Molecular docking studies and biological evaluation of 1,3,4-thiadiazole derivatives bearing Schiff base moieties as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2016.09.007

1,3,4-Thiadiazole derivatives bearing Schiff base moieties were designed, synthesized, and their tyrosinase inhibitory activities were evaluated. Some compounds displayed potent tyrosinase inhibitory activities, especially, 4-(((5-mercapto-1,3,4-thiadiazol-2-yl)-imino)methyl)-2-methoxy-phenol (14) exhibited superior inhibitory effect to the other compounds with an IC₅₀ value of 0.036?μM. The structure–activity relationships (SARs) were preliminarily discussed and docking studies showed compound 14 had strong binding affinity to mushroom tyrosinase. Hydroxy might be the active groups. The inhibition kinetics study revealed that compounds (13 and 14) inhibited tyrosinase by acting as uncompetitive inhibitors. The LD₅₀ value of the compound 14 was 5000?mg/kg.

Full text of DOI:10.1016/j.bioorg.2016.09.007

Bioorganic Chemistry 69 (2016) 29–36
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Molecular docking studies and biological evaluation of 1,3,4-thiadiazole
derivatives bearing Schiff base moieties as tyrosinase inhibitors
⇑
Junyuan Tang, Jinbing Liu , Fengyan Wu
Department of Biology and Chemical Engineering, Shaoyang University, Shao Shui Xi Road, Shaoyang 422100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2016
Revised 18 September 2016
Accepted 19 September 2016
Available online 20 September 2016
1,3,4-Thiadiazole derivatives bearing Schiff base moieties were designed, synthesized, and their tyrosi-
nase inhibitory activities were evaluated. Some compounds displayed potent tyrosinase inhibitory activ-
ities, especially, 4-(((5-mercapto-1,3,4-thiadiazol-2-yl)-imino)methyl)-2-methoxy-phenol (14) exhibited
superior inhibitory effect to the other compounds with an IC50 value of 0.036 lM. The structure–activity
relationships (SARs) were preliminarily discussed and docking studies showed compound 14 had strong
binding affinity to mushroom tyrosinase. Hydroxy might be the active groups. The inhibition kinetics
study revealed that compounds (13 and 14) inhibited tyrosinase by acting as uncompetitive inhibitors.
The LD50 value of the compound 14 was 5000 mg/kg.
Keywords:
1,3,4-Thiadiazole derivatives
Tyrosinase inhibitory activities
Inhibition kinetics
Ó 2016 Elsevier Inc. All rights reserved.
Docking studies
1. Introduction
in undesirable browning in fruits, vegetables, and beverages, which
impairs the color and sensory properties of these food products
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase,
EC 1.14.18.1, syn. polyphenol oxidase), as known as polyphenol
oxidase (PPO) possesses an active site where two copper ions bind
dioxygen in a form of the enzyme termed as oxy-tyrosinase [1].
The binuclear center together with three specific histidine residues
forms a square-pyramidal structure [2]. Tyrosinase is a key enzyme
in the biosynthesis of melanin pigment, and catalyzes two distinct
reactions involving molecular oxygen in the hydroxylation of
monophenols to o-diphenols (monophenolase), as well as oxida-
tion of o-diphenols to o-quinones (diphenolase) [3]. Therefore, it
plays a vital role in the processes of melanin formation in bacteria,
fungi, plants and mammals [4]. Because of the high reactivity of
tyrosinase, quinones could polymerize spontaneously to form high
molecular weight brown-pigments (melanins), which widely
spreads in skin, hair and eyes of mammals, or react with amino
acids and proteins to enhance brown color of the pigment pro-
duced [5]. Although melanin is important in preventing skin cancer
caused by overexposure to ultraviolent lights [6,7], excessive mel-
anin production and accumulation may lead to hyperpigmentation
such as melasma, freckles, ephelide, and solar lentigines [8].
Tyrosinase is also known to oxidize dopamine to form melanin in
the brain, and thus tyrosinase is implicated in the pathogenesis
of Parkinson’s disease and related neurodegenerative disorders
[9,10]. In food industry, tyrosinase catalyzes the reactions resulting
and may shorten shelf life, reduce market value, and lose nutri-
tional value during the postharvest handling processes [11]. One
way to avoid these negative effects resulted from tyrosinase is to
use inhibitors of this enzyme. Furthermore, in insects, tyrosinase
is associated with three biochemical processes, including scleroti-
zation of cuticle, defensive encapsulation and melanization of for-
eign organism, and wound healing [12,13]. These processes
provide potential targets for developing safer and effective tyrosi-
nase inhibitors as insecticides and ultimately for insect control. In
fact, tyrosinase inhibitors have been well appreciated in the fields
of cosmetics, medicine, food sciences and agriculture [14,15]. Most
of the recent researches have focused on below following fields:
first, finding new naturally occurring inhibitors of tyrosinase; sec-
ond, proceeding medicinal modifications or synthesis chemical
analogs of target inhibitors for better inhibitory activities and
lower side effects; third, investigating structure–activity and inhi-
bitory mechanism of tyrosinase inhibitors. By far, some natural and
synthetic tyrosinase inhibitors were reported, for example, hydro-
quinone, ascorbic acid, arbutin, kojic acid, aromatic aldehydes,
aromatic acids, aromatic alcohol, tropolone, and polyphenols
[16–19]. However, only handful molecules such as kojic acid,
arbutin, tropolone, and 1-phenyl-2-thiourea (PTU) are commer-
cially useful [20]. Moreover, it has been reported that arbutin
decomposes at room temperature (10% decomposition at 20 °C
for 15 days), while arbutin and tropolone have been demonstrated
not clinically efficacious when systematically analyzed in carefully
controlled studies [21]. As a result, there is still an urgent need for
⇑
Corresponding author.
