Synthesis and theoretical calculations of carbazole substituted chalcone urea derivatives and studies their polyphenol oxidase enzyme activity

Article abstract of DOI:10.3109/14756366.2012.688040

Synthesis of carbazole substituted chalcone urea derivatives and their polyphenol oxidase enzyme activity effects on the diphenolase activity of banana tyrosinase were evaluated. Tyrosinase has been purified from banana on an affinity gel comprised of Sep

Full text of DOI:10.3109/14756366.2012.688040

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(4): 808–815
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.688040
research arTIcLe
Synthesis and theoretical calculations of carbazole substituted
chalcone urea derivatives and studies their polyphenol
oxidase enzyme activity
Arleta Rifati Nixha1, Mustafa Arslan2, Yusuf Atalay3, Nahit Gençer4, Adem Ergün4, and Oktay Arslan4
1Chemistry Department, Faculty of Mathematical & Natural Sciences, University of Prishtina, Prishtina, Republic
of Kosova, 2Chemistry Department, Faculty of Arts and Sciences, Sakarya University, Sakarya, Turkey, 3Physics
Department, Faculty of Arts and Sciences, Sakarya University, Sakarya, Turkey, and 4Chemistry Department, Faculty of
Arts and Sciences, Balikesir University, Balikesir, Turkey
abstract
Synthesis of carbazole substituted chalcone urea derivatives and their polyphenol oxidase enzyme activity 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 results showed that most of the
compounds (3,4,5a,5d–h) inhibited and some of them (5c,5i–l) activated the tyrosinase enzyme activity. The
molecular calculations were performed using Gaussian software for the synthesized compounds to explain the
experimental results.
Keywords: Carbazole containing chalcone, urea, thiourea, tyrosinase inhibitors
Introduction
Chalcones are one of the major classes of natural prod-
ucts with widespread distribution in fruits, vegetables,
spices, tea and soy based foodstuff. ey have recently
received a great deal of attention due to their interest-
ing pharmacological activities5. Some natural prenyl-
ated chalcones showed potent tyrosinase inhibitory
activity. ree chalcones derivatives that are licuraside,
isoisoliquritin, and Licochalcone A were isolated from
the roots of the Glycyrrhiza species and competitively
inhibited the monophenolase activity of mushroom
tyrosinase6. Many biological activities have been attrib-
uted to this group, such as anticancer and antioxidant7,
antifungal8, antimicrobial9 and antibacterial10 activities.
A number of chalcone derivatives, have also been found
to inhibit several important enzymes in cellular systems,
including xanthine oxidase11, heme oxygenase12, protein
Polyphenol oxidase (PPO), sometimes referred to as phe-
nol oxidase, catecholase, phenolase, catechol oxidase,
or even tyrosinase, is considered to be an o-dipenol1.
PPO (EC 1.14.18.1), a multifunctional copper contain-
ing enzyme, is widely distributed in nature. It catalyzes
two distinct reactions of melanin synthesis: a hydroxyl-
ation of monophenols to o-diphenols (monophenolase
activity) and an oxidation of o-diphenols to o-quinones
(diphenolase activity), both using molecular oxygen2.
PPO is responsible, not only for melanisation in animals,
but also for browning in plant. e unfavourable enzy-
matic browning of fruits and vegetables generally results
in a loss of nutritional and commercial value3. An impor-
tant group is constituted by compounds structurally
analogous to phenolic substrate, which generally show
competitive inhibition with respect to this substrate,
although it can vary depending on enzyme source and
substrate used4.
tyrosine kinase13, quinone reductase14 and tyrosinase15,16
.
Nitrogen heterocycles have received a great deal of
attention in the literature because of biological properties.
Address for Correspondence: Nahit Gencer, Chemistry Department, Faculty of Arts and Sciences, Balikesir University, Cagis kampus,
Balikesir, 10145 Turkey. E-mail: ngencer@balikesir.edu.tr
(Received 28 February 2012; revised 11 April 2012; accepted 14 April 2012)
808

Enzyme activity and calculations of carbazole substituted chalcone ureas 809
Especially, among these heterocyclic systems, pyridine
Synthesis of carbazole substituted chalcone urea
derivatives was prepared according to Scheme 1.
containingcompoundshavebeenthesubjectofexpanding
research efforts in heteroaromatic and biological
chemistry17. e pyrido [2,3-d] pyrimidine heterocycles,
which are those annelated to a pyrimidine ring, are
important because of their wide range of biological18
and pharmaceutical applications (i.e. bronchodilators,
vasodilators) and their anti-allergic, cardiotonic,
antihypertensive, and hepatoprotective activities19.
