Synthesis of new glycyrrhetinic acid (GA) derivatives and their effects on tyrosinase activity

Article abstract of DOI:10.1016/j.bmc.2003.09.046

To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in the C-11 position, and the carboxylic function at the C-30 position was kept intact, reduced to an alcohol, or transformed to an aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30 carboxylic acid position of glycyrrhetinic acid. In subsequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC₅₀ value of 50 μM after 2 h reaction. IC₅₀ values of derivatives 4 and 16 were 120 and 170 μM, respectively. Further kinetic data indicated that these derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and activity suggest that GA derivative 5 might be a useful candidate for skin whitening. copy; 2003 Published by Elsevier Ltd.

Full text of DOI:10.1016/j.bmc.2003.09.046

Bioorganic & Medicinal Chemistry 11 (2003) 5345–5352
Synthesis of New Glycyrrhetinic Acid (GA) Derivatives and Their
Effects on Tyrosinase Activity
Soo-Jong Um,a,b Myoung-Soon Park,b Si-Ho Park,b Hye-Sook Han,b Youn-Ja Kwonb
and Hong-Sig Sinb,*
aDepartment of Bioscience & Biotechnology/Institute of Bioscience, Sejong University, Seoul 143-747, South Korea
bChebigen Inc., 305-B, Chungmugwan, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, South Korea
Received 5 July 2003; revised 23 September 2003; accepted 24 September 2003
Abstract—To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in
the C-11 position, and the carboxylic function at the C-30position was kept intact, reduced to an alcohol, or transformed to an
aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino
piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30carboxylic acid position of glycyrrhetinic acid. In sub-
sequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited
tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC50 value of 50 mM
after 2 h reaction. IC50 values of derivatives 4 and 16 were 120and 170 mM, respectively. Further kinetic data indicated that these
derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was
used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much
more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and
activity suggest that GA derivative 5 might be a useful candidate for skin whitening.
# 2003 Published by Elsevier Ltd.
Introduction
representative tyrosinase-suppressive melanogenic inhi-
bitors.10,11 Although these substances have long been
used as cosmetic skin-whitening agents, their instability
limits their broad usage. To increase stability, derivatives
of these compounds have recently been developed.12
Glycyrrhizin (GR), a triterpenoid saponin found in the
roots of licorice (Glycyrrhiza glabra), is composed of
one molecule of glycyrrhetinic acid (GA) and two
molecules of glucuronic acid. The biological activities of
GR are associated with 18b-GA, and include anti-
inflammatory,1 anti-viral,2 anti-allergic,3 and anti-tumor
activities.4,5 Moreover, GR and GA has been shown to
modulate melanogenesis in B16 melanoma cells.6
Stearyl glycyrrhetinate (SG), a derivative of GA, has
also been implicated in skin whitening, which has been
used as a sun care and sunscreen agent.7
In this study, we synthesized derivatives (3, 4, 5, 10, 13,
14, 15, and 16) starting from GA, another natural com-
pound, and compared their effects on tyrosinase activity
with those of vitamin C and kojic acid. In addition, we
described the structure and function relationship of the
GA derivatives.
Melanin synthesis, the main process involved in skin
pigmentation, is regulated largely by the melanogenic
enzyme tyrosinase.8 This is a bifunctional enzyme that
catalyzes the hydroxylation of tyrosine to DOPA and
promotes the oxidation of DOPA to DOPA-quinone.9
As natural compounds, vitamin C and kojic acid are
Results and Discussion
Chemistry
Although glycyrrhetinic acid (GA, 1) and stearyl gly-
cyrrhetinate (SG, 2) have been reported to have inhibi-
tory effects on melanogenesis, our initial assays indicated
that these compounds were not or weakly inhibitory on
mushroom tyrosinase activity. To increase inhibitory
potential of GA, we synthesized its derivatives for which
*Corresponding author. Tel.:+82-2-465-1691; e-mail: hssin@cheni
gen.com
0968-0896/$ - see front matter # 2003 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2003.09.046

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S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
drous THF readily afforded the amine 8 in 96% yields.
Subsequent coupling of GA and 8 with DCC/HOBt/
NMM followed by deprotection of N-Cbz compound
with 10% Pd/C and H2 provided glycyrrhetin amide 5
in 76% overall yield.
Conversion of GA 1 to the aldehyde 13 was achieved via
the diol 10, as shown in Scheme 3. GA was first reduced
18
the triol 9 with NaAlH2(OCH2CH2OCH3)2 in THF
and then catalytically hydrogenated with 10% Pd/C to
give 11-deoxo diol 10 in 60% yields. To selectively oxi-
dize the C-30position, the primary alcohol of 10 was
protected with TBDMSCl19 followed by acetylation of
the hydroxyl group on the C-3 position with AcCl/Py/
DMAP, and desilylation with TBAF gave 3-acetyl gly-
cyrrhetinol 11 in 62% yields. Subsequent oxidation20 of
11 was performed with PCC in CH2Cl2 to the aldehyde
12 in 85% yields, which was then hydrolyzed in the
presence of K2CO3/MeOH to the desired derivative 13
in 87% yields. The aldehyde 12 was also oxidized with
KMnO4 according to Masamune oxidation21 and
hydrolyzed under basic conditions to give 11-deoxo
glycyrrhetinic acid 14 in 87% yields. This compound
was then coupled with 4-fluorobenzylbromide using
K2CO3 as a reagent, to give fluoroglycyrrhetinate 15 in
92% yields. Subsequent coupling of 14 and 8 with DCC/
HOBt/NMM followed by deprotection of N-Cbz com-
pound with 10% Pd/C and H2 provided glycyrrhetin
amide 16 in 42% overall yield.
Scheme 1. Structures of glycyrrhetinic acid (GA, 1) and its derivatives
(2–5, 10, 13–16).
chemical modifications were mainly directed on ring C,
and on the carboxylic function at position 30( Scheme 1).
Our strategy for the synthesis of GA derivatives 3–5 is
illustrated in Scheme 2. The O-alkylation13 of GA and
4-fluorobenzyl bromide with K2CO3 (3 equiv) in DMF
at 80 ꢀC for 3 h gave fluorobenzyl glycyrrhetinate 3 in
95% yield. In addition, GA was coupled with 4-amino-
1-benzylpiperidine using EDCI/DMAP as a reagent,14
to prepare benzylpiperidine glycyrrhetin amide 4 in 40%
yield. Following our investigation of functionalization
at the piperidine ring, we synthesized compound 5 by
introducing an l-proline into the tert-N of synthetic 4.
