Discovery of 4-functionalized phenyl-O-β-d-glycosides as a new class of mushroom tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.09.018

A series of 4-functionalized phenyl-O-β-d-glycosides were designed, synthesized and evaluated as a new class of mushroom tyrosinase inhibitors. The results demonstrated that compounds 6a-13a bearing a thiosemicarbazide moiety exhibited potent activities with IC₅₀ values range from 0.31 to 52.8 μM. Particularly, compound 9a containing acetylated glucose moiety was found to be the most active molecule with an IC₅₀ value of 0.31 μM. SARs analysis suggested that (1) the thiosemicarbazide moiety remarkably contributed to the increase of inhibitory effects on tyrosinase; (2) the configuration and bond type of sugar moiety also played a very important role in determining their inhibitory activities. The inhibition kinetics and inhibition mechanism study revealed that compound 9a was reversible and competitive type inhibitor, whereas compound 13a was reversible and competitive-uncompetitive mixed-II type inhibitor.

Full text of DOI:10.1016/j.bmcl.2009.09.018

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6157–6160
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 4-functionalized phenyl-O-b-D-glycosides as a new class
of mushroom tyrosinase inhibitors
*
*
Wei Yi, Rihui Cao , Huan Wen, Qin Yan, Binhua Zhou, 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:
Received 23 June 2009
Revised 17 August 2009
Accepted 4 September 2009
Available online 10 September 2009
A series of 4-functionalized phenyl-O-b-
D
-glycosides were designed, synthesized and evaluated as a new
class of mushroom tyrosinase inhibitors. The results demonstrated that compounds 6a–13a bearing a thi-
osemicarbazide moiety exhibited potent activities with IC50 values range from 0.31 to 52.8 M. Particu-
larly, compound 9a containing acetylated glucose moiety was found to be the most active molecule with
an IC50 value of 0.31 M. SARs analysis suggested that (1) the thiosemicarbazide moiety remarkably con-
l
l
tributed to the increase of inhibitory effects on tyrosinase; (2) the configuration and bond type of sugar
moiety also played a very important role in determining their inhibitory activities. The inhibition kinetics
and inhibition mechanism study revealed that compound 9a was reversible and competitive type inhib-
itor, whereas compound 13a was reversible and competitive–uncompetitive mixed-II type inhibitor.
Ó 2009 Published by Elsevier Ltd.
Keywords:
4-Functionalized phenyl-O-b-
Tyrosinase inhibitors
SARs
D-glycosides
Kinetic analysis
Inhibition mechanism
Tyrosinase, a multifunctional type-3 copper-containing metal-
loenzyme, is widely distributed in microorganism, plants and ani-
mals with slightly different forms.1 It catalyzes two distinct
reactions of melanin biosynthesis: the o-hydroxylation of the ami-
of 8.4 mM, while its isomer
on mushroom tyrosinase activity.7 Recently, 4-hydroxyphenyl
b-maltoside (b-Ab- -G1) and 4-hydroxyphenyl b-maltotrioside
(b-Ab- -G2) were reported to exhibit more potent inhibitory ef-
fects on human tyrosinase than arbutin.8 More recently, we found
that the inhibitory effect of helicid, 4-formylphenyl-O-b- -allo-
pyranoside 6 (Fig. 1), and its analogues on mushroom tyrosinase
a-arbutin had no inhibitory effect
a
a
no acid
solase/monophenolase activity; EC 1.14.18.1), and subsequently
the oxidation of -DOPA to the corresponding o-dopaquinone (cat-
L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) (cre-
D
L
echolase/diphenolase activity; EC 1.10.3.1), which are the rate-lim-
iting steps in the pathway.2,3 Recent investigations showed that
the tyrosinase process was responsible not only for the undesired
enzymatic browning of fruits and vegetables but also for the for-
mation of various dermatological disorders, such as freckles, mel-
asma, ephelide and senile lentigines.4,5 Therefore, tyrosinase
inhibitors have become increasingly important in food industry
as well as in the medicinal and cosmetic products. In the last dec-
ades, a large number of naturally occurring and synthetic com-
pounds acting as tyrosinase inhibitors were reported,6–14 and the
most representative tyrosinase inhibitor so far is tropolone 1
N
OCH2CH3
OH
H3CO
O
S
OH
C
N
C
H3C
N
NH2
H
OH
IC50=0.4
1
μM
=0.3
2
μM
IC50=0.11
3
μ
M
IC50
OH
CHO
(Fig. 1) exhibiting an IC50 of 0.40 l
M.14 However, only few of them
OH
OH
H3C
H3C
S
are put into practical use due to their weak individual activities or
safety concerns. Undoubtedly, more efforts are still needed to
search and develop more effective and safe tyrosinase inhibitors.
