Arylamino containing hydroxamic acids as potent urease inhibitors for the treatment of Helicobacter pylori infection

Article abstract of DOI:10.1016/j.ejmech.2018.06.065

A novel series of aniline-containing hydroxamic acids were designed, synthesized and evaluated as anti-virulence agents for the treatment of gastritis and gastric ulcer caused by Helicobacter pylori. In vitro enzyme-based screen together with in vivo assays and structure?activity relationship (SAR) studies led to the discovery of three potent urease inhibitors 3-(3,5-dichlorophenylamino)–N-hydroxypropanamide (3a), 3-(2-chlorophenylamino)–N-hydroxypropanamide (3d) and 3-(2,4-dichlorophenylamino)–N-hydroxypropanamide (3n). Compounds 3a, 3d and 3n showed excellent urease inhibition with IC₅₀ values 0.043 ± 0.005, 0.055 ± 0.008 and 0.018 ± 0.002 μM, and significantly depressed gastritis developing at the dose of 32 mg/kg b. i.d with eradication rates of H. pylori reaching 92.3, 84.6 and 100%, respectively. Preliminary safety studies (acute toxicity in mice) disclosed that 3a, 3d and 3n was well-tolerated in KM mice with LD₅₀s of 2982.8, 3349.4 and 3126.9 mg/kg, respectively. Collectively, the data obtained in this study indicate that 3a, 3d and 3n, in particular 3n, could considered as promising candidates for the potential treatment of H. pylori caused gastritis and gastric ulcer, and hence merit further studies.

Full text of DOI:10.1016/j.ejmech.2018.06.065

Accepted Manuscript
Arylamino containing hydroxamic acids as potent urease inhibitors for the treatment of
Helicobacter pylori infection
Qi Liu, Wei-kang Shi, Shen-Zhen Ren, Wei-wei Ni, Wei-yi Li, Hui-min Chen, Pei Liu,
Jing Yuan, Xiao-su He, Jia-Jia Liu, Peng Cao, Pu-Zhen Yang, Zhu-Ping Xiao, Hai-
Liang Zhu
PII:
S0223-5234(18)30556-7
10.1016/j.ejmech.2018.06.065
EJMECH 10535
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 April 2018
Revised Date: 17 June 2018
Accepted Date: 29 June 2018
Please cite this article as: Q. Liu, W.-k. Shi, S.-Z. Ren, W.-w. Ni, W.-y. Li, H.-m. Chen, P. Liu, J. Yuan,
X.-s. He, J.-J. Liu, P. Cao, P.-Z. Yang, Z.-P. Xiao, H.-L. Zhu, Arylamino containing hydroxamic acids as
potent urease inhibitors for the treatment of Helicobacter pylori infection, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.06.065.
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ACCEPTED MANUSCRIPT
Arylamino Containing Hydroxamic Acids as Potent Urease Inhibitors
for the Treatment of Helicobacter Pylori Infection
Qi Liu, Wei-kang Shi, Shen-Zhen Ren, Wei-wei Ni, Wei-yi Li, Hui-min Chen, Pei Liu,
Jing Yuan, Xiao-Su He, Jia-Jia Liu, Peng Cao, Pu-Zhen Yang, Zhu-Ping Xiao and
Hai-Liang Zhu

ACCEPTED MANUSCRIPT
Arylamino Containing Hydroxamic Acids as Potent Urease Inhibitors for the
Treatment of Helicobacter Pylori Infection
Qi Liua, Wei-kang Shia, Shen-Zhen Renb, Wei-wei Nia, Wei-yi Lia, Hui-min Chena, Pei Liua,
Jing Yuana, Xiao-su Hea, Jia-Jia Liua, Peng Caoa, Pu-Zhen Yanga, Zhu-Ping Xiaoa,b,* and Hai-
Liang Zhua,b,*
aNational Demonstration Center for Experimental Chemistry Education, Hunan Engineering
Laboratory for Analyse and Drugs Development of Ethnomedicine in Wuling Mountains,
Jishou University, Jishou 416000, P. R. China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China
Abstract: A novel series of aniline-containing hydroxamic acids were designed, synthesized
and evaluated as anti-virulence agents for the treatment of gastritis and gastric ulcer caused
by Helicobacter pylori. In vitro enzyme-based screen together with in vivo assays and
structure−activity relationship (SAR) studies led to the discovery of three potent urease
inhibitors
3-(3,5-dichlorophenylamino)-N-hydroxypropanamide
(3a),
3-(2-
chlorophenylamino)-N-hydroxypropanamide (3d) and 3-(2,4-dichlorophenylamino)-N-
hydroxypropanamide (3n). Compounds 3a, 3d and 3n showed excellent urease inhibition
with IC50 values 0.043±0.005, 0.055 ± 0.008 and 0.018 ± 0.002 µM, and significantly
depressed gastritis developing at the dose of 32 mg/kg b.i.d with eradication rates of H. pylori
reaching 92.3, 84.6 and 100%, respectively. Preliminary safety studies (acute toxicity in mice)
disclosed that 3a, 3d and 3n was well-tolerated in KM mice with LD50s of 2982.8, 3349.4
and 3126.9 mg/kg, respectively. Collectively, the data obtained in this study indicate that 3a,
3d and 3n, in particular 3n, could considered as promising candidates for the potential
treatment of H. pylori caused gastritis and gastric ulcer, and hence merit further studies.
Corresponding author. Fax: +86-743-8563911; E-mail address:xiaozhuping2005@163.com
*Corresponding author. Fax: +86-25-83592672; E-mail address: zhuhl@nju.edu.cn
1

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Keywords: Arylamino containing hydroxamic acid; H. pylori urease inhibitor; Kinetics study;
gastritis and gastric ulcer; therapeutic efficacy.
2

ACCEPTED MANUSCRIPT
Arylamino Containing Hydroxamic Acids as Potent Urease Inhibitors for the
Treatment of Helicobacter Pylori Infection
Qi Liua, Wei-kang Shia, Shen-Zhen Renb, Wei-wei Nia, Wei-yi Lia, Hui-min Chena, Pei Liua,
Jing Yuana, Xiao-su Hea, Jia-Jia Liua, Peng Caoa, Pu-Zhen Yanga, Zhu-Ping Xiaoa,b,* and Hai-
Liang Zhua,b,*
aNational Demonstration Center for Experimental Chemistry Education, Hunan Engineering
Laboratory for Analyse and Drugs Development of Ethnomedicine in Wuling Mountains,
Jishou University, Jishou 416000, P. R. China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, P. R. China
Abstract: A novel series of aniline-containing hydroxamic acids were designed, synthesized
and evaluated as anti-virulence agents for the treatment of gastritis and gastric ulcer caused
by Helicobacter pylori. In vitro enzyme-based screen together with in vivo assays and
structure−activity relationship (SAR) studies led to the discovery of three potent urease
inhibitors
3-(3,5-dichlorophenylamino)-N-hydroxypropanamide
(3a),
3-(2-
chlorophenylamino)-N-hydroxypropanamide (3d) and 3-(2,4-dichlorophenylamino)-N-
hydroxypropanamide (3n). Compounds 3a, 3d and 3n showed excellent urease inhibition
with IC50 values 0.043±0.005, 0.055 ± 0.008 and 0.018 ± 0.002 µM, and significantly
depressed gastritis developing at the dose of 32 mg/kg b.i.d with eradication rates of H. pylori
reaching 92.3, 84.6 and 100%, respectively. Preliminary safety studies (acute toxicity in mice)
disclosed that 3a, 3d and 3n was well-tolerated in KM mice with LD50s of 2982.8, 3349.4
and 3126.9 mg/kg, respectively. Collectively, the data obtained in this study indicate that 3a,
3d and 3n, in particular 3n, could considered as promising candidates for the potential
treatment of H. pylori caused gastritis and gastric ulcer, and hence merit further studies.
Corresponding author. Fax: +86-743-8563911; E-mail address:xiaozhuping2005@163.com
*Corresponding author. Fax: +86-25-83592672; E-mail address: zhuhl@nju.edu.cn
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Keywords: Arylamino containing hydroxamic acid; H. pylori urease inhibitor; Kinetics study;
gastritis and gastric ulcer; therapeutic efficacy.
