Studies on depigmenting activities of dihydroxyl benzamide derivatives containing adamantane moiety

Article abstract of DOI:10.1016/j.bmcl.2008.12.106

Six diphenolic compounds containing adamantane moiety were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. The inhibitory activity of 4-adamantyl resorcinol 1 was similar to that of 4-n-butyl resorcinol in both assays. However, dihydroxyl benzamide derivatives 6a-e showed different inhibitory patterns. All derivatives significantly suppressed the cellular melanin formation without tyrosinase inhibitory activities. These behaviors indicated that the introduction of amide bond changes the binding mode of dihydroxyl groups to tyrosinase. Among derivatives, 6d (3,4-dihydroxyl compound) and 6e (2,3-dihydroxyl compound) showed stronger inhibitory activities (IC₅₀ = 1.25 μM and 0.73 μM, respectively) as compared to 4-n-butyl resorcinol (IC₅₀ = 21.64 μM) and hydroquinone (IC₅₀ = 3.97 μM). This study showed that the position of dihydroxyl substituent at aromatic ring is important for the intercellular inhibition of melanin formation, and also amide linkage and adamantane moiety enhance the inhibition.

Full text of DOI:10.1016/j.bmcl.2008.12.106

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1532–1533
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies on depigmenting activities of dihydroxyl benzamide derivatives
containing adamantane moiety
*
Ho Sik Rho , Heung Soo Baek, Soo Mi Ahn, Jae Won Yoo, Duck Hee Kim, Han Gon Kim
R & D Center, AmorePacific Corporation, 314-1, Bora-daong, Giheung-gu, Yongin-si, Kyounggi-do 449-729, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Six diphenolic compounds containing adamantane moiety were synthesized and evaluated as potent
inhibitors on tyrosinase activity and melanin formation in melan-a cells. The inhibitory activity of 4-ada-
mantyl resorcinol 1 was similar to that of 4-n-butyl resorcinol in both assays. However, dihydroxyl benz-
amide derivatives 6a–e showed different inhibitory patterns. All derivatives significantly suppressed the
cellular melanin formation without tyrosinase inhibitory activities. These behaviors indicated that the
introduction of amide bond changes the binding mode of dihydroxyl groups to tyrosinase. Among deriv-
atives, 6d (3,4-dihydroxyl compound) and 6e (2,3-dihydroxyl compound) showed stronger inhibitory
Received 16 August 2008
Revised 20 December 2008
Accepted 25 December 2008
Available online 1 January 2009
Keywords:
Depigmenting activity
Tyrosinase
Dihydroxyl benzamide
Adamantane moiety
activities (IC50 = 1.25
l
M
and 0.73
l
M, respectively) as compared to 4-n-butyl resorcinol
M). This study showed that the position of dihydroxyl
(IC50 = 21.64 M) and hydroquinone (IC50 = 3.97
l
l
substituent at aromatic ring is important for the intercellular inhibition of melanin formation, and also
amide linkage and adamantane moiety enhance the inhibition.
Ó 2008 Elsevier Ltd. All rights reserved.
Melanogenesis is the process by which the synthesis and distri-
bution of melanin within skin and hair follicles.1 Melanin has
important role in protecting the skin against the harmful effect
of UV-irradiation. However, increase in the levels of epidermal
melanin synthesis and uneven distribution can cause esthetic
problems such as freckles, melasma and aged spot. In melanocyte,
melanin are synthesized by the action of tyrosinase which cata-
lyzes the hydroxylation of tyrosine to DOPA and the oxidation of
DOPA to dopaquinone.2 Therefore, the inhibition of tyrosinase to
treat the pigmentation disorders has been a recent subject of many
studies. Certain polyphenolic compounds, containing resorcinol
moiety, showed potent depigmenting activities and their inhibi-
tory mechanisms were well investigated.3 Recently, the effects
and mechanism of 4-n-butyl resorcinol has been clearly eluci-
dated.4 The hypopigmentary effect of 4-n-butyl resorcinol results
from its direct inhibition of tyrosinase. The structure of 4-n-butyl
resorcinol consists of main two parts such as resorcinol moiety
and n-butyl group. n-Butyl group was introduced as a hydrophobic
moiety to increase the depigmenting activity of resorcinol. The
present work was designed to develop more potent depigmenting
agent containing diphenolic moiety such as catechol and resorcinol
(Fig. 1).
