In vitro inhibition of polyphenol oxidase by some new diarylureas

Article abstract of DOI:10.3109/14756366.2011.580743

A new series of N,N′-diarylureas (19) was synthesized. These compounds were investigated as inhibitors of polyphenol oxidase (PPO) which had been purified from banana by an affinity gel comprised of Sepharose 4B-l-tyrosine-p-amino benzoic acid. Ki values

Full text of DOI:10.3109/14756366.2011.580743

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(1): 125–131
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.580743
RESEARCH ARTICLE
In vitro inhibition of polyphenol oxidase by some new
diarylureas
Dudu Demir1, Nahit Gençer1, Oktay Arslan1, Hayriye Genç2, and Mustafa Zengin2
1Department of Chemistry, Balikesir University, Balikesir, Turkey and 2Department of Chemistry, Sakarya University,
Adapazari, Turkey
Abstract
A new series of N,N′-diarylureas (1–9) was synthesized. These compounds were investigated as inhibitors of
polyphenol oxidase (PPO) which had been purified from banana by an affinity gel comprised of Sepharose 4B-l-
tyrosine-p-amino benzoic acid. Ki values for (1), (2), (3), (5), (6), (7) and (8) were determined as 0.285, 17.97, 0.187,
0.108, 0.063, 0.044 and 0.047mM, respectively. Thus (2) was by far the most effective inhibitor. Interestingly, (4) and
(9) behaved as an activator of PPO in this study.
Keywords: Inhibition, enzymatic browning, polyphenoloxidase, diarylureas
Introduction
of sensory and nutritional quality and affects appear-
Polyphenol oxidase (PPO) (EC 1.14.18.1) is a copper-
containing enzyme, widely distributed in nature,
responsible for melanization in animals and brown-
ing in plants1,2. PPO also catalyzes the o-hydroxylation
of monophenols and the oxidation of o-diphenols to
o-quinones1. Enzymatic browning of fruits is related
to oxidation of phenolic endogenous compounds into
highly unstable quinones, which are later polymer-
ized to brown, red and black pigments3. e degree of
browning depends on the nature and amount of endog-
enous phenolic compounds, presence of oxygen, reduc-
ing substances and metallic ions, pH and temperature
and the activity of PPO, the main enzyme involved in
the reaction4. Enzymatic browning is also an economic
problem for processors and consumers1,5. At least five
causes of browning in processed and/or stored fruits
and plants are known: enzymatic browning of the
phenols, Maillard reaction, ascorbic acid oxidation,
caramelization and formation of browned polymers by
oxidized lipids6.
ance and organoleptic properties, inactivation of PPO
is desirable for preservation of foods8. Several methods
such as the addition of antioxidants and the exclusion
of oxygen as well as thermal processing have been
used to inhibit enzymatic browning. For inactivation of
PPO, thermal processing has limits like loss of sensory
and nutritional quality of food products. erefore,
high pressure treatment has been considered as an
alternative9,10
.
Ureasareveryimportantclassofcarbonylcompounds.
ey have extensive applications such as agrochemicals,
dyes for cellulose fibers, antioxidants in gasoline, resin
precursors and synthetic intermediates for fine chemi-
cals, pharmaceuticals, cosmetics and pesticides11–16
.
N,N′-diarylureas are valuable starting material used in
organic synthesis. ey have numerous applications
such as drugs, pesticides, herbicides, antioxidants and
anion-binding receptors17. Many urea derivatives are
very important compounds because of their biological
activities. In particular, several substituted ureas have
recently shown an inhibiting effect on HIV protease
Browning reactions are major causes of quality
loss during harvesting, post-harvest handling/storage
and processing of fruits, plants and vegetables in food
industry7. e enzymatic browning cause deterioration
enzyme11–13
.
In the present study, we have synthesized derivatives
of N,N′-diarylureas for evaluation as potential inhibitors
Address for Correspondence: Nahit Gençer, Biochemistry Division, Department of Chemistry, Balikesir University, Cagis Kampus, Balikesir
10145, Turkey. E-mail: ngencer@balikesir.edu.tr
(Received 07 January 2011; revised 11 April 2011; accepted 11 April 2011)
125

