Biological evaluation of coumarin derivatives as mushroom tyrosinase inhibitors

Article abstract of DOI:10.1016/j.foodchem.2012.07.055

A series of coumarin derivatives were synthesised and their inhibitory effects on the diphenolase activity of mushroom tyrosinase were evaluated. The results showed that some of the synthesised compounds exhibited significant inhibitory activities. Especially, 2-(1-(coumarin-3-yl)ethylidene) hydrazinecarbothioamide bearing thiose-micarbazide group exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 3.44 μM. The inhibition mechanism analysis of 2-(1-(coumarin-3-yl)-ethylidene) hydrazinecarbothioamide and 2-(1-(6-chlorocoumarin-3-yl)ethylidene)- hydrazinecarbothioamide demonstrated that the inhibitory effects of the compounds on the tyrosinase were irreversible. Preliminary structure activity relationships' (SARs) analysis suggested that further development of such compounds might be of interest.

Full text of DOI:10.1016/j.foodchem.2012.07.055

Food Chemistry 135 (2012) 2872–2878
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Biological evaluation of coumarin derivatives as mushroom tyrosinase inhibitors
a,
⇑
a
b
a
a
a
a
Jinbing Liu , Fengyan Wu , Lingjuan Chen , Liangzhong Zhao , Zibing Zhao , Min Wang , Sulan Lei
a
Department of Biology and Chemical Engineering, Shaoyang University, Shao Shui Xi Road, Shaoyang 422100, PR China
College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan 410004, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 April 2012
Received in revised form 23 May 2012
Accepted 3 July 2012
Available online 24 July 2012
A series of coumarin derivatives were synthesised and their inhibitory effects on the diphenolase activity of
mushroom tyrosinase were evaluated. The results showed that some of the synthesised compounds exhib-
ited significant inhibitory activities. Especially, 2-(1-(coumarin-3-yl)ethylidene)hydrazinecarbothioamide
bearing thiose-micarbazide group exhibited the most potent tyrosinase inhibitory activity with IC50 value
of 3.44 lM. The inhibition mechanism analysis of 2-(1-(coumarin-3-yl)-ethylidene)hydrazinecarbothioa-
mide and 2-(1-(6-chlorocoumarin-3-yl)ethylidene)-hydrazinecarbothioamide demonstrated that the
inhibitory effects of the compounds on the tyrosinase were irreversible. Preliminary structure activity rela-
tionships’ (SARs) analysis suggested that further development of such compounds might be of interest.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Coumarin derivatives
Tyrosinase inhibitors
Inhibition mechanism
1
. Introduction
to Parkinson’s and other neurodegenerative diseases (Zhu et al.,
2
011). In insects, tyrosinase is uniquely associated with three dif-
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase,
ferent biochemical processes, including sclerotisation of cuticle,
defensive encapsulation and melanisation of foreign organism,
and wound healing (Ashida & Brey, 1995). These processes provide
potential targets for developing safer and effective tyrosinase
inhibitors as insecticides and ultimately 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, arbutin, tropolone, and 1-
phenyl-2-thiourea (PTU) (Fig. 1) are used as therapeutic agents
and cosmetic products (Battaini, Monzani, Casella, Santagostini, &
Pagliarin, 2000).
Coumarin derivatives are an important class of compounds,
widely present in plants, including edible vegetables and fruits
(Curini, Cravotto, Epifano, & Giannone, 2006; Rai et al., 2010). Cou-
marin derivatives are of great interest due to their diverse struc-
tural features and versatile biological properties, such as anti-
inflammatory, antioxidant, vasorelaxant, cytotoxic, anti-HIV, anti-
tubercular and antimicrobial (Belluti et al., 2010; Chimenti, Bizzar-
ri, Bolasco, & Secci, 2010; Kostova, 2006; Neyts et al., 2009; Ostrov
et al., 2007; Roussaki, Kontogiorgis, Hadjipavlou-Litina, Hamilakis,
et al., 2010; Upadhyay & Mishra, 2010). In particular, their antibac-
terial, antifungal and anticancer activities make the compounds
attractive for further derivatisation and screening as novel thera-
peutic agents (Khode, Maddi, Aragade, Palkar, & Ronad, 2009).
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 phen-
ylthioureas, alkylthioureas and 1,3-bis-(5-methanesulfonylbu-
EC 1.14.18.1, syn. polyphenol oxidase), also known as polyphenol
oxidase (PPO), is a copper-containing monooxygenase that is
widely distributed in microorganisms, animals, and plants (Song
et al., 2006). Tyrosinase catalyses by involving molecular oxygen
in two distinct reactions: in the hydroxylation of monophenols
too-diphenols (monophenolase) and in the oxidation of o-diphe-
nols to o-quinones (diphenolase) (Chen, Liu, & Huang, 2003). Due
to the high reactivity, quinines could polymerise spontaneously
to form high molecular weight brown-pigments (melanins) or re-
act with amino acids and proteins to enhance brown colour of
the pigment produced (Matsuura, Ukeda, & Sawamura, 2006).
Hyperpigmentations, such as senile lentigo, melasma, freckles, and
pigmented acne scars are of particular concern to women. The
treatment usually involves the use of medicines or medicinal cos-
metics containing depigmenting agents or skin whitening agents
(
Tripathi et al., 1992). In clinical usage, tyrosinase inhibitors are
used for treatments of dermatological disorders related to melanin
hyperaccumulation and are essential in cosmetics for depigmenta-
tion (Schallreuter et al., 2009; Wood et al., 2009). For example, age
spots and freckle were caused by the accumulation of an excessive
level of epidermal pigmentation (Thanigaimalai et al., 2010).
Previous reports confirmed that tyrosinase was one of the main
causes of most fruits and vegetables quality loss during post har-
vest handling and processing, leading to faster degradation and
shorter shelf life (Yi et al., 2010). Tyrosinase has also been linked
⇑
Corresponding author. Tel./fax: +86 739 5431768.