OH
OH
OH
OH
H
N
OH
OH
4-n-butyl resorcinol
O
1
6a-e
Figure 1. Structure of 4-n-butyl resorcinol and diphenolic compounds containing
adamantane moiety 1 and 6a–e.
sized in one step through the reaction of adamantylation of resor-
cinol 2 using 1-adamantanol in CF3COOH at reflux condition
(Scheme 1).7
The synthetic pathways of dihydroxyl benzamide derivatives
containing adamantane moiety are shown in Scheme 2.
The starting materials, dihydroxy benzoic acids (3a–e) were re-
acted with acetic anhydride in the presence of Et3N and catalytic
amount of dimethylaminopyridine (DMAP) in THF to afford diacet-
oxy benzoic acids. Diacetoxy benzoic acids (4a–e) were treated
with ethylchloroformate and N-methyl morpholine in THF to con-
vert the carboxylic acids to an anhydrides. The anhydrides were re-
Considering cell permeability, we introduced adamantane moi-
ety5 expecting an increase in tyrosinase inhibitory activity and
enhancing cell penetration. 4-Adamantyl resorcinol 16 was synthe-
OH
OH
a
OH
OH
1
2
* Corresponding author. Tel.: +82 31 280 5805; fax: +82 31 281 8397.
E-mail address: thiocarbon@freechal.com (H.S. Rho).
Scheme 1. Reaction conditions; (a) 1-adamantanol, CF3COOH, reflux.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.106

H. S. Rho et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1532–1533
1533
detected (respectively, IC50 = 12.47
l
M and IC50 = 9.82
l
M). N-
OH
OH
OAc
OAc
a
b, c
Adamantyl-3,4-dihydroxy benzamide 6d showed a better inhibi-
tory activity (IC50 = 1.25 M). Among all the derivatives tested,
compound 6e containing 2,3-dihydroxyl groups exhibited stronger
inhibitory activity (IC50 = 0.73 M). Its activity was about five
times more potent than that of hydroquinone (IC50 = 3.97 M).
HO
HO
l
O
O
3a = 2,4-OH
3b = 3,5-OH
3c = 2,5-OH
3d = 3,4-OH
3e = 2,3-OH
l
4a = 2,4-OAc
4b = 3,5-OAc
4c = 2,5-OAc
4d = 3,4-OAc
4e = 2,3-OAc
l
Although five dihydroxyl benzamide derivatives (6a–e) had no
tyrosinase inhibitory activities, there were potent depigmenting
activities in cell based assay. Inhibition pathways of 6a–e may be
different from those of compound 1 and 4-n-butyl resorcinol. From
these result, compound 6d and 6e can be considered as a good can-
didate for depigmenting agent because their activities were more
potent than that of hydroquinone known to be the most powerful
depigmenting agent. Further studies on their depigmenting mech-
anism are underway in our laboratory.
OH
OH
OAc
OAc
H
H
N
d
N
O
O
6a = 2,4-OH
6b = 3,5-OH
6c = 2,5-OH
6d = 3,4-OH
6e = 2,3-OH
5a = 2,4-OAc
5b = 3,5-OAc
5c = 2,5-OAc
5d = 3,4-OAc
5e = 2,3-OAc
References and notes
Scheme 2. Reaction conditions: (a) acetic anhydride, Et3N, DMAP (cat), THF; (b)
ethylchloroformate, N-methylmorpholine, THF; (c) adamantamine.HCl, Et3N, DMF
(d) (d) KOH, H2O.
1. Spritz, R. A.; Hearing, V. J. Adv. Hum. Genet. 1994, 22, 1.
2. Marmol, V. D.; Beermann, F. FEBS Lett. 1996, 381, 165.
3. (a) Khatib, S.; Nerya, O.; Musa, R.; Shumuel, M.; Tamir, S.; Vaya, J. Bioorg. Med.
