Depigmenting activities of kojic acid derivatives without tyrosinase inhibitory activities

Article abstract of DOI:10.1016/j.bmcl.2012.04.046

We synthesized benzoate ester derivatives of kojic acid with and without adamantane moiety. Benzoate derivatives 2a-e that did not contain an adamantane moiety showed potent tyrosinase inhibitory activities. However, depigmenting activity was not noted in a cell-based assay. Contrasting results were obtained for benzoate derivatives (3a-e) containing an adamantane moiety. Compounds 3a-e showed potent depigmenting activities without tyrosinase inhibitory activities. To the best of our knowledge, this is the first study showing the depigmenting activities of kojic acid derivatives without tyrosinase inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2012.04.046

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4159–4162
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Depigmenting activities of kojic acid derivatives without tyrosinase
inhibitory activities
Jun-Cheol Cho a, Ho Sik Rho b, , Yung Hyup Joo b, Chang Seok Lee b, Jaekyoung Lee b, Soo Mi Ahn c,
⇑
Jung Eun Kim d, Song Seok Shin b, Young-Ho Park b, Kyung-Do Suh a, Soo Nam Park d,
⇑
a Division of Chemical Engineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea
b R&D Center, AmorePacific Corporation, Yongin 446-729, Republic of Korea
c Kyung Hee University Skin Biotechnology Center, Suwon 443-270, Republic of Korea
d 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:
We synthesized benzoate ester derivatives of kojic acid with and without adamantane moiety. Benzoate
derivatives 2a–e that did not contain an adamantane moiety showed potent tyrosinase inhibitory activ-
ities. However, depigmenting activity was not noted in a cell-based assay. Contrasting results were
obtained for benzoate derivatives (3a–e) containing an adamantane moiety. Compounds 3a–e showed
potent depigmenting activities without tyrosinase inhibitory activities. To the best of our knowledge, this
is the first study showing the depigmenting activities of kojic acid derivatives without tyrosinase inhib-
itory activities.
Received 5 November 2011
Revised 25 March 2012
Accepted 9 April 2012
Available online 16 April 2012
Keywords:
Depigmentation
Tyrosinase
Ó 2012 Published by Elsevier Ltd.
Hydroxylbenzoic acid
Kojic acid
Pigmentation is the process of melanin synthesis by melano-
cytes within the skin and hair follicles.1 The melanin synthesized
plays an important role in protecting the skin from the harmful ef-
fects of ultraviolet (UV) irradiation. However, excessive synthesis
and uneven distribution of melanin can lead to esthetic problems
such as melasma, lentigo, and age spots. Melanin synthesis is
mediated by several enzymes such as tyrosinase, tyrosinase related
protein-1, and tyrosinase related protein-2.2 Tyrosinase is involved
in the initial rate-limiting step of melanin synthesis. Tyrosinase,3 a
copper-containing enzyme, catalyzes the hydroxylation of tyrosine
to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA
to dopaquinone. Kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-
pyran-4-one 1),4 one of the metabolites used in fermentation by
Aspergillus species, is the most famous tyrosinase inhibitor. Kojic
acid inhibits tyrosinase via chelation of to copper in tyrosinase ac-
tive site. Kojic acid has been widely used as a depigmenting agent
in cosmetics because of its inhibitory effect on tyrosinase. How-
ever, its inhibitory activity is insufficient. A high dosage (above
2%) is required in cosmetic formulations. Therefore, many kojic
acid derivatives have been synthesized, usually by modifying the
C-2 hydroxyl group to form esters,5 ethers,6 sulfides,7 and peptide8
derivatives to increase tyrosinase inhibitory activity. Because of
these modifications, most of these derivatives showed higher
tyrosinase inhibitory and depigmenting activities than kojic acid
because of an increase in lipophilic character. During our ongoing
research for developing potent depigmenting agents, we noticed
an adamantane moiety9 in kojic acid derivatives because of the
lipophilic character of the moiety. In this study, we synthesized
benzoate esters of kojic acid with and without an adamantane moi-
ety. Tyrosinase inhibitory and depigmenting activities were inves-
tigated to determine the importance of the adamantane moiety
(Fig. 1).
