Synthesis, biological evaluation, and molecular docking studies of 2,5-substituted-1,4-benzoquinone as novel urease inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.07.002

A series of 2,5-substituted-1,4-benzoquinone (1-6) were prepared and structurally characterized by elemental analysis, IR spectra, ¹H and ¹³C NMR spectra, and single crystal X-ray determination. The urease inhibitory activities of the compounds against H. pylori urease were studied. Among the compounds, 2,5-bis(2-morpholin-4-ylethylamino)-[1,4]benzoquinone (2) shows the most effective activity with IC₅₀ value of 27.30 ± 2.17 μM. Docking simulation was performed to insert compound 2 into the crystal structure of H. pylori urease at the active site to determine the probable binding mode. As a result, compound 2 may be used as a potential urease inhibitor.

Full text of DOI:10.1016/j.bmc.2012.07.002

Bioorganic & Medicinal Chemistry 20 (2012) 4889–4894
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular docking studies
of 2,5-substituted-1,4-benzoquinone as novel urease inhibitors
⇑
Zhong-Lu You , Dong-Mei Xian, Mei Zhang, Xiao-Shan Cheng, Xiao-Fang Li
Department of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2,5-substituted-1,4-benzoquinone (1–6) were prepared and structurally characterized by ele-
mental analysis, IR spectra, 1H and 13C NMR spectra, and single crystal X-ray determination. The urease
inhibitory activities of the compounds against H. pylori urease were studied. Among the compounds, 2,5-
bis(2-morpholin-4-ylethylamino)-[1,4]benzoquinone (2) shows the most effective activity with IC50
Received 28 June 2012
Revised 1 July 2012
Accepted 2 July 2012
Available online 10 July 2012
value of 27.30 2.17 lM. Docking simulation was performed to insert compound 2 into the crystal struc-
ture of H. pylori urease at the active site to determine the probable binding mode. As a result, compound 2
may be used as a potential urease inhibitor.
Keywords:
Benzoquinone
Urease
Ó 2012 Elsevier Ltd. All rights reserved.
Inhibition
Crystal structure
Molecular docking
1. Introduction
inhibitory activities of the compounds were investigated from
both experimental and molecular docking study.
Urease (urea amidohydrolase; E.C.3.5.1.5) is a nickel-contain-
ing metalloenzyme that catalyzes the hydrolysis of urea to form
ammonia and carbamate.1,2 The resulting carbamate spontane-
ously decomposes to yield ammonia and carbon dioxide. High
concentration of ammonia arising from the reaction, as well as
the accompanying pH elevation, has important negative effects
in the fields of medicine and agriculture.3–6 Control of the activity
of urease through the use of inhibitors could counteract these
negative effects. In recent years, a variety of urease inhibitors
have been studied, including acetohydroxamic acid (AHA), humic
acid, 1,4-benzoquinone, and inorganic metal salts, etc.7–12 Among
the inhibitors, 1,4-benzoquinone and its derivatives have
been extensively studied for their inhibition on urease,13,14
however, no rational structure–activity relationships have
been achieved so far. As an extension of the work on the
exploration of effective urease inhibitors related to quinone deriv-
atives, in this paper, a series of 2,5-substituted-1,4-benzoquinon-
es, 2,5-bis(2-phenylaminoethylamino)-[1,4]benzoquinone (1),
2. Results and discussion
2.1. Chemistry
The compounds 1–6 were readily synthesized by the reaction of
1:2 molar ratio of hydroquinone with N-phenylethylene-1,2-dia-
mine, 2-morpholin-4-ylethylamine, cyclohexylamine, isopropyl-
amine, cyclopentylamine, and cyclopropylamine, respectively, in
OH
O
H
N
MeOH
r.t.
R
R
NH2
N
+
R
N
H
OH
O
N
2,5-bis(2-morpholin-4-ylethylamino)-[1,4]benzoquinone
(2),
(1) R =
(2) R =
O
2,5-biscyclohexylamino-[1,4]benzoquinone (3), 2,5-bisisopropyla-
mino-[1,4]benzoquinone (4), 2,5-biscyclopentylamino-[1,4]benzo-
quinone (5), and 2,5-biscyclopropylamino-[1,4]benzoquinone (6),
were synthesized and structurally characterized. The urease
(3) R =
(5) R =
(4) R =
(6) R =
⇑
Corresponding author. Tel.: +86 411 84216387.
