Synthesis of tyrosinase inhibitory kojic acid derivative

Article abstract of DOI:10.1002/ardp.200500213

Kojic acid derivative 2 was synthesized by joining two pyrone rings through an ethylene linkage by Horner-Emmons reaction of phosphonate 6 with aldehyde 7. The intermediates 6 and 7 were derived from kojic acid. The tyrosinase inhibitory activity of 2 was

Full text of DOI:10.1002/ardp.200500213

Arch. Pharm. Chem. Life Sci. 2006, 339, 111–114
Y. S. Lee et al.
111
Full Paper
Synthesis of Tyrosinase Inhibitory Kojic Acid Derivative
Yong Sup Lee1, Jang Hyun Park2, Min Hwan Kim1, Seon Hee Seo2, Hyoung Ja Kim1, 2
1 Kyung Hee East-West Pharmaceutical Research Institute and Department of Pharmaceutical Science,
College of Pharmacy, Kyung Hee University, Seoul, Korea
2 Division of Life Sciences, Korea Institute of Science and Technology, Seoul, Korea
Kojic acid derivative 2 was synthesized by joining two pyrone rings through an ethylene linkage
by Horner-Emmons reaction of phosphonate 6 with aldehyde 7. The intermediates 6 and 7 were
derived from kojic acid. The tyrosinase inhibitory activity of 2 was about 8 times more potent
(IC50 = 3.63 lM) than that of kojic acid (IC50 = 30.61 lM). Compound 2 also exhibited potent mela-
nin synthesis inhibitory activity (19.53% inhibition at 5 lg) indicating that the connection of
two pyrone rings of kojic acid through a suitable linker can be an useful strategy for identifica-
tion of potent tyrosinase inhibitiors.
Keywords: Tyrosinase inhibitor / Kojic acid / Melanin production / Skin-whitening /
Received: September 25, 2005; accepted: October 21, 2005
DOI 10.1002/ardp.200500213
Introduction
Melanogenesis is a physiological process resulting in the
production of melanin pigment, which plays an impor-
tant role in the prevention of sun-induced skin injury [1].
Although the melanin production in human skin is a
major defense mechanism against UV light, the accumu-
lation of an excess of epidermal pigmentation can cause
various hyperpigmentation disorders, such as melasma,
age spots, and sites of actinic damage.
Figure 1. Chemical structure of kojic acid 1 and the newly
synthesized derivative 2.
Tyrosinase (EC 1.14.18.1) is
a copper-containing
enzyme widely distributed in nature. It catalyzes two dis-
tinct reactions involving molecular oxygen in the mela-
nin synthesis, the hydroxylation of L-tyrosine to L-dopa
and the oxidation of L-dopa to dopaquinone. This dopa-
quinone is highly reactive and can polymerize sponta-
neously to form melanin in a series of reaction pathways
[2]. Accordingly, the regulation of melanin synthesis by
inhibition of tyrosinase to prevent hyperpigmentation
has been a recent subject of many studies [3].
and penicillium and has been used in many countries as a
skin-whitening agent because of its tyrosinase inhibitory
activity on melanin synthesis [4]. However, the inhibitory
activity of 1 is not sufficiently potent or unstable for sto-
rage for use in cosmetics. Accordingly, many semi-syn-
thetic kojic acid derivatives were synthesized usually by
the modification of C-7 hydroxyl group into ester [5]
hydroxyphenyl ether [6], glycoside [7], and amide deriv-
atives [8].
Kojic acid (1, Fig. 1) is one of the metabolites produced
by various fungal or bacterial strains such as aspergillus
In this study, we designed and synthesized a new kojic
acid derivative 2 (Fig. 1), which has two pyrone rings con-
nected by an ethylene linker. It was expected that com-
pound 2 has better tyrosinase inhibitory activity with
enhanced stability because it has two copper-chelating
pyrone rings and a chemically stable ethylene rather
than C-7 hydroxyl, ester or amide group.
Correspondence: Yong Sup Lee, PhD, Department of Pharmaceutical
Science, College of Pharmacy, Dongdaemoon-ku, 130-701 Seoul, Korea.
E-mail: kyslee@khu.ac.kr
Fax: +82 2 966-3885
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

112
Y. S. Lee et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 111–114
Scheme 1. Synthesis of kojic acid derivative.
Table 1. Tyrosinase and melanin synthesis inhibitory effects of
2.
