Synthesis, computational studies, tyrosinase inhibitory kinetics and antimelanogenic activity of hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl derivatives

Article abstract of DOI:10.1080/14756366.2019.1654468

The over expression of melanogenic enzymes like tyrosinase caused many hyperpigmentaion disorders. The present work describes the synthesis of hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl derivatives 3a–e and 5a–e as antimelanogenic agents. The tyrosinase inhibitory activity of synthesized derivatives 3a–e and 5a–e was determined and it was found that derivative 5c possesses excellent activity with IC₅₀ = 0.0089 μM compared to standard kojic acid (IC₅₀ = 16.69 μM). The presence of hydroxyl groups at the ortho and the para position of cinnamic acid phenyl ring in compound 5c plays a vital role in tyrosinase inhibitory activity. The compound 5d also exhibited good activity (IC₅₀ = 8.26 μM) compared to standard kojic acid. The enzyme inhibitory kinetics results showed that compound 5c is a competitive inhibitor while 5d is a mixed-type inhibitor. The mode of binding for compounds 5c and 5d with tyrosinase enzyme was also assessed and it was found that both derivatives irreversibly bind with target enzyme. The molecular docking and molecular dynamic simulation studies were also performed to find the position of attachment of synthesized compounds at tyrosinase enzyme (PDB ID 2Y9X). The results showed that all of the synthesized compounds bind well with the active binding sites and most potent derivative 5c formed stable complex with target protein. The cytotoxicity results showed that compound 5c is safe at a dose of 12 μg/mL against murine melanoma (B16F10) cells. The same dose of 5c was selected to determine antimelanogenic activity; the results showed that it produced antimelenogenic effects in murine melanoma (B16F10) cells. Based on our investigations, it was proposed that compound 5c may serve as a lead structure to design more potent antimelanogenic agents.

Full text of DOI:10.1080/14756366.2019.1654468

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, computational studies, tyrosinase
inhibitory kinetics and antimelanogenic activity of
hydroxy substituted 2-[(4-acetylphenyl)amino]-2-
oxoethyl derivatives
Muhammad Rafiq, Yasir Nazir, Zaman Ashraf, Hummera Rafique, Samina
Afzal, Amara Mumtaz, Mubashir Hassan, Anser Ali, Khurram Afzal,
Muhammad Rizwan Yousuf, Muhammad Saleem, Katarzyna Kotwica-
Mojzych & Mariusz Mojzych
To cite this article: Muhammad Rafiq, Yasir Nazir, Zaman Ashraf, Hummera Rafique, Samina
Afzal, Amara Mumtaz, Mubashir Hassan, Anser Ali, Khurram Afzal, Muhammad Rizwan
Yousuf, Muhammad Saleem, Katarzyna Kotwica-Mojzych & Mariusz Mojzych (2019) Synthesis,
computational studies, tyrosinase inhibitory kinetics and antimelanogenic activity of hydroxy
substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl derivatives, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 1-11, DOI: 10.1080/14756366.2019.1654468
To link to this article: https://doi.org/10.1080/14756366.2019.1654468
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 1562–1572
2
https://doi.org/10.1080/14756366.2019.1654468
RESEARCH ARTICLE
Synthesis, computational studies, tyrosinase inhibitory kinetics and
antimelanogenic activity of hydroxy substituted 2-[(4-acetylphenyl)amino]-2-
oxoethyl derivatives
a
ꢀ
b
ꢀ
b
c
d
e
Muhammad Rafiq , Yasir Nazir , Zaman Ashraf , Hummera Rafique , Samina Afzal , Amara Mumtaz ,
f#
g
d
h
i
Mubashir Hassan , Anser Ali , Khurram Afzal , Muhammad Rizwan Yousuf , Muhammad Saleem ,
j
k
Katarzyna Kotwica-Mojzych and Mariusz Mojzych
a
b
Department of Physiology & Biochemistry, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Punjab, Pakistan; Department of
c
d
Chemistry, Allama Iqbal Open University, Islamabad, Pakistan; Department of Chemistry, University of Gujrat, Gujrat, Pakistan; Faculty of Pharmacy,
Bahauddin Zakria University, Multan, Pakistan; Department of Chemistry, COMSAT University Islamabad, Abbottabad, Pakistan; Department of
Biology, College of Natural Sciences, Kongju National University, Gongju, Korea; Department of Zoology, Mirpur University of Science and
Technology (MUST), Mirpur, Pakistan; Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, Pakistan; Department of
Chemistry, University of Sargodha, Bhakkar, Pakistan; Department of Histology and Embryology with Experimental Cytology Unit, Medical University
of Lublin, Lublin, Poland; Department of Chemistry, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland
e
f
g
h
i
j
k
ABSTRACT
ARTICLE HISTORY
The over expression of melanogenic enzymes like tyrosinase caused many hyperpigmentaion disorders. The pre-
sent work describes the synthesis of hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl derivatives 3a–e
and 5a–e as antimelanogenic agents. The tyrosinase inhibitory activity of synthesized derivatives 3a–e and 5a–e
was determined and it was found that derivative 5c possesses excellent activity with IC50 ¼ 0.0089mM compared
to standard kojic acid (IC50 ¼ 16.69 mM). The presence of hydroxyl groups at the ortho and the para position of
cinnamic acid phenyl ring in compound 5c plays a vital role in tyrosinase inhibitory activity. The compound 5d
also exhibited good activity (IC50 ¼ 8.26 mM) compared to standard kojic acid. The enzyme inhibitory kinetics
results showed that compound 5c is a competitive inhibitor while 5d is a mixed-type inhibitor. The mode of
binding for compounds 5c and 5d with tyrosinase enzyme was also assessed and it was found that both deriva-
tives irreversibly bind with target enzyme. The molecular docking and molecular dynamic simulation studies
were also performed to find the position of attachment of synthesized compounds at tyrosinase enzyme (PDB
ID 2Y9X). The results showed that all of the synthesized compounds bind well with the active binding sites and
most potent derivative 5c formed stable complex with target protein. The cytotoxicity results showed that com-
pound 5c is safe at a dose of 12 mg/mL against murine melanoma (B16F10) cells. The same dose of 5c was
selected to determine antimelanogenic activity; the results showed that it produced antimelenogenic effects in
murine melanoma (B16F10) cells. Based on our investigations, it was proposed that compound 5c may serve as
a lead structure to design more potent antimelanogenic agents.
Received 23 May 2019
Revised 6 August 2019
Accepted 6 August 2019
KEYWORDS
Tyrosinase inhibition;
enzyme inhibitory kinetics;
molecular docking;
antimelanogenic activity;
cytotoxicity
GRAPHICAL ABSTRACT
CONTACT Mariusz Mojzych
Siedlce 08-110, Poland; Zaman Ashraf
mmojzych@yahoo.com
Department of Chemistry, Siedlce University of Natural Sciences and Humanities, 3-go Maja 54,
mzchem@yahoo.com Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
ꢀ
These authors contributed equally as first authors.
Mubashir Hassan Current Address: Institute of Molecular and Biotechnology, The University of Lahore, Pakistan.
#
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

2
M. RAFIQ ET AL.
1
. Introduction
reduce oxidative damage in human body and to downregulate
1
7–20
UV-induced melanogenesis
.
Melanogenesis is the process of melanin synthesis; most often
produced by cells called melanocytes . Melanocytes are dendritic
cells formed from melanoblasts which are unpigmented cells origi-
nating from embryonic neural crest cells . Melanocytes are found
in the basal layer of skin epidermis and hair follicles. The primary
function of melanocytes is the synthesis of melanin pigment. In
skin each melanocytes is surrounded by approximately 36 kerati-
For melanin inhibition, potent tyrosinase inhibitors and antioxi-
dant agents are always desirable. They can be obtained from a
variety of sources; however, safety concerns pose a big challenge
for their commercialization. There are a number of tyrosinase
inhibitors; hydroquinone and kojic acid and, potent antioxidants;
tert-butyl hydroxyanisole (BHA) and tert-butyl hydroxytoluene
1
,2
3
,4
(
BHT) which may show undesirable side effects; cytotoxicity,
5
,6
6,7
nocytes , to which they transfer their synthesized melanin
.
