Microwave-assisted synthesis and tyrosinase inhibitory activity of chalcone derivatives

Article abstract of DOI:10.1111/cbdd.12126

A series of chalcones and their derivatives were synthesized, and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesized compounds exhibited significant inhibitory activity, and four compounds exhibited more potent tyrosinase inhibitory activity than the reference standard inhibitor kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one). Specifically, 1-(-1-(4-methoxyphen- yl)-3-phenylallylidene)thiosemicarbazide (18) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 0.274 μm. The inhibition mechanism analysis of 1-(-1-(2,4-dihydroxyphenyl)-3-phenylallylidene) thiosemicarbazide (16) and 1-(-1-(4-methoxyphenyl)-3-phenylallylidene) thiosemicarbazide (18) demonstrated that the inhibitory effects of the two compounds on the tyrosinase were irreversible. Preliminary structure activity relationships' analysis suggested that further development of such compounds might be of interest.

Full text of DOI:10.1111/cbdd.12126

Chem Biol Drug Des 2013; 82: 39–47
Research Article
Microwave-Assisted Synthesis and Tyrosinase
Inhibitory Activity of Chalcone Derivatives
melasma, freckles, and pigmented acne scars, are of
Jinbing Liu*, Changhong Chen, Fengyan Wu and
Liangzhong Zhao
particular concern to women. Therefore, the regulation of
melanin synthesis by inhibiting the tyrosinase enzyme is a
current research topic in the context of preventing hyper-
pigmentation. The treatment usually involves the use of
medicines or medicinal cosmetics containing depigmenting
agents or skin-whitening agents (5,6). In clinical usage,
tyrosinase inhibitors are used for treatments of dermato-
logical disorders related to melanin hyperaccumulation and
are essential in cosmetics for depigmentation (7–11), such
as age spots and freckle, caused by the accumulation of
an excessive level of epidermal pigmentation (12,13).
Department of Biology and Chemical Engineering,
Shaoyang University, Shao Shui Xi Road, Shaoyang,
422100, China
*Corresponding author: Jinbing Liu, xtliujb@yahoo.com.cn,
jliu1238@umd.edu
A
series of chalcones and their derivatives were
synthesized, and their inhibitory effects on the dipheno-
lase activity of mushroom tyrosinase were evaluated.
The results showed that some of the synthesized com-
pounds exhibited significant inhibitory activity, and four
compounds exhibited more potent tyrosinase inhibitory
activity than the reference standard inhibitor kojic acid
(5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one). Specifi-
cally, 1-(-1-(4-methoxyphen- yl)-3-phenylallylidene)
thiosemicarbazide (18) exhibited the most potent tyrosi-
nase inhibitory activity with IC50 value of 0.274 lM. The
inhibition mechanism analysis of 1-(-1-(2,4-dihydroxy-
phenyl)-3-phenylallylidene) thiosemicarbazide (16) and
1-(-1-(4-methoxyphenyl)-3-phenylallylidene) thiosemic-
arbazide (18) demonstrated that the inhibitory effects of
the two compounds on the tyrosinase were irreversible.
Preliminary structure activity relationships’ analysis sug-
gested that further development of such compounds
might be of interest.
Previous reports confirmed that tyrosinase not only was
involved in melanism animals, but also was one of the
main causes of most fruits and vegetables quality loss dur-
ing postharvest handling and processing, leading to faster
degradation and shorter shelf life (14). Tyrosinase has also
been linked to Parkinson’s and other neurodegenerative
diseases (15). In insects, tyrosinase is uniquely associated
with three different biochemical processes, including scler-
otization of cuticle, defensive encapsulation and melaniza-
tion of foreign organism, and wound healing (16). These
processes provide potential targets for developing safer
and effective tyrosinase inhibitors as insecticides and ulti-
mately for insect control. Thus, the development of safe
and effective tyrosinase inhibitors is of great concern in the
medical, agricultural, and cosmetic industries. However,
only a few such as kojic acid (5-hydroxy-2-(hydroxymeth-
yl)-4H-pyran-4-one), arbutin, tropolone, ascorbic acid
derivatives, hydroxystilbene derivatives, and 1-phenyl-2-
thiourea are used as therapeutic agents and cosmetic
products (12,17).
Key words: chalcone derivatives, inhibition mechanism,
microwave-assisted synthesis, tyrosinase inhibitors
Received 2 December 2012, revised 26 January 2013 and
accepted for publication 14 February 2013
Chalcones (1,3-diaryl-2-propen-1-ones) constitute an
important class of natural products belonging to the flavo-
noids family of widespread occurrence in plants. They
have been reported to exhibit a wide range of pharmaceu-
tical effects including antioncogenetic, anti-inflammatory,
anti-ulcerative, antimalarial, antiviral, antifungal, and anti-
bacterial activities (18–20). Recently, chalcones were high-
lighted as a new class of tyrosinase inhibitor in several
publications (21,22). The literature survey revealed that
compounds with thiourea moieties have been reported to
demonstrate a wide range of pharmacological activities,
which include antibacterial, antifungal, anticonvulsant, and
tyrosinase inhibitory activity, such as phenylthioureas,
Tyrosinase(monophenol or o-diphenol, oxygen oxidoreduc-
tase, EC 1.14.18.1, syn. polyphenol oxidase), also known
as polyphenol oxidase (PPO), is
a copper-containing
monooxygenase that is widely distributed in microorgan-
isms, animals, and plants (1). Tyrosinase could catalyze
two distinct reactions involving molecular oxygen in the
hydroxylation of monophenols to o-diphenols (monophe-
nolase) and in the oxidation of o-diphenols to o-quinones
(diphenolase) (2). Due to the high reactivity, quinones
could polymerize spontaneously to form high-molecular
weight brown pigments (melanins) or react with amino
acids and proteins to enhance brown color of the pigment
produced (3,4). Hyperpigmentations, such as senile lentigo,
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12126
39

Liu et al.
