N-monoarylacetothioureas as potent urease inhibitors: synthesis, SAR, and biological evaluation

Article abstract of DOI:10.1080/14756366.2019.1706503

A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC₅₀ values of 0.16 ± 0.05 and 3.86 ± 0.10 μM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation (k_off=1.60 × 10^?3 s^?1) from the catalytic domain.

Full text of DOI:10.1080/14756366.2019.1706503

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
N-monoarylacetothioureas as potent urease
inhibitors: synthesis, SAR, and biological
evaluation
Wei-Yi Li, Wei-Wei Ni, Ya-Xi Ye, Hai-Lian Fang, Xing-Ming Pan, Jie-Ling He,
Tian-Li Zhou, Juan Yi, Shan-Shan Liu, Mi Zhou, Zhu-Ping Xiao & Hai-Liang Zhu
To cite this article: Wei-Yi Li, Wei-Wei Ni, Ya-Xi Ye, Hai-Lian Fang, Xing-Ming Pan, Jie-
Ling He, Tian-Li Zhou, Juan Yi, Shan-Shan Liu, Mi Zhou, Zhu-Ping Xiao & Hai-Liang Zhu
(2020) N-monoarylacetothioureas as potent urease inhibitors: synthesis, SAR, and biological
evaluation, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 404-413, DOI:
10.1080/14756366.2019.1706503
To link to this article: https://doi.org/10.1080/14756366.2019.1706503
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
View supplementary material
Submit your article to this journal
View Crossmark data
Published online: 27 Dec 2019.
View related articles
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 404–413
https://doi.org/10.1080/14756366.2019.1706503
RESEARCH PAPER
N-monoarylacetothioureas as potent urease inhibitors: synthesis, SAR, and
biological evaluation
Wei-Yi Lia, Wei-Wei Nia, Ya-Xi Yeb, Hai-Lian Fanga, Xing-Ming Pana, Jie-Ling Hea, Tian-Li Zhoua, Juan Yia,
Shan-Shan Liua, Mi Zhoua, 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
b
Ethnomedicine in Wuling Mountains, Jishou University, Jishou, PR China; State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University, Nanjing, PR China
ABSTRACT
ARTICLE HISTORY
Received 23 August 2019
Revised 10 December 2019
Accepted 14 December 2019
A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections
caused by urease-producing bacteria such as Helicobacter pylori. Here, we report a series of N-monosubsti-
tuted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound (b19)
was evaluated in detail and shows promising features for further development as an agent to treat H.
pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease
in intact cell were observed, which shows IC50 values of 0.16 0.05 and 3.86 0.10 mM, being 170- and 44-
fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the
monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible
KEYWORDS
Urease inhibitor;
N-monoarylacetothiourea;
Helicobacter pylori; SPR;
molecular dynamics
urease inhibitor, and displays nM affinity to urease with very slow dissociation (koff¼1.60 ꢀ 10ꢁ3
s
ꢁ1) from
the catalytic domain.
Introduction
urgently needed. We therefore have focussed our efforts on this
field for several years, and some potent urease inhibitors with
structural diversity were reported such as catechols, diarylethy-
Helicobacter pylori, a gram-negative and microaerophilic bacter-
ium, colonises the gastric mucosa of over 50% of the global popu-
lation1. In some individuals, chronic infection can induce a
significant inflammatory response, which triggers a loss of gastric
epithelial cells, resulting in gastric, duodenal ulcers and approxi-
mately 90% of cases of intestinal-type gastric carcinoma2. With
the help of urease (EC 3.5.1.5), a nickel dependent metalloenzyme
with an ability to catalyse the hydrolysis of urea to ammonia and
carbamates, H. pylori creates a local neutral environment for sur-
vival by continuously releasing ammonia into succus gastricus3. In
addition, Eaton et al. and Karita et al. demonstrated that urease-
negative mutant of the H. pylori strain was unable to colonise the
lenes, flavonoids, arylamides, and hydroxamic acids11–15
.
