Methoxy-substituted tyramine derivatives synthesis, computational studies and tyrosinase inhibitory kinetics

Article abstract of DOI:10.3390/molecules26092477

Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that displayed in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identifying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC₅₀ of 0.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC₅₀ of 2.1 nM compared to the positive control, kojic acid IC₅₀ 16700 nM. Results of human tyrosinase inhibitory activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6% and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhibition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (K_i 0.093 nM) and non-competitive inhibition for Ph6 (K_i 2.3 nM) revealed from Lineweaver–Burk plots. In silico docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further development of tyrosinase inhibitors.

Full text of DOI:10.3390/molecules26092477

molecules
Article
Methoxy-Substituted Tyramine Derivatives Synthesis,
Computational Studies and Tyrosinase Inhibitory Kinetics
Yasir Nazir 1 , Hummera Rafique , Naghmana Kausar , Qamar Abbas , Zaman Ashraf *,
,2
3
3
4
2,
Pornchai Rachtanapun 5
,6
, Kittisak Jantanasakulwong
5,6
and Warintorn Ruksiriwanich 1,5,7,*
1
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University,
Chiang Mai 50200, Thailand; ynchem@yahoo.com
Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan
Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan; humera_rafique@uoe.edu.pk (H.R.);
naghmana.kausar@uog.edu.pk (N.K.)
2
3
4
5
Department of Biology, College of Science, University of Bahrain, Sakhir 32038, Bahrain;
qamar.abbas.qau@gmail.com
Cluster of Agro Bio-Circular Green Industry (Agro BCG), Chiang Mai University,
Chiang Mai 50100, Thailand; pornchai.r@cmu.ac.th (P.R.); Jantanasakulwong.k@gmail.com (K.J.)
School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand
Cluster of Research and Development of Pharmaceutical and Natural Products Innovation for Human or
Animal, Chiang Mai University, Chiang Mai 50200, Thailand
6
7
*
Correspondence: zaman.ashraf@aiou.edu.pk (Z.A.); warintorn.ruksiri@cmu.ac.th (W.R.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Targeting tyrosinase for melanogenesis disorders is an established strategy. Hydroxyl-
substituted benzoic and cinnamic acid scaffolds were incorporated into new chemotypes that dis-
played in vitro inhibitory effects against mushroom and human tyrosinase for the purpose of identi-
fying anti-melanogenic ingredients. The most active compound 2-((4-methoxyphenethyl)amino)-2-
oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9), inhibited mushroom tyrosinase with an IC50 of
Citation: Nazir, Y.; Rafique, H.;
Kausar, N.; Abbas, Q.; Ashraf, Z.;
Rachtanapun, P.; Jantanasakulwong,
K.; Ruksiriwanich, W. Methoxy-
Substituted Tyramine Derivatives
Synthesis, Computational Studies
and Tyrosinase Inhibitory Kinetics.
Molecules 2021, 26, 2477. https://
doi.org/10.3390/molecules26092477
0
.059 nM, while 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) had an IC50 of 2.1 nM
compared to the positive control, kojic acid IC50 16700 nM. Results of human tyrosinase inhibitory
activity in A375 human melanoma cells showed that compound (Ph9) and Ph6 exhibited 94.6%
and 92.2% inhibitory activity respectively while the positive control kojic acid showed 72.9% inhi-
bition. Enzyme kinetics reflected a mixed type of inhibition for inhibitor Ph9 (K 0.093 nM) and
i
Academic Editor:
non-competitive inhibition for Ph6 (K 2.3 nM) revealed from Lineweaver–Burk plots. In silico
i
Constantinos Athanassopoulos and
Carlo Siciliano
docking studies with mushroom tyrosinase (PDB ID:2Y9X) predicted possible binding modes in
the catalytic site for these active compounds. Ph9 displayed no PAINS (pan-assay interference
compounds) alerts. Our results showed that compound Ph9 is a potential candidate for further
development of tyrosinase inhibitors.
Received: 21 March 2021
Accepted: 21 April 2021
Published: 23 April 2021
Keywords: tyramine derivatives; tyrosinase inhibitors; inhibitory activity; enzyme kinetics mecha-
nism; computational studies
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Tyrosinase is a copper containing metallo-enzymes and is widely distributed in nature,
from bacteria to plants and humans. It facilitates the o-hydroxylation of monophenols to
catechols following oxidation to quinones [1]. Its physiological function is the conversion
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Licensee MDPI, Basel, Switzerland.
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conditions of the Creative Commons
Attribution (CC BY) license (https://
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L-tyrosine into dopaquinone, which constitutes melanin biosynthesis in melanosomes [
L-tyrosine or phosphorylated isomers of L-3,4-dihydroxyphenylalanine (L-DOPA) serve
as key molecules in the regulation of the melanin biosynthesis [ ]. The color of hu-
man skin, hair and eyes is determined by the distribution patterns of melanin in ker-
atinocytes [ ]. Several factors such as UV exposure, -melanocyte-stimulating hormone
and melanocortin 1 receptor have been reported to modulate melanogenesis [ 10]. The
melanogenesis process is a potential cellular hazard and is limited to melanosomes in
2,3].
4–6
7,8
α
9,
4
.0/).
Molecules 2021, 26, 2477. https://doi.org/10.3390/molecules26092477
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2477
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melanocytes, which synthesize pigments and transfer them to recipient cells [11]. The aber-
rant proliferation of melanocytes arises from a type of skin cancer called melanoma [12,13].
Malignant melanocytes tend to display up-regulated melanogenesis and malfunctioning
melanosomes. Therefore, controlling a tyrosinase-dependent mechanism of melanogenesis
might be the basis for a potential anti-melanoma therapy. Specific tyrosinase inhibitors like
kojic acid, arbutin, azelaic acid, hydroquinone and electron-rich phenol and other chem-
ical scaffolds like resorcinol [14
–
16], biaryl,
β-phenyl-α,β-unsaturated carbonyl [17–20],
and imidazolethione-containing inhibitors [21
,
22] have been tested in the search for their
better tyrosinase ability to block the overproduction of melanin [23]. Thiosemicarbazone
derivatives have been reported to possess mushroom tyrosinase inhibitory activity [24].
Inulavosin and its benzo-derivatives effectively inhibited the melanogenesis of the cells [25].
Arbutin and kojic acid were among the most popular specific tyrosinase inhibitors for
topical application to the surface of the human skin because of beneficial effects on UV-
irritated and dry skin by reducing the UV impact before serious side effects came to limit
their human use [26]. Therefore, it is essential to find nontoxic skin-whitening agents for
meeting the social needs in terms of safe cosmetic practices and medical hyperpigmenta-
tion treatments.
It should be highlighted that targeting tyrosinase to treat melanogenesis disorders
is one of many possible approaches, due to the complex biochemical reaction involved
in the melanin synthesis [27]. Tyrosinase polymerizes phenolic substrate through oxida-
tive polymerization reactions by self-polymerization or cross-polymerization with other
compounds [28]. Rafiq et al., 2019 and Nazir et al., 2020 reported that cinnamic acid and
benzoic acid analogues possessing a substituted phenyl ring exhibited tyrosinase inhibitory
activity [29,30]. It has already been well established that phenolic compounds such as
caffeic acid, ferulic acid and p-coumaric acid play a vital role in tyrosinase inhibitory activ-
ity as the natural substrates tyrosine and L-DOPA also bear the phenolic hydroxyls [31].
Amides derived from coupling the coumaric acid and its derivatives with tyramine have
been synthesized and have been studied in different therapeutic areas such as anticancer
agents [32–34], antioxidants [35–38], and as selective N-methyl-D-aspartate (NMDA) re-
ceptor antagonists [39]. Roh et al., have reported melanogenesis-inhibitory effects of the
naturally occurring N-feruloyl serotonin and N-(p-coumaroyl) serotonin, isolated from
Safflower (Carthamus tinctorius L.) [40]. Hemp (Cannabis sativa L. var. sativa) contains a vast
number of constituents and the terpenoids and flavonoids present in Hemp extract may be
responsible for its tyrosinase inhibitory activity [41]. Tyrosinase inhibition activity of the
Hemp leaf extract (IC50 0.049
acid (IC50 0.005 mg/mL) [42]. Celastrus paniculatus seed oil (CPSO) has been reported to
exhibit more potent anti-tyrosinase activity (IC50 0.04 mg/mL) than 2-hydroxypropyl
cyclodextrin inclusion complex CPSO-HP CD (IC50 0.19 mg/mL) due to the presence of
±
0.02 mg/mL) is about 9.8 folds lower than standard kojic
β-
β
tocopherol [43]. The rice bran extract contains various saturated and unsaturated fatty
acids such as palmitic, stearic, linolenic and oleic acids and their ester derivatives. The
rice bran loaded in β-cyclodextrin at 1 mg/mL demonstrated the tyrosinase inhibitory
activity of 1.25 fold of the control and 0.52 folds of the standard theophylline [44]. The key
compounds, p-methylanisole, methyl salicylate, 2-phenyl ethyl alcohol, nerolidol, methyl
cinnamate, 3-hydroxy-4-phenyl-2-butanoate, cinnamyl alcohol and 2-phenyl ethyl ben-
zoate present in ethanol extract of the medicinal flower (Mimusops elengi L.) gave almost
no tyrosinase inhibition activity (IC50 > 200 mg/mL) at all concentrations in comparison
with kojic acid (IC50 0.049 mg/mL) in the supercritical carbon dioxide fluid extraction
(
scCO ) [45
,
46]. Moreover, quercetagetin found in the ethyl acetate (EA) extract of flowers
of African marigold (Tagetes erecta L.) exhibited tyrosinase inhibitory activity on L-tyrosine
IC50 89.31 g/mL), higher than – and –arbutins (IC50 157.77 and 222.35 g/mL) and
slightly higher (IC50 128.41 g/mL) than ellagic acid (IC50 151.1 g/mL) when using
2
(
µ
α
β
µ
µ
µ
L-DOPA as substrate [47]. In continuation of our research for finding potent tyrosinase
inhibitors, we synthesized methoxy-substituted tyramine-based amides by coupling dif-
ferent hydroxyl-substituted benzoic and cinnamic acids to explore their anti-tyrosinase