alkylthioureas, and 1,3-bis-(5-methanesulfonylbutyl)thio-
urea, displayed weak or moderate tyrosinase inhibitory
activity (23,24). More recently, our investigations also dem-
onstrated that thiosemicarbazide derivatives exhibited
potent inhibitory activities against mushroom tyrosinase
(25,26). Stimulated by these results, in continuation of our
research work on safe and effective tyrosinase inhibitors,
in the present investigation, we synthesized a series of
chalcones and their Schiff bases, and their inhibitory activi-
ties against mushroom tyrosinase were evaluated using
kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one)
as comparing substance. Meanwhile, the structure–activity
relationships of these compounds were also primarily dis-
cussed. Microwave-assisted synthesis is an efficient and
eco-friendly synthetic strategy and has now become a
powerful tool for green chemistry. Microwave irradiation
has been applied to organic reactions in the absence of
solvent or in the presence of a solid support, such as
clays, alumina, and silica, resulting in shorter reaction
times and better product yields than those obtained using
conventional heating (27–30). In this article, some com-
pounds were also synthesized by microwave irradiation.
with 10% HCl solution (10 mL) to form precipitate. The
precipitate was filtered and washed with appropriate
amount of water. The crude product was recrystallized
from 95% ethanol to afford substituted chalcone 1–12.
Method 2
Substituted acetophenone (5.0 mmol) and benzaldehyde
(5.0 mmol) were dissolved in ethanol (20 mL), and 30%
NaOH (1 mL) was added to the mixture. The reaction
mixture was stirred at room temperature. The completion of
the reaction was checked by TLC. The mixture was neu-
tralized with 10% HCl solution (10 mL) to form precipitate.
The precipitate was filtered and washed with appropriate
amount of water. The crude product was recrystallized
from 95% ethanol to afford substituted chalcone 1–12.
1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (1)
Yield 63.2%. Yellow solid, mp 78.3–80.3 °C; IR(KBr):
1736, 1653, 1549, 1333, 1217, 1151, 1084, 1030, 977,
827, 761/cm. 1HNMR (400 MHz, CDCl3) d 7.98(d, 2H,
J = 8.4 Hz, ph-H), 7.84(d, 1H, J = 15.6 Hz, =CH),
7.66–7.63 (m, 2H, ph-H), 7.50(d, 1H, J = 15.6 Hz, =CH),
7.49(d, 2H, J = 8.4 Hz, ph-H), 7.43–7.42(m, 3H, ph-H);
13CNMR (100 MHz, CDCl3) d 189.2, 145.3, 139.2, 136.5,
134.7, 130.7, 129.9, 129.1, 128.9, 128.5, 121.5.
Methods and Materials
Chemical reagents and instruments
Melting points (m.p.) were determined with WRS-1B melting
point apparatus, and the thermometer was uncorrected.
NMR spectra were recorded on Bruker 400 spectrometer-
sat 25 °C in CDCl3 or DMSO-d6. All chemical shifts (d) are
quoted in parts per million downfield from TMS, and cou-
pling constants (J) are given in hertz. Abbreviations used in
the splitting pattern were as follows: s = singlet, d = dou-
blet, t = triplet, q = quintet, m = multiplet; LC-MS spectra
were recorded using the LCMS-2010A. All reactions were
monitored by TLC (Merck Kieselgel 60 F254, Shanghai,
China), and the spots were visualized under UV light. Infra-
red (IR) spectra were recorded as potassium bromide pellets
on VECTOR 22 spectrometer.
1-(3-hydroxyphenyl)-3-phenylprop-2-en-1-one (2)
Yield 81.2%. Yellow solid, mp 128.6–130.3 °C; IR(KBr):
3208, 1648, 1570, 1445, 1346, 1259, 1188, 1030, 972,
856, 757/cm. 1HNMR (300 MHz, DMSO-d6) d 7.90–7.84
(m, 3H, ph-H), 7.75 (d, 1H, J = 15.6 Hz, =CH), 7.88 (d,
1H, J = 8.1 Hz, ph-H), 7.48–7.46 (m, 4H, ph-H), 7.41 (d,
1H, J = 15.6 Hz, =CH), 7.09 (d, 1H, J = 8.1 Hz, ph-H).
