Design and synthesis of novel hydroxypyridinone derivatives as potential tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.05.006

Two groups of novel hydroxypyridinone derivatives 6(a-e) and 12(a-c), were designed as potential tyrosinase inhibitors, and synthesized using kojic acid as a starting material. The tyrosinase inhibitory activity of these two groups was demonstrated to be potent, especially compounds 6e and 12a, whose IC₅₀ values for monophenolase activity were 1.95 μM and 2.79 μM, respectively. Both of these values are lower than that of kojic acid (IC₅₀ = 12.50 μM). Compounds 6e and 12a were investigated for the inhibitory effect on diphenolase activity. The results showed that the inhibitory mechanism of these two compounds was reversible and that the inhibitory type was a competitive-uncompetitive mixed-type. The values of IC₅₀ of 6e and 12a on the diphenolase activity of tyrosinase were determined to be 8.97 μM and 26.20 μM, respectively. The inhibitory constants (K_I and K_IS) of 6e were determined as 17.17 μM and 22.09 μM, respectively; and the K_I and K_IS values of 12a were 34.41 μM and 79.02 μM, respectively. Compound 6e showed a greater ability to reduce copper and a stronger copper chelating ability than kojic acid.

Full text of DOI:10.1016/j.bmcl.2016.05.006

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel hydroxypyridinone derivatives
as potential tyrosinase inhibitors
De-Yin Zhao a, Ming-Xia Zhang a, Xiao-Wu Dong b, Yong-Zhou Hu b, Xiao-Yan Dai a, Xiaoyi Wei c,
Robert C. Hider d, Jin-Chao Zhang e, Tao Zhou a,
⇑
a School of Food Science and Biotechnology, Zhejiang Gongshang University, Xiasha, Hangzhou, Zhejiang 310018, PR China
b Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
c College of Tourism & Food, Shanghai Business School, Shanghai 200235, PR China
d Division of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
e Chemical Biology Key Laboratory of Hebei Province, Hebei University, Baoding 071002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two groups of novel hydroxypyridinone derivatives 6(a–e) and 12(a–c), were designed as potential
tyrosinase inhibitors, and synthesized using kojic acid as a starting material. The tyrosinase inhibitory
activity of these two groups was demonstrated to be potent, especially compounds 6e and 12a, whose
Received 10 December 2015
Revised 20 April 2016
Accepted 3 May 2016
Available online xxxx
IC50 values for monophenolase activity were 1.95
lM and 2.79 lM, respectively. Both of these values
are lower than that of kojic acid (IC50 = 12.50 M). Compounds 6e and 12a were investigated for the
l
inhibitory effect on diphenolase activity. The results showed that the inhibitory mechanism of these
two compounds was reversible and that the inhibitory type was a competitive-uncompetitive mixed-
type. The values of IC50 of 6e and 12a on the diphenolase activity of tyrosinase were determined to be
Keywords:
3-Hydroxypyridinone
Tyrosinase inhibitor
Inhibitory mechanism
8.97
lM and 26.20
l
M, respectively. The inhibitory constants (KI and KIS) of 6e were determined as
M, respectively; and the KI and KIS values of 12a were 34.41 M and 79.02 M,
17.17
lM and 22.09
l
l
l
respectively. Compound 6e showed a greater ability to reduce copper and a stronger copper chelating
ability than kojic acid.
Ó 2016 Elsevier Ltd. All rights reserved.
Tyrosinase (EC 1.14.18.1), a multifunctional copper-containing
enzyme from the oxidase superfamily, is widely distributed in
mammals, plants, microorganisms, and insects.1 The active site of
tyrosinase is well conserved among the different species. Six his-
tidine residues, which are provided by a four helical bundle, coor-
dinate the two copper ions, which serve as the major cofactors in
the active site.2,3 Tyrosinase can catalyze the first and rate-limiting
principle, tyrosinase inhibitors have potential applications in the
agricultural, cosmetic and pharmaceutical industry.
