Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase

Article abstract of DOI:10.1016/j.bmcl.2011.09.024

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3- alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N′-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.

Full text of DOI:10.1016/j.bmcl.2011.09.024

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6824–6828
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural requirement of phenylthiourea analogs for their inhibitory activity
of melanogenesis and tyrosinase
a
a
a
a
b
c
Pillaiyar Thanigaimalai , Ki-Cheul Lee , Vinay K. Sharma , Cheonik Joo , Won-Jea Cho , Eunmiri Roh ,
c
a,
⇑
Youngsoo Kim , Sang-Hun Jung
College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764, Republic of Korea
College of Pharmacy, Chonnam National University, Kwangju, Republic of Korea
a
b
c
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 28 July 2011
Revised 6 September 2011
Accepted 7 September 2011
Available online 13 September 2011
Effect of a series of 1-phenylthioureas 1a–k and 1,3-disubstituted thioureas 2a–k were evaluated against
melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase
of 1-phenylthioureas 1a–k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in
melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-
diaryl or 1-phenyl-3-alkylthioureas, 2a–k, appears as melanogenic inhibitor without inhibition of tyros-
inase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing
explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N -
substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
0
1
1
-Phenylthioureas
,3-Disubstituted thioureas
Melaninogenic inhibitor
Tyrosinase inhibitor
Docking study
1
3–15
Pigmentation of mammals skin results from the production of
melanin by melanocytes through the process called melanogene-
sis. It protects the human skin from sun related injuries. Excess
accumulation or production of melanin in specific parts of the skin
characterizes diverse hyperpigmentary disorders such as melasma,
freckles, post-inflammatory melanoderma and age spot.1 Melanin
synthesis or melanogenesis is a complex pathway involving series
of chemical and enzymatic reactions.3 Among them, three en-
zymes such as tyrosinase, tyrosine related protein-1 (TRP-1) and
TRP-2 are involved in the transformation of tyrosine to melanin
hydroxylstilbine derivatives like resveratol
and methyl ester
of gentisic acid. Most of the inhibitors are structurally similar
to tyrosine or -DOPA, which act as suicide substrates of
1
6
L
1
7
tyrosinase.
Phenylthiourea (PTU, 1a) (Fig. 1) has long been known as a ref-
erence tyrosinase inhibitor with an IC50 value of 1.8
,2
18
19
lM. This
enzyme belongs to the type-3 copper protein group as a catechol
,4
20,21
oxidase.
More recently, we described the structural–activity
relationships (SARs) of series of phenylthiourea analogs (1a–k,
Fig. 1) against melanin formation in melanoma B16 cells.22 How-
ever, the mechanism of these analogs was not fully identified. In
the present report, we corroborated their mechanistic pathway
by experimenting on mushroom tyrosinase activity. In addition,
we designed, synthesized, and evaluated a series of 1,3-disubstut-
ed thioureas (2a–k), where the amino hydrogens in primary amine
5
pigments. Tyrosinase, a copper-containing membrane-bound gly-
coprotein, is the most critical and rate limiting enzyme that cata-
lyzes the first two steps in the biosynthetic pathway of melanin:
hydroxylation of tyrosine to
L-dihydroxyphenylalanine (L-DOPA)
and oxidation of
L
-DOPA to dopaquinone.6
Numerous tyrosinase inhibitors have been reported for their
use in cosmetics and pharmaceutical products as a way of prevent-
ing the over production of melanin. Hydroquinone is first discov-
ered as one of the most potent whitening agent but since its
introduction some adverse effects have been recognized.7 In re-
cent years, several tyrosinase inhibitors have been identified such
2
(3-NH ) of 1a were replaced with various substitutions. Further-
more, the molecular docking study demonstrated to get insight
in the mechanism of action with selected analogs of 1 and 2.
