Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis

Article abstract of DOI:10.1016/j.bmcl.2011.09.122

A novel kojic acid derivative containing a trolox moiety, (±)-5-hydroxy-4-oxo-4H-pyran-2-yl methyl 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylate (3a), was synthesized. The two biologically active compounds, namely, kojic acid and trolox, were conjug

Full text of DOI:10.1016/j.bmcl.2011.09.122

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7466–7469
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitory activity of novel kojic acid derivative containing trolox moiety
on melanogenesis
Soo Mi Ahn a, Ho Sik Rho b, , Heung Soo Baek b, Yung Hyup Joo b, Yong Deog Hong b, Song Seok Shin b,
⇑
Young-Ho Park b, Soo Nam Park c,
⇑
a Kyung Hee University Skin Biotechnology Center, Suwon, Republic of Korea
b AmorePacific R&D Center, Yongin 446-729, Republic of Korea
c Department of Fine Chemistry, Seoul National University of Science and Technology, Seoul 139-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 June 2011
Revised 14 September 2011
Accepted 29 September 2011
Available online 17 October 2011
A novel kojic acid derivative containing a trolox moiety, ( )-5-hydroxy-4-oxo-4H-pyran-2-yl methyl
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (3a), was synthesized. The two biologically active
compounds, namely, kojic acid and trolox, were conjugated via an ester bond as they are expected to
behave synergistically. The antioxidant activity and the tyrosinase inhibitory activity of this novel kojic
acid derivative on melanogenesis were evaluated. Compound 3a exhibited potent tyrosinase inhibitory
activity and radical scavenging activity. Limited structure–activity relationship (SAR) investigations indi-
cated that the tyrosinase inhibitory activities may originate from the kojic acid moiety, and the radical
scavenging activity may be due to the phenolic hydroxyl group of trolox. Compound 3a also exhibited
potent depigmenting activity in a cell-based assay. The limited SAR investigations revealed that the
depigmenting activity of 3a may be due to the synergistic activities of kojic acid and its trolox moiety.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Kojic acid
Trolox
Tyrosinase
DPPH
Depigmentation
A number of factors determine the color of mammalian skin and
hair. The most importantfactor is the degree and distribution of mel-
anin. Tyrosinase,1 a multi-functional copper-containing enzyme, is
widely distributed in fungi, plants, and animals. Tyrosinase cataly-
ses two distinct reactions of melanin synthesis: the hydroxylation
of monophenols and the oxidation of o-diphenols to o-quinones.
Both of these reactions depend on molecular oxygen.2 The produced
quinone is a highly reactive compound and can polymerize sponta-
neously to form melanin, which determines the color of mammalian
skin and hair. In addition, various types of stimulation, including
exposure to ultraviolet (UV) radiation, may cause damage to epider-
mal cells, leading to post-inflammatory hyperpigmentation.3 One of
the biggest reasons for hyperpigmentation is probably oxidative
stress caused by UV radiation. Recently, much research effort has fo-
cused on the regulation of tyrosinase as a method to control melano-
genesis. So far, a large number of naturally occurring and synthetic
tyrosinase inhibitors have already been reported.4 However, most
of them are either not potent enough to be of practical use or do
not comply with safety regulations imposed on cosmetic additives.