126 D. Demir et al.
of PPO that could be beneficial in the prevention of enzy-
matic browning.
General reaction
3
S
Materials and methods
HN
N
H
NO2
General procedure for the synthesis of
N,N′-diarylureas
Chemicals and solvents used in the study were obtained
from Sigma-Aldrich and Merck and were used without
further purification. Melting points were measured on
Barnstead/Electrothermal 9200 melting point appara-
N
X
O
S
R1
-H
-H
-H
-H
-H
-H
-F
R2
-H
-H
-H
-H
-I
R3
-CH3
-CH3
-NO2
-F
R4
-H
-H
-H
-H
-H
-H
-H
-H
-Cl
R5
1
2
3
4
5
6
7
8
9
-H
-H
-H
-H
-H
-H
-H
-H
-H
1
tus. H and 13C NMR spectra were recorded on a Varian
S
Mercury NMR at 300 and 75 MHz instrument in CDCl3,
respectively. IR spectra were obtained with Shimadzu IR
Prestige 21.
O
S
-H
S
-F
-H
1
S
-H
-H
O
S
-H
-H
-CF3
-Cl
-H
S
-H
HN
N
H
Purification of PPO
N
All purification steps were carried out at 25°C. e
extraction procedure was adopted from Wesche-
Ebeling and Montgomery18. e bananas were washed
with distilled water three times to prepare the crude
extract, 50 g of bananas were cut quickly into thin slices
and homogenized in a Waring blender for 2 min using
100 ml of 0.1 M phosphate buffer, pH 7.3 containing
5% poly(ethylene glycol) and 10 mM ascorbic acid.
After filtration of the homogenate through muslin, the
filtrate was centrifuged at 15,000g for 30 min, and the
supernatant was collected. A crude protein precipitate
was made by adding (NH4)2SO4 to 80% saturation. e
resulting precipitate was suspended in a minimum
volume of 5 mM phosphate buffer and then dialyzed
against 5 the same buffer overnight. e enzyme solu-
tion was then applied to the Sepharose 4B-tyrosine-p-
amino benzoic acid affinity column7, pre-equilibrated
with 5 mM phosphate buffer, pH 5.0. e affinity gel
was extensively washed with the same buffer before the
banana PPO (BPPO) was eluted with 1 M NaCl, 5 mM
phosphate, pH 7.0.
1-(isoquinolin-5-yl)-3-p-tolylurea (1): 1-isocyanato-
4-methylbenzene (6.94 mmol, 0.924 g) was dissolved
in toluene (10 ml) and added dropwise into the stirred
solution of 5-aminoisoquinoline (6.94 mmol, 1 g) in tet-
rahydrofuran (15 ml). e reaction mixture was stirred at
60°C overnight. e precipitate was collected by filtra-
tion and washed with acetone (5 ml) and dried under
vacuum at 40°C. Yield 95%; mp: 251–252°C; 1H NMR
(DMSO-d6): δ 2.2 (s, 3H), 7.0–7.1 (d, 2H), 7.36–7.39 (d,
2H), 7.56–7.59 (dd, 1H), 7.69–7.7 (d, 1H), 7.7–7.71 (d,
1H), 8.0–8.06 (d, 1H), 8.49–8.52 (d, 1H), 8.89 (d, 1H), 8.9
(s, 1H), 8.9 (s, 1H); 13C NMR (DMSO-d6): δ 21.05, 117.99,
118.97 (2C), 121.40, 121.74, 124.40, 129.96 (2C), 130.12,
130.85, 131.51, 135.54, 137.70, 148.86, 150.98, 153.55;
FT-IR ν (cm−1): 3140, 2966, 1595, 1541, 1498, 1444, 1332,
1263; (MH+): 276.1.
2
S
HN
N
H
BPPO activity
N
Enzyme activity was determined using catechol by mea-
suring the increase in absorbance at 420 nm19 in a Biotek
automated recording spectrophotometer. Enzyme activ-
ity was calculated from the linear portion of the curve.
One unit of PPO activity was defined as the amount of
enzyme that causes an increase in absorbance of 0.001
unit/min for 1 ml of enzyme at 25°C7.
1-(isoquinolin-5-yl)-3-p-tolylthiourea (2): e experi-
mental procedure is the same as described above.
1
Yield 90%; mp: 184–185°C; H NMR (DMSO-d6): δ 3.26
(s, 3H), 7.11–7.14 (d, 2H), 7.33–7.36 (d, 2H), 7.54–7.56
(d, 1H), 7.55–7.58 (d, 1H), 7.71–7.76 (t, 1H), 7.91–7.94
(d, 1H), 8.31–8.34 (d, 1H), 8.8 (s, 1H), 9.7 (s, 1H), 9.8
(s, 1H); 13C NMR (DMSO-d6): δ 21.23, 121.99, 125.05
(2C), 126.05, 126.16, 128.29, 129.62 (2C), 129.72, 132.67,
134.68, 136.37, 137.45, 148.99, 151.17, 182.14; FT-IR ν
(cm−1): 3134, 2972, 1595, 1541, 1498, 1444, 1332, 1263;
MS(MH+): 293.1.
Inhibition of BPPO activity
An aliquot of each inhibitor at various final concen-
trations was added to the standard reaction solution
immediately before the addition of enzyme extract.
Journal of Enzyme Inhibition and Medicinal Chemistry

