Kojyl thioether derivatives having both tyrosinase inhibitory and anti-inflammatory properties

Article abstract of DOI:10.1016/j.bmcl.2010.09.042

This study was conducted to examine the tyrosinase inhibitory and anti-inflammatory activities of kojic acid derivatives. A series of kojic acid derivatives containing thioether, sulfoxide, and sulfone linkages were synthesized. In the tyrosinase assay, k

Full text of DOI:10.1016/j.bmcl.2010.09.042

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6569–6571
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kojyl thioether derivatives having both tyrosinase inhibitory
and anti-inflammatory properties
Ho Sik Rho a, , Soo Mi Ahn a, Dae Sung Yoo c, Myung Kyoo Kim b, Dong Ha Cho c, Jae Youl Cho c,
⇑
⇑
a R & D Center, AmorePacific Corporation, Yongin 446-729, Republic of Korea
b Samkyung Costech Co., Ltd, Suwon 443-734, Republic of Korea
c College of Biomedical Science, Kangwon National University, Chuncheon 200-701, 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 30 June 2010
Revised 4 September 2010
Accepted 8 September 2010
Available online 21 September 2010
This study was conducted to examine the tyrosinase inhibitory and anti-inflammatory activities of kojic
acid derivatives. A series of kojic acid derivatives containing thioether, sulfoxide, and sulfone linkages
were synthesized. In the tyrosinase assay, kojyl thioether derivatives containing appropriate lipophilic
alkyl chains (pentane, hexane, and cyclohexane) showed potent inhibitory activity. However, sulfoxides
and sulfones exhibited decreased activity. Similar experimental results were obtained with inhibitory
activities of NO production being induced by LPS. The presence of thioether linkage and appropriated
lipophilic acid moiety was critical for the tyrosinase inhibitory and anti-inflammatory activities.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Kojyl thioether
Derivatives
Tyrosinase
Anti-inflammatory property
It is well known that 4-pyrones and their derivatives are
present in many natural materials and are biologically active.1
Kojic acid, 5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (1),
which is found in various traditional Japanese foods, is produced
from carbohydrate sources in an aerobic process by a variety of
microorganisms. Kojic acid has chelating activity2 and inhibits
tyrosinase,3 and polyphenoloxidase (PPO).4 Based on these effects,
kojic acid has been used as a depigmenting agent for cosmetics and
as a food additive to prevent enzymatic browning due to PPO. Kojic
acid has also been shown to scavenge free radicals and to prevent
photodamage.5 However, only a few studies on kojic acid and its
derivatives as NO inhibitors have been conducted. The biological
their structural importance for two biological activities. The syn-
thetic pathways are shown in Schemes 1 and 2.
Kojic acid derivatives (4a–h) were synthesized by the conden-
sation of kojyl chloride 3 with potassium salts of thiols. Kojic acid
1 was reacted with thionyl chloride to afford kojyl chloride 3. Kojyl
chloride 3 was reacted with potassium salts of thiols in DMF to
afford the corresponding thioether derivatives (4a–h). The thioe-
ther derivatives (4d, 4e, and 4h) were then reacted with MCPBA
(m-chloroperbenzoic acid) in methylene chloride to produce
sulfoxide derivatives (5d, 5e, and 5h). Finally, sulfone derivatives
(6d, 6e, and 6h) were obtained by the treatment of thioether deriv-
atives (4d, 4e, and 4h) with oxone in a mixture of MeOH/H2O.
The inhibitory activities of kojic acid derivatives against mush-
room tyrosinase were initially investigated and compared with
that of kojic acid. The results are shown in Table 1.
activities of kojic acid are due to its c-pyranone structure having
an enolic hydroxyl group. Thus, various kojic acid derivatives that,
are modified at the 2-position, have been developed to enhance
biological activities.6 Recently, we synthesized dimeric kojic acid
derivatives containing various chemical linkages including ester,
amide, and thioether. Among them, dithioether derivative (2)
exhibited the most potent inhibitory activities of tyrosinase7 and
NO production in LPS activated macrophages8 (Fig. 1).
Compound 4a, which contains an ethyl group, showed good
inhibitory activity (IC50 = 2.60 lM). Increases in the activities were
detected along with increasing chain length to compounds such as
n-propyl, n-butyl, and n-pentyl (Table 1, 4b–d). Compound 4d, 2-
(pentylthiomethyl)-5-hydroxy-4H-pyran-4-one, showed potent
We further investigated the structure–activity relationship of
kojic acid derivatives9 possessing sulfur linkages on the inhibition
of tyrosinase10 and NO production.11 In the present study, we syn-
thesized kojyl thioethers, sulfoxides, and sulfones and analyzed
inhibitory activity (IC50 = 0.097 lM). However, compounds with
greater chain lengths such as n-hexyl, n-heptyl, and n-octyl groups
had negative influence on inhibitory activities. The activity of
compound 4e containing a n-hexyl group (IC50 = 0.19 lM) was
lower than that of compound 4d containing a n-pentyl group.