Molecules 2021, 26, 2477
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activity and to recognize how these synthesized compounds interact with the active site
of the enzyme to search for more potent and novel compounds. These compounds were
explored for their effect as inhibitors of human melanocyte-tyrosinase and mushroom
tyrosinase and their ability as melanogenesis suppressers. The initial screening as an
anti-melanogenic agent was carried out using mushroom and human (melanoma cells)
tyrosinase. The in vitro tyrosinase inhibitory activity of the synthesized compounds (Ph1–
Ph10) was evaluated both experimentally and in silico. Due to the absence of a high-
resolution crystal structure of the human tyrosinase enzyme, mushroom tyrosinase has
frequently served as a model to assess the inhibitory activity of compounds with the fact
that both proteins share high sequence similarity in their active core region [48]. The in
silico molecular docking of all the synthesized compounds (Ph1–Ph10) was performed
against the mushroom tyrosinase crystal structure (PDB ID: 2Y9X) to determine the role of
different functionalities in the formation of a ligand–protein complex. All these compounds
(Ph1–Ph10) passed the PAINS (pan-assay interference compounds) filter, except for 2-((4-
methoxyphenethyl)amino)-2-oxoethyl 3,4-dihydroxybenzoate (Ph4) [49]. The most potent
derivatives, 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate (Ph5), 2-((4-
methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) and 2-((4-methoxyphenethyl)amino)-
2
-oxoethyl (E)-3-(2,4-dihydroxyphenyl) acrylate (Ph9) were selected for further characteri-
zation of their biological effects in tyrosinase inhibitory kinetics.
2. Results and Discussion
2.1. Synthesis of 4-Methoxyphenyl Ethyl Chloroacetamide (2)
The intermediate 2-(4-methoxyphenyl)ethan-1-aminechloroacetate(2) was synthesized,
purified by silica gel column and characterized by melting point and Fourier Transform
◦
Infrared (FTIR) spectroscopy. Melting point 99–101 C; reaction time, 3 h; yield, 85%;
Retardation factor (R ) 0.58 (n-hexane:ethyl acetate 3:1); FTIR, maximum wave number,
f
−
1
2
3
the maximum velocity (νmax)cm :3345 (-NH), 2943 (sp C-H), 2834 (sp C-H), 1656 (C=O
amide), 1589 (C=C aromatic), 1162 (C-O).
2.1.1. In Vitro Tyrosinase Inhibition Assay
In vitro mushroom tyrosinase inhibitory assay of all the synthesized inhibitors (Ph1–
Ph10) was performed and found that most of the title amide derivatives showed better
tyrosinase inhibition than reference drug kojic acid. The hydroxylated amides with cin-
namic acid moiety showed higher tyrosinase inhibition than those with benzoic acid
analogues. The IC50 value of inhibitor Ph9 bearing 2,4-dihydroxyl-substituted cinnamic
acid functionality was 0.000059
substituted benzoic acid moiety displayed an IC50 value of 0.231
compared with the standard drug kojic acid with IC50 16.7 M (Table 1). In vitro human
tyrosinase inhibition assay showed that compound Ph9 exhibited 94.59% inhibition at
g/mL (Table 1). This shows that the amide Ph9 has a greater potential to inhibit
µM, whereas the inhibitor Ph5 bearing 3,5-dihydroxy-
µM which is excellent as
µ
50 µ
human tyrosinase compared to standard kojic acid (72.94% inhibitory capacity). Bioassay
results revealed that the substitution of hydroxyl groups at cinnamic acid phenyl ring is the
decisive factor for inhibitory activity. Hence, we propose that substitution of the hydroxyl
group at ortho, para position on phenyl ring in Ph9 hampers the inhibitor to interact well
within the enzymatic pocket of tyrosinase. The kinetics and docking studies facilitated us
to explore the binding mode and amino acid residual interactions between the tyrosinase
and synthesized inhibitors.
2.1.2. Kinetics Mechanism
Among the synthesized Ph derivatives Ph5, Ph6 and Ph9 were selected to determine
their inhibition type and inhibition constants on mushroom tyrosinase activity. The po-
tential of these derivatives to inhibit the free enzyme and enzyme–substrate complex was
determined in terms of enzyme inhibitor (EI) and enzyme–substrate-inhibitor (ESI) con-
stants respectively. The kinetic studies of the enzyme by the Lineweaver–Burk plot of 1/V

