Synthesis, molecular docking and kinetic properties of β-hydroxy- β-phenylpropionyl-hydroxamic acids as Helicobacter pylori urease inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.07.047

Inhibition of urease results in Helicobacter pylori growth arrest in the stomach, promoting urease as promising targets for gastrointestinal ulcer therapy. Twenty hybrid derivatives of flavonoid scaffold and hydroxamic acid, β-hydroxy-β-phenylpropionylhydroxamic acids, were therefore synthesized and evaluated against H. pylori urease. Biological evaluation of these compounds showed improved urease inhibition exhibiting micromolar to mid-nanomolar IC₅₀ values. Most importantly, 3-(3-chlorophenyl)-3- hydroxypropionyl-hydroxamic acid (6g) exhibited high potency with IC ₅₀ of 0.083 ± 0.004 μM and K_i of 0.014 ± 0.003 μM, indicating that 6g is an excellent candidate to develop novel antiulcer agent. A mixture of competitive and uncompetitive mechanism was putatively proposed to understand the inconsistency between the crystallographic and kinetic studies for the first time, which is supported by our molecular docking studies.

Full text of DOI:10.1016/j.ejmech.2013.07.047

European Journal of Medicinal Chemistry 68 (2013) 212e221
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, molecular docking and kinetic properties of
b
-hydroxy-
b-
phenylpropionyl-hydroxamic acids as Helicobacter pylori urease
inhibitors
,
a
b
a
a
Zhu-Ping Xiao a , Zhi-Yun Peng , Jing-Jun Dong , Rui-Cheng Deng , Xu-Dong Wang ,
*
Hui Ouyang a, Pan Yang a, Juan He a, Yuan-Feng Wang a, Man Zhu a, Xiao-Chun Peng a,
,
c
,
a,b,
***
Wan-Xi Peng a
, Hai-Liang Zhu
**
a College of Chemistry and Chemical Engineering, 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
c School of Materials Science and Engineering, Central South University of Forestry and Technology, Changsha 410004, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of urease results in Helicobacter pylori growth arrest in the stomach, promoting urease as
promising targets for gastrointestinal ulcer therapy. Twenty hybrid derivatives of flavonoid scaffold and
hydroxamic acid, b-hydroxy-b-phenylpropionylhydroxamic acids, were therefore synthesized and eval-
uated against H. pylori urease. Biological evaluation of these compounds showed improved urease in-
hibition exhibiting micromolar to mid-nanomolar IC50 values. Most importantly, 3-(3-chlorophenyl)-3-
Received 31 March 2013
Received in revised form
3 July 2013
Accepted 5 July 2013
Available online 9 August 2013
hydroxypropionyl-hydroxamic acid (6g) exhibited high potency with IC50 of 0.083 ꢀ 0.004
mM and Ki
of 0.014 ꢀ 0.003
m
M, indicating that 6g is an excellent candidate to develop novel antiulcer agent. A
Keywords:
Hydroxamic acid
Helicobacter pylori urease
Inhibitor
mixture of competitive and uncompetitive mechanism was putatively proposed to understand the
inconsistency between the crystallographic and kinetic studies for the first time, which is supported by
our molecular docking studies.
Ó 2013 Elsevier Masson SAS. All rights reserved.
A mixture of competitive and uncompetitive
mechanism
1. Introduction
human population [7]. Most infected people are asymptomatic,
with moderate inflammation detectable only by biopsy and his-
Urease (urea amidohydrolase EC 3.5.1.5), a Ni-containing hy-
peractive metalloenzyme, is found in numerous bacteria, plants,
fungi, algae, and some invertebrates [1,2]. It accelerates the hy-
drolysis of urea to ammonia and carbon dioxide by at least 1014 over
the spontaneous reaction [3]. High concentration of ammonia
arising from the reaction, as well as the accompanying pH eleva-
tion, has important negative effects in the fields of medicine and
agriculture [4e6]. Helicobacter pylori is one of the most successful
human bacterial parasites, which colonizes more than half of the
tology. However, 15e20% of them would develop severe gastro-
duodenal pathologies, including stomach and duodenal ulcers,
adenocarcinomas and stomach lymphomas [4].
To colonize the gastroenteric tract, H. pylori dedicates several
genes to the biosynthesis of a cytosolic urease, which buffers the
pH of the bacterial immediate surroundings and creates a neutral
layer around its surface. H. pylori urease might cause damage to
the host cells through the production of ammonia, and help to
recruit neutrophils and monocytes in the inflamed mucosa and to
activate production of proinflammatory cytokines [8]. The
essential role of urease as a virulence factor is shown by the fact
that urease-defective H. pylori mutants cannot colonize the
stomach [9]. Therefore, strategies based on urease inhibition are
considered as a promising treatment for gastric and peptic ulcer
caused by H. pylori [10]. The great medicinal potential for urease
inhibitors is of high interest to researchers. Studies have been
performed on this field for over 60 years, and hundreds of
structures have been determined such as hydroxamic acids,
* Corresponding author. Tel.: þ86 743 8563911.
** Corresponding author. School of Materials Science and Engineering, Central
South University of Forestry and Technology, Changsha 410004, PR China. Tel.: þ86
73 85623301.
*** Corresponding author. State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, PR China. Tel.: þ86 25 8359 2572.
E-mail addresses: xiaozhuping2005@163.com (Z.-P. Xiao), pengwanxi@163.com
(W.-X. Peng), zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.047

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
213
Scheme 1. Reported urease inhibitors based on flavonoids.
phosphoramidates, urea derivatives, and polyphenols [11]. Out of
2. Result and discussions
them, phosphoramidates are the most active but they were not
marketed as drugs probably because of the rapid hydrolysis in the
low pH of gastric juice [12].
2.1. Chemistry
Hydroxamic acids are well known to form strong complexes
with a variety of transition metals, and their mechanism of inhi-
bition involves binding to the metal ions of the active site of en-
zymes. Hydroxamic acids have therefore attracted much attention
[13,14], and a large amount of hydroxamic acids were synthesized
as inhibitors against metalloenzymes such as urease [15e17], ma-
trix metalloproteinases [18], UDP-3-O-(R-3-hydroxymyristol)-N-
acetyl-glucosamine deacetylase [19] and histone deacetylase
(HDAC) [20]. The best studied urease inhibitor of hydroxamic acids
is acetohydroxamic acid (AHA), which was approved as Lithostat by
U.S. Food and Drug Administration in May, 1983. However, its
relatively moderate inhibitory activity requires rather large doses
(about 1000 mg/day for adults) [12]. Therefore, compounds with
high inhibitory activity and appropriate hydrolytic stability ur-
gently need to be discovered for the possible development of a
therapy for urease mediated bacterial infections. To this end, our
current efforts are focused on seeking novel urease inhibitors with
good hydrolytic stability.
