1. FULL PAPER
Inhibitory Kinetics of Azachalcones and their Oximes on Mushroom Tyrosinase:
A Facile Solid-state Synthesis
by Sini K. Radhakrishnan*, Ronald G. Shimmon, Costa Conn, and Anthony T. Baker
School of Chemistry and Forensic Science, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia
(phone: +61426687300; e-mail: sinikaranayil@gmail.com)
A solid-state-based mechanochemical process was used to synthesize novel azachalcones and their oximes as tyrosinase
inhibitors. Their inhibitory activities on mushroom tyrosinase using L-3,4-dihydroxyphenylalanine as a substrate were
investigated. Two of the novel oxime derivatives synthesized were seen to be more potent than the positive control, kojic
acid. Both the compounds 1b and 2b inhibited the diphenolase activity of tyrosinase in a dose-dependent manner with their
IC50 values of 15.3 and 12.7 lM, respectively. The kinetic analysis showed that their inhibition mechanism was reversible. Both
the novel oxime compounds displayed competitive inhibition with their Ki values of 5.1 and 2.5 lM, respectively. This method
minimizes waste disposal problems and provides a simple, efficient, and benign method for the synthesis of novel tyrosinase
inhibitors for use as skin-whitening agents or as anti-browning food additives.
Keywords: Azachalcones and their oximes, Tyrosinase inhibitors, Competitive inhibition, Grinding.
Introduction
Tyrosinase [EC 1.14.18.1], also known as, polyphenol oxi-
dase, a copper-containing metalloenzyme is responsible
for melanization in animals and browning in plants [1].
Melanin plays a crucial role against skin photocarcinogen-
esis [2]. However, an excessive accumulation of the pig-
ment could lead to serious esthetic issues. Alterations in
tyrosinase dysfunction could culminate with serious pig-
mentation disorders like melasma, chloasma, lentigo, age
spots, inflammatory hypermelanosis, and trauma-induced
hyperpigmentation [3]. In addition, tyrosinase is responsi-
ble for undesired enzymatic browning of fruits and veg-
etables that take place during senescence or damage in
postharvest handling, which makes the identification of
novel tyrosinase inhibitors extremely important. Enzy-
matic browning involves the enzyme tyrosinase that cat-
alyzes the ortho-hydroxylation of tyrosine (monophenol)
to 3,4-dihydroxy phenylalanine or L-3,4-dihydroxyphenyl-
alanine (L-DOPA, o-diphenol) and the oxidation of
L-DOPA to dopaquinone [4]. These quinone compounds
may irreversibly react with the amino and sulfhydryl
groups of proteins. The quinone–protein reaction
decreases the digestibility and inhibition of proteolytic
and glycolytic enzymes, and the bioavailability of essential
amino acids like cysteine and lysine. Thus, enzymatic
browning could culminate with discoloration and a
decline in the nutritional value of foods [5]. Recently, 4-
hexyl resorcinol has found to be quite effective for
browning control in fresh and dried fruit slices [6]. From
a structural perspective, tyrosinase belongs to a type-3 Cu
protein family known as the oxidoreductases harboring a
catalytic center [7 – 9]. Tyrosinase has two Cu ions in its
active site, which plays a vital role in its catalytic activity.
At the active site of tyrosinase, a dioxygen molecule binds
in side-on coordination between two Cu ions. Each of the
Cu ions is coordinated by three histidines in the protein
matrix [10]. The Cu-atoms participate directly in hydroxy-
lation of monophenols to diphenols (cresolase activity)
and in the oxidation of o-diphenols to o-quinones (cate-
chol oxidase activity) that enhance the production of the
brown color [11]. Therefore, chelation of tyrosinase Cu2+
by synthetic compounds or agents from natural sources
has been targeted as a way to inhibit or block tyrosinase
catalysis [12]. An alternative solution to inhibit tyrosinase
catalytic activity would be by effectively blocking access
to the active site of enzyme.
Despite reports of a large number of tyrosinase inhibi-
tors, only a few are used today because of their limita-
tions with regard to cytotoxicity, selectivity, and stability
[13 – 17]. This necessitates the need to develop new
tyrosinase inhibitors that could be used in the food indus-
try as well as in medicinal and cosmetic products but
without causing any adverse reactions. Previously, we
have reported the synthesis of hydroxy-substituted
azachalcone compounds with potential inhibitory effects
on mushroom tyrosinase activity [18]. Besides, our inter-
est for chelator agents led us to study the hydroxamic
acid group. Hydroxamate molecules, one of the major
classes of naturally occurring metal complexing agents,
DOI: 10.1002/cbdv.201500168 © 2016 Verlag Helvetica Chimica Acta AG, Z€urich
Chem. Biodiversity 2016, 13, 531 – 538 531

2. have been thoroughly studied as ligands for different
metal ions as Fe(III), Zn(II), and Cu(II)[19]. The chela-
tion involves the oxygen belonging to the =N–OH group
[20]. Also, oximes, popular in supramolecular chemistry
as strong hydrogen bond donors, could therefore be
involved in H-bond interactions with the amino acid resi-
dues of the enzyme [21]. On the basis of these findings,
we considered that it might be interesting to synthesize
oxime derivatives of the azachalcone compounds as
potential anti-browning food additives or even skin-
whitening agents.
Azachalcones are readily synthesized by the base-cata-
lyzed Claisen–Schmidt condensation of an aldehyde and
an appropriate ketone in a polar solvent like MeOH.
