(E)-2-Cyano-3-(substituted phenyl)acrylamide analogs as potent inhibitors of tyrosinase: A linear β-phenyl-α,β-unsaturated carbonyl scaffold

Article abstract of DOI:10.1016/j.bmc.2015.11.015

In this study, we synthesized (E)-2-cyano-3-(substituted phenyl)acrylamide (CPA) derivatives which possess a linear β-phenyl-α,β-unsaturated carbonyl scaffold and examined their inhibitory activities against tyrosinase. CPA analogs exerted inhibitory acti

Full text of DOI:10.1016/j.bmc.2015.11.015

Bioorganic & Medicinal Chemistry 23 (2015) 7728–7734
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
(
E)-2-Cyano-3-(substituted phenyl)acrylamide analogs as potent
inhibitors of tyrosinase: A linear b-phenyl-
scaffold
a,b-unsaturated carbonyl
a,y
a,y
a
a
a
a
Sujin Son , Haewon Kim , Hwi Young Yun , Do Hyun Kim , Sultan Ullah , Seong Jin Kim ,
b
a
a
b,
⇑
a,
⇑
Yeon-Jeong Kim , Min-Soo Kim , Jin-Wook Yoo , Pusoon Chun , Hyung Ryong Moon
a
College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
College of Pharmacy, Inje University, 197 Inje-ro, Gimhae, Gyeongnam 621-749, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized (E)-2-cyano-3-(substituted phenyl)acrylamide (CPA) derivatives which
possess a linear b-phenyl- ,b-unsaturated carbonyl scaffold and examined their inhibitory activities
against tyrosinase. CPA analogs exerted inhibitory activity against mushroom tyrosinase. Results from
the docking simulation indicated that CPA2 could bind directly to the active site of mushroom tyrosinase
and the binding affinity of CPA2 for tyrosinase might be higher than that of kojic acid, a well-known
potent tyrosinase inhibitor. In B16F10 cells, CPA2 significantly suppressed tyrosinase activity and
Received 5 October 2015
Revised 11 November 2015
Accepted 16 November 2015
Available online 17 November 2015
a
Keywords:
melanogenesis in a dose-dependent manner. At the concentration of 25
inhibitory activity comparable to that of kojic acid with no cytotoxic effect. Results from the present
study suggest that CPA2 bearing a linear b-phenyl- ,b-unsaturated carbonyl scaffold may be the
potential candidate for treatment of diseases associated with hyperpigmentation and that a linear
lM, CPA2 exhibited tyrosinase
(E)-2-Cyano-3-(substituted phenyl)
acrylamide
Tyrosinase inhibitor
Melanogenesis
a
b-phenyl-
a,b-unsaturated carbonyl scaffold might be closely related to potent tyrosinase inhibition.
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Tyrosinase (EC 1.14.18.1) is a multifunctional metalloenzyme
pheomelanin, brown to black neuromelanin, and mixed melanin
pigment of eu- and pheomelanin.
1
0,11
Although melanin has a photoprotective function in human
skin, hyperpigmentation of melanin in specific parts of the skin,
such as melasma, freckles, and lentigines, might cause an esthetic
and is classified into the type-3 copper protein family that contains
a coupled binuclear copper active site.1 Both monooxygenase and
oxidase activities of the enzyme come from the binding of dioxy-
gen to the two copper ions, each coordinately bonded with a
distinct set of three histidine residues within the active site.
Tyrosinase is widely distributed in bacteria, fungi, plants, and
animals,
biosynthesis: the hydroxylation of
phenylalanine ( -DOPA) and the subsequent oxidation of
to dopaquinone. In addition, mammalian tyrosinase has been
shown to catalyze a third reaction in melanogenesis: the oxidation
of 5,6-dihydroxyindole (DHI) to indole 5,6-quinone.9
,2
1
2,13
problem.
Moreover, undesirable enzymatic browning of fruits
and vegetables is one of the major problems in the agricultural
3
–5
14
and food fields.
Because tyrosinase is responsible for abnormal skin pigmenta-
tion and undesirable browning of foods, significant efforts have
been made to search for potent tyrosinase inhibitors. So far a huge
6
,7
and catalyzes two distinct reactions of melanin
-tyrosine to 3,4-dihydroxy-
-DOPA
L
L-
L
L
number of naturally occurring and synthetic tyrosinase inhibitors
8
15–19
have been reported.
However, their practical use is limited
due to their poor efficacy, safety concerns, low formulation stabil-
20,21
ity, and poor skin penetration.
There is still a considerable
Melanins, the end-products of complex multistep transforma-
demand for novel tyrosinase inhibitors that are safer and more
effective.
tions of
L-tyrosine, are polyphenol-like biopolymers and are repre-
sented by black to brown eumelanin, yellow to reddish-brown
Recently it was reported by the authors that the ‘b-phenyl-
a,b-
unsaturated carbonyl’ group can act as a good template showing
2
2
anti-tyrosinase activity. Compounds I–IV (Fig. 1), which possess
⇑
Corresponding authors. Tel.: +82 55 3203886 (P.C.), +82 51 5102815 (H.R.M.).
E-mail addresses: pusoon@inje.ac.kr (P. Chun), mhr108@pusan.ac.kr
this group, all exerted potent inhibitory activity against mushroom
22–25
tyrosinase and skin-whitening effects in vivo.
