Kinetic and in silico studies of novel hydroxy-based thymol analogues as inhibitors of mushroom tyrosinase

Article abstract of DOI:10.1016/j.ejmech.2015.05.031

The present studies reports the synthesis of hydoxylated thymol analogues (4a-e) and (6a-c) as mushroom tyrosinase inhibitors. The title compounds were obtained in good yield and characterized by FTIR, ¹H NMR, ¹³C NMR, Mass spectral

Full text of DOI:10.1016/j.ejmech.2015.05.031

European Journal of Medicinal Chemistry 98 (2015) 203e211
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Kinetic and in silico studies of novel hydroxy-based thymol analogues
as inhibitors of mushroom tyrosinase
,
,
Zaman Ashraf a b, Muhammad Rafiq a, Sung-Yum Seo a *, Kang Sung Kwon c,
Mustafeez Mujtaba Babar d, Najam-us-Sahar Sadaf Zaidi c
a Department of Biology, College of Natural Sciences, Kongju National University, Gongju 314-701, Republic of Korea
b Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
c Department of Chemistry, Chungnam National University Daejeon, 305-764, Republic of Korea
d Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12, Kashmir Highway, Islamabad, 44000, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The present studies reports the synthesis of hydoxylated thymol analogues (4aee) and (6aec) as
mushroom tyrosinase inhibitors. The title compounds were obtained in good yield and characterized by
FTIR, 1H NMR, 13C NMR, Mass spectral data and X-ray crystallography in case of compound (6a). The
inhibitory effects on mushroom tyrosinase and DPPH were evaluated and it was observed that 2-[5-
methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4-hydroxyphenyl)prop-2-enoate (6b) showed
Received 19 March 2015
Received in revised form
30 April 2015
Accepted 20 May 2015
Available online 21 May 2015
tyrosinase inhibitory activity (IC50 15.20
2-(propan-2-yl)phenoxy]-2-oxoethyl 3,4-dihydroxybenzoate (4d) exhibited higher antioxidant potential
(IC50 11.30 M) than standard ascorbic acid (IC50 24.20 M). The docking studies of synthesized thymol
mM) comparable to kojic acid (IC50 16.69 mM) while 2-[5-methyl-
Keywords:
Thymol analogues
Synthesis
Antioxidant activity
Mushroom tyrosinase inhibitors
Kinetic mechanism
Molecular docking
m
m
analogues was also performed against tyrosinase protein (PDBID 2ZMX) to compare the binding affinities
with IC50 values. The predicted binding affinities are in good agreement with the IC50 values as com-
pound (6b) showed highest binding affinity ꢀ7.1 kcal/mol. The kinetic mechanism analyzed by Line-
weavereBurk plots exhibited that compound (4d) and (6b) inhibit the enzyme by two different pathways
displayed mixed-type inhibition. The inhibition constants Ki calculated from Dixon plots for compounds
(4d) and (6b) are 34 mM and 25 mM respectively. It was also found from kinetic analysis that derivative
(6b) formed reversible enzyme inhibitor complex. It is propose on the basis of our investigation that title
compound (6b) may serve as lead structure for the design of more potent tyrosinase inhibitors.
© 2015 Published by Elsevier Masson SAS.
1. Introduction
as observed in melasma, freckles, lentigo senilis, and other forms of
melanin hyperpigmentation can be a serious aesthetic problem
Tyrosinase (EC 1.14.18.1) as a binuclear copper-containing met-
alloenzyme catalyzes two distinct reactions of melanin biosyn-
often causing emotional disturbance [5e7]. Taking into account the
key role of tyrosinase in melanin production, many tyrosinase in-
hibitors have found application in cosmetics and pharmaceutical
products [8e10]. A large number of tyrosinase inhibitors have been
reported but only a few are used because of their limitations with
regards to cytotoxicity, selectivity and stability. Thus, it is in great
need of developing new tyrosinase inhibitors without causing
adverse reactions.
thesis. It catalyzes
L
-tyrosine to L-3,4-dihydroxyphenylalanine (
L-
DOPA) and further oxidation of (
L-DOPA) to dopaquinone which
then transformed through several reactions into brown to black
melanin. The distribution patterns of melanin in the surrounding
keratinocytes determine the color of human skin [1,2]. Kadekaro
et al. and Petit et al. also reported that there are certain other fac-
tors such as UV exposure,
melanocortin 1 receptor and agouti-related protein which involved
in melanin biosynthesis [3,4]. Abnormal melanin production such
a
-melanocyte-stimulating hormone,
A number of cinnamic acid and benzoic acid analogues pos-
sessing hydroxy groups at position 3 and 4 of the phenyl ring have
been reported as antioxidants and mushroom tyrosinase inhibitors
[11,12]. Liu (2003) and Chen (2005) also reported that alkoxy
benzoic acids and hydroxy benzoic acids showed potent mushroom
tyrosinase inhibitory activity [13,14]. Thymol a naturally occurring
monoterpene phenol which is the main constituent of thyme
* Corresponding author. .
E-mail address: dnalove@kongju.ac.kr (S.-Y. Seo).
http://dx.doi.org/10.1016/j.ejmech.2015.05.031
0223-5234/© 2015 Published by Elsevier Masson SAS.

204
Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
possesses tyrosinase inhibitory activity [15] along with other
numerous pharmacological activities. As phenolic antioxidants,
thymol protects food qualities and organisms from damage induced
by oxidative stress. Thymol is used as meat preservatives or
flavoring agent in the food industry. It exhibited anti-inflammatory
effect in human neutrophils incubated [16] and also inhibits the
formation of lethal products through reactive nitrogen species [17].
Thymol displays antimicrobial [18e20] and wound-healing activity
[21] and is able to rise the levels of macrophage migration inhibi-
tory factor (MIF) in central nervous system [22]. Thymol increased
the in vitro fibroblast growth [23], effectively inhibited COX-1 [24]
and prevented inducible lymphocyte proliferation [25].
