Functionality study of chalcone-hydroxypyridinone hybrids as tyrosinase inhibitors and influence on anti-tyrosinase activity

Article abstract of DOI:10.1080/14756366.2020.1801669

In an attempt to synthesise new tyrosinase inhibitors, we designed and synthesised a series of chalcone-hydroxypyridinone hybrids as potential tyrosinase inhibitors adopting strategic modifications of kojic acid. All the newly synthesised compounds were characterised by NMR and mass spectrometry. Initial screening of the target compounds demonstrated that compounds 1a, 1d, and 1n had relatively strong inhibitory activities against tyrosinase monophenolase, with IC₅₀ values of 3.07 ± 0.85, 2.25 ± 0.8 and 2.75 ± 1.19 μM, respectively. The inhibitory activity against monophenolase was 6- to 8-fold higher than that of kojic acid. Compounds 1a, 1d, and 1n also showed inhibition of diphenolase, with IC₅₀ values of 17.05 ± 0.07, 11.70 ± 0.03 and 19.3 ± 0.28 μM, respectively. The inhibition kinetics of diphenolase indicates that compounds 1a and 1d induce reversible inhibition on tyrosinase. Finally, we found that copper coordination should be one of the important inhibitory mechanism of these compounds in tyrosinase.

Full text of DOI:10.1080/14756366.2020.1801669

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Functionality study of chalcone-hydroxypyridinone
hybrids as tyrosinase inhibitors and influence on
anti-tyrosinase activity
L. Ravithej Singh , Yu-Lin Chen , Yuan-Yuan Xie , Wei Xia , Xing-Wen Gong ,
Robert C. Hider & Tao Zhou
To cite this article: L. Ravithej Singh , Yu-Lin Chen , Yuan-Yuan Xie , Wei Xia , Xing-Wen Gong ,
Robert C. Hider & Tao Zhou (2020) Functionality study of chalcone-hydroxypyridinone hybrids as
tyrosinase inhibitors and influence on anti-tyrosinase activity, Journal of Enzyme Inhibition and
Medicinal Chemistry, 35:1, 1562-1567, DOI: 10.1080/14756366.2020.1801669
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1562–1567
https://doi.org/10.1080/14756366.2020.1801669
BRIEF REPORT
Functionality study of chalcone-hydroxypyridinone hybrids as tyrosinase inhibitors
and influence on anti-tyrosinase activity
L. Ravithej Singha, Yu-Lin Chenb, Yuan-Yuan Xiec, Wei Xiaa, Xing-Wen Gonga, Robert C. Hiderb and Tao Zhoua
b
aSchool of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang, PR China; Division of Pharmaceutical
c
Science, King’s College London, London, UK; College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, PR China
ABSTRACT
ARTICLE HISTORY
Received 22 February 2020
Revised 7 July 2020
Accepted 18 July 2020
In an attempt to synthesise new tyrosinase inhibitors, we designed and synthesised a series of chalcone-
hydroxypyridinone hybrids as potential tyrosinase inhibitors adopting strategic modifications of kojic acid.
All the newly synthesised compounds were characterised by NMR and mass spectrometry. Initial screening
of the target compounds demonstrated that compounds 1a, 1d, and 1n had relatively strong inhibitory
activities against tyrosinase monophenolase, with IC50 values of 3.07 0.85, 2.25 0.8 and 2.75 1.19 lM,
respectively. The inhibitory activity against monophenolase was 6- to 8-fold higher than that of kojic acid.
Compounds 1a, 1d, and 1n also showed inhibition of diphenolase, with IC50 values of 17.05 0.07,
11.70 0.03 and 19.3 0.28 lM, respectively. The inhibition kinetics of diphenolase indicates that com-
pounds 1a and 1d induce reversible inhibition on tyrosinase. Finally, we found that copper coordination
should be one of the important inhibitory mechanism of these compounds in tyrosinase.
KEYWORDS
Tyrosinase inhibitor;
hydroxypyridinone; chal-
cone analogue;
monophenolase;
diphenolase
1. Introduction
collagen peptides, sorghum peptides have also been found to
possess inhibitory effects on tyrosinase11,12
.
