Synthesis, structure-activity relationship analysis and kinetics study of reductive derivatives of flavonoids as Helicobacter pylori urease inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.03.016

In a continuing study for discovering urease inhibitors based on flavonoids, nineteen reductive derivatives of flavonoids were synthesized and evaluated against Helicobacter pylori urease. Analysis of structure-activity relationship disclosed that 4-deoxy analogues are more potent than other reductive products. Out of them, 4′,7,8-trihydroxyl-2-isoflavene (13) was found to be the most active with IC₅₀ of 0.85 μM, being over 20-fold more potent than the commercial available urease inhibitor, acetohydroxamic acid (AHA). Kinetics study revealed that 13 is a competitive inhibitor of H. pylori urease with a K_i value of 0.641 μM, which is well matched with the results of molecular docking. Biological evaluation and mechanism study of 13 suggest that it is a good candidate for discovering novel anti-gastritis and anti-gastric ulcer agent.

Full text of DOI:10.1016/j.ejmech.2013.03.016

European Journal of Medicinal Chemistry 63 (2013) 685e695
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structureeactivity relationship analysis and kinetics study
of reductive derivatives of flavonoids as Helicobacter pylori urease
inhibitors
,
b
,
a
b
a
a
a
Zhu-Ping Xiao a , Zhi-Yun Peng , Jing-Jun Dong , Juan He , Hui Ouyang , Yu-Ting Feng
*
, Chun-Lei Lu a, Wan-Qiang Lin a, Jin-Xiang Wang a, Yin-Ping Xiang a, Hai-Liang Zhu b
,
**
a College of Chemistry and Chemical Engineering, and Key Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou University, Jishou
416000, PR China
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In a continuing study for discovering urease inhibitors based on flavonoids, nineteen reductive de-
rivatives of flavonoids were synthesized and evaluated against Helicobacter pylori urease. Analysis of
structureeactivity relationship disclosed that 4-deoxy analogues are more potent than other reductive
products. Out of them, 40,7,8-trihydroxyl-2-isoflavene (13) was found to be the most active with IC50 of
Received 11 January 2013
Received in revised form
4 March 2013
Accepted 8 March 2013
Available online 16 March 2013
0.85
droxamic acid (AHA). Kinetics study revealed that 13 is a competitive inhibitor of H. pylori urease with a
Ki value of 0.641 M, which is well matched with the results of molecular docking. Biological evaluation
mM, being over 20-fold more potent than the commercial available urease inhibitor, acetohy-
m
Keywords:
3-Flavene
2-Isoflavene
Synthesis
and mechanism study of 13 suggest that it is a good candidate for discovering novel anti-gastritis and
anti-gastric ulcer agent.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Urease inhibitor
Mechanism
1. Introduction
functions of this enzyme, its inhibition by potent and specific
compounds could provide an invaluable addition for treatment of
Urease (E.C 3.5.1.5) is found in various living organisms,
including bacteria, fungi, higher plants, and some invertebrates
[1,2], which catalyzes the hydrolysis of urea to ammonia and carbon
dioxide, and is responsible for many microorganisms to use urea as
nitrogen source for growth. High concentration of ammonia arising
from these reactions causes an increase in pH, which permits
Helicobacter pylori to endure acidic pH of the stomach during
colonization. Urease therefore plays an important role in the
pathogenesis of gastric and peptic ulcer induced by H. pylori and is
known to be a major virulence factor [3]. Urease is also directly
involved in the development of infection stones, pyelonephritis,
hepatic coma and other disease states [4e6]. Due to diverse
infections, and secondary complexes such as gastritis and gastric
ulcer caused by H. pylori [3,7].
Several classes of compounds show significant inhibitory ac-
tivity against urease with hydroxamic acids being the best recog-
nized inhibitors [8,9] and with phosphoramidates being the most
active [10,11]. However, degradation of phosphoramidates at low
pH and teratogenicity of hydroxamic acids in rats prevent them
from using in vivo [12,13]. It is well known that structural diversity
and complexity within natural products are unique and the func-
tional complexity found in natural products will never be invented
de novo in a chemistry laboratory [14]. Over 75% of new chemical
entities submitted (1981e2004) were based on natural product
lead structures, indicating that the reliance on natural products is
so far the most-successful route for new drug discovering [15,16]. In
the past decades, exploration of urease inhibitors from natural
products has attracted much attention [3,17,18]. In 2001, Bae et al.
found daidzein having weak inhibitory activity against H. pylori
urease, which opened the door for urease inhibitor discovering
* Corresponding author. College of Chemistry and Chemical Engineering, and Key
Laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou
University, Jishou 416000, PR China. Tel./fax: þ86 743 8563911.
** Corresponding author. Tel.: þ86 25 8359 2572; fax: þ86 25 83592672.
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), zhuhl@nju.edu.cn
(H.-L. Zhu).
from flavonoids,
a large group of polyphenolic compounds
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.016

686
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
Table 1
naturally occurred in fruit, vegetables, nuts, seeds, flowers, and
bark [19e21]. Subsequently, (þ)-gallocatechin [22], quercetin [23],
datisdirin [24] and other flavonoids [25] were reported as potent
urease inhibitors. On the basis of these researches, our current ef-
forts are therefore focused on seeking novel urease inhibitors based
on flavonoids [13,26e29]. We have previously demonstrated that
substitution of a methylene group for the carbonyl group of a
deoxybenzoin (Scheme 1) resulted in a significant increase in ac-
tivity [26], which is also observed by comparison of the IC50 value of
(þ)-gallocatechin [22] with that of dihydromyricetin (Scheme 1)
[28]. These findings lead to a conception that reduction of flavonoid
would produce more potent urease inhibitor. In this paper, we
discuss our efforts to improve upon the enzyme inhibition of the 4-
ol and 4-deoxy series of flavonoids.
In vitro inhibitory activity data of the synthesized compounds against H. pylori
urease.
Entry
Structure
Yield
IC50, mM
1
91
328 ꢀ 35
43.7 ꢀ 3.8
4853 ꢀ 451
2
3
64
44
2. Results and discussion
2.1. Chemistry
Nineteen compounds (1e19) derived from flavonoids were ob-
tained via chemical reduction or catalytic hydrogenation (Table 1),
and eight of them were synthesized for the first time. All synthetic
compounds have been characterized by elemental analyses, MS,
and 1H NMR spectra. Out of them, compound 7 was further
confirmed by single crystal X-ray Crystallography (Fig. 1).
4
5
37
62
4172 ꢀ 367
Reduction of an isoflavone to a ring-opened product was ach-
ieved in dry THF by using LiAlH4 as reducing agent [30]. The a-
methyldeoxybenzoins 6 and 7 were therefore obtained from for-
mononetin and its methylated product, respectively (Scheme 2).