Molecules 2021, 26, 2477
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versus 1/[S] in the presence of different inhibitor concentrations gave a series of straight
lines as shown in Figures 1A, 2A and 3A. The results of Figures 1A and 3A showed that
compounds Ph5 and Ph9 intersected within the second quadrant. The analysis showed
m
that (Vmax) decreased with increasing Michaelis constant (K ) in the presence of increasing
concentrations of compounds Ph5 and Ph9 respectively. This behavior of compounds Ph5
and Ph9 indicated that it inhibits tyrosinase by two different pathways; competitively
forming enzyme inhibitor (EI) complex and interrupting enzyme–substrate-inhibitor (ESI)
complex in a non-competitive manner. The secondary plots of slope versus concentration
of compounds Ph5 and Ph9 showed EI dissociation constants (K ) Figures 1B and 3B,
i
0
while ESI dissociation constants (K ) were shown by secondary plots of intercept versus
i
concentration of compounds Ph5 and Ph9 Figures 1C and 3C. A lower value of K than
i
0
K pointed out stronger binding between enzyme and compounds Ph5 and Ph9 which
i
suggested preferred competitive over non-competitive manners (Table 2). Meanwhile, in
the case of compound Ph6, the Lineweaver–Burk plot gave a family of straight lines, all
of which intersected at the same point on the x-axis Figure 2A. The analysis showed that
m
1/Vmax decreased to a new value while that of K remains the same because of the increase
in the concentrations of compound Ph6. This behavior indicated that compound Ph6
inhibits tyrosinase non-competitively to form an enzyme inhibitor (EI) complex. The sec-
ondary plot of slope against the concentration of Ph6 showed EI dissociation constant (Ki)
Figure 2B. It is worth mentioning here that the enzyme inhibitory kinetics were performed
with a more efficient natural substrate L-DOPA (L-3,4-dihydroxyphenylalanine). Ph5 is a
3
,5-dihydroxyl-substituted benzoic acid derivative and Ph9 is 2,4-dihydroxyl-substituted
cinnamic acid derivative whereas, Ph6 is an unsubstituted cinnamic acid analogue. As
the substrate L-DOPA bears 3,4-dihydroxy substitution, this could be the reason that Ph5
and Ph9 bearing hydroxyl substituent gave mixed-type inhibition, whereas Ph6 bearing no
hydroxyl substituent was a non-competitive inhibitor.
Figure 1. Lineweaver–Burk plots for inhibition of tyrosinase in the presence of 2-((4-methoxyphenethyl)amino)-2-oxoethyl
,5-dihydroxybenzoate (Ph5). ( ) Concentrations of Ph5 were 0, 0.2 and 0.4 M, respectively. Substrate L-DOPA con-
centrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. ( ) The insets represent the plot of the slope and ( ) of the
3
A
µ
B
C
vertical intercepts versus inhibitor Ph5 concentrations to determine inhibition constants. The lines were drawn using linear
least-squares fit.

Molecules 2021, 26, 2477
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Table 1. The substitution pattern and inhibitory effects of Ph derivatives (Ph1–Ph10) on mushroom tyrosinase and human
tyrosinase (From melanoma cells).
Substitution Pattern
Mushroom Tyrosinase
Human Tyrosinase
(% Inhibition)
Compound
Yield (%)
IC50 ± SEM (µM)
R1
R2
Ph1
Ph2
3-OH
4.065 ± 0.154
0.463 ± 0.017
0.839 ± 0.0381
0.329 ± 0.021
0.231 ± 0.0078
0.00204 ± 0.000087
0.1042 ± 0.0038
0.923 ± 0.053
75.590 ± 5.653
73.049 ± 5.346
79.710 ± 6.61
82.17 ± 7.045
64.8 ± 3.84
92.24 ± 7.985
85.6 ± 6.041
86.27 ± 6.542
94.59 ± 8.071
79.23 ± 5.952
72.94 ± 5.125
78
82
76
71
76
84
75
78
76
82
4-OH
Ph3
2,4-di-OH
3,4-di-OH
3,5-di-OH
Ph4
Ph5
Ph6
-H
2-OH
Ph7
Ph8
4-OH
Ph9
2,4-di-OH
4-Cl
0.00005950 ± 0.00000225
0.4520 ± 0.0171
16.69 ± 2.6
Ph10
Kojic Acid
Ph means target phenolic products; Ph1: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3-hydroxybenzoate; Ph2: 2-((4-methoxyphen-
ethyl)amino)-2-oxoethyl 4-hydroxybenzoate; Ph3: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 2,4-dihydroxybenzoate; Ph4: 2-((4-methoxy-
phenethyl)amino)-2-oxoethyl 3,4-dihydroxybenzoate; Ph5: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate; Ph6: 2-
(
(4-methoxyphenethyl)amino)-2-oxoethyl cinnamate; Ph7: 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2-hydroxyphenyl)acrylate;
Ph8: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(4-hydroxyphenyl)acrylate; Ph9: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-
2,4-dihydroxyphenyl)acrylate; Ph10: 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(4-chlorophenyl)acrylate. R1 and R2 are specified
(
substitutents on the final products whereas, di is the number of substituents. SEM is standard error mean.
Figure 2. Lineweaver–Burk plots for inhibition of tyrosinase in the presence of 2-((4-methoxyphenethyl)amino)-2-oxoethyl
cinnamate (Ph6). (
A
) Concentrations of Ph6 were 0, 0.001 and 0.002
µM, respectively. Substrate L-DOPA concentrations
were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. (B) The insets represent the plot of the slope. The lines were drawn using
linear least-squares fit.

Molecules 2021, 26, 2477
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Figure 3. Lineweaver–Burk plots for inhibition of tyrosinase in the presence of 2-((4-methoxyphenethyl)amino)-2-
oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (Ph9). ( ) Concentrations of Ph9 were 0, 0.00003, 0.00006 and 0.00012 M,
respectively. Substrate L-DOPA concentrations were 0.125, 0.25, 0.5, 1 and 2 mM, respectively. ( ) The insets represent the
plot of the slope and ( ) of the vertical intercepts versus inhibitor Ph9 concentrations to determine inhibition constants. The
A
µ
B
C
lines were drawn using linear least-squares fit.
Table 2. Kinetic parameters of the mushroom tyrosinase for L-DOPA activity in the presence of different concentrations of
compound Ph5, Ph6 and Ph9.
Vmax
∆A/s)
0
Code
Dose (µM)
Km (mM)
Inhibition Type
K (µM)
i
K (µM)
i
(
1.251 × 10−
8.266 × 10−
5.242 × 10−
5
6
6
5
6
6
6
6
6
6
0
.0
0.181
0.238
0.263
0.232
0.232
0.238
0.192
0.227
0.238
0.25
Ph5
0.2
Mixed-Inhibition
0.219
0.475
-
0
.4
.0
−
0
1.272 × 10
7.060 × 10−
6.929 × 10−
4.848 × 10−
Ph6
Ph9
0
0
.001
.002
Non-competitive
0.0023
0
.0
−
0
0
0
.00003
.00015
.00012
4.666 × 10
-
Mixed-Inhibition
0.000093
4.285 × 10−
2.666 × 10−
0
V
max = maximal rate; K
m
= Michaelis constant; K = enzyme inhibitor constant; K = enzyme substrate inhibitor constant. Ph5: 2-((4-
i
i
methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate; Ph6: 2-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate; Ph9: 2-((4-
methoxyphenethyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate.
2.1.3. Molecular Docking Studies
Computational docking studies were undertaken with Maestro Schrodinger (11.2)
to predict the most favorable binding mode of the compounds (Ph1–Ph10) inside the
binding pocket of mushroom tyrosinase (PDB ID:2Y9X). The crystal structure of mushroom
tyrosinase (PDB ID:2Y9X) containing a tropolone ligand in the enzymatic pocket, was
retrieved from RCSB Protein Data Bank (RCSB PDB) (http://www.rcsb.org, accessed date

