Evaluation of kojyl benzoate derivatives as potential depigmenting agents in mouse B16/F1 melanoma cells

Full text of DOI:10.1002/bkcs.10772

Note
DOI: 10.1002/bkcs.10772
BULLETIN OF THE
Y. J. Choi et al.
KOREAN CHEMICAL SOCIETY
Evaluation of Kojyl Benzoate Derivatives as Potential Depigmenting
Agents in Mouse B16/F1 Melanoma Cells
†
†
‡,*
‡
§
Yeong Jin Choi, Sun Sang Kwon, Ho Sik Rho, Yong-Jin Kim, John Hwan Lee,
§
†,
*
Seong-Geun Oh, and Ji Man Kim
†Functional Material Laboratory, Department of Chemistry, Sungkyunkwan University, Suwon 16414,
Republic of Korea. *E-mail: jimankim@skku.edu
‡
R & D Center, AmorePacific Corporation, Yongin 17074, Republic of Korea.
*
E-mail: thiocarbon@hanmail.net
§
Department of Chemical Engineering, Hanyang University, Seoul 04763, Republic of Korea
Received November 6, 2015, Accepted February 23, 2016, Published online May 19, 2016
Keywords: Depigmenting activity, Kojyl benzoate, Tyrosinase, Melanogenesis
Human skin color is primarily determined by the synthesis
and distribution of melanin pigments, which are released by
melanocytes. Several enzymes in melanocytes have to be
be induced by reactive quinone intermediate of rhododen-
10
drol by tyrosinase.
Metal chelating compounds such as tropolone, hydro-
1
11
12
13a
activated to synthesize melanin. Among them, tyrosinase is
a key enzyme that catalyzes hydroxylation of L-tyrosine to
L-dopa and the subsequent oxidation of L-dopa to dopaqui-
none. Synthesized dopaquinone is a reactive intermediate
xamic acid, and kojic acid are also used as tyrosinase
inhibitors (Figure 2).
Kojic acid (8), 5-hydroxy-2-(hydroxymethyl)-4H-pyran-
4-one, is found in various traditional Japanese foods and, is
2
13b
for the synthesis of eumelanin and pheomelanin. There-
well known as a metal chelating agent. Several previous
fore, the classical target of depigmenting therapy is the
inhibition of tyrosinase. Tyrosinase is an oxidoreductase
containing dinuclear copper ions in active sites. Generally,
reports have shown kojic acid and its derivative (Figure 3)
14
as inhibitors of tyrosinase. The inhibitory mechanism is
the result of chelating activity of kojic acid into dinuclear
3
15
tyrosinase inhibitors are classified into two types. One is
phenolic compounds and the other is metal chelator. A4
numerous phenolic compounds, such as hydroquinone,
copper ions in the active site of tyrosinase. It is non com-
petitive inhibitor unlike phenolic compound. Recently, we
synthesized kojyl benzoate derivatives and evaluated their
5
6
7
14e
resveratrol, caffeic acid, and rucinol, have been devel-
oped as tyrosinase inhibitors (Figure 1). These compounds
demonstrated the inhibition of tyrosinase and thus interfer-
ing with the oxidation process in melanin synthesis.
Although phenolic compounds have strong depigmenting
tyrosinase inhibitory activities.
Among the tested com-
pounds, (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl 4-metho-
xybenzoate (10c) had potent inhibitory activity
(IC50 = 2.0 μM) in the mushroom tyrosinase assay.
a
14e
However, we could not evaluate their depigmenting activ-
ities in cell-based assay. There have been discrepant results
between tyrosinase inhibitory activity and the inhibition of
8
activities, there is also safety risk like leukoderma. Rhodo-
9
dendrol (5), 4-(4-hydroxyphenyl)-2-butanol, has been used
as a skin whitening agent in cosmetics until it was reported
to induce leukoderma accompanied by allergic contact der-
matitis in July 2013. The melanocyte toxicity appeared to
O
OH
OH O
O
O
OH
OH
N
H
HO
OH
Tropolone (6)
Hydroxamic acid (7)
Kojic acid (8)
O
HO
OH
HO
HO
HO
OH
Figure 2. Structures of metal chelating compounds.