However, treatment of the flavone analogue with LiAlH4 is quite
different. The ring-opened products 16, 17 and 18 were given in a
one-pot reaction from 5,7-dimethylated chrysin (Scheme 2), which
is a reaction reported for the first time. The presence of chalcone 16
indicates that dihydrochalcone 17 may be the reduction product of
16. As for compound 18, we putatively attributed to the demethy-
lation of 16 followed by hydrogenation or/and direct demethylation
of 17 with the help of Al(OAr)3, a Lewis acid yielded in situ from
LiAlH4.
3791 ꢀ 207
6
46
62
40
63
79
37
1050 ꢀ 94
2178 ꢀ 112
3867 ꢀ 237
92.9 ꢀ 8.7
358 ꢀ 31
We subsequently turned to hydrogenate flavones and iso-
flavones in the presence of Pd/C for preparing 4-ol analogues.
Tangeretin and methylated formononetin gave the hydrogenated
products 3 and 8 respectively, with the reduction of both carbone
carbon double bond and carbonyl group. However, when this sys-
tem was extended to apigenin and quercetin, flavonoids bearing
free hydroxyl groups, only carbonecarbon double bond was
reduced [31], forming compounds 1 and 2 respectively (Scheme 3).
This clearly indicated that the hydrogenation degree of a flavonoid
is strongly dependent on the number of the hydroxyl groups that it
7
8
9
10
11
1783 ꢀ 119
Scheme 1. Molecular structures of some derivatives of flavonoid.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
Table 1 (continued )
687
Table 1 (continued )
Entry
Structure
Yield
IC50
,
m
M
Entry
Structure
Yield
IC50, mM
12
13
14
55
86.2 ꢀ 5.9
0.85 ꢀ 0.06
22.1 ꢀ 2.0
22
e
4628 ꢀ 215 [28]
42
49
23
24
25
e
e
e
2003 ꢀ 115 [28]
3527 ꢀ 237
556 ꢀ 31 [28]
15
16
17
18
19
51
18
43
26
91
4.42 ꢀ 0.31
1220 ꢀ 105
1668 ꢀ 132
978 ꢀ 87
26
e
140 ꢀ 3 [26]
27
28
e
291 ꢀ 29 [28]
e
35.4 ꢀ 2.0 [28]
29
e
2185 ꢀ 163
33.2 ꢀ 1.8
30
31
e
e
302 ꢀ 37 [28]
18.2 ꢀ 1.6
20
21
e
138 ꢀ 12 [28]
AHA
bears, which may be caused by the adsorption of a flavonoid on
the surface of Pd/C being decreased with the increase of the
number of hydroxyl groups. It was reported that a flavanone is
stable to hydrogenation under neutral conditions but can be further
reduced under alkaline conditions and pressure [32]. Triggered
by this finding, Pd(OH)2/C was selected as catalyst to generate
e
11 ꢀ 0.9 [28]

688
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
doublet signals at about
d 2.7 (H4) with J about 17.0 and 3.0 Hz.
The single crystal structure of 7 clearly indicated it is the
reductive ring opening product of methylated formononetin. In
the 1H NMR spectrum of 7, the doublet signals at
(J ¼ 6.7 Hz) were assigned to methyl protons and that at
d
1.49
4.59
d
(J ¼ 6.9 Hz) to CH. Based on this assignment, compound 6 was
surely determined as the reductive ring opening product of
formononetin.
2.3. Urease inhibitory activity
It is well known, flavonoids are typically composed of two
phenolic rings and a pyrone ring, which are referred to as A, B and C
rings (Scheme 8). In this work, we attempted to modify ring C for
possible improvements in urease inhibitory activity. All the syn-
thetic compounds were evaluated for their inhibitory activities
against cell-free urease from H. pylori (Table 1). Reduction of D2,3
carbonecarbon double bond yielded compounds with a 2- to 4-fold
loss of activity (1 vs. 20, 2 vs. 21). On the contrary, replacement of
the 4-carbonyl group in tangeretin 22 with a methylene group (5)
produced some improvement in potency for inhibition of H. pylori
urease. Whereas it was quite surprise that 3-flavenes (14, 15 and
19), another kind of 4-deoxy analogues, show a significant increase
in activity when compared to their parent compounds (baicalein 27,
luteolin 28 and chrysin 30). These results clearly indicated that
maintenance of the sp2 hybridized C3 is very important to improve
the urease inhibitory activity. In the series of 3-flavenes, compound
4 must be mentioned. It showed a much less increase in activity
than others, proving to be approximately equipotent to the parent
compound (22), which may be attributed to its extremely high
hydrophobicity. To further understand the importance of the sp2
hybridized C3 in isoflavone series, some isoflavanes and isoflavenes
were next synthesized from the reduction of the corresponding
isoflavones. Converting isoflavones (23, 24 and 25) to the corre-
sponding isoflavanes (10, 12 and 9) resulted in a 2- to 6-fold in-
crease in potency. However it is quite different to 2-isoflavenes, 11
and 13 get a significant improvement of activity. It is worthy to note
Fig. 1. Molecular structure of 7.
4-methylene derivatives. As expected, isoflavones daidzein, for-
mononetin, and methylated formononetin were successfully con-
verted to the isoflavanes 9, 10, and 11, respectively (Scheme 4).
Contrary to isoflavones, tangeretin, baicalein, luteolin and chrysin
failed to furnish the corresponding isoflavanes, but generated 3-
flavenes 4, 14, 15 and 19 (Scheme 5) irrespective of the number of
the bearing hydroxyl groups. To our knowledge, this is the first
method for building the 3-flavene structure from flavone, adding
the last member into the method spectra for preparing all possible
patterns of C-ring.
In order to obtaine 2-flavenes and 2-isoflavenes, tangeretin was
selected to treat with LiAlH4eAlCl3, and compound 5 was formed in
a yield of 52% (Scheme 6). Obviously, this reductive system is not
suitable for hydroxylated species. Thus, we developed an expedient
route to yield 12 and 13 from formononetin and 7,8,40-isoflavone
(Scheme 7) based on the method for preparing 1,2-diarylethanes
[13,33].