C
N
C
O
O
HO
HO
N
NH2
HO
H
Ο
Ο
OH
OH
OH
b-Arbutin, hydroquinone-O-b-D-glucopyranoside 5 (Fig. 1), has
IC50=0.086
4
μM
IC50=8400
5
μM
been widely used as a whitening agent in cosmetics.6 It was re-
ported to show a diphenolase activity of tyrosinase with an IC50
IC50=2540
6
μM
Figure 1. Chemical structures of some known tyrosinase inhibitors tropolone (1),
3,4-dihydroxybenzaldehyde-O-ethyloxime (2), 1-(propan-2-ylidene)thiosemicar-
bazide (3), 1-[1-(4-methoxyphenyl)ethylidene]thiosemicarbazide (4), arbutin (5)
and helicid (6).
* Corresponding authors. Tel.: +86 20 84110918; fax: +86 20 84112245 (H.S.).
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), yjhxhc@mail.sysu.edu.cn
(H. Song).
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.09.018

6158
W. Yi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6157–6160
were superior to arbutin.9 These results indicated that the modi-
fication of glycosyl moiety of arbutin might facilitate their inhib-
itory effects on tyrosinase.
corresponding benzaldehyde glycosides 9–13 in 27–59% yield.
Hydrolyzation of 9 with sodium methoxide in methanol solution
gave compound 7 in 56% yield. Subsequently, the benzaldehyde
glycosides 6–13 reacted with thiosemicarbazide, hydroxylamine
and methoxyamine to produce Schiff base derivatives 6a–13a,
6b–13b and 6c–13c, respectively. All compounds synthesized were
characterized by chemical and spectral methods such as 1H NMR,
EI-MS and IR, and purities were confirmed by elemental
analysis.18,19
In addition, Ley and Bertram reported15 that benzaldoximes and
benzaldehyde-O-alkyloximes exhibited significant inhibitory activ-
ities against mushroom tyrosinase. Especially, the inhibitory effect
on tyrosinase of 3,4-dihydroxybenzaldehyde-O-ethyloxime
2
(Fig. 1) was comparable to tropolone. Our group also reported that
alkylidenethiosemicarbazides10 and 1-(1-arylethylidene)thiose-
micarbazides11 displayed remarkable inhibitory activities, and
the inhibitory effects on mushroom tyrosinase of 1-(propan-2-yli-
dene)thiosemicarbazide 3 (Fig. 1) and 1-[1-(4-methoxyphenyl)eth-
ylidene]thiosemicarbazide 4 (Fig. 1) were superior to tropolone.
These observations suggested that the thiosemicarbazide signifi-
cantly facilitated the increase of inhibitory effect on mushroom
tyrosinase.
The inhibition of our synthetic 4-functionalized phenyl-O-b-D-
glycosides on the diphenolase activity of mushroom tyrosinase
was investigated by usual procedure11 and compared with Arbutin.