,8
S
S
9
10
11
12
as azelaic acid, kojic acid, arbutin, ascorbic acid derivatives,
R
nN
H
NH2
N
H
N
R
R
1
1
a-k
2
a-k
⇑
Corresponding author. Tel.: +82 42 821 5939, cell: +82 10 2665 5939; fax: +82
2 823 6566.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
4
0
Figure 1. Structure of 1-phenythiourea and 1, 3- disubstituted thiourea derivatives
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.024

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6824–6828
6825
respect to PTU (1a, 85% inhibition at 10
l
M, IC50 = 1.5
l
M) as indi-
R
S
HN
R
6
(i)
cated in the activity of p-methyl (1b, 84% inhibition at 10
IC50 = 1.4 M) p-ethyl (1c, 87% inhibition at 10 M, IC50 = 0.8
p-tert-butyl (1d, 94% inhibition at 10 M, IC50 = 1.7 M) and
m-methyl phenylthiourea (1e, 98% inhibition at 10 M, IC50
0.60 M) in Table 1. Thus, the characteristics of SAR of these ana-
l
M,
R
1
N
5
C
S
N
H
N
R
l
l
l
M),
1
l
l
2
a-k
l
=
Scheme 1. Synthetic outline of 2a–k. Reagents and condition: (i) Acetonitrile, rt, 3–
h.
l
4
logs in the inhibition of tyrosinase seem to be the same as those
in the inhibition of melanogenesis in melanoma B16 cells.
In an attempt to confirm further, the o-substituted derivatives
Syntheses of compounds 2a–k23–33 were prepared as depicted
in the Scheme 1. Briefly, the treatment of phenylisothiocyanate
5) with appropriate amine (6) in acetonitrile at room temperature
(1f–h) were tested for their inhibitory activity against tyrosinase
as shown in Table 1. These o-substitutions on PTU abolished the
activity in melanogenesis regardless of the size of o-substituents.
However, their activity in the inhibition of tyrosinase was gradu-
ally decreased with increasing the size of o-substituents as
(
for 3–4 h produced the target compounds 2a–k.
The mushroom tyrosinase activity of all these derivatives 1a–k
and 2a–k was measured as reported in the previous study.
Briefly, oxidation of L-tyrosine by tyrosinase was spectrophoto-
metrically determined and the initial rate of dopachrome forma-
tion was determined as the increase of absorbance at wavelength
492/min) by using a Molecular Devices Micro-
M and the IC50 values for 1a–k
have taken as a mean from 3–5 independent experiments as shown
1
5,34
indicated in the o-methyl (1f, >100% inhibition at 10
.84 M), o-ethyl (1g, 31% inhibition at 10 M, IC50 = 27.1
and o-tert-butyl (1h, <10% inhibition at 10 M, IC50 >10
lM, IC50
=
0
l
l
l
lM)
lM).
Although there is slight discrepancy in SAR characteristics between
their inhibition of melanogenesis and inhibition of tyrosinase, still
the steric congestion with the o-substitution of phenyl ring of
phenylthioureas exerted the adverse effect in inhibition of both
melanogenesis and tyrosinase.
4
92 nm per min (
D
A
plate reader. The % inhibition at 10
l
in Table 1 and for 2a–k in Table 2.
To determine the melanin inhibitory activity, a series of com-
pounds 1a–k and 2a–k were assayed on melanoma B16 cells un-
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 standard. The % inhibition at 10 lM and the IC50 values were
The methylene unit insertion between phenyl and thiourea
remarkably reduces the inhibitory activity of tyrosinase as ob-
2
2
-MSH (100 nM).1
5,35,36
der the stimulus of
a
served in 1i (35% inhibition at 10
tion at 10 M, IC50 >30 M) and 1k (20% inhibition at 10
30 M). These results of 1i–k against tyrosinase are well corre-
lM, IC50 >30
l
M), 1j (18% inhibi-
(
l
l
lM, IC50
>
l
22
lated with their melanogenic inhibitory activity. From the above
studies, the SARs of phenylthioureas against mushroom tyrosinase
activity is parallel to results of inhibition of melanin formation in
taken as mean from 3–5 independent experiments as shown in
Tables 1 and 2.
