Synthesis and biological evaluation of helicid analogues as mushroom tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.056

A series of helicid analogues were synthesized and evaluated as tyrosinase inhibitors. The results demonstrated that some compounds had more potent inhibitory activities than arbutin (IC₅₀ 7.3 mM). In particular, compound 1c bearing 4,6-O-benzylidene substituent on the sugar moiety was found to be the most potent inhibitor with IC₅₀ value of 0.052 mM. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that helicid analogues were competitive inhibitors. The Circular dichroism spectra indicated that those compounds induced conformational changes of mushroom tyrosinase upon binding.

Full text of DOI:10.1016/j.bmcl.2008.10.056

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6490–6493
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of helicid analogues as mushroom
tyrosinase inhibitors
*
*
Wei Yi, Rihui Cao , Huan Wen, Qin Yan, Binhua Zhou, Yiqian Wan, Lin Ma, Huacan Song
School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of helicid analogues were synthesized and evaluated as tyrosinase inhibitors. The results dem-
onstrated that some compounds had more potent inhibitory activities than arbutin (IC50 7.3 mM). In par-
ticular, compound 1c bearing 4,6-O-benzylidene substituent on the sugar moiety was found to be the
most potent inhibitor with IC50 value of 0.052 mM. The inhibition kinetics analyzed by Lineweaver–Burk
plots revealed that helicid analogues were competitive inhibitors. The Circular dichroism spectra indi-
cated that those compounds induced conformational changes of mushroom tyrosinase upon binding.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 27 June 2008
Revised 8 September 2008
Accepted 13 October 2008
Available online 17 October 2008
Keywords:
Helicid analogues
Tyrosinase inhibitors
Competitive inhibitor
Inhibition kinetics
Circular dichroism
Tyrosinase (EC 1.14.18.1), a multifunctional copper-containing
enzyme, is widely distributed in nature. It catalyzes two distinct
reactions of melanin synthesis: the o-hydroxylation of monophe-
nols and the oxidation of o-diphenols to o-quinones. Quione is a
highly reactive compound and can polymerize spontaneously to
form melanin by a series of non-enzymatic reactions. Although
melanin is principally responsible for skin color and plays a crucial
role against skin photocarcinogenesis, abnormal melanin pigmen-
tation can cause some dermatological disorders associated with
freckles, melasma, ephelide and senile lentigines.1
Tyrosinase inhibitors are clinically useful for the treatment of
some dermatological disorders associated with melanin hyperpig-
mentation and also important in the cosmetic industry for whiten-
ing and depigmentation after sunburn.2–4 Many efforts have been
spent in the search for effective and safe tyrosinase inhibitors,
and a large number of naturally occurring and synthetic tyrosinase
inhibitors have already been reported.5–10 But some of their indi-
vidual activities are either not potent enough to be considered of
practical use or not compatible with safety regulations for food
and cosmetic additives. Therefore, it is still necessary to search
and develop novel tyrosinase inhibitors with potent activities
and lower side effects.
been used clinically as antalgic and hypnotic for a long time in
China and no obvious side effect has been reported.11,12 More
recently, our group reported that helicid analogues exhibited
potent acetylcholinesterase (AChE) inhibitory effects.13 Arbutin,
4-hydroxylphenyl-O-b-
D-glucopyranoside (Fig. 1), was reported
to show a dose-dependent inhibitory effect on the oxidation of
L
-DOPA catalyzed by mushroom tyrosinase with an IC50 of
8.4 mM.14 It has been widely used as a whitening agent in cosmet-
ics.15 By analogy with arbutin’s chemical structure, helicid and its
analogues are thought to be potential inhibitors of mushroom
tyrosinase.
It is well known that, within the structure of tyrosinase, there
are two copper ions in the active center of tyrosinase and a lipo-
philic long-narrow gorge near to the active center, and introducing
the proper lipophilic group into the inhibitor can exhibited potent
tyrosinase inhibitory activity. Therefore, in the present investiga-
tion, a series of helicid analogues bearing various lipophilic group
in glycosyl moiety were synthesized and their tyrosinase inhibitory
capacity were also evaluated. In addition, the inhibition kinetics
and mechanisms of action of the selected compounds were also
Helicid, 4-formylphenyl-O-b-D-allopyranoside (Fig. 1), was orig-
inally isolated as one of the main active constituents from Helicid
nilgrinica Bedd, a plant indigenous to western China, which has
OH
OH
O
O
OH
HO
HO
OH
O
HO
CHO
O
OH
Arbutin
OH
* Corresponding authors. Tel.: +86 20 84110918; fax: +86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), yjhxhc@mail.sysu.edu.cn
(H. Song).
Helicid
Figure 1. Chemical structures of helicid and arbutin.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.056

