N-[(Dihydroxyphenyl)acyl]serotonins as potent inhibitors of tyrosinase from mouse and human melanoma cells

Article abstract of DOI:10.1016/j.bmcl.2009.05.115

A series of N-acyl derivatives of tyramine, tryptamine, and serotonin were synthesized and tested on anti-melanogenic activity. The serotonin derivatives such as N-caffeoylserotonin (3) and N-protocatechuoylserotonin (9) were inhibitory to tyrosinase from

Full text of DOI:10.1016/j.bmcl.2009.05.115

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4178–4182
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-[(Dihydroxyphenyl)acyl]serotonins as potent inhibitors of tyrosinase
from mouse and human melanoma cells
*
Yoshimitsu Yamazaki , Yasuhiro Kawano, Akiko Yamanaka, Susumu Maruyama
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-acyl derivatives of tyramine, tryptamine, and serotonin were synthesized and tested on anti-
melanogenic activity. The serotonin derivatives such as N-caffeoylserotonin (3) and N-protocatechuoylse-
rotonin (9) were inhibitory to tyrosinase from mouse B16 and human HMV-II melanoma cells, while the
corresponding derivatives of tryptamine and 5-methoxytryptamine were almost inactive or less active
than the serotonin derivatives. The inhibitory activity of the serotonin derivatives increased with increas-
ing number of phenolic hydroxyl groups in the acyl moiety. Melanin formation in the culture of B16 cells
was suppressed by 3 and 9 with no cytotoxicity in the concentration range tested (IC50 = 15, 3 and
Received 6 April 2009
Revised 26 May 2009
Accepted 28 May 2009
Available online 11 June 2009
Keywords:
Tyrosinase
Inhibitor
Mouse
Human
Melanoma
Serotonin
Tryptamine
Phenolic acid
111 lM for 3, 9, and kojic acid, respectively). Thus the N-acylserotonin derivatives having a dihydroxy-
phenyl group are potential anti-melanogenic agents. Their inhibition of tyrosinase is primarily performed
through the 5-hydroxyindole moiety and further strengthened by the phenolic hydroxyl groups in the
acyl moiety.
Ó 2009 Elsevier Ltd. All rights reserved.
Tyrosinase inhibitors are important agents for treatment of skin
hyperpigmentation due to sun burning or aging, since the pigment,
melanin, is formed in a multistep process whose key steps are cat-
alyzed by the copper-containing enzyme tyrosinase (EC
1.14.18.1).1,2 Most of the inhibitors of tyrosinase are, therefore,
copper chelators3 or phenolic compounds structurally analogous
to the substrates, L-tyrosine and L-3,4-dihydroxyphenylalanine (L-
DOPA).4–6 Recently, polyhydroxycinnamamides7 and benzamides8
were reported as a new class of potent tyrosinase inhibitors, but
their structures were rather limited to
x-phenylalkylamides. Het-
erocyclic ring-containing alkylamides with anti-melanogenic
activity are rare except for N-coumaroyl- and N-feruloylserotonin
in safflower seeds.9 Although these structures are interesting with
regard to the indole intermediates in melanin biosynthesis,2 little
is known about the effect of the indole moiety on the anti-melano-
genic activity. Thus, we synthesized a series of N-acyl derivatives of
tryptamine, serotonin, and tyramine using caffeic, ferulic, vanillic,
protocatechuic, hydroxybenzoic, and benzoic acids to study the
structure–activity relationship (Fig. 1).
The synthesis was done by the acid chloride method, in which
the phenolic hydroxyl groups were protected by acetylation and,
after coupling with the amines, they were deprotected with hydra-
zine hydrate.10
Figure 1. Structures of the compounds.
The anti-melanogenic activity was tested on the inhibition of
the catecholase activity of tyrosinase extracted from mouse B16
* Corresponding author. Tel./fax: +81 29 861 6063.
E-mail address: y.yamazaki@aist.go.jp (Y. Yamazaki).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.115

Y. Yamazaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4178–4182
4179
Table 1
Anti-melanogenic activities of the synthesized compounds
Compounda
Mouse B16 melanoma cells
Melanin inhibition
Human HMV-II melanoma cell tyrosinase
Tyrosinase inhibition
Viability
Mb
IC50
(
lM)
% at 20
l
Mc
% at 100 l
Mb
b
b
No.