E-mail address: syuliujb@163.com (J. Liu).
http://dx.doi.org/10.1016/j.bioorg.2016.09.007
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

30
J. Tang et al. / Bioorganic Chemistry 69 (2016) 29–36
novel tyrosinase inhibitors with higher activity, better pharmacol-
ogy properties and lower side effect. Gratifyingly, it was reported
that phenyl thioureas and alkyl thioureas could exhibit weak to
moderate depigmenting activity [22], and the interaction with
the hydrophobic protein pocket is close to the binuclear copper
active site of tyrosinase. The sulfur atom of in the thioureas can
bind to both copper ions in the active site. Similarly, thiosemicar-
bazide derivatives and curcumin have potential to coordinate the
two copper ions in the active site of tyrosinase [23]. Inspired by
these precedent literatures, our research provides latest progress
in designing tyrosinase inhibitors.
We envisioned that thiadiazole, the hetero sulfur containing
aromatic ring, could improve lipophilicity, and the mesoionic
nature of thiadiazole makes this class of compounds to show good
tissue permeability [24,25]. The two-electron donor nitrogen sys-
tem (AN@CAS) as well as the hydrogen binding domain, may help
with pharmacodynamics of the molecule for better binding with
the receptors [26]. Plus, thiadiazoles are bioisosteres of pyrimidi-
nes, oxadiazoles, oxazoles, benzene and the derivatives have been
proved to display diverse range of biological and pharmacological
properties [27]. 1,3,4-thiadiazole, a unique structure, represents a
key motif in heterocyclic chemistry and medicinal chemistry
[28]. For instance, many drugs containing 1,3,4-thiadiazole moieties
are available on the market, e.g., acetazolamide, methazolamide
and sulfamethazole [29]. Recently, derivatives of 1,3,4-thiadiazole
have been reported for their antibacterial, antifungal, inflamma-
tory, antianxiety, and antitubercular activities [30]. Some scientists
have studied extensively on 1,3,4-thiadiazole derivatives as poten-
tial drugs to treat Alzheimer’s disease (AD) [31]. 1,3,4-Thiadiazole
derivatives bearing Schiff base moieties even display anticancer,
antibacterial, antidepressant, antidiabetic and antifungal activities
[32].
Lately, our studies indicated that thiosemicarbazide derivatives
exhibited potent inhibitory activities against mushroom tyrosi-
nase. Inhibition mechanism analysis showed that this series of
compounds could potentially bind to the binuclear active site of
tyrosinase [33]. 1,3,4-Thiadiazole-2(3H)-thiones exhibited potent
inhibitory activities against mushroom tyrosinase [34]. But to the
best of our knowledge, the tyrosinase inhibitory activities of the
1,3,4-thiadiazole Schiff base derivatives have never been reported
in the literatures. Combining the biological significance of 1,3,4-
thiadiazole and Schiff base moiety, we designed and synthesized
a library of 1,3,4-thiadiazole derivatives bearing Schiff base moi-
eties, and their inhibitory activities against mushroom tyrosinase
were evaluated using kojic acid as positive control. The struc-
ture–activity relationship (SAR) was analyzed and discussed. To
better elucidate the inhibition mechanism, the docking studies
were carried out and discussed as well.
China). Other chemicals were purchased from commercial suppli-
ers and were used without further purification.
2.1.1. Synthesis of 1,3,4-thiadiazole derivatives bearing Schiff base
moieties [30,35]
Thiosemicarbazide 18.228 g (0.2 mol) and anhydrous sodium
carbonate 10.6 g (0.1 mol) were suspended in 80 mL anhydrous
ethanol. The reaction mixture was stirred at RT and carbon disul-
phide 0.2 mol (15.228 g) was added dropwise in 1 h. The reaction
mixture was stirred under reflux for 4 h and monitored by TLC
until the reaction was completed. After completion of reaction,
the mixture was allowed to cool to room temperature, water
(100 mL) was then added to the mixture and neutralized by the
10% solution of hydrochloric acid, white precipitates of 2-amino-
5-mercapto-1,3,4-thiadiazole were generated.
2-Amino-5-mercapto-1,3,4-thiadiazole (5 mmol) was sus-
pended in 10 mL anhydrous ethanol and 6 mmol of different
aromatic aldehyde was added, using three drops concentrated sul-
furic acid as catalyst. The reaction mixture was stirred under reflux
for 8 h and monitored by TLC until the reaction was completed.
After completion of reaction, the mixture was allowed to cool
to room temperature, and then left overnight. Precipitates of
1,3,4-thiadiazole derivatives bearing Schiff base moieties were
generated. The solids were filtered, and washed with the mixture
of ethanol and hot water (1:1); dried solid was purified by crystal-
lization from anhydrous alcohol to afford the desired compound.
2.1.1.1. 2-(Benzylideneamino)-5-mercapto-1,3,4-thiadiazole (1). Yield
51.4%. Yellow solid, mp: 237–239.2 °C, IR (KBr) mmax 3310, 1567,
1476, 1376, 1312, 1121, 1051, 749, 711 cmꢀ1 1H NMR (DMSO-d6,
;
300 MHz) d 13.02 (s, 1H, SH), 8.62 (s, 1H, @CH), 7.91 (d, 2H,
J = 8.7 Hz, ph-H), 7.60 (t, 1H, J = 7.5 Hz, ph-H), 7.45 (t, 2H,
J = 7.5 Hz, ph-H); 13C NMR (DMSO-d6, 75 MHz) d 193.1, 180.9,
161.4, 136.1, 133.7, 129.9, 128.6; MS (ESI): m/z (100%) 222 (M+);
Anal. Calcd for C9H7N3S2: C, 48.85; H, 3.19; N, 18.99; found: C,
48.82; H, 3.18; N, 18.96.