Among the tested compounds, compound 4h, 2-(cyclohexylthiom-
ethyl)-5-hydroxy-4H-pyran-4-one, showed the most potent activ-
⇑
Corresponding authors. Tel.: +82 31 280 5805; fax: +82 31 281 8397 (H.S.R.).
E-mail addresses: thiocarbon@freechal.com (H.S. Rho), jaecho@kangwon.ac.kr
ity (IC50 = 0.087
lM); however, its IC50 was about 1/1100 that of
kojic acid (IC50 = 97.38
lM). To identify the structural importance
(J.Y. Cho).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.042

6570
H. S. Rho et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6569–6571
O
O
O
O
O
O
O
O
OH
S
S
S
HO
HO
OH HO
, Kojic acid
1
, Kojyl thioether derivative
2
4d
Figure 1. Structures of kojic acid and thioether derivatives.
sulfones had an effect on conformational changes, which caused
decreased inhibitory activities (Fig. 2).
After evaluating the tyrosinase inhibitory activity, we investi-
gated the anti-inflammatory activities of kojic acid derivatives
against NO production induced by LPS in macrophages and cyto-
toxicity.12 The results are shown in Table 2.
The inhibitory activity of NO production was found to be very
similar to inhibitory results of tyrosinase. Specifically, compounds
4d, 4e, and 4h showed inhibitory activities with IC50 values of
O
O
O
O
O
R
a
b
OH
Cl
S
HO
HO
HO
O
1
3
4a R = Ethyl
4b R = n-Propyl
4c R = n-Butyl
4d R = n-Pentyl
R = n-Hexyl
R = n-Heptyl
R = n-Octyl
4e
4f
4g
4h
61.98, 25.75, and 56.97 lM, respectively. However, sulfoxide deriv-
atives (5d, 5e, and 5h) and sulfone derivatives (6d, 6e, and 6h)
showed no inhibitory activity. Taken together these results suggest
that sulfide linkage and compounds with appropriate hydrophobic
chain lengths such as n-pentyl, n-heptyl, and cyclohexyl were
important for the inhibition of tyrosinase and NO production in
the structure of kojic acid derivatives.
R = Cyclohexyl
Scheme 1. Reagents and conditions: (a) SOCl2, DMF, rt; (b) potassium salt of thiols,
DMF, rt.
O
O
R
S
O
HO
a
O
O
R
5d R = n-Pentyl
5e R = n-Hexyl
5h R = Cyclohexyl
S
HO
b
O
R
S
O
O
4d R = n-Pentyl
4e R = n-Hexyl
4h R = Cyclohexyl
HO
O
6d R = n-Pentyl
6e R = n-Hexyl
6h R = Cyclohexyl
Scheme 2. Reagents and conditions: (a) MCPBA, methylene chloride, rt; (b) oxone,
MeOH/H2O, rt.
Figure 2. Energy minimized structures of 4e, 5e, and 6e. Calculations were
conducted using Spartan ‘04’ for Windows. The global minimum-energy structures
were obtained by molecular-mechanics computations and were further refined by
DFT computation (B3LYP at the 6-31G* level).
Table 1
Inhibitory activities of kojic acid derivatives on mushroom tyrosinase
a
a
Compounds
IC50
(lM)
Compounds
IC50 (lM)
Table 2
4a
4b
4c
4d
4e
4f
2.60
1.93
1.48
0.097
0.19
2.65
46.18
0.087
5d
5e
5h
6d
6e
6h
Kojic acid
79.87
73.75
79.03
76.60
49.15
69.01
87.38
NO inhibitory activities of kojic acid derivatives
a
Compounds
Inhibitory activity [IC50
(l
M)]
NO
Cytotoxicity
4a
4b
4c
4d
4e
4f
4g
4h
5d
5e
5h
6d
6e
6h
Kojic acid
>100
>100
>100
>100
>100
>100
>100
>100
98.30
>100
>100
>100
>100
>100
>100
>100
>100
>100
4g
4h
61.98
25.75
>100
>100
56.87
a
Values were determined from the logarithmic concentration–inhibition curves
and those presented are the mean values of the results of three experiments.
