Structure-activity relationship of naphthaldehydethiosemicarbazones in melanogenesis inhibition

Article abstract of DOI:10.1016/j.bmcl.2011.12.035

2-(Naphthalen-1-ylmethylene)hydrazinecarbothioamide (14, IC₅₀ = 1.1 μM) was discovered as a highly potent inhibitor of melanogenesis. To define the role of hydrogens (at N1 and N3) and sulfur in 14, a series of analogs 15a-p were synthesized an

Full text of DOI:10.1016/j.bmcl.2011.12.035

Bioorganic & Medicinal Chemistry Letters 22 (2012) 886–889
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship of naphthaldehydethiosemicarbazones
in melanogenesis inhibition
Pillaiyar Thanigaimalai a, Eeda Venkateswara Rao a, Ki-Cheul Lee a, Vinay K. Sharma 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, Republic of Korea
b College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
2-(Naphthalen-1-ylmethylene)hydrazinecarbothioamide (14, IC50 = 1.1 lM) was discovered as a highly
potent inhibitor of melanogenesis. To define the role of hydrogens (at N1 and N3) and sulfur in 14, a ser-
ies of analogs 15a–p were synthesized and evaluated for anti-melanogenic activity using melanoma B16
cells under the stimulus of a-MSH. It was observed that replacement of either of these hydrogens at N1 or
Received 19 October 2011
Revised 25 November 2011
Accepted 7 December 2011
Available online 13 December 2011
N3 by substituents increases the activity significantly. Conversely, concomitant substitutions decrease
the inhibitory potency. In addition, the presence of sulfur in thiosemicarbazone is essential for the
activity.
Keywords:
Melanogenesis inhibitor
2-(Naphthalen-1-ylmethylene)
hydrazinecarbothioamide
Skin whitening agent
Thiosemicarbazone
Ó 2011 Elsevier Ltd. All rights reserved.
Eumelanin and pheomelanin are the two basic types of melanin
pigments in human skin and hair. Although melanin has a major
role to protect the human skin from sun-related injuries, excess
accumulation of melanin in skin can causes diverse hyperpigmen-
tary disorders such as melasma, freckles and age spot.1,2 Melanin is
synthesized by a series of enzymatic catalyzed and chemical reac-
tions.3,4 Mainly, three melanocyte-specific enzymes such as tyros-
inase, tyrosine related protein-1 (TYRP-1) and TYRP-2 are involved
in the transformation of tyrosine to melanin pigments.5 It has been
reported that several factors affect the activity of these enzymes
and related proteins, which includes melanocyte stimulating hor-
mone (MSH), endothelin-1, basis fibroblast growth factor, and
UV-irradiation.6
1325 have been reported for the treatment of hyperpigmentation
disorders. However, due to adverse effects such as depigmenta-
tion,21 cytotoxicity,26 paradoxical hyperpigmentation10 dermatitis
and erythema,27 their use as cosmetic agents is being compromised.
Therefore, there is a need to find ideal hypopigmenting agents with
fewer or no side effects.
In the course of our studies to discover new hypopigmenting
agents, we have previously demonstrated the structural require-
ments of a series of phenylthiourea (PTU) and thiosemicarbazone
analogs28,29 as the subclass of synthetic melanogenesis inhibitors.
Accordingly,
p-planar connection to thiourea unit without steric
hindrance was identified as a main structural requirement in PTU
derivatives.28 Conversely, in case of thiosemicarbazones the aro-
matic group of thiosemicarbazones can be replaced with sterically
bulky carbocycles. Unlike PTU derivatives, steric bulkiness does
not hamper the activity of these thiosemicarbazones. Thus,
hydrophobicity of the benzylidene or cyclohexylmethylene group
on hydrazine of thiosemicarbazones is determinant factor for their
inhibitory activity in melanogenesis rather than planarity.29
However, the role of hydrogens at N1 and N3, and sulfur in
thiosemicarbazone has not been studied yet. In the present study,
we selected the compound 2-(naphthalen-1-ylmethylene)hydra-
Tyrosinase, a copper bearing multifunctional enzyme, is involved
in two rate limiting steps of melanin synthesis, that is, the hydrox-
ylation of tyrosine and oxidation of 2,4-dihydroxyphenylalanine
(L
-DOPA).7 Many of the presently known inhibitors act directly or
indirectly on this enzyme.8,9 Chemical agents including natural
products (Fig. 1A) such as hydroquinones 1,10 kojic acid 2,11 tropo-
lone 3,12 -minosine 4,13 hydroxylated stilbine derivatives 5,14,15
L
flavonols derivatives 6,16 methyl ester of gentisic acid 7,17,18 and
synthetic agents (Fig. 1B) such as N-phenylthiourea derivatives
8,19,20 kojic acid derivatives 9,21 oxadiazole 10,22 oxazolones 11,23
tetraketone derivative 1224 and 4,40-dihydroxybiphenyl compound
zinecarbothioamide (14, IC50 = 1.1 l
M, Fig. 2)29 as a lead, and inves-
tigated the structure–activity relationship by preparing a series of
analogs (15a–p) by replacing either of hydrogens or sulfur (Fig. 2)
and evaluating their anti-melanogenesis activity in melanoma B16
cells under the stimulus of
-MSH).
a-melanocyte stimulating hormone
⇑
Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
(a
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.035

