Synthesis and dual biological effects of hydroxycinnamoyl phenylalanyl/prolyl hydroxamic acid derivatives as tyrosinase inhibitor and antioxidant

Article abstract of DOI:10.1016/j.bmcl.2012.10.107

We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) derivatives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase activity and reduced melanin content in melanocytes Mel-Ab cell.

Full text of DOI:10.1016/j.bmcl.2012.10.107

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1136–1142
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and dual biological effects of hydroxycinnamoyl
phenylalanyl/prolyl hydroxamic acid derivatives as tyrosinase
inhibitor and antioxidant
Seon-Yeong Kwak a, Jin-Kyoung Yang a, Hye-Ryung Choi b, Kyung-Chan Park b, Young-Bu Kim c,
Yoon-Sik Lee a,
⇑
a School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea
b Department of Dermatology, Seoul National University, Bundang Hospital, Gyeonggi-do 464-707, Republic of Korea
c R&D Center, IPEERES Cosmetics Ltd, Gyeonggi-do, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported that caffeoyl-amino acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a
good antioxidant and tyrosinase inhibitor, in particular when caffeic acid was conjugated with proline
or amino acids having aromatic ring like phenylalanine. Here, various hydroxycinnamic acid (HCA) deriv-
atives were further conjugated with phenylalanyl hydroxamic acid and prolyl hydroxamic acid (HCA-
Phe-NHOH and HCA-Pro-NHOH) to study the structure and activity relationship as both antioxidants
and tyrosinase inhibitors. When their biological activities were evaluated, all HCA-Phe-NHOH and
HCA-Pro-NHOH exhibited enhanced antioxidant activity compared to HCA alone. Moreover, derivatives
of caffeic acid, ferulic acid, and sinapic acid inhibited lipid peroxidation more efficiently than vitamin
E analogue (Trolox). In addition, derivatives of caffeic acid and sinapic acid efficiently inhibited tyrosinase
activity and reduced melanin content in melanocytes Mel-Ab cell.
Received 18 September 2012
Accepted 24 October 2012
Available online 31 October 2012
Keywords:
Hydroxycinnamic acid
Hydroxamic acid
Antioxidant
Tyrosinase inhibitor
Hypo-pigmenting effect
Ó 2012 Elsevier Ltd. All rights reserved.
Reactive oxygen species (ROS) are generated by the reduction of
oxygen or oxidation of water molecules resulting in superoxide
cosmetic ingredients to delay skin aging process and food preser-
vatives to prevent food discoloration and deterioration.
Å
(O2ÅÀ, OOH), hydrogen peroxide (H2O2), peroxyl (ROOHÅ) and hy-
Hydroxycinnamic acids (HCAs) such as caffeic acid (CA; 3,4-
dihydroxy-cinnamic acid), p-coumaric acid (pCoA; 4-hydroxy-cin-
namic acid), ferulic acid (FA; 3-methoxy-4-hydroxy-cinnamic acid)
and sinapic acid (SA; 3,5-dimethoxy-4-hydroxy-cinnamic acid) are
well-known antioxidants, which are distributed in many plants
and found in fruits, tea, coffee, and wine.2,3 They exhibit strong
antioxidant activity because they can have resonance stabilized
phenoxy radicals after the hydrogen radical is abstracted due to
the phenolic nucleus and extended side chain conjugation.4 In par-
ticular, CA, pCoA and dihydrocaffeic acid (DHCA) possessing phenol
or catechol moiety have drawn attention due to their multiple bio-
logical and pharmaceutical activities.5 Various natural and syn-
thetic hydroxycinnamic acids analogues were reported and their
potential activities have been intensively investigated.6–10
droxyl (ÅOH) radicals. When ROS exists in moderate concentration,
it participates in physiological processes for desired cellular re-
sponses. In healthy bodies, the amount of ROS can be controlled
by antioxidant-mediated self-defense systems. For example, anti-
oxidants such as vitamin C, vitamin E and glutathione get rid of
free radical intermediates and terminate free radical-mediated
chain reactions to prevent cellular damage. However, overpro-
duced ROS leads to oxidative stress by disrupting the balance of
the cellular oxidation state. This results in a number of cellular
damage by oxidation of lipids, protein, DNA and RNA, which causes
the deconstruction of cell membranes, deactivation of enzymes,
acceleration of the aging process, and mediation of severe disease
states such as chronic lung diseases, diabetes, and neurogenerative
disorders.1 For these reasons, antioxidants that efficiently suppress
ROS activity have been intensively studied, not only for the preven-
tion of diseases but also for use in other applications such as
Melanocytes produce the dark pigment melanin through a com-
plex pathway in which tyrosinase (EC 1.14.18.1) plays a crucial
role. Tyrosinase is a metalloenzyme, which contains dinuclear
copper in the active site. It catalyzes both ortho-hydroxylation of
L
-tyrosine and the subsequent conversion of 3,4-dihydroxyphenyl-
⇑
Corresponding author. Present address: Department of Chemical and Biological
alanine (L-DOPA) to DOPA-quinone in the early stage of melano-
Engineering, Seoul National University, San 56-1, Daehak-dong, Gwanak-gu, Seoul
151-744, Republic of Korea. Tel.: +82 2 880 7080; fax: +82 2 876 9625.