1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (3)
Yield 67.2%. Yellow solid, mp 179.0–181.2 °C; IR(KBr):
1736, 1653, 1549, 1333, 1217, 1151, 1084, 1030, 977,
827, 761/cm. 1HNMR (400 MHz, CDCl3) d 7.98 (d, 2H,
J = 8.4 Hz, ph-H), 7.84 (d, 1H, J = 15.6 Hz, =CH),
7.66–7.63 (m, 2H, ph-H), 7.50 (d, 1H, J = 15.6 Hz, =CH),
7.49 (d, 2H, J = 8.4 Hz, ph-H), 7.43–7.42 (m, 3H, ph-H);
13CNMR (100 MHz, CDCl3) d 189.2, 145.3, 139.2, 136.5,
134.7, 130.7, 129.9, 129.1, 128.9, 128.5, 121.5.
Tyrosinase, L-3, 4-dihydroxyphenylalanine (L-DOPA), and
kojic acid were purchased from Sigma–Aldrich Chemical
Co (Shanghai, China). Other chemicals were purchased
from commercial suppliers and were dried and purified
when necessary.
The general procedures for the synthesis of
substituted chalcone compounds (1–12)
1-(2,4-dihydroxyphenyl)-3-phenylprop-2-en-1-one
(4)
Method 1
Yield 51.3%. Yellow solid, mp 139.0–141.4 °C; IR(KBr):
3237, 1752, 1628, 1549, 1491, 1441, 1358, 1225, 1142,
968, 860, 769/cm. 1HNMR (300 MHz, DMSO-d6) d 8.25
(d, 1H, J = 9.0 Hz, ph-H), 8.03 (d, 1H, J = 15.3 Hz, =CH),
7.93–7.90 (m, 2H, ph-H), 7.85 (d, 1H, J = 15.3 Hz, =CH),
7.49–7.47 (m, 3H, ph-H), 6.48 (d, 1H, J = 9.0 Hz, ph-H),
6.34 (s, 1H, ph-H).
To the mixture of substituted acetophenone (5.0 mmol)
and benzaldehyde (5.0 mmol), ethanol (20 mL) and 30%
NaOH in H2O (1 mL) were added. The reaction mixture
was taken in round-bottomed flask placed in a microwave
reactor and irradiated at 800W for 3 min. Then, this reac-
tion mixture was poured over crushed ice and acidified
40
Chem Biol Drug Des 2013; 82: 39–47

Synthesis and Activity of Chalcone Derivatives
1-(2-hydroxy-4-methoxyphenyl)-3-phenylprop-
2-en-1-one (5)
1-(4-bromophenyl)-3-phenylprop-2-en-1-one (10)
Yield 75.4%. Yellow solid, mp 100.5–102.7 °C; IR(KBr):
1736, 1648, 1557, 1391, 1337, 1213, 1155, 1072, 1030,
977, 827, 761/cm. 1HNMR (400 MHz, CDCl3) d 7.89 (d,
2H, J = 8.8 Hz, ph-H),7.83 (d, 1H, J = 15.6 Hz, =CH),
7.65–7.63 (m, 4H, ph-H), 7.49 (d, 1H, J = 15.6 Hz, =CH),
7.43–7.41 (m, 3H, ph-H); 13CNMR (100 MHz, CDCl3) d
189.3, 145.4, 136.9, 134.6, 131.9, 130.7, 130.0, 129.0,
128.5, 127.9, 121.4.
Yield 40.4%. Yellow solid, mp 87.7–99.0 °C; IR(KBr):
3370, 1595, 1483, 1242, 1163, 1105, 1076, 1022, 964,
1
860, 786/cm. HNMR (300 MHz, DMSO-d6) d 8.32 (d, 1H,
J = 9.0 Hz, ph-H), 8.06 (d, 1H, J = 15.3 Hz, =CH),
7.94–7.91 (m, 2H, ph-H), 7.86 (d, 1H, J = 15.3 Hz, =CH),
7.49–7.48 (m, 3H, ph-H), 6.60(d, 1H, J = 9.0 Hz, ph-H),
6.54 (s, 1H, ph-H), 3.86 (s, 3H, OCH3).
1-(4-methoxyphenyl)-3-phenylprop-2-en-1-one (6)
Yield 77.4%. White solid, mp 104.6–108.6 °C; IR(KBr):
2894, 1736, 1653, 1503, 1342, 1259, 1225, 1184, 1101,
1035, 968, 827, 761/cm. 1HNMR (400 MHz, CDCl3) d
8.06 (d, 2H, J = 8.8 Hz, ph-H),7.83 (d, 1H, J = 15.6 Hz,
=CH), 7.66–7.63 (m, 2H, ph-H), 7.56 (d, 1H, J = 15.6 Hz,
=CH), 7.42–7.41 (m, 3H, ph-H), 7.00 (d, 2H, J = 8.8 Hz,
ph-H), 3.89 (s, 3H, OCH3); 13CNMR (100 MHz, CDCl3) d
188.7, 163.4, 144.0, 135.1, 131.1, 130.8, 130.3, 128.9,
128.3, 121.9, 113.9, 55.5.
