1-(1-Arylethylidene)thiosemicarbazide derivatives: A new class of tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.10.102

A series of 1-(1-arylethylidene)thiosemicarbazide compounds and their analogues were synthesized and characterized by ¹H NMR, MS. Their tyrosinase inhibitory activities were investigated by an assay based on the catalyzing ability of tyrosinase

Full text of DOI:10.1016/j.bmc.2007.10.102

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1096–1102
1-(1-Arylethylidene)thiosemicarbazide derivatives: A new class
of tyrosinase inhibitors
Jinbing Liu, Wei Yi, Yiqian Wan, Lin Ma and Huacan Song*
School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China
Received 12 September 2007; revised 27 October 2007; accepted 29 October 2007
Available online 13 November 2007
Abstract—A series of 1-(1-arylethylidene)thiosemicarbazide compounds and their analogues were synthesized and characterized by
1H NMR, MS. Their tyrosinase inhibitory activities were investigated by an assay based on the catalyzing ability of tyrosinase for
the oxidation of L-DOPA, comparing with 4-methoxycinnamic acid and arbutin. The results showed that (1) all the synthesized
compounds could perform a significant inhibitory activity for tyrosinase; (2) for these compounds, the main active moiety interact-
ing with the center of tyrosinase would be thiosemicarbazo group; (3) the inhibitory activity was close related with thiosemicarbazide
moieties and the groups attached on the aromatic ring.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
activities or side effect. For example, 2-(4-hydroxyphen-
oxy)tetrahydro-6-(hydroxylmethyl)-2H-pyran-3,4,5-triol
(arbutin or arbutoside)36 (IC50 = 30 mM) and kojic
acid37 (IC50 = 23 lM) (Fig. 1) were not demonstrated
as their clinically efficient.38 So far tropolone is one of
the most strong tyrosinase-inhibitors reported (Fig. 1)
(IC50 = 0.4 lM),39 but the serious side effect limited its
use as medicine.
Tyrosinase(monophenol or o-diphenol, oxygen oxidore-
ductase, EC 1.14.18.1), also known as polyphenol oxi-
dase (PPO), is a copper-containing monooxygenase
that is widely distributed in microorganisms, animals,
and plants.1–6 Tyrosinase could catalyze two distinct
reactions involving molecular oxygen7–13 in the hydrox-
ylation of monophenols to o-diphenols (monopheno-
lase) and in the oxidation of o-diphenols to o-quinones
(diphenolase). Due to the high reactivity, quinones
could polymerize spontaneously to form high molecular
weight brown-pigments (melanins) or react with amino
acids and proteins to enhance brown color of the pig-
ment produced. In addition, tyrosinase is known to be
involved in the molting process of insect, and adhesion
of marine organisms.14–17
On the other hand, it was reported that phenyl thio-
ureas40 and alkyl thioureas41 could exhibit weak to
moderate depigmenting activity and Ley and Ber-
tram28 reported that benzaldoximes and benzalde-
hyde-o-alkyloximes
possess
higher
tyrosinase
inhibitory ability. Based on these reports, we hoped
that condensation products of acetophenone homo-
logues and thiosemicarbazide could show a high inhi-
bition activity, as sulfur atom and nitrogen atom are
able to complex the two copper atoms in the active
site of tyrosinase.
Therefore, there is an increasing significance to discover
effective tyrosinase inhibitors for clinical medicines, cos-
metic products, food industry as well as agricultural
purposes.18–24 Up to now, although a large number of
tyrosinase inhibitors were reported, most of them could
not be used now25–35 because of their lower individual
In order to look for highly potent tyrosinase inhibi-
tors, a series of 1-(1-arylethylidene)thiosemicarbazide
derivatives were synthesized and their inhibitory activ-
ities against mushroom tyrosinase were evaluated
using arbutin and 4-methoxycinnamic acid as compar-
ing substances. Meanwhile, the structure–activity rela-
tionships of these compounds were also primarily
discussed.
Keywords: Tyrosinase inhibitors; 1-(1-Arylethylidene)thiosemicarba-
zide; Analogues.