In vitro inhibition of polyphenol oxidase 127
Figure 1. Ki graphics of diarylureas on BPPO.
e concentration of inhibitor (diarylureas) giving
Results and discussion
50% inhibition was determined from a plot of residual
activity against inhibitor concentration, with 10 mM
catechol as substrate. e control was activity without
inhibitor.
e inhibition type of diarylureas was determined by
Lineweaver–Burk plots of 1/V versus 1/S at two inhibitor
concentrations (Figure 1). e inhibition constant, Ki,
© 2012 Informa UK, Ltd.

128 D. Demir et al.
reacts with disulphide bonds with PPO. is leads to the
change in tertiary structure of enzyme and inactivation.
e third process leading to PPO inhibition by bisul-
phate is via reduction of the intermediate quinones as
described for ascorbic acid27. e enzyme also seemed
to be sensitive to thiourea because PPO contains cop-
per as a co-factor, the irreversible inactivation of this
enzyme can be effected by substances (such as thiol
compounds thiourea, -hydroxyquinoline, etc.), which
remove copper from the active site of the enzyme29.
Because sulphur is much more polarizable than oxy-
gen, in this case, as already mentioned, a covalent bond
is formed by donation of one of the S lone pairs into
the empty 4s orbital of Cu. As sulphur is much “softer”
than oxygen it acts as a buffer of the polarization effects
caused by the metal cation, and these effects are not
transmitted to the rest of the molecule in a significant
amount. Hence, in this case, the conjugation of the near
aminogroup is much smaller than in urea and both C–N
bonds are almost equal30.
Table 1. e Ki values of diarylureas on BPPO.
Diarylureas
Ki (mM)
0.285
17.97
0.187
–
Inhibition type
Competitive
Non-competitive
Competitive
–
1
2
3
4
5
6
7
8
9
0.108
0.063
0.044
0.047
–
Competitive
Competitive
Competitive
Competitive
–
was deduced from the points of interception of the plots.
Depending on kinetic analysis, competitive inhibition
(1, 3, 5, 6, 7, 8) and uncompetitive inhibition (2) were all
seen in this study (Table 1). Surprisingly, neither (4) nor
(9) had much of an inhibiting effect, in any of the condi-
tions used. Ki values of 0.285, 17.97, 0.187, 0.108, 0.063,
0.044 and 0.047 mM were obtained for (1), (2), (3), (5),
(6), (7) and (8), respectively. Chilaka et al. reported that
thiourea was a good inhibitor of PPO, with low Ki value of
0.15 mM and inhibition of PPO was uncompetitive20. We
determined that (6), (7) and (8) were a better inhibitor
of PPO according to thiourea. Gulcin et al. reported that
sodium diethyl dithiocarbamate was the most effective
inhibitor (Ki: 1.79 × 10−6 mM) on nettle PPO21.
Declaration of interest
e work was financially supported by TUBITAK (TBAG-
110T133).
Several compounds reported as PPO inhibitors were
also shown to have inhibitors effect on the BPPO. e
results from inhibitor studies in other plant tissues
