1. Integrated kinetic studies and computational analysis on naphthyl
chalcones as mushroom tyrosinase inhibitors
Sini Radhakrishnan ⇑
, Ronald Shimmon, Costa Conn, Anthony Baker
School of Chemistry and Forensic Science, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia
a r t i c l e i n f o
Article history:
Received 19 May 2015
Revised 31 July 2015
Accepted 13 August 2015
Available online 18 August 2015
Keywords:
Chalcone oximes
Competitive inhibition
Tyrosinase
Docking
a b s t r a c t
Melanin helps to protect skin from the damaging ultraviolet radiation of the sun. Tyrosinase, the key
enzyme in melanogenesis is responsible for coloration of skin, hair and eyes. This enzyme is considered
to have a critical role in governing the quality and economics of fruits and vegetables, as tyrosinase activ-
ity can lead to spoilage through browning. Development of tyrosinase inhibitors is a promising approach
to combat hyperpigmentation conditions like ephelides, lentigo, freckles and post-inflammatory hyper-
pigmentation. In the present study, we have used a docking algorithm to simulate binding between
tyrosinase and hydroxy-substituted naphthyl chalcone oxime compounds and studied the inhibition of
tyrosinase. The results of virtual screening studies indicated that the estimated free energy of binding
of all the docked ligands ranged between À19.29 and À9.12 kcal/mol. Two of the oximes synthesized
were identified as competitive tyrosinase inhibitors and were found to be twice as potent as the control
kojic acid with their IC50 values of 12.22 lM and 19.45 lM, respectively. This strategy of integrating
experimental and virtual screening methods could give better insights to explore potent depigmentation
agents.
Ó 2015 Elsevier Ltd. All rights reserved.
The quantity of melanin is an important determinant of skin
color in humans but an excessive amount of it could be detrimen-
tal. Abnormal pigmentation is related to a variety of cosmetic and
clinical conditions including melasma, lentigo, age spots, inflam-
matory hypermelanosis and trauma-induced hyperpigmenta-
tion.1–3
Melanin is formed by a combination of enzymatically
catalyzed chemical reactions. The major rate limiting step in mel-
anin biosynthesis involves the enzyme tyrosinase [EC 1.14.18.1]
that catalyzes two different reactions of melanin biosynthesis,
the hydroxylation of L-tyrosine to L-DOPA (L-3,4-dihydroxy pheny-
lalanine) and oxidation of L-DOPA to DOPA quinone.4
In addition,
tyrosinase is responsible for undesired enzymatic browning of
fruits and vegetables that take place during senescence or damage
in post-harvest handling, which makes the identification of novel
tyrosinase inhibitors extremely important.5
Tyrosinase or polyphe-
nol oxidase (PPO) inhibitors have been used as herbicides to con-
trol weeds.6
It has also been suggested that tyrosinase may
contribute to the neurodegeneration associated with Parkinson’s
disease.7
Thus, studies on tyrosinase inhibitors have gained
immense significance mainly due to its wide range of applications
in cosmetics as a depigmentation agent and in agriculture for con-
trolling the quality and economics of fruits and vegetables.
From a structural perspective, tyrosinase belongs to a type-3
copper protein family known as the oxidoreductases harboring a
catalytic center.8–10
Tyrosinase has two copper ions in its active
site which play a vital role in its catalytic activity. At the active site
of tyrosinase, a dioxygen molecule binds in side-on coordination
between two copper ions. Each of the copper ions is coordinated
by three histidines in the protein matrix.11
The copper atoms par-
ticipate directly in hydroxylation of monophenols to diphenols
(cresolase activity) and in the oxidation of o-diphenols to o-qui-
nones (catechol oxidase activity) that enhance the production of
the brown color.12
Therefore, chelation of tyrosinase Cu2+
by syn-
thetic compounds or agents from natural sources has been tar-
geted as a way to inhibit or block tyrosinase catalysis.13
An
alternative solution to inhibit tyrosinase catalytic activity would
be by effectively blocking access to the active site of enzyme. Sev-
eral natural and synthetic tyrosinase inhibitors have been reported,
including aromatic aldehydes and acids, tropolone, arbutin, flavo-
noids, and kojic acid.14,15
However, many popular depigmenting
compounds either lack potency or produce undesirable side effects.