E-mail address: yslee@snu.ac.kr (Y.-S. Lee).
genesis, which is responsible for skin and hair color.11,12 The
DOPA-quinone yields melanin, which is a polymer comprising
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.107

S.-Y. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1136–1142
1137
a) Fmoc-Phe-OH or Fmoc-Pro-OH
a) Fmoc-NHOH, DIPEA
DCM/NMP (9:1, v/v)
48 h, rt
HATU, HOAt , DIPEA in NMP,
2 h, rt
H2N
O
Cl
b) 20% piperidine/NMP
3 min, 20 min, rt
b) 20% piperidine/NMP
3 min, 20 min, rt
Cl
R1
O
R2
HCA,
O
H2N
BOP, HOBt, DIPEA in NMP
HN
O
H
N
HN
O
R
R3
5 h, rt
R
O
O
R1
R2
O
N
H
N
R1
Cleavage cocktail
1 h, rt
N
H
OH
O
O
R3
R2
NH
HO
R3
HCA-Pro-NHOH
HCA-Phe-NHOH
Scheme 1. Solid phase synthesis of HCA-Phe-NHOH and HCA-Pro-NHOH. CA: R1 = R2 = OH, R3 = H; DHCA: R1 = R2 = OH, R3 = H, without the 2,3-double bond; pCoA:
R1 = R3 = H, R2 = OH; FA: R1 = OCH3, R2 = OH, R3 = H; SA: R1 = R3 = OCH3, R2 = OH.
numerous smaller component molecules such as 5,6-dihydroxyin-
dole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA),
through a series of enzymatic reactions, sequential oxidation and
oxidative cyclization.13 It participates in the enzymatic browning
of fruits and vegetables,14 and pigmentary skin diseases.15 There-
fore, studies on tyrosinase inhibitors have attracted a lot of interest
because tyrosinase inhibitors have broad applications in medicinal,
food and cosmetic industries.16–18 Numerous naturally existing
tyrosinase inhibitors have been discovered in plants, but have
mostly shown poor19 to moderate activity.20 Various copper
chelators including kojic acid (KA)21 and N-phenylthiourea (PTU)22
have also been reported as tyrosinase inhibitors. Of these, we
chose hydroxamic acid moiety for tyrosinase inhibitory activity
because hydroxamic acid group had strong metal chelating ability
with a wide range of metal ions such as Fe(III), Zn(II) and Cu(II).