1-(3-chlorophenyl)-3-phenylprop-2-en-1-one (11)
Yield 56.6%. Yellow solid, mp 95.9–97.0 °C; IR(KBr):
1771, 1648, 1599, 1325, 1283, 1205, 1047, 972, 856,
757/cm. 1HNMR (300 MHz, DMSO-d6) d 8.22 (s, 1H,
ph-H), 8.15 (d, 1H, J = 8.7 Hz, ph-H), 8.01 (d, 1H,
J = 15.6 Hz, =CH), 7.95–7.93 (m, 2H, ph-H), 7.82 (d, 1H,
J = 15.6 Hz, =CH), 7.75–7.73 (m, 2H, ph-H), 7.49–7.47
(m, 3H, ph-H).
1-(3-bromophenyl)-3-phenylprop-2-en-1-one (12)
Yield 67.2%. Yellow solid, mp 87.0–91.3 °C; IR(KBr):
1736, 1653, 1541, 1337, 1205, 1151, 1068, 981, 856,
757/cm. 1HNMR (400 MHz, CDCl3) d 8.14 (s, 1H, ph-H),
7.95 (d, 1H, J = 8.8 Hz, ph-H),7.84 (d, 1H, J = 15.6 Hz,
=CH), 7.72 (d, 1H, J = 8.8 Hz, ph-H), 7.66–7.64 (m, 2H,
ph-H), 7.48 (d, 1H, J = 15.6 Hz, =CH), 7.43–7.36 (m, 4H,
ph-H); 13CNMR (100 MHz, CDCl3) d 189.0, 145.7, 140.0,
135.6, 134.6, 131.5, 130.8, 130.2, 129.0, 128.6, 127.0,
123.0, 121.4.
1-(4-(methoxymethoxy)phenyl)-3-phenylprop-
2-en-1-one (7)
Yield 57.2%. Yellow solid; IR(KBr): 2897, 2843,
1731,1636, 1512, 1371, 1342, 1221, 1134, 1068, 1010,
956, 840, 790/cm. 1HNMR (400 MHz, CDCl3) d 8.04 (d,
2H, J = 8.8 Hz, ph-H),7.83 (d, 1H, J = 15.6 Hz, =CH),
7.66–7.63 (m, 2H, ph-H), 7.56 (d, 1H, J = 15.6 Hz, =CH),
7.42–7.41 (m, 3H, ph-H), 7.12 (d, 2H, J = 8.8 Hz, ph-H),
5.23 (s, 2H, OCH2), 3.50 (s, 3H, OCH3); 13CNMR
(100 MHz, CDCl3) d 188.9, 161.0, 144.2, 135.1, 132.0,
130.7, 130.4, 128.9, 128.4, 121.9, 115.9, 94.1, 56.3.
The general procedures for the synthesis of
substituted chalcone thiosemicarbazide Schiff
base compounds (13–21)
1-(4-fluorophenyl)-3-phenylprop-2-en-1-one (8)
Yield 56.5%. Yellow solid, mp 76.2–78.3 °C; IR(KBr):
1742, 1644, 1536, 1333, 1217, 1151, 1035, 981, 836,
Method 1
To the mixture of substituted chalcone (5.0 mmol) and thi-
osemicarbazide (5.0 mmol), ethanol (20 mL) and catalytic
amount of acetic acid were added. The reaction mixture
was taken in round-bottomed flask placed in a microwave
reactor and irradiated at 800W for 8 min. The reaction
mixture was allowed to cool to room temperature, and
precipitate was formed. The precipitate was filtered and
washed with appropriate amount of ethanol. The crude
product was recrystallized from 95% ethanol to afford
substituted chalcone Schiff base 13–21.
761/cm. 1HNMR (400 MHz, CDCl3)
J = 7.6 Hz, ph-H), 7.85 (d,
d
8.10 (d, 2H,
1H,
J = 15.6 Hz,
=CH),7.65–7.62 (m, 2H, ph-H), 7.66–7.64 (m, 2H, ph-H),
7.54 (d, 1H, J = 15.6 Hz, =CH), 7.44(d, 2H, J = 7.6 Hz,
ph-H),7.21–7.17(m, 4H, ph-H); 13CNMR (100 MHz, CDCl3)
d 188.9, 145.1, 134.8, 134.6, 131.2, 131.0, 130.6, 129.0,
128.5, 121.6, 115.8, 115.6.
1-(4-chlorophenyl)-3-phenylprop-2-en-1-one (9)
Yield 50.9%. Yellow solid, mp 97.1–98.3 °C; IR(KBr):
1736, 1653, 1549, 1333, 1217, 1151, 1084, 1030, 977,
827, 761/cm. 1HNMR (400 MHz, CDCl3) d 7.98 (d, 2H,
J = 8.4 Hz, ph-H), 7.84 (d, 1H, J = 15.6 Hz, =CH),
7.66–7.63 (m, 2H, ph-H), 7.50 (d, 1H, J = 15.6 Hz,
=CH), 7.49 (d, 2H, J = 8.4 Hz, ph-H), 7.43–7.42 (m, 3H,
d 189.2, 145.3,
139.2, 136.5, 134.7, 130.7, 129.9, 129.1, 128.9, 128.5,
121.5.