Numerous effects have been made to develop potent and safe
tyrosinase inhibitors from natural materials and synthetic meth-
ods.15–19 However, only a few are sufficiently potent for practical
use and comply with the general safety regulations. Thus, there
is a demand for novel tyrosinase inhibitors with superior activity
together with reduced side effects. Kojic acid (1), a metabolic pro-
duct of many species of Aspergillus and Penicillium moulds, has
been used as an ingredient in cosmetics and as an anti-browning
agent in foods that rapidly change colour by virtue of its apprecia-
ble anti-tyrosinase and antioxidant activities.20–23 Thus, modifica-
step of melanin formation, namely the hydroxylation of
L
-tyrosine
to -3-(3,4-dihydroxyphenyl)-alanine ( -DOPA) (monophenolase
L
L
activity) and also the subsequent oxidation of DOPA to dopaqui-
none (diphenolase activity).4 Dopaquinone is highly reactive and
can polymerize spontaneously to form melanin in a series of reac-
tion pathways.5 Therefore, tyrosinase plays an important role in
the pigmentation of skin,6,7 the browning of fruits and vegeta-
bles,8,9 wound healing,10 and cuticle formation in insects.11,12
Tyrosinase is also involved in neuromelanin formation in the brain
and neurodegeneration associated with Parkinson’s disease.13,14
Thus, tyrosinase inhibitors have attracted increasing attention. In
tion of kojic acid provides
a potential route for superior
tyrosinase inhibitors.24–27 Kojic acid has been demonstrated to
inhibit tyrosinase by chelating the copper ion normally present
in the active site of tyrosinase. Using the principle of bioisosterism,
we have changed the ‘O’ at position-1 in pyranone ring to ‘NH’,
synthesizing
a range of hydroxypyridinone–amino acid and
hydroxypyridinone–dipeptide conjugates. Their tyrosinase inhibi-
tory activity and mechanism of inhibition have been investigated.
⇑
Corresponding author. Tel.: +86 571 28008976; fax: +86 571 88905733.
E-mail address: taozhou@zjgsu.edu.cn (T. Zhou).
http://dx.doi.org/10.1016/j.bmcl.2016.05.006
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhao, D.-Y.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.006

2
D.-Y. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Hydroxypyridinone–
L
-amino acid conjugates (6) were synthe-
ure 1A, the inhibitory activity of compounds 6 follows the order:
6e > 6a > 6d > 6c > 6b. The IC50 values of compounds 6a–6e were
calculated to be 11.76, 28.71, 15.62, 12.48 and 1.95 lM respec-
tively. Among these five hydroxypyridinone–amino acid deriva-
tives, compound 6e was found to exhibit the strongest inhibition
against monophenolase activity of tyrosinase, being 6.4-fold more
sized starting from kojic acid (1) (Scheme 1). Benzylation of 5-
hydroxy group in kojic acid was achieved by the reaction of kojic
acid and benzyl chloride under basic condition to provide 2 in good
yield (80%). Condensation of 2 with ammonia generated 3 (74%),
which was then subjected to hydrogenation to remove benzyl
group, providing compound 4 (82%). Coupling of 4 with Cbz-
L
-
active than kojic acid (IC50 = 12.50
lM). Based on the data pre-
amino acid (5) via ester bond was carried out in the presence of
EDC and DMAP in DMF at room temperature, yielding product 6.28
The synthetic route of hydroxypyridinone–dipeptide conjugates
(12) is presented in Scheme 2. Compound 3 was treated with
thionyl chloride, followed by the reaction with aqueous ammonia,
providing amino group-containing compound 7. N-Benzyl oxygen
sented in Figure 1B, the IC50 values of compounds 12a–12c were
calculated to be 2.79, 6.93 and 14.26
lM, respectively. Compounds
90
A
80
70
60
50
40
30
20
10
carbonyl-
L
-phenylalanine (5a) reacted with methyl ester of
L-amino acid (8) in the presence of HCTU, generating compound
9, which were then hydrolyzed to produce compound 10. The cou-
pling of 10 with 7 was achieved in the presence of HCTU, yielding
compound 11, which were subjected to hydrogenation in the pres-
ence of Pd/C catalyst, providing hydroxypyridinone–dipeptide con-
jugates 12.29 All the compounds have been fully characterized by
1H NMR, 13C NMR and HRMS.