Kojic acid is currently applied as a cosmetic skin whitener and food
additive to prevent enzymatic browning.16
However, its use in cos-
metics has been limited, because of its instability during storage.17
In addition, kojic acid has been shown to promote thyroid and liver
http://dx.doi.org/10.1016/j.bmcl.2015.08.033
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding author. Tel.: +61 426687300.
E-mail address: Sini.KaranayilRadhakrishnan@student.uts.edu.au
S. Radhakrishnan).
Bioorganic & Medicinal Chemistry Letters 25 (2015) 4085–4091
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl

2. carcinogenicity in rodent models, leading to its ban in Switzer-
land.18
Although hydroquinone has been the mainstay treatment
for hyperpigmentation, its clinical potential has been complicated
due to its cytotoxic and mutagenic properties.19,20
This necessi-
tates the need for further investigation of potential tyrosinase inhi-
bitors that could be applied during food processing to curb
enzymatic browning and for the treatment of hyperpigmentation
disorders.
Chalcone, also known as phenyl styryl ketone is a privileged
structure with immense therapeutic potential. The ease of synthe-
sis, feasibility of oral administration and safety favor chalcones as
promising therapeutic agents. Chalcones are open-chain flavonoids
in which two aromatic rings are joined by a three carbon a,b-un-
saturated carbonyl system (1,3-diphenyl-2-propen-1-ones).18
The
presence in chalcones of a conjugated double bond and a com-
pletely delocalized p electron system reduces their redox poten-
tials and makes them prone to electron transfer reactions. The
a-b unsaturated bond enables the chalcone to act as a Michael
acceptor for nucleophilic species including glutathione (GSH) or
cysteine residues on proteins.21
As a part of our drug discovery pro-
gramme, we have hypothesized that the hydroxyl group of the
ligand may block the mushroom tyrosinase activity by binding to
the Cu atoms in the tyrosinase active site based on the fact that
previous findings had shown the role of hydroxyl groups in tyrosi-
nase inhibition.22
Oximes form an important class of organic com-
pounds having the general formula RR0
C@NAOH. They are
important precursors to functional groups such as amines, nitro
compounds and amides and also act as important ligands in the
formation of mono- and polynuclear metal complexes.23–26
Also,
oximes upon deprotonation, can act as strongly coordinating
ligands in metal-coordination chemistry.27,28
Classically, oximes are prepared by refluxing an alcoholic solu-
tion of a carbonyl compound with hydroxylamine hydrochloride
and pyridine.29
The excessive use of organic solvents, long reaction
times, high temperatures, and extensive work-up procedures,
make this solution-based synthetic method environmentally
stressful and expensive. This has been circumvented by employing
the use of a solvent free reaction that is not just eco-friendly but
also found to be much less time consuming with improved selec-
tivity and yields.30
The method makes use of local heat generated
by simply grinding the reactants and catalyzed by cheap and com-
mercially available CaO for driving the chemical reaction at room
temperature. Previously, several hydroxy-substituted 2-phenyl-
naphthalene derivatives were reported as potent tyrosinase inhibi-
tors. Among them, 4-(6-hydroxy-2-naphthyl)-1,3-bezendiol (HNB)
and 5-(6-hydroxy-2-naphthyl)-1,2,3-benzentriol (5HNB) were
found to inhibit mushroom tyrosinase activity.31,32
Hence, in our
continuous effort to search for potential tyrosinase inhibitors and
to develop a new template, we have attempted to design and syn-
thesize a series of novel hydroxy naphthyl substituted chalcone
oximes for application as depigmentation agents and as anti-
browning food additives. To confirm our hypothesis, we simulated
the docking between the ligands and tyrosinase and conducted
kinetic studies. The docking score for the ligand with receptor is
composed of various energy terms such as electrostatic energy,
van der Waals energy, and solvation energy.33
From the docking
results, we checked for possible hydrogen-bonding and non-bond-
ing interactions with the amino acid residues. For the control sim-
ulation, the docking simulation of kojic acid, a well-known
tyrosinase inhibitor, with tyrosinase was also performed (Fig. 1).
