3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease inhibitors: Synthesis, molecular docking and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2016.07.052

Helicobacter pylori urease is involved in several physiologic responses such as stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids were synthesized and evaluated for urease inhibition. Out of these compounds, 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active inhibitor with IC₅₀of 0.15?±?0.05?μM, showing a mixed inhibition with both competitive and uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and 0.12?μg·mL^?1for K_iand K_i′, respectively. The plasma protein binding assays suggested that d24 exhibited moderate binding to human and rabbit plasma proteins.

Full text of DOI:10.1016/j.bmc.2016.07.052

Accepted Manuscript
3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease in-
hibitors: Synthesis, molecular docking and biological evaluation
Wei-Kang Shi, Rui-Cheng Deng, Peng-Fei Wang, Qin-Qin Yue, Qi Liu, Kun-
Ling Ding, Mei-Hui Yang, Hong-Yu Zhang, Si-Hua Gong, Min Deng, Wen-
Run Liu, Qiu-Ju Feng, Zhu-Ping Xiao, Hai-Liang Zhu
PII:
S0968-0896(16)30569-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.07.052
BMC 13165
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 July 2016
22 July 2016
23 July 2016
Please cite this article as: Shi, W-K., Deng, R-C., Wang, P-F., Yue, Q-Q., Liu, Q., Ding, K-L., Yang, M-H., Zhang,
H-Y., Gong, S-H., Deng, M., Liu, W-R., Feng, Q-J., Xiao, Z-P., Zhu, H-L., 3-Arylpropionylhydroxamic acid
derivatives as Helicobacter pylori urease inhibitors: Synthesis, molecular docking and biological evaluation,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.052
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3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease
inhibitors: Synthesis, molecular docking and biological evaluation
Wei-Kang Shi,a Rui-Cheng Deng,a Peng-Fei Wang,b Qin-Qin Yue,a Qi Liu,a Kun-Ling Ding,a
Mei-Hui Yang,a Hong-Yu Zhang,a Si-Hua Gong,a Min Deng,a Wen-Run Liu,a Qiu-Ju Feng,a
Zhu-Ping Xiao,a  Hai-Liang Zhua, b
*
aCollege of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, P. R. China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.
R. China
Abstract: Helicobacter pylori urease is involved in several physiologic responses such as
stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of
urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused
our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids
were synthesized and evaluated for urease inhibition. Out of these compounds,
3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active
inhibitor with IC50 of 0.15±0.05 μM, showing a mixed inhibition with both competitive and
uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and
0.12 μg·mL-1 for Ki and Ki', respectively. The plasma protein binding assays suggested that d24
exhibited moderate binding to human and rabbit plasma proteins.
Keywords: 3-Arylpropionylhydroxamic acid; H. pylori urease inhibitor; Plasma protein binding;
Kinetics study; Non-linear fitting
 Corresponding author. E-mail address: xiaozhuping2005@163.com
* Corresponding author. E-mail address: zhuhl@nju.edu.cn
1

3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease
inhibitors: Synthesis, molecular docking and biological evaluation
Wei-Kang Shi,a Rui-Cheng Deng,a Peng-Fei Wang,b Qin-Qin Yue,a Qi Liu,a Kun-Ling Ding,a
Mei-Hui Yang,a Hong-Yu Zhang,a Si-Hua Gong,a Min Deng,a Wen-Run Liu,a Qiu-Ju Feng,a
Zhu-Ping Xiao,a  Hai-Liang Zhua, b
*
aCollege of Chemistry and Chemical Engineering, Jishou University, Jishou 416000, P. R. China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.
R. China
Abstract: Helicobacter pylori urease is involved in several physiologic responses such as
stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Thus, inhibition of
urease is taken for a good chance to treat H. pylori-caused infections, we have therefore focused
our efforts on seeking novel urease inhibitors. Here, a series of arylpropionylhydroxamic acids
were synthesized and evaluated for urease inhibition. Out of these compounds,
3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24) was the most active
inhibitor with IC50 of 0.15±0.05 μM, showing a mixed inhibition with both competitive and
uncompetitive aspects. Non-linear fitting of kinetic data gives kinetics parameters of 0.13 and
0.12 μg·mL-1 for Ki and Ki', respectively. The plasma protein binding assays suggested that d24
exhibited moderate binding to human and rabbit plasma proteins.
Keywords: 3-Arylpropionylhydroxamic acid; H. pylori urease inhibitor; Plasma protein binding;
Kinetics study; Non-linear fitting
 Corresponding author. E-mail address: xiaozhuping2005@163.com
* Corresponding author. E-mail address: zhuhl@nju.edu.cn
2

1 Introduction:
Helicobacter pylori (H. pylori) is one of the most successful human bacterial parasites, which
colonizes more than half of the human population.1 Most infected people would be asymptomatic
in their lifetime. Unfortunately, about 15-20% of them would develop into severe gastroduodenal
pathologies, such as stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas.2
For the past decades, the main recommendation treatments of H. pylori infection were that the
infected people regularly use proton pump inhibitors (PPI), bismuth compounds, and antibiotics
within a long period.3, 4 However, this recommendation was not safe for regular use of PPIs
which will cause false-negative results for gastric cancer diagnosis. In addition, the use of
antibiotics is prone to cause drug resistance.5, 6 Therefore, seeking novel and efficient drugs for
the treatment of gastrointestinal tract disease attracts extensive attention from worldwide
researchers.
Urease (urea amidohydrolase EC 3.5.1.5), a Ni-containing metalloenzyme, accelerates the
hydrolysis of urea to ammonia and carbon dioxide by at least 1014 over the spontaneous
reaction.7 With the help of a constitutive urease production, the excessive ammonia released
from urea is dissolved in succus gastricus, leading to an increase of pH value to create a friendly
environment for H. pylori survival.8, 9 The continuously produced ammonia was confidently
considered to enhance the permeability of gastric mucosa and to have toxicity for parietal cell,
which induces gastric inflammation and increases the risk of the development of duodenal and
gastric ulcers, gastric adenocarcinoma and gastric lymphoma.10, 11 Therefore, inhibition of urease
is taken for a good chance to treat infections caused by H. pylori.12, 13 In the past decades,
hundreds of urease inhibitors have been identified such as urea derivatives, hydroxamic acids,
phosphoramidates, and polyphenols.14 Among them, phosphoramidates are the most active but
3

they were not marketed as drugs probably because of the rapid hydrolysis in low pH of gastric
juice.15
Hydroxamic acid has a significant chelating property, being inclined to form strong complexes
with a variety of transition metals, and is usually involved in binding the metal ions of the active
site to inactivate the enzyme. Therefore, hundreds of hydroxamic acids were synthesized as
possible agents to treat H. pylori infections,16, 17 and acetohydroxamic acid (AHA) was the best
studied urease inhibitor.18 However, AHA shows only moderate activity and a relative large dose
(at least 1000 mg/d for adults) must be required.15 Therefore, highly potent urease inhibitors with
excellent hydrolytic stability are urgently required to be developed. Recently, we focused our
efforts on seeking novel urease inhibitors with structural diversity. Deoxybenzoins,19
deoxybenzoin oximes,20 1,2-diarylethanes,21 flavonoids,22, 23 flavenes,24 and furan-2(5H)-ones25
were therefore reported as potent urease inhibitors in our group. In order to impove activities of
urease inhibitors, we try to combine flavonoids with hydroxamic acid moiety to a molecule
(Scheme 1). Hydroxamic acid could combine the bottom of the active chamber and flavonoids
could integrate the top of the active chamber, through the binding mode, we might synthesis
mixed urease inhibitors and enhance urease inhibitory activities.
Scheme 1 Based flavonoids tricyclic skeleton urease inhibitor
4

