Design, synthesis of Cinnamyl-paeonol derivatives with 1, 3-Dioxypropyl as link arm and screening of tyrosinase inhibition activity in vitro

Article abstract of DOI:10.1016/j.bioorg.2020.104512

This study aimed to obtain tyrosinase inhibitors for treating hyperpigmentation. A series of cinnamyl ester analogues were designed and synthesized with cinnamic acid (CA) and peaonol compounds. The safety, melanin content and inhibitory effects of all ta

Full text of DOI:10.1016/j.bioorg.2020.104512

Bioorganic Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis of Cinnamyl-paeonol derivatives with 1, 3-Dioxypropyl as
link arm and screening of tyrosinase inhibition activity in vitro
Kai Tang1, Yi Jiang1, Huawei Zhang, Wenli Huang, Yundong Xie, Chong Deng*, Hongbo Xu,
Xiaomei Song, Hong Xu
Shaanxi University of Chinese Medicine, Xianyang 712046, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
This study aimed to obtain tyrosinase inhibitors for treating hyperpigmentation. A series of cinnamyl ester an-
alogues were designed and synthesized with cinnamic acid (CA) and peaonol compounds. The safety, melanin
content and inhibitory effects of all target compounds were evaluated. In the enzymatic activity test, the
Cinnamic acid
Peaonol
Tyrosinase inhibitors
Anti-melanin
Molecular docking
inhibitory rate of compounds 8, 13 and 14 had stronger inhibitory activity with the IC50 values of 20.7
μ
M, 13.98
μ
M and 15.16 μM, respectively than the positive drug kojic acid (IC50 with 30.83 μM). The cytotoxicity evalu-
ation showed that compounds 13 and 14 have higher safety than the other compounds to the proliferation of
B16F10 cells. The result of the melanocyte test supported that compound13 has stronger cellular tyrosinase
inhibitory activity than kojic acid and arbutin at 100 μM and 200 μM. The enzyme kinetics mechanism revealed
that compound 13 was a non-competitive inhibitor while compounds 8 and 14 were mixed inhibitors. For the
experiments of melanin content and tyrosinase activity in the B16F10 melanona cells, the inhibition rates of
compounds 8, 14 and 13 were with 19.62%, 20.59% and 23.83%, respectively. In addition, compound 13
revealed the highest inhibitory activity to tyrosinase in the melanocyte with inhibition rates of 23.83%, which
was better than kojic acid and arbutin (19.21% and 20.45%) at the same concentration. In the anti-
melanogenesis experiment, compounds 8 and 13 had better anti-melanin effects than kojic acid from 25 μM
to 100 μM. In summary, the results indicated that compounds 8, 13 and 14 had better tyrosinase inhibitory
activity and anti-melanogenesis activity. Especially, the compound 13 has potentiality to develop novel tyrosi-
nase inhibitors and whitening agents. The docking studies results revealed that the functional group of com-
pound 13 mostly depends on the phenolic hydroxyl moiety, and its hydroxyl group did not insert into the active
site of tyrosinase, which was in agreement with the results of the kinetics study.
1. Introduction
[4], which catalyze biosynthesis of melanin involved in two different
reactions [5] including from hydroxylation of monophenols to o-
Tyrosinase, as a multifunctional copper-containing oxidase, is widely
scattering in plants, animals and microorganisms innature, which be-
longs to the type 3 copper protein family [1,2]. It is also a polyphenol
oxidase, containing two copper atom located in the active site. The
activation of tyrosinase is closely related to the rate of melanogenesis
[3]. Melanin, one of the most widely distributed pigments in nature, is
responsible for color of human skin, animal fur and plant browning.
Tyrosinases, tyrosinase-related protein 1(TRP-1) and tyrosinase-related
protein 2(TRP-2), are well-known melanogenic limited enzymes, which
play an essential role in the regulation of melanogenesis. Among of
them, tyrosinase is the most important factor to regulate melanogenesis
diphenols and oxidation of o-diphenols to the corresponding o-quinones
[6]. In addition, the process of melanogenesis is confined to special
melanosomes in melanocytes, which synthesize pigments and transfer
them to the recipient cells. Eventually, a series of highly reactive qui-
nones prompt melaniogenesis, which is responsible for melanin of the
skin, hair and eyes. In conclusion, melaninis produced from oxidation
and polymerization of tyrosinase and plays irreplaceablevital role in
normal function of bioorganism [7–9]. However, excessive production
of melanin results in some pigmentation disorders, such as melanoma,
melasma, frecklesand post-inflammatory hyperpigmentation. Espe-
cially, melanoma is a form of skin cancer that arises from the aberrant
* Corresponding author.
E-mail address: 2051079@sntcm.edu.cn (C. Deng).
1
Kai Tang, Yi Jiang contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104512
Received 9 August 2020; Received in revised form 10 October 2020; Accepted 19 November 2020
Available online 24 November 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Kai Tang, Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2020.104512

K. Tang et al.
Bioorganic Chemistry xxx (xxxx) xxx
proliferation of melanocyte [10–13]. Furthermore, recent studies re-
ported that melanin involved in the substantianigra(SN) of humans
contributing to an important role in the brain neuromelanin develop-
ment, such as parkinson and other degenerative diseases [14–16].
Therefore, controlling a tyrosinase-dependent mechanism of melano-
genesis might be the basis for a potential anti-melanoma therapy, and
inhibition of melanogenesis is one of reasonable method to cure hy-
perpigmentation. As an essential and rate-limiting enzyme to regulate
melanogenesis, excessive activation of tyrosinase causes various
dermatological disorders. Hence, tyrosinase has been seen asan impor-
tant target for developing therapeutic agents of pigment disorders.
Tyrosinase inhibitors have been increasingly attention from researchers
to prevent or treat pigmentation disorders. In recent years, many articles
reported that a series of natural and synthesized products had inhibitory
activity of tyrosinase and anti-melaninogenic, such as vinca major and
its secondary metabolite [17,18], N-(4-methoxyphenyl)caffeic amide
[19], 3-heteroarylcoumarins [20], flavonoids [21], cinnamic acid ester
derivatives [22], 3-heteroarylcoumarins [23], carvacrol derivatives
[24],flavones [25],substituted vanllylcinnamate, and so on [26,27].
However, some well-knowninhibitors: kojicacid [28], azelaic acid
[29,30], arbutin [31], tropolone and hydroquinone [32,33]were limited
in medicine and cosmetics industry because of the side-effect such as
cytotoxicity, skin cancer, dermatitis and neurodegenerative disease
[2,20,28–34]. Therefore, those problems prompt us to search for safer,
more effective and cheaper tyrosinase inhibitors. Especially natural
products existed in Chinese medicinehas been increasingly paying
attention to researchers on account of low cytotoxicity and tyrosinase
inhibitory activity, such as caffeic acid and peaonol derivatives
[21,22,35,36], the structure of those compounds was showed Fig. 1. In
addition, paeonol and eugenolanaloges, a type of traditional skin–-
whitening agent [37–39], have moderate activity on the browning and
relevant enzymes of fresh-cut lettuce [40,41]. To search safer, more
effective and cheaper tyrosinase inhibitors, caffeic acid deriva-
tives were coupled with paeonol analoges by esterification reaction,
which was widely applied to synthesis drugs and food additive
[22,42–45].
2. Results and discussion
2.1. Synthetic procedures
Using caffeic acid analogues (d-f) as starting materials, the two
synthetic routes of the cinnamic acid ester derivatives have been showed
in scheme 1. Firstly, in order to successful link the cinnamic acid de-
rivative to the phenolic components to gain cinnamic acid esters struc-
ture, peaonol derivatives(a-c) have been etherified through Williamson
reaction to ascend the activity of reaction. In spite of the phenolic hy-
droxyl group in the two series components has a significant influence on
the subsequent reaction. Therefore, two synthetic routes were designed
to synthesis target compounds 1–1 shown in Scheme 2 and in Scheme 3.
Route 1: On the one hand, acetic anhydride was used to acetylate the
phenolic hydroxyl group in cinnamic acid derivatives d-f, and then
SOCl2 was used to make the acid chloride to enhance reaction activity;
on the other hand, 3-Bromo-1-propanol and phenolic derivatives were
used to synthesize ester products by Williamson reaction, finally two
kinds of compounds were linked to get twin drugs through esterification
reaction. Cinnamic acid analogues and peaonol derivatives were used as
raw materials to synthesize compounds 1–9 with triethylamin as cata-
lyst. Using the cinnamic acid derivatives 4e as starting material, com-
pounds 13 and 14 were obtained by the esterification.