21
dermatitis and skin cancer . Recently, methoxy phenyl thiourea,
Melanin is a pigment, primarily responsible for skin colour. It
retains antioxidative and photoscreening effect, therefore, pro-
vides skin photo-protection, prevents from injury and, absorbs
thiazole derivatives and kojic acid derivatives have been reported
22–24
as the tyrosinase inhibitor (Figure 1)
. Therefore, search for
safe and effective depigmenting agents is required which may
address these issues and serve as a better solution for treating
pigment related dermatological disorders.
8
and transforms harmful UV radiations into harmless heat . Despite
its advantages, increased production and accumulation of pigmen-
tations can cause skin problems such as freckles, age spots, post-
inflammatory hyperpigmentation, lentigo and melanoma. UV
which stimulates melanin synthesis is reported to cause gene
mutation, DNA damage, impaired immune system and cancer .
Pigmentation in metastatic melanoma patients results in short
overall and disease-free survival . Melanin content is correlated
with higher disease stage and seems to protect malignant mela-
nocytes from chemo-, radio- and photodynamic therapy
Therefore, inhibition of melanogenesis could be rational
approach for controlling metastatic melanoma, abnormal skin pig-
Keeping in view the importance of phenolic hydroxyl in tyrosin-
25,26
ase inhibition,
the present work is planned to synthesize
hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl deriva-
tives 3a–e and 5a–e to explore the role of different functionalities
in tyrosinase inhibitory activity. The enzyme inhibitory kinetics of
most potent compounds was determined by Line-weaver Burk
Plots and Dixon Plots. The reversibility of enzyme–inhibitor com-
plex was also determined. The computational molecular docking of
the synthesized compounds was performed against target protein
PDBID 2Y9X to predict the binding sites of these compounds in tar-
get protein. The molecular dynamic simulation was also performed
to check the stability of enzyme-inhibitor complex computationally.
The cytotoxicity and antimelanogenic activity of most potent
derivative 5c was also performed against murine melanoma
9
1
0
1
1,12
.
a
10
mentation and related disorders .
Tyrosinase is key an enzyme showing the rate limiting affect in
melanin biosynthesis. In cytosol, L-phenylalanine may be con-
verted to tyrosine by phenylalanine hydroxylase (PAH) in order to
serve as the substrate for tyrosinase . Tyrosinase also catalyses
the hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine
1
3
(B16F10) cells.
14
(L-DOPA) and L-DOPA to dopaquinones which under unregu-
lated conditions result in abnormal accumulation of melanin pig- 2. Results and discussion
1
5,16
ments
. Therefore, inhibition of tyrosinase is the simplest
2
.1. Chemistry
approach and tyrosinase inhibitors may be an attractive target to
achieve depigmentation.
The title compounds 3a–e and 5a–e were synthesized by the fol-
Additionally, antioxidants are reported critical for melanin lowing already reported method27,28 as shown in Schemes 1 and
inhibition. Reactive oxygen species (ROS) and free radicals are 2. 4-Acetyl aniline was reacted with chloroacetyl chloride in the
shown to contribute in numerous skin disorders including hyper- presence of triethyl amine to synthesized intermediate 1. The final
1
7
pigmentation . During hyperpigmentation, increased level of products 3a–e and 5a–e were synthesized by reacting substituted
hydrogen peroxide (H ) and other ROS is reported which may benzoic acids and cinnamic acids with intermediate 1. The forma-
increase the load of oxidative stress on melanocytes. Numerous tion of the final products was ascertained by their FTIR, HNMR
ROS scavengers and inhibitors of ROS generation are shown to and CNMR spectral data. The absorption for amide –NH stretch-
interfere with oxidation processes, therefore, frequently used to ing appeared at 3133–3156 cm
2 2
O
1
1
3
ꢁ
1
in FTIR spectra while amide
Figure 1. Structures of recently reported tyrosinase inhibitors.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
3
O
OH
O
O
H
NH2
H
N
Cl
2a-e
N
Cl
R
Cl
O
H C
R
3
H C
O
3
H C
O
i
ii
3
O
O
1
O
3a: R = 3-OH
3
3
3
3
b: R = 4-OH
c: R = 2,4-di-OH
d: R = 3,4-di-OH
e: R = 3,5-di-OH
ꢂ
Scheme 1. Synthesis of compounds 3a–e. Reagents and conditions: (i) CH
2
Cl
2
/Et
3
N, 0–5 C, reflux for 5 h; (ii) DMF/Et
3
N/KI, r.t., reflux for 24 h.
H
N
O
H
N
R
Cl
O
i
(
)n
O
+
R
H C
O
3
H C
O
(
)n
OH
3
O
O
1
4a-e
5a
5
5
5
5
b: R = 4-OH,
c: R = 2,4-di-OH, n = HC=CH
d: R = 4-Cl,
e: R = 4-OH,
n = HC=CH
n = HC=CH
n = CH -CH2
2
3
Scheme 2. Synthesis of compounds 5a–e. Reagents and conditions: (i) DMF/Et N/KI, r.t., reflux for 24 h.
ꢁ
1
1
carbonyl appeared at 1632–1656 cm . The amide –NH in HNMR
spectra appeared as a singlet at downfield region, rest of the sig-
nals are present in the acceptable regions.
Table 1. Tyrosinase inhibitory activity of the synthesized
compounds 3a–e and 5a–e.
Compounds
Mushroom tyrosinase inhibition IC50 (mM)
1
54.5 ± 3.1
324.6 ± 16.9
319.5 ± 31.9
145.2 ± 16.7
256.8 ± 23.4
165.9 ± 14.2
63.5 ± 2.5
3
3
3
a
b
c
2.2. Enzyme inhibitory kinetics
Hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl deriva-
tives 3a–e and 5a–e have been designed and synthesized to
explore their inhibitory effects on tyrosinase activity. Kojic acid a
clinically applied agent was used as a standard for comparison
purpose. The presence of phenolic hydroxyls is of special interest
because of their high tyrosinase inhibitory activity. It has been
exposed from our bioassay results that the major determining fac-
tor of inhibitory activity is the presence of phenolic hydroxyls at
cinnamic acid moiety (Table 1). Interestingly, compound 5c bear-
ing 2,4-dihydroxy substituted cinnamic acid moiety exhibited
excellent activity (IC50 ¼ 0.0089 mM) and is also more active than
standard kojic acid (IC50 ¼ 16.69 mM). The compound 5d also
exhibited good activity (IC50 ¼ 8.26 mM) compared to standard
kojic acid. The bioassay results proved that derivatives with
hydroxy substituted cinnamic acid moiety (5a–d) displayed greater
3d
3
5
5
e
a
b
97.3 ± 5.8
5c
5d
Kojic acid
0.0089 ± 0.0004
8.26 ± 1.7
16.69 ± 2.8
Dixon plots to determine the type of inhibition and inhibition
constant (KI).
From the kinetic analyses, the Lineweaver–Burk plot of 1/Vmax
versus 1/[S] in the presence of different concentrations of 5c
showed that K
m
value change while that of 1/Vmax remained the
same and in case of compound 5d both the Km and 1/Vmax
tyrosinase inhibitory activity compared to hydroxy substituted reached to new values as shown in Figures 2(a) and 3(a), respect-
benzoic acid derivatives (3a–e).
To understand the mechanism of mushroom tyrosinase inhib-
ition by compounds 5c and 5d, kinetic studies were conducted.
ively. This graphical representation showed that the compound 5c
is a competitive inhibitor and the compound 5d is a mixed-type
inhibitor. On the other hand, in the Dixon plot, the dissociation
Kinetic studies showed a concentration-dependent mushroom constant Ki value for 5c with 0.01 mM and for 5d with 6 mM was
tyrosinase inhibition by tested inhibitors. Constant and continual calculated from the plots for uninhibited enzyme and with differ-
monitoring of the reaction showed a marked decrease of the reac- ent concentrations of inhibitors as shown in Figures 2(b) and 3(b),
tion rate in the presence of the inhibitors, which ultimately indi- respectively.
cated the decrease of the final absorbance when compared with
The inhibitory mechanism of mushroom tyrosinase by com-
controls without inhibitor. The potency of inhibition exhibited by pounds 5c (in concentrations: 0.0, 0.0011, 0.0022, 0.0045, 0.0090
these compounds changed depending on the presence and pos- and 0.018 mM) and 5d (in concentrations: 0.0, 1.75, 3.5, 7.0, 14, 28
2
9
ition of substituents and on the class of compounds . Enzyme and 56 mM) were investigated. The plots of the remaining enzyme
inhibition kinetics was analysed by Lineweaver–Burk plot and activity versus the concentration of enzyme (1.25, 2.5 and 5 mg/

4
M. RAFIQ ET AL.
Figure 2. (a) Lineweaver–Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations 0.000, 0.0005, 0.0022, 0.0045,
.009 and 0.018 mM of compound 5c in the presence of different concentrations 0.062, 0.125, 0.25, 0.5, 1 and 2 mM, of L-DOPA. (b) Dixon plots for the inhibition of
0
the diphenolase activity of mushroom tyrosinase by various concentrations of compound 5c in the presence of different concentrations 0.125, 0.25, 0.5, 1 and 2 mM,
of L-DOPA.