OH
Resveratrol (2)
Caffeic acid (3)
Hydroquinone (1)
Resorcinolmoiety
OH
O
O
OH
HO
O
O
O
O
O
O
HO
OMe
HO
OH
HO
HO
Rucinol (4)
Rhododendrol (5)
10c
10i
Figure 1. Structures of phenolic compounds.
Figure 3. Structures of kojic acid and its derivatives.
Bull. Korean Chem. Soc. 2016, Vol. 37, 942–945
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Wiley Online Library
942

Note
BULLETIN OF THE
ISSN (Print) 0253-2964 | (Online) 1229-5949
KOREAN CHEMICAL SOCIETY
16
melanin content. In this research, we have evaluated the
depigmenting activities and cytotoxicities of kojyl benzoate
derivatives (10a–10l) in B16/F1 melanoma cells.
(8) with thionyl chloride produces kojyl chloride (9). Com-
pound 9 reacts with potassium salts of benzoic acids and
benzoic acids in dimethylformamide (DMF) at 110–120 C
ꢀ
The synthetic pathways for the kojyl benzoate deriva-
tives are shown in Scheme 1. The reaction of kojic acid
to give the corresponding ester derivatives (10a–10l). Spec-
1
6b
tral data of compounds were reported in a previous
14e
publication.
In this study, B16/F1 melanoma cells were used without
O
O
O
O
OH
Cl
O
O
α-MSH treatment, which act as stimulants of melanogen-
a
b
O
R
17
HO
HO
esis. Kojic acid was used as a positive control. Cell via-
HO
O
bility was tested for each compound at a concentration of
40 μM. The tyrosinase inhibitory activity, depigmenting
activity, and cytotoxicity for each compound are shown in
Table 1.
Kojic acid (8)
9
10a - 10l
Scheme 1. Reaction conditions: (a) thionyl chloride, dimethylfor-
mamide, room temperature; (b) potassium salts of benzoic acids,
ꢀ
benzoic acids in dimethylformamide, 110–120 C.
Table 1. Depigmenting activities of koyl benzoate derivatives (10a–10l) and kojic acid in B16/F1 melanoma without α-MSH treatment.
Inhibitory activity [IC ]
50
a
b
a
Compounds
R
Tyrosinase
Pigmentation
Survival of B16/F1
1
0a
3.2 μM
>40 μM
101.4% (40 μM)
1
0b
4.3 μM
2.0 μM
>40 μM
> 40 μM
103.2% (40 μM)
103.2% (40 μM)
N
1
0c
OMe
O
1
1
0d
0e
3.3 μM
2.8 μM
>40 μM
>40 μM
105.7% (40 μM)
95.0% (40 μM)
O
OH
OH
1
1
0f
3.3 μM
4.5 μM
>40 μM
>40 μM
98.8% (40 μM)
91.5% (40 μM)
0g
OH
1
0h
0i
OH
5.7 μM
3.5 μM
29.9 Æ 1.2 μM
37.7 Æ 1.1 μM
75.1% (40 μM)
95.5% (40 μM)
OH
OH
1
OH
1
0j
OH
4.4 μM
>40 μM
90.5% (40 μM)
OH
OH
1
0k
0l
3.3 μM
3.6 μM
30.3 Æ 1.5 μM
>40 μM
76.4% (40 μM)
103.9% (40 μM)
OH
OH
1
OH
Kojic acid
70.1 μM
380.6 Æ 1.9 μM
100.2% (2 mM)
a
Value is obtained from one experiment.
b
Values are obtained from three experiments.
