Synthesis and Evaluation of 2(3H)-Thiazole Thiones as Tyrosinase Inhibitors

Article abstract of DOI:10.1002/ardp.201200028

A series of 2(3H)-thiazole thiones 3-5 was synthesized and evaluated for tyrosinase inhibition and DPPH radical scavenging activities. Among them, 3-methyl-4-phenyl-2(3H)-thiazole thione (4a) showed good tyrosinase inhibitory activity, even better than that of the well-known tyrosinase inhibitor, namely, kojic acid. From the structure-activity point of view, although it was found that the phenolic hydroxyl group in prototype 3-5 might contribute to the scavenging activity against DPPH radicals, there was no correlation between the potency of tyrosinase inhibition and the presence of the phenolic moiety. The in silico ADME-Tox screening revealed that the drug-likeness and drug-score values of the most potent compound 4a were significantly higher than those of kojic acid. Copyright

Full text of DOI:10.1002/ardp.201200028

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
1
Full Paper
Synthesis and Evaluation of 2(3H)-Thiazole Thiones as
Tyrosinase Inhibitors
Saeed Emami1, Seyed Jalal Hosseinimehr1, Kami Shahrbandi1, Ahmad Ali Enayati2, and
Zahra Esmaeeli1
1 Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
2 Department of Medical Entomology, School of Public Health and Health Sciences Research Centre,
Mazandaran University of Medical Sciences, Sari, Iran
A series of 2(3H)-thiazole thiones 3–5 was synthesized and evaluated for tyrosinase inhibition and
DPPH radical scavenging activities. Among them, 3-methyl-4-phenyl-2(3H)-thiazole thione (4a) showed
good tyrosinase inhibitory activity, even better than that of the well-known tyrosinase inhibitor,
namely, kojic acid. From the structure–activity point of view, although it was found that the phenolic
hydroxyl group in prototype 3–5 might contribute to the scavenging activity against DPPH radicals,
there was no correlation between the potency of tyrosinase inhibition and the presence of the
phenolic moiety. The in silico ADME-Tox screening revealed that the drug-likeness and drug-score
values of the most potent compound 4a were significantly higher than those of kojic acid.
Keywords: ADME-Tox screening / Phenolic antioxidants / Radical scavenging activity / Thiazole / Thiazoline / Tyrosinase
inhibitors
Received: January 22, 2012; Revised: January 22, 2012; Accepted: March 15, 2012
DOI 10.1002/ardp.201200028
Introduction
inhibitors have become increasingly important in
the food industry as well as in medicinal and cosmetic
products.
Tyrosinase (monophenol monooxygenase), also known as
polyphenol oxidase, is a copper-containing multifunctional
oxidase widely found in animals, plants, and fungi. This
enzyme accepts many phenols and ortho-diphenols (catechols)
as substrates and catalyzes the aerobic hydroxylation of
monophenol and the oxidation of ortho-diphenol to the
corresponding ortho-quinone [1]. It is the key enzyme for
melanogenesis in mammals and causes the unfavorable
browning of fruits and vegetables [2, 3]. Therefore, the inhi-
bition of tyrosinase activity is important for controlling fruit
and vegetable browning, as well as in treating disorders
associated with overproduction of melanin, including
cutaneous hyperpigmentation, melasma, ocular retinitis
pigmentosa, post-inflammatory hyperpigmentation, solar
lentigo, and Addison’s disease [4–6]. Hence, tyrosinase
To date, several natural and synthetic tyrosinase inhibitors
have been described, the majority of which consist of a
phenol structure or metal chelating agents [7, 8]. However,
currently available tyrosinase inhibitors suffer from toxicity
and/or a lack of efficacy and insufficient penetrative ability.
For example, potentially active agents, such as kojic acid and
arbutin (Fig. 1), have not yet been demonstrated as clinically
efficient; moreover, serious side effects like carcinogenicity
limit their human use [9–11]. Obviously, more efforts are still
needed to design and develop potent tyrosinase inhibitors
with fewer side effects. A possible way to pursuit an effective
compound and to overcome some of these limitations could
be finding agents which may act through different mechan-
isms. An example of producing such agents is combining
tyrosinase inhibitors with efficient antioxidants. A number of
natural phenolic and methoxy phenolic antioxidants such
as p-coumaric acid, ferulic acid, and eugenol have been
reported to act as tyrosinase inhibitors (Fig. 2) [12, 13]. In
addition, tyrosinase inhibitory activity has been reported to
be associated with the thione-containing heterocycles (Fig. 3)
Correspondence: Dr. Saeed Emami, Department of Medicinal
Chemistry, Faculty of Pharmacy, Mazandaran University of Medical
Sciences, Sari, Iran.