6a
6b
6c
6d
Inhibitory activity of hydroxypyridinone derivatives 6 and 12
on monophenolase activity of mushroom tyrosinase was investi-
6e
kojic acid
gated using
-tyrosine as a substrate.30 The inhibitory effects on
L
0
10
20
30
40
50
mushroom tyrosinase increased with the increase of concentra-
tions of hydroxypyridinone derivatives (Fig. 1). As shown in Fig-
Concentration of inhibitor
(μM)
70
60
50
40
30
20
B
O
O
O
O
OH
OBn
OBn
OH
HO
HO
HO
HO
N
H
c
N
H
O
a
b
O
2
3
4
1
12a
12b
12c
6a: R = CH3
R
O
6b: R = CH2CH(CH3)2
6c: R = CHCH2CH3
Cbz
OH
OH
N
H
kojic acid
R
O
CH3
Cbz
O
5
N
N
6d: R = CH(CH3)2
H
H
d
O
H C
6e: R =
2
6 ( a-e )
0
10
20
30
40
50
60
70
Concentration of inhibitor (μM)
Scheme 1. Reagents and conditions: (a) BnCl, MeOH/H2O, 70 °C, 6 h, 80% yield; (b)
aqueous NH3 (37%), EtOH, reflux, overnight, 74% yield; (c) MeOH/H2O, H2 (30 psi),
5% Pd/C, rt, 8 h, 82% yield; (d) Cbz-
then rt overnight.
L
-amino acid (5), EDC, DMAP, DMF, ice-bath, 2 h;
Figure 1. Inhibition on monophenolase activity of tyrosinase. (A) Compound 6; (B)
compound 12.
O
O
OBn
OBn
H2N
HO
a
N
H
N
H
7
3
O
R
H
H3N
Cl
COOCH3
O
R
H
O
H
N
N
Cbz
O
N
OH
OH
Cbz
N
Cbz
7
N
R
8
H
H
O
O
c
d
b
9
5e
10
O
O
OH
H2C
12a: R=
OBn
O
R
O
R
H
H
N
H
N
H2N
N
N
N
H
Cbz
N
N
H
H
H
e
O
O
12b: R = CH2CH(CH3)2
12c: R = CH(CH3)2
12( a-c)
11
Scheme 2. Reagents and conditions: (a) (i) SOCl2, rt, 18 h; (ii) methanol, aqueous ammonia (37%), rt 2 h, then 40 °C 8 h, 69.5% yield; (b) DMF, HCTU, DIPEA, ice-bath 4–6 h,
over 93% yield; (c) MeOH/H2O, LiOH, ice-bath 6–8 h, hydrochloric acid; (d) DMF, HCTU, DIPEA, ice-bath 2 h, then rt overnight; (e) H2 (30 psi), Pd/C (5%), BnCl, EtOAc/MeOH
(1:1), rt 8–10 h.
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D.-Y. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
12a and 12b are 4.5 and 1.8-fold more active than kojic acid
against monophenolase activity of tyrosinase. Compound 12c
was found to have a lower inhibitory activity than kojic acid. Both
copper-binding affinity and lipophilicity are important factors
affecting the anti-tyrosinase activity. The copper-binding affinities
of the hydroxypyridinone derivative prepared in the present study
are anticipated to be similar due to the same chelating moieties
and similar electronic effect of substituted group at position-6 in
pyridine ring. For compound 12, the inhibitory effect decreased
with the decrease of hydrophobicity (ClogP for 12a, 12b and 12c
are calculated to be 0.12, À0.04 and À0.57),31 which is in good
agreement with previously published results.4,14 However, for
compound 6, the inhibitory effects do not follow the order of
hydrophobicity probably due to their high hydrophobicity (ClogP:
6a 2.86; 6b 4.17; 6c 4.14; 6d 3.64; 6e 4.32). With such a high
lipophilicity, these compounds are anticipated to readily enter
the hydrophobic pocket of tyrosinase. In such cases, it is reasonably
reckoned that the inhibitory activity mainly depends on the com-
plimentary fit between the compound and active site of enzyme,
which is affected by the substituting group and position on the
pyridinone ring. In our previous study, it was found that hydrox-
ypyridinone derivatives with a substitute at position-2 in pyridine
ring had hardly anti-tyrosinase activity, although they have supe-
rior copper-binding affinity and higher lipophilicity than kojic acid.