These com-
(
H.R. Moon).
y
pounds have a five-membered ring, and the carbonyl and a-carbon
These authors (S.S. and H.K.) contributed equally to this article.
http://dx.doi.org/10.1016/j.bmc.2015.11.015
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0

S. Son et al. / Bioorg. Med. Chem. 23 (2015) 7728–7734
7729
Figure 1. The rationale for the design of CPA derivatives with a linear b-phenyl-a,b-unsaturated carbonyl scaffold.
of the ‘b-phenyl-a,b-unsaturated carbonyl’ group constitute parts
Table 1
Inhibitory effects of derivatives CPA1–CPA12 and kojic acid on mushroom tyrosinase
activity
of this ring. In the beginning, an expansion of the five-membered
ring to a six-membered ring, such as, to barbituric and thiobarbi-
turic acids, was attempted to strengthen anti-tyrosinase activities
in vitro and in vivo. However, this ring expansion resulted in a
decrease of tyrosinase inhibition. As an alternative approach to
search for novel compounds possessing a powerful anti-tyrosinse
effect, the authors attempted to synthesize new compounds with
Compound
Inhibition (%)
CPA1
CPA2
CPA3–CPA12
Kojic acid
23.64 ± 0.31
44.45 ± 2.19
NI
39.35 ± 0.83
a ‘linear’ b-phenyl-
with a ring.
In this paper, we report the synthesis of (E)-2-cyano-3-(substi-
tuted phenyl)acrylamide derivatives CPA1–CPA12 carrying a linear
a
,b-unsaturated carbonyl scaffold, not fused
Mean ± standard errors are shown. Results are representative of at least three
independent experiments, which were conducted at a concentration of 25
denotes no inhibition.
lM. NI
b-phenyl-a,b-unsaturated carbonyl scaffold (Scheme 1) and their
inhibitory effects on tyrosinase activity.
2
2
. Results and discussion
.1. Synthesis of (E)-2-cyano-3-(substituted phenyl)acrylamide
derivatives
(E)-2-Cyano-3-(substituted phenyl)acrylamide derivatives,
CPA1–CPA12 were synthesized using a Knoevenagel reaction in
the presence of a catalytic amount of piperidine, as shown in
O
O
N
O
N
a
2
H N
H
Figure 2. Determination of the nature of tyrosinase inhibition. Inhibition type of
CPA2 was determined using Lineweaver–Burk plots. Results are mean values of 1/V,
H
2
N
+
R
R
the inverse of the increase in the absorbance per minute at the different
concentrations. The modified Michaelis–Menten equation was utilized: 1/Vmax
/K (1 + [S]/K ). denotes the velocity of the reaction; is the -tyrosine
concentration; K is the Michaelis constant; and K is the inhibition constant.
L-tyrosine
2-
cyanoacetamide
benzaldehydes
=
CPA1 - CPA12
1
m
i
V
S
L
m
i
Benzaldehydes:
O
O
O
O
O
O
OH
OMe
OEt
OH
H
H
H
H
H
H
H
Scheme 1. Refluxing 2-cyanoacetamide and a variety of appropri-
ately substituted benzaldehydes (1–12) in ethanol containing
piperidine gave (E)-isomers of 2-cyano-3-(variously substituted
phenyl)acrylamides as sole products, in a moderate to good yields.
The Knoevenagel condensation is known to primarily afford the
cyano group and the aryl group cis to each other. A previous study
of the Knoevenagel condensation of cyanoacetate with aryl aldehy-
des confirmed that the phenyl group is trans to the carboxyl group
OH
OH
OH
1
2
3
4
O
O
O
O
O
O
OMe
OH
OMe
OMe
H
H
H
H
OMe
OMe
OMe
5
6
7
8
1
3
based on C NMR long-range selective proton decoupling experi-
2
6
OMe
OMe
OMe
OH
Br
Br
ments. The authors confirmed the configuration of double bond
H
1
13
of the olefin products by comparing H and C NMR spectra with
27–29
OH
OH
those of authentic samples with (E)-geometry.
The spectral
OMe
9
OMe
10
Br
data of the olefin products were identical to those of authentic
samples, thus confirming that all compounds synthesized are
11
12
(E)-isomers.
Scheme 1. Reagents and conditions: (a) piperidine, ethanol, reflux.

7
730
S. Son et al. / Bioorg. Med. Chem. 23 (2015) 7728–7734
Figure 3. Docking simulation between tyrosinase and CPA2 or kojic acid. (a) Docking simulation between mushroom tyrosinase and CPA2 shown in magenta. (b) The
pharmacophore model generated using the LigandScout 3.1 program indicates possible hydrogen bonding interactions and hydrophobic interactions between tyrosinase
residues and CPA2. (c) Docking simulation between tyrosinase and kojic acid used as a positive control. (d) The pharmacophore model generated using the LigandScout 3.1
program indicates a possible hydrogen bonding interaction between tyrosinase residues and kojic acid. (e) Docking simulation between tyrosinase and L-tyrosine.
2
.2. Inhibitory effects of (E)-2-cyano-3-(substituted phenyl)
Figure 2, inhibition of mushroom tyrosinase resulted in a double-
acrylamide derivatives CPA1–CPA12 on mushroom tyrosinase
reciprocal plot, on which the four straight lines having different
inclination crossed the y-axis at similar points. This result showed
The effects of the synthesized compounds on the activity of
mushroom tyrosinase were studied. Kojic acid, that is a 10-fold
stronger tyrosinase inhibitor than arbutin,30 was used as a positive
control. Of the synthesized compounds, CPA1 and CPA2 showed
inhibitory effects while the others had no effect on the enzyme.
Compared with the effect of kojic acid against mushroom tyrosi-
nase, CPA2 exhibited a slightly greater effect and CPA1 showed a
less inhibitory effect (Table 1). An introduction of a hydroxyl group
into the 4-position of the phenyl ring endowed CPA1 with the inhi-
bitory ability against tyrosinase, whereas the additional substitu-
tions of an alkoxyl (OMe and OEt) or bromo group led the
corresponding compounds to lose the inhibitory activity (e.g.,
CPA3, CPA4 and CPA10–CPA12). It is notable that an additional
substitution with a hydroxyl group at 3-position of the phenyl ring
increased the inhibitory activity (e.g., CPA2), which might be
m
that K (Michaelis constant) values increased gradually as the con-
centration of CPA2 was increased while Vmax (maximum reaction
velocity) were not affected by CPA2 concentration. Therefore, it
is clear that CPA2 competitively inhibits mushroom tyrosinase in
a dose dependent manner.