Furthermore, because counteracting the oxidative stress caused by
UV radiation is a means to prevent skin damage, it is important to
design a multifunctional skin-whitening agent with both antioxida-
tive and anti-tyrosinase activities. Kojic acid,5 5-hydroxy-2-
(hydroxymethyl)-4H-pyran-4-one, is produced from carbohydrate
sources in an aerobic process by a variety of microorganisms. Previ-
ous research indicates that the tyrosinase inhibiting activity of kojic
acid may be due to its c-pyranone structure that contains an enolic
hydroxyl group. Kojic acid also exhibited free radical scavenging
activity and the prevention of photodamage.6 Trolox7 has also been
reported to be a potent radical scavengingagent, and its radical scav-
enging mechanism has been examined in detail. Recently, several
trolox analogues were developed to enhance radical scavenging
activity by conjugating trolox with additional antioxidant molecules
such as hydroxytyrosol and dopamine.8 Synergistic enhancing activ-
ities were detected in these compounds. Based on these findings, we
designed and synthesized a novel kojic acid derivative containing a
trolox moiety (compound 3a in Fig. 1). We evaluated the antioxidant
activity and the tyrosinase inhibiting and depigmenting effects on
melan-a cells. Scheme 1 shows the synthetic pathways for the novel
kojic acid derivative. The reaction of kojic acid with thionyl chloride
produces compound 4, which is conveniently converted to com-
pound 5 using dimethylsulfate and potassium carbonate in acetone
under reflux conditions. Chloride 4 reacts with potassium salts of
trolox, 4-hydroxybenzoic acid and 6-hydroxynathphoic acid in
dimethylformamide at 110–120 °C to give the corresponding ester
derivatives 3a–3c.9 Compound 5 reacts with potassium salts of trol-
ox in dimethylformamide at 110–120 °C to give the corresponding
ester derivatives 3d.9
⇑
Corresponding authors.
E-mail addresses: thiocarbon@freechal.com (H.S. Rho), snpark@seoultech.ac.kr
(S.N. Park).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.122

S. M. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7466–7469
7467
O
O
O
O
OH
O
O
O
O
O
HO
HO
HO
OH
OH
2, Trolox
3a
1, Kojic acid
Fig 1. Structures of kojic acid derivative containing trolox moiety.
O
O
O
O
OH
a
c
Cl
O
O
O
O
HO
HO
HO
HO
OH
OH
3a
O
4
O
O
1
, Kojic acid
O
3b
O
O
b
O
O
HO
OH
3c
O
O
O
Cl
c
O
O
O
O
MeO
O
5
MeO
OH
3d
Scheme 1. Reagents and conditions: (a) SOCl2, DMF, room temperature; (b) Me2SO4, K2CO3, acetone, reflux; (c) potassium salt of acids, DMF, 110–120 °C.
Compound 3a contains kojic acid and a trolox moiety. The trol-
ox structure is composed of a chroman ring and a phenolic hydro-
xyl group. To study the importance of the trolox structure for the
tested activities, we synthesized compounds 3b, 3c, and 3d and
compared their activities with that of compound 3a.
The inhibitory activities of the synthesized compounds on
mushroom tyrosinase10 was initially investigated and compared
with those of the mother compounds such as kojic acid and trolox
(Table 1). Kojic acid exhibited moderate tyrosinase inhibitory
activity (IC50 = 69.39 lM). Trolox exhibited no inhibitory activity.
However, compound 3a, synthesized by conjugating kojic acid
with trolox via an ester bond, exhibited potent inhibitory activity
against tyrosinase (IC50 = 5.36 lM). Compounds 3b and 3c, in
zene moieties, also exhibited potent inhibitory activity. When 5-
enolic hydroxyl group was protected by a methyl group,11 inhibi-
tory activity was lost completely (compound 3d). Next, we investi-
gated the radical scavenging activities. In the DPPH assay,12 two
compounds (3a and 3d) containing a trolox moiety exhibited rad-
ical scavenging activity. Compound 3d, in which enolic hydroxyl
groups were replaced by a methoxyl group, exhibited potent activ-
ity that was similar to that of trolox. These results indicated that
the tyrosinase inhibitory activities may have originated from the
kojic acid moiety and the radical scavenging activity may have
been due to the phenolic hydroxyl group in the trolox moiety.
