Structural requirements of (E)-6-benzylidene-4a-methyl-4,4a,5,6,7,8- hexahydronaphthalen-2(3H)-one derivatives as novel melanogenesis inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.02.060

Chalcone type compound 1a ((E)-6′-benzylidene-4a′-methyl- 4′,4a′,7′,8′-tetrahydro-3′H-spiro[[1,3] dithiolane-2,2′-naphthalen]-5′(6′H)-one) was discovered as an potent inhibitor in melanogenesis. To define its structure-activity relationship, a series of a

Full text of DOI:10.1016/j.bmcl.2011.02.060

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1922–1925
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural requirements of (E)-6-benzylidene-4a-methyl-4,4a,5,6,7,8-hexa-
hydronaphthalen-2(3H)-one derivatives as novel melanogenesis inhibitors
Pillaiyar Thanigaimalai a, Ki-Cheul Lee a, Vinay K. Sharma a, Eeda Vekateswara Rao a, Eunmiri Roh b,
Youngsoo Kim b, Sang-Hun Jung a,
⇑
a College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764, South Korea
b College of Pharmacy, Chungbuk National University, Cheongju 361-763, South Korea
a r t i c l e i n f o
a b s t r a c t
Chalcone type compound 1a ((E)-60-benzylidene-4a0-methyl-40,4a0,70,80-tetrahydro-30H-spiro[[1,3]dithio-
lane-2,20-naphthalen]-50(60H)-one) was discovered as an potent inhibitor in melanogenesis. To define its
structure–activity relationship, a series of analogs 1b–n, dithiolane truncated 2a–b and ring A removed
3a–e were prepared and evaluated. The electron donating substitution on the phenyl ring (ring C) rather
than an electron withdrawing group and dithiolane motif of 1 are needed for the activity enhancement.
Article history:
Received 1 January 2011
Revised 14 February 2011
Accepted 15 February 2011
Available online 18 February 2011
The scaffold containing both rings A and B associated with
of 1 was essential for antimelanogenesis.
a
,b-unsaturated system connected to phenyl
Keywords:
Chalcone
Melanogenesis
Melanoma B16 cells
Ó 2011 Elsevier Ltd. All rights reserved.
a-Melanocyte stimulating hormone
Melanin is the primary determinant of human skin and hair col-
O
B
O
O
or, and is heterogeneous biopolymer which plays an important role
in prevention of sun-related skin injury. Melanin is produced by
melanocytes through a process called melanogenesis in the basal
layer of epidermis.1 However, the excessive formation of melanin
in different specific parts of the human skin can causes diverse
hyperpigmentary disorders such as melasma, freckles, age spots
or liver spots, and actinic damage.2–7 The biosynthesis of melanin
involves series of enzymatic catalyzed and chemical reaction.
Among them, tyrosinase has attracted the much attention and it
plays important role in melanogenesis. Tyrosinase is a copper bear-
ing enzyme which widely distributed in nature. It catalyze two
step pathways involving molecular oxygen in the formation of
melanin; the hydroxylation of phenols to o-catechols, and the oxi-
dation of o-catechols to o-quinones, which further polymerize
spontaneously at physiological pH into melanin. Tyrosinase is also
associated to Parkinson and other neurodegenerative diseases, oxi-
dizing excess dopamine to DOPA quinone, highly reactive com-
pound that induce neuronal damage and cell death. Therefore,
the inhibition of tyrosinase is expected to provide effective thera-
peutic means for the treatment of diseases related to melanogene-
sis and dermatological disorders. Regarding this context, several
inhibitors of melanogenesis have been reported in the litera-
ture8–18 but their usage is being compromised due to their harmful
adverse effects and moreover only few of them are successfully
R1
S
A
C
A
B
R
C
B
R
O
S
1a-n
3a-e
2a-b
Chart 1. Target structures 1a–n, 2a–b and 3a–e for the antimelanogenesis activity.
Note = Substituents are indicated in Tables 1 and 2.
inhibiting the melanogenesis in melanoma cell or in skin model.
Therefore, the current therapies are considered to be inadequate
for these conditions. Taking this consideration into an account,
the search for new melanogenesis inhibitor from natural product
compounds and synthetic compounds with such activity is still
continues.19–21
Notably, chalcone derivatives are very versatile as physiologi-
cally active and common structure in numerous natural products
belonging to the flavonoid family.22–24 These compounds have
been reported to possess several biological activities, such as
cytotoxic,25 anti-malarial,26 antileishmanial,27anti-inflammatory,28
anti-HIV,29 antifungal30 and tyrosinase inhibitors.31 Having such
pharmacological activities, this scaffold has attracted medicinal
chemist and prompted us to investigate potent inhibitors of the
melaninogenesis. As we engaged to identify the new analog with
chalcone type derivatives, compound 1a (Chart
inhibition at 10 M, IC50 = 10.5 M) was discovered as melanogen-
esis inhibitor in melanoma B16 cells under the stimulant of
1 and 85%
l
l
⇑
Corresponding author.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.060

