Structural characteristics of thiosemicarbazones as inhibitors of melanogenesis

Article abstract of DOI:10.1016/j.bmcl.2010.08.114

A series of thiosemicarbazones 2(e-s) have been synthesized and studied their structure-activity relationship as melanogenesis inhibitor. Among them, (Z)-2-(naphthalen-1-ylmethylene)hydrazinecarbothioamide (2q, >100% inhibition at 10 μM, IC₅₀ = 1.1 μM, C log P = 3.039) showed the strongest inhibitory activity. Regarding their structure-activity relationship, the hydrophobic substituents regardless the position on phenyl ring of benzaldehyde thiosemicarbazones enhance the inhibitory activity. Furthermore, the aromatic group of benzaldehydethiosemicarbazones can be replaced with sterically bulky cyclohexyl. Thus, hydrophobicity of the aryl or alkyl group on hydrazine of thiosemicarbazones is determinant factor for their inhibitory activity in melanogenesis rather than planarity.

Full text of DOI:10.1016/j.bmcl.2010.08.114

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6794–6796
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural characteristics of thiosemicarbazones as inhibitors of melanogenesis
Ki-Cheul Lee a, Pillaiyar Thanigaimalai a, Vinay K. Sharma a, Min-Seok Kim a, Eunmiri Roh b,
Bang-Yeon Hwang 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:
Received 3 July 2010
Revised 23 August 2010
Accepted 24 August 2010
Available online 20 September 2010
A series of thiosemicarbazones 2(e–s) have been synthesized and studied their structure–activity
relationship as melanogenesis inhibitor. Among them, (Z)-2-(naphthalen-1-ylmethylene)hydra-
zinecarbothioamide (2q, >100% inhibition at 10 lM, IC50 = 1.1 lM, C log P = 3.039) showed the strongest
inhibitory activity. Regarding their structure–activity relationship, the hydrophobic substituents regard-
less the position on phenyl ring of benzaldehyde thiosemicarbazones enhance the inhibitory activity. Fur-
thermore, the aromatic group of benzaldehydethiosemicarbazones can be replaced with sterically bulky
cyclohexyl. Thus, hydrophobicity of the aryl or alkyl group on hydrazine of thiosemicarbazones is deter-
minant factor for their inhibitory activity in melanogenesis rather than planarity.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Thiosemicarbazones
Melanogenesis inhibitor
Hydrophobicity
Tyrosinase, also known as phenoloxidase (PO) a copper bearing
bifunctional enzyme is highly present in microorganism, animals,
and plants.1,2 It is the key enzyme involved in the synthesis of
melanin through the process called melanogenesis. Melanin
synthesized from the oxidation of tyrosine to dopaquinone by
tion for the search of good skin whitening agent, researchers have
been identified that phenylthiourea (PTU, Chart 1) as one of the
best tyrosinase inhibitors15 with an IC50 value of 1.8 M.16 More-
l
over, PTU was reported for the inhibition of tyrosinase enzyme that
belongs to catechol oxidase (a type-3 copper protein).17,18 The
crystal structure of phenylthiourea (PTU) bound tyrosinase has
not been explored yet. However, the catalytic sites of catechol oxi-
dase and tyrosinase have been known to be almost similar, so we
predict that binding mode of PTU at tyrosinase active site may be
similar to catechol oxidase. The binding of sulfur atom of this com-
pound to both copper ions in the active site of the enzyme19 and
the van der Waals interactions of phenyl group with the residues
lining the hydrophobic cavity (Phe 261, Ile 241, His 244) contribute
to the high affinity of the PTU to the enzyme.19
tyrosinase, which occurs in two steps: hydroxylation of
L-tyrosine
to -3,4-dihydroxyphenylalanine ( -DOPA) and then oxidation of
L
L
the latter to an o-quinone (dopaquinone).3 The first one is the
key step in melanogenesis because the reminder of the reaction
sequence can proceed spontaneously at physiological pH.4 Tyrosi-
nase play an important role in human neuromelanin formation in
the substantia nigra portion of the brain and it may also contribute
to Parkinson disease-related to neurodegeneration.5,6 In mammals,
tyrosinase is primarily responsible for skin pigmentation defects
and abnormalities such as flecks.7 In insects, tyrosinase is uniquely
associated with three different biochemical process, including
sclerotization of cuticle, defensive encapsulation and melanization
of foreign organism, and wound healing.8 It is possible that inhibi-
tion of PO could lead to abrogation of insect defense mechanisms
or abnormal body softening, both of which could be used in pest
control. Thus, tyrosinase plays a quite significant role in various
industries such as medicine,9 agriculture, and cosmetic products,10
primarily in relation to hyperpigmentation. The current available
therapies are considered to be inadequate for the skin treatment
despite many compounds have been reported as tyrosinase
inhibitor.11–14 Therefore, there is an urgent need for new molecules
which can effectively be used for the skin treatment. In continua-
H
H
N
NH2
N
NH2
R1
N
S
S
1
2 (a-s)
R1
=
R
N
R = H, CH3, OCH3, CH2CH3, C(CH3)3, Cl or OH.
⇑
Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
Chart 1. Antimelanogenesis agents: phenylthiourea
2(a–s).
1 and thiosemicarbazones
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.114