E-mail address: xtliujb@yahoo.com.cn (J. Liu).
0
308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.07.055

J. Liu et al. / Food Chemistry 135 (2012) 2872–2878
2873
Fig. 1. Chemical structure of known tyrosinase inhibitors.
tyl)thiourea, displayed weak or moderate tyrosinase inhibitory
activity (Ley & Bertram, 2001). More recently, our investigations
also demonstrated that thiosemicarbazide derivatives exhibited
potent inhibitory activities against mushroom tyrosinase (Liu, Yi,
Wan, Ma, & Song, 2008). During recent years, extensive studies
on the pharmacology of coumarin derivatives have been reported,
but the tyrosinase inhibitor activities of this kind of compounds
have hardly ever appeared in the literature. Stimulated by these re-
sults, in the present investigation, we synthesised a series of cou-
marin derivatives bearing thiosemicarbazide moieties or ester
groups, their inhibitory activities against mushroom tyrosinase
were evaluated using kojic acid as a comparing substance. Mean-
while, the structure–activity relationships of these compounds
were also primarily discussed. The aim of the present study was
the discovery of safe and efficient compounds as food additives
or food preservatives, which can offer a clue to the design and syn-
thesis of novel tyrosinase inhibitors.
(1C), 159.17 (1C), 155.27 (1C), 147.39 (1C), 134.33 (1C), 130.17
(1C), 124.92 (1C), 124.48 (1C), 118.20 (1C), 116.63 (1C), 30.49 (1C).
2.2.3. 6-Chloro-3-acetylcoumarin (2)
Yield 89.87%. Yellow solid, mp 213.1–213.9 °C. IR (KBr): 2946,
1726, 1611, 1469, 1412, 1355, 1278, 1134, 1082, 970, 829,
ꢀ1 1
765 cm
3
. H NMR (400 MHz, CDCl ): d 8.40(s, 1H, C@CH), 7.63
(dd, H, J = 8.5 Hz, 2.5 Hz, Ph-H), 7.60 (d, 1H, J = 2.5 Hz, Ph-H), 7.33
1
3
(d, 1H, J = 8.5 Hz, Ph-H), 2.72 (s, 3H, CH ); C NMR (100 MHz,
3
CDCl ) d 195.41 (1C), 158.57 (1C), 152.23 (1C), 145.39 (1C),
3
141.08 (1C), 132.12 (1C), 128.65 (1C), 127.56 (1C), 126.94 (1C),
118.02 (1C), 16.14 (1C).
2.2.4. 6-Bromo-3-acetylcoumarin (3)
Yield 94.10%. Yellow solid, mp 229.1–229.9 °C. IR (KBr): 3039,
1
7
732, 1675, 1608, 1550, 1415, 1351, 1233, 1201, 1063, 980, 832,
ꢀ1 1
68 cm
3
. H NMR (400 MHz, CDCl ): d 8.40 (s, 1H, C@CH), 7.78
(
(
dd, H, J = 8.5 Hz, 2.5 Hz, Ph-H), 7.74 (d, 1H, J = 2.5 Hz, Ph-H), 7.27
1
3
3
d, 1H, J = 8.5 Hz, Ph-H), 2.72 (s, 3H, CH ); C NMR (100 MHz,
2
. Materials and methods
CDCl
3
) d 195.41 (1C), 158.62 (1C), 152.71 (1C), 145.32 (1C),
1
1
40.77 (1C), 134.69 (1C), 131.12 (1C), 126.65 (1C), 120.88 (1C),
18.67 (1C), 116.18 (1C), 16.16 (1C).
2.1. Chemical reagents and instruments
Melting points (m.p.) were determined with WRS-1B melting
2
.3. The general procedure for the synthesis of substituted 2-(1-
point apparatus and the thermometer was uncorrected. NMR spec-
tra were recorded on Bruker 400 spectrometersat 25 °C in CDCl or
DMSO-d . All chemical shifts (d) are quoted in parts per million
(
coumarin-3-yl) ethylidene)hydrazinecarbothioamide compound
3
6
2.3.1. General
downfield from TMS and coupling constants (J) are given in hertz.
Abbreviations used in the splitting pattern were an follows: s = sin-
glet, d = doublet, t = triplet, q = quintet, m = multiplet, LC–MS spec-
tra were recorded using the LCMS-2010A. All reactions were
monitored by TLC (Merck Kieselgel 60 F254) and the spots were
visualised under UV light. Infrared (IR) spectra were recorded as
potassium bromide pellets on VECTOR 22 spectrometer.
The appropriate substituted 3-acetylcoumarin (10 mmol) was
dissolved in anhydrous ethanol (20 ml), thiosemicarbazide
10 mmol) and acetic acid (0.5 ml) were added to the above sys-
(
tem. The reaction mixture was refluxed for 6 h. The reaction was
monitored by TLC. After completion of the reaction, the reaction
mixture was cooled to room temperature. The appearing precipi-
tate was filtered and recrystallised from 95% alcohol to obtain
the corresponding substituted 2-(1-(coumarin-3-yl) ethyli-
dene)hydrazinecarbothioamide compound.
Tyrosinase, L-3, 4-dhydroxyphenylalanine (L-DOPA) and kojic
acid were purchased from Sigma–Aldrich Chemical Co. Other
chemicals were purchased from commercial suppliers and were
dried and purified when necessary.
2.3.2. 2-(1-(Coumarin-3-yl)ethylidene)hydrazinecarbothioamide (4)
Yield 85.38%. Yellow solid, mp 214.2–216.7 °C. IR (KBr): 3385,
2
.2. General procedures for the synthesis of substituted 3-
3
1
(
235, 3155, 1716, 1598, 1499, 1428, 1367, 1290, 1236, 1111,
acetylcoumarin
ꢀ1
1
069, 970, 861, 765 cm
.