Molecules 2021, 26, 2477
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15 March 2021). To determine the amino acids of the protein involved in binding with the
inhibitor, the selected poses of most active compounds Ph5, Ph6 and Ph9 were visualized.
Each inhibitor binds the protein in a different manner due to different substitution
patterns. The reference inhibitor kojic acid interacts tyrosinase binuclear catalytic site
with carbonyl group closer to cupper (400, 401) ions and side-chain hydroxyl is picking
up a hydrogen bond (1.73 Å) with amino acid residue Met280. The best ligand binding
poses of Ph5, Ph6 and Ph9 were selected based on binding energy (
−
5.339 Kcal/mol,
−
4.543 Kcal/mol and 7.93Kcal/mol) (Table 3), number and nature of amino acid units
−
involved in the binding, type of interaction and inter-molecular distance between the
ligand atoms and the amino acid residues. For the most active compound Ph9 (green
in Figure 4d), both the ortho phenolic and para methoxy phenyl moieties are picking up
H-bond interactions (2.93 Å, 3.04 Å) with side-chain residues Asn260 and Asn81 with
bond lengths of 2.93 Å and 3.04 Å, respectively. The para phenolic group is interacting by
Cu400 (3.68 Å) and Cu401 (3.10 Å) and this ring is further stabilized by π–π stacking with
His85, His263 and His259. The amide hydrogen of Ph9 is predicted to form a hydrogen
bond (3.36 Å) with the nitrogen of the neighboring His244. For the most active compound
Ph5 from benzoic acid derivatives (Figure 4b), the para methoxy moiety is picking two
strong H-bond interactions with side-chain residues Asn81 (2.07 Å) and His85 (2.31 Å)
respectively and this ring is additionally stabilized by π–π stacking with His85. The amide
hydrogen of the inhibitor Ph5 interacts through a hydrogen bond (2.40 Å) with the nitrogen
of the neighboring His244. The large size and planner shape of Ph6 prevents it from
entering the narrow binuclear copper-binding site and bounds to a shallow area at the
surface of the enzyme pocket. However, a hydrogen bond (2.31 Å) is observed between
the amide carbonyl and side-chain Asn81. Both para methoxy phenyl and cinnamic acid
phenyl groups form π–π interaction with residues His85 and Phe192, respectively. The
described binding mode would allow the substrate to enter the pocket, providing a useful
non-competitive model. Our in vitro enzyme inhibitory assay showed a non-competitive
inhibition for Ph6. The Ph6 is an unsubstituted cinnamic acid derivative and does not bear
any hydroxyl substituent which is very important in ligand binding through hydrogen
bonding, Coulomb and van der Waals interactions (Figure 4c). His259, His263, His296,
His61, His85, His94 are conserved residues with Cu (400) and Cu (401) ions in the core
region. The residues that played an important role in stabilizing the protein inhibitor
complex are Asn260, Asn81, His85, His244 and Met280 and these findings are in accordance
with the studies by Pintus et al. [50].
Table 3. Docking energies of synthesized amide derivatives (Ph1–Ph10) against mushroom tyrosinase,
PDB ID 2Y9X.
Compound
Docking Energy (Kcal/mol), PDB ID (2Y9X)
Ph1
Ph2
Ph3
Ph4
Ph5
Ph6
Ph7
Ph8
Ph9
−5.492
−4.070
−5.850
−6.830
−5.339
−4.543
−5.890
−5.511
−7.930
−5.516
−4.149
Ph10
Kojic Acid
Ph means target phenolic products; Ph1: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3-hydroxybenzoate;
Ph2: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 4-hydroxybenzoate; Ph3: 2-((4-methoxyphenethyl)amino)-
2
-oxoethyl 2,4-dihydroxybenzoate; Ph4: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,4-dihydroxybenzoate;
Ph5: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate; Ph6: 2-((4-methoxyphenethyl)amino)-2-
oxoethyl cinnamate; Ph7: 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2-hydroxyphenyl)acrylate; Ph8: 2-((4-
methoxyphenethyl)amino)-2-oxoethyl(E)-3-(4-hydroxyphenyl)acrylate; Ph9: 2-((4-methoxyphenethyl)amino)-
2
-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate; Ph10: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(4-
chlorophenyl)acrylate.

Molecules 2021, 26, 2477
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Figure 4.
Ball and stick representation of the docking pose of (a) Kojic acid (a-brick
red), 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate (Ph5), (
methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6), ( )-yellow and 2-((4-methoxyphenethyl)
amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (Ph9), ( )-green in the active site of mush-
room tyrosinase (2Y9X). The inhibitor binds in the active site closer to the Cu . Amino acid residue
b)-magenta, 2-((4-
c
d
2
+
2
+
forming the binding pocket of the tyrosinase is labeled in stick representation. Cu ions are shown
in brown and labelled.
3. Materials and Methods
3.1. Chemistry
All chemicals used for the synthesis of compounds (Ph1–Ph10) were purchased from
Sigma Aldrich (St. Louis, MO, USA). Melting points were determined using a Digimelt
MPA 160, USA apparatus and are reported uncorrected. The FTIR spectra were recorded
−
1
with Shimadzu FTIR–8400S spectrometer (Kyoto, Japan, υ, cm ). Elemental Analysis (C,
H) was carried out on a Flash 2000 series elemental analyzer with thermal conductivity
detector (TCD) system (Thermo Flash 2000 elemental analyzer, Thermo Fisher Scientific
1
13
Inc., Waltham, MA, USA) and results are with
±
0.3%. The H NMR and C NMR spectra
(
DMSO-d ) were recorded using a Bruker 400 MHz spectrometer (Bruker, Freemont, CA,
6
USA). Chemical shifts (δ) are reported in ppm downfield from the internal standard tetra
methyl silane (TMS). The purity of the compounds was checked by thin layer chromatogra-
phy (TLC) on silica gel plate using n-hexane and ethyl acetate as mobile phase. Mushroom
tyrosinase, L-DOPA and 2-(4-methoxyphenyl)ethan-1-amine were purchased from Sigma
Aldrich (St. Louis, MO, USA). Stock solutions of the reducing substrates were prepared in
phosphate buffer (20 mM, pH 6.8).
3
.2. General Procedure for the Synthesis of Title Compounds (Ph1–Ph5) and (Ph6–Ph10)
The intermediate 2-(4-methoxyphenyl)ethan-1-aminechloroacetate ( ) was synthe-
sized by following the Sidhu et al., method with some modifications [51]. The 2-(4-
methoxyphenyl) ethan-1-amine ( ) (0.01 mol) was dissolved in anhydrous dichloromethane
DCM) (25 mL) and triethylamine (C H ) N (0.01 mol) was added slowly. The resulting
2
1
(
2
5 3
◦
solution was then cooled in an ice salt mixture to 0 to
−
5 C. The chloroacetyl chloride
(0.01 mol) in dry DCM was added drop-wise to the reaction mixture with constant stir-
ring over a period of 1h maintaining the temperature constant. The reaction mixture was
then stirred at room temperature for a further 5 h, washed with 5% HCl, and 5% sodium