Route 2: TBDMSCl was used to protect the hydroxyl group in ferulic
acid under alkaline conditions to obtain 4e; then compound 4e reacted
with SOCl2 to produce the acyl chloride, which was mixed with paeonol
analogues to produces esters 10–12. Finally, TBDMS group was removed
from compounds 10 and 11 by Tetrabutylammonium fluride(1.0 mol/L
in THF solution) to get the target compounds 13 or 14 as shown in
Scheme 3.
2.2. Mushroom tyrosinase inhibition evaluation
The inhibitory activity of compounds 1–14 were evaluated on the
diphenolase activity of mushroom tyrosinase using L-dopa as the sub-
strate and Kojic acid as a standard. The inhibitory activity of compounds
1-14was screened at the concentrations of 0.2 mM, 0.4 mM, 0.6 mM, 0.8
mM and 1 mM, respectively. The inhibition percentage and IC50 values
were calculated and the inhibition curves have been obtained by
Graphpad Prism 8.0 software. The experimental results and the struc-
tures of compounds 1–14 were listed in Table 1. Firstly, all target
compounds, except for compounds 2, 3, 7 and 9, exhibited inhibitory
activity on mushroom tyrosinase with the inhibitory rates of over 60% at
the concentration of 1 mM, and the inhibition rates of compounds 8, 13,
and 14 were 78.8%, 82.4% and 80.6%, respectively.
In this study, 14 cinnamic-paeonol ester analogues were synthesized
via esterification reaction with caffeic acid derivatives and paeonol an-
alogues. The inhibitory activity and kinetic parameters of all target
compounds were calculated by mushroom tyrosinase tests and the
compound 8, 13 and 14 were screened out preliminary. Secondly, the
safety, anti-melanogenesis activity and cell tyrosinase inhibitory effects
of compound 8, 13 and 14 were estimated with murine melanoma cell
line (B16F10). Finally, computational analysis was carried out the in-
teractions with the catalytic binding site between inhibitors and amino
acid residue. We hope this study could pave the way for developing
novel effective tyrosinase inhibitors, searching for new whitening agents
and anti-browning agents for food products and medical industry.
In addition, the concentration-effect curve of compounds 8, 13 and
14 were tested at 10 different concentrations, the inhibition curves were
showed in Fig. 2. In order to comparetothe parent compound, the
O
O
R1
HO
O
HO
OH
OH
O
OH
a. Paeonol
R2
paeonol derivatives
b. Eugenol
c. 2,4-dihydroxycetophenone
R3
R4
COOH
O
COOH
HO
HO
COOH
COOH
HO
f. Caffeic acid
Cinnamic acid derivatives
d. Cinnamic acid
e. Ferulic acid
Fig. 1. The structures of lead compounds: the paeonol and cinnamic derivatives.
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K. Tang et al.
Bioorganic Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis routes of intermediate compounds 1a-1c, 3d-3f and 4e. Reagents and conditions: (a) acetone, K2CO3, BrC3H6OH, reflux, 6 h; (b) (CH3CO)2O, 4
h, reflux; (c) SOCl2, DCM, reflux, 5 h; (d) TBDMSCl, Et3N, DCM, 0 ◦C to rt, 4 h; (e) SOCl2, DCM, reflux, 5 h.
Scheme 2. Synthesis routes of intermediate compounds 1a-1c, 3d-3f and 4e. Reagents and conditions: (a) Pyridine, DCM(dried by MgSO4), ꢀ 10 ◦C or rt, overnight.
inhibitory activity was evaluated at the same concentration. The results
showed that the IC50 values of the parent compounds were from 154.6
suggested that the inhibitory activity of these compounds were signifi-
cantly improved. Especially, the IC50 values of compounds 8, 13 and 14
μ
M to 1135.8
μ
M, the IC50 values of the 14 compounds were lower than
were 20.7 μM, 13.9 μM and 15.1 μM, respectively. Among these target
the parent compound except for compound 2, 3, 7 and 9, which
compounds, 1–9 and 10–14 were two series of cinnamic acid ester
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K. Tang et al.
Bioorganic Chemistry xxx (xxxx) xxx
Scheme 3. The synthetic route of novel tyrosinase inhibitors 13–14 based on natural product scaffolds. Reagents and conditions: (a) Tetrabulammonium fluoride, rt,
2 h.
Table 1
The inhibition percentage of target compounds 1–14 on enzymatic-based tyrosinase activity at the concentration of 1 mM.
Compound
R1
R2
R5
R6
Inhibition (%)
75.8 ± 1.73
54.6 ± 0.88
49.2 ± 2.96
75.1 ± 2.81
70.6 ± 2.41
75.3 ± 1.59
58.4 ± 1.61
78.8 ± 2.42
59.5 ± 2.86
65.2 ± 2.59
69.8 ± 2.71
59.6 ± 1.56
82.4 ± 1.36
80.6 ± 1.70
71.1 ± 2.57
68.8 ± 2.42
45.7 ± 2.25
70.2 ± 1.17
56.4 ± 1.47
45.1 ± 2.46
81.8 ± 1.84
IC50
1
2-COCH3
2-COCH3
2-COCH3
4-COCH3
4-COCH3
2-OCH3
2-OCH3
4-COCH3
2-OCH3
2-COCH3
4-COCH3
2-OCH3
4-COCH3
2-OCH3
5-OCH3
OCH3
OCOCH3
H
OCOCH3
OCOCH3
H
130.5 ± 5.55
411.8 ± 29.5
226.1 ± 20.86
85.72 ± 18.53
138.6 ± 16.33
83.4–169
2
5-OCH3
3
5-OCH3
4
3-OH
OCH3
OCOCH3
OCOCH3
OCH3
H
OCOCH3
OCOCH3
OCOCH3
OCOCH3
H
5
3-OH
6
4-CH2CH = CH2
4-CH2CH = CH2
3-OH
7
214.6 ± 4.77
20.7 ± 3.51
411.8 ± 21.01
120.2 ± 18.53
25.37 ± 7.56
126.7 ± 11.7
13.98 ± 5.44
15.16 ± 4.763
154.6 ± 20.44
169.2 ± 16.87
>1000
8
9
4-CH2CH = CH2
5-OCH3
H
H
10
OCH3
OCH3
OCH3
OCH3
OCH3
TBDMS-
TBDMS-
TBDMS-
OH
11
3-OH
12
4-CH2CH = CH2
3-OH
13
14
5-COCH3
OH
Ferulic
Caffeic acid
Cinnamic
Peaonol
Eugenol
336.1 ± 17.02
566.4 ± 28.25
>1000
2,4-Dihydroxyacetophenone
kojic
30.83 ± 7.56
Notes: Values represent the mean ± standard deviation (SD) of mushroom tyrosinase inhibition experiments (n = 3); In the result of IC50 values, the concentration of
these compounds were 0.2, 0.4, 0.6, 0.8 and 1.0 mM respectively (n = 3).
compounds, and the difference among themwasthe number of phenolic
hydroxyl groups. With the increasing of the phenolic hydroxyl group
number, the IC50 values were decreasing and inhibitory rates were
improved. The higher the number of phenolic hydroxyl groups, the
smaller the IC50 and the stronger the inhibitory activity. The activity
decreased significantly when the corresponding phenolic hydroxyl
group was replaced.
The study on structure–activity relationship indicated that the
phenolic hydroxyl group may be the important active group for the in-
hibition of tyrosinase activity, which was closely connected with the
number of phenolic hydroxyl groups and result shows: di-phenols >
mono-phenols > non-phenol compounds. The inhibitory activity of
compounds 13 and 14 was 2.3 and 2 times than the positive drug kojic
acid respectively.
2.3. The inhibition mechanism study
The inhibition mechanism of compound 8, 13 and 14 were investi-
Fig. 2. The concentration-inhibition curve of tyrosinase inhibition of 8, 13, 14
gated based on the results of inhibition type and kinetic study, the
and kojic acid.
method is similar with 4.2.2, but the concentration of L-dopa and
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K. Tang et al.
Bioorganic Chemistry xxx (xxxx) xxx
mushroom tyrosinase with slight modification. The relationship be-
tween enzyme activity and its concentration in the presence of com-
pound 8, 13 and 14 plots of the remaining enzyme activity versus the
enzyme concentration at the different inhibitor concentrations given a
set of straight line, which all pass through the origin (Fig. 3. 8-a, 13-a
and 14-a). Increasing the inhibitors concentration reduced the slope of
the line, indicating that the inhibitor reduced the activity of the enzyme.
