Antioxidation and tyrosinase inhibition of polyphenolic curcumin analogs

Article abstract of DOI:10.1271/bbb.110547

A series of polyphenolic curcumin analogs were synthesized and their inhibitory effects on mushroom tyrosinase and the inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical formation were evaluated. The results indictated that the analogs posses

Full text of DOI:10.1271/bbb.110547

Biosci. Biotechnol. Biochem., 75 (12), 2351–2358, 2011
Antioxidation and Tyrosinase Inhibition of Polyphenolic Curcumin Analogs
1;
1
1
1
Zhi-Yun DU,1;2; Yong-Fu JIANG, Zhi-Kai TANG, Rong-QingyMO, Gui-Hua XUE,
*
*
y
Yu-Jing LU,1 Xi ZHENG,1 Chang-Zhi DONG,2; and Kun ZHANG
1;
1Laboratory of Natural Medicine and Green Chemistry, School of Light Industry and Chemical Engineering,
Guandgong University of Technology, Guangzhou 510006, China
2
´
ITODYS/CNRS UMR 7086, Universite Paris Diderot Paris 7, 75205 Paris Cedex 13, France
Received July 25, 2011; Accepted August 22, 2011; Online Publication, December 7, 2011
[doi:10.1271/bbb.110547]
A series of polyphenolic curcumin analogs were
synthesized and their inhibitory effects on mushroom
tyrosinase and the inhibition of 1,1-diphenyl-2-picryl-
hydrazyl (DPPH) free radical formation were evaluated.
The results indictated that the analogs possessing m-
diphenols and o-diphenols exhibited more potent inhib-
itory activity on tyrosinase than reference compound
rojic acid, and that the analogs with o-diphenols
exhibited more potent inhibitory activity of DPPH
free-radical formation than reference compound vita-
min C. The inhibition kinetics, analyzed by Linewea-
ver–Burk plots, revealed that compounds B2 and C2
bearing o-diphenols were non-competitive inhibitors,
while compounds B11 and C11 bearing m-diphenols were
competitive inhibitors. In particular, representative
compounds C2 and B11 showed no side effects at a dose
of 2,000 mg/kg in a preliminary evaluation of acute
toxicity in mice. These results suggest that such
polyphenolic curcumin analogs might serve as lead
compounds for further design of new potential tyrosi-
nase inhibitors.
not compatible with safety regulations for medicines and
cosmetics. The potentially active inhibitors, such as
kojic and arbutin, have not yet been demonstrated to
be clinically efficient inhibitors.12,13) Hydroquinone, a
widely used skin-lightening agent, is still controversial
as to its biosafety due to its cytotoxic and mitogenic
properties.14) Hence, it is still necessary to search for and
develop novel tyrosinase inhibitors with potent activities
and smaller side effects.
In addition, antioxidant therapies are increasingly
recognized as a potential strategy to prevent many
diseases, including inflammation, acute CNS injury,
cardiovascular disease, and asthma. Applications of
antioxidants as preservatives in skin-protective ingre-
dients in cosmetics are also receiving increasing
attention and interest.15,16) Developing tyrosinase inhib-
itors with potent antioxidation may be a feasible way to
balance skin brightening and side effects.
Curcumin (diferuloylmethane, Scheme 1), a well-
known antioxidant polyphenol from the rhizome of
Curcuma longa Linn, is a major ingredient of turmeric.
It is used in the therapy of inflammatory and infectious
diseases in ayurvedic medicine.17) Many studies indicate
that curcumin has cancer preventive,18) anti-inflamma-
tory,19) antioxidative,20) and antiviral activities.21) In
addition, the safety of curcumin is evident from its
consumption for centuries at levels of up to 100 mg/d by
people in many countries.22) These beneficial properties
have attracted numerous efforts for the development of
curcumin as a safe therapeutic agent. Curcumins have
been approved as additives in cosmetics and as a natural
yellow colorants and antioxidants for many years.23)
Recently, it was found to be beneficial in treating certain
skin diseases,24) and one of its derivatives, tetrahydro-
curcumin, has been recommended for use in cosmetics
as a lighting agent.25) Furthermore, Lee et al. reported
recently that some curcumin analogs exhibited inhib-
itory activity against tyrosinase.26) This attracted our
interest to further study of the inhibitory effect on
tyrosinase and the antioxidation of curcumin analogs.
In this study, considering the important roles of
polyphenolic hydroxyls in certain reported tyrosinase
inhibitors,27–30) a series of curcumin analogs bearing
polyphenols were synthesized (Scheme 2), and their
inhibitory activities against tyrosinase and free-radical
Key words: tyrosinase inhibition; antioxidation; poly-
phenolic curcumin analogs; inhibition
kinetics
Tyrosinase (EC 1.14.18.1), also known as polyphenol
oxidase, is a multifunctional copper-containing enzyme
widely distributed in nature.1) It catalyzes two distinct
reactions of melanin biosynthesis: the o-hydroxylation
of monophenols to o-phenols (monophenolase activity),
and the oxidation of o-phenols to o-quinones (dipheno-
lase activity).2) These quinones are highly reactive
compounds that can polymerize spontaneously to form
melanins, which determine the color of mammalian skin
and hair.3) Various dermatological disorders, such as age
spots, freckle, melasma, and ephelide, are caused by the
accumulation of excessive levels of epidermal pigmen-
tation.4) Hence, tyrosinase inhibitors have become
increasingly important in medication5–7) and the cos-
metics of industry8,9) to prevent hyperpigmentation.
In recent years, a large number of naturally occur-
ring and synthetic tyrosinase inhibitors have been
reported,4,10,11) but some of these inhibitors are either
not potentially active enough to be of practical use, or
y
To whom correspondence should be addressed. Kun ZHANG, Tel: +86-20-39322235; Fax: +86-20-39322231; E-mail: kzhang@gdut.edu.cn;
Chang-Zhi DONG, Tel: +33-01-57276868; Fax: +33-01-57277263; E-mail: dong@univ-paris-diderot.fr
These authors contributed equally to this work.
