Evaluation of new chalcone derivatives as polyphenol oxidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.09.130

A newly series of 4-(phenylurenyl)chalcone (4a-j) and 4′- (phenylurenyl/thiourenyl)chalcone (9a-l) derivatives were synthesized and their inhibitory effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose 4B-l-tyrosine-p-aminobenzoic acid. The result showed that 4a-j inhibited the PPO enzyme activity. Conversely, 9a-h and 9i-l showed activator effect on tyrosinase enzyme activity.

Full text of DOI:10.1016/j.bmcl.2011.09.130

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7479–7482
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of new chalcone derivatives as polyphenol oxidase inhibitors
Fatih Sonmez a, Sedat Sevmezler a, Alparslan Atahan b, Mustafa Ceylan c, Dudu Demir d, Nahit Gencer d,
,
⇑
Oktay Arslan d, Mustafa Kucukislamoglu a
a Department of Chemistry, Faculty of Art and Sciences, Sakarya University, Sakarya 54140, Turkey
b Department of Chemistry, Faculty of Art and Sciences, Duzce University, Duzce 81620, Turkey
c Department of Chemistry, Faculty of Art and Sciences, Gaziosmanpasa University, Tokat 60150, Turkey
d Department of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir 10145, Turkey
a r t i c l e i n f o
a b s t r a c t
A newly series of 4-(phenylurenyl)chalcone (4a–j) and 40-(phenylurenyl/thiourenyl)chalcone (9a–l)
derivatives were synthesized and their inhibitory effects on the diphenolase activity of banana tyrosinase
were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sepharose
Article history:
Received 27 July 2011
Revised 27 September 2011
Accepted 27 September 2011
Available online 19 October 2011
4B-
L-tyrosine-p-aminobenzoic acid. The result showed that 4a–j inhibited the PPO enzyme activity.
Conversely, 9a–h and 9i–l showed activator effect on tyrosinase enzyme activity.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Chalcones
Tyrosinase inhibitors
Chalcones (1,3-diaryl-2-propen-1-ones) one of the major classes
of natural products with widespread distribution in fruits, vegeta-
bles, spices, tea and soy based foodstuff have been recently the sub-
jects of great interest for their interesting pharmacological
activities.1 They contain two aromatic rings with an unsaturated
chain. Many biological activities have been attributed to this group,
such as antitumoral,2 anticancer and antioxidant,3 antifungal,4 anti-
mitotic,5 chemoprotective,6 anti-inflammatory,7,8 antimicrobial,9
anti-nociceptive10 and antibacterial11 activities. A number of chal-
cone derivatives have also been found to inhibit several important
enzymes in cellular systems, including xanthine oxidase,12 aldose
reductase,13 heme oxygenase,14 protein tyrosine kinase,15,16 qui-
none reductase17 and tyrosinase.15 Additionally, the radioiodinated
chalcones have been useful amyloid imaging agents for detecting b-
amyloid plaques in the brain of Alzheimer’s disease.18 Reactions of
4-aminochalcones with isocyanates give unsymmetrically substi-
tuted urea derivatives which are linked to a series of biological
activities including antiglycating,19 MCH-R1 antagonists,20 P2Y1
receptor antagonists,21 heparanase inhibitors,22 anti-HIV,23 cyto-
static and antioxidant,24 and proliferation inhibitors25 properties.
Tyrosinase (monophenol or o-diphenol, oxygen oxidoreductase,
EC 1.14.18.1), also known as polyphenol oxidase (PPO), is a
copper-containing monooxygenase that is widely distributed in
microorganisms, animals, and plants.26 Tyrosinase could catalyze
two distinct reactions involving molecular oxygen in the hydroxyl-
ation of monophenols to o-diphenols (monophenolase) and in the
oxidation of o-diphenols to o-quinones (diphenolase).27 Due to the
high reactivity, quinines could polymerize spontaneously to form
higher molecular weight brown pigments (melanins) or react with
amino acids and proteins to enhance brown color of the pigment
produced.28,29 Previous reports confirmed that tyrosinase not only
was involved in melanising in animals, but also was one of the
main causes of most fruits and vegetables quality loss during post
harvest handling and processing, leading to faster degradation and
shorter shelf life.30 Recently, investigation demonstrated that var-
ious dermatological disorders, such as age spots and freckle, were
caused by the accumulation of an excessive level of epidermal pig-
mentation.31,32 Tyrosinase has also been linked to Parkinson’s and
other neurodegenerative diseases.33 In insects, tyrosinase is un-
iquely associated with three different biochemical processes,
including sclerotization of cuticle, defensive encapsulation and
melanisation of foreign organism, and wound healing.34 These pro-
cesses provide potential targets for developing safer and effective
tyrosinase inhibitors as insecticides and ultimately for insect con-
trol. Thus, the development of safe and effective tyrosinase inhib-
itors is of great concern in the medical, agricultural, and cosmetic
industries. However, only a few such as kojic acid, arbutin, tropo-
lone, and 1-phenyl-2-thiourea are used as therapeutic agents and
cosmetic products.32,35
In this study series of 10 new 4-(phenylurenyl)chalcone (4a–j)
and 4 known10 and 8 new 40-(phenylurenyl/thiourenyl)chalcone
(9a–l) derivatives were synthesized and the effect of them on
tyrosinase have been evaluated. The 4-nitrochalcones 2a–d,
prepared by the condensing various acetophenones and 4-nitro-
benzaldehyde with NaOH as base, were reducted with tin(II)
⇑
Corresponding author. Tel.: +90 266 612 1278; fax: +90 266 612 1215.
E-mail address: ngencer@balikesir.edu.tr (N. Gencer).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.130