Thus, the diphenolase activity of compounds 8, 13 and 14 on tyrosinase
was reversible type inhibition.
result suggested that compounds 8 and 14 were both mixed-type in-
hibitors and indicated that they bind not only with the free enzyme but
also with the enzyme-substrate complex. This complex could prevent the
free oxygen molecule taking part in the oxidation of o-diphenols and
cause tyrosinase to lose catalytic ability. The inner mechanism of in-
hibitor had been discussed through the results of molecular docking.
2.4. Cytotoxicity of compounds 8, 13 and 14 in B16F10 cells
The kinetic data of compound 8, 13 and 14 on the L-dopa oxidation
were expressed by Lineweaver-Burk double-reciprocal plots. For com-
pound 13, the plots of 1/v versus 1/[S]to give a group of straight lines
with different slopes that intersect the vary point on the X-axis (Fig. 3.
13-b), suggesting that the Km remains the same, while Vmax decreases
with increasing concentrations of compound 13. The results indicated
that the inhibition type of compound 13 was non-competitive with a Ki
The cell viability of compounds 8, 13 and 14 was measured and
evaluated using the B16F10 cell line. These compounds acted on the cells
at concentrations of 25, 50, 100, and 200 μM for 24 h. The arbutin and
kojic acid were used in positive groups and the experimental results
were showed in Fig. 5. Compound 8 with the values of 79.13% showed
that a certain cytotoxicity compared with compounds 13 and 14 with the
values of 85.96% and 88.47% at the average level of 200 μM. Com-
value of 12.3
μ
M, which means that the binding site of 13 with tyrosi-
pounds 13 and 14 were safer than kojic acid and arbutin at the con-
nase is not the active site. This result also reflects that the compound 13
reduces the activity of tyrosinase whether or not it is already bound to
the substrate. A series of straight lines intercept at the point in the sec-
ond quadrant that compounds 8 and 14 were mixed type inhibitors with
centration in the range of 100 μM and the values were 91.99% and
91.96%, respectively. Furthermore, single-factor analysis showed that
compounds 13 and 14 were not significant difference with the control
group in the cell survival rate at the concentration lower than 100 μM (P
a Ki value of 88.49
17.54
μ
M and 12.30
μM, and Kis value of 238.09
μ
M and
< 0.05), indicating compounds 13 and 14 have high safety.
μM, respectively (Fig. 3, Table 2 and Fig. 4). Meanwhile, this
Fig. 3. Determination of the inhibition mechanism on diphenolase activity mushroom tyrosinase by compound 8, 13 and 14: The Fig. 8-a, 13-a and 14-a represent
effects of tyrosinase concentrations on its diphenolase activity at different concentrations of inhibitors. The concentrations of compound 8 for curves were 0, 15, 31,
62 and 125 μM, compound 13 and 14 for curves were 0, 7, 15, 31 and 62 μM espectively; Lineweaver-Burk plots (8-b, 13-b and 14-b) for mushroom tyrosinase with L-
dopa as substrate in the presence of compound 8, 13 and 14. The concentrations of compound 8 and 13 for curves were 0, 15, 31, 62 and 125
curves were 0, 7, 15, 31 and 62 M espectively.
μM, compound 14 for
μ
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Bioorganic Chemistry xxx (xxxx) xxx
Fig. 3. (continued).
at the 200
μM and 100
μ
M (P < 0.05). The compound 14 showed a
Table 2
moderate inhibitory activity at 100 μM, but compound 8 and 13 showed
The inhibition mechanism, Ki and Kis values of compounds 8, 13 and 14.
strong tyrosinase inhibitory activity with inhibition rate of 20.59% and
23.83%, respectively. Especially compound 13 have the highest inhibi-
tory activity, which was higher than that of kojic acid and arbutin
(19.21% and 20.45%) at the same concentration level, and have a
certain degree of concentration dependence. In addition, the cytotox-
Compounds
Inhibition type
Ki (
μ
M)
Kis (μM)
8
Mixed type
88.49
6.81
12.3
238.09
17.18
17.54
13
14
Non-competitive
Mixed type
icity of compound 8 at 100 μM was higher than that of compounds 13
and 14, indicating that the tyrosinase inhibitory activity has no direct
relationship with cytotoxicity, it also proved that compounds 13 and 14
were indeed stronger and safe cell tyrosinase inhibitor.
2.5. The anti-melanogenesis activity of compounds 8, 13 and 14 in
B16F10 cells
In summary, the comprehensive results of the mushroom tyrosinase
activity inhibition experiment and relevant cell experiments showed
that compound 13 has a higher inhibitory activity on B16F10 cells
tyrosinase, and the cytotoxicity was lower than compounds 8 and 14. In
addition, compounds 13 and 14 have lower cytotoxicity than kojic acid
In the model group, 1 μM α-MSH (melanin-promoting hormone) was
added to enhance the melanin content and stabilize the melanin content
in melanocytes. Kojic acid and arbutin were used as positive control.
Results of anti-melanogenesis inhibition experiments showed that the
three compounds 8, 13 and 14 were significantly different with the
and compounds 8 at 100
μM. Among the cell tyrosinase inhibitory ac-
model group at 200
μ
M and showed a certain degree of concentration
tivity, compound 13 exhibited the strongest inhibitory activity at the
dependence. The
α
-MSH model group showed extremely significant
concentration of 50 μM and 100 μM. In the conclusion, compound 13
statistical differences with the control group. Compounds 8, 13 and 14
suppressed the melanin production with the inhibition ratio values of
19.62%, 23.83%, and 20.59%, respectively. Among which compound 13
had the highest anti-melanogenic activity (Fig. 6).
displayed better activity than other inhibitors in cellular tyrosinase
inhibitory activity experiment.
2.7. Antioxidant activity
2.6. Cell tyrosinase inhibitory activity:
Tyrosinase catalyzes biosynthesis of melanin involved in oxidation of
o-diphenols to the corresponding o-quinones [6]. Therefore, the anti-
oxidant activities of compound 8, 13, 14 were measured with Vc as a
positive control and the results were presented in Fig. 8 in the form of
DPPH radical scavenging activity. The half maximal effective concen-
trations (EC50) of compound 13 and Vc were calculated by Graph Pad
The inhibitory effects of compounds 8, 13 and 14 on the tyrosinase
activity of B16F10 cells were showed in Fig. 7. The control group was
normal B16F10 cells, and the experiment results showed that three
compounds had significant differences compared with the control group
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Bioorganic Chemistry xxx (xxxx) xxx
Fig. 4. The determination of the inhibition constant Ki and Kis: The Fig. 8-c, 13-c and 14-c represent the plot of slope versus the concentration of 8, 13 and 14 for the
determination of the inhibition constant Ki respectively. The Fig. 8-d, 13-d and 14-d represent the plot of intercept versus the concentration of 8, 12 and 13 for the
determination of the inhibition constant Kis respectively.
Prism 8.0.2, and also were listed in Fig. 8. As shown in Fig. 8, obviously,
the DPPH radical scavenging activity of compound 13 was higher than
those of compound 8 and 14 with EC50 of 0.14 mmol/L. The EC50 of
compound 8 and 14 were not determined under 4 mmol/L due to their
poor DPPH radical scavenging activity. Compound 13 has two phenolic
hydroxyl groups, compound 8 and 14 just have one hydroxyl group,
perhaps the introduction of the second hydroxyl group resulted in the
increase in DPPH radical scavenging activity of Compound 13. Vc has
been widely applied as an antioxidant and a positive control in antiox-
idant experiment because of its excellent antioxidant activities. In this
study, the EC50 of Vc and compound 13 were 0.03, 0.14 mmol/L,
The active site 5 (-29.76; ꢀ 10.28; ꢀ 42.21) was screened out and
12.0000 was selected as the radius of activity site. It is repositioned in
the cavity of the enzyme protein to obtain the ligand molecule and the
enzyme amino acid residue. Finally, two-dimensional diagram of the
interaction was showed in Fig. 9, group B. The lowest energy confor-
mation of the enzyme inhibitor complex has been calculated and pre-
dicted through intermolecular hydrogen bond donors and interactions.