After testing the tyrosinase inhibitory and radical scavenging
activities, we determined the potency of the inhibition of melanin
formation.13 The results are shown in Table 2. Kojic acid inhibited
melanin synthesis at high concentration (IC50 = 1.60 mM). Trolox
which the chroman moiety was replaced by naphthalene and ben-
Table 1
Inhibitory activities of kojic acid derivatives against mushroom tyrosinase and DPPH
radicals
Table 2
Depigmenting activities of kojic acid derivatives
Compounds
Inhibitory activitya
Tyrosinase (IC50
69.39
>200
Compounds
Inhibitory activitya
Depigmentation (IC50
1.60 mM
)
DPPH (IC50
)
)
Cytoxicity (IC50
3.50 mM
)
Kojic acid
Trolox
3a
3b
3c
l
M
1.45 mM
Kojic acid
Trolox
3a
3b
3c
lM
10.08
11.61
l
l
M
M
>300
17.70
>200
>200
>200
l
l
M
M
300
lM
5.36
9.65
5.51
l
l
l
M
M
M
56.47 lM
>200
>200
l
M
lM
lM
lM
>200
>200
>200
lM
lM
lM
3d
>200
lM
19.78
lM
3d
lM
a
a
Values were determined from logarithmic concentration-inhibition curves and
are the means of three experiments.
Values were determined from logarithmic concentration-inhibition curves and
are the means of three experiments.

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S. M. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7466–7469
Fig 2. Docked conformation of compounds 3a and kojic acid in the binding site of tyrosinase: (A) compound 3a, (B) kojic acid.
relationship investigations, we conclude that kojic acid and trolox
moieties are both important for depigmenting activity. Overall,
these results suggest that compound 3a shows promise as a poten-
tially effective skin depigmenting agent. Further studies on its
mechanism of action in melanogenesis are underway.
Table 3
Docking scores and the contributions of interactions
Compounds
Surflex-Dock scorea
Polar contribution
3a
Kojic acid
6.56
4.81
3.22
2.19
a
Surflex-Dock scores were presents the binding affinity in units of ÀlogKd.
Acknowledgments
Murine melan-a melanocytes were originally derived from
C57BL/6J (black, a/a) mice, a kind gift from Prof. Dorothy C. Bennett
(St. George’s Hospital, London, UK).
did not exhibit depigmenting activity at non-cytotoxic concentra-
tions. Compound 3a exhibited potent depigmenting activity
(IC50 = 17.70 lM), and its activity was approximately 90 times
more potent than that of kojic acid. Interestingly, compound 3b,
in which chroman moiety is replaced by naphthalene moiety,
exhibited no inhibitory activity against the melanin synthesis.
Compound 3c also exhibited no inhibitory activity. From these re-
sults, we conclude that the phenolic hydroxyl group of the chro-
man ring plays an important role in depigmenting activity.
However, compound 3d, which contains trolox and methyl pro-
tected kojic acid moieties exhibited no depigmenting activity.
These results indicate that depigmenting activity may be due to
synergistic activities between the trolox and kojic acid moieties.
In addition, primary tyrosinase binding to the active site may orig-
inate from the kojic acid moiety and secondary hydrophobic bind-
ing may be due to the chroman ring of trolox. Furthermore, the
antioxidant activity of trolox also has a positive effect on depig-
menting activity.
References and notes
1. Marmol, V. D.; Beermann, F. FEBS Lett. 1996, 381, 165.
2. Sanchez-Ferrer, A.; Rodriguez-Lopez, J. N.; Garcia-Canavas, F.; Garcia-Carmona,
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3. Gilchrest, B. A.; Park, H. Y.; Eller, M. S.; Yaar, M. Photochem. Photobiol. 1996, 63,
1.
4. (a) Solano, F.; Briganti, S.; Picardo, M.; Ghanem, G. Pigment Cell Res. 2006, 19,
550; (b) Brigati, S.; Camera, E.; Picardo, M. Pigment Cell Res. 2003, 16, 101; (c)
Rho, H. S.; Baek, H. S.; Woo, J. W.; Kim, D. H.; Kim, H. G. Bull. Korean Chem. Soc.
2009, 30, 475; (d) Kim, K. D.; Song, M. H.; Yum, E. K.; Jeon, O. S.; Ju, Y. W.;
Chang, M. S. Bull. Korean Chem. Soc. 2010, 31, 181; (e) Dooley, T. P.; Gadwood, R.