Chem. 2005, 13, 433; (b) Shimizu, K.; Kondo, R.; Sakai, K.; Takeda, N.; Nagahata,
T.; Oniki, T. Lipids 2001, 36, 1321; (c) Shimizu, K.; Kondo, R.; Sakai, K. Planta
Med. 2000, 66, 11; (d) Nerya, O.; Vaya, J.; Musa, R.; Izrael, S.; Ben-Arie, R.; Tamir,
S. J. Agric. Food Chem. 2003, 51, 1201.
acted immediately adamantamine.HCl to produce the correspond-
ing amide derivatives (5a–e). The hydrolysis of compounds (5a–e)
afforded desired final products, dihydroxyl benzamides (6a–e).8
Above six synthesized compounds were evaluated for mush-
room tyrosinase activity9 and their ability to inhibit melanin for-
mation by a murine melanocytes cell line (Melan-a).10 Their
activities were compared with 4-n-butyl resorcinol and hydroqui-
none (Table 1).
4. Kim, D. S.; Kim, S. Y.; Park, S. H.; Choi, Y. G.; Kwon, S. B.; Kim, M. K.; Na, J. I.;
Youn, S. W.; Park, K. C. Biol. Pharm. Bull. 2005, 12, 2216.
5. (a) Rho, H. S.; Baek, H. S.; Lee, B. S.; Kim, J. H.; Kim, D. H.; Chang, I. S. Bull. Korean
Chem. Soc. 2006, 27, 115; (b) Baek, H. S.; Rho, H. S.; Yoo, J. W.; Ahn, S. M.; Lee, J.
Y.; Lee, J.; Kim, M. K.; Chang, I. S. Bull. Korean Chem. Soc. 2008, 29, 43.
6. Compound 1: 1H NMR (300 MHz, DMSO-d6): d 8.98 (s, 1H), 8.89 (s, 1H), 6.80 (d,
1H, J = 8.4 Hz), 6.22 (s, 1H), 6.10 (d, 1H, J = 8.4 Hz), 1.99 (s, 9H), 1.69 (s, 6H). IR
vmax (KBr) 3486, 3390, 2903, 1614 cmÀ1. FABMS: (m/e) 259 [M+H]+.
7. Shokova, E.; Tafeenko, V.; Kovalev, V. Tetrahedron Lett. 2002, 43, 5153.
8. We previous reported dihydroxyl benzamide derivatives 6a–6e on Korean
Patent 0,643,514, 2005 and 0,740,575, 2007. Compound 6a: 1H NMR (300 MHz,
DMSO-d6): d 12.62 (s, 1H), 10.07 (s, 1H), 7.73 (d, 1H, J = 8.7 Hz), 7.65 (s, 1H),
6.21 (m, 2H), 2.05 (s, 9H), 1.65 (s, 6H). IR vmax (KBr) 3429, 3221, 2904, 1650,
1583 cmÀ1. FABMS: (m/e) 302 [M+H]+. Compound 6b: 1H NMR (300 MHz,
DMSO-d6): d 9.36 (s, 2H), 7.34 (s, 1H), 6.57 (s, 2H), 6.29 (s, 1H), 2.02 (s, 9H), 1.63
(s, 6H). IR vmax (KBr) 3430, 3221, 2907, 1652, 1584 cmÀ1. FABMS: (m/e) 302
[M+H]+. Compound 6c: 1H NMR (300 MHz, DMSO-d6): d 11.14 (s, 1H), 8.93 (s,
1H), 8.04 (s, 1H), 7.26 (s, 1H), 6.71 (m, 2H), 2.05 (s, 9H), 1.65 (s, 6H). IR vmax
Consistent with previous reports, 4-n-butyl resorcinol induced a
strong inhibition of the tyrosianse activity (IC50 = 0.15
strong activity was also detected in melan-a cells
(IC50 = 21.64 M). When the n-butyl group was replaced by ada-
mantane moiety, the resulting compound 1 showed slightly lower
tyrosinase inhibitory activity (IC50 = 0.90 M) but more potent
depigmenting activity (IC50 = 8.82 M) in cell based assay. These
lM). Its
l
l
l
results indicate that adamantane group enhances the tyrosinase
inhibitory activity and cell penetration of resorcinol like n-butyl
group. Encouraged by this result, we synthesized five dihydroxyl
benzamide derivatives containing adamantane moiety and evalu-
ated their activities. Dihydroxy benzoic acids were conjugated with
adamantamine. Surprisingly, in tyrosinase assay, N-adamantyl-2,4-
dihydroxy benzamide 6a showed no inhibitory activity
(KBr) 3429, 3222, 2905, 1651, 1580 cmÀ1
.
FABMS: (m/e) 302 [M+H]+.