Thiourea derivatives, substrate analogues of urease, were
reported as potential urease inhibitors16–18. In chemical structure,
they are all N,N0-disubstituted thioureas, resulting in a different
binding mode from that of urea18,19, which may be caused by the
high hindrance around thiourea score. Considering that the urea
binding pocket is narrow and small, we believe that the thiourea
moiety of N-monosubstituted thioureas could reach the bottom of
the urea-binding pocket and chelate the two Ni atoms due to a
tinny head. Based on this hypothesis and as a continuation of ure-
ase inhibitor screening, a series of N-arylacetothioureas were
designed and synthesised. Some resulted compounds showed
excellent potency against H. pylori urease.
gastric mucosa under the acidic conditions of the stomach4,5
.
Therefore, urease is considered as a virulence factor playing an
essential role for establishment of H. pylori colonisation in human.
Therefore, urease inhibitors could serve as drugs for treating H.
pylori caused disease such as gastritis and peptic ulcers6.
In the past decades, thousands of urease inhibitors have been
reported, and they were designed exclusively by either attacking
Materials and methods
Biology materials
the metallocenter or mimicking the substrate of ureases7–10
.
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 sterile and defibrinated blood were from Hyclone
However, urease has a highly specific substrate urea, which makes
it very challenging for the development of urease inhibitors. In
spite of enormous efforts being made, only acetohydroxamic acid
(AHA) has so far been approved by FDA for the treatment of urin-
ary tract infections. Novel urease inhibitors with high potency are (Logan, UT).
CONTACT Zhu-Ping Xiao
xiaozhuping2005@163.com; Hai-Liang Zhu
zhuhl@nju.edu.cn
National Demonstration Center for Experimental Chemistry
Education, Hunan Engineering Laboratory for Analyse and Drugs Development of Ethnomedicine in Wuling Mountains, Jishou University, Jishou 416000, PR China
Supplemental data for this article is available online at here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
405
Bacteria
min. The relative amount of inhibitor bound to the urease was
determined by measuring the net increase in refractive index over
time compared to control running buffer. There is an inline sub-
traction of reference surface during the run. This change is usually
reported in response units (RU). Sensograms were processed and
analysed using TraceDrawer software. The binding curves were fit
to determine the equilibrium dissociation constant (KD).
H. pylori (ATCC 43504; American Type Culture Collection,
Manassas, VA) was grown in Brucella broth supplemented with
10% sheep sterile and defibrinated blood for 24 h at 37 ꢂC under
microaerobic conditions (5% O2, 10% CO2, and 85% N2), as our
previously described literature13,14
.
Preparation of H. pylori urease
Enzyme kinetic study
For urease inhibition assays, 50 mL broth cultures (2.0 ꢀ 108
CFU/mL) were centrifuged (5000ꢀg, 4 ꢂC) to collect the bacteria,
and after washing twice with phosphate-buffered saline (pH 7.4),
the H. pylori precipitation was stored at –80 ꢂC 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 (15,000ꢀg, 4 ꢂC), 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 ꢂC until use in the experiment.
Based on the indophenol method, the velocity of ammonia pro-
duction (V) was measured in the presence of concentration gra-
dients of urea ([S]) for every specific concentration of selected
compounds ([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 (Equation (1)). Subsequently, the
ꢀ
ꢁ
ꢀ
ꢁ
½Iꢃ
½Iꢃ
resulted fitting constants (VK 1 þ K or
1 þ K0 ) and [I] were
1
Vmax
m
max
i
i
linearly fitted to give kinetic parameters Ki and K0 , respectively.
i
The experimental assay was performed against a pure urease
(Jack bean urease) for the consideration of precision.