Tyrosinase is a copper-containing metalloenzyme widely distrib-
uted in animals, plants, and microorganisms and is a key and rate-
limiting enzyme for melanogenesis1. It is isolated from diverse
resources and its structural properties are well documented2. A di-
copper centre is the common structural unit of the active site
which is found in all species. Each copper ion is coordinated by
three histidine residues. Further, antiferromagnetic coupling of the
two copper ions renders the active site EPR inactive3. During the
tyrosinase-catalysed oxidation process, the binuclear metal centre
activates an O2 molecule and subsequently oxidises substrates
such as L-tyrosine and L-DOPA to catecholates and benzoquinones,
respectively4. As tyrosinase plays a fundamental role in the mela-
nogenesis process, the abnormal function of this enzyme poses
challenges in a range of socio-economic areas such as agriculture,
food, and the pharmaceutical industry5. Tyrosinase regulates pig-
mentation, and abnormal expression can lead to various pig-
mented diseases such as freckles and age spots6. If the expression
is insufficient, skin diseases such as those associated with albinism
and vitiligo will occur. In fruits and vegetables, tyrosinase causes
browning during post-harvest processing, transportation, and stor-
age, affecting its quality and commercial value7.
Development of useful tyrosinase inhibitors, possessing potent
commercial utility has been limited to date due to poor solubility,
low shelf-life, and safety concerns13. Kojic acid, a secondary
metabolite produced by Aspergillus and Penicillium moulds, pro-
vides a promising starting point for the synthesis of new tyrosin-
ase inhibitors; the a-hydroxy ketone functionality plays an
important role in tyrosinase inhibition14,15. Based on previous
results, relating to hydroxypyridinones16–19, we have functionalised
kojic acid to form new derivatives in attempt to identify more
potent tyrosinase inhibitors.
As many chalcones possess tyrosinase inhibitory activities (IC50
value for tyrosinase inhibition of 20,40,4-trihydroxychalcone is
8.1 lM)20,21, it was decided to investigate the properties of chal-
cone analogues (1) which contain either a pyranone or pyridinone
ring (Figure 1).
2. Materials and methods
2.1. Chemistry
1H NMR and 13 C NMR spectra were recorded on a Bruker Avance
400 spectrometer (Bruker Corp., Karlsruhe, Germany) with TMS as
an internal standard. Electrospray ionisation (ESI) mass spectra
were obtained by infusing samples into an LCQ Deca XP ion trap
instrument (ThermoFinnigan, SanJose, CA). High resolution mass
There are many tyrosinase inhibitors originating from both nat-
ural sources and chemical synthesis8. Kojic acid, vitamin C, cyst-
eine, and arbutin are inhibitors isolated from natural sources.
Although ideal in many respects, kojic acid has not found wide
application due to its metabolic instability9. Benzaldehyde, 4-halo- spectra (HRMS) were determined on Waters QTOF micro (Waters,
benzoic acids, and 4-chlorosalicylic acid are tyrosinase inhibitors of USA). Lyophilised powder of mushroom tyrosinase (ꢀ1000 U/mg)
synthetic origin10. In addition, extracts from animals and fish, was purchased from Sigma. Kojic acid, L-tyrosine, and L-DOPA of
CONTACT Tao Zhou
taozhou@zjgsu.edu.cn
School of Food Science and Biotechnology, Zhejiang Gongshang University, 18 Xuezheng Street, Xiasha,
Hangzhou, Zhejiang, 310018, PR China
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1563
For pKa determinations, a cuvette path length of 10 mm was used
while for metal stability constant determinations, a cuvette path
length of 50 mm was used. All instruments were interfaced to a
computer and controlled by an in-house programme. An auto-
mated titration adopted the following strategy: the pH of a solu-
tion was increased by 0.1 pH unit by the addition of KOH solution
(0.1 M) from the autoburette. The pH readings were judged to be
stable if their values varied by less than 0.01 pH unit after a set
incubation period. For pKa determinations, an incubation period of
1.5 min was adopted; for copper(II) stability constant determina-
tions, an incubation period of 3 min was adopted. The cycle was
repeated until the defined end point pH value was achieved.
Titrations were carried out in the solution with molar ratio of
DMSO: H2O being 0.2: 1 due to the solubility issue of samples
and/or the corresponding copper(II) complexes. Under this condi-
tion, the pH metre readings are shifted, compared to the aqueous
solution. All the titration data were analysed with the
HypSpec2014 programme (http://www.hyperquad.co.uk/)26,27. The
speciation plot and pM values were calculated with the HYSS pro-
gramme28. Based on deferiprone titrations, pCu2þ at pH 7.6,
O
O
O
X
OH
HO
OH
OH
R2
1 (X=O or NR1)
2',4',4-trihydroxychalcone
Figure 1. Structure of a typical chalcone and designed molecules 1.
analytical grade were purchased from Aladdin chemicals,
Shanghai, China. All other chemicals were of AR grade and used
without any further purification. 5-Benzyloxy-2-hydroxymethyl-
pyran-4-one was prepared according to previous report22.