Figure 3. (a) Lineweaver–Burk plots for the inhibition of the diphenolase activity of mushroom tyrosinase by various concentrations 0.00, 1.75, 3.5, 7, 14 and 28 mM of
compound 5d in the presence of different concentrations 0.125, 0.25, 0.5, 1 and 2 mM, of L-DOPA. (b) Dixon plots for the inhibition of the diphenolase activity of
mushroom tyrosinase by various concentrations of compound 5d in the presence of different concentrations 0.125, 0.25, 0.5, 1 and 2 mM, of L-DOPA.
Figure 4. Relationship between the catalytic activity of tyrosinase and various concentrations of compound 5c shown in (a) and 5d shown in (b).
mL) at different inhibitor concentrations for the catalysis of L- compound 5d and the same conclusion was drawn that 5d was
DOPA gave a series of parallel straight lines with the same slopes irreversible inhibitor (Figure 4(b)). These results suggested that
indicating that the inhibitory effect of 5c on the tyrosinase was both the compounds effectively inhibited the enzyme by binding
3
0
irreversible (Figure 4(a)). Identical research was carried out for to its binuclear active site irreversibly .

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
5
2
.3. Cytotoxicity and antimelanogenic activity
images which showed no effect on cell morphology after incuba-
tion with 5c (Figure 6).
To investigate the effect of compound 5c on cellular melanin syn-
thesis, we first assessed the safe treatment dose of tested deriva-
tive 5c using MTT cell viability assay. For viability assay, murine
melanoma (B16F10) cells were incubated with various concentra-
tions of compound 5c for 24 h and then MTT assay was performed
as described in the methods section. The result showed concen-
tration dependent 5c effect on cell viability as shown in Figure 5.
The 5c compound showed significant toxic effect (33.7% com-
pared to control normalized to 100%) at 24 mg/ml concentrations
however no significant effect at 12 mg/ml concentration was
observed. Therefore, we selected 12 mg/ml concentration (safe
concentration) of 5c for further analysis. Later, we took cellular
For melanin measurement, B16F10 cells were incubated in cell
culture medium containing various concentrations of 5c for 24 h.
Interestingly, cellular melanin analysis showed decrease of melanin
content with increasing concentration of 5c (Figure 4). The signifi-
cant decrease of cellular melanin content was observed at 12 mg/
ml where we got 82% melanin content as compared to control
normalized to 100% as given in Figure 7. The melanoma cells are
widely used for melanin investigations and various studies have
shown the effect of compound concentration on melanogene-
31–35
sis
. In conclusion, our results indicate pronounced ability of
5c compound to inhibit melanin synthesis in cells in vitro which
may show promising future in the field of cosmetics or medicine.
Figure 5. Cell viability measurements. The murine melanoma (B16F10) cells were Figure 7. Intracellular melanin measurements. The murine melanoma (B16F10)
incubated with indicated concentrations of 5c, and the cell viability was meas- cells were incubated with indicated concentrations of 5c, and the cellular melanin
ured by MTT assay. The data was expressed as a percentage of the control (nor- content was detected. The data were expressed as a percentage of control (nor-
malized to 100%) with mean ± standard deviation and was analysed using malized to 100%) with mean ± standard deviation and, and were analysed using
ꢀ
ꢀ
ꢀ
Student’s t-tests, p < 0.05.
Student’s t-tests, p < 0.005.
Figure 6. The B16F10 cells morphology. The cells were incubated with (a) cell culture medium, (b) 3 mg/ml, (c) 6 mg/ml, and (d) 12 mg/ml concentrations of 5c for 24 h
and then cell images were taken by inverted fluorescent microscope (Nikon, ECLIPSE, Ti, Tokyo, Japan).

6
M. RAFIQ ET AL.
Table 2. Chemo-informatics evaluation of synthesized compounds.
Properties
3a
313
3b
313
3c
329
3d
329
3e
329
5a
323
5b
339
5c
355
5d
357
5e
341
MW
HBA
HBD
LogP
LogS
PSA
MR
Den.
ST
5
2
5
2
6
3
6
3
6
3
4
1
5
2
6
3
4
1
5
2
2.16
61.44
75.69
83.77
1.33
59.2
33.21
Yes
2.16
64.12
75.69
83.77
1.33
59.2
33.21
Yes
1.78
124.89
92.24
85.66
1.41
66.6
33.95
Yes
1.78
85.62
91.17
85.66
1.41
66.6
33.95
Yes
1.90
92.75
93.31
85.66
1.41
66.6
33.95
Yes
3.18
6.53
57.48
92.42
1.24
52.7
36.33
Yes
2.92
9.53
75.09
94.30
1.31
58.8
37.88
Yes
2.54
23.64
91.64
96.18
1.38
65.5
38.13
Yes
3.89
0.80
57.48
97.31
1.31
53.7
38.57
Yes
2.43
75.09
75.09
92.59
1.28
55.7
36.7
Yes
PZ
Lipinski rule
MW: molecular weight (g/mol); HBA: hydrogen bond acceptor; HBD: hydrogen bond donor; LogP: lippophilicity of partition coefficient; LogS:
3
lippopilicity of water (mg/L); PSA: polar surface area; MR: molar refractivity; Den: density (g/cm ±0.06); ST: surface tension (dyne/cm ±3.0);
3
24
3
PZ: polarizability (cm ±0.5 ꢃ 10 ); MR: molar refractivity (cm ± 0.3); ST: surface tension.
2.4. Structural assessment of target protein
Table 3. Docking results of synthesized compounds
using PyRx
Mushroom tyrosinase (Agaricusbisporus) is a class of oxidoreduc-
tase copper containing protein comprises 391 amino acids. The
structure architecture of mushroom tyrosinase showed that it con-
sists of 39% helices (154 residues) and 14% b sheets (57 residues)
and 46% coils (180 residues). The X-ray diffraction study confirmed
Compounds
Energy values (kcal/mol)
3
3
3
3
a
b
c
ꢁ7.6
ꢁ7.7
ꢁ7.6
ꢁ7.4
ꢁ7.6
ꢁ7.1
ꢁ6.9
ꢁ7.9
ꢁ6.4
ꢁ7.1
d
_
its resolution 2.78A, R-value 0.238 and unit cell crystal dimensions
like length and angles of coordinates. The unit cell length values
3e
5
5
5
5
a
b
c
were observed for a ¼ 103.84, b ¼ 104.82 and c ¼ 119.36 with
ꢂ
ꢂ
ꢂ
angles 90 , 110.45 and 90 for all a, b and c dimensions, respect-
ively. The Ramachandran plots and values indicated that 95.90%
of residues were in favoured regions and 100.0% residues were
lies in allowed regions (Figure S1). This selected Ramachandran
graph values showed the good accuracy of phi (u) and psi (w)
angles among the coordinates of receptor molecules and most of
residues plunged in acceptable region.
d
5e
derivatives. The graphical depiction of 5c docking complex is
mentioned in Figure 8.