A
N
IC50
(l
M)
% at 100
l
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
C
C
C
C
F
F
P
P
Y
T
S
M
T
S
Y
T
S
S
S
T
S
T
S
S
—
—
15
2
73
1
4
29
6
ne
ne
15
ne
>30
23
ne
ne
3
84**
54**
99
30
14
55 10
6
4
28 11
—
—
216 10
—
—
41**
102
100
88**
69**
99
4
3
7
5
2
5
9
9
6
3
5
1
5
3
7
2
5
13
48
9
2
7
P
73
9
48
50
32
0
31
0
22
23
22
62
55
43
39
16
0
9
1
23
18
15
54
52
D
V
H
H
B
B
A
S
42 13
211 13
—
233 32
—
333 29
311 13
550 93
15
101
93
94
>30
>30
>30
ne
>30
>30
>30
13
100
85**
96
93*
101
100
99d
17
18
Kojic acid
5HI
68
61 11
3
111
a
A and N represent the acyl and amine moieties, respectively: C = caffeoyl, F = feruloyl, P = protocatechuoyl, D = 2,4-dihydroxybenzoyl, V = vanilloyl, H = p-hydroxybenzoyl,
B = benzoyl, A = acetyl, Y = tyramine, T = tryptamine, S = serotonin, M = 5-methoxytryptamine, and 5HI = 5-hydroxyindole.
b
Values are means or means sd (standard deviation), n = 3–5. ‘—’ = too large to be determined. ne = not evaluated for cytotoxicity.
Values are means of 8 or 9 wells. and represent p < 0.01 and p < 0.05 versus control by Student’s t-test, respectively.
At 200 lM.
c
**
*
d
melanoma cells using
for each compound was calculated from the enzyme activity in the
presence of the compound at a concentration of 100 M (Table 1).
L
-DOPA as the substrate.11 Percent inhibition
slightly active or inactive in suppressing the medium darkening,
and other compounds (1, 2, 4, 7, 8, and 14) showed cytotoxicity
in the concentration range of 10–30 lM (Table 1) so that their
l
A noteworthy fact was that all N-acylserotonin derivatives (3, 6, 9–
11, 13, 15, and 16) inhibited the tyrosinase, while the tryptamine
derivatives (2, 5, 8, 12, and 14) were inactive or weakly active
inhibitors under the same experimental condition. Furthermore,
serotonin (17) and 5-hydroxyindole (18) were active and a 5-
methoxyindole derivative (4) was almost inactive in this assay.12
These results indicate that the 5-hydroxyl group in the indole moi-
ety plays an essential role in inhibiting tyrosinase. The function of
this hydroxyl group needs the indole ring, since the tyramine
derivatives (1 and 7) were less active than the corresponding sero-
tonin derivatives (3 and 9) (Table 1) and 5-hydroxyindan and 6-
hydroxyquinoline were slightly active and inactive, respectively
(data not shown). The inhibitory activity was also affected by the
structure of the acyl moiety. The IC50 of the N-acylserotonins de-
creases in the order of 15 > 13 P 6 P 11 > 9 P 10 > 3, that is, the
order of increasing number of their hydroxyl groups. This correla-
tion implies that the inhibitory activity is strengthened by the phe-
nolic hydroxyl group(s) in the acyl moiety.
The serotonin derivatives also inhibited human tyrosinase in
HMV-II melanoma cells13 (Table 1). In this case, N-caffeoyl- (2),
N-feruloyl- (5), and N-protocatechuoyltryptamine (8) showed
weak inhibitory activity, but other tryptamine derivatives were
inactive. The tyramine derivatives 1 and 7 were stronger than
the tryptamine derivatives 2 and 8, but weaker than the corre-
sponding serotonin derivatives 3 and 9, respectively. These facts
indicate that the catechol or guaiacol moiety also contributes to
the inhibition of human tyrosinase, while they are less effective
than the 5-hydroxyindole moiety.
anti-melanogenic activities were not accurately evaluated. Thus,
the serotonin derivatives with a catechol or guaiacol moiety were
active, the corresponding tryptamine derivatives were cytotoxic,
and other simpler compounds (except 18) were neither cytotoxic
nor active in suppressing the cellular melanogenesis.