The binding affinity of the enzyme inhibitor complex during the docking
process was calculated, not only taking into consideration the best score
indicating the matching energy, but also including the best conforma-
tion to achieve geometric matching and eliminating adverse in-
teractions. Small molecule inhibitors and tyrosine results of acid enzyme
affinity scores showed in Table 3.
respectively, which showed that compound 13 was
antioxidant.
a potential
Furthermore, the type of functional groups determined the in-
teractions in the enzyme-inhibitor complexes. The results of docking
were also basically kept up with the rule of structure–activity relation-
ship. The phenolic hydroxyl group in cinnamyl derivatives contributes
to the binding affinity of the enzyme-inhibitor complexes. It was not
doubted that the number of phenolic hydroxyl groups and ester bonds
played essential roles in interacting protein. The result of docking score
showed that compound 8, 13 and 14 performed better than the original
ligand as kojic acid and arbutin. Among of those compounds, the com-
pound 13 exerted the best interaction with the protein, which was partly
different with the result of the enzyme inhibition test. Generally, a
2.8. Molecular docking analyses
As compounds 8, 13 and 14 exhibited better mushroom tyrosinase
inhibition than the other cinnamyl-paeonol derivatives, Cellular tyros-
inase inhibitory activity molecular docking was introduced to predict
the docking conformation of these three compounds(Fig. 9 group A and
group B). The construction of molecular and the analyses of docking
were possessed as delineated between 2Y9X and the active compounds
8, 13, 14, kojic acid and arbutin in the subsequent section. Kojic acid and
arbutin were used reference to using the same active site and method.
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Bioorganic Chemistry xxx (xxxx) xxx
Fig. 4. (continued).
Fig. 5. Cytotoxicity of compounds 8, 13 and 14; Notes: n = 3,*P < 0.05,**: P < 0.01, (with control group).
hydrogen bond was found between kojic acid and Arbutin with Gln74,
Tyr98 and Tyr78 (Fig. 9-B Compound kojic acid and arbutin). Other
interactions were also discovered including Van der Waals force be-
tween inhibitor with Leu 77, His76, Thr324 (Fig. 9-B Compound kojic
acid). On the one hand, this result revealed that compound 13 has not
interacted with copper ions through polar contacts, but it has interaction
with Gln48, Asp10, Glv68 through affection of Hydrogen bonds, the
interactions of Pi-Anlon with Asp10 was also formed (Fig. 9-B Com-
pound 14).
On the other hand, hydrogen bonds was found, which came from the
ester group with Lys70 to compound 8 between protein and ligand, and
ester group interacts with Lys5 and Met325 through electrostatic
attraction benzene ring (Fig. 9-B Comopund 8). Moreover, the com-
pound 13 showed potential interaction with the active site, which
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Bioorganic Chemistry xxx (xxxx) xxx
Fig. 6. Melanin production inhibitory effect of compounds 8, 11, 13 and 14 in B16F10 melanoma cells. Notes: n = 3, ##: P < 0.01(compared with control group), **:
P < 0.01, ***: P < 0.001, ****: P < 0.0001 (compared with model group);
Fig. 7. Cellular tyrosinase inhibitory activity of compounds 8, 13 and 14 in B16 F10 melanoma cells. Notes: n = 3, *: P < 0.05, **: P < 0.01, ***: P < 0.001(compared
with model group).
provided evidence for the result of stronger inhibition activity of
tyrosinase. On the basis of molecular docking, the result indicated that
the basic moiety of arginine showed electron donating property. The
covalent H has easily to form H-bond with electrophilic O atom in the
ester group and phenolic hydroxyl group. The docking result revealed
that the phenolic hydroxyl could enhance the binding affinity of the
enzyme-inhibitor complexes. Compared with compounds 8 and 14, both
just with one phenolic hydroxyl, compound 13 has two phenolic hy-
droxyls and exhibited the best tyrosinase inhibition activity among these
three compounds.
hydroxyl moiety. The hydroxyl group of compound 13 did not insert into
the active site of tyrosinase, which was in agreement with the results of
the kinetics study.
3. Conclusions
Fourteen cinnamic ester derivatives have been designed and syn-
thesized as tyrosinase inhibitors by ester reaction. The result of mush-
room tyrosinase test exhibited that compounds 8, 13 and 14 have better
inhibition at 1 mM than kojic acid and the lower IC50 values. Especially,
The kinetics study showed that compound 13 was a non-competitive
inhibitor, while compounds 8 and 14 were a mixed-type inhibitor. The
docking studies of the compound 14 contribute to explain the difference
to a degree. The results also revealed that the functional group of the
cinnamic-phenol ester derivatives mostly depends on the phenolic
compound 13 with IC50 value of 13.9 μM showed tyrosinase inhibitory
activity 2.2 times better than that of kojic acid with IC50 value of 30.83
μ
M. In addition, inhibition on tyrosinase and melanin content indicated
that compound 13 displayed better activity than other inhibitors in the
B16F10 melanoma cells, which the cell viability was higher than
9

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Bioorganic Chemistry xxx (xxxx) xxx
Fig. 8. DPPH radical scavenging capacities of compounds 8, 13, 14, Vc and the EC50 of Vc and compound 13.
compound 8 and kojic acid at 100
activity, compound 13 exhibited the strongest inhibitory activity at the
concentration of 50 and 100 M. Furthermore, kinetic mechanism sug-
μ
M. In the cell tyrosinase inhibitory
mixture was refluxed for 5 h at 75 ◦C. Then the solution was heated to
85 ◦C for evaporation. After cooling to room temperature, and then
concentrated under reduced pressure to obtain yellow solution 3d
(yield: 75–85%);
μ
gested that the competitive mode of compound 13 was non-competitive
manner. In the antioxidant experiment, compound 13 exhibited a
certain extent antioxidant ability. Molecular docking analysis performed
that the main type of interactive bind with active site was Hydrogen
bond. Summarizing these results, compound 13 was proved to be most
potential tyrosinase inhibitor and anti-melanogenesis agents in these 14
derivatives.
4.1.2.2. (E)-4-(3-chloro-3-oxoprop-1-en-1-yl)-2-methoxyphenyl acetate
(compunds 3e). Compound e replaced compounds d as material, others
synthesis procedures was similar with compound 3d, which was yellow
solid.
Compound e replaced compounds d as material, others synthesis
procedures was similar with compound 3d, which was a yellow solid.
4. Experimental section
4.1.2.3. (E)-4-(3-chloro-3-oxoprop-1-en-1-yl)-1,2-phenylene
diacetate
4.1. Chemistry
(Compound 3f). Compound f replaced compounds d as material, others
procedures as the same as compound 3d, which was a yellow solid.
4.1.1. General procedures for synthesis of compounds 1a-c:
4.1.3. (E)-3-(4-((tert-butyldimethylsilyl)oxy)-3-methoxyphenyl)acryloyl
chloride (compounds 4e)
4.1.1.1. 1-(2-(3-hydroxypropoxy)-4-methoxyphenyl)ethan-1-ketone
(compound 1a). 3-Bromo-1-propanol (2.0 mM, 2 eq) was added to a
solution of compound a (1.0 mM, 1 eq) in acetone solution, and then
added extra dry K2CO3 (0.6 mM, 0.6 eq), the mixture was stirred under
reflux at 76 ◦C for 6 h. The reaction was monitored by TLC, the mixture
was concentrated under reduced pressure after reaction finished. Then
the residue was dissolved in Ethyl acetate, washed with brine. The slight
yellow organic layer was dried with MgSO4, and then concentrated
under reduced pressure to give a bright yellow oil as an intermediate,
which was purified by flash chromatography to gain 1a as a light yellow
oil (Yield: 60%).
Tert-butyldimethylsilyl chloride (6 mM, 2 eq) was added to a sus-
pension of compounds e (3 mM, 1 eq) in 100 mL DCM at 0 ◦C, and then
NEt3 (3 mM, 1 eq)was added slowly. The mixture was stirred at room
temperature for 4 h after 1 h. The mixture was washed with water and
brine respectively. The organic layer was dried with MgSO4, and then
concentrated under reduced pressure to give a bright yellow oil as an
intermediate, which was purified by flash chromatography to get 4e as a
white power (Yield: 91.6%).
4.1.3.1. (E)-3-(2-acetyl-5-methoxyphenoxy) propyl 3-(3-methoxy-4-
(prop-1-en-2-yloxy) phenyl) acrylate (compound 1). The residue 1a (0.7
mM, 1 eq) was dissolved in dry DCM, added 2d (1 mM, 1.3 eq) at 0 ◦C,
and then added NEt3 (0.7 mM, 1 eq), which was stirred at room tem-
perature overnight, the reaction system was monitored by TLC. The
mixture was washed with 1 M HCl solution, saturated NaHCO3 and brine
respectively. The organic layer was dried with MgSO4, and concentrated
to give a crude product, which was purified by chromatography on silica
gel using petroleum ether/ethyl acetate (2:1, v:v) as original eluent to
get the etherification product 1.