However, the traditional synthesis of their oxime deriva-
tives involves refluxing an aqueous/alcoholic solution of
the corresponding azachalcone compound with hydroxy-
lamine hydrochloride and base, followed by solvent
extraction to obtain the pure product [22][23]. The exces-
sive 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 green chemistry that is not only
eco-friendly but also found to be much less time consum-
ing with improved selectivity and yields [24]. The current
study employs the use of green chemistry where the
desired azachalcones were prepared by grinding together
equivalent amounts of the appropriate ketones and differ-
ent aldehydes in the presence of solid pulverized NaOH
at room temperature. Focusing on our interest toward
green chemistry, the use of calcium oxide for the synthe-
sis of oximes of azachalcones was examined and it was
found that the reaction proceeded smoothly to give oxi-
mes in quantitative yields. Thus, we have developed a
novel, quick, environmentally safe, and clean method for
the synthesis of oximes of azachalcones (Scheme 1) utiliz-
ing pestle and mortar under solvent-free conditions. The
method makes use of local heat generated by simply
grinding the reactants, and is catalyzed by cheap and
commercially available CaO for driving the chemical
reaction at room temperature. Most importantly, this
method minimizes waste disposal problems and provides
a rather benign protocol for the synthesis of potent tyrosi-
nase inhibitors to be targeted for their use as anti-brown-
ing food additives. The structures of the compounds
synthesized were confirmed by 1
H-NMR, 13
C-NMR,
FTIR, and HR-MS. Assays were performed with L-DOPA
as the substrate, using kojic acid, a well-known strong
tyrosinase inhibitor as the positive control. We have fur-
ther investigated the kinetic parameters and inhibition
mechanisms of active tyrosinase inhibitor compounds.
Results and Discussion
Effect of Compounds on Tyrosinase Activity: Inhibition
Kinetics
The present work is targeted toward the synthesis of
tyrosinase inhibitors by solid-state synthesis. In the first
step, simple grindstone chemistry was utilized to synthe-
size azachalcones. The parent azachalcones were prepared
by grinding together equivalent amounts of the appropri-
ate ketones and different aldehydes in the presence of
solid pulverized NaOH at room temperature. In the sec-
ond step, the azachalcones on reaction with hydroxy-
lamine hydrochloride and calcium oxide was converted
into corresponding oximes which were isolated and
recrystallized from AcOEt. This method emphasizes the
effectiveness of CaO in oxime synthesis under grinding
conditions without any rearrangement of a,b-double bond.
All the synthesized compounds were characterized by
spectroscopic data. The azachalcones (1a – 6a) synthe-
sized exhibited moderate tyrosinase inhibitory activities
with the exception of compounds 7a and 8a that had low
inhibition. This indicates that a 20
-OH group substituted
on ring A appears to be an important design element in
achieving enhanced tyrosinase inhibition. The presence of
a pyridinyl skeleton resulted in an improved tyrosinase
effect. The pyridinyl N-atom of substituted azachalcone
derivatives can possibly get protonated at physiological
pH or might be available to coordinate the Cu-atom
Scheme 1. General method for synthesis of azachalcones and their oxime derivatives.
532 Chem. Biodiversity 2016, 13, 531 – 538
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3. existing in the tyrosinase active site. Replacement of the
phenyl group in ring A with a naphthyl group brought
about a decrease in tyrosinase inhibitory activity as seen
with compounds 4a – 6a. This could be possibly due to
steric hindrance caused by the presence of the bulky
naphthyl group that could hinder the binding of the
ligand with the active site of the enzyme.
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 [25]. Briefly, all the synthesized com-
pounds were screened for the diphenolase inhibitory
activity of mushroom tyrosinase inhibition activity using
L-DOPA as substrate [26]. It was interesting to note that
all of the oxime derivatives synthesized (1b – 8b) had
good tyrosinase inhibitory activities when compared with
the positive control, kojic acid. In particular, compounds
1b and 2b exhibited the greatest inhibition of the
L-DOPA oxidase activity of mushroom tyrosinase. These
compounds were found to be more potent than the posi-
tive control, kojic acid (IC50: 37.3 lM). The structures of
the non-cyclic moieties of our molecules have similar fea-
tures to those of hydroxamic acids and hydroxamates
which are good chelating agents. Tyrosinase inhibition of
compounds 1b – 8b depended on the competency of the
hydroxamate moiety to chelate with the dinuclear Cu-
active site in the active site of enzyme [27]. There is a
possibility of the oxime moiety to coordinate with the Cu
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 sub-
sequently leading to an inhibition in mushroom tyrosinase
activity. Also, oximes, popular in supramolecular chem-
istry as strong H-bond donors, could therefore be
involved in H-bond interactions with the amino acid resi-
dues of the enzyme [28]. This could, in turn, help the
compound to have a better fit to the catalytic pocket of
the enzyme tyrosinase. Compound 7b with a 2,4-
dimethoxy substituent and compound 8b with a
p-dimethyl substituent showed moderate tyrosinase inhibi-
tions of 52.5% and 50.6%, respectively. The inhibitory
activities of these newly synthesized compounds were
compared to that of kojic acid as a reference standard
and the results are shown in Tables 1 and 2.
Inhibition Mechanism of the Selected Compounds on
Mushroom Tyrosinase
To explore the mechanism of active inhibitors, we con-
ducted a study of the kinetic behavior of tyrosinase activ-
ity in the presence of active inhibitors 1b and 2b. The
kinetic and inhibition constants were obtained by the
method previously described [29]. We used L-DOPA as
substrate for the effect of inhibitor compounds on the oxi-
dation of L-DOPA by tyrosinase (diphenolase activity).
The result showed that both compounds could inhibit the
diphenolase activity of tyrosinase in a dose-dependent
manner. With increasing concentrations of inhibitors, the
remaining enzyme activity decreased exponentially. The
inhibitor concentrations leading to 50% activity lost (IC50)
for compounds 1b and 2b were estimated to be 15.3 and
12.7 lM, respectively. The plots of the remaining enzyme
activity vs. the concentration of enzyme at different inhibi-
tor concentrations gave a family of straight lines, which all
passed through the origin. The presence of inhibitor did
not reduce the amount of enzyme, but just resulted in the
inhibition of enzyme activity. The results showed that both
the compounds 1b and 2b were reversible inhibitors of
mushroom tyrosinase for oxidation of L-DOPA (Fig. 1).