Results and discussion
Chemistry
Compound
Tyrosinase
inhibition IC50, [lM] melanin synthesis
% Inhibition of
% Survival of B16F10
melanoma cell
Kojic acid derivative 2 was synthesized by joining two
pyrone rings by Horner-Emmons reaction of phospho-
nate with aldehyde components, both of which can be
derived from kojic acid as shown in Scheme 1. The C-5
hydroxyl group of 1 was selectively protected with the
reaction of p-methoxybenzyl chloride (PMB-Cl) to afford 3
in 81% yield [9]. The C-7 hydroxyl group of 3 was con-
verted to bromide 5 via mesylation of alcohol followed by
bromination of the resulting mesylate 4 with the reac-
tion of sodium bromide in DMF in 58% yield for two steps
[10]. Arbuzov reaction of 5 with trimethyl phosphite in
toluene at reflux temperature gave phosphonate 6 in
79% yield.
For the preparation of aldehyde 7, PMB-protected kojic
acid 3 was used again. Compound 3 was oxidized with
manganese oxide in chloroform to afford 7 in 75% yield.
The Horner-Emmons reaction of phosphonate 6 with
aldehyde 7 with NaH in DMF gave PMB-protected dimmer
8, which was subjected to hydrolysis condition by treat-
ment of trifluoroacetic acid (TFA) in CH2Cl2 to provide
kojic acid derivative 2 in 78% yield.
2
3.63
32.12 (at 12.5 lg)
19.53 (at 5 lg)
12.91 (at 2.5 lg)
16.37 (at 200 lg)
93.38 (at 12.5 lg)
96.59 (at 5 lg)
98.50 (at 2.5 lg)
93.96 (at 200 lg)
Kojic acid
30.61
nase inhibitory activity of 2 was about 8 times more
potent (IC50 = 3.63 lM) than that of kojic acid (IC50
=
30.61 lM). Compound 2 also exhibited superior melanin
synthesis inhibitory activity (19.53% inhibition at 5 lg) at
a nontoxic concentration compared to kojic acid (16.36%
inhibition at 200 lg) indicating that joining of two pyr-
one rings of kojic acid through a suitable linker can be
another strategy for identification of potent tyrosinase
inhibitiors. However, further structural modification of
2 is needed for improvement of pharmacokinetic proper-
ties, since the solubility of 2 was low in working solution
during assay procedures.
This research was supported by a research grant of Kyung Hee
University in 2004.
Biological activity
The resulting kojic acid derivative 2 was assayed on inhi-
bition of tyrosinase as shown in Table 1 and the activity
data of kojic acid were included as standards for compar-
ison. Compound 2 was also tested on the melanin synth-
esis using B16F10 melanoma cell to investigate the
effects of tyrosinase inhibitory activities on melanin pro-
duction. The cytotoxicities of 2 and kojic acid on mela-
noma cell were examined using MTT assay. The tyrosi-
Experimental
Chemistry
1H-NMR and 13C-NMR spectra were recorded on a Gemini Varian-
200 (200 and 50 MHz, respectively; Varian Inc., Palo Alto, CA,
USA). Analytical thin layer chromatographies (TLC) were carried
out by precoated silica gel (E. Merck Kiesegel 60F254 layer thick-
ness 0.25 mm; Merck, Darmstadt, Germany). Flash column chro-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 111–114
Tyrosinase Inhibitory Kojic Acid Derivative
113
matographies were performed with Merck Kiesegel 60 Art 9385
(230–400 mesh). All solvents used were purified according to
standard procedures.
red for 6 h. After cooling to r.t., the reaction mixture was filtered
through Celite 545. The filtrate was concentrated and diluted
with EtOAc. The insoluble solid was removed by filtration, again
through Celite 545. The filtrate was concentrated and solidified
with EtOAc and n-hexane to afford 7 as a light yellowish solid
(3.0 g, 75%). 1H-NMR (CDCl3) d 9.63 (s, 1H, –CHO), 7.66 (s, 1H, pyr-
one-H6), 7.31 (d, 2H, J = 8.51 Hz, Ph-H2,6), 6.97 (s, 1H, pyrone-H3),
6.89 (d, 2H, J = 8.51 Hz, Ph-H3,5), 5.05 (s, 2H, Ph–CH2 –), 3.80 (s,
3H, OCH3); 13C-NMR(CDCl3) d 184.3, 174.6, 160.3, 156.0, 149.4,
142.1, 130.1, 127.5, 120.3, 114.5, 72.2, 55.6.