Inhibitory effect of 6e and 12a on diphenolase activity of mush-
increase of inhibitory effect. Under the same conditions, the inhibi-
tory effect of compound 6e was better than that of compound 12a.
In the cases of both 6e and 12a, the increasing rate of o-quinone
formation became slower with increasing reaction time, indicating
that the inhibitory effect decreased. In addition, the reaction pro-
cess catalyzed by the diphenolase activity of tyrosinase had no
lag time.
As indicated in Figure 3, the relative activity of enzyme
decreased with increasing concentration of inhibitor, indicating
that the activity of enzyme was inhibited in a dose dependent
manner. The IC50 values of compounds 6e and 12a were calculated
to be 8.97 and 26.20 lM, respectively. Thus, 6e is more effective
than 12a in the inhibition on diphenolase activity of tyrosinase.
The inhibitory mechanism of 6e and 12a on mushroom tyrosi-
nase was investigated using -DOPA as a substrate. For both com-
L
pounds, investigation on the relationship between enzyme
activity and its concentration in the presence of compounds 6e
and 12a indicated that the plots of the remaining enzyme activity
versus the concentration of enzyme at different inhibitor concen-
trations gave a family of straight lines, which all passed through
the origin (Fig. 4). This result is similar to that reported by Chen
et al.32 Increase of inhibitor concentration resulted in descent of
the slope of the line, indicating that the presence of inhibitor
resulted in the inhibition of enzyme activity.33 Thus, the inhibition
of both compounds 6e and 12a on diphenolase activity of
tyrosinase is reversible.
room tyrosinase was investigated using
L
-Dopa as a substrate. The
kinetic courses of the oxidation of -Dopa by mushroom tyrosinase
L
The kinetic data of the inhibition of -DOPA oxidation by 6e and
L
in the presence of different concentrations of compound 6e or 12a
were investigated. As shown in Figure 2, the formation of o-qui-
none increased with time. The absorbance values reduced with
increasing concentration of compounds 6e and 12a, indicating an
12a were expressed in Lineweaver–Burk double-reciprocal plots
(Fig. 5A–I, B–I).33 The plots of 1/v versus 1/[S] gave a group of
straight lines with different slopes that intercept in the second
quadrant, indicating that both compounds 6e and 12a can bind
not only with free enzyme but also with the enzyme–substrate
complex, namely, both 6e and 12a were competitive–uncompeti-
tive mixed type inhibitors. The equilibrium constant of inhibitor
for binding with free enzyme (KI) was obtained from a plot of slope
(Km/Vm) versus the concentration of the inhibitor (Fig. 5A-II and B-
II), and with enzyme–substrate complex (KIS) was obtained from a
plot of the vertical intercept (1/Vm) versus the concentration of the
inhibitor (Fig. 5A-III and B-III). The KI and KIS values of 6e were
0.16
1
6e
0.14
A
0.12
0.10
2
0.08
determined to be 17.17
tor constants (KI and KIS) of 12a were determined as 34.41
79.02 M, respectively. In both cases, the KIS value is larger than
lM and 22.09
lM, respectively. The inhibi-
3
lM and
0.06
l
4
0.04
the KI value, indicating that the affinity of the inhibitors for free
enzyme is greater than that for the enzyme–substrate complex.
In order to further investigate the inhibitory mechanism of
hydroxypyridinone derivatives, copper reduction capacity34 and
copper chelating ability35 of compound 6e were determined. The
capacity of the reducing cupric ion to cuprous ion by 6e at different
concentrations is shown in Figure 6A. The increases in absorbance
5
6
0.02
0
100
200
300
400
500
600
Time (sec)
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
1
12a
B
2
100
6e
12a
3
4
5
6
80
60
40
20
0
100
200
300
400
500
600
Time (sec)
0
5
10
15
20
25
30
35
[I] (μM)
Figure 2. Inhibition kinetics on diphenolase activity of tyrosinase. (A) 6e; (B) 12a.