Lineweaver–Burk Analysis of Tyrosinase Inhibition by
inhibitor Compounds 1b and 2b
We measured the reaction rates in the presence of active
inhibitors with various concentrations of L-DOPA as a
substrate. As the concentrations of active inhibitors 1b
and 2b increased, Km values gradually increased, but Vmax
values did not change, thereby indicating the inhibitors
act as competitive inhibitors of mushroom tyrosinase
(Fig. 2). The inhibition kinetics were illustrated by Dixon
plots, which were obtained by plotting 1/V vs. [I] with
varying concentrations of substrate. Dixon plots gave a
family of straight lines passing through the same point at
the second quadrant, giving the inhibition constant (Ki)
Table 1. Tyrosinase inhibition effects of azachalcones (1a – 8a)
Compound Yield [%] Inhibition ratea
)
[%] at 50 lM
1a 48.2 59.2 Æ 0.2
2a 85.5 55.9 Æ 0.1
3a 42.3 48.9 Æ 0.4
4a 52.2 49.5 Æ 1.7
5a 82.5 59.2 Æ 2.2
6a 57.8 42.7 Æ 0.5
7a 88.0 12.3 Æ 0.2
8a 85.5 16.9 Æ 1.3
a
) Values indicate mean Æ SE for three determinations.
Table 2. Substitution pattern and tyrosinase inhibition effects of oxi-
mes of azachalcone compounds (1b – 8b)
Compound Yield [%] Inhibition ratea
)
[%] at 50 mM
1b 37.5 77.5 Æ 1.1
2b 65.5 80.6 Æ 0.5
3b 52.7 69.8 Æ 1.2
4b 49.5 58.2 Æ 1.2
5b 80.2 62.6 Æ 0.3
6b 50.5 57.5 Æ 0.7
7b 63.5 52.5 Æ 1.5
8b 69.6 50.6 Æ 0.6
Kojic acid 42.8 Æ 0.2
a
) Values indicate mean Æ SE for three determinations.
Chem. Biodiversity 2016, 13, 531 – 538 533
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4. (Fig. 3). The Ki value estimated from this Dixon plot was
5.1 and 2.5 lM, respectively for the compounds 1b and 2b.
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).
Conclusion
In this paper, we synthesized novel azachalcone oxime
compounds, and studied their inhibition on the dipheno-
lase activity of mushroom tyrosinase. The solvent-free
synthesis is particularly attractive because it incorporates
the principles of green chemistry. Azachalcone oxime
compounds 1b and 2b were found to be significantly more
potent than kojic acid with their IC50 values of 15.3 and
12.7 lM, respectively. Both the compounds exhibited
reversible competitive inhibition. In a nutshell, the pre-
sent study showed that the synthesized compounds can be
used as a template for the development of potent agents
in food preservation and cosmetics.
Experimental Part
General
Tyrosinase, L-DOPA, and kojic acid were purchased from
Sigma–Aldrich Chemical Co. (Castle Hill, NSW,
Fig. 1. The inhibitory mechanisms of compounds 1b and 2b on mush-
room tyrosinase were reversible. The concentrations of inhibitor used
for curves 0 – 4 are 0, 0.25, 0.5, 1.0, and 2.0 lM, respectively.
Fig. 2. Lineweaver–Burk plots for inhibition of compounds 1b and 2b
against mushroom tyrosinase for the catalysis of L-DOPA. The inhibi-
tor concentrations were 0, 0.05, 0.10, and 0.15 mM. The final enzyme
concentration was 2.9 lg/ml.
Fig. 3. Dixon plot for the inhibitory effect of compounds 1b and 2b
on L-DOPA oxidation catalyzed by mushroom tyrosinase. The inhibi-
tor concentrations were 0, 0.5, and 2.0 lM, respectively. The L-DOPA
concentrations were 100, 200, and 300 lM.
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5. Australia). TLC: Kieselgel 60F254 (Merck, Victoria, Aus-
tralia) and the spots were visualized under UV light.
M.p.: WRS-1B melting point apparatus (Shanghai, P. R.
China); uncorrected. IR spectra: Thermo Scientific NICO-
LET 6700 FT-IR spectrometer (Victoria, Australia); ṽ in
cmÀ1
. NMR spectra: Agilent 500 spectrometer (Victoria,
Australia) at 25° in CDCl3 or (D6)DMSO; d in ppm rel.
to Me4Si as internal standard, J in Hz. HR-MS: Agilent
Technologies 6520 LC/MS-QTOF; in m/z.
General Method for the Synthesis of Azachalcone
Derivatives (1a – 8a)
A mixture of the appropriate aromatic ketone (2 mmol),
aromatic aldehyde (2 mmol), and NaOH (7 mmol) was
thoroughly ground with a pestle in an open mortar at
room temperature for 2 – 3 min until the mixture turned
into a melt. The initial syrupy reaction mixture solidified
within 3 – 5 min. The grinding continued for 5 – 10 min
more, and the reaction was monitored by TLC. The solid
was washed with cold water to remove the sodium
hydroxide and recrystallized from MeOH to give the cor-
responding chalcone derivative.
(2E)-1-(2-Hydroxyphenyl)-3-(pyridin-2-yl)prop-2-en-1-
one (1a). M.p. 94 – 95°. IR (KBr): 3421, 3047, 2361, 1715,
1592, 1559, 1347, 1311, 1233, 1209, 983, 817, 756. 1
H-
NMR (500 MHz, CDCl3): 12.56 (s, OH); 8.66 (d, J = 9.0,
H–C(2)); 7.96 (d, J = 9.0, H–C(5)); 7.91 (t, J = 7.5, H–C
(4)); 7.89 (d, J = 15.5, Hb); 7.74 (d, J = 15.5, Ha); 7.52 (t,
J = 8.5, H–C(40
)); 7.38 (t, J = 8.0, H–C(3)); 7.34 (d, H–C
(60
)); 7.03 (d, J = 7.5, H–C(30
)); 6.96 (t, J = 9.0, H–C(50
)).
13
C-NMR (125 MHz, (D6)DMSO): 195.2 (C=O); 152.6 (C
(6)); 126.1 (C(5)); 137.5 (C(4)); 124.9 (C(3)); 154.2 (C(2));
166.1 (C(60
)); 121.4 (C(10
)); 121.6 (C(50
)); 139.2 (C(40
));
122.4 (C(30
)); 132.3 (C(20
));122.2 (vinylic); 144.6 (vinylic).
HR-MS: 226.0869 ([M+H]+
; calc. 226.0868).