In the present study, we have synthesized derivatives
of carbazole substituted chalcone urea for evaluation as
potential inhibitors of PPO that could be beneficial in the
prevent enzymatic browning.
Acetylation of 9-ethylcarbazole (2)
3-Acetyl-9-ethylcarbazole (2) necessary for the subsequent
Claisen condensation was synthesized20 in one step start-
ing from 9-ethyl-carbazole (1) (7.80 g, 40 mmol) and acetyl
chloride (7 mL, 100 mmol). e Friedel – Crafts acylation is
commonly catalyzed by BiCl3 (20.50 g, 65 mmol).
General procedure for the synthesis of chalcones (3)
e most useful method used to prepare chalcones is the
condensation of acetophenones with benzaldehydes.
Equimolar portions of 3-acetyl-9-ethylcarbazole (3.97
g, 16.77 mmol) and 4-nitrobenzaldehyde (2.48 g, 16.41
mmol) were dissolved in 30 mL of methanol. A 30 mL ali-
quot of 20% aqueous potassium hydroxide solution was
then slowly added dropwise to the reaction mixture. e
reaction solution was allowed to stir at room temperature
for 24 h. e solution was extracted with AcOEt with an
aqoues phase. Organic phase washed three times with
water, dried with anhydrous magnesium sulphate and
evaporated under reduced pressure.
Materials and methods
General procedure of the carbazole substituted
chalcone urea derivatives
Melting points were taken on a Yanagimoto micro-
melting point apparatus and are uncorrected. IR spectra
were measured on a SHIMADZU Prestige-21 (200 VCE)
spectrometer. 1H and 13C NMR spectra were measured on
spectrometer at VARIAN Infinity Plus 300 and at 75 Hz,
1
respectively. H and 13C chemical shifts are referenced to
the internal deuterated solvent. e elemental analysis
was carried out with a Leco CHNS-932 instrument Flash
column chromatography was performed using Merck sil-
ica gel 60 (230–400 mesh ASTM). All chemicals was pur-
chased from Merck, Alfa Easer, Sigma-Aldrich and Fluka.
1-(9-Ethyl-9H-carbazol-3-yl)-3-(4-nitro-phenyl)-propenone (3)
Recrystallized from ether to give as light yellow crystals.
Yield 69.2%, m.p. 231°C; IR (KBr, υ, cm–1): 3070 (C=C, aro-
matic), 1660 (C=O), 1589 (NO2); 1HNMR (300 MHz, CDCl3,
Scheme 1. Synthesis of carbazole substituted chalcone urea derivatives.
© 2013 Informa UK, Ltd.

810 A. R. Nixha et al.
δ, ppm): 8.85 (1H, s, = CH), 7.81-7.82 (1H, d, = CH), 7.33–8.30
(10H, m, - Ar-H), 7.27–7.31 (1H, t, = CH), 4.39–4.46 (2H, q,
- CH2), 1.46–1.61 (3H, t, - CH3). Anal. Calcd. for C23H18N2O3:
C, 74.58; H, 4.90; N, 7.56. Found: C, 74.40; H, 4.974; N, 7.492.
1-{4-[3-(9-Ethyl-9H-carbazol-3-yl)-3–oxo-propenyl]-phenyl}–3
-(4-nitro-phenyl)-thiourea (5c)
Recrystallized from acetone/hexane to give as light brown
powder. Yield 85.3%, m.p. 215.5°C; IR (KBr, υ, cm–1,): 3225
(-NH), 3058 (C=C, aromatic), 1179 (C=S), 1591 (NO2);
1HNMR (300 MHz, DMSO-d6, δ, ppm): 10.12 (1H, s, -NH),
10.01 (1H, s, -NH), 8.80 (1H, s, = CH), 8.07–8.16 (4H, m,
- Ar-H), 7.81–7.84 (1H, d, = CH), 7.21–7.74 (11H, m, -
Ar-H), 4.35–4.38 (2H, q, - CH2), 1.35–1.40 (3H, t, - CH3).