Chalcones were synthesized by the base catalyzed Claisen–Sch-
midt condensation of an aldehyde and an appropriate ketone in a
polar solvent like methanol (Scheme 1). In the second step, these
naphthyl chalcones on reaction with hydroxylamine hydrochloride
were converted into their corresponding oximes which were iso-
lated and recrystallized from ethylacetate. This method
emphasizes the effectiveness of CaO in oxime synthesis (Scheme 2)
under grinding conditions without any rearrangement of a,b-dou-
ble bond. The structures of the compounds were confirmed by 1
H
NMR, 13
C NMR, FTIR and HRMS. The signals for phenolic hydroxyl
protons appeared between 11 and 13 ppm and were indicated by
1
H NMR spectra. The signals for aromatic hydrogens are between
6.85 and 8.75 ppm. The signals for vinylic protons appeared
between 9.5 and 13 ppm which confirms their trans conforma-
tions. Chemical shift values further confirm an anti-isomer confor-
mation of the oximes. Assays were performed with L-DOPA as the
substrate, using kojic acid, a well-known tyrosinase inhibitor as the
positive control.
The parent naphthyl chalcones (1a–1g) showed poor or low
tyrosinase inhibitory activities (Table 1). However, it was interest-
ing to note that the oxime derivatives synthesized (2a–2e) had bet-
ter tyrosinase inhibitory activities when compared with the
positive control kojic acid, whereas the oxime compounds 2f and
2g had slightly decreased inhibition. In particular, compounds 2b
and 2c exhibited the greatest inhibition of L-DOPA oxidase activity
of mushroom tyrosinase with their IC50 values of 12.22 lM and
19.45 lM, respectively. These compounds were found to be more
potent than the positive control, kojic acid (IC50; 23.72 lM). Some
possible structure–activity relationships could be inferred from
tyrosinase inhibitory assay results: The pyridinyl nitrogen atom
in compounds 2a–2c can possibly get protonated at physiological
pH or might be available to coordinate the copper atom existing
in the tyrosinase active site. Additionally, some research groups
have reported hydroxamate derivatives to possess tyrosinase inhi-
bitory activities.35
The structures of the non-cyclic moieties of our
molecules have similar features to those of hydroxamic acids and
hydroxamates which are good chelating agents. There is a possibil-
ity of the oxime moiety to coordinate with the copper metal at the
active site of mushroom tyrosinase thereby preventing electron
transfer by the metal ion. This could decrease the enzymes ability
to oxidize the substrate subsequently leading to an inhibition in
mushroom tyrosinase activity. This was in agreement with the
experimental data. It has been suggested that the presence of a
hydroxyl group and of an electron donator group in the phenol ring
is a primary requirement for effective action as an alternative sub-
strate of tyrosinase. The hydroxyl groups in compounds carry out
the nucleophilic attack on the coppers of the tyrosinase active site
and are directly involved in transferring protons during catalysis,
which resulted in inactivation of tyrosinase. Also, the presence of
an ortho–para directing methoxy group in compound 2d could be
accounted to its better tyrosinase inhibitory potential. The elec-
tron-donating groups increase the electron density of ring B
through a resonance donating effect and higher electron density
binds copper ions more effectively in the active site of enzyme.
Thus, the inhibition mechanism of novel substituted hydroxy chal-
cone oximes 2a–2g might involve binding to the copper active site
of mushroom tyrosinase.