>100
>100
>100
>100
>100
>100
of sulfide linkages, we oxidized the sulfur functional group of the
compounds (4d, 4e, and 4h) and evaluated their inhibitory activi-
ties. Interestingly, sulfoxide derivatives (5d, 5e, and 5h) and
sulfone derivatives (6d, 6e, and 6h) exhibited decreased activity.
In tyrosinase assays, the sulfide linkage is a critical factor for inhi-
bition activity. Without the sulfide group, the kojic acid moiety
may not bind tightly to the active site or to other essential parts
of tyrosinase. The additional oxygen groups in the sulfoxides and
89.41
a
Values were determined from logarithmic concentration–inhibition curves and
are the means of three experiments.

H. S. Rho et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6569–6571
6571
0.90 (m, 6H). 13C NMR (125 MHz, DMSO-d6): d 173.7, 165.3, 145.6, 139.6, 111.9,
43.1, 32.9, 30.8, 25.31, 25.21. FABMS: (m/e) 241 [M+H]+. Compound 5e: 1H NMR
(300 MHz, DMSO-d6): d 9.23 (s, 1H), 8.08 (s, 1H), 6.39 (s, 1H), 4.17 (d, 1H,
J = 13.2 Hz), 3.95 (d, 1H, J = 13.2 Hz), 2.83 (m, 2H), 1.65 (m, 2H), 1.38–1.27 (m,
6H), 0.86 (t, 3H, J = 6.3 Hz). 13C NMR (125 MHz, DMSO-d6): d 173.2, 158.9,
145.9, 140.1, 115.2, 54.0, 51.1, 30.7, 27.6, 21.89, 21.82, 13.8. FABMS: (m/e) 259
[M+H]+. Compound 5h: 1H NMR (300 MHz, DMSO-d6): d 9.21 (s, 1H), 8.09 (s,
1H), 6.41 (s, 1H), 4.13 (d, 1H, J = 13.2 Hz), 3.96 (d, 1H, J = 13.2 Hz), 2.72 (m, 1H),
1.98–0.72 (m, 10H). 13C NMR (125 MHz, DMSO-d6): d 173.2, 159.4, 145.9,
140.1, 115.2, 57.8, 51.7, 26.0, 25.0, 24.8, 24.5, 23.6. FABMS: (m/e) 257 [M+H]+.
Compound 6e: 1H NMR (300 MHz, DMSO-d6): d 9.32 (s, 1H), 8.11 (s, 1H), 6.49 (s,
1H), 4.58 (s, 2H), 3.26 (m, 2H), 1.69 (m, 2H), 1.50–1.22 (m, 6H), 0.86 (t, 3H,
J = 6.3 Hz). 13C NMR (125 MHz, DMSO-d6): d 173.3, 156.1, 146.1, 140.4, 116.2,
55.7, 52.4, 30.6, 27.2, 21.7, 21.0, 13.7. FABMS: (m/e) 275 [M+H]+. Compound 6h:
1H NMR (300 MHz, DMSO-d6): d 9.23 (s, 1H), 8.07 (s, 1H), 6.43 (s, 1H), 4.51 (s,
2H), 3.09 (m, 1H), 2.02 (m, 2H), 1.80 (m, 2H), 1.60–0.95 (m, 6H). 13C NMR
(125 MHz, DMSO-d6): d 173.3, 156.1, 146.1, 140.4, 116.4, 60.5, 53.0, 24.64,
24.39, 24.27. FABMS: (m/e) 273 [M+H]+.
In conclusion, we synthesized a series of kojyl thioether deriva-
tives (4a–h), sulfoxide derivatives (5d, 5e, and 5h), and sulfone
derivatives (6d, 6e, and 6h). Compound 4h, 2-(cyclohexylthiometh-
yl)-5-hydroxy-4H-pyran-4-one, showed the most potent activity in
the tyrosinase assay (IC50 = 0.087
omethyl)-5-hydroxy-4H-pyran-4-one, showed the most potent
activity in the NO assay (IC50 = 25.75 M). Analysis of the structure–
lM). Compound 4e, 2-(hexylthi-
l
activity relationship revealed that sulfide linkage and appropriate
hydrophobic moieties such as n-pentyl, n-heptyl, and cyclohexyl are
important for higher inhibitory activity in both tyrosinase and NO
production assays.
References and notes
10. Measurements of mushroom tyrosinase activity: mushroom tyrosinase,
1. (a) Gerwick, W. H. J. Nat. Prod. 1989, 52, 252; (b) Yamamura, S.; Nishiyama, S.
Bull. Chem. Soc. Jpn. 1997, 70, 2025; (c) Rossi, M. H.; Yoshida, M.; Maia, G. S.