(2E)-1-(2-Hydroxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-
one (2a). M.p. 86 – 88°. IR (KBr): 3400, 1646, 1315, 1591,
1646, 1492, 825 and 689, 755 and 689. 1
H-NMR
(500 MHz, CDCl3): 12.55 (s, 1 H, OH); 8.7 (s, 2 H); 7.89
(d, 1 H, Ha, J = 12.5); 7.79 (d, 2 H, H(2), and H(6),
J = 10.5); 7.55 (d, 1 H, J = 8.0); 7.53 (t, 1 H, H(40
),
J = 9.0); 7.51 (dd, 1 H, H(50
), J = 8.0); 7.48 (dd, 1 H, H
(5), J = 8.5); 7.06 (d, 1 H, Hb, J = 12.5); 6.99 (t, 1 H, H
(30
), J = 9.5). 13
C-NMR (125 MHz, (D6)DMSO): 193.1
(C=O); 119.7 (C(10
)); 129.7 (C(20
)); 118.8 (C(30
)); 136.9 (C
(40
)); 119.0 (C(50
)); 163.7 (C(60
)); 150.6 (C(2)); 142.1 (C
(6)); 124.4 (C(5)); 129.7 (C(3)); 131.4 (C(4)); 123.2
(vinylic); 144.4 (vinylic). HR-MS: 226.0862 ([M+H])+
;
calc. 226.0868).
(2E)-1-(2-Hydroxyphenyl)-3-(pyridin-4-yl)prop-2-en-1-
one (3a). M.p. 139 – 140°. IR (KBr): 3445, 3034, 1710,
1583, 1568, 1376, 1349, 1318, 1216, 809, 752. 1
H-NMR
(500 MHz, CDCl3): 12.66 (s, 1 H, OH); 8.88 (s, 2 H, H
(30
), and H(50
), J = 15.5); 7.96 (d, 1 H, Ha, J = 11.0); 7.71
(d, 2 H, H(20
), and H(60
), J = 9.0); 7.54 (t, 1 H, H(40
),
J = 7.5); 7.46 (d, 1 H, H(4), J = 9.5); 7.44 (t, 1 H, H(6),
J = 7.5); 7.05 (d, 1 H, Hb, J = 11.0); 6.95 (t, 1 H, H(3),
J = 8.0); 6.89 (dd, 1 H, H(5), J = 8.0). 13
C-NMR
(125 MHz, (D6)DMSO): 195.7 (C=O); 153.2 (C(2) and C
(6)); 122.3 (C(3) and C(5)); 132.3 (C(20
)); 121.7 (C(30
));
139.6 (C(40
)); 121.4 (C(50
)); 124.8 (C(10
)); 166.3 (C(60
));
144.5 (C(4)); 144.7 (vinylic); 127.2 (vinylic). ESI-MS:
226.1 ([M+H]+
).
(2E)-1-(3-Hydroxynaphthalen-2-yl)-3-(pyridin-2-yl)prop-
2-en-1-one (4a). M.p. 109 – 110°. IR (KBr): 3360, 3350,
2924, 2723. 1712, 1652, 1629, 1350, 1312, 1246, 1215, 822,
751. 1
H-NMR (500 MHz, CDCl3): 13.50 (s, 1 H, OH);
8.79 (dd, 1 H, H(3), J = 9.0); 8.01 (dd, 1 H, J = 9.5, H(90
));
7.92 (dd, 1 H, H(6), J = 10.5); 7.85 (dd, 1 H, H(40
), J = 7.5);
7.82 (d, 1 H, Ha, J = 13.5); 7.79 (dd, 1 H, H(5), J = 10.0);
7.57 (d, 1 H, H(4), J = 8.5); 7.52 (dd, 1 H, H(60
), J = 9.5);
7.50 (dd, 1 H, J = 8.0, H(80
)); 7.39 (t, 1 H, H(70
), J = 7.0);
7.25 (d, 1 H, H(30
), J = 15.5); 7.18 (d, 1 H, Hb, J = 13.5). 13
C-
NMR (125 MHz, (D6)DMSO): 193.1 (C=O); 114.0 (C(10
));
162.5 (C(20
)); 122.1 (C(30
)); 136.2 (C(40
)); 127.4 (C(50
)); 130.2
(C(60
)); 124.5 (C(70
)); 128.2 (C(80
)); 124.8 (C(90
)); 131.6 (C
(100
)); 140.1 (vinylic); 128.5 (vinylic); 150.2 C(1); 148.2 (C3);
124.5 (C(4)); 135.9 (C(5)); 120.8 (C(6)). HR-MS: 276.1034
([M+H]+
; calc. 276.1024).
(2E)-1-(3-Hydroxynaphthalen-2-yl)-3-(pyridin-3-yl)prop-
2-en-1-one (5a). M.p. 99 – 101°. IR (KBr): 3375, 3325,
3250, 1715, 1655, 1355, 1265, 1242, 1210, 815, 756. 1
H-
NMR (500 MHz, CDCl3): (13.65 (s, 1 H, OH); 9.22 (s,
1 H, H(2)); 8.80 (d, 1 H, H(4), J = 7.5); 8.27 (d, 1 H, H
(6), J = 10.5); 8.12 (dd, 1 H, H(90
), J = 8.5); 7.90 (dd, 1 H,
J = 7.5, H(40
)); 7.82 (d, 1 H, Ha, J = 10.5); 7.59 – 7.65 (m,
1 H, H(60
), J = 9.0); 7.52 (dd, 1 H, H(80
), J = 9.0); 7.48
(dd, 1 H, H(5), J = 9.0); 7.35 (t, 1 H, H(70
), J = 7.5); 7.14
(d, 1 H, Hb, J = 10.5); 7.08 (d, 1 H, H(30
), J = 15.0).
13
C-NMR (125 MHz, (D6)DMSO): 193.4 (C=O); 114.0 (C
(10
)); 162.7 (C(20
)); 119.1 (C(30
)); 135.7 (C(40
)); 127.2
(C(50
)); 126.3 (C(60
)); 123.5 (C(70
)); 128.0 (C(80
)); 125.9
(C(90
)); 130.7 (C(100
)); 142.5 (vinylic); 128.2 (vinylic);
131.2 C(1); 150.9 (C(2)); 155.1 (C(4)); 125.9 (C(5)); 136.0
(C(6)). HR-MS: 276.1042 ([M+H]+
; calc. 276.1024).