E-mail: sd_emami@yahoo.com
Fax: 0098 151 3543084
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S. Emami et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
OH
Results and discussion
O
O
OH
OH
HO
HO
Chemistry
O
HO
The general synthetic strategy employed to prepare the tar-
get compounds was based on the cyclocondensation reaction
of substituted dithiocarbamate and a-bromoacetophenone
derivatives, as outlined in Scheme 1. The commercially avail-
able starting acetophenones 1a–c were subjected to a-bromi-
nation with CuBr2 in refluxing CHCl3–EtOAc to yield a-
bromoacetophenones 2a–c [18]. On the other hand, conden-
sation of methylamine with carbon disulfide in the presence
of potassium acetate gave the corresponding dithiocarba-
mate salt, which was treated with a-bromoacetophenones
2a–c in MeOH to afford an intermediate products 3a–c which
can be isolated and have been shown to be 4-hydroxythiazo-
line-2-thione derivatives. Subsequently, dehydration of 3a–c
with diluted hydrochloric acid furnished 2(3H)-thiazole thi-
ones 4a–c in a good yield. The 4-phenyl analogs 5a and b were
prepared by the reaction of corresponding a-bromoacetophe-
none with triethylammonium N-phenyldithiocarbamate [19]
in acetone and subsequent dehydration in 80% H2SO4.
O
OH
Kojic acid
Arbutin
Figure 1. Examples of currently available tyrosinase inhibitors.
including 1,3,4-oxadiazole-2(3H)-thiones [14], imidazolidine-
2-thiones, tetrahydropyrimidine-2(1H)-thiones [15], and 3,4-
dihydroquinazoline-2(1H)-thiones [16, 17]. Therefore, we
introduced the 2(3H)-thiazole thione moiety into the para-
position of the phenolic ring instead of the alkenyl side chain
of natural antioxidants, ferulic acid, and eugenol (Fig. 4).
Herein, we describe the synthesis, tyrosinase inhibitory,
and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging
activities of 4-aryl-2(3H)-thiazole thiones. Hydroxy or
methoxy substituents on the phenyl ring were also changed
to explore the structure–activity relationship (Fig. 4).
O
O
CH2
H3CO
HO
H3CO
HO
OH
OH
HO
p-Coumaric acid
Eugenol
Ferulic acid
Figure 2. Structures of natural phenolic antiox-
idants exhibiting tyrosinase inhibitory activity.
S
O
S
R
N
N
N
R2
NH
N
R1
N
n
m
N
H
S
m = 1 or 2
_
N
H
n = 1 3
3,4-Dihydroquinazoline-
Imidazolidine-2-thiones &
tetrahydropyrimidine-
2(1H)-thiones
1,3,4-Oxadiazole-2(3H)-
Figure 3. Thione-containing heterocyclic
compounds reported to act as tyrosinase
inhibitors.
2(1H)-thiones
thiones
Thione-containing
heterocycle
Scaffold found in
ferulic acid and eugenol
R1
S
R1
S
S
N
N
H3CO
HO
S
R1 = Me, Ph
R
2 = H, 4-OH, 3-OMe-4-OH
R2
_
Designed compounds
2(3H) thiazole thiones
Figure 4. Designed 2(3H)-thiazole thiones as
tyrosinase inhibitors.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
2(3H)-Thiazole Thiones as Tyrosinase Inhibitors
3
S
S
S
H3C
H3C
N
N
S
c
R
R
OH
b
_
_
3a c
4a c
O
O
Br
a
R
R
_
d
_
2a c
1a c
S
a: R = H
S
S
Ph
Ph
b: R = 4-OH
N
_
N
c: R = 3-OMe 4-OH
S
e
OH
R
R
5a,b
Reagents and conditions: (a) CuBr2, CHCl3-EtOAc, reflux; (b) methylamine solution 40%, CS2, CH3COOK, MeOH;
(c) hydrochloric acid 0.5%, MeOH, reflux; (d) triethylammonium N-phenyldithiocarbamate, acetone; (e) H2SO4 80%.
Scheme 1. Synthesis of 3-methyl- or 3-phenyl-4-aryl-2(3H)-thiazole thiones (4a–c and 5a and b).
Biological activity
119.9 and 147.8 mM and were more potent than the reference
drug kojic acid with IC50 of 170.2 mM. Moreover, N-phenyl-
thiazole thione analog 5b showed significant inhibitory
activity (IC50 ¼ 354.8 mM).
Tyrosinase inhibitory activities of synthesized compounds
were determined according to the literature with some modi-
fications [20, 21]. Briefly, the inhibition activities of the syn-
thesized compounds were tested on mushroom tyrosinase
using L-tyrosine as substrate and kojic acid as a standard
inhibitor.
From the structure–activity point of view, it could be
noticed that dehydration of compounds 3a and b (resulting
in compounds 4a and b) improves the inhibitory activity.
Surprisingly, non-phenolic N-methyl compound 4a showed
the highest inhibitory activity, while the inhibitory activity
of phenolic N-phenyl analog 5b was more than that of cor-
responding non-phenolic compound 5a. By comparing the
IC50 values of N-methyl derivatives 4a and b with N-phenyl
analog 5a and b, it is revealed that the methyl group is more
favorable for activity. Introduction of a methoxy group on
position 3 of phenolic compounds did not improve the
activity; even in the case of 4b it dramatically diminished
the activity.
Primarily, compounds were screened for their inhibitory
activities at different concentrations in the range of
3–150 mg/mL (results were not shown). The resulting percent-
age of inhibition for compounds 3–5 at 30 mg/mL concen-
tration were selected for comparison and are summarized in
Table 1. Also, the IC50 value (inhibitory concentration for 50%
inhibition, mM) of the examined compounds is presented in
Table 1.