1. Introduction:
Urease (EC 3.5.1.5), a nickel dependent metalloenzyme with an ability to catalyze the
hydrolysis of urea to ammonia and carbamates,[1] exists in plants, fungi and bacteria,[2] being
well known as an important virulence factor in the pathogenesis of several diseases.[3,4]
Helicobacter pylori, a microaerophilic bacterium with high urease activity, is known to be
one of the most successful human bacterial parasites in gastrointestinal track, and colonizes
more than half of the human population.[5] On one hand, urease excreted by H. pylori buffers
the pH to allow the bacterium survive in the acidic environment of the stomach;[6] on the
other hand, urease serves as a virulence factor to cause about 15-20% of infected people
developing into severe gastroduodenal pathologies, such as stomach and duodenal ulcers,
adenocarcinomas and stomach lymphomas.[7,8] In 1991, Eaton demonstrated that urease-
negative H. pylori mutants were unable to colonize the stomach of gnotobiotic piglets,[9]
suggesting that urease activity is indispensable for survival of H. pylori in the gastric
microenvironment. Therefore, the strategy based on urease inhibition is considered as a
promising treatment for infections caused by urease-containing becteria.[10] In the past
decades, the great medicinal potential for urease inhibitors attracts extensive attention from
worldwide researchers,[11-14] and hundreds of structures such as hydroxamic acids,
phosphorodiamidates, imidazoles, thiobarbituric acids, thioureas and flavonoids have been
identified.[15-21] In mechanism, a hydroxamic acid strongly binds to nickel ions of the active
site to form a strong complex with urease, and have therefore attracted much more
attention.[22-23] Up to now, hydroxamic acids are the best recognized urease inhibitors, with a
marketed drug acetohydroxamic acid (AHA) which was approved by U.S. Food and Drug
Administration in May, 1983.
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In the past several years, we focused our efforts on seeking novel urease inhibitors, and
flavonoids,[21,24] deoxybenzoins,[25] deoxybenzoin oximes,[26] 1,2-diarylethanes,[27-28] furan-
2(5H)-ones[29] and 3-arylpropionyldroxamic acids[30] were identified. 3-(3-Chlorophenyl)-3-
hydroxypropionylhydroxamic acid (CPH), found as the most active with IC50 of 0.083±0.004
µM in our group, is a good lead for further modifications. Molecular docking of CPH
revealed that phenyl moiety binds to the surface of urease, and hydroxamic acid moiety binds
to both nickel ions in the active site. These two functional sections are linked by a carbon
chain arm.[30] Meanwhile, consideration of CPH structurally containing a chiral centre and S-
/R-isomers not showing significant difference in activity,[31] we replaced the chiral center
COH with NH for synthetic convenience and structural diversity (Figure 1). A series of 2-(N-
arylamino)acetohydroxamic acids (2) and 3-(N-arylamino)propionylhydroxamic acids (3)
were designed and synthesized. Biological evaluations revealed that the modification led to a
marked improvement in potency.
2. Results and dissussion
2.1. Chemistry
Direct replacement of the chiral center COH in the CPH skeleton with NH furnishes the first
series (2), and the synthetic route illustrated in Scheme 1. N-Alkylation of the selected aniline
4 with ethyl bromoacetate offered compound 5, which was amidated with NH2OH·HCl to
give 2-(N-arylamino)acetohydroxamic acids (2).[32]
Replacement of ethyl bromoacetate with ethyl 3-bromopropionate was beyond our
expectations, and was suffered a very poor yield for preparing the corresponding N-alkylation
intermediate (6). Therefore, 4 was converted to 6 by nucleophilic addition of 4 to ethyl
acrylate in the presence of terfluoroacetic acid (TFA) (Scheme 2).
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2.2. Inhibitory activity against cell-free urease
In the process of designing novel non-phosphoramide urease inhibitors, we prepared and
evaluated a series of aryl-containing hydroxamic acids.[30,2] The structure-activity relationship
(SAR) analysis and molecular dockings suggested that the arm linkage is important for the
binding location of the aryl moiety. In light of this observation, our modification strategy first
focused on the replacement of the branching point with NH, yielding the series 1 (2a-2w), as
shown in Figure 1. Newly synthesized β-aminoacetohydroxamic acids (2a-2w) were initially
evaluated for inhibition activity against extracted H. pylori urease, in parallel with AHA as a
positive reference (Table 1). With a bromo group on the benzene ring, compounds 2k, 2l and
2v showed significant inhibition activity with IC50 of 0.86±0.03, 2.01±0.05 and 12.3±0.9 µM,
being 2- to 24-fold more potent than the reference compound AHA. Replacement of the
bromine atom in 2k, 2l or 2v with Cl, Me, F, NO2 or OMe resulted in compounds (2i, 2j, 2u;
2s; 2a; 2n; 2d, 2t, 2c) less potency in the order of decreasing hydrophobicity, exhibiting IC50
values of 1.15-69.8µM. Interestingly, when both meta-positions were replaced by Cl, Me, F
and OMe, the resulting compounds (2m, 2b, 2r and 2e) showed high potency with IC50
values of 0.13-5.11µM. These observations suggest that hydrophobic substituents in benzene
ring generally possess more favorable inhibitory activity. Molecular docking disclosed that
benzene moiety establishes favorable hydrophobic interactions with backbone residues of
urease (Figure 2A). Out of these compounds, 2m was the most potent in this series, but a
little less potent than the lead CPH.
In order to improve both the binding affinity and efficiency, 2m was selected as a lead and
we took a close look at urease-2m interactions. No significant hydrogen bonding interactions
could be observed between NH of aniline moiety and near residues (Asn 168, Ala 169, Ala
365 and Cys 321), while this NH group is located near hydrophobic side chains of Ala 169
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and Ala 365 (Figure 2A and 2B), indicating that a lipophilic CH2 group could be inserted for
possibly improving activity of compound 2m. This hypothesis was further confirmed by the
binding model of 3a (Figure 2A). Based on this consideration, 3a was designed and
synthesized, showing 3-fold improvement of potency (IC50=0.043µM).
Next, we designed molecules 3b-3q to investigate the importance of the number of
substituent and their positions on the phenyl ring, which were evaluated and summarized in
Table 2. As expected, propionyl derivatives (3b-3q) generally show more potency than acetyl
anaguoes (2a-2w) with a maximum of up to 3670-fold increase. This may be attributed to the
intermolecular interactions built by NH and ethylene group. As an example, the most active
compound 3n interacts through its NH group with Cys321, His322 and Arg338 (Figure 3B),
forming significant hydrogen bonding interactions which were not observed in 2m binding
model (Figure 2B). The binding complex of 3n-urease was further stabilized by hydrophobic
interactions of ethylene group with Ala169, Ala365 and Met366 (Figure 3B). In comparison
with 3a, the phenyl group of 3n is closer to the hydrophobic areas of the active pocket
(Figure 3A). Compounds 3c-3f, bearing an alkoxy group or a halogen atom, displayed equal
or improved potency compared to 3b. In the mono-substitued series, 3d carrying o-chloro
was the most potency with IC50 of 0.055±0.08 µM. Introduction of additional chloro group at
the other ortho-position or either of the two meta-positions in 3d produces compounds (3j, 3h
and 3o) with a moderate to marked loss in activity. However, introduction chloro groups at
both ortho- and para-positions gave the most active compound 3n (IC50=0.018 ± 0.002µM)
in all tested compounds, exhibiting an over 1500-fold potency increase compared to the
positive control AHA.
2.3. Inhibitory activity against urease in intact cell
While the above data demonstrates activity in a cell-free system, in order to validate the
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potential use of these compounds in proof of concept animal models, compounds with
IC50<1.0 µM (2h, 2k, 2m, 3a, 3d-3f, 3h, 3i and 3n) were selected to be incubated with H.
pylori for evaluating the efficiency in cell-based models by using AHA as positive control.
The results are shown in Table 1 and 2. Assayed compounds suppressed urease in intact cell
with IC50 values ranging from 2.23 to 48.1 µM, showing much higher potency than AHA
(IC50=172±7 µM). However, a 46- to 110-fold increase of IC50 values was observed in
comparison with those against cell-free urease. The decrease of potency may be caused by
the barrier of cell membrane that the compound must transfer and a metabolic loss of the
compound in the presence of H. pylori cells. The most active compound, 3n with 2,4-dichloro
substitution pattern on the benzene ring, would be a promising urease inhibitor deserving
further research as an antiulcer and infectious stone agent.
