Design and synthesis of thiourea derivatives with sulfur-containing heterocyclic scaffolds as potential tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.03.013

Tyrosinase is a key enzyme during the production of melanins in plants and animals. A class of novel N-aryl-N′-substituted phenylthiourea derivatives (3a-i, 6a-k) were designed, synthesized and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed some 4,5,6,7-tetrahydro-2-[[(phenylamino)thioxomethyl]amino]-benzo[b]thiophene-3-carboxylic acid derivatives (3a-i) exhibited moderate inhibitory potency on diphenolase activity of tyrosinase. When the scaffold of 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid was replaced with 2-(1,3,4-thiadiazol-2-yl)thio acetic acid, the inhibitory activity of compounds (6a-k) against tyrosinase was improved obviously; especially, the inhibitory activity of compound 6h (IC₅₀ = 6.13 μM) is significantly higher than kojic acid (IC₅₀ = 33.3 μM). Moreover, the analysis on inhibition mechanism revealed that compound 6h might plays the role as a noncompetitive inhibitor.

Full text of DOI:10.1016/j.bmc.2016.03.013

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of thiourea derivatives with sulfur-containing
heterocyclic scaffolds as potential tyrosinase inhibitors
⇑
Pingping Liu, Chen Shu, Lujie Liu, Qingchun Huang, Yanqing Peng
Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyrosinase is a key enzyme during the production of melanins in plants and animals. A class of novel
N-aryl-N0-substituted phenylthiourea derivatives (3a–i, 6a–k) were designed, synthesized and their inhi-
bitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed
some 4,5,6,7-tetrahydro-2-[[(phenylamino)thioxomethyl]amino]-benzo[b]thiophene-3-carboxylic acid
derivatives (3a–i) exhibited moderate inhibitory potency on diphenolase activity of tyrosinase. When
the scaffold of 4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid was replaced with 2-(1,3,4-thiadi-
azol-2-yl)thio acetic acid, the inhibitory activity of compounds (6a–k) against tyrosinase was improved
Received 22 January 2016
Revised 1 March 2016
Accepted 4 March 2016
Available online xxxx
Keywords:
Tyrosinase
Inhibition
Thiourea derivatives
Thiadiazol
obviously; especially, the inhibitory activity of compound 6h (IC50 = 6.13
kojic acid (IC50 = 33.3 M). Moreover, the analysis on inhibition mechanism revealed that compound 6h
might plays the role as a noncompetitive inhibitor.
lM) is significantly higher than
l
Ó 2016 Published by Elsevier Ltd.
Thiophene
1. Introduction
health issues, tyrosinase was also used as a main target in research
of skin-whitening products. As a commonly used inhibitor, kojic
acid has been banned as cosmetic ingredient in many countries
because of its serious side effects.6 However, as a phenolic com-
pound, kojic acid (Scheme 1, a) is unstable under aerobic condition,
especially in solutions. Hence, more efforts should be made in the
development of effective and stable novel tyrosinase inhibitors
with lower side effects.
A given structure of two copper ions in the active center of
tyrosinase and a lipophilic long-narrow gorge near to the active
center provided ideal models to develop original tyrosinase inhibi-
tor. Over the past decades, a broad spectrum of natural and syn-
thetic compounds, such as Resveratrol (Scheme 1, b),7 chalcone
derivatives (Scheme 1, c),8 kojic acid and derivatives,9 thiocar-
bonyl-containing benzylidene derivatives (Scheme 1, d),10
thiosemicarbazone analogues (Scheme 1, e)11 and phenylthiourea
derivatives (Scheme 1, f),12 have been described to inhibit
tyrosinase.
Most of those compounds have inherent function in chelating
metal ions which plays a critical role in the inhibition of tyrosinase.
As be well known, sulfur-containing moieties, such as thiocarbonyl
in thioureas or thiosemicarbazones, sulfur atoms on heterocycles,
can chelate transition metal ions.11f,g,i In the present work, initially,
we designed and synthesized a serial of novel N-aryl-N0-substituted
phenyl thiourea derivatives (Scheme 1, 3a–i) in order to discover
efficient tyrosinase inhibitors. Thiophene scaffold was introduced
into the structure because sulphur-containing heterocycles were
Tyrosinase (polyphenol oxidase, EC 1.14.18.1),1 a multifunc-
tional copper-containing polyphenol oxidative enzyme widely dis-
tributed in plants, microorganisms, fungi and animals, catalyzes
two types of oxidative reactions: hydroxylation of monophenols
to o-diphenols, which are oxidized to the corresponding
o-quinones.2 Quinones can polymerize non-enzymatically to mela-
nin as the most important natural biopolymer responsible of
pigmentation and the color and patterns of mammalian skin. It
had also been reported that the tyrosinase might be relevant to
melanoma.3 In agriculture areas, tyrosinase is responsible for the
undesired enzymatic browning of fruits and vegetables that take
place during senescence or damage at the time of post-harvest
handling. The browning pigments lead to organoleptic and nutri-
tional modifications, thus depreciating the quality of the food
product.4
Tyrosinase inhibitors were widely used in agriculture and food
industry, as well as in medicinal and cosmetic products due to pre-
venting oxidization and decreasing the excessive accumulation of
pigmentation.5 Many tyrosinase inhibitors are widely used in cos-
metic products for whitening and depigmentation after sunburn.