The capacity of target compounds 3–5 to scavenge the
stable free radical DPPH was monitored according to the
literature method [22], using 2,6-di-tert-butyl-4-methylphenol
(butylated hydroxytoluene, BHT) as a standard. Mean values
of the three-independent samples were calculated for each
compound. The results of preliminary screening are
expressed as percentage of inhibition at the concentration
of 250 mM against DPPH compared with the control values.
Also, the IC50 values for compounds 3–5 are compared with
those of standard BHT and presented in Table 1.
Phenolic compounds 3c, 4c, and 5b inhibited DPPH
radicals by 50% at a concentration of 372 ꢀ 22, 347 ꢀ 45,
and 104.75 ꢀ 1.6 mM, respectively, whereas the remaining
compounds showed a very weak DPPH radical scavenging
activity (IC50 >400 mM). Based on the percentage of inhi-
bition (Table 1), it can be concluded that the appreciable
inhibition showed by 3c, 4c, 5b, and also by 3b and 4b was
probably due to the presence of the free OH group in the aryl
moiety, which can donate hydrogen atoms. After donating a
hydrogen atom, the compounds exist in their radical form
Results showed that compounds 4a and b exhibited good
inhibition on mushroom tyrosinase with the IC50 values of
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S. Emami et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
Table 1. Tyrosinase inhibitory and DPPH radical scavenging activities of compounds 3–5
Compound Chemical structure
Tyrosinase inhibitory activity
DPPH radical scavenging activity
% Inhibition at 30 mg/mL
IC50 (mM)
% Inhibition at 250 mM
2.86 ꢀ 0.18
IC50 (mM)
>400
S
H3C
N
3a
14.9
>400
>400
>400
S
OH
S
H3C
N
3b
5.5
5.0
8.47 ꢀ 1.76
>400
S
OH
HO
S
H3C
N
3c
45.23 ꢀ 0.75
372 ꢀ 22
S
H3CO
HO
OH
S
S
H3C
N
4a
4b
119.9
147.8
1.1 ꢀ 0.88
>400
>400
66.0
58.2
S
H3C
N
14.52 ꢀ 2.21
S
HO
S
H3C
N
4c
1.7
>400
44.76 ꢀ 1.46
347 ꢀ 45
S
H3CO
HO
S
S
Ph
N
5a
5b
7.2
>400
9.91 ꢀ 0.08
>400
S
Ph
N
27.2
354.8
59.97 ꢀ 0.71
104.75 ꢀ 1.6
S
HO
H3C
H3C
CH3
OH
BHT
–
–
52.43 ꢀ 1.15a)
82.3 ꢀ 0.4
CH3
CH3
CH3
H3C
O
O
OH
Kojic acid
39.3b)
170.2
–
–
HO
BHT, butylated hydroxytoluene.
a)
% Inhibition at the concentration of 100 mM.
Kojic acid was tested at 15 mg/mL.
b)
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
2(3H)-Thiazole Thiones as Tyrosinase Inhibitors
5
Table 2. Toxicity risks and physicochemical properties of compounds 3–5 in comparison with kojic acid, predicted by OSIRIS Property
Explorer
Compound Chemical structure
Toxicity risks
Physicochemical properties
Mutagenic Tumorigenic Irritant Reproductive effect
CLogP
LogS
MW
S
H3C
N
3a
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
2.97
ꢁ1.64
225
S
OH
S
H3C
N
3b
2.67
2.56
ꢁ1.34
ꢁ1.36
241
271
S
OH
HO
S
H3C
N
3c
S
H3CO
HO
OH
S
S
H3C
N
4a
4b
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þ)
3.41
3.12
ꢁ2.81
ꢁ2.51
207
223
S
H3C
N
S
HO
S
H3C
N
4c
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
3.01
ꢁ2.53
253
S
H3CO
HO
S
S
Ph
N
5a
5b
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(ꢁ)
(þ)
4.67
4.37
ꢁ4.66
ꢁ4.36
269
285
S
Ph
N
S
HO
HO
O
O
OH
Kojic acid
(þ)
(þ)
(þ)
(ꢁ)
ꢁ0.94
ꢁ1.04
142
and the electron conjugation effect in the structure stabilizes
the radical which favors the reaction. The presence of the
electron-donating methoxy group at the ortho-position of the
hydroxy group enhanced the stabilization of the resulting
oxygen-centered radical. As a result, methoxy phenolic com-
pounds 3c and 4c were more active than the corresponding
phenolic compounds 3b and 4b, respectively.
As described previously, tyrosinase catalyzes hydroxylation
of monophenols to ortho-phenols and further oxidizes them
to ortho-quinone using oxygen [1]. Obviously, antioxidants
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S. Emami et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
or compounds with redox properties may inhibit the oxi-
dation process without interacting with the tyrosinase [11].
The compounds described in this report were evaluated
for their free radical scavenging and tyrosinase inhibition
activities. The results showed that compound 4a has
the highest tyrosinase inhibitory activity with no DPPH
free radical scavenging activity. Thus, there is no evidence
for direct correlation between free radical scavenging
activity and tyrosinase inhibition in these series of
compounds.
risks in a handy value to predict the molecule’s overall drug
potential (http://www.organic-chemistry.org/prog/peo/).
According to our theoretical toxicity evaluation of the
mutagenic, tumorigenic, irritant, and reproductive profiles
of compounds 3–5 (Table 2), they did not show any potential
toxic effect with the exception of compounds 4b and 5b,
which showed reproductive effects. In contrast, the currently
used drug, kojic acid, presented a high in silico toxicity risk
including mutagenic, tumorigenic, and irritant effects.