The N-hydroxy phenylacrylamide scaffold (7), an analog of 6, has
been employed extensively by many groups to generate potent
HDAC inhibitors and has resulted in at least four clinical programs
[27]. This clearly indicates that there is a great promise to get potent
urease inhibitors with medicinal potential from derivatives
of 6. Based on this conception, twenty
b-hydroxy-b-phenyl-
propionylhydroxamic acids (6ae6t) were synthesized for screen of
urease inhibitors and they were all reported for the first time.
Scheme 3 outlines methods where an aromatic aldehyde (8) was
reacted with ethyl bromoacetate in THF via Reformatsky reaction to
generate substituted ethyl 3-aryl-3-hydroxylpropionate (9). Sub-
sequently, the ester (9) was converted to the corresponding
hydroxamic acid (6) via hydroxylamine in methanolic sodium
methoxide [28]. Compound 6u was synthesized by direct hydrox-
yamination of ethyl 4-(N,N-dimethyl)benzoate (10) (Scheme 4).
Compounds 6ae6t were obtained in a racemic form due to a chiral
center at C-3 position; fortunately, the chiral center is not included
in the pharmacophore (C(O)NHOH). Therefore, the racemic mixture
was not separated for urease inhibitory assays, which would not
disturb us to discover inhibitors with a difference of at least one
order of magnitude.
Very recently, we have reported flavonoids [21], deoxybenzoins
[22], deoxybenzoin oximes [23], 1,2-diarylethanes [24], 3-flavenes
and 2-isoflavenes [25] as urease inhibitors (Scheme 1), in which
quercetin (1, IC50 ¼ 11.2 ꢀ 0.9
diol (2, IC50 ¼ 1.5 ꢀ 0.2 M) and 7,8,4-trihydroxyl-3-isoflavene (3,
IC50 ¼ 0.85 ꢀ 0.06 M) showed significant inhibitory activity
mM), 4-(4-fluorophenethyl)phen-1,2-
m
2.2. Urease inhibitory activity
m
against H. pylori urease (Scheme 1). To the best of our knowledge,
compound 3 is the first acid stable urease inhibitor with IC50 value
being in the nanomolar range. This opened the door for discov-
ering potent urease inhibitors with a medicinal potential based on
flavonoids. By comparison of the active compound structures, a
scaffold (4) shown as Scheme 2 would be extracted. In consider-
ation of the high binding affinity between hydroxamic acids and
transition metal ions [26], it is a clever strategy that combination
of a hydroxamic acid moiety with an active compound for urease
inhibitor discovering. On the other hand, hydroxamic acid moiety
(5) could mimic a ring structure (Scheme 2). Therefore, a chime-
rical urease inhibitor (6) was designed by mimics of ring A in
We investigated substitutions to the aromatic portion in an
effort to find more potent compound in comparison with our pre-
viously reported compounds (1, 2 and 3). The results of the inhi-
bition assays against H. pylori urease for the synthetic compounds
are displayed in Table 1. In comparison with compound 6a, intro-
duction of a single hydroxyl or methoxy group on the phenyl ring in
any position generates compounds 6b, 6e and 6f, and 6j with a less
than 5-fold decrease in potency. Whereas, when a fluorine atom
was introduced, the resulted compounds (6d, 6h and 6m) show a
moderate increase in activities. Unexpectedly, chlorine in these
positions was found to be much more potent as illustrated by
compounds 6g, 6c and 6l. These three compounds are the most
structure 4 with a hydroxamic acid moiety and ring C with a b-
active in their own series, showing IC50
s
of 1.19
ꢀ
0.08,
hydroxylcarbonyl moiety. In the present paper, we reported the
synthesis of 6 with varying substituents on the benzene ring and
the evaluation of their inhibitory activity using purified H. pylori
urease.
0.083 ꢀ 0.004 and 3.39 ꢀ 0.22
m
M respectively. This emphasized
the importance of a hydrophobic substituent in improving potency.
However, replacement of the chloro substituent with a more hy-
drophobic substitution group, such as trifluoromethyl or benzyloxy
Scheme 2. Design of the hybrid hydroxamic acid (6).

214
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
Scheme 3. Preparation of compounds 6ae6t. Reagents and conditions: (a) ethyl bromoacetate, Zn, THF, reflux, 1h, 45e90%; (b) hydroxylamine hydrochloride, NaOMe, MeOH, 0 ꢁC,
51e94%.
group, resulted in 2- to 18-fold loss of activity, illustrating that the
hydrophobic area cannot comfortably accommodate these larger
groups. In an effort to further understand the observed results,
cLogPs of the synthesized compounds were calculated by Chem-
Draw Ultra 11.0 (Table 1), and the plots of IC50 vs cLogP are shown as
Fig. 1. These plots clearly revealed that chloro group is the most
suitable substituent for improving potency with regard to steric-
hindrance and hydrophobic properties, and both a hydrophilic
group and a steric-hindrance group on the phenyl ring in any po-
sition is detrimental to the activity. This is also supported by
compared IC50s of 6g vs 6r and 6d vs 6s.
AHA and compounds with relative low IC50 values against cell-
free urease (6c, 6g-i, 6p-r and 6t) were selected for inhibiting
urease activity in intact H. pylori. As shown in Table 1, the IC50
values of the test compounds against urease in intact cell were 3- to
9-fold higher than those for extracted urease. The higher IC50s may
reflect the permeability barrier of the cell surface to the compounds
by H. pylori. Another interesting finding of this assay is that 6g and
6p also significantly inhibited the urease activity in intact cell. This
indicates that 6g and 6p would be a potential urease inhibitor de-
serves further research.