4.1.1.2. 1-(2-hydroxy-5-(3-hydroxypropoxy)phenyl)ethan-1-ketone
(compunds1b). The synthesis method was similar with compound 1a,
which was a white crystal (Yield: 85%).
4.1.1.3. 3-(4-allyl-2-methoxyphenoxy)propan-1-ol (compunds1c). The
synthesis method was similar with compound 1a, which was a light oil
(Yield: 72%).
4.1.2. General procedures for synthesis of compounds 3d-f:
White crystal. m.p. 110–111 ◦C. Yield: 56%; 1H NMR (400 MHz,
CDCl3) δ: 7.85 (d, J = 8.7 Hz, 1H), 7.66 (t, J = 10.9 Hz, 1H), 7.12 (d, J =
8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.53 (d, J = 8.7, 1H), 6.46 (d, J =
10.9 Hz, 1H), 6.38 (d, J = 15.9 Hz, 1H), 4.44 (t, J = 6.2 Hz, 2H), 4.18 (t,
J = 6.1 Hz, 2H), 3.86 (t, J = 6.7 Hz, 6H), 2.61 (s, 3H), 2.32 (s, 3H),
2.19–2.16(m, 2H); 13C NMR (100 MHz, CDCl3) δ: 197.71, 168.97,
166.82, 164.54, 160.22, 151.45, 141.53, 132.91, 117.94, 111.26,
4.1.2.1. Cinnamoyl chloride (compounds 3d). Excess acetic anhydride
was added to compound d (5 mM, 1 eq), and the mixture was refluxed
for 5 h at 140 ◦C. The mixture was concentrated under reduced pressure
after reaction finished to gain immediate. SOCl2 (5 mL, 15 mM) was
added to a solution of the immediate (3.0 mM) in DCM (20 mL), and the
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Group A
Compound 14
Compound 8
Kojic
Compound 13
Arbutin
Fig. 9. Ligand-protein interaction of compounds 8, 13, 14, kojic acid and arbutin with the active site of mushroom tyrosinase (2Y9X) has been fulfilled by Discovery
Studio 2019. These two groups pictures displayed interaction of compound 8, 13, 14, kojic acid and arbutin with the enzyme, respectively. The pictures in group A
show the 3D docking of ligands in the active binding pocket with the hydrophobic effect area displayed; The pictures in group B showed the 2D interaction pattern
between the ligands and animo acid residue, the bottom of these figures in group B represents the important interactions type between the ligand atoms and the
amino acid residues of the protein.
105.18, 99.06, 65.14, 61.43, 55.99, 55.66, 32.27, 28.78, 20.78; IR
6.36 (s, 1H), 4.43 (t, J = 6.2 Hz, 3H), 4.17 (t, J = 6.1 Hz, 3H), 3.84 (d, J
= 1.0 Hz, 3H), 2.61 (d, J = 1.0 Hz, 3H), 2.30 (d, J = 2.4 Hz, 3H), 2.27 (m,
J = 6.1 Hz, 2H), 2.17 (s, 2H); 13C NMR (100 MHz, CDCl3) δ197.93,
177.73, 166.58, 164.56, 160.23, 143.35, 142.50, 132.92, 124.10,
122.87, 118.93, 105.26, 99.02, 65.09, 61.48, 55.67, 32.27, 28.73,
20.76; IR(cmꢀ 1)2955.64, 1780.24, 1750.35, 1630.24, 1580.64,
1496.51, 1410.38, 1382.36, 1366.35, 1010.94, 820.64, 704.52, 650.32.
HRMS (ESI, positive) m/z calcd for C25H26O9 ([M]+): 469.4740, found:
469.3763.
(cmꢀ 1
) 2976.23, 2331.06, 1720.48, 1605.26, 1580.56, 1551.26,
1414.64, 1279.38, 1246.35, 1225.36, 1030.18, 1002.64, 750.42,
716.24. HRMS (ESI, positive) m/z calcd for C24H26O8 [M]+:442.4640,
found: 441.3437.
4.1.3.2. (E)-4-(3-(3-(2-acetyl-5-methoxyphenoxy) propoxy)-3-oxoprop-1-
en-1-yl)-1, 2-phenylene diacetate(compound 2).. Compound 2e replaced
compounds 2d as material, others procedures as the same as compound
1.
White crystal. m.p. 126–127 ◦C. Yield: 71%; 1H NMR (400 MHz,
CDCl3) δ7.86 (d, J = 1.0 Hz, 1H), 7.83 (d, J = 1.0 Hz, 1H), 7.65 (s, 1H),
7.61 (s, 1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.36 (s, 1H), 7.26 (s, 1H), 7.23 (s,
1H), 7.21 (s, 1H), 6.53 (d, J = 10.0 Hz, 2H), 6.45 (s, 2H), 6.40 (s, 1H),
4.1.3.3. 3-(2-acetyl-5-methoxyphenoxy) propyl cinnamate (compound
3).. Compound 2f replaced compounds 2d as material, others proced-
ures as the same as compounds 1.
White crystal (EtOAc). m.p. 75–76 ◦C. Yield: 65%; 1H NMR (400
11

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Group B
Compound 8
Compound 14
Compound 13
Kojic
Arbutin
Fig. 9. (continued).
MHz, CDCl3) δ 7.90 (d, J = 10.4 Hz, 1H), 7.70 (d, J = 15.9 Hz, 1H), 7.57
(d, J = 31.1 Hz, 2H), 7.41 (d, J = 18.9 Hz, 1H), 7.27 (s, 1H), 6.87 (s, 1H),
6.68 (d, J = 18.2 Hz, 2H), 4.42 (d, J = 5.9 Hz, 2H), 4.17 (d, J = 5.9 Hz,
1H), 2.40 – 2.15 (m, 2H), 1.60 (d, J = 9.3 Hz, 2H), 1.25 (s, 2H); 13C NMR
(100 MHz, CDCl3) δ195.99, 167.03, 165.32, 162.99, 145.23, 134.09,
129.09, 128.56, 123.68, 116.90, 109.62, 65.05, 61.11, 29.70, 28.59; IR
(cmꢀ 1): 2941.43, 2890.54, 2364.27, 2327.87, 1968.56, 1642.92,
1603.43, 1564.35, 1415.99, 1258.56, 1206.17, 1058.54, 961.62,
832.27, 711.23, 613.01.HRMS (ESI, positive) m/z calcd for C21H22O5
([M]+): 354.4020, found: 353.3917.
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4.1.3.7. (E)-3-(4-allyl-2-methoxyphenoxy)
propyl
3-(4-acetoxy-3-
Table 3
The docking score of 1–14 compounds inhibitors with mushroom tyrosianse:
methoxyphenyl) acrylate (compound 7).. Compound 1b replaced com-
pounds 1a as material, others procedures as the same as compounds 1.
White solid. m.p. 79–80 ◦C. Yield: 68%; 1H NMR (400 MHz, CDCl3)
δ7.64 (d, J = 16.0 Hz, 1H), 7.26 (s, 1H), 7.10 (s, 2H), 7.05 (d, J = 8.0 Hz,
1H), 6.84 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 6.1 Hz, 1H), 6.38 (d, J = 16.0
Hz, 1H), 5.95 (d, J = 16.8, 6.7 Hz, 1H), 5.08 (d, J = 17.0 Hz, 2H), 4.42 (t,
J = 5.1 Hz, 2H), 4.14 (t, J = 6.3 Hz, 2H), 3.86 (d, J = 7.8 Hz,3H), 3.33 (d,
J = 6.7 Hz, 1H), 2.24 (dd, J = 8.1, 4.5 Hz, 2H), 1.57(s, 1H); 13C NMR
(100 MHz, CDCl3) δ168.97, 168.89,151.42, 149.51, 146.58, 144.21,
141.44, 137.73, 133.42, 133.30, 123.34, 120.49, 118.28, 115.79,
Compounds
Dock Score
Compounds
Dock Score
1
72.4421
83.1585
75.2711
85.2159
87.4696
75.0121
90.6535
92.1343
58.9965
86.4831
89.1886
12
61.7
2
13
103.89
97.34
36.02
37.38
55.14
55.45
58.29
52.01
67.22
54.52
3
14
4
Ferulic
5
Caffeic acid
6
Cinnamic
7
Paeonol
8
Eugenol
9
Kojic
113.51, 112.38, 111.22, 65.82, 61.65, 55.99, 39.92, 28.78; IR(cmꢀ 1
)
10
11
Arbutin
2′,4′-Dihydroxyacetophenone
2920.34, 2360.96, 1725.26, 1606.95, 1552.89, 1417.52, 1319.77,
1247.40, 1175.28, 1139.46, 1064.98, 995.54, 786.96, 710.54, 610.51.