Bull. Korean Chem. Soc. 2016, Vol. 37, 942–945
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
943

BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
Compounds containing no-hydroxyl groups in the benzo-
Table 2. Depigmenting activities of koyl benzoate derivatives
(10h–10l) and kojic acid in B16/F1 melanoma with α-MSH
treatment.
ate group (compounds 10a–10d) had no inhibitory activity.
There were discrepancies between tyrosinase inhibitory
activity and the inhibition of melanin content. Mono-
hydroxyl containing compounds (10e–10g) also showed no
inhibitory activity. The positions of the mono-hydroxyl
group in the benzoate group had no effect on the com-
pound’s inhibitory activities. Next, we evaluated several
compounds (10h–10l) with the di-hydroxyl groups at vari-
ous positions. Compound 10h (2,3-dihydroxy) had the
highest inhibitory activity (IC50 = 29.9 Æ 1.2 μM). How-
ever, it conferred a slight cytotoxicity a concentration of
Survival of
B16/F1
a
b
Compounds
10h
R
Pigmentation
OH
33.4 Æ 1.3 μM
84.9% (40 μM)
OH
OH
1
0i
OH
38.9 Æ 0.9 μM
96.0% (40 μM)
1
0j
OH
>40 μM
101.0% (40 μM)
4
0 μM. Compound 10k (3,4-dihydroxy) had a similar
depigmenting activity (IC50 = 30.3 Æ 1.5 μM) with a slight
cytotoxicity. The depigmenting activities and cytotoxicities
of 10h and 10k may be due to the catechol moiety. Com-
pound 10i (2,4-dihydroxy) had a similar depigmenting
activity (IC50 = 37.7 Æ 1.1 μM). However, at a concentra-
tion of 40 μM, it did not induce cytotoxicity. Although
compound 10j (2,5-dihydroxy) and compound 10l (3,5-
dihydroxy) had dihydroxyl groups, their IC50 were greater
than 40 μM. Based on these results, we concluded that
depigmenting activity and cytotoxicity are closely related to
the position of the dihydroxyl groups in the benzoate
group. The catechol moiety (2,3-dihydroxy and 3,4-dihy-
droxy) resulted in both depigmenting activity and cytotox-
icity. In the resorcinol moiety, the 2,4-dihydroxy position
yielded a desirable result, conferring depigmenting activity
without cytotoxicity. After evaluation of the depigmenting
activity in B16/F1 melanoma cells without α-MSH treat-
ment, we evaluated the activities of dihydroxyl compounds
OH
OH
1
0k
33.8 Æ 1.4 μM
>40 μM
79.2% (40 μM)
98.8% (40 μM)
OH
OH
1
0l
OH
Kojic acid
454.4 Æ 2.7 μM
98.7% (2 mM)
a
Values are obtained from three experiments.
Value is obtained from one experiment.
b
Experimental Section
Synthesis of (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl
Benzoate (10a). To a stirred solution of kojyl chloride
9 (4.80 g, 30.0 mmol) in DMF (100 mL) under N potas-
sium salt of benzoic acid (4.79 g, 30.0 mmol) with benzoic
acid (2.73 g, 2.25 mmol) was added. The reaction mixture
was stirred for 2 h at 110–120 C, after which DMF was
evaporated in vacuo. The residue was extracted with ethyl
acetate (500 mL), and then was washed with water. The
organic layer was dried with anhydrous MgSO and con-
4
centrated to give a crude product. The resultant was puri-
fied by crystallization from ethyl acetate–hexane to give
10a (5.37 g) in 73% yields. H NMR (300 MHz, DMSO-
d ): δ 9.29 (s, 1H), 8.11 (s, 1H), 8.02 (m, 2H), 7.71 (m,
2
(
10h–10l) with α-MSH stimulation (Table 2).