Compounds 3a–3d, 4a–4e, 6a–6d and 7a–7d were prepared
according to previous reports16,17,23,24. General procedure for the
synthesis of compounds 1a–1o and their physical and spectro-
scopic data are presented in Supplementary Materials.
2.2. Tyrosinase inhibition assay
2.2.1. Monophenolase and diphenolase inhibition assay
denoted as pCu2þ7.6, with [ligand]total ¼ 10 mM and [Metal]total
¼
Monophenolase and diphenolase activity was measured as
described previously by monitoring the dopachrome absorbance
at 475 nm17. L-Tyrosine and L-DOPA were used as reaction sub-
strate for monophenolase and diphenolase assays, respectively. In
the reaction, L-tyrosine (50 mL, 2 mM) or L-DOPA (50 mL, 0.5 mM),
90 mL of pH 6.8 phosphate buffer and 5 mL volume of different
concentrations of final compounds (1a–1o) in DMSO were mixed
and incubated at 30 ꢁC. Finally, 20 units of enzyme was quickly
added to the reaction mixture and incubated at 30 ꢁC for 10 min.
The absorbance at 475 nm was recorded with a microplate reader.
During these incubations, the final concentration of DMSO was
limited to 3% by volume. The assay was carried out in triplicate
and DMSO was used as control17.
1 mM, were calculated as close approximations for pCu2þ at pH 7.4
in the aqueous solution. Analytical grade reagent materials were
used in the preparation of all solutions.
2.4. Molecular docking study
Molecular docking was performed by using CDOCKER module
embedded in Discovery Studio 2.5 software (Accelrys Software,
Inc., San Diego, CA, USA)17. The X-ray crystal structure of tyrosin-
ase from Agaricus bisporus (PDB ID: 2Y9X) was retrieved from the
Protein Data Bank (http://www.rcsb.org/pdb). All crystallographic
water molecules and ions were removed from the protein struc-
ture. 3 D structure of compound 1d was generated in Chem3D
Ultra 8.0, and conformations were generated by using a modified
CHARMm force field. The obtained conformations were then
docked into the binding site of tyrosinase. The docked conform-
ation with the lowest energy was used for the analysis of bind-
ing mode.
2.2.2. Inhibition kinetics on diphenolase activity of tyrosinase
The same experimental protocol described above was adapted for
inhibition kinetics, with different inhibitor concentration. However
the absorbance was measured at 1 min reaction intervals for
twelve minutes16.
2.5. Statistical analysis
2.2.3. Reversibility of inhibitory effect of compounds on tyrosinase
Measurement method was substantially the same to that
described above, changing the added concentration of the
enzyme solution, different concentrations of inhibitor on mush-
room tyrosinase L-DOPA as reaction substrate16.
All the experiments were performed in triplicate. The data were
statistically analysed using MS-Excel and GraphPad Prism
6 software.
3. Results and discussion
2.3. pKa and copper(II) stability constants determination
3.1. Chemistry
An automated titration system used in the study consists of a
Metrohm 765 Dosimat autoburette, a Mettler Toledo MP230 pH
metre with SENTEK pH electrode (P11), and an HP 8453 UV-visible
spectrophotometer with a Hellem quartz flow cuvette being circu-
lated through by a Gilson Mini-plus #3 pump—speed capability
(20 ml/min). A potassium chloride electrolyte solution (0.1 M) was
used to maintain the ionic strength. The temperature of the test
solutions was maintained in a thermostatic jacketed titration ves-
sel at 25 ꢁC ( 0.1 ꢁC) using a Fisherbrand Isotemp water bath. The
pH electrodes were calibrated using GLEE25 with data obtained by
The synthetic route and complete reaction conditions for the
preparation of compounds 1a–1o are illustrated in Scheme 1. The
benzyl protection of kojic acid with BnCl in presence of sodium
hydroxide and methanol:water (1:1) as reaction solvent at 70 ꢁC
gave the intermediate compound 2. Further, refluxing of inter-
mediate 2 with different aliphatic amines in ethanol gave the
intermediates (3a–3d) in good yield. The corresponding aldehydes
(4a–4e) were afforded by the selective oxidation of compounds
3a–3d with active MnO2, in moderate yield. The phosphonium
salts (7a–7d) were synthesised by reaction of a-bromo acetophe-
titrating a volumetric standard HCl (0.1 M) in KCl (0.1 M) with KOH nones (6a–6d) with triphenylphosphine in dichloromethane in
(0.1 M) under an argon gas atmosphere in the vessel. The solution excellent yield. Finally, the target hybrid compounds (1a–1o) were
under investigation was stirred vigorously during the experiment. obtained using the Wittig reaction by the condensation of