E-mail address: youzhonglu@yahoo.com.cn (Z.-L. You).
Scheme 1. Synthesis of the compounds.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.002

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Z.-L. You et al. / Bioorg. Med. Chem. 20 (2012) 4889–4894
Figure 1. A perspective view of the molecular structure of 1 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level.
Figure 2. A perspective view of the molecular structure of 2 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level.
Figure 3. A perspective view of the molecular structure of 3 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level.
Figure 4. A perspective view of the molecular structure of 4 with the atom labeling scheme. Thermal ellipsoids are drawn at the 30% probability level.
methanol, at room temperature (Scheme 1), with high yields (over
90%) and purity. The color of the reaction mixtures was turned
from colorless to red. The compounds have been characterized by
elemental analyses, IR spectra, 1H and 13C NMR spectra. Structures
of the compounds 1–4 were further confirmed by single crystal X-
ray crystallography (CCDC—840475 for 1, 840476 for 2, 840477 for
3, and 840478 for 4). We have tried to prepare the well-shaped sin-
gle crystals of the compounds 5 and 6, but only thin schistose-
shaped products were formed, which were not suitable for single
crystal X-ray diffraction.
2.2. Structure description of the compounds 1–4
Single crystal structural X-ray crystallography reveals that the
compounds 1–4 are 2,5-substituted-1,4-benzoquinones. The substi-
tute groups are N-phenylethyl for 1 (Fig. 1), 2-morpholin-4-ylethyl

Z.-L. You et al. / Bioorg. Med. Chem. 20 (2012) 4889–4894
4891
Table 1
Hydrogen bond distances (Å) and bond angles (°) for the compounds 1–4
ues of 41.50 3.22 and 35.36 4.03
l
M, respectively. It should be
notable that the IC50 values of 2 and 5 are even lower than the AHA
(46.27 0.73 M).
l
D–HÁ Á ÁA
d(D–H)
d(HÁ Á ÁA)
d(DÁ Á ÁA)
Angle (D–HÁ Á ÁA)
From the results, it can be seen that the flexible substituted
groups with suitable sizes such as 2-morpholin-4-ylethyl, cyclo-
hexyl, and cyclopentyl are preferred units for the urease inhibition.
However, the substituted groups with rigid or very small sizes in
the compounds can severely decrease the urease inhibitory
activities.
1
N2–H2AÁ Á ÁO1i
0.90(1)
0.90(1)
2.22(3)
2.31(2)
2.963(5)
3.156(4)
140(3)
158(3)
N1–H1Á Á ÁO1i
2
N1–H1Á Á ÁO1
0.90(1)
2.18(2)
2.628(2)
110(2)
3
N2–H2Á Á ÁO1ii
0.90(1)
0.90(1)
2.15(2)
2.36(2)
2.946(3)
3.203(3)
146(3)
157(3)
N1–H1Á Á ÁO2iii
2.4. Molecular docking study
4
N1–H1Á Á ÁO1iv
0.90(1)
2.16(2)
3.018(2)
158(2)
The molecular docking study was performed to investigate the
binding effects between the compounds and the active sites of
the H. pylori urease. In the X-ray structure available for the native
H. pylori urease, two nickel atoms were coordinated by His136,
His138, Kcx219, His248, His274, Asp362 and water molecules,
while in the AHA-inhibited urease, the water molecules were re-
placed by AHA.11 Figures 6–8 are the binding models for the com-
pounds 2, 3, and 5, respectively, in the enzyme active site of the
urease. The docking scores are À4.75 for 1, À5.34 for 2, À6.17 for
3, À3.96 for 4, À5.88 for 5, and À4.16 for 6. As a comparison, the
docking score for the AHA is À5.01. The values of the docking
scores are roughly agreed with the inhibitory activities observed
from the experiment, indicating the results are believable.
From the docking results, it can be seen that compound 1 is lo-
cated far away from the active site of the urease, as a result of the
large bulk of the compound. The molecule of 1 is bind with the ure-
ase through two N–HÁ Á ÁO hydrogen bonds. For 2, the molecule is
located at the active site of the urease. There form two hydrogen
bonds among the amino NH groups and the quinone O atoms of
the compound with the Asp223 and His221 residues of the urease
(Fig. 6). For 3 and 5, the binding models are similar to each other.