H NMR (400 MHz, DMSO-d
), 7.94 (s, 1H,
), 7.76 (d, J = 8.5 Hz, H, Ph-H), 7.65 (t, 1H, J = 8.5 Hz, Ph-H),
.44 (d, 1H, J = 8.5 Hz, Ph-H), 7.38 (t, 1H, J = 8.5 Hz, Ph-H), 2.38 (s,
6
) d 10.61
s, 1H, NH), 8.46 (s, 1H, C@CH), 8.39 (s, 1H, NH
2
2.2.1. General
NH
2
Piperidine (5 mol%) was added to the mixture of substituted sal-
7
3
(
(
icylaldehyde (1 mmol) and ethylacetoacetate (1.1 mmol) in dry
CH CN (10 ml), then the reaction mixture was stirred for about
h at the room temperature. The progress of the reaction was
monitored by TLC. After completion of the reaction, formed precip-
itate was collected by filtration and washed with cold CH CN, then
dried under vacuum to provide the substituted 3-acetylcoumarin.
13
3 6
H, CH ); C NMR (100 MHz, DMSO-d ) d 179.72 (1C), 159.56
3
1C), 154.07 (1C), 146.39 (1C), 142.55 (1C), 132.83 (1C), 129.56
1C), 126.25 (1C), 125.20 (1C), 119.39 (1C), 116.41 (1C), 16.47 (1C).
4
3
2
.3.3. 2-(1-(6-Chlorocoumarin-3-
yl)ethylidene)hydrazinecarbothioamide (5)
Yield 87.68%. Yellow solid, mp 232.0–232.9 °C. IR (KBr): 3417,
3235, 3138, 1732, 1707, 1588, 1502, 1479, 1451, 1428, 1287,
2
.2.2. 3-Acetylcoumarin (1)
Yield 76%. Yellow solid, mp 120.7–122.1 °C. IR (KBr): 3078,
026, 1723, 1675, 1598, 1556, 1441, 1406, 1351, 1297, 1220,
198, 1162, 1098, 967, 762 cm
ꢀ1 1
1233, 1204, 1130, 1092, 954, 925, 871, 832, 772 cm
(400 MHz, DMSO-d ) d 10.49 (s, 1H, NH), 8.44 (s, 1H, NH
(s, 1H, C@CH), 8.44 (s, 1H, NH ), 7.82 (s, H, Ph-H), 7.68 (dd, 1H,
J = 8.5 Hz, 2.5 Hz, Ph-H), 7.47 (d, 1H, J = 8.5 Hz, Ph-H), 2.45 (s, 3H,
.
H NMR
3
1
8
6
2
), 8.41
ꢀ
1 1
.
H NMR (400 MHz, CDCl
3
): d
2
.49 (s, 1H, C@CH), 7.65–7.63 (m, 2H, Ph-H), 7.37–7.32 (m, 2H,
13
13
Ph-H), 2.71 (s, 3H, CH
3
); C NMR (100 MHz, CDCl
3
) d 195.41
3 6
CH ); C NMR (100 MHz, DMSO-d ) d 179.31 (1C), 158.64 (1C),

2
874
J. Liu et al. / Food Chemistry 135 (2012) 2872–2878
1
1
51.96 (1C), 145.37 (1C), 140.63 (1C), 131.79 (1C), 128.35 (1C),
27.63 (1C), 126.82 (1C), 120.40 (1C), 117.97 (1C), 15.87 (1C).
d 8.43 (s, 1H, C@CH), 7.76–7.71 (m, 2H, Ph-H), 7.27 (d, J = 8.8 Hz,
1H, Ph-H), 4.45 (q, J = 7.2 Hz, 2H, CH ), 1.43 (t, J = 7.2 Hz, 3H,
) d 162.59 (1C), 155.89 (1C),
2
1
3
3 3
CH ); C NMR (100 MHz, CDCl
2
.3.4. 2-(1-(6-Bromocoumarin-3-yl)ethylidene)
153.92 (1C), 146.94 (1C), 136.88 (1C), 131.49 (1C), 119.49 (1C),
119.32 (1C), 118.50 (1C), 117.30 (1C), 62.18 (1C), 14.17 (1C).
hydrazinecarbothioamide (6)
Yield 89.15%. Yellow solid, mp 231.9–232.9 °C. IR (KBr): 3404,
3
1
d
222, 3135, 1720, 1588, 1495, 1467, 1422, 1236, 1233, 1204,
2
.4.5. Ethyl 7-hydroxycoumarin-3-carboxylate (10)
Yield 51.04%. Yellow solid, mp 177.5–179.9 °C. IR (KBr): 3462,
ꢀ1 1
101, 1060, 957, 861, 826, 775 cm
) d 10.60 (s, 1H, NH), 8.44 (s, 1H, NH
), 7.80 (s, H, Ph-H), 7.68 (dd, 1H, J = 8.5 Hz, 2.5 Hz, Ph-
.
H NMR (400 MHz, DMSO-
6
2
), 8.40 (s, 1H, C@CH), 7.90
2
1
1
6
3
1
1
991, 1745, 1678, 1614, 1444, 1380, 1297, 1242, 1217, 1140,
(
s, 1H, NH
H), 7.41 (d, 1H, J = 8.5 Hz, Ph-H), 2.24 (s, 3H, CH
100 MHz, DMSO-d ) d 179.30 (1C), 158.58 (1C), 152.63 (1C),
45.32 (1C), 140.71 (1C), 134.53 (1C), 130.93 (1C), 126.73 (1C),
20.80 (1C), 118.35 (1C), 116.14 (1C), 15.86 (1C).