K.-C. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6794–6796
6795
Table 1
H
H
H
Inhibitory activity of thiosemicarbazones 2a–s against melanogenesis in melanoma
B16 cells
N
NH2
N
NH2
R1
O
R1
N
H2N
S
S
H
3 (e-s)
4
2 (e-s)
N
NH2
R1
N
S
Scheme 1. Synthesis of thiosemicarbazone analogs. Reagents and conditions: (i)
heat, 20 min, water/ethanol (1:1). Note: R1 = substituents are located in Table 1.
2(a-s)
Entry
No.
Compd
No.
R1
Inhibition at
10
M (%)b
IC50
C log Pk
In our previous study,20 we defined the structural requirements
of phenylthiourea derivatives and some benzaldehydethiosemicar-
bazones 2a–d for their inhibitory activities against melanogenesis
in melanoma B16 cells. Interestingly, some of the compounds such
as ethyl and tert-butyl derivatives of benzaldehydethiosemicarbaz-
one showed very potent activity among the thiourea derivatives.
l
valuesj
1
2
3
2aa
2ba
2ca
C6H4
p-(CH3)C6H4
p-
(CH2CH3)C6H4
p-
<10
64
>100
>10
6.8
3.4
1.865
2.364
2.893
4
2da
>100
2.7
3.691
(C(CH3)3)C6H4
p-(Cl)C6H4
p-(OCH3)C6H4
p-(OH)C6H4
m-(CH3)C6H4
m-(Cl)C6H4
m-
(OCH3)C6H4
m-(OH) C6H4
o-(CH3)C6H4
o-
(CH2CH3)C6H4
o-(Cl)C6H4
o-(OCH3)C6H4
o-(OH)C6H4
Naphthyl
Cyclohexyl
Pyridyl
2eb
2fb
2gb
2hb
2ic
92
42
16
>100
>100
40
4.2
15.1
17.5
2.8
2.4
15.4
2.802
1.920
1.198
2.364
2.802
1.920
Along with that, the
p-planar structure to thiourea unit without
5
6
7
8
9
steric hindrance in PTU derivatives is required for potent inhibitory
activity. However, in case of thiosemicarbazones, a hydrophobic
substituent at p-position is essential for the same. Thus, the de-
tailed structure–activity relationship of thiosemicarbazones to-
ward their inhibition of melanogenesis in melanoma B16 cells is
required. Therefore, in the current study we prepared a series of
thiosemicarbazones 2e–s as shown in Chart 1 and evaluated their
inhibitory activity against the formation of melanin in melanoma
B16 cells.
10
2jb
11
12
13
2kb
2ld
2m
28
>100
>100
24
3.4
2.7
1.198
2.364
2.899
14
15
16
17
18
19
2ne
2of
2pg
2qh
2r
>100
52
24
>100
81
15
1.4
16.1
21
1.1
3.4
2.802
1.920
1.198
3.039
1.987
1.088
All substituted thiosemicarbazones 2 were synthesized by the
treatment of appropriate benzaldehyde with thiosemicarbazide
as shown in Scheme 1.20,21 Briefly; a hot (40–50 °C) solution of
an appropriate aldehyde 3e–s in ethanol (30 mL) was added to
the pre-heated (40–50 °C) solution of thiosemicarbazide 4 in water
(30 mL) and allowed to stir for 10–15 min at same temperature
(40–50 °C). The reaction mixture was immediately cooled about
to 5–10 °C and resulting title compound (2e–s)22–29was filtered.
All the above-synthesized derivatives were tested for their inhibi-
tory activity of melanogenesis in melanoma B16 cell line30 as
shown in Table 1 rather than tyrosinase itself since the purpose
of these inhibitors is to reduce the formation of melanin.
2si
>30
Note: a2(a–d)20
,
b2(e–h, j–k)19
,
c2i23
,
d2l24
,
e2n25
,
f2o26
,
g2p27
,
h2q28 and i2s29 are
j
consistent with references. IC50 values are taken as mean from three independent
experiments. kC log P values are calculated by Chemdraw ultra 9.0v.
(28% inhibition at 10
showed the poor activity.
Unlike in PTU derivatives,20 the o-methyl substituted thiosemi-
carbazone 2l (>100% inhibition at 10 M, IC50 = 3.4 M,
C log P = 2.364) showed almost comparable activity to p-(2b) and
m-(2h) substituted derivatives. These results encourage us to go
for detail study regarding the substituent effect at o-position of
thiosemicarbazones on melanogenesis inhibition. Thus, some more
analogs with bulky substituents were synthesized such as o-ethyl
lM, IC50 = 24 lM, C log P = 1.198) also
As we discussed earlier,20 the hydrophobic bulky substituent at
p-position of benzaldehyde thiosemicarbazones play an important
role in enhancing the activity of thiosemicarbazones. As compared
to simple benzaldehyde thiosemicarbazone 2a (<10% inhibition at
l
l
10
lM, IC50 >0 lM, C log P = 1.865), the other bulky group contain-
ing compounds such as 2b (64% inhibition at 10
l
M, IC50 = 6.8
M, IC50 = 3.4
M, IC50 = 2.7
lM,
lM,
lM,
(2m, >100% inhibition at 10
and o-chloro analogs (2n, >100% inhibition at 10
l
M, IC50 = 2.7
l
M, C log P = 2.893)
C log P = 2.364), 2c (100% inhibition at 10
l
lM, IC50 = 1.4 M,
l
C log P = 2.893) and 2d (100% inhibition at 10
l
C log P = 2.802). As indicated in Table 1, they also turned up with
good inhibition against melanogenesis as potent as p and m bulky
substituted analogs. This outcome supports that the substituents at
o-position did not disturb the ‘clamp structure’ (H–N–C–N–H)31 of
thiourea unit which has been known as essential structure for
activity. However, the electron donating substituents at o-position
C log P = 3.69) showed enhanced activity. However, the detailed
study is required about the role of hydrophobic and hydrophilic
groups at that position. Therefore, in the first set of experiment,
we introduced the hydrophobic or hydrophilic groups at the same
position as shown in halogen analog 2e (92% inhibition at 10
IC50 = 4.2 M, C log P = 2.802), methoxy analog 2f (42% inhibition
at 10 M, IC50 = 15.1 M, C log P = 1.902) and hydroxyl analog 2g
(16% inhibition at 10 M, IC50 = 17.5 M, C log P = 1.198). Hydro-
lM,
l
as shown in 2o (52% inhibition at 10
l
M, IC50 = 16.1
M, IC50 = 21
l
M,
M,
l
l
C log P = 1.920) and 2p (24% inhibition at 10
l
l
l
l
C log P = 1.198) exhibited very low inhibition similarly as we dis-
cussed above in case of p- and m-positions.
phobic substituted chloro analog enhanced the activity however,
polar substituents like methoxy and hydroxy reduced the activity.
These results imply that the bulky hydrophobic groups at p-posi-
tion of benzaldehydethiosemicarbazones effectively enhance the
inhibitory activity of melanogenesis on melanoma B16 cells.
After confirming the role of substitution at p-position of thio-
semicarbazone, we shifted our focus toward m-substitution effect
of thiosemicarbazones on activity. Interestingly, the hydrophobic
substituents at m-position as shown in 2h (>100% inhibition at
The above findings suggest that the hydrophobic or bulky sub-
stituents at p- or m- or o-position play an important role for the
increment of the activity of thiosemicarbazones. Conversely, the
hydrophilic or electron donating groups at p- or m- or o-position
of thiosemicarbazones were not favorable for the inhibition of
melanogenesis.
To find the optimum size of aromatic group in thiosemicarba-
zones for their inhibitory activity, we extended the phenyl ring
in 2a to naphthyl moiety. Surprisingly, the naphthyl analog 2q
10
10
l
l
M, IC50 = 2.8
M, IC50 = 2.4
l
M, C log P = 2.364) and 2i (>100% inhibition at
lM, C log P = 2.802) showed almost same activity
(>100% inhibition at 10 lM, IC50 = 1.1 lM, C log P = 3.039) showed
very strong inhibition than 2a. This result indicated that naphthyl
as p-substituted derivatives. As indicated in p-derivatives 2f and
2g, the electron donating substituent at m-position such as 2j
group is more beneficial than a phenyl group due to its larger plane
(40% inhibition at 10 lM, IC50 = 15.4 lM, C log P = 1.920) and 2k