They are well filled in the active pocket of the urease. For 3, there
form three hydrogen bonds among the amino NH groups and the
quinone O atoms of the compound with the Arg338, Met317 and
His322 residues of the urease (Fig. 7). For 5, there form two hydro-
gen bonds among the amino NH groups and the quinone O atoms
of the compound with the Met317 and His322 residues of the ure-
ase (Fig. 8). For the molecules of 4 and 6, even though their sizes
are much smaller than those of the other four compounds, they
are located far away from the active site of the urease. The mole-
cule of 4 is bind with the urease through two N–HÁ Á ÁO hydrogen
bonds. The molecule of 6 is bind with the urease with no obvious
hydrogen bonds. The results of the molecular docking study could
explain the activities of the compounds against H. pylori urease.
Symmetry codes: (i) 1Àx, 1Ày, Àz; (ii) x, 1/2Ày, 1/2 + z; (iii) x, 1/2Ày, À1/2 + z; (iv)
1/2 + x, 1/2Ày, 1/2 + z.
80
60
40
20
Null
Null
Null
0
1
2
3
4
5
6
7
Compounds
Figure 5. Inhibition rates of the tested materials to the urease. Compound
7
represents AHA.
for 2 (Fig. 2), cyclohexyl for 3 (Fig. 3), and isopropyl for 4 (Fig. 4).
There are crystallographic inversion centers in the compounds 1,
2, and 4, which are located at the midpoints of the quinone groups.
The dihedral angle between the quinone ring and the benzene ring
in 1 is 77.8(3)°. The morpholine rings in 2 and the cyclohexyl rings
in 3 are in chair conformation. The distances between the C and O
atoms of the quinone groups in the compounds are range from
1.23 to 1.24 Å, confirming them as typical double bonds. Close
examination of the structures of the compounds reveals that the
bond lengths are comparable to each other, and also comparable
to those observed in the substituted quinone derivatives.15–17 The
crystal structures of the compounds are stabilized by hydrogen
bonds (Table 1).
3. Conclusion
The present study reports the synthesis, structures and urease
inhibitory activities of a series of 2,5-substituted-1,4-benzoqui-
nones. Three of the compounds have effective urease inhibition.
Among the compounds, 2 may be used as a potential urease inhib-
itor, which deserves further study. The urease inhibitory activities
and the molecular docking studies of the compounds against H. py-
lori urease indicate that suitable size and flexibility of the substi-
tuted groups of the benzoquinone rings are important factors for
the exploration of effective urease inhibitors.
2.3. Pharmacology
The measurement of Helicobacter pylori urease inhibitory activ-
ity was carried out for three parallel times. The percents of inhibi-
tion at the concentration of 100 lM for the compounds against
urease are 81.63 3.18 (2), 62.71 2.82 (3), and 68.79 5.91 (5).
There are no or very weak inhibition observed for compounds 1,
4, and 6 at the same condition. The AHA was used as a reference7
with the percent of inhibition of 85.21 5.27 at the concentration
4. Experimental protocols
4.1. General
of 100
the most effective activity with the IC50 value of 27.30 2.17
Compounds 3 and 5 also show effective activity with the IC50 val-
l
M. The results are depicted in Figure 5. Compound 2 shows
Starting materials, reagents and solvents with AR grade were
purchased from commercial suppliers and used without further
l
M.

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Z.-L. You et al. / Bioorg. Med. Chem. 20 (2012) 4889–4894
Figure 6. 3D (left) and 2D (right) binding mode of 2 with H. pylori urease. Hydrogen bonds are shown as dashed lines.
Figure 7. 3D (left) and 2D (right) binding mode of 3 with H. pylori urease. Hydrogen bonds are shown as dashed lines.
purification. Elemental analyses were performed on a Perkin-Elmer
240C elemental analyzer. The IR spectra were recorded on a Jasco
FT/IR-4000 spectrometer as KBr pellets in the 4000–400 cmÀ1 re-
gion. Single crystal structural X-ray diffraction was carried out at
a Bruker SMART 1000 CCD area diffractometer equipped with
13C NMR (DMSO-d6): 40.8, 92.1, 111.9, 115.9, 128.9, 148.2, 151.1,
177.3. IR data (KBr, cmÀ1): 3397 (w), 3276 (m, sh), 1639 (vw),
1602 (m), 1550 (s), 1497 (s), 1443 (s), 1369 (w), 1301 (m), 1257
(w), 1179 (w), 1130 (w), 1096 (w), 875 (w), 816 (w), 753 (m),
698 (w), 670 (w), 515 (vw), 463 (vw). Single crystals of 1 suitable
for X-ray diffraction were obtained by recrystallization of the prod-
uct in methanol.