Keeping in view the importance of these structural features we
have synthesized the hydroxylated thymol analogues having ben-
zoic acid and cinnamic acid moieties in order to discover their
tyrosinase inhibitory potential to offer a source for the develop-
ment of new effective tyrosinase inhibitors. The antioxidant activity
was also carried out as most of the clinically used tyrosinase in-
hibitors kojic acid, arbutin, kaempferol, hydroquinone, etc. all
possess antioxidant activity. In addition to evaluate the tyrosinase
inhibitory potential and antioxidant activity of synthesized hy-
droxylated thymol derivatives molecular docking was carried out to
predict the position of the synthesized compounds in the active site
of the 3D structure of tyrosinase (PDB ID 2ZMX). The investigation
of the binding interactions during docking analysis between ligand-
protein functionalities is important to elucidate the possible mo-
lecular mechanism [26,27].
nucleophilic substitution at intermediate (2) with hydroxy
substituted benzoic acids (3aee) and substituted and unsubstituted
cinnamic acids (5aec) respectively. All of the synthesized com-
pounds have been characterized by FTIR, 1HNMR, 13CNMR and Mass
spectroscopic data. Compound (6a) yielded single crystals, suitable
for X-ray diffraction studies. The X-ray diffraction analysis also
confirmed the formation of the desired product and this is an
ambiguous evidence of the structure.
2.2. Bioassay for tyrosinase inhibitory activity
Hydroxylated thymol analogues have been designed to evaluate
their inhibitory effects on mushroom tyrosinase activity. Kojic acid
a competitive tyrosinase inhibitor was used as standard for com-
parison purpose. The hydroxylated tyrosinase inhibitors as
described in preceding section are of special interest because of
their high activity (IC50 < 10 mM). Novel thymol analogues (4aee)
and (6aec) have been synthesized by incorporation of hydroxyl-
ated benzoic acids and cinnamic acids. The synthesis of mono and
di-hydroxylated derivatives with different position of eOH at
phenyl ring was carried out to explore the role of multiple hydroxyl
groups in tyrosinase inhibition. It has been exposed from our
bioassay results (Table 1) that the major determining factor of
inhibitory activity is the position and not the number of the hy-
droxyl groups. Interestingly, compound (4d) bearing 3,4-dihydroxy
substituted benzoic acid moiety showed higher activity (IC50
45.0
mM) than (4c) and (4e) having IC50 56.1 and 220.9 mM
respectively. The derivatives (4c) and (4e) possess 2,4- and 3,5-
dihydroxy substituted benzoic acid residues respectively. In case
of compound (4d) two hydroxy groups are present on adjacent
positions of phenyl ring; this impedes the molecule to interact well
2. Results and discussion
2.1. Chemistry
with enzyme. This structural feature can be well correlated with L-
The title compounds (4aee) and (6aec) were synthesized by
following the already reported method [28] with some modifica-
tion shown in Schemes 1 and 2. The thymol chlroacetyl derivative
(2) was synthesized by esterification of phenolic eOH group of
thymol with chloroacetyl chloride in the presence of (C2H5)3N and
anhydrous methylene chloride as solvent. The presence of ester
carbonyl stretching at 1723 cmꢀ1 and disappearance of the eOH
stretching in FTIR spectra confirmed the formation intermediate
(2). The final products (4aee) and (6aec) were prepared by simple
DOPA which is used as substrate for tyrosinase enzyme during
bioassay. Thus compound (4d) because of close structural similar-
ities with L-DOPA is more active among the dihydroxylated thymol
analogues. Table 1 presented the IC50 values of the synthesized
thymol analogues and it was observed that kojic acid is more active
than all of the synthesized thymol analogues except compound
(6b). Compound (6b) showed excellent tyrosinase inhibitory ac-
tivity with IC50 15.2
mM and is so inhibitor as kojic acid (IC50
16.69
mM). The synthesized thymol derivatives (6a), (6b) and (6c)
CH3
CH3
O
(C2H5)3N/CH2Cl2
0 to -5°C
O
+
HO
H3C
O
Cl
Cl
Cl
CH3
H3C
CH3
(1)
(2)
CH3
O
CH3
OH
O
+
(C2H5)3N/KI
DMF
O
O
O
Cl
R
O
R
H3C
CH3
O
(3a-3e)
H3C
CH3
(2)
R=
(4a-4e)
R is same as in (3a-3e)
3a = 3-OH
3b = 4-OH
3c = 2,4-di-OH 3d = 3,4-di-OH
3e = 3,5-di-OH
Scheme 1. Synthesis of thymol analogues (4aee).

Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
205
Table 1
Tyrosinase inhibitory activity and free radical scavenging activity of the synthesized thymol analogues (4aee) and (6aec).
Codes
Tyrosinase inhibition Activity
% Inhibition (25 g/mL)
DPPH Activity
m
IC50 SEM
m
M
% Inhibition (500
m
g/mL)
IC50 SEM mM
a
a
a
4a
4b
4c
4d
4e
6a
6b
6c
48
33
68
55
5
42
93
77
1
2
2
3
79.3 5.3
91.5 9.4
56.1 5.9
45.0 1.5
220.9 11.6
100.8 14.3
15.2 0.42
45.2 4.3
3
2
1
1
1
3
a
11.30 2.09
a
2
9
8
5
2
3
2
a
a
3
4
2
50
6
1
1344.0 18.76
a
a
Kojic acid
Ascorbic acid
100
16.69 2.8
a
a
97
24.20 1.93
a
Not determined.
all possess cinnamic acid moiety but varied in substitution pattern
on phenyl ring. The derivative (6a) have unsubstituted cinnamic
acid residue while compound (6b) and (6c) possess 4-hydroxyl and
4-chloro cinnamic acid moiety respectively. Interestingly, presence
of 4-hydroxyl group on cinnamic acid phenyl ring in compound
(6b) led to a dramatic increase in tyrosinase inhibitory activity. The
concentrations of compounds. This behavior of compounds (4d)
and (6b) indicated that it inhibits tyrosinase by two different
pathways and show mixed type inhibition. On the other hand in
Dixon plot, slope obtained from the plots for uninhibited enzyme
and with different concentrations of inhibitors (4d) and (6b) was
consistent with the characteristic patterns of mixed-type inhibition
compound (6a) (IC50 100.8
mM) is 6.6 times less active while (6c)
with Ki value 34 mM and 25 mM as shown in Figs. 1e2(b)
(IC50 45.2 M) is 2.97 times less active than the most potent de-
m
respectively.
rivative (6b). Based upon our results we propose that thymol
analogue (6b) may serve as a lead structure for the design of more
potent tyrosinase inhibitors.