*

2352
Z.-Y. DU et al.
scavenging properties on DPPH were evaluated. In
addition, the inhibition mechanism and preliminary
acute toxicity of the potent active compounds were also
investigated, with the aim of developing novel tyrosi-
nase inhibitors with potent activities, together with
smaller side effects.
B1{11, C1{11, D1{7, E1{10, and F1{10) were subjected to
tyrosinase inhibitory assay by measuring the oxidation
of L-DOPA according to a published protocol.29) As
Fig. 1 indicates, the remaining enzyme activity rapidly
decreased with increasing concentrations of compound
C11. Enzyme inhibition data were expressed as IC50
values as summarized in Table 1. Kojic acid, a com-
mercial whitening ingredient, was used as reference
compound.
Results and Discussion
Chemistry
Based on the data shown in Table 1, it was found that
natural curcuminoids 1–3 showed potent inhibitory
activities against tyrosinase, and that compound 3
exhibited the highest inhibitory activity, with an IC50
value of 33.50 mM, among curcuminoids. In the synthetic
curcumin analogs, compounds with o-diphenols (A2-F2)
and m-diphenols (A11-C11) exhibited much higher
inhibitory activities than others. Compound C11
(IC50 ¼ 0:65 mM) was the strongest inhibitor of all the
observed compounds, and it was much stronger than the
reference kojic acid (IC50 ¼ 28:59 mM).
Natural curcuminoids (1–3) were isolated from
turmeric, and 19 new compounds and 41 known
compounds were synthesized through condensation of
the appropriate aromatic aldehydes and cyclohexanone
(A1{11), cyclopentanone (B1{11), acetone (C1{11), tetra-
hydrothiopyran-4-one (D1{10), tetrahydropyran-4-one
(E1{10), and tetrahydropyran-4-one (F1{10) under acidic
conditions at room temperature following published
procedures.31) These polyphenolic curcumin analogs
were synthesized by a one-step procedure without
phenolic hydroxy protection, and the final products
were purified by recrystallization. The structures were
determined using various spectroscopic methods, in-
1
cluding H NMR and EI-MS, and purity was confirmed
by elemental analysis.
Inhibitory effects on mushroom tyrosinase
Natural curcuminoids (1, 2, and 3) isolated from
Curcuma longa and synthetic curcumin analogs (A1{11
,
Fig. 1. Effects of Compound C11 on Mushroom Tyrosinase for the
Catalysis of L-DOPA.
Scheme 1. Chemical Structures of Curcuminoids.
Scheme 2. Synthesis of Curcumin Analogs.

Tyrosinase Inhibition of Curcumin Analogs
2353
Table 1. Antioxidative and Tyrosinase Inhibitory Effects of Curcumin Analogs
Inhibition (IC50/mM)a
Inhibition (IC50/mM)a
Compound
Compound
DPPH
Tyrosinase
DPPH
Tyrosinase
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
>300
16:82 ꢀ 0:87
48:40 ꢀ 2:06
70:10 ꢀ 3:65
NIb
64:48 ꢀ 3:42
2:16 ꢀ 0:58
68:26 ꢀ 3:96
>200
D1
D2
D3
D4
D5
D6
D7
E1
>300
29:26 ꢀ 1:42
65:18 ꢀ 3:72
71:56 ꢀ 3:83
NI
171:60 ꢀ 8:02
11:32 ꢀ 1:60
>200
>200
NI
NI
>200
>200
53:68 ꢀ 1:68
73:80 ꢀ 3:98
>300
85:10 ꢀ 4:32
103:20 ꢀ 5:12
>300
>200
>200
47:28 ꢀ 2:17
1:36 ꢀ 0:68
62:37 ꢀ 3:84
>200
193:22 ꢀ 7:43
>200
>200
>300
E2
E3
18:80 ꢀ 0:51
56:90 ꢀ 3:18
53:70 ꢀ 2:32
NI
216:81 ꢀ 11:36
50:60 ꢀ 3:23
>300
0:93 ꢀ 0:22
41:87 ꢀ 1:38
1:24 ꢀ 0:22
56:40 ꢀ 2:89
>200
E4
E5
NI
>200
>200
5:79 ꢀ 0:12
48:90 ꢀ 1:69
59:20 ꢀ 3:01
NI
E6
E7
49:06 ꢀ 1:87
82:78 ꢀ 4:67
>300
E8
E9
177:11 ꢀ 5:78
>200
>200
NI
>200
>200
>300
>300
>300
17:90 ꢀ 0:54
78:06 ꢀ 3:28
>300
E10
F1
72:60 ꢀ 3:68
2:64 ꢀ 0:83
79:80 ꢀ 4:77
>200
172:32 ꢀ 5:11
>200
>200
F2
21:10 ꢀ 0:86
53:90 ꢀ 2:37
52:20 ꢀ 2:13
NI
>300
F3
124:62 ꢀ 6:78
47:10 ꢀ 4:33
>300
F4
0:78 ꢀ 0:13
31:26 ꢀ 1:32
1:19 ꢀ 0:21
41:25 ꢀ 1:73
116:44 ꢀ 3:76
NI
F5
NI
>200
F6
58:70 ꢀ 2:86
83:64 ꢀ 3:54
>300
6:73 ꢀ 0:18
40:20 ꢀ 2:03
65:72 ꢀ 2:87
NI
F7
>200
>200
F8
F9
>300
>300
>200
>200
F10
1
38:40 ꢀ 1:65
96:4 ꢀ 5:40
>300
176:32 ꢀ 5:61
162:83 ꢀ 3:81
102:43 ꢀ 2:53
>200
21:62 ꢀ 1:21
37:80 ꢀ 1:67
34:64 ꢀ 1:53
21:14 ꢀ 0:82
ND
94:73 ꢀ 3:76
53:03 ꢀ 2:17
33:50 ꢀ 1:26
NDc
2
3
>300
186:42 ꢀ 8:21
Vc
Kojic acid
>200
0:65 ꢀ 0:12
28:59 ꢀ 1:30
48:24 ꢀ 3:78
aValues are means with standard deviation for three different experiments.
bNI, No Inhibition.
cND, no data.
Compared with the inhibitory activities of curcumin
analogs bearing 4-phenolic hydroxyls (A1-E1), the
activities of these compounds decreased when R3 and
R5 were replaced by bromine, methoxyl, or t-butyl.