2.4. Kinetics of urease inhibition by compounds 2m, 3a and 3n
To gain information on the mechanism of inhibition, the potent inhibitor 2m, 3a and 3n were
selected for a kinetic study. Data are reported in Fig. 4-6 as plots by non-linear fitting of
experimental data against Eq (1), and kinetic constants (Ki and Kiʹ) were obtained by linear
fitting of calculated values (b and c) vs [I] against Eq (2) and Eq (3) (Table 3). The present
work establishes that inhibition of urease by arylamino containing hydroxamic acid is fully
reversible, in agreement with our recent report.[30] As shown in Table 3, Ki is not equal to Kiʹ,
suggesting a dual-site binding mode. This may be attributed to a typical hydroxamic acid
being presented as an equilibrium mixture of molecule and anion forms under physiological
condition, which therefore supported that the novel hydroxamic acid acts as a mixed-type
mechanism. Recently, based on this equilibrium hypothesis, our molecular docking model
gave a possible explain for the mixed type inhibition mechanism of aryl derivative
hydroxamic acids.[30] It is to be noted that Byung-Ha Oh et al. reported the co-crystal
structure of H. pylori urease with AHA in 2001,[33] revealing that AHA is a specific inhibitor
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of urease. In the past decades, kinetic studies disclosed that the aliphatic hydroxamic acids,
including AHA, were noncompetitive inhibitors, while the aryl derivatives were of a mixed
type.[34]
[S]
V =
b =
c =
(1)
(2)
(3)
b + c[S]
Km
Vm
[I]
(1+
)
Ki
1
[I]
(1+
)
Vm
Ki '
2.5. Acute Toxicity Study in KM Mice
An acute toxicity study of compounds 3a, 3d and 3n was conducted in vivo in KM mice. The
tested animals recieved a single oral dose of test compounds at 500, 1000, 2000, 3000 and
5000 mg/kg (suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) solution).
Compounds 3a, 3d and 3n have been found to be compatible with potential in vivo studies
showing LD50 of 2982.8, 3349.4 and 3126.9 mg/kg, respectively (Table 4). As the mice
administered a single dose up to 1000 mg/kg of test compounds remained alive and appeared
healthy during a subsequent 14 day observation. This acute toxicity study indicated that 3a,
3d and 3n was well-tolerated in KM mice.
2.6. Compound evaluation in mice model of H. pylori
To further screen for compounds with potent urease inhibitory activity and tolerable toxicity
in vivo, therapeutic efficacy was studied using an H. pylori infected mouse model.
Compounds 3a, 3d and 3n were therefore selected to be assayed and results are shown in
Table 5 and Figure 7-8. Significant gastric mucosa injury was observed in untreated mice,
while this was barely observable in treated mice. On the contrary, the market urease inhibitor,
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AHA, only partially reduced the gastric mucosa injury (Figure 7-8). Oral 3n treatment
significantly reduced the survival of H. pylori with the eradication rate of up to 92.3% at 16
mg/kg, while compound 3a and 3d required a higher dose, 32 and 64 mg/kg, to achieve the
same level of efficacy. In comparison with the excellent therapeutic efficacy of 3a, 3d and 3n,
the positive control AHA exhibited very low eradication rate (44.4%) at 32 mg/kg (Table 5).
Together, our data clearly demonstrated that 3a, 3d and 3n had potent activity against H.
pylori in vivo.
3. Conculsions
In all, we have synthesized a series of aniline containing hydroxamic acids, which were tested
for inhibitory activity against H. pylori urease. Generally, compounds with a three-carbon
arm show higher potency than those with only two-carbon against H. pylori urease both in
cell and in cell-free extracts. Many compounds show excellent activity against extracted H.
pylori urease with micromolar to low nanomolar IC50 values, with top three active
compounds (3a, 3d and 3n) show IC50s of 43±5, 55 ±8 and 18 ± 2 nM. They also show good
potency against H. pylori urease in intact cell with IC50 values having about 100-fold increase.
Kinetic studies of three selected compounds (2m, 3d and 3n) suggested a mixed type of
inhibition mechanism. Meanwhile, the molecular docking of these compounds supports their
potency against H. pylori urease and confirms that they inhibited urease with a mixture of
competitive and uncompetitive mechanism. Efficacy studies in H. pylori-infected KM mice
demonstrated that compounds 3a, 3d and 3n were markedly depressed gastritis developing at
the dose of 32 mg/kg b.i.d with H. pylori eradication rate of 92.3, 84.6 and 100%,
respectively. It is worth mentioning that the dose used in the in vivo anti-H. pylori infection
experiment is much lower than LD50s. Our findings indicate that these arylamino containing
hydroxamic acids can potentially be applied for the treatment of gastritis and gastric ulcer
caused by H. pylori.
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4. Experimental section
4.1. Biology materials
Protease inhibitors (Complete, Mini, EDTA-free) were purchased from Roche Diagnostics
GmbH (Mannheim, Germany) and Brucella broth was from Becton Dickinson and Company
(Sparks, MD). Sheep serum was from Hyclone (Utah, American).
4.2. Bacteria
H. pylori (ATCC 43504; American Type Culture Collection, Manassas, VA) was grown in
Brucella broth supplemented supplemented with 10% sheep sterile and defibrinated blood for
24 h at 37 °C under microaerobic conditions (5% O2, 10% CO2, and 85% N2), as our
previously described literatures.[24-27]
4.3. Preparation of H. pylori urease
For urease inhibition assays, 50 mL broth cultures (2.0×108 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 -80 °C for 8 h, and then 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.
4.4. Measurement of urease inhibitory activity
The assay mixture, containing 25 mL (10U) of H. pylori urease which was replaced by 25 mL
of cell suspension (4.0×107 CFU/mL) for the urease assay of intact cells and 25 mL of the
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test compound, was pre-incubated for 1.5 h at room temperature in a 96-well assay plate.
Urease activity was determined by measuring ammonia production using the indophenol
method as described by Weatherburn.[35]
4.5. Kinetic study
Based on the indophenol method, the velocity of ammonia production (V) was measured in
the presence of concentration gradients of urea ([S]) for every specific concentration of 2m,
3a and 3n ([I]). Nonlinear fitting curves to data of V and [S] were used to determine the type
of enzyme inhibition based on the general kinetics equation (Eq. 1). Consequently, resulted
data of b (Eq. 2) or c (Eq. 3) and [I] were treated as a linear fitting to give the inhibitory
constants Ki and Ki′, which were determined from the intersection on the X-axis of the
corresponding fitting line. All experiments were conducted in triplicate.
4.6. Protocol of docking study
Molecular docking of compounds 2m, 3a and 3n into the structure of H. pylori urease
complex structure was carried out using SYBYL-X version 2.1.1 software suite (Tripos, Inc.,
St. Louis, MO).[36] The X-ray structure of urease from H. pylori was downloaded from the
Protein Data Bank (PDB code: 1e9y)[33] and was modified by adding hydrogen atoms and
removing water as well as cocrystallized substrate (AHA). The active site was defined as all
the amino acid residues confined within a 5 Å radius sphere centered about AHA, and the
composite structure without original ligand was utilized as the in silico model for docking
studies. Default parameters and values within the minimization dialogue were used except
where otherwise mentioned. The docked conformations of ligands were evaluated and ranked
using Surflex-Dock and four scoring functions implemented in the CSCORE software
module within the SYBYL-X environment. The CSCORE module allowed consensus scoring
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that integrated multiple well-known scoring functions such as ChemScore, D-Score, G-Score
and PMF-Score to evaluate docked ligand conformations.
4.7. Acute toxicity experiment.
Male and female adult KM mice (18−22 g) were obtained from the Changsha Silaikejingda
Animal Co. Ltd (Hunan, China) and housed under pathogen-free conditions with a 12 h
light/dark cycle. Food and water were given ad libitum throughout the experiment. The in
vivo acute toxicity of 3a, 3d or 3n in KM mice was assessed by a single intragastrical
injection at various doses (n=10, mice/group, 200 µL per mouse). Compound 3a, 3d or 3n
was dissolved in 0.5 % CMC-Na solution and the controls received injections of vehicle
alone. The mortality was recorded for 14 days after administration. The 50% lethal dose
(LD50) was estimated by probit analysis using SPSS version 16.0.