*
Corresponding author. Tel.: +86 20 84110918; fax: +86 20 8411
2245; e-mail: yjhxhc@mail.sysu.edu.cn
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.102

J. Liu et al. / Bioorg. Med. Chem. 16 (2008) 1096–1102
1097
O
O
OH
OH
OH
O
O
OH
H3CO
CH=CHCO2H
HO
HO
HO
O
OH
4-methoxycinnamic acid
tropolone
kojic acid
arbutin
Figure 1. Structures of some known tyrosinase inhibitors.
2. Results and discussion
2.1. Chemistry
of 1-(1-arylethylidene)thiosemicarbazide compounds
and their analogues against tyrosinase were summarized
in Table 1.
According to the general procedure shown in Scheme 1,
the synthesis of a series of 1-(1-arylethylidene)thiosemi-
carbazide compounds and their analogues could be car-
ried out easily in anhydrous alcohol, using acetic acid as
catalyst, just by the condensation of methyl ketone or
aldehyde with thiosemicarbazide in the molecular ratio
of 1:1 or 1:2. The reaction mixture was refluxed for
24 h and then cooled to room temperature, the formed
precipitate was separated by filtration. If necessary, the
target compounds could be purified by recrystallization
from 95% alcohol. The yields for these reactions are
from fair to good (65–86%), and all the compounds were
The IC50 value of 4-methoxycinnamic acid, determined
in this work, is 0.41 mM, the reported IC50 value of 4-
methoxycinnamic acid is 0.34 mM and 0.43 mM;6,43
therefore, both the values reported and determined in
this work are close to each other, which means that
the protocol employed in this work could be reasonable.
Tropolone (in Fig. 1), as tyrosinase inhibitor, could
show an IC50 value of 0.4 lM.39 It could be found from
Table 1 that compounds 1, 2, 3, 6, 9, 11, and 17 all
showed a little higher inhibiting ability against tyrosi-
nase than tropolone. Meantime, the IC50 values of com-
pounds 4, 7, 13, 14 are close to that of tropolone.
Therefore, it is worth for these synthesized compounds
to be investigated further.
1
characterized by H NMR, MS.
2.2. Inhibiting activity
For evaluating the tyrosinase inhibitory activity, all the
synthesized compounds were subjected to tyrosinase
inhibition assay with L-DOPA as substrate, according
to the typical assay protocol developed by Hearing.42
The tyrosinase inhibitory activity of arbutin and 4-meth-
oxycinnamic acid was ever reported,6,36 therefore, they
were selected as comparing substances. The IC50 values
For the relationships between the structure and the
activity of these synthesized compounds, some results
could be obtained from the data listed in Table 1:
(1) For the 1-(1-phenylethylidene)thiosemicarbazide
compounds, the substituents attached on the benzene
ring could change the inhibition activity. Methyl, hydro-
S
O
R
S
N
N
NH2
+
H
R
CH3
H2NHN
NH2
H3C
1-14
R =: 1, phenyl; 2, 4-methylphenyl; 3, 4-hydroxyphenyl; 4, 2,4-dihydroxyphenyl;
5, 2,4,6-trihydroxyphenyl; 6, 4-fluorophenyl; 7, 4-bromophenyl;
8, 4-isopropylphenyl; 9, 4-methoxyphenyl; 10, 2-pyrazinyl; 11, 2-thiophenyl;
12, 3-pyridinyl; 13, (4-methoxyphenyl)methyl; 14, 2-(4-hydroxyphenyl)ethyl.
H
O
S
N
N
NH2
R
+
S
R
H2NHN
Ph
NH2
Ph
15,16
R =: 15, hydroxy(phenyl)methyl; 16, benzoyl.
H
S
N
N
NH NH2
S
N
H2N
O
O
+
H3C
CH3
H2NHN
S
NH2
H3C
CH3
17
Scheme 1. Synthesis of 1-(1-arylethylidene)thiosemicarbazide compounds and their analogues.