The IC50 values of all obtained compounds were summarized in Ta-
ble 1. The benzaldehyde glycosides 6–13 were inactive to mush-
room tyrosinase at the concentration of 200 lM (data not
shown). Similarly, compounds 6b–13b and 6c–13c bearing
hydroxylamine and methoxamine moiety, respectively, also exhib-
ited no inhibitory effects on mushroom tyrosinase at the concen-
Inspired by these results, a series of novel 4-functionalized phe-
nyl-O-b-D-glycosides bearing thiosemicarbazide, oxime and meth-
yloxime were designed, synthesized and evaluated as a new class
of mushroom inhibitors. The purpose of this investigation was to
investigate the inhibitory effects on mushroom tyrosinase of 4-
functionalized phenyl-O-b-D-glycosides, with the ultimate aim of
developing novel potent tyrosinase inhibitors. To the best of our
knowledge, this is the first time to report that the inhibitory effect
tration of 200 lM. As predicted, compounds 6a–13a bearing a
Table 1
Inhibitory effects on mushroom tyrosinase of 4-functionalized phenyl-O-b-D-glyco-
sides as compared with arbutin
a
Compounds
IC50
(l
M)
Compounds
IC50 (lM)
of 4-functionalized phenyl-O-b-D-glycosides on the diphenolase
6a
7a
8a
9a
10a
11a
12a
13a
6b
7b
8b
9b
Arbutin
3.61 0.55
2.96 0.62
3.41 0.28
0.31 0.12
0.41 0.09
52.8 2.2
36.5 3.8
0.65 0.21
>200
10b
11b
12b
13b
6c
7c
8c
9c
10c
11c
12c
13c
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
activity of mushroom tyrosinase. We hope that these findings
may lead to the discovery of therapeutically potent agents against
clinically dermatological disorders including hyperpigmentation as
well as skin melanoma.
The synthesis of compounds 6–13, 7a–c and 9a–c has been de-
scribed in our previous report.16 The procedure for the preparation
of compounds 6a–13a, 6b–13b and 6c–13c was outlined in
Scheme 1. Briefly, helicid 6 reacted with acetyl anhydride in the
presence of anhydrous pyridine to provide 4-formylphenyl
>200
>200
>200
(2,3,4,6-tetra-O-acetyl)-b-D-allopyranoside 8. In the presence of
7300 600b
NaOH and tetrabutylammonium bromide (TBAB), the correspond-
ing glycosyl bromide, prepared according to the methods described
elsewhere,17 reacted with 4-hydroxybenzaldehyde to afford the
a
IC50 = mean SEM. SEM: standard error of mean.
Values in the literature2,7 is 5.3–8.4 mM.
b
OHC
OR
iii
6-13
ii
i
S
HO
N
H3CO
N
N
H2N
OR
OR
OR
N
H
6a-13a
OH
6b-13b
OH
6c-13c
OAc
O
O
O
8
R=
AcO
BzO
HO
HO
HO
6
9
R=
R=
7 R=
OAc
OH
OH
OAc
OBz
OH
OAc
AcO
AcO
OAc
O
O
O
AcO
AcO
11 R=
10 R=
13 R=
OAc
OBz
OAc
OBz
OAc
O
OAc
OAc
O
O
AcO
O
BzO
BzO
OAc
AcO
12 R=
OBz
OAc
Scheme 1. Synthesis of 4-functionalized phenyl-O-b-
D-glycosides 6a–13a, 6b–13b and 6c–13c. Reagents and conditions: (i) H2NHCSNH2/EtOH/reflux, 5–12 h; (ii)
NH2OHÁHCl/EtOH, pH 6–7, 45 °C, 2–6 h; (iii) NH2OCH3ÁHCl/EtOH, pH 6–7, 45 °C, 2–6 h.

W. Yi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6157–6160
6159
thiosemicarbazide moiety demonstrated significant tyrosinase
inhibitory effects with IC50 values range from 0.31 to 52.8 M.
These results suggested that the thiosemicarbazide moiety con-
with an IC50 value in the nanomolar range, and compound 9a was
l
found to be the most active molecule with an IC50 value of
0.31 lM comparable to tropolone (IC50 = 0.40 l
M),14 one of the
tributed to the increase of inhibitory effects.
well-known tyrosinase inhibitor. All these observations indicated
that (1) the configuration and bond type of sugar moiety played a
very important role in determining inhibitory activities; (2) the
lipophilic property of acetylated sugar moiety facilitated the inhib-
itory potency.