2.2. Structure description
that 2-isoflavene 13 with IC50 of 0.85 ꢀ 0.06
mM is over 160-fold
Compounds 4, 14, 15 and 19 were reported for the first time
and their structures were identified as 3-flavene through 1H NMR
more potent than its parent compound 26, being the most active
of all tested compounds. This contrasted sharply with that of 2-
flavene 5, which may be also caused by the difference of hydro-
phobicity. Consistent with our observation in naturally occurred
flavonoids, an appropriately hydroxylated flavonoid skeleton is a
key structural characteristic for an active compound [28], which
gives a reasonable explanation for 13 having much more potent
than 11. The above mentioned results also disclosed that replace-
ment of 4-carbonyl group with CH2 or CH group is beneficial to both
flavone and isoflavone for enhancement of urease inhibitory
spectra. The double doublet signals at about
d 5.16 to 5.59 were
assigned to H2 with J about 12.6 and 3.0 Hz, indicating the vicinal
coupling of H2eH3 and the long-range coupling of H2eH4. On
the other hand, H3 is adjacent to H2 and H4. The double doublet
signals at about d 3.0 with J about 17.0 and 12.7 Hz were therefore
attributed to H3. Furthermore, over 14 Hz of coupling constant
clearly suggests the cis-coupled protons of H3 and H4. The as-
signments made above were further confirmed from the double
Scheme 2. Formation of open-ring products from flavonoids.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
689
Scheme 3. Hydrogenation of some flavonoids by using Pd/C as catalyst.
activity. This hypothesis was indirectly confirmed by the activities
of 4-flavonol 3 and 4-isoflavonol 8 with no improvement as
compared to their parent compounds.
especially the synthetic analogues of flavonoids such as catechol
derivatives [27], has not been done so far. To gain information on the
inhibition mechanism of 4-deoxyflavonoids, the most potent in-
hibitor 13 was therefore selected for a kinetics study. The type of
inhibition was elucidated from the analysis of LineweavereBurk
plots, in which Km was decreased in the presence of 13 leaving un-
affected Vmax (Fig. 2). This pattern indicated a pure competitive in-
hibition, which means that compound 13 competes for the same
binding site as the substrate urea. Unexpectively, this is in contrast
with the non-competitive inhibition mechanism of quercetin,
revealing that removal of 4-oxygen might cause the change of
mechanism. The Ki value was calculated directly from Dixon plots
(Fig. 3), and was confirmed by a plotting of the slopes of the
LineweavereBurk plot vs. the concentration of 13 (Fig. 4) [36].
With the respect to the reductive ring opening products of fla-
vonoids,
a-methyldeoxybenzoins (6 and 7), chalcone (16) and
dihydrochalcone (17) displayed only a slight increase in potency.
2.4. Kinetics of urease inhibition by 2-isoflavene 13
In the past ten years, several natural flavonoids were documented
as potent urease inhibitors, including flavones [24], isoflavones [34],
C-glycosylflavonoids [35] and flavonoid glycosides [23]. Although
quercetin, a typical flavone, has recently been determined as a
reversible inhibitor of H. pylori urease with a non-competitive inhi-
bition mechanism by our group [28], the inhibition mechanism
of other flavonoids such as isoflavone and C-glycosylflavonoid,
The obtained Ki value was 0.146
mg/mL (0.641 mM), demonstrating
the strong affinity of 13 towards H. pylori urease.
Scheme 4. Hydrogenation of isoflavones by using Pd(OH)2/C as catalyst.

690
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
Scheme 5. Hydrogenation of flavones by using Pd(OH)2/C as catalyst.
ꢀ
2.5. Molecular docking
atom of the carboxyl group of Asp165 (distance ¼ 2.184 A). The
binding pose of 13 is therefore stabilized through the attachment of
ring B to the protein backbone. The above mentioned hydrogen
bond network strongly indicated that a high affinity of 13 to the
target enzyme is acquired, and rationalizes its potent activity
observed in biological assays.
In an effort to elucidate the competitive inhibition mechanism
revealed by the kinetics study, molecular docking of the most active
compound (13) into the crystal structure of H. pylori urease (entry
1E9Y in the Protein Data Bank) was performed by the AutoDock
program, and the best possible binding modes are shown in Fig. 5.
As expected by the LineweavereBurk plots, 2-isoflavene (13) is
nicely bound to the region of the urea binding pocket. In detail, the
compound is oriented with its benzopyran moiety in proximity to
urea binding cavity, letting ring B locate at the mouth of the cavity.
The channel to the active site for urea is therefore blocked off
(Fig. 6). The 7-hydroxyl group of 13 strongly interacts with the
enzyme, providing the main binding affinity for ligandeurease
complex. The O atom as acceptor receives two strong hydrogen
bonding interactions from the NH2 groups of Arg338 at distances of
3. Conclusion
In summary, we established a structureeactivity relationship
between nineteen reductive derivatives of flavonoids and their
inhibitory activity against H. pylori urease. Briefly, 4-deoxy ana-
logues are more potent than other reductive products, with
3-flavenes and 2-isoflavenes being the most active in their own
series. Among the assayed compounds, 2-isoflavene 13 with IC50 of
0.85 ꢀ 0.06
mM is the most active, showing over 20-fold more
ꢀ
ꢀ
1.915 A and 1.947 A, as well as a relative weak interaction from NH
potent than the positive control, acetohydroxamic acid (AHA). Ki-
netics studies revealed that the most active compound 13 is a
reversible inhibitor with a pure competitive inhibition mechanism,
which is rationalized by the molecular docking study. The high
potency and unequivocal inhibition mechanism of 13 against
H. pylori urease suggests that this compound deserves to be further
researched as a good candidate to treat gastritis and gastric ulcer.
ꢀ
group of His322 at a distance of 3.494 A. This hydroxyl group, on the
other hand, is used as a donor, forming OeH/N and OeH/O
hydrogen bonding interactions with His248 and Glu222 respec-
tively. In addition, 8-hydroxyl group hydrogen bonds to the O atom
ꢀ
of the carboxyl group of Asp223 (distance ¼ 2.220 A) and to the NH
ꢀ
in imidazole ring of His322 (distance ¼ 1.983 A), which further
anchors 13 at the active site. It is worth of note that ring B is also of
primary importance for the interactions network of compound 13.
Two strong hydrogen bonds are observed between 40-hydroxyl
4. Experimental section
ꢀ
group and the NH2 of Asn168 (distance ¼ 2.121 A) as well as the O
4.1. Materials
Protease inhibitors (Complete mini EDTA-free) were purchased
from Roche Diagnostics GmbH (Mannheim, Germany) and Brucella
broth was from BectoneDickinson (Cockeysville, MD). Horse serum
was from Hyclone (Utah, American).
4.2. Bacteria
H. pylori (ATCC 43504; American Type Culture Collection,
Scheme 6. Reduction of tangeretin by LiAlH4eAlCl3 system.