Anal. Calcd. for C30H24N4O3S: C, 69.21; H, 4.65; N, 10.76; S,
6.16. Found : C, 68.18; H, 4.35; N, 10.75; S, 5.19.
General procedure for the synthesis of amino chalcones (4)
A mixture of 3 (1.1 g, 3.24 mmol), SnCl2 (3.66 g, 16.22
mmol) and tetrahydrofuran (30 mL) was stirred under
reflux for 4 h. When the reaction completed, the solvent
was evaporated under reduced pressure and the reaction
mixture was diluted with water (15 mL), adjusted to pH
= 10 with 1M sodium hydroxide solution, extracted with
ethyl acetate (3 × 25 mL). e organic phase was dried
over MgSO4 and filtered.
1-{4-[3-(9-Ethyl-9H-carbazol-3-yl)-3-oxo-propenyl]-phenyl}-3-
(2-fluoro-phenyl)-thiourea (5d)
Recrystallized from ethyl acetate/hexane to give as light
brown powder. Yield 88.6%, m.p. 252°C; IR (KBr, υ, cm–1):
3329 (-NH), 3019 (C=C, aromatic), 1194 (C=S), 1178 (C-F);
1HNMR (300 MHz, DMSO-d6, δ, ppm): 10.24 (1H, s, -NH),
9.69 (1H, s, -NH), 9.16 (1H, s, = CH), 8.14–8.18 (1H, d, = CH),
7.30–8.37 (15H, m, - Ar-H), 4.50–4.52 (2H, q, - CH2), 1.33–
1.36 (3H, t, - CH3). Anal. Calcd. for C30H24FN3OS: C, 73.00; H,
4.90; N, 8.51; S, 6.50. Found : C, 71.89; H, 4.45; N, 8.15; S, 5.29.
3-(4-Amino-phenyl)-1-(9-ethyl-9H-carbazol-3-yl)-propenone (4)
Recrystallized from ether to give as dark red crystals.
Yield 50%, m.p. 152°C; IR (KBr, υ, cm–1): 1589 (- NH2),
1
3052 (C=C, aromatic), 1624 (C=O); HNMR (300 MHz,
CDCl3, δ, ppm): 9.17 (1H, s, = CH), 8.39-8.42 (1H, d, = CH),
8.30-8.33 (1H, d, = CH), 7.92–7.97 (1H, d, = CH), 7.32–7.80
(5H, m, - Ar-H), 7.51–7.56 (1H, t, = CH), 7.30–7.35 (1H, t,
= CH), 6.71–6.74 (2H, d, = CH), 4.48–4.50 (2H, q, -CH2),
4.47 (2H, s, -NH2), 1.33–1.36 (3H, t, -CH3). Anal. Calcd. for
C23H20N2O: C, 81.15; H, 5.92; N, 8.23. Found: C, 79.25; H,
6.03; N, 7.56.
1-(4-Chloro-phenyl)-3-{4-[3-(9-ethyl-9H-carbazol-3-yl)-3-oxo-
propenyl]-phenyl}-thiourea (5e)
Recrystallized from acetone/hexane to give as orange
powder. Yield 77.6%, m.p. 163.9°C; IR (KBr, υ, cm–1): 3262
(-NH), 2981 (C=C, aromatic), 2260 (-N=C=S), 748 (C-Cl);
1HNMR (300 MHz, DMSO-d6, δ, ppm): 10.16 (1H, s, -NH),
10.09 (1H, s, -NH), 9.16 (1H, s, = CH), 8.37–8.40 (1H, d,
= CH), 8.31–8.32 (1H, d, = CH), 8.28–8.29 (1H, d, = CH),
7.92–7.95 (1H, d, =CH), 7.30–7.80 (12H, m, - Ar-H), 4.45–
4.54 (2H, q, - CH2), 1.31–1.38 (3H, t, - CH3). Anal. Calcd. for
C30H24ClN3OS: C, 70.64; H, 4.74; N, 8.24; S, 6.29. Found : C,
70.04; H, 4.24; N, 7.24; S, 5.19.
General procedure for synthesis of ureas and thioureas
A solution of 3-(4-amino-phenyl)-1-(9-ethyl-9H-carba-
zol-3-yl)-propenone (4) (0.19 g, 0.559 mmol) and isocya-
nate or isothiocyanate derivatives (0.096 g, 0.704 mmol)
in dry DMF (2 mL) was stirred at 40°C for 6 h, quenched
with water and filtered.