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 886–889
A: Natural Products
887
O
O
O
OH
R1
OH
HO
N
OH
OH
NH2
1
O
O
a) Hydroquinone R = -OH
2
. Kojic acid
3
. Tropolone
R2
4.
L-Minosine
b) Arbutin R = -O(β)-Glucose
R6
R3
R4
OH O
O
OH
OH
R1
R1
R5
HO
O
HO
7.
R2
OH
6
. Flavonols
Gentisic acid
5.
Hydroxylated stilbenes,
a) Quercetin R1 = R2 = -OH
a) Resveratrol
R1 = R2 = R3 = -OH
b) Kaempferol R1 = -H, R2 = -OH
b) Oxyresverotrol R1 = R2 = R3 = R5 = -OH
1
2
c
) Apigenine R = R = -H
R1 = R2 = R5 = R6 = -OH
c
) Gnetol
B: Synthetic Agents
R1
N
O
O
N
N
R
N
HO
R2
O
O R
O
X
N
Br
8
. PTU analogs,
9.Kojic acid derivatives
R = various alkyl or alkoxy
10.Oxodiazole derivative
R, R1 = H, CH3
R2 = H, OH or OMe
X = O or S
NH2
O
O
N
O
HO
OH
O
O
O
12.Tetraketone derivative
13.4,4'-Dihydroxybiphenyl
11.Oxazolone derivative
analog
Figure 1. Known natural (A) and synthetic (B) tyrosinase inhibitors.
H
O
X
S
X
X
R1
H2N
R1
N
H
N
N
(i)
N
R1
N
N
H
N
3
1
N
N
+
N
N
R3 R2
R3 R2
H
R3 R2
15a-p
14, IC50 = 1.1 μM
15a-n p
,
16
17
R2 = H,
R
,
or
S
S
N
CH3
(ii)
R = H, CH3, Cl or OCH3
N
N
CH3
N
N
N
R1 = R3 = H, CH3 or CH2CH3.
X = O or S
H
CH3
CH3 CH3
15m
15o
Figure 2. The structures of lead 14 and their newly designed melanogenesis
inhibitors 15a–p.
Scheme 1. Synthesis of melanogenesis inhibitors 15a–p. Regents and condition: (i)
ethanol/water (1:1), reflux, 50 min; (ii)CH3I/KOH, rt. Note: Substituents (R1, R2, R3
and X) are indicated in Table 1.
Compounds 15a–p were prepared as depicted in Scheme 1.
Briefly, a hot solution of aldehyde 16 in ethanol was added to the
pre-hot solution of appropriate thiosemicarbazide 1730–34 in water
(equal volume of ethanol) and this mixture was allowed to stir for
nearly 50 min at 50–60 °C. The reaction mixture was then cooled to
5–10 °C to precipitate and the resulting thiosemicarbazone com-
pounds 15a–n were filtered.35–40 Compound 15o was synthesized
by the treatment of 15m with methyl iodide in presence of KOH
and tetra-butyl ammonium bromide in tetrahydrofuran at room
temperature for overnight.41 All thiosemicarbazones 15a–o were
then purified by column chromatography to give the pure
compounds. Semicarbazone 15p was prepared using the same
procedure used for the preparation of 15a with semicarbazide in-
stead of thiosemicarbazide.
As described previously,42,43 biological assay was performed to
determine the ability of compounds 15a–p to inhibit the formation
of melanin in melanoma B16 cells under the stimulus of
a-MSH
(100 nM) for 3 day incubation. Melanoma B16 cells (CRL6323)
were obtained from ATCC (Manassas, USA). Amounts of melanin
released into the culture media were determined by measuring
absorbance values at 405 nm with synthetic melanin as the stan-
dard. Data for % inhibition at 10
Table 1.
lM and IC50 values are shown in