Recently, salicylaldehyde derivatives were reported to exhibit moderate urease inhibitory
activities,26 while analogues of salicylic acid (1) (Scheme 2) was reported to display excellent
urease inhibitory activities.27 Inspiration of these findings, we combined the pharmacophore
benzoic acid moiety in salicylic acid and Flavonoids tricyclic structure and hydroxamic acid
moiety in AHA to establish a novel scaffold (2) for urease inhibitor discovery (Scheme 2). Based
on this hypothesis, some derivatives (3) were synthesized and evaluated as urease inhibitors
(Scheme 2). The initial results revealed a significant improvement of the potency against H.
pylori urease in comparison with both parents. It is to be noted that our data confirmed the
excellent potency of compounds bearing the salicylic fragment with IC50 in the micromolar range
against H. pylori urease.18 As a continuation of this effort, a series of 2 with N-methyl moiety or
salicylic core were synthesized for further understanding of structure-activity relationships
(SAR). The molecular docking and enzyme kinetics studies were also herein reported.
Scheme 2 Compound structures.
2 Materials and methods
2.1 Chemistry
All chemicals (reagent grade) were purchased from Aldrich (U.S.A), Greatwall Chemical
Reagent Co., Ltd (China) and Sinopharm Chemical Reagent Co., Ltd (China). Separation of the
5

compounds by column chromatography was carried out with silica gel 60 (200-300 mesh ASTM,
E. Merck). The quantity of silica gel was 20-50 times according to the weight charged on the
column. Then, the eluates were monitored by TLC. Melting points (uncorrected) were measured
on a XT4 MP apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained from the
1
Mariner System 5304 mass spectrometer. All H NMR and selected 13C NMR spectra were
recorded on the Bruker AV-400 spectrometer at 25 °C with TMS and solvent signals being
allotted as internal standards. Chemical shifts were reported in ppm (δ). Elemental analyses were
performed on a CHN-O-Rapid instrument being within ± 0.4 % of the theoretical values.
2.1.1 General procedure for preparation of compounds b23-b24
Two equivalent of anhydrous potassium carbonate and benzyl chloride were added into a
solution of compound b21 or b22 (15 mmol) in 60mL of DMSO and catalytic amount of
potassium iodide (1.6 nmol) were then added under nitrogen atmosphere. The resulted mixture
was stirred for 2-3 h at 72 °C. After cooling, the residue was poured into 60 mL of distilled water,
which was extracted thrice with 300 mL of AcOEt. The combined organic layer was washed
twice with 40 mL of saturated salt water and dried over MgSO4. Subsequently, the solvent was
removed under reduced pressure. The resulted residue was then purified by column
chromatography on silica gel to give b23 or b24 in an excellent yield.
2.1.2 General procedure for preparation of compounds c1-c24
An appropriately substituted benzaldehyde (10 mmol) and newly activated zinc (20 mmol) were
added into anhydrous THF (18 mL), which was heated to reflux for 10 min under stirring. After
the mixture was cooled to 42-45°C, 10 mmol of ethyl bromoacetate in anhydrous THF (7 mL)
was added under nitrogen atmosphere within 30 min. The reaction was monitored by TLC, and
6

the mixture was partitioned between water and AcOEt. The organic layer was washed twice with
saturated NH4Cl, saturated NaHCO3 and saturated brine respectively. After drying over MgSO4,
solvent was removed under reduced pressure. The result residue was purified by a column
chromatography on silica gel to yield compounds c1-c24 (Scheme 3).
Scheme 3 Synthetic route of 3-hydroxy-3-phenylpropionyl-hydroxamic acids.
2.1.3 General procedure for preparation of compounds d1-d24
20 mmol of sodium methoxide and 10 mmol of hydroxylamine hydrochloride or N-methyl
hydroxylamine hydrochloride were added into absolute methanol (20 mL) at room temperature,
which was stirred for 10 min, and followed by the addition of compound c (5 mmol). After
completion of the reaction (monitored by TLC), the mixture was diluted with 20 mL of ethanol.
Removal of the precipitation gave a slight yellow solution, which was condensed under reduced
pressure. The residue was purified by a column chromatography on silica gel (AcOEt-petroleum
as eluent) to furnish compound d (Scheme 3).
2.1.3.1 3-(2-chlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d1)
1
White powder, 62%, mp 113-115°C, H NMR (CDCl3): 2.89-3.05 (m, 2H); 3.35 (s, 3H);
5.51-5.61 (m, 1H); 6.64 (bs, 2H); 7.23-7.37 (m, 3H); 7.63-7.70 (m, 1H); EIMS m/z 229 (M+).
Anal. Calcd for C10H12ClNO3: C, 52.30; H, 5.27; Cl, 15.44; N, 6.10; Found: C, 52.38; H, 5.26;
7

Cl, 15.42; N, 6.11.
2.1.3.2 3-(2-methoxyphenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d2)
White powder, 33%, mp 108-111°C, 1H NMR (DMSO-d6): 2.48-2.52 (m, 1H); 2.64-2.72 (m, 1H);
3.12 (s, 3H); 3.78 (s, 3H); 5.06 (bs, 1H); 5.30-5.33 (m, 1H); 6.93 (d, J=4.8Hz, 1H); 6.95 (d,
J=6.1Hz, 1H); 7.22 (t, J=7.7Hz, 1H); 7.45 (d, J=7.3Hz, 1H); 9.77 (bs, 1H); 13C NMR (100 MHz,
DMSO-d6): 36.10; 55.73; 63.60; 110.86; 120.67; 126.53; 128.19; 133.95; 155.73; 171.61; EIMS
m/z 225 (M+). Anal. Calcd for C11H15NO3: C, 58.66; H, 6.71; N, 6.22; Found: C, 58.60; H, 6.72;
N, 6.23.
2.1.3.3 3-(3-fluorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d3)
1
White powder, 39%, mp 84-86°C, H NMR (DMSO-d6): 2.60 (dd, J=15.4Hz, J=4.6Hz, 1H);
2.81 (dd, J=15.4Hz, J=8.2Hz, 1H); 3.09 (s, 3H); 5.00-5.05 (m, 1H); 5.42 (d, J=4.2Hz, 1H); 7.05
(t, J=8.9Hz, 1H); 7.17 (t, J=12.9Hz, 1H); 7.19 (t, J=8.2Hz, 1H); 7.36 (dd, J=14.1Hz, J=4.6Hz,
1H); 9.85 (s, 1H); EIMS m/z 213 (M+). Anal. Calcd for C10H12FNO3: C, 56.33; H, 5.67; F, 8.91;
N, 6.57; Found: C, 56.44; H, 5.66; F, 8.90; N, 6.56.
2.1.3.4 3-(3-chlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d4)
1
White powder, 58%, mp 86-88°C, H NMR (DMSO-d6): 2.60 (dd, J=15.5Hz, J=5.0Hz, 1H);
2.80 (dd, J=15.4Hz, J=8.0Hz, 1H); 3.08 (s, 3H); 4.98-5.03 (m, 1H); 5.45 (d, J=4.6Hz, 1H);
7.30-7.9 (m, 4H); 9.86 (s, 1H); EIMS m/z 229 (M+). Anal. Calcd for C10H12ClNO3: C, 52.30; H,
5.27; Cl, 15.44; N, 6.10; Found: C, 52.25; H, 5.27; Cl, 15.46; N, 6.11.
2.1.3.5 3-(3-bromophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d5)
1
White powder, 51%, mp 81-83°C, H NMR (DMSO-d6): 2.61 (dd, J=15.4Hz, J=4.9Hz, 1H);
2.81 (dd, J=15.4Hz, J=8.1Hz, 1H); 3.09 (s, 3H); 4.98-5.03 (m, 1H); 5.44 (d, J=4.3Hz, 1H); 7.28
(t, J=7.7Hz, 1H); 7.34 (d, J=7.6Hz, 1H); 7.43 (d, J=7.7Hz, 1H); 7.54 (s, 1H); 9.85 (s, 1H); EIMS
8