The bonding analysis of all compounds against mushroom
tyrosinase showed that 5c compound is directly interacts with
active residues of targeted protein. The structure activity rela-
2.5. Chemo-informatics properties and Lipinski’s rule
The predicted chemo-informatics properties such as molar volume, tionship (SAR) analysis shows that 5c builds five hydrogen
density, polarizability and surface tension were evaluated by com- bonds at specific residues (HIS263, ASN260, HIS85, GLU322 and
putational approach. Literature study established a standard value ALA323) against the target protein. The carbonyl oxygen of
for molar refractivity (40–130), molecular weight (160–480) and benzene ring of 5c interacts with HIS263and ASN260 positions
36
number of atoms (20–70) . Results showed that 5c predicted val- having bonds lengths 3.26 Å and 2.69 Å. The ester oxygen of
ues are much better than standard values and all other synthe- the same compound also interacts with GLU322 with bonding
sized compounds. Moreover, the Lipinski’s rule of five (RO5) says distance 2.90 Å. The hydroxyl groups of the same compounds
nothing about specific chemistry or structural features found in form bonds with HIS85 and ALA323 with bonds length 2.25 Å
drugs or non-drugs. The computational results showed that 5c and 2.75 Å, respectively. Literature study also justified that these
possesses 6 HBA, 3 HBD and 2.54 LogP values which significantly interacted residues are significant in the downstream signalling
3
8
justified its drug like behaviour. Moreover, their molecular weight pathways . The graphical representations of all other
(
(
355.11 g/mol) was also much better than standard value interacted compounds (3a–3e, and 5a, 5b, 5d and 5e) against
<5000 g/mol). The RO5 justifies that molecules with poor absorp- target protein are mentioned in Figures S2–S3 (see
tion are more likely to have more than 5 HBD, MWT over 500,
logP over 5 and more than 10 HBA. However there are plenty of
examples available for RO5 violation amongst the existing drugs .
In overall results, these predicted values justify the significance of
Supplementary materials).
Moreover, comparative analysis showed that 5c has good
binding energy values (ꢁ7.90 kcal/mol) as compared other deriv-
atives. The other candidate compounds (3a–3e) also possess
good binding energy values ꢁ7.60 kcal/mol, ꢁ7.70 kcal/mol,
3
7
5c
synthesized compound as
a good candidate molecule
(Table 2).
ꢁ
7.60 kcal/mol, ꢁ7.40 kcal/mol and ꢁ7.60 kcal/mol, while 5a, 5b,
5
d and 5e showed ꢁ7.10 kcal/mol, ꢁ6.90 kcal/mol, ꢁ6.40 kcal/
mol and ꢁ7.10 kcal/mol binding energy values, respectively
Table 3). Here, it is noticeable that most of the candidate com-
The docked complexes of all the synthesized compounds pounds bind in the active binding regions but little fluctuated
3a–3e and 5a–5e) were analysed on the basis of lowest bind- with their conformational positions. The comparative binding
2.6. Molecular docking
(
(
ing energy values and hydrogen bonding analyses. Results analysis and bioassay study showed that 5c was the most
showed that 5c was the most active compound with best bind- active and significant compound as compared to other synthe-
ing energy value (–7.90 kcal/mol) compared to others sized compounds.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
7
Figure 8. Docking interaction between 5c and target protein. The 5c is mentioned in green colour with embedded oxygen functional groups in red and nitrogen in
purple colours. The target protein is highlighted in line ribbon format with light grey colour. The active binding site amino acids are highlighted in red colour. Two
copper ions are also mentioned in black colour. Five hydrogen bonds were observed between 5c and receptor amino acids like HIS263, ASN260, HIS85, GLU322 and
_
_
_
_
_
_
ALA323 with bonding distances 3.26 A, 2.69 A, 2.25 A, 2.90 A and 2.75 A, respectively. The black dotted lines show the binding distance in angstrom (A).
3
. Conclusion
amides 3a–e were obtained by treating intermediate 1 with
hydroxy substituted benzoic acids 2a–e (0.01 mole), triethyl amine
The hydroxy substituted 2-[(4-acetylphenyl)amino]-2-oxoethyl
derivatives 3a–e and 5a–e with various hydroxyl moieties have
been synthesized to explore their role in tyrosinase inhibitory
activity. The analogue 5c showed excellent activity with
IC ¼0.0089 mM better than standard kojic acid IC ¼16.69 mM.
(
(
0.01 mol) and potassium iodide (0.01 mol) in dimethylformamide
DMF, 10 ml) at room temperature, while compounds 5a–e were
synthesized by reacting intermediate 1 with acids 4a–e under the
same conditions. After the completion of reaction, the mixture
was poured into ice cold water with stirring and extracted with
ethyl acetate (4 ꢃ 20 ml). The combined organic layer was washed
with dilute hydrochloric acid (5%), sodium carbonate solution (5%)
and finally with aqueous NaCl solution. The organic layer was
dried over anhydrous magnesium sulphate, filtered and the solv-
ent was removed under reduced pressure to afford the crude title
5
0
50
The enzyme inhibitory kinetics results confirmed that compounds
5c, a competitive inhibitor, and 5d, a mixed type inhibitor. Both
compounds 5c and 5d inhibit the enzyme by irreversible mode of
binding. The computational molecular dynamic simulation results
revealed that synthesized compounds bind well with the active
binding sites and most potent derivative 5c formed stable com-
plex with target protein. The cytotoxicity evaluation performed amides 3a–e and 5a–e purified by silica gel column
against murine melanoma (B16F10) cells showed that most potent chromatography.
derivative 5c is nontoxic at a dose of 12 mg/mL. The same dose of
5c was selected to determine antimelanogenic activity; the results
showed that it produced antimelenogenic effects in murine mel-
anoma (B16F10) cells. It is proposed that analogue 5c may serve
as lead structure to design highly potent antimelanogenic agents.
2
-[(4-Acetylphenyl)amino]-2-oxoethyl 3-hydroxybenzoate (3a)
ꢂ
Solid; reaction time, 24 h; yield 76%; melting point 166–168 C; R
f
ꢁ1
0
3
1
.52 (n-hexane:ethyl acetate 2:1), FTIR ꢀmax cm : 3336 (ꢁOH),
2
3
132 (ꢁNH), 2936 (sp C–H), 2835(sp C–H), 1731 (C¼O ester),
656 (C¼O amide), 1596 (C¼C aromatic), 1147 (C–O, ester); ESI-
4
. Experimental
þ
1
MS: m/z 336 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
d: 10.63 (s, 1H, ꢁNH), 9.90 (s, 1H, ꢁOH), 7.94 (d, J ¼ 8.2 Hz, 2H, H-
4
.1. Synthesis of title compounds 3a–e and 5a–e
-Actylaniline (0.01 mol) was reacted with chloroacetyl chloride
0.01 mol) in the presence of triethylamine (0.01 mol) in anhydrous
1
1
7, H-19), 7.74 (d, J ¼ 8.2 Hz, 2H, H-16, H-20), 7.48–7.43 (m, 2H, H-
, H-6), 7.35 (t, J ¼ 2.4 Hz, 1H, H-3), 7.09–7.07 (m, 1H, H-5), 4.96 (s,
4
(
1
3
ꢂ
2H, H-11, ꢁCH ), 2.54 (s, 3H, H-23, ꢁCH ); C NMR (100 MHz,
dichloromethane (20 ml) at 0 to ꢁ5 C. The reaction mixture was
stirred at room temperature for 4 h, extracted with ethyl acetate,
washed with dilute hydrochloric acid and 5% sodium hydroxide
2
3
DMSO
,
ppm) d: 196.98 (C-21, C¼O, ketone), 166.47 (C-12, C¼O,
amide), 165.91 (C-8, C¼O, ester), 158.03 (C-4), 143.28 (C-15), 132.4
solution. The organic layer was removed under reduced pressure (C-18), 130.8 (C-2), 130.4 (C-6), 130 (C-17,19), 121.1 (C-1), 120.6 (C-
to afford the intermediate
as solid with2 melting point 5), 120.2 (C-4), 118.9 (C-16,20), 116.3 (C-3), 60.6 (C-11, –CH ), 26.9
: C, 65.17; H, 4.97; Found C,
1
2
ꢂ
ꢁ1
3
1
54–155 C; FTIR ꢀmax cm : 3178 (–NH), 2922 (sp C–H), 2812 (sp
(C-23, –CH ); Anal Calcd For C17H15NO
3 5
C–H), 1634 (C¼O amide) and 1599 (C ¼ C aromatic). The title 65.14; H, 4.95.

8
M. RAFIQ ET AL.
2
-[(4-Acetylphenyl)amino]-2-oxoethyl 4-hydroxybenzoate (3b)
196.98 (C-22, C¼O,ketone), 166.50 (C-13, C¼O, amide), 165.96 (C-
9, C¼O, ester), 159.06 (C-3,5), 143.27 (C-16), 132.46 (C-19), 131.25
ꢂ
Solid; reaction time, 24 h; yield 82%; melting point 184–186 C; R
f
ꢁ
1
0
3
1
.53 (n-hexane:ethyl acetate 2:1), FTIR ꢀmax cm : 3365 (–OH), (C-18,20), 130.03 (C-1), 118.97 (C-17,21), 108.1 (C-4), 107.86 (C-2,6),
2
3
133 (–NH), 2983 (sp C–H), 2885(sp C–H), 1715 (C¼O ester), 63.57 (C-12, –CH ), 26.90 (C-23, –CH ); Anal Calcd For C H NO :
2
3
17 15
5
648 (C¼O amide), 1599 (C¼C aromatic), 1152 (C–O, ester); ESI- C, 62.00; H, 4.55; Found C, 61.97; H, 4.51.