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J. Liu et al. / Bioorg. Med. Chem. 16 (2008) 1096–1102
Table 1. Inhibitory activity of 1-(1-arylethylidene)thiosemicarbazide against tyrosinase
Compound
IC50 (lM)
1
0.34
2
0.27
3
0.31
4
0.58
5
22.0
6
0.17
7
0.52
8
1.0
Compound
IC50 (lM)
9
0.11
10
0.88
11
0.14
12
0.82
13
0.42
14
0.54
15
55.5
17
0.15
Compound
IC50 (mM)
16a
0.10a
Thiosemicarbazideb
2.00b
1-(Thiophen-2-yl)-ethanone
0.15
Acetophenone
0.85
Compound
1-(4-Fluorophenyl)-
ethanone
1-(4-
4-Methoxycinnamic
acidd
Arbutine
Methoxyphenyl)-
ethanonec
2.00c
IC50 (mM)
0.99
0.41d
10.4e
a The concentration of 0.10 (mM) corresponding to inhibition percentage, determined in this work, is 47%.
b The concentration of 2.00 (mM) corresponding to inhibition percentage, determined in this work, is 39.9%.
c The concentration of 2.00 (mM) corresponding to inhibition percentage, determined in this work, is 47.2%.
d The reported IC50 values of 4-methoxycinnamic acid is 0.34–0.43 mM.6,43
e For arbutin, the concentration of 10.40 (mM) corresponding to inhibition percentage, determined in this work, is 30%. The reported IC50 value of
arbutin was more than 30 mM.36
xyl, fluoro, and methoxy on the 4-positon of benzene
ring could enhance the activity, but bromo and isopro-
His 90
pyl could decrease the activity. Although hydroxyl on
4-position could enhance the activity, with the increase
of the number of hydroxyl on benzene ring, the activity
of 1-(1-phenylethylidene)thiosemicarbazide compounds
would decrease.
His 250
His 81
His 57
CuA
O
CuB
His 282
O
(2) The increase in the number of thiosemicarbazido
group in one molecule could not obviously enhance
the activity, although the thiosemicarbazido group
could be the main active position in these synthesized
compounds. For example, the IC50 value against tyrosi-
nase for 1 is 0.34 lM, for 17 is 0.15 lM.
His 254
Figure 2. The active center structure of tyrosinase.
between arylethylidenethiosemicarbazide compounds
and tyrosinase when both substrates were mixed to-
gether in solution. In addition, sulfur atom and nitrogen
atom, especially sulfur atom, of arylethylidenethiosemic-
arbazide compounds and copper ion of tyrosinase could
be the center of complexation. The structures of the
complexes are suggested as shown in Figure 3, based
on the active center structure of tyrosinase.
(3) When the benzene ring of phenylethylidenethiose-
micarbazide molecule was changed by heterocyclic ring,
the obtained ethylidenethiosemicarbazide compounds
could still show a stronger activity against tyrosinase.
For example, the IC50 value for 11 is 0.14 lM.
(4) When thiosemicarbazido group and benzene ring are
separated by one or two methene groups, the change on
the inhibitory activity of the obtained ethylidenethiose-
micarbazide compounds is not significant.
In the complex, the active center of tyrosinase could
coordinate with two arylethylidenethiosemicarbazide
molecules at the same time in two opposite directions.
It is suggested that the intramolecule hydrogen bond
was formed between hydrogen atom on 3-N and nitro-
gen atom (2-N) in arylethylidenethiosemicarbazide mol-
ecule, which are beneficial to decreasing molecular
energy state.
(5) The inhibitory activity of corresponding compounds
would be decreased obviously when the methyl of 1-(1-
phenylethylidene)thiosemicarbazide compounds was
changed into hydroxy(phenyl)methyl or benzoyl.
According to the structure of arylethylidenethiosemicar-
bazide molecules, there would be two forms (Fig. 3,
form A and form B) to form complexes with tyrosinase.