5-(4-Methoxybenzyloxy)-2-hydroxymethyl-4H-pyran-4-
one 3
To a stirred solution of kojic acid (2, 4.75 g, 33.4 mmol) and
K2CO3 (9.68 g, 70.0 mol) in dry DMF (70 mL) was added PMB-Cl
(5.66 mL, 41.7 mmol), then the reaction mixture was heated
under refluxed for 1 h at 808C. The solvent was removed under
reduced pressure and the precipitate was filtered, washed with
water and ethyl acetate to afford 3 as a white solid (7.04 g, 81%).
1H-NMR (DMSO-d6) d 8.16 (s, 1H, pyrone-H6), 7.36 (d, 2H, J = 8.5 Hz,
Ph-H2,6), 6.96 (d, 2H, J = 8.5 Hz, Ph-H3,5), 6.33 (s, 1H, pyrone-H3),
5.70 (t, 1H, J = 5.8 Hz, –OH), 4.87 (s, 2H, Ph–CH2 –), 4.30 (s, 2H, J =
5.8 Hz, –CH2OH), 3.77 (s, 3H, OCH3).
1,2-trans-Bis(5-hydroxy-4H-pyran-4-one-2-yl)ethene 2
To a stirred solution of 6 (200 mg, 0.56 mmol) and NaH (45 mg,
1.12 mmol) in dry THF (35 mL) was added a solution of 7
(131.1 mg, 0.50 mmol) in THF (5 mL) dropwise under N2 atmo-
sphere at 08C. After stirring at r.t. for 1 h, the reaction mixture
was poured into water. The resulting precipitate was filtered
and washed with water and diethyl ether. The filtered cake was
collected and dried in vacuo to give PMB-protected kojic acid
derivative 8 as a white solid (226 mg, 83%), which was used in
the next step without further purification. To a stirred solution
of 8 (100 mg, 0.20 mmol) in CH2Cl2 (15 mL) was slowly added tri-
fluoracetic acid (0.09 mL, 1.20 mmol) at r.t. The reaction mixture
was stirred for 30 min and the solvent was removed by evapora-
tion. The resulting solid was washed with diethyl ether and
dried in vacuo to afford 2 as a yellow solid (48 mg, 94%). 1H-NMR
(DMSO-d6) d 8.11 (1H, s, pyrone-H2), 7.15 (1H, s, –CH=CH–), 6.72
(1H, s, pyrone-H5); 13C-NMR (DMSO-d6) d 174.2, 159.1, 146.7,
139.9, 126.1, 115.1.
(5-(4-Methoxybenzyloxy)-4-oxo-4H-pyran-2-yl)methyl
methanesulfonate 4
To a stirred solution of 3 (300 mg, 1.44 mmol) in CH2Cl2 (12 mL)
was added triethylamine (0.19 mL, 1.37 mmol) and methanesul-
fonyl chloride (0.09 mL, 1.19 mmol) at 08C. After stirring for 2 h
at room temperature (r.t.), the mixture was diluted with EtOAc
and washed with brine. The organic layer was dried by MgSO4,
concentrated, and solidified with ether and n-hexane to afford 4
1
as a white solid (312 mg, 80%). H-NMR (DMSO-d6) d 8.28 (s, 1H,
pyrone-H6), 7.35 (d, 2H, J = 8.5 Hz, Ph-H2,6), 6.95 (d, 2H, J = 8.5 Hz,
Ph-H3,5), 6.58 (s, 1H, pyrone-H3), 5.15 (s, 2H, –CH2OSO2 –), 4.87 (s,
2H, Ph–CH2 –), 3.76 (s, 3H, OCH3), 3.31 (s, 3H, –SO2CH3).
Biology
5-(4-Methoxybenzyloxy)-2-bromomethyl-4H-pyran-4-one
5
Mushroom tyrosinase inhibition assay
Tyrosinase activity was determined by the method described by
Tomita et al. [11] with slight modification and kojic acid was
used as a positive control. Briefly, to a 96-well plate was added
50 lL 0.1 M phosphate buffer (pH 6.8), 50 lL of L-tyrosine solu-
tion (0.3 mg/mL in water), 5 lL of tyrosinase (Sigma, 26103
units/mL in buffer; http://www.Sigma-Aldrich.com) and 40 lL of
water were mixed in a micro-tube and then 5 lL of the test sub-
stance were added (B). After incubation at 378C for 10 min, the
amount of dopa produced in the reaction mixture was measured
at 475 nm. The inhibitory activity of sample was expressed as
the concentration, which inhibits 50% of the enzyme activity
(IC50). The same solution without test substance (A) was also pre-
pared and the UV absorbance was measured at 475 nm. The %
inhibition was calculated using the formula [(A–B)/A]6100.