HRMS (ESI, positive) m/z calcd for C25H28O7 [M]+: 440. 4920, found:
439.4391.
4.1.3.4. (E)-3-(4-acetyl-3-hydroxyphenoxy)
propyl
3-(4-acetoxy-3-
methoxyphenyl) acrylate (compound 4). Compound 1c replaced com-
pounds 1a as material, others procedures as the same as compound 1.
White crystal. m.p. 143–144 ◦C. Yield: 65%; 1HNMR (400 MHz,
CDCl3) δ12.74 (s, 1H), 7.66 (s, 1H), 7.64 (d, J = 1.2 Hz, 1H), 7.63(t, 1H),
7.17 (s, 1H), 7.13 (d, J = 1.9 Hz, 1H), 7.10 (dd, J = 3.8, 1.8 Hz, 2H), 7.06
(s, 1H), 7.04 (s, 1H), 6.45 (d, J = 2.5 Hz, 1H), 6.44 – 6.42 (m, 2H), 6.40
(s, 1H), 6.36 (s, 1H), 4.40 (t, J = 6.2 Hz, 2H), 4.14 (t, J = 6.2 Hz, 2H),
3.88 (s, 6H), 2.55 (s, 3H), 2.23(s, 3H), 2.19 – 2.16 (m, 2H); 13CNMR
(100 MHz, CDCl3) δ202.77, 168.98, 166.86, 165.34, 165.28, 151.44,
141.51, 132.45, 121.40, 114.01, 108.15, 101.31, 64.80, 61.22, 55.99,
28.49, 26.39, 20.79; IR (cmꢀ 1): 2962.35, 2889.58, 2359.82, 1757.29,
1715.34, 1632.21, 1509.73, 1377.54, 1327.59, 1253.68, 1189.7,
1170.32, 1160.73, 1063.45, 998.03, 844.99, 819.08, 598.06; HRMS
4.1.3.8. 3-(3-acetyl-4-hydroxyphenoxy) propyl cinnamate (compound
8).. Compound 1c replaced compounds 1a as material, others proced-
ures as the same as synthesis compounds 3.
White crystal (EtOAc/ petroleum ether).m.p: 117 ◦C; Yield: 64%; 1H
NMR (400 MHz, CDCl3) δ 7.92–7.86 (dd, J = 15.9 Hz, 2H), 7.7 (d, J =
12.0 Hz, 1H), 7.6 (d, J = 6.0 Hz, 2H), 7.54–7.52 (s, J = 6.0 Hz, 2H), 7.43
(d, J = 3.0 Hz, 1H), 4.38 (t, J = 12 Hz, 2H), 4.13 (t, J = 9.0 Hz, 2H), 3.81
(s, 3H), 2.54 (s, 3H), 2.34 – 2.09 (m, 2H); 13C NMR (100 MHz, CDCl3)
δ196.00, 167.03, 165.33, 162.9, 151.43, 147.77, 145.00, 134.37,
130.98, 129.01, 128.55, 117.85, 116.92, 112.46, 109.63, 65.04, 61.10,
29.70, 28.58; IR: 3414.76, 2910.35, 2360.96, 1720.21, 1606.95,
1552.89, 1415.66, 1319.77, 1247.40, 1175.28, 1138.83, 1062.98,
995.54, 766.96, 710.38, 620.15. ESI-MS: 339.38HRMS (ESI, positive)
m/z calcd for C20H20O5 ([M]+): 340.3750, found: 339.3834.
(ESI, positive) m/z calcd for
C
24H24O8 [M]+: 428.4370, found:
427.4297.
4.1.3.5. (E)-4-(3-(3-(4-acetyl-3-hydroxyphenoxy)propoxy)-3-oxoprop-1-
en-1-yl)-1, 2-phenylene diacetate(compound 5).. Compound 1c replaced
compounds 1a as material, others procedures as the same as compounds
2. White crystal. m.p. 88–89 ◦C. Yield: 73%; 1H NMR (400 MHz, CDCl3)
δ12.74 (s, 1H), 7.66 – 7.58 (m, 2H), 7.41 (d, J = 2.1 Hz, 1H), 7.39 (d, J =
2.1 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.23 (s, 2H), 7.22 (d, J = 8.4 Hz,
1H), 6.45 (d, J = 2.5 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 6.38 (d, J = 8.4
Hz, 1H), 4.39 (t, J = 6.2 Hz, 2H), 4.13 (t, J = 6.2 Hz, 2H), 2.56 (s, 3H),
2.32 – 2.29 (m, 6H), 2.25 – 2.16 (m, 2H), 1.57 (s, 2H); 13C NMR (100
MHz, CDCl3) δ 202.81, 168.30, 166.65, 165.34, 165.27, 143.26, 142.49,
132.48, 124.07, 119.01, 108.12, 101.35, 64.78, 61.33, 28.45, 26.35,
20.80, 20.76; IR (cmꢀ 1) 3412.74, 3072.89, 2963.33, 2899.11, 2621.23,
1770.16, 1701.95, 1630.20, 1559.66, 1420.12, 1375.31, 1251.94,
1195.70, 1054.45, 998.21, 899.14, 827.47, 710.46, 601.39. ESI-MS:
HRMS (ESI, positive) m/z calcd for C24H24O9 ([M]+): 456.4470,
found: 455.4372.
4.1.3.9. 3-(4-allyl-2-methoxyphenoxy) propyl cinnamate (compound 9)..
Compound 1c replaced compounds 1a as material, others procedures as
the same as synthesis compounds 3.
White solid (EtOAc/ petroleum ether). m.p: 115 ◦C; Yield: 77%; 1H
NMR (400 MHz, CDCl3) δ7.69 (d, J = 16.0 Hz, 1H), 7.53 (s, 2H), 7.39 (t,
2H), 6.85 (d, J = 8.6 Hz, 1H), 6.72 (s, 2H), 6.44 (d, J = 16.0 Hz, 1H),
5.95 (m, J = 5.5 Hz, 1H), 5.08 (d, J = 18.6 Hz, 2H), 4.42 (t, J = 6.2 Hz,
2H), 4.14 (t, J = 6.3 Hz, 2H), 3.85(s, 3H), 3.34 (d, J = 6.6 Hz, 2H),
2.30–2.19 (m,2H), 1.57 (s,1H); 13C NMR (100 MHz, CDCl3) δ196.84,
167.51, 165.05, 163.48, 152.23, 147.72, 145.67, 134.69, 132.14, 131.5,
130.92, 129.5, 128.4, 127.90, 124.81, 117.50, 116.93, 112.56, 61.05,
29.98. IR (cmꢀ 1): 2966.26, 2359.96, 1771.99, 1706.48, 1629.35,
1554.31, 1417.05, 1249.95, 1228.36, 1009.59, 837.03, 813.41, 711.19,
608.22. HRMS (ESI, positive) m/z calcd for C22H24O4 ([M]+): 352.4300,
found: 351.3562.
4.1.3.10. (E)-3-(2-acetyl-5-methoxyphenoxy) propyl 3-(4-((tert-butyldi-
methylsilyl)oxy)-3- methoxyphenyl)acrylate (compound 10).. The solu-
tion of acetic anhydride (25 mM, 10 eq) was added to a solution of
compound 4 (1.5 mM, 1 eq) in DCM, and the mixture was refluxed for 2
h. SOCl2 (0.15 M) was added to a solution of Compound2a (1.0 mM) in
DCM (20 mL), and the mixture was refluxed for 5 h at 75 ◦C. Then the
solution was heated to 85 ◦C for evaporation. After cooling to room
temperature, and then concentrated under reduced pressure to obtain
yellow solution, which went straight to the next reaction. Compounds
1a-c (2.5 mM, 1.7 eq) was added at 0 ◦C, and then added NEt3 (5 mM, 2
eq). The reaction was stirred at room temperature overnight and
monitored by TLC. The mixture was washed with water and brine
respectively. The organic layer was dried over MgSO4, and then
concentrated under reduced pressure to give a bright yellow oil as an
intermediate. The obtained residue was purified by silica gel chroma-
tography using petroleum ether/ethyl acetate (1:1, v: v) as original
eluent to give compounds 10–12 (Yield: 65–75%).
4.1.3.6. (E)-4-(3-(3-(4-allyl-2-methoxyphenoxy) propoxy)-3-oxoprop-1-
en-1-yl)-1, 2-phenylene diacetate (compound 6).. Compound 1b
replaced compounds 1a as material, others procedures as the same as
compounds 2.