Of all glycoside thiosemicarbazides 6a–13a, compounds 6a
(IC50 = 3.61
l
M) and 7a (IC50 = 2.96
lM) bearing a b-D-allopyrano-
side and b-
D-glucopyranoside moiety, respectively, demonstrated
similar inhibitory effects on mushroom tyrosinase, whereas, their
tetra-O-acetyl congeners 8a (IC50 = 3.41 M) and 9a
(IC50 = 0.31 M) displayed distinct inhibitory activities, and com-
pound 9a exhibited 10-fold inhibitory potential comparable to
compound 8a. Obviously, tetra-O-aetyl-b- -glucopyranoside moi-
ety was more favourable. Replacement of tetra-O-acetyl-b- -gluco-
pyranoside of compound 9a with tetra-O-aetyl-b-
galactopyranoside moiety gave compound 10a (IC50 = 0.41 M),
l
The inhibition mechanism on the mushroom tyrosinase by 9a
and 13a for the oxidation of L-DOPA was investigated. Figure 2
l
showed the relationship between enzyme activity and concentra-
tion in the presence of different concentrations of 9a and 13a,
respectively. The plots of the remaining tyrosinase activity versus
the concentrations of tyrosinase in the presence of different con-
centrations of 9a and 13a gave a family of straight lines, which
all passed through the origin. Increasing the concentration of 9a
and 13a resulted in the decreasing slope of the lines, indicating
that the inhibition of 9a and 13a on the mushroom tyrosinase
was reversible. Previous literature20 reported that 1-phenylthio-
urea was defined as an irreversible inhibitor of tyrosinase, and sug-
gesting that it could exhibit strong affinity for copper ions in the
active centre. Our recent investigation11 also indicated that the
inhibitory effect of 1-(1-phenylethylidene)thiosemicarbazide was
an irreversible inhibitor. Obviously, the inhibition mechanism of
9a and 13a was different from 1-phenylthiourea and 1-(1-phenyl-
ethylidene)thiosemicarbazide. By analogy with their chemical
structures, these results suggested that the introduction of sugar
moiety might affect the binding mode of the substrate and the ac-
tive site of tyrosinase.
D
D
D
-
l
which exhibited comparative inhibitory potency. Unfortunately,
replacement of acetyl substituents of compound 9a with benzoyl
groups to afford compound 11a exhibited sharply decreased inhib-
itory effect with IC50 value of 52.8
lM. Interestingly, compound
12a (IC50 = 36.5 M) bearing a tri-O-benzoyl-b-
l
D
-xylopyranoside
moiety had more potent inhibitory activity than compound 11a.
To further investigate the inhibitory effect of the molecular
dimension of glycosides, compound 13a bearing a hepta-O-acetyl-
b-
As shown in Figure 3, compound 13a also exhibited significant
inhibitory potency with IC50 value of 0.65 M. Particularly, com-
D-lactoside was examined for its tyrosinase inhibitory activity.
l
pounds 9a, 10a and 13a exhibited remarkable inhibitory activities
1
A
The kinetic behaviours of 9a and 13a on the mushroom tyrosi-
0.30
nase, during the oxidation of
L-DOPA, were determined from
0.25
0.20
3
2
80
A
70
0.15
2
60
50
40
30
20
10
0.10
3
0.05
0.00
1
0
2
4
6
8
10
12
[E] (μg/mL)
0
-14 -12 -10 -8 -6 -4 -2
0
2
4
6
8
10 12 14
1/[S](m M -1
)
B 0.30
1
3
2
0.25
0.20
0.15
0.10
0.05
0.00
40
30
20
10
0
B
1
2
3
-14 -12 -10 -8 -6 -4 -2
-10
0
2
4
6
8
10 12 14
1/[S](mM-1)
-20
-30
-40
0
1
2
3
4
5
6
7
8
9
10
[E] (μg/mL)
Figure 2. Effects of concentrations of tyrosinase on its activity for the catalysis of
-DOPA at different concentrations. A and B represented the inhibition effectory of
compounds 4a and 6a, respectively. The concentrations of compound 4a for curves
1–3 are 0, 1.20 and 2.40 M, respectively. And the concentrations of compound 6a
for curves 1–3 are 0, 1.02 and 2.04 M, respectively.