Accelrys Discovery studio 4.5 suite was utilized to simulate
binding between the active site of mushroom tyrosinase and sub-
stituted hydroxy naphthyl chalcone oximes. To model the tyrosi-
nase structure, we used the crystal structure of Agaricus bisporus
(mushroom) tyrosinase (PDB ID: 2Y9X) A chain. The results of vir-
tual screening studies indicated that the estimated CDOCKER
energy of all the docked ligands ranged between À19.29 and
À9.12 kcal/mol. Figure 1 shows selected docked conformations of
compounds 2a–2g along with the positive control, kojic acid in
the tyrosinase binding site. Additionally, we searched for hydrogen
bonding interactions between mushroom tyrosinase and the inhi-
bitor compounds or kojic acid (Table 2). Among all the compounds
docked, compound 2b showed the lowest estimated free energy of
binding which correlated well with the experimental results. We
searched for tyrosinase residues that might bind to compound 2b
4086 S. Radhakrishnan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4085–4091

3. and found that the most important binding residues for interaction
with 2b are His259, Phe264, Val248, Ala286, His61, His244, His85,
Ser282, Val299, Arg95, Leu303, His263, Asn260 and Val283. The
residues could have a key function and effect on binding affinity.
The NH–OH group was actively involved in hydrogen bonding
interactions in compounds 2a–2c, both as donor (via the O–H moi-
ety) and as hydrogen bond acceptor (via the N@CAOH moiety).
Also, oximes, popular in supramolecular chemistry as strong
hydrogen bond donors, could therefore be involved in hydrogen
bond interactions with the amino acid residues of the enzyme.36
This could in turn, help the compound to have a better fit to the
catalytic pocket of the enzyme tyrosinase.
Coordinate bonds make a major contribution to the binding of
an inhibitor in the active site. There was a coordination between
Cu401 and the pyridinyl nitrogen atom of compound 2c at a dis-
tance of 2.61 Å. Formation of a complex between a ligand and
the copper ion in the active site of mushroom tyrosinase could pre-
vent electron transfer by the metal ion. Moreover, the binding of
the inhibitor via a coordinate bond will ensure that access to the
active site by the substrate is effectively blocked. This could curb
the enzymes ability to oxidize the substrates subsequently leading
to an inhibition in mushroom tyrosinase. The catalytic pocket of
the enzyme is hydrophobic. Hydrophobic p–p stacking interac-
tions were seen with His263. For kojic acid, it was observed that
Figure 1. Docking result of compounds 2a–2g and the reference compound, kojic acid, in the tyrosinase catalytic pocket. Ligands 2a–2g are displayed as ball and stick while
the core amino acid residues are displayed as stick. The green dotted lines show the hydrogen bond interactions and the purple lines show the non-bonding interactions. The
ochre balls represent the copper ions.
S. Radhakrishnan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4085–4091 4087

4. the compound forms hydrogen bonds with Asn260 and also a p–p
interaction with His283.
In the present study, tyrosinase extracted from the edible
mushroom Agaricus bisporus is used due to its easy availability
and high homology with the mammalian enzyme that renders it
well suited as a model for studies on melanogenesis.37
We mea-
sured the reaction rates in the presence of active inhibitors 2b
and 2c with various concentrations of L-DOPA as a substrate.38
As
the concentrations of inhibitors increased, Km values gradually
increased, but Vmax values did not change, thereby indicating the
inhibitors to act as competitive inhibitors of mushroom tyrosinase.
The absorbance variations from these studies were used to gener-
ate Lineweaver–Burk plots to determine the inhibition type (Fig. 2).
Dixon plots gave a family of straight lines passing through the
same point at the second quadrant, giving the inhibition constant
(Ki) (Fig. 3). The Ki value estimated from this Dixon plot was
6.46 lM, and 7.82 lM for the compounds 2b and 2c, respectively.
A comparison of the Km and Ki values of the compounds with that
of kojic acid revealed that they possess much higher affinity to
tyrosinase than kojic acid (Table 3).