Shifting of the fluoro or methoxy group from the C-3 (6h or 6f)
to the C-2 (6d or 6b) position resulted in a very slight decrease in
enzyme activity, while moving to the C-4 positions, as in com-
pounds 6m and 6j, respectively, resulted in over 2-fold loss of ac-
tivity. However, the case for chlorine is quite different. Both C-2
2.3. Kinetics of urease inhibition by compounds 6g and 6p
Since Kobashi [29] first demonstrated in 1962 that hydroxamic
acids were highly specific inhibitors of plant and bacterial original
urease, many workers have extended the observations. 2001,
Byung-Ha Oh et al. reported the crystal structure of H. pylori urease
complexed with acetohydroxamic acid (AHA) [30], revealing that
AHA may be a competitive inhibitor of H. pylori urease by binding at
its active site. However, kinetic studies showed that the aliphatic
isomer (6c with IC50 of 1.19 ꢀ 0.08
mM) and C-4 isomer (6l with IC50
of 3.39 ꢀ 0.22 M) are much less active than C-3 isomer 6g, with
m
14- and 40-fold decrease in potency, respectively. Meanwhile, 6g
was the most active analog against H. pylori urease with activity to
double digit nM numbers, showing at least a 330-fold improvement
of potency compared to the commercially available urease inhibi-
tor, AHA. This revealed that the hydrophobic 3-chloro group gets
the best match to the enzyme active site among the tested com-
pounds. Combining 3-chloro group and another substituent led to
several quite active analogs (6r, 6t, 6p and 6q) with IC50s in the
Table 1
In vitro inhibitory activity data of the synthesized compounds against H. pylori
urease.
Entry
Compounds
cLogP
IC50 (mM)
Cell-free urease
Urease in intact cell
range of single digit mM to submicromolar. However, introducing
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
2.142
2.061
2.855
2.285
1.475
2.061
2.855
2.285
3.025
2.061
3.829
2.855
2.285
3.025
2.307
3.448
3.448
2.485
2.344
2.972
2.307
15.6 ꢀ 1.3
38.5 ꢀ 2.7
1.19 ꢀ 0.08
5.60 ꢀ 0.34
131 ꢀ 9
NDa
the second substituent at C-2, C-4 and C-5 position is not beneficial
to potency improvement, resulting in a 2- to 15-fold decrease in
activity. In general, a higher cLogP also results in a lower IC50 for the
multi-substituted analogs. On the basis of these findings, other
possible active compounds with IC50 in the nanomolar range may
be hit in the 3,5-disubstituted derivatives. In addition, a derivative
NDa
9.21 ꢀ 0.83
NDa
NDa
23.2 ꢀ 1.7
0.083 ꢀ 0.004
3.99 ꢀ 0.21
1.22 ꢀ 0.05
75.1 ꢀ 5.2
61.5 ꢀ 3.9
3.39 ꢀ 0.22
8.01 ꢀ 0.57
7.89 ꢀ 0.71
7.13 ꢀ 0.64
0.23 ꢀ 0.01
0.37 ꢀ 0.02
1.28 ꢀ 0.07
11.0 ꢀ 0.9
1.04 ꢀ 0.06
77.9 ꢀ 7.1
27.6 ꢀ 2.5
NDa
0.36 ꢀ 0.03
13.3 ꢀ 1.2
6.81 ꢀ 0.53
NDa
of phenylhydroxamic acid, 6u, with IC50 of 77.9 ꢀ 7.1
m
M was
9
around 10 times less active than the corresponding the
b
-phenyl-
10
11
12
13
14
15
16
17
18
19
20
21
22
propionylhydroxamic acid analog (6o), emphasizing the impor-
tance of the ethylene link for an active compound.
NDa
NDa
NDa
NDa
NDa
0.91 ꢀ 0.07
1.42 ꢀ 0.22
11.3 ꢀ 1.9
NDa
6s
6t
6u
AHA
8.90 ꢀ 0.91
NDa
110 ꢀ 13
Scheme 4. Preparation of compound 6u. Reagents and conditions: (a) hydroxylamine
a
hydrochloride, NaOMe, MeOH, 0 ꢁC, 2 h, 54%.
No determination.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
215
from the active site, assisted by the movement of His
a
322, and the
155
135
115
95
para
mobile flexible flap then opens. Based on this catalytic mechanism,
a model was built for understand the inconsistency between the
crystallographic and kinetic studies (Fig. 4). We assumed that
hydroxamic acid inhibits the urease activity in a mixture of
competitive and uncompetitive mechanism. According to the
equilibrium of species in the enzyme reaction (Scheme 5), the
enzyme kinetic equation for this mixed mechanism can be
expressed as:
meta
ortho
75
55
Vmax½Sꢃ
35
ꢀ
ꢁ
ꢀ
ꢁ
V ¼
(1)
½Iꢃ
½Iꢃ
i
Km 1 þ K
þ
1 þ K0 ½Sꢃ
i
15
Among equation (1): Km ¼ kꢂ1 þ k2=k1, Vmax ¼ k2½Eꢃ0,
Ki ¼ kꢂi=ki, Ki0 ¼ kꢂ0 i=ki0.
-5
1.4 1.6 1.8
2
2.2 2.4 2.6 2.8
cLogP
3
3.2 3.4 3.6 3.8
4
Obviously, when Ki is equal to Ki0, equation (1) turns into the
noncompetitive kinetic equation (2):
Fig. 1. The relationship of IC50s and cLogPs of benzene moieties.
Vmax½SꢃKi
V ¼
(2)
ðKm þ ½SꢃÞ þ ðKi þ ½SꢃÞ
hydroxamic acids, including AHA, were noncompetitive inhibitors,
while the aryl derivatives were of a mixed type [31]. These para-
doxical results encouraged us to study the inhibition mechanism of
obtained compounds. Therefore, the mechanism inhibition of
Therefore, some hydroxamic acids are kinetically noncompeti-
tive inhibitors, and others are acted as mixed competitive in-
hibitors. Although our inhibition model is quite different from the
typical mixed type, they are kinetically identical. We called it as an
apparent mixed competitive mechanism.