When only one side of p-phenolic hydroxyl, R3 or R5,
was replaced by methoxyl (A3-F3), the inhibitory
activities decreased slightly, whereas, when both sides
of p-phenolic hydroxyl, R3 and R5, were replaced by
non-hydroxylic groups, the inhibitory activities decreased
sharply. In addition, the activities of curcumin analogs
bearing 2-phenolic hydroxyls (A9-F9) were much
weaker than those of curcumin analogs bearing 4-
phenolic hydroxyls (A1-F1). In a comparison between
compounds A10-C10 and compounds A11-C11, only
4-phenolic hydroxyls was replaced by methoxyls, and
the activities of A10-C10 almost dispeared, whereas the
activities of A11-C11 were the strongest of all the
compounds. It suggested that the 4-phenolic hydroxyls
in curcumin analogs play a crucial role in inhibitory
activity against tyrosinase. Nerya and his coworkers also
found that the OH group substation at the para position
of the chalcone benzene ring was the major factor
affecting inhibitory potency.31)
with acetone linkers (series C, compounds C1{11)
exhibited the strongest activities, and those of com-
pounds with cyclopentanone (series B, compounds
B1{11), tetrahydropyran-4-one (series E, compounds
E1{10), cyclohexanone (series A, compounds A1{11),
tetrahydrothiopyran-4-one (series F, compounds F1{10
)
and piperidin-4-one (D1{7) were weaker in that order,
which hints that the smaller bulk of these linkers
exhibited stronger activity than the others.
Inhibition mechanism of the selected compounds as to
mushroom tyrosinase
Lineweaver-Burk plots of the inhibition kinetics of
tyrosinase for the selected compounds, B2, C2, B11, and
C11, are shown in Fig. 2. Figure 2A and B are for
selected compounds B2 and C2 with o-diphenols, and
the straight lines crossing the x axis at the same point
show unchanged Km and decreased Vmax with increasing
inhibitor concerntrations, which is typical for non-
competitive inhibition. In Fig. 2C and D, for selected
compounds B11 and C11 with m-diphenols, the straight
lines crossing the y axis at the same point show
unchanged Vmax and decreased Km with increasing
inhibitor concentrations, which is typical for competitive
inhibition. The inhibition constants Ki (Table 2) were
calculated from the plots of the slope versus the
concentration of selected compounds.
In addition, the different ketone linkers between two
aromatic rings of curcumin analogs showed different
active trend, although there was no big difference
between different linkers. The activities of compounds

2354
Z.-Y. DU et al.
A
B
C
D
Fig. 2. Lineweaver–Burk Plots of the Inhibition of Selected Compounds B2, C2, B11, and C11 on Mushroom Tyrosinase for the Catalysis of
L-DOPA.
The concentrations of B2 C2, B11, and C11 for curves 1–3 were 0, 0.4 mM, 0.8 mM; 0, 0.3 mM, 0.6 mM; 0, 0.6 mM, 1.2 mM; 0, 0.6 mM, and 1.2 mM
respectively.
Table 2. Kinetics and Inhibition Constants of Compounds B2, C2,
B11, and C11 in the Activity of Mushroom Tyrosinase
Most competitive inhibitors of tyrosinase have mo-
lecular structures that closely resemble that of the
products of the various steps. Base on the structures
Compound
Inhibition type
Inhibition onstant (Ki) (mM)
of synthetic curcumin analogs, the compounds with
o-diphenols were similar to the substrate L-DOPA, but
the mechanisms of curcumin analogs B2 and C2 with
o-diphenols showed non-competitive inhibition, and
compounds B11 and C11 with m-diphenols showed
competitive inhibition, which indicates that curcumin
analogs with o-diphenols combined with both the free
enzyme and the enzyme-substrate complex, and that
curcumin analogs with m-diphenols combined only with
free enzyme molecule. Baek et al. have reported that
tyrosinase inhibitors 1,3-diphenylpropanes with m-
diphenols showed competitive inhibition,32) and Song
et al. have reported that 3,5-dihydroxystilbenes with
m-diphenols also exihibited competitive inhibition.33)
The details of the inhibition mechanisms of curcumin
analogs as to tyrosinase call for further study.
B2
C2
Non-competetive
Non-competetive
Competetive
0.50
0.45
0.37
0.24
B11
C11
Competetive
activities of DPPH radical scavenging, whereas the
introduction of bromines weakens their activities. In
addition, in a comparison with diffenent linkers between
the two aromatic rings, cylopentanone (series B) and
actone (series C) showed stronger DPPH radical
scavenging properties than the others.
Evaluation of acute toxicity in mice
Safety is a primary consideration for tyrosinase
inhibitors, especially for those materials used in med-
icine and cosmetic products. In this study, C2 and B11
were selected as representative compounds to evaluate
toxicity in mice. Clinical symptoms were measured for
14 d after a single oral gavage administration in accord
with the OECD 423 Guideline. The results indictated
that none of the mice after administration at a dose of
2,000 mg/kg of body weight per d showed any mortal-
ity, and autopsy of the animals at the end of the
experimental period (14 d) revealed no apparent changes
Effects on free radical scavenging
Table 1, columns 2 and 5, shows the DPPH radical
scavenging activities of curcumin analogs. Compounds
with o-diphenols (A2-F2) showed more active DPPH
radical scavenging than the other curcumin analogs.
Generally, in a comparision within the same series
of curcumin analogs, the introduction of methoxyl and
t-butyl to the adjacent of 4-phenols strengthens the

Tyrosinase Inhibition of Curcumin Analogs
2355
Fig. 3. Proposed Binding Modes of C2 (A) and C11 (B) in the Active Site of the Tyrosinase (PDB accession code 2ZWE).
The inhibitor molecules are colored yellow for carbon atoms. The broken lines show hydrogen-bonding, and the solid lines show the distance
of metal-coordination interactions. The docking models were generated using Surflex-Dock.
in any organs. This indicates that compounds C2 and B11
had no acute toxicity at 2,000 mg/kg in mice.
analogs with m-diphenols were competitive. The dock-
ing study that the analogs with m-diphenols and those
with o-diphenols were different. The primary evaluation
of acute toxicity showed that C2 and B11 were not
toxic to the mice. These results suggest that with the
advantage of easily synthesizable small molecules,
polyphenolic curcumin analogs can be developed as
pharmacological tyrosinase inhibitors.
