Structure-based modification of 3-/4-aminoacetophenones giving a profound change of activity on tyrosinase: From potent activators to highly efficient inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.02.013

In this study, we developed 3-/4-aminoacetophenones and their structure-based 3-/4-aminophenylethylidenethiosemicarbazide derivatives, respectively, as novel tyrosinase activators and inhibitors. Notably, all the obtained thiosemicarbazones displayed more potent tyrosinase inhibitory activities than kojic acid. Especially, compound 7k was found to be the most active tyrosinase inhibitor with IC₅₀ value of 0.291 1/4M. The structure-activity relationships (SARs) analysis showed that: (1) the amine group was absolutely necessarily for determining the tyrosinase activation activity; (2) the introduction of thiosemicarbazide group played a very vital role in transforming tyrosinase activators into tyrosinase inhibitors; (3) the phenylethylenethiosemicarbazide moiety was crucial for determining the tyrosinase inhibitory activity; (4) the type of acyl group had no obvious effect on the inhibitory activity; (5) the position of amide substituent on the phenyl ring influenced the tyrosinase inhibitory potency. Moreover, the inhibition mechanism and inhibition kinetics study revealed that compound 7k was reversible and non-competitive inhibitor, and compound 8h was reversible and competitive-uncompetitive mixed-II type inhibitor.

Full text of DOI:10.1016/j.ejmech.2015.02.013

European Journal of Medicinal Chemistry 93 (2015) 255e262
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Structure-based modification of 3-/4-aminoacetophenones giving a
profound change of activity on tyrosinase: From potent activators to
highly efficient inhibitors
,
,
, b, *
Ao You a, Jie Zhou a b, Senchuan Song a, Guoxun Zhu a, Huacan Song a **, Wei Yi a
a School of Chemistry and Chemical Engineering, Sun Yat-Sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
b VARI/SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we developed 3-/4-aminoacetophenones and their structure-based 3-/4-
aminophenylethylidenethiosemicarbazide derivatives, respectively, as novel tyrosinase activators and
inhibitors. Notably, all the obtained thiosemicarbazones displayed more potent tyrosinase inhibitory
activities than kojic acid. Especially, compound 7k was found to be the most active tyrosinase inhibitor
Received 5 November 2014
Received in revised form
16 January 2015
Accepted 10 February 2015
Available online 12 February 2015
with IC50 value of 0.291 mM. The structure-activity relationships (SARs) analysis showed that: (1) the
amine group was absolutely necessarily for determining the tyrosinase activation activity; (2) the
introduction of thiosemicarbazide group played a very vital role in transforming tyrosinase activators
into tyrosinase inhibitors; (3) the phenylethylenethiosemicarbazide moiety was crucial for determining
the tyrosinase inhibitory activity; (4) the type of acyl group had no obvious effect on the inhibitory
activity; (5) the position of amide substituent on the phenyl ring influenced the tyrosinase inhibitory
potency. Moreover, the inhibition mechanism and inhibition kinetics study revealed that compound 7k
was reversible and non-competitive inhibitor, and compound 8h was reversible and competitive-
uncompetitive mixed-II type inhibitor.
Keywords:
Aminophenylethylidenethiosemicarbazide
compounds
Tyrosinase activator
Tyrosinase inhibitor
SARs
Inhibition mechanism
Inhibitory kinetics
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
produce melanin, which was responsible for color of mammals-
skin and -hair [2]. Moreover, tyrosinase was also found to be
Tyrosinase (EC 1.14.18.1) is a multifunctional oxygenase which is
widely distributed in nature, structurally belonging to the type-3
copper protein family [1]. It is also well known that tyrosinase
catalyzes two distinct reactions as the rate-limiting step of melanin
biosynthesis. The first one is the hydroxylation of L-tyrosine to L-
3,4-dihydroxyphenylalanine (L-DOPA), and the second is the
oxidation of L-DOPA to ortho-quinone (dopaquinone). The latter
was highly reactive and could further oxidized spontaneously to
involved in the molting process of insects [3], the browning of fruits
and vegetables [4], and the happen of Parkinson's and other
neurodegenerative diseases [5].
Driven by their biologically applied power, tyrosinase activators/
inhibitors have become increasingly important in agriculture and
food industry, as well as in medicinal and cosmetic products. For
example, tyrosinase activators could protect human skin from UV
irradiation damage via stimulating the synthesis of melanin [6]. In
contrast, tyrosinase inhibitors could be used as skin-whitening
agents for the treatment of some dermatological disorders associ-
ated with melanin hyperpigmentation [7]. As a result, so far a huge
number of natural and synthetic compounds acting as tyrosinase
activators/inhibitors have been extensively reported [8,9]. Howev-
er, most of them are not potent enough to put into practical use due
to their weak individual activities or safety concerns. Undoubtedly,
it is still necessary to develop new tyrosinase activators/inhibitors
with improved therapeutic profiles.
Abbreviations: SARs, structure-activity relationships; L-DOPA, L-3,4-
dihydroxyphenylalanine; THSG, 2,3,5,40-tetrahydroxystilbene-2-O-
b-D-glucoside;
IC50, the half maximal inhibitory concentration; DMSO, dimethyl sulfoxide.
* Corresponding author. VARI/SIMM Center, Center for Structure and Function of
Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China.
** Corresponding author.
E-mail addresses: yjhxhc@mail.sysu.edu.cn (H. Song), yiwei.simm@simm.ac.cn
(W. Yi).