2.1.1.2. 2-((2-Fluorobenzylidene)amino)-5-mercapto-1,3,4-thiadia-
zole (2). Yield 16%. Yellow solid, mp: 242.6–244.1 °C, IR (KBr) mmax
3223, 2995, 1562, 1491, 1382, 1313, 1267, 1208, 1108, 1051, 942,
754, 671 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 13.18 (s, 1H, SH),
;
8.83 (s, 1H, @CH), 8.25 (d, 1H, J = 7.5 Hz, ph-H), 7.54 (t, 1H,
J = 8.1 Hz, ph-H), 7.34 (d, 1H, J = 7.5 Hz, ph-H), 7.29 (t, 1H,
J = 8.1 Hz, ph-H); 13C NMR (DMSO-d6, 75 MHz) d 187.9, 180.8,
157.4, 136.9, 131.7, 129.4, 125.2, 122.1, 116.8; MS (ESI): m/z
(100%) 240 (M+); Anal. Calcd for C9H6FN3S2: C, 45.17; H, 2.53; N,
17.56; found: C, 45.15; H, 2.53; N, 17.52.
2.1.1.3. 2-((3-Chlorobenzylidene)amino)-5-mercapto-1,3,4-thiadia-
zole (3). Yield 59.9%. Yellow solid, mp: 253.4–255.3 °C, IR (KBr)
mmax 3284, 1570, 1510, 1373, 1309, 1259, 1188, 1105, 1047, 738,
2. Materials and methods
677 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 13.12 (s, 1H, SH), 8.86
;
2.1. Chemistry
(s, 1H, @CH), 8.12 (s, 1H, ph-H), 7.96 (d, 1H, J = 7.8 Hz, ph-H),
7.80 (d, 1H, J = 6.9 Hz, ph-H), 7.45 (t, 1H, J = 8.7 Hz, ph-H); 13C
NMR (DMSO-d6, 75 MHz) d 192.1, 180.5, 161.4, 136.5, 134.2,
131.2, 130.4, 127.9, 127.0; MS (ESI): m/z (100%) 256 (M+); Anal.
Calcd for C9H6ClN3S2: C, 42.27; H, 2.36; N, 16.43; found: C,
42.25; H, 2.35; N, 16.42.
Melting points (m.p.) were determined on SGW-X4 melting
point apparatus and the thermometer was uncorrected. NMR spec-
tra were recorded on Bruker 300 spectrometers at 25 °C in DMSO-
d6 using TMS as internal standard for protons. Chemical shift val-
ues are mentioned in d (ppm) and coupling constants (J) are given
in Hz. Mass-spectrometric (MS) data is reported in m/z using the
LCMS- 2010A. The reaction progress was monitored by TLC (Merck
Kieselgel 60 F254). The chromatograms were visualized under UV
254–365 nm and iodine. Infrared (IR) spectra were recorded on
2.1.1.4. 2-((4-Chlorobenzylidene)amino)-5-mercapto-1,3,4-thiadia-
zole (4). Yield 30.2%. Yellow solid, mp: 222.1–224.5 °C, IR (KBr)
mmax 3275, 1563, 1488, 1376, 1313, 1259, 1185, 1094, 1051,
1015, 783, 744 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 13.18 (s,
;
VECTOR 22 spectrometer in KBr pellets and are reported in cmꢀ1
Tyrosinase, L-3,4-dhydroxyphenylalanine (L-DOPA) and kojic acid
were purchased from Sigma–Aldrich Chemical Co (Shanghai,
.
1H, SH), 8.75 (s, 1H, @CH), 7.95 (d, 2H, J = 6.9 Hz, ph-H), 7.71 (d,
2H, J = 6.9 Hz, ph-H); 13C NMR (DMSO-d6, 75 MHz) d 192.1, 180.9,
161.4, 139.3, 134.8, 131.5, 129.3; MS (ESI): m/z (100%) 256 (M+);

J. Tang et al. / Bioorganic Chemistry 69 (2016) 29–36
31
Anal. Calcd for C9H6ClN3S2: C, 42.27; H, 2.36; N, 16.43; found: C,
42.28; H, 2.34; N, 16.45.
2.1.1.11.
2-((3,5-Dimethoxybenzylidene)amino)-5-mercapto-1,3,4-
thiadiazole (11). Yield 44.8%. Yellow solid, mp: 227–229 °C, IR
(KBr) mmax 3339, 3082, 2926, 1609, 1557, 1463, 1325, 1264, 1200,
1155, 1059, 971, 845, 754 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
2.1.1.5. 2-((3,4-Dichlorobenzylidene)amino)-5-mercapto-1,3,4-thiadi-
azole (5). Yield 67.8%. Yellow solid, mp: 223.3–225.7 °C, IR (KBr)
mmax 3404, 1566, 1469, 1376, 1309, 1264, 1190, 1103, 1053, 746,
13.19 (s, 1H, SH), 8.67 (s, 1H, @CH), 7.08 (s, 2H, ph-H), 6.61 (s,
1H, ph-H), 3.76 (s, 6H, 2CH3); 13C NMR (DMSO-d6, 75 MHz) d
186.8, 180.9, 168.7, 161.4, 138.7, 107.5, 104.2, 55.58; MS (ESI):
m/z (100%) 282 (M+); Anal. Calcd for C11H11N3O2S2: C, 46.96; H,
3.94; N, 14.93; found: C, 46.95; H, 3.96; N, 14.97.