Conversion of 4 to its derivative 5 did not proceed by
straightforward reduction and subsequent coupling.
The preparation of intermediate 8 in four steps started
from 4-amino-1-benzylpiperidine 6. Thus, the protec-
tion of 6 with 1 M NaOH and (Boc)2O afforded N-Boc
compound in quantitative yield. Subsequent debenzyla-
tion15 of its crude product with 10% Pd/C and H2 fol-
lowed by coupling of N-Cbz proline with DCC/HOBt/
NMM16 provided the Boc-Cbz amide 7 in 82% overall
yield. Further treatment with HCl etherate17 in anhy-
Effects on tyrosinase activity
The inhibition of our synthetic GA derivatives on
mushroom tyrosinase was initially investigated and
compared with those of vitamin C and kojic acid. As
shown in Figure 1, the tyrosinase activity, determined
using l-DOPA as a substrate, was not much changed by
incubating with 100 mM GA or its derivatives for 10
min, whereas was severely inhibited by adding same
concentrations of vitamin C or kojic acid. However,
reactions for 120min reversed the inhibition pattern.
Scheme 2. Synthesis of GA derivatives 3–5 and intermediates 6–8: (a) 4-Fluorobenzyl bromide, K2CO3, DMF, 80ꢀC, 3 h; (b) 4-Amino benzylpiperidine,
EDCI, DMAP, DMF, overnight; (c) 8, DCC, HOBt, NMM, CH2Cl2, rt; 4 h; (d) 10% Pd/C, H2, MeOH, 1 atm, rt, 3 h; (e) (Boc)2O, 1 M NaOH, t-BuOH,
rt, overnight; (f) 10% Pd/C, H2, MeOH, 1 atm, rt, overnight; (g) N-Cbz-proline, DCC, HOBt, NMM, CH2Cl2, rt, 4 h; (h) HCl etherate, THF, 0 ꢀC, 2 h.

S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
5347
Scheme 3. Synthesis of GA derivatives 10 and 13–16: (a) NaAlH2(OCH2 CH2OCH3)2, THF, rt, 4 h; (b) 10% Pd/C, H2, EtOH, 1 atm, rt, overnight;
(c) TBDMCl, imidazole, DMF, rt, 2 h; (d) AcOCl, Py., DMAP, CH2Cl2, 0 ꢀC, rt, 4 h; (e) TBAF, THF, 0 ꢀC, rt, overnight; (f) PCC, CH2Cl2, 0 ꢀC, rt,
2 h; (g) KMnO4, 1.25 M NaH2PO4, 0 ꢀC, rt, 2 h; (h) K2CO3, MeOH, 24 h; (i) 4-Fluorobenzylbromide, K2CO3, DMF, 80 ꢀC, 3 h; (j) 14, DCC, HOBt,
NMM, CH2Cl2, rt; 4 h; (k) 10% Pd/C, H2, MeOH, 1 atm, rt, 3 h.
Although GA and some derivatives (2, 3, 10, 13, 14, and
15) were still inactive, other GA derivatives (4, 5, and
16) strongly inhibited tyrosinase activity, whereas vita-
min C and kojic acid were no longer inhibitory. Inter-
estingly, kojic acid increased tyrosinase activity, which
will be discussed late.
together, both piperidine and ketone groups are related
to inhibitory function against tyrosinase.
In subsequent experiments, we investigated the effects of
reaction time on the tyrosinase activity. For this pur-
pose, we incubated mushroom tyrosinase with 100 mM
of GA derivatives 4, 5, 16, vitamin C, and kojic acid for
different time points. As shown in Figure 2, the inhibi-
tory potentials of GA derivatives increased gradually,
whereas those of vitamin C and kojic acid were highest
at early time points (20and 10min, respectively), but
afterwards became sharply decreased with time. To rule
out any possibility that the inhibition may result from
quenching the absorbance generated by a reaction
between tyrosinase and L-DOPA, similar reactions were
performed in the absence of tyrosinase. Although data
are not shown, no changes in absorbance were detected
at 475 nm, implying that the observed inhibition is spe-
cific for tyrosinase. Therefore, these results suggest that
our GA derivatives could be nonclassical and slow-
binding inhibitors, which are different from a classical
and fast-binding inhibitor like vitamin C and kojic acid.
After the kinetics on the slow-binding inhibition of
enzyme reaction were well-defined,22 a similar mode of
inhibition was also found by other groups using tyr-
osinase and its inhibitors such as m-coumaric acid and
l-mimosine.23,24 However, our experimental conditions
cannot exclude the possibility that such inhibition may
result from the secondary reaction of the product by our
GA derivatives. Of interest, kojic acid appeared to
The inhibitory potentials of GA derivatives 4, 5, 16 were
further determined by measuring their IC50 values, the
concentrations showing 50% inhibition. The IC50 values
of GA derivatives, vitamin C, and kojic acid at different
reaction times are summarized in Table 1. At 10min,
only vitamin C and kojic acid showed strong, dose-
dependent suppressive effects on tyrosinase activity. At
60min, the inhibitory potentials of vitamin C and kojic
acid were markedly reduced, whereas those of GA deri-
vatives began to become evident. At 120min, the pat-
tern of inhibition was completely reversed. Moreover, at
reaction times longer than 2 h, only GA derivatives
showed inhibitory effects (data not shown). Why are
only derivatives 4, 5 and 16 active and derivative 5 the
most active? Structurally, derivative 4 contains benzyl-
piperidine linked to GA via amide bond. Other two
compounds 5 and 16 also contain piperidines that cou-
pled to l-proline instead of benzyl group. Therefore,
piperidine appears to be important for the inhibition
activity. The difference between 5 and 16 is present in
the ring C, where compound 5 contains ketone. Since
compound 5 is more active than 16, a ketone in the ring
C may also contribute on the increased activity. Taken

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S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
Table 1. IC50 values of GA derivatives on tyrosinase activity
Derivatives
IC50 (mM)a
10min
60min
120min
Glycyrrhetinic acid (GA)
GA derivative 4
GA derivative 5
GA derivative 16
Vitamin C
>500b
>500
>500
>500
>500
100
80
>500
70
35
100
>500
>500
150
0
3030
60
Kojic acid
>500
aIC50 values were determined by interpolation of the dose–response
curves obtained by DOPA-oxidase assays using 5 mM of l-DOPA at
three different time points as described in the Experimental.
b>500 means undetectable in our concentration range tested.