m/z 273 (M+). Anal. Calcd for C10H12BrNO3: C, 43.82; H, 4.41; Br, 29.15; N, 5.11; Found: C,
43.75; H, 4.41; Br, 29.17; N, 5.12.
2.1.3.6 3-(3-methoxyphenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d6)
1
White powder, 62%, mp 79-80°C, H NMR (DMSO-d6): 2.57 (dd, J=15.4Hz, J=4.2Hz, 1H);
2.79 (dd, J=15.4Hz, J=8.6Hz, 1H); 3.10 (s, 3H); 3.75 (s, 3H); 4.95-5.05 (m, 1H); 5.27 (bs, 1H);
6.79 (dd, J=7.4Hz, J=2.2Hz, 1H); 6.90-6.93 (m, 2H); 7.23 (t, J=8.1Hz, 1H); 9.83 (s, 1H); EIMS
m/z 225 (M+). Anal. Calcd for C11H15NO3: C, 58.66; H, 6.71; N, 6.22; Found: C, 58.73; H, 6.70;
N, 6.21.
2.1.3.7 3-(4-fluorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d7)
1
White powder, 32%, mp 78-80°C, H NMR (DMSO-d6): 2.59 (dd, J=15.4Hz, J=4.8Hz, 1H);
2.81 (dd, J=15.4Hz, J=8.2Hz, 1H); 3.09 (s, 3H); 5.00-5.03 (m, 1H); 5.33 (s, 1H); 7.13 (t,
J=8.9Hz, 2H); 7.38 (dd, J=8.2Hz, J=5.9Hz, 2H); 9.84 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
36.07; 42.34; 68.80; 115.13 (d, J=20.8Hz); 128.19 (d, J=8.0Hz); 142.18; 161.59 (d, J=240.5Hz);
171.07; EIMS m/z 213 (M+). Anal. Calcd for C10H12FNO3: C, 56.33; H, 5.67; F, 8.91; N, 6.57;
Found: C, 56.27; H, 5.66; F, 8.92; N, 6.58.
2.1.3.8 3-(4-chlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d8)
1
White powder, 39%, mp 95-96°C, H NMR (CDCl3): 2.69-2.78 (m, 2H); 3.00-3.04 (bs, 1H);
3.34 (s, 3H); 5.15-5.25 (m, 1H); 7.37 (s, 4H); EIMS m/z 229 (M+). Anal. Calcd for C10H12ClNO3:
C, 52.30; H, 5.27; Cl, 15.44; N, 6.10; Found: C, 52.35; H, 5.26; Cl, 15.42; N, 6.11.
2.1.3.9 3-(4-bromophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d9)
1
White powder, 32%, mp 97-99°C, H NMR (DMSO-d6): 2.58 (dd, J=15.4Hz, J=4.9Hz, 1H);
2.80 (dd, J=15.4Hz, J=8.0Hz, 1H); 3.08 (s, 3H); 4.96-5.01 (m, 1H); 5.38 (d, J=4.1Hz, 1H); 7.31
(d, J=8.2Hz, 2H); 7.50 (d, J=8.4Hz, 2H); 9.84 (s, 1H); EIMS m/z 273 (M+). Anal. Calcd for
9

C10H12BrNO3: C, 43.82; H, 4.41; Br, 29.15; N, 5.11; Found: C, 43.75; H, 4.42; Br, 29.18; N,
5.12.
2.1.3.10 3-(4-methoxyphenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d10)
1
White powder, 48%, mp 101-103°C, H NMR (CDCl3): 2.94 (dd, J=16.4Hz, J=10.4Hz, 1H);
3.04 (dd, J=16.4Hz, J=7.7Hz, 1H); 3.24 (s, 3H); 3.85 (s, 3H); 5.42-5.47 (m, 1H); 6.96 (d,
J=8.7Hz, 2H); 7.37 (d, J=8.7Hz, 2H); 13C NMR (100 MHz, DMSO-d6): 36.08; 55.46; 69.03;
113.82; 127.44; 138.02; 158.62; 171.31; EIMS m/z 225 (M+). Anal. Calcd for C11H15NO3: C,
58.66; H, 6.71; N, 6.22; Found: C, 58.62; H, 6.72; N, 6.23.
2.1.3.11 3-(4-(dimethylamino)phenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d11)
1
White powder, 41%, mp 72-73°C, H NMR (CDCl3): 2.66 (dd, J=16.2Hz, J=3.6Hz, 1H); 2.78
(dd, J=16.2Hz, J=9.4Hz, 1H); 2.87 (bs, 1H); 2.93 (s, 6H); 3.70 (s, 3H); 5.02-5.06 (m, 1H); 6.70
(d, J=7.5Hz, 1H); 7.23 (d, J=7.8Hz, 1H); EIMS m/z 238 (M+). Anal. Calcd for C12H18N2O3: C,
60.49; H, 7.61; N, 11.76; Found: C, 60.57; H, 7.60; N, 11.74.
2.1.3.12 3-(4-(benzyloxy)phenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d12)
1
White powder, 45%, mp 124-126°C, H NMR (CDCl3): 2.92-2.99 (m, 1H); 3.02-3.08 (m, 1H);
3.26 (s, 3H); 3.33 (bs, 1H); 5.11 (bs, 1H); 5.13 (s, 2H); 5.43-5.48 (m, 1H); 7.04 (d, J=8.7Hz, 2H);
7.38 (d, J=8.6Hz, 2H); 7.43-7.48 (m, 5H); 1H); 3.36 (s, 3H); 5.14-5.19 (m, 1H); 7.26 (d,
13
J=8.2Hz, 1H); 7.47 (d, J=8.0Hz, 1H); 7.55 (s, 1H); C NMR (100 MHz, CDCl3): 31.67; 40.04;
70.10; 79.18; 115.20; 127.48; 128.13; 128.18; 128.69; 129.24; 136.66; 159.44; EIMS m/z 301
(M+). Anal. Calcd for C17H19NO4: C, 67.76; H, 6.36; N, 4.65; Found: C, 67.69; H, 6.37; N, 4.65.
2.1.3.13 3-(2,3-dichlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d13)
1
White powder, 63%, mp 119-121°C, H NMR (CDCl3): 2.90-3.16 (m, 2H); 3.37 (s, 3H);
5.52-5.63 (m, 1H); 6.85 (bs, 2H); 7.24-7.33 (m, 1H); 7.43 (dd, J=8.8Hz, J=2.0Hz, 1H); 7.60 (dd,
10