Manassas, VA) was grown in Brucella broth supplemented with 10%

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
691
Scheme 7. Reduction of formononetin and 40,7,8-isoflavone by NaBH4.
heat-inactivated horse serum for 24 h at 37 ꢁC under microaerobic
4.5. Measurement of urease inhibitory activity
conditions (5% O2, 10% CO2, and 85% N2), as our previously
described [26e28].
The assay mixture, containing 25
mL (10 U) of H. pylori urease
and 25 L of the test compound, was pre-incubated for 1.5 h at
m
room temperature in a 96-well assay plate. Urease activity was
determined by measuring ammonia production using the indo-
phenol method as described by Weatherburn [38].
4.3. Preparation of H. pylori urease
For urease inhibition assays, 50 mL broth cultures
(2.0 ꢂ 108 CFU/mL) were centrifuged (5000 g, 4 ꢁC) to collect the
bacteria, and after washing twice with phosphate-buffered saline
(pH 7.4), the H. pylori precipitation was stored at ꢃ80 ꢁC. H. pylori
was returned to room temperature, and after addition of 3 mL of
distilled water and protease inhibitors, sonication was performed
for 60 s. Following centrifugation (15,000 g, 4 ꢁC), the supernatant
was desalted through Sephadex G-25 column (PD-10 columns,
Amersham Pharmacia Biotech, Uppsala, Sweden). The resultant
crude urease solution was added to an equal volume of glycerol and
stored at 4 ꢁC until use in the experiment.
4.6. Kinetics study
LineweavereBurk plots of 1/absorbance vs. 1/urea were used to
determine the type of enzyme inhibition. Urease inhibition was
measured by varying the concentration of urea in the presence of
different concentrations of 13. Inhibitory constants (Ki) were
determined as the intersection on the X-axis of the plots of the
slopes vs. different concentrations of inhibitor, in which the slopes
obtained from the LineweavereBurk lines, or directly measured
from Dixon plots as the intersection of four lines. All experiments
were conducted in triplicate.
4.4. Urease activity determination
4.7. Protocol of docking study
One unit of urease activity is defined as the amount of enzyme
needed to liberate 1.0 mmol of NH3 from urea per min at pH 7.0 at
The automated docking studies were carried out using Auto-
Dock version 4.2. First, AutoGrid component of the program
25 ꢁC. According to the enzymatic assays of SigmaeAldrich [37], the
activity of the obtained H. pylori urease was determined. Briefly,
1 mL of 0.5 M urea with 0.05% (w/v) bovine serum albumin solution
was pipetted into a test tube and warmed to 25 ꢁC. Then, 0.20 mL of
urease solution containing 0.02 M sodium phosphate buffer (pH 7.0
at 25 ꢁC) was added. The obtained mixture was incubated at 25 ꢁC
for exactly 5 min with magnetic stirring, and followed by addition
of 0.20 mL of indicator (4.0 mg/mL of p-nitrophenol solution),
which was titrated immediately with 0.1 M standardized HCl until
the colour turns from yellow to colourless. As for blank, only dif-
ference is that the urease solution was added after incubation. The
urease activity was calculated based on the following formula:
concentration of
70
60
50
40
30
20
10
0
compound 13
19.5
9.77
4.88
2.44
µg•
µg•
µg•
µg•
mL-1
mL-1
mL-1
mL-1
0:1 mmol=mL ꢂ
D
VHClmL ꢂ 1000
m
mol=mmol
U=mL urease ¼
5 min ꢂ 0:2 mL
-0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
1/urea mM-1
-10
Fig. 2. Double-reciprocal LineweavereBurk plot of the inhibition of the H. pylori urease
activity by compound 13.
Scheme 8. The three-ring structure of flavonoid.

692
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
Fig. 5. Binding mode of compound 13 with H. pylori urease. For clarity, only interacting
residues were labelled. Hydrogen bonding interactions are shown in dash. The figure
was made using PyMol.
root-mean-square deviation (RMSD) were clustered together and
the results of the most favourable free energy of binding were
selected as the resultant complex structures.
Fig. 3. The Dixon plot of the reciprocal of the initial velocities vs. various concentra-
tions of compound 13 at fixed substrate concentrations.
pre-calculates a three-dimensional grid of interaction energies
4.8. Crystallographic studies
based on the macromolecular target using the AMBER force field.
ꢀ
ꢀ
The cubic grid box of 62 A size (x, y, z) with a spacing of 0.375 A and
grid maps were created representing the catalytic active target site
region where the native ligand was embedded. Then automated
docking studies were carried out to evaluate the binding free en-
ergy of the inhibitor within the macromolecules. The GALS search
algorithm (genetic algorithm with local search) was chosen to
search for the best conformers. The parameters were set using the
software 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 50 randomly placed individuals, a maximum
number of 2.5 ꢂ106 energy evaluations, and a maximum number of
2.7 ꢂ 104 generations. A mutation rate of 0.02 and a crossover rate
X-ray single-crystal diffraction data for compound 7 was
collected on a Bruker SMART APEX CCD diffractometer at 296(2) K
ꢀ
using MoK
a
radiation (
l
¼ 0.71073 A) by the
u scan mode. The
program SAINT was used for integration of the diffraction profiles.
All the structures were solved by direct methods using the SHELXS
program of the SHELXTL package and refined by full-matrix least-
squares methods with SHELXL [39]. All non-hydrogen atoms of
compound 7 were refined with anisotropic thermal parameters. All
hydrogen atoms were generated theoretically onto the parent
atoms and refined isotropically with fixed thermal factors.
4.9. Chemistry
ꢀ
of 0.8 were chosen. Results differing by less than 0.5 A in positional
All chemicals (reagent grade) used were purchased from Aldrich
(U.S.A) and Sinopharm Chemical Reagent Co., Ltd (China). Melting
points (uncorrected) were determined on an XT4 MP apparatus
(Taike Corp., Beijing, China). EI mass spectra were obtained on a
Waters GCT mass spectrometer, and 1H NMR spectra were recorded
Fig. 4. The secondary replot of the LineweavereBurk plot, slopes of the Lineweavere
Burk plot vs. various concentrations of compound 13.
Fig. 6. Binding mode of compound 13 with H. pylori urease. The enzyme is shown as
surface; while 13 docked structures are shown as sticks.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
693
on a Bruker AV-300 spectrometer at 25 ꢁC with TMS and solvent
signals allotted as internal standards. Chemical shifts were reported
in ppm (d). Elemental analyses were performed on a CHN-O-Rapid
(dd, J ¼ 12.8 Hz, J ¼ 2.9 Hz, 1H); 5.87 (s, 2H); 7.14e7.25 (m, 5H);
EIMS m/z 256 (Mþ). Anal. Calcd for C15H12O4: C, 70.31; H, 4.72;
Found: C, 70.25; H, 4.73.
instrument and were within ꢀ0.4% of the theoretical values.