urease by two selected b-hydroxy-b-phenylpropionylhydroxamic
acids (6g and 6p) was investigated in a kinetic inhibition with a
LineweavereBurke plot. Double reciprocal plots of the data dis-
closed that 6g and 6p are mixed competitive inhibitors instead of
the expected competitive inhibitors with respect to the substrate
urea (Fig. 2A and Fig. 3A). The Ki value was calculated from a
plotting of the slopes of the LineweaverꢂBurk plot versus the
concentration of the inhibitor (Figs. 2B and 3B) [32]. The obtained Ki
2.4. Molecular docking
In an effort to further elucidate the mixed inhibition mechanism
revealed by the kinetic study and to confirm our assumed inhibition
model (Scheme 5), we performed molecular docking studies. The
most active compound (6g) was selected to dock with H. pylori
urease (entry 1E9Y in the Protein Data Bank) by the AutoDock
program. As above mentioned, 6g was formed as a raceme. Thus,
docking studies were performed on both possible configurations (R
and S) to evaluate if one or both enantiomers could be actually
interacting with the biological target. The binding results of R-6g
and S-6g are shown as both enzyme surface (Fig. 5A and B) and
cartoon modes (Figure 6A and B, 7A and 7B), respectively. On one
hand, the binding models revealed that R-6g and S-6g nearly fill
equivalent spaces, suggesting that they should display a similar
potency against H. pylori urease. On the other hand, the models
disclosed that both R-6g and S-6g showed two best possible
binding modes, respectively. One is well filled in the active pocket
values for 6g and 6p were 0.014 ꢀ 0.003 and 0.045 ꢀ 0.007
mM,
demonstrating the strong affinities of them toward H. pylori urease.
The crystallographic studies of urease revealed that there is a
flexible flap, a helix-turn-helix motif composed of residues
a313e
a
346 [33], over the active site [30]. According to Benini et al., the
highly mobile flap is in the open conformation can allow extensive
access of the substrate to the active site having the best fit [34].
Urea subsequently replaces the water molecules in the native
urease active site and bridges the two metal ions, which induces a
change in the conformation of the flexible flap back to a closed
position by rearranging the Alaa365. In the closed conformation,
CeN bond of the urea molecule is broken and urea collapses into
ammonia and a Ni-bound carbamate. Finally, ammonia is released
A
B
3500
140
µ
0
M
µ
µ
µ
0.39
0.93
1.40
M
M
M
3000
2500
2000
1500
1000
500
120
100
80
60
40
20
0
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
-0.015 -0.010 -0.005 0.000
0.005
0.010
1/urea mM
0.015
0.020
concentration of 6g
µ
M
-20
Fig. 2. (A) LineweaverꢂBurk plot for the inhibition of urease by 6g. Data were obtained in three different experiments with triplicates. (B) The plots of the slopes from the
LineweavereBurk lines vs. different concentrations of inhibitor.

216
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
A
B
Fig. 3. (A) LineweaverꢂBurk plot for the inhibition of urease by 6p. Data were obtained in three different experiments with triplicates. (B) The plots of the slopes from the
LineweavereBurk lines vs. different concentrations of inhibitor.
(defined as mode A, Figs. 5A, 6A and B) and the other is tightly
anchored at beside the entrance of the active site (defined as mode
B, Figs. 5B, 7A and B).
the interaction network is nearly consistent with that of S-6g. This
binding mode undoubtedly confirms the characteristic of kineti-
cally competitive inhibition of 6g.
In the three-dimensional models (Figs. 6A and 7A) of the S-6g, it
is important to note that the acylhydroxamic acid moiety (C(O)
NHOH) is of primary importance for its interactions network. In
mode A (Fig. 6A), S-6g binds the active site in an anion form (C(O)
NHOꢂ), in which the oxygen ion bridges the two nickel ions with
In mode B, S-6g and R-6g were docked with H. pylori urease in a
neutral molecule form (Fig. 7A and B). By comparison with mode A,
S-6g or R-6g is located under the flexible flap (residues a313ea346)
over the active site, with the benzene ring filling in the gap between
Cys321, Ala365 and Met366 and with the acylhydroxamic acid
moiety forming an extensive hydrogen bonding network. S-6g
using the oxygen of hydroxamic acid moiety (NHOH) as an acceptor
ꢀ
metaleligand distance of 2.060 and 2.175 A, respectively. The
carbonyl oxygen shows a hydrogen bonding interaction with the
ꢀ
ꢀ
NH2 group of His221 at a distance of 3.293 A and a metaleligand
receives a hydrogen bonding interaction (3.590 A) from Gly367 and
ꢀ
ꢀ
interaction with a distance of 3.205 A, NH group of S-6g was found
as a donor forms two hydrogen bond with Gln364 (3.728 A) and
ꢀ
to strongly hydrogen bond with the carbonyl group of Ala365 at a
Ala365 (2.973 A). While R-6g using the same oxygen as an acceptor
ꢀ
ꢀ
distance of 2.354 A. The same NH group interacted with the both
receives two hydrogen bonding interactions (3.484 and 3.555 A)
ꢀ
oxygens of Asp362 at distances of 2.104 and 3.135 A, respectively. In
from Gly367 and Ala365, and as a donor forms only one hydrogen
ꢀ
addition to the tightly bound acylhydroxamic acid moiety, S-6g is
bond with Met366 (2.012 A). A strong hydrogen bonding interac-
ꢀ
further anchored to the active site by a strong hydrogen bond
tion with a distance of 2.443 A was observed between the carbonyl
ꢀ
(2.279 A) between its
b
-hydroxyl group and Asn168. The benzene
group of S-6g and the back bone NH of Met366. As for R-6g, this
ꢀ
ring is sandwiched in between Cys321, Ala365 and Met366 through
hydrogen bonding interaction and hydrophobic in-
hydrogen bond is lightly weak (2.525 A).
b
-Hydroxyl group of S-6g
a SeHꢄ
p
or R-6g tightly anchor the flexible flap of the enzyme by two
hydrogen bonding interactions built by Met317 and Val320. The
teractions within a distance of 3.5 Å. Fig. 6B depicts the 3D model of
the interaction between R-6g and the target, which revealed that
movable flap is further fixed by the SeHꢄp contacts occurred
Fig. 4. The assumed mechanism mode mixed by competitive and uncompetitive inhibition.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
217
excellent potency of 6g and 6p against H. pylori urease suggests that
they deserve to be further researched as a good candidate to treat
gastritis and gastric ulcer. More detailed studies of the pharmaco-
logical profiles of these promising compounds are underway in our
lab. Meanwhile, using 6g and 6p as leads, continued efforts are also
underway to search more potent urease inhibitors.
4. Experimental section
Scheme 5. The equilibrium of species in the enzyme reaction for the assumed inhi-
bition mechanism.