J=8.6Hz, J=1.7Hz, 1H); EIMS m/z 263 (M+). Anal. Calcd for C10H11Cl2NO3: C, 45.48; H, 4.20;
Cl, 26.85; N, 5.30; Found: C, 45.45; H, 4.20; Cl, 26.87; N, 5.29.
2.1.3.14 3-(2,4-dichlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d14)
1
White powder, 63%, mp 127-129°C, H NMR (DMSO-d6): 2.45-2.50 (m, 1H); 2.75 (dd,
J=16.0Hz, J=9.4Hz, 1H); 3.10 (s, 3H); 5.32-5.35 (m, 1H); 5.57 (d, J=4.4Hz, 1H); 7.45 (dd,
13
J=8.4Hz, J=2.0Hz, 1H); 7.55 (d, J=1.8Hz, 1H); 7.61 (d, J=8.4Hz, 1H); 9.86 (s, 1H); C NMR
(100 MHz, CDCl3): 35.73; 37.48; 67.05; 127.72; 128.31; 129.14; 131.71; 134.01; 138.51; 165.17;
EIMS m/z 263 (M+). Anal. Calcd for C10H11Cl2NO3: C, 45.48; H, 4.20; Cl, 26.85; N, 5.30; Found:
C, 45.53; H, 4.19; Cl, 26.83; N, 5.29.
2.1.3.15 3-(2,4-dichlorophenyl)-3-hydroxypropionylhydroxamic acid (d15)
1
White powder, 68%, mp 163-166°C, H NMR (DMSO-d6): 2.12 (dd, J=14.2Hz, J=9.6Hz, 1H);
2.27 (dd, J=14.2Hz, J=2.7Hz, 1H); 5.25-5.30 (m, 1H); 5.66 (d, J=4.7Hz, 1H); 7.46 (dd, J=8.4Hz,
J=1.6Hz, 1H); 7.56 (d, J=1.6Hz, 1H); 7.59 (d, J=8.4Hz, 1H); 8.80 (s, 1H); 10.41 (s, 1H); EIMS
m/z 249 (M+). Anal. Calcd for C9H9Cl2NO3: C, 43.22; H, 3.63; Cl, 28.35; N, 5.60; Found: C,
43.19; H, 3.63; Cl, 28.37; N, 5.61.
2.1.3.16 3-(2,6-dichlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d16)
1
White powder, 57%, mp 116-117°C, H NMR (CDCl3): 2.92-2.99 (m, 1H); 3.02-3.08 (m, 1H);
3.36 (s, 1H); 3.43 (s, 3H); 6.02 (dd, J=8.8Hz, J=3.0Hz, 1H); 7.22 (t, J=7.9Hz, 1H); 7.35 (s, 1H);
7.37 (d, J=7.8Hz, 1H); EIMS m/z 263 (M+). Anal. Calcd for C10H11Cl2NO3: C, 45.48; H, 4.20; Cl,
26.85; N, 5.30; Found: C, 45.43; H, 4.21; Cl, 26.88; N, 5.31.
2.1.3.17 3-(3,4-dichlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d17)
1
White powder, 66%, mp 108-109°C, H NMR (DMSO-d6): 2.65-2.71 (m, 1H); 2.81-2.96 (m,
1H); 3.36 (s, 3H); 5.14-5.19 (m, 1H); 7.26 (d, J=8.2Hz, 1H); 7.47 (d, J=8.0Hz, 1H); 7.55 (s, 1H);
11

13C NMR (100 MHz, CDCl3): 35.85; 39.76; 40.91; 69.39; 125.03; 127.77; 130.62; 131.81;
132.81; 142.72; 172.14; EIMS m/z 263 (M+). Anal. Calcd for C10H11Cl2NO3: C, 45.48; H, 4.20;
Cl, 26.85; N, 5.30; Found: C, 45.43; H, 4.21; Cl, 26.87; N, 5.30.
2.1.3.18 3-(3,4-bis(benzyloxy)phenyl)-3-dihydroxy-N-methylpropionylhydroxamic acid
(d18)
1
White powder, 44%, mp 54-56°C, H NMR (DMSO-d6): 2.55-2.63 (m, 2H); 3.58 (s, 3H);
4.85-4.90 (m, 1H); 5.11 (s, 4H); 5.42 (d, J=4.8Hz, 1H); 6.86 (d, J=8.3Hz, 1H); 7.00 (d, J=8.2Hz,
1H); 7.10 (s, 1H); 7.29-7.48 (m, 10H); EIMS m/z 407 (M+). Anal. Calcd for C24H25NO5: C,
70.74; H, 6.18; N, 3.44; Found: C, 70.63; H, 6.19; N, 3.44.
2.1.3.19
3-(4-(benzyloxy)-3-methoxyphenyl)-3-dihydroxy-N-methylpropionylhydroxamic
acid (d19)
1
White powder, 51%, mp 137-139°C, H NMR (DMSO-d6): 2.53-2.57 (m, 1H); 2.79 (dd,
J=15.2Hz, J=8.4Hz, 1H); 3.08 (s, 3H); 3.76 (s, 3H);4.93-4.96 (m, 1H); 5.05 (s, 2H); 5.20 (d,
J=4.4Hz, 1H); 6.82 (d, J=8.4Hz, 1H); 6.95 (d, J=8.4Hz, 1H); 6.97 (s, 1H); 7.33 (t, J=7.0Hz, 1H);
7.39 (t, J=7.0Hz, 2H); 7.44 (d, J=6.8Hz, 2H); EIMS m/z 331 (M+). Anal. Calcd for C18H21NO5:
C, 65.24; H, 6.39; N, 4.23; Found: C, 65.30; H, 6.38; N, 4.23.
2.1.3.20 3-(3,4-dimethoxyphenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d20)
White powder, 52%, mp 146-147°C, 1H NMR (CDCl3): 2.93-3.09 (m, 2H); 3.27 (s, 3H); 3.94 (s,
3H); 3.96 (s, 3H); 5.43-5.48 (m, 1H); 6.92 (d, J=8.2Hz, 1H); 6.97 (d, J=1.7Hz, 1H); 7.00 (dd,
J=8.2Hz, J=1.8Hz, 1H); EIMS m/z 255 (M+). Anal. Calcd for C12H17NO5: C, 56.46; H, 6.71; N,
5.49; Found: C, 56.50; H, 6.70; N, 5.48.
2.1.3.21 3-(5-chloro-2-hydroxyphenyl)-3-hydroxy-N-methylpropionylhydroxamic acid (d21)
12