6796
K.-C. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6794–6796
zones. Thus hydrophobicity of the aryl or alkyl group on hydrazine
of thiosemicarbazones is determinant factor for their inhibitory
activity in melanogenesis rather than planarity.
S
H
N
N
NH2
N
H
NH2
N
S
2a, IC50 >10 µM,
C log P = 1.865
Acknowledgments
2q, IC50 = 1.1 µM,
C log P = 3.039
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.
lncreament of
liphophilicity
poor transfer
good transfer
Supplementary data
Figure 1. The correlation of inhibitory activity of 2a and 2q with their liphophilicity
on melanoma B16 cell membrane.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.114.
size. In addition, the naphthyl group also increases the hydropho-
bicity. Thus to confirm the necessity of aromatic group, phenyl
group of 2a was replaced with bulky cyclohexyl group as shown
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In conclusion, we have design, synthesized and tested a series of
thiosemicarbazones 2(e–s) as inhibitors of melanogenesis for
defining their structure–activity relationship (SAR) studies. As a re-
sult (Z)-2-(naphthalen-1-ylmethylene)hydrazinecarbothioamide
(2q) proved the best melanogenesis inhibitor among synthesized
compounds. Regarding SAR, we observed the two important points.
Substituent’s with highly hydrophobic property regardless the po-
sition of phenyl ring of the thiosemicarbazones is more important
for enhancement of the activity than the hydrophilic one. In addi-
tion, the aromatic group of thiosemicarbazones can be replaced
with sterically bulky carbocycles. Unlike PTU derivatives, steric
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