667 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 13.15 (s, 1H, SH), 8.82
;
(s, 1H, @CH), 7.97 (s, 1H, ph-H), 7.75 (d, 1H, J = 8.4 Hz, ph-H),
7.65 (d, 1H, J = 8.4 Hz, ph-H); 13C NMR (DMSO-d6, 75 MHz) d
189.7, 181.3, 159.8, 136.8, 135.1, 134.5, 133.2, 131.3, 128.4; MS
(ESI): m/z (100%) 291 (M+); Anal. Calcd for C9H5Cl2N3S2: C, 37.25;
H, 1.74; N, 14.48; found: C, 37.28; H, 1.76; N, 14.49.
2.1.1.12. 2-(((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)phenol
(12). Yield 51%. Yellow solid, mp: 252–254 °C, IR (KBr) mmax
3434, 3072, 2875, 1607, 1571, 1528, 1482, 1374, 1276, 1199,
1146, 1067, 761 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 14.53 (s,
;
2.1.1.6.
4-Chloro-2-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)
1H, SH), 11.15 (s, 1H, OH), 8.88 (s, 1H, @CH), 7.87 (d, 1H,
J = 7.8 Hz, ph-H), 7.53 (t, 1H, J = 7.2 Hz, ph-H), 7.02–6.96 (m, 2H,
ph-H); 13C NMR (DMSO-d6, 75 MHz) d 191.7, 186.5, 166.1, 160.8,
135.8, 130.1, 119.9, 119.5, 117.2; MS (ESI): m/z (100%) 238 (M+);
Anal. Calcd for C9H7N3OS2: C, 45.55; H, 2.97; N, 17.71; found: C,
45.57; H, 2.98; N, 17.73.
methyl)phenol (6). Yield 53%. Yellow solid, mp: 247.3–250.1 °C, IR
(KBr) mmax 3440, 3067, 2862, 1599, 1561, 1517, 1470, 1231, 1173,
1063, 804, 711 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 14.58 (s,
;
1H, SH), 11.27 (s, 1H, OH), 8.80 (s, 1H, @CH), 7.84 (s, 1H, ph-H),
7.54 (d, 1H, J = 8.7 Hz, ph-H), 7.05 (d, 1H, J = 8.7 Hz, ph-H); 13C
NMR (DMSO-d6, 75 MHz) d 189.7, 180.7, 164.0, 158.7, 135.0,
127.8, 123.6, 121.1, 119.5; MS (ESI): m/z (100%) 272 (M+); Anal.
Calcd for C9H6ClN3OS2: C, 39.78; H, 2.23; N, 15.46; found: C,
39.76; H, 2.25; N, 15.48.
2.1.1.13.
4-(((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)ben-
zene-1,3-diol (13). Yield 57.6%. Yellow solid, mp: 240–243 °C, IR
(KBr) mmax 3305, 3139, 1635, 1587, 1516, 1438, 1319, 1241, 1213,
;
1171, 1113, 1059, 973, 864, 797 cmꢀ1 1H NMR (DMSO-d6,
2.1.1.7.
4-Bromo-2-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)
300 MHz) d 14.37 (s, 1H, SH), 11.40 (s, 1H, OH), 10.73 (s, 1H,
OH), 8.70 (s, 1H, @CH), 7.71 (d, 1H, J = 6.9 Hz, ph-H), 7.65 (s, 1H,
ph-H), 6.34 (d, 1H, J = 6.9 Hz, ph-H); 13C NMR (DMSO-d6,
75 MHz) d 190.9, 185.9, 165.7, 164.4, 163.2, 133.2, 119.9, 109.3,
102.3; MS (ESI): m/z (100%) 254 (M+); Anal. Calcd for C9H7N3O2S2:
C, 42.68; H, 2.79; N, 16.59; found: C, 42.66; H, 2.82; N, 16.57.
methyl)phenol (7). Yield 51.5%. Yellow solid, mp: 253.0–255.1 °C,
IR (KBr) mmax 3420, 3058, 2858, 1606, 1555, 1512, 1467, 1347,
1269, 1171, 1062, 965, 828, 729 cmꢀ1
;
1H NMR (DMSO-d6,
300 MHz) d 14.57 (s, 1H, SH), 11.28 (s, 1H, OH), 8.78 (s, 1H,
@CH), 7.97 (s, 1H, ph-H), 7.61 (d, 1H, J = 8.7 Hz, ph-H), 7.00 (d,
1H, J = 8.7 Hz, ph-H); 13C NMR (DMSO-d6, 75 MHz) d 189.6, 180.7,
163.9, 159.3, 137.7, 130.9, 124.2, 121.7, 110.9; MS (ESI): m/z
(100%) 317 (M+); Anal. Calcd for C9H6BrN3OS2: C, 34.19; H, 1.91;
N, 13.29; found: C, 34.18; H, 1.93; N, 13.32.
2.1.1.14.
4-(((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)-2-
methoxyphenol (14). Yield 63.2%. Yellow solid, mp: 219.5–
221.8 °C, IR (KBr) mmax 3275, 1606, 1565, 1512, 1382, 1322, 1272,
1205, 1101, 1053, 928, 753 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
2.1.1.8. 2-((4-(Tert-butyl)benzylidene)amino)-5-mercapto-1,3,4-thia-
diazole(8). Yield66.1%.Whitesolid,mp214.2–216.5 °C,IR(KBr)mmax
3395, 2960, 2868, 1689, 1607, 1567, 1499, 1379, 1315, 1264, 1185,
14.41 (s, 1H, SH), 10.34 (s, 1H, OH), 8.55 (s, 1H, @CH), 7.55 (s,
1H, ph-H), 7.42 (d, 1H, J = 8.7 Hz, ph-H), 6.99 (d, 1H, J = 8.7 Hz,
ph-H), 3.86 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) d 190.9,
186.2, 167.9, 152.9, 148.1, 128.6, 126.7, 115.6, 111.1, 55.5; MS
(ESI): m/z (100%) 268 (M+); Anal. Calcd for C10H9N3O2S2: C,
44.93; H, 3.39; N, 15.72; found: C, 44.96; H, 3.38; N, 15.73.