Hydroxamic acids supposedly inhibit the metalloenzyme by metal
chelation and hydrogen bonding, or through the formation of a
charge transfer complex in the enzyme.23 Recently, several reports
showed that hydroxamate derivatives had tyrosinase inhibitory
activity.24,25
In our previous work, we showed that caffeoyl-amino
acidyl-hydroxamic acid (CA-Xaa-NHOH) acted as both a good
antioxidant and tyrosinase inhibitor, in particular, caffeoyl-
prolyl-hydroxamic acid (CA-Pro-NHOH) was capable of inhibiting
tyrosinase activity as well enhancing antioxidant activity, and caf-
feoyl aromatic amino acid hydroxamic acid such as caffeoyl-
phenylalanyl-hydroxamic acid (CA-Phe-NHOH) exhibited good
tyrosinase inhibitory activity.26 Here, we further studied on these
bioactive compounds by conjugating phenylalanyl-hydroxamic
acid and prolyl-hydroxamic acid with various hydroxycinnamic
acids (HCA-Phe-NHOH and HCA-Pro-NHOH). In addition, we dem-
onstrated that our compounds were effective in the cell system.
All HCA-Pro-NHOH and most of HCA-Phe-NHOH were obtained
with high purity (93ꢀ99%) (Scheme 1). Some HCA-Phe-NHOH
100
90
80
70
60
50
40
30
20
10
0
Figure 1. DPPH radical scavenging activity of HCA-Phe-NHOH, HCA-Pro-NHOH (black solid bars) and HCA (white solid bars). Conditions: [Antioxidant]/[DPPH] (mol/
mol) = 0.25 for CA, DHCA, and their derivatives; 0.50 for pCoA, FA, SA, and their derivatives. The reaction was performed for 10 min at 25 °C. Each experiment was performed
in triplicate and averaged. The values are given as the mean standard error.

1138
S.-Y. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1136–1142
Figure 2. Antioxidant activity of HCA, HCA-Phe-NHOH, HCA-Pro-NHOH and Trolox (positive control) on lipid peroxidation for 48 h. The absorbance was measured at 500 nm
by ferric thiocyanate method. Conditions: lipid peroxidation proceeded in emulsified system. The final concentration of each antioxidant was 90 lM; reaction was carried out
at 50 °C under the dark. Each experiment was performed in triplicate and averaged. The values are given as the mean standard error.
obtained with moderate purity (ꢀ80%) were further purified by
semi-preparative RP-HPLC. They were identified by mass
spectrometry.27
The results of lipid peroxidation inhibition were quite different
when compared to those of DPPH RSA in hydrophilic environment.
All of HCA-Phe-NHOH derivatives showed better lipid peroxidation
inhibition activity than HCA-Pro-NHOH derivatives (Fig. 2). CA-
Phe-NHOH and CA-Pro-NHOH significantly enhanced antioxidant
activity compared to CA. FA-Pro-NHOH and SA-Pro-NHOH also
exhibited enhanced antioxidant activity, and FA-Phe-NHOH and
SA-Phe-NHOH showed excellent antioxidant activity for 48 h.
The order of lipid peroxidation inhibition activity was FA-Phe-
NHOH ꢁ SA-Phe-NHOH > FA-Pro-NHOH ꢁ CA-Phe-NHOH ꢁ CA-
Pro-NHOH > SA-Pro-NHOH > Trolox (Fig. 2a, b, and c). DHCA and
pCoA derivatives showed inferior lipid peroxidation inhibition
activity to Trolox, even though they had enhanced activity com-
pared to DHCA or pCoA alone (Fig. 2d and e).
Next, we evaluated tyrosinase inhibition activity of HCA-Phe-
NHOH and HCA-Pro-NHOH derivatives.33 They revealed from mod-
erate to good tyrosinase inhibitory activity, although none of HCAs
showed tyrosinase inhibitory activity when L-DOPA was used as a
substrate. We hypothesized that amino acidyl-hydroxamic acid
had tyrosinase inhibitory activity and the antioxidant group HCA
First, we evaluated antioxidant activity by DPPH radical scav-
enging test28,29 and lipid peroxidation inhibition test.30,31 CA and
DHCA has strong DPPH radical scavenging activity (RSA) because
the ortho-dihydroxy group can generate stabilized phenoxy radical
by hydrogen bond after donating hydrogen radical.32 Since pCoA,
FA and SA had relatively weak DPPH RSA compared to CA, we per-
formed DPPH RSA test under two different assay conditions to
compare the conjugation effect of amino acidyl-hydroxamic acid.