To summarize, in this Letter, experimental kinetics and compu-
tational analysis were used to study the inhibition of tyrosinase by
novel naphthyl chalcone oxime compounds as tyrosinase inhibi-
tors. In terms of developing effective tyrosinase inhibitors, the
presence of an NHOH moiety in the chalcone framework appears
important as this group can coordinate the copper ions. Docking
simulation studies yielded crucial information concerning the
operation of the inhibitor in the binding pocket of tyrosinase. Com-
pounds 2b and 2c were identified as competitive inhibitors in a
OH
O
Z
Y
X
b
OH
N
Z
Y
X
OH
OH
O R
1
R
2
R
3
R
4
b
OH
N R
1
R
2
R
3
R
4
OH
1a: X = N; Y = CH; Z = CH
1b: X = CH; Y = N; Z = CH
1c: X = CH; Y = CH; Z = N
2a: X = N; Y = CH; Z = CH
2b: X = CH; Y = N; Z = CH
2c: X = CH; Y = CH; Z = N
1d: R1
& R3
= O-Me; R2
& R4
= H
1e: R1
& R4
= O-Me; R2
& R3
= H
1f: R2
& R3
= O-Me; R1
& R4
= H
1g: R2
& R4
= O-Me; R1
& R3
= H
2d: R1
& R3
= O-Me; R2
& R4
= H
2e: R1
& R4
= O-Me; R2
& R3
= H
2f: R2
& R3
= O-Me; R1
& R4
= H
2g: R2
& R4
= O-Me; R1
& R3
= H
Scheme 2. Synthesis of oximes of naphthyl chalcones. Reagents and conditions: (b) NH2OH/HCl, CaO.
Fig. 1 (continued)
O
CH3
OH
+
O
HR
a
O
OH
R
R = aromatic group
Scheme 1. (General method for synthesis of naphthyl-chalcones 1a–1h). Reagents
and conditions: (a) MeOH, NaOH, 0 °C, 24 h.
4088 S. Radhakrishnan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4085–4091

5. kinetic study and were found to be significantly more potent than
kojic acid. An ortho–para directing group on ring B was favorable
for better tyrosinase inhibition. These studies confirm that
tyrosinase inhibition is complex and that it is the interaction of
several substituents and the structural framework that leads to
effective binding at the active site causing inhibition of enzyme
function.
Notes: Chemical reagents and instruments: Melting points (mp)
were determined with WRS-1B melting point apparatus and the
thermometer was uncorrected. NMR spectra were recorded on Agi-
lent 400 spectrometer at 25 °C in CDCl3 or DMSO-d6. All chemical
shifts (d) are quoted in parts per million downfield from TMS and
coupling constants (J) are given in Hz. Abbreviations used in the
splitting pattern were as follows: s = singlet, d = doublet, t = triplet,
m = multiplet. HRMS spectra were recorded using the Agilent
Technologies 6520 LC/MS-QTOF. All reactions were monitored by
TLC (Merck Kieselgel 60 F254) and the spots were visualized under
UV light. Infrared (IR) spectra were recorded on Thermo Scientific
NICOLET 6700 FT-IR spectrometer. Tyrosinase, L-3,4-dihydrox-
yphenylalanine (L-DOPA) and kojic acid were purchased from
Sigma–Aldrich Chemical Co.
General method for the synthesis of naphthyl chalcone derivatives
(1a–1h): To a stirred solution of 20
-hydroxy-10
-acetonaphthone
(1 mM) and an appropriate substituted aldehyde (1 mM) in 25 ml
methanol, was added pulverized NaOH (2 mM) and the mixture
was stirred at room temperature for 24–36 h. The reaction was
monitored by TLC using n-hexane/ethyl acetate (7:3) as mobile
phase. The reaction mixture was cooled to 0 °C (ice-water bath)
and acidified with HCl (10% v/v aqueous solution) to afford total
precipitation of the compounds. In most cases, a yellow precipitate
was formed, which was filtered and washed with 10% aqueous HCl
solution. In the cases where an orange oil was formed, the mixture
was extracted with CH2Cl2, the extracts were dried (Na2SO4) and
the solvent was evaporated to give the respective chalcones (1a–
1h).
General method for the synthesis of oximes of naphthyl chalcone
compounds (2a–2g): A mixture of hydroxy naphthyl chalcone
(1 mmol), hydroxylamine hydrochloride (1.2 mmol) and CaO
(0.6 mmol) was ground in a mortar with a pestle. On completion
of the reaction as monitored by TLC, ethylacetate (2 Â 10 mL)
was added to the reaction mixture and filtered to separate the
CaO. The filtrate was concentrated down to approximately 6 mL
and then poured into crushed ice to obtain the product as a precip-
itate. The precipitate was filtered and dried in high vacuum to yield
the pure oxime.