Method 2
To the mixture of substituted chalcone (5.0 mmol) and thi-
osemicarbazide (5.0 mmol), ethanol (20 mL) and catalytic
amount of acetic acid were added. The reaction mixture
was refluxed at 80 °C over a silicone oil bath for 6–24 h.
The completion of the reaction was checked by TLC. The
reaction mixture was allowed to cool to room temperature,
and precipitate was formed. The precipitate was filtered
ph-H); 13CNMR (100 MHz, CDCl3)
Chem Biol Drug Des 2013; 82: 39–47
41

Liu et al.
and washed with appropriate amount of ethanol. The
crude product was recrystallized from 95% ethanol to
afford substituted chalcone Schiff base 13–21.
1H, J = 15.6 Hz, =CH), 7.46–7.37 (m, 3H, ph-H), 6.60 (d,
1H, J = 15.6 Hz, =CH), 6.59–6.54 (m, 2H, ph-H), 3.77 (s,
3H, OCH3). MS (ESI): m/z (100%) 326 (M-1).
1-(-1-(2-hydroxyphenyl)-3-phenylallylidene)
thiosemicarbazide (13)
1-(-1-(4-methoxyphenyl)-3-phenylallylidene)
thiosemicarbazide (18)
Yield 42.9%. Yellow solid, mp 201.3–207.6 °C; IR(KBr):
3395, 1648, 1557, 1499, 1283, 1217, 1113, 1076, 985,
881, 761/cm. 1HNMR (300 MHz, DMSO-d6) d 10.44 (s,
1H, NH), 8.64 (s, 1H, NH), 8.16 (s, 1H, NH), 8.13 (d, 1H,
J = 9.0 Hz, ph-H), 7.96 (d, 1H, J = 15.6 Hz, =CH), 7.70
(d, 2H, J = 8.7 Hz, ph-H), 7.43–7.41 (m, 4H, ph-H), 7.28
(d, 1H, J = 15.6 Hz, =CH), 6.46 (d, 1H, J = 9.0 Hz, ph-H),
6.34 (s, 1H, ph-H).
Yield 28.7%. Yellow solid, mp 158.6–165.7 °C; IR(KBr):
1595, 1483, 1242, 1163, 1105, 1076, 1022, 964, 860,
786/cm. 1HNMR (300 MHz, DMSO-d6) d 10.96 (s, 1H,
NH), 9.92 (s, 1H, NH), 8.53 (s, 1H, NH), 7.77 (d, 1H,
J = 15.6 Hz, =CH), 7.74–7.72 (m, 2H, ph-H), 7.61 (d, 2H,
J = 8.7 Hz, ph-H),7.41 (d, 1H, J = 15.6 Hz, =CH),
7.39–7.36 (m, 3H, ph-H), 7.01 (d, 2H, J = 8.7 Hz, ph-H),
3.81 (s, 3H, OCH3). MS (ESI): m/z(100%) 312 (M + 1).
1-(-1-(3-hydroxyphenyl)-3-phenylallylidene)
1-(-1-(4-fluorophenyl)-3-phenylallylidene)
thiosemicarbazide (14)
thiosemicarbazide (19)
Yield 63.1%. Yellow solid, mp 178.7–180.1 °C; IR(KBr):
3378, 1615, 1570, 1487, 1433, 1234, 1159, 1076, 968,
827, 757/cm. 1HNMR (300 MHz, DMSO-d6) d 11.07 (s,
1H, NH), 9.54 (s, 1H, NH), 8.46 (s, 1H, NH), 7.79 (d, 1H,
J = 15.6 Hz, =CH), 7.75–7.72 (m, 3H, ph-H), 7.47
(d, 1H, J = 8.1 Hz, ph-H), 7.41–7.37 (m, 4H, ph-H), 7.24
(d, 1H, J = 15.6 Hz, =CH), 7.01 (d, 1H, J = 8.1 Hz, ph-H).
Yield 32.9%. Yellow solid, mp 168.9–171.0 °C; IR(KBr):
1603, 1487, 1333, 1279, 1221, 1159, 1122, 1075, 964,
873, 761/cm. 1HNMR (400 MHz, DMSO-d6) d11.09 (s,
1H, NH), 8.86 (s, 1H, NH), 8.32 (s, 1H, NH), 7.82 (d, 1H,
J = 15.6 Hz, =CH), 7.75 (d, 2H, J = 7.5 Hz, ph-H),
7.71–7.68 (m, 2H, ph-H), 7.49–7.47 (m, 2H, ph-H), 7.47
(d, 1H, J = 15.6 Hz, =CH), 7.39–7.25 (m, 3H, ph-H);
13CNMR (100 MHz, DMSO-d6) d 196.4, 179.4, 147.6,
140.1, 136.3, 131.9, 129.8, 129.1, 128.5, 121.6, 119.2,
115.7. MS (ESI): m/z (100%) 300 (M + 1).
1-(-1-(4-hydroxyphenyl)-3-phenylallylidene)
thiosemicarbazide (15)
Yield 37.9%. Yellow solid, mp 200.1–204.5 °C; IR(KBr):
3341, 1603, 1487, 1333, 1279, 1221, 1159, 1122, 964,
873, 836, 761/cm. 1HNMR (300 MHz, DMSO-d6) d 10.90
(s, 1H, NH), 9.76 (s, 1H, NH), 8.58 (s, 1H, NH), 8.02 (d,
2H, J = 8.4 Hz, ph-H),7.75 (d, 1H, J = 15.0 Hz, =CH),
7.74–7.20 (m, 2H, ph-H), 7.49 (d, 1H, J = 15.0 Hz, =CH),
7.49–7.47 (m, 3H, ph-H), 7.00 (d, 2H, J = 8.4 Hz, ph-H).