The ratio of CA and DHCA derivatives to DPPH was 0.25, and the
ratio of pCoA, FA, SA and their derivatives to DPPH was 0.5. As
shown in Figure 1, only CA-Pro-NHOH showed significantly
enhanced DPPH RSA, whereas SA-Pro-NHOH exhibited slightly en-
hanced DPPH RSA, and FA derivatives just maintained the activity
of FA itself. In the case of DHCA, its strong activity for releasing
hydrogen radical seemed to be a little hindered by conjugation
with amino acidyl-hydroxamic acid. pCoA and its derivatives did
not show any sufficient DPPH RSA.

S.-Y. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1136–1142
1139
100
90
80
70
60
50
40
30
20
10
0
50 µM
100 µM
Figure 3. Mushroom tyrosinase inhibitory activity of HCA-Phe-NHOH and HCA-Pro-NHOH at 50
inhibition activity was determined after treating 50 M (gray solid bars) or 100 M (black solid bars) of each sample with
(100 g/ml), and incubating at 25 °C for 10 min. Each experiment was performed in triplicate and averaged. The values are given as the mean standard error.
lM (gray solid bars) and 100
lM (black solid bars). Conditions: % Tyrosinase
l
l
L-DOPA (2.5 mM) and mushroom tyrosinase
l
100
90
80
70
60
50
40
30
20
10
0
CA-Phe-NHOH
CA-Pro-NHOH
pCoA-Phe-NHOH
pCoA-Pro-NHOH
Kojic acid
0
20
40
60
80
100
Concentration (µM)
Figure 4. Tyrosinase inhibition activity of CA derivatives, pCoA derivatives, and kojic acid. Conditions: % Tyrosinase inhibition activity was determined after treating each
sample with -DOPA (2.5 mM) and mushroom tyrosinase (100 g/ml), and incubating at 25 °C for 10 min. Each enzyme reaction was performed for 10 min at 25 °C. Each
experiment was performed in triplicate and averaged. The values are given as the mean standard error.
L
l
delayed
of Ac-Phe-NHOH and Ac-Pro-NHOH was only 6% and 5%,
respectively, even at a relatively high concentration of 100 M. In
addition, as shown in Figure 3, tyrosinase inhibition activity of
HCA-Phe-NHOH and HCA-Pro-NHOH was more dependent on the
kinds of antioxidant group, even though HCA-Phe-NHOH seemed
to exhibit 5–20% higher tyrosinase inhibitory activity than HCA-
Pro-NHOH. This means that HCA plays an important role in binding
to tyrosinase as well as in reducing substrate oxidation. CA moiety
may act as a substrate analogue because the 3,4-dihydroxyphenyl
L-DOPA oxidation. However, tyrosinase inhibitory activity
l
Binding group to
hydrophobic
pocket
O
H
N
HO
HO
Structural
similarity to
substrate
N
H
OH
O
Chelating group to
dinuclear copper
CA-Phe-NHOH
ring is similar to the natural substrate L-DOPA. FA and SA deriva-
tives exhibited poor tyrosinase inhibition activity because the
methoxy group next to para-hydroxyl group may have introduced
steric hindrance when approaching to the active site of tyrosinase.
In addition, FA and SA moiety have less structural similarity to the
natural substrate and exhibited less reducing activity than CA in
hydrophilic environment.
We also measured tyrosinase inhibition activity of DHCA and
pCoA derivatives to prove the effect of CA moiety on tyrosinase
inhibition. pCoA-Phe-NHOH and pCoA-Pro-NHOH showed good
tyrosinase inhibition activity probably because pCoA had similar
O
H2N
O
O
N
H
O
O
OH
Cu (II)
Cu (II)
KA-Phe-NH2
Figure 5. Comparison of possible tyrosinase inhibition mechanism between CA-
Phe-NHOH and KA-Phe-NH2.

1140
S.-Y. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1136–1142
120
100
80
60
40
20
0
different in each assay system. We could only comprehensively
measure the activity of these compounds in the melanocytes be-
cause the cell system contained both hydrophilic and hydrophobic
environments and biological melanin synthetic pathway was af-
fected by both tyrosinase and antioxidant.