Moreover, tyrosinase inhibitors could be used in the control of
the molting process of insect.6 With the increasing concern on
⇑
Corresponding author. Fax: +86 21 64252603.
E-mail address: yqpeng@ecust.edu.cn (Y. Peng).
http://dx.doi.org/10.1016/j.bmc.2016.03.013
0968-0896/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Liu, P.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.013

2
P. Liu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
OH
O
O
Considering carboxylic acids have relatively better solubility in
OH
aqueous phase, moreover, some carboxylic acids themselves were
reported showed inhibitory effect on tyrosinase.13 Hence, carboxy-
lates 2 were then transformed to corresponding carboxylic acids 3
by hydrolysis in alkaline solution.
HO
HO
HO
OH
b (resveratrol)
a (kojic acid)
To our delight, some of these compounds exhibit inhibitory effect
on tyrosinase, although the activities were lower than kojic acid.
Encouraged by these results, we decided to extend the structural
diversity by the replacement of the thiophene scaffold of 3 with
1,3,4-thiadiazol containing thioether-type side chain. Subsequently,
N-phenyl-N0-[5-(carboxymethylthio)-1,3,4-thiadiazol-2-yl] thiourea
derivatives (6a–k) were synthesized as represented in Scheme 2.
In comparison with 3, the inhibitory activities of compounds 6
against tyrosinase were distinctly enhanced. To explore the
inhibition mechanism of these compounds, the inhibitory kinetics
of compound 6h was also studied in detail.
OH HO
OH
O
NH
HN
OH
O
HO
S
c
d
OMe
O
OMe
H
N
NH2
N
S
O
MeO
HN
S
N
NH2
Cl
e
f
2. Experimental section
2.1. General
COOH
NH
H
N
H
N
S
NH
R
N
S
S
S
COOH
S
Melting points were measured on BUCHI B-450 melting point
apparatus. 1H and 13C NMR spectra were recorded on a Bruker AM-
400 (400 MHz) spectrometer. Massspectral analyses were performed
on an Agilent mass spectrometer under electron spray ionization.
Mushroom tyrosinase (EC 1.14.18.1) was purchased from Worthing-
ton Biochemical Corporation. All other reagents were commercial
products with analytical grade purity and used as received.
6a-k
3a-i
R
Scheme 1.
expected to be effective in chelating with copper ion. At thebeginning
of this work, 4,5,6,7-tetrahydro-2-[[(substitutedphenylamino)
thioxomethyl]amino]-benzo[b]thiophene-3-carboxylate derivatives
(2a–i) were synthesized by the designed route (Scheme 2).
Unfortunately, it was very difficult to dissolve these compounds in
aqueous buffer solution containing enzyme due to their poor
solubility so the activity of compounds 2a–i could not be evaluated.
2.2. Chemistry
2.2.1. Synthesis of compounds 1, 2a–i and 3a–i
Compound 1 was prepared through standard Gewald reac-
tion.14 4.4 g (0.05 mol) of morpholine was added dropwise into a
stirred solution of cyclohexanone (4.91 g, 0.05 mol), methyl
cyanoacetate (4.95 g, 0.05 mol) and sulfur (1.92 g, 0.06 mol) in
35 mL ethanol. On completion, the mixture was refluxed for fur-
ther 3 h. After cooling to room temperature, the precipitate was
separated by filtration and recrystallized from ethanol to give 1
as pale yellow powders (8.6 g, 82% yield), mp 128.2–129.4 °C
(lit.15 128–130 °C), MS (GC–MS): m/z, 211 [M+H]+, 179, 151, 125,
91, 77, 65, 53.
COOMe
COOMe
N
a
NH2
+
O
S
Gewald reaction
S8
1
b
COOH
NH
To the stirred solution of compound 1 (0.22 g, 2 mmol) in 3 mL
absolute ethanol, 2 mmol of aryl isothiocyanate was added and
refluxed for 13–16 h under the atmosphere of nitrogen. The solvent
was removed by rotary evaporation and the crude product was
washed with cold ethanol, dried, and recrystallized from ethanol
to afford compounds 2a–i.
COOMe
NH
c
S
S
NH
NH
S
R
S
R
3a-i
2a-i
10 mL of aqueous NaOH solution (20% wt) was added into the
stirred solution of compounds 2a–i in ethanol (5 mL). The resulting
mixture was refluxed for 2 h. Subsequently, the solvent was
removed by rotary evaporation, and the residue was added
20 mL of water followed by extracted with dichloromethane
(10 mL). The water layer was neutralized with diluted hydrochloric
acid to afford crude product as a precipitate which was then col-
lected by filtration. Recrystallization from ethanol gave purified
compounds 3a–i.
H2N
H2N
N
N
S
H
N
H2N
N
N
d
e
NH2
S
S
S
SH
4
5
COOH
f
H
N
H
N
N
S
N
R
S
S
2.2.1.1.
4,5,6,7-Tetrahydro-2-[[(phenylamino)thioxomethyl]
Yield
amino]-benzo[b]thiophene-3-carboxylic acid (3a).