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1922–1925
1923
O
O
H
(i)
R1
O
S
S
+
R1
S
S
4
1k
1a-l 1n
5
,
2 N, HCl
1m
(ii)
O
R
O
2
2a
: R = CH3
2b: R = OCH3
Scheme 1. Synthesis of the target structures 1 and 2. Regents and condition: (i) KOH, R1-CHO (ii) NCS, silver nitrate. Note = substituents (R1) are indicated in Table 1.
presence of potassium hydroxide in 90% ethanolic solution under
refluxing condition.33 Subsequently, the conversion of compounds
1 to corresponding compounds 2 by the treatment of N-chlorosuc-
cinamide and silver nitrate.34 The compounds 3 were prepared
according to known procedure35–39 as indicated in Scheme 2.
For all synthesized analogs 1, 2, 3 and 4, the ability to inhibit the
formation of melanin from melanoma B16 cells was determined
O
O
O
(i)
H
+
R
R
5
6
3
under stimulus of
a
-MSH (100 nM) for 3 day incubation.12 The
Scheme 2. Synthesis of target structure 3. Regents and condition: (i) KOH, ethanol/
water (90:10), reflux, 12 h; Note = substituents (R) are indicated in Table 2.
melanoma B16 cells (CRL6323) was obtained from ATCC (Manas-
sas, USA). Amounts of melanin released into the culture media
were determined by measuring absorbance values at 405 nm with
synthetic melanin as the standard. Data for % inhibition at 10 lM
and IC50 values are mean values from 3 to 5 separate experiments
as shown in Tables 1 and 2.
In order to explore the structure activity relationship of 1a (85%
at 10 lM, C log P = 4.609) in the inhibitory melanogenesis activity,
a series of analogs 1b–n, 2a–b and 3a–e were evaluated. Therefore,
to investigate the effect of substitution on phenyl ring of 1a, the
various substituents were studied as shown in Table 1. Primarily,
the hydrophobic substituents at p-position like methyl (1b,
a
-melanocyte stimulating hormone (a-MSH) by random screening
process. Interestingly, compound 1a has approximately 8 or 12
times more potent in inhibition than kojic acid and arbutin. There-
fore in our present study, we synthesized a series of analogs of 1a
for exploring their preliminary structure–activity relationship on
the inhibitory activity of
a-MSH-induced melanogenesis in mela-
noma B16 cell line. The general chemical structures of lead 1a with
their analogs 1b–n, 2a–b and 3a–e have been indicated in Chart 1.
The synthesis of compounds 1, 2 was outlined in Scheme 1. The
expected compound 1 was obtained by the aldol condensation
reaction of 432 with various commercial aldehydes 5 in the
>100% inhibition at 10 lM, IC50 = 17.1 lM, C log P = 5.108), ethyl
Table 1
Inhibitory activity of 1a–n for their antimelanogenesis in melanoma B16 cell line
O
R1
S
S
1a-n
Entry No
Compound No
R1
% of Inhibition at 10a
(l
M)
IC50 value (
lM)
C log Pb value
1
2
3
4
5
6
7
8
9
10
11
12
13
14
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
C6H5
4-(CH3)C6H5
85
>100
80
83
35
62
85
75
>100
43
10.5
17.1
18.5
13.5
>30
25.5
11.5
16.5
3.5
>30
6.0
13.5
4.3
4.609
5.108
5.637
6.435
5.322
5.915
5.783
5.063
4.528
4.267
4.043
6.239
3.942
4.744
4-(CH2CH3)C6H5
4-(C(CH3)3)C6H5
4-(Cl)C6H5
3,4-(Cl)2C6H5
1-Naphthyl
(–CH@CH)C6H5
4-(OCH3)C6H5
3,4-(OCH3)2C6H5
4-(OCH2OCH3)C6H5
4-(OCH2Ph)C6H5
4-(OH)C6H5
92
80
96
65
1m
1n
Arbutin
4-(N(CH3)3)C6H5
7.6
120
a
The percentage of inhibition values obtained as a mean from 3 to 5 independent experiments.
C log P values are obtained from Chemdraw 11.0 V.
b