*
HCA-Phe-NHOH, HCA-Pro-NHOH and HCA did not affect on cell
viability, while only CA showed cytotoxicity at 100
lM in Mel-Ab
cells37 (Fig. 6).
As shown in Figure 7, CA-Pro-NHOH, SA-Phe-NHOH and SA-Pro-
NHOH inhibited melanogenesis as much as arbutin, which is used
as a hypo-pigmenting agent in the cosmetic industry. HCAs and FA-
Phe-NHOH hardly reduced melanin contents, and FA-Pro-NHOH
did not sufficiently inhibit melanogenesis.38 In cell system, HCA-
Pro-NHOH showed similar or 10–30% higher anti-melanogenesis
activity than HCA-Phe-NHOH. This is clrealy shown in FA-Phe-
NHOH and FA-Pro-NHOH.
Interestingly, SA-Phe-NHOH and SA-Pro-NHOH showed hypo-
pigmenting effect in melanocyte, even though sufficient tyrosinase
inhibition activity was not observed. This is probably due to differ-
ent cell permeability of each molecule and the different tyrosinase
structures in the assay system because mushroom tyrosinase was
used in vitro tube test and mouse melanocyte was used for cell as-
say system. Moreover, there is a possibility that SA-Phe-NHOH and
SA-Pro-NHOH can affect not only tyrosinase inhibition and retar-
dation of substrate oxidation, but also other pathways related to
receptor-mediated melanogenesis in melanocytes. To understand
the role of SA derivatives in melanocytes more clearly, further
study is needed.
In summary, various HCA-Phe-NHOH and HCA-Pro-NHOH
derivatives were prepared and their antioxidant and tyrosinase
inhibition activity was compared to those of CA-Phe-NHOH and
CA-Pro-NHOH. All of HCA-Phe-NHOH and HCA-Pro-NHOH showed
enhanced antioxidant activity in lipid peroxidation test, in particu-
lar, CA derivatives, FA derivatives and SA derivatives surpassed
trolox. In the structure-tyrosinase inhibition activity study, HCA-
Phe-NHOH showed 5–20% higher tyrosinase inhibition activity
than HCA-Pro-NHOH, and HCA moiety played an important role
in both binding to tyrosinase and reducing substrate. Among them,
CA derivatives exhibited good tyrosinase inhibition activity due to
substrate similarity and strong reducing activity.
Figure 6. Effect of HCA-Phe-NHOH, HCA-Pro-NHOH, HCA and arbutin on cell
viability in Mel-Ab cells. The cells were treated with samples (100 M) containing
l
new serum-free media and incubated for 24 h. CCK-8 solution was added and cells
were incubated for another 2 h at 37 °C. The amount of water-soluble formazan
generated by the activity of dehydrogenase in cells was measured by optical density
at 450 nm. Each experiment was performed in triplicate and averaged. The values
are given as the mean standard error. ⁄P <0.01
structure to the natural substrate L-tyrosine. However, their tyros-
inase inhibition activity was inferior to CA derivatives because CA
had stronger antioxidant activity than pCoA in hydrophilic envi-
ronment (Figs. 1 and 4). Interestingly, DHCA derivatives did not
sufficiently inhibit tyrosinase activity, even though DHCA had
3,4-dihydroxyphenyl ring like CA and exerted higher antioxidant
activity than CA in hydrophilic environment. This is most likely
due to the fact that presence of the conjugated double bond makes
the molecule less flexible and leads to form a stable Schiff base
with a primary amino group of tyrosinase.34,35
CA-Phe-NHOH and CA-Pro-NHOH showed significantly higher
tyrosinase inhibition activity than FA and SA derivatives. In partic-
ular, CA-Phe-NHOH showed excellent tyrosinase inhibition activity
(IC50 = 4.9
(IC50 = 4.2
l
l
M), which was comparable to KA-Phe-NH2
M), the strongest tyrosinase inhibitor reported in our
lab to date36 (Fig. 5).