Similar results were obtained under conditions of α-MSH
ꢀ
stimulation. Compound 10h (2,3-dihydroxy) and compound
10k (3,4-dihydroxy) had depigmenting activities with slight
cytotoxicities. Compound 10i (2,4-dihydroxy) had depig-
menting activity (IC50 = 38.9 Æ 0.9 μM) without cytotox-
icity at a concentration of 40 μM. On the other hand, kojic
acid inhibited melanin synthesis at a high concentration
1
(
IC50 = 454.4 Æ 2.7 μM). The depigmenting activity of
kojic acid was dramatically enhanced by the ester modifica-
tion of benzoic acid containing 2,3-dihydroxy, 3,4-dihy-
droxy, and 2,4-dihydroxy groups.
6
13
1H), 7.58 (m, 2H), 6.55 (s, 1H), 5.22 (s, 2H). C-NMR
(125 MHz, DMSO-d ): 173.5, 164.9, 161.4, 146.0, 139.9,
33.7, 129.3, 128.8, 128.7, 112.6, 61.9. FABMS, m/e
6
1
In conclusion, we evaluated the depigmenting activities
of kojyl benzoate derivatives in B16/F1 melanoma cells
without and with α-MSH treatment. Depigmenting activity
and cytotoxicity were influenced by the position of dihy-
droxyl groups in the benzoate group. The catechol moiety
+
245.1 [M–H] .
Cell Culture. The B16/F1 melanoma cell lines were
obtained from Korean Cell Line Bank (Seoul, Korea). Cells
were cultured in Dulbecco’s modified Eagle medium
(DMEM) containing fetal bovine serum (FBS, 10%), peni-
cillin (100 U/mL), and streptomycin (0.1 mg/mL) at 37 C
in a humidified atmosphere of 5% CO .
2
Measurements of Cell Viability. Viability of cultured
cells was determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide [MTT] method). Cells were
added into each well in 96-well plates, and cultured for
24 h. After kojic acid derivatives treatment, 100 μL of
(2,3-dihydroxy and 3,4-dihydroxy) had depigmenting activ-
ꢀ
ity along with a slight cytotoxicity. However, the 2,4-
dihydroxy positions (resorcinol moiety) had depigmenting
activity without cytotoxicity. Together, these results suggest
that compound 10i (2,4-dihydroxy) is a potentially effective
skin depigmenting agent. Further studies on the mechanism
of kojyl benzoate derivatives in melanogenesis are
underway.
Bull. Korean Chem. Soc. 2016, Vol. 37, 942–945
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
944

BULLETIN OF THE
Note
KOREAN CHEMICAL SOCIETY
MTT (5 mg/mL in PBS) was added to each well. Cells
were incubated at 37 C for 30 min, and dimethyl sulfoxide
10. a) S. Ito, M. Ojika, T. Yamashita, K. Wakamatsu, Pigm. Cell
Melanoma Res. 2014, 27, 744; b) S. Ito, M. Okura,
Y. Nakanishi, M. Ojika, K. Wakamatsu, T. Yamashita, Pigm.
Cell Melanoma Res. 2015, 28, 295.
ꢀ
(DMSO) was added to dissolve the formazan crystals. The
absorbance was measured at 560 nm with a microplate
reader (Molecular Devices Co., Sunnyvale, CA, USA).
1
1. S. Takahashi, T. Kamiya, T. Abe, S. Tanuma, Bioorg. Med.
Chem. 2010, 18, 8112.
4
Measurements of Melanin Content. The cells (2 × 10
1
2. a) H. S. Baek, H. S. Rho, S. M. Ahn, D. H. Kim, I. S. Chang,
Bull. Korean Chem. Soc. 2008, 29, 43; b) H. S. Rho,
H. S. Baek, S. M. Ahn, D. H. Kim, H. G. Kim, Bull. Korean
Chem. Soc. 2009, 30, 475.
cells/mL) were seeded into 24-well plates and kojic acid
derivatives were added in triplicate. The medium was chan-
ged daily and after 4 days of culture, the cells were lysed
with 0.1 mL of 1 N NaOH. Then, 100 μL of each crude
cell extract was transferred into 96-well plates. The relative
melanin content was measured at 400 nm with a microplate
reader (Molecular Devices Co., Sunnyvale, CA, USA).