2.4. In silico docking simulation of tyrosinase with CPA2 and
kojic acid
To explore whether CPA2 directly inhibits tyrosinase by binding
to the active site of the enzyme, the authors have performed a
docking study using AutoDock vina, which is the most commonly
used due to its automated docking capability. The docking simula-
tion was successful (Fig. 3). The docking score between the ligand
and the receptor is represented by various energy terms such as
electrostatic energy, van der Waals energy, and the salvation
energy. The binding energy of CPA2 was ꢀ7.2 kcal/mol while that
of kojic acid, a positive control, was ꢀ5.4 kcal/mol. The docking
simulation provided corroborating evidence that the binding affin-
ity of CPA2 for tyrosinase might be higher than that of kojic acid.
This result implies that the inhibitory effect of CPA2 on tyrosinase
activity is attributed to the binding affinity of CPA2 for tyrosinase
attributed to the structural similarity of CPA2 to L-dopa, one of nat-
ural tyrosinase substrates.
2
.3. Determining the nature of tyrosinase inhibition
The Lineweaver–Burk plot was utilized to elucidate the type of
inhibition of CPA2 against mushroom tyrosinase. As depicted in

S. Son et al. / Bioorg. Med. Chem. 23 (2015) 7728–7734
7731
Figure 4. The effect of CPA2 on B16F10 cell viability. Cell viabilities at different
concentrations of CPA2 are expressed as mean percentage viabilities versus
untreated control cells. Results are representative of at least three independent
experiments.
Figure 6. The effect of CPA2 on cellular tyrosinase activity. B16F10 cells were
treated with -MSH and exposed to different concentrations of CPA2 or kojic acid
for 24 h. Tyrosinase activities are expressed as mean percentage inhibitions versus
control cells treated with -MSH alone. The asterisk denotes statistical significance
a
a
between control cells and cells exposed to CPA2 or kojic acid: ⁄⁄⁄, p <0.001. Results
are representative of at least three independent experiments.
respectively, have great potential to interact with tyrosinase inhi-
bitors which compete with tyrosinase substrates at the enzyme
active site.
2
.5. Cell viability assay
The effect of CPA2 on B16F10 cell viability was assessed by a
MTT assay (Fig. 4). After 24 h treatment with 5, 10, or 25 lM of
CPA2, cell viabilities were 99.6%, 98.6%, and 96.9% relative to
untreated controls. This results imply that CPA2 is non-cytotoxic
3
1
at concentrations of 625
lM.
2
.6. Melanin content assay
The inhibitory effect of CPA2 on
in B16F10 cells was evaluated by measuring intracellular melanin
as described in experimental section. Melanin contents in CPA2/
MSH co-treated cells were significantly small compared to those in
cells treated with -MSH alone. Furthermore, CPA2 inhibited mel-
anin production at 0–25 M in a dose-dependent manner and at
25 M the inhibitory potency of CPA2 was slightly greater than
that of kojic acid (Fig. 5).
a-MSH-induced melanogenesis
a
-
Figure 5. The effect of CPA2 on
were treated with -MSH and exposed to different concentrations of CPA2 or kojic
acid for 24 h. Melanin contents are expressed as mean percentage inhibitions versus
control cells treated with -MSH alone. The asterisk denotes statistical significance
a-MSH-mediated melanogenesis. B16F10 cells
a
a
l
a
l
between control cells and cells exposed to CPA2 or kojic acid: ⁄⁄⁄, p <0.001. Results
are representative of at least three independent experiments.
2
.7. Cellular tyrosinase activity assay
and the binding between CPA2 and tyrosinase may be more ener-
getically favored than the kojic acid-to-tyrosinase binding. More-
The effect of CPA2 on cellular tyrosinase activity was examined
a-MSH-stimulated B16F10 cells. CPA2 significantly reduced
tyrosinase activity in a dose-dependent manner (Fig. 6). Assess-
ment of tyrosinase activity demonstrated a similar trend to the
melanin content assay. This result implies that the anti-melano-
genic effect of CPA2 is attributed to its ability to suppress tyrosi-
nase activity.
in
over, compared with
tyrosinase, the binding affinity of CPA2 was higher than that of
tyrosine: the binding energy of
-tyrosine was ꢀ5.8 kcal/mol. The
L-tyrosine, one of the natural substrates of
L
-
L
authors searched for amino acid residues of tyrosinase interacting
with CPA2 using LigandScout 3.1. As shown in Figure 3b, Met280
residue of tyrosinase was mainly responsible for the hydrogen
bonding interaction with CPA2. The phenolic 4-hydroxyl group of
CPA2 served as a hydrogen bond donor and the carbonyl oxygen
of the Met280 was hydrogen-bonded to the phenolic hydroxyl
group. In addition, the phenyl ring of CPA2 was responsible for
hydrophobic interactions with two amino acid residues, Phe264
and Val283. On the other hand, there was one hydrogen bonding
interaction between kojic acid and tyrosinase via Asn260 (Fig. 3d).