When we performed in vitro tube tests to evaluate tyrosinase
inhibition activity and antioxidant activity of HCA-Phe-NHOH
and HCA-Pro-NHOH, the most active compound was found to be
CA-Pro-NHOH, SA-Pro-NHOH and SA-Phe-NHOH reduced
melanin contents in Mel-Ab cells by 30%, on the other hand,
120
100
*
*
*
*
*
*
80
60
40
20
0
Figure 7. Effect of HCA-Phe-NHOH, HCA-Pro-NHOH, HCA and arbutin on melanogenesis in Mel-Ab cells. The inhibitors were treated at the concentrations of 100 lM for
4 days. The inhibitor-treated cells were dissolved in 1 mL of 1 N NaOH at 100 °C for 30 min, and centrifuged for 20 min at 16,000g, after which the optical densities of the
supernatants were measured at 400 nm. Each experiment was performed in triplicate and averaged. The values are given as the mean standard error. ⁄P <0.01.

S.-Y. Kwak et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1136–1142
1141
loading level was 1.0 mmol/g, which was determined by Fmoc titration. After
treating with 20% piperidine/N-methyl-2-pyrrolidone (NMP) for 30 min to
remove Fmoc groups, Fmoc-L-Pro-OH or Fmoc-L-Phe-OH (2 equiv) was coupled
CA-Phe-NHOH, FA-Pro-NHOH, and FA-Phe-NHOH did not suffi-
ciently reduce melanin synthesis. Overall, CA-Pro-NHOH was the
best promising bioactive compound acting as an excellent antiox-
idant in both hydrophilic and hydrophobic environments, and a
good tyrosinase inhibitor at the same time. This study could be a
good starting point for further refinement of potentially useful bio-
active compounds in the fields of medicine, agriculture, and cos-
metic industry.
to the resin with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3,-tetramethyl uronium
hexafluorophosphate methanaminium (HATU), 1-hydroxy-7-azabenzotriazole
(HOAt), and DIPEA (4 equiv) for 1.5 h at room temperature. After removing
Fmoc groups by 20% piperidine/NMP, HCA (2 equiv) was coupled to the amino
acid anchored resin with benzotriazole-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate (BOP; 2 equiv), hydroxybenzotriazole
(HOBt; 2 equiv) and DIPEA (3 equiv) for 5 h. The final product was cleaved
from the resin by 30% trifluoroacetic acid (TFA)/DCM (v/v) for 1 h. The resin
was filtered, and the filtrate was concentrated in high vacuum, followed by
precipitation with cold diethyl ether. The resulting HCA-Phe-NHOH and HCA-
Pro-NHOH were identified by QUATTRO Triple Quardrupole Tandem mass
Acknowledgments
spectrometer (Micromass
& Waters, Milford, MA, USA) at National
This work was supported by a grant from IPEERES Cosmetic Ltd
and WCU (World Class University) program, Korea Science and
Engineering Foundation, Ministry of Education, Science and Tech-
nology (R32-2010-000-10213-0).
Instrumentation Center for Environmental Management (NICEM): CA-Phe-
NHOH (m/z calcd: 343.1 [M+H]+; found: 343.0), CA-Pro-NHOH (m/z calcd:
293.1 [M+H]+; found: 293.1), DHCA-Phe-NHOH (m/z calcd: 345.1 [M+H]+;
found: 345.1), DHCA-Pro-NHOH (m/z calcd: 295.1 [M+H]+; found: 295.1),
pCoA-Phe-NHOH (m/z calcd: 327.1 [M+H]+; found: 327.1), pCoA-Pro-NHOH
(m/z calcd: 277.1 [M+H]+; found: 277.0), FA-Phe-NHOH (m/z calcd: 357.1
[M+H]+; found: 357.1), FA-Pro-NHOH (m/z calcd: 307.1 [M+H]+; found: 307.0),
SA-Phe-NHOH (m/z calcd: 387.1 [M+H]+; found: 387.0), SA-Pro-NHOH (m/z
calcd: 337.1 [M+H]+; found: 337.1). Their purities were analyzed by RP-HPLC
(Thermo Scientific Spectra System AS300; Thermo-Fisher, Waltham, MA, USA)
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16. Friedman, M. J. Agric. Food Chem. 1996, 44(3), 631.