1105, 1052, 931, 835, 761 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
13.25 (s, 1H, SH), 8.83 (s, 1H, @CH), 7.66 (d, 2H, J = 8.4 Hz, ph-H),
7.48 (d, 2H, J = 8.4 Hz, ph-H), 1.35 (s, 9H, 3CH3); 13C NMR (DMSO-
d6, 75 MHz) d 192.2, 180.7, 155.7, 152.9, 131.3, 129.7, 126.8, 34.8,
31.2; MS (ESI): m/z (100%) 278 (M+); Anal. Calcd for C13H15N3S2: C,
56.28; H, 5.45; N, 15.15; found: C, 56.29; H, 5.44; N, 15.12.
2.1.1.15.
4-(((5-Mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)-2,6-
dimethoxyphenol (15). Yield 52.2%. Yellow solid, mp: 267–
268.5 °C, 1H NMR (DMSO-d6, 300 MHz) d 14.42 (s, 1H, SH), 9.77
(s, 1H, OH), 8.53 (s, 1H, @CH), 7.31 (s, 2H, ph-H), 3.84 (s, 6H,
2CH3); 13C NMR (DMSO-d6, 75 MHz) d 191.1, 186.0, 168.1, 148.1,
142.0, 124.4, 107.6, 56.2; MS (ESI): m/z (100%) 298 (M+); Anal.
Calcd for C11H11N3O3S2: C, 44.43; H, 3.73; N, 14.13; found: C,
44.45; H, 3.76; N, 14.13.
2.1.1.9. 2-((2-Nitrobenzylidene)amino)-5-mercapto-1,3,4-thiadiazole
(9). Yield 22.7%. Yellow solid, mp: 229.7–232.2 °C, IR (KBr) mmax
3221, 2987, 1610, 1562, 1519, 1352, 1309, 1265, 1215, 1188, 1110,
1055, 947, 856, 781, 733 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
13.19 (s, 1H, SH), 9.36 (s, 1H, @CH), 8.16 (d, 1H, J = 8.1 Hz, ph-H),
7.92 (d, 1H, J = 7.5 Hz, ph-H), 7.79 (t, 1H, J = 8.4 Hz, ph-H), 7.67 (t,
1H, J = 7.8 Hz, ph-H); 13C NMR (DMSO-d6, 75 MHz) d 189.9, 180.1,
158.6, 149.5, 134.2, 133.7, 130.8, 128.2, 124.9; MS (ESI): m/z
(100%) 267 (M+); Anal. Calcd for C9H6N4O2S2: C, 40.59; H, 2.27; N,
21.04; found: C, 40.58; H, 2.28; N, 21.06.
2.1.1.16. 2-((2,4,6-Trimethoxybenzylidene)amino)-5-mercapto-1,3,4-
thiadiazole (16). Yield 86%. Yellow solid, mp: 263–265 °C, IR (KBr)
mmax 3084, 2890, 1599, 1520, 1463, 1384, 1325, 1276, 1239,
1113, 1064, 767 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d 14.29 (s,
;
1H, SH), 8.69 (s, 1H, @CH), 6.31 (s, 2H, ph-H), 3.90 (s, 6H, 2CH3),
3.82 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz) d 190.8, 185.8,
167.2, 163.4, 161.3, 104.5, 91.7, 56.3, 55.7; MS (ESI): m/z (100%)
312 (M+); Anal. Calcd for C12H13N3O3S2: C, 46.29; H, 4.21; N,
13.49; found: C, 46.31; H, 4.23; N, 13.52.
2.1.1.10. 2-((2-Methoxybenzylidene)amino)-5-mercapto-1,3,4-thiadi-
azole (10). Yield 38.3%. Yellow solid, mp: 148.2–151.3 °C, IR (KBr)
mmax 3328, 2934, 2835, 1564, 1491, 1373, 1312, 1252, 1175,
1116, 1051, 933, 802, 754 cmꢀ1 1H NMR (DMSO-d6, 300 MHz) d
;
13.17 (s, 1H, SH), 9.32 (s, 1H, @CH), 7.69 (d, 1H, J = 8.1 Hz, ph-H),
7.52 (t, 1H, J = 8.4 Hz, ph-H), 7.03 (d, 1H, J = 8.4 Hz, ph-H), 6.99 (t,
1H, J = 7.8 Hz, ph-H); 13CNMR (DMSO-d6, 75 MHz) d 189.9, 181.2,
157.4, 155.5, 135.9, 131.0, 128.6, 122.4, 121.0, 56.0; MS (ESI): m/
z (100%) 252 (M+); Anal. Calcd for C10H9N3OS2: C, 47.79; H, 3.61;
N, 16.72; found: C, 47.82; H, 3.63; N, 16.75.
2.1.1.17. 2-(((5-(Benzylthio)-1,3,4-thiadiazol-2-yl)imino)methyl)-4-
chlorophenol (17). 2-Amino-5-mercapto-1,3,4-thiadiazole 3.99 g
(0.03 mol) was added to a 50 mL flask, and the solution of KOH
(1.683 g, 0.03 mol) in 10 mL was added under stirring at room

32
J. Tang et al. / Bioorganic Chemistry 69 (2016) 29–36
temperature. Halogenated hydrocarbon (0.1 mol) was added by
dropwise in 0.5 h with vigorous stirring. The reaction mixture
was stirred at room temperature and checked by TLC (petroleum
ether: ethyl acetate 1:1). When reaction was complete, the yellow
precipitate of crude thiadiazole sulfide was formed. This was
filtered, washed with water to neutrality, and crystallized from
95% ethanol.