4.1. Biology 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).
between Cys321 and the benzene ring of S-6g or R-6g, while the
benzene ring is stabled by the hydrophobic interactions from
Met366 and Ala365. S-6g or R-6g in the current pose, like a support
frame, blocks the flap to move. According to Benini et al., the flap
must be in a closed conformation, before CeN bond of the urea
molecule was broken and urea collapsed into ammonia and a Ni-
bound carbamate. Therefore, urease will not complete the cata-
lytic hydrolysis of urea without the movement of this flap, leading
to an inactive enzyme. This inactive urease complex will be formed
only after a urea molecule is in the active site, which provides a
rational explanation for the kinetically uncompetitive inhibition
characteristic of hydroxamic acids.
4.2. Bacteria
H. pylori (ATCC 43504; American Type Culture Collection,
Manassas, VA) was grown in Brucella broth supplemented with 10%
heat-inactivated horse serum for 24 h at 37 ꢁC under microaerobic
conditions (5% O2, 10% CO2, and 85% N2), as our previously
described [21e25].
It is well known that hydroxamic acid is a weak acid (pKa of 8 for
AHA). Therefore, a typical hydroxamic acid is presents as an equi-
librium mixture of molecule and anion forms under physiological
condition. Our molecular docking modes disclosed that both
molecule and anion of hydroxamic acid are all potent inhibitors of
H. pylori urease, but show different actions of kinetics. This makes
hydroxamic acid inhibits H. pylori urease in an apparent mixed
mechanism.
4.3. Preparation of H. pylori urease
For urease inhibition assays, 50 mL broth cultures
(2.0 ꢅ 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 ꢂ80 ꢁ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 (15,000ꢅ 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.
3. Conclusion
A total of twenty hybrids of flavonoid scaffold and hydroxamic
acid,
b-hydroxy-b-phenyl-propionylhydroxamic acids, were syn-
thesized, tested for urease activity, and docked to H. pylori urease.
Many compounds exhibit micromolar to low micromolar IC50
values in the in vitro H. pylori urease inhibition assay. Kinetic studies
of two representative derivatives (6g and 6p) suggested a mixed
type of inhibition mechanism. While, the molecular docking of the
4.4. Measurement of urease inhibitory activity
The assay mixture, containing 25 mL (10U) of H. pylori urease
most active compound, 6g (IC50 ¼ 0.083 ꢀ 0.004
m
M), further
which was replaced by 25
for the urease assay of intact cells and 25 mL of the test compound,
m
L of cell suspension (4.0 ꢅ 107 CFU/mL)
revealed that the two possible enantiomers of 6g may display a
similar potency against H. pylori urease and inhibit urease with a
mixture of competitive and uncompetitive mechanism. The
was pre-incubated for 1.5 h at room temperature in a 96-well assay
plate. Urease activity was determined by measuring ammonia
Fig. 5. Binding modes of compound 6g with H. pylori urease. The enzyme is shown as surface; while 6g is shown as sticks. (A) 6g docked as molecule forms (R-6g, yellow; S-6g,
salmon); (B) 6g docked as anion forms (R-6g, cyan; S-6g, yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)

218
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
Fig. 6. Binding modes of compound 6g with H. pylori urease. The enzyme is shown as cartoon; while 6g is shown as sticks. (A) S-6g docked as anion form; (B) R-6g docked as anion
form.
production using the indophenol method as described by Weath-
erburn [35].
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 ꢅ 106
energy evaluations, and a maximum number of 2.7 ꢅ 104 genera-
tions. A mutation rate of 0.02 and a crossover rate of 0.8 were
4.5. Kinetic study
LineweavereBurk plots of 1/absorbance versus 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 6g or 6p. 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. All experiments were
conducted in triplicate.
ꢀ
chosen. Results differing by less than 0.5 A in positional root-mean-
square deviation (RMSD) were clustered together and the results of
the most favorable free energy of binding were selected as the
resultant complex structures.
4.7. Chemistry
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
on a Bruker AV-300 spectrometer at 25 ꢁC with TMS and solvent
signals allotted as internal standards. Chemical shifts were reported
4.6. Protocol of docking study
The automated docking studies were carried out using Auto-
Dock version 4.2. First, AutoGrid component of the program pre-
calculates a three-dimensional grid of interaction energies 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 for mode
in ppm (d). Elemental analyses were performed on a CHNeO-Rapid
instrument and were within ꢀ0.4% of the theoretical values.
ꢀ
A (85 A for mode B) 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 energy of the inhibitor within the mac-
romolecules. The GALS search algorithm (genetic algorithm with
local search) was chosen to search for the best conformers. The
4.7.1. General procedure for the preparation of compounds 9ae9t
Zinc powder is treated with 2% HCl to give activated zinc. After
the suspension of activated zinc (10 mmol) in dry THF (8 mL) is
heated to reflux, the heating is stopped, and a solution of ethyl
Fig. 7. Binding modes of compound 6g with H. pylori urease. The enzyme is shown as cartoon; while 6g is shown as sticks. (A) S-6g docked as molecule form; (B) R-6g docked as
molecule form.

Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
219
bromoacetate (6 mmol) and appropriate substituted benzaldehyde
(8, 5 mmol) in dry THF (5 mL) is added within 30 min. After being
heated to reflux for 0.5e3 h, the mixture is cooled and magnetically
stirred for 15 min with saturated NH4Cl (20 mL). The aqueous phase
is extracted with AcOEt (2 ꢅ 50 mL) and the combined organic
phases are washed with a 5% NaHCO3 solution (2 ꢅ 20 mL). After
drying over MgSO4, concentration under reduced pressure fur-
nishes a residue, which is purified over a silica gel column eluting
with AcOEt-petroleum ether to yield an ester 9.
(dd, J ¼ 13.9 Hz, J ¼ 9.6 Hz, 1H); 2.27 (dd, J ¼ 14.1 Hz, J ¼ 2.9 Hz, 1H);
3.78 (s, 1H); 5.15 (d, J ¼ 4.6 Hz, 1H); 5.22e5.29 (m, 1H); 6.90e6.96
(m, 2H); 7.21 (t, J ¼ 6.9 Hz, 1H); 7.41 (d, J ¼ 7.3 Hz, 1H); 8.71 (s, 1H);
10.31 (s, 1H); 13C NMR (DMSO-d6): 41.4, 55.8, 64.0, 110.9, 120.7,
126.5, 128.3, 133.8, 155.8, 167.8; EIMS m/z 211 (Mþ). Anal. Calcd for
C10H13NO4: C, 56.86; H, 6.20; N, 6.63; Found: C, 56.91; H, 6.20;
N, 6.61.