2
ꢀ
1 1
034, 970, 842, 797 cm
H, OH), 8.51 (s, 1H, C@CH), 8.08 (d, J = 8.8 Hz, 1H, Ph-H), 6.87–
.84 (m, 2H, Ph-H), 4.43 (t, J = 7.2 Hz, 2H, CH ), 1.42 (t, J = 7.2 Hz,
) d 162.81 (1C), 156.00 (1C),
3
. H NMR (400 MHz, CDCl ) d 11.06 (s,
13
3
); C NMR
(
1
1
6
2
1
3
3 3
H, CH ); C NMR (100 MHz, CDCl
53.62 (1C), 147.83 (1C), 134.30 (1C), 129.55 (1C), 128.94 (1C),
19.63 (1C), 119.27 (1C), 118.65 (1C), 61.87 (1C), 14.51 (1C).
2
3
.4. The general procedures for the synthesis of substituted coumarin-
-carboxylic acid ester compounds
2.4.6. Coumarin-3-carboxylic acid (11)
2.4.1. General
Yield 69.76%. White solid, mp 203.6–204.3 °C. IR (KBr): 3052,
The substituted coumarin-3-carboxylic acid ethyl esters
1745, 1691, 1614, 1489, 1431, 1371, 1223, 1207, 1037, 983, 826,
ꢀ1 1
(
Scheme 2) were prepared by Knoevenagel reaction. To a mixture
of diethyl malonate (1 mmol) and the appropriate salicylaldehyde
1 mmol) in ethanol (10 ml) was added piperidine (5 mol %) and
800, 768 cm
3
. H NMR (400 MHz, CDCl ) d 12.32 (s, 1H, COOH),
9.00 (s, 1H, C@CH), 7.82–7.76 (m, 2H, Ph-H), 7.51–7.47 (m, 2H,
1
3
(
3
Ph-H); C NMR (100 MHz, CDCl ) d 164.09 (1C), 162.39 (1C),
the reaction mixture was stirred at the room temperature. The pro-
gress of the reaction was monitored by TLC. After completion of the
reaction, the solvent was removed under vacuum and the residue
was purified by chromatography. The obtained substituted couma-
rin-3-carboxylic acid ethyl ester (2 mmol) was dissolved in 10%
NaOH (50 ml), then 3 N HCl (50 ml) was added the mixture. The
suspension was filtered and dried under vacuum to provide substi-
tuted coumarin-3-carboxylic acid. The coumarin-3-carboxylic acid
154.56 (1C), 151.48 (1C), 135.76 (1C), 130.49 (1C), 126.25 (1C),
118.45 (1C), 117.20 (1C), 114.89 (1C).
2.4.7. 6-Chlorocoumarin-3-carboxylic acid (12)
Yield 93.18%. White solid, mp > 300 °C. IR (KBr): 3110, 3046,
1
1
1
7
755, 1726, 1617, 1582, 1479, 1409, 1374, 1342, 1249, 1210,
ꢀ
1 1
150, 1079, 967, 880, 791 cm
6
. H NMR (400 MHz, DMSO-d ) d
3.41 (s, 1H, COOH), 8.70 (s, 1H, C@CH), 8.05 (s, 1H, Ph-H), 7.78–
(
2 mmol) was added to thionyl chloride (30 ml), the reaction mix-
13
.75 (m, 1H, Ph-H),7.50 (d, J = 9.2 Hz, 1H, Ph-H);
C NMR
ture was refluxed for about 2 h, the thionyl chloride was removed
under vacuum. The desired substituted coumarin-3-carbonyl chlo-
ride was obtained. To a mixture of substituted coumarin-3-car-
bonyl chloride (2 mmol) and toluene (20 ml) or ethyl ether
(
6
100 MHz, DMSO-d ) d 164.71 (1C), 164.52 (1C), 158.09 (1C),
1
1
46.88 (1C), 134.18 (1C), 132.57 (1C), 116.47 (1C), 113.25 (1C),
11.48 (1C), 102.19 (1C).
(
20 ml) was added dropwise appropriate alcohol (2 mmol) and
then the reaction mixture was refluxed for about 12 h. The solvent
was removed under vacuum and the residue was purified by
chromatography.
2.4.8. 6-Bromocoumarin-3-carboxylic acid (13)
Yield 67.45%. White solid, mp 208.3–210.3 °C. IR (KBr): 3436,
3049, 1764, 1681, 1611, 1553, 1476, 1415, 1364, 1242, 1204,
ꢀ
1 1
1
140, 1066, 964, 874, 804 cm
13.41 (s, 1H, COOH), 8.70 (s, 1H, C@CH), 8.05 (s, 1H, Ph-H), 7.78–
7.75 (m, 1H, Ph-H),7.50 (d, J = 9.2 Hz, 1H, Ph-H);13
NMR
(100 MHz, DMSO-d ) d 164.71 (1C), 164.52 (1C), 158.09 (1C),
6
. H NMR (400 MHz, DMSO-d ) d
2.4.2. Ethyl coumarin-3-carboxylate (7)
Yield 85.06%. Yellow solid, mp 93.1–96.2 °C. IR (KBr): 3055,
C
2
1
972, 1764, 1697, 1608, 1559, 1479, 1447, 1371, 1297, 1242,
6
ꢀ
1 1
204, 1130, 1028, 983, 916, 797 cm
.
H NMR (400 MHz, CDCl
3
)
146.88 (1C), 134.18 (1C), 132.57 (1C), 116.47 (1C), 113.25 (1C),
111.48 (1C), 102.19 (1C).
d 8.52 (s, 1H, C@CH), 7.66–7.60 (m, 2H, Ph-H), 7.37–7.30 (m, 2H,
Ph-H), 4.44 (q, J = 7.2 Hz, 2H, CH ), 1.43 (t, J = 7.2 Hz, 3H, CH );
) d 162.97 (1C), 157.94 (1C), 155.08
2
3
1
3
C NMR (100 MHz, CDCl
3
2.4.9. 7-Hydroxycoumarin-3-carboxylic acid (14)
(
(
1C), 148.50 (1C), 134.30 (1C), 129.46 (1C), 124.80 (1C), 118.28
1C), 117.82 (1C), 116.71 (1C), 61.91 (1C), 14.15 (1C).