Nowadays, the crystallographic structure of tyrosinase from
http://dx.doi.org/10.1016/j.ejmech.2015.02.013
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

256
A. You et al. / European Journal of Medicinal Chemistry 93 (2015) 255e262
Table 1
three different species [10e12] has been established, enabling a
close look at its three-dimensional structure and also opening up
the way for next structure-based design of new and potent tyros-
inase inhibitors. Within the structure, there are two copper ions in
the active center of tyrosinase and a lipophilic long-narrow gorge
near to the active center. In addition, recently a wide range of thi-
osemicarbzone derivatives have been developed as highly potent
tyrosinase inhibitors, most of which came from Chen [13] and our
group's contribution [14]. Further investigation revealed that the
introduction of thiosemicarbazide group was absolutely necessary
for determining the tyrosinase inhibitory activity because it was
able to efficiently complex the two copper ions in the active center
of tyrosinase. Motivated by these, we speculated that, the intro-
duction of both thiosemicarbazide group and a proper hydrophobic
subunit to the 3-/4-aminoacetophenones scaffold, might form po-
tential interactions with the dicopper active center and the hy-
drophobic pocket of tyrosinase, thus to deliver an improved
compound activity.
The structures and IC50 values of 1-(4-aminophenyl)ethylidenethiosemicarbazide
(3) and 1-(4-amidophenyl)ethylidenethiosemicarbazide compounds (7a-n).
Entry
Amide group structure
IC50 (mmol/L)
3
eNH2
eNHCOCH3
eNHCOCF3
eNHCOCCl3
2.62
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
Ref.
0.508
1.336
0.406
0.372
0.644
0.814
0.492
0.762
0.356
0.498
0.291
0.610
1.142
0.578
eNHCOC2H5
eNHCOC3Hꢁ7 n
eNHCOC3Hꢁ7 iso
eNHCOC4Hꢁ9ꢁnn
eNHCOC5H1ꢁ1n
eNHCOC6H13
eNHCOOCH3
eNHCOC6H5
eNHCOC6H4F-p
eNHCOC6H4OCH3-p
eNHCOCH2C6H5
Kojic acid
28.5
In order to confirm the hypothesis and considering the impor-
tance of amide moiety in modern medicinal chemistry, here two
series of novel 3-/4-aminophenylethylidenethiosemicarbazide de-
rivatives were designed, synthesized and their inhibitory effects on
mushroom tyrosinase were evaluated. Meanwhile, the parent 3-/4-
aminoacetophenones were also investigated. Furthermore, the
SARs were discussed and the inhibition mechanism and the
inhibitory kinetics of selected compounds were studied. We hope
that the findings can lead to the discovery of highly efficient
pharmacological agents for the treatment of tyrosinase-related
disorders.
Table 2
The structures and IC50 values of 1-(3-aminophenyl)ethylidenethiosemicarbazide
(4) and 1-(3-amidophenyl)ethylidenethiosemicarbazide compounds (8a-i).
Entry
Amido group structure
IC50 (mmol/L)
4
eNH2
eNHCOCH3
eNHCOCF3
6.11
4.12
8a
8b
8c
8d
8e
8f
8g
8h
8i
5.760
3.62
1.54
4.41
1.69
2.202
1.53
1.79
eNHCOC3Hꢁ7 iso
eNHCOC4Hꢁ9 n
eNHCOC4Hꢁ9ꢁtnert
eNHCOC5H1ꢁ1n
eNHCOC6H13
eNHCOC6H5
eNHCOCH2C6H5
2. Results and discussion
2.1. Synthesis
2.2. Biological activity
The synthesis of 5a-n, 6a-i, 7a-n and 8a-i was summarized in
Scheme 1, and the chemical structure of the corresponding sub-
stituent at the phenyl ring was given in Tables 1 and 2. Briefly, the
amide-substituted acetophenones 5a-n and 6a-i were synthesized
smoothly through the classical amidation procedure by using 1 and
2 as the starting materials. Subsequently, the condensation of the
corresponding 1, 2, 5a-n and 6a-i with thiosemicarbazide could be
readily carried out in anhydrous alcohol using acetic acid as catalyst
to provide the desired phenylethylidenethiosemicarbazide de-
rivatives 3, 4, 7a-n and 8a-i in good to excellent yields.
2.2.1. Effects of 1e4, 7a-n and 8a-i on the diphenolase activity of
mushroom tyrosinase
In China, several naturally occurring tyrosinase activators are
put into practical use [15]. For example, 2,3,5,40-tetrahydrox-
ystilbene-2-O-
that activated tyrosinase by 12e126% at
0.12e75.0 g/L [15a], was originally isolated as one of the main
b
-D-glucoside (THSG), a potent tyrosinase activator
a
concentration of
m
active constituents from a traditional Chinese herb-Radix Polygoni
Multiflori, which has been used clinically to treat hypopigmentary
Scheme 1. Synthesis of 1-(aminophenyl)ethylidenethiosemicarbazone compounds (3 and 4) and their derivatives (7aen and 8aei). Reagents and reaction conditions: a, thio-
semicarbazide, anhydrous ethanol, 50e80 ꢀC; b, acyl chloride, triethylamine, anhydrous DMF, ice-bath.

A. You et al. / European Journal of Medicinal Chemistry 93 (2015) 255e262
257
skin disease for a long time. To our delight, our initial investigation
showed that, at a concentration of 100e1000 m/L, 1 and 2
IC50 value of 0.291 mM.
m
From the data of the tyrosinase inhibitory activities of our
synthetic 3-/4-aminophenylethylidenethiosemicarbazides and
their derivatives, the following SARs results could be derived:
exhibited potent activation effects on mushroom tyrosinase with
the activation ratio of 39e138% and 36e102%, respectively, as
determined by dose-dependent activation curve analysis (Fig. 1).
The results suggested that compounds 1 and 2 could serve as po-
tential candidates for the treatment of hypopigmentary skin dis-
ease and further development of such compounds might be of
interest. By analogy with the chemical structures of 1 and 2, it
revealed that the amino group at the position-4 of the phenyl ring
was more beneficial than that at the position-3 of the phenyl ring.