þ
1
MS: m/z 336 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
d: 10.55 (s, 1H, –NH), 10.42 (s, 1H, –OH), 7.96 (d, J ¼ 7.2 Hz, 2H, H-
1
7, H-19), 7.88 (d, J ¼ 4.4 Hz, 2H, H-1, H-3), 7.73 (d, J ¼ 6.8 Hz, 2H, 2-[(4-Acetylphenyl)amino]-2-oxoethyl cinnamate (5a)
ꢂ
H-16, H-20), 6.89 (d, J ¼ 4.4 Hz, 2H, H-5, H-6), 4.92 (s, 2H, H-11, Solid; reaction time, 24 h; yield 78%; melting point 164–166 C; R
f
13
ꢁ1
–
1
5
1
6
6
CH
2
), 2.54 (s, 3H, H-22, –CH
3
); C NMR (100 MHz, DMSO
,
ppm); d 0.52 (n-hexane:ethyl acetate 2:1), FTIR ꢀ
cm : 3144 (–NH),
max
2
3
96.98 (C-21, C¼O, ketone), 166.73 (C-12, C¼O, amide), 165.69 (C- 2918 (sp C–H), 2820 (sp C–H), 1728 (C¼O ester), 1650 (C¼O
), 162.76 (C-8, C¼O, ester), 143.29 (C-15), 132.43 (C-18), 132.26 (C- amide), 1593 (C¼C aromatic), 1146 (C–O, ester); ESI-MS: m/z 346
þ
1
,3), 130.01 (C-17,19), 120.17 (C-2), 118.98 (C-16,20), 115.89 (C-4,6), [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm) d: 10.54 (s,
3.27 (C-11, –CH
5.17; H, 4.97; Found C, 65.13; H, 4.93.
2 3 5
), 26.9 (C-22, –CH ); Anal Calcd For C17H15NO : C,
1
1
H, H-14, –NH), 7.96 (d, J ¼ 2.0 Hz, 2H, H-18, H-20), 7.75 (m, 5H, H-
7, H-21, H-7, H-6, H-2), 7.46 (m, 3H, H-3, H-4, H-5), 6.78 (d,
J ¼ 16.0 Hz, 1H, H-8), 4.86 (s, 2H, H-12, –CH
2
), 2.54 (s, 3H, H-23,
ppm) d: 196.98 (C-22, C¼O,
ketone), 166.56 (C-13, C¼O, amide), 166.27 (C-9, C¼O, ester),
45.91 (C-7), 143.26 (C-16), 134.4 (C-19), 132.46 (C-1), 131.16 (C-
3,5), 130.01 (C-4), 129.8 (C-2,6), 119.02 (C-17,21), 117.86 (C-8),
3.19 (C-12, –CH ), 26.90 (C-23, –CH ); Anal Calcd For C H NO :
1
3
–
3 ,
CH ); C NMR (100 MHz, DMSO
2
-[(4-Acetylphenyl)amino]-2-oxoethyl 2,4-dihydroxybenzoate (3c)
ꢂ
Solid; reaction time, 24 h; yield 73%; melting point 227–229 C; R
f
1
ꢁ
1
0
3
1
.47 (n-hexane:ethyl acetate 2:1), FTIR ꢀmax cm : 3354 (–OH),
2
3
155 (–NH), 2951 (sp C–H), 2856(sp C–H), 1728 (C¼O ester),
637 (C¼O amide), 1597 (C¼C aromatic), 1158 (C–O, ester); ESI-
6
2
3
19 17
4
þ
1
C, 70.58; H, 5.01; Found C, 70.53; H, 4.98.
MS: m/z 352 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
d: 10.59 (s, 1H, H-7, –OH),10.54 (s, 1H, H-8, –OH), 10.46 (s, 1H, H-
1
5, –NH), 7.96 (d, J ¼ 8.4 Hz, 2H, H-18, H-20), 7.76 (d, J ¼ 8.4 Hz, 2H,
2
-[(4-Acetylphenyl)amino]-2-oxoethyl (E)-3-(4-hydroxyphenyl)acry-
H-17, H-21), 7.73 (d, J ¼ 8.8 Hz, 1H, H-3), 6.43 (dd, J 8.8 Hz,
1
¼
late (5b)
J
2
¼2.0 Hz, 1H, H-4), 6.34 (d, J ¼ 2.0 Hz, 1H, H-6), 4.96 (s, 2H, H-12,
ꢂ
13
Solid; reaction time, 24 h; yield 72%; melting point 226–228 C; R
f
–
1
9
1
1
CH ), 2.54 (s, 3H, H-23, –CH ); C NMR (100 MHz, DMSO ppm) d:
2
3
,
ꢁ1
0
3
1
.45 (n-hexane:ethyl acetate 2:1), FTIR ꢀ
cm : 3366 (–OH),
max
96.98 (C-22, C¼O, ketone), 168.66 (C-13, C¼O, amide), 166.36(C-
, C¼O, ester), 164.95 (C-5), 163.12 (C-1), 143.18 (C-16), 132.58 (C-
9), 132.51 (C-3), 130.03 (C-19,20), 119.01 (C-17,21), 108.97 (C-4),
2
3
173 (–NH), 2981 (sp C–H), 2867 (sp C–H), 1732 (C¼O ester),
639 (C¼O amide), 1593 (C¼C aromatic), 1147 (C–O, ester); ESI-
þ
1
MS: m/z 362 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
d: 10.57 (s, 1H, H-15, –NH), 10.11 (s, 1H, H-7, –OH), 7.95 (d,
J ¼ 2.0 Hz, 2H, H-19,21), 7.74 (d, J ¼ 2.0 Hz, 2H, H-18, H-22), 7.63 (m,
2 3
04.33 (C-2), 103.07 (C-6), 63.37 (C-12, –CH ), 26.91 (C-23, –CH );
Anal Calcd For C H NO : C, 62.00; H, 4.55; Found C, 61.95;
H, 4.52.
1
7
15
5
3
H, H-2, H-6, H-8), 6.83 (d, J ¼ 8.4 Hz, 2H, H-3, H-5), 6.52 (d,
J ¼ 16.0 Hz, 1H, H-9), 4.82 (s, 2H, H-13, –CH
2
), 2.54 (s, 3H, H-25,
1
3
2
-[(4-Acetylphenyl)amino]-2-oxoethyl 3,4-dihydroxybenzoate (3d)
–CH ); C NMR (100 MHz, DMSO ppm) d: 196.99 (C-23, C¼O,
3
,
ꢂ
Solid; reaction time, 24 h; yield 70%; melting point 205–207 C; R
f
ketone), 166.74 (C-14, C¼O, amide), 166.64 (C-10, C¼O, ester),
ꢁ
1
0
3
1
.46 (n-hexane:ethyl acetate 2:1), FTIR ꢀ
cm : 3314 (–OH),
max
160.56 (C-4), 146.13 (C-8), 143.32 (C-17), 132.42 (C-20), 130.96 (C-
2
3
156 (–NH), 2934 (sp C–H), 2843 (sp C–H), 1730 (C¼O ester),
2
1
C
,6), 129.99 (C-19,21), 125.46 (C-1), 118.99 (C-18,22), 116.31 (C-9),
13.87 (C-3,5), 63.0 (C-13, –CH ), 26.91 (C-25, –CH ); Anal Calcd For
642 (C¼O amide), 1592 (C¼C aromatic), 1148 (C–O, ester); ESI-
2
3
þ
1
MS: m/z 352 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
19 5
H17NO : C, 67.25; H, 5.01; Found C, 67.21; H, 4.97.