1
White powder, 47%, mp 120-122°C, H NMR (DMSO-d6): 2.50-2.57 (m, 1H); 2.62-2.69 (m,
1H); 3.10 (s, 3H); 5.20-5.23 (m, 2H); 6.77 (d, J=8.5Hz, 1H); 7.07 (dd, J=8.5Hz, J=2.7Hz, 1H);
7.32 (s, 1H); 9.81 (bs, 2H); EIMS m/z 245 (M+). Anal. Calcd for C10H12ClNO4: C, 48.89; H, 4.92;
Cl, 14.43; N, 5.70; Found: C, 48.81; H, 4.92; Cl, 14.44; N, 5.71.
2.1.3.22 3-(5-chloro-2-hydroxyphenyl)-3-hydroxypropionylhydroxamic acid (d22)
1
White powder, 49%, mp 117-119°C, H NMR (DMSO-d6): 2.04 (dd, J=14.0Hz, J=9.7Hz, 1H);
2.32 (dd, J=14.1Hz, J=2.8Hz, 1H); 5.17 (d, J=9.1Hz, 1H); 5.35 (d, J=3.4Hz, 1H); 6.77 (d,
J=8.6Hz, 1H); 7.07 (dd, J=8.5Hz, J=2.7Hz, 1H); 7.29 (d, J=2.6Hz, 1H); 8.76 (s, 1H); 9.76 (s,
1H); 10.34 (s, 1H); 13C NMR (100 MHz, DMSO-d6): 63.59; 69.83; 114.15; 127.46; 127.63;
128.18; 128.90; 137.42; 153.46; EIMS m/z 231 (M+). Anal. Calcd for C9H10ClNO4: C, 46.67; H,
4.35; Cl, 15.31; N, 6.05; Found: C, 46.64; H, 4.36; Cl, 15.33; N, 6.06.
2.1.3.23 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxy-N-methylpropionylhydroxamic acid
(d23)
White powder, 62%, mp 154-156°C, 1H NMR (DMSO-d6): 2.87-2.95 (m, 2H); 3.13 (s, 3H); 5.12
(s, 2H); 5.41-5.50 (m, 1H); 6.91 (d, J=8.6Hz, 1H); 7.25 (dd, J=8.6Hz, J=2.4Hz, 1H);
7.38-7.45(m, 5H); 7.61 (d, J=2.1Hz, 1H); EIMS m/z 335 (M+). Anal. Calcd for C17H18ClNO4: C,
60.81; H, 5.40; Cl, 10.56; N, 4.17; Found: C, 60.77; H, 5.41; Cl, 10.58; N, 4.17.
2.1.3.24 3-(2-benzyloxy-5-chlorophenyl)-3-hydroxypropionylhydroxamic acid (d24)
1
White powder, 66%, mp 122-124°C, H NMR (DMSO-d6): 2.08 (dd, J=13.2Hz, J=9.7Hz, 1H);
2.37 (d, J=13.8Hz, 1H); 5.16 (s, 2H); 5.33-5.36 (m, 1H); 5.43 (d, J=4.4Hz, 1H); 7.06(d, J=8.8Hz,
1H); 7.25 (dd, J=8.7Hz, J=2.7Hz, 1H); 7.32 (d, J=7.2Hz, 1H); 7.38 (d, J=7.6Hz, 1H); 7.40 (d,
J=7.2Hz, 2H); 7.49 (d, J=7.3Hz, 2H); 8.78 (s, 1H); 10.35 (s, 1H); EIMS m/z 321 (M+). Anal.
Calcd for C16H16ClNO4: C, 59.73; H, 5.01; Cl, 11.02; N, 4.35; Found: C, 59.80; H, 5.00; Cl,
13

11.00; N, 4.35.
2.3. Measurement of urease inhibitory activity
2.3.1. Preparation of H. Pylori
H. pylori 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), which has been
reported in the literatures.19-22, 24
2.3.2. Preparation of H. pylori urease
The bacteria were collected through 50 mL broth cultures (2.0×108 CFU/mL) which were
centrifuged (5000 g, 4 °C) by medical centrifuge. After washing twice with phosphate-buffered
saline (pH 7.4), the H. pylori precipitation was prepared which was then added into 3 mL
distilled water and protease inhibitors under sonication for 1 min. The supernatant was desalted
through Sephadex G-25 column. Subsequently, centrifugation (12,000 g, 4 °C) was performed.
Finally, the residue was added into an equal volume of glycerol and stored at 4 °C for the
following testing.
2.3.3. Activity testing
Reaction mixtures comprising 25 μL (10U) of H. pylori urease and 25 μL of the test compound,
was pre-incubated for 50 min at 37 °C in a 96-well assay plate. Urease activity was determined
by measuring the absorbance of ammonia production and using the indophenol method described
by Weatherburn.28
2.4. 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 d22 or d24
([I]). Nonlinear fitting curves to data of V and [S] were used to determine the type of enzyme
14

inhibition based on the general kinetics equation (eq. 1). For a specific [I], a (eq. 2), b (eq. 3) and
c (eq. 4) are constants, equation 1 was therefore converted to eq. 5 to perform curve fitting.
Consequently, data of b or c 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. The results of nonlinear fitting were shown in Fig. 1A and Fig. 1B.
Fig. 1. (A) Each velocity (V) was nonlinearly fitted against the concentrations of urea [S] in the
presence of a specific concentration of compound d22. (B) Each velocity (V) was nonlinearly
fitted against the concentrations of urea [S] in the presence of a specific concentration of
compound d24.
Vmax [S]
[I]
Km (1 )  (1
Ki
V 
(1)
[I]
Ki '
)[S]
a Vmax
(2)
(3)
[I]
b  Km (1
)
Ki
[I]
c 1
(4)
Ki '
15

ax
(5)
y 
b  cx
Where Km is the Michaelis constant, Vmax is the maximal velocity, Ki is the dissociation constant
for the dissociation of the urease-inhibitor complex into free enzyme and inhibitor, and Ki′ is the
dissociation constant for the dissociation of the urease-urea-inhibitor complex into enzyme-urea
complex and free inhibitor.
2.5. Protocol of docking study
The automated docking studies were carried out by using AutoDock version 4.2. First, AutoGrid
component of the program pre-calculated a three-dimensional grid of interaction energies based
on the macromolecular target by using the AMBER force field. A grid box of 58×58×58 Å size
(x, y, z) with a spacing of 0.375 Å for the mode of d22 (d21, d24) and grid maps were created,
which represent 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 macromolecules. The genetic algorithm with local search (GA-LS) was
chosen to search the best conformers. The parameters were set by 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 7.5×106 energy evaluations, and a maximum number of 2.7×104 generations. A
mutation rate of 0.02 and a crossover rate of 0.8 were chosen. Results differing by less than 0.5
Ǻ 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.
2.6. Plasma Protein Binding
The plasma protein binding of d22 or d24 was estimated in triplicate with ultra-filtration method
by using millipore Amicon® centrifugal filter devices (Billerica, MA, USA). Human plasma or
16