MoKa radiation, and the structures were solved by direct method
using SHELXTL package.18 The crystallographic data for the com-
pounds are summarized in Table 2.
4.2.2. Synthesis of 2,5-bis(2-morpholin-4-ylethylamino)-
[1,4]benzoquinone (2)
4.2. Synthesis
Hydroquinone (1.0 mmol, 0.11 g) and 2-morpholin-4-ylethyl-
amine (1.0 mmol, 0.13 g) were mixed in methanol, and stirred at
room temperature for 1 h. The solution was changed from colorless
to red. The methanol was evaporated to obtain red crystalline
product of 2, which was washed with methanol, and dried in air.
Yield: 95%. Anal. Calcd for C18H28N4O4: C, 59.3; H, 7.7; N, 15.4.
Found: C, 59.2; H, 7.8; N, 15.2%. 1H NMR (DMSO-d6): 2.40 (t, 8H),
2.51 (s, 2H), 3.24 (t, 4H), 3.36 (t, 4H), 3.56 (t, 8H), 5.27 (s, 2H).
13C NMR (DMSO-d6): 38.8, 52.9, 55.3, 66.1, 92.1, 150.9, 177.2. IR
data (KBr, cmÀ1): 3355 (m, sh), 1646 (s), 1613 (s), 1495 (m),
1454 (m), 1354 (m), 1334 (m), 1293 (m), 1143 (m), 1115 (s),
4.2.1. Synthesis of 2,5-bis(2-phenylaminoethylamino)-
[1,4]benzoquinone (1)
Hydroquinone (1.0 mmol, 0.11 g) and N-phenylethylene-1,2-
diamine (1.0 mmol, 0.14 g) were mixed in methanol, and stirred
at room temperature for 1 h. The solution was changed from color-
less to red. The methanol was evaporated to obtain red crystalline
product of 1, which was washed with methanol, and dried in air.
Yield: 93%. Anal. Calcd for C22H24N4O2: C, 70.2; H, 6.4; N, 14.9.
Found: C, 70.0; H, 6.4; N, 15.0%. 1H NMR (DMSO-d6): 3.22 (t, 4H),
3.30 (t, 4H), 3.33 (s, 2H), 5.31 (s, 2H), 6.56 (m, 6H), 7.08 (t, 4H).

Z.-L. You et al. / Bioorg. Med. Chem. 20 (2012) 4889–4894
4893
Figure 8. 3D (left) and 2D (right) binding mode of 5 with H. pylori urease. Hydrogen bonds are shown as dashed lines.
Table 2
Crystallographic and experimental data for the compounds 1À4
Compound
1
2
3
4
Formula
Mr
C
22H24N4O2
C18H28N4O4
364.4
C18H26N2O2
302.4
C12H18N2O2
222.3
376.4
T (K)
298(2)
298(2)
298(2)
298(2)
Crystal shape/color
block/red
0.18 Â 0.17 Â 0.15
Orthorhombic
Pbca
7.729(6)
7.928(6)
Block/red
0.20 Â 0.20 Â 0.18
Monoclinic
P21/c
17.118(1)
5.131(1)
11.124(1)
107.987(3)
929.3(2)
2
Block/red
0.23 Â 0.20 Â 0.20
Monoclinic
P21/c
14.225(2)
8.970(2)
13.369(2)
99.348(9)
1683.3(5)
4
Block/red
Crystal size (mm3)
0.23 Â 0.20 Â 0.20
Monoclinic
C2/c
10.783(2)
9.109(2)
12.867(3)
106.472(15)
1212.0(4)
4
Crystal system
Space group
a (Å)
b (Å)
c (Å)
31.60(2)
b (°)
V (Å3)
1936(2)
4
1.291
Z
Dc (g cmÀ3
)
1.302
0.093
1.193
0.078
1.218
0.084
l
(Mo-K
a
) (mmÀ1
)
0.085
F(000)
800
392
656
480
Reflections collected
Unique reflections
Observed reflections (I P 2
Parameters
12,573
1770
741
5413
2033
1372
122
9218
3449
2163
205
3436
1297
805
78
r
(I))
133
Restraints
2
1
2
1
Min. and max. transmission
0.985, 0.987
1.023
0.0725, 0.1210
0.1913, 0.1643
0.142, À0.116
0.982, 0.983
1.025
0.0430, 0.1012
0.0689, 0.1181
0.133, À0.149
0.982, 0.985
1.009
0.0847, 0.2165
0.1192, 0.2484
0.419, À0.261
0.981, 0.984
1.059
0.0481, 0.1217
0.0850, 0.1413
0.135, À0.150
Goodness-of-fit on F2
R1, wR2 [I P 2
r
(I)]a
R1, wR2 (all data)a
Large diff. peak and hole (eÅÀ3
)
a
2
R1 = FoÀFc/Fo, wR2 = [ w(Fo ÀFc2)/ w(Fo2)2]1/2
.