Among the above extracellular melanin formation inhibitors, N-
protocatechuoylserotonin (9) showed the lowest IC50 and was
about 40-fold stronger than kojic acid, the usual anti-melanogenic
agent. However, as shown in Figure 2A, the intracellular melanin
accumulation in B16 cells was not suppressed by this compound
in a concentration range of 2–40
compound 9 (20–40 M) suppressed by 20% the intracellular mel-
anin content, but cytotoxicity was found at the concentration of
40 M (Fig. 2B). Other serotonin derivatives 3 and 6 were also inac-
lM. In the case of HMV-II cells,
l
l
tive in suppressing the intracellular melanin accumulation in B16
cells (data not shown).
The inhibition mechanism of 9 with B16 tyrosinase was kineti-
cally studied. N-Acetylserotonin (16) and protocatechuamide (19)
were also studied for comparison.15 The Lineweaver–Burk plots
obtained with different concentrations of 9 intersect the X-axis at
very close points (Fig. 3A) and the same is found for 16 (Fig. 3B),
while those for 19 cross on the Y-axis (Fig. 3C). These results indi-
cate that 9 and 16 apparently inhibit the enzyme noncompetitively
(apparent Ki = 64
petitively (Ki = 3.7 mM) in the
was preincubated with 16 without or with a small amount of
l
M and 240
l
M, respectively) and 19 does com-
L
-DOPA oxidation. Next, the enzyme
L-
DOPA. The residual activity of the enzyme preincubated only with
16 was independent of the preincubation, while it decreased with
The anti-melanogenic activity was further tested in the mouse
B16 melanoma cell culture.14 Five serotonin derivatives (3, 6, 9,
10, and 11) showed the dose-dependent suppression in the med-
ium darkening due to melanin formation. The IC50 values are sum-
marized in Table 1. No cytotoxicity was found with these
compounds by the tetrazolium reduction assay at the concentra-
the preincubation time when both 16 and L-DOPA were present in
the mixture (Fig. 3D).
Mushroom tyrosinase is widely used for screening of anti-mel-
anogenic agents.3,4 We tested the effect of the present compounds
on mushroom tyrosinase. The initial rates (% of the control,
mean sd, n = 3) were 268 8 for 3, 191 10 for 9, and 36 2 for
tions below 30
l
M.14 Compounds 5, 12, 13, and 15–17 were
kojic acid at a concentration of 100 lM, showing that 3 and 9 acti-

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Y. Yamazaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4178–4182
Figure 2. Anti-melanogenic activity and cytotoxicity of compound 9 in cell cultures. (A) Mouse B16 melanoma cells. (B) Human HMV-II melanoma cells. Values (% control)
are the means SEM (standard error of the mean, n = 3–4) in a representative experiment that was repeated with comparable results. s, cell viability; N, extracellular
melanin; and d, intracellular melanin.
Figure 3. B16 tyrosinase reactions in the presence of compounds 9, 16, and 19 with
L–B plots for 16 (0 (s), 30 (d), and 100 M (N)); and (C) L–B plots for 19 (0 (s), 300 (d), 1000 (N), and 2500
16 without (s) or with -DOPA (d). The mixtures prepared by adding 10 l ethanol containing 20 mM 16 and 50
buffer (pH 6.8) lacking or containing 0.125 mM -DOPA were preincubated at 37 °C for 5, 10, 20, or 30 min and then mixed with 0.47 ml of the phosphate buffer containing
5.3 mM -DOPA for the enzyme activity measurement. The control activity (=100) was obtained after 31 min preincubation without 16. Every point is the mean sd (n = 3).
L
-DOPA. (A) Lineweaver–Burk (L–B) plots for 9 (0 (s), 30 (d), 50 (N), and 70
M (j)). (D) shows the effect of preincubation of the enzyme with
l of the enzyme solution into 0.47 ml 50 mM phosphate
lM (j)); (B)
l
l
L
l
l
L
L
Plot lines in A, B, and C are drawn by least square linear regression.
vated mushroom tyrosinase in contrast to the strong inhibition of
B16 and HMV-II enzymes. Mushroom tyrosinase is reported to be
activated by various kinds of compounds.2 It is not clear how 3
and 9 affect this enzyme, but it should be mentioned that the assay
with mushroom tyrosinase might create a misleading expectation
for the activity of compounds against mammalian tyrosinases.