1564
L. R. SINGH ET AL.
O
O
OBn
OBn
iii
O
HO
i
i
N
N
O
O
O
R1
OBn
R1
OH
i
4a-4d
R1
3a-3d
HO
HO
O
O
i
i
2
i
OBn
Kojic Acid
3a,4a = H
O
3b,4b = Methyl
3c,4c = Ethyl
3d,4d = n-Butyl
O
4e
O
O
O
R2
PPh3
Br
Br
v
iv
5a,6a,7a = H
R2
5b,6b,7b = 3,4-dimethoxy
5c,6c,7c = 2,4-dimethoxy
5d,6d,7d = 4-flouro
R2
R2
7a-d
6a-d
O
5a-d
O
O
O
OBn
a) vi
b) vii
PPh3
Br
X
+
OH
R2
OHC
X
4a-e
R2
1a-o
7a-d
R2
X
R2
X
1a 4-fluoro
NCH3
1i
H
NCH3
1b 2-hydroxy,4-methoxy NCH3
1j
3,4-dihydroxy
2-hydroxy,4-methoxy NC4H9
4-fluoro
NC4H9
1c
2,4-dimethoxy
O
NH
NC2H5
NC2H5
1k
1l
1m
1n
1o
1d 4-fluoro
NC4H9
NH
NH
1e
1f
H
H
3,4-dihydroxy
3,4-dimethoxy
2-hydroxy,4-methoxy NH
1g 2-hydroxy,4-methoxy NC2H5
1h 4-fluoro NC2H5
Scheme 1. Synthetic route of compounds 1. Reagents and conditions: (i) BnCl, NaOH, MeOH/H2O, 70 ꢁC, 6 h, 80% yield; (ii) appropriate amines, EtOH, reflux, overnight,
(iii) MnO2, 1,4-dioxane, (iv) Br2, CHCl3, rt, 10 min; (v) PPh3, CH2Cl2, 30min, (vi) tert-BuOK, dry THF, (vii) BBr3, DCM, 0 ꢁC to rt, 2 h, 55–72% yield.
phosphonium salts 7 with aldehydes (4a–4e) in presence of tert-
BuOK in dry THF and subsequent deprotection of benzyl group
with BBr3 in dichloromethane at 0 ꢁC. All the newly synthesised
compounds were characterised using 1H NMR, 13 C NMR
and HRMS.
Table 1. Inhibition of compounds (1a–1o) (50 mM) on monophenolase activity
of mushroom tyrosinase under the conditions of 30 ꢁC and pH 6.8.
Compounds
Inhibition (%)
R2
X
clogPa
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
85.3
66.7
50.3
89.5
63.3
78.8
71.4
57.8
72.8
69.4
64.6
50.3
66.7
87.6
63.3
75.4
4-F
NCH3
NCH3
O
1.60
1.41
1.51
1.53
1.81
0.84
1.78
1.97
1.43
2.31
2.84
3.03
1.36
1.01
1.34
ꢂ0.89
2-OH, 4-OCH3
2,4-dimethoxy
4-F
H
3,4-dihydroxyl
2-OH, 4-OCH3
4-F
H
3,4-dihydroxyl
2-OH, 4-OCH3
4-F
H
3,4-dimethoxy
2-OH, 4-OCH3
–
NH
NC2H5
NC2H5
NC2H5
NC2H5
NCH3
NC4H9
NC4H9
NC4H9
NH
3.2. Inhibitory effect of compounds 1a–1o on
monophenolase activity
All the final compounds (1a–1o) were screened for monopheno-
lase inhibitory activity at 50 lM (Table 1). Four compounds (1a,
1d, 1f, and 1n) were found to possess a stronger inhibitory activ-
ity on monophenolase activity of mushroom tyrosinase than kojic
acid under the conditions defined in Table 1. The inhibition rate
of 1a, 1d, 1f, and 1n was 85.3%, 89.5%, 78.8% and 87.6% at
50 lM, respectively, which was higher than that of kojic acid
(75.4%). These four compounds were tested at different concen-
trations to realise IC50 values (Figure 2); 1a 3.07 0.85 lM, 1d
2.25 0.80 lM, 1f 8.11 2.67 lM, and 1n 2.75 1.19 lM. The inhibi-
tory effects of 1a, 1d, 1f, and 1n were 5.7-, 7.8-, 2.2-, and 6.4-fold
higher than that of kojic acid (IC50 ¼ 17.55 1.91 lM). Compounds
1a, 1d, and 1n possess obviously superior monophenylase inhibi-
tory activity to that of 20,40,4-trihydroxychalcone (IC50 ¼ 8.1 lM,
inhibition rate is 67% at 50 lM)20, while the activity of 1f is close
1l
1m
1n
1o
NH
NH
–
Kojic acid
aThe clogP values were calculated from website: http://www.molinspiration.com/
cgi-bin/properties.