showed thiol reagents as the most effective inhibitors
for those enzymes22,23. Reducing agents, antioxidants
and enzymatic inhibitors prevent browning chemically
by reducing the o-quinones. e effect of these reduc-
ing agents can be considered as temporary because
these compounds are oxidized irreversibly by reaction
with pigment intermediates, endogenous enzymes
and metals such as copper. Among sulphur-containing
agents, l-cysteine is an effective compound to prevent
enzymic browning. Direct inhibition of PPO by cysteine
through the formation of stable complexes with cop-
per has also been proposed24. Halim and Montgomery
showed in a series of publications that Cys can inhibit
enzymic browning of pear juice concentrate more effec-
tively than sulphite25. Kahn used Cys to inhibit brown-
ing of cut or pureed avocados and bananas26. Among
the tested anti-browning reagent, the most effective
ones were dithiothreitol and sodium metabisulphite23.
e action of sulphite in the prevention of enzymatic
browning can usually be explained by several pro-
cesses. One is the action on o-quinones. e formation
of quinone–sulphite complexes prevents the quinone
polymerization27. A further action of metabisulphite
on PPO is directly on the enzyme structure leading to
the inactivation of PPO. Golan-Goldhirsh and Whitaker
and Embs and Markakis found that during pre-incuba-
tion of PPO with sulphite (dithiothreitol, glutathione),
there was a gradual loss in the ability of the enzyme to
cause browning27,28. It has been suggested that sulphite
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Appendix
1H, 13C NMR and IR spectral of diarylureas
4
O
HN
N
H
F
N
1-(isoquinolin-5-yl)-3-(4-nitrophenyl)thiourea (3): e experimental procedure is the same as described above. Yield
92%; mp: 264–265°C; 1H NMR (DMSO-d6): δ 3.3 (s, 3H), 7.1–7.13 (d, 1H), 7.13–7.16 (d, 1H), 7.48–7.51 (d, 1H), 7.49–7.52 (d,
1H), 7.56–7.6 (d, 1H), 7.7–7.72 (d, 1H), 7.2–7.5 (d, 1H), 8–8.02 (d, 1H), 8.48–8.51 (d, 1H), 8.8 (s, 1H), 8.9 (s, 1H), 9 (s, 1H);
13C NMR (DMSO-d6): δ 115.93, 116.23, 118.41, 120.59 (2C), 121.46, 121.96, 124.66, 130.10 (2C), 130.93, 135.40, 136.65,
148.85, 151.01, 153.67; FT-IR ν (cm−1): 3140, 2972, 1597, 1541, 1498, 1444, 1332, 1263; MS(MH+): 324.07.
5
S
HN
N
H
I
N
1-(4-fluorophenyl)-3-(isoquinolin-5-yl)urea (4): e experimental procedure is the same as described above. Yield 85%;
mp: 182–183°C; 1H NMR (DMSO-d6): δ 7.55–7.57 (d, 1H), 7.58–7.61 (d, 1H), 7.75–7.78 (t, 1H), 7.81–7.89 (d, 2H), 7.92–7.99
(d, 1H), 8.19–8.22 (d, 2H), 8.34–8.37 (d, 1H), 8.92 (s, 1H), 10.3 (s, 1H), 10.5 (s, 1H); 13C NMR (DMSO-d6): δ 122.17, 122.69,
124.99, 125.80, 126.17, 128.74, 129.79, 132.56, 135.83, 143.17, 146.94, 148.97, 151.35, 182.09; FT-IR ν (cm−1): 3140, 2972,
1597, 1541, 1498, 1444, 1332, 1263; MS(MH+): 281.1.
© 2012 Informa UK, Ltd.