N-Feruloylserotonin (6) was found to be an tyrosinase inhibi-
tor,9 and was studied on anti-atherogenic property.16 The related
tryptamine derivative 5 was recently shown to regulate the pro-
other than 6, 1, and 18 has been reported in any study concerning
anti-melanogenic activity. The tyramine derivative 1 was the
strongest one in the panel for human tyrosinase inhibitors by
Okombi et al.,7 yet the corresponding serotonin derivative (3) is
stronger than 1.
The present study has revealed that N-acylserotonins and their
nucleus, 5-hydroxyindole (18), are good inhibitors against mouse
and human melanoma tyrosinase, while N-acyltryptamines are
all poor inhibitors. Among the N-acylserotonins, N-caffeoylseroto-
nin (3) is the strongest inhibitor, and N-caffeoyl- (1) and N-proto-
catechuoyltyramine (7) are moderate or weak inhibitors of the
tyrosinases. These facts indicate that the tyrosinase inhibition is
duction of cytokines such as adiponectin17 and TNF-a 18
The caf-
.
feoyl analogue, 2,19 and the benzoyl analogues, 1420 and 15,21
also appeared in the literature, but none of the present compounds

Y. Yamazaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4178–4182
4181
primarily performed by the 5-hydroxyindole or catechol moiety.
Tyrosinase contains two copper atoms in a binuclear complex,
and this complex is thought to be the catalytic center.1,22 The cat-
echol hydroxyl groups of the substrate are bound to the copper
atoms so that a catechol-containing inhibitor would compete for
the copper center with the substrate. The competitive inhibition
by protocatechuamide (19) is consistent with this mechanism.
Thus, the question is why the inhibitory activities of the cate-
chol-containing tryptamine derivatives (2 and 8) are so weak as
compared with the corresponding serotonin derivatives (3 and
9). The 5-hydroxyl group on the indole ring should be capable of
binding to the copper atom, as suggested by the UV spectral
change of serotonin with copper ions.23 This expectation is sup-
ported by the fact that 5-hydroxyindole (18) and serotonin show
inhibitory activity. If a compound such as 3 and 9 has both 5-
hydroxyindole and catechol moieties, the 5-hydroxyindole moiety
would occupy the copper center prior to the catechol group, and
the latter would stabilize the whole binding through some interac-
tion with amino acid residue(s) of the enzyme. The catechol group
also can bind to the copper center, and in this case 5-hydroxyl
group on the indole ring may act to strengthen the binding. How-
ever, this orientation (copper binding by the catechol group) would
be abortive for the tryptamine derivatives 2 and 8 probably be-
cause of a steric hindrance by the indole ring that lacks a hydroxyl
group to compensate for the disadvantage in binding energy by an
interaction (e.g., hydrogen bond formation) to the enzyme. This
role of the hydroxyl group at the opposite site against the copper
binding moiety is possibly played by the phenolic groups in other
N-acylserotonins as well. Therefore, the number of the phenolic
hydroxyl group of compounds 3, 6, 9, 10, 11, 13, and 15 may pos-
itively correlate with the inhibitory activity. The difference be-
tween the activities of the catechol- and resorcinol-containing
derivatives (9 and 10) suggests that the positions of phenolic hy-
droxyl groups also affect the inhibition. 5-Hydroxyindole (18) itself
inhibited B16 tyrosinase as strongly as kojic acid (Table 1), but 3 or
10 was stronger than 18. This fact indicates that the activity of the
nucleus 5-hydroxyindole can be increased by the introduction of a
suitable side chain with phenolic groups. The comparison between
the serotonin and tyramine derivatives (1 vs 3; 7 vs 9) indicates
that the NH group of the indole ring also plays an important role
in the present inhibition, presumably by an interaction with the
copper center or with the polypeptide around it. Of course, there
is another possibility that the serotonin derivatives bind to the en-
zyme at a site remote from the copper center and thereby inhibit
the enzyme via an allosteric effect. The noncompetitive-type inhi-
bition by 9 and 16 might support this possibility. However, non-
competitive inhibition can be caused by another mechanism
whereby the inhibitor binds to the enzyme in a form different from
those receiving the substrate.24,25 Tyrosinase takes three forms for
copper states in the catalytic cycle, that is, the oxidized (met) form,
the oxygenated (oxy) form, and the reduced (red) form. In the cat-
echol oxidation, the substrate binds to the met and oxy forms. If an
inhibitor binds only to the red form, the inhibition is apparently
noncompetitive with respect to the substrate, as demonstrated
by iodide24 and cyanide ions.25 The fact that the preincubation of
stronger than 3 in the suppression of culture darkening. However,
no compound significantly suppressed the melanin accumulation
in B16 cells. To these cells, the serotonin derivatives were not cyto-
toxic. On the other hand, the corresponding tryptamine derivatives
(of the structures with more surface-active property) showed
strong cytotoxicity. These facts imply a poor membrane permeabil-
ity of the polar serotonin derivatives. Improvement of this point is
also to be addressed in future.