½ ꢃ
1 þ K0
Vmax
S
ꢀ
ꢁ
ꢀ
ꢁ
V ¼
(1)
Measurement of urease inhibitory activity
½ ꢃ
½ ꢃ
I
I
½ ꢃ
S
Km 1 þ K
þ
i
i
Urease activity was determined in the 96-well assay plate by
measuring ammonia production using the indophenol method as
described by Weatherburn20. Briefly, to each well, 50 mL of urea
(10 mM) in phosphate buffer solution was added in a mixture of
25 mL (10 U) of H. pylori urease and 25 mL of the test compound,
which was incubated at 37 ꢂC for 0.5 h. Fifty mL of phenol reagent
(containing 127 mM phenol and 0.168 mM sodium nitroprusside)
and 50 mL of alkali reagent (containing 125 mM NaOH and
0.168 mM NaOCl) were added in turn. The resulted mixture was
incubated at 37 ꢂC for another 0.5 h for colouration developing.
The increasing absorbance was measured at 630 nm after 50 min
using a microplate reader (Molecular Devices, San Jose, CA).
Percentage inhibitions were calculated from the following formula
(Equation (1)). Experiments were performed in triplicate and AHA
was used as reference drug, and the IC50 value was determined as
the concentration of compound that give 50% inhibition of max-
imal activity. As for the urease assay of intact cells, 25 mL (10 U) of
H. pylori urease was replaced by 25 mL of cell suspension
(4.0 ꢀ 107 CFU/mL).
Protocol of docking study
Molecular docking of compounds b19 into the structure of H.
pylori urease complex structure was carried out using SYBYL-X ver-
sion 2.1.1 software suite (Tripos, Inc., St. Louis, MO)23. The X-ray
structure of urease from H. pylori was downloaded from the
Protein Data Bank (PDB code: 1e9y)24 and was modified by adding
hydrogen atoms and removing water as well as cocrystallised sub-
strate (AHA). The active site was defined as all the amino acid resi-
dues confined within a 5 Å radius sphere centred about AHA, and
the composite structure without original ligand was utilised as the
in silico model for docking studies. Default parameters and values
within the minimisation dialogue were used except where other-
wise mentioned. The docked conformations of ligands were eval-
uated 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 that
integrated multiple well-known scoring functions such as
ChemScore, D-Score, G-Score, and PMF-Score to evaluate docked
ligand conformations.
Determination of minimal inhibitory concentrations
The minimum inhibitory concentration (MIC) values were deter-
mined using the broth microdilution protocol according to the
Molecular dynamic simulations protocol
methods
of
the
Clinical
and
Laboratory
Standards
Institute (CLSI)21.
Molecular dynamics was performed using Desmond 4.2 with the
standard RESPA integration and 2 fs time step. The TIP3P water
model was used and exhibited well when combining with the
OPLS force field. Each simulation system including the prepared
protein, the inhibitor, and several Cl– added to achieve charge
neutrality was immersed in a cubic box (10 Å). First, all the pre-
pared systems were minimised until a gradient threshold (25 kcal/
mol/Å) was reached by the steepest-descent (SD) method, and
then coupled to the Berenson thermostat with 300 K reference
temperature and the Berendsen barostat with 1.01325 bar refer-
ence pressure. The calculation of long-range electrostatics was
Ligand affinity study
The binding kinetics of selected compounds were assayed via sur-
face plasmon resonance (SPR) using an OPEN SPR instrument
(Nicoya Lifesciences, Kitchener, Canada). First, urease dissolved
(50 mg/mL) in PBS buffer (1 mM KH2PO4, 155 mM NaCl, 3 mM
Na2HPO4-12H2O, pH 7.4), was immobilised to a CM5 chip using a
standard amine coupling procedure22. Then, SPR measurements
were carried out in PBS, and stock solutions were diluted in the
same buffer. Data were collected with OpenSPR control software. based on the Particle-Mesh Ewald method. The cut-off for cou-
Experiments were performed by monitoring the refractive index lomb interaction was set at 9.0 Å. After equilibrated, all the sys-
changes as a function of time under constant flow rate of 20 mL/ tems began to run in the NPT ensemble for 6 ns. The relative

406
W.-Y. LI ET AL.