2.4. Reversible inhibition
The most potent derivative (6b) was selected to ascertain the
formation of reversible enzyme-Inhibitor complex between tyros-
inase and inhibitor (6b). The plots of initial rate verses enzyme
2.3. Kinetic mechanism
concentrations (4, 6, 8, 10, 15 and 20 mg/mL) for compound (6b) all
The inhibitory mechanism of the most active compounds (6b)
consisted of straight lines passing through the origin. Increasing the
inhibitor concentration resulted in a decrease in the slope of the
lines, which indicate that the enzyme undergoes a reversible in-
hibition [30] (Fig. 3).
and (4d) on mushroom tyrosinase for the oxidation of L-DOPA was
studied. Kinetic studies showed a concentration dependent inhi-
bition of mushroom tyrosinase by thymol derivatives (4d) and (6b).
Continuous monitoring of the reaction showed a marked decrease
in reaction rate in the presence of the inhibitors, which is ulti-
mately, indicated the decrease in the final absorbance when
compared with controls containing no inhibitor. The potency of
inhibition exhibited by these compounds varied depending on the
presence of type and position of the functionalities on the aromatic
rings [29]. Inhibition kinetics was analyzed by Lineweaver-Burk
plot and Dixon plots to determine the type of inhibition and inhi-
bition constant (Ki). The results of Figs. 1e2(a) showed that in case
of compounds (4d) and (6b) by increasing the concentration of
2.5. DPPH radical scavenging assay
Thymol derivatives were evaluated for the DPPH free radical
scavenging ability as excessive melanogenesis generates free radi-
cals which start unwanted reactions. The thymol derivative (4d)
exhibited excellent radical scavenging activity when compared
with the standard ascorbic acid. The IC50 value of compound (4d) is
11.30 mM while IC50 value of ascorbic acid is 24.20 mM. The presence
substrate (
L
-DOPA) gave family of straight lines, which intersected
of ortho disubstituted phenyl ring in compound (4d) play signifi-
cant role in radical scavenging activity as it showed more potent
radical scavenging activity than ascorbic acid. All of the other
within the second quadrant. The analysis showed that Vmax
decreased with changing Km in the presence of increasing
Fig. 1. a) Lineweaver-Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (4d) in the presence of different
concentrations of L-DOPA. b) Dixon plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (4d) in the presence of
different concentrations of L-DOPA (0.062, 0.125, 0.25, 0.5 and 1 mM, respectively).

206
Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
Fig. 2. a) Lineweaver-Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (6b) in the presence of different
concentrations of L-DOPA. b) Dixon plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations of compound (6b) in the presence of
different concentrations of L-DOPA (0.062, 0.125, 0.25, 0.5 and 1 mM, respectively).
the stability of ligandetarget complex. There is hydrogen bond
between 4-hydroxy group of derivative (4c) and amino acid THY37
of the target protein with bond length 1.84 Å (Fig. 6). The binding
energy of thymol analogue (4c) is ꢀ6.5 kcal/mo. The presence of
phenolic hydroxyl on acid moiety in the synthesized compounds is
the determining factor of binding energies. Another important
structural feature is the cinnamic acid moiety. It was observed that
when phenolic hydroxyl is present on para position of the cinnamic
acid residue then ligand has maximum binding affinity with the
receptor protein. The experimentally determined IC50 values also
confirmed the docking results as compound (6b) showed the
maximum inhibition of the mushroom tyrosinase activity. The
partition coefficient values (log P) of the synthesized compounds
were calculated by ChemSketch ACD labs 2012 to verify the drug-
likeness properties of the compounds. The most potent de-
rivatives (6b) and (4d) have partition coefficient values 4.92 0.33
and 4.97 0.32 respectively which are not beyond the limits of (log
P) values.
Fig. 3. Effects of concentrations of mushroom tyrosinase on its activity for the catalysis
of L-DOPA at different concentrations of compound (6b).
3. Conclusion
thymol derivatives showed little activity even at high concentration
(500 mg/mL) Table 1.
The hydroxylated thymol analogues (4aee) and (6aec) having
substituted benzoic acids and cinnamic acids moieties have been
synthesized and fully characterized. The computational studies
investigation revealed that thymol analogue (6b) possesses
maximum binding affinity (ꢀ7.1 kcal/mol) with the target protein
(PDBID 2ZMX). The wet lab results are in good agreement with the
calculated docking scores. The most potent activity against mush-
room tyrosinase was exhibited by the compound (6b) having IC50
2.6. Docking studies
The in silico docking of the synthesized thymol derivatives was
performed against crystal structure of tyrosinase protein (PDBID
2ZMX) to compare their experimental IC50 with binding affinities.