Figure 1. Effect of GA derivatives on tyrosinase activity. To measure
tyrosinase activity, the DOPA-oxidase assay was employed in the
presence of 1 mM of L-DOPA as described in the Experimental.
Assays were performed with 100 mM of inhibitors and GA derivatives
at two different time points: (A) 10min; (B) 120min. Relative activity
(%) means percentage of relative absorbance at 475 nm in the presence
versus in the absence of inhibitor. Symbols indicated as follows: C,
control; VC, vitamin C; KA, kojic acid; 1, Stearyl glycyrrhetinate
(SG); 2, glycyrrhetinic acid (GA); 3–16; GA derivatives.
increase tyrosinase activity after 30min reaction. Since
this effect is specific for tyrosinase as described above,
we assume that DOPA-quinone and/or DOPA-chrome,
reaction products of tyrosinase may cooperate with
kojic acid, which leads to shift equilibrium to the acti-
vation of tyrosinase under an excess condition of
substrate, l-DOPA.
Interestingly, our data appear to be opposite to what
was reported by other group.25 In this report, they sug-
gested that glycyrrhizin (GR), composed of one mole-
cule of GA and two molecules of glucuronic acid,
increases the cellular melanin content and tyrosinase
activity. However, they found that GA had no or
weakly inhibited melanogenesis, suggesting that glu-
curonic acid may have a certain role in stimulating
melanogenesis. In addition, another group has reported
that glabridin, a main constituent of the hydrophobic
fraction of licorice extracts, shows inhibitory effects on
melanogenesis.26 In our study, GR was not analyzed
and GA had no clear effect on tyrosinase activity as
reported. Therefore, it is still possible that some GA
derivatives may inhibit tyrosinase activity. As indicated
in our study, GA derivatives with piperidine and ketone
groups showed the inhibitory activity against tyrosinase.
Figure 2. Comparison of the time-dependent responses of GA deriva-
tives (4, 5, and 16), vitamin C, and kojic acid. The DOPA-oxidase
assay was employed in the presence of 5 mM of l-DOPA as described
in the Experimental. Assays were performed with 100 mM of GA
derivatives (A), and vitamin C and kojic acid (B) by increasing reaction
time. Data are expressed as a percentage (%) inhibition that means
level of inhibition in the presence of inhibitor.

S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
5349
Conclusions
to room temperature. The crude was treated with H2O
(200 mL), the aqueous layer was extracted with EtOAc
(2ꢁ20mL). The joined organic extracts were washed
with H2O (100 mL), brine (50 mL), dried (Na2SO4) and
evaporated. Purification by flash chromatography (n-
hexane–EtOAc=1:3) afforded 3 as a white powder
(0.35 g, 95%). Mp 142–144 ꢀC; 1H NMR (400 MHz,
CDCl3) : d 7.36 (m, 2H), 7.07 (m, 2H), 5.57 (s, 1H), 5.11
(dd, J=12.30, 3.87 Hz, 2H), 3.21 (m, 1H), 2.80 (m, 1H),
2.33 (s, 1H), 2.18 (m, 4H), 1.65 (m, 6H), 1.60(s, 3H),
1.36 (s, 3H), 1.34 (m, 8H), 1.15 (s, 3H), 1.12 (s, 3H), 1.10
(s, 3H), 1.01 (m, 2H), 0.81 (s, 3H), 0.74 (s, 3H); 13C
NMR (100 MHz, CDCl3): d 200.12, 176.15, 169.00,
132.00, 131.96, 130.29, 130.21, 128.50, 115.61, 115.40,
78.69, 65.45, 61.77, 60.37, 54.91, 48.19, 45.32, 43.93,
43.14, 41.01, 39.11, 37.60, 37.04, 32.72, 31.73, 31.12,
28.41, 28.21, 28.07, 27.27, 26.41, 23.33, 18.63, 17.45,
16.35, 15.59,14.18; MS: m/z (100%)=601 ([M+Na]+,
100), 579 (M+, 13), 269 (13), 105 (8) ; HREIMS m/z
578.3773 (calcd for C37H51FO4 578.3771).
In this study, we synthesized some derivatives glycyr-
rhetinic acid (GA) and determined their anti-tyrosinase
activities. Of the GA derivatives synthesized, com-
pounds 4, 5, and 16 inhibited l-DOPA xidation of
mushroom tyrosinase, whereas its parental GA and
other derivatives including commercially available SG
were not active in inhibition. Our analysis on structure
and function relationship suggests that both piperidine
and ketone groups are critical for the inhibitory activity
of GA derivatives against tyrosinase. Of note, the inhi-
bition was time-dependent, suggesting that these com-
pounds could be slow-binding inhibitors as reported for
other compounds like m-coumaric acid and l-mimo-
sine. In our study, vitamin C and kojic acid, broadly
used whitening agents in cosmetic formulation, strongly
inhibited the tyrosinase activity for 20and 10min,
respectively. Afterwards, their activities sharply
decreased with time, indicating that these compounds
are very unstable during prolonged reaction. Among
three GA derivatives, compound 5 was most active with
IC50 of 35 mM at 120-min reaction. In addition, time-
course experiments indicated that compound 5 could be
much more stable than vitamin C and kojic acid. Taken
together, GA derivative 5 might be a useful candidate as
a skin-whitening agent for cosmetic use, although its
cytotoxicity remains to be determined in normal skin
cells and in animals.
10-Hydroxy - 2,4a,6a,6b,9,9,12a - heptamethyl - 13 - oxo -
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10, 11,12,12a,12b,13,14b-ei-
cosahydro-picene-2-carboxylic acid (1-benzyl-piperidin-4-
yl)-amide (4). A mixture of 18b-glycyrrhetinic acid
(0.30 g, 0.64 mmol), EDCI (0.37 g, 1.91 mmol) and
DMAP (0.08 g, 0.64 mmol) was added DMF (15 mL).