(2E)-1-(3-Hydroxynaphthalen-2-yl)-3-(pyridin-4-yl)prop-
2-en-1-one (6a). M.p. 92 – 94°. IR (KBr): 3479, 3362, 3250,
1718, 1690, 1325, 1265, 1220, 1217, 769, 747. 1
H-NMR
(500 MHz, CDCl3): (13.20 (s, 1 H, OH); 8.82 (dd, 2 H, H
(3), and H(5), J = 11.5); 8.15 (dd, 1 H, H(90
), J = 7.5); 7.87
(dd, 1 H, H(40
), J = 7.0); 7.84 (d, 1 H, Ha, J = 12.0); 7.79
(dd, 2 H, H(2), and H(6), J = 9.5); 7.75 – 7.82 (t, 1 H, H
Table 3. Effect on mushroom tyrosinase activity and kinetic analysis
of compounds
Compound Type of inhibition IC50 [mM] Ki
a
) [mM]
1b Competitive 15.3 Æ 1.0 5.1
2b Competitive 12.7 Æ 0.8 2.5
Kojic acid Competitive 37.3 Æ 0.3 8.8
a
) Values were measured at 5 mM of active compounds and Ki is the
(inhibitor constant).
Chem. Biodiversity 2016, 13, 531 – 538 535
© 2016 Verlag Helvetica Chimica Acta AG, Z€urich www.cb.wiley.com

6. (60
), J = 9.5); 7.49 (dd, 1 H, H(80
), J = 9.5); 7.37 (t, 1 H, H
(70
), J = 8.0); 7.22 (d, 1 H, Hb, J = 12.0); 7.19 (d, 1 H, H
(30
), J = 9.5). 13
C-NMR (125 MHz, (D6)DMSO): 194.4
(C=O); 117.2 (C(10
)); 163.7 (C(20
)); 119.2 (C(30
)); 136.8
(C(40
)); 128.4 (C(50
)); 130.6 (C(60
)); 124.5 (C(70
)); 128.4
(C(80
)); 122.9 (C(90
)); 132.7 (C(100
)); 142.2 (vinylic); 127.2
(vinylic); 142.2 (C(1)); 122.5 (C(2) and C(6)); 150.5 (C(3)
and C(5)). HR-MS: 276.1014 ([M+H]+
; calc. 276.1024).
(2E)-3-(2,4-Dimethoxyphenyl)-1-(pyridin-2-yl)prop-2-en-
1-one (7a). M.p. 130 – 132°. IR (KBr): 3017, 2954, 1669,
1640, 1405, 1355, 1265, 1175, 805, 672. 1
H-NMR (500 MHz,
CDCl3): 8.73 (dd, 1 H, H(3), J = 10.5); 8.29 (dd, 1 H, H(6),
J = 8.0); 7.92 (d, 1 H, Ha, J = 14.5); 7.85 (t, 1 H, H(5),
J = 9.5); 7.72 (dd, 1 H, H(60
), J = 9.0); 7.45 – 7.52 (dd, 1 H,
H(4), J = 11.5); 7.15(d, 1 H, Hb, J = 14.5); 6.54 (d, 1 H, H
(50
), J = 8.5); 6.46 (s, 1 H, H(30
)); 3.91 (s, 3 H); 3.86 (s,
3 H). 13
C-NMR(125 MHz, (D6)DMSO): 199.7 (C=O);
153.5 (C(1)); 121.2 (C(6)); 135.9 (C(5)); 127.2 (C(4)); 148.9
(C3); 55.6 (Me); 162.9 (C(10
)); 97.7 (C(30
)); 166.5 (C(40
));
107.2 (C(50
)); 130.2 (C(60
)); 144.2 (vinylic); 128.2 (vinylic).
ESI-MS: 270.1 ([M+H]+
).
(2E)-3-[4-(Dimethylamino)phenyl]-1-(pyridin-2-yl)prop-
2-en-1-one (8a). M.p. 120 – 122°. IR (KBr): 3372, 3055,
3005, 2922, 1870, 1659, 1149, 1054, 802, 679. 1
H-NMR
(500 MHz, CDCl3): 8.73 (dd, 1 H, H(3), J = 9.5); 8.03 (dd,
1 H, H(6), J = 8.5); 7.89 (d, 1 H, Ha, J = 11.0); 7.83 (t,
1 H, H(5), J = 9.5); 7.62 (dd, 2 H, H(20
), and H(40
),
J = 9.5); 7.47 (m, 1 H, H(4), J = 10.5); 7.19 (d, 1 H, Hb,
J = 11.0); 6.68 (m, 2 H, H(30
), and H(50
), J = 11.5). 13
C-
NMR (125 MHz, (D6)DMSO): 198.2 (C=O); 153.2 C(1);
121.0 (C(6)); 136.9 (C(5)); 127.7 (C(4)); 147.2 (C3); 39.8 (-
Me); 125.9 (C(10
)); 110.7 (C(30
) and C(50
)); 130.9 (C(2) and
C(60
)); 157.2 (C(40
)); 141.3 (vinylic); 127.5 (vinylic). ESI-
MS: 253.1 ([M+H]+
).
General Method for the Synthesis of Oximes of
Azachalcone Compounds (1b – 8b)
A mixture of azachalcone (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, AcOEt (2 9 10 ml) was
added to the reaction mixture and filtered to separate the
CaO. The filtrate was concentrated down to approxi-
mately 6 ml and then poured into crushed ice to obtain
the product as a precipitate. The precipitate was filtered
and dried in high vacuum to yield the pure oxime.