4.8. Compound evaluation in mice model of H. pylori.
KM mice (18−22 g) were obtained from the Changsha Silaikejingda Animal Co. Ltd (Hunan,
China) and housed under pathogen-free conditions with a 12 h light/dark cycle. Food and
water were given ad libitum throughout the experiment. KM mice were received an
intragastrical injection of NaHCO3 solution (0.2 mol/L 0.25 mL) after were fasted for 4 h.
Fifteen min later they were then intragastrically injected with H. pylori (0.4 mL,1
×109cfu/mL). They were infected with H. pylori once a day for a week. Three weeks later, H.
pylori infected mice were randomly divided into fifteen groups (n=13): one model group,
three 3a groups (32 mg/kg b.i.d, 64 mg/kg b.i.d, 128 mg/kg b.i.d), three 3d groups (32 mg/kg
b.i.d, 64 mg/kg b.i.d, 128 mg/kg b.i.d), three 3n groups (16 mg/kg b.i.d, 32 mg/kg b.i.d, 64
mg/kg b.i.d) and one acetohydroxamic acid group (32 mg/kg b.i.d). Compounds were
dissolved in 0.5 % CMC-Na solution. Mice were intragastrically received 0.6 mL of
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compound for two doses at 8:00 a.m. and 6:00 p.m. every day. The treatment lasted for 14
days. Mice were killed and each stomach was divided into two parts. One part was fixed with
4% paraformaldehyde, embedded in paraffin, and cut into 3.0-µm-thick sections for analysis
of gastric mucosal lesion. The tissue sections were deparaffinized, and hematoxylin and eosin
(HE) stain was applied to the sections. The other part was homogenized for H. pylori
determination according to a previous report.[37]
4.9. 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 a XT4
MP apparatus (Taike Corp., Beijing, China). EI mass spectra were obtained on Waters GCT
mass spectrometer, and 1H NMR spectra were recorded on a Bruker AV-400 spectrometer at
25 °C with TMS and solvent signals allotted as internal standards. Chemical shifts were
reported in ppm (δ). Elemental analyses were performed on a Foss Heraeus CHN-O-Rapid
instrument and were within ± 0.4% of the theoretical values.
4.9.1. General procedure for the preparation of compounds 5
To a solution of a selected aniline (5 mmol) in dry DMSO or acetone (25 mL) was added an
equivalent of anhydrous K2CO3, which was heated to reflux. Then, 4.5 mmol of ethyl
bromoacetate in dry DMSO or acetone (5 mL) was added dropwise over 30 min. The reaction
mixture was refluxed for 3-8 h (monitored by TLC), and was poured into distilled water (20
mL). The mixture was extracted with EtOAc (3×50 mL), dried overMgSO4, filtered, and
concentrated in vacuo. The crude product was purified by flash silica chromatography to
afford 5.
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4.9.2. General procedure for preparation of compounds 6
Anhydrous TFA (3-5 drops) was added into appropriately substituted aniline (10 mmol) and
then heated to 100 °C. To this mixture was added 3 mL of ethyl acrylate (EA) within 10min,
and the mixture was stirred at 100 °C for 24 h. After this period the EA was removed under
vacuum. The resulted residue was purified by silica gel chromatography to yield compound 6.
4.9.3. General procedure for the preparation of compounds 2a-2w and 3a-3q
A mixture of sodium methoxide (10 mmol) and hydroxylamine hydrochloride (6 mmol) in
anhydrous methanol was well stirred for 30 min. To this mixture was added compound 5 or 6
(3 mmol) was added, and the mixture was stirred at room temperature for 1-3h. The reaction
was quenched with ice (30 g) and neutralized with dilute HCl, which was extracted with ethyl
acetate (3 × 60 mL). The organic layers were collected, dried over sodium sulfate, filtered,
and evaporated under vacuum. The residure was purified by flash silica chromatography
(methanol-dichloromethane) to afford pure product (2 or 3) as a white solid.
4.9.3.1. 2-(4-Fluorophenylamino)-N-hydroxyacetamide (2a)
White power, 43%, mp 127-128 °C, 1H NMR (400 MHz, DMSO-d6): 3.55 (d, J=6.0Hz, 2H);
5.81 (t, J=6.2 Hz, 1H); 6.55 (dd, J=8.9 Hz, J=4.4 Hz, 2H); 6.93 (t, J=8.9 Hz, 2H); 8.83 (s,
1H); 10.59 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 45.34; 113.53 (d, J=7.4Hz); 115.60 (d,
J=21.9Hz); 145.49; 155.02 (d, J=231.2Hz); 167.37; EIMS m/z 184 (M+). Anal. Calcd for
C8H9FN2O2: C 52.17; H 4.93; F 10.32; N 15.21; found: C 52.23; H 4.92; F 10.31; N 15.22.
4.9.3.2. 2-(3,5-Dimethylphenylamino)-N-hydroxyacetamide (2b)
1
Light yellow power, 46%, mp 145-147°C, H NMR(400 MHz, DMSO-d6): 2.14(s, 6H);
3.54(d, J=6.1 Hz, 2H); 5.63(t, J=6.3 Hz, 1H); 6.18(s, 2H); 6.22(s, 1H); 8.80(s, 1H);10.52 (s,
15

ACCEPTED MANUSCRIPT
1H); 13C NMR(100 MHz, DMSO-d6): 21.73; 45.04; 110.82; 118.78; 138.01; 148.75; 167.60;
EIMS m/z 194 (M+). Anal. Calcd for C10H14N2O2: C 61.84; H 7.27; N 14.42; found: C 61.78;
H 7.28; N 14.44.
4.9.3.3. N-Hydroxy-2-(2-methoxyphenylamino)acetamide (2c)
1
White power, 41%, mp 145-146°C, H NMR(400 MHz, DMSO-d6): 3.62(d, J=5.9 Hz, 2H);
3.79(s, 3H); 5.13(t, J=6.0 Hz, 1H); 6.42(dd, J=7.8 Hz, J=1.5 Hz, 1H); 6.60(td, J=7.7 Hz,
J=1.6 Hz, 1H); 6.78(td, J=7.6 Hz, J=1.3 Hz, 1H); 8.82(dd, J=7.9 Hz, J=1.3 Hz, 1H); 8.87(s,
1H); 10.61(s, 1H); 13C NMR(100 MHz, DMSO-d6): 44.61; 55.75; 109.97; 110.20; 116.83;
121.41; 137.94; 146.99; 167.32; EIMS m/z 196 (M+). Anal. Calcd for C9H12N2O3: C 55.09; H
6.16; N 14.28; found: C 55.12; H 6.15; N 14.26.
4.9.3.4. N-Hydroxy-2-(3-methoxyphenylamino)acetamide (2d)
Light yellow power, 48%, mp 125-126°C, 1H NMR (400 MHz, DMSO-d6): 3.57 (d, J=6.1 Hz,
2H); 3.67 (s, 3H); 5.86 (t, J=6.2 Hz, 1H); 6.10-6.22 (m, 3H); 6.97 (d, J=8.0 Hz, 1H); 9.57 (bs,
2H); 13C NMR(100 MHz, DMSO-d6): 45.47; 55.80; 109.54; 111.08; 116.47; 121.85; 137.22;
145.88; 167.49; EIMS m/z 196 (M+). Anal. Calcd for C9H12N2O3: C 55.09; H 6.16; N 14.28;
found: C 55.12; H 6.15; N 14.26.
4.9.3.5. 2-(3,5-Dimethoxyphenylamino)-N-hydroxyacetamide (2e)
White power, 56%, mp 123-124°C, 1H NMR (400 MHz, DMSO-d6): 3.54 (d, J=6.1 Hz, 2H);
3.65 (s, 6H); 5.76 (s, 3H); 5.89 (t, J=6.2 Hz, 1H); 8.85 (s, 1H); 10.59 (s, 1H); 13C NMR (100
MHz, DMSO-d6): 45.04; 56.13; 110.97; 118.43; 138.11; 148.72; 167.63; EIMS m/z 226 (M+).