The common amino acid residues (Met280, Ser282, Val283 and
Ala286) found within a 3 Å distance from CPA2 and kojic acid,
3. Conclusions
In present study, (E)-2-cyano-3-(substituted phenyl)acrylamide
derivatives possessing a linear
a,b-unsaturated carbonyl scaffold
were designed and successfully synthesized. Of the synthesized
derivatives, two compounds CPA1 and CPA2 exerted inhibitory
activity against mushroom tyrosinase. Our docking simulation
results demonstrated that CPA2 is able to bind directly to the

7
732
S. Son et al. / Bioorg. Med. Chem. 23 (2015) 7728–7734
active site of mushroom tyrosinase with higher binding affinity
than kojic acid and -tyrosine and our kinetic study indicated that
CPA2 inhibits tyrosinase in a competitive manner. Additionally, in
B16F10 melanoma cells CPA2 significantly suppressed cellular
tyrosinase activity and melanogenesis in a dose-dependent man-
4.2.1.3.
mide (CPA3).
(E)-2-Cyano-3-(4-hydroxy-3-methoxyphenyl)acryla-
Yellowish solid; reaction time, 24 h; yield,
84.4%; H NMR (500 MHz, DMSO-d ) d 10.23 (s, 1H, OH), 8.04
(s, 1H, vinylic H), 7.71, 7.59 (each s, 2H, NH ), 7.65 (d, 1H,
J = 2.0 Hz, 2 -H), 7.44 (dd, 1H, J = 8.0, 2.0 Hz, 6 -H), 6.92 (d, 1H,
L
1
6
2
0
0
0
13
ner and was as effective as kojic acid at 25
lM with no cytotoxic
3 6
J = 8.5 Hz, 5 -H), 3.80 (s, 3H, CH ); C NMR (100 MHz, DMSO-d )
effect. Collectively, CPA2 bearing a linear b-phenyl-
a
,b-unsatu-
d 164.0, 152.1, 151.5, 148.4, 126.6, 123.9, 118.1, 116.6, 113.9,
102.1, 56.2; LRMS (ESI-) m/z 217 (MꢀH) .
ꢀ
rated carbonyl scaffold may be the potential candidate for a new
therapeutic drug for treatment of diseases associated with hyper-
pigmentation and for skin-whitening agents. This research indi-
cates that a ‘linear’ b-phenyl-a,b-unsaturated carbonyl scaffold
also plays an essential role in showing anti-melanogenic effect
4
(
.2.1.4. (E)-2-Cyano-3-(3-ethoxy-4-hydroxyphenyl)acrylamide
CPA4). Yellow solid; reaction time, 12 h; yield, 56.4%; 1H NMR
(
7
(
2
400 MHz, DMSO-d
.70, 7.58 (each s, 2H, NH
dd, 1H, J = 8.0, 1.6 Hz, 6 -H), 6.91 (d, 1H, J = 8.0 Hz, 5 -H), 4.02 (q,
2 3 2 3
H, J = 6.8 Hz, CH CH ), 1.33 (t, 3H, J = 6.8 Hz, CH CH ); C NMR
6
) d 10.15 (s, 1H, OH), 8.00 (s, 1H, vinylic H),
0
2
), 7.61 (d, 1H, J = 1.6 Hz, 2 -H), 7.39
through the direct inhibition of tyrosinase enzyme.
0
0
13
4
4
. Experimental section
.1. Materials
(
1
100 MHz, DMSO-d
18.1, 116.6, 114.8, 102.1, 64.5, 15.2; LRMS (ESI-) m/z 231 (MꢀH) .
6
) d 164.0, 152.3, 151.5, 147.5, 126.7, 123.9,
ꢀ
4
.2.1.5.
(E)-2-Cyano-3-(3-hydroxy-4-methoxyphenyl)acryla-
CPA1–CPA12
Mushroom tyrosinase, 3-(4-hydroxyphenyl)-
-(3,4-dihydroxyphenyl)- -alanine
hydroxymethyl-4H-4-pyranone (kojic acid), 3-(4,5-dimethyl-2-
were
synthesized
in
L
our
laboratory.
mide (CPA5). Greenish yellow solid; reaction time, 6 h; yield,
-alanine (L-tyrosine),
1
7
9.2%; H NMR (400 MHz, DMSO-d
6
) d 9.54 (s, 1H, OH), 7.95
), 7.48 (d, 1H,
3
L
(L
-DOPA),
5-hydroxy-2-
(
s, 1H, vinylic H), 7.74, 7.60 (each s, 2H, NH
2
0
0
J = 1.6 Hz, 2 -H), 7.35 (dd, 1H, J = 8.4, 1.6 Hz, 6 -H), 7.06 (d, 1H,
J = 8.8 Hz, 5 -H), 3.82 (s, 3H, CH ); C NMR (100 MHz, DMSO-d )
3 6
d 163.9, 152.4, 151.1, 147.4, 125.3, 125.3, 117.7, 116.2, 112.7,
thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide
(MTT),
13
0
alpha-melanocyte-stimulating hormone (a-MSH), phenylmethyl-
sulfonyl fluoride (PMSF), 4-(1,1,3,3-tetramethylbutyl)phenyl-
polyethylene glycol (TritonTM X-100), dimethyl sulfoxide (DMSO),
ethyl alcohol, potassium dihydrogen phosphate, potassium
hydrogen phosphate, 2-cyanoacetamide, piperidine, ethyl alcohol
and several benzaldehydes were purchased from Sigma–Aldrich
ꢀ
1
03.2, 56.4; LRMS (ESI-) m/z 217 (MꢀH) .
4.2.1.6. (E)-2-Cyano-3-(4-methoxyphenyl)acrylamide (CPA6).