Molecules 2021, 26, 2477
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hydroxide solution. The organic layer was washed with saturated aqueous NaCl, dried
over anhydrous magnesium sulfate, filtered and the solvent was removed under reduced
pressure. The crude product was purified by silica gel column to afford the corresponding
2-(4-methoxyphenyl)ethan-1-aminechloroacetate (2).
The substituted benzoic acids (3a–e) (0.01 mol), triethylamine (0.01 mol) and potassium
iodide (KI) (0.01 mol) were mixed in dimethyl formamide (DMF) (25 mL) and stirred at
room temperature. The 2-(4-methoxyphenyl)ethan-1-amine chloroacetate intermediate (2)
was then added to the reaction mixture slowly and stirred the reaction mixture at the same
temperature overnight (Scheme 1). The final products (Ph1–Ph5) were then extracted with
ethyl acetate (3
×
25 mL). The combined ethyl acetate layer was then washed with 5%
HCl, 5% sodium carbonate and finally with brine solution. The organic layer was dried
over anhydrous magnesium sulfate, filtered and the solvent was removed under reduced
pressure to afford the crude products (Ph1–Ph5). The title compounds (Ph1–Ph5) were
purified by silica gel column chromatography (n-hexane: ethyl acetate 3:1). The same
procedure was used for the preparation of compounds (Ph6–Ph10) Scheme 2.
Scheme 1. Synthesis of compounds (4a
–
e
); Reagents and conditions. (i) CH Cl / (C H ) N, 0– 5 °C,
−
2
2
2
5 3
reflux for 5 h; (ii) DMF/(C H ) N/KI, r.t. stirring for 24 h.
2
5 3
Scheme 2. Synthesis of compounds (Ph6–Ph10); (ii) DMF/(C H ) N/KI, r.t. Stirring for 24 h.
2
5 3
3.3. Spectral Characterization of Synthesized Compounds (Ph1–Ph10)
2
8
2
-((4-methoxyphenethyl)amino)-2-oxoethyl 3-hydroxybenzoate (Ph1) solid; melting point,
◦
−1
6–88 C; R 0.54 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3378 (O-H), 3245 (-NH),
f
2
3
967 (sp C-H), 2856 (sp C-H), 1729 (C=O ester), 1645 (C=O amide), 1592 (C=C aromatic),
1
1187 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
9.86 (s, 1H,-OH), 8.27 (t, J = 4.4 Hz,
1
H,-NH), 7.46 (dd J = 6.4, 8 Hz, 1H), 7.42 (t, J = 4 Hz, 1H), 7.13 (d, J = 8.8 Hz, 2H), 7.07
(
3
dd, J = 6.4, 8 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 4 Hz, 1H), 4.04 (s, 2H,-CH ),
2
1
3
.72 (s, 3H,-OCH ), 3.28 (d,d, J = 6, 7.6 Hz, 2H,-CH ), 2.67 (t, J = 7.6 Hz, 2H,-CH ),
C
3
2
2
NMR (100 MHz, DMSO, ppm);
δ
167.95 (C=O, amide), 166.92 (C=O, ester), 158.19, 157.95,
1
4
31.63, 131.54, 131.49, 130.96, 120.98, 120.65, 116.43, 114.24, 63.39 (-CH ), 55.45 (-OCH ),
2
3
1.39 (-CH ), 34.44 (-CH ) (Figure S1). Elem. Anal. Calc. for C18H19NO5; C, 65.64; H, 5.82;
2
2
Found; C, 65.66; H, 5.84.

Molecules 2021, 26, 2477
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2
9
2
-((4-methoxyphenethyl)amino)-2-oxoethyl 4-hydroxybenzoate (Ph2) solid; melting point,
◦
−1
3–95 C; R 0.53 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3364 (O-H), 3278 (-NH),
f
2
3
956 (sp C-H), 2867 (sp C-H), 1723 (C=O ester), 1652 (C=O amide), 1597 (C=C aromatic),
1
1
178 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
10.39 (s, 1H,-OH), 8.26 (t, J = 4.4 Hz,
1
H,-NH), 7.88 (d, J = 4.8 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H6.88 (d, J = 8.4 Hz, 2H), 6.84 (d,
J = 4.8 Hz, 2H), 4.03 (s, 2H,-CH ), 3.72 (s, 3H,-OCH ), 3.28 (d,d, J = 6, 7.6 Hz, 2H,-CH ), 2.67
2
3
2
(
t, J = 7.6 Hz, 2H,-CH2), 13C NMR (100MHz, DMSO, ppm);
δ
166.23 (C=O, amide), 165.48,
1
62.67 (C=O, ester), 158.16, 132.32, 131.64, 130.06, 120.31, 115.75, 114.24, 63.05 (-CH ), 55.45
2
(
-OCH ), 43.1 (-CH ), 34.43 (-CH ) (Figure S2). Elem. Anal. Calc. for C18H19NO5; C, 65.64;
3 2 2
H, 5.82; Found; C, 65.62; H, 5.80.
-((4-methoxyphenethyl)amino)-2-oxoethyl 2,4-dihydroxybenzoate (Ph3) solid; melting
2
◦
2
−1
point, 122–124 C; R 0.51 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3343 (O-H), 3276
f
3
(-NH), 2944 (sp C-H), 2865 (sp C-H), 1725 (C=O ester), 1639 (C=O amide), 1601 (C=C
1
aromatic), 1184 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
10.53 (s, 1H,-OH), 8.27 (t,
J = 4.4 Hz, 1H,-NH), 7.74 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 5.2 Hz, 2H), 6.84 (d, J = 5.2 Hz, 2H),
6
.83 (dd, J = 5.2, 2.0 Hz, 1H), 6.32 (d, J = 2.0 Hz, 1H), 4.03 (s, 2H,-CH ), 3.72 (s, 3H,-OCH ),
2 3
13
3.28 (d,d, J = 6, 7.6 Hz, 2H,-CH ), 2.67 (t, J = 7.6 Hz, 2H,-CH ), C NMR (100MHz, DMSO,
2
2
ppm); δ168.68 (C=O, amide), 166.79 (C=O, ester), 166.23, 164.86, 158.19, 131.54, 131.49,
1
31.24, 130.09, 130.06, 108.79, 104.42, 63.37 (-CH ), 55.45 (-OCH ), 40.62 (-CH ), 34.43(-CH )
2 3 2 2
(
2
Figure S3). Elem. Anal. Calc. for C18H19NO6; C, 62.60; H, 5.55; Found; C, 62.62; H, 5.57.
-((4-methoxyphenethyl)amino)-2-oxoethyl 3,4-dihydroxybenzoate (Ph4) solid; melting
◦
−1
point, 112–114 C; R 0.55 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3367 (O-H), 3249
(
aromatic), 1159 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
f
2
3
-NH), 2923 (sp C-H), 2865 (sp C-H), 1728 (C=O ester), 1642 (C=O amide), 1589 (C=C
1
δ
9.88 (s, 1H, H-7, -OH),
7
2
7
.98 (d, J = 5.2 Hz, 2H), 7.75 (d, J = 5.2 Hz, 2H), 7.43 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.4,
.0 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 4.04 (s, 2H,-CH ), 3.72 (s, 3H,-OCH ), 3.28 (d,d, J = 6,
2
3
13
.6 Hz, 2H,-CH ), 2.67 (t, J = 7.6 Hz, 2H,-CH ), C NMR (100 MHz, DMSO, ppm); δ166.78
2
2
(
C=O, amide), 165.8 (C=O, ester), 151.26, 145.6, 143.32132.42, 130.01, 122.69, 120.39, 118.96,
1
17.01, 115.85, 63.26 (-CH ), 55.45 (-OCH ), 40.62 (-CH ), 34.43 (-CH ) (Figure S4). Elem.
2
3
2
2
Anal. Calc. for C18H19NO6; C, 62.60; H, 5.55; Found; C, 62.59; H, 5.54.
-((4-methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate (Ph5) solid; melting
2
◦
2
−1
point, 132–134 C; R 0.53 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3354 (O-H), 3246
f
3
(-NH), 2934 (sp C-H), 2843 (sp C-H), 1732 (C=O ester), 1640 (C=O amide), 1592 (C=C
1
aromatic), 1160 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
9.67 (s, 2H, -OH), 8.25 (t,
J = 4.4 Hz, 1H,-NH), 7.14 (d, J = 2.8 Hz, 2H), 6.88 (s, 2H), 6.84 (d, J = 2.8 Hz, 2H), 6.48 (t,
J = 2 Hz, 1H), 4.06 (s, 2H, -CH ), 3.72 (s, 3H,-OCH ), 3.28 (d,d, J = 6, 7.6 Hz, 2H,-CH ), 2.67
2
3
2
(
t, J = 7.6 Hz, 2H,-CH2), 13C NMR (100 MHz, DMSO, ppm);
δ
167.96 (C=O, amide), 165.81
(
C=O, ester), 158.19, 158.17, 131.49, 130.06, 129.75, 114.24, 107.94, 107.85, 63.32 (-CH ), 55.44
2
(
-OCH ), 40.84 (-CH ), 34.43 (-CH ) (Figure S5). Elem. Anal. Calc. for C18H19NO6; C,
3 2 2
6
2
2.60; H, 5.55; Found; C, 62.61; H, 5.56.
-((4-methoxyphenethyl)amino)-2-oxoethyl cinnamate (Ph6) solid; melting point, 103–
◦
−1
2
1
05 C; R 0.61 (n-hexane:ethyl acetate 3:1);FTIR νmax cm : 3297 (-NH), 2932 (sp C-H),
f
3
2
843 (sp C-H), 1726 (C=O ester), 1651 (C=O amide), 1597 (C=C aromatic), 1186 (C-O,
1
ester); H NMR (400 MHz, DMSO, ppm):
δ
8.27 (t, J = 4.4 Hz, 1H,-NH), 7.76 (d, J = 2.8 Hz
,
2
H), 7.73 (d, J = 16 Hz, 1H), 7.45 (d, J = 4.4 Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 6.86 (d,
J = 4.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 16 Hz, 1H), 4.03 (s, 2H,-CH ), 3.72
2
1
3
(
(
s, 3H,-OCH ), 3.28 (d,d, J = 6, 7.6 Hz, 2H,-CH ), 2.67 (t, J = 7.6 Hz, 2H,-CH ), C NMR
100 MHz, DMSO, ppm); δ167.03 (C=O, amide), 166.23 (C=O, ester), 158.19, 145.57, 134.45,
3
2
2
1
31.62, 131.49, 131.10, 130.06, 128.88, 118.09, 114.24, 62.85 (-CH ), 55.45 (-OCH ), 40.79
2 3
(
-CH ), 34.43 (-CH ) (Figure S6). Elem. Anal. Calc. for C20H21NO4; C, 70.78; H, 6.24;
2 2
Found; C, 70.80; H, 6.26.