86%, white solid, mp 241.3–241.7 °C. 1H NMR (400 MHz, DMSO-
d6) d: 7.48–7.44 (m, 2H, Ar-H), 7.40–7.37 (m, 1H, Ar-H), 7.21 (d,
J = 7.6 Hz, 2H, Ar-H), 2.73–2.68 (m, 4H, 2 ꢀ CH2), 1.79–1.71 (m,
4H, 2 ꢀ CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 179.72,
162.12, 154.50, 144.53, 136.37, 134.23, 134.13, 133.54, 133.22,
6a-k
COOH
Scheme 2. Synthesis of compounds 3a–i, 6a–k. Reagents and conditions: (a)
morpholine, EtOH, reflux; (b) aryl isothiocyanates, ethanol, reflux; (c) ethanol, 20%
NaOH, reflux; (d) CS2, NaOH, ethanol, reflux; (e) ClCH2COOH, NaOH, H2O, 60 °C; (f)
aryl isothiocyanates, Et3N, DMF, CH3CN, 50 °C.
Please cite this article in press as: Liu, P.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.013

P. Liu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
121.35, 30.05, 29.19, 27.70, 26.78 ppm; MS (LC–MS): m/z 315.1
[Mꢁ17]+. Anal. Calcd for C16H16N2O2S2: C, 57.81; H, 4.85; N, 8.43.
Found: C, 57.89; H, 4.92; N, 8.38.
2.73–2.68 (m, 4H, 2 ꢀ CH2), 1.78–1.71 (m, 4H, 2 ꢀ CH2) ppm; 13C
NMR (100 MHz, DMSO-d6) d: 174.15, 156.69, 149.29, 140.57,
132.86, 131.06, 130.39, 129.18, 128.35, 128.09, 128.06, 116.04,
24.72, 23.87, 22.38, 21.46 ppm; MS (LC–MS): m/z 349.0 [MꢁH]+.
Anal. Calcd for C16H15FN2O2S2: C, 54.84; H, 4.31; N, 7.99. Found:
C, 54.92; H, 4.36; N, 7.94.
2.2.1.2. 4,5,6,7-Tetrahydro-2-[[(4-methylphenylamino)thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3b).
acid
Yield 76%, white solid, mp 345.5–345.8 °C. 1H NMR
(400 MHz, DMSO-d6) d: 13.63 (s, 1H, COOH), 7.25 (d, J = 6.8 Hz,
2H, Ar-H), 7.07 (d, J = 7.2 Hz, 2H, Ar-H), 2.72–2.68 (m, 4H,
2 ꢀ CH2), 2.36 (s, 3H, CH3), 1.77–1.71 (m, 4H, 2 ꢀ CH2) ppm; 13C
NMR (100 MHz, DMSO-d6) d: 175.08, 157.43, 149.67, 137.75,
137.17, 131.62, 129.92, 129.15, 128.75, 116.57, 25.31, 24.43,
22.94, 22.02, 21.25 ppm; MS (LC–MS): m/z 329.1 [MꢁOH]+. Anal.
Calcd for C17H18N2O2S2: C, 58.93; H, 5.24; N, 8.09. Found: C,
59.02; H, 5.31; N, 8.02.
2.2.1.8. 4,5,6,7-Tetrahydro-2-[[(3-chlorophenylamino) thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3h).
acid
Yield 81%, white solid, mp 326.2–326.5 °C. 1H NMR
(400 MHz, DMSO-d6) d: 13.73 (s, 1H, COOH), 7.51–7.45 (m, 2H,
Ar-H), 7.41 (s, 1H, Ar-H), 7.23 (d, J = 7.2 Hz, 1H, Ar-H), 2.72–2.68
(m, 4H, 2 ꢀ CH2), 1.78–1.71 (m, 4H, 2 ꢀ CH2) ppm; 13C NMR
(100 MHz, DMSO-d6) d: 174.15, 156.70, 149.29, 140.58, 132.87,
131.05, 130.40, 129.18, 128.37, 128.09, 128.06, 116.05, 24.72,
23.88, 22.39, 21.46 ppm; MS (LC–MS): m/z 349.0 [Mꢁ17]+. Anal.
Calcd for C16H15ClN2O2S2: C, 52.38; H, 4.12; N, 7.64. Found: C,
52.46; H, 4.19; N, 7.56.
2.2.1.3.
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3c).
Yield 86%, white solid, mp 342.6–343.1 °C. 1H NMR
4,5,6,7-Tetrahydro-2-[[(4-fluorophenylamino)thioxo-
acid
(400 MHz, DMSO-d6) d: 13.69 (s, 1H, COOH), 7.28 (d, J = 6.8 Hz,
4H, Ar-H), 2.73–2.68 (m, 4H, 2 ꢀ CH2), 1.79–1.71 (m, 4H,
2 ꢀ CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 175.05, 157.42,
149.75, 136.00, 135.97, 131.69, 131.61, 128.85, 116.59, 116.36,
116.13, 25.29, 24.43, 22.94, 22.02 ppm; MS (LC–MS): m/z 333.1
[Mꢁ17]+. Anal. Calcd for C16H15FN2O2S2: C, 54.84; H, 4.31; N,
7.99. Found: C, 54.92; H, 4.38; N, 7.93.
2.2.1.9. 4,5,6,7-Tetrahydro-2-[[(3,4-difluorohenylamino) thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3i).