1071 (w), 1026 (w), 945 (w), 911 (w), 859 (w), 809 (m), 770 (w),
593 (m), 469 (vw). Single crystals of 2 suitable for X-ray diffraction
were obtained by recrystallization of the product in methanol.
(d, 2H). 13C NMR (DMSO-d6): 24.2, 24.9, 30.9, 50.7, 92.1, 149.8,
177.3. IR data (KBr, cmÀ1): 3272 (m, sh), 1638 (m), 1590 (s),
1563 (s), 1493 (s), 1449 (m), 1343 (m), 1297 (s), 1250 (m), 1208
(m), 1143 (w), 1091 (w), 976 (w), 889 (w), 869 (w), 815 (m), 743
(w), 669 (m), 575 (w), 504 (w), 425 (w). Single crystals of 3 suitable
for X-ray diffraction were obtained by recrystallization of the prod-
uct in methanol.
4.2.3. Synthesis of 2,5-biscyclohexylamino-[1,4]benzoquinone
(3)
Hydroquinone (1.0 mmol, 0.11 g) and cyclohexylamine
(1.0 mmol, 0.10 g) were mixed in methanol, and stirred at room
temperature for 1 h. The solution was changed from colorless to
red. The methanol was evaporated to obtain red crystalline product
of 3, which was washed with methanol, and dried in air. Yield: 93%.
Anal. Calcd for C18H26N2O2: C, 71.5; H, 8.7; N, 9.3. Found: C, 71.3; H,
8.7; N, 9.2%. 1H NMR (DMSO-d6): 1.15 (m, 2H), 1.37 (m, 8H), 1.59
(d, 2H), 1.67 (d, 4H), 1.77 (d, 4H), 2.51 (m, 2H), 5.32 (s, 2H), 7.25
4.2.4. Synthesis of 2,5-bisisopropylamino-[1,4]benzoquinone
(4)
Hydroquinone (1.0 mmol, 0.11 g) and isopropylamine
(1.0 mmol, 0.06 g) were mixed in methanol, and stirred at room
temperature for 1 h. The solution was changed from colorless to
red. The methanol was evaporated to obtain red crystalline product

4894
Z.-L. You et al. / Bioorg. Med. Chem. 20 (2012) 4889–4894
of 4, which was washed with methanol, and dried in air. Yield: 87%.
Anal. Calcd for C12H18N2O2: C, 64.8; H, 8.2; N, 12.6. Found: C, 65.1;
H, 8.1; N, 12.7%. 1H NMR (DMSO-d6): 1.16 (d, 12H), 3.63 (m, 2H),
5.27 (s, 2H), 7.29 (d, 2H). 13C NMR (DMSO-d6): 21.1, 43.6, 92.2,
149.9, 177.4. IR data (KBr, cmÀ1): 3210 (m, sh), 1637 (m), 1592
(s), 1559 (s), 1470 (s), 1446 (m), 1351 (m), 1303 (s), 1223 (m),
1207 (m), 1164 (w), 1139 (w), 967 (m), 872 (w), 841 (w), 765
(w), 732 (w), 516 (w), 415 (w). Single crystals of 4 suitable for X-
ray diffraction were obtained by recrystallization of the product
in methanol.
