65.4%; mp: 150–154 °C. IR (KBr): 3302 (OH), 1644 (C@O),
3042 (Ar CAH), 1592, 1582, 1440 (C@C), 1225 (CAO) cmꢂ1 1H
:
NMR (CDCl3): d 12.7 (s, 1H, OH), 6.49–6.51 (m, 3H, C6H3), 7.34
Fig. 2. The ORTEP diagram of compound 9.

M.N. Akhtar et al. / Journal of Molecular Structure 1085 (2015) 97–103
101
melanin is a serious problem for human skin. Naturally occurring
compounds flavonoids, stilbenes and resorcinols have been known
as potential tyrosinase inhibitors [19]. Chalcones 1–10 were pre-
pared via Claisen–Schmidt condensation reaction by using our
designed compounds 20 or 20,40-dihydroxy acetophenone with
Results and discussion
Melanin an important pigment is widely distributed in bacteria,
fungi, plants and animals, which play a vital protective role against
skin photocarcinogenesis, however the abnormal production of
Compound 3
Compound 4
Compound 6
Ring
A
Ring
B
Ring
A
Ring
B
Ring
B
Ring
A
Compound 9
Compound 5
Compound 7
Ring
A
Ring
B
Ring
B
Ring
A
Ring
B
Ring
A
Compound 2
Compound 1
Kojic acid 11
Ring
B
Ring
A
Ring
B
Ring
A
Compound 10
Ring
B
Ring
A
Fig. 3. 2D pictorial diagram showing worthy interactions of active binding site of tyrosinase enzyme (PDB code 1WX2) with all compounds.

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M.N. Akhtar et al. / Journal of Molecular Structure 1085 (2015) 97–103
Fig. 4. Dock pose of all compounds in the active site of tyrosinase enzyme (PDB code 1WX2).
corresponding benzaldehyde in methanol. The compounds were
characterized by detailed spectroscopic techniques and evaluation
for tyrosinase inhibitions studies [20] (Fig. 1, Table 1).
Chalcones (1–10) were synthesized via a Claisen–Schmidt con-
densation reaction. A substituted methyl ketone and respective
aldehydes were dissolved in methanol in addition of KOH pallets
as discussed in experimental section. The synthetic compounds
were characterized with help of IR, UV, EI-MS, 1H NMR, 13C NMR
and single X-ray crystallographic techniques. The IR spectra of
(11) (positive control) with IC50 value at 12.01 0.11 lM (Table 1).
Among them, compounds 1–3 and 9 were found to be the most
active as tyrosinase inhibitors, which showed the 90–97% inhibi-
tion comparable with standard kojic acid (11). Judging from the
structures of 1–3 the compound 3 possessing 4-hydroxyl substi-
tuted group was found to be most active tyrosinase inhibitor pos-
sibly showed strong chelation with binuclear copper containing
tyrosinase enzymes. While compounds flavokawain B (1) and fla-
vokawain A (2) might be coupled or chelated with the binuclear
copper active site. It is well established that substituted resorcinol
moiety act as potential inhibitor of tyrosinase [19] and position of
hydroxy group attached to the ring A is important for tyrosinase
inhibition [10,21]. Tyrosinase is use mono-, di-, and trihydroxyphe-
nols as substrates as it is evident in literature that the monohydr-
chalcones showed
a peak at 1640–1648 (C@O), 1560–1588
(C@CA@O), 1460–1478 (C@C, Ar) and 1200–1000 (CAO) cmꢂ1
.
The 1H NMR spectra of synthetic chalcones appeared at d 12.0–
14.4 (1, 2, 4, 5, 6, 7, 9, 10) were due the -chelated OH with carbonyl
group. Two characteristics protons appeared at d 8.0–7.89 (d,
J = 15.5 Hz) and 7.8–7.5 (d, J = 15.5) were due the b and
a
(E) dou-
oxyphenols (p-cresol and tyrosine), dihydroxyphenols (catechol, L-
ble bond protons, respectively. The aromatic protons appeared at d
8.0–6.0 of both ring A & B in the 1H NMR spectroscopy. All data of
compounds were compared with the previously published data of
chalcones [22,23]. While the structure of compound 9 was con-
firmed by single X-ray crystallography as shown in Fig. 2 and data
presented in Table 2.
The chalcone derivatives 1–10 and kojic acid (11) (positive con-
trol) were examined for their inhibitory activity in vitro [21]. The
finding results of compounds 1–3 exhibited significant percentage
inhibition of tyrosinase with 90.61 0.42–97.36 0.36% as shown
dopa, catechin, and chlorogenic acid), and trihydroxyphenols
(pyrogallol), indicating the potential use of monohydroxyphenols
[22]. However, in our findings position of hydroxyl group at 40
(compound 3) is slightly more selective than 20-hydroxyl position
(compound 2, 3, 4, 9) possibly electronically less crowded and che-
lated strongly with copper ions for tyrosinase inhibition [23–25].
These results showed that presence of hydroxy groups at 20 or 20,
40 positions may also contributed significantly for tyrosinase
inhibition.
in Table 1. Compound
97.36 0.36% with IC50 = 14.20 012
with kojic acid (11) IC50 value of 12.01 0.11
derivatives, 4-7 and 9 exhibited a significant inhibition of tyrosi-
nase with IC50 values 15.61 0.31–14.23 0.41 M. However, the
compounds 8 and 10 showed a lower inhibition (Table 1). Com-
pounds 1-7 and 9 were further evaluated for their inhibition by
oxidative hydroxylation of diphenolase activity. Compound 1, 2,
3 and 9 were identified as potent tyrosinase inhibitors based on
their low IC50 values 14.38 0.12, 14.26 0.08, 14.20 0.03 and
3
showed potential inhibition by
M, which is comparable
M. Other chalcones
Docking analysis and SARs
l
l
Docking results showed that most of the compounds establish
ionic bridge between His 305 on enzymes and carbonyl oxygen
atom of the ligands particularly with compounds 3, 4, 5, 9 and kojic
acid. Additionally, these compounds forms bond with copper ion
exhibited batter IC50 value. Generally, all compounds establish
one polar contact with either one of the amino acid including
Glu 49, Tyr 295, Gln 303, Val 306 and Arg 308 by involving back-
bone and side chain atoms as acceptor. Fig. 3 summarizes all the
interactions of each compound in the binding site of tyrosinase
enzyme (PDB code 1WX2). Compound 3 contains a hydroxyl group
l
14.23 0.41
lM, respectively (Table 1). These compounds were
promising and comparable with the inhibitory effect of kojic acid

M.N. Akhtar et al. / Journal of Molecular Structure 1085 (2015) 97–103
103
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Natural tyrosinase inhibitors could be better sources for depig-
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activity relationship of these chalcones strongly supported the
finding that 4-hydroxy chalcone or resorcinols could be potential
tyrosinase inhibitors and added a new compounds for depigmenta-
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Acknowledgment
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This project was supported by the Universiti Malaysia Pahang
(Grant Nos. RDU 120373 and RDU 120389).
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