C.; Kilgore, K.; Thomasco, L. M. Skin Pharmacol. 1994, 7, 188; (f) Kim, D. S.; Kim,
S. Y.; Park, S. H.; Choi, Y. G.; Kwon, S. B.; Kim, M. K.; Na, J. I.; Youn, S. W.; Park, K.
C. Biol. Pharm. Bull. 2005, 12, 2216.
5. Ohyama, Y.; Mishima, Y. Fragrance J. 1990, 6, 53.
6. Mitani, H.; Koshiishi, I.; Sumita, T.; Imanari, T. Eur. J. Pharmacol. 2001, 411, 169.
7. (a) Peus, D.; Meves, A.; Pott, M.; Beyerle, A.; Pittelkow, M. R. Free Radic. Bio. Med.
2001, 30, 425; (b) Shimizu, K.; Kondo, R.; Sakai, K.; Takeda, N.; Nagahata, T.;
Oniki, T. Lipids 2001, 36, 1321; (c) Wu, J.; Sugiyama, H.; Zeng, L.-H.; Mickle, D.;
Wu, T.-W. Biochem. Cell. Biol. 1998, 76, 661.
To explain tyrosinase binding of compound 3a, we conducted a
modeling study.14 Figure 2 shows the selected docked conforma-
tion of compounds 3a and kojic acid. The docking results from
the crystal structure of tyrosinase in the modeling study agreed
well with the observed in vitro data, which indicated that com-
8. Palozza, P.; Simone, R.; Picci, N.; Buzzoni, L.; Ciliberti, N.; Natangelo, A.;
Manfredini, S.; Vertuani, S. Free Radic. Bio. Med. 2008, 44, 1452.
9. Synthesis of compound 3a. To a stirred solution of kojyl chloride 2 (3.50 g,
21.8 mmol) in DMF (80 mL) under N2 was added potassium salt of trolox
(6.28 g, 21.8 mmol) with trolox (4.09 g, 16.3 mmol). The reaction mixture was
stirred for 2 h at 110 °C–120 °C, after which DMF was evaporated in vacuo. The
residue was extracted with ethyl acetate (500 mL), washed with water. The
organic layer was dried with anhydrous MgSO4 and concentrated to give a
crude product. The resultant was purified by crystallization from ethyl acetate–
hexane to give 3a (5.71 g) in 70% yields. 1H NMR (300 MHz, DMSO-d6): d 9.19
(s, 1H), 7.91 (s, 1H), 7.41 (s, 1H), 6.22 (s, 1H), 4.90 (s, 2H), 2.51 (m, 1H), 2.30 (m,
2H), 2.00 (s, 3H), 1.97 (s, 3H), 1.91 (s, 3H), 1.80 (m, 1H), 1.50 (s, 3H). FABMS: (m/
e) 373 [MÀH]+. Compound 3b. Yield 68%. 1H NMR (300 MHz, DMSO-d6): d 10.22
(s, 1H), 9.23 (s, 1H), 8.59 (s, 1H), 8.12 (s, 1H), 8.02 (d, 1H, J = 8.1 Hz), 7.84 (d, 1H,
J = 8.1 Hz), 7.79 (d, 1H, J = 8.1 Hz), 7.19 (m, 2H), 6.59 (s, 1H), 5.24 (s, 2H).
FABMS: (m/e) 311 [MÀH]+. Compound 3c. Yield 69%. 1H NMR (300 MHz, DMSO-
d6): d 10.19 (br s, 1H), 9.32 (br s, 1H), 8.10 (s, 1H), 7.87 (d, 2H, J = 8.4 Hz), 6.88
(d, 2H, J = 8.4 Hz), 6.51 (s, 1H), 5.15 (s, 2H). FABMS: (m/e) 261 [MÀH]+.
Compound 3d. Yield 75%. 1H NMR (300 MHz, DMSO-d6): d 7.98 (s, 1H), 7.47 (s,
1H), 6.22 (s, 1H), 4.96 (d, 1H, J = 14.1 Hz), 4.93 (d, 1H, J = 14.1 Hz), 3.62 (s, 3H),
2.51 (m, 1H), 2.30 (m, 2H), 2.03 (s, 3H), 2.01 (s, 3H), 1.95 (s, 3H), 1.80 (m, 1H),
1.54 (s, 3H). FABMS: (m/e) 387 [MÀH]+.
pound 3a (IC50 = 5.36
lM) was a potent inhibitor of tyrosinase
while kojic acid (IC50 = 69.39
l
M) exhibited moderate tyrosinase
inhibitory activity. The docked score of 3a indicated tight binding
to the active site of tyrosinase compared with kojic acid 1. The re-
sults are shown in Table 3.