To a stirred solution of 4 (300 mg, 0.88 mmol) in DMF (15 mL)
was added NaBr (226 mg, 2.20 mmol) at r.t. After further stirring
for 50 min, the reaction mixture was poured into water and
extracted by EtOAc. The organic layer was dried over MgSO4, con-
centrated, and solidified with ether and n-hexane to afford 5 as
an ivory colored solid (194 mg, 72%). 1H-NMR (DMSO-d6) d 8.25 (s,
1H, pyrone-H6), 7.35 (d, 2H, J = 8.5 Hz, Ph-H2,6), 6.96 (d, 2H, J = 8.5
Hz, Ph-H3,5), 6.57 (s, 1H, pyron-H3), 4.86 (s, 2H, Ph–CH2 –), 4.55 (s,
2H, -CH2Br), 3.76 (s, 3H, OCH3).
Dimethyl(5-(4-methoxybenzyloxy)-4-oxo-4H-pyran-2-
yl)methylphosphonate 6
To a solution of 5 (300 mg, 0.92 mmol) in toluene (50 mL) was
added trimethyl phosphite (1.08 mL, 9.22 mmol) and the mix-
ture was heated at reflux. After 86 h, excess trimethyl phosphite
was removed by simple distillation and the resulting oily resi-
due was purified by flash column chromatography (CH2Cl2/
CH3OH = 20:1) to afford 6 as a white solid (258 mg, 79%). 1H-NMR
(DMSO-d6) d 8.18 (s, 1H, pyrone-H6), 7.33 (d, 2H, J = 8.6 Hz, Ph-
H2,6), 6.98 (d, 2H, J = 8.6 Hz, Ph-H3,5), 6.32 (s, 1H, pyron-H3), 4.85
(s, 2H, Ph–CH2 –), 3.76 (s, 3H, -OCH3), 3.65 and 3.70 (two s, each
3H, OCH3), 3.42 (d, 2H, J = 21.6 Hz, CH2PO–).
Cell culture
B16F10 mouse melanoma cells were purchased from the Ameri-
can Type Culture Collection. The cells were grown in DMEM
(Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal
bovine serum (FBS, Gibco BRL) and penicillin/streptomycin
(100 IU/mL and 100 lg/mL, respectively, Sigma). The cells were
maintained in a humidified incubator with 5% CO2 at 378C. All
tested compounds were prepared in DMSO.
Determination of melanin contents
5-Hydroxy-4-oxo-4H-pyran-2-carbaldehyde 7
Melanin contents were determined according to Hosoi et al. [12]
with modification and performed in triplicate at least twice. On
day 1, a total of 86104 cells were added to 60-mm plates, and
were incubated at 378C in 5% CO2 incubator. On day 2, each
A mixture of 3 (4.0 g, 15.3 mmol) and manganese (IV) oxide
(2.65 g, 30.6 mmol) suspended in CHCl3 (100 mL) was heated at
reflux for 12 h. An additional amount of manganese (IV) oxide
(2.65 g, 30.6 mmol) was treated and the mixture was further stir-
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Y. S. Lee et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 111–114
10 lL of test samples in DMSO were added to the plate, which
was then incubated at 378C for 72 h in a CO2 incubator. After
being washed with PBS, the cells were lysed with 1 mL of 1 N
NaOH and 200 lL portions of crude cell extract were transferred
to 96-well plates. Melanin content was determined at 405 nm.
Effect of the test samples on melanin content was expressed as
the percent inhibition of the value obtained in B16F10 mouse
melanoma cells cultured with DMSO alone (control).
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MTT assay was performed according to a micro-culture MTT
method [13]. Briefly, B16F10 mouse melanoma cell suspension
was poured into a 96-well plate (103 cells/well) and the cells were
allowed to completely adhere to the plate overnight. Then, each
test samples were added to the plate, which was then incubated
at 378C for 24 h in a CO2 incubator. After incubating, 20 lL of
MTT solution (2 mg/mL) was added to each well and incubated
for 4 h and then the supernatant was removed. The formazan
dye was solubilized by adding 150 lL DMSO to each well, fol-
lowed by gentle shaking. The optical density of the resulting
supernatant was measured at 540 nm using an ELISA reader
(Molecular Devices Corporation, Sunnyvale, CA, USA).
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