The concentrations of inhibitors (6e and 12a) for curves 1–6 were 0.00, 4.17, 8.33,
16.67, 25.00 and 33.33
lM, respectively.
Figure 3. Inhibitory effect of 6e and 12a on the diphenolase activity of tyrosinase.
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4
D.-Y. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
1.2
100
80
60
40
20
0
1
6e
A
1.0
6e
0.8
2
kojic acid
3
4
0.6
0.4
0.2
A
5
6
0
5
10
15
20
25
30
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Amount of enzyme (U)
Concentration (mM)
100
80
60
40
20
100
80
60
40
20
0
1
2
12a
B
3
4
5
6
6e (pH=7.4)
Kojic acid (pH=7.4)
6e (pH=5.0)
Kojic acid (pH=5.0)
B
0
5
10
15
20
25
30
0
1
2
3
4
5
6
Amount of enzyme (U)
Concentration (mM)
Figure 4. Determination of the inhibitory mechanism of 6e (A) and 12a (B) on
mushroom tyrosinase. The concentrations of inhibitors for curves 1–6 were 0.00,
Figure 6. Copper reducing capacity (A) and copper chelating ability (B) of
compound 6e and kojic acid.
4.17, 8.33, 16.67, 25.00 and 33.33
l
M, respectively.
value at 483 nm indicate the formation of cuprous ion. The
absorbance increased with increasing concentration of compound
6e up to 0.15 mM, thereafter the absorbance remained unchanged,
indicating that all cupric ions were reduced to cuprous ion. In the
case of kojic acid, the absorbance at 483 nm increased with the
increase of its concentration until 0.3 mM, indicating that com-
pound 6e possesses a stronger copper reducing capability than
kojic acid. It is well known that tyrosinase exists in three isoforms,
namely, oxy-tyrosinase [Cu(II)Cu(II)O2], met-tyrosinase [Cu(II)Cu
(II)], and deoxy-tyrosinase [Cu(I)Cu(I)].36 Met-tyrosinase is
reduced by reductant to deoxy-tyrosinase, which is then oxidized
by oxygen, forming oxy-tyrosinase capable of catalyzing mono-
or diphenol oxidation.37 Copper in the active site of tyrosinase
plays a key role in browning reaction. Reduction of cupric ion to
cuprous ion at the active site of tyrosinase by compound 6e could
convert tyrosinase into the deoxy form. Thus, it is suggested that
compound 6e could inhibit the dopachrome formation by reducing
met-tyrosinase to deoxy-tyrosinase.
As shown in Figure 6B, at low concentrations (<1 mM), the cop-
per chelating ability of compound 6e and kojic acid both increased
with increasing concentration (P <0.05), after which the increasing
rate rapidly slowed down and then reached a maximum. For com-
pound 6e, at pH 5.0 and 7.4, copper chelating abilities were deter-
mined to be 91.34 0.28% and 83.34 0.29% at 0.952 mM, and
97.52 0.31% and 88.92 0.33% at 2.857 mM, respectively; while
for kojic acid, at pH 5.0 and 7.4, they were 82.64 0.25% and
74.35 0.27% at 0.952 mM, reaching a maximum of 90.10 0.25%
and 80.08 0.22% at 3.81 mM, indicating superior copper chelating
ability of 6e to that of kojic acid. Tyrosinase contains a coupled bin-
uclear copper center in its active site. The two cupric ions, bound
10
0.6
0.4
0.2
0.0
II
5
A
I
8
4
3
6
4
2
0
5
10 15 20 25 30 35
[I] (μM)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
III
2
1
-6 -4 -2
0
2
4
6
8
10 12
0
5
10 15 20 25 30 35
1/[S] (mM)-1
[I] (μM)
0.20
0.15
0.10
0.05
0.00
B
3.0
II
5
4
3
I
2.5
2.0
1.5
1.0
0.5
0
5
10 15 20 25 30 35
2
1
[I] (μM)
0.6
0.4
0.2
0.0
III
-6 -4 -2
0
2
4
6
8
10 12
0
5
10 15 20 25 30 35
1/[S] (mM)-1
[I] (μM)
Figure 5. Lineweaver–Burk plots (A–I and B–I) of mushroom tyrosinase with L-
DOPA as a substrate in the presence of 6e (A) and 12a (B). A-II and B-II represent the
plot of slope versus the concentration of 6e and 12a for determining the inhibition
constants KI. A-III and B-III represent the plot of intercept versus the concentration
of 6e and 12a for determining the inhibition constants KIS
.