Molecular docking study
Recently, the crystallographic structure of tyrosinase
was determined. The three-dimensional structure of
tyrosinase make it possible to gain a better understand-
ing of the tyrosinase inhibition mechanism. Considering
that compounds C2 and C11 exhibited potent inhibition
against tyrosinase and that their inhibition mechanisms
were different, we selected them to study the interaction
mode by docking. Although the structure of mushroom
tyrosinase has not yet been determined, we borrowed the
crystallographic data because there is high homology for
the active center of most tyrosinases of different origins.
It was found that C2 and C11 both formed a ꢀ-ꢀ stack
between one of aromatic rings of the ligands and His
194 of tyrosinase, and multiple hydrogen bonds between
the phenolic hydroxyls of the compounds and the
residues of the active site of tyrosinase (Fig. 3). In
Fig. 3A, C2 with o-diphenols formed four hydrogen
bonds with Gly 183, Trp 184 and Ser 206, and the
distances between the 3,4-phenolic hydroxyls of the
Experimental
Reagents and general procedures. Melting points were determined
on a Yanagimoto micro-melting apparatus (MP 500D), and are
uncorrected. The 1H NMR spectra were measured on a 300 mhz NMR
Spectrometer (Varian Gemini-2000) using DMSO as solvent, unless
otherwise specified. Chemical shifts for 1H NMR (300 MHz) were
expressed as ppm with TMS as internal standard. Multiplicities were
recorded as singlet, broad singlet, doublet, triplet, quartet, and
multiplet. Mass spectra were obtained on
a mass spectrometer
(LCMS-2010A). Elemental analyses were performed on a elemental
analyzer (Perkin Elmer 240C). Curcuminoids (1–3) were isolated from
an extract of Curcuma longa, and compounds A1{7, B1{7, C1{6, and
D1{7, were synthesized, purified and characterized as previously
described.34,35) Tyrosinase and L-3,4-dihydroxy phenylalanine (L-
DOPA) were purchased from Sigma–Aldrich (St. Louis, Mo). Other
˚
compound and dinuclear copper ions were 3.09 A and
chemicals
were
purchased
from
commercial
suppliers
˚
3.83 A respectively (Fig. 3A). C11 with m-diphenols
and were dried and purified when necessary. The water used was
re-distillated and ion-free.
formed seven hydrogen bonds, with Ser 206, Ala 202,
Asn 191, Asn 188, Thr 203, and Ser 146, and the
distances between the 2,4-phenolic hydroxyls and the
General procedure for the synthesis of curcumin analogs. Sixty
curcumin analogs (series A, B, C, D, E, and F) were synthesized as
˚
˚
dinuclear copper ions were 4.54 A and 5.01 A respec-
tively (Fig. 3B). The different interactions of C2 and C11
with tyrosinase might explain why the inhibitory avtivity
of the curcumin analogs with m-diphenols was stronger
than that of the curcumin analogs with o-diphenols.
Their modes of inhibition of tyrosinase were entirely
different.
previously described, with modifications.34,35)
A mixture of the
appropriate aldehyde (0.01 mol) and the ketone (0.005 mol) was
dissolved in glacial acetic acid or ethanol saturated with anhydrous
hydrogen chloride and this was heated in a water bath at 25–30 ꢁC
for 2 h. After standing for 2–5 d, the mixture was treated with cold
water and filtered. The solid obtained was then washed and dried. The
crude product was recrystallized from appropriate solvents (methanol
or ethanol).
Conclusion
2,6-Bis(3,5-dibromo-4-hydroxylbenzylidene)cyclohexanone (A8).
Yield 83%. mp 238–239 ꢁC. 1H NMR (CDCl3, 300 MHz) ꢁ (ppm):
10.32 (brs, 2H, –OH), 7.77 (s, 4H, ArH), 7.46 (s, 2H, –CH=C–), 2.85
(m, 4H, –CH2–C–CH2–), 1.75 (m, 2H, C–CH2–C). LC–MS (m=z): 621
½M ꢂ 1ꢃꢂ. Anal. Calc. for C20H14Br4O3: C 38.62, H 2.27. Found: C
38.59, H 2.30.
In this study, a series of polyphenolic curcumin
analogs were synthesized, and their effects on mush-
room tyrosinase inhibition and free-radical scaveraging
were evaluated. The results indicated that the analogs
possessing m- or o-diphenols exhibited more potent
activity inhibition of tyrosinase than the others. Analysis
of the inhibition kinetics revealed that the analogs with
o-diphenols were non-competitive inhibitors, while the
2,6-Bis(2-hydroxybenzylidene)cyclohexanone (A9). Yield 75%. mp
147–149 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 9.87 (brs, 2H, –
OH), 7.79 (s, 2H, ArH), 7.30 (s, 2H, –CH=), 7.18 (m, 2H, ArH), 6.89
(d, J ¼ 8:1 Hz, 2H, ArH), 6.83 (d, J ¼ 8:1 Hz, 2H, ArH), 2.81 (m, 4H,

2356
Z.-Y. DU et al.
–CH2–C–CH2–), 1.67 (m, 2H, C–CH2–C). LC–MS (m=z): 305
½M ꢂ 1ꢃꢂ. Anal. Calc. for C20H18O3: C 78.41, H 5.92. Found:
C 78.21, H 6.01.
2H, ArH), 3.74 (s, 6H, OCH3). LC–MS (m=z): 325 ½M ꢂ 1ꢃꢂ. Anal.
Calc. for C19H18O5: C 69.93, H 5.56. Found: C 69.74, H 5.60.
1,5-Bis(2,4-dihydroxybenzylidene)penta-1,4-3-one (C11). Yield 42%.
mp >300 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 10.08 (brs, 2H,
–OH), 9.80 (brs, 2H, –OH), 7.62 (d, J ¼ 15:9, 2H, –CH=C–), 7.40
(d, J ¼ 8:1, 2H, ArH), 6.48 (d, J ¼ 15:9 Hz, 2H, –CH=C–), 6.40
(d, J ¼ 8:1 Hz, 2H, ArH), LC–MS (m=z): 299 ½M ꢂ 1ꢃꢂ. Anal. Calc.
for C17H14O5: C 68.45, H 4.73. Found: C 68.24, H 4.80.