When the coordination between copper ion and sulfur
atom of arylethylidenethiosemicarbazide molecule oc-
curred, the intermolecule hydrogen bond would be
formed between hydrogen atom on 3-N (form A) or
on 2-N (form B) and two oxygen atoms located between
two copper ions, which would make the coordination
between copper ion and sulfur atom become more tight,
and consequently, which could make the free oxygen
molecule decrease its reaction ability and even unable
to take part in the hydroxylation with monophenols
2.3. Inhibiting mechanism
It was reported that the structure of tyrosinase was
determined44 (shown in Fig. 2), there are two copper
ions in the active center of tyrosinase and it was deduced
that there is a lipophilic long-narrow gorge near to the
active center.45
There are sulfur atom and nitrogen atom in the molecule
of arylethylidenethiosemicarbazide compounds and
their analogues, and sulfur atom and nitrogen atom
could exhibit strong affinity for copper ion. Therefore,
it could be supposed that complexes would be formed

J. Liu et al. / Bioorg. Med. Chem. 16 (2008) 1096–1102
1099
His 81
His 81
His 57
His 57
His 90
CuA
CuA
R1
S
His 90
R2
3
H
C
N
S
R1
R2
N
1
H
H
O
H
C
H
O
N
C
O
N
H
N
H
O
2
N
N
3
1
H
2
2
N
H
N
R2
R1
1
N
C
3
N
3
N
2
R2
H
S
H
S
CuB
1
CuB
H
R1
His 250
His 250
His 254
His 254
His 282
His 282
A
B
Figure 3. The proposed structure of the formed complexes between tyrosinase and synthesized compounds.
and in the oxidation with o-diphenols, as the free oxygen
molecule was surrounded closely by two copper ions
from tyrosinase and two hydrogen atoms from two aryl-
should exhibit strong affinity for copper atom of tyros-
inase. However, 1-(thiophen-2-yl)ethanone just ex-
pressed a weaker inhibiting ability against tyrosinase
at the same condition, which means that the sulfur
atom of 1-(thiophen-2-yl)ethanone possesses a weaker
affinity or a weaker coordination ability than that of
thiosemicarbazido group of compounds 11, 1-(1-(thio-
phen-2-yl)-ethylidene)thiosemicarbazide, although both
sulfur atoms of compounds 11 could possess chance
to take part in the complexation at the same time.
The reason could be that there is a larger bulk inhibi-
tion for the coordination between copper ion and sul-
fur atom of thiophene; meantime, there is a strong
competition from the coordination between copper
ion and sulfur atom of thiosemicarbazido group.
Therefore, the thiosemicarbazido group could be the
important active position in the molecule of arylethyl-
idenethiosemicarbazide compounds as tyrosinase inhib-
itor, and the major form of the complexation of
tyrosinase with compound 11 is supposed as B, instead
of A (Fig. 4).
ethylidenethiosemicarbazide molecules.
tyrosinase would lose its catalyzing ability.
Therefore,
As for the complex form A and form B (Fig. 3), which
one is the best beneficial form? Based on the molecular
energy state, form A should be the best one. For the
form A, comparing with form B, larger substituted-ben-
zene ring (R1) and methyl (or H) (R2) could depart from
tyrosinase for a longer distance, and consequently, the
bulk inhibition from R1 and R2 would decrease, which
would decrease the energy of the complex and which is
just beneficial to the formation of the complexes be-
tween arylethylidenethiosemicarbazide and tyrsoinase.
In order to confirm this conclusion, four compounds,
1-(4-
acetophenone,
1-(4-fluorophenyl)ethanone,
methoxyphenyl)ethanone, and 1-(thiophen-2-yl)-etha-
none, which are corresponding to the starting materials
of compounds 1, 6, 9, and 11 were selected and their
inhibiting abilities against tyrosinase determined at the
same condition, their IC50 value are all more than
0.15 mM. At the same time, when the concentration of
thiosemicarbazide is 2.00 mM, the corresponding inhibi-
tion percentage, determined in this work, is only 39.9%.
As to the lower IC50 value of thiosemicarbazide, com-
paring with that of arylethylidenethiosemicarbazide
compounds and their homologues, the following two
main reasons could be suggested here: (1) Because thi-
osemicarbazide was hydrophilic and tyrosinase was
lipophilic and all the tyrosinase inhibition assays were
determined almost in phosphate buffer, there was a
less chance for thiosemicarbazide and tyrosinase to
On the other hand, there is a sulfur atom in molecule
of 1-(thiophen-2-yl)-ethanone, and this sulfur atom
His 81
His 81
His 57
His 90
H2N
CuA
H
S
C
H
0
His 9
CuA
H
N
S
S
S
H3C
S
N
H
H
H
C
O
N
O
S
H
N
N
H3C
CH3
O
O
H
N
N
N
N
C
N
CuB
H
H
S
S
CuB
C
CH3
H
H
NH2
His 250
His 250
His 282
His 254
B
A
His 282
Figure 4. The proposed structures of the complexation of tyrosinase with arylethylidenethiosemicarbazide compounds and 1-(1-(thiophen-2-
yl)ethylidene)thiosemicarbazide.