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D.-Y. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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28. General procedure for preparation of 6: To a solution of compound 4 (5 mmol)
with three histidine residues, are directly involved in the different
catalytic activities. When compound 6e enters the active site of the
tyrosinase, it could outcompete with the histidine residues for
the binding of cupric ion, thereby resulting in a loss of activity.
The copper chelating activity of 6e was in accordance with its
tyrosinase inhibitory activity (Fig. 1). Therefore, copper chelation
of 6e is one of the important mechanisms for the inhibition of
tyrosinase.
and Cbz-L-amino acid (5) (5.5 mmol) in DMF (20 mL) was added 1-ethyl-(3-
dimethyllaminopropyl) carbodiimine hydrochloride (EDC, 5.5 mmol) and 4-
dimethylaminopyridine (DMAP, 0.55 mmol). The resulting solution was stirred
on an ice bath for 2 h, then at room temperature overnight. After removal of
the solvent, the residue was dissolved in CH2Cl2 (60 mL), and washed with
saturated NaHCO3 solution twice followed by brine, and dried over anhydrous
Na2SO4. After filtration, the filtrate was concentrated, then the residue was
purified by silica gel column chromatography (MeOH/CH2Cl2 1:10), providing
compound 6 as off-white solids. The silica gel column must be pretreated with
maltol to remove iron completely. Data for 6e: Yield 71.3%. 1H NMR (500 MHz,
DMSO-d6) d: 2.92–3.11 (m, 2H, CH2), 4.39 (m, 1H, CH), 4.98 (s, 2H, CH2), 5.21 (s,
2H, CH2), 7.15 (s, 1H, C3-H in pyridinone), 7.20–7.35 (m, 10H, Ph), 7.89 (d,
J = 6.5 Hz, 1H, NH), 8.08 (s, 1H, C6-H in pyridinone). 13C NMR (125 MHz, DMSO-
d6) d: 36.80, 55.93, 62.00, 65.98, 112.39, 127.00, 127.30, 128.03, 128.26, 128.68,
128.77, 129.59, 137.30, 137.67, 142.81, 145.32, 156.46, 161.71, 171.73. ESI-
HRMS: m/z, calcd for C23H23N2O6 ([M+H]+) 423.1551, found 423.1542.
In order to understand the interaction mode of inhibitor binding
to tyrosinase, the molecular docking of compound 6e to Agaricus
bisporus tyrosinase38 was carried out using Accelrys Discovery Stu-
dio 2.5 software. As shown in Figure 7, the hydroxypyridinone ring
of compound 6e can insert into the bottom of active pocket in
tyrosinase, the 3-hydroxy group and 4-carbonyl group coordinate
with copper ion. Two benzyl groups stretch out with a T shape,
forming
a stable combined conformation via hydrophobic
interaction with hydrophobic area in the active pocket of tyrosi-
nase, indicating a good deal of complimentary fit between com-
pound 6e and active site of tyrosinase.
In summary, a range of hydroxypyridinone derivatives were
synthesized in the present study. Among them, 6e was found to
exhibit the highest tyrosinase inhibitory activity. 6e exhibited
reversible and mixed type inhibition on mushroom tyrosinase.
Chelating copper at the active site of tyrosinase is an important
mechanism for the inhibition of tyrosinase. Thus it could find
application in medicine, cosmetic and agriculture areas.