Under the experimental conditions, kojic acid was reported
to have a high-sensitizing potential and to potentially cause
irritant contact dermatitis [30]. Furthermore, this compound
was demonstrated to have some mutagenic properties, at
least in some strains of bacteria [31, 32].
The tyrosinase inhibitory action of kojic acid is attributed
to its ability to chelate copper ions in the tyrosinase active
site [23]. Also, previous studies found that hydroxyl groups in
the benzene ring of flavonoids played an important role in
enzyme inhibition, while methylated and glycosylated flavo-
noids exhibited less inhibitory effects [24]. In the absence of
the phenolic hydroxyl group, compound 4a containing the
thione moiety may be capable of acting as a metal chelator in
the tyrosinase active site. This suggestion can be supported by
several reports describing thione-containing compounds as
tyrosinase inhibitors, where the sulfur atom was found to
coordinate with both copper ions in the tyrosinase active site
[14–17, 25, 26].
Interestingly, our data showed that all compounds had a
positive drug-likeness (1.7–3.64) and drug-score (0.37–0.91)
values were significantly greater than those of kojic acid
(0.36 and 0.17, respectively, Fig. 5). The positive values of
drug-likeness point out that the compound contains predom-
inantly better fragments, which are frequently present in
traded drugs but not in the non-drug-like collection of com-
mercially available Fluka chemicals (http://www.organic-
chemistry.org/prog/peo/).
In silico ADME-Tox screening
Several years ago, when some clinical trials failed due to poor
ADME-Tox characters of drug candidates, new approaches
for early ADME-Tox profiling were rapidly evolved [27]. A
promising drug candidate should be ‘‘drug-like’’, which
means that it should have the optimized ADME-Tox profiles
similar to known drugs [28]. In general, absorption, trans-
port, protein affinity, toxicity, and metabolic stability of a
lead compound depend on many factors including hydro-
phobicity, electronic distribution, hydrogen bonding,
molecule size, and flexibility. Thus, by evaluating these fac-
tors ADME-Tox profiles of lead compounds can be predicted
[29]. In this work, to investigate the overall potential
of designed compounds to be qualified as a drug, the com-
pounds 3–5 were subjected to an in silico ADME-Tox screening
by evaluating their theoretical drug-likeness and drug-score
in comparison with kojic acid, a commercial tyrosinase
inhibitor. The drug-likeness and drug-score were predicted
using OSIRIS Property Explorer (http://www.organic-chemistry.
org/prog/peo/). OSIRIS involves the database of traded drugs
and supposedly non-drug-like Fluka compounds to assess the
occurrence frequency of each fragment in the individual
molecule. It allows drawing chemical structures and calcu-
lating on-the-fly various drug-relevant properties including
ClogP, logS, MW, and drug-likeness. OSIRIS Property Explorer
also estimated the risks of side effects, such as mutagenicity
and tumorigenicity. To calculate the overall drug score,
OSIRIS combines ClogP, logS, MW, drug-likeness, and toxicity
Figure 5. Calculated drug-likeness and drug-score values for
designed compounds 3–5 compared to kojic acid, the commercial
tyrosinase inhibitor, using OSIRIS Property Explorer.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
2(3H)-Thiazole Thiones as Tyrosinase Inhibitors
7
NMR spectra were recorded using a Bruker 500 spectrometer,
and chemical shifts are expressed as d (parts per million, ppm)
with tetramethylsilane (TMS) as an internal standard. The mass
spectra were run on an Agilent 6410. Merck silica gel 60 F254
plates were applied for analytical TLC.
Detailed ADME-Tox properties of the most potent com-
pound 4a were investigated using I-Lab 2.0. This is an online
ADME-Tox web server introduced by ACD/Labs team (https://
ilab.acdlabs.com/iLab2/), predicting pharmacokinetics and
pharmacodynamics features of compounds. Toxicity of a
given molecule including acute toxicity, genotoxicity, and
organ specific health effects can also be investigated using
this server. The ADME-Tox results showed that the compound
4a has an oral bioavailability of more than 70%, i.e., good
solubility, stability, and passive absorption. Compound 4a
does not undergo significant first-pass metabolism and acts
as a non-substrate and non-inhibitor of P-glycoprotein.
Estrogen receptor binding affinity estimation predicted no
binding to estrogen receptor alpha for compound 4a. This
compound also had a low probability for inhibition of hERG.
The predicted oral LD50 value of 4a in mouse was found to be
240 mg/kg. Also, compound 4a has a very low probability for
genotoxicity as predicted by Ames test. In general, the pre-
dicted pharmacokinetics, pharmacodynamics, and toxicity
profiles for compound 4a appear to be favorable for future
lead optimization.
General procedure for the synthesis of 4-hydroxy-3-methyl-
4-phenylthiazolidine-2-thione derivatives (3a–c)
To a stirred solution of potassium acetate (1.96 g, 20 mmol) in
methanol (12 mL), which was cooled in an ice bath, 40% meth-
ylamine (1.54 g, 20 mmol) was added. The temperature was
maintained below 108C throughout the reaction. A solution of
carbon disulfide (1.24 mL, 20 mmol) in methanol (1.5 mL) was
added dropwise to the stirred mixture over 15 min. After 2.5 h, a
solution of a-bromoacetophenone (10 mmol) in methanol was
added dropwise to the cold mixture, and stirring was continued
for 1.5 h. Then, water (10 mL) was added and the resulting solids
were separated by suction filtration and dried under reduced
pressure at room temperature to give pure 3a–c.