1-{4-[3-(9-Ethyl-9H-carbazol-3–yl)-3-oxo-propenyl]-phenyl}-
3-(4-fluoro-phenyl)-urea (5a)
Recrystallized from ethyl acetate/hexane to give as light
brown powder. Yield 88.89%, m.p. 285.3°C; IR (KBr, υ,
cm–1): 3292 (-NH), 1707 (C=O), 1178 (C-F), 831 (C-H, aro-
1-{4-[3-(9-Ethyl-9H-carbazol-3-yl)-3–oxo-propenyl]-phenyl}-
3-phenyl-thiourea (5f)
1
Recrystallized from eter/ethyl acetate/hexane to give
as orange powder. Yield 91%, m.p. 171.8°C; IR (KBr, υ,
cm–1): 3327 (-NH), 3053 (C=C, aromatic), 2187 (-N=C=S);
1HNMR (300 MHz, DMSO-d6, δ, ppm): 10.07 (1H, s, -NH),
10.01 (1H, s, -NH), 9.15 (1H, s, = CH), 8.13–8.17 (1H, d, =
CH), 7.16–8.38 (16H, m, - Ar-H), 4.50–4.52 (2H, q, - CH2),
1.33–1.36 (3H, t, - CH3). Anal. Calcd. for C30H25N3OS: C,
75.76; H, 5.30; N, 8.84; S, 6.74. Found : C, 75.06; H, 4.50;
N, 7.94; S, 5.74.
matic); HNMR (300 MHz, DMSO-d6, δ, ppm): 8.67 (1H,
s, -NH), 8.35 (1H, s, -NH), 8.14 (1H, s, = CH), 8.03-,Ar-H),
4.21–4.28 (2H, q, - CH2), 1.26–1.31 (3H, t, - CH3). Anal.
Calcd. for C30H24FN3O2: C, 75.46; H, 5.07; N, 8.80. Found:
C, 74.06; H, 4.67; N, 7.90.
1-{4-[3-(9–Ethyl-9H-carbazol-3-yl)-3-oxo-propenyl]-phenyl}-
3-(4-nitro-phenyl)-urea (5b)
Recrystallized from eter/acetone/hexane to give as brown
powder. Yield 85.9%, m.p. 172.4°C; IR (KBr, υ, cm–1): 3252
(-NH), 3050 (C=C, aromatic), 1707 (C=O), 1501 (NO2);
1HNMR (300 MHz, DMSO-d6, δ, ppm): 9.56 (1H, s, -NH),
9.26 (1H, s, -NH), 9.16 (1H, s, = CH), 8.37–8.40 (1H, d, =
CH), 8.30–8.31 (1H, d, = CH), 8.27–8.28 (1H, d, = CH),
8.21–8.24 (1H, d, = CH), 7.95–8.10 (1H, d, = CH), 7.57–7.91
(9H, m, - Ar-H), 7.30–7.33 (1H, t, = CH), 7.52–7.54 (1H, t,
=CH), 4.52–4.54 (2H, q, - CH2), 1.34–1.39 (3H, t, - CH3).
Anal. Calcd. for C30H24N4O4 : C, 71.42; H, 4.79; N, 11.10.
Found: C, 70.40; H, 4.19; N, 10.25.
1-{4-[3-(9-Ethyl-9H–carbazol-3-yl)-3-oxo-propenyl]-phenyl}-
3-(3-iodo-phenyl)-thiourea (5g)
Recrystallized from acetone/hexane to give as brown
powder. Yield 76.9%, m.p. 207.8°C; IR (KBr, υ, cm–1):
3307 (-NH), 3048 (C=C, aromatic), 1192 (C=S), 622 (C-I);
1HNMR (300MHz, DMSO-d6, δ, ppm): 10.22 (1H, s, -NH),
10.07 (1H, s, -NH), 9.16 (1H, s, = CH), 8.37–8.40 (1H, d,
= CH), 8.29-8.31 (1H, d, = CH), 8.14–8.19 (1H, d, = CH),
7.50–7.99 (11H, m, - Ar-H), 7.30–7.35 (1H, t, = CH),
Journal of Enzyme Inhibition and Medicinal Chemistry

Enzyme activity and calculations of carbazole substituted chalcone ureas 811
7.13–7.19 (1H, t, = CH), 4.51–4.53 (2H, q, - CH2), 1.34–1.38
(3H, t, -CH3). Anal. Calcd. for C30H24IN3OS: C, 59.90; H, 4.02;
N, 6.99; S, 5.33. Found : C, 58.73; H, 3.48; N, 6.598; S, 4.18.
bananas were cut quickly into thin slices and homog-
enized 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 col-
lected. 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 phos-
phate buffer and then dialyzed against 5 the same buffer
overnight. e enzyme solution was then applied to the
Sepharose 4B-tyrosine-p-amino benzoic acid affinity
column22, 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 1M NaCl, 5 mM phosphate, pH 7.0.