1100
J. Liu et al. / Bioorg. Med. Chem. 16 (2008) 1096–1102
interact to form complex; (2) The presence of substi-
tuted-aromatic ring in arylethylidenethiosemicarbazide
compounds would be the main beneficial reason, be-
cause it was deduced that there is a lipophilic long-
narrow gorge near to the active center45 and the lipo-
philic aromatic ring of arylethylidenethiosemicarbazide
compounds could easily get close to the gorge, which
would make the thiosemicarbazido group of arylethyl-
idenethiosemicarbazide compounds get close to the ac-
tive center of tyrosinase much more easily. In other
words, lipophilic moiety of arylethylidenethiosemicar-
bazide compounds could bring the thiosemicarbazido
group to the site close to the active center of tyrosi-
nase, which was beneficial for the coordination be-
tween the active center of tyrosinase and the
thiosemicarbazido group of thiosemicarbazide com-
pounds. It is the stronger coordination between the
active center of tyrosinase and arylethylidenethiose-
micarbazide compounds than that between the active
center of tyrosinase and thiosemicarbazide, arylethyl-
4. Experimental
4.1. Equipments and reagents
Melting points (mp) were determined with WRS-1B
melting point apparatus and are uncorrected. NMR
spectra were recorded on Mercury-Plus300 spectrome-
ters at 25 ꢁC using CDCl3 or DMSO-d6 as a solvent.
All chemical shifts (d) are quoted in ppm downfield
from TMS and coupling constants (J) are given in
Hz. LC–MS spectra were recorded using the LCMS-
2010A. All reactions were monitored by TLC (Merck
Kieselgel 60 F254) and the spots were visualized under
UV light. The appropriate aldehyde or ketone, thio-
semicarbazide, and 4-methoxycinnamic acid were pur-
chased from Darui Chemical Co. (ShangHai, China),
arbutin was obtained from Brillian Biochemical Co.
(Beijing, China). The other commercially available re-
agents and solvents were used without further purifica-
tion. Mushroom tyrosinase and L-DOPA were
purchased from Sigma Chemical Co. (Sigma-Aldrich
China, Beijing, China).
idenethiosemicarbazide compounds would show
a
stronger inhibiting ability than thiosemicarbazide
against tyrosinase.
4.2. Synthesis of thiosemicarbazides
3. Conclusions
4.2.1. The general procedures for the synthesis of 1-(1-
arylethylidene)thiosemicarbazide compounds. The appro-
priate aldehyde or ketone (10 mmol) was dissolved in
In this study, a series of 1-(1-arylethylidene)thiosemi-
carbazide compounds and their analogues were syn-
thesized and characterized by 1H NMR, MS. Their
inhibitory activities against tyrosinase were studied
based on the catalyzing ability of tyrosinase for the
oxidation of L-DOPA, using arbutin and 4-methoxy-
cinnamic acid as comparing substrate. The following
conclusions would be obtained: (1) 1-(1-arylethylid-
ene)thiosemicarbazide compounds could perform a
significant inhibitory activity for tyrosinase; seven
compounds could show stronger inhibiting ability than
reported tropolone, four compounds showed nearly
same IC50 values with tropolone. (2) The form of
the interaction between these compounds and tyrosi-
nase could be supposed as in Figure 3 (form A) in
which the formation of hydrogen bond between
hydrogen atom on 3-N (form A) and free oxygen mol-
ecule located between two copper ions of tyrosinase
would make the free oxygen molecule decrease its
reaction ability and even unable to take part in the
hydroxylation and in the oxidation. (3) For these
investigated compounds, it was suggested that the
main active moiety interacting with the center of
tyrosinase would be thiosemicarbazido group. (4)
The liposoluble group of arylethylidenethiosemicarbaz-
ide compounds would make the interaction between
the active center of tyrosinase and thiosemicarbazido
moiety become much easier, which would make tyros-
inase lose its catalyzing ability. (5) Although thiose-
micarbazido group could be the main active part,
the increase in the number of thiosemicarbazido group
could not obviously increase their activity. (6) The
changes of the substituent on benzene ring would ex-
ert an influence on the inhibitory activity. Meanwhile,
when benzene was replaced by heterocyclic ring, the
corresponding activity would have little change.
anhydrous
ethanol(10 mL),
thiosemicarbazide
(10 mmol) and acetic acid (0.5 mL) were added to the
above solution. The reaction mixture was refluxed for
24 h and then was cooled to room temperature. The
appearing precipitate was filtered and recrystallized
from 95% alcohol to obtain the corresponding 1-(1-
arylethylidene)thiosemicarbazide compounds.