The reaction mixture was added 4-aminobenzylpiper-
idine (0.14 g, 0.70 mmol) and stirred for overnight at
room temperature. The crude was treated with H2O
(200 mL), the aqueous layer was extracted with EtOAc
(2ꢁ50mL). The joined organic extracts were washed
with H2O (100 mL), brine (50 mL), dried (Na2SO4) and
evaporated. Purification by flash chromatography (10%
MeOH–EtOAc) afforded 4 as a white powder (0.16 g,
Experimental
The 18b-glycyrrhetinic acid was purchased from Sigma
Chemical Co. (Sigma-Aldrich Korea, Seoul, Korea). Dry
CH2Cl2, DMF were obtained by distillation from CaH2.
THF was distilled from sodium, benzophenone. The
other commercially available reagents and solvents were
used without further purification. All reactions were
conducted under an Ar atmosphere, except for those
reactions utilizing water as a solvent. They were mon-
itored by TLC (Merck Kieselgel 60, F254). All the pro-
ducts prepared were purified by flash column
chromatography on silica gel 60 (Merck, 230–400
mesh). The 1H NMR and 13C NMR spectra were
recorded on a JEOL JNM EX-400 using CDCl3 or
CD3OD as a solvent. All chemical shifts (d) are quoted
in ppm downfield from TMS and coupling constants (J)
are given in Hz. Low-resolution mass spectra were
measured on a Agilent 1100 LC/MSD (API-ES) mass
spectrometer and High-resolution electron impact mass
spectra were obtained on VG Auto-spec Ultma mass
spectrometer (EI 70eV.)
ꢀ
1
40%); mp 122–125 C; H NMR (400 MHz, CDCl3): d
7.26 (m, 5H), 5.62 (s, 1H), 5.57 (s, 1H), 5.11 (m, 1H),
4.10(m, 1H), 3.49 (s, 2H), 3.22 (m, 1H), 2.80(m, 4H),
2.32 (s, 1H), 2.12 (m, 4H), 1.91 (m, 4H), 1.79 (m, 7H),
1.44 (m, 6H), 1.13 (s, 3H), 1.12 (s, 3H), 1.10(s, 3H), 1.04
(m, 2H), 1.00 (s, 3H), 0.93 (s, 3H), 0.80 (s, 3H), 0.79 (s,
3H); 13C NMR (100 MHz, CDCl3): d 199.98, 174.93,
169.13, 138.19, 129.06, 128.43, 128.20, 127.04, 78.72,
62.92, 61.81, 60.37, 54.95, 52.27, 48.17, 46.27, 48.27,
45.33, 43.36, 43.19, 41.83, 39.19, 39.13, 37.43, 37.06,
32.75, 32.30, 32.11, 31.86, 31.48, 29.50, 28.51, 28.11,
27.26, 26.45, 26.37, 23.32, 21.04, 18.63, 17.46, 16.32,
15.58; MS: m/z (100%)=643 (M+, 100), 425 (12), 174
(63), 91 (74); HREIMS m/z 642.4765 (calcd for
C42H62N2O3 642.4760).
10-Hydroxy - 2,4a,6a,6b,9,9,12a - heptamethyl - 13 - oxo -
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10, 11,12,12a,12b,13,14b-ei-
cosahydro-picene-2-carboxylic acid [1-(pyrrolidine-2-
carbo nyl)-piperidin-4-yl]-amide (5). To a solution of 8
(1.25 g, 3.77 mmol) in CH2Cl2 (60mL) was added drop
by drop NMM (0.87 mL, 7.92 mmol) at 0 ꢀC. After
10-Hydroxy - 2,4a,6a,6b,9,9,12a - heptamethyl - 13 - oxo -
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10, 11,12,12a,12b,13,14b-ei-
cosahydro-picene-2-carboxylic acid 4-fluoro-benzyl ester
(3). A mixture of 18b-glycyrrhetinic acid (0.30 g, 0.64
mmol), K2CO3 (0.26 g, 1.91 mmol) and 4-fluorobenzyl-
bromide (0.13 g, 0.70 mmol) was added DMF (15 mL).
The reaction mixture was heated 3 h at 80 ꢀC and cooled
ꢀ
stirring for 10min at 0 C, a reaction mixture was added
18b-glycyrrhetinic acid (2.66 g, 5.65 mmol) and DCC (1.16
ꢀ
g, 5.65 mmol) at 0 ꢀC. After stirring for 10min at 0 C, the
reaction mixture warmed up room temperature and stirred
for 4 h. The appearing precipitated was filtered off and fil-
trate was evaporated in vacuo. Purification by flash

5350
S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
chromatography (CH2Cl2–MeOH=45:1!40:1) affor-
ded coupling compound as a white powder (2.36 g,
80%). Continuously, to a solution of coupling com-
pound (2.36 g, 3.01 mmol) in MeOH (30 mL) was
added Pd/C (10%, 0.23 g) at room temperature.
After stirring for 3 h under H2, a reaction mixture
was filtered with Celite pad and evaporated. Purifica-
tion by recrystallization (MeOH/CH2Cl2) afforded 5
as a white powder (1.86 g, 95%, overall yield: 76%);
mp 182–185 ꢀC; 1H NMR (400 MHz, CDCl3): d 5.61
(d, J=3.7 Hz,1H), 5.48 (m, 1H), 4.58 (d, J=3.7 Hz,
1H), 4.04 (m, 1H), 3.88 (m, 2H), 3.16 (m, 3H), 2.71
(m, 3H), 2.32 (s, 1H), 2.20(m, 6H), 1.56 (m, 12H),
1.36 (s, 6H), 1.43 (m, 5H), 1.12 (s, 6H), 1.10(m,
4H), 0.99 (s, 3H), 0.85 (m, 2H), 0.80 (s, 6H); 13C
NMR (100 MHz, CDCl3): d 200.00, 175.13, 172.37,
162.6, 128.47, 78.63, 61.78, 57.98, 52.91, 48.06, 46.56,
45.34, 43.93, 43.73, 43.38, 43.20, 41.77, 41.58, 39.13,
37.41, 36.02, 34.63, 32.60, 31.84, 31.55, 31.42, 31.24,
30.88, 29.43, 29.01, 28.56, 28.09, 26.87, 26.42, 25.24,
22.62, 17.44, 15.09, 14.09, 11.41; MS: m/z
(100%)=650 (M+, 100), 306 (15); HREIMS m/z
649.4820(calcd for C 40H63N3O4 649.4819).