Yield 93.58%. Yellow solid, mp 263.4–265.3 °C. IR (KBr): 3158,
2
1
834, 1726, 1710, 1611, 1572, 1499, 1454, 1396, 1367, 1322,
ꢀ1 1
278, 1220, 1137, 1028, 993, 852, 794 cm
) d 12.79 (s, 1H, COOH),11.22 (s, 1H, OH), 8.69 (s, 1H,
C@CH), 7.76 (d, J = 8.8 Hz, 1H, Ph-H), 6.88 (dd, J = 8.8 Hz, 1H, Ph-
. H NMR (400 MHz,
2.4.3. Ethyl 6-chlorocoumarin-3-carboxylate (8)
DMSO-d
6
Yield 88.00%. White solid, mp 173.7–176.5 °C. IR (KBr): 3068,
2
1
1
972, 1752, 1697, 1614, 1556, 1470, 1364, 1287, 1239, 1207,
1
3
H), 6.77 (d, J = 8.8 Hz, 1H, Ph-H); C NMR (100 MHz, DMSO-d
1
1
6
) d
64.69 (1C), 164.48 (1C), 158.08 (1C), 157.46 (1C), 149.88 (1C),
32.49 (1C), 114.53 (1C), 112.95 (1C), 111.08 (1C), 102.09 (1C).
ꢀ1 1
021, 996, 839, 791 cm
H, C@CH), 7.59 (s, 1H, Ph-H), 7.57 (d, J = 8.4 Hz, 1H, Ph-H), 7.32
), 1.42 (t,
) d 162.62 (1C),
3
. H NMR (400 MHz, CDCl ) d 8.43 (s,
(
d, J = 8.4 Hz, 1H, Ph-H), 4.44 (q, J = 7.2 Hz, 2H, CH
2
1
3
3 3
J = 7.2 Hz, 3H, CH ); C NMR (100 MHz, CDCl
1
1
1
56.00 (1C), 153.44 (1C), 147.08 (1C), 134.11 (1C), 130.10 (1C),
28.41 (1C),119.50 (1C), 118.79 (1C), 118.23 (1C), 62.18 (1C),
4.16 (1C).
2.4.10. Methyl coumarin-3-carboxylate (15)
Yield 89.65%. White solid, mp 156.8–158.7 °C. IR (KBr): 3046,
2946, 1758, 1694, 1614, 1559, 1470, 1431, 1355, 1300, 1242,
ꢀ1
1
1
204, 1085, 1005, 832, 794 cm
3
. H NMR (400 MHz, CDCl ) d
2
.4.4. Ethyl 6-bromocoumarin-3-carboxylate (9)
Yield 67.45%. White solid, mp 171.0–172.3 °C. IR (KBr): 3068,
8.47 (s, 1H, C@CH), 7.61–7.58 (m, 2H, Ph-H), 7.33–7.26 (m, 2H,
Ph-H), 3.96 (s, 3H, CH ); C NMR (100 MHz, CDCl ) d 169.25
3 3
1
3
2
1
972, 1752, 1710, 1614, 1595, 1550, 1467, 1409, 1364, 1284,
(1C), 155.8 (1C), 147.69 (1C), 147.63 (1C), 134.29 (1C), 134.26
(1C), 130.19 (1C), 128.49 (1C),118.75 (1C), 118.26 (1C), 53.04 (1C).
ꢀ1 1
236, 1207, 1018, 989, 858, 791 cm
3
. H NMR (400 MHz, CDCl )

J. Liu et al. / Food Chemistry 135 (2012) 2872–2878
2875
1009, 919, 861, 797 cm 1. 1H NMR (500 MHz, CDCl
ꢀ
2.4.11. Methyl 6-bromocoumarin-3-carboxylate (16)
3
) d 9.87 (s,
Yield 86.50%. Yellow solid, mp 176.8–178.9 °C. IR (KBr): 3039,
1H, OH), 8.49 (s, 1H, C@CH), 7.64–7.59 (m, 2H, Ph-H), 7.35–7.30
(m, 1H, Ph-H), 4.43 (t, J = 6.5 Hz, 2H, CH ), 1.80–1.74 (m, 2H,
CH ), 1.41–1.36 (m, 4H, 2CH ), 0.93 (t, J = 7.0 Hz, 3H, CH
NMR (125 MHz, CDCl ) d 163.15 (1C), 156.59 (1C), 155.18 (1C),
148.33 (1C), 134.23 (1C), 129.46 (1C), 124.77 (1C), 118.51 (1C),
117.90 (1C), 116.76 (1C), 66.08 (1C), 28.26 (1C), 28.01 (1C), 22.43
(1C), 13.96 (1C).
2
1
8
1
(
(
972, 1748, 1697, 1617, 1588, 1556, 1473, 1435, 1377, 1287,
2
ꢀ1
1
13
236, 1204, 999, 880, 816, 794 cm . H NMR (400 MHz, CDCl
3
) d
2
2
3
);
C
.47 (s, 1H, C@CH), 7.76–7.20 (m, 2H, Ph-H), 7.27 (d, J = 8.8 Hz,
3
13
H, Ph-H), 3.97 (s, 3H, CH
3
); C NMR (100 MHz, CDCl
3
) d 163.26
1C), 155.91 (1C), 154.00 (1C), 147.50 (1C), 137.06 (1C), 131.56
1C), 119.29 (1C), 119.13 (1C), 118.54 (1C), 117.40 (1C), 53.06 (1C).