acid
Yield 91%, white solid, mp 343.4–343.8 °C. 1H NMR
(400 MHz, DMSO-d6) d: 13.74 (s, 1H, COOH), 7.57–7.48 (m, 2H, Ar-H),
7.16–7.14 (m, 1H, Ar-H), 2.73–2.68 (m, 4H, 2 ꢀ CH2), 1.77–1.71 (m,
4H, 2 ꢀ CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 174.30, 156.71,
149.27, 135.88, 131.06, 128.40, 126.56, 126.54, 118.97, 118.78, 117.52,
117.34, 116.02, 24.72, 23.87, 22.38, 21.45 ppm; MS (LC–MS): m/z
351.0 [Mꢁ17]+. Anal. Calcd for C16H14F2N2O2S2: C, 52.16; H, 3.83; N,
7.60. Found: C, 52.24; H, 3.91; N, 7.54.
2.2.1.4. 4,5,6,7-Tetrahydro-2-[[(4-chlorophenylamino)thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3d).
acid
Yield 81%, white solid, mp 352.6–352.9 °C. 1H NMR
(400 MHz, DMSO-d6) d: 7.52 (d, J = 7.6 Hz, 2H, Ar-H), 7.28 (d,
J = 7.2 Hz, 2H, Ar-H), 2.73–2.68 (m, 4H, 2 ꢀ CH2), 1.79–1.71 (m,
4H, 2 ꢀ CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 174.24,
156.72, 149.24, 138.15, 132.52, 131.05, 130.97, 128.91, 128.32,
116.02, 24.72, 23.86, 22.37, 21.44 ppm; MS (LC–MS): m/z 349.0
[Mꢁ17]+. Anal. Calcd for C16H15ClN2O2S2: C, 52.38; H, 4.12; N,
7.64. Found: C, 52.44; H, 4.21; N, 7.58.
2.2.2. Synthesis of 4, 5 and 6a–k
Carbon disulfide (7.6 g, 0.1 mol) was added dropwise into an
ice-cooled solution of thiosemicarbazide (4.55 g, 0.05 mol) and
NaOH (2.4 g, 0.06 mol) in ethanol (20 mL). On completion, the mix-
ture was stirred for 0.5 h at room temperature, and then refluxed
for further 5 h. After cooling, 90 mL of water was added and the
insoluble solid was filtered off. The filtrate was neutralized with
hydrochloric acid and the precipitates were collected by filtration.
Recrystallization of crude product from aqueous ethanol provided
pure product as pale yellow crystals in 76% yield. Mp 232.1–
232.8 °C (lit.16 230–232 °C), MS (LC–MS): m/z 133.1 [M+], 117.1,
105.1, 91.1, 74.1, 56.0.
The mixture of 2-amino-5-mercapto-1,3,4-thiadiazole 4 (5.33 g,
0.04 mol), chloroacetic acid (3.78 g, 0.04 mol) and NaOH (3.2 g,
0.08 mol) in 50 mL water was heated at 60 °C for 4 h. After cooling
to room temperature, the solution was neutralized with
hydrochloric acid. The precipitate was filtered and then recrystal-
lized from aqueous ethanol (5:1) to obtain purified 5 as pale yellow
crystalline solid. Yield 75%, mp 238.4–239.1 °C; 1H NMR (400 MHz,
DMSO-d6): d 12.82 (s, 1H, COOH), 7.28 (s, 2H, NH2), 3.90 (s, 2H,
CH2) ppm; MS (EI): m/z 191.1 [M+] (28), 147.0 (44), 133.1 (100),
114.0 (38), 74.1 (19).
2.2.1.5. 4,5,6,7-Tetrahydro-2-[[(4-bromophenylamino)thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3e).
acid
Yield 75%, white solid, mp 317.1–317.5 °C. 1H NMR
(400 MHz, DMSO-d6) d: 7.65 (d, J = 8.8 Hz, 2H, Ar-H), 7.20 (d,
J = 8.4 Hz, 2H, Ar-H), 2.72–2.67 (m, 4H, 2 ꢀ CH2), 1.78–1.71 (m,
4H, 2 ꢀ CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 174.16,
156.70, 149.29, 138.63, 131.90, 131.34, 131.05, 128.37, 121.10,
116.03, 24.73, 23.88, 22.39, 21.46 ppm; MS (LC–MS): m/z 395.0
[Mꢁ15]+. Anal. Calcd for C16H15ClN2O2S2: C, 46.72; H, 3.68; N,
6.81. Found: C, 46.78; H, 3.76; N, 6.74.
2.2.1.6. 4,5,6,7-Tetrahydro-2-[[(2-methylphenylamino)thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3f).
acid
Yield 79%, white solid, mp 325.8–326.1 °C. 1H NMR
(400 MHz, DMSO-d6) d: 13.71 (s, 1H, COOH), 7.31–7.26 (m, 3H,
Ar-H), 7.12 (d, J = 6.8 Hz, 1H, Ar-H), 2.73–2.68 (m, 4H, 2 ꢀ CH2),
2.02 (s, 3H, CH3), 1.78–1.71 (m, 4H, 2 ꢀ CH2) ppm; 13C NMR
(100 MHz, DMSO-d6) d: 173.63, 156.33, 149.42, 138.15, 135.13,
131.06, 130.38, 128.71, 128.43, 128.21, 126.72, 115.81, 24.72,
23.88, 22.35, 21.44, 16.87 ppm; MS (LC–MS): m/z 329.1 [Mꢁ17]+.