Figure 3. Lineweaver–Burk plots for inhibition of helicid analogues on mushroom
tyrosinase for the catalysis of -DOPA. A and B represented inhibition effect of
compounds 4a and 6a, respectively. The concentrations of compound 4a for curves
1–3 are 0, 0.06 and 0.12 M, respectively. And the concentrations of compound 6a
for curves 1–3 are 0, 0.05 and 0.10 M, respectively.
L
L
l
l
l
l

6160
W. Yi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6157–6160
Lineweaver–Burk double reciprocal plots. The results (Fig. 3A)
showed that the plots of 1/V versus 1/[S] gave a family of straight
lines with different slopes that intersected one another in the Y-
axis. The value of Vm remained the same and the value of Km in-
creased with the concentrations of 9a, indicating that it was a com-
petitive inhibitor of mushroom tyrosinase. Interestingly, Figure 3B
showed that the inhibition behaviour of 13a was different from
that of 9a. The Lineweaver–Burk double reciprocal plots in the
presence of 13a yielded a family of straight lines with different
slopes that intersected one another in the third quadrant, indicat-
References and notes
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Boumendjel, A. Bioorg. Med. Chem. Lett. 2006, 16, 2252.
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ing that it was
inhibitor.
a competitive–uncompetitive mixed-II type
Walker and Wilson21 reported that tyrosinase had two distinct
sites of combination for the binding of the substrate and the inhib-
itor. Recently, the crystallographic structure of tyrosinase has been
established,22 enabling a close look at its three-dimensional struc-
ture and a better understanding of its mechanism of action. This
three-dimensional structure revealed that one site of combination
was the binuclear copper ions active centre and the other site of
combination was the hydrophobic enzyme pocket active site
adjoining the binuclear copper ions active site, which is composed
of some bioactive amino acids, such as Arg 55, Trp 184, Glu 182, Ile
42, His 190 and Ala 202.23 The inhibition kinetics of 9a and 13a led
us to hypothesize that 9a interacted not only with the binuclear
copper ions active site, but also with the hydrophobic protein do-
main surrounding the binuclear copper active site. The sulfur atom
of the thiosemicarbazide moiety chelate the binuclear copper of
tyrosinase, and such interaction acted as a bridge to link the acet-
ylated glucose moiety and the hydrophobic protein pocket, which
facilitated the acetylated glucose moiety to approach the hydro-
phobic protein pocket. Such interactions resulted in inhibiting
14. Kahn, V.; Andrawis, A. Phytochemistry 1985, 24, 905.
15. Ley, J. P.; Bertram, H. J. Bioorg. Med. Chem. 2001, 9, 1879.
16. Wen, H.; Lin, C. L.; Que, L.; Ge, H.; Ma, L.; Cao, R. H.; Wan, Y. Q.; Peng, W. L.;
Wang, Z. H.; Song, H. C. Eur. J. Med. Chem. 2008, 43, 166.
17. (a) Flecher, H. G.; Hudson, C. S. J. Am. Chem. Soc. 1947, 69, 921; (b) Ness, R. K.;
Flecher, H. G.; Hudson, C. S. J. Am. Chem. Soc. 1951, 73, 959.