Notably, incorporation of the acetyl group onto the amine moiety of
(1) Compounds 1 and 2 possessed potent tyrosinase activation
activities, while their thiosemicarbazone derivatives showed
highly efficient tyrosinase inhibitory potency. The results
showed that the introduction of thiosemicarbazide group led
to a remarkable change on tyrosinase potency. Furthermore,
it suggested that the phenylethylenethiosemicarbazide
moiety played a vital role in determining the tyrosinase
inhibitory activity, which further supported previous reports
by Chen and our groups [13,14].
(2) As shown in Tables 1e2, it was observed that the thio-
semicarbazone compounds bearing amide-substituent on
the phenyl ring all showed better tyrosinase inhibitory ac-
tivities than their corresponding parent compounds 3 and 4,
suggesting that the introduction of proper hydrophobic
substituent facilitated tyrosinase inhibitory activity, which
was in agreement with our structure-based design, and in
the present investigation, the eNHCOC6H5 group was
optimal.
1 to give 5a, showing no activity at the concentration of 100 mmol/L.
The results suggested that the amine group was absolutely neces-
sarily for determining the tyrosinase activation potency.
Next,
the
inhibition
of
synthetic
3-/4-
aminophenylethylidenethiosemicarbazides 3e4 and their de-
rivatives 7a-n and 8a-i on the diphenolase activity of mushroom
tyrosinase was investigated. Here the well-known tyrosinase in-
hibitor kojic acid was used as the standard reference. The IC50
values of all obtained compounds and kojic acid were summarized
in Tables 1 and 2. As predicted, these structure-based compounds
all displayed more efficient tyrosinase inhibitory activities (the
range of IC50 values, for 7a-n: from 0.291 to 1.336
m
M; for 8a-i: from
(3) In general, 4-amidophenylethylidenethiosemicarbazide de-
rivatives 7a-n showed significant inhibitory activities on
tyrosinase and their IC50 values were of the same order of
magnitude. The similar results were also obtained from 3-
amidophenylethylidenethiosemicarbazide derivatives 8a-i.
These findings revealed that the type of acyl group had no
obvious effect on the inhibitory potency. Moreover, it hinted
that the COeNH part might play an important role in
improving tyrosinase inhibitory activity since previous
literature have revealed that the nitrogen and oxygen atoms
of tyrosinase inhibitors could form the potential H-bonding
interactions with the sulfhydryl, amino, carboxyl or hydroxyl
groups in the active site of tyrosinase [7b,14d,16].
1.53 to 5.76 M). Especially, com-
m
M) than kojic acid (IC50 ¼ 28.5
m
pound 7k was found to be the most active tyrosinase inhibitor with
(4) Compared with 7a-i and 8a-i, the results showed that the
compounds bearing an amide group at the position-4 of the
phenyl ring displayed more profound tyrosinase inhibitory
activities than the ones bearing an amide group at position-3
of the phenyl ring. It suggested that the position of the
substituent influenced the tyrosinase inhibitory potency, and
in the investigation, the amide group at the position-4 of the
phenyl ring was more efficacious for the inhibitory activity. It
was consistent with previous reports by Kubo [17], Jimenez
[18].
The above SAR results were summarized in Fig. 2.
2.2.2. Inhibition mechanism of selected compounds 7k and 8h on
mushroom tyrosinase
Inspired by the above results, next we were sought to explore
the inhibition mechanism of compounds 7a-n and 8a-i. As illus-
trated in Fig. 3, the inhibition mechanism on mushroom tyrosinase
by compounds 7k and 8h for the oxidation of L-DOPA was studied.
The results showed that, the plots of the remaining tyrosinase ac-
tivity versus the concentrations of tyrosinase in the presence of
different concentrations of 7k and 8h gave a family of straight lines,
which all passed through the origin point. Increasing the concen-
tration of 7k and 8h resulted in a decrease in the slope of the line,
revealing that the inhibition of 7k and 8h on tyrosinase was
reversible. It should be noted that, previous reports from Eicken
[19] and our groups [14a] have defined 1-phenylthiourea and 1-(1-
phenylethylidene)thiosemicarbazide as the irreversible inhibitors
of tyrosinase. Obviously, the inhibition mechanism of 7k and 8h
Fig. 1. Dose-dependent activation effects of 1 and 2 on mushroom tyrosinase.

258
A. You et al. / European Journal of Medicinal Chemistry 93 (2015) 255e262
Promoting inhibitory-activity group
The introduction of acyl group enhanced the inhibitory activity, while
its type had no obvious infulence on impoving the inhibitory activity
R
O
Activation-activity key group
The amine group was absolutely necessarily for determining the
tyrosinase activation activity
NH
The position of substituents on the phenyl ring influenced the tyrosinase
inhibitory potency, and the position-4 was optimal
Inhibitory-activity key group
H3C
N
a)The introduction of thiosemicarbazide group played a very vital role
in transforming activator into inhibitor
b)The thiosemicarbazone moiety was crucial for the tyrosinase
inhibitory activity
HN
S
H2N
Fig. 2. SARs analysis.
was different from 1-phenylthiourea and 1-(1-phenylethylidene)
thiosemicarbazide. By analogy with their chemical structures, it
suggested that the introduction of acyl moiety might affect the
binding mode of the substrate and the active site of tyrosinase.