Matoba et al. published recently the crystallographic structure of
tyrosinase (PDBID 2ZMX) which enables us to predict the binding
affinities of thymol derivatives. The compound (6b) showed highest
binding affinity with the target protein having binding energy
value ꢀ7.1 kcal/mol. The experimentally determined tyrosinase
inhibitory activity results are in good agreement with the predicted
binding affinities by in silico docking. The 4ehydroxy group of
cinnamic acid phenyl ring in compound (6b) formed hydrogen
15.2 mM. The presence of phenolic eOH at para-position on cin-
namic acid phenyl ring play very significant role in tyrosinase in-
hibition activity. The kinetic analysis of compound (6b) shown that
it is mixed type inhibitor with Ki value 25 mM and formed reversible
drugereceptor complex with mushroom tyrosinase. It was
concluded from our results that the title compound (6b) may serve
as lead structure for the design of more potent tyrosinase
inhibitors.
bonds with GLU67 having distance 1.99 Å. The
present in the same compound between phenyl ring of thymol
moiety and PHE34 having distance 3.81 Å (Fig. 4).
p-p stacking also
The phenolic hydroxyls present at positions 3 and 4 in com-
pound (4d) formed hydrogen bonds with SER96 having bond
lengths 2.36 Å and 2.89 Å respectively (Fig. 5) The hydrogen of 3-
hydroxyl in the same compound also interact with VAL95 having
distance 2.40 Å between two interacting groups. The ester carbonyl
oxygen in compound (4d) also formed hydrogen bond with ARG140
having bond length 2.40 Å. Table 2 presented the calculated bind-
ing energies of the synthesized thymol analogues and it determines
4. Experimental
All chemicals used for the synthesis of compounds were pur-
chased from Sigma Chemical Co. Melting points were determined
using a Digimelt MPA 160, USA melting point apparatus and are
reported uncorrected. The FTIR spectra were recorded with Shi-
madzu FTIRe8400S spectrometer (Kyoto, Japan,
spectra were performed on an Agilent 6460 Series Triple
y
, cmꢀ1). Mass

Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
207
Fig. 4. The interactions of compound (6b) with the active site of mushroom tyrosinase (PDBID 2ZMX) generated by using Discovery Studio 4.1.0. The a) shows the two dimensional
interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The b) shows the three-dimensional
docking of the compound in the binding pocket. Dashed lines indicate bond distances between interacting functionalities of the ligand and receptor.
Fig. 5. The interactions of compound (4d) with the active site of mushroom tyrosinase (PDBID 2ZMX) generated by using Discovery Studio 4.1.0. The a) shows the two dimensional
interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The b) shows the three-dimensional
docking of the compound in the binding pocket. Dashed lines indicate bond distances between interacting functionalities of the ligand and receptor.
was 350 ꢁC. Nitrogen was used as a desolvation gas (flow 600 L/h).
Elemental Analysis (C, H) were carried out on a Flash 2000 series
Table 2
Average binding affinity and Log P values of the synthesized thymol analogues
(4aed) and (6aec).
elenatal analyzer with TCD detector system and results are with
0.3%. The 1H NMR and 13C NMR spectra (CDCl3) and (DMSO-d6)
Compound
Average binding affinity (kcal/mol)
Log pa
were recorded using a Bruker 400 MHz spectrometer. Chemical
shifts (d) are reported in ppm downfield from the internal standard
tetramethylsilane (TMS). The purity of the compounds was checked
by thin layer chromatography (TLC) on silica gel plate using n-
hexane and ethyl acetate as mobile phase. The procedure for the
synthesis of the desired compounds is depicted in Schemes 1 and 2.
4a
4b
4c
4d
4e
6a
6b
6c
5.8
6.4
6.5
6.6
6.5
5.6
7.1
5.7
5.17 0.30
5.14 0.30
5.20 0.33
4.97 0.32
4.88 0.32
5.45 0.30
4.92 0.33
5.98 0.33
a
Log p calculated from ChemSketch ACD labs 2012.
4.1. Reagents
Quadrupole instrument (Agilent). The ionization was achieved by
electrospray ionization in the positive ion mode (ESIþ) and nega-
tive ion mode (ESI-). The capillary voltage was set to 4.0 kV. The
source temperature was 120 ꢁC, and the desolvation temperature
Mushroom tyrosinase was purchased from Sigma (USA); L-DOPA
and thymol were purchased from Sigma (USA). Stock solutions of
the reducing substrates were prepared in phosphate buffer (20 mM,
pH 6.8).

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Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
Fig. 6. The interactions of compound (4c) with the active site of mushroom tyrosinase (PDBID 2ZMX) generated by using Discovery Studio 4.1.0. The a) shows the two dimensional
interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein. The b) shows the three-dimensional
docking of the compound in the binding pocket. Dashed lines indicate bond distances between interacting functionalities of the ligand and receptor.
CH3
O
CH3
OH
R
O
O
+
(C2H5)3N/KI
DMF
R
O
O
Cl
O
(5a-5c)
H3C
CH3
O
(2)
H3C
CH3
R=
5a = -H
5c = -Cl
(6a-6c)
5b = -OH
R is same as in (5a-5c)
Scheme 2. Synthesis of thymol analogues (6aec).
4.1.1. Synthesis of thymol chloroacetyl derivative (2)
column chromatography (n-hexane: ethyl acetate 4:1). The same
procedure was used for the preparation of compounds (6aec)
Scheme 2.
A mixture of thymol (1) (0.01 mol), triethylamine (0.01 mol) in
anhydrous dichloromethane (25 mL) was cooled in an ice salt
mixture to 0 to ꢀ5 ꢁC. To this reaction mixture chloroacetyl chloride
(0.01mol) in dry dichloromethane was added drop wise with
constant stirring over a period of 1 h maintaining the temperature
constant. The reaction mixture was then stirred at room tempera-
ture for further 5 h, washed with 5% HCl, and 5% sodium hydroxide
solution. The organic layer was washed with saturated aqueous
NaCl, dried over anhydrous magnesium sulfate, filtered and solvent
was removed under reduced pressure. The crude product was pu-
rified by silica gel column to afford the corresponding thymol
chloroacetyl derivative (2). Colorless oil; reaction time, 6 h; yield,
84%; Rf 0.58 (n-hexane: ethyl acetate 3:1), FTIR nmax cmꢀ1: 2923
(sp2 CeH), 2843 (sp3 CeH), 1723 (C]O ester), 1599 (C]C aro-
matic), 1148 (CeO, ester).