7480
F. Sonmez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7479–7482
chloride in ethanol.18 The 4-aminochalcones were reacted with
isocyanates to get product 4-(phenylurenyl)chalcone (4a–j)
(Scheme 1)25 at high yields but synthesis of 4-(phenylthioure-
nyl)chalcones with 4-aminochalcone and isothiocyanate were
unsuccessful. The syntheses of 4-(phenylurenyl/tiourenyl)acetoph-
enones 7a–d were obtained by reaction of p-aminoacetophenone
with phenylisocyanate or phenylisothiocyanate derivatives. The
subsequent treatment of these derivatives and substituted
benzaldehydes with NaOH, in methanol/DMSO, at room tempera-
ture, (Scheme 2)36 were given 40-(phenylurenyl/thiourenyl)chal-
cone (9a–l).
relating to NH stretch, absorptions in 1606–1650 cmꢀ1 from
,b-unsaturated carbonyl moiety stretch and absorptions in
a
1654–1710 cmꢀ1 from urea carbonyl moiety stretching. The 1H
NMR spectra for all the synthesized urea and thiourea compounds
show signals between 8.65 and 10.12 ppm relating to hydrogens
attached to the nitrogen. The signals for aromatic hydrogens are
between 6.55 and 8.15 ppm. The vinylic protons are in this same
regions. Through the 13C NMR data, a sign can be seen about
187.53–189.72 ppm, relating to chalcone carbonyl. This is followed
by the sign about 152.50–153.04 ppm for urea carbonyl and
179.67–179.79 ppm for thiourea carbonyl.
All new compounds were characterized by 1H NMR, 13C NMR, IR
and MS. In the infrared spectra of compounds 4a–j and 9a–l, it was
possible to observe the absorptions between 3273 and 3410 cmꢀ1
All purification steps were carried out at 25 °C. The extraction
procedure was adopted from Wesche-Ebeling & Montgomery.37
The enzyme was purified by Sepharose 4B-tyrosine-p-aminoben-
O
H
O
O
O
SnCl2.2H2O
NO2
EtOH, reflux
NO2
R1
/NaOH
rt
EtOH
R1
R1
NH2
(aq)
2a-d
1a-d
3a-d
R2
THF, TEA
4a
H
H
4f
Cl
4b
H
4"-F
4g
4c
H
4d
H
4e
R1
R2
Cl
4"-F
4j
N
C
O
reflux
4"-CH3 3"-OCH3
4h
CH3
4"-F
O
2'
A
2
4i
CH3
1'
1
3'
3
R1
Cl
OCH3
B
H
N
2''
5''
H
6'
6
1''
6''
R1 4'
4
N
3''
R2 4"-CH3 3"-OCH3
4"-CH3 4"-CH3
5
5'
R2
O
4''
4a-j
Scheme 1. Synthesis of 4-(phenylurenyl)chalcone derivatives.
O
O
NCX
R1
X
toluene
reflux
+
R1
H2N
N
N
H
H
5
6a-d
7a-d
O
H
EtOH
/NaOH(aq)
9a
O
9b
O
H
Cl
9h
O
9c
O
H
9d
O
H
9e
O
9f
O
rt
8a-d
R2
X
R1
H
CH3 CH3
O
2
3''
2'
R1
R2 Br
CH3 OCH3 Br
9i
S
Cl
9l
S
3'
4'
1'
1
4''
5''
3
2''
1''
X
A
B
5
9g
O
9j
S
H
9k
S
N
N
R2
4
6'
6
5'
6''
H
H
X
9a-l
R1 CH3 CH3
H
CH3 CH3
R2 CH3 OCH3 Cl OCH3 Cl OCH3
Scheme 2. Synthesis of 40-(phenylurenyl)chalcone and 40-(phenylthiourenyl)chalcone derivatives.