4.9.2.3. 30,40,5,7-Tetrahydroxy-3-flavene (15). Light yellow powder,
yield 51%, mp 224e226 ꢁC, 1H NMR (CDCl3): 2.62 (dd, J ¼ 17.0 Hz,
J ¼ 3.0 Hz, 1H); 2.76 (dd, J ¼ 17.0 Hz, J ¼ 12.6 Hz, 1H); 5.19 (dd,
J ¼ 12.8 Hz, J ¼ 2.8 Hz, 1H); 5.82 (d, J ¼ 1.8 Hz, 1H); 5.99 (d,
J ¼ 2.0 Hz, 1H); 6.04 (s, 1H); 6.53 (d, J ¼ 8.4 Hz, 1H); 6.88 (dd,
J ¼ 8.6 Hz, J ¼ 2.2 Hz, 1H); 6.96 (d, J ¼ 1.8 Hz, 1H); 7.58 (s, 1H); EIMS
m/z 272 (Mþ). Anal. Calcd for C15H12O5: C, 66.17; H, 4.44; Found: C,
66.08; H, 4.45.
4.9.1. General procedure for the preparation of compounds 1e3 and
8
To a solution of flavonoid (4 mmol) in dry tetrahydrofuran
(15 mL) or N,N-dimethylformamide (15 mL) was added 20e400 mg
of 10% Pd/C in a hydrogen atmosphere at room temperature. The
resulted mixture was stirred for 3e6 h, and the catalyst was
removed by filtration. Concentration under reduced pressure fur-
nished a residue, which was purified over a silica gel column
eluting with EtOAcepetroleum ether.
4.9.2.4. 5,7-Dihydroxy-3-flavene (19). White powder, yield 91%, mp
199e200 ꢁC, 1H NMR (DMSO-d6): 2.78 (dd, J ¼ 17.2 Hz, J ¼ 3.2 Hz,
1H); 3.26 (dd, J ¼ 17.0 Hz, J ¼ 12.6 Hz, 1H); 5.59 (dd, J ¼ 12.6 Hz,
J ¼ 2.7 Hz,1H); 5.90 (d, J ¼ 1.8 Hz,1H); 5.93 (d, J ¼ 2.0 Hz,1H); 7.36e
7.47 (m, 3H); 7.52 (d, J ¼ 6.6 Hz, 2H); 10.83 (s, 1H); 12.13 (s, 1H);
EIMS m/z 240 (Mþ). Anal. Calcd for C15H12O3: C, 74.99; H, 5.03;
Found: C, 74.92; H, 5.03.
4.9.1.1. 40,5,7-Trihydroxydihydroflavanone (1). White powder, yield
91%, mp 222e224 ꢁC, 1H NMR (DMSO-d6): 4.01 (t, J ¼ 6.6 Hz, 1H);
4.53 (d, J ¼ 6.6 Hz, 2H); 5.89 (d, J ¼ 1.5 Hz, 2H); 6.72 (d, J ¼ 8.6 Hz,
2H); 7.07 (d, J ¼ 8.4 Hz, 2H); 9.41 (s, 1H); 10.84 (s, 1H); 12.19 (s, 1H);
EIMS m/z 272 (Mþ). Anal. Calcd for C15H12O5: C, 66.17; H, 4.44;
Found: C, 66.22; H, 4.43.
4.9.2.5. 40,7-Dihydroxyflavane (9). White powder, yield 63%, mp
157e159 ꢁC, 1H NMR (CDCl3): 2.62 (d, J ¼ 9.3 Hz, 2H); 2.77e2.89 (m,
1H); 3.65 (t, J ¼ 10.4 Hz, 1H); 5.90 (dd, J ¼ 10.4 Hz, J ¼ 2.9 Hz, 1H);
6.07 (d, J ¼ 2.4 Hz, 1H); 6.12 (dd, J ¼ 8.2 Hz, J ¼ 2.4 Hz, 1H); 6.55 (d,
J ¼ 8.4 Hz, 2H); 6.61 (d, J ¼ 8.0 Hz, 1H); 6.79 (d, J ¼ 8.4 Hz, 2H); EIMS
m/z 242 (Mþ). Anal. Calcd for C15H14O3: C, 74.36; H, 5.82; Found: C,
74.43; H, 5.81.
4.9.1.2. 3,30,40,5,7-Pentahydroxyflavanone (2). White powder, yield
64%, mp over 300 ꢁC, 1H NMR (CDCl3): 4.26 (d, J ¼ 11.6 Hz, 1H); 4.71
(d, J ¼ 11.0 Hz, 1H); 5.86 (d, J ¼ 1.8 Hz, 1H); 6.02 (d, J ¼ 2.0 Hz, 1H);
6.56 (d, J ¼ 8.6 Hz, 1H); 7.25 (dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz, 1H); 7.39 (d,
J ¼ 2.0 Hz, 1H); 8.15 (s, 1H); 9.79 (s, 1H); 11.84 (s, 1H); EIMS m/z 304
(Mþ). Anal. Calcd for C15H12O7: C, 59.21; H, 3.98; Found: C, 59.35;
H, 3.97.
4.9.2.6. 7-Hydroxy-40-methoxyflavane (10). White powder, yield
79%, mp 158e160 ꢁC, 1H NMR (CDCl3): 2.93 (d, J ¼ 8.6 Hz, 2H); 3.11e
3.23 (m, 1H); 3.81 (s, 3H); 3.96 (t, J ¼ 10.4 Hz, 1H); 4.29 (dd,
J ¼ 10.4 Hz, J ¼ 2.9 Hz, 1H); 5.04 (s, 1H); 6.36 (d, J ¼ 2.2 Hz, 1H); 6.39
(dd, J ¼ 8.0 Hz, J ¼ 2.4 Hz, 1H); 6.89 (d, J ¼ 8.4 Hz, 2H); 6.93 (d,
J ¼ 8.0 Hz, 1H); 7.16 (d, J ¼ 8.6 Hz, 2H); EIMS m/z 256 (Mþ). Anal.
Calcd for C16H16O3: C, 74.98; H, 6.29; Found: C, 74.87; H, 6.30.
4.9.1.3. 40,5,6,7,8-Pentamethoxy-4-flavanol (3). White powder, yield
44%, mp 132e133 ꢁC, 1H NMR (CDCl3): 1.94e2.04 (m, 1H); 2.74e
2.83 (m, 2H); 3.77 (s, 3H); 3.84 (s, 6H); 3.86 (s, 3H); 3.93 (s, 3H);
4.96 (d, J ¼ 8.0 Hz, 1H); 6.91 (d, J ¼ 8.6 Hz, 2H); 7.34 (d, J ¼ 8.6 Hz,
2H); EIMS m/z 376 (Mþ). Anal. Calcd for C20H24O7: C, 63.82; H, 6.43;
Found: C, 63.71; H, 6.44.