2-(4-Amino-piperidine-1-carbonyl)-pyrrolidine-1-car-
boxylic acid benzyl ester (8). To a solution of 13 (1.32 g
3.06 mmol) in CH2Cl2 (13 mL) was added 1.0M solu-
tion HCl in diethylether (200 mL) at 0 ꢀC. After stirring
for 10min at 0 ꢀC, a reaction mixture was warmed up
room temperature and stirred for 2 h. The reaction
mixture was concentrated to give 8 as white powder
solid (0.97 g, 96%); mp 79–82 ꢀC; H NMR (400 MHz,
1
DMSO-d6): d 7.33 (m, 5H), 5.97 (m, 2H), 4.72 (m, 1H),
4.18 (m, 1H), 3.82 (m, 1H), 3.42 (m, 4H), 3.07 (m, 2H),
2.89 (m, 2H), 2.20(m, 1H), 1.82 (m, 4H), 0.86 (m, 2H);
MS: m/z (100%)=332 (M+, 100), 288 (18); HREIMS
m/z 331.1896 (calcd for C18H25N3O3 331.1895).
11 - Hydroxymethyl - 4,4,6a,6b,8a,11,14b - heptamethyl -
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10, 11,12,12a,14,14a,14b-ei-
cosahydro-picene-3,14-diol (9). To a solution of 18b-gly-
cyrrhetinic acid (3.00 g, 6.37 mmol) in THF (63 mL)
was added drop by drop NaAl(OCH2CH2OCH3)2H2
ꢀ
(8.90mL, 31.87 mmol) at 0 C. After stirring for 10min
at 0 ꢀC, a reaction mixture was warmed up room tem-
perature and stirred for 4 h. The reaction quenched with
1 N HCl (aq) and extracted with EtOAc. The extracts
were washed with brine, dried (Na2SO4) and evaporated
in vacuo. Purification by recrystallization (EtOAc–n-
hexane) afforded 9 as a white powder (1.84 g, 63%); mp
232–235 ꢀC; 1H NMR (400 MHz, CDCl3): d 5.30(d,
J=3.80Hz, 1H), 4.30(t, J=4.20Hz, 1H), 3.45 (m, 3H),
3.21 (m, 1H), 1.95 (m, 4H), 1.54 (m, 9H), 1.40(s, 3H),
1.34 (m, 8H), 0.99 (m, 2H), 1.17 (s, 3H), 1.14 (s, 3H),
0.96 (s, 3H), 0.82 (s, 3H), 0.78 (s, 3H),0.69 (s, 3H); MS:
m/z (100%)=481 ([M+Na]+, 100), 268 (17), 186 (8),
105 (37); HREIMS m/z 458.3760(calcd for C 30H50O3
458.3759).
2-(4-tert-Butoxycarbonylamino-piperidine-1-carbonyl)-
pyrrolidine-1-carboxylic acid benzyl ester (7). A mixture
of 1 M NaOH (11.56 mL, 11.56 mmol) and t-BuOH (5
mL) was stirred at room temperature for 30min. 4-
Amino benzylpiperidine (1.07 mL, 5.26 mmol) was
added dropwise to a reaction mixture. Di-tert-dibu-
tylcarbonate (1.26 g, 5.78 mmol) was added to a reac-
tion mixture. The reaction was stirred at room
temperature for overnight. The reaction mixture was
acidified with 1 N HCl (<pH1), extracted EtOAc (2ꢁ30
mL) and washed with H2O (500 mL), Brine, dried with
Na2SO4, filtered, concentrated to give N-BOC com-
pound as white powder solid (1.50g, 98%). To a solu-
tion of N-BOC compound (1.46 g, 11.10mmol) in
MeOH (110 mL) was added Pd/C (10%, 0.15 g) at room
temperature. After stirring for overnight under H2, the
reaction mixture was filtered with Celite pad and evap-
orated. Purification by recrystallization (MeOH/
CH2Cl2) afforded debenzylation compound as a white
powder (0.96 g, 95%). Continuously, to a solution of N-
CBZ-Proline (1.19 mmol, 4.79 mmol) in CH2Cl2 (50mL)
were added HOBt (0.78 g, 5.75 mmol) and DCC (1.19 g,
5.75 mmol) at 0 ꢀC. The solution was stirred for 30min
at 0 ꢀC. To this mixture was added debenzylation com-
pound (0.96 g, 4.79 mmol) in dry DMF (5 mL) and
NMM (1.10 mL, 10.06 mmol), stirred 4 h at room tem-
perature. The crude was treated with H2O (300 mL), the
aqueous layer was extracted with EtOAc (2ꢁ50mL).
The joined organic extracts were washed with H2O (150
11 - Hydroxymethyl - 4,4,6a,6b,8a,11,14b - heptamethyl -
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10, 11,12,12a,14,14a,14b-ei-
cosahydro-picen-3-ol (10). To a solution of 9 (1.84 g,
4.01 mmol) in EtOH (20 mL) was added Pd/C (10%,
0.27 g) at room temperature. After stirring for overnight
under H2, a reaction mixture was filtered with Celite
pad and evaporated. Purification by flash chromato-
graphy (n-hexane–EtOAc=4:1!2:1) and recrystalliza-
tion (EtOAc–n-hexane) afforded 10 as a white powder
(1.70g, 95%); mp 241–244 ꢀC; 1H NMR (400 MHz,
CDCl3): d 5.18 (t, J=3.46 Hz, 1H), 3.50(dd, J=10.63,
8.66 Hz, 2H), 3.22 (m, 1H), 2.07 (m, 4H), 1.69 (m, 7H),
1.41 (m, 12H), 1.14 (s, 3H), 0.99 (s, 3H), 0.96 (s, 3H),
0.93 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H),0.79 (s, 3H); ); 13C
NMR (100 MHz, CDCl3): d 146.83, 125.88, 77.89,
68.25, 62.57, 56.51, 47.59, 45.72, 44.72, 43.99, 43.78,
42.21, 39.12, 38.48, 36.56, 33.72, 32.63, 32.46, 29.81,
27.88, 27.47, 27.56, 27.52, 25.20, 24.45, 19.63, 18.57,
16.65, 15.98, 15.51; MS: m/z (100%)=465 ([M+Na]+,
100), 252 (20), 170 (12), 89 (45); HREIMS m/z 442.3815
(calcd for C30H50O2 442.3811).
mL), brine (80mL), dried (Na SO4) and evaporated.