W. Yi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6490–6493
6491
CPh3
O
OH
O
O
OH
O
HO
CHO
O
O
HO
CHO
O
HO
Ph
CHO
O
v
i
iii
O
OH
OH
1
OH
OH
O
1a
H3C CH3
1d
ii
iv
OPO(OCH3)2
OH
O
O
O
O
O
CHO
O
CHO
HO
O
CHO
AcO
OH
OH
OH
OH
OAc
OAc
1e
1b
1c
Scheme 1. Synthesis of compounds 1a–e. Reagents and conditions: (i) C(Ph)3Cl, helicid, pyridine, rt, 20 h, 68%; (ii) a—Ac2O, pyridine; b—I2, NaS2O3, rt, 12 h, 59%; (iii) helicid,
I2, NaS2O3, rt, 4 h, 56%; (vi) POCl3, Et3N, CHCl2, CH3OH, CH3ONa, 0 °C, 2 h, 83%; (v) helicid, benzaldehyde, dimethyl acetal, 4-methylbenzenesulfonic acid, DMF, 50 °C, 12 h, 70%.
OH
O
OAc
O
HO
HO
CHO
O
AcO
AcO
CHO
O
OH
2d
OAc
2a
AcO
AcO
OAc
OAc
O
O
CHO
HO
O
CHO
HO
O
AcO
2b
OAc
OAc
2
O
O
CHO
O
CHO
O
BzO
OH
OBz
OH
2e
OBz
2c
Figure 2. Synthesis of compounds 2a–e.
investigated. To the best of our knowledge, this is the first report
about inhibitory effects of helicid analogues on the diphenolase
activity of mushroom tyrosinase.
Table 1
Inhibitory effects on mushroom tyrosinase of helicid and its analogues as compared
with the standard inhibitors.
The synthesis of helicid analogues 1a–e and 2a–e was summa-
rized in Scheme 1 and Figure 2. The reaction of helicid with chlo-
rotriphenylmethane in anhydrous pyridine gave the expected
derivatives 1a in 68% yield. Compound 1a was first acetylated then
selectively deprotected to provide the target compound 1b. Helicid
was reacted with benzaldehyde and dimethyl acetal in anhydrous
DMF in the presence of 4-methylbenzenesulfonic acid to afford the
derivative 1c in good yield. Compound 1d was prepared from the
starting material helicid and anhydrous acetone using iodine as
catalyst. The synthesis of compound 1e was carried out by reacting
compound 1d with POCl3 in anhydrous dichloromethane, followed
by esterification with anhydrous methanol in the presence of so-
dium methoxide. The preparation of compounds 2a–e has been de-
scribed in our previous report.7 The structures of the synthesized
compounds were determined by different spectroscopic tech-
niques, like 1H NMR, EIMS and IR, and purities were confirmed
by elemental analysis.
The inhibition of our synthetic helicid analogues and helicid on
the mushroom tyrosinase was investigated by usual procedure16
and compared with those of 4-methoxycinnamic acid and arbutin.
The IC50 value of all compounds investigated is summarized in Ta-
ble 1. As shown in Table 1, parent compounds helicid 1 and 4-
hydroxybenzaldehyde 2 exhibited weak inhibitory activities with
IC50 of 2.54 and 1.12 mM, respectively. Compounds 1a–b and 2a–
c had no clear inhibitory effect on tyrosinase activity at concentra-
tion of 3.0 mM, while compounds 1c–e and 2d–e demonstrated
potent inhibitory activity with IC50 value of less than 1 mM. Inter-
estingly, compound 1c bearing 4,6-O-benzylidene substituent on
the sugar moiety showed the most potent inhibitory effect on
mushroom tyrosinase with IC50 value of 0.052 mM. Preliminary
structure–activity relationships (SARs) analysis indicated that (1)
a
Compound
IC50 (mM)
Inhibition rate (%) at 0.1 mM
1
1a
1b
1c
1d
1e
2
2a
2b
2c
2d
2e
2.54
>3.0
>3.0
0.052
0.62
0.43
1.12b
>3.0
>3.0
>3.0
0.94
0.28
7.3c
10
Ni
Ni
77
13
24
15
Ni
5.0
Ni
30
42
Ni
22
Arbutin
4-Methoxycinnamic acid
0.41d
a
Assay performed using mushroom tyrosinase. Values are means of three dif-
ferent experiments.
Values in the literature4 is 1.2 mM.
b
Values in the literature14,17 is 5.3–8.4 mM.
c
Values in the literature17 is 0.42 mM.
d
introducing 4,6-O-benzylidene (compound 1c), 2,3-O-isopropyli-
dene (compound 1d) and 6-O-dimethyl phosphate (compound
1e) into sugar moiety facilitated inhibitory activity on tyrosinase,
These results confirmed that the lipophilic property might play a
vital role in determining their inhibitory activities. Among these
compounds, 4,6-O-benzylidene was more favorable; (2) the config-
uration of sugar moiety affected greatly inhibitory activity on
mushroom tyrosinase, compound 2d bearing a b-
side (IC50 = 0.94 mM) was more active than helicid 1 bearing a b-
-allopyranoside (IC50 = 2.54 mM), which suggested that the bind
type in sugar moiety can make the inhibitory activities of tyro-
D-glucopyrano-
D