2,6-Bis(2-hydroxy-4-methoxybenzylidene)cyclohexanone (A10). Yield
72%, mp 206–208 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 9.80
(brs, 2H, –OH), 7.21 (d, J ¼ 8:1 Hz, 2H, ArH), 6.73 (s, 2H, –CH=),
6.61 (d, J ¼ 8:1 Hz, 2H, ArH), 6.47 (s, 2H, ArH), 3.68 (s, 6H, –OCH3),
2.81 (m, 4H, –CH2–C–CH2–), 1.74 (m, 2H, C–CH2–C). LC–MS (m=z):
366. Anal. Calc. for C22H22O5: C 72.12, H 6.05. Found: C 72.21,
H 6.08.
3,5-Bis(4-hydroxybenzylidene)-tetrahydropyran-4-one (D1). Yield
82%. mp 226–228 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 10.03
(brs, 2H, –OH), 7.55 (s, 2H, –CH=), 7.28 (d, J ¼ 8:1, 4H, ArH), 6.85
(d, J ¼ 8:1, ArH), 4.85 (s, 4H, –CH2–O–CH2–). LC–MS(m=z): 307
½M ꢂ 1ꢃꢂ. Anal. Calc. for C19H16O4: C 74.01, H 5.23. Found: C 74.00,
H 5.24.
2,6-Bis(2,4-dihydroxybenzylidene)cyclohexanone (A11). Yield 51%,
mp >300 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 10.00 (s, 2H,
–OH), 9.80 (s, 2H, –OH), 7.16 (d, J ¼ 8:1 Hz, 2H, ArH), 6.33 (s, 2H,
–CH=), 6.26 (d, J ¼ 8:1 Hz, 2H, ArH), 6.19 (s, 2H, ArH), 2.80 (m, 4H,
–CH2–C–CH2–), 1.72 (m, 2H, C–CH2–C). LC–MS (m=z): 338. Anal.
Calc. for C22H22O5: C 70.99, H 5.36. Found: C 70.83, H 5.42.
3,5-Bis(3,4-dihydroxybenzylidene)-tetrahydropyran-4-one (D2). Yield
90%. mp >300 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 9.50 (brs,
2H, –OH), 9.18 (brs, 2H, –OH), 7.45 (s, 2H, –CH=), 6.81 (s, 2H, ArH),
6.76 (m, 4H, ArH), 4.83 (s, 4H, –CH2–O–CH2–). LC–MS (m=z): 339
½M ꢂ 1ꢃꢂ. Anal. Calc. for C19H16O6: C 67.05, H 4.74. Found: C 67.01,
H 4.79.
2,6-Bis(3,5-dibromo-4-hydroxylbenzylidene)cyclopentanone (B8).
Yield 85%. mp >300 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm):
10.55 (brs, 2H, –OH), 8.07 (s, 4H, ArH), 7.32 (s, 2H, –CH=C–), 3.05
(s, 4H, –CH2–CH2–). LC–MS (m=z): 607 ½M ꢂ 1ꢃꢂ. Anal. Calc. for
C19H12Br4O3: C 37.54, H 1.99. Found: C 37.49, H 2.02.
3,5-Bis(4-hydroxy-3-methoxybenzylidene)-tetrahydropyran-4-one
(D3). Yield 82%. mp 226–228 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 9.54 (brs, 2H, –OH), 7.52 (s, 2H, –CH=), 7.07 (s, 2H, ArH),
7.00 (m, 2H, ArH), 6.83 (m, 2H, ArH), 4.98 (s, 4H, –CH2–O–CH2–),
3.81 (s, 6H, OCH3). LC–MS (m=z): 367 ½M ꢂ 1ꢃꢂ. Anal. Calc. for
2,5-Bis(2-hydroxybenzylidene)cyclopentanone (B9). Yield 81%. mp
224–226 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 10.09 (brs, 2H,
–OH), 7.77 (s, 2H, –CH=C–), 7.53 (d, J ¼ 8:1 Hz, 2H, ArH), 7.22
(m, 4H, ArH), 6.88 (m, 4H, ArH), 3.01 (s, 4H, –CH2–CH2–). LC–MS
(m=z): 291 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C19H16O3: C 78.06, H 5.52.
Found: C 77.93, H 5.46.
C21H20S6: C 68.47, H 5.47. Found: C 68.45, H 5.49.
3,5-Bis(3,5-ditertbutyl-4-hydroxylbenzylidene)-tetrahydropyran-4-one
2,5-Bis(2-hydroxy-4-methoxybenzylidene)cyclopentanone (B10). Yield
74%. mp 212–213 ꢁC. 1H NMR (MSDO-d6, 400 MHz) ꢁ (ppm): 10.23
(s, 2H, –OH), 7.74 (s, 2H, ArH), 7.50 (d, J ¼ 8:1 Hz, 2H, –C=CH–),
6.51 (d, J ¼ 8:1 Hz, 4H, ArH), 3.76 (s, 6H, –OCH3), 2.96 (s, 4H,
–CH2–CH2–). LC–MS (m=z): 351 ½M ꢂ 1ꢃꢂ. Anal. Calc. for
C21H20O5: C 71.58, H 5.72. Found: C 71.45, H 5.55.
(D4). Yield 65%. mp 236–238 ꢁC. 1H NMR (DMSO, 300 MHz)
ꢁ (ppm): 7.59 (brs, 2H, –OH), 7.51 (s, 2H, –C=), 7.13 (s, 4H,
–ArH), 4.90 (s, 4H, –CH2–O–CH2–), 1.49 (s, 36H, C–CH3). LC–MS
(m=z): 531 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C35H48O4: C 78.91, H 9.08.
Found: C 78.92, H 9.13.
3,5-Bis(3,4-dimethoxybenzylidene)-tetrahydropyran-4-one
(D5).