Anal. Calcd for C10H14N2O4: C 53.09; H 6.24; N 12.38; found: C 53.06; H 6.25; N 12.39.
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4.9.3.6. 2-(2,3-Dichlorophenylamino)-N-hydroxyacetamide (2f)
Light yellow power, 47%, mp 147-149°C, 1H NMR (400 MHz, DMSO-d6): 3.73 (d, J=5.8 Hz,
2H); 5.96 (t, J=5.3 Hz, 1H); 6.50 (d, J=8.2 Hz, 1H); 6.83 (d, J=7.6 Hz, 1H); 7.14 (t, J=8.1 Hz,
1H); 8.92 (s, 1H); 10.68 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 45.41; 109.36; 116.25;
117.80; 128.37; 132.29; 146.11; 168.02; EIMS m/z 234 (M+). Anal. Calcd for C8H8Cl2N2O2:
C 40.88; H 3.43; Cl 30.16; N 11.92; found: C 40.91; H 3.43; Cl 30.13; N 11.93.
4.9.3.7. 2-(2,4-Dichlorophenylamino)-N-hydroxyacetamide (2g)
1
White power, 50%, mp 145-146°C, H NMR(400 MHz, DMSO-d6): 3.71 (d, J=5.8 Hz, 2H);
5.77 (t, J=6.0 Hz, 1H); 6.55 (t, J=8.9 Hz, 1H); 7.20 (dd, J=8.8 Hz, J=3.1 Hz, 1H); 7.38 (d,
J=3.5 Hz, 1H); 9.86 (s, 2H); 13C NMR (100 MHz, DMSO-d6): 44.34; 112.76; 118.97; 119.99;
128.21; 128.63; 143.38; 166.64; EIMS m/z 234 (M+). Anal. Calcd for C8H8Cl2N2O2: C 40.88;
H 3.43; Cl 30.16; N 11.92; found: C 40.85; H 3.43; Cl 30.18; N 11.93.
4.9.3.8. 2-(3,4-Dichlorophenylamino)-N-hydroxyacetamide (2h)
Light yellow power, 55%, mp 116-118°C, 1H NMR (400 MHz, DMSO-d6): 3.60 (d, J=5.9 Hz,
2H); 6.45 (t, J=5.8 Hz, 1H); 6.55 (dd, J=8.8 Hz, J=2.7 Hz, 1H); 6.73 (d, J=2.4 Hz, 1H); 7.25
13
(d, J=8.8 Hz, 1H); 9.81 (bs, 2H); C NMR (100 MHz, DMSO-d6): 44.93; 112.53; 118.75;
120.06; 128.29; 128.47; 143.62; 167.04; EIMS m/z 234 (M+). Anal. Calcd for C8H8Cl2N2O2:
C 40.88; H 3.43; Cl 30.16; N 11.92; found: C 40.92; H 3.43; Cl 30.16; N 11.91.
4.9.3.9. 2-(3-Chlorophenylamino)-N-hydroxyacetamide (2i)
Light yellow power, 42%, mp 112-114°C, 1H NMR (400 MHz, DMSO-d6): 3.59 (d, J=6.0 Hz,
2H); 6.25 (t, J=5.8 Hz, 1H); 6.52 (d, J=8.0 Hz, 1H); 6.57 (d, J=8.0 Hz, 1H); 6.58 (s, 1H);
13
7.07 (t, J=8.2 Hz, 1H); 8.88 (s, 1H); 10.62 (s, 1H); C NMR (100 MHz, DMSO-d6): 45.06;
17

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110.54; 117.32; 119.08; 128.15; 129.70, 144.08, 166.88; EIMS m/z 200 (M+). Anal. Calcd
for C8H9ClN2O2: C 47.89; H 4.52; Cl 17.67; N 13.96; found: C 47.92; H 4.52; Cl 17.65; N
13.95.
4.9.3.10. 2-(4-Chlorophenylamino)-N-hydroxyacetamide (2j)
White power, 57%, mp 106-108°C, 1H NMR (400 MHz, DMSO-d6): 3.57 (d, J=6.1 Hz, 2H);
6.10 (t, J=6.0 Hz, 1H); 6.56 (d, J=8.8 Hz, 2H); 7.09 (d, J=8.8 Hz, 2H); 9.03 (s, 1H); 10.10 (s,
2H); 13C NMR (100 MHz, DMSO-d6): 45.12; 107.55; 114.69; 131.80; 148.17; 167.06; EIMS
m/z 200 (M+). Anal. Calcd for C8H9ClN2O2: C 47.89; H 4.52; Cl 17.67; N 13.96; found: C
47.84; H 4.52; Cl 17.69; N 13.97.
4.9.3.11. 2-(3-Bromophenylamino)-N-hydroxyacetamide (2k)
White power, 48%, mp 102-103°C, 1H NMR (400 MHz, DMSO-d6): 3.58 (d, J=6.2 Hz, 2H);
6.27 (t, J=6.2 Hz, 1H); 6.55 (dd, J=8.3 Hz, J=2.2 Hz, 1H); 6.70 (dd, J=8.0 Hz, J=1.8 Hz, 1H);
6.71 (d, J=2.5 Hz, 1H); 7.01 (t, J=7.9 Hz, 1H); 9.55 (s, 2H); 13C NMR (100 MHz, DMSO-d6):
44.93; 110.58; 117.21; 119.15; 128.09; 129.64, 144.18, 167.20; EIMS m/z 244 (M+). Anal.
Calcd for C8H9BrN2O2: C 39.21; H 3.70; Br, 32.60; N 11.43; found: C 39.25; H 3.70; Br,
32.56; N 11.44.
4.9.3.12. 2-(4-Bromophenylamino)-N-hydroxyacetamide (2l)
White power, 55%, mp 101-102°C, 1H NMR (400 MHz, DMSO-d6): 3.58 (d, J=6.0 Hz, 2H);
6.14 (t, J=6.2 Hz, 1H); 6.52 (d, J=8.6 Hz, 2H); 7.21 (d, J=8.7 Hz, 2H); 9.66 (s, 2H); 13C
NMR (100 MHz, DMSO-d6): 44.68; 107.36; 114.72; 131.74; 148.12; 167.14; EIMS m/z 244
(M+). Anal. Calcd for C8H9BrN2O2: C 39.21; H 3.70; Br, 32.60; N 11.43; found: C 39.27; H
3.70; Br, 32.57; N 11.42.
18

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4.9.3.13. 2-(3,5-Dichlorophenylamino)-N-hydroxyacetamide (2m)
Light yellow power, 60%, mp 121-122°C, 1H NMR (400 MHz, DMSO-d6): 3.62 (d, J=6.2 Hz,
2H); 6.56 (d, J=1.8 Hz, 2H); 6.62 (t, J=6.3 Hz, 1H); 6.64 (d, J=1.8 Hz, 1H); 8.90 (s, 1H);
10.60 (s, 1H); 13C NMR(100 MHz, DMSO-d6): 45.11; 110.78; 118.93; 138.10; 148.69;
167.82; EIMS m/z 234 (M+). Anal. Calcd for C8H8Cl2N2O2: C 40.88; H 3.43; Cl 30.16; N
11.92; found: C 40.84; H 3.43; Cl 30.19; N 11.93.
4.9.3.14. N-Hydroxy-2-(3-nitrophenylamino)acetamide (2n)
Yellow power, 45%, mp 119-121°C, 1H NMR (400 MHz, DMSO-d6): 3.71 (d, J=6.0 Hz, 2H);
6.72 (t, J=6.2Hz, 1H); 7.01 (dd, J=8.0 Hz, J=2.3 Hz, 1H); 7.28-7.38 (m, 2H); 7.40 (dd, J=7.7
Hz, J=2.1 Hz, 1H); 8.94 (s, 1H); 10.73 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 44.32;
105.95; 110.87; 119.21; 130.29; 149.18; 149.98; 166.77; EIMS m/z 211 (M+). Anal. Calcd
for C8H9N3O4: C 45.50; H 4.30; N 19.90; found: C 45.55; H 4.30; N 19.88.