1924
P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1922–1925
Table 2
Inhibitory activity of 2a–b, 3a–e and 4 for their antimelanogenesis in melanoma B16 cell line
Entry No
Compound No
Structurea
% of inhibition at 10b
(lM)
IC50 value (lM)
C log Pc value
O
O
1
2a
60
23
3.312
O
2
2b
72
6.0
2.552
O
O
O
3
4
3a35
<10
<10
>30
>30
3.727
4.226
O
O
3b
5
6
7
3c36
3d37
3e39
<10
<10
<10
>30
>30
>30
4.440
3.646
3.06
Cl
O
O
O
OH
O
8
432
<10
>30
2.266
S
S
a
b
c
The substituents of 3a–e are directly indicated with complete structure.
The percentage of inhibition values obtained as a mean from 3 to 5 independent experiments.
C log P values are obtained from Chem Draw 11.0 V.
(1c, 80% inhibition at 10
lM, IC50 = 18.5
lM, C log P = 5.637) and
C log P = 4.528) was markedly increased the antimelanogenesis
tert-butyl group (1d, 83% inhibition at 10
l
M, IC50 = 13.5 M,
l
activity. This result encouraged us to prove further, hence the fol-
C log P = 6.435) were introduced. However, the activity does not al-
tered with the respect of increasing hydrophobic bulky group on
the phenyl ring of these analogs, which indicates that the hydro-
phobic substituent should not be required to enhance the activity.
lowing 3,4-dimethoxy (1j, 43% inhibition at 10
C log P = 4.267), methoxymethoxy (1k, 92% inhibition at 10
IC50 = 6.0
10 M, IC50 = 13.5
tion at 10 M, IC50 = 4.3
amino (1n, 66% inhibition at 10
derivatives were prepared and evaluated for their inhibitory activ-
ity. As results, 1k, 1m and 1h were exhibited comparable activity to
each other and more potent than 1a. From this study, it could be
suggested that the electron donating property of the substituents
should be favorable to achieve the inhibitors with high potential.
However, the inactivity of compound 1j bearing with 3,4-dime-
thoxy may due to serious steric hindrance of methoxy at 3-position,
which hampers the binding to the putative receptor. Considering
the C log P values of this series 1a–n, their hydrophobicity do not af-
fect the activity as indicated in Table 1.
l
M, IC50 >30
l
l
M,
M,
l
M, C log P = 4.043), benzyloxy (1l, 80% inhibition at
M, C log P = 6.239), hydroxyl (1m, 86% inhibi-
M, C log P = 3.942) and N,N1-dimethyl-
M, IC50 = 7.6 M, C log P = 4.744)
l
l
In addition to that, the p-chloro (1e, 35% inhibition at 10
>30 M, C log P = 5.322) and 3,4-dichloro group (1f 62% inhibition
at 10 M, IC50 = 25.5 M, C log P = 5.915) substitutions were
lM, IC50
l
l
l
l
l
l
l
noticeably dropped the activity. This suggests that the substituents
with electron withdrawing property must have the adverse effect
for the activity of 1a. In the next set of experiment, to examine
the effect of size of this planar moiety, phenyl (ring C), was re-
placed with naphthyl group (1g, 85% inhibition at 10
lM,
IC50 = 11.5 M, C log P = 5.783), which results in dramatic loss of
l
activity. Therefore the enlargement of aromatic ring in this region
is obviously unfavorable for the effect on anti-melanogenesis activ-
ity. Since
the extended conjugation with one more vinyl as shown in 1h (75%
inhibition at 10 M, IC50 = 16.5 M, C log P = 5.063) was demon-
a,b-unsaturated system in chalcone has an essential role,
In the continuation of SAR study, the beneficial role of dithio-
lane motif of 1a was explored by comparison of 2a (60% inhibition
l
l
strated. However, it seems that such elongation does not contrib-
at 10
lM, IC50 = 23
lM, C log P = 3.312) and 2b (72% inhibition at
ute for the activity improvement.
10 M, IC50 = 6.0
l
lM, C log P = 2.552) with their corresponding
Thus in the next set of experiment, the electron donating groups
on phenyl ring (ring C) were introduced. As a result, the activity of
p-methoxy analog (1i, >100% inhibition at 10 lM, IC50 = 3.5 lM,
analogs 1b and 1i. As results, the 2a and 2b were nearly twofold
reduce the activity. This implies that the bulky dithiolane moiety
must be highly favorable for the activity enhancement.

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1922–1925
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ing a series of monocyclic ,b-unsaturated ketones such as 3a
(<10% inhibition at 10 M, IC50 >30 M), 3b (<10% inhibition at
10 M, IC50 >30 M), 3c (<10% inhibition at 10 M, IC50 >30 M),
3d (<10% inhibition at 10 M, IC50 >30 M) and 3e (<10% inhibition
at 10 M, IC50 >30 M). None of them showed any inhibition,
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which implies that the ring A is very important for the inhibitory
activity of 1a. Additionally, to prove the structural necessity of ben-
zylidene system of 1a, the activity of compound 4 (<10% inhibition
at 10
edly, the result was end up with no inhibition even at 100
centration. This strongly suggests that the activity was mainly
lM, IC50 >30
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M, C log P = 2.266) was examined. As expect-
l
M con-
mediated by both rings A and B associate with
a,b-unsaturated
system in 1.
In summary, the compound 1a was discovered as novel melano-
genesis inhibitor in melanoma B16 cells under the stimulant of
a-
MSH. In order to improve the activity, a series of analogs such as
1b–n, 2a–b and 3a–e were evaluated for their preliminary struc-
ture–activity relationship (SAR) study. Accordingly the following
structural requirements could be considered for the activity of 1;
(i) the electron donating group on phenyl ring (ring C) rather than
electron withdrawing group, (ii) the variation in hydrophobicity
(C log P) does not have an impact on the activity, (iii) the both rings
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a,b-unsaturated system were mainly
important for the activity and (iv) dithiolone moiety must be
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This work was supported by a Grant (R01-2007-000-20099-0)
and Priority Research Centers Program (2009-0093815) through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology.
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Supplementary data (experimental procedure) associated with
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j.bmcl.2011.02.060.
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