2.2.3. Inhibitory types of selected compounds 7k and 8h on
mushroom tyrosinase
To further insight into the inhibition mechanism, finally we
determined the inhibitory types of selected compounds 7k and 8h
from LineweavereBurk double reciprocal plots. In the presence of
7k, the kinetics of the enzyme was shown in Fig. 4A. The results
displayed that the plots of 1/V versus 1/[S] gave three straight lines
with different slopes, they intersected on the horizontal axis. With
the increase of the concentration of compound 7k, the value of Vm
descended but the values of Km remained same, indicating that 7k
was a non-competitive tyrosinase inhibitor. The result showed that
7k could bind, not only with free enzyme, but also with the enzy-
meesubstrate complex [1c,20]. Very interestingly, the results from
Fig. 4B showed that the inhibition behavior of 8h was different from
Fig. 3. Determination of the inhibitory effects of 7k and 8h on mushroom tyrosinase
for the catalysis of L-DOPA (A and B). The concentrations of 7k for curves 1e4 were 0.0,
Fig. 4. Determination of the inhibitory types of selected compounds 7k and 8h on
mushroom tyrosinase for the catalysis of L-DOPA (A and B). Concentrations of 7k and
0.1, 0.3 and 0.5
mM, respectively. The concentrations of 8h for curves 1e4 were 0.0, 1.0,
1.5 and 2.0 M, respectively.
m
8h for curves 1e3 were 0.1, 0.3, 0.5 mM and 1.0, 1.5, 2.0 mM, respectively.

A. You et al. / European Journal of Medicinal Chemistry 93 (2015) 255e262
259
that of 7k. As shown in Fig. 4B, the LineweavereBurk double
reciprocal plots in the presence of 8h yielded a family of straight
lines with different slopes that intersected one another in the third
quadrant. With the increase of the concentration of 8h, the values
of Vm and Km all descended, suggesting that 8h was a competitive-
uncompetitive mixed-II type inhibitor. The result revealed that 8h
could only bind with free enzyme [14c].
4.2.1.1. 1-(4-Aminophenyl)ethylidenethiosemicarbazide
(3).
Yellowish powder, yield 72%, m.p. 182e183 ꢀC. 1H NMR (300 MHz,
DMSO-d6)
d
: 9.97 (s, 1H), 8.11 (s, 1H), 7.73 (s, 1H), 7.62 (d, J ¼ 7.3 Hz,
2H), 6.54 (d, J ¼ 8.4 Hz, 2H), 5.45 (s, 2H), 2.19 (s, 3H). 13C NMR
(75 MHz, DMSO-d6):
d 178.0, 150.1, 149.0, 127.8, 124.6, 113.1, 13.6.
ESI-MS m/z ¼ 209.1 [M þ 1]þ.
4.2.1.2. 1-(3-Aminophenyl)ethylidenethiosemicarbazide
(4).
Yellowish solid product, yield 69%, m.p.184e186 ꢀC. 1H NMR
3. Conclusion
(300 MHz, DMSO-d6):
d: 10.22 (s, 1H), 8.32 (s, 1H), 7.71 (s, 1H), 7.11
(s, 1H), 7.06e6.99 (m, 2H), 6.66e6.55 (m, 1H), 5.06 (s, 2H), 2.23 (s,
In summary, we have defined for the first time 3-/4-
aminoacetophenones as the potent tyrosinase activators.
Structure-based modification toward them resulted in a remark-
able change of the potency on tyrosinase (from potent activators to
highly efficient inhibitors). The results from the biological evalua-
tion on the diphenolase activitiy of mushroom tyrosinase showed
3H). 13C NMR (75 MHz, DMSO-d6)
d: 178.8, 148.7, 148.4, 138.2, 128.7,
115.0, 114.5, 111.8, 14.1. ESI-MS m/z ¼ 209.1 [M þ 1]þ.
4.2.2. General procedure for the synthesis of 7a-n and 8a-i
(i) To a solution of aminoacetophenones (5.0 mmol) in anhy-
drous DMF (10 mL) were added 1.0 equiv of the corre-
sponding acyl chloride (5.0 mmol) and 1.2 equiv of
triethylamine (6.0 mmol) at 0 ꢀC in ice-bath under argon
atmosphere, and the reaction was allowed to stir until all the
starting material had disappeared. Then, the resulting
mixture was acidified with 1 M HCl to PH < 7 and extracted
with ethyl acetate (3 times). The obtained organic phase was
washed with 5% NaHCO3 (2 times) and brine (2 times), dried
over Na2SO4 and concentrated in vacuo to give the in-
termediates 5a-n and 6a-i, which was used directly for the
next step.
(ii) To the solution of appropriate 5a-n and 6a-i (5.0 mmol) in
anhydrous ethanol (20 mL), thiosemicarbazide (5.0 mmol)
and acetic acid (0.5 mL) were added. The reaction mixture
was stirred at 50e80 ꢀC for 3 h and then cooled to room
temperature. The precipitate solid was filtered, washed with
ether, and purified by recrystallization from 95% ethanol to
afford desired compounds7a-n and 8a-i.
that
all
the
designed
3-/4-
aminophenylethylidenethiosemicarbazide derivatives have more
potent tyrosinase inhibitory activities than the well-known tyros-
inase inhibitor kojic acid. Especially, compound 7k exhibited the
most potent tyrosinase inhibitory activity with IC50 value of
0.291 mM. The SARs analysis indicated that: (1) the amine group
was absolutely necessarily for determining the tyrosinase activa-
tion activity; (2) the introduction of thiosemicarbazide group
played a very vital role in transforming tyrosinase activators into
tyrosinase inhibitors; (3) the phenylethylenethiosemicarbazide
moiety was crucial for determining the tyrosinase inhibitory ac-
tivity; (4) the type of acyl group had no obvious effect on the
inhibitory activity, thus suggesting that the COeNH part might play
an important role in improving tyrosinase inhibitory activity; (5)
the position of amide substituent on the phenyl ring influenced the
tyrosinase inhibitory potency, and in the investigation, the
position-4 was optimal. Moreover, the inhibition mechanism and
inhibition kinetics study revealed that compound 7k was reversible
and non-competitive inhibitor, and compound 8h was reversible
and competitive-uncompetitive mixed-II type inhibitor. Taken
together, these results suggested that these compounds could serve
as a class of promising candidates for further development of drug
for the treatment of tyrosinase-related disorders.