2
.4.12. Methyl 7-hydroxycoumarin-3-carboxylate (17)
Yield 93.58%. White solid, mp 116.2–117.2 °C. IR (KBr): 3055,
2.5. Tyrosinase assay
2
1
956, 1745, 1694, 1614, 1563, 1451, 1361, 1313, 1268, 1246,
217, 1153, 996, 919, 797 cm . H NMR (400 MHz, CDCl ) d 9.96
3
The spectrophotometric assay for tyrosinase was performed
ꢀ1 1
according to the method reported by our groups with some slight
modifications (Liu et al., 2008; Yi et al., 2010). Briefly, all the syn-
thesised compounds were screened for the diphenolase inhibitory
(
s, 1H, OH), 8.55 (s, 1H, C@CH), 7.66–7.60 (m, 2H, Ph-H), 7.36–
13
7
1
1
5
3 3
.32 (m, H, Ph-H), 3.95 (s, 3H, CH ); C NMR (100 MHz, CDCl ) d
63.69 (1C), 156.64 (1C), 155.23 (1C), 149.04 (1C), 134.42 (1C),
29.52 (1C), 124.85 (1C), 118.00 (1C), 117.85 (1C), 116.78 (1C),
2.86 (1C).
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
2.4.13. Pentyl coumarin-3-carboxylate (18)
Yield 53.28%. Yellow solid, mp 133.4–134.5 °C. IR (KBr): 3055,
2
1
956, 2927, 1764, 1691, 1620, 1563, 1470, 1415, 1383, 1351,
2
5 °C. Then the L-DOPA (0.5 mM) was added to the reaction mix-
ꢀ
1 1
294, 1246, 1207, 1085, 989, 829, 788 cm
) d 8.71 (s, 1H, C@CH), 8.08 (d, J = 8.8 Hz, 1H, Ph-H),
.79–7.58 (m, 2H, Ph-H), 7.50 (d, J = 8.8 Hz, 1H, Ph-H), 4.72 (t,
J = 6.8 Hz, 2H, CH ), 2.52–2,50 (m, 2H, CH ), 1.73–1.66 (m, 2H,
CH ), 1.37–1.31 (m, 2H, CH ), 0.92 (t, J = 6.8 Hz, 3H, CH
NMR (100 MHz, DMSO-d ) d 162.95 (1C), 156.05 (1C), 153.65
1C), 147.81 (1C), 134.31 (1C), 129.61 (1C), 128.93 (1C), 119.67
1C), 119.37 (1C), 118.69 (1C), 65.81 (1C), 28.25 (1C), 27.95 (1C),
2.24 (1C), 14.31 (1C).
. H NMR (400 MHz,
ture 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
concentration 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 per-
cent of inhibition of tyrosinase reaction was calculated as
following:
DMSO-d
7
6
2
2
1
3
2
2
3
);
C
6
(
(
2
Inhibition rate ð%Þ ¼ ½ðB—SÞ=Bꢁ ꢂ 100
Here, the B and S are the absorbances for the blank and samples.
Kojic acid was used as reference standard inhibitors for
comparison.
2.4.14. Isopropyl 6-chlorocoumarin-3-carboxylate (19)
Yield 67.45%. Yellow solid, mp 82.1–82.6 °C. IR (KBr): 3046,
2
1
CDCl
982, 2959, 1748, 1604, 1563, 1473, 1457, 1374, 1297, 1249,
ꢀ
1 1
210, 1137, 1101, 1005, 922, 864, 797 cm
) d 8.38 (s, 1H, C@CH), 7.60–7.57 (m, 2H, Ph-H), 7.32 (d,
J = 8.8 Hz, 1H, Ph-H), 5.30–5.24 (m, 1H, CH), 1.40 (d, J = 6.4 Hz,
. H NMR (400 MHz,
3
3
. Results and discussion
1
3
6
1
1
3 3
H, 2CH ); C NMR (100 MHz, CDCl ) d 162.00 (1C), 155.97 (1C),
3
.1. Synthesis
53.41 (1C), 146.56 (1C), 134.09 (1C), 130.08 (1C), 128.47 (1C),
20.00 (1C), 118.91 (1C), 118.23 (1C), 69.99 (1C), 21.76 (2C).
According to the general procedure shown in Scheme 1, substi-
tuted coumarin was synthesised by Knoevenagel condensation. In
presence of piperidine as catalyst, the reaction of substituted sali-
cylaldehyde and ethyl acetoacetate was carried out at room tem-
perature. In present of acetic acid as catalyst, the obtained
substituted coumarin was further converted into it is thiosemicar-
bazide derivative. The yields of these compounds were from mod-
erate to good (Chimenti et al., 2010; Chimenti, Bizzarri, Bolasco,
Secci, & Chimenti, 2007; Liu et al., 2008; Wanare et al., 2010).
According to the general procedure shown in Scheme 2, the substi-
tuted coumarin-3-carboxylate ethyl ester was obtained from
substituted salicylaldehyde and diethyl malonate by the Knoeve-
nagel condensation. The substituted coumarin-3-carboxylate ethyl
ester was further converted into the target compound by the
hydrolysis reaction, chlorination reaction, esterification reaction
in turn (Chimenti et al., 2009; Khode et al., 2009).
2
.4.15. Isopropyl 6-bromocoumarin-3-carboxylate (20)
Yield 69.75%. White solid, mp 217.3–218.8 °C. IR (KBr): 3065,
2
1
CDCl
972, 1745, 1710, 1611, 1563, 1473, 1409, 1367, 1290, 1246,
ꢀ
1 1
207, 1101, 1005, 970, 877, 829, 794 cm
) d 8.37 (s, 1H, C@CH), 7.75–7.70 (m, 2H, Ph-H), 7.27 (d,
J = 8.8 Hz, 1H, Ph-H), 5.30–5.24 (m, 1H, CH), 1.40 (d, J = 6.4 Hz,
. H NMR (400 MHz,
3
1
3
6
1
1
3 3
H, 2CH ); C NMR (100 MHz, CDCl ) d 161.99 (1C), 155.93 (1C),
53.90 (1C), 146.41 (1C), 136.76 (1C), 131.45 (1C), 119.95 (1C),
19.38 (1C), 118.25 (1C), 117.30 (1C), 70.02 (1C), 21.78 (2C).