1-(3,5-Dichloro-phenyl)-3-{4-[3-(9-ethyl-9H-carbazol-3-yl)-3-
oxo-propenyl]-phenyl}-thiourea (5h)
Recrystallized from acetone/hexane to give as brown
powder. Yield 64.1%, m.p. 201.4°C; IR (KBr, υ, cm–1): 3294
(-NH), 3077 (C=C, aromatic), 2039 (-N=C=S), 750 (C-Cl);
1HNMR (300 MHz, DMSO-d6, δ, ppm): 9.47 (1H, s, -NH),
9.41 (1H, s, -NH), 8.71 (1H, s, =CH), 6.99–8.10 (15H, m, -
Ar-H), 4.23–4.30 (2H, q, - CH2), 1.28–1.34 (3H, t, - CH3).
Anal. Calcd. for C30H23Cl2N3OS: C, 66.18; H, 4.26; N, 7.72;
S, 5.89. Found : C, 65.28; H, 3.56; N, 6.92; S, 4.89.
1-{4-[3-(9-Ethyl-9H-carbazol-3-yl)-3-oxo-propenyl]-
phenyl}-3-(3–fluoro-phenyl)-thiourea (5i)
Recrystallized from ethyl acetate/hexane to give as dark
orange powder. Yield 71.9%, m.p. 184.2°C; IR (KBr, υ, cm–1):
3319 (-NH), 2037 (-N=C=S), 1192 (C-F), 748 (C-H, aro-
matic); 1HNMR (300 MHz, DMSO-d6, δ, ppm): 10.17 (1H,
s, -NH), 10.12 (1H, s, -NH), 10.06 (1H, s, = CH), 9.13 (1H,
s, = CH), 8.28–8.29 (1H, d, = CH), 8.25–8.26 (1H, d, = CH),
8.10–8.15 (1H, d, = CH), 7.89–7.93 (1H, d, = CH), 6.91–7.74
(11H, m, - Ar-H), 4.46–4.53 (2H, q, - CH2), 1.31–1.36 (3H,
t, - CH3). Anal. Calcd. for C30H24FN3OS: C, 73.00; H, 4.90;
N, 8.51; S, 6.50. Found : C, 68.71; H, 4.07; N, 8.45; S, 6.79.
BPPO activity
Enzyme activity was determined; using catechol, by mea-
suring the increase in absorbance at 420 nm23, 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’s min−1 for 1 ml of enzyme at 25°C.
Inhibition of BPPO activity
e inhibition type of diarylureas was determined by
Lineweaver-Burk plots of 1/V vs. 1/S at two inhibitor
concentrations. e inhibition constant, Ki, was deduced
from the points of interception of the plots.
1-(2,4-Dichloro-phenyl)–3-{4-[3-(9-ethyl-9H-carbazol-3-yl)-3-
oxo-propenyl]-phenyl}-thiourea (5j)
Recrystallized from ether/acetone/hexane to give as
brown powder. Yield 83.6%, m.p. 193.8°C; IR (KBr, υ,
cm–1): 3329 (-NH), 3038 (C=C, aromatic), 1194 (C=S), 789
(C-Cl); 1HNMR (300 MHz, DMSO-d6, δ, ppm): 10.34 (1H,
s, -NH), 9.69 (1H, s, -NH), 9.16 (1H, s, = CH), 8.37–8.40
(1H, d, = CH), 8.30–8.32 (1H, d, = CH), 8.14–8.19 (1H, d, =
CH), 7.95–7.97 (1H, d, = CH), 7.30–7.89 (11H, m, - Ar-H),
4.51–4.53 (2H, q, - CH2), 1.33–1.36 (3H, t, - CH3). Anal.
Calcd. for C30H23Cl2N3OS: C, 66.18; H, 4.26; N, 7.72; S, 5.89.