4.1.2.1. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3-
hydroxybenzoate (4a). solid; reaction time, 24 h; yield, 82%;
melting point, 74e76 ꢁC; Rf 0.52 (n-hexane:ethyl acetate 2:1), FTIR
nmax cmꢀ1: 3079 (OeH), 3025 (sp2 CeH), 2975 (sp3 CeH),1728 (C]
O ester), 1588 (C]C aromatic), 1158 (CeO, ester); ESI-MS: m/z 351
[M þ 23] (M þ Na)þ; 1H NMR (CDCl3,
ppm): 7.72 (d, J ¼ 8.0 Hz, 1H,
d
H-6), 7.58 (s, 1H, H-2), 7.34 (dd, J ¼ 8.4, 7.6 Hz, 1H, H-5), 7.23 (d,
J ¼ 7.6 Hz, 1H, H-4), 7.08 (d, J ¼ 2.8 Hz, 1H, H-30), 7.05 (d, J ¼ 7.6 Hz,
1H, H-40), 6.90 (s, 1H, H-60), 5.13 (s, 2H, -CH2), 3.03 (sept, 1H,
J ¼ 6.8 Hz, H-100), 2.33 (s, 3H, H-300), 1.37 (s, 1H, -OH), 1.21 (d,
J ¼ 6.8 Hz, 6H, H-200); 13C NMR (CDCl3, d ppm); 166.7 (C]O ester),
165.7 (C]O, ester), 155.7 (C-3), 147.2 (C-10), 136.9 (C-20), 136.7 (C-
50), 130.3 (C-6), 129.8 (C-2), 127.5 (C-1), 126.5 (C-30), 122.4 (C-40),
122.3 (C-60), 120.7 (C-4), 116.5 (C-5), 61.2 (eCH2), 29.7 (C-100), 27.0
(C-300), 23.0 (C-200); Anal Calcd For C19H20O5: C, 69.51; H, 6.10; Found
C, 69.48; H, 6.17.
4.1.2. Synthesis of thymol analogues (4aee) and (6aec)
A mixture of thymol chloroacetyl derivative (2) (0.01 mol), hy-
droxy substituted benzoic acids (3aee) (0.01 mol), triethyl amine
(0.01 mol), potassium iodide (0.01 mol) in dimethyl formamide
(25 mL) was stirred overnight at room temperature (Scheme 1). The
reaction mixture was poured into finely crushed ice with stirring
and extracted with ethyl acetate (4 ꢂ 25 mL). The combined organic
layer was washed with 5% HCl, 5% sodium hydroxide and finally
with aqueous NaCl solution. The organic layer was dried over
anhydrous magnesium sulfate, filtered and the solvent was
removed under reduced pressure to afford the crude products
(4aee). The title compounds (4aee) were purified by silica gel
4.1.2.2. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 4-
hydroxybenzoate (4b). Solid; reaction time, 24 h; yield, 80%;
melting point, 83e85 ꢁC; Rf 0.50 (n-hexane:ethyl acetate 2:1), FTIR
nmax cmꢀ1: 3126 (OeH), 2913 (sp2 CeH), 2852 (sp3 CeH), 1705 (C]
O ester), 1589 (C]C aromatic), 1148 (CeO, ester); ESI-MS: m/z 351
[M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6,
d
ppm): 7.99 (dd, J ¼ 6.0,
2.4 Hz, 2H, H-2, H-6), 7.28 (d, J ¼ 2.8 Hz, 1H, H-20), 7.22 (dd, J ¼ 5.2,
2.4 Hz, 1H, H-40), 7.05 (d, J ¼ 7.6, 1H, H-60), 6.85 (dd, J ¼ 6.0, 2.4 Hz,

Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
209
2H, H-3, H-5), 5.11 (s, 2H, -CH2), 3.57 (s, 1H, -OH), 3.04 (sept,
6.89 (s, 1H, H-600), 6.56 (d, J ¼ 16.0 Hz, 1H, H-1), 5.02 (s, 2H, -CH2),
3.03 (sept, J ¼ 6.8 Hz, 1H, H-1000), 2.34 (s, 3H, H-3000), 1.19 (d,
J ¼ 6.8 Hz, 1H, H-100), 2.32 (s, 3H, -H-300)), 1.21 (d, J ¼ 3.6 Hz, 6H, H-
200); 13C NMR (DMSO-d6,
d
ppm); 167.2 (C]O ester), 165.7 (C]O,
J ¼ 6.8 Hz, 6H, H-2000); 13C NMR (DMSO-d6,
d ppm); 166.7 (C]O
ester), 160.5 (C-4), 147.2 (C-10), 136.9 (C-20), 136.7 (C-50), 132.3 (C-3,
C-5), 127.5 (C-1), 126.5 (C-30), 122.4 (C-40), 121.2 (C-60), 115.3 (C-2,C-
6), 60.9 (eCH2), 29.7 (C-100), 27.0 (C-300), 23.0 (C-200); Anal Calcd For
ester), 166.1 (C]O, ester), 147.3 (C-100), 146.4 (C-2), 136.9 (C-200),
134.1 (C-500), 130.6 (C-20,C-60), 128.9 (C-30, C-50), 128.2 (C-40), 127.5
(C-10), 126.5 (C-300), 122.4 (C-400), 116.6 (C-600), 60.6 (eCH2), 27.1 (C-
1000), 23.0 (C-3000), 20.7 (C-2000); Anal Calcd For C21H22O4: C, 74.56; H,
6.51; Found C, 74.45; H, 6.69.
C
19H20O5: C, 69.51; H, 6.10; Found C, 69.57; H, 6.02.