The synthetic pathways are shown in Schemes 1 and 2. Benzo-
ate ester derivatives of kojic acid (2a–e) were synthesized by the
condensation of kojyl chloride (5) with potassium salts of benzoic
acids.10 Kojic acid (1) was reacted with thionyl chloride to afford
kojyl chloride (5). Kojyl chloride (5) was then reacted with potas-
sium salts of benzoic acids in N,N-dimethylformamide (DMF) to af-
ford the corresponding ester derivatives (2a–e).
Adamantyl benzoic acids were synthesized by the reaction of
adamantylation of benzoic acid by using 1-adamantanol in trifluo-
roacetic acid (CF3COOH) under reflux condition.11 Adamantyl ben-
zoic acids were converted to potassium salts. Kojyl chloride (4) was
reacted with potassium salts of adamantyl benzoic acids in DMF to
afford the corresponding ester derivatives (3a–e).
Synthesized compounds were evaluated for mushroom tyrosi-
⇑
nase activity.12 The inhibition of tyrosinase was tested using
L
Corresponding authors.
E-mail addresses: thiocarbon@freechal.com (H.S. Rho), snpark@seoultech.ac.kr
-tyrosine or L-DOPA as substrate. Their anti-tyrosinase activities
(S.N. Park).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.04.046

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J.-C. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4159–4162
O
O
O
O
HO
O
O
O
O
O
OH
HO
OH
HO
OH
O
Kojic acid (1)
2b
3b
Figure 1. Structures of kojic acid and benzoate ester derivatives.
O
O
O
O
a
b
HO
Cl
O
R
OH
OH
OH
O
O
O
5
1, Kojic acid
2a, R = 4-OMe
2b, R = 4-OH
2c, R = 2,4-OH
2d, R = 2-OH, 4-OMe
2e, R = 2,4-OMe
Scheme 1. Reaction conditions: (a) thionyl chloride, DMF, room temperature; (b) potassium salts of benzoic acids, DMF, 110–120 °C.
O
O
O
O
O
O
OH
R
R
a
b, c
OH
OH
R
7a, R = 4-OMe
7b, R = 4-OH
3a, R = 4-OMe
3b, R = 4-OH
3c, R = 2,4-OH
6a, R = 4-OMe
6b, R = 4-OH
7c, R = 2,4-OH
7d, R = 2-OH, 4-OMe
7e, R = 2,4-OMe
6c, R = 2,4-OH
6d, R = 2-OH, 4-OMe
6e, R = 2,4-OMe
3d, R = 2-OH, 4-OMe
3e, R = 2,4-OMe
Scheme 2. Reaction conditions: (a) 1-adamantanol, CF3COOH, reflux; (b) potassium hydroxide, methanol, room temperature; (c) kojyl chloride, DMF, 110–120 °C.
were compared with those of hydroquinone and kojic acid (Table
1).
depigmenting activities at non-cytotoxic concentrations. The
compound (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl 4-methoxy-
5-adamantyl benzoate (3a) showed potent depigmenting activity
Compounds 2a–e did not contain an adamantane moiety and
exhibited potent inhibitory activities against tyrosinase using
L
-
(IC50 = 3.42
When the methoxy group was replaced by a hydroxyl group
(3b), similar inhibitory activity (IC50 = 3.81 M) was observed
with lower cytotoxicity (IC50 = 27.64 M). Introduction of 2-hydro-
xy group (3c and 3d) slightly decreased the inhibitory activity
(IC50 = 8.03 M and IC50 = 8.92 M, respectively). 2,4-Dimethoxy
lM) with moderate cytotoxicity (IC50 = 19.01 lM).
tyrosine. In case of using -DOPA, similar results were obtained.