In silico docking between tyrosinase and target compounds: To fur-
ther understand the binding modes of synthesized compounds
with mushroom tyrosinase, molecular docking studies of com-
pounds 2a–2g were performed using Discovery Studio 4.5 (Accel-
rys, San Diego, CA, USA). To model the tyrosinase structure, we
used the crystal structure of Agaricus bisporus (mushroom) tyrosi-
nase (PDB ID: 2Y9X) A chain. Site Identification by Ligand Compet-
itive Saturation (SILCS) technique has been used. The method
involves simultaneously incorporating the ligands and doing
Table 2
Tyrosinase inhibition effects of naphthyl chalcones (2a–2g)
Compound Tyrosinase inhibition at 50 lM*
(%) CDOCKER energy (kcal/mol) Hydrogen bond prevalence (ligand-residue) Hydrogen bond distance (Å)
2a 58.2 ± 0.32 À12.62 N–OH. . .Oe2 (Glu256) 1.96
(His244) Hd2. . .N–OH 2.78
2b 74.9 ± 0.75 À19.29 N–OH. . .Ne2 (His244) 2.01
2c 70.5 ± 0.70 À13.51 N–OH. . .O (Asn260) 2.49
(Asn260) Ha. . .OH–N 2.47
2d 52.3 ± 0.14 À11.95 2-OMe. . .O (Gly281) 2.39
2e 56.4 ± 0.18 12.36 (His259) He1. . .5-OMe 2.50
2-OMe. . .O (Gly281) 2.75
2f 32.2 ± 0.22 9.12 (Arg268) HH12. . .20
-OH 1.96
(His259) He1. . .3-OMe 3.03
(His259) He. . .4-OMe 2.43
3-O-Me. . .Oe (Asn260) 2.35
4-O-Me. . .Oe2 (Glu256) 2.55
2g 49.6 ± 0.44 À9.89 20
-OH. . .Ne2 (His244) 2.50
*
Chalcone derivatives were synthesized according to the details in Scheme 2. Values indicate means ± SE for three determinations.
-150
-100
-50
0
50
100
150
200
250
300
-4 -2 0 2 4 6
1/V(µM/min)-1
1/S (mM/L)
0 mM
0.15 mM
0.30 mM
0.60 mM
2b
-150
-100
-50
0
50
100
150
200
250
300
-4 -2 0 2 4 6
1/V(µM/min)-1
1/S (mM/L)
2c
Figure 2. Lineweaver–Burk plots for inhibition of active compounds 2b and 2c
against mushroom tyrosinase for the catalysis of L-DOPA. The inhibitor concentra-
tions were 0, 0.15, 0.30 and 0.60 mM. The final enzyme concentration was 2.9 lg/
ml.
Table 1
Tyrosinase inhibition effects of parent naphthyl chalcones (1a–1g)
Compound Tyrosinase inhibition at 50 lM*
(%)
CDOCKER energy
(kcal/mol)
1a 27.4 ± 0.14 À11.12
1b 32.2 ± 0.44 À10.65
1c 28.2 ± 0.57 À11.22
1d 30.5 ± 0.56 À9.50
1e 37.6 ± 0.44 À11.15
1f 22.4 ± 0.77 À10.22
1g 18.7 ± 0.17 À8.64
Kojic acid 47.2 ± 0.55 À11.69
*
Chalcone derivatives were synthesized according to the details in Scheme 1.
Values indicate means ± SE for three determinations.
S. Radhakrishnan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4085–4091 4089

6. ‘Molecular Dynamics simulation’. It is an in silico free energy-
based competition assay that generates three-dimensional proba-
bility maps of fragment binding (Frag Maps) indicating favorable
fragment–protein interactions. The scores for docking simulation
are approximate binding energies. The docking score for the ligand
with receptor is composed of various energy terms such as electro-
static energy, van der Waals energy, and solvation energy. From
the docking results, we checked for possible hydrogen-bonding
and non-bonding interactions with the amino acid residues. For
the control simulation, the docking simulation of kojic acid, a
well-known tyrosinase inhibitor, with tyrosinase was also per-
formed. Discovery Studio automatically generates 2-dimentional
ligand interaction diagrams that use distinctive colors and con-
tours to identify the protein–ligand interactions and to study the
properties of the enzyme catalytic pocket.