freshly collected rabbit plasma and the tested compound were incubated for 20 min at 37 ºC.
Samples in triplicate were placed in centrifugal filter devices and centrifuged at 5000 r/min for
15 min at 37 ºC. Initial plasma samples and ultra-filtrates were diluted 8-fold and then subjected
to an ultraviolet-visible spectrophotometer. The non-specific binding (NBS) to the filtration
membrane was estimated by spiked equivalent concentration in phosphate buffer saline (PBS)
and further processing the samples similar to the plasma samples. The plasma protein binding
was determined by using the eq. 6:
Cf
% Protein binding  (1-
(1 NBS))100
(6)
Co
Where C0 is the dosing concentration of the compound; Cf is the concentration in the filtered
matrix; and NBS is calculated as 1-(Cp,f/Cp,uf) where Cp,f is the average concentration in the
filtered PBS and Cp,uf is the average concentration in the unfiltered PBS. Student’s t-test of
significance was applied to compare the effect of concentration on plasma protein binding of
tested compounds.
3. Results and discussion
3.1. Chemistry
Twenty-four 3-hydroxy-3-phenylpropionylhydroxamic acids were synthesized and the synthetic
routes were outlined in Scheme 3. With some modifications, β-hydroxy esters (c1-c24) were
formed from corresponding aldehydes (b1-24) with ethyl bromoacetate in the presence of Zn
according to the classical Reformatsky reaction. Specifically, significant improvement of the
yield was achieved when anhydrous THF was selected as solvent in comparison with the
previously reported processes.29 The resulted esters (c1-c24) were then converted to the desired
hydroxamic acids (d1-d24) in the presence of hydroxylamine hydrochloride (N-methyl
17

hydroxylamine hydrochloride) and sodium methoxide in methanol.30 These hydroxamic acid
derivatives were all reported for the first time, and fully characterized by elementary analyses,
EIMS and 1H NMR together with some selected 13C NMR.
3.2. Measurement of urease inhibitory activity
Recently, we focus our efforts on improving the activity of urease inhibitors bearing hydroxamic
acid moiety.18, 31 To explore the binding pocket dimensions and its steric tolerance in amide
moiety, a series of N-methyl-3-hydroxy-3-phenylpropionylhydroxamic acids was therefore
designed and synthesized. All compounds described in this work were evaluated for inhibitory
activity against H. pylori urease (Table 1). Many N-methyl analogs were weak or inactive
inhibitors of the enzyme with IC50 values over 50 μM, and only one compound d23
(IC50=26.2±2.1 μM) showed a compared potency to the positive AHA in this series. These results
revealed that N-methyl is not tolerance, possibly by interfering with the binding of hydroxamic
acid to the target site. This claim was supported by the following observations: removal of the
methyl
group
of
hydroxamic
acid
segment
gave
3-(3-chlorophenyl)-3-hydroxypropionylhydroxamic acid,18 d15, d22 and d24, and showed a 30-
to 800-fold increase in activity. Therefore, no substituent might be introduced for improvement.
18

Table 1 Structure and inhibitory activity (IC50) against H. pylori urease of d1-d24
structure
entry
IC50(μM)
R
R1
Cl
OMe
H
R2
H
R3
H
R4
H
d1
d2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
56.6±1.6
84±3.5
H
H
H
d3
F
H
H
71.2±4.2
48±1.4
d4
H
Cl
Br
OMe
H
H
H
d5
H
H
H
78.2±3.6
89.2±3.9
77.2±1.5
87.4±2.4
97.6±5.7
328±15
d6
H
H
H
d7
H
F
H
d8
H
H
Cl
H
d9
H
H
Br
H
d10
d11
d12
d13
d14
d15
d16
d17
d18
d19
d20
d21
d22
d23
d24
AHA
H
H
OMe
N(CH3)2
OBn
H
H
H
H
H
287±27
H
H
H
198±12
Cl
Cl
Cl
Cl
H
Cl
H
H
95.8±5.7
367±32
Cl
H
H
Cl
H
13.4±0.9
77.9±0.5
90.4±4.6
376±19
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
Cl
H
Cl
OBn
OMe
OMe
Cl
Cl
Cl
Cl
---
Cl
H
OBn
OBn
OMe
H
H
H
H
258±17
H
H
405±29
H
OH
OH
OBn
OBn
---
30.5±0.4
1.10±0.08
26.2±2.1
0.15±0.05
23.8±1.5
H
H
CH3
H
H
H
H
H
---
---
---
19

As for SAR of the phenyl moiety, change of Cl from the meta-position (d4) to the ortho- or
para-position led to analogues (d1 or d8) with a slight loss of activity. Potency against H. pylori
urease was also decreased when 3-Cl or 4-Cl was replaced with F, Br, OMe, N(Me)2 or OBn, but
a similar downward tendency was not observed in derivatives substituted at the 2-position. In
details, introduction of a chloride atom at the ortho-position (d4 vs d13, d1 vs d16) resulted in a
moderate loss in potency, and a further loss was found for the conversion of Cl to OMe group
(d1 vs d2). On the contrary, introduction of OH group at the ortho-position (d4 vs d21) led to a
slight increase in activity, and protection of OH group as OBn group further caused a 2- to 7-fold
27
improvement. This is consistent with the previous observations,26,
and confirmed the
importance of salicylic scaffold for urease inhibition. Among the obtained compounds, d22 and
d24 (Scheme 4) showed excellent inhibitory activity against H. pylori urease with IC50 values of
1.10±0.08 and 0.15±0.05 μM respectively, being over 20-fold more potent than the positive
control AHA.
Scheme 4 Compound structures of d22 and d24.
3.3 Kinetic of urease inhibition by compound d22 and d24
Based on results of enzyme assays, compounds d22 and d24, showed the potential inhibitory
activity against H. pylori urease, were selected for kinetic studies. It is worth noting that urea
20

was found to show substrate inhibition against H. pylori urease (Fig. 2). Substrate inhibition
refers to inhibition of enzyme activity at high substrate concentrations. It will lead to a velocity
curve rising to a maximum as the substrate concentration increased, and then descending either
to zero or to a non-zero asymptote.32 Although many enzymes, such as dehydrogenases, kinases,
methyltransferases and hydroxylases, were reported to be inhibited by their own substrates,
substrate inhibition of H. pylori urease as well as other urease was reported for the first time. The
substrate inhibition constant (Ksi) was derived by nonlinear least-square fitting the substrate
inhibition equation (eq 7) to the data of reaction rates versus substrate concentrations.33 In
particular, nonlinear fitting of the substrate-velocity data also yielded the kinetic parameter Km
with a value for urea of 3.11 mM in agreement with the previously reported values.34, 35 As
shown in Fig. 2, urea showed the substrate inhibition phenomenon at the concentration higher
than 4 mM with Ksi value of 6.47±1.78 mM. Therefore, kinetic studies in the presence of d22 or
d24 were performed at the concentration lower than 4 mM to avoid the visible substrate
inhibition.
Fig. 2 Substrate inhibition of H. pylori urease
21