Phytochemistry 1997, 45, 1263; (d) Bransova, J.; Brtko, J.; Uher, M.; Novotny, L.
Int. J. Biochem. Cell Biol. 1995, 27, 701.
2. Yuen, V. G.; Caravan, P.; Gelmini, L.; Glover, N.; Mcneill, J. H.; Setyawati, I. A.;
Zhou, Y.; Orvig, C. J. Inorg. Biochem. 1997, 73, 109.
L
-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 (2100 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
l
l
l
L
a
microplate reader (Bio-Rad 3550, Richmond, 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).
3. Ohyama, Y.; Mishima, Y. Fragrance J. 1990, 6, 53.
4. Chen, J. S.; Wei, C. I.; Rolle, R. S.; Otwell, W. S.; Balaban, M. O.; Marshall, M. R. J.
Agric. Food Chem. 1991, 39, 1396.
11. Measurements of NO production: RAW264.7 cells (1 Â 106 cells/ml) were
5. Mitani, H.; Koshiishi, I.; Sumita, T.; Imanari, T. Eur. J. Pharmacol. 2001, 411, 169.
6. (a) Kobayashi, Y.; Kayahara, H.; Tasada, K.; Nakamura, T.; Tanaka, H. Biosci.
Biotechnol. Biochem. 1995, 59, 1745; (b) Kobayashi, Y.; Kayahara, H.; Tadasa, K.;
Tanaka, H. Bioorg. Med. Chem. Lett. 1996, 6, 1303; (c) Kadokawa, J.; Nishikura, T.;
Muraoka, R.; Tagaya, H.; Terada, Y.; Fukuoka, N. Synth. Commun. 2003, 33, 1081;
(d) Lee, Y. S.; Park, J. H.; Kim, M. H.; Seo, S. H.; Kim, H. J. Arch. Pharm. Chem. Life
Sci. 2006, 339, 111; (e) Rho, H. S.; Baek, H. S.; You, J. W.; Kim, S.; Lee, J. Y.; Kim,
D. H.; Chang, I. S. Bull. Korean Chem. Soc. 2007, 28, 471.
preincubated with kojic acid derivatives for 30 min and continuously activated
with LPS (1
l
g/ml) for 24 h. Nitrite in culture supernatants was measured by
l of Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthyl]-
l samples of
the medium for 10 min at room temperature. OD at 570 nm (OD570) was
measured using Spectramax 250 microplate reader (Molecular Devices,
adding 100
l
ethylenediamine dihydrochloride in 5% phosphoric acid) to 100
l
a
Sunnyvale, CA, USA). A standard curve of NO was made with sodium nitrite.
12. MTT assay: After the preincubation of RAW264.7 cells (1 Â 106 cells/ml) for
7. Rho, H. S.; Baek, H. S.; Ahn, S. M.; Kim, D. H.; Chang, I. S. Bull. Korean Chem. Soc.
2008, 29, 1569.
18 h, kojyl thioether derivatives (0–100 lM) were added to the cells and
incubated for 24 h. The cytotoxic effect of kojyl thioether derivatives was then
evaluated by a conventional MTT assay. At 3 h prior to culture termination,
10 ll of the MTT solution (5 mg/ml in a phosphate buffered-saline, pH 7.4)
were added and the cells were continuously cultured until termination. The
incubation was halted by the addition of 15% sodium dodecyl sulfate into each
8. Yoo, D. S.; Lee, J.; Choi, S. S.; Rho, H. S.; Cho, D. H.; Shin, W. C.; Cho, J. Y.
Pharmazie 2010, 65, 261.
9. The data of selected compounds: compound 4e: 1H NMR (300 MHz, DMSO-d6):
d 9.13 (s, 1H), 8.05 (s, 1H), 6.37 (s, 1H), 3.63 (s, 2H), 2.53 (m, 2H), 1.51 (m, 2H),
1.24 (m, 6H), 0.87 (t, 3H, J = 6.3 Hz). 13C NMR (125 MHz, DMSO-d6): d 173.6,
164.8, 145.6, 139.6, 112.0, 32.3, 31.2, 30.7, 28.6, 27.7, 21.9, 13.8. FABMS: (m/e)
243 [M+H]+. Compound 4h: 1H NMR (300 MHz, DMSO-d6): d 9.12 (s, 1H), 8.04
(s, 1H), 6.39 (s, 1H), 3.67 (s, 2H), 2.69 (m, 1H), 1.90 (m, 2H), 1.65 (m, 2H), 1.51–
well, solubilizing formazan. The absorbance at 570 nm (OD570–630
measured by a Spectramax 250 microplate reader.
) was