4.9.3.15. 2-(2,5-Dichlorophenylamino)-N-hydroxyacetamide (2o)
Light yellow power, 47%, mp 141-142°C, 1H NMR (400 MHz, DMSO-d6): 3.74 (d, J=5.7 Hz,
2H); 5.94 (t, J=6.0 Hz, 1H); 6.56 (d, J=2.3 Hz, 1H); 6.64 (dd, J=8.4 Hz, J=2.3 Hz, 1H); 7.27
(d, J=8.4 Hz, 1H); 8.95 (s, 1H); 10.69 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 44.34;
112.76; 118.97; 119.99; 128.21; 128.63; 143.38; 166.64; EIMS m/z 234 (M+). Anal. CalCd
for C8H8Cl2N2O2: C 40.88; H 3.43; Cl 30.16; N 11.92; found: C 40.91; H 3.43; Cl 30.14; N
11.90.
4.9.3.16. N-Hydroxy-2-(phenylamino)acetamide (2p)
White power, 46%, mp 124-125°C, 1H NMR (400 MHz, DMSO-d6): 3.58 (d, J=6.1 Hz, 2H);
19

ACCEPTED MANUSCRIPT
6.85 (t, J=6.2 Hz, 1H); 6.34-6.78 (m, 3H); 7.08 (t, J=6.9 Hz, 2H); 9.80 (s, 2H); 13C NMR(100
MHz, DMSO-d6): 44.91; 112.80; 116.75; 129.25; 148.76; 167.53; EIMS m/z 166 (M+). Anal.
Calcd for C8H10N2O2: C 57.82; H 6.07; N 16.86; found: C 57.77; H 6.07; N 16.88.
4.9.3.17. 2-(2,6-Dichlorophenylamino)-N-hydroxyacetamide(2q)
White power, 51%, mp 131-132°C, 1H NMR (400 MHz, DMSO-d6): 3.88 (d, J=5.8 Hz, 2H);
5.30 (t, J=5.7 Hz, 1H); 6.84 (t, J=8.0 Hz, 1H); 7.32 (d, J=8.0 Hz, 2H); 8.94 (s, 1H); 10.60 (s,
13
1H); C NMR(100 MHz, DMSO-d6): 44.98; 110.89; 118.83; 138.27; 148.53; 167.19; EIMS
m/z 234 (M+). Anal. Calcd for C8H8Cl2N2O2: C 40.88; H 3.43; Cl 30.16; N 11.92; found: C
40.85; H 3.43; Cl 30.18; N 11.93.
4.9.3.18. 2-(3,5-Difluorophenylamino)-N-hydroxyacetamide (2r)
White power, 43%, mp 138-140°C, 1H NMR (400 MHz, DMSO-d6): 3.60 (d, J=6.1 Hz, 2H);
6.22 (dd, J=10.8 Hz, J=2.3 Hz, 2H); 6.26 (dd, J=9.8 Hz, J=2.3 Hz, 1H); 6.62 (t, J=6.2 Hz,
13
1H); 8.90 (s, 1H); 10.64 (s, 1H); C NMR (100 MHz, DMSO-d6): 43.07; 90.63 (t, J=26.1
Hz); 94.76 (d, J=28.1 Hz); 151.72 (t, J=13.4 Hz); 164.09 (dd, J=241.5 Hz; J=16.4 Hz);
167.19; EIMS m/z 202 (M+). Anal. Calcd for C8H8F2N2O2: C 47.53; H 3.99; F 18.80; N
13.86; found: C 47.57; H 3.99; F 18.78; N 13.85.
4.9.3.19. N-Hydroxy-2-(p-tolylamino)acetamide (2s)
Light yellow power, 50%, mp 139-140°C, 1H NMR (400 MHz, DMSO-d6): 2.15 (s, 3H); 3.54
(d, J=6.1 Hz, 2H); 5.60 (t, J=6.2 Hz, 1H); 6.47 (dd, J=8.3 Hz, 2H); 6.89 (d, J=8.0 Hz, 2H);
13
8.80 (s, 1H); 10.53 (s, 1H); C NMR (100 MHz, DMSO-d6): 20.99; 45.16; 107.31; 114.80;
131.71; 148.19; 167.05; EIMS m/z 180 (M+). Anal. Calcd for C9H12N2O2: C 59.99; H 6.71; N
15.55; found: C 59.97; H 6.71; N 15.56.
20

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4.9.3.20. N-Hydroxy-2-(4-methoxyphenylamino)acetamide (2t)
White power, 48%, mp 139-140°C, 1H NMR (400 MHz, DMSO-d6): 3.52 (d, J=6.2 Hz, 2H);
3.64 (s, 3H); 5.42 (t, J=6.3 Hz, 1H); 6.52 (d, J=8.9 Hz, 2H); 6.72 (d, J=8.9 Hz, 2H); 8.80 (s,
13
1H); 10.54 (s, 1H); C NMR (100 MHz, DMSO-d6): 44.92; 55.14; 107.43; 114.60; 131.82;
148.06; 167.79; EIMS m/z 196 (M+). Anal. Calcd for C9H12N2O3: C 55.09; H 6.16; N 14.28;
found: C 55.06; H 6.17; N 14.29.
4.9.3.21. 2-(2-Chlorophenylamino)-N-hydroxyacetamide (2u)
1
White power, 57%, mp 121-123°C, H NMR(400 MHz, DMSO-d6): 3.69 (d, J=5.9 Hz, 2H);
5.56 (t, J=5.9 Hz, 1H); 6.55 (dd, J=8.2 Hz, J=2.5 Hz, 1H); 6.62 (td, J=7.6 Hz, J=1.5 Hz, 1H )
7.12 (td, J=7.7 Hz, J=1.5 Hz, 1H); 7.26 (dd, J=7.9 Hz, J=1.5 Hz, 1H); 8.89 (s, 1H); 10.66 (s,
1H); 13C NMR (100 MHz, DMSO-d6): 44.36; 111.87; 117.56; 118.53; 128.43; 129.39,
144.14, 166.92; EIMS m/z 200 (M+). Anal. Calcd for C8H9ClN2O2: C 47.89; H 4.52; Cl 17.67;
N 13.96; found: C 47.92; H 4.52; Cl 17.66; N 13.95.
4.9.3.22. 2-(2-Bromophenylamino)-N-hydroxyacetamide (2v)
White power, 48%, mp 103-105°C, 1H NMR (400 MHz, DMSO-d6): 3.69 (d, J=5.7 Hz, 2H);
5.42 (t, J=5.6 Hz, 1H); 6.53-6.59 (m, 2H); 7.18 (td, J=8.2 Hz, J=1.5 Hz, 1H); 7.42 (dd, J=7.8
Hz, J=1.5 Hz, 1H); 8.91 (s, 1H); 10.67 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 45.30;
112.05; 117.94; 118.89; 128.36; 129.32, 144.27, 167.11; EIMS m/z 244 (M+). Anal. Calcd
for C8H9BrN2O2: C 39.21; H 3.70; Br, 32.60; N 11.43; found: C 39.25; H 3.70; Br, 32.58; N
11.42.
4.9.3.23. 2-(3,4-Dihydroquinolin-1(2H)-yl)-N-hydroxyacetamide (2w)
21

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White power, 53%, mp 132-134 °C, 1H NMR (400 MHz, DMSO-d6): 1.89 (p, J=6.2 Hz, 2H);
2.68 (t, J=6.2 Hz, 2H); 3.34-3.37 (m, 2H); 3.76 (s, 2H); 6.42 (d, J=8.2 Hz, 1H); 6.50 (t, J=7.3
Hz, 1H); 6.88 (d, J=7.2 Hz, 1H); 6.93 (t, J=7.5 Hz, 1H); 8.81(s, 1H); 10.56 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): 22.14; 28.07; 47.71; 49.08; 110.99; 115.78; 122.40; 127.46; 129.43;
144.98; 168.06; EIMS m/z 206 (M+). Anal. Calcd for C11H14N2O2: C 64.06; H 6.84; N 13.58;
found: C 64.09; H 6.84; N 13.56.