1
White solid; reaction time, 24 h; yield, 85.2%;
500 MHz, DMSO-d
H NMR
(
6
) d 8.09 (s, 1H, vinylic H), 7.94 (d, 2H,
J = 8.5 Hz, 2 -H, 6 -H), 7.78, 7.64 (each s, 2H, NH ), 7.11 (d, 2H,
); C NMR (100 MHz,
) d 163.8, 163.3, 150.8, 133.1, 125.1, 117.7, 115.5, 103.6,
(
St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM),
0
0
2
fetal bovine serum (FBS), trypsin, phosphate buffer solution (PBS),
penicillin, and streptomycin were obtained from Gibco Life Tech-
nologies Inc. (Carlsbad, CA, USA). All chemicals and solvents were
of analytical grade. The B16F10 mouse melanoma cell line was
kindly provided by Professor Hae Young Chung (College of
Pharmacy, Pusan National University, Republic of Korea).
0
0
13
J = 8.5 Hz, 3 -H, 5 -H), 3.83 (s, 3H, CH
DMSO-d
3
6
ꢀ
5
6.3; LRMS (ESI-) m/z 201 (MꢀH) .
4.2.1.7.
(E)-2-Cyano-3-(3,4-dimethoxyphenyl)acrylamide
(CPA7). White solid; reaction time, 24 h; yield, 88.5%; 1H NMR
(
400 MHz, CDCl
3
) d 8.21 (s, 1H, vinylic H), 7.68 (d, 1H, J = 2.0 Hz,
0
0
4
4
.2. Method
2 -H), 7.45 (dd, 1H, J = 8.4, 2.0 Hz, 6 -H), 6.92 (d, 1H, J = 8.4 Hz,
5
CH
0
-H), 6.27, 6.01 (each s, 2H, NH
2 3
), 3.94 (s, 3H, CH ), 3.92 (s, 3H,
1
3
.2.1. General procedure for the synthesis of (E)-2-cyano-3-
3
); C NMR (100 MHz, CDCl
3
) d 162.8, 153.9, 153.7, 149.5,
(
substituted phenyl)acrylamide derivatives (CPA1–CPA12,
Scheme 1)
A solution of 2-cyanoacetamide (100 mg, 1.19 mmol) and an
127.7, 124.9, 118.1, 111.7, 111.2, 99.7, 56.4, 56.2; LRMS (ESI-)
m/z 231 (MꢀH) .
ꢀ
appropriate benzaldehyde (1.0 equiv) in ethanol (1.0 mL) was
refluxed in the presence of piperidine (0.035 mL, 0.35 mmol),
cooled, and then water was added. Precipitates generated were fil-
tered and washed with water and in some cases organic solvents
4.2.1.8.
(E)-2-Cyano-3-(2,4-dimethoxyphenyl)acrylamide
(CPA8). Yellowish solid; reaction time, 24 h; yield, 91.4%; 1
H
NMR (400 MHz, DMSO-d
6
) d 8.31 (s, 1H, vinylic H), 8.05 (d, 1H,
), 6.69 (dd, 1H, J = 8.8,
), 3.83
) d 165.3, 163.7, 161.2,
45.4, 130.3, 118.0, 113.9, 107.3, 102.8, 99.0, 56.8, 56.4; LRMS
0
J = 8.8 Hz, 6 -H), 7.70, 7.60 (each s, 2H, NH
2.0 Hz, 5 -H), 6.65 (d, 1H, J = 2.0 Hz, 3 -H), 3.85 (s, 3H, CH
3
(s, 3H, CH ); C NMR (100 MHz, DMSO-d
3 6
1
2
0
0
(
methylene chloride and/or ethyl acetate) to give pure (E)-2-
1
3
cyano-3-(substituted phenyl)acrylamide products, CPA1–CPA12.
ꢀ
4
.2.1.1. (E)-2-Cyano-3-(4-hydroxyphenyl)acrylamide (CPA1).
(ESI-) m/z 231 (MꢀH) .
1
Yellow solid; reaction time, 12 h; yield, 86.3%;
400 MHz, DMSO-d ) d 10.53 (br s, 1H, OH), 8.01 (s, 1H, vinylic
H), 7.83 (d, 2 H, J = 8.8 Hz, 2 -H, 6 -H), 7.71, 7.58 (each s, 2H,
H NMR
(
6
4.2.1.9. (E)-2-Cyano-3-(3,4,5-trimethoxyphenyl)acrylamide
(CPA9). White solid; reaction time, 12 h; yield, 90.7%; H NMR
(400 MHz, CDCl
6.30, 5.95 (each s, 2H, NH
0
0
1
0
0
13
0
0
NH
2
), 6.90 (d, 2H, J = 8.8 Hz, 3 -H, 5 -H); C NMR (100 MHz,
3
) d 8.21 (s, 1H, vinylic H), 7.23 (s, 2H, 2 -H, 6 -H),
), 3.93 (s, 3H, CH ), 3.89 (s, 6H,
DMSO-d
LRMS (ESI-) m/z 187 (MꢀH) .
6
) d 164.0, 162.4, 151.1, 133.5, 123.6, 118.0, 116.9, 102.1;
2
3
ꢀ
13
2ꢁCH
3
); C NMR (100 MHz, CDCl
26.9, 117.7, 108.5, 101.5, 61.3, 56.5; LRMS (ESI-) m/z 261 (MꢀH) .
3
) d 162.3, 154.2, 153.5, 142.8,
ꢀ
1
4
(
.2.1.2.