2-Amino-5-sulfide-1,3,4-thiadiazole (5 mmol) was suspended
in 10 mL anhydrous ethanol and 6 mmol of aromatic aldehyde
was added, using tetrabutylammonium bromide (TBAB) as cata-
lyst. The reaction mixture was stirred under reflux for 8 h and
monitored by TLC (petroleum ether: ethyl acetate 4:1) until the
reaction was complete. After completion of reaction, the mixture
was allowed to cool to room temperature, and then left overnight.
Precipitates of 1,3,4-thiadiazole sulfide derivative bearing Schiff
base moieties were produced. The solid that separated out was fil-
tered, washed with the mixture of ethanol and hot water (1:1);
dried solid was purified by crystallization from anhydrous alcohol
to afford the desired compound.
2.4. In silico docking simulation of tyrosinase with compounds 14 [38–
40]
For docking simulations, we used the AutoDock4.2 program.
Among the many tools available for in silico protein-ligand dock-
ing, AutoDock4.2 is the most commonly used due to its automated
docking capability. The 3D structure of tyrosinase was used the
crystal structure of Agaricus bisporus (PDB ID: 2Y9X) without
homology modeling. We conducted simulations of the docking of
tyrosinase to the compounds 14. To prepare the compound for
the docking simulation, the following steps was performed: (1)
conversion of 2D structures into 3D structures, (2) calculation of
charges, and (3) addition of hydrogen atoms using the ChemOffice
program (http://www.cambridgeoft.com).
2.5. Acute toxicity [41]
Acute toxicity experiments were carried out using KM mice of
both sexes weighing 18–22 g. The food and water were provided
according to EPA OPPTS Harmonized Test guideline. All animals
were provided by Qinlongshan Laboratory animal center SCXK
(Yu) 2005-0001. Various doses of the compound 14 were given
by intragastric administration to the healthy KM mice. After the
intragastric administration of the compound, mice were observed
continuously for any gross behavioral changes and deaths, and
intermittently for 1 week, the LD50 values can be obtained.
Yield 49.92%. Yellow solid, mp: 148.2–150.7 °C, 1H NMR (CDCl3,
500 MHz) d 11.79 (s, 1H, OH), 8.95 (s, 1H, CH), 7.44–7.30 (m, 7H,
Ph-H), 7.00 (d, J = 8.6 Hz, 1H, Ph-H), 4.57 (s, 2H, CH2); 13C NMR
(CDCl3, 125 MHz) d 170.4, 166.7, 164.8, 160.2, 135.6, 132.6,
132.3, 129.3, 128.9, 128.1, 124.6, 119.4, 118.9, 38.4; MS (ESI): m/
z (100%) 362 (M+). Anal. Calcd for C16H12ClN3OS2: C, 53.11; H,
3.34; N, 11.61; found: C, 53.09; H, 3.36; N, 11.63.
2.2. Tyrosinase activity assay
3. Results and discussion
Tyrosinase inhibition assays were performed according to the
method reported by our group [36]. Briefly, 1,3,4-thiadiazole
derivatives were tested for diphenolase inhibitory activity of
tyrosinase using L-DOPA (dihydroxyphenylalanine) as substrate.
All the compounds were dissolved in DMSO and its final concentra-
tion in the reaction mixture was 2.0%. Thirty units of mushroom
tyrosinase (0.5 mg/ml) were first pre-incubated with the com-
pounds, in 50 mM phosphate buffer (pH 6.8), for 10 min at 25 °C.
Then the L-DOPA (0.5 mM) was added to the reaction mixture
and the enzyme reaction was continuously monitored by measur-
ing the change in absorbance at 475 nm of formation of the DOPA
chrome for 1 min. The measurement was performed in triplicate
for each concentration and averaged before further calculation.
The activity was expressed as the sample concentration that gave
a 50% inhibition in the enzyme activity (IC50). The percent of inhi-
bition of tyrosinase was calculated as follows:
3.1. Chemistry
The synthesis of 1,3,4-thiadiazole derivatives was shown in
Scheme 1. As depicted in Scheme 1, 2-amino-5-mercapto-1,3,4-th
iadiazole can be synthesized by the condensation reaction of
thiosemicarbazide with carbon disulphide in ethanol using anhy-
drous sodium carbonate as catalyst under reflux conditions. Thia-
diazole sulfide was synthesized from 2-amino-5-mercapto-1,3,4-
thiadiazole and halogenated hydrocarbon. 1,3,4-thiadiazole
derivatives bearing Schiff base moieties were prepared by con-
densing 2-amino-5-mercapto-1,3,4-thiadiazole or 5-(benzyla
mino)-1,3,4-thiadiazole-2-thiol with different aromatic aldehydes
in ethanol using concentrated sulfuric acid as catalyst under reflux
conditions. The resulting products were obtained in moderate
yields. All the target compounds were characterized spectroscopic
methods.
Inhibition rate ð%Þ ¼ ½ðB ꢀ SÞ=Bꢁ ꢂ 100
3.2. Biological activity
Here, the B and S were the absorbances for the blank and samples.
All the experiments were carried out at least in triplicate and aver-
aged. Kojic acid was used as the positive control.