Compound 6d: 1H NMR (300 MHz, DMSO-d6): d 9.18 (bs, 1H), 9.02 (bs, 1H),
7.19 (s, 1H), 7.17 (s, 1H), 7.10 (d, 1H, J = 8.1 Hz), 6.71 (d, 1H, J = 8.1 Hz). 2.03 (s,
9H), 1.63 (s, 6H). IR vmax (KBr) 3428, 3220, 2903, 1650, 1582 cmÀ1. FABMS: (m/
e) 302 [M+H]+. Compound 6e: 1H NMR (300 MHz, DMSO-d6): d 12.20 (s, 1H),
9.21 (s, 1H), 7.92 (s, 1H), 7.33 (d, 1H, J = 1.5 Hz), 6.89 (d, 1H, J = 1.5 Hz), 6.64 (m,
1H), 2.07 (s, 9H), 1.65 (s, 6H). IR vmax (KBr) 3427, 3223, 2901, 1650, 1580 cmÀ1
.
FABMS: (m/e) 302 [M+H]+.
9. Measurements of mushroom tyrosinase activity: Mushroom tyrosinase,
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 U/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-
(IC50 > 200 lM). All other derivatives (6b, 6c, 6d and 6e), having
different dihydroxyl positions, also showed no inhibitory activities.
Introduction of amide linkage caused a negative influence on the
binding to tyrosinase. However, unexpected results were obtained
in melan-a cells. Compound 6a exhibited potent depigmenting
activity (IC50 = 34.15 lM). When 3,5-dihydroxyl, and 2,5-dihydr-
oxyl derivatives (6b and 6c) were tested, increased activities were
l
l
l
l
L
a
microplate reader (Bio-Rad 3550, Richnmond, CA, U.S.A.) 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).
10. Measurements of melanin content and cell viability: Melanin content and cell
number were measured in melan-a melanocytes. Murine melan-a melanocytes
were originally derived from C57BL/6 J (black, a/a) mice, a kind gift from Prof.
Dorothy C. Bennett (St. George’s Hospital, London, U.K.). The melanin content
Table 1
Depigmenting activities of 4-n-butyl resorcinol and diphenolic compounds containing
adamantane moiety 1 and 6a–6e
was measured using the method reported by Hosoi et al. with
a slight
modification.11 The cells (2 Â 105 cells/ml) were seeded into 24-well plates and
the test compounds were added in triplicate. The medium was changed daily
and after 4 d of culture, the cells were lysed with 1 ml of 1 N NaOH. Then
a
a
Compound
Tyrosinase IC50
M)
Pigmentation IC50
(lM)
% Survival of melan-
a cell
(
l
200 ll of each crude cell extract was transferred into 96-well plates. The
4-n-Butyl
0.15
21.64
98.21 (80
l
M)
relative melanin content was measured at 400 nm with a microplate reader
(Molecular Devices). Cell viability was determined using a modified crystal
violet assay.12 After removing the medium from each well, the cells were
washed with PBS and stained with 0.1% crystal violet in 10% ethanol for 5 min
at room temperature. The cells were then rinsed four times with distilled
water, and crystal violet retained by adherent cells was extracted with 95%
ethanol at room temperature for 10 min. Crystal violet absorption was
measured at 590 nm (Molecular Devices Co., Sunnyvale, CA, U.S.A.).
11. (a) Hosoi, J.; Abe, E.; Suda, T.; Kuroki, T. Cancer Res. 1985, 45, 1474; (b) Cho, Y.;
Kim, K.-H.; Shim, J.-S.; Hwang, J.-K. Biol. Pharm. Bull. 2008, 31, 986.
12. Dooley, T. P.; Gadwood, R. C.; Kilgore, K.; Thomasco, L. M. Skin Pharmacol. 1994,
7, 188.
resorcinol
Compound 1
6a (2,4-OH)
6b (3,5-OH)
6c (2,5-OH)
6d (3,4-OH)
6e (2,3-OH)
Hydroquinone
0.90
8.82
34.15
12.47
9.82
1.25
0.73
3.97
91.73 (20
99.24 (50
91.33 (30
99.10 (20
95.23 (30
96.17 (20
87.27 (10
lM)
lM)
lM)
lM)
lM)
lM)
lM)
>200
>200
>200
>200
>200
9.81
a
Values were determined from logarithmic concentration-inhibition curves and
are given as means of three experiments.