CPA2). Dark yellow solid; reaction time, 24 h; yield, 45.4%;
NMR (400 MHz, DMSO-d
) d 9.69 (br s, 2H, 2ꢁOH), 7.90 (s, 1H,
), 7.49 (d, 1H, J = 2.0 Hz,
-H), 7.23 (dd, 1 H, J = 8.4, 2.0 Hz, 6 -H), 6.83 (d, 1H, J = 8.4 Hz,
(E)-2-Cyano-3-(3,4-dihydroxyphenyl)acrylamide
1
H
4.2.1.10. (E)-2-Cyano-3-(4-hydroxy-3,5-dimethoxyphenyl)acry-
lamide (CPA10). Yellow solid; reaction time, 4 h; yield, 54.0%;
6
1
vinylic H), 7.68, 7.54 (each s, 2H, NH
2
H NMR (500 MHz, CDCl
3
) d 8.20 (s, 1H, vinylic H), 7.28 (s, 2H,
), 6.07 (s, 1H, OH), 3.94 (s,
); C NMR (100 MHz, DMSO-d ) d 163.8, 151.9, 148.5,
0
0
0
0
0
2
5
1
2 -H, 6 -H), 6.23, 5.64 (each s, 2H, NH
2
13
13
-H); C NMR (100 MHz, DMSO-d
6
) d 164.2, 151.5, 151.3, 146.4,
6H, 2ꢁCH
3
6
25.9, 123.9, 118.0, 116.7, 116.6, 101.6; LRMS (ESI-) m/z 203
141.2, 122.6, 118.2, 109.1, 102.4, 56.7; LRMS (ESI-) m/z 247
ꢀ
ꢀ
(
MꢀH) .
(MꢀH) .

S. Son et al. / Bioorg. Med. Chem. 23 (2015) 7728–7734
7733
4
.2.1.11. (E)-3-(3-Bromo-4-hydroxyphenyl)-2-cyanoacrylamide
4.2.5. Cell culture
B16F10 murine melanoma cells were cultured in DMEM supple-
mented with 10% heat-inactivated fetal bovine serum (FBS) and
1
(
CPA11). Yellow solid; reaction time, 30 h; yield, 64.2%;
H
NMR (500 MHz, DMSO-d
J = 2.0 Hz, 2 -H), 8.01 (s, 1H, vinylic H), 7.82 (dd, 1H, J = 9.0,
6
) d 11.42 (br s, 1H, OH), 8.15 (d, 1H,
0
penicillin/streptomycin (100 IU/50
5% CO atmosphere.
lg/mL) at 37 °C in a humidified
0
2
5
1
.0 Hz, 6 -H), 7.75, 7.65 (each s, 2H, NH
2
), 7.07 (d, 1H, J = 8.5 Hz,
2
0
13
-H); C NMR (100 MHz, DMSO-d
6
) d 163.6, 158.9, 149.7, 135.8,
32.3, 124.9, 117.6, 117.4, 110.8, 103.6; LRMS (ESI-) m/z 264
4.2.6. Cell viability assay
Cell viability was determined by an MTT assay as previously
ꢀ
(
MꢀH) .
3
5
described. B16F10 cells were seeded in a 24-well plate at a den-
4
sity of 5 ꢁ 10 cells/well and allowed to adhere overnight at 37 °C
4
.2.1.12.
(E)-2-Cyano-3-(3,5-dibromo-4-hydroxyphenyl)acry-
2
in a humidified 5% CO atmosphere. The next day, cells were trea-
lamide (CPA12). Yellowish solid; reaction time, 3 days; yield,
1
0
0
ted with different concentrations of CPA2 (0, 5, 10, or 25 lM) and
7
5.4%; H NMR (500 MHz, DMSO-d
6
) d 8.15 (s, 2H, 2 -H, 6 -H),
1
3
incubated under the same conditions. After 24 h of incubation,
MTT solution (0.5 mg/mL) was added to each well and the plate
was incubated for 2 h at 37 °C. After removing supernatants, for-
8
.02 (s, 1H, vinylic H), 7.79, 7.72 (each s, 2H, NH
2
); C NMR
(
100 MHz, DMSO-d
6
) d 163.2, 155.1, 148.3, 134.8, 126.5, 117.2,
ꢀ
1
12.6, 105.7; LRMS (ESI-) m/z 342 (MꢀH) .
mazan crystals were dissolved in 200 lL of EtOH/DMSO (1:1) and
transferred to a 96-well ELISA plate. Well absorbances were read
at 570 nm using an ELISA reader. All experiments were conducted
in triplicate.
4
.2.2. Inhibitory effect of (E)-2-cyano-3-(substituted phenyl)
acrylamide derivatives CPA1–CPA12 on mushroom tyrosinase
The inhibitory effects of the synthesized compounds on mush-
room tyrosinase were investigated as previously described with
4
.2.7. Melanin content assay
3
2
minor modifications.
L of each compound (5
tration) was mixed with 170
mM -tyrosine solution, 50 mM potassium phosphate buffer
pH 6.5), and distilled water (10:10:9, v/v/v). Subsequently, 20
In each well of a 96-well microplate,
M, 10 M, or 25 M as a final concen-
L of substrate solution containing
Inhibitory effect on melanin biosynthesis was assessed using a
1
0
l
l
l
l
melanin content assay as previously described with slight modifi-
l
36
cation. Briefly, B16F10 cells were seeded in a 24-well plate
1
(
L
4
(
5 ꢁ 10 cells/well) and allowed to adhere for 24 h at 37 °C in a
lL
humidified 5% CO
with 1 -MSH and different concentrations of CPA2 (0, 5, 10,
or 25 M) or kojic acid (25 M) for 24 h. After being rinsed in
PBS, cells in each well were lysed by incubation for 1 h at 60 °C
in 200 L of 1 N NaOH. Lysates were transferred to a 96-well ELISA
2
atmosphere. The cells were then co-treated
of mushroom tyrosinase solution (1000 U/mL) was added to each
well of the plate and the plate was then incubated. After an incu-
bation period of 30 min at 25 °C, the absorbances of dopachrome
produced in each well were measured at 450 nm using an ELISA
reader (Tecan, Austria). Kojic acid was used as a reference com-
pound. All experiments were performed in triplicate. Tyrosinase
inhibition rates were calculated using:
l
l
M a
l
l
plate and well absorbances were measured at 405 nm using an
ELISA reader. Absorbances of CPA2 and kojic acid were expressed
as mean percentage inhibitions versus control cells with no inhibi-
tor. All experiments were performed in triplicate.