L
The hydrophobic benzoate increased the inhibitory activity of kojic
acid. However, the numbers and positions of the phenolic hydroxyl
groups in benzoate had only a minor effect on their inhibitory
activities. In contrast to the results for the compounds (2a–e), com-
pounds 3a–e containing an adamantane moiety did not show
l
l
l
l
compound 3e also showed potent inhibitory activity. In the case
of compounds 3a–e, tyrosinase inhibitory activity was not directly
related with depigmenting activity. These differences can be
explained by two hypotheses. The first hypothesis is related to
difference between the amino acid sequences of mushroom tyros-
inase and murine tyrosinase.15 The second hypothesis is related to
differences in the depigmenting mechanism due to the addition of
the adamantane moiety to the kojic acid derivatives. To the best of
our knowledge, this is the first study on the depigmenting
activities of kojic acid derivatives without tyrosinase inhibitory
activities. To determine the pharmacophore for the depigmenting
activity of compounds 3a–e, we synthesized new derivatives that
did not contain the chelating part of the kojic acid moiety. The
5-enolic hydroxyl group of kojic acid was protected by a methyl
group. The synthetic pathway is shown in Scheme 3. On analyzing
the cytotoxicity IC50/depigmentation inhibition IC50, we selected
compounds 3a and 3b and modified their structures. Kojyl chloride
tyrosinase inhibitory activities (IC50 >200 lM) in both substrates
conditions. The bulky adamantane group caused steric hindrance
that changed the binding to the active site of tyrosinase.13
After testing for tyrosinase inhibitory activities, we evaluated
their depigmenting activities in Melan-a cells14 (Table 2)
Interestingly, in the cell-based assay, compounds 2a–e did not
show inhibitory activities (IC50 >40
lM) or cytotoxicity (IC50
>80 M). Tyrosinase inhibitory activity was not directly related
l
with depigmenting activity. Next, we evaluated compounds (3a–
e) containing an adamantane moiety in the aromatic ring. Recently,
introduction of an adamantane moiety was found to increase the
depigmenting activity of resorcinol.9a These results indicate that
an adamantane group has some positive effect on depigmenting
activity by changing the lipophilic parameter. Unexpected results
were obtained in the cell-based assay. Although compounds 3a–e
did not have tyrosinase inhibitory activities, they showed potent

J.-C. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4159–4162
4161
Table 1
(5) was treated with dimethyl sulfate in K2CO3/acetone under re-
flux condition to afford a 5-methoxy kojyl chloride (8). 5-Methoxy
kojyl chloride (8) was reacted with potassium salts of adamantyl
benzoic acids in DMF to afford the corresponding ester derivatives
(4a and 4b).
The depigmenting activities are shown in Table 3. As expected,
tyrosinase inhibitory activities were not observed during the treat-
ment of compounds 4a and 4b because of the loss of a free enolic
hydroxyl group. However, compounds 4a and 4b showed potent
Anti-tyrosinase activities of hydroquinone, kojic acid and benzoate ester derivatives
a
Compounds
Substrate
Tyrosinase inhibition (IC50
)
(
lM)
Hydroquinone
Kojic acid
10.02
65.08
187.20
2.03
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
-Tyrosine
-Tyrosine
-DOPA
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
-Tyrosine
-DOPA
2.05
2.80
-Tyrosine
-DOPA
depigmenting activities (IC50 = 4.78 lM and IC50 = 3.87 lM, respec-
1.80
tively) at non-cytotoxic concentrations. Their activities and
cytotoxicity were very similar to those of compounds 3a and 3b.
These results indicate that depigmenting activity may not be due
to kojic acid moiety, which can bind to the active site of tyrosinase.
Furthermore, the adamantane moiety has a critical and positive ef-
fect on depigmenting activity.
In conclusion, we synthesized benzoate ester derivatives of
kojic acid with and without an adamantane moiety and evaluated
their tyrosinase inhibitory and depigmenting activities. Com-
pounds 3a–e containing an adamantane moiety showed potent
depigmenting activities without tyrosinase inhibitory activities.
The compounds 4a and 4b, in which the enolic hydroxyl moiety
of kojic acid was protected by a methyl group, also showed potent
depigmenting activities. From these results, we conclude that a
benzoate moiety containing an adamantane group is a more
important depigmenting pharmacophore than kojic acid. Further
studies on the mechanism of action of this moiety in melanogene-
sis are underway.
3.51
-Tyrosine
-DOPA
3.61
10.64
20.08
3.23
-Tyrosine
-DOPA
-Tyrosine
-DOPA
4.80
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
-Tyrosine
-DOPA
-Tyrosine
-DOPA
-Tyrosine
-DOPA
-Tyrosine
-DOPA
-Tyrosine
-DOPA
a
Values were determined from logarithmic concentration–inhibition curves and
are given as the mean values of data from three experiments.