1-(-1-(3-chlorophenyl)-3-phenylallylidene)
thiosemicarbazide (20)
Yield 45.8%. Yellow solid, mp 175.2–178.3 °C; IR(KBr):
1595, 1565, 1466, 1321, 1279, 1134, 1072, 968, 865,
836, 753/cm. 1HNMR (300 MHz, DMSO-d6) d 11.13 (s,
1H, NH), 9.12 (s, 1H, NH), 8.57 (s, 1H, NH), 8.37 (s, 1H,
ph-H), 8.12 (d, 1H, J = 8.7 Hz, ph-H), 8.09 (d, 1H,
J = 15.6 Hz, =CH), 7.78–7.74 (m, 2H, ph-H), 7.56 (d, 1H,
J = 16.0 Hz, =CH), 7.51–7.49 (m, 2H, ph-H), 7.46–7.42
(m, 3H, ph-H).
1-(-1-(2,4-dihydroxyphenyl)-3-phenylallylidene)
thiosemicarbazide (16)
Yield 32.1%. Yellow solid, mp 185.1–188.9 °C; IR(KBr):
3361, 1599, 1516, 1495, 1445, 1288, 1230, 1163, 1101,
997, 831, 699/cm. 1HNMR (300 MHz, DMSO-d6) d 10.28
(s, 1H, NH), 8.34(s, 1H, NH), 8.33 (d, 1H, J = 8.4 Hz, ph-
H), 8.11(s, 1H, NH), 7.57 (d, 2H, J = 8.4 Hz, ph-H), 7.47
(d, 1H, J = 15.6 Hz, =CH), 7.42–7.40 (m, 2H, ph-H),
7.34–7.30 (m, 2H, ph-H), 7.01 (d, 1H, J = 15.6 Hz, =CH),
7.01–6.96 (m, 2H, ph-H). MS (ESI): m/z(100%) 312 (M-1).
1-(-1-(3-bromophenyl)-3-phenylallylidene)
thiosemicarbazide (21)
Yield 39.4%. Yellow solid, mp 159.7–162.2 °C; IR(KBr):
1595, 1491, 1445, 1317, 1292, 1205, 1122, 1064, 968,
873, 757/cm. 1HNMR (300 MHz, DMSO-d6) d 11.16 (s,
1H, NH), 8.40 (s, 1H, NH), 8.14 (s, 1H, NH), 7.99 (s, 1H,
ph-H), 7.88 (d, 1H, J = 8.7 Hz, ph-H),7.80 (d, 1H,
J = 15.9 Hz, =CH), 7.60–7.30 (m, 2H, ph-H), 7.64 (d, 1H,
J = 8.7 Hz, ph-H), 7.44–7.40 (m, 4H, ph-H), 6.81 (d, 1H,
J = 15.9 Hz, =CH).
1-(-1-(2-hydroxy-4-methoxyphenyl)-3-
phenylallylidene)thiosemicarbazide (17)
Yield 17.5%. Yellow solid, mp 116.5–122.6 °C; IR(KBr):
3407, 1599, 1487, 1433, 1259, 1196, 1155, 1097, 831,
695/cm. 1HNMR (300 MHz, DMSO-d6) d 10.34 (s, 1H,
NH), 8.24 (s, 1H, NH), 8.21 (d, 1H, J = 8.7 Hz, ph-H),
8.03 (s, 1H, NH), 7.56 (d, 2H, J = 6.9 Hz, ph-H), 7.47 (d,
Assay of the diphenolase activity
The spectrophotometric assay for tyrosinase was per-
formed according to the method reported by our groups
42
Chem Biol Drug Des 2013; 82: 39–47

Synthesis and Activity of Chalcone Derivatives
with some slight modifications (14,25). Briefly, all the syn-
thesized compounds were screened for the diphenolase
inhibitory activity of tyrosinase using L-DOPA as substrate.
All the compounds were dissolved in DMSO. The final
concentration of DMSO in the test solution was 2.0%.
Phosphate buffer, pH 6.8, was used to dilute the DMSO
stock solution of test compounds. Thirty units of mush-
room tyrosinase (0.5 mg/mL) were first pre-incubated with
the compounds, 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
monitored by measuring the change in absorbance at
475 nm of formation of the DOPA chrome for 1 min. The
measurement was performed in triplicate for each concen-
tration and averaged before further calculation. IC50 value,
a concentration giving 50% inhibition of tyrosinase activity,
was determined by interpolation of the dose–response
curves. The percent of inhibition of tyrosinase reaction was
calculated as following:
afford substituted chalcone. Chalcone Schiff bases were
synthesized from corresponding chalcone and thiosemic-
arbazide in present of acetic acid as catalyst. The rapid,
highly yielding, and eco-friendly microwave-assisted
organic syntheses were also used to synthesize substi-
tuted chalcones and their Schiff bases. The majority of
microwave-assisted reactions were conducted in polar,
highly microwave absorbing solvent. The yields of these
compounds are from moderate to good, and all the target
compounds were characterized by IR, NMR. Some target
compounds were also characterized by MS.