ꢁ
2,5-Bis(2,4-dihydroxybenzylidene)cyclopentanone (B11). Yield 52%.
mp >300 ꢁC. 1H NMR (MSDO-d6, 300 MHz) ꢁ (ppm): 10.00 (s, 2H,
–OH), 9.80 (s, 2H, –OH), 7.69 (s, 2H, –CH=C–), 7.37 (d, J ¼ 8:7, 2H,
arom), 6.38 (s, 2H, arom), 6.32 (d, J ¼ 8:7 Hz, 2H, arom), 2.92 (s, 4H,
–CH2–CH2–). ESI-MS (m=z): 325 (½M þ 1ꢃþ). Anal. Calc. for
Yield 62%. mp 134–135 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
(ppm): 7.78 (s, 2H, –CH=CH–), 6.91 (dd, J ¼ 8:1, 4H, arom), 6.87
(s, 2H, arom), 4.96 (s, 4H, –CH2–O–CH2–), 3.93 (s, 6H, –OCH3), 3.91
(s, 6H, –OCH3). LC–MS (m=z): 625 ½M þ 1ꢃþ. Anal. Calc. for
C23H24O6: C 69.68, H 6.10. Found: C 69.61, H 6.13.
C
19H16O5: C 70.36, H 4.97. Found: C 70.25, H 5.04.
3,5-Bis(4-hydroxy-3,5-dimethoxybenzylidene)-tetrahydropyran-4-one
(D6). Yield 82%. mp 226–228 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 9.03 (brs, 2H, –OH), 7.58 (s, 2H, –CH=), 6.70 (s, 4H, ArH),
4.95 (s, 4H, –CH2–O–CH2–), 3.81 (s, 12H, OCH3). LC–MS (m=z): 411
½M ꢂ 1ꢃꢂ. Anal. Calc. for C23H24O8: C 64.48, H 5.65. Found: C 64.43,
H 5.68.
1,5-Bis(3-bromo-4-hydroxy-5-methoxyphenyl)penta-1,4-dien-3-one
(C7). Yield 85%. mp 171–172 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 10.05 (brs, 2H, –OH), 7.63 (d, J ¼ 15:9, 2H, –CH=C–), 7.54
(d, J ¼ 8:1 Hz, 2H, ArH), 7.39 (d, J ¼ 8:1 Hz, 2H, ArH), 7.20
(d, J ¼ 15:9, 2H, –CH=C–), 3.90 (s, 6H, OCH3). LC–MS (m=z): 481
½M ꢂ 1ꢃꢂ. Anal. Calc. for C19H16Br2O5: C 47.14, H 3.33. Found:
C 47.10, H 3.28.
3,5-Bis(3-bromo-4-hydroxy-5-methoxybenzylidene)-tetrahydropyran-
4-one (D7). Yield 45%. mp 171–172 ꢁC. 1H NMR (DMSO-d6, 300
MHz) ꢁ (ppm): 10.09 (brs, 2H, –OH), 7.55 (s, 2H, –CH=), 7.16 (s, 2H,
ArH), 7.03 (s, 2H, ArH), 4.91 (s, 4H, –CH2–O–CH2–), 3.88 (s, 6H,
–OCH3). LC–MS (m=z): 525 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C21H18Br2O6:
C 47.94, H 3.45. Found: C 47.87, H 3.49.
1,5-Bis(3,5-dibromo-4-hydroxylphenyl)penta-1,4-diene-3-one (C8).
Yield 92%. mp 280–281 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm):
10.53 (brs, 2H, –OH), 8.02 (s, 4H, ArH), 7.65 (d, J ¼ 15:9 Hz, 2H,
–CH=C–), 7.25 (d, J ¼ 15:9 Hz, 2H, –C=CH–). LC–MS (m=z): 581
½M ꢂ 1ꢃꢂ. Anal. Calc. for C17H10Br4O3: C 35.09, H 1.73. Found:
C 35.06, H 1.74.
3,5-Bis(3,5-dibromo-4-hydroxylbenzylidene)-tetrahydropyran-4-one
(D8). Yield 62%. mp 134–135 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 10.50 (br, 2H, OH); 7.61 (s, 4H, ArH), 7.50 (s, 2H, CH=CH);
4.86 (s, 4H, –CH2–O–CH2–). LC–MS (m=z): 623 ½M ꢂ 1ꢃꢂ. Anal.
Calc. for C19H12Br4O4: C 36.58, H 1.94. Found: C 36.52, H 1.98.
1,5-Bis(2-hydroxyphenyl)penta-1,4-dien-3-one (C9). Yield 81%. mp
224–226 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 10.21 (brs, 2H,
–OH), 7.90 (d, J ¼ 15:9 Hz, 2H, –CH=C–), 7.66 (d, J ¼ 8:1 Hz, 2H,
ArH), 7.24 (m, 4H, ArH), 6.87 (m, 4H, ArH). LC–MS (m=z): 265
½M ꢂ 1ꢃꢂ. Anal. Calc. for C17H14O3: C 76.68, H 5.30. Found: C 76.56,
H 5.23.
3,5-Bis(2-hydroxybenzylidene)-tetrahydropyran-4-one (D9). Yield
74%. mp 167–168 ꢁC. 1H NMR (DMSO, 300 MHz) ꢁ (ppm): 10.09
(brs, 2H, –OH), 7.89 (s, 2H, ArH), 7.24 (m, 2H, –ArH), 7.07 (m, 2H,
–ArH), 6.91 (m, 2H, –ArH), 6.85 (m, 2H, –ArH), 4.79 (s, 4H, –CH2–
O–CH2–). LC–MS (m=z): 307 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C19H16O4: C
74.01, H 5.23. Found: C 74.00, H 5.25.