Vmax [S]
V 
(7)
1
Km [S]
[S]2
Ksi
Where Km is the Michaelis constant, Vmax is the maximal velocity, and Ksi is the substrate
inhibition constant.
In the past decades, hundreds of hydroxamic acid derivatives were determined as urease
inhibitors. Investigation of the kinetics of inhibition by several hydroxamic acids on urease by
using Lineweaver-Burk plots revealed that some inhibited noncompetitively and others
competitively.36, 37 Based on the same method, 3-hydroxy-3-phenylpropiohydroxamic acids,
novel hydroxamic acid derivatives found in our group, were determined as an inhibitor with
mixed inhibition mechanism, exhibiting both competitive and uncompetitive aspects.18 However,
data fitting gave an uncompetitive Ki (Ki′) with negative value. In comparison of the linear
regression analysis (the Lineweaver-Burk analysis) with the non-linear fitting, the standard
deviations for the non-linear fitting are lower than for the Lineweaver-Burk analysis. Mason
claimed that while the Lineweaver-Burk plot is useful for quick evaluations of data, it raises the
risks of biasing kinetic parameters and hiding inhibitory kinetics, or even mislead investigators.38
Enzyme kinetic data (V and (S)) of d22 and d24 were consequently fitted with non-linear
least-squares fitting based on eq. 5 (Fig. 1A and Fig. 1B). A linear regression analysis was
subsequently used to fit [I] and the resulted b or c from non-linear fitting (Fig. 3A, Fig. 3B, Fig.
4A and Fig. 4B), giving the Ki parameters of 1.11 μg·mL-1 and 0.13 μg·mL-1, and Ki′ parameters
of 0.28 μg·mL-1 and 0.12 μg·mL-1 for d22 and d24 respectively. Ki values revealed that the
binding affinity of d24 is higher than d22, which is consistent with that of IC50 suggested.
22

Fig. 3. (A) The constants b from the corresponding V-S plot were plotted against concentrations
of compound d22. (B) The constant c from the corresponding V-S plot were plotted against
concentrations of compound d22.
Fig. 4. (A) The constants b from the corresponding V-S plot were plotted against concentrations
of compound d24. (B) The constants c from the corresponding V-S plot were plotted against
concentrations of compound d24.
3.4. Molecular docking
For further understand the SAR observed in enzyme assays, compounds d21, d22 and d24 were
selected to be performed molecular docking studies. The binding results are displayed in Fig. 5
23

and Fig. 6. In the three-dimensional model shown as surface (Fig. 5), it is important to note that
the three representative inhibitors (d21, d22 and d24) adopt nearly identical orientations within
the enzyme active site, and the hydroxamic moiety reaches the bottom of the active pocket,
where AHA is occupied in enzyme complex 1E9Y. In docking models of d22 and d24 (Fig. 6A
and Fig. 6C), the O atom in NHO- moiety coordinates two nikel ions in a bidentate fashion and
establishes a hydrogen bond with His-221, while the H atom in NHO- moiety builds three
hydrogen bonds with COO- of Asp-362 and CO of Ala-365, respectively. However, in the
docking model of d21, the NMeO- moiety coordinates only one nikel ion and only the hydrogen
bond with His-221 is maintained (Fig. 6B). As an evidence for the observation in enzyme assays,
replacement of NH of hydroxamic moiety with NMe leads to a significant loss of potency.
Obviously, 5-chlorosalicylic scaffold makes stabilizing hydrophobic contacts with residues
Met-317 and Met-366 and is also importance for the ligand-binding affinity. In comparison with
d22, benzyl group of d24 forms additional hydrophobic contacts with the enzyme surface mainly
established by Asn-168 and Ala-169, which may probably contribute to the slight increase in
potency of compound d24. Overall, the molecular docking study could give a qualitative
interpretation of the observed SAR in biological assays.
24

Fig. 5. Binding modes of compounds d21, d22 and d24 with H. pylori urease. The enzyme is
shown as surface; while d21 is shown as yellow sticks, and d22 as purple and d24 as green.
25

Fig. 6. Binding modes of compounds d21, d22 and d24 with H. pylori urease with the enzyme
shown cartoonand the ligand as sticks: (A) compound d21; (B) compound d22; (C) compound
d24.
3.5. Plasma Protein Binding
Plasma protein binding (PPB) of drugs is a significant parameter in pharmacokinetics studies,
which influences the distribution, metabolism and excretion of the drug in vivo.39, 40 Therefore,
determination of plasma protein binding is important in drug discovery and development, and
PPB has become a common assay in drug discovery organizations.41 In spite of the critical nature
of PPB, PPB itself has no relevance to efficacy in most cases and should not be optimized
through structural modification or used to develop structure-PPB relationships.42, 43 Bearing these
considerations in mind, PPB was measured for the two most active compounds d22 and d24, and
the results are summarized in Table 2. Both compounds exhibited moderate binding to human
and rabbit plasma proteins (from 37.4±1.6 to 52.8±2.7%). In comparison with d22, a relatively
low protein binding was observed for d24, which has to do with its high lipophilicity.44
26

Table 2 Plasma Protein Binding (%) of the synthetic compounds d22 and d24
compounds
human
52.8±2.7
37.4±1.6
rabbit
45.2±2.6
43.8±2.2
d22
d24
4. Conclusions
In the present study, arylpropionylhydroxamic acid derivatives have been developed leading to
the identification of compounds d22 and d24, which showed IC50 values of 1.10±0.08 and
0.15±0.05 μM with 20-fold more potent than the positive control AHA. The PPB assay revealed
that compound d24 exhibited moderate binding to human and rabbit plasma proteins. These data
suggested that d24 could be used as a lead for a structural improvement to obtain a more potent
drug for the treatment of H. pylori-caused disease. It is to be noted that urea was found to exhibit
substrate inhibition phenomenon against H. pylori urease for the first time, and showed
significant inhibition at the concentration higher than 4 mM. Based on this finding, the kinetic
studies for d24 revealed a mixed inhibition with both competitive and uncompetitive aspects.
Non-linear fitting of kinetic data gave these two kinetics parameters of 0.13 and 0.12 μg·mL-1,
respectively. SAR analysis and molecular docking disclosed that substitution of NMe for NH in
the hydroxamic moiety led to a significant loss of potency and replacement of OH with
benzyloxy group in salicylic scaffold resulted in an increase in potency.
Acknowledgments
The work was financed by grants from National Natural Science Foundation (grant No.
81273382) of China, by a Project supported by Hunan Provincial Natural Science Foundation of
27