d: 10.56 (s, 1H, H-14, –NH), 9.88(s, 1H, H-7, –OH), 9.44 (s, 1H, H-8,
–
1
1
OH), 7.98 (d, J ¼ 5.2 Hz, 2H, H-18, H-20), 7.75 (d, J ¼ 5.2 Hz, 2H, H-
7, H-21), 7.43 (d, J ¼ 2.0 Hz, 1H, H-2), 7.40 (dd, J1 ¼ 8.4, J ¼2.0 Hz, 2-[(4-Acetylphenyl)amino]-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl)a-
H, H-6), 6.86 (d, J ¼ 8.0 Hz, 1H, H-5), 4.86 (s, 2H, H-12, –CH ), 2.51
2
2
crylate (5c)
1
3
(s, 3H, H-23, –CH
3
); C NMR (100 MHz, DMSO
,
ppm) d: 196.98 (C-
ꢂ
Solid; reaction time, 24 h; yield 70%; melting point 236–238 C; R
f
2
2, C¼O, ketone), 166.78 (C-13, C¼O, amide), 165.8(C-9, C¼O,
ꢁ1
0
3
1
.43 (n-hexane:ethyl acetate 2:1), FTIR ꢀ
cm : 3366 (–OH),
max
ester), 151.26(C-4), 145.6 (C-3), 143.32 (C-16), 132.42 (C-19), 130.01
C-18,20), 122.69 (C-1), 120.39(C-6), 118.96 (C-17,21), 117.01 (C-
2
3
154 (–NH), 2959 (sp C–H), 2844(sp C–H), 1726 (C¼O ester),
654 (C¼O amide), 1592 (C¼C aromatic), 1157 (C–O, ester); ESI-
(
5
),115.85 (C-2), 63.26 (C-12, –CH ), 26.90 (C-23, –CH ); Anal Calcd
þ
1
2
3
MS: m/z 378 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
5
For C17H15NO : C, 62.00; H, 4.55; Found C, 61.96; H, 4.52.
d: 10.48 (s, 1H, H-8, –OH), 10.22 (s, 1H, H-16, –NH), 9.93 (s, 1H, H-7,
–
OH), 7.96 (d, J ¼ 2.0 Hz, 2H, H-20, H-22), 7.85 (d, J ¼ 16.0 Hz, 1H,
H-9), 7.73 (d, J ¼ 2.0 Hz, 2H, H-19, H-23), 7.46 (d, J ¼ 8.4 Hz, 1H, H-
6), 6.5 (d, J ¼ 16.0 Hz, 1H, H-10), 6.38 (s, 1H, H-3), 6.3 (d, J ¼ 8.4 Hz,
2
-([(4-Acetylphenyl)amino]-2-oxoethyl 3,5-dihydroxybenzoate (3e)
ꢂ
solid; reaction time, 24 h; yield 74%; melting point 249–251 C; R
f
1
3
ꢁ
1
1H, H-5), 4.78 (s, 2H, H-14, –CH ), 2.54 (s, 3H, H-25, –CH );
C
0
3
1
.45 (n-hexane:ethyl acetate 2:1), FTIR ꢀmax cm : 3365 (–OH),
2
3
2
3
NMR (100 MHz, DMSO ppm) d: 196.98 (C-24, C¼O, ketone), 167.22
145 (–NH), 2949 (sp C–H), 2862 (sp C–H), 1744 (C¼O ester),
623 (C¼O amide), 1601 (C¼C aromatic), 1147 (C–O, ester); ESI-
,
(
C-15, C¼O, amide), 166.91(C-11, C¼O, ester), 161.69 (C-2), 159.21
þ
1
(C-4), 143.32 (C-18), 141.94 (C-9), 132.41 (C-21), 131.07 (C-6), 130
MS: m/z 352 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
d: 10.57 (s, 1H, H-14, –NH), 9.69 (s, 2H, H-7, H-8 –OH), 7.97 (d, (C-20,22), 118.99 (C-19,23), 116.31 (C-9), 113.01 (C-10), 112.59 (C-1),
), 25.6 (C-25, –CH );
: C, 64.22; H, 4.78; Found C, 64.18;
J ¼ 1.6 Hz, 2H, H-18, H-20), 7.74 (d, J ¼ 1.6 Hz, 2H, H-17, H-21), 6.90 108.38 (C-5), 102.96 (C-3), 62.86 (C-14, –CH
2
3
(
–
d, J ¼ 2.0 Hz, 2H, H-2, H-6), 6.49 (s, 1H, H-4), 4.92 (s, 2H, H-12, Anal Calcd For C19
H17NO
6
13
2 3 ,
CH ), 2.54 (s, 3H, H-23, –CH ); C NMR (100 MHz, DMSO ppm) d: H, 4.73.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
9
2
-[(4-Acetylphenyl)amino]-2-oxoethyl-(E)-3-(4-chlorophenyl)acrylate concentrations 0.0, 1.75, 3.5, 7, 14 and 28 mM, respectively, were
(
5d)
used. Substrate L-DOPA concentration was among 0.0625–2 mM in
all kinetic study. Pre-incubation and measurement time was the
.50 (n-hexane:ethyl acetate 2:1), FTIR ꢀmax cm : 3136 (–NH), same as discussed in mushroom tyrosinase inhibition assay proto-
ꢂ
solid; reaction time, 24 h; yield 78%; melting point 161–163 C; R
0
2
f
ꢁ
1
2
3
923 (sp C–H), 2855 (sp C–H), 1719 (C¼O ester), 1648 (C¼O col. The formation of DOPAchrome was continuously monitored at
amide), 1591 (C¼C aromatic), 1150 (C–O, ester); ESI-MS: m/z 382.5 475 nm for 5 min at a 30 s interval in the microplate reader after
þ
1
[
1
M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm) d: 10.52 (s, addition of enzyme. The inhibition type on the enzyme was
H, H-15, –NH), 7.96 (d, J ¼ 1.6 Hz, 2H, H-19, H-21), 7.82 (d, assayed by Lineweaver–Burk plots of inverse of velocities (1/V)
ꢁ1
J ¼ 1.6 Hz, 2H, H-18, H-22), 7.75 (m, 3H, H-2, H-6, H-8), 7.51 (d, versus inverse of substrate concentration 1/[S] mM , and the
J ¼ 6.4 Hz, 2H, H-3, H-5), 6.84 (d, J ¼ 16.0 Hz, 1H, H-9), 4.85 (s, 2H, inhibition constant Ki was determined by the Dixon plot of 1/V
13
H-13, –CH
2
), 2.54 (s, 3H, H-25, –CH
3
); C NMR (100 MHz, DMSO
,
versus inhibitor concentrations.
ppm) d: 196.97 (C-23, C¼O, ketone), 166.5 (C-14, C¼O, amide),
1
2
66.14 (C-10, C¼O, ester), 144.48 (C-8), 143.24 (C-17), 135.69 (C-
4
.4. Cytotoxicity and cell morphology
To test the safety level of 5c compound, MTT assay (3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) was per-
0), 133.37 (C-4), 132.47 (C-1), 130.69 (C-19,21), 130 (C-2,6), 129.48
(C-3,5), 119.01 (C-18,22), 118.69 (C-9), 60.21 (C-13, –CH ), 26.89 (C-
2
2
6
5, –CH
3 4
); Anal Calcd For C19H18NO : C, 63.42; H, 5.00; Found C,
3.38; H, 4.95.
43
5
formed . The murine melanoma (B16F10) cells (1.5 ꢃ 10 cells/ml)
were seeded in 24 well plates (SPL Korea) with DMEM medium at
ꢂ
3
7 C and 5% CO
2
incubator. Test compound 5c was dissolved in
2
-[(4-Acetylphenyl)amino]-2-oxoethyl
3-(4-hydroxyphenyl)propa-
DMSO and diluted with culture medium to get the final concen-
trations of 0 (control), 6, 12 and 24 mg/ml, and then added to the
cells for 24 h further incubation. The MTT assay was then con-
ducted, and results were calculated in percentage of control. In
addition, cellular images were taken by inverted fluorescent micro-
scope (Nikon, ECLIPSE, Tokyo, Japan) to access the 5c effect on
cell morphology.