29. General procedure for preparation of 12: To a suspension of compound 11
(2 mmol), BnCl (2.4 mmol) in methanol (20 mL)/ethyl acetate (20 mL) was
added 5% Pd/C (5% weight of compound 11). Hydrogenation was carried out
under 30 psi H2 for 8 h at room temperature. After filtration to remove the
catalyst, the filtrate was concentrated, small amount of diethyl ether was
added, allowed to stand in a fridge. Hydrochloride salts of compound 12 were
obtained as white powder by filtration. Data for 12a: Yield 86.5%. 1H NMR
(500 MHz, DMSO-d6) d: 2.95–3.13 (m, 4H, CH2, CH2), 4.04 (m, 1H, CH), 4.37–
4.48 (m, 2H, CH2), 4.54 (m, 1H, CH), 7.13 (s, 1H, C3-H in pyridinone), 7.19–7.31
(m, 10H, Ph), 8.10 (s, 1H, C6-H in pyridinone), 8.26 (br, 3H, NH+3), 9.01 (t,
J = 6.0 Hz, 1H, NH), 9.05 (d, J = 8.0 Hz, 1H, NH). 13C NMR (125 MHz, DMSO-d6) d:
19.06, 37.23, 37.63, 53.87, 55.18, 56.53, 111.12, 126.32, 126.92, 127.52, 128.66,
128.90, 129.69, 130.08, 135.33, 137.92, 146.66, 146.84, 161.83, 168.50, 171.49.
ESI-HRMS: m/z, calcd for C24H27N4O4 ([M+H]+) 435.2027, found 435.2014.
30. Determination of inhibitory activity of hydroxypyridinone derivatives on
Acknowledgements
The authors acknowledge the financial support for this work
provided by Science Technology Department of Zhejiang Province
of China (No. 2013C24006) and the National Natural Science
Foundation of China (No. 20972138).
mushroom tyrosinase:
monophenolase activity assay and
for the diphenolase activity assay.
L
-Tyrosine (2 mM) was used as the substrate for the
-DOPA (0.5 mM) was used as the substrate
-tyrosine (1 mL) or -DOPA (1 mL),
L
L
L
phosphate buffer (1.8 mL, pH 6.8) and different concentrations of inhibitor
(0.1 mL in DMSO) were mixed and incubated at 30 °C. Then, an aqueous
solution of mushroom tyrosinase (0.1 mL, 200 U/mL) was added to the above
solution and mixed quickly. The reaction system (totally 3 mL) was incubated
at 30 °C for 10 min. Absorption and kinetic measurements were carried out on
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.05.
006.
a
Shimadzu UV3600 UV–Vis spectrophotometer at 475 nm. Controls
containing the same amount of DMSO without inhibitor, were routinely
carried out. All the measurements were performed in triplicate. The inhibition
rate of tyrosinase was calculated according to the following formula:
Inhibition rate (%) = [1 À (OD3 À OD4)/(OD1 À OD2)] Â 100
References and notes
where OD1 was the absorbance value without inhibitor, OD2 was the
absorbance value without substrate and inhibitor, OD3 was the absorbance
value of the experimental group, OD4 was the absorbance value without
substrate.
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6
D.-Y. Zhao et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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free cupric ion concentrations were obtained from a standard curve of free
cupric ion concentration (0–0.1 mM) vs absorbance ratio. The difference
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the concentration of chelated cupric ion. Assays were performed in triplicate.
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(TMM) (5 mM) were prepared in hexamine buffer (10 mM, pH 5.0 and 7.4)
containing 10 mM KCl. Chelator solutions (1–12 mM) were prepared by
diluting the stock solution (in DMF) with the same buffer. CuSO4 (l.0 mL)
was mixed with a chelator solution (1.0 mL), followed by the addition 0.1 mL of
TMM. The absorbance of the resulting reaction mixture was recorded at
460 nm and 530 nm after incubation for 10 min at room temperature.
According to the calculated absorbance ratios of A460/A530, the corresponding
Please cite this article in press as: Zhao, D.-Y.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.006