J. Song et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6943–6946
6945
Figure 2. Dose dependency of melanogenesis inhibitory activities and cell viability data of selected compounds 8e, 8h and 9b. The bar graphs represent the percent inhibition
relative to the positive control ( -MSH only) and the line graphs indicate the number of viable cells. Cell counts were performed on a trypsinized cell suspension with a
a
hemocytometer. Cell viability was evaluated by trypan blue staining.
regulated by microphthalmia-associated transcription factor
(MITF).12 Further studies are required to determine whether the
molecular target of this compound lies upstream or downstream
of MITF.
In summary, we have identified cyclohepta[d]isoxazole deriva-
tives as novel melanogenesis inhibitors. Compound 8e, 8h and 9b
exhibited approximately 85- to 126-fold potency over kojic acid
without any detectable cytotoxicity. Additional investigation with
compound 9b revealed that its activity was associated with an
inhibition of tyrosinase expression without affecting the catalytic
activity of tyrosinase. We believe that those chemicals regulating
tyrosinase expression could be utilized to elucidate the molecular
mechanism of melanogenesis as a chemical tool. Further optimiza-
tion and mechanism studies are ongoing and will be reported in
due course.
Figure 3. Western blot analysis of whole cell lysates. The fold increase represents
the relative expression levels of tyrosinase corrected for the amount of actin, as
determined by band densitometry.
group to the meta position (8f) led to a complete loss of activity.
Replacement of the propyl group of 8a with the methyl group gave
way to the corresponding analogue 8b, which did not retain any
activity at all. We turned our attention to the piperazine ring in
the A region. One-carbon expansion of the piperazine ring of 8a
afforded the corresponding 13b, which retained notable activity
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2012-
0001386).
at 10 lM, but exhibited weak activity at 1 lM. A significant drop
in melanogenic activity was observed in another homopiperazine
derivative 13a. Insertion of one carbon between the A and B
regions (11) resulted in a loss of activity. IC50 values of the three
compounds (8e, 8h, and 9b)10 were determined comparable to
kojic acid in the mouse melanoma B16F10 cell line. The three com-
pounds showed significant inhibition of melanogenesis in a dose-
dependent manner without cytotoxicity (Fig. 2). para-Methoxy
derivative 8e showed a 126-fold potency over kojic acid, an excel-
lent whitening agent (IC50 = 0.67 and 84.54, respectively), and phe-
nol derivative 8h and para-ethoxy derivative 9b exhibited activity
References and notes
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10. Spectral data for compound 8e, 8h and 9b. For 8e: 1H NMR (CDCl3, 600 MHz) d
6.91 (d, 2H, J = 9.0 Hz), 6.85 (d, 2H, J = 9.0 Hz), 3.93 (t, 2H, J = 5.1 Hz), 3.77 (s,
3H), 3.76 (t, 2H, J = 5.1 Hz), 3.14 (t, 2H, J = 5.1 Hz), 3.06 (t, 2H, J = 5.1 Hz), 2.91 (t,
approximately 85-fold over kojic acid (IC50 = 1.01 lM and 0.99 lM,
respectively). These data indicate that small para-alkoxyl substitu-
ents provided potent melanogenesis inhibitory activity in this ser-
ies. Other changes of A and B region negatively affect activity for
melanogenesis. It appears that further optimization should focus
on isoxazole moiety.
We carried out biochemical experiments to investigate the
mechanism of action of this series. Described compounds did not
directly inhibit tyrosinase (data not shown). Compound 9b turned
out to regulate tyrosinase expression. A distinct decrease of tyros-
inase expression was observed after treatment of compound 9b
using western blot analysis (Fig. 3). Expression level of tyrosinase
2H, J = 6.0 Hz), 2.54 (t, 2H, J = 6.0 Hz), 1.81–1.86 (m, 2H), 1.69–1.77 (m, 4H); 13
C
NMR (CDCl3, 150 MHz): 172.02, 160.55, 157.29, 154.50, 145.16. 119.06, 116.07,
114.54, 55.55, 51.61, 51.06, 47.13, 42.15, 30.45, 27.95, 25.57, 22.43; LRMS (EI)
m/z 355 [M]+; For 8h: 1H NMR (CDCl3, 600 MHz) d 6.82 (d, 2H, J = 9.0 Hz), 6.76
(d, 2H, J = 9.0 Hz), 5.78 (br s, 1H), 3.92 (t, 2H, J = 5.1 Hz), 3.73 (t, 2H, J = 5.1 Hz),
in cells in presence of
in the absence of -MSH. Compound 9b inhibited significantly
tyrosinase expression for 48 h. Expression of tyrosinase is mainly
a-MSH was 1.6-fold higher than that in cells
a