China (grant No. 2015JJ2116) and a Graduate Research Innovation Project supported by Jishou
University (grant No. JGY201637).
References
1. Parsonnet, J. Aliment Pharmacol Ther 1995, 9, 4.
2. Chey, W. D.; Murthy, U.; Linscheer, W.; Barish, C.; Riff, D.; Rubin, H.; Safdi, M.; Schwartz, H.; Shah, U.;
Wruble, L. The American journal of gastroenterology 1998, 93, 16.
3. Chey, W.; Chathadi, K.; Montague, J.; Ahmed, F.; Murthy, U. The American journal of gastroenterology 2001,
96, 1028.
4. Hunter, P. EMBO reports 2012, 13, 680.
5. Simmons, K. J.; Chopra, I.; Fishwick, C. W. Nature Reviews Microbiology 2010, 8, 501.
6. Lodhi, M. A.; Nawaz, S. A.; Iqbal, S.; Khan, K. M.; Rode, B. M.; Choudhary, M. I. Bioorganic & medicinal
chemistry 2008, 16, 3456.
7. Sachs, G.; Weeks, D. L.; Wen, Y.; Marcus, E. A.; Scott, D. R.; Melchers, K. Physiology 2005, 20, 429.
8. Stingl, K.; Altendorf, K.; Bakker, E. P. Trends in microbiology 2002, 10, 70.
9. Maroney, M. J.; Ciurli, S. Chemical reviews 2013, 114, 4206.
10. Algood, H. M. S.; Cover, T. L. Clinical microbiology reviews 2006, 19, 597.
11. Modolo, L. V.; de Souza, A. X.; Horta, L. P.; Araujo, D. P.; de Fátima, Â. Journal of advanced research 2015,
6, 35.
12. Saeed, A.; Tehseen, Y.; Rafique, H.; Furtmann, N.; Bajorath, J.; Flörke, U.; Iqbal, J. MedChemComm 2014, 5,
1371.
13. Azizian, H.; Nabati, F.; Sharifi, A.; Siavoshi, F.; Mahdavi, M.; Amanlou, M. Journal of molecular modeling
2012, 18, 2917.
14. Kosikowska, P.; Berlicki, Ł. Expert opinion on therapeutic patents 2011, 21, 945.
15. Zhang, Y.; Feng, J.; Jia, Y.; Wang, X.; Zhang, L.; Liu, C.; Fang, H.; Xu, W. Journal of medicinal chemistry
2011, 54, 2823.
16. Neelarapu, R.; Holzle, D. L.; Velaparthi, S.; Bai, H.; Brunsteiner, M.; Blond, S. Y.; Petukhov, P. A. Journal of
medicinal chemistry 2011, 54, 4350.
17. Xiao, Z. P.; Peng, Z. Y.; Dong, J. J.; Deng, R. C.; Wang, X. D.; Ouyang, H.; Yang, P.; He, J.; Wang, Y. F.; Zhu,
M. European journal of medicinal chemistry 2013, 68, 212.
18. Xiao, Z. P.; Shi, D. H.; Li, H. Q.; Zhang, L. N.; Xu, C.; Zhu, H. L. Bioorganic & medicinal chemistry 2007, 15,
3703.
19. Li, H. Q.; Xiao, Z. P.; Yan, T.; Lv, P. C.; Zhu, H. L. European journal of medicinal chemistry 2009, 44, 2246.
20. Xiao, Z. P.; Ma, T. W.; Fu, W. C.; Peng, X. C.; Zhang, A. H.; Zhu, H. L. European journal of medicinal
chemistry 2010, 45, 5064.
21. Xiao, Z. P.; Wang, X. D.; Peng, Z. Y.; Huang, S.; Yang, P.; Li, Q. S.; Zhou, L. H.; Hu, X. J.; Wu, L. J.; Zhou, Y.
Journal of agricultural and food chemistry 2012, 60, 10572.
22. Xiao, Z. P.; Peng, Z. Y.; Dong, J. J.; He, J.; Ouyang, H.; Feng, Y. T.; Lu, C. L.; Lin, W. Q.; Wang, J. X.; Xiang,
Y. P. European journal of medicinal chemistry 2013, 63, 685.
23. Peng, Z. Y.; Wang, X. D.; Feng, Y. T.; He, J.; Xiao, Z. P. Journal of Jishou University (Natural Sciences
Edition) 2012, 6, 021.
24. Wang, X. D.; Wei, W.; Wang, P. F.; Yi, L. C.; Shi, W. K.; Xie, Y. X.; Wu, L. Z.; Tang, N.; Zhu, L. S.; Peng, J.
Bioorganic & medicinal chemistry 2015, 23, 4860.
25. Kubo, J.; Lee, J. R.; Kubo, I. Journal of Agricultural and Food Chemistry 1999, 47, 533.
26. Mao, W. J.; Lv, P. C.; Shi, L.; Li, H. Q.; Zhu, H. L. Bioorganic & medicinal chemistry 2009, 17, 7531.
27. Weatherburn, M. Analytical chemistry 1967, 39, 971.
28. Zou, X. L.; Du, G. F.; Sun, W. F.; He, L.; Ma, X. W.; Gu, C. Z.; Dai, B. Tetrahedron 2013, 69, 607.
29. Kalin, J. H.; Butler, K. V.; Akimova, T.; Hancock, W. W.; Kozikowski, A. P. Journal of medicinal chemistry
2012, 55, 639.
30. Deng, R. C.; Wei, W.; Wang, X. D.; Xiao, Z. P. Chinese Journal of New Drugs 2014, 23, 2189.
28

31. Jang, E. H.; Park, S. A.; Chi, Y. M.; Lee, K. S. Biochemical and biophysical research communications 2015,
461, 487.
32. Sun, H.; Zhou, X.; Zhang, X.; Wu, B. Journal of agricultural and food chemistry 2015, 63, 6001.
33. Fishbein, W. N.; Daly, J. E. Experimental Biology and Medicine 1970, 134, 1083.
34. Fishbein, W. N. Journal of Biological Chemistry 1969, 244, 1188.
35. Park J. B.; Imamura L.; Kobashi K.; Itoh H.; Miyazaki T.; Horisaki T. Biological and Pharmaceutical Bulletin
1995, 18, 208.
36. Fishbein, W. N.; Carbone, P. P. Journal of Biological Chemistry 1965, 240, 2407.
37. Mason, G. F.; Lai, J. C. Metabolic brain disease 2000, 15, 133.
38. Mehvar, R. American Journal of Pharmaceutical Education 2005, 69, 103.
39. Wagner, J. G. Pharmacology & therapeutics 1981, 12, 537.
40. Di, L.; Umland, J. P.; Trapa, P. E.; Maurer, T. S. Journal of pharmaceutical sciences 2012, 101, 1327.
41. Riccardi, K.; Cawley, S.; Yates, P. D.; Chang, C.; Funk, C.; Niosi, M.; Lin, J.; Di, L. Journal of pharmaceutical
sciences 2015, 104, 2627.
42. Liu, X.; Chen, C.; ECA Hop, C. Current topics in medicinal chemistry 2011, 11, 450.
43. Fernández, S.; Giglio, J.; Rey, A. M.; Cerecetto, H. Bioorganic & medicinal chemistry 2012, 20, 4040.
44. Stepanić, V.; Žiher, D.; Gabelica-Marković, V.; Jelić, D.; Nunhuck, S.; Valko, K.; Koštrun, S. European journal
of medicinal chemistry 2012, 47, 462.
Supporting Information:
Additional Supporting Information may be found in the online version of this article:
Appendix. 1H NMR Spectra of reported compounds.
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