Anal. Calcd for C17H18N2O2S2: C, 58.93; H, 5.24; N, 8.09. Found: C,
59.01; H, 5.32; N, 8.04.
0.95 g (0.005 mol) of 2-[(5-amino-1,3,4-thiadiazol-2-yl)thio]
acetic acid 5 and Et3N (1.01 g, 0.01 mol) was dissolved in 5 mL
DMF and 20 mL CH3CN. Subsequently, aryl isothiocyanate
(0.005 mol) was added slowly into above-mentioned solution under
the atmosphere of nitrogen. The resulting mixture was heated for
5 h at 50 °C and CH3CN was removed by rotary evaporation. After
cooling, 20 mL of water was added and the insoluble impurities
were isolated by filtration. The filtrate was neutralized with
hydrochloric acid. The crude product thus precipitated was filtered
and recrystallized from ethanol to afford desired compounds 6a–k.
2.2.1.7. 4,5,6,7-Tetrahydro-2-[[(3-fluorophenylamino) thioxo-
methyl]amino]-benzo[b]thiophene-3-carboxylic
(3g).
acid
Yield 76%, white solid, mp 321.2–321.3 °C. 1H NMR
2.2.2.1. N-Phenyl-N0-[5-(carboxymethylthio)-1,3,4-thiadiazol-2-
(400 MHz, DMSO-d6) d: 13.73 (s, 1H, COOH), 7.51–7.45 (m, 2H,
Ar-H), 7.41 (d, J = 7.2 Hz, 1H, Ar-H), 7.23 (d, J = 6.8 Hz, 1H, Ar-H),
yl]thiourea (6a).
Yield 59%, yellow solid, mp 211.7–212.1 °C.
1H NMR (400 MHz, CDCl3) d: 10.49 (s, 1H, COOH), 9.79 (s, 1H, NH),
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4
P. Liu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9.06 (s, 1H, NH), 7.66 (d, J = 7.6 Hz, 2H, Ar-H), 7.34 (t, J = 8.0 Hz, 2H,
Ar-H), 7.13 (t, J = 7.2 Hz, 1H, Ar-H), 4.07 (s, 2H, CH2) ppm;
13C NMR (100 MHz, CDCl3) d: 182.64, 169.32, 153.70, 139.34,
128.94, 128.45, 124.40, 122.74, 118.92, 34.88 ppm; HRMS
2.2.2.8.
methylthio)-1,3,4-thiadiazol-2-yl] thiourea (6h).
N-[3-(Trifluoromethyl)phenyl]-N0-[5-(carboxy-
Yield 61%,
yellow solid, mp 229.1–229.3 °C. 1H NMR (400 MHz, DMSO-d6) d:
10.51 (s, 1H, COOH), 9.16 (s, 1H, NH), 7.56 (s, 1H, Ar-H), 7.47 (d,
J = 8.0 Hz, 1H, Ar-H), 7.35 (d, J = 8.0 Hz, 1H, Ar-H), 6.97–7.01 (m,
1H, Ar-H), 3.94 (s, 2H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6)
d: 183.20, 169.77, 149.72, 139.81, 138.85, 128.58, 127.76, 126.46,
125.15, 125.06, 122.35, 120.68, 119.65, 115.26, 36.20 ppm; HRMS
(ESI-MS) calcd. for
324.9891.
C
11H9N4O2S3 [MꢁH]+:324.9888, Found:
2.2.2.2. N-(4-Methylphenyl)-N0-[5-(carboxymethyl thio)-1,3,4-
thiadiazol-2-yl]thiourea (6b). Yield 52%, brownish solid,
(ESI-MS): calcd. for
392.9761.
C
12H8F3N4O2S3 [MꢁH]+:392.9767, found:
mp 238.1–238.3 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.40 (s, 1H,
COOH), 9.98 (s, 1H, NH), 9.03 (s, 1H, NH), 7.50 (d, J = 7.8 Hz, 2H,
Ar-H), 7.14 (d, J = 8.0 Hz, 2H, Ar-H), 4.08 (s, 2H, CH2), 2.28 (s, 3H,
CH3) ppm; 13C NMR (100 MHz, DMSO-d6) d: 181.95, 169.55,
156.77, 136.76, 130.23, 128.91, 123.02, 119.24, 35.98, 20.54 ppm;
HRMS (ESI-MS): calcd. for C12H11N4O2S3 [MꢁH]+: 339.0044, found:
339.0042.
2.2.2.9. N-(2,4-Difluorophenyl)-N0-[5-(carboxymethyl-thio)-1,3,
4-thiadiazol-2-yl]thiourea (6i). Yield 56%, yellow solid, mp
215.1–215.8 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.19 (s, 1H,
COOH), 8.96 (s, 1H, NH), 7.59 (d, J = 6.8 Hz, 1H, Ar-H), 7.28–7.32
(m, 1H, Ar-H), 7.08 (s, 1H, Ar-H), 4.14 (s, 2H, CH2) ppm; 13C NMR
(100 MHz, DMSO-d6) d: 190.23, 169.75, 167.13, 159.07, 152.99,
138.15, 125.66, 115.08, 111.56, 104.39, 35.73 ppm; HRMS (ESI-
MS): calcd. for C11H9F2N4O2S3 [M+H]+:362.9856, found: 362.9836.