130 D. Demir et al.
6
F
S
HN
N
H
N
1-(3-iodophenyl)-3-(isoquinolin-5-yl)thiourea (5): e experimental procedure is the same as described above. Yield
1
89%; mp: 185–186°C; H NMR (DMSO-d6): δ 7.0–7.13 (t, 1H), 7.45–7.48 (d, 1H), 7.49–7.51 (d, 1H), 7.52–7.56 (d, 1H),
7.52–7.59 (d, 1H), 7.7–7.75 (t, 1H), 7.92–7.94 (d, 1H), 7.97 (s, 1H), 8.30–8.34 (d, 1H), 8.9 (s, 1H), 9.9 (s, 1H), 10.0 (s, 1H); 13
C
NMR (DMSO-d6): δ 94.55, 122.14, 124.13, 125.95, 126.18, 128.55, 129.81, 131.00, 132.61, 132.87, 133.75, 135.99, 141.66,
149.00, 151.28, 182.12; FT-IR ν (cm−1): 3145, 2966, 1597, 1541, 1498, 1444, 1332, 1263; MS(MH+): 405.26.
7
F
S
HN
N
H
N
1-(3-fluorophenyl)-3-(isoquinolin-5-yl)thiourea (6): e experimental procedure is the same as described above. Yield
86%; mp: 186–187°C; 1H NMR (DMSO-d6): δ 6.8–7.0 (t, 1H), 7.2–7.3 (d, 1H), 7.34–7.4 (t, 1H), 7.5 (s, 1H), 7.51–7.53 (d, 1H),
7.51–7.54 (d, 1H), 7.78–7.81 (t, 1H), 7.95–8.0 (d, 1H), 8.34–8.39 (d, 1H), 8.9 (s, 1H), 10.0 (s, 1H), 10.01 (s, 1H); 13C NMR
(DMSO-d6): δ 120.12, 122.11, 125.95, 126.20, 128.51, 129.77, 130.58, 130.71, 132.65, 136.08, 141.90, 142.04, 148.97, 151.27,
160.83, 182.10; FT-IR ν (cm−1): 3140, 2966, 1595, 1541, 1498, 1444, 1332, 1263; MS(MH+): 297.07.
8
F
F
F
S
HN
N
H
N
1-(2-fluorophenyl)-3-(isoquinolin-5-yl)thiourea (7): e experimental procedure is the same as described above. Yield
92%; mp: 184–185°C; 1H NMR (DMSO-d6): δ 7.1–7.2 (m, 2H), 7.2–7.26 (d, 1H), 7.5–7.6 (m, 2H), 7.56–7.59 (d, 1H), 7.73–7.78
(t, 1H), 7.95–7.98 (d, 1H), 8.25–8.37 (d, 1H), 8.9 (s, 1H), 9.5 (s, 1H), 10.09 (d, 1H); 13C NMR (DMSO-d6): δ 116.212, 116.479,
121.912, 124.615, 125.990, 126.173, 127.910, 128.025, 128.494, 129.292, 129.602, 132.526, 135.955, 148.940, 151.048,
182.962; FT-IR ν (cm−1): 3140, 2972, 1597, 1541, 1498, 1444, 1332, 1263; MS(MH+): 297.07.
9
Cl
S
HN
N
H
Cl
N
1-(isoquinolin-5-yl)-3-(3-(trifluoromethyl)phenyl)thiourea (8): e experimental procedure is the same as described
above. Yield 96%; mp:186–187°C; 1H NMR (DMSO-d6): δ 7.4 (s, 1H), 7.5–7.56 (t, 1H), 7.58–7.59 (d, 1H), 7.61–7.62 (d, 1H),
7.9–8.0 (t, 1H), 8.36–8.39 (d, 1H), 8.9 (s, 1H), 10.0 (s, 1H), 10.2 (s, 1H); 13C NMR (DMSO-d6): δ 120.91, 121.55, 122.18,
122.951, 125.92, 126.20, 128.47, 128.59, 129.38, 129.80, 129.86, 130.13, 132.61, 135.83, 141.14, 148.96, 151.29; FT-IR ν
(cm−1): 3140, 2972, 1597, 1541, 1498, 1444, 1332, 1263; MS(MH+): 347.36.
R2
R1
R3
R4
X
X
NH2
R1
R4
C
N
H
R2
R3
N
HN
R5
+
N
Toluene
Tetrahydrofuran
60°C
R5
N
overnight
Journal of Enzyme Inhibition and Medicinal Chemistry

In vitro inhibition of polyphenol oxidase 131
1-(3,5-dichlorophenyl)-3-(isoquinolin-5-yl)thiourea (9): e experimental procedure is the same as described above.
Yield 96%; mp:170–171°C; 1H NMR (DMSO-d6): δ 339 (s, 3H), 7.3 (s, 1H), 7.5–7.58 (d, 1H), 7.58–7.60 (d, 1H), 7.6 (s, 2H),
7.78–7.81 (t, 1H), 7.98–8.0 (d, 1H), 8.3–8.34 (d, 1H), 8.9 (s, 1H), 10.0 (s, 1H), 10.2 (s, 1H); 13C NMR (DMSO-d6): δ 122.206,
122.634, 124.230, 125.883, 126.219, 128.746, 129.816, 132.584, 134.088, 135.775, 142.782, 149.001, 151.334, 182.180; FT-IR
ν (cm−1): 3140, 2972, 1597, 1541, 1498, 1444, 1332, 1263; MS(MH+): 347.01.
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