½
The inhibitionð%Þ ¼ ½1 ꢀ ðA=BÞꢂ ꢁ 100ꢂ
where ‘A’ represents the absorbance of the test compound and ‘B’
represents the absorbance of control with no test compound.
4
.2.8. Cellular tyrosinase activity assay
Tyrosinase activity was determined by measuring the rate of
L
-DOPA oxidation as previously described with slight modifica-
4
.2.3. Determination of type of tyrosinase inhibition exhibited
by CPA2
Kinetic analysis was conducted to determine the type of inhibi-
tion exhibited by CPA2 on mushroom tyrosinase. In each well of a
6-well plate, 10 L of CPA2 (0, 5, 10, or 25 M [final concentra-
tions]) was mixed with 170 L of -tyrosine solution (0.5, 1, 2, or
mM [final concentrations]). Following the addition of 20 L of
mushroom tyrosinase solution (1000 U/mL) to each well, the assay
mixture was incubated at 25 °C for 30 min and amounts of dopa-
chrome produced in each well were then monitored using an ELISA
reader at 450 nm. All experiments were carried out in triplicate at a
constant temperature of 25 °C. A Lineweaver–Burk plot was uti-
lized to determine inhibition type.
37
tions. Briefly, B16F10 cells were seeded in a 24-well plate at a
4
density of 5 ꢁ 10 cells/well and allowed to adhere overnight
2
2
4 h at 37 °C in a humidified 5% CO atmosphere. Cells were then
treated with 1
0, 5, 10, or 25
the same conditions. After 24 h of incubation, cells were washed
twice with PBS and lysed with 100 L/well of lysis buffer
containing 90 L of 50 mM phosphate buffer (pH 6.8), 5 L of 1%
Triton X-100, and 5 L of 0.1 mM PMSF. Cells were then frozen at
80 °C for 30 min. After being thawed, lysates were centrifuged
at 12,000g for 30 min at 4 °C. The supernatants (80 L) so obtained
were placed in a 96-well ELISA, mixed with 20 L of 10 mM
-DOPA, and incubated for 30 min at 37 °C. Absorbance of each well
l
l
M
a
-MSH and different concentrations of CPA2
9
l
l
(
M) or kojic acid (25 M), and incubated under
l
l
L
4
l
l
l
l
l
ꢀ
l
l
L
was measured at 500 nm. Tyrosinase inhibitory activity was
calculated by the following equation:
4
.2.4. In silico docking simulation of tyrosinase with CPA2 and
kojic acid
AutoDock vina was used for the in silico protein–ligand docking
simulation. The 3D structure of Agaricus bisporus tyrosinase (PDB
½
Inhibitionð%Þ ¼ ½ðA ꢀ BÞ ꢀ ðC ꢀ DÞꢂ=ðA ꢀ BÞ ꢁ 100ꢂ
where ‘A’ is the absorbance of the blank (without test compound)
after incubation, ‘B’ is the absorbance of the blank (without test
compound) before incubation, ‘C’ is the absorbance of test com-
pound after incubation, and ‘D’ is the absorbance of test compound
before incubation.
3
3,34
ID: 2Y9X) and a systemic search technique were used.
The
binding site of tyrosine in the crystal structure of the tyrosinase
enzyme was used as a docking pocket. Docking simulations were
performed between tyrosinase and CPA2 or kojic acid. To prepare
compounds for docking simulation, (1) 2D structures were con-
verted into 3D structures, (2) charges were calculated, and (3)
hydrogen atoms were added using the ChemOffice program
4
.2.9. Statistical analysis
All experiments were performed in triplicate, and results are
(http://www.cambridgesoft.com). LigandScout 3.1 was used to
presented as mean ± standard errors (SE). One way analysis of vari-
ance (ANOVA) followed by Tukey’s test was used to determine the
significances of intergroup differences among all groups (GraphPad
predict possible hydrogen bonding residues between compounds
and tyrosinase and to identify pharmacophores.

7
734
S. Son et al. / Bioorg. Med. Chem. 23 (2015) 7728–7734
Software, SanDiego, CA, USA). The analysis was performed using
GraphPad Software (SanDiego, CA) and p-values of <0.05 were con-
sidered statistically significant.
17. Jeong, J. Y.; Liu, Q.; Kim, S. B.; Jo, Y. H.; Mo, E. J.; Yang, H. H.; Song, D. H.; Hwang,
B. Y.; Lee, M. K. Molecules 2015, 20, 8730.
1
1
2
8. Saewan, N.; Thakam, A.; Jintaisong, A.; Kittigowitana, K. Int. J. Pharm. Pharm. Sci.
014, 6, 270.
9. Akhtar, M. N.; Sakeh, N. M.; Zareen, S.; Gul, S.; Lo, K. M.; Ul-Haq, Z.; Shah, S. A.
A.; Ahmad, S. J. Mol. Struct. 2015, 1085, 97.
2
Acknowledgments
0. Hermanns, J. F.; Piérard-Franchimont, C.; Piérard, G. E. Int. J. Cosmet. Sci. 2000,
2
2, 67.
This work was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
21. Parvez, S.; Kang, M.; Chung, H. S.; Cho, C.; Hong, M. C.; Shin, M. K.; Bae, H.
Phytother. Res. 2006, 20, 921.
22. Kim, H. R.; Lee, H. J.; Choi, Y. J.; Park, Y. J.; Woo, Y.; Kim, S. J.; Park, M. H.; Lee, H.
W.; Chun, P.; Chung, H. Y.; Moon, H. R. MedChemComm 2014, 5, 1410.