2.2.2.3.
thiadiazol-2-yl]thiourea (6c).
N-(4-Fluorophenyl)-N0-[5-(carboxymethylthio)-1,3,4-
Yield 42%, yellow solid, mp
247.6–247.8 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.47 (s, 1H,
COOH), 9.64 (s, 1H, NH), 9.13 (s, 1H, NH), 7.28–7.15 (m, 4H, Ar-
H), 3.89 (s, 2H, CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d:
181.10, 169.63, 155.30, 149.53, 137.69, 135.08, 130.31, 128.22,
126.14, 36.09 ppm; HRMS (ESI-MS): calcd. for C11H10FN4O2S3 [M
+H]+: 344.9950, found: 344.9934.
2.2.2.10.
1,3,4-thiadiazol-2-yl]thiourea (6j).
N-(3,4-Difluorophenyl)-N0-[5-(carboxymethyl-thio)-
Yield 48%, yellow solid,
mp 248.4–249.1 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.60 (s, 1H,
COOH), 9.97 (s, 1H, NH), 9.33 (s, 1H, NH), 7.36 (s, 1H, Ar-H),
7.12–7.15 (m, 1H, Ar-H), 6.92 (d, J = 8.4 Hz, 1H, Ar-H), 4.02 (s, 2H,
CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 188.76, 166.71,
150.52, 148.09, 144.85, 135.81, 124.62, 118.01, 116.15, 108.95,
2.2.2.4.
thiadiazol-2-yl]thiourea (6d).
N-(4-Chlorophenyl)-N0-[5-(carboxymethylthio)-1,3,4-
Yield 54%, yellow solid, mp
36.41 ppm; HRMS(ESI-MS): calcd. for
362.9856, found: 362.9836.
C
11H9F2N4O2S3 [M+H]+:
204.8–205.2 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.12 (s, 1H,
COOH), 9.55 (s, 1H, NH), 8.83 (s, 1H, NH), 7.52 (d, J = 7.2 Hz, 2H,
Ar-H), 7.02 (d, J = 7.6 Hz, 2H, Ar-H), 4.07 (s, 2H, CH2) ppm; 13C
NMR (100 MHz, DMSO-d6) d: 181.43, 169.97, 149.86, 138.02,
135.41, 130.64, 128.55, 126.47, 36.41 ppm; HRMS (ESI-MS): calcd.
for C11H10ClN4O2S3 [MꢁH]+: 358.9498, found: 358.9496.
2.2.2.11. N-(3-Chloro-4-fluorophenyl)-N0-[5-(carboxymethylthio)-
1,3,4-thiadiazol-2-yl] thiourea (6k). Yield 55%, yellow solid,
mp 240.2–240.4 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.45 (s, 1H,
COOH), 9.55 (s, 1H, NH), 8.83 (s, 1H, NH), 7.53 (s, 1H, Ar-H), 6.87 (s,
1H, Ar-H), 6.53 (s, 1H, Ar-H), 4.07 (s, 2H, CH2); 13C NMR (100 MHz,
DMSO-d6) d: 182.64, 171.54, 163.34, 136.84, 131.45, 128.40,
122.74, 118.92, 116.15, 109.58, 36.34 ppm; HRMS (ESI-MS): calcd.
for C11H9ClFN4O2S3 [M+H]+: 378.9560, found: 378.9575.
2.2.2.5. N-(2-Methoxyphenyl)-N0-[5-(carboxymethylthio)-1,3,4-
thiadiazol-2yl]thiourea (6e).
Yield 55%, yellow solid, mp
202.4–202.5 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.81 (s, 1H,
COOH), 9.76(s, 1H, NH), 9.56 (s, 1H, NH), 7.91 (d, J = 6.8 Hz, 1H,
Ar-H), 6.90–7.18 (m, 3H, Ar-H), 4.04 (s, 2H, CH2), 3.81 (s, 3H,
OCH3) ppm; 13C NMR (100 MHz, DMSO-d6) d: 179.54, 169.68,
162.58, 156.87, 149.45, 127.42, 125.07, 120.42, 120.14, 111.61,
55.88, 35.71 ppm; HRMS (ESI-MS): calcd. for C12H13N4O3S3 [M
+H]+: 357.0150, found: 357.0153.
2.3. Tyrosinase activity assay
The inhibitory activity of test compounds against tyrosinase was
investigated using previously described procedure17 with slight
modifications. Briefly, the compounds 3a–i and 6a–k were assessed
for the diphenolase inhibitory activity against tyrosinase using
L-DOPA as substrate. Stock solutions of the test compounds were
prepared in DMSO; further dilutions were done in DMSO to yield
various concentrations using phosphate buffer (pH = 6.8). The
2.2.2.6.
thiadiazol-2-yl]thiourea (6f).