RMSD and root mean square fluctuation (RMSF) were calculated
N-(3,4-Dichlorophenylaceto)urea (b3). White powder, 58%, mp:
based on the analysis of the MD trajectories by the VMD (version 202.1–203.4 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.75 (s, 2H,
1.9.1) and xmgrace tool.
CH2); 7.28 (dd, J ¼ 2.1 Hz, J ¼ 10.3 Hz, 1H, Ar); 7.57 (d, J ¼ 2.0 Hz,
1H, Ar); 7.59 (d, J ¼ 8.0 Hz, 1H, Ar); 9.44 (s, 1H, NH2); 9.51 (s, 1H,
NH2); 11.33 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): 41.61;
130.12; 130.46; 130.87; 131.21; 132.08; 135.91; 171.9; 181.99.
N-(3-Hydroxyphenylaceto)urea (b4). White powder, 51%, mp:
160.3–161.5 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.60 (s, 2H,
CH2); 6.65 (d, J ¼ 7.1 Hz, 1H, Ar); 6.69 (d, J ¼ 8.5 Hz, 1H, Ar); 6.70 (s,
1H, Ar); 7.10 (t, J ¼ 7.8 Hz, 1H, Ar); 9.38 (s, 1H, OH); 9.41 (s, 1H,
NH2); 9.58 (s, 1H, NH2); 11.26 (s, 1H, NH); 13C NMR (150 MHz,
DMSO-d6): 42.87; 114.35; 116.60; 120.34; 129.80; 136.16; 157.77;
172.85; 182.18.
N-(3-Methoxyphenylaceto)urea (b5). White powder, 59%, mp:
184.5–186.1 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.67 (s, 2H,
CH2); 3.73 (s, 3H, OCH3); 6.83 (dd, J ¼ 8.3 Hz, J ¼ 2.8 Hz, 1H, Ar);
6.85 (d, J ¼ 7.5 Hz, 1H, Ar); 6.88 (t, J ¼ 1.8 Hz, 1H, Ar); 7.23 (td,
J ¼ 7.9 Hz, J ¼ 1.6 Hz, 1H, Ar); 9.42 (s, 1H, NH2); 9.57 (s, 1H, NH2);
11.28 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): 42.89; 55.44;
112.73; 115.63; 121.99; 129.87; 136.31; 159.66; 172.71; 182.16.
N-(2-Fluorophenylaceto)urea (b6). White powder, 60%, mp:
157.2–159.0 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.75 (s, 2H,
CH2); 7.14 (t, J ¼ 9.1 Hz, 1H, Ar); 7.19 (t, J ¼ 7.5 Hz, 1H, Ar); 7.24 (s,
1H, NH2); 7.29 (t, J ¼ 7.7 Hz, 1H, Ar); 7.36 (dd, J ¼ 14.1 Hz, J ¼ 7.2 Hz,
1H, NH2); 9.04 (s, 1H, NH2); 9.81 (s, 1H, NH); 13C NMR (150 MHz,
DMSO-d6): 37.53; 115.88 (d, J ¼ 21.5 Hz); 119.49 (d, J ¼ 15.7 Hz);
124.80 (d, J ¼ 3.7 Hz); 130.22 (d, J ¼ 8.2 Hz); 131.55 (d, J ¼ 3.6 Hz);
160.94 (d, J ¼ 246.8 Hz); 170.31; 181.98.