In conclusion, this study shows that 5-hydroxyindole ring is a
promising component for tyrosinase inhibitors in combination
with a suitable pendant group such as the catechol-containing acyl
group.
References and notes
1. Briganti, S.; Camera, E.; Picardo, M. Pigment Cell Res. 2003, 16, 101.
2. Seo, S.-Y.; Sharma, V. K.; Sharma, N. J. Agric. Food Chem. 2003, 51, 2837.
3. (a) Khan, M. T. H. Pure Appl. Chem. 2007, 79, 2277; (b) Criton, M.; Le Mellay-
Hamon, V. Bioorg. Med. Chem. Lett. 2008, 18, 3607.
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Jia, Q. Chem. Pharm. Bull. 2008, 56, 1292; (c) Hyun, S. K.; Lee, W.-H.; Jeong, D.
M.; Kim, Y.; Choi, J. S. Biol. Pharm. Bull. 2008, 31, 154.
5. Efdi, M.; Ohguchi, K.; Akao, Y.; Nozawa, Y.; Koketsu, M.; Ishihara, H. Biol. Pharm.
Bull. 2007, 30, 1972.
6. Liu, S. H.; Pan, I. H.; Chu, I. M. Biol. Pharm. Bull. 2007, 30, 1135.
7. Okombi, S.; Rival, D.; Bonnet, S.; Mariotte, A.-M.; Perrier, E.; Boumendjel, A.
Bioorg. Med. Chem. Lett. 2006, 16, 2252.
8. Cho, S. J.; Roh, J. S.; Sun, W. S.; Kim, S. H.; Park, K. D. Bioorg. Med. Chem. Lett.
2006, 16, 2682.
9. (a) Nagatsu, A.; Zhang, H. L.; Mizukami, H.; Okuyama, H.; Sakakibara, J.;
Tokuda, H.; Nishino, H. Nat. Prod. Lett. 2000, 14, 153; (b) Roh, J. S.; Han, J. Y.;
Kim, J. H.; Hwang, J. K. Biol. Pharm. Bull. 2004, 27, 1976.
10. 3-(3,4-Dihydroxyphenyl)-N-[2-(5-hydroxyindol-3-yl)ethyl]-2-propenamide
(3) was synthesized by coupling serotonin hydrochloride with 3-(3,4-
diacetoxyphenyl)-2-trans-propenoyl chloride in dimethylformamide. The
reaction product was worked up in the usual way, treated with hydrazine
hydrate for deprotection,17 and purified by silica gel column chromatography
(Wako gel C-300). The main product was eluted in 75–100% ethyl acetate/
benzene fractions. These fractions were combined, and concentrated to give a
residue, which was crystallized from ethyl acetate/hexane as fine needles, 60%
yield, mp 138–140 °C and 174–175 °C (with multiple phase transitions), Anal.