(
Grant no. NRF-2013R1A1A2009949) and by the National Research
Foundation of Korea (NRF) grant funded by the Korean government
MSIP) (Grant no. 2009-0083538).
23. Ha, Y. M.; Kim, J.-A.; Park, Y. J.; Park, D.; Kim, J. M.; Chung, K. W.; Lee, E. K.; Park,
J. Y.; Lee, J. Y.; Lee, H. J.; Yoon, J. H.; Moon, H. R.; Chung, H. Y. Biochim. Biophys.
Acta 2011, 1810, 612.
(
24. Ha, Y. M.; Kim, J.-A.; Park, Y. J.; Park, D.; Choi, Y. J.; Kim, J. M.; Chung, K. W.;
Han, Y. K.; Park, J. Y.; Lee, J. Y.; Moon, H. R.; Chung, H. Y. MedChemComm 2011,
2
, 542.
References and notes
2
5. Kim, S. H.; Ha, Y. M.; Moon, K. M.; Choi, Y. J.; Park, Y. J.; Jeong, H. O.; Chung, K.
W.; Lee, H. J.; Chun, P.; Moon, H. R.; Chung, H. Y. Arch. Pharm. Res. 2013, 36,
1
.
Della Longa, S.; Ascone, I.; Bianconi, A.; Bonfigli, A.; Castellano, A. C.; Zarivi, O.;
Miranda, M. J. Biol. Chem. 1996, 271, 21025.
Aguilera, F.; McDougall, C.; Degnan, B. M. BMC Evol. Biol. 2013, 13, 96.
Lewis, E. A.; Tolman, W. B. Chem. Rev. 2004, 104, 1047.
1
189.
6. Cho, H.; Iwashita, T.; Hamaguchi, M.; Oyama, Y. Chem. Pharm. Bull. 1991, 39,
341.
2
2
2.
3.
4.
3
7. Saxena, N.; Pandey, J.; Tamarkar, A.; Srivastava, A. K.; Tripathi, R. P. Trends
Carbohydr. Res. 2009, 1, 29.
8. Nitsche, C.; Steuer, C.; Klein, C. D. Bioorg. Med. Chem. 2011, 19, 7318.
9. Kaupp, G.; Naimi-Jamal, M. R.; Schmeyers, J. Tetrahedron 2003, 59, 3753.
0. Tsutomu, K.; Susumu, T.; Hidetaka, N.; Yoshiyuki, O. R.; Takao, H. Planta Med.
Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. J. Biol. Chem.
2
006, 281, 8981.
2
2
3
5.
6.
7.
8.
Ramsden, C. A.; Riley, P. A. Bioorg. Med. Chem. 2014, 22, 2388.
van Gelder, C. W.; Flurkey, W. H.; Wichers, H. J. Phytochemistry 1997, 45, 1309.
Fairhead, M.; Thöny-Meyer, L. N. Biotechnology 2012, 29, 183.
Kobayashi, T.; Vieira, W. D.; Potterf, B.; Sakai, C.; Imokawa, G.; Hearing, V. J. J.
Cell Sci. 1995, 108, 2301.
2003, 69, 457.
3
3
1. Kong, N.; Jiang, T.; Zhou, Z.; Fu, J. Dent. Mater. 2009, 25, 1371.
2. Hyun, S. K.; Lee, W. H.; Jeong, D. M.; Kim, Y.; Choi, J. S. Biol. Pharm. Bull. 2008,
9
.
Körner, A.; Pawelek, J. Science 1982, 217, 1163.
3
1, 154.
1
0. Slominski, A.; Tobin, D. J.; Shibahara, S.; Wortsman, J. Physiol. Rev. 2004, 84,
155.
3
3
3
3. Moustakas, D. T.; Lang, P. T.; Pegg, S.; Pettersen, E.; Kuntz, I. D.; Brooijmans, N.;
Rizzo, R. C. J. Comput. Aided Mol. Des. 2006, 20, 601.
4. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
5. Riss, T.; Moravec, R. A.; Niles, A. L.; Benink, H. A.; Worzella, T. J.; Minor, L. In
Assay Guidance Manual; Eli Lilly & Company and the National Center for
Advancing Translational Sciences, 2013.
6. Chen, L. G.; Chang, W. L.; Lee, C. J.; Lee, L. T.; Shih, C. M.; Wang, C. C. Biol. Pharm.
Bull. 2009, 32, 1447.
1
1
1. d’Ischia, M.; Wakamatsu, K.; Napolitano, A.; Briganti, S.; Garcia-Borron, J. C.;
Kovacs, D.; Meredith, P.; Pezzella, A.; Picardo, M.; Sarna, T.; Simon, J. D.; Ito, S.
Pigm. Cell Melanoma Res. 2013, 26, 616.
1
1
1
1
2. Im, S.; Kim, J.; On, W. Y.; Kang, W. H. Br. J. Dermatol. 2002, 46, 165.
3. Brenner, M.; Hearing, V. J. Photochem. Photobiol. 2008, 84, 539.
4. Kim, Y. J.; Uyama, H. Cell. Mol. Life Sci. 2005, 62, 1707.
5. Ashraf, Z.; Rafiq, M.; Seo, S.-Y.; Kwon, K. S.; Babar, M. M.; Sadaf Zaidi, N.-U.-S.
Eur. J. Med. Chem. 2015, 98, 203.
3
3
7. Bae, S. J.; Ha, Y. M.; Kim, J. A.; Park, J. Y.; Ha, T. K.; Park, D.; Chun, P.; Park, N. H.;
Moon, H. R.; Chung, H. Y. Biosci., Biotechnol., Biochem. 2013, 77, 65.
1
6. Lu, T.-M.; Ko, H.-H.; Ng, L.-T.; Hsieh, Y.-P. Chem. Biodivers. 2015, 12, 963.