2
.4.16. Isopropyl 7-hydroxycoumarin-3-carboxylate (21)
Yield 61.36%. White solid, mp 204.7–205.9 °C. IR (KBr): 3062,
2
1
975, 1742, 1704, 1620, 1563, 1473, 1419, 1364, 1290, 1246,
ꢀ1 1
207, 1105, 1009, 967, 832, 791 cm
3
. H NMR (400 MHz, CDCl )
d 9.98 (s, 1H, OH), 8.44 (s, 1H, C@CH), 7.64–7.59 (m, 2H, Ph-H),
7
.34–7.30 (m, 1H, Ph-H), 5.28–5.23 (m, 1H, CH), 1.39 (d,
13
J = 6.5 Hz, 6H, 2CH
3
); C NMR (100 MHz, CDCl
3
) d 162.34 (1C),
3.2. Tyrosinase inhibitory activity
1
1
2
56.70 (1C), 155.05 (1C), 148.03 (1C), 134.21 (1C), 129.48 (1C),
24.82 (1C), 118.66 (1C), 117.87 (1C), 116.69 (1C), 69.63 (1C),
1.80 (2C).
For evaluating the tyrosinase inhibitory activity, all the synthes-
ised compounds were subjected to tyrosinase inhibition assay with
L-DOPA as substrate, according to the method reported by our
2
.4.17. Pentyl 7-hydroxycoumarin-3-carboxylate (22)
Yield 56.18%. Yellow solid, mp 53.2–55.0 °C. IR (KBr): 3046,
949, 1764, 1723, 1604, 1447, 1367, 1294, 1242, 1207, 1124,
groups with some slight modifications. The tyrosinase inhibitory
activities of kojic acid was ever reported, therefore, it was selected
as comparing substance. The IC50 values of coumarin derivatives
2

2
876
J. Liu et al. / Food Chemistry 135 (2012) 2872–2878
Scheme 1. Synthesis of coumarin compounds bearing thiosemicarbazide moieties. Reagents and conditions: (I) piperidine, acetonitrile, RT, 1–4 h. (II) Acetic acid, ethanol,
reflux, 6 h.
Scheme 2. Synthesis of the substituted 2-oxo-2H-coumarin-3-carboxylate esters. Reagents and conditions: (i) piperidine, ethanol, RT. (ii) 10% NaOH/3 N HCl. (iii) Thionyl
chloride, reflux. (iv) Appropriate alcohol/toluene or ethyl ether, reflux.
against tyrosinase were summarised in Table 1, and IC50 values of
all these compounds were determined from logarithmic concen-
tration–inhibition curves and given as means of three experiments.
Our results showed that compounds 3–6 and 20 exhibited po-
tent inhibition on mushroom tyrosinase with IC50 values ranged
exhibited more potent inhibitory activities than the 3-acetylcou-
marin, with the increase of lipophilicity of the compound, the
inhibitory activities increased gradually. These results showed that
the inhibitory activities of 3-acetylcoumarin and it is halogen
substituted analogues ralated to the lipophilicity. However, 2-(1-
(coumarin-3-yl)ethylidene) hydrazinecarbothioamide showed the
most potent inhibitory activity in all their homologues, and with
the increase of the radius of halogen atom, the inhibitory activities
decreased gradually. In addition, the inhibitory activities of the 2-
(1-(coumarin-3-yl)ethylidene)hydrazinecarbothioamide and it is
homologues were more potent than the inhibitory activities of 3-
acetylchroman-2-one and it is homologues. This result might be
related to different inhibitory mechanism. Since the 2-(1-(couma-
rin-3-yl)ethylidene) hydrazinecarbothioamide and it is homo-
logues mainly depended on chelation of the sulfur atom with the
active centre of tyrosinase, the increase of the the radius of halogen
atom might cause stereohindrance for the inhibitors approaching
the active site of the enzyme. However, substituted 3-acety-
lcoumarins are similar to benzaldehyde-type inhibitors, the tyros-
inase inhibitory mechanism of this type of inhibitors, come from
from 3.44 to 195.03 lM. Especially, compounds 4–6 bearing thio-
semicarbazide showed more potent inhibitory activities than the
other compounds. In addition, compound 4 demonstrated more
potent inhibitory activity than the reference standard inhibitor
kojic acid. Most of the coumarin-3-carboxylic acid analogues
showed low or no activities against tyrosinase. The results may
be related to the structure of tyrosinase, which contained a type-
3
copper centre with a coupled dinuclear copper active site in
the catalytic core. Tyrosinase inhibition of compounds 4–6 de-
pended on the competency of the sulfur atom to chelate with the
dicopper nucleus in the active site, and tyrosinase would lose its
catalysing ability after forming complex (Gerdemann, Eicken, &
Krebs, 2002).
When comparing to the tyrosinase inhibitory activities of
substituted 3-acetylcoumarin, compounds with halogen atom

J. Liu et al. / Food Chemistry 135 (2012) 2872–2878
2877
Table 1
Tyrosinase inhibitory activities and yields of the synthesised compounds.
Compounds
Yield (%)
CLog Pd
IC50
(l
M)a
Percent of inhibitionc
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
76
0.9114
1.7644
1.9144
1.5506
2.4036
2.5536
1.91
–
–
8.3%
42.45%
89.87
94.10
85.38
87.68
89.15
85.06
88.00
67.45
51.04
69.76
93.18
67.45
93.58
89.65
86.50
93.58
53.28
67.45
69.75
61.36
56.18
195.03
3.44
34.32
114.68
–
–
–
–
–
–
–
–
–
–
–
–
NAb
2.623
2.773
23.2%
12.88%
b
0
1
2
3
4
5
6
7
8
9
0
1
2
2.17114
1.54175
2.26375
2.41375
1.43245
1.381
2.244
1.64214
3.497
2.932
3.082
2.48014
3.75814
NA
43.38%
38.57%
29.68%
48.4%
33.33%
b
NA
NA
b
33.32%
15.62%
–
Fig. 2. The effect of concentrations of tyrosinase on its activity for the catalysis of
DOPA at different concentration of compound 4. The concentrations of compound 4
for curves 1–4 are 0, 25, 50 and 100 mol/l, respectively.