Found : C, 64.80; H, 3.84; N, 7.15; S, 4.70.
results and discussion
Carbazole containing amino, nitro and urea chalcone
derivatives were synthesized and examined effect on
polyphenol oxidase enzyme. N-ethyl-3-acetylcarbazole
(2)waspreparedwithN-ethylcarbazoleandacetylchloride
by ZnCl2 ctalayst. Carbazole containing nitro (3) and
amino (4) chalcones prepared by the condensing various
acetophenones and 4-nitrobenzaldehyde with NaOH as
base, were reducted with tin (II) chloride in ethanol.e
4-aminochalcones were reacted with isocyanates and
isothiocyanates to get the products (5a–l) at high yields.
1-{4-[3-(9-Ethyl-9H–carbazol–3- yl)–3-oxo-propenyl]-phenyl}-
3-(3-trifluoromethyl-phenyl)-thiourea (5k)
Recrystallized from acetone/hexane to give as orange pow-
1
der. Yield 73.7%, m.p.199.4°C; IR (KBr, υ, cm–1): 3316 (-NH),
e prepared compounds were characterized by H
1
3058 (C=C, aromatic), 1175 (C=S), 1106 (C-CF3); HNMR
NMR, 13C NMR, IR and elemental analysis. e hydro-
gens attached to the nitrogen rezonans synthesized urea
and thiourea compounds between 8.50 and 10.40 ppm
and was indicated from the 1H NMR spectra. e signals
for aromatic hydrogens and vinylic protons are between
6.55 and 8.15 ppm. From the 13C NMR spectra,chalcone
and urea carbonyl are seen at around 188 and 150 ppm
respectively. In the infrared spectra of compounds 5a–k,
it was possible to observe the absorptions between 3250
and 3450 cm−1 relating to NH stretch, absorptions in
1600–1650 cm−1 from α, β-unsaturated carbonyl moiety
stretch and absorptions in 1650–1750 cm–1 from urea car-
bonyl moiety stretching.
(300 MHz, DMSO-d6, δ, ppm): 10.30 (1H, s, -NH), 10.21 (1H,
s, -NH), 9.15 (1H, s, = CH), 8.37–8.39 (1H, d, = CH), 8.31–8.32
(1H, d, = CH), 8.28–8.29 (1H, d, = CH), 7.49–8.19 (13H, m, -
Ar-H), 7.30–7.35 (1H, t, = CH), 4.51–4.54 (2H, q, - CH2), 1.34–
1.39 (3H, t, - CH3). Anal. Calcd. for C31H24F3N3OS: C, 68.49; H,
4.45; N, 7.73; S, 5.90. Found : C, 69.06; H, 4.37; N, 7.55; S, 5.12.
Purification of PPO
All purification steps were carried out at 25°C. e extrac-
tion procedure was adopted from Wesche-Ebeling &
Montgomery21. e bananas were washed with distilled
water three times to prepare the crude extract, 50 g of
© 2013 Informa UK, Ltd.

812 A. R. Nixha et al.
Figure 1. Ki graphics of chalcone urea derivatives on BPPO.
For evaluating the tyrosinase inhibitory activity, all
the prepared compounds were subjected to tyrosinase
inhibition assay with catechol as substrate. e results
showed that most of the compounds (3, 4, 5a, 5c–g)
inhibited and some of them (5b, 5h–k) activated the
tyrosinase enzyme activity. Ki values were calculated
from inhibition curves obtained with ureas (Figure 1). Ki
values of 19.97, 14.96, 24.96, 29.91, 7.47, 9.96, 7.48, 19.95
µM were obtained for ((3), (4), (5a), (5c), (5d), (5e),
(5f), and (5g) respectively (Table 1). According to Ki val-
ues, (5d) was the most effective inhibitor for BPPO.
Several compound reported as PPO inhibitors were
also shown to have inhibitors effect on the BPPO. Among
the tested anti-browning reagent, the most effective
ones were dithiotreithol and sodium metabisulfite24. e
action of sulfite in the prevention of enzymatic browning
can usually be explained by several processes. One is the
action on o-quinones. e formation of quinone-sulfite
complexes prevents the quinone polymerization. e
action of sulfite in the prevention of enzymatic browning
can usually be explained by several processes. One is the
action on o-quinones. e formation of quinone-sulfite
complexes prevents the quinone polymerization25. e
enzyme also seemed to be sensitive to thiourea since
PPO contains copper as a co-factor, the irreversible
Table 1. Ki inhibition constants, dipol moment and ΔEHOMO-LUMO
(eV) values of the compounds.