4.7.2.7. 3-(3-Chlorophenyl)-3-hydroxypropionylhydroxamic
acid
(6g). White powder, 76%, mp 125e127 ꢁC, 1H NMR (DMSO-d6):
2.26e2.40 (m, 2H); 4.80e6.00 (bs, 1H); 4.97 (dd, J ¼ 8.0 Hz,
J ¼ 5.1 Hz, 1H); 7.12e7.24 (m, 3H); 7.33 (s, 1H); 7.81 (s, 1H); 10.32
(bs, 1H); 13C NMR (DMSO-d6): 43.0, 69.3, 125.0, 126.1, 127.3, 130.5,
133.3, 148.4, 167.0; EIMS m/z 215 (Mþ). Anal. Calcd for C9H10ClNO3:
C, 50.13; H, 4.67; Cl, 16.44; N, 6.50; Found: C, 50.18; H, 4.66; Cl,
16.42; N, 6.50.
4.7.2. General procedure for the preparation of compounds 6ae6u
To a stirred solution of sodium methoxide (10 mmol) and
6 mmol of hydroxylamine hydrochloride at 0 ꢁC is added an ester (9
or 10, 3 mmol). The reaction is monitored by TLC, and upon
completion the reaction mixture is brought to pH 7e8 with 1 M
hydrochloric acid. The resulted mixture is extracted thrice with
AcOEt, and the combined organic layer washes with brine. After
drying over MgSO4, removement of the solvent under reduced
pressure give a slight yellow oil. Compound 6 is obtained after
purification with column chromatography on silica gel, eluting with
AcOEt-petroleum ether.
4.7.2.8. 3-(3-Fluorophenyl)-3-hydroxypropionylhydroxamic
acid
(6h). White powder, 65%, mp 115e117 ꢁC, 1H NMR (DMSO-d6):
2.20e2.33 (m, 2H); 4.92e4.99 (m, 1H); 5.52 (d, J ¼ 4.6 Hz, 1H); 7.05
(t, J ¼ 9.0 Hz, 1H); 7.14 (t, J ¼ 7.3 Hz, 1H); 7.16 (d, J ¼ 12.2 Hz, 1H);
7.35 (dd, J ¼ 13.0 Hz, J ¼ 7.5 Hz, 1H); 8.74 (s, 1H); 10.34 (s, 1H); EIMS
m/z 199 (Mþ). Anal. Calcd for C9H10FNO3: C, 54.27; H, 5.06; F, 9.54;
N, 7.03; O, 24.10; Found: C, 54.34; H, 5.05; F, 9.52; N, 7.02.
4.7.2.1. 3-Hydroxy-3-phenylpropionylhydroxamic acid (6a). White
powder, 52%, mp 156e158 ꢁC, 1H NMR (DMSO-d6): 2.26e2.41 (m, 2H);
3.7e4.5 (bs, 1H); 5.06 (dd, J ¼ 8.0 Hz, J ¼ 5.1 Hz, 1H); 7.48e7.56 (m,
5H); 7.94 (s, 1H); 10.36 (bs, 1H); EIMS m/z 181 (Mþ). Anal. Calcd for
C9H11NO3: C, 59.66; H, 6.12; N, 7.73; Found: C, 59.63; H, 6.12; N, 7.74.
4.7.2.9. 3-Hydroxy-3-(3-trifluoromethylphenyl)propionylhydroxamic
acid (6i). White powder, 62%, mp 126e128 ꢁC, 1H NMR (DMSO-d6):
2.30e2.38 (m, 2H); 5.01e5.07 (m, 1H); 5.62 (d, J ¼ 4.6 Hz, 1H);
7.52e7.64 (m, 3H); 7.68 (s, 1H); 8.75 (s, 1H); 10.36 (s, 1H); 13C NMR
(DMSO-d6): 43.0, 69.2, 122.6, 122.64, 124.1, 124.2, 129.6, 130.5, 147.2,
166.9; EIMS m/z 249 (Mþ). Anal. Calcd for C10H10F3NO3: C, 48.20; H,
4.04; F, 22.87; N, 5.62; Found: C, 48.12; H, 4.05; F, 22.89; N, 5.61.
4.7.2.2. 3-Hydroxy-3-(2-methoxyphenyl)propionylhydroxamic acid
(6b). White powder, 54%, mp 124e126 ꢁC, 1H NMR (CDCl3): 2.25
(dd, J ¼ 13.5 Hz, J ¼ 9.0 Hz, 1H); 2.48 (dd, J ¼ 13.4 Hz, J ¼ 2.5 Hz,1H);
4.43e4.50 (m, 1H); 5.17 (d, J ¼ 6.5 Hz, 1H); 7.14 (t, J ¼ 7.5 Hz, 1H);
7.27 (t, J ¼ 7.7 Hz, 2H); 7.48 (d, J ¼ 7.1 Hz, 1H); 7.85 (s, 1H); 10.38 (bs,
1H); 13C NMR (DMSO-d6): 41.7, 55.5, 63.7, 115.6, 124.8, 128.2, 129.3,
132.5, 158.2, 167.0; EIMS m/z 211 (Mþ). Anal. Calcd for C10H13NO4: C,
56.86; H, 6.20; N, 6.63; Found: C, 56.92; H, 6.19; N, 6.62.
4.7.2.10. 3-Hydroxy-3-(4-methoxyphenyl)propionylhydroxamic acid
(6j). White powder, 73%, mp 113e114 ꢁC, 1H NMR (DMSO-d6):
2.19e2.35 (m, 2H); 3.73 (s, 1H); 4.85e4.92 (m, 1H); 5.25 (d,
J ¼ 4.4 Hz, 1H); 6.88 (d, J ¼ 8.6 Hz, 2H); 7.24 (d, J ¼ 8.8 Hz, 2H); 8.70
(s, 1H); 10.32 (s, 1H); 13C NMR (DMSO-d6): 43.3, 55.5, 69.5, 113.9,
127.4, 137.7, 158.7, 167.4; EIMS m/z 211 (Mþ). Anal. Calcd for
C10H13NO4: C, 56.86; H, 6.20; N, 6.63; Found: C, 56.78; H, 6.21;
N, 6.64.