2-[(1E,2E)-N-Hydroxy-3-(pyridin-2-yl)prop-2-enimidoyl]
phenol (1b). M.p. 139 – 140°. IR (KBr): 3440, 3325, 3047,
2361, 1685, 1592, 1559, 1347, 1309, 1233, 1209, 992, 789,
756. 1
H-NMR (500 MHz, CDCl3): 12.65 (s, 1 H–OH);
8.78 (s, 1 H); 7.96 (t, 1 H, H(5), J = 9.0); 7.91 (t, 1 H,
H(4), J = 8.5); 7.89 (q, 2 H, H(40
), and H(50
), J = 8.5);
7.87 (d, 1 H, Hb, J = 16.0); 7.74 (d, 1 H, Ha, J = 15.5);
7.66 (dd, 1 H, H(6), J = 9.0); 7.48 (dd, 1 H, H(3),
J = 7.5); 7.37 (d, 1 H, H(60
), J = 9.5); 6.98 (dd, 1 H, H
(30
), J = 8.0). 13
C-NMR (125 MHz, (D6)DMSO): 159.8
(C=N); 152.3 (C(6)); 126.2 (C(5)); 137.3 (C(4)); 124.4
(C3); 165.6 (C(60
)); 121.4 (C(10
)); 121.6 (C(50
)); 139.2 (C
(40
)); 122.4 (C(30
)); 132.3 (C(20
)); 122.5 (vinylic); 144.7
(vinylic). HR-MS: 241.0952 ([M+H]+
; calc. 241.0977).
2-[(1E,2E)-N-Hydroxy-3-(pyridin-3-yl)prop-2-enimidoyl]
phenol (2b). M.p. 144 – 146°. IR (KBr): 3445, 3320, 3247,
1660, 1580, 1528, 1355, 1309, 1318, 1216, 786, 771. 1
H-
NMR (500 MHz, CDCl3): 12.20 (s, 1 H, OH); 9.38 (s,
1 H); 8.79 (s, 2 H, H(30
) and H(50
), J = 15.0); 7.89 (d,
1 H, Ha, J = 13.0); 7.75 (d, 2 H, H(20
) and H(60
), J = 9.5);
7.52 (t, 1 H, H(40
), J = 9.0); 7.42 (t, 1 H, H(6), J = 7.5);
7.26 (d, 1 H, H(4), J = 10.0); 7.05 (d, 1 H, Hb, J = 13.0);
6.82 (dd, 1 H, H(5), J = 8.5); 6.55 (t, 1 H, H(3), J = 8.5).
13
C-NMR (125 MHz, (D6)DMSO): 160.2 (C=N); 152.5 (C
(2) and C(6)); 121.4 (C(3) and C(5)); 132.3 (C(20
)); 120.6
(C(30
)); 140.6 (C(40
)); 121.4 (C(50
)); 125.7 (C(10
)); 162.3
(C(60
)); 144.5 (C(4)); 144.4 (vinylic); 128.2 (vinylic). HR-
MS: 241.0992 ([M+H]+
; calc. 241.0977).
2-[(1E,2E)-N-Hydroxy-3-(pyridin-4-yl)prop-2-enimidoyl]
phenol (3b). M.p. 137 – 139°. IR (KBr): 3425, 3340, 3035,
2347, 1652, 1590, 1552, 1345, 1300, 1222, 1210, 991, 783,
751. 1
H-NMR (500 MHz, CDCl3): 12.25 (s, 1 H–OH); 8.78
(dd, 2 H, H(3), and H(5), J = 15.5); 8.70 (s, 1 H); 7.99 (d,
1 H, Ha, J = 11.5); 7.74 (dd, 2 H, H(2), and H(6), J = 9.0);
7.59 (t, 1 H, H(40
), J = 10.5); 7.51 (dd, 1 H, H(60
), J = 7.5);
7.10 (d, 1 H, Hb, J = 11.5); 6.97 (dd, 1 H, H(30
), J = 9.0); 6.89
(dd, 1 H, H(50
), J = 8.0). 13
C-NMR (125 MHz, (D6)DMSO):
159.9 (C=N); 153.4 (C(2) and C(6)); 122.41 (C(3) and C(5));
132.3 (C(20
)); 121.8 (C(30
)); 139.8 (C(40
)); 121.4 (C(50
)); 124.8
(C(10
)); 166.4 (C(60
)); 144.5 (C(4)); 144.7 (vinylic); 127.2
(vinylic). HR-MS: 241.0933 ([M+H]+
; calc. 241.0977).
3-[(1E,2E)-N-Hydroxy-3-(pyridin-2-yl)prop-2-enimidoyl]
naphthalen-2-ol (4b). M.p. 149 – 151°. IR (KBr): 3360,
3322, 3248, 1652, 1629, 1342, 1276, 1215, 782, 751. 1
H-NMR
(500 MHz, CDCl3): 13.52 (s, 1 H, OH); 9.31 (s, 1 H); 8.81
(dd, 1 H, H(3), J = 9.5); 8.07 (dd, 1 H, H(90
), J = 9.0); 7.94
(dd, 1 H, H(6), J = 10.5); 7.85 (d, 1 H, Ha, J = 10.5); 7.82
(dd, 1 H, H(40
), J = 7.0); 7.80 (dd, 1 H, H(5), J = 9.5); 7.75
(dd, 1 H, H(60
), J = 10.5); 7.54 (d, 1 H, H(4), J = 8.5); 7.52
(dd, 1 H, H(80
), J = 8.5); 7.23 (d, 1 H, H(30
), J = 15.0); 7.32
(t, 1 H, H(70
), J = 7.5); 7.10 (d, 1 H, Hb, J = 10.5). 13
C-
NMR (125 MHz, (D6)DMSO): 163.5 (C=N); 114.1 (C(10
));
164.0 (C(20
)); 120.1 (C(30
)); 136.7 (C(40
)); 128.6 (C(50
));
130.2 (C(60
)); 123.6 (C(70
)); 128.3 (C(80
)); 124.0 (C(90
));
131.7 (C(100
)); 142.1 (vinylic); 128.1 (vinylic); 151.3 C(1);
150.2 (C3); 124.6 (C(4)); 137.9 (C(5)); 122.0 (C(6)). HR-MS:
291.1132 ([M+H]+
; calc. 291.1133).