Dipol
ΔEHOMO-LUMO
(eV)
Compound Activity
Ki (µM) moment (D)
3
4
inhibitor
inhibitor
inhibitor
activator
inhibitor
inhibitor
inhibitor
inhibitor
inhibitor
activator
activator
activator
activator
19.97
14.96
24.96
—
29.91
7.47
9.96
7.48
19.95
—
7.8467
5.9614
4.4375
3.4041
7.2517
7.1944
4.8791
4.3063
—*
3.9318
3.2735
2.0184
1.4482
0.24562
0.25245
0.23121
0.22925
0.23055
0,27405
0.24775
0.25965
—*
0.22423
0.22948
0.22959
0.22934
5a
5b
5c
5d
5e
5f
5g
5h
5i
—
—
—
5j
5k
*Could not be calculated by the Gaussian.
Journal of Enzyme Inhibition and Medicinal Chemistry

Enzyme activity and calculations of carbazole substituted chalcone ureas 813
Figure 2. Calculated geometric structures of the some compounds using HF method with 6-31G basis set.
inactivation of this enzyme can be effected by sub-
(5b, 5h–5k) did not inhibit tyrosinase at 50 µM, as well
as show activator effects on tyrosinase enzyme. e
other compounds (3,4, 5a, 5c–5g) showed enzyme
inhibitory effects. Carbazole substituted chalcones
(3 and 4) are precursor for the final products which
are carbazole substituted chalcone urea derivatives.
e compounds (3) and (4) are good inhibitory effect
on tyrosinase enzyme. at can be explained either
HOMO-LUMO energy differences or electron releasing
and electron withdrawing groups on the phenyl ring.
e compound (4) is better inhibitory effect than (3)
because HOMO-LUMO energy differences of 4 (0.25245
eV) higher than 3 (0.24562 eV). In addition to this,
amine group on the phenyl ring donates electron to
the carbonyl group of chalcone and higher electron
density binds copper ions more effectively in the active
cite of enzyme. Urea derivatives (5a, 5c–5g) have
enzyme inhibitory effect due to HOMO-LUMO energy
differences. Generally, differentiation between HOMO
and LUMO reflects the intensity of electron affinity, and
stances (such as thiol compounds thiourea, -hydroxy-
quinoline, etc.), which remove copper from the active
site of the enzyme26. Chilaka et al. reported that thiourea
was a good inhibitor of PPO, with low Ki values of 0.15
mM and inhibition of PPO was uncompetitive27. We
were determined that (3), (4), (5a), (5c), (5d), (5e),
(5f), and (5g) was a better inhibitor of PPO according
to thiourea. Gulcin et al. reported that sodium diethyl
dithiocarbamate was the most effective inhibitor (Ki:
1.79 nM) on nettle PPO28. It was reported that the
enzyme inhibited with diarylureas15 and phenylthio-
urea29, propanoic acid30, alkyldithiocarbonates31 and
coumarin schiff-bases32, n-alkyl dithiocarbamate com-
pounds33, sodium diethyl dithiocarbamate28.
In order to understand and explain the experimental
resultsobtained,molecularcalculationswereperformed
using Gaussian software34a,b
, for the synthesized
compounds and some of the molecular structures are
shown in Figure 2. Some of the prepared compounds
© 2013 Informa UK, Ltd.

814 A. R. Nixha et al.
lower differentiation suggests higher electron affinity35.
e calculated HOMO-LUMO energy differences of the
compounds((5a, 5c–5g) showed a linear relationship
for higher inhibitory effects with increasing ΔEHOMO-LUMO
values which are higher than 0.23eV and the compounds
(5b, 5h–5k) which have ΔEHOMO-LUMO values lower than
0.23eV showed enzyme activity effects. Among the urea
compounds, 5d (Ki, 7.47 µM and ΔEHOMO-LUMO, 0.27405
eV) is the most inhibition effect. 5c (Ki, 29.91 µM and
ΔEHOMO-LUMO, 0.23055 eV) is the least inhibition effect.
is relation probably to be explained that the higher
ΔEHOMO-LUMO differences of the molecule increases
enzyme inhibition activity for possibility of binding
to copper ions in the active cite of enzyme. Moreover,
the dipole moments of the molecules are higher than
4.0 shows enzyme inhibitory effect. e results are
shown in Table 1.
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Declaration of interest
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