4.2.2.1. 1-(4-Acetamidophenyl)ethylidenethiosemicarbazide
Yellowish solid product, yield 88%, m.p. 228e229 ꢀC. 1H NMR
(300 MHz, DMSO-d6) : 10.17 (s, 1H), 10.07 (s, 1H), 8.26 (s, 1H), 7.88
(d, J ¼ 8.1 Hz, 3H), 7.60 (d, J ¼ 7.3 Hz, 2H), 2.26 (s, 3H), 2.06 (s, 3H).
(7a).
d
13C NMR (75 MHz, DMSO-d6)
d: 178.6, 168.4, 147.5, 140.2, 132.0,
4. Experimental section
127.1, 118.2, 24.0, 13.6. ESI-MS m/z ¼ 251.2 [M þ 1]þ. Anal. Calcd for
4.1. General
C
11H14N4OS: C, 52.78; H, 5.64; N, 22.38; found: C, 52.73; H, 5.59; N,
22.50.
Melting points were determined on a WRS-1B digital instru-
ment without correction. NMR spectra were recorded on a Varian
4.2.2.2. 1-(4-Trifloroacetamidophenyl)ethylidenethiosemicarbazide
(7b). Solid product, yield 92%, m.p.170 ꢀC (dec.). 1H NMR (300 MHz,
Mercury-Plus 300 spectrometer in DMSO-d6. All chemical shifts (d)
were quoted in parts per million and coupling constants (J) were
given in Hertz. Mass spectra were obtained from VG ZAB-HS, LCMS-
2010A or LCQ DECA XP spectrometer. All commercially available
reagents and solvents were used without further purification.
Mushroom tyrosinase (specific activity of the enzyme is 6680 U/
mg) and L-DOPA (L-3, 4-dhydroxyphenylalanine) were purchased
from Sigma Chemical Co.
DMSO-d6)
d: 11.37 (s, 1H), 10.26 (s, 1H), 8.33 (s, 1H), 8.02 (d,
J ¼ 8.7 Hz, 3H), 7.72 (d, J ¼ 8.7 Hz, 2H), 2.31 (s, 3H). 13C NMR
(75 MHz, DMSO-d6):
d 178.8, 154.7, 154.2, 146.9, 137.1, 134.6, 127.3,
120.4, 13.7. ESI-MS m/z ¼ 305.1 [M þ 1]þ.
4.2.2.3. 1-(4-Trichloroacetamidophenyl)ethylidenethiosemicarbazide
(7c). Yellowish crystal product, yield 80%, m.p. 195 ꢀC (dec.). 1H
NMR (300 MHz, DMSO-d6) d: 10.95 (s, 1H),10.23 (s, 1H), 8.30 (s, 1H),
4.2. Chemistry
8.00 (d, J ¼ 8.8 Hz, 3H), 7.70 (d, J ¼ 8.8 Hz, 2H), 2.29 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) d: 178.8, 159.6, 147.0, 138.0, 134.3, 127.2, 120.6,
4.2.1. General procedure for the synthesis of 3e4
92.9, 13.7. ESI-MS m/z ¼ 353.2 [M þ 1]þ.
To the solution of appropriate aminoacetophenones (5 mmol) in
anhydrous ethanol (30 mL), thiosemicarbazide (5 mmol) and acetic
acid (0.5 mL) were added. The reaction mixture was stirred at
50e80 ꢀC for 3 h and then cooled to room temperature. The pre-
cipitate solid was filtered and washed with ethanol to afford pure
target compounds 3e4.
4.2.2.4. 1-(4-Propionamidophenyl)ethylidenethiosemicarbazide (7d).
Yellowish solid product, yield 86%, m.p. 221e222 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d: 10.16 (s, 1H), 9.99 (s, 1H), 8.25 (s, 1H), 7.88
(d, J ¼ 8.8 Hz, 3H), 7.61 (d, J ¼ 8.8 Hz, 2H), 2.40e2.28 (m, 2H), 2.26 (s,
3H), 1.08 (t, J ¼ 7.5 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d: 178.6,

260
A. You et al. / European Journal of Medicinal Chemistry 93 (2015) 255e262
172.1, 147.5, 140.3, 131.9, 127.2, 118.3, 29.5, 13.7, 9.5. ESI-MS m/
z ¼ 265.2 [M þ 1]þ.
4 . 2 . 2 . 1 2 . 1 - ( 4 - ( 4 - F l o r o b e n z a m i d o p h e n y l ) ) e t h y l -
idenethiosemicarbazide (7l). Solid product, yield 87%, m.p.
201e202 ꢀC. 1H NMR (300 MHz, DMSO-d6)
d: 10.34 (s, 1H), 10.15 (s,
1H), 8.24 (s, 1H), 8.10e7.97 (m, 2H), 7.93 (d, J ¼ 8.6 Hz, 3H), 7.79 (d,
4.2.2.5. 1-(4-Butyramidophenyl)ethylidenethiosemicarbazide
Yellowish solid product, yield 82%, m.p. 217e218 ꢀC. 1H NMR
(300 MHz, DMSO-d6) : 10.16 (s, 1H), 10.00 (s, 1H), 8.25 (s, 1H), 7.88
(7e).
J ¼ 8.2 Hz, 2H), 7.36 (t, J ¼ 8.4 Hz, 2H), 2.29 (s, 3H). 13C NMR
(75 MHz, DMSO-d6) d: 179.2, 165.0, 148.0, 140.6, 133.2, 131.0, 130.9,
d
127.6, 120.1, 115.9, 115.7, 14.2. ESI-MS m/z ¼ 331.4 [M þ 1]þ.