F. Sonmez et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7479–7482
7481
Table 1
effect on tyrosinase enzyme. Generally, differentiation between
HOMO and LUMO reflects the intensity of electron affinity, and
lower differentiation suggests higher electron affinity.40 Molecular
calculations were performed using Gaussian software.41 The expla-
nation of the different effect of 4a–j and 9a–l on tyrosinase en-
zyme, we performed quantum chemical calculations of 4i and 9g
structures to calculate HOMO and LUMO energy levels. HOMO–
LUMO energy differences of 4i (0.23709 eV) were lower than 9g
(0.25085 eV). These results were compatible with the inhibition ef-
Inhibitory effect of 4-(phenylurenyl) chalcone derivatives on banana tyrosinase
activities
Entry
Catechol
Type of inhibition (Ki, lM)
IC50
(
l
M)
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.172 ( 0.005)
0.177 ( 0.003)
0.241 ( 0.007)
0.244( 0.006)
0.133 ( 0.003)
0.238 ( 0.004)
0.152 ( 0.002)
0.217 ( 0.005)
0.134 ( 0.005)
0.289 ( 0.009)
Competitive (0.037)
Competitive (0.125)
Competitive (0.057)
Competitive (0.240)
Competitive (0.117)
Competitive (0.129)
Competitive (0.059)
Competitive (0.117)
Competitive (0.067)
Competitive (0.022)
fect of 4i (IC50 = 0.134 lM) and 9g (not active). We also calculated
planarity of 4i and 9g. Our result showed a clear relation between
the planar character of 4-(phenylurenyl) chalcones and the
potency of these compounds as tyrosinase inhibitors. The ELUMO
and the delocation of these orbital, probably favored by the
planarity of the molecule, were correlated with the inhibitory
efficiency (Fig. 1).
4j
zoic acid affinity column.38 Enzyme activity was determined; using
catechol, by measuring the increase in absorbance at 420 nm
according to the method Espin et al.39 For evaluating the tyrosinase
inhibitory activity, all the synthesized compounds were subjected
to tyrosinase inhibition assay with catechol as substrate. The result
showed that 4-(phenylurenyl)chalcone derivatives (4a–j) inhibited
the PPO enzyme activity. On the other hand, 40-(phenylure-
nyl)chalcone (9a–h) and 40-(phenylthiourenyl)chalcone deriva-
tives (9i–l) showed activator effect on PPO enzyme activity.
The IC50 values and inhibition constants of 4a–j analogues
against PPO were summarized in Table 1. We have determined
the IC50 values of 0.133–0.289 mM for the inhibition of banana
PPO. It was determined that they are all competitive inhibitors.
The result may be related to the structure of tyrosinase contained
a type-3 copper center with a coupled dinuclear copper active site
in the catalytic core.
The present investigation reported that 4-(phenylurenyl)chal-
cone derivatives (4a–j) had potent inhibitory effects on the diphe-
nolase activity of banana tyrosinase. Interestingly, compound 4e
was found to be the most potent inhibitor with IC50 value of
0.133 l
M. On the other hand, 40-(phenylurenyl)chalcone deriva-
tives (9a–l) were totally inactive. Different effects of these com-
pounds were explained with quantum calculation method for
structure of 4i and 9g which containing same group. Results
showed good correlation intensity of electron affinity and inhibi-
tion of tyrosinase enzyme.
The enzyme PPO, a copper containing enzyme ubiquitously
present in plants, has been studied as a model oxidizing enzyme
since it contains metal ion (copper) and utilizes molecular
oxygen.26 Flavonoids, especially chalcones like butein, have been
reported to possess a copper chelation activity.42 The inhibition
of PPO may be due to the chelation of copper, which is present
in the active site of PPO.
As the concentration of inhibitors raised, the residual enzyme
activity drastically decreased. All inhibitors manifested a similar
relationship between the enzyme activity and enzyme concentra-
tion. From the progress curve obtained, compounds (4a–j) showing
solid lines below the line of enzyme activity have indicated for en-
zyme inhibition. In each case, the type of inhibition was deduced
from Lineweaver–Burk double reciprocal plots. The inhibitory con-
stants were calculated from secondary plots of apparent Km,
against inhibitor concentration. Good straight lines were obtained,
and a typical example is shown in Figure 1 for 4-(phenylurenyl)
chalcone 4a.
L-cycteine is an effective compound to prevent enzymatic
browning. Direct inhibition of polyphenol oxidase by cystein
through the formation of stable complexes with copper has also
been proposed.43 The enzyme also seemed to be sensitive to thio-
urea since PPO contains copper as a co-factor, the irreversible inac-
tivation of this enzyme can be effected by substances (such as thiol
compounds thiourea, -hydroxyquinoline, etc.), which remove cop-
per from the active site of the enzyme.44
Detailed information regarding the synthesis, spectroscopic
characterization and biological evaluation of all compounds pre-
sented in this Letter can be found in the Supplementary data
provided.
On the other hand, 40-(phenylurenyl)-chalcone derivatives (9a–
l) did not inhibit tyrosinase at 50 lM, as well as showed activator
4i
9g
Energy: -1101.81915184 a.u.
Dipole Moment: 4.1305 Debye
HOMO: -0.29432 eV
Energy: -1101.81848948 a.u.
Dipole Moment: 6.0978 Debye
HOMO: -0.30556 eV
LUMO: -0.05569 eV
LUMO: -0.04168 eV
ΔEHOMO-LUMO= - 0.23863 eV
ΔEHOMO-LUMO= -0.26388 eV
Figure 1. Calculated geometric structures of 4i and 9g using HF method with 6-31G basis set.

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