4-Hydroxy-3-methyl-4-phenylthiazolidine-2-thione (3a)
Yield 96%; mp 126–1288C; IR (KBr, cmꢁ1): 3253, 2988, 2940, 1445,
1381, 1216, 1199, 1102, 1072, 1004, 941, 867, 770, 739, 703, 646;
1H NMR (500 MHz, DMSO-d6) d 2.86 (s, 3H, CH3), 3.59 (s, 2H,
thiazolidine), 7.37–7.43 (m, 3H, phenyl), 7.44–7.48 (m, 2H, phe-
nyl), 7.71 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) d 32.18 (CH3),
42.52 (C-5 thiazolidine), 99.60 (C-4 thiazolidine), 125.11 (C-2 and
C-6 phenyl), 128.73 (C-3 and C-5 phenyl), 140.96 (C-1 phenyl),
194.34 (C-2 thiazolidine). MS (m/z, %) 225 (Mþ, 9), 152 (27), 120
(11), 105 (100), 91 (18), 77 (73), 72 (13), 51 (29), 45 (16). Anal. Calcd.
for C10H11NOS2: C, 53.30; H, 4.92; N, 6.22. Found: C, 53.09; H,
4.68; N, 6.34.
Conclusions
In conclusion, a series of 2(3H)-thiazole thiones 3–5 was
synthesized and evaluated for tyrosinase inhibition and
DPPH radical scavenging activities. Among them, 3-methyl-
4-phenyl-2(3H)-thiazole thione (4a) showed a good tyrosinase
inhibitory activity, even better than that of the well-known
tyrosinase inhibitor kojic acid. Moreover, the in silico ADME-
Tox study demonstrated that the drug-likeness and drug-
score values of the most potent compound 4a are signifi-
cantly higher than kojic acid. From the structure–activity
point of view, it was found that phenolic hydroxyl group in
prototype 3–5 might contribute to the scavenging activity
against DPPH radical, whereas, there was no correlation
between the potency of tyrosinase inhibition and the pres-
ence of a phenolic moiety. Tyrosinase inhibitors have been
reported to have important role in controlling fruit and
vegetable browning, and in treating disorders associated
with overproduction of melanin. Accordingly, the results
of this study would support the development of new agents
in the food industry as well as in medicinal and cosmetic
products.
4-Hydroxy-4-(4-hydroxyphenyl)-3-methylthiazolidine-
2-thione (3b)
Yield 73%; mp 148–1508C; IR (KBr, cmꢁ1): 3478, 3193, 1599, 1516,
1439, 1379, 1303, 1220, 1102, 1000, 943, 838, 865. 1H NMR
(500 MHz, DMSO-d6) d 2.83 (s, 3H, CH3), 3.54 (s, 2H, thiazolidine),
6.80 (d, 2H, J ¼ 8.58 Hz, H-3 and H-5 phenyl), 7.18 (d, 2H,
J ¼ 8.58 Hz, H-2 and H-6 phenyl), 7.51 (s, 1H, OH), 9.64 (s, 1H,
OH phenolic). 13C NMR (125 MHz, DMSO-d6) d 32.99 (CH3), 43.54
(C-5 thiazolidine), 100.59 (C-4 thiazolidine), 116.16 (C-3 and C-5
phenyl), 127.43 (C-2 and C-6 phenyl), 132.18 (C-1 phenyl), 158.56
(C-4 phenyl), 194.85 (C-2 thiazolidine). MS (m/z, %) 241 (Mþ, 11),
223 (45), 168 (73), 150 (18), 136 (18), 122 (45), 121 (100), 107 (36),
93 (91), 73 (54), 65 (82), 45 (29). Anal. Calcd. for C10H11NO2S2: C,
49.77; H, 4.59; N, 5.80. Found: C, 49.90; H, 4.44; N, 5.75.
4-Hydroxy-4-(4-hydroxy-3-methoxyphenyl)-
3-methylthiazolidine-2-thione (3c)
Yield 72%; mp 125–1278C; IR (KBr, cmꢁ1): 3542, 3203, 1606, 1515,
1465, 1432, 1384, 1270, 1226, 1146, 1105, 1025, 990, 863, 793,
716, 617. 1H NMR (500 MHz, DMSO-d6) d 2.85 (s, 3H, NCH3),
3.51 (d, 1H, J ¼ 12.11 Hz, H-5a, thiazolidine), 3.60 (d, 1H,
J ¼ 12.11 Hz, H-5b thiazolidine), 3.78 (s, 3H, OCH3), 6.76
(d, 1H, J ¼ 8.20 Hz, H-6 phenyl), 6.81 (d, 1H, J ¼ 8.23 Hz, H-5
phenyl), 6.94 (s, 1H, H-2 phenyl), 7.52 (s, 1H, OH alcoholic), 9.20
(s, 1H, OH phenolic). 13C NMR (125 MHz, DMSO-d6) d 33.09 (N-CH3),
43.51 (C-5 thiazolidine), 56.52 (OCH3), 100.57 (C-4 thiazolidine),
110.41 (C-2 phenyl), 116.19 (C-5 phenyl), 118.79 (C-6 phenyl),
Experimental
Chemistry
All starting materials, reagents and solvents were purchased
from Merck Co. and were used without further purification.