Molecules 2021, 26, 2477
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2
-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(2-hydroxyphenyl)acrylate (Ph7) solid;
◦
−1
melting point, 111–113 C; R 0.55 (n-hexane:ethyl acetate 3:1);FTIR νmax cm : 3343 (O-H),
3
f
2
3
267 (-NH), 2949 (sp C-H), 2845 (sp C-H), 1731 (C=O ester), 1654 (C=O amide), 1593
1
(
C=C aromatic), 1186 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
10. 38 (s,1H, -OH),
8
.12 (t, J = 5.6 Hz, 1H, -NH), 7.93 (d, J = 16 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.26 (t,
J = 7.2 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.94 (d, J= 7.6 Hz, 1H), 6.87 (t, J = 7.6 Hz, 1H), 6.84
d, J = 8.4 Hz, 2H), 6.7 (d, J = 16 Hz, 1H), 4.04 (s, 2H,-CH ), 3.72 (s, 3H,-OCH ), 3.28 (d,d,
(
2
3
13
J = 6, 7.6 Hz, 2H, -CH ), 2.67 (t, J = 7.6 Hz, 2H -CH ), C NMR (100 MHz, DMSO, ppm);
2
2
δ
167.17 (C=O, amide), 166.64 (C=O, ester), 158.19, 157.40, 141.28, 132.34, 130.06, 129.75,
1
29.52, 121.12, 119.91, 117.18, 116.69, 114.24, 62.74 (-CH ), 55.4 (-OCH ), 40.8 (-CH ), 34.44
2
3
2
(
-CH ) (Figure S7). Elem. Anal. Calc. for C20H21NO5; C, 67.59; H, 5.96; Found; C, 67.61;
2
H, 5.98.
-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(4-hydroxyphenyl)acrylate (Ph8) solid;
2
◦
−1
melting point, 119–121 C; R 0.53 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3371
f
2
3
(O-H), 3267 (-NH), 2976 (sp C-H), 2859 (sp C-H), 1733 (C=O ester), 1655 (C=O amide),
1
1
598 (C=C aromatic), 1177 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
10.06 (s, 1H,-
OH), 8.26 (t, J = 5.2 Hz, 1H,-NH), 7.63 (d, J = 16 Hz, 1H), 7.59 (d, J = 2 Hz, 2H), 7.14 (d,
J = 3.2 Hz, 2H), 6.86 (d, J = 3.2 Hz, 2H), 6.81 (d, J = 2 Hz, 2H), 6.46 (d, J = 16 Hz, 1H),
4
2
.03 (s, 2H,-CH ), 3.72 (s, 3H,-OCH ), 3.27 (d,d J = 6,7.6 Hz, 2H, -CH ), 2.66 (t, J = 7.6 Hz,
2
3
2
H, -CH2), 13C NMR (100MHz, DMSO, ppm);
δ167.18 (C=O, amide), 166.23 (C=O, ester),
1
5
60.45, 158.19, 145.81, 131.64, 130.87, 130.06, 125.52, 116.29, 114.24, 114.14, 62.65 (-CH2),
5.42 (-OCH ), 41.27 (-CH ), 34.67 (-CH ) (Figure S8). Elem. Anal. Calc. for C20H21NO5;
3
2
2
C, 67.59; H, 5.96; Found; C, 67.58; H, 5.95.
-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (Ph9) solid;
2
◦
−1
melting point, 175–177 C; R 0.51 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3365 (O-H),
3
aromatic), 1178 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
f
2
3
276 (-NH), 2959 (sp C-H), 2869 (sp C-H), 1730 (C=O ester), 1647 (C=O amide), 1603 (C=C
1
δ
10.22 (s, 1H, -OH), 9.93 (s,
1H,-OH), 8.27 (t, J = 5.2 Hz, 1H,-NH), 7.82 (d, J = 16 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.14
(d, J = 2.4, 2H), 6.86 (d, J = 2.4 Hz, 2H), 6.47 (d, J = 16 Hz, 1H), 6.39 (d, J = 2 Hz, 1H), 6.31 (d,
J = 2 Hz, 1H), 4.04 (s, 2H,-CH2), 3.72 (s,3H,-OCH ), 3.27 (d,d J = 6,7.6 Hz, 2H,-CH ), 2.67
3
2
(
t, J = 7.6 Hz, 2H,-CH2), 13C NMR (100MHz, DMSO, ppm);
δ167.36 (C=O, amide), 166.24
(C=O, ester), 161.60, 159.18, 158.19, 158.16, 141.72, 131.66, 131.07, 129.98, 129.75, 114.24,
1
12.91, 108.34, 62.52 (-CH ), 55.44 (-OCH ), 43.10 (-CH ), 34.68 (-CH ) (Figure S9). Elem.
2
3
2
2
Anal. Calc. for C20H21NO6; C, 64.68; H, 5.70; Found; C, 64.70; H, 5.72.
-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(4-chlorophenyl)acrylate (Ph10) solid;
2
◦
−1
melting point, 107–109 C; R 0.58 (n-hexane:ethyl acetate 3:1); FTIR νmax cm : 3278
f
2
3
(-NH), 2956 (sp C-H), 2887(sp C-H), 1727 (C=O ester), 1657 (C=O amide), 1598 (C=C
1
aromatic), 1180 (C-O, ester); H NMR (400 MHz, DMSO, ppm):
δ
8.27 (t, J = 5.2 Hz, 1H -NH),
7
6
.8 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 16 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 2 Hz, 2H),
.87 (d, J= 2 Hz, 2H), 6.74 (d, J = 16 Hz, 1H), 4.03 (s, 2H, -CH ), 3.72(s, 3H, -OCH ), 3.28
2
3
13
(d,d J = 7.6,6.0 Hz, 2H,-CH ), 2.67 (t, J = 7.6 Hz, 2H, -CH ), C NMR (100 MHz, DMSO,
2
2
ppm); δ166.98 (C=O, amide), 166.23 (C=O, ester), 158.17, 144.14, 135.60, 133.43, 131.49,
1
30.61, 130.06, 129.49, 118.94, 114.24, 62.89 (-CH ), 55.42 (-OCH ), 41.27 (-CH ), 34.43 (-CH )
2 3 2 2
(Figure S10). Elem. Anal. Calc. for C20H20ClNO4; C, 64.26; H, 5.39; Found; C, 64.24;
H, 5.37.
3.4. Mushroom Tyrosinase Inhibition Assay
In vitro mushroom tyrosinase (Sigma Aldrich, St. Louis, MO, USA) inhibition assay
was performed, following our already reported method [52]. In detail, 140
buffer (20 mM, pH 6.8), 20 L of mushroom tyrosinase (30 U/mL) and 20
inhibitor solution were placed in the wells of a 96-well microplate. After pre-incubation
for 10 min at room temperature, 20 L of L-DOPA (3,4-dihydroxyphenylalanine, Sigma
µL of phosphate
µ
µL of the
µ
Aldrich, St. Louis, MO, USA) (0.85 mM) was added and the assay plate was further
◦
incubated at 25 C for 20 min. Afterward the absorbance of dopachrome was measured