13. a) Y. Ohyama, Y. Mishima, Fragrance J. 1990, 6, 53; b)
S. Emami, S. J. Hosseinimehr, S. M. Taghdisi,
S. Akhlaghpoor, Bioorg. Med. Chem. Lett. 2007, 17, 45.
1
4. a) H. S. Rho, H. S. Baek, S. M. Ahn, D. H. Kim, I. S. Chang,
Bull. Korean Chem. Soc. 2008, 29, 1569; b) H. S. Rho,
H. S. Baek, S. M. Ahn, D. H. Cho, J. S. Hwang, Bull. Korean
Chem. Soc. 2010, 31, 2375; c) H. S. Rho, S. M. Ahn,
D. S. Yoo, M. K. Kim, D. H. Cho, J. Y. Cho, Bioorg. Med.
Chem. Lett. 2010, 20, 6569; d) S. M. Ahn, H. S. Rho,
H. S. Baek, Y. H. Joo, Y. D. Hong, S. S. Shin, Y. H. Park,
S. N. Park, Bioorg. Med. Chem. Lett. 2011, 21, 7466; e)
H. S. Rho, C. S. Lee, S. M. Ahn, Y. H. Park, S. N. Park, Bull.
Korean Chem. Soc. 2011, 32, 4411.
References
1
2
3
. P. A. Piley, Pigm. Cell Res. 2003, 16, 548.
. S. Osaki, Arch. Biochem. Biophys. 1963, 100, 378.
. S. Halaouli, M. Asther, J. C. Sigoillot, M. Hamdi,
A. Lomascolo, J. Appl. Microbiol. 2006, 100, 219.
. C. A. Ramsden, P. A. Riley, Bioorg. Med. Chem. Lett. 2014,
4
2
4, 2463.
1
5. a) J. Cabanes, S. Chazarra, F. Garcia-Carmona, J. Pharm.
Pharmacol. 1994, 46, 982; b) C. Gerdemann, C. Eicken,
B. Krebs, Acc. Chem. Res. 2002, 35, 183.
5
6
. H. Satooka, I. Kubo, Bioorg. Med. Chem. 2012, 20, 1090.
. S. Kudugunti, H. Thorsheim, M. Yousef, L. Guan,
M. Moridani, Chem. Biol. Interact. 2011, 192, 243.
. D. S. Kim, S. Y. Kim, S. H. Park, Y. G. Choi, S. B. Kwon,
M. K. Kim, J. L. Na, S. W. Youn, K. C. Park, Biol. Pharm.
Bull. 2005, 28, 2216.
1
6. a) H. S. Rho, H. S. Baek, S. M. Ahn, J. W. Yoo, D. H. Kim,
H. G. Kim, Bioorg. Med. Chem. Lett. 2009, 19, 1532; b)
J. C. Cho, H. S. Rho, H. S. Baek, S. M. Ahn, B. Y. Woo,
Y. D. Hong, J. W. Cheon, J. M. Heo, S. S. Shin, Y. H. Park,
K. D. Suh, Bioorg. Med. Chem. Lett. 2012, 22, 2004.
7
8
. J. Seneschal, K. Boniface, K. Ezzedine, A. Taieb, Exp. Der-
matol. 2014, 23, 879.
1
7. J. H. Jang, J. H. Lee, H. K. Shin, Y. H. Choi, J. D. Lee,
B. T. Choi, Int. J. Mol. Med. 2010, 25, 287.
9
. H. Akazawa, T. Akihisa, Y. Taguchi, N. Banno, R. Yoneima,
K. Yasukawa, Biol. Pharm. Bull. 2006, 29, 1970.
Bull. Korean Chem. Soc. 2016, Vol. 37, 942–945
© 2016 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
945