White crystal (EtOAc).m.p. 74–75 ◦C. Yield: 60%; 1H NMR (400
MHz, CDCl3) δ 7.63 (d, J = 16.0 Hz, 1H), 7.41 (dd, J = 8.5 Hz, 1H), 7.39
(d, J = 8.5 Hz, 1H), 7.35 (d, J = 16.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H),
6.83 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 6.4 Hz, 1H), 6.38 (d, J = 6.4 Hz,
1H), 5.95 (m, J = 6.7 Hz, 1H), 5.07 (t, J = 14.5 Hz, 1H), 4.17 (t, 2H),
3.82 (d, J = 14.5 Hz, 9H), 3.32 (s, 1H), 2.79 (s, 1H), 2.30 (s, 3H), 2.11 –
1.97 (m, 2H), 1.63(s, 2H), 1.25 – 1.22 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 168.27, 168.20, 166.67, 146.56, 142.97, 137.74, 133.27,
120.50, 119.25, 115.78, 113.49, 112.34, 65.78, 61.74, 55.97, 39.93,
28.75, 20.81, 20.77; IR (cmꢀ 1) 2941.43, 2890.54, 2364.27, 2327.87,
1968.56, 1642.92, 1603.43, 1564.35, 1415.99, 1258.56, 1206.17,
1058.54, 961.62, 832.27, 711.23, 613.01. HRMS (ESI, positive) m/z
calcd for C26H28O8 ([M]+): 468.5020, found: 467.4419.
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White crystal. m.p. 80–81 ◦C. 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J
4.1.3.14. (E)-3-(3-acetyl-4-hydroxyphenoxy) propyl ꢀ 3-(4-hydroxy-3-
methoxyphenyl) acrylate (compound 14).. 1 mL NH4F of THF solution (1
mol/L, 1.5 eq) was added to a solution of the compound 10 (0.2 mmol, 1
eq) in THF (20.0 mL), other procedure were accordance with the syn-
thesise of the compound 13. Product as white powder have gain even-
tually (Yield: 76%).
= 8.8 Hz, 1H), 7.63 (d, J = 15.9 Hz, 1H), 7.26 (s, 1H), 7.03 (dd, J = 5.4,
2.0 Hz, 3H), 6.84 (d, J = 8.7 Hz, 1H), 6.53 (dd, J = 8.8, 2.3 Hz, 1H), 6.46
(d, J = 2.3 Hz, 1H), 6.29 (d, J = 15.9 Hz, 1H), 4.42 (t, J = 6.2 Hz, 2H),
4.18 (t, J = 6.2 Hz, 2H), 3.84 (d, J = 4.1 Hz, 8H), 2.61 (d, J = 2.5 Hz,
3H), 2.36 – 2.20 (m, 2H), 1.58 (d, J = 1.2 Hz, 1H), 1.01–0.95 (m, 9H);
13C NMR (100 MHz, CDCl3) δ 202.75, 167.30, 165.38, 165.30, 151.26,
147.69, 145.32, 132.43, 127.36, 123.00, 121.12, 115.46, 113.99,
110.76, 108.17, 101.31, 64.85, 60.96, 55.50, 28.55, 26.38, 25.73,
18.58; IR (cmꢀ 1): 3891.33, 2855.73, 2362.10, 1700.70, 1610.32,
1552.48, 1415.97, 1272.62, 904.53, 842.09, 710.08. HRMS (ESI, posi-
tive) m/z calcd for C27H38O6Si ([M]+): 498.6910, found: 497.6240.
White powder (CH3OH).m.p. 152–153 ◦C; Yield: 35%; 1H NMR (400
MHz, CDCl3) δ12.76 (s, 1H), 7.65 (s, 1H), 7.62 (d, J = 1.0 Hz, 1H), 7.03
(m, J = 6.0 Hz, 2H), 6.84 (d, J = 8.2 Hz, 1H), 6.45 (d, J = 3 Hz, 1H), 6.44
(s,1H), 6.30 (d, 1H), 4.40 (d, J = 6.4 Hz, 2H), 4.15 (d, J = 6.3 Hz, 2H),
3.93 (s, 3H), 3.33 (s,3H), 2.70 – 2.48 (m, 2H), 2.19 (s, 3H), 2.04 (s, 1H),
1.31 – 1.21 (m, 2H); 13C NMR (100 MHz, CDCl3) δ197.63, 197.61,
194.74, 164.61, 164.60, 164.46, 133.25, 120.62, 105.07, 105.06,
105.03, 100.0, 99.04, 66.69, 55.65, 55.62, 32.01, 30.99; IR: 3585.91,
3414.30, 1653.24, 1630.48, 1582.63, 1430.25, 1120.56, 840.65,
730.28, 708.51, 620.65. HRMS (ESI, positive) m/z calcd for C22H24O7
([M]+): 400.4270, found: 399.1528.
4.1.3.11. (E)-3-(2-acetyl-5-methoxyphenoxy) propyl-3-(4-((tert-butyldi-
methylsilyl) oxy)-3-methoxyphenyl) acrylate (compound 11).. The com-
pound 1c was replaced with the compounds 1a, others synthesis
peocedures as the same as the synthesis of 10.
White crystal (EtOAc) m.p. 79–80 ◦C. Yield: 66%; 1H NMR (400
MHz, CDCl3) δ12.74 (s, 1H), 7.62 (s, 1H), 7.61 (d, 1H), 7.02 (s, 2H), 6.84
(d, J = 8.6 Hz, 1H), 6.45 (d, J = 8.5 Hz, 2H), 6.29 (d, J = 15.9 Hz, 1H),
4.39 (t, J = 6.2 Hz, 2H), 4.14 (t, J = 6.2 Hz, 2H), 3.83 (s, 3H), 2.55 (s,
3H), 2.27– 2.10 (m, 2H), 1.57(s, 2H), 0.99 (s, 9H); 13C NMR (100 MHz,
CDCl3) δ173.23, 165.21, 132.37, 128.22, 122.48, 120.80, 115.46,
114.19, 111.31, 107.65, 100.64, 65.04, 60.98, 55.58, 26.31, 25.65; IR
(cmꢀ 1): 3238.39, 2944.03, 2891.54, 2855.82, 2622.35, 2359.74,
1701.44, 1608.34, 1553.95, 1416.34, 1272.61, 1152.55, 905.83,
842.02, 710.98, 612.37. HRMS (ESI, positive) m/z calcd for C27H36O6Si
([M]+): 484.6640, found: 483.5265.
4.2. Tyrosinase activity assay
4.2.1. Anti-tyrosinase activity
The measurement of tyrosinase (EC 1.14.18.1) inhibition was
possessed according to the method of Nazir with minor modification
[46,47]. Mushroom tyrosinase (Sigma-aldrich, 1000 Umgꢀ 1), L-3, 4-
dihydroxyphenylalanine (L-dopa), dimethyl sulfoxide (DMSO) were
purchased from Sigma Chemical Co (St. Louis, Mo, USA). All of com-
pounds were dissolved in DMSO, which tested for di-phenolase inhibi-
tory activity of tyrosinase by L-dopa as substrate.Thetest controls the
final concentration of DMSO in the solution was 2.0%. Firstly, 160
μ
L of
L of mushroom tyrosinase (final con-
L of the inhibitor solution were com-
phosphate buffer (pH 6.86), 20
μ
4.1.3.12. (E)-3-(4-allyl-2-methoxyphenoxy) propyl ꢀ 3-(4-((tert-butyldi-
methylsilyl)oxy)-3- methylphenyl)acrylate (compound 12).. The com-
pound 1b was replaced with the compounds 1a, others synthesis
peocedures as the same as the synthesis of 10.
centration: 50U/mLꢀ 1) and 10
μ
bined in a centrifuge tube. After pre-incubation for 10 min at room
temperature, 10 μL L-dopa (final concentration: 0.4 mM) was added to
White crystal (EtOAc). m.p. 155–156 ◦C. Yield: 61%; 1H NMR (400
MHz, CDCl3) δ12.54 (s, 1H) , 7.61 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 2.0
Hz, 0H), 7.39 (d, J = 2.0 Hz, 1H), 7.36 (d, J = 1.9 Hz, 1H), 7.22 (d, J =
8.4 Hz, 1H), 4.39 (t, J = 6.2 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.86 (s,
3H), 2.54 (s, 3H), 2.31 (d, J = 2.5 Hz, 2H), 2.21 (s, 1H), 2.21 (s, 1H),
1.58 (s, 1H); 13C NMR (100 MHz, CDCl3) δ198.75, 165.39, 160.89,
132.41, 121.19, 106.45, 98.15, 66.89, 60.95, 55.76, 32.09, 31.31,
25.63; IR (cmꢀ 1): 2861.34, 256.19, 1745.26, 1680.59, 1671.64,
1630.29, 1601.34, 1580.61, 850.32, 790.25, 751.38, 680.24. HRMS
(ESI, positive) m/z calcd for C29H40O5Si ([M]+): 496.7190, found:
495.7261.
the mixture to examine immediately. The enzyme reaction was moni-
tored for 1 min by measuring the change in absorbance at 475 nm
corresponding to the formation of dopachrome. Inhibition of tyrosinase
reaction was calculated as follows: inhibition ratio (I%) = [(As-Ab)/ (Ac-
Ab)] × 100, whereAb and As are the absorbance of the blank and
samples.