6492
W. Yi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6490–6493
sianse change obviously; (3) the ribose moiety (compound 2e) was
more preferable than glucose (compound 2d and helicid 1). These
results indicated that the increase of hydroxyl group might cause
stereo-hindrance for the inhibitors approaching to the active site
of the enzyme.
induced conformational changes of mushroom tyrosinase upon
binding.
Safety is a primary consideration for tyrosinase inhibitors,
especially for those materials used in food and cosmetic prod-
ucts. Compounds 1c and 2d were selected to evaluate acute tox-
icity in mice. Clinical symptom was measured for 20 days after
the oral single gavage administration of 1200 mg/kg. The results
showed that all of the mice after administration at dose of
1200 mg/kg body weight/day did not show any mortality, and
autopsy of the animals at the end of the experimental period
(20 days) revealed no apparent changes in any organs. The re-
sults indicated that the compound 1c and 2d were safe at dose
of 1200 mg/kg in mice.
The kinetic behavior of helicid and its analogues 1c and 2d on
the mushroom tyrosinase, during the oxidation of L-DOPA, were
determined from Lineweaver–Burk double reciprocal plots. The
Lineweaver–Burk plots from Figure 3 indicated that they were
competitive inhibitors of the mushroom tryrosinase. These results
suggested that helicid and its analogues may only bind with the
free enzyme.
The influence of helicid 1 and its analogues 1c and 2d on the
secondary structure of mushroom tyrosinase was also investi-
gated by Circular dichroism spectroscopy.18,19 The results were
In conclusion, the present investigation reported the effects of
helicid analogues on the diphenolase activity of mushroom
summarized in Table 2. As shown in Table 2, the
a-helical con-
tyrosinase for the oxidation of L-DOPA. SARs analysis indicated
tent of tyrosinase was 48.4% in the absence of inhibitor and the
b-sheet was not found. However, the b-sheet was noticed in the
presence of inhibitors, and its contents were increased gradually
with the increase of the concentration of the inhibitors. On the
other hand, it was observed that the content of rigidity structure
that the nature of substituents on sugar moiety, the configura-
tion and type of sugar moiety affected inhibitory activity on
mushroom tyrosinase. The inhibition kinetics analyzed by
Lineweaver–Burk plots revealed that helicid and its analogues
were competitive inhibitors. Preliminary acute toxicity assay
demonstrated that helicid analogues were safe as tyrosinase
inhibitors. All these data suggested that these molecules might
be served as candidates for further development of drug for
the treatment of dermatological disorders.
(a-helix + b-sheet) of tyrosinase was increased while that of loop
structure (b-turn + random) of tyrosinase was decreased accom-
panying the increasing of concentration of helicid and its ana-
logues. These results indicated that helicid and its analogues
22
60
A
B
20
55
50
45
40
35
30
25
20
15
10
5
18
16
14
12
10
8
6
4
2
0
0
-5
-4
-3
-2
-1
0
4
7
-5
-4
-3
-2
-1
0
1
3
4
5
6
7
1
3
5
2
6
2
1/[S](mM-1
)
1/[S](mM-1)
C
20
18
16
14
12
10
8
6
4
2
0
-6
-5
-4
-3
-2
-1
0
1
4
5
7
2
6
3
1/[S](mM-1)
Figure 3. Lineweaver–Burk plots for inhibition of helicid analogues on mushroom tyrosinase for the catalysis of
compounds 1, 1c and 2d, respectively.
L-DOPA. (A), (B),. and (C) represents inhibition effectory of

W. Yi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6490–6493
6493
Table 2
The influence of helicid and its analogues on the secondary structure of tyrosinase.
Compound
Concentration (lM)
a-Helix (%)
b-Sheet (%)
a
-Helix+b-Sheet (%)
b-Turn (%)
Random (%)
b-Turn+Random (%)
1
0
80
160
48.4
33.9
40.3
0
19.3
20.5
48.4
53.2
60.8
18.6
17.1
13.5
33.0
29.7
25.7
51.6
46.8
39.2
1c
80
160
62.2
49.2
17.0
29.5
79.2
78.7
8.5
7.1
12.3
14.2
20.8
21.3
2d
80
160
28.4
31.1
33.4
34.7
61.8
65.8
10.8
9.5
27.4
24.7
38.2
34.2
5. Kobayashi, Y.; Kayahara, H.; Tadasa, K.; Tanaka, H. Bioorg. Med. Chem. Lett. 1996,
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