noate (5e)
ꢂ
Solid; reaction time, 24 h; yield 74%; melting point 234–236 C; R
f
ꢁ
1
0
3
1
.45 (n-hexane:ethyl acetate 2:1), FTIR ꢀmax cm : 3356 (–OH),
2
3
149 (–NH), 2944 (sp C–H), 2856(sp C–H), 1721 (C¼O ester),
643 (C¼O amide), 1590 (C¼C aromatic), 1147 (C–O, ester); ESI-
þ
1
MS: m/z 364 [M þ 23] (M þ Na) ; H NMR (400 MHz, DMSO, ppm)
d: 10.43 (s, 1H, H-15, –NH), 9.18 (s, 1H, H-7, –OH), 7.95 (d,
J ¼ 2.0 Hz, 2H, H-19,21), 7.71 (d, J ¼ 2.0 Hz, 2H, H-18, H-22), 7.05 (d,
J ¼ 2.0 Hz, 2H, H-2, H-6), 6.68 (d, J ¼ 2.0 Hz, 2H, H-3, H-5), 4.70 (s,
4.5. Melanin content measurement
2
H, H-13, –CH ), 2.78 (t, J ¼ 7.6 Hz, 2H, H-8), 2.66 (t, J ¼ 1.2 Hz, 2H,
13
2
5
H-9), 2.54 (s, 3H, H-25, –CH
3
); C NMR (100 MHz, DMSO
,
ppm) d: For melanin measurement, melanoma (1.5 ꢃ 10 ) cells were
ꢂ
1
1
(
96.99 (C-23, C¼O, ketone), 172.49 (C-10, C¼O, ester), 166.52 (C- seeded in cell culture plates for 24 h at 37 C and 5%
4, C¼O, amide), 156.10 (C-4), 143.20 (C-17), 132.47 (C-20), 130.90 CO
incubator. Then cells were incubated with various concentra-
2
C-2,6), 129.59 (C-19,21), 119.10 (C-18,22), 115.58 (C-3,5), 63.00 (C- tions of 5c in cell culture medium for further 24 h. Later, assay for
3, –CH ), 35.66 (C-9), 29.83 (C-8), 26.90 (C-25, –CH
); Anal Calcd melanin measurement was performed following already reported
For C19 : C, 66.86; H, 5.57; Found C, 66.80; H, 5.52.
1
2
3
4
4
H
19NO
5
method . Briefly, the cells were trypsinised and centrifuged for
5
6
min at 1000 rpm. The cell pellet was dissolved in 1 N NaOH at
0 C for 1 h and melanin content was measured spectrophoto-
ꢂ
4
.2. Anti-tyrosinase activity
metrically at 405 nm wavelengths using a microplate reader
BioTek, synergy HT, Winooski, VT).
(
The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co., ST.
Louis, MO) was used for in vitro bioassays as described previously
39,40
with some modifications
. Briefly, 140 mL of phosphate buffer
4.6. Computational studies
(20 mM, pH 6.8), 20 mL of mushroom tyrosinase (30 U/mL) and
2
0 mL of the inhibitor solution were placed in the wells of a 96- 4.6.1. Retrieval of protein structure from PDB
well micro plate. After pre-incubation for 10 min at room tempera- The crystal structure of mushroom tyrosinase (PDBID: 2Y9X) was
ture, 20 mL of 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
retrieved from the Protein Data Bank (PDB) (http://www.rcsb.org).
The energy minimization of target protein was carried out by
employing conjugate gradient algorithm and Amber force field in
ꢂ
4
5
4
92 nm using a micro plate reader (OPTI Max, Tunable). Kojic acid
UCSF Chimera 1.10.1 . The stereo-chemical properties,
was used as a reference inhibitor and for negative tyrosinase
inhibitor phosphate buffer was used instead of the inhibitor solu-
tion. The extent of inhibition by the test compounds was
expressed as the percentage of concentration necessary to
achieve 50% inhibition (IC50). Each concentration was analysed in
three independent experiments run in triplicate. The IC50 values
were determined by the data analysis and graphing software
Origin 8.6, 64-bit.
46
Ramachandran graph and values of mushroom tyrosinase were
47
assessed by Molprobity server , while the hydrophobicity graph
48
was generated by Discovery Studio 4.1 Client . The protein archi-
tecture and statistical percentage values of helices, beta-sheets,
49
coils and turns were accessed by VADAR 1.8 .
4.6.2. Candidate structures
The synthesized compounds 3a–e and 5a–e were sketched in
drawing ACD/ChemSketch tool. The designed ligand molecules
were further visualized and minimized by UCSF Chimera 1.10.1 in
4.3. Kinetic analysis of the inhibition of mushroom tyrosinase
A series of experiments were performed to determine the inhib- PDB format. The drug assessment properties of these compounds
41,42
ition kinetics by following method
. Inhibitor 5c with concen- were access by various computational tools like Molinspiration
trations 0.0, 0.0005, 0.0022, 0.0045, 0.009, 0.018 mM and 5d with (http://www.molinspiration.com/) and Molsoft (http://www.molsoft.

1
0
M. RAFIQ ET AL.
com/). The Osiris Property Explorer (http://www.organic-chemistry.
org/prog/peo/) an online tool was used to calculate their possible
melanomas: selective review and hypothesis. Anticancer Res
1998;18:3709–15.
tumorigenic or mutagenic risks and drug-likeness values. Lipinski’s 12. Bro z_ yna AA, J oꢀ ꢀz wicki W, Roszkowski K, et al. Melanin content
rule of five was justified by using Molsoft and Molinspiraion tools.
Furthermore, different molecular properties such as molar refrac-
in melanoma metastases affects the outcome of radiother-
apy. Oncotarget 2016;7:17844.
tivity, density, surface tension and polarizability were also 13. Videira IF, Moura DF, Magina S. Mechanisms regulating mel-
accessed by chemsketch online.
anogenesis. An Bras Dermatol 2013;88:76–83.
1
4. Cooksey CJ, Garratt PJ, Land EJ, et al. Evidence of the indir-
ect formation of the catecholic intermediate substrate
responsible for the autoactivation kinetics of tyrosinase. J
Biol Chem 1997;272:26226–35.
4
.6.3. Molecular docking
Molecular docking experiment was employed on all synthesized
ligand molecules 3a–e and 5a–e against mushroom tyrosinase
1
1
5. Hearing VJ, Jim ꢀe nez M. Analysis of mammalian pigmentation
at the molecular level. Pigment Cell Res 1989;2:75–85.
6. Fairhead M, Th o€ ny-Meyer L. Bacterial tyrosinases: old
enzymes with new relevance to biotechnology. New
Biotechnol 2012;29:183–91.
7. B u€ y u€ kokuro gꢁ lu ME, G u€ l c¸ in I, Oktay M, K u€ frevio gꢁ lu OI. In vitro
50
using diverse PyRx tool . The grid box parametric dimension val-
_
ues (X ¼ 61.0781, Y ¼ 56.3001 and Z ¼ 63.1015) with spacing 1.0A
were adjusted to attain the finest binding conformational pose of
protein-ligand molecules. The maximum docking poses (100 num-
bers of run) for each docking were adjusted to obtain the best
docking complex with good conformational pose. All compounds
were docked separately against crystal structure of mushroom
tyrosinase and the obtained docked complexes were further eval-
uated on lowest binding energy (kcal/mol) values using Discovery
Studio (4.1) and UCSF Chimera 1.10.1.
1
antioxidant properties of dantrolene sodium. Pharmacol Res
2
001;44:491–4.
1
8. Shahidi F, Janitha PK, Wanasundara PD. Phenolic antioxi-
dants. Crit Rev Food Sci Nutr 1992;32:67–103.
_
€_
1
9. G u€ l c¸ in I, Oktay M, K u€ frevio gꢁ lu OI, Aslan A. Determination of
antioxidant activity of lichen Cetraria islandica (L) Ach. J
Ethnopharmacol 2002;79:325–9.
Disclosure statement
2
2
2
2
0. Yasui H, Sakurai H. Age-dependent generation of reactive
oxygen species in the skin of live hairless rats exposed to
UVA light . Exp Dermatol 2003;12:655–61.
1. Ito N, Hirose M, Fukushima S, et al. Studies on antioxidants:
their carcinogenic and modifying effects on chemical car-
cinogenesis. Food Chem Toxicol 1986;24:1071–82.
2. Ashraf Z, Rasool R, Hassan M, et al. Synthesis, bioevaluation
and molecular dynamic simulation studies of dexibuprofe-
n–antioxidant mutual prodrugs. Int J Mol Sci 2016;17:2151.
3. (a) Larik FA, Saeed A, Channar PA, et al. Design, synthesis,
kinetic mechanism and molecular docking studies of novel
No potential conflict of interest was reported by the authors.
References
1
.
Bonaventure J, Domingues MJ, Larue L. Cellular and molecu-
lar mechanisms controlling the migration of melanocytes
and melanoma cells. Pigment Cell Melanoma Res 2013;26:
316–25.
2
3
.
.