4.4. Docking simulations
Molecular docking study of the compounds into the 3D X-ray
structure of the H. pylori urease (entry 1E9Y in the Protein Data
Bank) was carried out by using the AutoDock version 4.2. First,
AutoGrid component of the program precalculates a 3D grid of
interaction energies based on the macromolecular target using
the AMBER force field. The cubic grid box of 100 Â 100 Â 60 Å3
points in x, y, and z direction with a spacing of 0.375 Å and grid
maps were created representing the catalytic active target site re-
gion where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the biding free energy
of the inhibitor within the macromolecules. The GALS search algo-
rithm (genetic algorithm with local search) was chosen to search
for the best conformers. The parameters were set using the soft-
ware ADT (AutoDockTools package, version 1.5.4) on PC which is
associated with AutoDock 4.2. Default settings were used with an
initial population of 100 randomly placed individuals, a maximum
number of 2.5 Â 106 energy evaluations, and a maximum number
of 2.7 Â 104 generations. A mutation rate of 0.02 and a crossover
rate of 0.8 were chosen. Give overall consideration of the most
favorable free energy of biding and the majority cluster, the results
were selected as the most probable complex structures.
4.2.5. Synthesis of 2,5-biscyclopentylamino-[1,4]benzoquinone
(5)
Hydroquinone (1.0 mmol, 0.11 g) and cyclopentylamine
(1.0 mmol, 0.08 g) were mixed in methanol, and stirred at room
temperature for 1 h. The solution was changed from colorless to
red. The methanol was evaporated to obtain red crystalline product
of 5, which was washed with methanol, and dried in air. Yield: 92%.
Anal. Calcd for C16H22N2O2: C, 70.0; H, 8.1; N, 10.2. Found: C, 70.2;
H, 8.0; N, 10.4%. 1H NMR (DMSO-d6): 1.53–1.69 (m, 12H), 1.90 (m,
4H), 3.75 (m, 2H), 5.27 (s, 2H), 7.30 (d, 2H). 13C NMR (DMSO-d6):
23.7, 31.6, 53.3, 92.8, 150.5, 177.3. IR data (KBr, cmÀ1): 3261 (m,
sh), 1637 (m), 1592 (s), 1567 (s), 1491 (s), 1449 (m), 1344 (m),
1296 (s), 1245 (m), 1207 (m), 1136 (w), 1087 (w), 971 (w), 873
(w), 854 (w), 821 (m), 745 (w), 657 (m), 573 (w), 511 (w), 443 (w).
Acknowledgments
4.2.6. Synthesis of 2,5-biscyclopropylamino-[1,4]benzoquinone
(6)
This work was financially supported by the Natural Science
Foundation of China (Project No. 20901036), and by the Distin-
guished Young Scholars Program of Higher Education of Liaoning
Province (Grant No. LJQ2011114).
Hydroquinone (1.0 mmol, 0.11 g) and cyclopropylamine
(1.0 mmol, 0.06 g) were mixed in methanol, and stirred at room
temperature for 1 h. The solution was changed from colorless to
red. The methanol was evaporated to obtain red crystalline product
of 6, which was washed with methanol, and dried in air. Yield: 87%.
Anal. Calcd for C12H14N2O2: C, 66.0; H, 6.5; N, 12.8. Found: C, 65.9;
H, 6.5; N, 13.0 %. 1H NMR (DMSO-d6): 0.63 (m, 4H), 0.75 (m, 4H),
2.45 (m, 2H), 5.46 (s, 2H), 7.72 (d, 2H). 13C NMR (DMSO-d6): 6.2,
24.2, 94.0, 152.3, 177.9. IR data (KBr, cmÀ1): 3215 (m, sh), 1636
(m), 1592 (s), 1556 (s), 1472 (s), 1445 (m), 1351 (m), 1301 (s),
1222 (m), 1207 (m), 1163 (w), 1139 (w), 972 (m), 867 (w), 845
(w), 751 (w), 737 (w), 532 (w), 426 (w).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.002.
References and notes
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until use in the experiment. The mixture, containing 25
lL of the
H. pylori urease and 25 L of the test compound, was pre-incubated
l
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for 3 h at room temperature in a 96-well assay plate. Urease activ-
ity was determined by measuring ammonia production using the
indophenol method as that described by Weatherburn.20