17. Mosher, D. B.; Pathak, M. A.; Fitzpatrick, T. B. In Dermatology in General
Medicine; Fitzpatrick, T. B., Eisen, A. Z., Wolff, K., Freedberg, I. M., Austen, K. F.,
Eds.; McGraw-Hill Press: New York, 1983; pp 205–225.
29. Yamaguchi, T.; Takamura, H.; Matoba, T.; Terao, J. Biosci. Biotechnol. Biochem.
1998, 62, 1201.
30. Lipid peroxidation inhibition test: Linoleic acid (0.284 g) and 0.284 g of Tween 20
were mixed together in 0.1 M sodium phosphate buffer (pH 7.0) to prepare
50 mM linoleic acid emulsion. The linoleic acid emulsion (2.5 mL), 2.0 mL of
the phosphate buffer, 0.5 mL of water, and 0.5 mL of test samples dissolved in
methanol or methanol alone were mixed in a glass vial (10 mL-volume). The
total reaction volume was fixed at 5.5 mL, and the final concentration of test
samples was 90 lM. These glass vials containing reaction mixture were tightly
18. Maeda, K.; Fukuda, M. J. Soc. Cosmet. Chem. 1991, 42(6), 361.
19. Song, K. K.; Huang, H.; Han, P.; Zhang, C. L.; Shi, Y.; Chen, Q. X. Biochem. Biophys.
Res. Commun. 2006, 342, 1147.
capped by a rubber septum, and then kept at 50 °C under the dark for 48 h.
Ferric thiocyanate assay31 was used with a slight modification to evaluate
antioxidant activity of test samples. Aliquots of the reaction mixture (25
were taken out at specific intervals, and mixed with 1.175 mL of 75% ethanol,
25 L of 20 mM ferrous chloride in 3.5% HCl, and 25 L of 30% ammonium
lL)
20. Shimizu, K.; Kondo, R.; Sakai, K. Planta Med. 2000, 66, 11.
21. Battaini, G.; Monzani, E.; Casella, L.; Santagostini, L.; Pagliarin, R. J. Biol. Inorg.
Chem. 2000, 5(2), 262.
l
l
thiocyanate. The absorbance was measured at 500 nm after 3 min, when the
color development by ferric thiocyanate complex reached maximum. Each
experiment was performed in triplicate and averaged.
22. Klabunde, T.; Eicken, C.; Sacchettini, J. C.; Krebs, B. Nat. Struct. Mol. Biol. 1998,
5(12), 1084.
23. Rich, P. R.; Wiegand, N. K.; Blum, H.; Moore, A. L.; Bonner, W. D., Jr. Biochim.
Biophys. Acta 1978, 525, 325.
24. Criton, M.; Le Mellay-Hamon, V. Bioorg. Med. Chem. Lett. 2008, 18(12), 3607.
25. Baek, H. S.; Rho, H. S.; Yoo, J. W.; Ahn, S. M.; Lee, J. Y.; Kim, M. K.; Kim, D. H.;
Chang, I. S. Bull. Korean Chem. Soc. 2008, 29, 43.
26. Kwak, S. Y.; Lee, S.; Choi, H. R.; Park, K. C.; Lee, Y. S. Bioorg. Med. Chem. Lett.
2011, 21, 5155.
31. Buege, J. A.; Aust, S. D. Methods Enzymol. 1978, 52C, 302.
32. Son, S.; Lewis, B. A. J. Agric. Food Chem. 2002, 50, 468.
33. Tyrosinase inhibition test: Tyrosinase inhibition activity was carried out with
DOPA as a substrate. Two hundred fifty microliters of phosphate buffer (0.1 M,
pH 6.8), 250 L of 2.5 mM -DOPA, 200 L of water, and 25 L of inhibitor
L-
l
L
l
l
dissolved in DMSO or DMSO alone were mixed together in an Eppendorf tube
(1.5 mL-volume). The reaction mixture was incubated at 25 °C for 10 min after
treating with 25 lL of aqueous mushroom tyrosinase solution (100 lg/mL),
27. Solid phase synthesis of HCA-Phe-NHOH and HCA-Pro-NHOH: Hydroxylamine
hydrochloride (834 mg, 12 mmol) was dissolved in 40 mL of aqueous
sodium hydrogen carbonate (2.2 g, 26 mmol), and cooled to 5 °C. N-(9-
fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu, 4.0 g, 12 mmol)
dissolved in 40 mL ethyl acetate was added drop wise to the rapidly stirred
hydroxylamine solution in an ice-bath and stirred for 4 h at room temperature.