L-
131.05
–
–
13.9%
l
b
NA
Kojic acid
23
a
Values were determined from logarithmic concentration–inhibition curves (at
least eight points) and are given as means of three experiments.
b
Not active at 200
Percent of inhibition of tyrosinase reaction at the 200
Value of CLog P was obtained by ChemBioDraw Ultra 12.0.
l
M concentration.
c
lM.
d
the ability to form a Schiff base with a primary amino group in the
enzyme (Kubo et al., 1999). The increase of lipophilicity might be
benefited to the formation of Schiff base.
As shown in Table 1, coumarin-3-carboxylic acid and all of the
substituted coumarin-3-carboxylic acids exhibited certain inhibi-
tory activities against tyrosinase at the concentration of 200 lM.
However some of the coumarin-3-carboxylic acid esters and the
substituted coumarin-3-carboxylic acid esters lost their inhibitory
activities against tyrosinase. The results indicated that the carboxyl
group might be effective group to the interaction of compound
with the active site of tyrosinase. From Table 1, comparing the
ethyl esters of coumarin-3-carboxylic acid and their substituted
analogues, only the ethyl esters of coumarin-3-carboxylic acid with
halogen atom showed weak inhibitory activities at the concentra-
Fig. 3. The effect of concentrations of tyrosinase on its activity for the catalysis of
DOPA at different concentration of compound 5. The concentrations of compound 5
for curves 1–4 are 0, 25, 50 and 100 mol/l, respectively.
L-
l
tion of 200
acid, only the methyl coumarin-3-carboxylate exhibited inhibitory
activity at the concentration of 200 M. The isopropyl ester of cou-
marin-3-carboxylic acid and it is substituted analogues had certain
inhibitory activity against tyrosinse, especially, isopropyl 6-bromo-
coumarin-3-carboxylate with the IC50 value of 131.05 lM showed
the most potent activity than the other ester compounds. For the
pentyl ester compounds, the inhibitory percent of pentyl couma-
rin-3-carboxylate is 33.32% at the concentration of 200 lM, and
pentyl 7-hydroxycoumarin-3-carboxylate exhibited no inhibitory
activity at the same concentration. These results suggested that
the ester group structure might be an important factor for the
improvement of inhibitory activity.
lM. Among the methyl esters of coumarin-3-carboxylic
l
fects of compound 4, 5 on the tyrosinase were irreversible,
suggesting that substituted 2-(1-(coumarin-3-yl)ethylidene)-
hydrazinecarbothioamide compounds effectively inhibited the en-
zyme 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 centre of tyrosinase and a
lipophilic long-narrow gorge near to the active centre. Compound
4 or 5 could exhibit strong affinity for copper ions in the active cen-
tre and form a reversible non-covalent complex with the tyrosi-
nase, and this then reacts to produce the covalently modified
‘‘dead-end complex’’ (Tsou, 1988).
The present investigation reported that coumarin derivatives
had potent inhibitory effects on the diphenolase activity of mush-
room tyrosinase. Interestingly, compound 4 was found to be the
3.3. Inhibitory mechanism
The inhibitory mechanisms of the selected compounds 4, 5 on
most potent inhibitor with IC50 value of 3.44 lM. Preliminary
mushroom tyrosinase for the oxidation of
L
-DOPA were deter-
structure activity relationships (SARs) analysis indicated that (1)
thiosemicarbzide moiety might play an important role in deter-
mining their inhibitory activities, because of the sulfur atom could
chelate with the dicopper nucleus in the active site, and tyrosinase
would lose its catalysing ability after forming complex; (2) car-
boxyl group might be effective group to the interaction of
mined. Figs. 2 and 3 showed the relationship between enzyme
activity and concentration in the presence of different concentra-
tions of the above-mentioned compounds. The results showed that
the plots of V versus [E] gave a family of parallel straight lines with
the same slopes. These results demonstrated that the inhibitory ef-

2
878
J. Liu et al. / Food Chemistry 135 (2012) 2872–2878
compound with the active site of tyrosinase; (3) the ester group
structure might be an important factor for the improvement of
inhibitory activity. The inhibition mechanism analysis of com-
pounds 4, 5 demonstrated that the inhibitory effects of the two
compounds on the tyrosinase were irreversible.
Ley, J. P., & Bertram, H. (2001). Hydroxy- or methoxy-substituted benzaldoximes
and benzaldehyde-O-alkyloximes as tyrosinase inhibitors. Bioorganic and
Medicinal Chemistry, 9, 1879–1885.
Liu, J. B., Yi, W., Wan, Y. Q., Ma, L.,
&
Song, H. C. (2008). 1-(1-
new class of tyrosinase
Arylethylidene)thiosemicarbazide derivatives:
A
inhibitors. Bioorganic and Medicinal Chemistry, 14, 1096–1102.
Matsuura, R., Ukeda, H., & Sawamura, M. (2006). Tyrosinase inhibitory activity of
citrus essential oils. Journal of Agricultural Food Chemistry, 54, 2309–2313.
Neyts, J., De Clercq, E., Singha, R., Chang, Y. H., Das, A. R., Chakraborty, S. K., et al.
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2009). Synthesis and evaluation of vancomycin aglycon analogues that bear
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This work was financially supported by the Foundation of Edu-
cation Department of Hunan Province, China (09B091), and the Key
Subject Foundation of Shaoyang University (2008XK201).
Ostrov, D. A., Hernández Prada, J. A., Corsino, P. E., Finton, K. A., Le, N., & Rowe, T. C.
2007). Discovery of novel DNA gyrase inhibitors by high-throughput virtual
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Rai, U. S., Isloor, A. M., Shetty, P., Vijesh, A. M., Prabhu, N., & Isloor, S. (2010). Novel
chromeno [2,3-b]-pyrimidine derivatives as potential anti-microbial agents.
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