22
melanoma B16 cells. Therefore, it can be concluded that the inhi-
In the first attempt, we focused on tyrosinase inhibitory activity
of the previously reported thioureas 1a–k for the inhibition of mel-
anin formation. As shown in Table 1, PTU (1a) and p- or m-substi-
tuted phenylthioureas (1b–e) have shown very strong potency in
the tyrosinase inhibitory activity as well as melanogenesis inhibi-
tory activity. On comparing with our previous result of melanogen-
bition of melanogenesis of phenylthioureas 1a–k in melanoma B16
cell line is a consequence of the inhibition of tyrosinase catalytic
activity. In addition, this result proves that the direct p-planar con-
nection to thiourea unit in N-phenylthioureas is absolutely re-
quired for their inhibitory activity in tyrosinase and eventually
melanogenesis. Although the crystal structure of PTU bound tyros-
inase has not been known, reported crystal structure of PTU bound
2
2
2
2
ic inhibition, the tyrosinase inhibition of p- and m-hydrophobic
substitution of phenylthioureas shows negligible differences with
20,21
catechol oxidase highly supports this structural characteristics.
The sulfur atom of this compound binds to both copper ions in the
active site of the enzyme and the phenyl group interacts with the
Table 1
2
1
hydrophobic cavity.
Since the binding mode of N’ site of PTU has not been explored,
we investigated the role of amino hydrogens (3-NH ) of PTU (1a)
1
-Phenylthioureas (1a–k), melanin formation and tyrosinase inhibition
S
2
nN
H
NH2
through their replacement with alkyl or phenyl substitutions. Thus,
a series of analogs, 2a–k, were synthesized and initially tested
against melanin formation in melanoma B16 cell line. The methyl
R
1
a-k
derivative 2a (30% inhibition at 10
low activity compared to the PTU. However, the phenyl substitu-
tion retains the activity as shown in 2b (88% inhibition at 10 M,
IC50 = 4.5 M). Therefore, introduction hydrophobic group on para
lM, IC50 = 35 lM) shows very
Entry No.a
R
n Inhibition of melanin B16 Inhibition of mushroom
a
cells
tyrosinase
l
%
at 10
Inhibitionb IC50
(
l
M) % Inhibitionb IC50
(l
M)
l
(
l
M)
(at 10
lM)
position of the phenyl ring of 2b was expected to increase the
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
–H
4-CH
4-CH
0 84
0 83
0 88
0 95
0 78
0 <10
0 <10
0 <10
1 14
2 16
3 18
54
5.4
5.5
4.9
3.7
85
84
87
94
98
>100
31
<10
41
18
1.5
1.4
0.8
1.7
0.6
0.84
27.1
>100
25.4
>100
64.8
70.4
activity. Accordingly, the derivatives with p-methyl (2c, 79% inhi-
3
2
bition at 10
lM, IC50 = 6.3
lM) or p-chloro (2d, >100% inhibition
CH
3
at 10 M, IC50 = 3.4
l
l
M) were studied. However, these substitu-
4-C(CH
)
3 2
tions did not influence the activity of 2b. Conversely, the increasing
hydrophilicity in this position of 2b resulted in dropping the activ-
3-CH
2-CH
2-CH
2-C(CH
–H
3
3
2
5.0
>10
>10
>10
>10
>10
>10
15.8
CH
3
ity as indicated in p-methoxy 2e, (59% inhibition at 10
IC50 = 8.2 M) or p-hydroxy analog 2f, (24% inhibition at 10
IC50 = 20.7
l
l
M,
M,
)
3 2
l
l
M). Therefore, phenyl substitution was considered to
j
k
–H
–H
be optimal in 2b.