Molecules 2021, 26, 2477
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at 475 nm using a microplate reader (OPTIMax, Tunable, Sunnyvale, CA, USA). All of
the compounds were dissolved in DMSO and blank tests were performed with solvent
DMSO. The IC50 values were reported after subtracting the value of DMSO. Kojic acid
was used as a reference inhibitor (positive control). The amount of inhibition by the test
compounds was expressed as the percentage of concentration necessary to achieve 50%
inhibition (IC ). Each concentration was analyzed in three independent experiments. The
50
IC50 values were determined by the data analysis and graphing software Origin 8.6, 64-bit.
The tyrosinase %age inhibition was determined by using the following formula:
Inhibition (%) = [(B − S)/B] × 100
(1)
Here, the absorbance of the blank and inhibitor are represented by B and S respectively.
3.5. Human Tyrosinase Inhibition Assay
3.5.1. Cell Culture and Preparation of Tyrosinase
A375 human melanoma cells were obtained from American Type Culture Collection
(
ATCC; Rockville, MD, USA). A375 cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM; Invitrogen, Burlington, ON, Canada) supplemented with L-glutamine,
0% (v/v) fetal bovine serum (FBS; Invitrogen), 50 g/mL streptomycin (Sigma Aldrich,
1
µ
St. Louis, MO, USA), 50 units/mL penicillin (Sigma Aldrich) and supplemented with
◦
2
00
µ
M of L-tyrosine for tyrosinase induction. Cell cultures were incubated at 37 C, in a
humidified atmosphere of 5% CO . Cells were scraped out from the tissue culture plate
2
◦
with phosphate-buffer saline (PBS) and were homogenized at 4 C in PBS. The homogenate
was centrifuged at 1000
mixture was centrifuged at 10,000
was used for the measurement of the inhibitory effects.
×
g for 10 min. The precipitate was sonicated in PBS on ice and the
×
g for 30 min. The supernatant containing tyrosinase
3.5.2. Human Tyrosinase Inhibition Assay
The tyrosinase inhibitory activity of the synthesized amides (Ph1–Ph10) was deter-
mined by following the already reported method with few modifications [53
assay reaction mixture (200 L) contained 3.3 mM L-DOPA in 0.33 M phosphate buffer
pH 7.0) and the enzyme in the presence and absence of inhibitors. Fifteen or twenty units
,54]. The
µ
(
of tyrosinase were used to determine the % inhibition. The reaction mixture was incubated
◦
at 37 C for 10 min and the absorbance was recorded at 475 nm using a microplate reader
(OPTI Max, Tunable). One unit of the enzyme was defined as the amount of enzyme which
increases the absorbance value by 0.001 at 475 per minute under the same conditions as
described above.
3.6. Kinetic Analysis of the Inhibition of Tyrosinase
A series of experiments were performed to calculate the inhibitory kinetics of com-
pounds Ph5, Ph6 and Ph9 by following the already reported method [55]. The compounds
concentrations are: 0, 0.2 and 0.4 M for Ph5; 0, 0.001 and 0.002 M for Ph6; 0, 0.00003,
M for Ph9. The concentration of substrate L-DOPA was between
.125 to 2 mM in all kinetic studies. Pre-incubation time and measurement time were
µ
µ
0
0
.00006, and 0.00012 µ
the same as discussed in mushroom tyrosinase inhibition assay protocol. Maximal initial
velocity was determined from initial linear portion of absorbance up to five minutes after
addition of enzyme at a 30 s interval. The inhibition type on the enzyme was determined by
Lineweaver–Burk plots of inverse velocities (1/V) versus the inverse of substrate concen-
−
1
tration 1/[S] mM . The EI-dissociation constant K was determined by the secondary plot
i
0
of 1/V versus inhibitor concentrations while ESI-dissociation constant K was determined
i
by intercept versus inhibitor concentrations.