4.9.3.24. 3-(3,5-Dichlorophenylamino)-N-hydroxypropanamide (3a)
White power, 52%, mp 131-132 °C, 1H NMR (400 MHz, DMSO-d6): 2.29 (t, J=6.5 Hz, 2H);
3.48 (q, J=6.2 Hz, 2H); 6.56 (d, J=1.8 Hz, 2H); 6.62 (t, J=6.0 Hz, 1H); 6.64 (d, J=1.8 Hz,
1H); 8.85 (s, 1H); 10.54 (s, 1H); 13C NMR(100 MHz, DMSO-d6): 32.01; 44.95; 110.86;
118.87; 138.09; 148.68; 167.53; EIMS m/z 248 (M+). Anal. Calcd for C9H10Cl2N2O2: C,
43.40; H, 4.05; Cl, 28.47; N, 11.25; found: C, 43.43; H, 4.05; Cl, 28.46; N, 11.24.
4.9.3.25. N-Hydroxy-3-(phenylamino)propanamide (3b)
White power, 49%, mp 115-117 °C, 1H NMR (400 MHz, DMSO-d6): 2.23 (t, J=7.1 Hz, 2H);
3.23 (q, J=6.7 Hz, 2H); 5.54 (t, J=5.9 Hz, 1H); 6.53 (t, J=7.3 Hz, 1H); 6.56 (d, J=7.8 Hz, 2H);
13
7.07 (t, J=7.8 Hz, 2H); 8.76 (s, 1H); 10.43 (s, 1H); C NMR(100 MHz, DMSO-d6): 32.77;
44.05; 112.60; 116.62; 129.39; 148.97; 168.01; EIMS m/z 180 (M+). Anal. Calcd for
C9H12N2O2: C 59.99; H 6.71; N 15.55; found: C 59.95; H 6.72; N 15.57.
4.9.3.26. N-Hydroxy-3-(2-methoxyphenylamino)propanamide (3c)
Light yellow power, 48%, mp 101-102 °C, 1H NMR (400 MHz, DMSO-d6): 2.27 (t, J=6.6 Hz,
2H); 3.27 (q, J=6.4 Hz, 2H); 3.76 (s, 3H); 4.88 (t, J=5.9 Hz, 2H); 6.55(d, J=7.3 Hz, 1H);
6.56(d, J=7.0 Hz, 1H); 6.78 (t, J=8.0 Hz, 1H); 6.79 (t, J=7.9 Hz, 1H); 8.81 (s, 1H); 10.49 (s,
22

ACCEPTED MANUSCRIPT
13
1H); C NMR(100 MHz, DMSO-d6): 32.44; 44.67; 55.71; 109.86; 110.27; 116.80; 121.45;
137.91; 146.90; 167.73; EIMS m/z 210 (M+). Anal. Calcd for C10H14N2O3: C 57.13; H 6.71;
N 13.33; found: C 57.11; H 6.72; N 13.34.
4.9.3.27. 3-(2-Chlorophenylamino)-N-hydroxypropanamide (3d)
Light yellow power, 42%, mp 101-102 °C, 1H NMR (400 MHz, DMSO-d6): 2.30 (t, J=6.8 Hz,
2H); 3.35 (q, J=6.4 Hz, 2H); 5.39 (t, J=5.8 Hz, 1H); 6.60 (td, J=7.6 Hz, J=1.4 Hz, 1H); 6.74
(dd, J=8.2 Hz, J=1.4 Hz, 1H); 7.15 (td, J=7.7 Hz, J=1.5 Hz, 1H); 7.25 (dd, J=7.8 Hz, J=1.5
Hz, 1H); 8.82 (s, 1H); 10.51 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 32.18; 44.79; 111.80;
117.51; 118.59; 128.37; 129.42, 144.21, 167.12; EIMS m/z 214 (M+). Anal. Calcd for
C9H11ClN2O2: C 50.36; H 5.17; Cl 16.52; N 13.05; found: C 50.31; H 5.17; Cl 16.53; N
13.06.
4.9.3.28. 3-(4-Fluorophenylamino)-N-hydroxypropanamide (3e)
1
Light yellow power, 58%, mp: 100-102 °C, H NMR (400 MHz, DMSO-d6): 2.22 (t, J=7.1
Hz, 2H); 3.20 (t, J=7.1 Hz, 2H); 5.50 (s, 1H); 6.55 (dd, J=9.0 Hz, J=4.3 Hz, 2H); 6.92 (t,
13
J=8.8 Hz, 2H); 8.75 (s, 1H); 10.43 (s, 1H); C NMR (100 MHz, DMSO-d6): 33.01; 45.09;
113.57 (d, J=7.2Hz); 115.66 (d, J=22.4Hz); 145.43; 155.07 (d, J=232.7Hz); 167.44; EIMS
m/z 198 (M+). Anal. Calcd for C9H11FN2O2: C 54.54; H 5.59; F 9.59; N 14.13; found: C
54.57; H 5.59; F 9.58; N 14.12.
4.9.3.29. 3-(2-Bromophenylamino)-N-hydroxypropanamide (3f)
Light yellow power, 53%, mp 121-123 °C, 1H NMR (400 MHz, DMSO-d6): 2.30 (t, J=6.7 Hz,
2H); 3.34 (q, J=6.5 Hz, 2H); 5.27 (t, J=5.8 Hz, 1H); 6.54 (td, J=7.6 Hz, J=1.5 Hz, 1H);
6.72(dd, J=8.2 Hz, J=1.5 Hz, 1H); 7.19 (td, J=7.9 Hz, J=1.5 Hz, 1H); 7.41 (dd, J=7.8 Hz,
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13
J=1.5 Hz, 1H); 8.82 (s, 1H); 10.52 (s, 1H); C NMR (100 MHz, DMSO-d6): 32.07; 40.05;
109.18; 111.76; 117.78; 129.17; 132.71; 145.24; 168.18; EIMS m/z 258 (M+). Anal. Calcd
for C9H11BrN2O2: C 47.12; H 4.28; Br 30.84; N 10.81; found: C 47.16; H 4.28; Br 30.82; N
10.80.
4.9.3.30. 3-(3,5-Dimethylphenylamino)-N-hydroxypropanamide (3g)
1
White power, 55%, mp 127-128 °C, H NMR (400 MHz, DMSO-d6): 2.14 (s, 6H); 2.21 (t,
J=7.1 Hz, 2H); 3.21 (q, J=6.7 Hz, 2H); 5.35 (t, J=5.9 Hz, 1H); 6.18 (s, 2H); 6.19 (s, 1H); 8.74
(s, 1H); 10.42 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 21.75; 32.80; 39.86; 110.52; 118.27;
138.11; 148.93; 168.15; EIMS m/z 208 (M+). Anal. Calcd for C11H16N2O2: C 63.44; H 7.74;
N 13.45; found: C 63.48; H 7.73; N 13.43.
4.9.3.31. 3-(2,5-Dichlorophenylamino)-N-hydroxypropanamide (3h)
White power, 48%, mp: 123-124 °C, 1H NMR (400 MHz, DMSO-d6): 2.28 (t, J=6.8 Hz, 2H);
3.36(q, J=6.7 Hz, 2H); 5.74 (t, J=5.8 Hz, 1H); 6.61 (dd, J=8.4 Hz, J=2.4 Hz, 1H); 6.76 (d,
J=2.4 Hz, 1H); 7.26 (d, J=8.4 Hz, 1H); 8.82 (s, 1H); 10.52 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 32.15; 44.97; 112.71; 118.92; 119.98; 128.26; 128.55; 143.42; 166.99; EIMS m/z
248 (M+). Anal. Calcd for C9H10Cl2N2O2: C 43.40; H 4.05; Cl 28.47; N 11.25; found: C
43.43; H 4.05; Cl 28.45; N 11.24.
4.9.3.32. 3-(3,4-Dichlorophenylamino)-N-hydroxypropanamide (3i)
White power, 43%, mp 123-125 °C, 1H NMR (400 MHz, DMSO-d6): 2.21 (t, J=6.9 Hz, 2H);
3.24 (q, J=6.6 Hz, 2H); 6.13 (t, J=5.9 Hz, 1H); 6.55 (dd, J=8.8 Hz, J=2.8 Hz, 1H); 6.74 (d,
J=2.7 Hz, 1H); 7.25 (d J=8.7 Hz, 1H); 8.76 (s, 1H); 10.43 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 32.30; 45.11; 112.82; 118.93; 119.92; 128.27; 128.66; 143.34; 167.04; EIMS m/z
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248 (M+). Anal. Calcd for C9H10Cl2N2O2: C 43.40; H 4.05; Cl 28.47; N 11.25; found: C
43.37; H 4.05; Cl 28.49; N 11.26.