1,5-Bis(2-hydroxy-4-methoxyphenyl)penta-1,4-dien-3-one (C10). Yield
68%. mp 149–151 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 10.28
(brs, 2H, –OH), 7.82 (d, J ¼ 15:9, 2H, –CH=C–), 7.60 (d, J ¼ 8:1,
2H, ArH), 7.10 (d, J ¼ 15:9 Hz, 2H, –CH=C–), 6.46 (d, J ¼ 8:1 Hz,

Tyrosinase Inhibition of Curcumin Analogs
2357
3,5-Bis(2-hydroxy-4-methoxybenzylidene)-tetrahydropyran-4-one
(D10). Yield 65%. mp >300 ꢁC. 1H NMR (DMSO, 300 MHz) ꢁ (ppm):
9.89 (brs, 2H, –OH), 7.32 (d, J ¼ 8:1 Hz, 2H, ArH), 6.73 (s, 2H,
–CH=), 6.61 (d, J ¼ 8:1 Hz, 2H, ArH), 6.47 (s, 2H, ArH), 4.38 (s, 4H,
–CH2–O–CH2–), 3.71 (s, 6H, –OCH3). LC–MS (m=z): 367 ½M ꢂ 1ꢃꢂ.
Anal. Calc. for C22H20O6: C 68.47, H 5.47. Found: C 68.41, H 5.52.
(s, 2H, –CH=), 6.64 (d, J ¼ 8:1 Hz, 2H, ArH), 6.47 (s, 2H, ArH), 3.71
(s, 6H, –OCH3), 3.58 (s, 4H, –CH2–S–CH2–). LC–MS (m=z): 383
½M ꢂ 1ꢃꢂ. Anal. Calc. for C21H20O5S: C 65.61, H 5.24. Found:
C 65.55, H 5.30.
Tyrosinase inhibition assay.
A spectrophotometric assay for
tyrosinase was performed according to a method reported36) by Yi W
et al. with little modification. Measurement was performed in triplicate
for each concentration. IC50 values were determined by interpolation
of the dose-response curves.The inhibition types of the inhibitors were
determined by Lineweaver–Burk plots and a replot of enzyme activity
versus the reciprocal of the substrate L-DOPA concentration.
3,5-Bis(4-hydroxybenzylidene)-tetrahydrothiopyran-4-one (E1). Yield
95%. mp >300 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm): 9.95 (brs,
2H, –OH), 7.51 (s, 2H, –CH=), 7.38 (d, J ¼ 8:1 Hz, 4H, ArH), 6.83
(d, J ¼ 8:1 Hz, 4H, ArH), 3.92 (s, 4H, –CH2–S–CH2–). LC–MS (m=z):
323 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C19H16O3S: C 70.35, H 4.97. Found:
C 70.31, H 5.03.
DPPH radical scavenging assay. The Free radical scavenging
activities of curcum analogs were assayed using A stable DPPH,
following standard method37) with little modification. Briefly, 1 mL of
0.1 mM DPPH radical solution was mixed with 3 mL of various
concentrations of curcumin analogs or vitamin C dissolved in
methanol. The mixture was vortexed vigorously and left for 30 min
at 40 ꢁC in the dark. For baseline control, 3 mL of methanol was used.
The absorbance was measured at 517 nm.
3,5-Bis(3,4-dihydroxybenzylidene)-tetrahydrothiopyran-4-one (E2).
Yield 90%. mp >300 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm):
9.43 (brs, 2H, –OH), 9.14 (brs, 2H, –OH), 7.41 (s, 2H, –CH=), 6.92
(s, 2H, ArH), 6.83 (d, J ¼ 8:1 Hz, 2H, ArH), 6.80 (d, J ¼ 8:1 Hz, 2H,
ArH), 3.93 (s, 4H, –CH2–S–CH2–). LC–MS (m=z): 355 ½M ꢂ 1ꢃꢂ.
Anal. Calc. for C19H16O5S: C 64.03, H 4.53. Found: C 64.01, H 4.55.
3,5-Bis(4-hydroxy-3-methoxybenzylidene)-tetrahydrothiopyran-4-one
(E3). Yield 82%. mp 98–100 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 9.64 (brs, 2H, –OH), 7.57 (s, 2H, –CH=), 7.00 (s, 4H, ArH),
6.85 (m, 4H, ArH), 3.89 (s, 4H, –CH2–S–CH2–), 3.81 (s, 6H, OCH3).
LC–MS(m=z): 383 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C21H20O5S: C 65.61, H
5.24. Found: C 65.60, H 5.26.
Acute toxicity study in mice. The acute toxicity of C2 and B11 was
examined according to the OECD 423 Guideline for Testing of
Chemicals of Acute Oral Toxicity–Acute Toxic Class Method (OECD,
2001).38) Since the available information suggested that curcumin
analogs have low toxicity potential and that mortality was unlikely, the
test procedure was applied with a starting dose of 2,000 mg/kg of body
mass. Male and female Chinese Kun Ming (KM) mice (n ¼ 12, six
males and six females, 7 weeks old, 18–22 g), obtained from Center for
Animal Tests of Sun Yat-sen University, China, were housed in the
university-approved animal facility in rooms maintained at 22 ꢀ 2 ꢁC,
at 55–60% humidity under a 12-h photoperiod. After 5 d of adaptation
to laboratory conditions, the mice were divided into four equal groups
(controls, male and female, and treated, male and female, three animals
in each group, equal body masses) and given by gavage either a single
dose of 2,000 mg of sample (dispersed in 3% Tween 80 aqueous
solution)/kg of body mass and an equal volume of distilled water
containing 3% Tween 80 for the control groups, using a suitably
graduated syringe and a stainless steel intubation cannula. The mice
had free access to distilled water and a commercial standard diet. They
were observed individually at least once during the first 30 min after
dosing, and periodically during the first 24 h, with special attention
during the first 4 h, and daily thereafter, for a total of 14 d. All
observations were systematically recorded and records were main-
tained for each animal. The individual body masses of animals were
determined shortly before the test substance was administered and
weekly thereafter. All the animals were sacrificed at the end of the
observation period and subjected to necropsy.
3,5-Bis(3,5-ditertbutyl-4-hydroxylbenzylidene)-tetrahydrothiopyran-
4-one (E4). Yield 65%. mp 248–249 ꢁC. 1H NMR (DMSO, 300 MHz)
ꢁ (ppm): 7.54 (brs, 2H, –OH), 7.42 (s, 2H, –C=), 7.23 (s, 4H, –ArH),
3.98 (s, 4H, –CH2–S–CH2–), 1.40 (s, 36H, C–CH3). LC–MS (m=z):
547 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C35H48O3S: C 76.60, H 8.82. Found: C
76.58, H 8.79.