28
30
c
To prove the necessity of this additional planar phenyl substitu-
tion for the activity of 2b, phenyl group in 2b was replaced with
bulkier cyclohexyl moiety as shown in 2g (>100% inhibition at
Kojic acid
a
Compounds 1a–k was reported as melanogenesis inhibitors in the previous
study.22
b
10
lM, IC50 = 2.4 lM), which shows nearly two-fold more potent
%
of inhibition values taken as a mean from 3–5 independent experiments.
c
Kojic acid used as a positive control.
melanogenic inhibition than 2b. Thus, the increments of bulkiness

6
826
P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6824–6828
Table 2
Structures of 2a–k, melanin formation and tyrosinase inhibition
S
R
N
H
N
R
1
2
a-k
Entry No
R
R1
Inhibition of melanin B16 cellsa
Inhibition of mushroom tyrosinasea
%
Inhibitiona (at 10
l
M)
IC50
35
(l
M)
% Inhibitiona (at 10
l
M)
IC50 (lM)
2
a23
b24
CH3
–H
–H
30
88
<10
>100
>100
2
4.5
6.3
<10
<10
2
2
2
2
c25
d26
e27
f28
–H
–H
–H
–H
79
>100
>100
>100
>100
CH3
>100
59
3.4
<10
<10
<10
Cl
O
8.2
24
20.7
OH
2
2
2
g29
h30
i31
–H
–H
–H
>100
91
2.4
4.7
2.2
<10
<10
<10
>100
>100
>100
>100
2
2
j32
–H
>100
1.8
<10
>100
k
30
54
19.6
15.8
<10
30
>100
70.4
Kojic acidb
a
%
of inhibition values taken as a mean from 3–5 independent experiments.
b
Kojic acid used as a positive control.
and hydrophobicity on N’-position of 2a are more important than
planarity of phenyl ring. Therefore, we inserted the methylene
units between thiourea (3-NH) and phenyl units in 2b. As a result,
the activity was gradually enhanced by increasing the length of
methylene unit as shown in methylene (2h, 91% inhibition at
537 inhibit the biosynthesis of tyrosinase for melanogenic inhibi-
tion in melanoma B16 cell line under the stimulation of -MSH
Thus, di-substi-
tuted thioureas 2a–k are considered to be analogs of 4 and non-
tyrosinase related melanogenic inhibitor.
a
3
6,37
without direct inhibition of tyrosinase (Fig. 2).
1
0
l
M, IC50 = 4.7
l
M), ethylene (2i, >100 inhibition at 10
l
l
M,
M,
In addition, the binding models of 1e and 2b with tyrosinase en-
zyme are simulated using Surflex-Dock in Sybyl version 8.1.1 by Tri-
pos Associates, operating under Red Hat Linux 4.0 with an IBM
computer (Intel Pentium 4, 2.8 GHz CPU, 1 GB memory). A docking
study of synthesized ligands was carried out with the X-ray crystal-
lographic structure of tyrosinase (Bacillus megaterium)–kojic acid
complex (PDB ID:3nq1) obtained from Sendovski, M. et al.38 Ini-
tially, the structure of inhibitors were drawn in to the Sybyl package
with standard bond lengths, and angles and minimized using a
conjugate gradient method. The Gasteiger-Huckel charge, with a
distance-dependent dielectric function, was applied for the minimi-
zation of the molecule. After performing Surflex-Dock, the scores of
10-docked conformers were ranked in a molecular spreadsheet. The
conformer was again speculated the binding mode regarding the
detailed interactions to the amino acids in the cavity.
IC50 = 2.2
IC50 = 1.8
l
l
M) and propylene (2j, >100 inhibition at 10
M) analogs. Nevertheless, when both hydrogens (3-
NH
the melanogenic inhibition was dramatically decreased as shown
in analog 2k (30% inhibition at 10 M, IC50 = 19.6 M). This result
indicated that one of these hydrogens must be free to participate
in hydrogen bonding of thiourea unit in 2.