N-(3-Fluorophenyl)-N0-[5-(carboxymethylthio)-1,3,4-
Yield 59%, yellow solid, mp
214.9–215.4 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.62 (s, 1H,
COOH), 10.07 (s, 1H, NH), 9.34 (s, 1H, NH), 7.54–7.58 (m, 2H, Ar-
H), 7.45–7.46 (m, 1H, Ar-H), 7.36–7.38 (m, 1H, Ar-H), 4.07 (s, 2H,
CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 188.25, 169.29,
163.27, 153.74, 141.13, 140.20, 130.55, 129.98, 119.23, 114.96,
34.74 ppm; HRMS (ESI-MS): calcd. for C11H10FN4O2S3 [M+H]+:
344.9950, found: 344.9934.
200
lL reaction system included 50
l
L diluted solution of test com-
pound, 50
lL phosphate buffer, 50
lL L-DOPA and 50 L tyrosinase
l
solution. The enzyme reaction was monitored by measuring the
change in absorbance at 475 nm for 5 min at 30 °C. The measure-
ment was completed in triplicate for each concentration and aver-
aged before further calculation. The phosphate buffer containing
2.0% DMSO instead of the diluted solution was used as the negative
control. The kojic acid was used as a positive control.
The determination of inhibition kinetics was performed by mod-
ification of the above-mentioned method: for each of four different
inhibitor concentrations (0, 3.67, 6.35, and 12.70 lM, respectively).
L-DOPA concentration was varied (0.25, 0.5, 1.0, 2.0, 4.0 and
6.0 mM). The inhibition type on the enzyme was assayed by Line-
weaver–Burk plots, and the inhibition constant was determined by
the second plots of the apparent Km/Vm (slope of Lineweaver–Burk
double reciprocal lines) versus the concentration of compound 6h.
2.2.2.7.
thiadiazol-2-yl]thiourea (6g).
N-(3-Chlorophenyl)-N0-[5-(carboxymethylthio)-1,3,4-
Yield 52%, yellow solid, mp
175.4–175.6 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.62 (s, 1H,
COOH), 10.07 (s, 1H, NH), 9.32 (s, 1H, NH), 7.81–7.86 (m, 1H, Ar-
H), 7.35–7.46 (m, 2H, Ar-H), 7.15–7.18 (m, 1H, Ar-H), 4.09 (s, 2H,
CH2) ppm; 13C NMR (100 MHz, DMSO-d6) d: 183.71, 169.53,
153.99, 150.06, 141.05, 132.98, 130.71, 123.59, 118.91, 115.50,
36.30 ppm; HRMS (ESI-MS): calcd. for C11H10ClN4O2S3 [MꢁH]+:
358.9498, found: 358.9496.
Please cite this article in press as: Liu, P.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.013

P. Liu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
of tyrosinase. Considering thiadiazole, oxadiazole and triazole rings
monodentate binding to the active site dicopper center of tyrosi-
nase,11f dihydro-asparagusic acid having inhibitory effect on
tyrosinase,18 we designed a serial of compounds 6a–k with the
scafford 2-((1,3,4-thiadiazol-2-yl)thio)acetic acid connected to
thiourea pharmacophore which were prospected to be efficient
in chelating with the dicopper active center. These compounds
could be prepared readily by the reaction between 2-[(5-amino-
1,3,4-thiadiazol-2-yl)thio]acetic acid and isothiocyanates.
Inhibitory activity against tyrosinase (diphenolase) of compounds
6a–k was investigated. As shown in Table 2, all of N-phenyl-N0-[5-
(carboxymethylthio)-1,3,4-thiadiazol-2-yl]thiourea derivatives dis-
played potent tyrosinase inhibitory activities. Especially, the
3. Results and discussion
3.1. Synthesis
As shown in Scheme 2, 2-amino-4,5,6,7-tetrahydro-benzo[b]
thiophene-3-carboxylate (1) was synthesized by Gewald reaction.
Compound 1 reacted with aryl isothiocyanates under nitrogen
atmosphere to afford compounds 2a–i, hydrolysis of 2a–i to afford
compounds 3a–i in good yields. The structures of the products 3a–i
were identified by their 1H NMR, 13C NMR spectrum and mass
spectrum data. Their 1H NMR spectrum showed a broad signal at
d 13.7 (COOH).
Aryl isothiocyanates hydrolyzed in the presence of water and
led to complex byproducts. Hence, the solvents employed must
be pre-dried, and the reaction should be carried out under the
atmosphere of nitrogen.
The reaction of 2-amino-5-mercapto-1,3,4-thiadiazole (4) and
chloroacetic acid could be carried out in aqueous phase to afford
2-[(5-amino-1,3,4-thiadiazol-2-yl)thio] acetic acid (5). The hydrol-
ysis of chloroacetic acid became a serious problem at elevated tem-
peratures; thus the reaction temperature should be controlled at
about 60 °C to avoid the side reaction. The compound (5) reacted
with aryl isothiocyanates at 50 °C for 5 h to afford compounds
6a–k in moderate yield. The structures of the products 6a–k were
supported by their 1H NMR, 13C NMR spectrum and mass data.