18. General procedures for the synthesis of compounds 6a–13a: The appropriate
compounds 6–13 (1 mmol) were dissolved in anhydrous ethanol (15 mL),
thiosemicarbazide (1 mmol) was added to the above solution. The reaction
mixture was refluxed for 5–12 h and cooled to room temperature. The
precipitate was filtered, washed with ethyl ether, and recrystallized from
95% alcohol to give compounds 6a–13a in 56–91% yields. Compound 8a: White
solid powder, yield 79%, mp 177–178 °C. IR (KBr, cmÀ1
) m: 3452, 3301, 1753,
1579, 1503, 1409, 1226, 1092, 1046, 714; 1H NMR (DMSO-d6, 300 MHz) d:
10.04 (s, 1H, NH), 7.89 (s, 1H, –CH@N–), 7.61 (d, J = 8.7 Hz, 2H, Ar-H), 7.19 (s,
1H, NH), 7.04 (d, J = 8.7 Hz, 2H, Ar-H), 6.49 (s, 1H, NH), 5.75 (t, J = 3.0 Hz, 1H,
CH), 5.42 (d, J = 8.1 Hz, 1H, CH), 5.17 (dd, J = 8.1 Hz, 3.1 Hz, 1H, CH), 5.07 (dd,
J = 9.6 Hz, 2.7 Hz, 1H, CH); 4.28 (d, J = 4.2 Hz, 1H, CH), 4.27–4.23 (m, 2H, CH),
2.18, 2.10, 2.06, 2.05 (4 Â s, 4 Â 3H, 4 Â CH3CO); 13C NMR (DMSO-d6, 75 MHz)
d: 178.5, 171.3, 171.2, 170.3, 158.3, 143.2, 129.8, 129.6, 158.3, 117.4, 96.5, 70.8,
69.7, 68.1, 66.7, 62.6, 21.3, 21.2; ESI-MS: m/z 526 (M+1, 33.5), 548 (M+Na, 100).
Anal. Calcd for C22H27N3O10S: C, 50.28; H, 5.18; N, 8.00. Found: C, 49.95; H,
4.91; N, 7.75.
the combination of the substrate L-DOPA and the binuclear copper
active site, whereas the large size of lactoside moiety of 13a hin-
dered it to approach the activity centre of enzyme, and compound
13a could only bind with free enzyme.
In conclusion, the present investigation reported for the first
time the inhibitory effects of 4-functionalized phenyl-O-b-
sides on the diphenolase activity of mushroom tyrosinase for the
oxidation of -DOPA. Compound 9a bearing acetylated glucose
moiety was found to be the most active molecule with an IC50 va-
lue of 0.31 M. SARs analysis suggested that (1) the thiosemicarba-
D-glyco-
L
19. General procedures for the synthesis of compounds 6b–13b and 6c–13c. To the
solution of compounds 6–13 (1 mmol) in 15 mL of ethanol was added
hydroxylamine hydrochloride or methoxylamine hydrochloride (1 mmol)
which had been adjusted with 2 M NaOH to pH 6–7. The mixture was stirred
for 6 h at 45 °C. After being cooled, the formed precipitate was filtered off,
washed with ether and recrystallized from 95% ethanol to afford compounds
6b–13b and 6c–13c in 46–85% yields. Compound 6c: White solid powder, yield
l
zide moiety contributed to the increase of inhibitory effects on
tyrosinase; (2) the configuration and bond type of sugar moiety
also played a very important role in determining their inhibitory
activities. The inhibition kinetics and inhibition mechanism study
revealed that compound 9a was reversible and competitive type
inhibitor, whereas compound 13a was reversible and competi-
tive–uncompetitive mixed-II type inhibitor. All these data sug-
gested that these molecules might be served as candidates for
further development of drug for the treatment of dermatological
disorders.
66%, mp 83–84 °C. IR (KBr, cmÀ1
) m: 3355, 2930, 1608, 1509, 1236, 1046, 916,
539; 1H NMR (DMSO-d6, 300 MHz) d: 8.15 (s, 1H, –CH@N–), 7.53 (d, J = 8.7 Hz,
2H, Ar-H), 7.03 (d, J = 8.7 Hz, 2H, Ar-H), 5.15 (d, J = 7.2 Hz, 1H), 3.93–3.91 (m,
1H, CH Â 2), 3.86 (s, 3H, OCH3), 3.69–3.65 (m, 1H, CH), 3.46–3.40 (m, 3H,
CH Â 3); 13C NMR (DMSO-d6, 75 MHz) d: 159.8, 149.0, 129.1, 126.5, 117.2, 99.5,
75.4, 72.5, 71.0, 67.7, 62.2, 61.8; ESI-MS: m/z 314 (M+1, 100). Anal. Calcd for
C14H19NO7: C, 53.67; H, 6.11; N, 4.47. Found: C, 53.38; H, 6.32; N, 4.55.
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The authors thank the Natural Science Foundation of Guang-
dong Province, China (2004B30101007), and Kunming Baker Nor-
ton Pharmaceutical Co. Ltd for financial support on this study.