4.2.2. 1-(1-Phenylethylidene)thiosemicarbazide (1). Yield
86%; mp 132–134 ꢁC; H NMR (300 MHz, DMSO-d6):
d 8.95 (1H, br s, NH), 7.70 (2H, d, J = 1.2 Hz, phH),
7.48 (1H, br s, NH2), 7.41 (3H, m, J = 1.2 Hz, phH),
6.53 (1H, br s, NH2), 2.31 (3H, s, CH3). MS (ESI): m/
z (100%) = 194 (M+1).
1
4.2.3. 1-(1-p-Tolylethylidene)thiosemicarbazide (2). Yield
78%; mp 158–160 ꢁC; H NMR (300 MHz, DMSO-d6):
d 8.77 (1H, br s, NH), 7.61 (2H, d, J = 7.5 Hz, phH),
7.36 (1H, br s, NH2), 7.21 (2H, d, J = 7.5 Hz, phH),
6.42 (1H, br s, NH2), 2.39 (3H, s, COCH3), 2.29 (3H,
s, ph-CH3). MS (ESI): m/z (100%) = 208 (M+1).
1
4.2.4. 1-(1-(4-Hydroxyphenyl)ethylidene)thiosemicarba-
zide (3). Yield 73%; mp 208–209 ꢁC; 1H NMR
(300 MHz, DMSO-d6): d 10.02 (1H, br s, NH), 9.69
(1H, s, OH), 8.13 (1H, br s, NH2), 7.77 (2H, d,
J = 8.7 Hz, phH), 7.72 (1H, br s, NH2), 6.75 (2H,d,
J = 8.7 Hz, phH), 2.22 (3H, s, CH3). MS (ESI): m/z
(100%) = 208 (Mꢀ1).
4.2.5. 1-(1-(2,4-Dihydroxyphenyl)ethylidene)thiosemicar-
bazide (4). Yield 68%; mp 186–187 ꢁC; 1H NMR
(300 MHz, DMSO-d6): d 12.57 (1H, s, OH), 10.57
(1H, s, OH), 9.72 ( 1H, br s, NH), 7.74 (1H, s, NH2),
7.70 (1H, s, NH2), 7.36 (H, d, J = 8.4 Hz, phH), 6.37

J. Liu et al. / Bioorg. Med. Chem. 16 (2008) 1096–1102
1101
(1H, d, J = 8.4 Hz, phH), 6.23 (1H, s, phH), 2.25 (3H, s,
CH3). MS (ESI): m/z (100%) = 224 (Mꢀ1).
4.2.14. 1-(1-(4-Methoxyphenyl)propan-2-ylidene)thiosem-
icarbazide (13). Yield 88%, mp 122–123 ꢁC; H NMR
(300 MHz, DMSO-d6): d 8.50 (1H, br s, NH), 7.26
(1H, br s, NH2),7.10 (2H, d, J = 8.7 Hz, phH), 6.86
(2H, d, J = 8.7 Hz, phH), 6.34 (1H, br s, NH2), 3.80
(3H, s, OCH3), 3.50 (2H, s, CH2), 1.84 (3H, s, CH3).
MS (ESI): m/z (100%) = 238 (M+1).
1
4.2.6. 1-(1-(2,4,6-Trihydroxyphenyl)ethylidene)thiosemi-
carbazide (5). Yield 65%; mp 211–213 ꢁC; 1H NMR
(300 MHz, DMSO-d6): d 12.18 (2H, s, OH), 10.32
(1H, s, OH), 9.72 (1H, br s, NH), 7.73 (1H, br s,
NH2), 7.36 (1H, br s, NH2), 5.78 (2H, s, phH),
2.54 (3H, s, CH3). MS (ESI): m/z (100%) = 240
(Mꢀ1).