The reaction was monitored by TLC (ethyl acetate/hexane = 1:1, Rf = 0.4). After
the water layer was removed, the organic layer was washed with saturated
aqueous potassium hydrogen sulfate and brine. This organic extract was
concentrated in high vacuum, and then N-Fmoc protected hydroxylamine
(Fmoc-NHOH) was obtained as a white crystalline solid after trituration in
hexane and stored overnight (80% yield). Its structure was identified
by 1H NMR (JNM-LA300 spectrometer, JEOL Ltd, Tokyo, Japan): (dH, CDCl3)
4.21 (1H, t, Fmoc CH), 4.32 (2H, d, Fmoc CH2), 7.28–7.43, 7.68, 7.86 (8H, m,
Fmoc Ar. CH), 8.77 (1H, s, NH), 9.75 (1H, br s, OH). Fmoc-NHOH (2 equiv) was
coupled to 2-chlorotrityl chloride (CTC) resin (1.43 mmol/g) with N,N’-
diisopropylethylamine (DIPEA; 4 equiv) in dichloromethane (DCM) for 48 h.
Fmoc-NHOH loaded CTC resin was treated with 10% DIPEA/methanol (v/v) to
block the remaining chloride groups. The resulting resin was filtered, and its
after which the UV absorbance was measured at 475 nm. The percentage of
tyrosinase inhibition activity was calculated as% inhibition = (1ÀA/B) Â 100,
where A represents the absorbance of reaction mixture containing inhibitor,
and B represents the absorbance of reference reaction mixture containing
DMSO instead of inhibitor. Each experiment was performed in triplicate. The
values are given as the mean standard error.
34. Iwai, K.; Kishimoto, N.; Kakino, Y.; Mochida, K.; Fujita, T. J. Agric. Food Chem.
2004, 52, 4893.
35. Kubo, I.; Kinst-Hori, I. J. Agric. Food Chem. 1999, 47, 4574.
36. Noh, J. M.; Kwak, S. Y.; Seo, H. S.; Seo, J. H.; Kim, B. G.; Lee, Y. S. Bioorg. Med.
Chem. Lett. 2009, 19, 5586.
37. Cell viability test: The cell viability was tested using Cell Counting Kit-8 (CCK-8;
CK04, Dojindo, Kumamoto, Japan). Mel-Ab cells (2 Â 103 per well) were seeded
into 96-well plates. Culture media were replaced with serum-free DMEM after
24 h, and incubated for another 24 h. Then, cells were treated with samples
(100
lM) containing new serum-free media and incubated for 24 h. CCK-8
solution was added and cells were incubated for another 2 h at 37 °C. The

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estimated. When the samples were treated at the concentrations of 100
amount of water-soluble formazan generated by the activity of dehydrogenase
l
M for
in cells was measured by optical density at 450 nm using SpectraMax Plus
Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
38. Melanogenesis inhibition assay: Mel-Ab cells were cultured in DMEM with 10%
FBS, 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), 1 nM cholera toxin
4 days, the inhibitors-treated cells produced less amount of melanin than the
cells without inhibitors. The treated cells were then dissolved in 1 mL of 1 N
NaOH at 100 °C for 30 min, and centrifuged for 20 min at 16,000g, after which
the optical densities of the supernatants were measured at 400 nm. Each
experiment was performed in triplicate and averaged.
(CT), 50
lg/mL of streptomycin, and 50 U/mL penicillin at 37 °C in 5% CO2.
Effects of the inhibitors on melanin formation in Mel-Ab cell line were