coefficients (clogP) influence the monophenylase inhibitory activ-
ity (Table 1). The four most active inhibitors possessed clogP val-
ues equal to or less than 1.6 and they also have strong electron
withdrawing functions on the benzene ring, i.e. F, OH or OMe.
to that of 20,40,4-trihydroxychalcone. The substituents on position- The inductive effect will dominate for F and OMe substituents.
1 (X) and on the benzene ring (R2) (Scheme 1), and hydrophobi- When the pyridinone ring N is replaced by an oxygen (1c), low
city of these molecules reflected by their calculated partition inhibitory activity resulted. When the clogP value was greater

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1565
Figure 2. Inhibitory effect of 1a, 1d, 1f, and 1n on the monophenolase activity
of mushroom tyrosinase. The assays were performed at 30 ꢁC and pH 6.8.
Figure 4. Determination of the inhibitory reversibility of 1a (a) and 1d (b) on
mushroom tyrosinase. The concentrations of inhibitors for curves were 0.00,
3.125, 6.25, 12.5, and 25.00 mM, respectively. The assays were performed at 30 ꢁC
and pH 6.8.
process catalysed by the diphenolase activity of tyrosinase had no
lag time. This result is in agreement with those of previous
reports18,19
.
The inhibition of compounds 1a, 1d, and 1n on the dipheno-
lase activity of tyrosinase increased with increasing concentration
of inhibitor. The IC50 values of 1a, 1d, and 1n were determined to
be 17.1 0.07 mM, 11.7 0.03 mM, and 19.3 0.28 respectively.
The inhibitory reversibility of 1a and 1d on mushroom tyrosin-
ase was investigated using L-DOPA as a substrate. For both com-
pounds, investigation on the relationship between enzyme activity
and its concentration in the presence of compounds 1a and 1d
indicated that the plots of the remaining enzyme activity versus
the concentration of enzyme at different inhibitor concentrations
gave a family of straight lines, which all passed through the origin
(Figure 4). 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. Thus, the inhibition
of both compounds 1a and 1d on diphenolase activity of tyrosin-
ase is reversible.
Figure 3. Inhibitory effect of different concentrations of 1a (a) and 1d (b) on the
diphenolase activity of tyrosinase. The assays were performed at 30 ꢁC and
pH 6.8.
than 1.6, there was an appreciable reduction in inhibitory potency
(1c, 1 g, 1 h, 1j, 1k, 1 l). The 2-hydroxy-4-methoxybenzene aro-
matic rings (1 b, 1o) and unsubstituted benzene (1e, 1 m) were
also found to lack potent inhibitory properties. Thus the presence
of 4-fluoro- or 3,4-dimethoxy benzene in the structure leads to
optimal activity together with either a NH or NMe in the pyridi-
none ring.