The 17 synthesized compounds were assessed using tyrosinase
inhibition assay with kojic acid as a positive control, and the IC50
values against tyrosinase were recorded in Table 1. As we expected,
15 compounds exhibited potent inhibition activities against mush-
room tyrosinase, with IC50 values at single digit or sub-micromolar
level, better than the reference inhibitor kojic acid. The other com-
pounds showed some inhibitory activities against tyrosinase.
Pleasingly, compound 14 exhibited the most potent tyrosinase
2.3. Kinetic analysis of the inhibition of tyrosinase
A series of experiments were performed to determine the inhi-
bition kinetics by following method [37]. Inhibitor (13) with con-
centrations 0, 0.025, 0.05, 0.075, 0.1
concentrations 0, 0.005, 0.01, 0.015 and 0.02
used. Substrate L-DOPA concentration was among 1014–304
all kinetic study. Preincubation and measurement time was the
same as discussed in mushroom tyrosinase inhibition assay proto-
col. The inhibition type on the enzyme was assayed by Linewea-
vere Burk plots of inverse of velocities (1/V) versus inverse of
substrate concentration 1/[S] mMꢀ1, and the inhibition constants
were determined by the secondary plots of the apparent 1/Vm
versus the concentrations of the inhibitors.
l
l
M, and (14) with
M, respectively were
M in
inhibitory activity with IC50 value of 0.036 lM. The results showed
l
that the substitution pattern of the benzene ring has a great influ-
ence in determining activities against tyrosinase. From the result of
Table 1, if the hydrogens on benzene ring were substituted with
halogens, all of the compounds showed better inhibitory activities
than compound 1 (49.246 lM). Compound 3 exhibited lower inhi-
bitory activity than compound 4. However, the dihalogen substi-
tuted compound 5 showed lower inhibitory activity than the
compound 4. The results showed that the type, position and

J. Tang et al. / Bioorganic Chemistry 69 (2016) 29–36
33
Scheme 1. The synthesis of 1,3,4-thiadiazole derivatives bearing Schiff base moieties.
Table 1
orine, better inhibitory activity was achieved than other halogen
substituted compounds. The IC50 value of compound 2 was
Tyrosinase inhibitory activities of the synthesized compounds.
Compounds
R
IC50 (l
M)a SD
0.765
4.753
l
l
M, the IC50 values of the two compounds 3 and 4 were
M and 2.460 M, respectively. This result may be because
l
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Phenyl
2-Fluorophenyl
3-Chlorophenyl
4-Chlorophenyl
3,4-Dichlorophenyl
5-Chloro-2-hydroxylphenyl
5-Bromo-2-hydroxylphenyl
4-Bu-phenyl
49.246 0.184
0.765 0.077
4.753 0.109
2.460 0.155
3.621 0.142
1.718 0.135
1.971 0.131
1.390 0.118
0.671 0.093
0.733 0.083
5.353 0.136
0.785 0.064
0.255 0.065
0.036 0.002
0.473 0.096
0.907 0.142
247.350 3.782
23.6
fluorine atom has smaller atomic radius and greater electro-
negativity. Moreover, comparing to the tyrosinase inhibitory activ-
ity of compounds 3, 4 and 6, when the hydroxyl group was intro-
duced onto the benzene ring, an elevated inhibitory activity
could be observed. These results showed that hydroxyl group
may be beneficial to increasing the inhibitory activity of this kind
of compounds with halogen atom at benzene ring. From Table 1,
the IC50 value of compound 10 with a methoxyl substituted ben-
2-Nitrophenyl
2-Methoxylphenyl
3,5-Dimethoxylphenyl
2-Hydroxylphenyl
2,4-Dihydroxylphenyl
4-Hydroxyl-3-methoxylphenyl
3,5-Dimethxoyl-4-hydroxylphenyl
2,4,6-Trimethoxylphenyl
5-Chloro-2-hydroxylphenyl
–
zene ring (2-position) was 0.733
disubstituted methoxyl groups (3,5-position) exhibited some
tyrosinase inhibitory activity with an IC50 value of 5.353 M, it
lM. However, compound 11 with
l
showed lower inhibitory activity than the compound 10. Interest-
ingly, the IC50 of compound 16 with trisubstituted methoxyl
groups (2,4,6-position) was 0.907 lM. This result might be
17
Kojic acid
explained that the inhibitory activity methoxyl substituted com-
pound might be related to the position of methoxyl group.
For compounds 12 and 13, the inhibitory activity increased as
the number of hydroxyl group increased. This result showed the
hydroxyl group number might have some influences on the inhibi-
tory activity. Compound 14 was the best inhibitor among all the
a
Inhibitor concentration (mean of three independent experiments) required for
50% inactivation of tyrosinase.
number of halogen atom at the benzene ring might modulate the
activities. When the hydrogen of benzene ring was replaced by flu-

34
J. Tang et al. / Bioorganic Chemistry 69 (2016) 29–36
Fig. 1. (A) Kinetic inhibition analysis of compound 13 against the oxidation of L-DOPA by tyrosinase. The mode of inhibition type exhibited through Lineweavere-Burk plot
with different concentrations of compound 13. The inhibitor concentrations of compound 13 for Curves 1–5 were 0, 0.025, 0.05, 0.075 and 0.1
versus concentration of compound 13 was used to determine the inhibition constant KI.
lM. (B) The plot of intercept
compounds, but in contrast, with the increased number of the
methoxyl groups, the inhibitory activity of the compound 15 was
decreased. This result might be related to the steric hindrance.