Melting points were determined in open glass capillaries using
a Bibby Stuart Scientific SMP3 apparatus and are uncorrected.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
S. Emami et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
132.69 (C-1 phenyl), 147.82 (C-4 phenyl), 148.43 (C-3 phenyl), 195.09
(C-2 thiazolidine). MS (m/z, %) 271 (Mþ, 17), 238 (15), 198 (41), 150.4
(100), 123 (40), 77 (9), 72 (38). Anal. Calcd. for C11H13NO3S2: C, 48.69;
H, 4.83; N, 5.16. Found: C, 48.60; H, 5.01; N, 4.98.
ring, the minimum volume of water (0.5 mL) to cause solution
was added and the cold solution was stirred for 15 min again.
After evaporation of acetone under reduced pressure, the residue
was washed with water and dissolved in 80% sulfuric acid. After
0.5 h, water (20 mL) was added and the precipitated thione 5a
and b was filtered and washed with water.
General procedure for the synthesis of 3-methyl-4-aryl-
2(3H)-thiazole thione derivatives (4a–c)
A mixture of 4-hydroxy-3-methyl-4-phenylthiazolidine-2-thione
derivatives 3a–c (500 mg), 0.5% hydrochloric acid (15 mL) and
methanol (5 mL) was refluxed for 0.5 h. On cooling, crystals were
formed which then were collected by filtration.
3,4-Diphenylthiazole-2(3H)-thione (5a)
Yield 93%; mp 149–1518C; IR (KBr, cmꢁ1): 3050, 1591, 1487, 1445,
1342, 1316, 1282, 1238, 1051, 905, 771, 753, 736, 696. 1H NMR
(500 MHz, CDCl3)
d 6.67 (s, 1H, thiazoline), 7.11 (d, 2H,
J ¼ 7.81 Hz, phenyl), 7.21–7.33 (m, 5H, phenyl), 7.37–7.44
(m, 3H, phenyl). 13C NMR (125 MHz, DMSO-d6) d 110.00 (C-5 thiaz-
oline), 128.19 (C-40 phenyl), 128.90 (C-4 phenyl), 128.97 (C-2, C-20,
C-6 and C-60 phenyl), 129.02 (C-3, C-30, C-5 and C-50 phenyl), 130.41
(C-10 phenyl), 137.73 (C-1 phenyl), 144.14 (C-4 thiazoline), 188.97
(C-2 thiazoline). MS (m/z, %) 269 (Mþ, 100), 268 (93), 167 (16), 149
(49), 134 (95), 121 (18), 104 (27), 91 (94), 77 (87), 69 (36), 57 (27), 51
(53), 43 (24). Anal. Calcd. for C15H11NS2: C, 66.88; H, 4.12; N, 5.20.
Found: C, 66.82; H, 4.01; N, 5.39.
3-Methyl-4-phenylthiazole-2(3H)-thione (4a)
Yield 94%; mp 127–1298C; IR (KBr, cmꢁ1): 3079, 1487, 1447, 1340,
1232, 1180, 1118, 959, 931, 772, 747, 703, 622. 1H NMR (500 MHz,
CDCl3) d 3.62 (s, 3H, CH3), 6.54 (s, 1H, thiazoline), 7.37–7.41
(m, 2H, phenyl), 7.52–7.56 (m, 3H, phenyl). 13C NMR (125 MHz,
DMSO-d6) d 36.00 (CH3), 109.22 (C-5 thiazoline), 128.85 (C-2 and
C-6 phenyl), 129.19 (C-3 and C-5 phenyl), 129.69 (C-4 phenyl),
130.53 (C-1 phenyl), 144.40 (C-4 thiazoline), 186.82 (C-2 thiaz-
oline). MS (m/z, %) 207 (Mþ, 100), 206 (53), 167 (16), 149 (59), 147
(44), 134 (85), 130 (18), 107 (29), 89 (41), 77 (47), 57 (46), 43 (35).
Anal. Calcd. for C10H9NS2: C, 57.93; H, 4.38; N, 6.76. Found: C,
58.09; H, 4.36; N, 6.99.
4-(4-Hydroxyphenyl)-3-phenylthiazole-2(3H)-thione (5b)
Yield 67%; mp 169–1718C; IR (KBr, cmꢁ1): 3253, 1655, 1574, 1511,
1366, 1320, 1288, 1202, 1173, 991, 841, 815, 693. 1H NMR
(500 MHz, CDCl3)