4.1.2.3. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 2,4-
dihydroxybenzoate (4c). Solid; reaction time, 24 h; yield, 78%;
melting point, 103e105 ꢁC; Rf 0.46 (n-hexane:ethyl acetate 2:1),
FTIR nmax cmꢀ1: 3093 (OeH), 2924 (sp2 CeH), 2852 (sp3 CeH),1710
(C]O ester), 1602 (C]C aromatic), 1159 (CeO, ester); ESI-MS: m/z
4.1.2.7. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4-
hydroxyphenyl)prop-2-enoate (6b). Solid; reaction time, 24 h;
yield, 80%; melting point, 117e119 ꢁC; Rf 0.48 (n-hexane:ethyl ac-
etate 2:1), FTIR nmax cmꢀ1: 3103 (eOH), 2945 (sp2 CeH), 2864 (sp3
CeH), 1726 (C]O), 1601 (C]C aromatic), 1122 (CeO, ester); ESI-
367 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6,
d ppm): 7.82 (d,
J ¼ 8.0 Hz, 1H, H-6), 7.23 (d, J ¼ 8.0 Hz, 1H, H-5), 7.08 (d, J ¼ 0.8 Hz,
1H, H-60), 6.89 (s, 1H, H-3), 6.38 (d, J ¼ 2.4 Hz, 1H, H-30), 6.36 (dd,
J ¼ 6.4, 2.4 Hz, 1H, H-40), 5.12 (s, 2H, -CH2), 3.02 (sept, J ¼ 7.2 Hz, 1H,
H-100), 2.34 (s, 3H, H-300), 1.32 (s, 2H, -OH), 1.22 (d, J ¼ 6.8 Hz, 6H, H-
MS: m/z 377 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6,
d ppm):
7.83 (d, J ¼ 16.0 Hz, 1H, H-2), 7.57 (dd, J ¼ 4.0, 2.0 Hz, 2H, H-20, 60),
7.42 (dd, J ¼ 4.0, 2.4 Hz, 2H, H-30, 50), 7.24 (d, J ¼ 6.8 Hz, 1H, H-300),
7.07 (d, J ¼ 8.0 Hz, 1H, H-400), 6.90 (s, 1H, H-600), 6.57 (d, J ¼ 16.0 Hz,
1H, H-1), 5.03 (s, 2H, -CH2), 3.04 (sept, J ¼ 6.8 Hz, 1H, H-1000), 2.34 (s,
3H, H-3000), 1.23 (d, J ¼ 6.8 Hz, 6H, H-2000); 13C NMR (DMSO-d6,
200); 13C NMR (DMSO-d6,
d ppm); 169.0 (C]O ester), 166.8 (C]O
ester), 163.9 (C-2), 162.7 (C-4), 147.1 (C-10), 136.9 (C-20), 136.8 (C-50),
132.1 (C-6), 127.6 (C-40), 126.6 (C-30), 122.3 (C-60), 108.2 (C-3), 104.8
(C-5), 103.1 (C-1), 60.9 (eCH2), 29.7 (C-100), 27.0 (C-300), 23.0 (C-200);
Anal Calcd For C19H20O6: C, 66.28; H, 5.81; Found C, 66.16; H, 5.73.
d
ppm); 167.3 (C]O ester), 166.6 (C]O, ester), 158.1 (C-1000), 147.2
(C-2), 146.3 (C-200), 136.9 (C-500), 130.2 (C-30, C-50), 127.5 (C-4 ), 126.8
(C-10), 126.5 (C-300), 122.4 (C-400), 115.9 (C-20,C-60), 113.7 (C-600), 56.6
(eCH2), 29.1 (C-1000), 27.1 (C-3000), 23.0 (C-2000); Anal Calcd For
4.1.2.4. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3,4-
dihydroxybenzoate (4d). Solid; reaction time, 24 h; yield, 75%;
melting point, 159e161 ꢁC; Rf 0.42 (n-hexane:ethyl acetate 2:1),
FTIR nmax cmꢀ1: 3126 (OeH), 2965 (sp2 CeH), 2827 (sp3 CeH), 1712
(C]O ester), 1604 (C]C aromatic), 1128 (CeO, ester); ESI-MS: m/z
C21H22O5: C, 71.19; H, 6.21; Found C, 71.28; H, 6.13.
4.1.2.8. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-(4-
chlorophenyl)prop-2-enoate (6c). solid; reaction time, 24 h; yield,
84%; melting point, 82e84 ꢁC; Rf 0.52 (n-hexane:ethyl acetate 2:1),
FTIR nmax cmꢀ1: 3923 (sp2 CeH), 2854 (sp3 CeH), 1728 (C]O
ester), 1627 (C]C aromatic), 1162 (CeO, ester); ESI-MS: m/z 395
367 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6,
d ppm): 7.43 (d,
J ¼ 2.4 Hz, 1H, H-2), 7.39 (dd, J ¼ 6.0, 2.4 Hz, 1H, H-6), 7.25 (d,
J ¼ 8.0 Hz, 1H, H-5), 7.07 (d, J ¼ 0.8 Hz, 1H, H-50), 6.96 (d, J ¼ 7.6 Hz,
1H, H-30), 6.87 (dd, J ¼ 8.4, 0.8 Hz, 1H, H-40), 5.14 (s, 2H, -CH2), 3.36
(s, 2H, -OH), 2.98 (sept, J ¼ 6.8 Hz, 1H, H-100), 2.26 (s, 3H, H-300), 1.11
[M þ 23] (M þ Na)þ; 1H NMR (DMSO-d6,
d ppm): 7.75 (d,
(d, J ¼ 6.8 Hz, 6H, H-200); 13C NMR (DMSO-d6,
d
ppm); 167.6 (C]O
J ¼ 16.0 Hz, 1H, H-2), 7.42 (d, J ¼ 8.8 Hz, 2H, H-20, 60), 7.24 (d,
J ¼ 8.0 Hz, 1H, H-300), 7.09 (d, J ¼ 7.2 Hz, 1H, H-400), 6.89 (s, 1H, H-600),
6.83 (d, J ¼ 8.8 Hz, 2H, H-30, H-50), 6.38 (d, J ¼ 16.0 Hz, 1H, H-1), 5.03
(s, 2H, -CH2), 3.03 (sept, J ¼ 6.8 Hz,1H, H-1000), 2.33 (s, 3H, H-3000),1.19
ester), 165.7 (C]O ester), 151.5 (C-3), 147.4 (C-4), 145.6 (C-10), 137.2
(C-20), 136.7 (C-50), 127.7 (C-6), 126.9 (C-40), 122.8 (C-30), 122.7 (C-
60),119.8 (C-2),116.9 (C-5),115.9 (C-1), 61.4 (eCH2), 26.7 (C-100), 23.3
(C-300), 20.7 (C-200); Anal Calcd For C19H20O6: C, 66.28; H, 5.81;
Found C, 66.39; H, 5.89.