2
Purification by flash chromatography (MeOH–
CH2Cl2=30:1!25:1) afforded 7 as a white powder
(1.82 g, 88%, overall yield: 82%); mp 142–142 ꢀC; H
1
NMR (400 MHz, CDCl3): d 7.33 (m, 5H), 5.08 (dd,
J=12.16, 11.9 Hz, 2H), 4.60(m, 2H), 3.90(m, 1H), 3.61
(m, 4H), 2.03 (m, 8H), 1.44 (s, 9H), 1.31 (m, 2H); MS:
m/z (100%)=454 ([M+Na]+, 100), 332 (81), 288 (10);
HREIMS m/z 431.2421 (calcd for C23H33N3O5
431.2420).
Acetic acid 11-hydroxymethyl-4,4,6a,6b,8a,11,14b-hepta-
methyl-1,2,3,4,4a,5,6,6a,6b, 7,8,8a,9,10,11,12,12a,14,14a,
14b-eicosahydro-picen-3-yl ester (11). To a solution of
10 (0.97 g, 2.09 mmol) in DMF (20 mL) was added
TBDMSCl (0.63 g, 4.18 mmol) and imidazole (0.57 g,
8.37 mmol) at 0 ꢀC. After stirring for 10min at 0 ꢀC, a

S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
5351
reaction mixture was warmed up room temperature and
stirred for 2 h. The crude was treated with 1 N HCl (200
mL), the aqueous layer was extracted with diethylether
(2ꢁ40mL). The joined organic extracts were washed
with H2O (100 mL), brine (50 mL), dried (Na2SO4) and
evaporated. Purification by flash chromatography (n-
hexane–EtOAc=12:1) afforded TBDMS protection
compound as a colorless oil (1.05 g, 90%). To a solution
of TBDMS protection compound (1.05 g, 1.88 mmol) in
CH2Cl2 (20mL) was added acetylchloride (.016 mL,
2.25 mmol), pyridine (0.30 mL, 4.13 mmol) and DMAP
(7 mg, 0.37 mmol) at 0 ꢀC. After stirring for 10min at
0 ꢀC, a reaction mixture was warmed up room tem-
perature and stirred for 4 h. The crude was treated with
1 N KHSO4 (200 mL), the aqueous layer was extracted
with EtOAc (2ꢁ40mL). The joined organic extracts
were washed with H2O (100 mL), brine (50 mL), dried
(Na2SO4) and evaporated. Purification by flash chro-
matography (n-hexane–EtOAc=30:1) afforded acetyl
compound as a white powder (0.84 g, 75%). Con-
tinuously, to a solution of acetyl compound (0.84 g, 1.41
mmol) in THF (14 mL) was added 1.0M TBAF (2.5
mL, 2.52 mmol) at 0 ꢀC. After stirring for 10min at
0 ꢀC, a reaction mixture was warmed up room tem-
perature and stirred for overnight. The reaction mixture
was concentrated and the residue was purified by flash
EtOAc (2ꢁ20mL). The joined organic extracts were
washed with H2O (100 mL), brine (50 mL), dried
(Na2SO4) and evaporated. Purification by flash chro-
matography (n-hexane–EtOAc=5:1) afforded 13 as a
white powder (0.08 g, 87%); mp 235–237 ꢀC; H NMR
1
(400 MHz, CDCl3): d 5.18 (t, J=3.57 Hz, 1H), 3.23 (m,
1H), 3.22 (m, 1H), 1.75 (m, 6H), 1.60(m, 5H), 1.37 (m,
5H), 1.25 (m, 5H), 1.16 (s, 3H), 0.99 (s, 3H), 0.90 (s,
3H), 0.85 (m, 2H), 0.83 (s, 6H), 0.79 (s, 3H), 0.77 (s,
3H); 13C NMR (100 MHz, CDCl3): d 206.30, 148.23,
127.08, 69.25, 65.59, 57.57, 47.49, 45.52, 44.22, 43.75,
43.45, 42.51, 39.42, 38.40, 36.45, 33.82, 32.53, 32.42,
28.88, 28.27, 27.87, 27.84, 27.52, 25.20, 24.85, 19.93,
18.77, 16.75, 15.68, 15.41; MS: m/z (100%)=463
([M+Na]+, 100), 236 (21), 159 (15), 73 (19); HREIMS
m/z 440.3651 (calcd for C30H48O2 440.3654).
10-Hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,
6,6a,6b,7,8,8a,9,10,11,12, 12a,12b,13,14b-eicosahydro-
picene-2-carboxylic acid (14). To a solution of 12 (0.23
g, 0.49 mmol) in mixture solvent (t-BuOH/
CH2Cl2/1.25 M NaH2PO4=7.5 mL/1 mL/6 mL) was
added 1.0M KMnO 4 (2.06 mL, 1.96 mmol) at 0 ꢀC.
After stirring for 1 h at 0 ꢀC, a reaction mixture was
warmed up room temperature and stirred for 30min.
The reaction quenched with satd Na2SO3 (aq) and
extracted with diethylether. The extracts were washed
with brine, dried (Na2SO4) and evaporated in vacuo.
The residue was purified by flash chromatography (n-
hexane–EtOAc=5:1) afforded acid compound as a
white powder (0.16 g, 70%). Continuously, to a solution
of acid compound (0.16 g, 0.33 mmol) in MeOH (3 mL)
was added K2CO3 (0.46 g, 3.30 mmol) at room tem-
perature. The reaction was stirred for 24 h. The crude
was treated with H2O (200 mL), the aqueous layer was
extracted with EtOAc (2ꢁ50mL). The joined organic
extracts were washed with H2O (150mL), brine (50
mL), dried (Na2SO4) and evaporated. Purification by
flash chromatography (n-hexane–EtOAc=5:1) afforded
14 as a white powder (0.13 g, 87%, overall yield: 61%);
chromatography
(n-hexane–EtOAc=4:1)
afforded
compound 11 as a white powder (0.63 g, 92%, overall
yield: 62%); mp 210–215 ꢀC; 1H NMR (400 MHz,
CDCl3): d 5.18 (t, J=3.40Hz, 1H), 4.50(m, 1H), 3.49
(dd, J=10.60, 8.70 Hz, 2H), 2.05 (s, 3H), 1.94 (m, 4H),
1.66 (s, 3H), 1.62 (m, 5H), 1.57(s, 3H), 1.32 (m, 9H),
1.21 (s, 3H), 1.06 (m, 2H), 1.01 (s, 3H), 0.90 (s, 3H), 0.87
(s, 3H), 0.86 (s, 3H), 0.83 (s, 3H); MS: m/z (100%)=507
([M+Na]+, 100), 268 (18), 105 (12); HREIMS m/z
484.3913 (calcd for C32H52O3 484.3916).