4.9.1.4. 40,7-Dimethoxy-4-flavanol (8). White powder, yield 40%,
mp 125e127 ꢁC, 1H NMR (CDCl3): 2.34e2.38 (m, 1H); 3.09 (d,
J ¼ 8.3 Hz, 2H); 3.79 (d, J ¼ 8.0 Hz, 1H); 5.92 (d, J ¼ 1.8 Hz, 1H); 6.65
(d, J ¼ 8.4 Hz, 1H); 6.88 (d, J ¼ 8.6 Hz, 2H); 7.16 (dd, J ¼ 8.4 Hz,
J ¼ 2.0 Hz, 1H); 7.30 (d, J ¼ 8.6 Hz, 2H); EIMS m/z 286 (Mþ). Anal.
Calcd for C17H18O4: C, 71.31; H, 6.34; Found: C, 71.39; H, 6.34.
4.9.2.7. 40,7-Dimethoxyflavane (11). White powder, yield 37%, mp
116e118 ꢁC, 1H NMR (CDCl3): 2.75 (d, J ¼ 8.4 Hz, 2H); 3.06e3.18 (m,
1H); 3.69 (s, 3H); 3.73 (s, 3H); 3.91 (t, J ¼ 10.4 Hz, 1H); 4.31 (dd,
J ¼ 10.6 Hz, J ¼ 2.8 Hz, 1H); 6.25 (d, J ¼ 2.0 Hz, 1H); 6.30 (dd,
J ¼ 8.2 Hz, J ¼ 2.2 Hz,1H); 6.84 (d, J ¼ 8.4 Hz, 2H); 6.93 (d, J ¼ 8.0 Hz,
1H); 7.19 (d, J ¼ 8.4 Hz, 2H); EIMS m/z 270 (Mþ). Anal. Calcd for
C17H18O3: C, 75.53; H, 6.71; Found: C, 75.46; H, 6.72.
4.9.2. General procedure for the preparation of compounds 4, 14, 15,
19, 9, 10 and 11
4.9.3. Preparation of 40,5,6,7,8-pentamethoxy-2-flavene (5)
To a solution of flavonoid (4 mmol) in dry tetrahydrofuran
(15 mL) or N,N-dimethylformamide (15 mL) was added 100 mg of
20% Pd(OH)2/C in a hydrogen atmosphere at room temperature. The
resulted mixture was stirred for 4e8 h, and the catalyst was
removed by filtration. Concentration under reduced pressure fur-
nished a residue, which was purified over a silica gel column
eluting with EtOAcepetroleum ether.
A solution of tangeretin (1.49 g, 4 mmol) in dry tetrahydrofuran
(20 mL) was dropped during 20 min into a stirred solution of
aluminium chloride (1.6 g, 12 mmol) and lithium aluminium with
hydride (0.23 g, 6 mmol) in dry tetrahydrofuran (25 mL) at 0 ꢁC.
Tetrahydrofuran was removed under reduced pressure, then the
obtained residue was decomposed with wet EtOAc followed by
dropwise addition of water on an ice-water bath. The mixture was
extracted with EtOAc. After removal of the solvent, the resulting
residue was purified over a silica gel column eluting with EtOAce
petroleum ether (V:V ¼ 1:12), and gave 5 as colourless crystal in a
yield of 62%. Mp 137e138 ꢁC, 1H NMR (DMSO-d6): 3.38 (d, J ¼ 3.7 Hz,
2H); 3.76 (s, 3H); 3.78 (s, 6H); 3.83 (s, 6H); 5.60 (t, J ¼ 3.5 Hz, 1H);
6.99 (d, J ¼ 8.8 Hz, 2H); 7.63 (d, J ¼ 8.8 Hz, 2H); EIMS m/z 358 (Mþ).
Anal. Calcd for C20H22O6: C, 67.03; H, 6.19; Found: C, 66.94; H, 6.20.
4.9.2.1. 40,5,6,7,8-Pentamethoxy-3-flavene (4). White powder, yield
37%, mp 109e110 ꢁC, 1H NMR (CDCl3): 2.83 (dd, J ¼ 16.7 Hz,
J ¼ 2.9 Hz, 1H); 3.03 (dd, J ¼ 16.7 Hz, J ¼ 12.8 Hz, 1H); 3.83 (s, 6H);
3.85 (s, 3H); 3.90 (s, 3H); 4.05 (s, 3H); 5.38 (dd, J ¼ 12.8 Hz,
J ¼ 2.7 Hz, 1H); 6.94 (d, J ¼ 8.6 Hz, 2H); 7.39 (d, J ¼ 8.4 Hz, 2H); EIMS
m/z 358 (Mþ). Anal. Calcd for C20H22O6: C, 67.03; H, 6.19; Found: C,
67.16; H, 6.18.
4.9.4. General procedure for the preparation of compounds 6, 7, 16,
17 and 18
A solution of flavonoid (5 mmol) in dry tetrahydrofuran
(20 mL) was dropped into a stirred solution of lithium aluminium
4.9.2.2. 40,5,6,7,8-Pentamethoxy-3-flavene (14). Light yellow pow-
der, yield 49%, mp 149e151 ꢁC, 1H NMR (CDCl3): 2.55 (dd,
J ¼ 17.0 Hz, J ¼ 3.1 Hz,1H); 2.83 (dd, J ¼ 17.2 Hz, J ¼ 12.8 Hz,1H); 5.15

694
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
hydride (10 mmol) in dry tetrahydrofuran (20 mL) at room tem-
perature. Concentration under reduced pressure furnished a res-
idue, which was decomposed with wet EtOAc followed by
dropwise addition of water on an ice-water bath. The mixture was
extracted with EtOAc. After removal of the solvent, the resulting
residue was purified over a silica gel column eluting with EtOAce
petroleum ether.
Acknowledgements
The work was financed by grants from National Natural Science
Foundation (grant Nos. 81273382) of China, by a Project supported
by Hunan Provincial Natural Science Foundation of China (grant No.
11JJ3113), by a grant from National Natural Science Foundation
(Youth Science Funds, No. 31100421) of China and by aid program
for Science and Technology Innovative Research Team (Chemicals
of Forestry Resources and Development of Forest Products) in
Higher Educational Institutions of Hunan Province.
4.9.4.1. 2,4-Dihydroxyl-40-methoxyl-
a
-methyldeoxybenzoin
(6).