Calcd for C19H18N2O4: C, 67.45, H, 5.36, N, 8.28. Found: C, 66.92; H, 5.47; N,
8.00. 1H NMR (270 MHz, acetone-d6) d: 2.909 (2H, t, J = 7 Hz, CH2), 3.591 (2H,
m, CH2), 6.454 (1H, d, J = 15 Hz, CH@CH), 6.703 (1H, dd, J = 8 and 2 Hz, H-60),
6.829 (1H, d, J = 8 Hz, H-5), 6.930 (1H, dd, J = 8 and 2 Hz, H-6), 7.034 (1H, d,
J = 2 Hz, H-40), 7.076 (1H, d, J = 2 Hz, H-2), 7.094 (1H, s, H-20), 7.193 (1H, d,
J = 8 Hz, H-70), 7.325 (1H, br s, NH), 7.439 (1H, d, J = 15 Hz, CH@CH), and 9.758
(1H, br s, H-10). 3,4-Dihydroxy-N-[2-(5-hydroxyindol-3-yl)ethyl]benzamide (9)
was synthesized in the same way as described above using serotonin
hydrochloride and 3,4-diacetoxybenzoyl chloride. The final product (9) was
crystallized from ethyl acetate/benzene as fine needles, 18% yield, mp 194–
196 °C, FAB-MS m/z 313 ([M+H]+) (calcd C17H16N2O4 = 312), 1H NMR (270 MHz,
acetone-d6) d: 2.952 (2H, t, J = 8 Hz, CH2), 3.621 (2H, m, NCH2), 6.700 (1H, dd,
J = 9 and 2 Hz, H-60), 6.831 (1H, d, J = 8 Hz, H-5), 7.055 (1H, d, J = 2 Hz, H-40),
7.101 (1H, br s, H-20), 7.189 (1H, dd, J = 9 and 1 Hz, H-70), 7.287 (1H, dd, J = 8
and 2 Hz, H-6), 7.447 (1H, d, J = 2 Hz, H-2), 7.579 (1H, br s, NH), and 9.723 (1H,
br s, H-10). Other compounds (1, mp 211–212 °C; 2, mp 186–187 °C; 4, mp
211–213 °C; 6, mp 104–111 °C; 7, mp 218–221 °C; 8, mp 151–152 °C; 10, mp
118–123 °C and 227–231 °C; 11, mp 212–214 °C; 12, mp 200–201 °C; 13, mp
134–168 °C; 14, mp 141–142 °C (lit.20 mp 141–142 °C); 15, mp 200–201 °C;
and 19, mp 212–214 °C (lit.26 mp 212 °C)) were similarly prepared and
identified by elemental analysis, HR-MS, or FAB-MS and/or 1H NMR. 5 was
prepared in our previous work.17 Compounds 16 and 18 were purchased from
Tokyo Chemical Industry (Tokyo, Japan) and 17 (hydrochloride) was from
Wako Pure Chemical Industries (Osaka, Japan).
11. B16 melanoma cells were grown in DMEM medium containing 15% FBS.
Tyrosinase was extracted from the cells by the published method.5 Assay
solutions were prepared by mixing 0.94 ml 50 mM phosphate buffer (pH 6.8)
the enzyme with 16 in the presence of L-DOPA enhanced the inhi-
bition time-dependently (Fig. 3D) suggests that the red form is in-
volved in this inhibition, because the red form is generated only
during the catalytic process. However, further study is needed to
clarify the inhibitory mechanism.
The N-acylserotonins are good inhibitors for the cell-free
tyrosinase and melanogenesis in the culture medium. These two
inhibitory activities show a similar tendency concerning the
structure–activity relationship, but there are some exceptions.
For instance, N-protocatechuoylserotonin (9), which is weaker
than N-caffeoylserotonin (3) for the cell-free enzyme, is very much
containing 2.6 mM
compound unless otherwise stated. The reaction was started by adding the
enzyme solution (50 to the
l, containing 2.5–3 mU tyrosinase activity27
L-DOPA and 10 ll ethanol containing 0–30 mM test
l
)
substrate solutions prewarmed to 37 °C, and the dopachrome formation at
37 °C for 5 min was determined by measuring absorbance at 475 nm
(e
= 3700 Mꢀ1 cmꢀ1).27 Experiments with tyrosinase extracted from HMV-II
cells13 (Dai-Nippon-Sumitomo Pharmaceuticals Inc., Osaka, Japan) and
mushroom tyrosinase (Sigma) were done in the same way as described above.