4.2.15. 1-(4-(4-Hydroxyphenyl)butan-2-ylidene)thiosemi-
carbazide (14). Yield 71%, mp154–155 ꢁC; 1H NMR
(300 MHz, DMSO-d6): d 9.87 (1H, br s, NH), 8.50
(1H, s, OH), 7.12 (1H, br s, NH2), 7.02 (2H, d,
J = 6.3 Hz, phH), 6.77 (2H, d, J = 6.3 Hz, phH), 6.22
(1H, br s, NH2), 2.80 (2H, t, CH2), 2.60 (2H, t, CH2),
1.89 (3H, s, CH3). MS (ESI): m/z (100%) = 236 (Mꢀ1).
4.2.7. 1-(1-(4-Fluorophenyl)ethylidene)thiosemicarbazide
1
(6). Yield 82%, mp 154–156 ꢁC; H NMR (300 MHz,
DMSO-d6): d 10.17 (1H, br s, NH), 8.24 (1H, br s,
NH2), 7.98 (2H, d, J = 6.9 Hz, phH), 7.78 (1H, br s,
NH2), 7.20 (2H,d, J = 6.9 Hz, phH), 2.28 (3H, s, CH3).
MS (ESI): m/z (100%) = 212 (M+1).
4.2.16. 1-(2-Hydroxy-1,2-diphenylethylidene)thiosemicar-
bazide (15). Yield 58%, mp 178–179 ꢁC; 1H NMR
(300 MHz, DMSO-d6): d 11.44 (1H, br s, NH), 8.40
(1H, br s, NH2), 7.97 (1H, br s, NH2), 7.90 (t, 2H,
phH), 7.36 (m, 8H, phH), 7.29 (s, 1H, OH), 6.27 (d,
1H, CH). MS (ESI): m/z (100%) = 286 (M+1).
4.2.8. 1-(1-(4-Bromophenyl)ethylidene)thiosemicarbazide
1
(7). Yield 79%, mp 190–192 ꢁC; H NMR (300 MHz,
DMSO-d6): d 10.08 (1H, br s, NH), 8.28 (1H, br s,
NH2), 7.97 (1H, br s, NH2), 7.89 (2H, d, J = 7.8 Hz,
phH), 7.55 (2H, d, J = 7.8 Hz, phH), 2.27 (3H, s,
CH3). MS (ESI): m/z (100%) = 273 (M+1).
4.2.17. 1-(2-Oxo-1,2-diphenylethylidene)thiosemicarba-
zide (16). Yield 62%, mp 203–204 ꢁC; 1H NMR
(300 MHz, DMSO-d6): d 11.87 (1H, br s, NH), 8.20
(1H, br s, NH2), 7.39 (m, 10H, phH), 6.75 (1H, br s,
NH). MS (ESI): m/z (100%) = 284 (M+1).
4.2.9. 1-(1-(4-Isopropylphenyl)ethylidene)thiosemicarba-
zide (8). Yield 68%, mp 99–100 ꢁC; 1H NMR
(300 MHz, CDCl3): d 8.77 (1H, br s, NH),7.64 (2H,d,
J = 8.1 Hz, phH), 7.37 (1H, br s, NH2), 7.25 (2H, d,
J = 8.1 Hz, phH),6.47 (1H, br s, NH2), 2.94 (m, 1H,
CH), 2.29 (3H, s, CH3), 1.28 (d, 6H, 2CH3). MS
(ESI): m/z (100%) = 236 (M+1).
4.2.18. 1-(1,4-Diacetylphenyl)dithiosemicarbazide (17).
1
Yield 82%, mp > 280 ꢁC; H NMR (300 MHz, CDCl3):
d 10.22 (2H, br s, NH), 8.28 (2H, br s, NH2), 7.94 (2H,
br s, NH2), 2.30 (6H, s, 2· CH3). MS (ESI): m/z
(100%) = 309 (M+1).