(d, J ¼ 6.8 Hz, 6H, H-2000); 13C NMR (CDCl3,
d ppm); 166.6 (C]O
ester), 165.9 (C]O, ester), 147.2 (C-100), 144.9 (C-2), 136.9 (C-200),
136.6 (C-500), 132.6 (C-20,C-60), 129.4 (C-30, C-50), 129.2 (C-40), 127.5
(C-10), 126.5 (C-300), 122.4 (C-400), 117.2 (C-600), 60.8 (eCH2), 27.0 (C-
1000), 23.0 (C-3000), 20.7 (C-2000); Anal Calcd For C21H21O4Cl: C, 67.65;
H, 5.64; Found C, 67.51; H, 5.73.
4.1.2.5. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl 3,5-
dihydroxybenzoate (4e). Solid; reaction time, 24 h; yield, 82%;
melting point, 110e112 ꢁC; Rf 0.46 (n-hexane:ethyl acetate 2:1),
FTIR nmax cmꢀ1: 3125 (OeH), 2924 (sp2 CeH), 2852 (sp3 CeH),1709
(C]O ester), 1600 (C]C aliphatic) 1603 (C]C aromatic), 1132
(CeO, ester); ESI-MS: m/z 367 [M þ 23] (M þ Na)þ; 1H NMR (DMSO-
4.2. Mushroom tyrosinase inhibition assay
d6,
d
ppm): 7.26 (d, J ¼ 8.0 Hz,1H, H-30), 7.06 (d, J ¼ 8.0 Hz,1H, H-40),
6.89 (m, 3H, H-2, H-4, H-6), 6.48 (s, 1H, H-60), 5.17 (s, 2H, -CH2), 3.47
The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.)
was used for in vitro bioassays as described previously with some
(s, 2H, -OH), 2.99 (sept, J ¼ 6.8 Hz, 1H, H-100), 2.27 (s, 3H, H-300), 1.10
(d, J ¼ 6.8 Hz, 6H, H-200); 13C NMR (DMSO-d6,
d
ppm); 169.0 (C]O
modifications [31,32]. Briefly, 140
pH 6.8), 20 L of mushroom tyrosinase (30 U/mL) and 20
inhibitor solution were placed in the wells of a 96-well micro plate.
After pre-incubation for 10 min at room temperature, 20 L of
mL of phosphate buffer (20 mM,
ester), 166.9 (C]O ester), 163.9 (C-3, C-5), 162.8 (C-10), 147.1 (C-20),
136.9 (C-50), 132.1 (C-2, C-6), 127.7 (C-40), 126.6 (C-30), 122.2 (C-60),
108.2 (C-4), 104.7 (C-1), 60.9 (eCH2), 29.7 (C-100), 27.0 (C-300), 23.0
(C-200); Anal Calcd For C19H20O6: C, 66.28; H, 5.81; Found C, 66.12;
H, 5.91.
m
mL of the
m
L-
DOPA (3,4-dihydroxyphenylalanine) (0.85 mM) was added and the
plate was further incubated at 25 ꢁC for 20 min. Subsequently the
absorbance of dopachrome was measured at 475 nm using a micro
plate reader (OPTI Max, Tunable). Kojic acid was used as a reference
inhibitor and for negative tyrosinase inhibitor phosphate buffer
was used instead of the inhibitor solution. The extent of inhibition
by the test compounds was expressed as the percentage of con-
centration necessary to achieve 50% inhibition (IC50). Each con-
centration was analyzed in three independent experiments. The
IC50 values were determined by the data analysis and graphing
software Origin 8.6, 64-bit.
4.1.2.6. 2-[5-Methyl-2-(propan-2-yl)phenoxy]-2-oxoethyl (2E)-3-
phenylprop-2-enoate (6a). Solid; reaction time, 24 h; yield, 86%;
melting point, 94e96 ꢁC; Rf 0.54 (n-hexane:ethyl acetate 2:1), FTIR
nmax cmꢀ1: 3918 (sp2 CeH), 2820 (sp3 CeH), 1723 (C]O ester),
1593 (C]C aromatic), 1146 (CeO, ester); ESI-MS: m/z 361 [M þ 23]
(M þ Na)þ; 1H NMR (DMSO-d6,
d
ppm): 7.80 (d, J ¼ 16.0 Hz, 1H, H-
2), 7.50 (dd, J ¼ 4.8, 2.0 Hz, 2H, H-20, 60), 7.39e7.41 (m, 3H, H-30, H-
40, H-50), 7.25 (d, J ¼ 4.8 Hz, 1H, H-300), 7.09 (d, J ¼ 6.4 Hz, 1H, H-400),

210
Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
4.3. Free radical scavenging assay
5.3. Grid parameters
Radical scavenging activities was determined by modifying
method of [33] by 2,2-diphenyl-1- picrylhydrazyl (DPPH) assay. The
For the docking of ligand molecules to the tyrosinase protein
structure, search space coordinates were provided to the AutoDock
Vina using AutoDock Tools. The dimensions of the grid box were set
in a manner to ensure that the ligand could bind to all the potential
binding sites of the protein and, hence, provide the best binding
conformation. Default values provided by the program were
retained for the rest of the parameters. The number of grid points in
xyz was set to the spacing value equivalent to 1.0 Å and the grid
center to ꢀ5, ꢀ15 and 18.