Tyrosinase inhibition assay: Mushroom tyrosinase, L-DOPA and
tested samples were prepared by dissolving in 50 mM Na2HPO4–
NaH2PO4 buffer (pH 6.8). Reaction mixtures containing 50 lL of
2 mmol LÀ1
of L-DOPA, 50 lL of phosphate buffer and 50 lL of dif-
ferent concentrations (0.5 lM, 1.0 lM, 2.5 lM and 5.0 lM) of
tested compounds were added in 96 well microtiter plates,
followed by the addition of 50 lL of 0.2 mg mLÀ1
of mushroom
tyrosinase. Then, the absorbance variations accompanying the oxi-
dation of the substrate (L-DOPA) were recorded using a Flex Station
3 micro plate reader at 475 nm under a constant temperature of
37 °C. Average velocity (v) of the oxidation of the substrate (L-
DOPA) was determined from the linear slope of curve. Kojic acid
was used as positive control. The % inhibition of tyrosinase activity
was calculated as follows:
% inhibition ¼ ½1 À ðAbssample À AbsblankÞ=AbscontrolŠ Â 100
where Abssample, Absblank and Abscontrol are the absorbance of the
experimental sample, the blank and the control, respectively. IC50
indicates the concentration of the sample when the enzyme activity
is inhibited by 50%.
Determination of the inhibition type of compounds 2b and 2c
on mushroom tyrosinase.
Mushroom tyrosinase (50 lL; 0.2 mg mLÀ1
) was incubated with
50 lL of various concentrations of enzyme substrate and 50 lL of
phosphate buffer, and then 50 lL of different concentrations of
tested samples were simultaneously added to the reaction mix-
tures. The absorbance variations from these studies were used to
generate Lineweaver–Burk plots to determine the inhibition type.
The kinetic parameter (Km) of the tyrosinase activity was calcu-
lated by linear regression from Lineweaver–Burk plots. For the type
of enzyme inhibition and the inhibition constant (Ki) for an
enzyme–inhibitor complex, the mechanisms were analyzed by
Dixon plot, which is a graphical method [plot of 1/enzyme velocity
(1/V) vs inhibitor concentration (I) with varying concentrations of
the substrate].
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.08.
033.
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-40
-30
-20
-10
0
10
20
30
40
50
-40 -30 -20 -10 0 10 20 30
1/v(µM/min)-1
[inhibitor] µM
2b
-40
-30
-20
-10
0
10
20
30
40
50
-40 -30 -20 -10 0 10 20 30
1/v(µM/min)-1
[inhibitor] µM
2c
Figure 3. Dixon plot for the inhibitory effect of compounds 2b and 2c on L-DOPA
oxidation catalyzed by mushroom tyrosinase. The inhibitor concentrations were 0,
10 and 20 lM. The L-DOPA concentrations were 100, 200 and 300 lM.
Table 3
Effect on mushroom tyrosinase activity and kinetic analysis of compounds
Compound Type of inhibition$
IC50
*
(lM) Ki
#
(lM)
2a Competitive 20.25 ± 0.55 11.05
2b Competitive 12.22 ± 0.37 6.46
2c Competitive 19.45 ± 0.66 7.82
2d Competitive 22.96 ± 0.14 10.60
2e Competitive 21.88 ± 0.44 9.40
2f Competitive 42.27 ± 0.22 22.95
2g Competitive 52.85 ± 0.30 28.60
Kojic acid — 23.72 ± 0.48 11.20
*
(IC50): refers to the concentration of compound that caused 50% inhibition.
#
Values were measured at 5 lM of active compounds and Ki is the (inhibitor
constant).
$
Lineweaver–Burk plot of mushroom tyrosinase: Data are presented as mean
values of 1/V, which is the inverse of the increase in absorbance at wavelength
492 nm/min (DA492/min), for three independent tests with different concentra-
tions of L-DOPA as the substrate.
4090 S. Radhakrishnan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4085–4091

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