888
P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 886–889
Table 1
Melanogenesis inhibitory activity of 15a–p in melonama B16 cell line
X
N
R1
N
N
R3 R2
15a-p
N–R3
Compound No:
N–R1
N–R2
X
% of Inhibition at 10 (
l
M)a
IC50 (lM)
15a37
15b
–CH3
–CH2CH3
–Ph
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–CH3
–H
–CH3
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–CH3
–CH3
–H
–H
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
S
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
76
2.00
1.10
0.50
0.58
1.90
0.86
0.45
0.83
0.49
0.92
0.27
0.29
0.28
1.60
8.60
15c38
15d
–Ph (4-CH3)
–Ph(4-Cl)
–Ph(4-OCH3)
–Ph(3-CH3)
–Ph(3-OCH3)
–Ph(2-CH3)
–Ph(2-OCH3)
–CH2Ph
–C6H11
–CH3
–H
–CH3
–H
15e
15f39
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p40
1429
Kojic acid
Arbutin
<10
100
>100
1.10
70
–H
180
Note: Substituent.
a
IC50 values are taken as a mean from 3–5 independent experiments.
As shown in the Table 1, both kojic acid and arbutin were eval-
uated in our assay as control. The initial objective of our study was
to investigate the role of hydrogens at N1 of 14. Accordingly,
replacement of one of the hydrogens by a methyl group as indi-
other hand, replacement of planar phenyl ring with more bulky
benzyl or cyclohexyl group resulted in 2–5-fold activity improve-
ment as shown in the analogs 15k (>100% inhibition at 10
l
M,
IC50 = 0.27 M) and 15l (>100% inhibition at 10 M, IC50 = 0.29 l
l
l
M).
cated in the analog 15a (>100% inhibition at 10
exhibited comparable activity to 14. Further, we introduced some
more substituents like ethyl (15b, >100% inhibition at 10
l
M, IC50 = 2.0
l
M)
This enhancement of the activity might be originated from the
increment of bulkiness or basicity at terminal nitrogen (N1) of 14.
In the next experiment, we demonstrated the inhibitory activity
by replacing both hydrogens of 14 at N1. The resulting N,N-di-
l
l
M,
M,
IC50 = 1.1
IC50 = 0.5
l
l
M) or phenyl (15c, >100% inhibition at 10
M) and observed that phenyl analog 15c had the most
methyl analog 15m (>100% inhibition at 10 lM, IC50 = 0.28 lM)
potent activity, among them. Therefore, replacement of N1-hydro-
gen by bulkier phenyl moiety is more appreciable for the activity
enhancement.
In an attempt to further improve the potency, we then examined
the effect of substituents on phenyl ring of 15c. Accordingly, analog
associate with p-substituent methyl (15d, >100% inhibition at
showed nearly 5 and 10 fold more potent activity than 14 and
mono-methyl-substituted 15a, respectively. This outcome is again
emphasizing the fact that the increment of hydrophobicity is
important for the activity. Further, the necessity of N3-hydrogen
in 14 was also explored by replacing with methyl substituent as
shown in the analog 15n (>100% inhibition at 10
IC50 = 1.60 M). This equipotent activity to 14 indicated that
N3-hydrogen can also be replaced with substituent.
lM,
10
l
M, IC50 = 0.58
l
M) or chloro (15e, >100% inhibition at 10
l
l
M,
M,
l
IC50 = 1.90
IC50 = 0.86
l
l
M) or methoxy (15f, >100% inhibition at 10
M) exhibited equipotent activity to 15c. This similar
We then investigated the substituents for all hydrogens at a same
time. As a result, the activity was dropped to low level as indicated in
fashion was also observed in the case of m- and o-substituted ana-
logs as m-methyl (15g, >100% inhibition at 10
l
M, IC50 = 0.45
M, IC50 = 0.83
M, IC50 = 0.49
M, IC50 = 0.92
l
l
M),
M),
the analog 15o (76% inhibition at 10 lM, IC50 = 8.60 lM). This low
m-methoxy (15h, >100% inhibition at 10
l
level of activity may occur from the steric congestion of the substit-
uents at both the nitrogens N1 and N3, which block the binding abil-
ity of thiosemicarbazide moiety. On the other hand, the activity was
totally mediated by the thiosemicarbazide moiety in 14. As shown
in the Table 1, an analog associated with semicarbazide unit in
o-methyl (15i, >100% inhibition at 10
o-methoxy (15j, >100% inhibition at 10
l
l
l
M), and
lM). From
these studies, it was clearly visualized that the substituent on phe-
nyl ring of 15c does not have any impact on the activity. On the
Effective hydrophobic
binding
Essential
S
1
N
H
N
H
N
H
3
Substitutions at N1 or N3: Favorable
Concurrent substitutions at N1and N3: Unfavorable
14
Figure 3. Structure–activity relationships of lead 14.

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 886–889
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the melanogenesis inhibitory activity.
In conclusion, a series of semicarbazides 15a–p were designed,
synthesized, and evaluated in for their anti-melanogenesis activity
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Acknowledgment
This work was supported by a Priority Research Centers Pro-
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