(d, J ¼ 8.8 Hz, 3H), 7.62 (d, J ¼ 8.9 Hz, 2H), 2.30 (t, J ¼ 7.4 Hz, 2H),
2.26 (s, 3H), 1.69e1.54 (m, 2H), 0.92 (t, J ¼ 7.4 Hz, 3H). 13C NMR
4 . 2 . 2 . 1 3 . 4 - ( 4 - M e t h o x y b e n z a m i d o p h e n y l ) e t h y l -
idenethiosemicarbazide (7m). Solid product, yield 85%, m.p.
(75 MHz, DMSO-d6) d: 178.6, 171.3, 147.5, 140.3, 132.0, 127.11, 118.3,
18.4, 13.7,13.6. ESI-MS m/z ¼ 279.2 [M þ 1]þ.
202e203 ꢀC. 1H NMR (300 MHz, DMSO-d6)
d
: 10.15 (d, J ¼ 7.7 Hz,
2H), 8.23 (s, 1H), 7.93 (t, J ¼ 8.7 Hz, 5H), 7.79 (d, J ¼ 7.9 Hz, 2H), 7.05
4.2.2.6. 1-(4-iso-Butyramidophenyl)ethylidenethiosemicarbazide
(d, J ¼ 7.4 Hz, 3H), 3.83 (s, 3H), 2.53 (s, 1H), 2.28 (s, 3H). 13C NMR
(7f). Solid product, yield 88%, m.p. 216e217 ꢀC. 1H NMR (300 MHz,
(75 MHz, DMSO-d6) d:178.7, 164.9, 162.9, 147.5, 140.4, 132.4, 129.8,
DMSO-d6)
d: 10.16 (s, 1H), 9.96 (s, 1H), 8.25 (s, 1H), 7.89 (d,
127.0, 126.5, 119.5, 113.6, 55.4, 13.7. ESI-MS (m/z) ¼ 343.4 [M þ 1]þ.
J ¼ 8.8 Hz, 3H), 7.63 (d, J ¼ 8.8 Hz, 2H), 2.60 (m, 1H), 2.26 (s, 3H), 1.10
(d, J ¼ 6.8 Hz, 6H). 13C NMR (75 MHz, DMSO-d6)
d: 178.6, 175.3,
4.2.2.14. 1-(4-Phenylacetamidophenyl)ethylidenethiosemicarbazide
147.5140.4, 131.9, 127.1, 118.4, 35.0, 19.5, 13.7. Anal. Calcd for
(7n). Solid product, yield 84%, m.p. 211e212 ꢀC. 1H NMR (300 MHz,
C
13H18N4OS: C, 56.09; H, 6.52; N, 20.13; found: C, 56.04; H, 6.40; N,
20.41. ESI-MS m/z ¼ 279.2 [M þ 1]þ.
DMSO-d6)
d: 10.32 (s, 1H), 10.17 (s, 1H), 8.25 (s, 1H), 7.90 (d,
J ¼ 8.8 Hz, 3H), 7.62 (d, J ¼ 8.8 Hz, 2H), 7.34 (d, J ¼ 4.4 Hz, 4H),
7.29e7.23 (m, 1H), 3.66 (s, 2H), 2.26 (s, 3H). 13C NMR (75 MHz,
4.2.2.7. 1-(4-Pentanamidophenyl)ethylidenethiosemicarbazide (7g).
Yellowish solid product, yield 87%, m.p. 213e214 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d: 10.18 (s, 1H), 10.01 (s, 1H), 8.27 (s, 1H), 7.89
DMSO-d6) d: 178.61, 169.2, 147.5, 140.1, 135.8, 132.2, 129.1, 128.3,
127.2, 126.5, 118.4, 43.3, 13.7. ESI-MS m/z ¼ 327.3 [M þ 1]þ.
(d, J ¼ 8.8 Hz, 3H), 7.62 (d, J ¼ 8.8 Hz, 2H), 2.33 (t, J ¼ 7.4 Hz, 2H),
4.2.2.15. 1-(3-Acetamidophenyl)ethylidenethiosemicarbazide (8a).
Brown powder product, yield 88%, m.p. 204e205 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d: 10.35 (s, 1H), 9.99 (s, 1H), 8.41 (s, 1H), 7.95
2.27 (s, 3H), 1.65e1.52 (m, 2H), 1.40e1.25 (m, 2H), 0.90 (t, J ¼ 7.3 Hz,
3H). 13C NMR (75 MHz, DMSO-d6)
d: 178.6, 171.4, 147.5, 140.3, 132.0,
127.1, 118.3, 36.1, 27.1, 21.8, 13.7, 13.7. Anal. Calcd for C14H20N4OS: C,
57.51; H, 6.89; N, 19.16; found: C, 57.25; H, 6.77; N, 19.52. ESI-MS m/
z ¼ 293.3 [M þ 1]þ.
(s, 1H), 7.78 (s, 1H), 7.73 (d, J ¼ 8.0 Hz, 1H), 7.63 (d, J ¼ 8.0 Hz, 1H),
7.33 (t, J ¼ 8.0 Hz, 1H), 2.31 (s, 3H), 2.08 (s, 3H). 13C NMR (75 MHz,
DMSO-d6) d: 178.9,168.4, 147.7, 139.2, 138.1,128.6,121.3, 120.0,117.1,
24.0, 14.1. ESI-MS m/z ¼ 251.2 [M þ 1]þ.
4.2.2.8. 1-(4-Hexanamidophenyl)ethylidenethiosemicarbazide (7h).