assay solution consisted of 100
picrylhydrazyl (DPPH), 20 L of increasing concentration of test
compounds and the volume was adjusted to 200 L in each well
mL of (150 mM) 2,2-diphenyl-1-
m
m
with DMSO. This reaction mixture was then incubated for 30 min at
room temperature. Ascorbic acid (Vitamin C) was used as a refer-
ence inhibitor. The assay measurements were carried out by using a
micro plate reader (OPTI Max, Tunable) at 517 nm. The reaction rates
were compared and the percent inhibition due to the presence of
tested inhibitors was calculated. Each concentration was analyzed
in three independent experiments run in triplicate. The concen-
tration required for 50% decrease in the absorbance of a control
solution of DPPH was expressed as IC50. The IC50 values were
determined by the Data analysis and graphing software Origin 8.6,
64-bit.
5.4. Docking analysis and visulaization of binding conformations
Docking analysis was carried out using AutoDock Vina which
uses an iterated local search algorithm by employing the search
space parameters provided by the user. The output was obtained in
the form of binding energies (Kcal/mol) and the best binding con-
formations. The binding conformations with the lowest energy
coefficients were selected and visualized in Discovery Studio (4.1.0)
[40]. Spatial (3D) and linear (2D) interaction maps were studied in
order to determine the amino acids involved in the ligand-protein
interactions.
4.4. Kinetic analysis of the inhibition of tyrosinase
A series of experiments were performed to determine the in-
hibition kinetics by following method [34]. Inhibitor (4d) with
concentrations 0, 9, 18, 36.2, 72.5
mM, and (6b) with concentrations
0, 7, 14 and 28 M, respectively were used. Substrate
m
L-DOPA con-
centration was among 0.0625e2 mM in all kinetic study. Pre-
incubation and measurement time was the same as discussed in
mushroom tyrosinase inhibition assay protocol. Formation of
DOPAchrome was continuously monitored at 475 nm for 5 min at a
30s interval in the microplate reader after addition of enzyme. The
inhibition type on the enzyme was assayed by LineweavereBurk
plots of inverse of velocities (1/V) versus inverse of substrate con-
centration 1/[S] mMꢀ1, and the inhibition constant Ki was deter-
mined by Dixon plot of 1/V versus inhibitor concentrations. The
reversible kinetics of the enzyme inhibitor complex was also
determined for different concentration (0.0, 2.2, 4.4, 8.8 and
5.5. Crystallographic data collection for (6a)
Single crystal X-ray diffraction data set for (6a) was collected at
room temperature up to a max 2.04
SMART CCD area-detector diffractometer using monochromatic
q
of ca. 28.22ꢁ on a Bruker
MoK
a
radiation,
l
¼ 0.71069 Å. The structure was solved by direct
method [41] and refined by full matrix least squares on F2 with
anisotropic displacement parameters for non-H atoms using
SHELXL-2013 [42]. All hydrogen atoms were located from differ-
ence Fourier maps and refined as riding on their parent atoms.
17.5
m
M) of derivatives (6b) versus the enzyme concentration (4, 6,
5.6. Crystal structure description
8, 10, 15 and 20
m
g/mL).
The colorless single crystals of compound (6a) suitable for X-ray
diffraction were grown from ethyl acetate using the slow evapo-
ration technique. Diffraction intensity data were collected with
5. Molecular docking
5.1. Preparation of target protein
FR590 MACH3 single crystal diffractometer using Mok
a mono-
chromatic radiation (
l
¼ 0.71073 Å) at room temperature (296 K).
Docking analysis of the thymol analogues (4aee) and (6aec)
against the tyrosinase enzyme was performed in order to compare
the relative affinity of the chemicals against the enzyme. For this
purpose, AutoDock Vina (1.1.2) was utilized which provides an ac-
curate and efficient means to determine the protein-ligand binding
mode predictions [35]. The tertiary structure of the protein (PDB ID
2ZMX) was retrieved from RCSB Protein DataBank Site [36,37].
Bound ligand and solvent molecules were removed from the pro-
tein structure using the Chimera Visualization software (1.8) [38].
The protein was saved in the pdb format. AutoDock Tools (1.5.6)
was then used to protonate the protein tertiary structure and finally
save it in the dockable file format (pdbqt).
The compound (6a) crystallizes in orthorhombic crystal system
with Pna21 space group and detailed structure refinement data and
crystallographic data are presented in (Table S1) as supplementary
material. The bond length of carbonyl
is less than carbonyl -bond between C(2)-O(18) due to
delocalization in C(5)-O(19) -bond. As these -electrons are in
conjugation with the -electrons of cinnamic acid double bond
p-bond between C(5)-O(19)
p
p-electrons
p
p
p
between C(6)-C(7). The detailed bond lengths and bond angle of
atomic coordinates for all atoms are tabulated as (Table S2 & S3).
The additional data for the molecule (6a) are alternatively available
from the Cambridge Crystallographic Data Centre as CCDC 1054911.
An ORTEP drawing of the compound (6a) with the atomic
numbering scheme is shown in Figure S1 (Supplementary
material).
5.2. Ligand preparation
Thymol analogues were drawn in the ChemSketch software
(11.02). After the 3-D optimization, the ligand structures were
saved in the mol format. For file format conversion, ArgusLab (4.0.1)
was used and the ligands were saved in the pdb format [39]. These
files were then retrieved in the AutoDock Tools which was used to
observe the ligand geometry and bond flexibility. The ligands were
then saved in the pdbqt format.
Acknowledgements
This work (Grants No.C0036335) was supported by Business for
Cooperative R & D between Industry, Academy, and Research
Institute and funded by Korea Small and Medium Business
Administration in 2012.

Z. Ashraf et al. / European Journal of Medicinal Chemistry 98 (2015) 203e211
Chem. 56 (2008) 3989e3996.
211
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