Acetic acid 11-formyl-4,4,6a,6b,8a,11,14b-heptamethyl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a, 9,10,11,12,12a,14,14a,14b-ei-
cosahydro-picen-3-yl ester (12). To a solution of 11
(0.13 g, 0.27 mmol) in CH2Cl2 (5 mL) was added PCC
(0.09 g, 0.40 mmol) at 0 ꢀC. After stirring for 1 h at 0 ꢀC,
a reaction mixture was warmed up room temperature
and stirred for 1 h. The reaction mixture was filtered
with Celite pad and evaporated. Purification by flash
chromatography (n-hexane–EtOAc=14:1) afforded 12
mp 289–231 ꢀC; H NMR (400 MHz, CD3OD): d 5.15
1
(t, J=3.44 Hz, 1H), 3.01 (m, 1H), 1.85 (m, 8H), 1.55 (m,
8H), 1.35 (m, 5H), 0.93 (m, 2H), 1.05 (s, 3H), 1.01 (m,
3H), 0.88 (s, 3H), 0.85 (s, 6H), 0.67 (s, 3H), 0.66 (s, 3H);
13C NMR (100 MHz, CDCl3): d 181.08, 145.23, 125.08,
68.25, 64.59, 56.87, 47.38, 45.32, 45.22, 43.85, 43.52,
42.51, 39.82, 38.25, 36.25, 33.72, 32.83, 32.22, 28.79,
28.37, 27.97, 27.79, 27.72, 25.30, 24.75, 19.89, 18.87,
16.87, 15.78, 15.41; MS: m/z (100%)=479 ([M+Na]+,
100), 413 (53), 381 (31), 148 (22); HREIMS m/z
456.3605 (calcd for C30H48O3 456.3603).
as a white powder (0.10 g, 85%); mp 212–215 ꢀC; H
1
NMR (400 MHz, CDCl3): d 9.45 (s, 1H), 5.20(t,
J=3.37 Hz, 1H), 4.50(m, 1H), 2.07 (s, 3H), 1.92 (m,
4H), 1.67 (s, 3H), 1.63 (m, 5H), 1.53(s, 3H), 1.35 (m,
9H), 1.23 (s, 3H), 1.08 (m, 2H), 1.02 (s, 3H), 0.90 (s,
3H), 0.84 (s, 3H), 0.86 (s, 3H), 0.83 (s, 3H); MS: m/z
(100%)=505 ([M+Na]+, 100), 268 (15), 191 (8), 105
(12); HREIMS m/z 482.374 (calcd for C32H50O3
482.3759).
10-Hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,
5,6,6a,6b,7,8,8a,9,10,11,12, 12a,12b, 13,14b-eicosahydro-
picene-2-carboxylic acid 4-fluoro-benzyl ester (15) fol-
lowed by the method of compound 3. Yield; 96% as a
white powder; mp 243–245 ꢀC; 1H NMR (400 MHz,
CDCl3): d 7.34 (m, 2H), 7.04 (m, 2H), 5.18 (t, J=3.43
Hz, 1H), 5.13 (dd, J=12.30, 9.96 Hz, 2H), 3.22 (m, 1H),
1.92 (m, 6H), 1.63 (m, 9H), 1.28 (m, 8H), 1.12 (s, 6H),
0.99 (s, 3H), 0.94 (m, 3H), 0.93 (s, 3H), 0.76 (s, 3H), 0.72
(s, 3H); 13C NMR (100 MHz, CDCl3): d 176.23, 149.00,
132.02, 131.86, 130.49, 130.45, 128.48, 115.63, 115.38,
Acetic acid 11-formyl-4,4,6a,6b,8a,11,14b-heptamethyl-
1,2,3,4,4a,5,6,6a,6b, 7,8,8a,9, 10,11,12,12a,14,14a,14b-
eicosahydro-picen-3-yl ester (13). To a solution of 12
(0.10 g, 0.26 mmol) in MeOH (5 mL) was added K2CO3
(0.30 g, 2.20 mmol) at room temperature. A reaction
mixture was stirred for 24 h. The crude was treated with
H2O (200 mL), the aqueous layer was extracted with

5352
S.-J. Um et al. / Bioorg. Med. Chem. 11 (2003) 5345–5352
78.89, 65.25, 60.37, 54.71, 48.39, 45.82, 44.52, 43.93,
43.14, 41.21, 39.32, 38.60, 36.54, 33.82, 32.83, 32.52,
29.81, 28.88, 28.37, 27.56, 26.59, 25.20, 23.45, 19.63,
18.35, 16.85, 15.58, 15.18; MS: m/z (100%)=587
([M+Na]+, 100), 565 (M+, 13), 255 (13), 91 (8);
HREIMS m/z 564.3975 (calcd for C37H53FO3
564.3979).
lic of Korea. S.J.U. was supported by BK21 project
from Ministry of Education & Human Resources
Development.
References and Notes
1. Finney, R. S. H.; Tarknoy, A. L. J. Pharm. Pharmacol.
1960, 12, 49.
2. Pompei, R.; Flore, O.; Marcialis, M. A.; Pani, A.; Loddo,
B. Nature 1979, 281, 689.
3. Tanaka, S.; Uno, C.; Akimoto, M.; Tabata, M.; Honda, C.;
Kamisako, W. Planta Med. 1991, 57, 527.
4. Nishino, H.; Kitagawa, K.; Iwashima, A. Carcinogenesis
1984, 5, 1529.
