Structural requirement(s) of N-phenylthioureas and benzaldehyde thiosemicarbazones as inhibitors of melanogenesis in melanoma B 16 cells

Article abstract of DOI:10.1016/j.bmcl.2010.02.067

In order to define the structural requirements of phenylthiourea (PTU), a series of thiourea and thiosemicarbazone analogs were prepared and evaluated as inhibitors of melanogenesis in melanoma B16 cells. The most potent analog was 2-(4-tert-butylbenzylidene)hydrazinecarbothioamide (1u) with an IC₅₀ value of 2.7 μM in inhibition of melanogenesis. The structure for potent inhibitory activity of these derivatives are required with the direct connection of π-planar structure to thiourea without steric hinderance in PTU derivatives and the hydrophobic substituent at para position in case of semicarbazones.

Full text of DOI:10.1016/j.bmcl.2010.02.067

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2991–2993
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural requirement(s) of N-phenylthioureas and benzaldehyde
thiosemicarbazones as inhibitors of melanogenesis in melanoma B 16 cells
P. Thanigaimalai a, Tuan Anh Le Hoang a, Ki-Cheul Lee a, Seong-Cheol Bang a, Vinay K. Sharma a,
Cheong-Yong Yun b, 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, South Korea
b College of Pharmacy, Chungbuk National University, Cheongu 361-763, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to define the structural requirements of phenylthiourea (PTU), a series of thiourea and thiosem-
icarbazone analogs were prepared and evaluated as inhibitors of melanogenesis in melanoma B16 cells.
The most potent analog was 2-(4-tert-butylbenzylidene)hydrazinecarbothioamide (1u) with an IC50
Received 21 December 2009
Revised 25 January 2010
Accepted 17 February 2010
Available online 20 February 2010
value of 2.7
lM in inhibition of melanogenesis. The structure for potent inhibitory activity of these deriv-
atives are required with the direct connection of
p
-planar structure to thiourea without steric hinderance
in PTU derivatives and the hydrophobic substituent at para position in case of semicarbazones.
Keywords:
Ó 2010 Published by Elsevier Ltd.
Tyrosinase inhibitor
Melanogenesis inhibitor
Phenylthiourea and benzaldehyde
thiosemicarbazone
In mammals, melanin production is primary responsible for the
skin color, and melanin plays vital role in the absorption of free
radicals formed in cytoplasm, in protecting human skin from the
harmful UV-radiation and in scarvanging chemicals.1 The biosyn-
thesis of melanin (melanogenesis) is a complex pathway involving
enzymatic and chemical reaction in melanocyte cells.2–5 The mel-
enzyme belongs with catechol oxidase to the type-3 copper protein
group.21,22 Crystal structure of PTU bound catechol oxidase was re-
ported.21,22 The sulfur atom of this compound binds to both copper
ions in the active site of the enzyme.22 In addition to that interac-
tion with the dicopper center, the van der Waals interactions be-
tween the residues lining the hydrophobic cavity (Phe 261, Ile
241, His 244) contribute to the high affinity of the PTU to the en-
zyme.22 Although the crystal structure of PTU bound tyrosinase
has not been known, the binding mode of this compound would
be very much similar22 since the active site23 of tyrosinase is highly
conserved and has similar hydrophobic cavity with Tyr 98.
To our knowledge, there have been no reports concerning about
the structure–activity relationship (SAR) of phenylthiourea ana-
logs. Therefore, we evaluated a series of phenylthiourea derivatives
1a–q and thiosemicarbazones 1r–u for their inhibitory activity and
studied SAR on melanogenesis. The general structures of PTU and
thiosemicarbazone analogs were shown in Chart 1.
anin synthesized from the conversion of simple amino acid,
L-tyro-
sine, into 3,4-dihydroxyphenylalanine -DOPA) and the
(L
subsequent oxidation to form dopaquinone by catalyzing the
tyrosinase, the membrane bound copper bearing phenolase, which
is the critical enzyme for melanin biosynthesis in melanosomes
produced by melanocytes.6–8 In man, defects in the enzyme are re-
lated to a number of pathologies such as oculoutaneous albinism.
However, the excessive formation and an abnormal aggregation
of melanin in different specific parts can cause diverse hyperpig-
mentary disorders such as melasma, freckles, age spot, and post-
inflammatory melanoderma.9,10 Therefore, the regulation of mela-
nin synthesis by inhibiting the tyrosinase enzyme is a current re-
search topic in the context of preventing hyperpigmentation. In
this regard, diverse tyrosinase inhibitors have been actively discov-
ered such as kojic acid,11 arbutin,12 ascorbic acid derivatives,13
hydroxylstilbine derivatives like resveratol14–16 and methyl ester
of genistic acid.17,18
The thiourea derivatives 1a–q are obtained from benzoyl isothi-
ocyanate and commercially available amines as illustrated in
Scheme 1. Briefly, the intermediate benzoyl isothiocyanate 3 was
S
H
n
N
NH2
N-Phenylthiourea (1a, PTU) has long been known as reference
N
N
NH2
R
R
inhibitor19 against tyrosinase with an IC50 value of 1.8 M.20 This
l
H
S
1(r-u)
1(a-q)
n = 0, 1, 2, 3 or 4.
* Corresponding author. Tel.: +82 42 821 5939; fax: +82 42 823 6566.
E-mail address: jungsh@cnu.ac.kr (S.-H. Jung).
R = H, CH3, CH2CH3, C(CH3)3, Cl, CH2OH or OCH3
Chart 1. General structure of phenyl thiourea and thiosemicarbazone analogs.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.02.067