3,5-Bis(3,4-dimethoxybenzylidene)-tetrahydrothiopyran-4-one (E5).
Yield 62%. mp 134–135 ꢁC. 1H NMR (DMSO-d6, 300 MHz) ꢁ (ppm):
7.73 (s, 2H, CH=CH), 7.03 (dd, J ¼ 8:1, 2H, arom), 6.93 (s, 2H,
arom), 6.91 (dd, J ¼ 8:1, 2H, arom), 3.96 (s, 4H, –CH2–S–CH2–), 3.93
(s, 6H, –OCH3), 3.91 (s, 6H, –OCH3). LC–MS (m=z): 413 ½M þ 1ꢃþ.
Anal. Calc. for C23H24O5S: C 66.97, H 5.86. Found: C 66.86, H 5.90.
3,5-Bis(4-hydroxy-3,5-dimethoxybenzylidene)-tetrahydrothiopyran-
4-one (E6). Yield 82%. 149–151 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 8.93 (brs, 2H, –OH), 7.53 (s, 2H, –CH=), 6.80 (s, 4H, ArH),
4.03 (s, 4H, –CH2–S–CH2–), 3.80 (s, 12H, –OCH3). LC–MS (m=z):
411 ½M ꢂ 1ꢃꢂ. Anal. Calc. for C23H24O7S: C 62.15, H 5.44. Found:
C 62.13, H 5.47.
3,5-Bis(3-bromo-4-hydroxy-5-methoxybenzylidene)-tetrahydrothio-
pyran-4-one (E7). Yield 48%. mp 224–225 ꢁC. 1H NMR (DMSO-d6,
300 MHz) ꢁ (ppm): 10.09 (brs, 2H, –OH), 7.48 (s, 2H, –CH=), 7.26
(s, 2H, ArH), 7.11 (s, 2H, ArH), 3.99 (s, 4H, –CH2–S–CH2–), 3.87
(s, 6H, –OCH3). LC–MS (m=z): 541 ½M ꢂ 1ꢃꢂ. Anal. Calc. for
C21H18Br2O5S: C 46.52, H 3.35. Found: C 46.43, H 3.40.
Molecular modeling. To date, all attempts to determine exper-
imentally the X-ray structure of human tyrosinase have failed. To
overcome this problem, a homology model was made with the crystal
structure of a bacterium tyrosinase taken from Streptomyces casta-
neoglobisporus as template (PDB accession code 2ZWE). The crystal
structure of tyrosinase 2ZWE was a complex with caddie protein
ORF378 which bound in the active site of tyrosinase, and hence the
3,5-Bis(3,5-dibromo-4-hydroxylbenzylidene)-tetrahydrothiopyran-4-
one (E8). Yield 95%. mp 240–242 ꢁC. 1H NMR (DMSO-d6, 300 MHz)
ꢁ (ppm): 10.47 (br, 2H, OH); 7.82 (s, 4H, ArH), 7.45 (s, 2H, –CH=C–),
3.92 (s, 4H, –CH2–S–CH2–). LC–MS (m=z): 639 ½M ꢂ 1ꢃꢂ. Anal.
Calc. for C19H12Br4O3S: C 35.66, H 1.89. Found: C 35.60, H 1.91.
ORF378 was removed according to the method reported by Matoba Y
39)
et al.
For a docking study, all water molecules were removed,
AMBER charges were assigned, the orientations of the side chain
amides were corrected, and hydrogen atmos were added and their
positions were optimized by energy minimization using the AMBER7
FF99 force field. Compounds C2 and C11 were selected and built using
Sybyl 8.0 (Tripos, St. Louis, MO). After sketching of the molecules,
3,5-Bis(2-hydroxybenzylidene)-tetrahydrothiopyran-4-one (E9). Yield
74%. mp 171–172 ꢁC. 1H NMR (DMSO, 300 MHz) ꢁ (ppm): 9.98 (brs,
2H, –OH), 7.73 (s, 2H, –CH=), 7.27 (d, J ¼ 8:1, 2H, –ArH), 7.12
(m, 2H, –ArH), 6.90 (d, J ¼ 8:1, 2H, ArH), 6.84 (m, 2H, –ArH), 3.87
(s, 4H, –CH2–S–CH2–). LC–MS (m=z): 323 ½M ꢂ 1ꢃꢂ. Anal. Calc. for
Gasteiger-Huckel partial charges were automatically assigned. Energy
¨
evaluations were made using the Triopos force-filed. Geometry
optimizations were performed using 20 iterations of simplex followed
by 500 steps of steepest descent protocol and then 2,000 steps of
Powell algorithm minimization. The molecular energies of all com-
C
19H16O3S: C 70.35, H 4.97. Found: C 70.30, H 5.06.
pounds always converged within a gradient displacement criterion of
2
˚
0.001 kcal/A . Docking calculations were performed with Surflex-
3,5-Bis(2-hydroxy-4-methoxybenzylidene)-tetrahydrothiopyran-4-one
(E10). Yield 57%. mp 220–222 ꢁC. 1H NMR (DMSO, 300 MHz)
Dock on the Red Hat workstation. ‘‘Protomol’’ of Surflex-Dock was
used to guide molecular docking. The protomol was defined by setting
ꢁ (ppm): 9.85 (brs, 2H, –OH), 7.26 (d, J ¼ 8:1 Hz, 2H, ArH), 6.98

2358
Z.-Y. DU et al.
˚
the threshold value and the bloat value at 0.66 and 2 A respectively.
The binding pocket of tyrosinase has been defined with the residues
surrounding the dicopper ions, since they are implied in the recognition
of the L-DOPA substrate.39) The values of additional starting
conformations per molecule and maximum number of poses per ligand
were both expanded to 40 to increase the accuracy of the binding
mode. Other parameters were based on the software default setting.
Then the ligands were docked into the active side of tyrosinase under
the same conditions.
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This work was supported financially by National
Natural Science Foundation of China (no. 21042003), the
Science and Technology Planning Project of Guangdong
Province (2007A020300007-10), the Natural Science
Foundation of Guangdong Province (S2011010004967),
and the Guangdong Provincial Higher Education 211
Project (Batch 3) Foundation.
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