However, compound 15 exhibited better inhibitory activity than
the compound 11. The result also showed that hydroxyl group
may be beneficial to increasing the inhibitory activity. From the
Table 1, based on the inhibitory activity of compounds 6 and 17,
when the thiol group was etherified by the benzyl chloride, the
inhibitory activity dropped drastically. Thus, free thiol group could
be an active group in the inhibition. In addition, the o-dopaquinone
can react with the thiol group of the inhibitor [42].
with the same slopes. Meanwhile, as the inhibitor concentration
increased, the values of both km and Vm decreased, but the ratio
of km/Vm remained unchanged. The results indicated that both
3.3. Inhibitory mechanism
Among the tested target compounds, compound 13 and 14
showed superior inhibitory activities to the other compounds,
and therefore, we carried out the kinetic analysis of the two com-
pounds for tyrosinase inhibition using L-DOPA as the substrate.
The Lineweaver–Burk plots for the inhibition of tyrosinase by the
two compounds were obtained with various concentrations of
the two compounds and the substrate (Figs. 1 and 2). The inhibi-
tion types on the enzyme by compounds 13 and 14 for oxidation
of LDOPA were studied as shown in Figs. 1 and 2, respectively.
The plots of 1/v versus 1/[S] gave a family of parallel straight lines
Fig. 3. The effect of concentrations of tyrosinase on its activity for the catalysis of L-
DOPA at different concentration of compound 9. The concentrations of compound 9
for curves 1–4 are 0, 0.25, 0.5 and 1 lM, respectively.
Fig. 2. (A) Kinetic inhibition analysis of compound 14 against the oxidation of L-DOPA by tyrosinase. The mode of inhibition type exhibited through Lineweavere-Burk plot
with different concentrations of compound 14. The inhibitor concentrations of compound 14 for Curves 1–5 were 0, 0.005, 0.01, 0.015 and 0.02
versus concentration of compound 14 was used to determine the inhibition constant KI.
lM. (B) The plot of intercept

J. Tang et al. / Bioorganic Chemistry 69 (2016) 29–36
35
compounds 13 and 14 were uncompetitive inhibitors. The inhibi-
tion constants (KI) of the two compounds 13 and 14 were obtained
from a plot of the vertical intercept (1/Vm) versus the concentra-
ꢀ5.94 kcal/mol, it proved that compound 14 formed stable drug
receptor complex with tyrosinase. The phenolic hydroxyl present
at position 4 in compound 14 can interact with the copper atom
(Cu 401) of tyrosinase (Fig. 4). This result showed hydroxyl group
might be activity group. Van der Waals force is also the interaction
between compound 14 and tyrosinase. The 1,3,4-thiadiazole ring
formed pi-lone pair to GLY281 with bond length of 2.91 Å. The
tion of the inhibitors as 0.075 lM and 0.038 lM, respectively.
The catalytic constants (kcat) of the two compounds 13 and 14 were
0.61 sꢀ1 and 0.83 sꢀ1. kcat/km values of the two compounds 13 and
14 were 1.27 mMꢀ1 sꢀ1 and 1.08 mMꢀ1 sꢀ1. The inhibitory mecha-
nism of the compound 9 with no hydroxyl group on mushroom
tyrosinase for the oxidation of L-DOPA was determined (Fig. 3).
The result displayed that the plots of V versus [E] gave a family
of parallel straight lines with the same slopes, thus compound 9
was irreversible inhibitor.
p-
p
stacking present in the compound 14 between phenyl ring
and LYS180 having distance 1.82 Å (Fig. 4). ALA286 and VAL283
can form -alkyl interaction present phenyl ring having bond
p
lengths 4.36 Å and 4.04 Å respectively. ASN260 and HIS259 can
form hydrogen bonds with methoxy of the compound. There is
no significant interaction between mercapto and tyrosinase.
3.4. In silico docking between tyrosinase and compounds 14
3.5. Acute toxicity
The binding affinities of the inhibitors to the receptor tyrosinase
were calculated using computational docking studies. The lower
value showed the more stable complex formed between the ligand
and target protein. The binding energy of compound 14 was
To evaluate acute toxicities of the synthesized compounds,
compound 14 was selected to test the acute toxicity. The LD50
value for acute toxicity of compound 14 in mice after intragastric
administration was obtained. Test result indicated the LD50 value
of compound 14 was 5000 mg/kg. It showed that compound 14
oral dose commonly used in clinical treatment should be safe.
The result might be beneficial to search and develop novel tyrosi-
nase inhibitor with better activity together with lower side effects.
4. Conclusion
In summary, we have developed a series of 1,3,4-thiadiazole
derivatives bearing Schiff base moieties as novel tyrosinase inhibi-
tors. The results showed that many of them exhibited remarkable
tyrosinase inhibitory activities with IC50 values at single digit or
sub-micromolar level. Compound 14 was found to be the best
tyrosinase inhibitor in this series with the IC50 value of 0.036 lM.
Structure-activity relationship (SAR) analysis indicated that (1)
free thiol group and hydroxyl group could be active groups in the
inhibition; (2) the steric hindrance could be an unfavorable factor;
(3) substituted group was introduced onto the benzene ring, an
elevated inhibitory activity could be observed; (4) the position
and numbers of hydroxyl groups might play an important role in
the increase of inhibitory activity. Moreover, the inhibition kinetics
study revealed that compounds 13 and 14 exhibited such inhibi-
tory effects on tyrosinase by acting as the uncompetitive inhibitors.
Docking studies showed compound 14 combined strongly with
mushroom tyrosinase. Hydroxyl group interact with the copper
atom of tyrosinase, this result showed that the group was active
groups. The acute toxicity of compound 14 was evaluated, LD50
value of the compound was 5000 mg/kg. It indicated that com-
pound 14 oral dose commonly used in clinical treatment should
be safe.
Acknowledgments
We thank the innovation team of Shaoyang University for fund-
ing this project. This work was also financially supported by the
Foundation of Education Department of Hunan Province, China
(15A172).
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