Yellowish solid product, yield 81%, m.p. 178e179 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d: 10.17 (s, 1H), 10.00 (s, 1H), 8.26 (s, 1H), 7.88
4.2.2.16. 1-(3-Trifloroacetamidophenyl)ethylidenethiosemicarbazide
(8b). Solid product, yield 93%, m.p.168 ꢀC (dec.). 1H NMR (300 MHz,
(d, J ¼ 8.8 Hz, 3H), 7.62 (d, J ¼ 8.8 Hz, 2H), 2.31 (t, J ¼ 7.4 Hz, 2H),
DMSO-d6)
(d, J ¼ 8.0 Hz, 2H), 7.75 (d, J ¼ 8.0 Hz,1H), 7.44 (t, J ¼ 8.0 Hz,1H), 2.31
(s, 3H). 13C NMR (75 MHz, DMSO-d6)
: 179.0, 154.8, 154.3, 146.9,
d: 11.27 (s, 1H), 10.38 (s, 1H), 8.43 (s, 1H), 8.07 (s, 1H), 7.82
2.26 (s, 3H), 1.66e1.53 (m, 2H), 1.36e1.23 (m, 4H), 0.88 (t, J ¼ 6.9 Hz,
3H). 13C NMR (75 MHz, DMSO-d6)
d: 178.6, 171.4, 147.5, 140.3, 132.0,
d
127.1, 118.3, 36.4, 30.9, 24.7, 21.9, 13.8, 13.7. ESI-MS m/z ¼ 307.3
138.5, 136.2, 128.9, 123.9, 121.8, 119.3, 14.0. ESI-MS m/z ¼ 305.2
[M þ 1]þ.
[M þ 1]þ.
4.2.2.9. 1-(4-Heptanamidophenyl)ethylidenethiosemicarbazide (7i).
Solid product, yield 83%, m.p. 187e188 ꢀC. 1H NMR (300 MHz,
4.2.2.17. 1-(3-iso-Butyramidophenyl)ethylidenethiosemicarbazide
(8c). Solid product, yield 85%, m.p. 198e199 ꢀC. 1H NMR (300 MHz,
DMSO-d6)
d: 10.16 (s, 1H), 10.00 (s, 1H), 8.25 (s, 1H), 7.88 (d,
DMSO-d6) d: 10.34 (s, 1H), 9.86 (s, 1H), 8.41 (s, 1H), 7.99 (s, 1H), 7.75
J ¼ 8.8 Hz, 3H), 7.61 (d, J ¼ 8.7 Hz, 2H), 2.31 (t, J ¼ 7.4 Hz, 2H), 2.26 (s,
(d, J ¼ 6.0 Hz, 2H), 7.63 (d, J ¼ 7.9 Hz, 1H), 7.32 (t, J ¼ 8.0 Hz, 1H),
3H), 1.57 (d, J ¼ 6.9 Hz, 2H), 1.27 (s, 6H), 0.85 (d, J ¼ 6.6 Hz, 3H). 13
C
2.68e2.54 (m, 1H), 2.30 (s, 3H), 1.12 (d, J ¼ 6.8 Hz, 6H). 13C NMR
NMR (75 MHz, DMSO-d6) d: 178.6, 171.4, 147.5, 140.3, 132.0, 127.1,
(75 MHz, DMSO-d6) d: 179.0, 175.3, 147.7, 139.4, 138.1, 128.6, 121.3,
118.3 36.42, 31.0, 28.31, 25.0, 22.0, 13.9, 13.7. ESI-MS m/z ¼ 321.3
119.9, 117.1, 35.0, 19.5, 14.1. Anal. Calcd for C13H18N4OS: C, 56.09; H,
6.52; N, 20.13; found: C, 55.86; H, 6.40; N, 20.39. ESI-MS m/
z ¼ 279.2 [M þ 1]þ.
[M þ 1]þ.
4 . 2 . 2 . 1 0 . 1 - ( 4 - M e t h o x y f o r m a m i d o p h e n y l ) e t h y l -
idenethiosemicarbazide (7j). Yellowish solid product, yield 71%,
4.2.2.18. 1-(3-Pentanamidophenyl)ethylidenethiosemicarbazide (8d).
Solid product, yield 88%, m.p. 198e199 ꢀC, 1H NMR (300 MHz,
DMSO-d6) d: 10.32 (s, 1H), 9.90 (s, 1H), 8.39 (s, 1H), 7.94 (s, 1H), 7.72
m.p. 217e218 ꢀC. 1H NMR (300 MHz, DMSO-d6)
d
: 10.10 (s, 1H), 9.76
(s, 1H), 8.20 (s, 1H), 7.84 (d, J ¼ 7.8 Hz, 3H), 7.45 (d, J ¼ 8.1 Hz, 2H),
3.66 (s, 3H), 2.24 (s, 3H). 13C NMR (75 MHz, DMSO-d6)
: 178.6,
d
(d, J ¼ 7.5 Hz, 2H), 7.61 (d, J ¼ 7.7 Hz, 1H), 7.30 (t, J ¼ 7.9 Hz, 1H),
153.8, 147.6, 140.1, 131.5, 127.3, 117.4, 51.7, 13.7. ESI-MS m/z ¼ 267.3
2.40e2.20 (m, 5H), 1.64e1.48 (m, 2H), 1.38e1.23 (m, 2H), 0.89 (t,
[M þ 1]þ.
J ¼ 7.3 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d: 179.0, 171.4, 147.7,
139.3, 138.1, 128.6, 121.3, 119.9, 117.0, 36.1, 27.2, 21.8, 14.1, 13.7. ESI-
4.2.2.11. 1-(4-Benzamidophenyl)ethylidenethiosemicarbazide (7k).
MS m/z ¼ 293.4 [M þ 1]þ.