In the next experiment, to prove the mechanism of melanogenic
inhibition of 2a–k, inhibitory activity against tyrosinase was
measured. Unexpectedly, none of these inhibited the tyrosinase
catalytic activity as shown in the Table 2. From this result, we
can hypothesize that these compounds act through a pathway dif-
ferent from the inhibition of tyrosinase activity to prevent melano-
2
) were replaced by bulky substituents like isopropyl groups,
l
l
3
6
genesis. Previously, we reported that compounds 4 (JSH-18) and

P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6824–6828
6827
H C
3
N
HN
Ring opening
N
H
S
N
S
H
4
(JSH-18)
2i
Melanin formation inhibition: IC = 0.8 µM
Melanin formation inhibition: IC = 2.2 µM
50
50
Tyrosinase inhibition: IC50 >100 µM
Tyrosinase inhibition: IC >100 µM
50
Truncated unit
Truncated unit
NH2
NH
Ring opening
N
H
S
N
H
S
5
1a (PTU)
Melanin formation inhibition: IC = 15.8 µM
Melanin formation inhibition: IC = 5.4 µM
50
50
Tyrosinase inhibition: IC50 >100 µM
Tyrosinase inhibition: IC = 1.5 µM
50
Figure 2. The SARs correlation diagram has cyclic and open-cyclic thioureas for the inhibition on melanin formation in B16 and tyrosinase enzyme
In the binding model of 1e (Fig. 3), the thiourea unit took the
position in the narrow cavity to near Copper metal and the amino
groups had H-bonds with Glu195 and Asn 205, which were consid-
ered as an important amino acids in the active site. The benzene
ring located in the hydrophobic region and methyl group of 1e
had hydrophobic interactions with Met 61, and Met 184 and the
backbone structure of 1e as shown in the Figure 3. On the other
hand, the 1,3-diphenylthiourea, 2b showed low docking scores
than mono-substituted one. As depicted in the Figure 4, the mole-
cule was located very far from the active site due to bulkiness of
either phenyl ring. From these studies, it can be suggested that
2
the 3-NH group must be free for the tyrosinase inhibition.
Effect of a series of 1-phenylthioureas 1a–k and 1,3-disubsti-
tuted thioureas 2a–k were evaluated against melanin formation
in melanoma B16 cell line and mushroom tyrosinase. From the
consistency in results of 1-phenylthioureas 1a–k in inhibitory
activity of tyrosinase with melanogenic inhibition, it was proved
that the melanogenic inhibition in melanoma B16 cells of
1
-phenylthioureas is due to their inhibition of tyrosinase. How-
ever, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a–k appears as
non-tyrosinase related melanogenic inhibitor. In addition, the
molecular docking study of 1e and 2b to binding pocket of
tyrosinase has given the convincing explanation regarding the
Figure 3. Docking model of the compound 1e in the active site.
Figure 4. Molcad docking models have the compound 1e (A) and 2b (B) in the active site.

6
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P. Thanigaimalai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6824–6828
necessity of direct connection of planar phenyl to thiourea unit
without N’-substitution of phenylthioureas 1 as tyrosinase inhibi-
tor and 2 as non-tyrosinase inhibitor. Thus, the 1,3-disubstituted
thioureas 2a–k might act through a different mechanism from
the inhibition of tyrosinase activity to reduce the melanogenesis
in melanoma B16 cells. The detailed mechanism of actions directed
towards the identification of molecular target of these 1,3-disubsti-
tuted thioureas as hypopigmenting agents are ongoing and will be
reported in due course.
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.
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3
3
5
acetonitrile (10 mL), added corresponding amine 6 and the resulting mixture
was allowed to stir for 3–4 h at room temperature. The reaction mixture was
evaporated under reduced pressure and resulting solid was poured in to water.
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2
.
3
4
.
.
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2
SO4. The organic layer was concentrated and the crude product
was purified using column chromatography. 2k: Yield 94%; viscous solid;
1
993, 207, 33.
R
1
2
1
C
f
= 0.45 (Ethyl acetate/hexane; 1:4), IR (neat): 3200, 3163, 1665, 1600, 1554,
1
5
.
.
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(CH(CH
H, Ar-H), 7.52 (t, J = 8.0 Hz, 2H, Ar-H), 7.81 (bs, 1H, NH). HRMS calcd for
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3 2
) )), 6.00 (bs, 1H, NH), 7.13 (t, J = 8.0 Hz, 2H, Ar-H), 7.28 (t, J = 7.6 Hz,
6
13
20 2
H N
7
8
.
.
3
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L
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