4.9.3.33. 3-(2,6-Dichlorophenylamino)-N-hydroxypropanamide (3j)
White power, 47%, mp 103-105 °C, 1H NMR (400 MHz, DMSO-d6): 2.23 (t, J=6.8 Hz, 2H);
3.48 (q, J=6.7 Hz, 2H); 4.86 (t, J=6.6 Hz, 1H); 6.87 (t, J=8.0 Hz, 1H); 7.34 (dd, J=8.0 Hz,
13
J=1.5 Hz, 2H); 8.79 (s, 1H); 10.48 (s, 1H); C NMR(100 MHz, DMSO-d6): 32.06; 44.92;
110.95; 118.76; 138.23; 148.35; 167.20; EIMS m/z 248 (M+). Anal. Calcd for C9H10Cl2N2O2:
C 43.40; H 4.05; Cl 28.47; N 11.25; found: C 43.45; H 4.05; Cl 28.45; N 11.24.
4.9.3.34. N-Hydroxy-3-(4-nitrophenylamino) propanamide (3k)
Yellow power, 42%, mp 159-160 °C, 1H NMR (400 MHz, DMSO-d6): 2.28 (t, J=6.7 Hz, 2H);
3.40 (q, J=6.6 Hz, 2H); 6.65 (d, J=9.3 Hz, 2H); 7.35 (t, J=5.8 Hz, 1H); 7.99 (d, J=9.2 Hz,
2H); 8.82 (s, 1H); 10.49 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 32.27; 45.12; 107.44;
114.89; 134.21; 149.38; 167.14; EIMS m/z 225 (M+). Anal. Calcd for C9H11N3O4: C 48.00; H
4.92; N 18.66; found: C 47.97; H 4.92; N 18.67.
4.9.3.35. N-Hydroxy-3-(3-nitrophenylamino) propanamide (3l)
Yellow power, 49%, mp 130-131 °C, 1H NMR (400 MHz, DMSO-d6): 2.26 (t, J=6.1 Hz, 2H);
3.33 (q, J=6.2 Hz, 2H, δ 3.34 containing the HOD signal ); 6.44 (t, J=5.8 Hz, 1H); 6.99 (d,
J=2.3 Hz, 1H); 7.20-7.52 (m, 3H); 9.70 (s, 2H); 13C NMR (100 MHz, DMSO-d6): 32.32;
39.45; 105.63; 110.26; 118.67; 130.39; 149.30; 149.97; 167.86; EIMS m/z 225 (M+). Anal.
Calcd for C9H11N3O4: C 48.00; H 4.92; N 18.66; found: C 48.04; H 4.92; N 18.65.
4.9.3.36. N-Hydroxy-3-(2-nitrophenylamino) propanamide (3m)
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1
Red power, 51%, mp 144-145 °C, H NMR (400 MHz, DMSO-d6): 2.40 (t, J=6.4 Hz, 2H);
3.59 (q, J=6.3 Hz, 2H); 6.70 (t, J=8.1 Hz, 1H); 7.08 (d, J=8.1 Hz, 1H); 7.55 (t, J=7.3 Hz, 1H);
13
8.07 (d, J=8.0 Hz, 1H); 8.27 (t, J=5.8 Hz, 1H); 9.00 (s, 1H); 10.62 (s, 1H); C NMR (100
MHz, DMSO-d6): 32.19; 41.45; 105.90; 110.33; 118.72; 130.35; 149.37; 149.95; 167.28;
EIMS m/z 225 (M+). Anal. Calcd for C9H11N3O4: C 48.00; H 4.92; N 18.66; found: C 47.97;
H 4.92; N 18.67.
4.9.3.37. 3-(2,4-Dichlorophenylamino)-N-hydroxypropanamide (3n)
White power, 40%, mp: 111-113 °C, 1H NMR (400 MHz, DMSO-d6): 2.29 (t, J=6.9 Hz, 2H);
3.34 (q, J=6.5 Hz, 2H), 5.58 (t, J=6.6 Hz, 1H); 6.74 (d, J=8.8 Hz, 1H); 7.19 (dd, J=8.8 Hz,
J=2.4 Hz, 1H); 7.35 (d, J=2.0 Hz, 1H); 8.84 (s, 1H); 10.52 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): 32.05; 40.24; 112.50; 118.83; 119.52; 128.34; 128.67; 143.42; 168.10; EIMS m/z
248 (M+). Anal. Calcd for C9H10Cl2N2O2: C 43.40; H 4.05; Cl 28.47; N 11.25; found: C
43.45; H 4.05; Cl 28.45; N 11.24.
4.9.3.38. 3-(2,3-Dichlorophenylamino)-N-hydroxypropanamide (3o)
White power, 47%, mp 154-144 °C, 1H NMR (400 MHz, DMSO-d6): 2.29 (t, J=6.0 Hz, 2H);
3.37 (q, J=6.0 Hz, 2H); 5.76 (t, J=5.6 Hz, 1H); 6.71 (d, J=8.3 Hz, 1H); 6.80 (d, J=7.9 Hz,
13
1H); 7.15 (t, J=8.1 Hz, 1H); 8.85 (s, 1H); 10.54 (s, 1H); C NMR (100 MHz, DMSO-d6):
32.06; 40.05; 109.91; 116.00; 117.25; 128.94; 132.10; 146.06; 168.06; EIMS m/z 248 (M+).
Anal. Calcd for C9H10Cl2N2O2: C 43.40; H 4.05; Cl 28.47; N 11.25; found: C 43.36; H 4.05;
Cl 28.49; N 11.26.
4.9.3.39. 3-(3,5-Difluorophenylamino)-N-hydroxypropanamide (3p)
White power, 51%, mp 102-103 °C, 1H NMR (400 MHz, DMSO-d6): 2.22 (t, J=6.3 Hz, 2H);
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3.25 (q, J=6.5 Hz, 2H); 6.19 (t, J=8.2 Hz, 1H); 6.21 (d, J=9.6 Hz, 1H); 6.34 (t, J=6.2 Hz, 1H);
13
8.81 (s, 1H); 10.47 (s, 1H); C NMR (100 MHz, DMSO-d6): 32.40; 39.45; 90.52 (t, J=26.7
Hz); 94.92 (d, J=27.7 Hz); 151.63 (t, J=13.9 Hz); 164.01 (dd, J=240.7 Hz; J=16.7 Hz);
167.82; EIMS m/z 216 (M+). Anal. Calcd for C9H10F2N2O2: C 50.00; H 4.66; F 17.58; N
12.96; found: C 50.06; H 4.66; F 17.56; N 12.95.
4.9.3.40. 3-(3,4-Dihydroquinolin-1(2H)-yl)-N-hydroxypropanamide (3q)
1
Light yellow power, 57%, mp 218-220 °C, H NMR (400 MHz, DMSO-d6): 1.83 (quint,
J=6.3 Hz, 2H); 2.20 (t, J=7.1 Hz, 2H); 2.65 (t, J=6.4 Hz, 2H); 3.21 (t, J=5.6 Hz, 2H); 3.47 (t,
J=7.1 Hz, 2H); 6.47 (t, J=7.3 Hz, 1H); 6.58 (d, J=8.2 Hz, 1H); 6.86 (d, J=7.3 Hz, 1H); 6.96 (t,
13
J=7.8 Hz, 1H); 8.76 (s, 1H); 10.46 (s, 1H); C NMR (100 MHz, DMSO-d6): 22.19; 28.00;
29.72; 47.66; 49.05; 110.93; 115.84; 122.42; 127.40; 129.36; 144.93; 168.16; EIMS m/z 220
(M+). Anal. Calcd for C12H16N2O2: C 65.43; H 7.32; N 12.72; found: C 65.46; H 7.32; N
12.71.
Acknowledgments
The work was financed by grants from National Natural Science Foundation (grant No.
81273382) of China, by a Graduate Research Innovation Project supported by Jishou
University (grant No. JGY201655, No. Jdy17019). The authors acknowledge the
contributions of the following scientists to the manuscript: Xiangxi Autonomous Prefecture
People’s Hospital (led by Min-Jing Chen, and Dong-Ping Zhou), Jishou University (Xian-Hui
Li), Hunan University (Li-Jun Wu).
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