2992
P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2991–2993
O
O
S
O
n
n
(ii)
NH2
Cl
N
H
N
H
NCS
(i)
R
R
5(a-p)
4(a-p)
2
3
(iii)
n = 0, 1, 2 ,3 or 4
S
n
N
H
NH2
R
1 (a-p)
Scheme 1. Synthesis of phenylthiourea analogs. Reagents and conditions: (i) NH4SCN, 60 °C, 15 min; (ii) 60 °C, 30 min; (iii) 5% NaOH, 70 °C, 20 min. Note, R = substituents are
located in Table 1.
prepared from benzoyl chloride 2 by treating with ammoniumthi-
ocyanate at reflux for 30 min in acetonitrile.24 Intermediate 5 was
obtained by treating 3 with amines 4 at reflux for 30 min and then
was hydrolyzed by hot 5% NaOH for 20 min to give the desired
compounds 1a–q.24–28 The substituted semithiocarbazones 1r–u
synthesized by the treatment of appropriate benzaldehyde 6a–d
with semicarbazide 7 as shown in Scheme 2.29,30 All the above-syn-
thesized derivatives were tested for their inhibitory activity of
melanogenesis in melanoma B16 cell line16 as shown in Table 1
rather than tyrosinase itself since the purpose of these inhibitors
is to reduce the formation of melanin.
at this position such as ethyl (1j: >10% inhibition at 10
IC50 = >10 M, C log P = 1.733), tert-butyl (1k: >10% inhibition at
10 M, IC50 = >10 M, C log P = 2.571), methoxy (1l: >10% inhibi-
tion at 10 M, IC50 = >10 M, C log P = 0.847) and hydroxymethyl
derivative (1m: >10% inhibition at 10 M, IC50 = >10 M,
C log P = À0.853). These compounds also did not turn up with
any inhibition. The above findings lead us to predict that the C-2
substituted phenyl ring may hinder the complex formation be-
tween thiourea unit and the metallic center of the active site in en-
zyme due to the steric hindrance.
In addition to confirm the importance of the direct connection
of phenyl to thiourea of PTU, methylene units were inserted be-
tween them. The resulting compounds 1n–q did not exhibit any
inhibition against melanogenesis. This implies that increasing
bulkiness with sp3 carbons in this position may prevent the access
lM,
l
l
l
l
l
l
l
As reported earlier,20 PTU (1a) showed moderate inhibition
(84% inhibition at 10 lM, IC50 = 5.4 lM, C log P = 0.745) against
melanogenesis. In order to explore the role of substituents on the
phenyl ring of PTU, we introduced various substituents at different
positions of phenyl ring. The p-methyl substituted analog 1b (83%
of thiourea group into the active site and
p, p stacking of phenyl
inhibition at 10
rable activity with 1a. Thus, increasing the bulkiness at this posi-
tion such as 4-ethylphenylthiourea, 1c (88% inhibition at 10 M,
IC50 = 4.9 M, C log P = 1.773), 4-tert-butylphenylthiourea, 1d
(95% inhibition at 10 M, IC50 = 3.7 M, C log P = 2.571) and 4-
chlorophenylthiourea, 1e (84% inhibition at 10 M, IC50 = 4.0 M,
l
M, IC50 = 5.5
lM, C log P = 1.244) showed compa-
group.23 Thus, direct connection of planar phenyl to thiourea is
critical for activity.
l
For more detailed studies regarding p system of PTU deriva-
l
tives, thiosemicarbazone derivatives 1r–u were synthesized as