d 4.30 (s, 1H, thiazoline), 6.85 (d, 2H,
J ¼ 7.90 Hz, phenyl), 7.00–7.90 (m, 5H, phenyl), 7.84 (d, 2H,
J ¼ 8.10 Hz, phenyl), 10.47 (br s, OH). MS (m/z, %) 286 (Mþ, 7),
283 (12), 226 (24), 225 (52), 182 (5), 167 (14), 135 (37), 121 (56), 93
(43), 77 (100). Anal. Calcd. for C15H11NOS2: C, 63.13; H, 3.89; N,
4.91. Found: C, 63.40; H, 3.77; N, 4.93.
4-(4-Hydroxyphenyl)-3-methylthiazole-2(3H)-thione (4b)
Yield 77%; mp 211–2138C; IR (KBr, cmꢁ1): 3400, 1610, 1505, 1435,
1337, 1274, 1219, 1105, 964, 834, 767, 644. 1H NMR (500 MHz,
DMSO-d6) d 3.48 (s, 3H, CH3), 6.88 (d, 2H, J ¼ 8.05 Hz, H-3 and H-5
phenyl), 6.92 (s, 1H, thiazoline), 7.33 (d, 2H, J ¼ 7.90 Hz, H-2 and
H-6 phenyl), 9.94 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) d 36.78
(CH3), 108.90 (C-5 thiazoline), 116.42 (C-3 and C-5 phenyl), 122.04
(C-1 phenyl), 131.60 (C-2 and C-6 phenyl), 145.64 (C-4 thiazoline),
159.55 (C-4 phenyl), 187.43 (C-2 thiazoline). MS (m/z, %) 223 (Mþ,
76), 163 (20), 150 (30), 134 (20), 121 (100), 93 (20), 69 (24), 57 (31),
43 (21). Anal. Calcd. for C10H9NOS2: C, 53.78; H, 4.06; N, 6.27.
Found: C, 53.71; H, 4.00; N, 6.34.
Biological activity
Tyrosinase inhibition assay
All the synthesized compounds were dissolved in DMSO to a
concentration of 5 mg/mL. Phosphate buffer pH 6.8 was used to
dilute the DMSO stock solution of test compound. Test samples
(20 mL) were thoroughly mixed with a solution of L-tyrosine
(2 mM in potassium phosphate buffer, 80 mL) in 96-well micro-
plates. Then, mushroom tyrosinase (1000 U/mL, 20 mL) was
added to initiate the reaction. The assay mixture was incubated
at 258C for 30 min. After incubation, the increase in absorbance
was determined as the optical density at 492 nm in a microplate
reader (Bio-TEK Instruments Inc., USA). The same solution with-
out test substance was also prepared and the absorbance was
measured at 492 nm. The percent of inhibition was calculated
using the formula [(A ꢁ B)/A] ꢂ 100, where A is the absorbance
with solvent instead of test sample and B is the absorbance with
the dilute sample. IC50 value, the concentration giving 50%
inhibition of tyrosinase activity, was determined by interp-
olation of the dose–response curve [20, 21].
4-(4-Hydroxy-3-methoxyphenyl)-3-methylthiazole-2(3H)-
thione (4c)
Yield 68%; mp 180–1818C; IR (KBr, cmꢁ1): 3257, 3101, 1613, 1595,
1510, 1453, 1417, 1326, 1270, 1244, 1201, 1179, 1103, 1027, 971,
851, 799, 764, 753, 666. 1H NMR (500 MHz, DMSO-d6) d 3.50 (s, 3H,
NCH3), 3.81 (s, 3H, OCH3), 6.87–6.95 (m, 3H, phenyl), 7.08 (s, 1H,
thiazoline), 9.48 (s, 1H, OH). 13C NMR (125 MHz, DMSO-d6) d 36.86
(N-CH3), 56.71 (OCH3), 108.99 (C-5 thiazoline), 114.25 (C-2 phenyl),
116.39 (C-5 phenyl), 122.29 (C-6 phenyl), 123.10 (C-1 phenyl),
145.75 (C-4 thiazoline), 148.51 (C-4 phenyl), 148.89 (C-3 phenyl),
187.42 (C-2 thiazoline). MS (m/z, %) 253 (Mþ, 65), 251 (100), 238
(16), 192 (8), 175 (16), 132 (20), 104 (22), 50 (11). Anal. Calcd.
for C11H11NO2S2: C, 52.15; H, 4.38; N, 5.53. Found: C, 51.93; H,
4.58; N, 5.47.
DPPH radical scavenging assay
Several concentrations of methanolic solutions of compounds
were prepared. The compound solution (1.0 mL) was added to the
methanolic DPPH solution (3.0 mL, 0.1 mM) and the mixture was
kept in the dark for 15 min. The absorbance at 517 nm was then
measured by a UV/Visible spectrophotometer. The percent scav-
enging activity was calculated using the following formula:
Inhibition (%) ¼ 100 ꢂ (Abscontrol ꢁ Abscompound)/Abscontrol. The
DPPH radical scavenging activity of compounds was expressed as
General procedure for the synthesis of 3-phenyl-4-aryl-
2(3H)-thiazole thione derivatives (5a and b)
Triethylammonium N-phenyldithiocarbamate [19] (270 mg,
1 mmol) suspended in acetone (7 mL) was stirred and cooled
in an ice bath. Then, a-bromoacetophenone 2a and b (1 mmol)
was added portion-wise over a 30 min period. After 1 h of stir-
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–9
2(3H)-Thiazole Thiones as Tyrosinase Inhibitors
9
IC50 obtained from the linear regression plot between concen-
trations of test compound and percent inhibitions [22].
[14] K. W. Lam, A. Syahida, Z. Ul-Haq, M. B. Abdul Rahman, N. H.
Lajis, Bioorg. Med. Chem. Lett. 2010, 20, 3755–3759.