In conclusion, we synthesized a novel kojic acid derivative
containing a trolox moiety, ( )-5-hydroxy-4-oxo-4H-pyran-2-yl)-
methyl
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate
(3a). This compound was synthesized by conjugating kojic acid
and trolox via an ester linkage and its inhibitory activity on melano-
genesis was evaluated. Compound 3a exhibited potent tyrosinase
inhibitory activity and radical scavenging activity. In the cell-based
assay, compound 3a decreased the melanin content efficiently with-
out cell cytotoxicity. From the results of limited structure–activity

S. M. Ahn et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7466–7469
7469
10. Measurements of mushroom tyrosinase activity: Mushroom tyrosinase,
tyrosine were purchased from Sigma Chemical. The reaction mixture for
mushroom tyrosinase activity consisted of 150 l of 0.1 M phosphate buffer
(pH 6.5), 3 l of sample solution, 8 l of mushroom tyrosinase (2,100 unit/ml,
0.05 M phosphate buffer at pH 6.5), and 36 l of 1.5 mM -tyrosine. Tyrosinase
activity was determined by reading the optical density at 490 nm on
L
-
were seeded into 24-well plates and the test compounds were added in
triplicate. The medium was changed daily and after 4 d of culture, the cells
l
were lysed with 1 ml of 1 N NaOH. Then 200
transferred into 96-well plates. The relative melanin content was measured at
400 nm with microplate reader (Molecular Devices). Cell viability was
ll of each crude cell extract was
l
l
l
L
a
a
determined using a modified crystal violet assay. After removing the medium
from each well, the cells were washed with PBS and stained with 0.1% crystal
violet in 10% ethanol for 5 min at room temperature. The cells were then rinsed
four times with distilled water, and crystal violet retained by adherent cells
was extracted with 95% ethanol at room temperature for 10 min. Crystal violet
absorption was measured at 590 nm (Molecular Devices Co., Sunnyvale, CA,
USA).
microplate reader (Bio-Rad 3550, Richnmond, CA, USA) after incubation for
20 min at 37 °C. The inhibitory activity of the sample was expressed as the
concentration that inhibits 50% of the enzyme activity (IC50).
11. (a) Rho, H. S.; Baek, H. S.; Ahn, S. M.; Yoo, J. W.; Kim, D. H.; Kim, H. G. Bull.
Korean Chem. Soc. 2009, 30, 475; (b) Rho, H. S.; Baek, H. S.; Ahn, S. M.; Kim, M.
K.; Ghimeray, A. K.; Cho, D. H.; Hwang, J. S. Bull. Korean Chem. Soc. 2010, 31,
2375.
12. DPPH radical-scavenging assay was carried out using the following procedure.
The reaction mixture containing various concentrations of the test samples and
DPPH methanolic solution (0.2 mM) was incubated at room temperature for
30 min and the absorbance was measured at 517 nm. The scavenging activity
was expressed as a percent compared to control DPPH solution (100%).
13. Measurements of melanin content and cell viability: Melanin content and cell
number were measured in melan-a melanocytes. The cells (2 Â 105 cells/ml)
14. Molecular modeling study was carried out on linux system using SYBYL-X
version 1.2. To prepare the tyrosinase structure, the crystal structure of the oxy
form of S. castaneoglobisporus tyrosinase was taken from the Protein Data
Bank (PDB cod 1 wx2) because there is no crystal structure of mushroom
tyrosinase published yet. The caddie protein and water molecules were
removed. Hydrogen atoms were added to the enzyme using the SYBYL. For the
molecular docking method, Surflex-Dock version 2.5 was used using standard
parameters and allowing the hydrogen of protein movement.