shown in Table 1. The compounds 1r and 1u were reported for
their inhibitory activity against tyrosinase from Pieris rapae (Lepi-
doptera) larvae,31 but with very weak inhibition. Accordingly,
l
l
l
l
C log P = 1.764) also exhibited potent inhibition. To examine the ef-
fect of hydrophilic or highly electron donating group, we prepared
benzaldehyde thiosemicarbazone (1r, >10% inhibition at 10
l
M,
the hydroxyl analog 1f (70% inhibition at 10
C log P = 0.073) and the methoxy compound 1g (80% inhibition at
10 M, IC50 = 5.6 M, C log P = 0.847), which also exhibited compa-
rable activity to 1a. These data suggest that variation of the bulki-
ness or lipophilicity of substituents at p-position of phenyl ring of
PTU hardly affect the inhibitory activity, which indicates that these
groups do not participate in the interaction of phenylthioureas 1a–
g with the active site of tyrosinase.
Substitution at m-position as shown in 1h (78% inhibition at
10 lM, IC50 = 5.0 lM, C log P = 1.244) gave almost similar effect
on the activity as p-substitution. Thus, m-substitution of PTU dose
not disrupt the binding mode of PTU to the active site.
Interestingly, o-methyl substituted compound 1i (>10% inhibi-
tion at 10 lM, IC50 = >10 lM, C log P = 1.244) did not show any
inhibition. This result made us more curious regarding the substi-
tution at o-position and its effect on inhibition in melanogenesis.
Thus, we synthesized compounds with bulkier group substitutions
l
M, IC50 = 6.3
l
M,
IC50 = >10 lM, C log P = 1.865) did not show any activity in our bio-
l
l
Table 1
Inhibitory activity of thiones 1a–u against melanogenesis in melanoma B16 cells
Compd
R
n
Inhibition at 10
l
M
IC50 values (l
M) C log Pb
1aa
1ba
1ca
1da
1ea
1fa
1ga
1ha
1ia
4-H
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
—
—
—
—
84
83
88
95
84
70
80
78
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
64
5.4
5.5
4.9
3.7
4.0
6.3
5.6
5.0
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6.8
0.745
1.244
1.213
2.571
1.764
0.073
0.847
1.244
1.244
1.213
2.571
0.847
À0.853
1.074
1.403
1.782
2.311
1.865
2.364
2.893
3.691
4-CH3
4-CH3CH2
4-C(CH3)3
4-Cl
4-OH
4-OCH3
3-CH3
2-CH3
2-CH3CH2
2-C(CH3)3
2-OCH3
2-CH2OH
H
H
H
H
H
1ja
1ka
1la
1m
1na
1o
1p
1q
O
H
H
N
NH2
N
NH2
1ra
1sa
1ta
1ua
(i)
H
H2N
N
4-CH3
4-CH3CH2
4-C(CH3)3
S
S
R
R
100
100
3.4
2.7
7
6(a-d)
1(r-u)
a
Analytical data of compounds 1a,24 1b,24 1c,28 1d,26 1e,24 1f,25 1g,24 1h,24 1j,27
Scheme 2. Synthesis of benzaldehyde thiosemicarbazone analogs. Reagent and
conditions: (i) heat, 20 min, H2O–ethanol. Note, R = substituents are located in
Table 1.
1k,26 1l,24 1n,25 1r,29 1s,29 1t,29 and 1u,30 are consistent with references.
b
C log P values were calculated by Chemdraw 9.0.

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2991–2993
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Supplementary data associated with this article can be found, in
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