Cytotoxicity assay
The stock solutions of the selected compounds (250 mg/mL, in
PBS) were prepared in medium. L-02 cells and P69 cells were
grown in medium (90% RPMI-1640 medium, 10% foetal bovine
serum, 1% penicillin/streptomycin) and maintained at 37 ꢂC in a
humidified atmosphere containing 5% CO2, respectively. Cells
were seeded in 96-well cell culture plates. On the day when seed-
ing, the cells were exposed to 250 mg/mL of compounds and fur-
ther cultured for 72 h at 37 ꢂC. Cell proliferation was then
determined using the thiazolyl blue tetrazolium bromide
(MTT) assay.
Chemistry
All chemicals (reagent grade) used were purchased from
Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Melting
points (uncorrected) were determined on a XT4 MP apparatus
(Taike Corp., Beijing, China). EI mass spectra were obtained on
Agilent 6120 mass spectrometer, and 1H NMR spectra were
recorded on a Bruker AV-600 spectrometer at 25 ꢂC with TMS and
solvent signals allotted as internal standards. Chemical shifts were
reported in ppm (d). Elemental analyses were performed on a
Foss Heraeus CHN-O-Rapid instrument and were within 0.4% of
the theoretical values.
N-(3-Chlorophenylaceto)urea (b7). White powder, 59%, mp:
136.6–137.4 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.74 (s, 2H,
CH2); 7.25 (dt, J ¼ 7.2 Hz, J ¼ 1.6 Hz, 1H, Ar); 7.32–7.36 (m, 2H, Ar);
7.38 (t, J ¼ 1.8 Hz, 1H, Ar); 9.44 (s, 1H, NH2); 9.54 (s, 1H, NH2); 11.32
(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): 42.26; 127.36; 128.66;
129.82; 130.64; 133.30; 137.26; 172.25; 182.05.
N-(4-Hydroxyphenylaceto)urea (b8). White powder, 57%, mp:
177.1–178.3 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.56 (s, 2H,
CH2); 6.70 (d, J ¼ 8.0 Hz, 1H, Ar); 7.07 (d, J ¼ 8.0 Hz, 1H, Ar); 9.32 (s,
1H, OH); 9.39 (s, 1H, NH2); 9.58 (s, 1H, NH2); 11.20 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 40.03; 115.62; 124.96; 130.74; 156.77;
173.39; 182.23.
General procedure for the preparation of compounds b
A selected arylacetic acid (10 mmol) was solved in SOCl2 (50 mL),
which was heated to 90 ꢂC and stirred for 1–2 h until the reaction
was completed. The crude product (a1–a29) was furnished after
removal of SOCl2 under reduced pressure. To the resulting resi-
due, 50 mL of toluene was added and stirred for 30 min at room
temperature. Thiourea (40 mmol) was subsequently added, and
the reaction solution was heated to 100 ꢂC for 1.5–2 h. After the
end of the reaction was established by TLC, the solvent was
removed under vacuum, and excess saturated NaHCO3 solution
was added. The resulted mixture was extracted with ethyl acetate,
dried over MgSO4, filtered, and concentrated under vacuum. The
product was purified by a silica gel column using ethyl acetate
and petroleum ether as eluent to afford compound b (b1–b29) in
moderate to high yield as white powder (all the 1H NMR and 13C
NMR Spectra of Compoundsb1-29 in Supporting Information).
N-(3-Trifluoromethylphenylaceto)urea (b9). White powder, 51%,
1
mp: 204.3–205.8 ꢂC, H NMR (600 MHz, DMSO-d6) d (ppm): 3.86 (s,
2H, CH2); 7.57 (d, J ¼ 7.5 Hz, 1H, Ar); 7.60 (d, J ¼ 7.8 Hz, 1H, Ar);
7.64 (d, J ¼ 7.0 Hz, 1H, Ar); 7.68 (s, 1H, Ar); 9.44 (s, 1H, NH2); 9.53
(s, 1H, NH2); 11.36 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6):
42.28; 124.12 (q, J ¼ 3.8 Hz); 124.67 (q, J ¼ 272.2 Hz); 126.63 (q,
J ¼ 3.8 Hz); 129.46 (q, J ¼ 31.5 Hz); 129.82; 134.18; 136.23;
172.22; 182.03.