4.7.2.3. 3-(2-Chlorophenyl)-3-hydroxypropionylhydroxamic
acid
(6c). White powder, 67%, mp 181e183 ꢁC, 1H NMR (CDCl3): 2.27
(dd, J ¼ 14.1 Hz, J ¼ 9.4 Hz, 1H); 2.51 (dd, J ¼ 14.4 Hz, J ¼ 2.7 Hz, 1H);
4.43e4.52 (m, 1H); 5.38 (d, J ¼ 6.9 Hz, 1H); 7.21 (t, J ¼ 7.5 Hz, 1H);
7.30 (t, J ¼ 7.7 Hz, 2H); 7.50 (d, J ¼ 7.1 Hz, 1H); 7.81 (s, 1H); 10.40 (bs,
1H); EIMS m/z 215 (Mþ). Anal. Calcd for C9H10ClNO3: C, 50.13; H,
4.67; Cl, 16.44; N, 6.50; Found: C, 50.18; H, 4.66; Cl, 16.42; N, 6.51.
4.7.2.11. 3-(4-Benzyloxyphenyl)-3-hydroxypropionylhydroxamic acid
(6k). White powder, 60%, mp 124e129 ꢁC, 1H NMR (DMSO-d6):
2.21e2.35 (m, 2H); 4.18 (s, 2H); 4.83e4.91 (m, 1H); 5.32 (d,
J ¼ 4.6 Hz,1H); 6.86 (d, J ¼ 8.7 Hz, 2H); 7.27 (d, J ¼ 8.8 Hz, 2H); 7.47e
7.55 (m, 5H); 8.65 (s, 1H); 10.35 (s, 1H); EIMS m/z 287 (Mþ). Anal.
Calcd for C16H17NO4: C, 66.89; H, 5.96; N, 4.88; Found: C, 66.96; H,
5.95; N, 4.87.
4.7.2.4. 3-(2-Fluorophenyl)-3-hydroxypropionylhydroxamic
acid
(6d). White powder, 92%, mp 138e140 ꢁC, 1H NMR (CDCl3): 2.26
(dd, J ¼ 14.0 Hz, J ¼ 9.2 Hz, 1H); 2.47 (dd, J ¼ 14.2 Hz, J ¼ 2.8 Hz, 1H);
4.41e4.49 (m, 1H); 5.23 (d, J ¼ 7.0 Hz, 1H); 7.22 (t, J ¼ 7.4 Hz, 1H);
7.33 (t, J ¼ 7.6 Hz, 2H); 7.48 (t, J ¼ 7.5 Hz, 1H); 7.85 (s, 1H); 10.36 (bs,
1H); 13C NMR (DMSO-d6): 43.0, 69.3, 125.0, 126.1, 127.3, 130.5, 133.3,
148.4, 167.0; EIMS m/z 199 (Mþ). Anal. Calcd for C9H10FNO3: C,
54.27; H, 5.06; F, 9.54; N, 7.03; Found: C, 54.19; H, 5.06; F, 9.56;
N, 7.05.
4.7.2.12. 3-(4-Chlorophenyl)-3-hydroxypropionylhydroxamic
acid
(6l). White powder, 62%, mp 159e162 ꢁC, 1H NMR (DMSO-d6):
2.20e2.36 (m, 2H); 4.90e5.00 (m, 1H); 5.47 (d, J ¼ 4.6 Hz, 1H);
7.32e7.37 (m, 4H); 8.73 (bs, 1H); 10.34 (bs, 1H); 13C NMR (DMSO-
d6): 43.1, 69.2, 128.1, 128.5, 131.8, 144.8, 167.0; EIMS m/z 215 (Mþ).
Anal. Calcd for C9H10ClNO3: C, 50.13; H, 4.67; Cl, 16.44; N, 6.50;
Found: C, 50.18; H, 4.67; Cl, 16.42; N, 6.49.
4.7.2.5. 3-Hydroxy-3-(3-hydroxyphenyl)propionylhydroxamic acid
(6e). White powder, 70%, mp 163e165 ꢁC, 1H NMR (DMSO-d6):
2.27e2.35 (m, 2H); 4.10e5.10 (m, 1H); 4.88 (dd, J ¼ 8.0 Hz,
J ¼ 5.1 Hz,1H); 6.59 (dd, J ¼ 7.3 Hz, J ¼ 2.0 Hz,1H); 6.73 (d, J ¼ 7.7 Hz,
1H); 6.77 (s, 1H); 7.02 (t, J ¼ 7.9 Hz, 1H); 7.66 (s, 1H); 10.25 (bs, 1H);
EIMS m/z 197 (Mþ). Anal. Calcd for C9H11NO4: C, 54.82; H, 5.62; N,
7.10; Found: C, 54.77; H, 5.61; N, 7.12.
4.7.2.13. 3-(4-Fluorophenyl)-3-hydroxypropionylhydroxamic
acid
(6m). White powder, 53%, mp 130e132 ꢁC, 1H NMR (DMSO-d6):
2.19e2.35 (m, 2H); 4.91e4.97 (m, 1H); 5.42 (d, J ¼ 4.6 Hz, 1H); 7.13
(d, J ¼ 8.8 Hz, 2H); 7.35 (dd, J ¼ 8.7 Hz, J ¼ 5.6 Hz, 2H); 8.72 (s, 1H);
10.33 (s, 1H); EIMS m/z 199 (Mþ). Anal. Calcd for C9H10FNO3: C,
54.27; H, 5.06; F, 9.54; N, 7.03; Found: C, 54.20; H, 5.01; F, 9.56;
N, 7.02.
4.7.2.6. 3-Hydroxy-3-(3-methoxyphenyl)propionylhydroxamic acid
(6f). White powder, 94%, mp 150e151 ꢁC, 1H NMR (DMSO-d6): 2.07

220
Z.-P. Xiao et al. / European Journal of Medicinal Chemistry 68 (2013) 212e221
4.7.2.14. 3-Hydroxy-3-(4-trifluorophenyl)propionylhydroxamic acid
(6n). White powder, 68%, mp 158e160 ꢁC, 1H NMR (DMSO-d6):
2.40e2.46 (m, 2H); 4.00e4.80 (bs, 1H); 5.11e5.16 (m, 1H); 7.52e
7.60 (m, 4H); 7.75 (s, 1H); 10.39 (s, 1H); EIMS m/z 249 (Mþ). Anal.
Calcd for C10H10F3NO3: C, 48.20; H, 4.04; F, 22.87; N, 5.62; Found: C,
48.28; H, 4.04; F, 22.85; N, 5.61.
Acknowledgment
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 and 13JJ2030), 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.