3-[(1E,2E)-N-Hydroxy-3-(pyridin-3-yl)prop-2-enimidoyl]
naphthalen-2-ol (5b). M.p. 154 – 156°. IR (KBr): 3400,
3320, 3250, 1655, 1690, 1352, 1280, 1215, 790, 751. 1
H-NMR
(500 MHz, CDCl3): 13.72 (s, 1 H, OH); 9.35 (s, 1 H); 9.11
(s, 1 H, H(2)); 8.85 (d, 1 H, H(4), J = 8.0); 8.20 (d, 1 H, H
(6), J = 11.5); 8.01 (dd, 1 H, H(90
), J = 8.0); 7.99 (dd, 1 H,
H(40
), J = 7.75); 7.85 (d, 1 H, Ha, J = 12.5); 7.79 (dd, 1 H,
H(60
), J = 12.0); 7.54 (dd, 1 H, H(80
), J = 8.0); 7.52 (dd,
1 H, H(5), J = 9.0); 7.38 (t, 1 H, H(70
), J = 7.5); 7.28 (d,
1 H, J = 15.5, H(30
)); 7.12 (d, 1 H, Hb, J = 12.5). 13
C-NMR
536 Chem. Biodiversity 2016, 13, 531 – 538
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7. (125 MHz, (D6)DMSO): 162.1 (C=N); 114.5 (C(10
)); 163.9
(C(20
)); 119.9 (C(30
)); 136.9 (C(40
)); 128.2 (C(50
)); 129.7 (C
(60
)); 123.5 (C(70
)); 128.2 (C(80
)); 123.9 (C(90
)); 131.9 (C
(100
)); 142.27 (vinylic); 128.05 (vinylic); 131.6 C(1); 151.2 (C
(2)); 154.6 (C(4)); 124.9 (C(5)); 136.0 (C(6)). HR-MS:
291.1149 ([M+H]+
; calc. 291.1133).
3-[(1E,2E)-N-Hydroxy-3-(pyridin-4-yl)prop-2-enimidoyl]
naphthalen-2-ol (6b): M.p. 132 – 134°. IR (KBr): 3462,
3350, 3310, 3249, 1625, 1690, 1322, 1276, 1225, 1215, 769,
742. 1
H-NMR (500 MHz, CDCl3): 13.59 (s, 1 H, OH); 9.30
(s, 1 H); 8.89 (m, 2 H, H(3), and H(5), J = 10.5); 8.09 (dd,
1 H, H(90
), J = 7.5); 7.92 (dd, 1 H, H(40
), J = 7.5); 7.81 (d,
1 H, Ha, J = 11.5); 7.74 (dd, 2 H, H(2), and H(6), J = 9.0);
7.72 (m, 1 H, H(60
), J = 9.5); 7.52 (dd, 1 H, H(80
), J = 9.0);
7.35 (t, 1 H, H(70
), J = 8.5); 7.22 (d, 1 H, H(30
), J = 11.5);
7.15 (d, 1 H, Hb, J = 11.5). 13
C-NMR (125 MHz, (D6)
DMSO): 164.1 (C=N); 114.0 (C(10
)); 163.4 (C(20
)); 119.2 (C
(30
)); 136.9 (C(40
)); 128.0 (C(50
)); 129.6 (C(60
)); 123.5 (C
(70
)); 128.0 (C(80
)); 122.9 (C(90
)); 131.7 (C(100
)); 142.1
(vinylic); 126.2 (vinylic); 141.6 C(1); 122.2 (C(2) and C(6));
151.6 (C(3) and C(5)). HR-MS: 291.1142 ([M+H]+
; calc.
291.1133).
(1E,2E)-3-(2,4-Dimethoxyphenyl)-N-hydroxy-1-(pyridin-
2-yl)prop-2-en-1-imine (7b). M.p. 103 – 105°. IR (KBr):
3455, 3330, 3025, 2970, 1620, 1424, 1352, 1260, 1120,
1165, 855, 672. 1
H-NMR (500 MHz, CDCl3): 8.70 (dd,
1 H, H(3), J = 11.0); 8.27 (dd, 1 H, H(6), J = 8.5); 7.95
(d, 1 H, Ha, J = 14.5); 7.89 (t, 1 H, H(5), J = 9.0); 7.45
(dd, 1 H, H(4), J = 10.5); 7.79 (dd, 1 H, H(60
), J = 9.5);
7.22 (d, 1 H, Hb, J = 14.5); 6.50 (d, 1 H, H(50
), J = 8.0);
6.49 (s, 1 H, H(30
)); 3.89 (s, 3 H); 3.82 (s, 3 H). 13
C-
NMR (125 MHz, (D6)DMSO): 160.7 (C=N); 151.3 C(1);
120.2 (C(6)); 137.6 (C(5)); 125.9 (C(4)); 149.1 (C3); 57.1
(Me); 160.9 (C(10
)); 98.2 (C(30
)); 162.1 (C(40
)); 102.2 (C
(50
)); 131.2 (C(60
)); 142.4 (vinylic); 129.1 (vinylic). HR-
MS: 285.1220 ([M+H]+
; calc. 285.1239).
4-[(1E,3E)-3-(Hydroxyimino)-3-(pyridin-2-yl)prop-1-
en-1-yl]-N,N-dimethylaniline (8b). M.p. 104 – 106°. IR
(KBr): 3465, 3369, 3042, 2965, 2452, 1640, 1252, 1164,
1090, 862, 651. 1
H-NMR (500 MHz, CDCl3): 8.77 (dd,
1 H, H-3, J = 8.5); 8.07 (dd, 1 H, H(6), J = 9.5); 7.92 (dd,
2 H, H(20
), and H(40
), J = 10.5); 7.89 (t, 1 H, H(5),
J = 9.0); 7.82 (d, 1 H, Ha, J = 12.0); 7.51 (dd, 1 H, H(4),
J = 11.5); 7.20 (d, 1 H, Hb, J = 12.0); 6.72 (dd, 2 H, H(30
),
and H(50
), J = 15.5). 13
C-NMR(125 MHz, (D6)DMSO):
169.5 (C=N); 150.6 C(1); 127.5 (C(6)); 136.2 (C(5)); 128.3
(C(4)); 145.0 (C3); 42.2 (-Me); 125.2 (C(10
)); 111.3 (C(30
)
and C(50
)); 135.9 (C(2) and C(60
)); 159.6 (C(40
)); 140.9
(vinylic); 129.4 (vinylic). HR-MS: 268.1412 ([M+H]+
; calc.