Borovansky J, Riley PA, editors. Melanins and melanosomes:
biosynthesis, structure. Physiological and pathological func-
tions. New York: John Wiley & Sons; 2011.
Lei TC, Virador V, Yasumoto KI, et al. Stimulation of melano-
blast pigmentation by 8-methoxypsoralen: the involvement
of microphthalmia-associated transcription factor, the pro-
tein kinase a signal pathway, and proteasome-mediated
degradation. J Invest Dermatol 2002;119:1341–9.
1
-pentanoyl-3-arylthioureas as inhibitors of mushroom tyro-
sinase and free radical scavengers. Eur J Med Chem 2017;
41:273.; (b) Saeed A, Ali Mahesar P, Ali Channar P, et al.
1
Synthesis, Molecular docking studies of Coumarinyl-pyrazo-
linyl substituted thiazoles as non-competitiveinhibitors of
mushroom tyrosinase. Bioorg Chem 2017;74:187.
2
2
4. Ashooriha M, Khoshneviszadeh M, Khoshneviszadeh M, et al.
4
.
Sviderskaya EV, Hill SP, Balachandar D, et al. Agouti signaling
protein and other factors modulating differentiation and
proliferation of immortal melanoblasts. Dev Dynamics 2001;
1
,2,3-Triazole-based kojic acid analogs as potent tyrosinase
inhibitors: design, synthesis and biological evaluation.
Bioorg Chem 2019;82:414–22.
221:373–9.
5. Menezes JCJMDS, Kamat SP, Cavaleiro JAS, et al. Synthesis
and antioxidant activity of long chain alkyl hydroxycinna-
mates. Eur J Med Chem 2011;46:773–7.
5.
6.
7.
8.
9.
Seiberg M. Keratinocyte–melanocyte interactions during
melanosome transfer. Pigment Cell Res 2001;14:236–42.
Lin JY, Fisher DE. Melanocyte biology and skin pigmentation.
Nature 2007;445:843.
Delevoye C. Melanin transfer: the keratinocytes are more
than gluttons. J Invest Dermatol 2014;134:877–9.
Meredith P, Riesz J. Radiative relaxation quantum yields for
synthetic eumelanin. Photochem Photobiol 2004;79:211–6.
26. Miliovsky M, Svinyarov I, Mitrev Y, et al. A novel one-pot syn-
thesis and preliminary biological activity evaluation of cis-
restricted polyhydroxy stilbenes incorporating protocate-
chuic acid and cinnamic acid fragments. Eur J Med Chem
2013;66:185–92.
Vandamme M, Robert E, Pesnel S, et al. Antitumor effect of 27. Ashraf Z, Rafiq M, Seo S-Y, et al. Synthesis, kinetic mechan-
plasma treatment on U87 gliomaxenografts: preliminary
results. Plasma Process Polym 2010;7:264–73.
0. Bro z_ yna AA, J oꢀ ꢀz wicki W, Carlson JA, Slominski AT.
ism and docking studies of vanillin derivatives as inhibitors
of mushroom tyrosinase. Bioorg Med Chem 2015;23:
5870–80.
1
Melanogenesis affects overall and disease-free survival in 28. Ashraf Z, Rafiq M, Seo S-Y, et al. Kinetic and in silico studies
patients with stage III and IV melanoma. Hum Pathol 2013;
4:2071–4.
of novel hydroxy-based thymol analogues as inhibitors of
mushroom tyrosinase. Eur J Med Chem 2015;98:203–11.
4
1
1. Slominski A, Paus RA, Mihm MC. Inhibition of melanogenesis 29. Chen Z, Cai D, Mou D, et al. Design, synthesis and biological
as an adjuvant strategy in the treatment of melanotic evaluation of hydroxy-or methoxy-substituted 5-benzylidene

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
11
(thio) barbiturates as novel tyrosinase inhibitors. Bioorg Med 40. Wang Q, Qiu L, Chen XR, et al. Inhibitory effects of phloridzin
Chem 2014;22:3279–84.
dihydrate on the activity of mushroom (Agaricus bisporus)
3
0. Huang HC, Chang TY, Chang LZ, et al. Inhibition of melano-
genesis versus antioxidant properties of essential oil
extracted from leaves of Vitex negundo Linn and chemical
composition analysis by GC-MS. Molecules 2012;17:3902–16.
1. Lee J, Jung E, Park J, et al. Glycyrrhizin induces melanogene-
sis by elevating a cAMP level in B16 melanoma cells. J
Invest Dermatol 2005;124:405–11.
tyrosinase. Bioorg Med Chem 2007;15:1568–71.
4
4
1. Chiari ME, Vera DM, Palacios SM, Carpinella MC. Tyrosinase
inhibitory activity of a 6-isoprenoid-substituted flavanone
isolated from Daleaelegans. Bioorg Med Chem 2011;19:
3
3
3
474–82.
2. Rafiq M, Saleem M, Hanif M, et al. Synthesis, structural eluci-
dation and bioevaluation of 4-amino-1, 2, 4-triazole-3-thio-
ne’s Schiff base derivatives. Arch Pharmacal Res 2016;39:
2. Kim A, Yang Y, Lee MS, et al. NDRG2 gene expression in
B16F10 melanoma cells restrains melanogenesis via inhib-
ition of Mitf expression. Pigment Cell Melanoma Res 2008;
1
61–71.
4
3. Mosmann T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 1983;65:55–63.
4. Chung KW, Park YJ, Choi YJ, et al. Evaluation of in vitro and
in vivo anti-melanogenic activity of a newly synthesized
strong tyrosinase inhibitor (E)-3-(2, 4 dihydroxybenzylidene)
pyrrolidine-2, 5-dione (3-DBP). Biochim Biophys Acta
(BBA)–General Subjects 2012;1820:962–9.
21:653–64.
3
3. Hamid MA, Sarmidi MR, Park CS. Mangosteen leaf extract
increases melanogenesis in B16F1 melanoma cells by stimu-
lating tyrosinase activity in vitro and by up-regulating tyro-
sinase gene sinase gene expression. Int J Mol Med 2012;29:
4
209–17.
3
3
3
3
4. Jung GD, Yang JY, Song ES, Park JW. Stimulation of melano-
genesis by glycyrrhizin in B16 melanoma cells. Exp Mol Med
2001;33:131.
45. Pettersen EF, Goddard TD, Huang CC, et al. UCSF Chimera-a
visualization system for exploratory research and analysis. J
Comput Chem 2004;25:1605–12.
5. Kadam RU, Roy N. Recent trends in drug-likeness prediction:
a comprehensive review of in silico methods. Indian J
Pharm Sci 2007;69:609.
6. Tian S, Wang J, Li Y, et al. The application of in silico drug-
likeness predictions in pharmaceutical research. Adv Drug
Deliv Rev 2015;86:2–10.
7. Chen WC, Tseng TS, Hsiao NW, et al. Discovery of highly
potent tyrosinase inhibitor, T1, with significant anti-melano-
genesis ability by zebrafish in vivo assay and computational
molecular modeling. Sci Rep 2015;5:7995.
4
4
4
6. Lovell SC, Davis IW, Arendall WB, et al. Structure validation
by Calpha geometry: phi,psi and Cbeta deviation. Proteins
2
003;50:437–50.
7. Chen VB, Arendall WB, Headd JJ, et al. MolProbity: all-atom
structure validation for macromolecular crystallography. Acta
Crystallograph Sect D: Biol Crystallogr 2010;66:12–21.
8. Pramanik S, Roy K. Exploring QSTR modeling and toxico-
phore mapping for identification of important molecular
features contributing to the chemical toxicity in Escherichia
coli. Toxicol In Vitro 2014;28:265–72.
9. Willard L, Ranjan A, Zhang H, et al. VADAR: a web server for
quantitative evaluation of protein structure quality. Nucleic
Acids Res 2003;31:3316–9.
3
3
8. Ashraf Z, Rafiq M, Nadeem H, et al. Carvacrol derivatives as
mushroom tyrosinase inhibitors; synthesis, kinetics mechan-
ism and molecular docking studies. PLoS One 2017;12:
e0178069.
9. Ashraf Z, Rafiq M, Seo SY, et al. Design, synthesis and bioe-
valuation of novel umbelliferone analogues as potential
4
mushroom tyrosinase inhibitors. J Enzyme Inhib Med Chem 50. Dallakyan S, Olson AJ. Small-molecule library screening by
2015;30:874–83. docking with PyRx. Meth mol biol. 2015;1263:243–50.