Table 2
Depigmenting activities of hydroquinone, kojic acid and benzoate ester derivatives
a
a
Compounds
Depigmentation (IC50
4.21
1.64 mM
)
Cytotoxicity (IC50
15.00
3.53 mM
)
Cytotoxicity (lM) /depigmentation (lM)
Hydroquinone
Kojic acid
lM
l
M
3.56
—
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
>40
>40
>40
>40
>40
3.42
3.81
8.03
8.92
4.00
l
l
l
l
l
M
M
M
M
M
M
M
M
M
M
>80
>80
>80
>80
>80
19.01
27.64
27.42
26.78
17.58
l
l
l
l
l
M
M
M
M
M
—
—
—
—
—
l
l
l
l
l
l
M
5.55
7.25
3.41
3.00
4.39
l
l
l
l
M
M
M
M
a
Values were determined from logarithmic concentration–inhibition curves and are given as the mean values of data from three experiments. —: not analyzed.
O
O
O
O
O
a
b
Cl
Cl
O
OH
OMe
R
OMe
O
O
5
8
4a, R = 4-OMe
4b, R = 4-OH
Scheme 3. Reaction conditions: (a) dimethylsulfate, K2CO3/acetone, reflux; (b) potassium salts of adamantyl benzoic acids, DMF, 110–120 °C.
Table 3
Depigmenting activities of compounds (4a and 4b)
a
a
Compounds
Depigmentation (IC50
)
Cytotoxicity (IC50
)
Cytotoxicity (lM) /depigmentation (lM)
4a
4b
4.78
3.87
31.28
23.84
6.54
6.16
a
Values were determined from logarithmic concentration–inhibition curves and are given as the mean value of data from three experiments.

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J.-C. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4159–4162
was dried with anhydrous MgSO4 and concentrated to give a crude product.
Acknowledgments
The resultant was purified by crystallization from ethyl acetate–hexane to give
2b (5.50 g) in 70% yields. 1H NMR (300 MHz, DMSO-d6): d 10.08 (s, 1H), 9.25 (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),
5.14 (s, 2H). 13C NMR (125 MHz, DMSO-d6): 173.5, 164.7, 162.4, 161.8, 146.0,
139.8, 131.7, 119.1, 115.4, 112.3, 61.4. ESI MS, m/e 261.2 [MÀH]+. Compound
3b: Yield 66%. 1H NMR (300 MHz, DMSO-d6): d 10.20 (s, 1H), 9.22 (s, 1H), 8.10
(s, 1H), 7.76 (s, 1H), 7.69 (d, 1H, J = 8.4 Hz), 6.89 (d, 1H, J = 8.4 Hz), 6.48 (s, 1H),
5.14 (s, 2H), 2.06 (s, 9H), 1.72 (s, 6H). 13C NMR (125 MHz, DMSO-d6): 173.5,
165.1, 161.9, 161.3, 145.9, 139.8, 135.7, 128.9, 128.1, 118.8, 116.4, 112.2, 61.2,
36.4, 36.1, 28.1. ESI MS, m/e 395.2 [MÀH]+. Compound 4b: Yield 72%. 1H NMR
(300 MHz, DMSO-d6): d 10.40 (s, 1H), 8.16 (s, 1H), 7.76 (s, 1H), 7.69 (d, 1H,
J = 8.4 Hz), 6.89 (d, 1H, J = 8.4 Hz), 6.44 (s, 1H), 5.15 (s, 2H), 3.65 (s, 3H), 2.06 (s,
9H), 1.72 (s, 6H). 13C NMR (125 MHz, DMSO-d6): 172.4, 165.1, 161.9, 161.4,
148.1, 139.5, 135.7, 128.9, 128.1, 118.8, 116.4, 113.0, 61.1, 56.1, 36.4, 36.1,
28.1. ESI MS, m/e 395.2 [MÀH]+.
Murine melan-a melanocytes were originally derived from
C57BL/6J (black, a/a) mice, a kind gift from Professor Dorothy C.
Bennett (St. George’s Hospital, London, UK).
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(pH 6.5), 3 l of sample solution, 8 l of mushroom tyrosinase (2100 unit/ml,
0.05 M phosphate buffer at pH 6.5), and 36 l of 1.5 mM -tyrosine or -DOPA.
L-
L
l
l
l
l
L
L
Tyrosinase activity was determined by reading the optical density at 490 nm
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extracted with ethyl acetate (500 ml), washed with water. The organic layer
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
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a
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