10-Hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-1,2,3,4,4a,5,
6,6a,6b,7,8,8a,9,10,11,12, 12a, 12b,13,14b-eicosahydro-
picene-2-carboxylic acid [1-(pyrrolidine-2-carbonyl)-pi-
peridin-4-yl]-amide (16) followed by the method of com-
pound 5. Yield; 42% as a white powder; mp 188–
191 ꢀC; H NMR (400 MHz, CDCl3): d 5.41 (m, 1H),
1
5. Yi, H.; Nakashima, I.; Isobe, K. Am. J. Clin. Med. 1996,
24, 271.
5.38 (m, 1H), 4.45 (m, 1H), 5.13 (m, 2H), 4.04 (m, 1H),
3.88 (m, 2H), 3.16 (m, 3H), 2.71 (m, 3H), 2.32 (s, 1H),
2.20(m, 6H), 1.56 (m, 12H), 1.36 (s, 6H), 1.33 (m, 5H),
1.11 (s, 6H), 1.09 (m, 4H), 0.89 (s, 3H), 0.75 (m, 2H),
0.63 (s, 6H); 13C NMR (100 MHz, CDCl3): d 175.13,
172.37, 128.47, 78.63, 61.78, 57.98, 52.91, 48.06, 46.56,
45.34, 43.93, 43.73, 43.38, 43.20, 41.77, 41.58, 39.13,
37.41, 36.02, 34.63, 33.50, 32.60, 31.84, 31.55, 31.42,
31.24, 30.88, 29.43, 29.01, 28.56, 28.09, 26.87, 26.42,
25.24, 22.62, 17.44, 15.09, 14.09, 11.41; MS: m/
z(100%)=636 (M+, 100), 291 (17); HREIMS m/z
635.5028 (calcd for C40H65N3O3 635.5026).
6. Abe, H.; Ohya, N.; Yamamoto, K. F.; Shibuya, T.; Arichi,
S.; Odashima, S. Eur. J. Cancer Clin. Oncol 1987, 23, 1549.
7. (a) Deckner, G. E.; SaNogueira, J. P., Jr.; Zukowski, J. M.
US Patent 5 968 528 1999. (b) Randall, W. B.; Prida, C. L. US
Patent 1999, 5 939 082.
8. Garcia-Borron, J. C.; Solano, F. Pigment Cell Res. 2002,
15, 162.
9. Riley, P. A. Pigment Cell Res. 1993, 6, 182.
10. Hansson, C.; Rorsman, H.; Rosengren, E. Acta Derm.
Venereol 1981, 61, 147.
11. Cabanes, J.; Chazarra, S.; Garcia-Carmona, F. J. Pharm.
Pharmacol. 1994, 46, 982.
12. Morisaki, K.; Ozaki, S. Chem. Pharm. Bull. (Tokyo)
1996, 44, 1647.
Tyrosinase assay
13. (a) Chung, Y. J.; Kim, Y.-C.; Park, H.-J. Bull. Kor. Chem.
Soc. 2002, 23, 1481. (b) den Branden, S. V.; Compernolle, F.;
Hoornaert, G. J. J. Chem. Soc., Perkin Trans. 1 1992, 1035.
14. Sin, H. S.; Kwon, Y. J.; Han, H. S.; Park, S. H.; Park,
M. S.; Rho, Y. S.; Um, S. J. Bull. Kor. Chem. Soc. 2002, 23,
1806.
15. Saito, S.; Nagase, S.; Kawase, M.; Nagamura, Y. Eur. J.
Med. Chem. 1996, 31, 557.
16. Tierney, M. T.; Grinstaff, M. W. J. Org. Chem. 2000, 65,
5355.
17. Saari, W. S.; Schwering, J. E.; Lyle, P. A.; Smith, S. J.;
Engelhardt, E. L. J. Med. Chem. 1990, 33, 97.
18. Takahashi, K.; Shibata, S.; Yano, S.; Harada, M.; Saito, H.;
Tamura, Y.; Kumagai, A. Chem. Pharm. Bull. 1980, 28, 3449.
19. Gopinath, R.; Patel, B. K. Org. Lett. 2000, 2, 4177.
20. Furstner, A.; Mlynarski, J.; Albert, M. J. Am. Chem. Soc.
¨
2002, 124, 10274.
21. Abiko, A.; Roberts, J. C.; Takemasa, T.; Masamune, S.
Tetrahedron Lett. 1986, 27, 4537.
22. Morrison, J. E. Trends Biochem. Sci. 1982, 7, 102.
23. Cabanes, J.; Garcia-Carmona, F.; Garcia-Canovas, F.;
Iborra, J. L.; Lozano, J. A. Biochim. Biophys. Acta 1984, 790, 101.
24. Cabanes, J.; Garcia-Canovas, F.; Tudela, J.; Lozano, J. A.;
Garciaa-Carmona, F. Phytochemistry 1987, 26, 917.
25. Jung, G. D.; Yang, J. Y.; Song, E. S.; Park, J. W. Exp.
Mol. Med. 2001, 33, 131.
The DOPA-oxidase assay measured the second major
catalytic reaction of tyrosinase, the conversion of
DOPA to DOPA-chrome via DOPA-quinone. This
reaction was performed in the chromogenic appearance
of DOPA-chrome from 1 or 5 mM DOPA at 475 nm.
Forty microliters of 5 mM DOPA, 100 mL of 100 mM
phosphate buffer (pH 6.8), and 10 mL of the same buffer
with GA derivatives, vitamin C, or kojic acid were
added to a 96-well plate, and then 40 mL of mushroom
tyrosinase (0.01 mg/mL) (Sigma Chemical Co., St.
Louis, MO, USA) were mixed. Following incubation at
37 ꢀC for the indicated time, the absorbance of reaction
mixture was determined at wavelength 475 nm. Dose-
response curves were obtained by performing assays in
the presence of increasing concentrations of inhibitors
(0, 1.6, 3.2, 6.3, 12.5, 25, 50, 100, 200 mM). IC50 value, a
concentration giving 50% inhibition of tyrosinase
activity, was determined by interpolation of the dose–
response curves.
Acknowledgements
This study was supported by a grant 10009583 Ministry
of Commerce, Industry and Energy (MOCIE), Repub-
26. Yokota, T.; Nishio, H.; Kubota, Y.; Mizoguchi, M. Pig-
ment Cell Res. 1998, 11, 355.