Molecules 2021, 26, 2477
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3.7. Molecular Docking Studies
We studied all the synthesized compounds (Ph1–Ph10) using molecular docking
simulations to determine possible binding orientations of the compounds in the crystal
structure of the mushroom tyrosinase. For this purpose, we used the crystal structure of
the Agaricus bisporus tyrosinase (PDB:2Y9X) from the RCSB Protein Data Bank (RCSB PDB).
2
Y9X is a crystal structure of mushroom tyrosinase complex with tropolone. Tropolone
was removed from the active site and all compounds (Ph1–Ph10) were docked in the active
site. All ligands were prepared by LigPrep (Schrödinger) in their neutral form and their
conformation optimized in the OPLS-3 force field. The protein structure was prepared by
using one of the four monomers from the PDB entry and using the Protein Preparation
(
Schrödinger) for adding hydrogens and by removing all water molecules beyond 5.0 Å
and setting protonation states appropriate for pH 7. The receptor grid box was defined as
0 Å. Docking was performed with Glide (Schrödinger) using extra precision (XP) with
2
default settings and glide scoring function, reporting the 15 top ranked poses for each
ligand. Visual inspection of the binding pose and generation of figure was done with
Maestro (Schrödinger) [50].
3.8. Structure–Activity Relationship (SAR)
In the present study novel tyrosinase inhibitors (Ph1–Ph10) containing amide and ester
functionalities were synthesized and evaluated for their tyrosinase inhibitory potential
against mushroom and melanoma cells human tyrosinase. Mushroom tyrosinase being
commercially available has served almost in all studies conducted on tyrosinase inhibition
so far. Phenolics are the largest groups in tyrosinase inhibitors and several polyphenols
are accepted as substrates by tyrosinase, depending upon the presence and position of
substituent [56]. Cinnamic acid has been reported to possess broad physiological actions
involving tyrosinase inhibitory activity [57]. The synthesized compounds (Ph6–Ph10) are
all cinnamate esters/amides but differ in the substitution pattern on the cinnamate phenyl
ring. Compound (Ph6) bears an unsubstituted cinnamic acid moiety while compound (Ph7),
(Ph8) and (Ph9) possess 2-hydroxy, 4-hydroxy and 2,4-dihydroxy-substituted cinnamic
acid component, respectively. The tyrosinase inhibitory activity of 2,4-dihydroxy cinnamic
acid is already reported [58] and its incorporation as an ester/amide in compound Ph9
resulted in an increased inhibitory potential. The replacement of hydrogen from the para
position of the cinnamic acid phenyl ring in Ph6 with a 2,4-dihydroxyls in Ph9 led to a
significant increase in tyrosinase inhibitory activity (Table 1).
It was found that inhibitor Ph9 bearing dihydroxy-substituted cinnamic acid moiety
is the most promising compound with an inhibitory potential (IC50 0.00006
µM) much
better than the standard kojic acid (IC50 16.7 M). The enhanced inhibitory effect of Ph9
µ
is potentially due to the presence of two hydroxyl groups at ortho and para positions of
phenyl ring of cinnamic acid moiety. Whereas the Ph6 bearing an unsubstituted cinnamic
acid moiety also displayed an excellent enzyme inhibitory activity (IC50 0.00204
inhibitor Ph5 with two hydroxyls at 3,5 positions of benzoic acid moiety also exhibited
excellent tyrosinase inhibition (IC50 0.231 M) (Figure 5). The bioassay results proved that
µM). The
µ
derivatives with hydroxy-substituted cinnamic acid moiety (Ph7-Ph9) displayed greater
tyrosinase inhibitory activity compared to hydroxy-substituted benzoic acid derivatives
(Ph1-Ph5). It has already been demonstrated that phenolic hydroxyls play a key role in
tyrosinase inhibition activity as the natural substrates L-tyrosine and L-DOPA also bear the
phenolic hydroxyls [59]. Here, we report that the ortho and para-hydroxyls of Ph9 seem to
perform a similar role in tyrosinase inhibitory activity.

Molecules 2021, 26, 2477
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Figure 5. Structure–activity relationship (SAR) for the synthesized compounds (Ph1–Ph10),
promising compounds from both series (Ph1–Ph5 and Ph6–Ph10) are represented. Ph1: 2-((4-
methoxyphenethyl)amino)-2-oxoethyl 3-hydroxybenzoate; Ph2: 2-((4-methoxyphenethyl)amino)-
2-oxoethyl 4-hydroxybenzoate; Ph3: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 2,4-dihydroxy-
benzoate; Ph4: 2-((4-methoxyphenethyl)amino)-2-oxoethyl 3,4-dihydroxybenzoate; Ph5: 2-((4-
methoxyphenethyl)amino)-2-oxoethyl 3,5-dihydroxybenzoate; ; Ph7: 2-((4-methoxyphenethyl)amino)
-2-oxoethyl (E)-3-(2-hydroxyphenyl)acrylate; Ph8: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-
(
4-hydroxyphenyl)acrylate; Ph9: 2-((4-methoxyphenethyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxy-
phenyl)acrylate; Ph10: 2-((4-methoxyphenethyl)amino)-2-oxoethyl (E)-3-(4-chlorophenyl)acrylate.
4. Conclusions
The mushroom and human tyrosinase inhibitors Ph1–Ph10 with excellent tyrosinase
inhibitory potential compared to the reference kojic acid are presented in the current work.
A series of compounds Ph1–Ph10 containing ester/amide functionalities were synthesized
and evaluated to check their inhibitory potential against tyrosinase enzyme. Simple
nucleophilic substitution synthetic routes are adopted to synthesize the desired compounds
in good and excellent yield (71–84%). The mushroom (IC ) and human tyrosinase (%
50
age) enzyme inhibitory activities showed that the compound Ph9 (IC50 0.00005950
µ
M,
M, 92.24%) bearing
µM, 64.8%) containing 3,5-
9
4.59%) with 2,4-dihydroxycinnamic acid moiety, Ph6 (IC50 0.00204 µ
unsubstituted cinnamic acid and inhibitor Ph5 (IC50 0.231
dihydroxybenzoic acid unit showed excellent enzyme inhibitory potential, much better
than the standard kojic acid (IC50 16.7 M, 72.94%). The presence of ortho and para hydroxyl
µ
substituents on phenyl ring and p-methoxy-substituted benzene ring on the other side
of Ph9 played a vital role in enhanced tyrosinase inhibitory activity. Kinetic mechanism
reflected the mixed type of inhibition for inhibitors Ph9 (K 0.000093
µ
M), Ph5 (K 0.219
µ
M)
M) revealed from Lineweaver–Burk
plots. In silico molecular docking studies showed that the predicted binding affinity of
the synthesized compound Ph9 is excellent with a binding energy 7.93 Kcal/mol. It can
i
i
and non-competitive inhibition for Ph6 (K 0.0023
µ
i
−
be concluded from our results that the inhibitor Ph9 is highly potent and may be a model
structure for the development of novel and potent melanogenic inhibitors.
Supplementary Materials: The NMR data and spectral data are available online.
Author Contributions: Conceptualization, Z.A. and W.R.; methodology, Q.A.; software, Y.N.; valida-
tion, H.R., and Z.A.; formal analysis, N.K.; investigation, K.J.; resources W.R. and P.R.; data curation,
Y.N. and Z.A.; writing—original draft preparation, Y.N. and W.R.; writing—review and editing,
Z.A. and W.R.; visualization, Y.N. and Z.A.; supervision Z.A., W.R.; project administration, Z.A.
and W.R.; funding acquisition, W.R. All authors have read and agreed to the published version of
the manuscript.

Molecules 2021, 26, 2477
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Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in insert article and
Supplementary Material here.
Acknowledgments: The authors gratefully acknowledge the Chiang Mai University, Chiang Mai,
Thailand. The current research was partially supported by Chiang Mai University.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Zaman Ashraf may provide samples upon request.
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