Tyrosinase inhibitory activity
For evaluating the tyrosinase inhibitory activity, all the syn-
thesized compounds were subjected to tyrosinase inhibi-
tion assay with L-DOPA as substrate, according to the
method reported by our groups with some slight modifica-
tions. The tyrosinase inhibitory activity of kojic acid
(5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one) was ever
reported; therefore, it was selected as comparing sub-
stance. Figure 1 showed that the remaining enzyme activ-
ity rapidly decreased with the increasing concentrations of
compound 18. The IC50 values of substituted chalcones
and their Schiff bases against tyrosinase were summarized
in Table 1, and IC50 values of all these compounds were
determined by interpolation of the dose–response curves
and given as means of three experiments.
Inhibition rateð%Þ ¼ ½ðB ꢀ SÞ=Bꢁ ꢂ 100
Here, the B and S are the absorbances for the blank and
samples. Kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-
pyran-4-one) was used as reference standard inhibitor for
comparison.
Results and Discussion
Our results showed that most of compounds exhibited
potent inhibition on mushroom tyrosinase with IC50 values
ranged from 0.274 to 148.41 lM. Specifically, compounds
14–21 bearing thiosemicarbazide showed more potent
inhibitory activities than the other compounds. In addition,
compounds 16–19 demonstrated more potent inhibitory
Synthesis
In the present investigation, substituted chalcones have
been prepared by the Claisen–Schmidt condensation of
substituted acetophenone and benzaldehyde (Scheme 1).
The crude product was recrystallized from 95% ethanol to
Scheme 1: Synthesis of the substituted chalcones and their thiosemicarbazide Schiff base.
Chem Biol Drug Des 2013; 82: 39–47
43

Liu et al.
200 lM, but it still showed higher activity against tyrosinase
than 1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (1).
The results showed substituted chalcone Schiff bases
exhibited more potent inhibitory activities than correspond-
ing substituted chalcones. Most of the chalcone analogs
showed low activities against tyrosinase. The results
showed that the group of thiosemicarbazide played impor-
tant role in increasing activities against tyrosinase. These
may be related to the structure of tyrosinase contained a
type-3 copper center with a coupled dinuclear copper
active site in the catalytic core. Tyrosinase inhibition of
compounds 14–21 depended on the competency of the
sulfur atom to chelate with the dicopper nucleus in the
active site, and tyrosinase would lose its catalyzing ability
after forming complex (31).
From the tyrosinase inhibitory activities of compounds 1–3,
although these compounds show low activities, we can
find 1-(2-hydroxyphenyl)-3- phenylprop-2-en-1-one (3)
exhibited more potent inhibitory activity than the other
compounds. This suggested that hydroxyl group at the
position of benzene ring might play an important role in
determining this kind of compounds inhibitory activities.
This may be related to the steric hindrance. Comparing to
the tyrosinase inhibitory activities of compounds 1–4, com-
pound 4 exhibited the most potent tyrosinase inhibitory
activity with IC50 value of 110.22 lM. This result showed
that the number of hydroxyl group may be an important
effecting factor of inhibitory activities. However, from the
Table 1, we can find that compound 6 (CLogP = 3.84)
Figure 1: Effect of compound 18 on the diphenolase activity of
mushroom tyrosinase for the catalysis of L-DOPA at 25 °C.
Table 1: Tyrosinase inhibitory activities of the synthesized com-
pounds
Compounds
CLogPa
IC50 (lM)b
Percent of inhibitionc
1
3.96
3.50
3.50
3.45
4.03
3.84
3.35
3.84
4.41
4.56
4.41
4.56
4.92
4.92
4.92
4.42
5.00
5.28
5.29
5.86
6.01
11.2
2
18.35
29.06
75.2
3
4
110.22
141.80
5
31.5
6
54.11
13.26
40.2
40.25
37.22
65.91
NAd
47.97
59.14
71.61
95.49
95.58
98.64
96.59
85.68
76.78
11.2
7
exhibited higher activity than the compound
3
8
(CLogP = 3.50). This suggested that methoxyl group at
the 4-position of benzene ring might be beneficial to
improve this kind of compounds inhibitory activities. This
may be related to the lipophilicity. 1-(3-bromophenyl)-3-
phenylprop-2-en-1-one (12) showed the most potent
inhibitory activity in all the halogen substituted chalcone.
Comparing the inhibitory activities of compounds 8–10
with halogen atom at the 4th position of benzene ring and
compounds 11–12 with halogen atom at the 3th position
of benzene ring, with the increase in the radius of halo-
gen atom, the inhibitory activities decreased gradually.