The 1H NMR spectrum of compound 5 shows a signal at d 7.28
(s, 2H, NH2). The 1H NMR spectrum of compound 6 showed three
broad signals at d 11 ꢂ 9 (COOH, NH, NH). Their structures were
confirmed by their mass spectra which showed peaks correspond-
ing to their molecular ion.
obtained compounds 6c (IC50 = 20.9
(IC50 = 20.1 M) and 6j (IC50 = 20.5
tyrosinase inhibitory activities than the reference inhibitor kojic acid
(IC50 = 33.3 M).
lM), 6h (IC50 = 6.13
lM), 6i
l
lM) exhibited more potent
l
These results showed that the introduction of 2-[(5-amino-
1,3,4-thiadiazol-2-yl)thio] acetic acid substructure was crucial for
presenting the inhibitory effect on tyrosinase.The data in Table 2
shows that compounds including fluorine atoms at phenyl ring
exhibited more potent tyrosinase inhibitory activities.
3.2.2. Inhibitory mechanism of compound 6h on mushroom
tyrosinase
Finally we determined the inhibitory type of selected com-
pound 6h from Lineweaver–Burk double reciprocal plots. In the
presence of 6h, the kinetics of the enzyme was shown in Figure 1a.
The results displayed that the plots of 1/V versus 1/[S] gave four
straight lines with different slopes and intersected on the horizon-
tal axis. When the concentration of compound 6h was elevated, the
Vmax value decreased, whereas the Km value was unchanged, sug-
gesting compound 6h is regarded as a noncompetitive inhibitor
3.2. Biological activity
of mushroom tyrosinase. The inhibition constant (KI = 8.13 lM)
was obtained by plotting the slope values versus the concentration
of the compound 6h, as shown in Figure 1b.
Many thiourea derivatives were known to be competitive inhi-
bitors of tyrosinase due to their chelating ability to copper ions at
the active sites. Interestingly, the Lineweaver–Burk analysis
revealed a non-competitive behavior of compound 6h. Although
the title compounds were designed initially as copper-chelating
competitive inhibitors, the experimental results reveal that they
might act through allosteric effects.
3.2.1. Inhibitory effects on the diphenolase activity of tyrosinase
The inhibition of as-synthesized 4,5,6,7-tetrahydro-2-[[(sub-
stitutedphenylamino)thioxomethyl]amino]-benzo[b]thiophene-3-
carboxylic acids 3a–i on the tyrosinase was investigated and
compared with kojic acid at 75 lM. As shown in Table 1, part
of these compounds exhibited inhibitory activity on tyrosinase.
However, their activities were lower than kojic acid.
We speculated that the carboxyl group neighbor thiourea phar-
macophore might retard the binding of inhibitor to the active site
Table 1
Structures and inhibitory activity against tyrosinase (diphenolase) of compounds 3a–i
at 75 lM
Table 2
Structures and inhibitory activity against tyrosinase (diphenolase) of compounds 6a–
k
COOH
NH
R
H
N
H
N
N
S
NH
R
N
S
S
S
COOH
3a-i
S
6a-k
Compounds No.
R
% inhibition
Compounds No.
R
IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
H
4-CH3
4-F
4-Cl
4-Br
2-CH3
3-F
3-Cl
3,4-Di-F
Kojic acid
53.3 0.5a
6a
6b
6c
6d
6e
6f
6g
6h
H
4-CH3
4-F
4-Cl
2-OCH3
3-F
3-Cl
3-CF3
2,4-Di-F
3,4-Di-F
3-Cl-4-F
Kojic acid
27.7 1.2
54.4 0.8
20.9 1.2
26.3 0.5
27.1 0.6
31.3 0.8
59.7 0.5
6.13 0.3
20.1 1.2
20.5 0.8
33.9 1.2
33.3 1.1
NIb
44.3 1.5
NI
31.7 1.3
42.1 0.5
44.8 0.3
NI
3i
44.7 0.3
88.5 0.3
6i
6j
6k
Positive control
a
SEM: standard error of the mean.
NI: no inhibitory activity.
Positive control
b
Please cite this article in press as: Liu, P.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.013

6
P. Liu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
needed on human tyrosinase or melanocytes to support the poten-
tial of these compounds for human-directed applications.
Acknowledgment
Financial support from National Key Technology R&D Program
(2011BAE06B02) is gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.03.013.
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Figure 1. Determination of the inhibitory type and inhibition constant of com-
pound 6h. The concentrations of 6 h for curves 1–4 were 12.70, 6.35, 3.67 and 0 lM,
respectively. (a) Lineweaver–Burk plots for inhibition of compound 6h on the
activity of tyrosinase. (b) The plot of slope versus the concentration of compound
6h for determining the inhibition constant KI.
4. Conclusions
In summary, on the basis of the initial thiofuran-type lead com-
pound identified from screening a small focused library, we have
discovered 5-(carboxymethylthio)-1,3,4-thiadiazol-2-yl thiourea
as a novel scaffold for tyrosinase inhibitors. The newly synthesized
compounds exhibit a remarkable inhibitory effect on the dipheno-
lase activity of tyrosinase, therefore are promising for their uses as
tyrosinase inhibitors. The results showed that several compounds,
especially the compound 6h, had higher inhibitory activity against
tyrosinase in comparison with the widely used tyrosinase inhibitor
kojic acid. The inhibition mechanism studies revealed that the
compounds prepared in this study exhibited inhibitory effect on
tyrosinase by acting as the noncompetitive inhibitor.
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believe that sulfur-containing heterocyclic thiourea derivatives
can serve as a valuable prototype for further development. The
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optimization of thiourea-based inhibitors might provide a new
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Please cite this article in press as: Liu, P.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.013