268.1450).
Enzyme Activity Assay
The activity assay used 3 ml of reaction medium contain-
ing 0.5 mM L-DOPA in 50 mM Na2HPO4–NaH2PO4 buf-
fer (pH 6.8). The final concentration of tyrosinase was
3.33 mg/ml. The substrate reaction progress curve was
analyzed to obtain the reaction rate constants. The reac-
tion was carried out at 30 °C and pH 6.8.
Determination of the Inhibition Type of Compounds
1b and 2b on Mushroom Tyrosinase
Inhibitors were first dissolved in DMSO and used for the
test after a 30-fold dilution. The final concentration of
DMSO in the test solution was 3.33%. Mushroom tyrosi-
nase (50 ll; 0.2 mg/ml) was incubated with 50 ll of vari-
ous concentrations of enzyme substrate and 50 ll of
phosphate buffer, and then 50 ll of different concentra-
tions of tested samples were simultaneously added to the
reaction mixtures. The measurement was performed in
triplicate for each concentration and averaged before fur-
ther calculation. 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 calculated 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/en-
zyme velocity (1/V) vs. inhibitor concentration (I) with
varying concentrations of the substrate.
REFERENCES
[1] A. Sanchez-Ferrer, J. N. Rodriguez-Lopez, F. Garcia-Canavas,
F. Garcia-Carmona, Biochim. Biophys. Acta 1995, 1247, 1.
[2] M. Brenner, V. J. Hearing, Photochem. Photobiol. 2008, 84, 539.
[3] S. K. Fistarol, P. H. Itin, J. Dtsch. Dermatol. Ges. 2010, 8, 187.
[4] C. J. Cooksey, P. J. Garratt, E. J. Land, S. Pavel, C. A. Rams-
den, P. A. Riley, N. P. M. Smit, J. Biol. Chem. 1997, 272, 26226.
[5] M. V. Martinez, J. R. Whitaker, Trends Food Sci. Tech. 1995, 6,
195.
[6] A. J. McEvily, R. Iyneger, Crit. Rev. Food Sci. Nutr. 1992, 32,
253.
[7] K. E. Van Holde, K. I. Miller, H. Decker, J. Biol. Chem. 2001,
276, 15563.
[8] S. Naish-Byfield, P. A. Riley, Pigment Cell Res. 1998, 11, 127.
[9] E. J. Land, C. A. Ramsden, P. A. Riley, Acc. Chem. Res. 2003,
36, 300.
[10] Y. Matoba, T. Kumagai, A. Yamamoto, H. Yoshitsu, M.
Sugiyama, J. Biol. Chem. 2008, 281, 8981.
[11] H. Decker, F. Tuczek, Trends. Biochem. Sci. 2000, 25, 392.
[12] N. Radhakrishnan, S. Ashok, V. Kavitha, G. Rameshkumar, A.
Gnanamani, J. Chem. Pharm. Res. 2013, 5, 320.
[13] A. Karioti, A. Protopappa, N. Megoulas, H. Skaltsa, Bioorg.
Med. Chem. 2007, 15, 2708.
[14] S. Okombi, D. Rival, S. Bonnet, A. M. Mariotte, E. Perrier, A.
Boumendjel, Bioorg. Med. Chem. Lett. 2006, 16, 2252.
[15] G. Tocco, A. Fais, G. Meli, M. Begala, G. Podda, M. B. Fadda,
M. Corda, O. A. Attanasi, P. Filippone, S. Berretta, Bioorg.
Med. Chem. Lett. 2009, 19, 36.
[16] H. Satooka, I. Kubo, Bioorg. Med. Chem. 1090, 2012, 20.
[17] S. K. Radhakrishnan, R. G. Shimmon, C. Conn, A. T. Baker,
Bioorg. Med. Chem. Lett. 2015, 25, 1753.
[18] M. Criton, V. Mellay-Hamon, Bioorg. Med. Chem. Lett. 2008,
18, 3607.
Chem. Biodiversity 2016, 13, 531 – 538 537
© 2016 Verlag Helvetica Chimica Acta AG, Z€urich www.cb.wiley.com

8. [19] H. S. Baek, H. S. Rho, J. W. Yoo, S. M. Ahn, J. Y. Lee, J. Lee,
M. K. Kim, D. K. Kim, I. S. Chang, Bull. Korean Chem. Soc.
2008, 29, 43.
[20] C. B. Aaker€oy, J. Desper, B. A. Helfrich, P. Metrangolo, T.
Pilati, G. Resnati, A. Stevenazzi, Chem. Commun. 2007, 41,
4236.
[21] H. J. Lindner, S. Goettlicher, Acta Crystallogr. Sect. B 1969, 25,
832.
[22] C. B. Aaker€oy, N. Schultheiss, A. Rajbanshi, J. Desper, C.
Moore, Cryst. Growth Des. 2008, 9, 432.
[23] P. Anastas, J. Warner, ʻGreen Chemistryʼ, New York, Oxford
University Press, 1998, p. 30.
[24] M. T. H. Khan, Top Heterocycl. Chem. 2007, 9, 119.
[25] K. K. Song, Q. X. Chen, Q. Wang, L. Qiu, H. Huang, J.
Enzyme Inhib. Med. Chem. 2005, 20, 239.
[26] C. Gerdemann, C. Eicken, B. Krebs, Acc. Chem. Res. 2002, 35,
183.
[27] J. N. Low, L. M. N. B. F. Santos, C. F. R. A. C. Lima, P.
Branda~o, L. R. Gomes, Eur. J. Med. Chem. 2010, 1, 61.
[28] Y. J. Zhu, L. Qiu, J. J. Zhou, H. Y. Guo, Y. H. Hu, Z. C. Li,
Q. Wang, Q. X. Chen, B. Liu, J. Enzyme Inhib. Med. Chem.
2010, 25, 798.
[29] Q. X. Chen, K. K. Song, L. Qiu, X. D. Liu, H. Huang, H. Y.
Guo, Food Chem. 2005, 91, 269.
Received May 29, 2015
Accepted October 5, 2015
538 Chem. Biodiversity 2016, 13, 531 – 538
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