4.2.2. Kinetic analysis of the inhibition of mushroom tyrosinase
A series of diluted inhibitor solutions were prepared (8: 0, 15, 31, 62
and 125 μM, 13: 0, 7, 15, 31, and 62 μM, 14: 0, 15, 31, 62 and 125 μM).
The L-dopa concentration was 0.4 mM and the concentration of tyrosi-
nase were 0.0, 8.3, 16.7 and 33.3 U mLꢀ 1, respectively. Substrate L-dopa
concentration range from 0.2 to 1.6 mM in all kinetic study. Pre-
incubation and measurement time wasthe same as discussed in mush-
room tyrosinase inhibition assay protocol. The formation of L-dopa
chrome was continuously monitored at 475 nm for 5 min at a 30 s in-
terval in the microplate reader after the addition of enzyme. Inhibition
type on the enzyme was assayed by Lineweaver–Burk plots of inverse of
velocities (1/V) versus inverse of substrate concentration 1/[S], and the
inhibition constant KIwas determined by the Dixon plot of 1/V versus
inhibitor concentrations.
4.1.3.13. (E)-3-(2-acetyl-5-methoxyphenoxy)
propyl-3-(4-hydroxy-3-
methoxyphenyl) acrylate (compound 13).. 1 mL NH4F of THF solution
(1 mol/L, 1.5 eq) was added to a solution of the compound 11 (0.2
mmol, 1 eq) in THF (20.0 mL), and the mixture was stirred for 4 h at
room temperature. The reaction mixture was concentrated, which was
washed with saturated NaHCO3, H2O and brine respectively. The
organic layer was dried with MgSO4, which was purified by chroma-
tography to give 13 as a pale yellow powder and white powder
respecticely (Yield: 70.6%).
White powder (CH3OH).m.p. 127–128 ◦C; Yield: 54%;1H NMR (400
MHz, CDCl3) δ12.79(s, 1H), 7.64 (s, 1H), 7.62 (d, J = 6.0 Hz, 1H), 7.08
(dd, J = 6.0 Hz, 2 Hz, 1H), 7.03 (d, 1H), 6.92 (s, J = 6.0 Hz, 1H), 6.46 (d,
J = 3.0 Hz, 1H), 6.43 (d, J = 3.0 Hz, 1H),4.38 (t, J = 9.0 Hz, 2H), 4.13 (t,
4.2.3. Cytotoxicity of compounds 8, 13 and 14
B
16F10 melanoma cell (1.5× 105 cells/L) line obtained from (Cell
Bank of the Chinese Academy of Science, China) was cultured at 37 ◦C
under 5% CO2 atmosphere and was used to determine the cytotoxicity.
The B16F10 cells (104 × cells/well) were seeded in Dulbecco’s modified
J = 9.0 Hz,2H), 3.92 (s, 3H),2.20 (m, J = 6.2 Hz, 2H), 2.55 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ195.96, 167.01, 165.29, 162.98, 151.50,
147.76, 134.36, 129.08, 128.55, 123.72, 117.84, 112.45, 109.63, 64.79,
61.10, 28.10, 26.50, 26.12, 20.68; IR (cmꢀ 1) 3434.96, 3238.39,
2955.44, 2893.19, 2856.26, 1699.01, 1628.08, 1509.92, 1469.06,
1280.11, 1265.24, 1180.25, 1020.38, 903.50. HRMS (ESI, positive) m/z
calcd for C21H22O7 ([M]+): 386.4000, found: 385.3173.
Eagle’s medium (DMEM) containing 100 IU/mL penicillin, and 100 μg/
mL streptomycin, supplemented with 10% FBS, under 5% CO2 humid-
ified atmosphere at 37 ◦C. The cell viability assay was evaluated by the
conversion of MTT method to a purple formazan precipitate as previ-
ously described. After 24 h, the various concentrations of compounds
were subsequently added and incubated for 24 h. The survival rate was
14

K. Tang et al.
Bioorganic Chemistry xxx (xxxx) xxx
calculated from plotted results using cells untreated by test compounds
as reference with some slight modifications [48]
structures files were built with Discovery Studio 2019 software, and
Ledock software was used for the docking. All the ligand and water
atoms were removed. The copper ions in the active site were kept.
Hydrogen atoms were auto-added by the protein prepared on Ledock
software to obtain protein file format. The final conformation and psf
topology file were extracted for docking. Ligand molecular files are
performed screened and prepared through the rules of Lipiski five. The
native ligand was re-docked to investigate the validity of the docking
protocol. The docking site was considered using the position of the
tyrosinase inhibitor and the radius active sites were set to ꢀ 29.76;-
10.28; ꢀ 42.21 dimensions. In order to verify the results of docking, the
known tyrosinase inhibitors, such as arbutin and kojic acid (KA) were
also docked to the molecule. As the results present (Fig. 8), the docked
conformation of tropolonewas well superposed to the conformation in
the crystal structure (RSMD < 1.0A).
4.2.4. Determine the cellular melanin content;
Melanin content assay was performed to evaluate the inhibitory ef-
fect of compounds 8, 11, 13and 14 (25, 50, 100, and 200 µM) on
melanogenesis using the standard method with slight modification [49].
Briefly, control group containing 1 × 105 cells per well, and treated
group with 1 × 105 cells containing 1 µM
α-MSH and various concen-
trations of compounds 8, 11, 13, 14, kojic and A(25, 50, 100, and 200
µM) for 48 h. The medium was removed and then 1% Triton X-100
mixed in 10 mM phosphate buffered saline was added. The mixture was
frozen at ꢀ 80 ◦C and thawed at room temperature. A freshly substrate
(2 mM L-dopa) was preparedand then added to the supernatant and
incubated. The absorbance of each well was subsequently read at 475
nm.
Melanincontent(%) = [(As ꢀ Ab)/(Ac ꢀ Ab)]100
Declaration of Competing Interest
The As,Ab and Acwere the absorbance of test compounds, the blank
and control group respectively.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.2.5. Determination of cellular tyrosinase activity:
Tyrosinase inhibition assays of cinnamyl-phenol ester derivatives 8,
11, 13 and 14 were performed as previously described with slight
modification [50,51]. In brief, B16F10 cells were inoculated at 5 × 104
cells per well in 96-well plates and incubated for 24 h in a humidified 5%
Acknowledgements
The authors would like to thank Shaanxi Province Key Technologies
R&D Program (S2017-ZDYF-YBXM-SF-0639), Shaanxi University of
Chinese Medicine Innovative team (2019-YL12) and Shaanxi University
of Chinese Medicine Program (303-123890013) for financial support of
this work.
CO2 atmosphere at 37 ◦C. The cells were then treated with
α
-MSH (1 µM)
and kojic acid (25 µM) or
α-MSH (1 µM) and compounds8, 11, 13 and 14
(0, 5, 10, or 25 µM) and re-cultured for 24 h under the same conditions.
Cells were then washed with PBS buffer, lysed with lysis buffer (100 µL
containing 1% Triton X-100 (5 µL), 0.1 mM PMSF (5 µL), and 50 mM PBS
(pH 6.8, 90 µL), frozen at ꢀ 80 ◦C for 30 min, centrifuged at 12,000 rpm
for 30 min at 4 ◦C, transferred to 96-well plates, treated with L-dopa
(total volume 100 µL; 80 µL of supernatant and 20 µL of 10 mM L-dopa),
and incubated for 30 min at 37 ◦C. Tyrosinase inhibitions were deter-
mined by measuring optical densities at 475 nm in a Tecan, Man-
nedorfmicroplate reader. The experiment was performed in triplicate.
Tyrosinase activity (I%) = [(As-Ab)/ (Ac-Ab)]× 100
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104512.
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