[15] P. Thanigaimalai, K.-C. Lee, S.-C. Bang, J.-H. Lee, C.-Y. Yun, E.
Roh, B.-Y. Hwang, Y. Kim, S.-H. Jung, Bioorg. Med. Chem. 2010,
18, 1135–1142.
This work was supported by a grant from the Research Council of
Mazandaran University of Medical Sciences, Sari, Iran. A part of this
work was related to the PharmD thesis of Kami Shahrbandi (Faculty of
Pharmacy, Mazandaran University of Medical Sciences).
[16] P. Thanigaimalai, V. K. Sharma, K.-C. Lee, C.-Y. Yun, Y. Kim,
S.-H. Jung, Bioorg. Med. Chem. Lett. 2010, 20, 4771–4773.
[17] P. Thanigaimalai, K.-C. Lee, S.-C. Bang, J.-H. Lee, C.-Y. Yun,
E. Roh, B.-Y. Hwang, Y. Kim, S.-H. Jung, Bioorg. Med. Chem.
2010, 18, 1555–1562.
The authors have declared no conflict of interest.
[18] S. Emami, A. Foroumadi, M. Falahati, E. Lotfali, S.
Rajabalian, S.-A. Ebrahimi, S. Farahyar, A. Shafiee, Bioorg.
Med. Chem. Lett. 2008, 18, 141–146.
References
[19] S. Emami, A. Foroumadi, Chin. J. Chem. 2006, 24, 791–794.
[1] J. R. Whitaker, Polyphenol oxidase. In: Food Enzymes, Structure
and Mechanism (Ed.: D. W. S. Wong), Chapman & Hall,
New York 1995, pp. 271–307.
[20] V. J. Hearing, Methods in Enzymology, Vol. 142, Academic Press,
New York 1987, p. 154.
´
[2] I. Kubo, I. Kinst-Hori, S. K. Chaudhuri, Y. Kubo, Y. Sanchez,
T. Ogura, Bioorg. Med. Chem. 2000, 8, 1749–1755.
[21] T.-S. Chang, H.-Y. Ding, H.-C. Lin, Biosci. Biotechnol. Biochem.
2005, 69, 1999–2001.
[3] M. Perez-Gilabert, F. Garcia-Carmona, Biochem. Biophys. Res.
Commun. 2001, 285, 257–261.
[22] T. Hatano, H. Kagawa, T. Yasuhara, T. Okuda, Chem. Pharm.
Bull. 1988, 36, 2090–2097.
[4] P. Grimes, J. J. Nordlund, A. G. Pandya, S. Taylor, M. P.
Rendon, P. Ortonne, J. Am. Acad. Dermatol. 2006, 54,
S255–S256.
[23] G. Battaini, E. Monzani, L. Casella, L. Santagostini, R.
Pagliarin, J. Biol. Inorg. Chem. 2000, 5, 262–268.
[24] L. P. Xie, Q. X. Chen, H. Huang, H. Z. Wang, R. Q. Zhang,
Biochemistry (Moscow) 2003, 68, 487–491.
[5] A. G. Pandya, I. L. Guevara, Dermatol. Clin. 2000, 18, 91–98.
[6] D. T. Hartong, E. L. Berson, T. P. Dryja, Lancet 2006, 368,
1795–1809.
[25] T. Klabunde, C. Eicken, J. C. Sacchettini, B. Krebs, Nat. Struct.
Biol. 1998, 5, 1084–1090.
[7] A. M. Mayer, Phytochemistry 1987, 26, 11–20.
[26] J. Liu, F. Wu, L. Chen, J. Hu, L. Zhao, C. Chen, L. Peng, Bioorg.
Med. Chem. Lett. 2011, 21, 2376–2379.
[8] S. Passi, M. Nazzaro-Porro, Br. J. Dermatol. 1981, 104, 659–665.
´
[27] A. de la Nuez, R. Rodrıguez, Biotecnol. Aplicada 2008, 25, 97–
110.
[9] S. Briganti, E. Camera, M. Picardo, Pigm. Cell. Res. 2003, 16,
101–110.
[28] C. A. Lipinski, Drug Discovery Today: Technol. 2004, 1, 337–341.
[10] K. I. Nihei, I. Kubo, Bioorg. Med. Chem. Lett. 2003, 13, 2409–
2412.
[29] D. C. Young, Computational Drug Design, John Wiley & Sons
Inc, Hoboken, New Jersey 2009.
[11] S. Y. Seo, V. K. Sharma, N. Sharma, J. Agric. Food Chem. 2003,
51, 2837–2853.
[30] T. Piamphongsant, Int. J. Dermatol. 1998, 37, 897–903.
´
´
´
[12] C. Gomez-Cordoves, B. Bartolome, W. Vieira, V. M. Virador,
J. Agric. Food Chem. 2001, 49, 1620–1624.
[31] C. I. Wei, T. S. Huang, S. Y. Fernando, K. T. Chung, Toxicol. Lett.
1991, 59, 213–220.
[13] E. T. Arung, E. Matsubara, I. W. Kusuma, E. Sukaton,
K. Shimizu, R. Kondo, Fitoterapia 2011, 82, 198–202.
[32] X.-H. Gao, L. Zhang, H. Wei, H.-D. Chen, Clin. Dermatol. 2008,
26, 367–374.
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www.archpharm.com