Solid product, yield 82%, m.p. 140 ꢀC (dec.). 1H NMR (300 MHz,
DMSO-d6)
d
: 10.38 (s, 1H), 10.20 (s, 1H), 8.29 (s, 1H), 8.03e7.90 (m,
4.2.2.19. 1-(3-tert-Pentanamidophenyl)ethylidenethiosemicarbazide
5H), 7.83 (d, J ¼ 8.9 Hz, 2H), 7.68e7.49 (m, 3H), 2.30 (s, 3H). 13C NMR
(8e). Solid product, yield 84%, m.p. 220e221 ꢀC. 1H NMR (300 MHz,
(75 MHz, DMSO-d6)
d: 178.7, 165.6, 147.4, 140.2, 134.8, 132.6, 131.7,
DMSO-d6)
(d, J ¼ 7.8 Hz, 2H), 7.64 (d, J ¼ 7.8 Hz,1H), 7.31 (t, J ¼ 8.0 Hz,1H), 2.30
(s, 3H), 1.24 (s, 9H). 13C NMR (75 MHz, DMSO-d6)
: 178.9, 176.5,
147.8, 139.3, 137.9, 128.3, 121.4, 120.9, 118.2, 27.2, 14.1. Anal. Calcd for
d: 10.31 (s, 1H), 9.21 (s, 1H), 8.40 (s, 1H), 8.01 (s, 1H), 7.80
128.4, 127.7, 127.1, 119.5, 13.7. Anal. Calcd for C16H16N4OS: C, 61.52;
H, 5.16; N, 17.93; found: C, 61.31; H, 5.07; N, 18.04. ESI-MS m/
z ¼ 313.2 [M þ 1]þ.
d

A. You et al. / European Journal of Medicinal Chemistry 93 (2015) 255e262
261
C14H20N4OS: C, 57.51; H, 6.89; N, 19.16; found: C, 57.59; H, 6.74; N,
reported by our groups [22] with slight modifications. Briefly, all
the synthesized compounds were screened for the diphenolase
inhibitory activity of tyrosinase using L-DOPA as substrate. All
thiosemicarbazone compounds were dissolved in DMSO and their
final concentration in DMSO was 2.0%. Phosphate buffer, pH 6.8,
was used to dilute the DMSO stock solution of test compounds.
Thirty units of tyrosinase (0.5 mg/mL) was first pre-incubated with
the samples, in 50 mM phosphate buffer (pH ¼ 6.8), for 10 min at
25 ꢀC, L-DOPA solution (0.5 mM) was added to the mixture and the
enzyme reaction was monitored by measuring the change in
absorbance at 475 nm of formation of the DOPA chrome for 1 min.
The measurement was performed in triplicate for each concentra-
tion and averaged before further calculation. IC50 value was
determined by interpolation of the doseeresponse curves. As a
control, the IC50 value of kojic acid was also determined at the same
conditions.
19.42. ESI-MS m/z ¼ 293.2 [M þ 1]þ.
4.2.2.20. 1-(3-Hexanamidophenyl)ethylidenethiosemicarbazide (8f).
Solid product, yield 83%, m.p. 189e190 ꢀC. 1H NMR (300 MHz,
DMSO-d6) d: 10.34 (s, 1H), 9.90 (s, 1H), 8.40 (s, 1H), 7.95 (s, 1H), 7.74
(d, J ¼ 6.6 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.31 (t, J ¼ 8.0 Hz, 1H),
2.37e2.25 (m, 5H), 1.67e1.54 (m, 2H), 1.39e1.20 (m, 4H), 0.88 (t,
J ¼ 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d: 179.0, 171.3, 147.7,
139.3, 138.1, 128.6, 121.2, 119.8, 117.0, 36.4, 30.9, 24.7, 21.9, 14.1, 13.8.
Anal. Calcd for C15H22N4OS: C, 58.79; H, 7.24; N, 18.28; found: C,
58.74; H, 7.15; N, 18.48. ESI-MS m/z ¼ 307.3 [M þ 1]þ.
4.2.2.21. 1-(3-Heptanamidophenyl)ethylidenethiosemicarbazide
(8g). Powder product, yield 85%, m.p. 127e128 ꢀC. 1H NMR
(300 MHz, DMSO-d6) d: 10.33 (s, 1H), 9.90 (s, 1H), 8.40 (s, 1H), 7.94
(s, 1H), 7.73 (d, J ¼ 6.7 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.30 (t,
J ¼ 8.0 Hz, 1H), 2.36e2.25 (m, 5H), 1.64e1.54 (m, 2H), 1.35e1.23 (m,
Acknowledgments
6H), 0.87 (t, J ¼ 6.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6)
d: 179.0,
171.3, 147.7, 139.3, 138.1, 128.6, 121.2, 119.8, 117.0, 36.4, 31.0, 28.3,
This work was supported by the Science and Technology Project
of Guangdong Province, China (2009B060700048), Shanghai
Municipal Natural Science Foundation (15ZR1447800) and the
Fundamental Research Funds for the Central Universities.
25.0, 22.0, 14.1, 13.9. ESI-MS m/z ¼ 321.3 [M þ 1]þ.
4.2.2.22. 1-(3-Benzamidophenyl)ethylidenethiosemicarbazide (8h).
Powder product, yield 84%, m.p. 210e211 ꢀC. 1H NMR (300 MHz,
DMSO-d6) d: 10.37 (s, 1H), 10.27 (s, 1H), 8.43 (s, 1H), 8.18 (s, 1H), 7.96
Appendix A. Supplementary data
(t, J ¼ 9.5 Hz, 3H), 7.81 (s, 1H), 7.70 (d, J ¼ 7.7 Hz, 1H), 7.56 (d,
J ¼ 7.9 Hz, 3H), 7.38 (t, J ¼ 8.0 Hz,1H), 2.33 (s, 3H). 13C NMR (75 MHz,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.02.013.
DMSO-d6) d: 179.0, 165.6, 147.6, 139.1, 138.1, 134.8, 131.6, 128.6,
128.4, 127.6, 122.0, 121.2, 118.5, 14.1. Anal. Calcd for C16H16N4OS: C,
61.52; H, 5.16; N, 17.93; found: C, 61.20; H, 5.05; N, 18.12. ESI-MS m/
z ¼ 313.2 [M þ 1]þ.
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