Comparing to the inhibitory activities of all the thiosemicar-
bazide Schiff bases, 1-(-1-(4-methoxyphenyl)-3-phenylally-
lidene)thiosemicarbazide (18) exhibited the most potent
tyrosinase inhibitory activity with IC50 value of 0.274 lM.
From the tyrosinase inhibitory activities of compounds
13–18, the same result can be also obtained, hydroxyl
group at the position of benzene ring and the number
hydroxyl group might play an important role in determining
this kind of Schiff base compounds inhibitory activities,
and methoxyl group at the 4th position of benzene ring
might be beneficial to improve this kind of Schiff base
compounds inhibitory activities. Comparing to the tyrosi-
nase inhibitory activities of compounds 19–21, the result
9
10
11
12
117.18
13
14
148.41
77.87
19.79
18.81
0.274
7.56
23.91
52.68
23
15
16
17
18
19
20
21
Kojic acid
aValue of CLogP was obtained by ChemBioDraw Ultra 12.0.
bValues were determined from logarithmic concentration–inhibition
curves (at least seven points) and are given as means of three
experiments.
cPercent of inhibition of tyrosinase reaction at the 200 lM.
dNot active at 200 lM concentration.
activity than the reference standard inhibitor kojic acid,
and
1-(-1-(4-methoxyphenyl)-3-phenylallylidene)thiose-
micarbazide (18) exhibited the most potent tyrosinase
inhibitory activity with IC50 value of 0.274 lM. 1-(-1-
(2-hydroxyphenyl)-3-phenylallylidene)thiosemicarbazide (13)
bearing thiosemicarbazide exhibited low tyrosinase inhibi-
tory activity, and the percent of inhibitory is 47.97% at the
showed
that
1-(-1-(4-fluorophenyl)-3-phenylallylidene)
thiosemicarbazide (19) have better activity than the other
two compounds; with the increase in the radius of halogen
44
Chem Biol Drug Des 2013; 82: 39–47

Synthesis and Activity of Chalcone Derivatives
atom, the inhibitory activities also decreased gradually.
According to the ClogP values of compounds 19–21, the
inhibitory activities of these compounds may be also
related to the lipophilicity.
Inhibitory mechanism
The inhibitory mechanisms of the selected 1-(-1-(2,4-di-
hydroxyphenyl)- 3-phenylallylidene)thiosemicarbazide (16)
and 1-(-1- (4-methoxyphenyl)-3-phenylallylidene)thiosemic-
arbazide (18) on mushroom tyrosinase for the oxidation of
L-DOPA were determined (32). Figures 2 and 3 showed
the relationship between enzyme activity and concentra-
tion in the presence of different concentrations of the
above-mentioned compounds. The results displayed that
the plots of V versus [E] gave a family of parallel straight
lines with the same slopes. These results demonstrated
that the inhibitory effects of compounds 16, 18 on the
tyrosinase were irreversible, suggesting that thiosemicar-
bazide Schiff bases of substituted chalcone compounds
effectively inhibited the enzyme by binding to its binuclear
active site irreversibly. The result may be related to the
structure of tyrosinase; within the structure, there are two
copper ions in the active center of tyrosinase and a lipo-
philic long-narrow gorge near to the active center. Com-
pounds 16 or 18 could exhibit strong affinity for copper
ions in the active center and form a reversible non-cova-
lent complex with the tyrosinase, and this then reacts to
produce the covalently modified ‘dead-end complex’
(13,33–35).
Figure 3: The effect of concentrations of tyrosinase on its activity
for the catalysis of L-DOPA at different concentration of
compound 18. The concentrations of compound 18 for curves
1–4 are 0, 0.2, 0.3 and 0.5 lM, respectively.
inhibitors of tyrosinase for defining their structure–activity
relationship (SAR) studies. As a result, 1-(-1-(4-methoxyphe-
nyl)-3-phenylallylidene)thiosemicarbazide (18) proved the
best tyrosinase inhibitor among synthesized compounds.
Preliminary structure activity relationships (SARs) analysis
indicated that (i) thiosemicarbazide moiety might play an
important role in determining their inhibitory activities,
because of the sulfur atom could chelate with the dicopper
nucleus in the active site, and tyrosinase would lose its cata-
lyzing ability after forming complex; (ii) methoxyl group at the
4th position of benzene ring might be beneficial to improve
the inhibitory activities of tyrosinase; (iii) hydroxyl group at
the position of benzene ring and the number hydroxyl group
might affect inhibitory activity. The inhibition mechanism
analysis of 1-(-1-(2,4-dihydroxyphenyl) -3-phenylallylidene)
thiosemicarbazide (16) and 1-(-1- (4-methoxyphenyl)-3-
phenylallylidene)thiosemicarbazide (18) demonstrated that
the inhibitory effects of the two compounds on the tyrosi-
nase were irreversible.
Conclusions
In conclusion, we have design, synthesized, and tested a
series of chalcones and their thiosemicarbazones as
Acknowledgments
This work was financially supported by the Foundation of
Hunan Provincial Science
& Technology Department,
China (2012FJ4350), the Key Subject Foundation of Sha-
oyang University (2008XK201) and the Hunan Province
University Students Research Fund (Xiang Jiao Tong
[2010]244-320).
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