4.2.10. 1-(1-(4-Methoxyphenyl)ethylidene)thiosemicarba-
zide (9). Yield 83%, mp 175–176 ꢁC; 1H NMR
(300 MHz, CDCl3): d 9.04 (1H, br s, NH),7.57 (2H, d,
J = 8.7 Hz, phH), 7.27 (1H, br s, NH2), 7.26 (1H, br s,
NH2), 7.25 (2H, d, J = 8.7 Hz, phH), 3.67 (3H, s,
OCH3), 2.16 (3H, s, CH3). MS (ESI): m/z
(100%) = 224 (M+1).
4.3. Tyrosinase assay
Tyrosinase inhibition assays were performed according
to the method developed by Hearing with slight modi-
fication.42 Briefly, all the synthesized compounds were
screened for the o-diphenolase inhibitory activity of
tyrosinase using L-DOPA as substrate. All the active
inhibitors from the preliminary screening were sub-
jected to IC50 studies. All the synthesized compounds
were dissolved in DMSO to a concentration of 2.5%.
Phosphate buffer, pH 6.8, was used to dilute the
DMSO stock solution of test compound. Thirty units
of mushroom tyrosinase (28 nM) was first pre-incu-
bated with the compounds, in 50 nM phosphate buffer
(pH 6.8), for 10 min at 25 ꢁC. Then the L-DOPA
(0.5 mM) was added to the reaction mixture and the
enzyme reaction was monitored by measuring the
change in absorbance at 475 nm of formation of the
DOPAchrome for 10 min. Dose–response curves were
obtained by performing assays in the presence of
increasing concentrations of inhibitors (0, 1.6, 3.2,
6.3, 12.5, 25, 50, 100, 200 lM ). IC50 value, a concen-
tration giving 50% inhibition of tyrosinase activity, was
determined by interpolation of the dose–response
curves. The percent of inhibition of tyrosinase reaction
was calculated as follows:
4.2.11. 1-(1-(Pyrazin-2-yl)ethylidene)thiosemicarbazide
1
(10). Yield 67%, mp 190–191 ꢁC; H NMR (300 MHz,
DMSO-d6): d 10.43 (1H, br s, NH), 9.62 (1H, s, pyrazine
proton), 8.58 (2H, d, pyrazine protons), 8.44 (1H, br s,
NH2), 8.28 (1H, br s, NH2), 2.36 (3H, s, CH3). MS
(ESI): m/z (100%) = 196 (M+1).
4.2.12. 1-(1-(Thiophen-2-yl)ethylidene)thiosemicarbazide
1
(11). Yield 65%, mp 146–147 ꢁC; H NMR (300 MHz,
DMSO-d6): d 8.86 (1H, br s, NH), 7.35 (1H, d, thio-
phen), 7.31 (1H, d, thiophene proton), 7.28 (1H, br s,
NH2), 7.04 (1H, t, thiophene proton), 6.59 (1H, br s,
NH2), 2.31 (3H, s, CH3). MS (ESI): m/z (100%) = 200
(M+1).
4.2.13. 1-(1-(Pyridin-3-yl)ethylidene)thiosemicarbazide
1
(12). Yield 70%, mp 212–214 ꢁC; H NMR (300 MHz,
DMSO-d6): d 10.29 (1H, br s, NH), 9.08 (1H, s, pyridine
proton), 8.54 (1H, d, pyridine proton), 8.31 (1H, d, pyr-
idine proton), 8.29 (1H, br s, NH2), 8.05 (1H, br s,
NH2), 7.40 (1H, t, pyridine proton), MS (ESI): m/z
(100%) = 195 (M+1).
percent inhibition ð%Þ ¼ ½ðB ꢀ SÞ=Bꢁ ꢂ 100:

1102
J. Liu et al. / Bioorg. Med. Chem. 16 (2008) 1096–1102
21. Hollman, P. C. H.; Gaag, M. V. D.; Mengelers, M. J. B.
Free Radic. Biol. Med. 1996, 21, 703.
Here, the B and S are the absorbances for the blank and
samples, respectively. 4-Methoxycinnamic acid and
arbutin were used as standard inhibitors for the
tyrosinase.
22. Mosher, D. B.; Pathak, M. A.; Zpatrick, T. B.. In Update:
Dermatology in General Medicine; Fitzpatrick, A. Z.,
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Eds.; McGraw-Hill: New York, 1983; Vol. 1, p 205.
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