4.7.2.15. 3-Hydroxy-3-[(4-N,N-dimethylamino)phenyl]propionylhy-
droxamic acid (6o). White powder, 65%, mp 152e154 ꢁC, 1H NMR
(DMSO-d6): 2.23e2.38 (m, 2H); 3.68 (s, 6H); 4.92e4.98 (m, 1H);
5.43 (d, J ¼ 4.6 Hz, 1H); 6.86 (d, J ¼ 8.7 Hz, 2H); 7.23 (d, J ¼ 8.6 Hz,
2H); 7.80 (s, 1H); 10.34 (s, 1H); EIMS m/z 224 (Mþ). Anal. Calcd for
C11H16N2O3: C, 58.91; H, 7.19; N, 12.49; Found: C, 58.84; H, 7.20;
N, 12.52.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.07.047.
4.7.2.16. 3-(2,3-Dichlorophenyl)-3-hydroxypropionylhydroxamic
acid (6p). White powder, 80%, mp 112e113 ꢁC, 1H NMR (DMSO-d6):
2.18e2.26 (m, 2H); 4.88e4.94 (m, 1H); 5.42 (d, J ¼ 4.6 Hz, 1H); 6.81
(d, J ¼ 8.7 Hz, 1H); 7.03 (t, J ¼ 8.5 Hz, 1H); 7.21 (d, J ¼ 8.8 Hz, 1H);
7.82 (s, 1H); 10.31 (s, 1H); 13C NMR (DMSO-d6): 42.8, 68.8, 126.7,
128.3, 129.8, 130.8, 131.2, 146.9, 166.8; EIMS m/z 249 (Mþ). Anal.
Calcd for C9H9Cl2NO3: C, 43.22; H, 3.63; Cl, 28.35; N, 5.60; Found: C,
43.28; H, 3.63; Cl, 28.33; N, 5.59.
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4.7.2.17. 3-(3,4-Dichlorophenyl)-3-hydroxypropionylhydroxamic
acid (6q). White powder, 82%, mp 135e137 ꢁC, 1H NMR (DMSO-d6):
2.25e2.41 (m, 2H); 4.96e5.04 (m, 1H); 5.46 (d, J ¼ 4.4 Hz, 1H); 6.83
(d, J ¼ 8.8 Hz, 1H); 7.17 (d, J ¼ 8.6 Hz, 1H); 7.31 (s, 1H); 7.76 (s, 1H);
10.36 (s, 1H); EIMS m/z 249 (Mþ). Anal. Calcd for C9H9Cl2NO3: C,
43.22; H, 3.63; Cl, 28.35; N, 5.60; Found: C, 43.15; H, 3.64; Cl, 28.37;
N, 5.61.
4.7.2.18. 3-(5-Chloro-2-hydroxyphenyl)-3-hydroxypropionylhydroxa
mic acid (6r). White powder, 77%, mp 92e93 ꢁC, 1H NMR (DMSO-
d6): 2.31 (dd, J ¼ 14.6 Hz, J ¼ 8.8 Hz, 1H); 2.52 (dd, J ¼ 14.6 Hz,
J ¼ 3.8 Hz, 1H); 3.50e4.50 (bs, 1H); 5.23 (dd, J ¼ 8.8 Hz, J ¼ 3.7 Hz,
1H); 6.76 (d, J ¼ 8.6 Hz,1H); 6.99 (dd, J ¼ 8.6 Hz, J ¼ 2.7 Hz, 2H); 7.31
(d, J ¼ 2.6 Hz,1H); 7.89 (s,1H); 9.38 (bs,1H); 10.38 (bs,1H); 13C NMR
(DMSO-d6): 40.8, 64.3, 116.9, 122.9, 126.4, 127.5, 134.4, 152.8, 167.6;
EIMS m/z 231 (Mþ). Anal. Calcd for C9H10ClNO4: C, 46.67; H, 4.35; Cl,
15.31; N, 6.05; Found: C, 46.61; H, 4.35; Cl, 15.32; N, 6.06.
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4.7.2.19. 3-(5-Fluoro-2-methoxyphenyl)-3-hydroxypropionylhydroxa
mic acid (6s). White powder, 87%, mp 127e129 ꢁC, 1H NMR (DMSO-
d6): 2.30e2.50 (m, 2H); 3.78 (s, 3H); 4.20e5.10 (bs, 1H); 5.24 (t,
J ¼ 6.4 Hz, 1H); 6.58 (dd, J ¼ 9.0 Hz, J ¼ 2.2 Hz, 1H); 6.69 (d,
J ¼ 8.6 Hz,1H); 7.41 (t, J ¼ 8.7 Hz,1H); 7.83 (s,1H); 10.38 (bs,1H); 13C
NMR (DMSO-d6): 41.8, 56.0, 63.5, 101.7, 110.6, 124.2, 128.8, 158.8,
160.1, 167.1; EIMS m/z 229 (Mþ). Anal. Calcd for C10H12FNO4: C,
52.40; H, 5.28; F, 8.29; N, 6.11; Found: C, 52.48; H, 5.27; F, 8.27;
N, 6.10.
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03.016.
4.7.2.20. 3-(3,5-Dichloro-2-hydroxyphenyl)-3-hydroxypropionyl
hydroxamic acid (6t). White powder, 51%, mp 129e131 ꢁC, 1H NMR
(DMSO-d6): 2.10e2.30 (m, 1H); 2.30e2.48 (m, 1H); 5.21 (bs, 1H);
7.26 (s, 1H); 7.35 (s, 1H); 8.8 (bs, 1H); 10.35 (bs, 1H); EIMS m/z 265
(Mþ). Anal. Calcd for C9H9Cl2NO4: C, 40.63; H, 3.41; Cl, 26.65; N,
5.26; Found: C, 40.55; H, 3.41; Cl, 26.68; N, 5.27.
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4.7.2.21. 4-Dimethylaminophenylhydroxamic
acid
(6u). White
powder, 54%, mp 148e150 ꢁC, 1H NMR (DMSO-d6): 2.93 (s, 6H);
2.30e2.48 (m, 1H); 6.70 (d, J ¼ 8.8 Hz, 2H); 7.39 (d, J ¼ 8.8 Hz, 2H);
7.96 (s, 1H); 10.64 (s, 1H); EIMS m/z 180 (Mþ). Anal. Calcd for
C9H12N2O2: C, 59.99; H, 6.71; N, 15.55; Found: C, 59.90; H, 6.71; N,
15.57.

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