White powder, yield 46%, mp 86e88 ꢁC, 1H NMR (CDCl3): 1.49 (d,
J ¼ 6.8 Hz, 3H); 3.76 (s, 3H); 4.59 (q, J ¼ 6.6 Hz, 1H); 6.30 (dd,
J ¼ 9.0 Hz, J ¼ 2.0 Hz, 1H); 6.34 (d, J ¼ 2.0 Hz, 1H); 6.84 (d, J ¼ 8.6 Hz,
2H); 7.21 (d, J ¼ 8.4 Hz, 2H); 7.69 (d, J ¼ 8.8 Hz, 1H); 12.89 (s, 1H);
EIMS m/z 272 (Mþ). Anal. Calcd for C16H16O4: C, 70.57; H, 5.92;
Found: C, 70.53; H, 5.92.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.03.016.
4.9.4.2. 2-Hydroxyl-4,40-dimethoxyl-
a
-methyldeoxybenzoin
(7).
References
Colourless crystal, yield 62%, mp 74e75 ꢁC, 1H NMR (CDCl3): 1.50 (d,
J ¼ 6.8 Hz, 3H); 3.76 (s, 3H); 3.79 (s, 3H); 4.59 (q, J ¼ 6.8 Hz, 1H);
6.35 (dd, J ¼ 8.8 Hz, J ¼ 2.0 Hz, 1H); 6.38 (d, J ¼ 2.0 Hz, 1H); 6.84 (d,
J ¼ 8.4 Hz, 2H); 7.21 (d, J ¼ 8.4 Hz, 2H); 7.69 (d, J ¼ 9.0 Hz, 1H); 12.92
(s, 1H); EIMS m/z 286 (Mþ). Anal. Calcd for C17H18O4: C, 71.31; H,
6.34; Found: C, 71.38; H, 6.33.
[1] M.A.S. Aslama, S. Mahmood, M. Shahid, A. Saeed, J. Iqbal, Eur. J. Med. Chem. 46
(2011) 5473e5479.
[2] M. Arfan, H. Amin, I. Khan, M.R. Shah, H. Shah, A.Z. Khan, S.M.A. Halimi,
N. Khan, W.A. Kaleem, M. Qayum, A. Shahidullah, M.A. Khan, Med. Chem. Res.
21 (2012) 2454e2457.
[3] K.S.T. Ramsay, P. Wafo, Z. Ali, A. Khan, O.O. Oluyemisi, B.P. Marasini, I.A. Khan,
N.T. Bonaventure, M.I. Choudhary, Atta-ur-Rahman, Fitoterapia 83 (2012)
204e208.
[4] B. Krajewska, R. van Eldik, M. Brindell, J. Biol. Inorg. Chem. 17 (2012) 1123e1134.
[5] q. Berlicki, M. Bochno, A. Grabowiecka, A. Bia1as, P. Kosikowska, P. Kafarski,
Amino Acids 42 (2012) 1937e1945.
[6] Z.-L. You, D.-H. Shi, J.-C. Zhang, Y.-P. Ma, C. Wang, K. Li, Inorg. Chim. Acta 384
(2012) 54e61.
[7] D.C. Menezes, E. Borges, M.F. Torres, J.P. Braga, Chem. Phys. Lett. 548 (2012)
85e89.
[8] K. Kobashi, J. Hase, K. Uehare, Biochim. Biophys. Acta 65 (1962) 380e383.
[9] B. Krajewska, J. Mol. Catal. B Enzymatic 59 (2009) 9e21.
[10] Z. Amtul, Atta-ur-Rahman, R.A. Siddiqui, M.I. Choudhary, Curr. Med. Chem. 9
(2002) 1323e1348.
[11] M. Kot, W. Zaborska, K. Orlinska, J. Enzym. Inhib. Med. Chem. 16 (2001)507e516.
[12] M.J. Dominguez, C. Sanmartin, M. Font, J.A. Palop, S. San Francisco, O. Urrutia,
F. Houdusse, J. Garcia-Mina, J. Agric. Food Chem. 56 (2008) 3721e3731.
[13] Z.-P. Xiao, T.-W. Ma, W.-C. Fu, X.-C. Peng, A.-H. Zhang, H.-L. Zhu, Eur. J. Med.
Chem. 45 (2010) 5064e5070.
4.9.4.3. 2-Hydroxyl-4,6-dimethoxylchalcone (16). Light yellow pow-
der, yield 18%, mp 85e88 ꢁC, 1H NMR (CDCl3): 3.84 (s, 3H); 3.92 (s,
3H); 5.97 (d, J ¼ 2.2 Hz, 1H); 6.12 (d, J ¼ 2.4 Hz, 1H); 7.38e7.41 (m,
3H); 7.59e7.62 (m, 2H); 7.78 (d, J ¼ 15.6 Hz, 1H); 7.91 (d, J ¼ 15.8 Hz,
1H); 14.29 (s, 1H); EIMS m/z 284 (Mþ). Anal. Calcd for C17H16O4: C,
71.82; H, 5.67; Found: C, 71.76; H, 5.68.
4.9.4.4. 2-Hydroxyl-4,6-dimethoxyldihydrochalcone (17). White pow-
der, yield 43%, mp 108e110 ꢁC, 1H NMR (CDCl3): 2.99 (t, J ¼ 7.3 Hz,
2H); 3.32 (t, J ¼ 7.3 Hz, 2H); 3.82 (s, 3H); 3.83 (s, 3H); 5.92 (d,
J ¼ 2.2 Hz, 1H); 6.07 (d, J ¼ 2.2 Hz, 1H); 7.18e7.33 (m, 5H); 14.02 (s,
1H); EIMS m/z 286 (Mþ). Anal. Calcd for C17H18O4: C, 71.31; H, 6.34;
Found: C, 71.37; H, 6.33.
[14] F. von Nussbaum, M. Brands, B. Hinzen, S. Weigand, D. Häbich, Angew. Chem.
Int. Ed. 45 (2006) 5072e5129.
[15] F.E. Koehn, G.T. Carter, Nat. Rev. Drug Discov. 4 (2005) 206e220.
[16] D.J. Newman, G.M. Cragg, K.M. Snader, J. Nat. Prod. 66 (2003) 1022e1037.
[17] S. Firdous, N.H. Ansari, I. Fatima, A. Malik, N. Afza, L. Iqbal, M. Lateef, Arch.
Pharm. Res. 35 (2012) 1133e1137.
[18] N. Brigitte, T.S. Valerie, V.J. Catherine, S.A. Muhammad, L. Mehreen, I. Lubna,
A. Nigaht, N.A. Ephrem, J. Enzyme Inhib. Med. Chem. (2012), http://dx.doi.org/
10.3109/14756366.2012.719025.