12. Tyrosinase inhibition by 5-hydroxyindole was also reported by others: Seiji, M.
Federation Proc. 1961, 20, 6; Pomerantz, S. H. J. Biol. Chem. 1963, 238, 2351.
13. Sugimoto, K.; Nishimura, T.; Nomura, K.; Sugimoto, K.; Kuriki, T. Biol. Pharm.
Bull. 2004, 27, 510.

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Y. Yamazaki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4178–4182
14. B16 melanoma cells were cultured in 96-well plates (1.5 ꢁ 104 cells in 0.2 ml
phosphate buffer (pH 6.8) were as follows: 258 (13.2) and 286 (11.2) for 9, 278
(5.7) and 294 (4.9) for 16, and 256 (9.2) and 290 (5.4) for 19.
medium per well) for 1 day, and 2 ll ethanol solution containing 0–10 mM test
compound was added to each well. The plates were incubated for 2 days and
the absorbance of the medium at 415 nm was measured with a plate reader.
16. Koyama, N.; Kuribayashi, K.; Seki, T.; Kobayashi, K.; Furuhata, Y.; Suzuki, K.;
Arisaka, H.; Nakano, T.; Amino, Y.; Ishii, K. J. Agric. Food Chem. 2006, 54, 4970.
17. Yamazaki, Y.; Kawano, Y.; Uebayasi, M. Life Sci. 2008, 82, 290.
18. Yamazaki, Y.; Kawano, Y.; Uebayasi, M. J. Clin. Biochem. Nutr. 2008, 43, 413.
19. Park, J. B.; Chen, P. J. Chromatogr., B 2007, 852, 398.
20. Noland, W. E.; Hartman, P. J. J. Am. Chem. Soc. 1954, 76, 3227.
21. Gozzo, A.; Lesieur, D.; Duriez, P.; Fruchart, J. C.; Teissier, E. Free Radical Biol.
Med. 1999, 26, 1538.
22. Decker, H.; Schweikardt, T.; Tuczek, F. Angew. Chem., Int. Ed. 2006, 45, 4546.
23. Hadi, N.; Malik, A.; Azam, S.; Khan, N. U.; Iqbal, J. Toxic. in Vitro 2002, 16, 669.
24. Tepper, A. W. J. W.; Bubacco, L.; Canters, G. W. J. Biol. Chem. 2002, 277, 30436.
25. Wilcox, D. E.; Porras, A. G.; Hwang, Y. T.; Lerch, K.; Winkler, M. E.; Solomon, E. I.
J. Am. Chem. Soc. 1985, 107, 4015.
The medium was aspirated off, and cell viability was assayed with
a
tetrazolium reagent (CellTiter 96Ò, Promega). Intracellular melanin content
was measured by the published method,6 but with some modifications. Cells
were cultured in 24-well plates (7.5 ꢁ 104 cells in 1 ml medium per well) for
1 day, and ethanol solutions (2–6 ll) of test compounds were added to the
wells. After 2 days incubation, cells of each well were collected by a scraper,
washed with a phosphate-buffered saline, and then homogenized in 0.2 ml 1 N
NaOH by sonication. Absorbance at 415 nm of the homogenates was measured
to evaluate the intracellular melanin content.
15. Phenolic compounds are often inhibitors as well as substrates for tyrosinase.
However, the mixtures of 100
B16 tyrosinase in the assay condition omitting
l
M 9, 100
l
M 16, or 100 or 2500
lM 19 with
L-DOPA showed no change in
26. Barger, G. J. Chem. Soc. 1908, 93, 563.
the UV–vis spectra (230–610 nm) during an incubation for 60 min, suggesting
that oxidation of these compounds by the tyrosinase is, if occurring, negligible
27. (a) Jiménez, M.; Chazarra, S.; Escribano, J.; Cabanes, J.; García-Carmona, F. J.
Agric. Food Chem. 2001, 49, 4060; (b) Cabanes, J.; García-Carmona, F.; García-
Cánovas, F.; Iborra, J. L.; Lozano, J. A. Biochim. Biophys. Acta 1984, 790, 101.
in this kinetic study. The spectral data [kmax nm (e
mMꢀ1 cmꢀ1)] in the