3.4. pKa values and copper(II) affinity constant of 1n
It is well known that the main inhibitory mechanism of kojic acid
on tyrosinase involves chelation of copper in the active site in
tyrosinase. Thus, for the purpose of exploring the inhibitory mech-
anism of compounds 1 on tyrosinase, the pKa values of 1n and its
affinity for copper(II) were determined. The proton equilibria of 1n
are presented in Scheme 2. Using the spectrophotometric titration
method, the three pKa values of 1n obtained from nonlinear least-
squares regression analysis were found to be 3.3, 8.8, and 12.0,
which correspond to the 4-oxo functional group, the 5-hydroxyl
group, and 1-NH, respectively. As a bidentate, 1n can form two
3.3. Inhibitory kinetics and reversibility on diphenolase activity
of tyrosinase
The inhibitory kinetic courses of mushroom tyrosinase in the pres-
ence of different concentrations of compounds 1a and 1d was
investigated by monitoring their inhibition on diphenolase inhibi-
tory activity using L-Dopa as a substrate. The formation of o-quin-
one generated by the oxidation of L-Dopa increased with time,
and the absorbance values reduced with increasing concentration species of copper complexes, CuLH and CuL2H2 (assuming 1-NH
of compounds 1a and 1d (Figure 3). In addition, the reaction group does not deprotonate when forming the complexes with

1566
L. R. SINGH ET AL.
OH
O
O
O
pKa1
OH
OH
O
pKa2
O
R
N
H
I
R
N
H
R
N
H
R
N
H
III
II
pKa3
O
N
O
R
IV
Scheme 2. Proton equilibria and resonance structure of 1n.
20.9 and 40.4, respectively. The pCu2þ value is a more suitable
parameter to reflect the copper affinity of ligands, which is meas-
ured under the conditions of [Cu]total¼10ꢂ6M, [L]total¼10ꢂ5M, pH
7.4. The pCu2þ value of 1n was calculated from the pKa values
and the log stability constants of its copper complexes using
HYSS programme, being 9.9, which is greater than that of kojic
acid (pCu2þ¼7.3)29. Thus, copper chelation is undoubtedly an
important inhibitory mechanism on tyrosinase.
100
80
60
40
20
0
Cu
CuLH
CuL H
2 2
3.5. Molecular docking study
In order to understand the interaction mode of inhibitor binding
to tyrosinase, the molecular docking of compound 1d to Agaricus
bisporus tyrosinase was performed. As shown in Figure 6, com-
pound 1d binds to the active site of tyrosinase at the bottom of
hydrophobic cavity of the receptor. The 4-oxy is located between
Cu1 and Cu2 with a distance of 2.47 and 2.13 Å, respectively. The
distance between Cu1 and the oxygen in 5-hydroxy group is
2.53 Å. Thus, the 4-oxy and 5-hydroxy group on the pyridinone
ring can coordinate with Cu1, forming a 5-membered chelating
ring; the 4-oxy can also coordinate with Cu2. Compound 1d inter-
acts with the side chain of Val283 and Val 248 via hydrophobic
contacts, and interacts with the copper ligands His 61 and His263
via pi–pi stack, indicating the formation of a stable conformation.
3
5
7
9
pH
Figure 5. Speciation plots of copper ion in the presence of 1n.
4. Conclusions
Tyrosinase is an essential enzyme in melanogenesis metabolic pro-
cess in microorganisms, plants, and animals, and thus is an attract-
ive target for the discovery of novel anti-tyrosinase agents. In the
present study, we have synthesised and screened a series of new
chalcone analogues containing hydroxypyridinone moiety. Using
enzyme inhibition assays, we have identified compound 1a, 1d,
and 1n as potential lead molecules. The inhibitory effect of com-
pounds 1a and 1d on diphenolase activity of mushroom tyrosin-
ase activity is reversible. These findings support the previous
proposal that kojic acid modification is a promising strategy for
developing new potential tyrosinase inhibitors30,31
Figure 6. Configuration for the interaction of compound 1d with tyrosinase.
copper). The distribution of these two complexes varies with the
pH (Figure 5). The log stability constants of these two complexes
Disclosure statement
(CuLH and CuL2H2), log b1 and log b2, were determined to be The authors declare no conflict of interest, financial or otherwise.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1567
with Fe3þ, Al3þ, Cu2þ and Zn2þ, and in vivo bioassays. J
Inorg Biochem 2019;193:152–65.
Funding
The authors are grateful to the Zhejiang Provincial Natural Science
Foundation of China [No. LY17B020001], and School of Food
Science and Biotechnology, Zhejiang Gongshang University for
financial support and constant encouragement.
16. Li DF, Hu PP, Liu MS, et al. Design and synthesis of hydroxy-
pyridinone-L-phenylalanine conjugates as potential tyrosin-
ase inhibitors. J Agric Food Chem 2013;61:6597–603.
17. Shao LL, Wang XL, Chen K, et al. Novel hydroxypyridinone
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