4.9.4.5. 2,6-Dihydroxyl-4-methoxyldihydrochalcone
(18). White
powder, yield 26%, mp 172e173 ꢁC, 1H NMR (DMSO-d6): 2.89 (t,
J ¼ 7.9 Hz, 2H); 3.29 (t, J ¼ 7.9 Hz, 2H); 3.74 (s, 3H); 5.96 (s, 2H);
7.16e7.31 (m, 5H); 12.33 (s, 1H); EIMS m/z 272 (Mþ). Anal. Calcd for
C
16H16O4: C, 70.57; H, 5.92; Found: C, 70.49; H, 5.93.
[19] Z.-P. Xiao, Z.-Y. Peng, M.-J. Peng, W.-B. Yan, Y.-Z. Ouyang, H.-L. Zhu, Mini-rev.
Med. Chem. 11 (2011) 169e177.
4.9.5. General procedure for the preparation of compounds 12 and 13
To a stirred solution of flavonoid (5 mmol) in ethanol (30 mL)
was added sodium borohydride (10 mmol) at 0 ꢁC in a nitrogen
atmosphere. After decomposition with dropwise addition of water,
the mixture was extracted with EtOAc. After removal of the solvent,
the resulting residue was purified over a silica gel column eluting
with EtOAcepetroleum ether.
[20] N.C. Cook, S. Samman, J. Nutr. Biochem. 7 (1996) 66e76.
[21] R. Slimestad, T. Fossen, I.M. Vågen, J. Agric. Food Chem. 55 (2007) 10067e10080.
[22] S. Matsubara, H. Shibata, F. Ishikawa, T. Yokokura, M. Takahashi, T. Sugimura,
Biochem. Biophys. Res. Commun. 310 (2003) 715e719.
[23] S. Shabana, A. Kawai, K. Kai, K. Akiyama, H. Hayashi, Biosci. Biotechnol. Bio-
chem. 74 (2010) 878e880.
[24] M. Ahmad, N. Muhammad, M. Ahmad, M. Arif Lodhi, N. Jehan, Z. Khan,
R. Ranjit, F. Shaheen, M.I. Choudhary, J. Enzym. Inhib. Med. Chem. 23 (2008)
386e390.
4.9.5.1. 7-Hydroxyl-40-methoxyl-2-isoflavene (12). White powder,
yield 55%, mp 185e187 ꢁC, 1H NMR (DMSO-d6): 3.77 (s, 3H); 4.92 (s,
2H); 6.11 (d, J ¼ 2.4 Hz, 1H); 6.27 (dd, J ¼ 8.2 Hz, J ¼ 2.4 Hz, 1H); 6.41
(d, J ¼ 8.0 Hz, 1H); 6.73 (s, 1H); 6.81 (d, J ¼ 8.6 Hz, 2H); 7.35 (d,
J ¼ 8.6 Hz, 2H); 8.46 (s, 1H); EIMS m/z 254 (Mþ). Anal. Calcd for
[25] A. Muhammad, I. Anis, A. Khan, B.P. Marasini, M.I. Choudhary, M.R. Shah, Arch.
Pharm. Res. 35 (2012) 431e436.
́
́
[26] Z.-P. Xiao, D.-H. Shi, H.-Q. Li, L.-N. Zhang, C. Xu, H.-L. Zhu, Bioorg. Med. Chem.
́
15 (2007) 3703e3710.
[27] H.-Q. Li, Z.-P. Xiao, Y. Luo, T. Yan, P.-C. Lv, H.-L. Zhu, Eur. J. Med. Chem. 44
(2009) 2246e2251.
[28] Z.-P. Xiao, X.-D. Wang, Z.-Y. Peng, S. Huang, P. Yang, Q.-S. Li, L.-H. Zhou, X.-J. Hu,
L.-J. Wu, Y. Zhou, H.-L. Zhu, J. Agric. Food Chem. 60 (2012) 10572e10577.
[29] Z.-P. Xiao, F. Zhang, Y.-C. Wang, S.-F. Yi, X.-C. Peng, H.-L. Zhu, Chin. J. Struct.
Chem. 30 (2011) 330e335.
[30] A. Salakka, K. Wahala, J. Chem. Soc. Perkin Trans. I (Org. Biomol. Chem.) 18
(1999) 2601e2604.
[31] K. Pihlaja, P. Tähtinen, K.D. Klika, T. Jokela, A. Salakka, K. Wähälä, J. Org. Chem.
68 (2003) 6864e6869.
C
16H14O3: C, 75.57; H, 5.55; Found: C, 75.63; H, 5.54.
4.9.5.2. 40,7,8-Trihydroxyl-2-isoflavene (13). White powder, yield
42%, mp 202e205 ꢁC, 1H NMR (DMSO-d6): 4.99 (s, 2H); 6.32 (d,
J ¼ 8.0 Hz, 1H); 6.44 (d, J ¼ 8.0 Hz, 1H); 6.71 (s, 1H); 6.76 (d,
J ¼ 8.4 Hz, 2H); 7.32 (d, J ¼ 8.8 Hz, 2H); 8.37 (s,1H); 9.01 (s,1H); 9.55
(s, 1H); EIMS m/z 256 (Mþ). Anal. Calcd for C15H12O4: C, 70.31; H,
4.72; Found: C, 70.26; H, 4.73.
[32] H.G. Krishnamurty, S. Sathyanarayana, Synth. Commun. 19 (1989) 119e123.
[33] C.K. Lau, C. Dufresne, P.C. Bélanger, S. Piétré, J. Scheigetz, J. Org. Chem. 51
(1986) 3038e3043.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 63 (2013) 685e695
695
[34] E.-A. Bae, M.J. Han, D.-H. Kim, Planta Med. 67 (2001) 161e163.
[35] S. Perveen, A.M. El-Shafae, A. Al-Taweel, G.A. Fawzy, A. Malik, N. Afza, M. Latif,
L. Iqbal, J. Asian Nat. Prod. Res. 13 (2011) 799e804.
[37] Sigma. https://www.safcglobal.com/technical-documents/protocols/biology/
enzymatic-assay-of-urease-from-jack-beans.html, 1995.
[38] M.W. Weatherburn, Anal. Chem. 39 (1967) 971e974.
[36] K.-F. Chan, I.L.K. Wong, J.W.Y. Kan, C.S.W. Yan, L.M.C. Chow, T.H. Chan, J. Med.
Chem. 55 (2012) 1999e2014.
[39] G.M. Sheldrick, SHELXTL-97, Program for Crystal Structure Solution and
Refinement, University of Göttingen, Göttingen, Germany, 1997.