N-Phenylacetourea
(b1).
White
powder,
47%,
mp:
N-(3,4-Dimethoxyphenylaceto)urea (b10). White powder, 50%,
110.9–112.1 ꢂC, 1H NMR (600 MHz, CDCl3) d (ppm): 3.71 (s, 2H,
CH2); 7.28 (d, J ¼ 6.8 Hz, 2H, Ar); 7.35 (t, J ¼ 7.3 Hz, 1H, Ar); 7.39 (t,
J ¼ 7.3 Hz, 2H, Ar); 7.44 (s, 1H, NH2); 9.29 (s, 1H, NH2); 9.88 (s, 1H,
NH); 13C NMR (150 MHz, CDCl3): 43.97; 128.06; 129.22; 129.38;
132.19; 171.72; 182.01; MS (ESI) m/z 195 (M þ H)þ; Anal. Calcd for
C9H10N2OS: C, 55.65; H, 5.19; N, 14.42; S, 16.51; found: C, 55. 59; H,
5.19; N, 14.44; S, 16.53.
1
mp: 146.3–147.6 ꢂC, H NMR (600 MHz, DMSO-d6) d (ppm): 3.61 (s,
2H, CH2); 3.72 (s, 3H, OCH3); 3.73 (s, 3H, OCH3); 6.81 (dd, J ¼ 8.2 Hz,
J ¼ 2.0 Hz, 1H, Ar); 6.89 (d, J ¼ 8.3 Hz, 1H, Ar); 6.90 (d, J ¼ 2.0 Hz,
1H, Ar); 9.40 (s, 1H, NH2); 9.58 (s, 1H, NH2); 11.23 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 42.44; 55.89; 55.96; 112.26; 113.65;
121.87; 127.15; 148.29; 148.98; 173.10; 182.19.
N-(4-Methoxyphenylaceto)urea (b11). White powder, 56%, mp:
143.5–144.7 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.62 (s, 2H,
CH2); 3.73 (s, 3H, OCH3); 6.89 (d, J ¼ 8.6 Hz, 2H, Ar); 7.21 (d,
J ¼ 8.6 Hz, 2H, Ar); 9.40 (s, 1H, NH2); 9.58 (s, 1H, NH2); 11.25 (s, 1H,
NH); 13C NMR (150 MHz, DMSO-d6): 41.98; 55.49; 114.28; 126.76;
130.84; 158.72; 173.22; 182.21.
N-(3-Bromophenylaceto)urea (b2). White powder, 53%, mp:
207.4–208.8 ꢂC, 1H NMR (600 MHz, CDCl3) d (ppm): 3.58 (s, 2H,
CH2); 6.98 (s, 1H, NH); 7.13 (dt, J ¼ 7.3 Hz, J ¼ 1.3 Hz, 1H, Ar); 7.20
(t, J ¼ 7.8 Hz, 1H, Ar); 7.37 (t, J ¼ 1.9 Hz, 1H, Ar); 7.42 (ddd,
J ¼ 8.0 Hz, J ¼ 2.0 Hz, J ¼ 1.1 Hz, 1H, Ar); 8.63 (s, 1H, NH2); 9.70 (s,
1H, NH2); 13C NMR (150 MHz, CDCl3): 42.21; 121.94; 129.04; 130.25;
130.95; 132.69; 137.54; 172.26; 182.04.
N-(4-Bromophenylaceto)urea (b12). White powder, 53%, mp:
174.9–176.2 ꢂC, 1H NMR (600 MHz, CDCl3) d (ppm): 3.56 (s, 2H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
407
CH2); 6.99 (s, 1H, NH); 7.07 (d, J ¼ 8.3 Hz, 2H, Ar); 7.44 (d, J ¼ 8.3 Hz, 11.40 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): 40.23; 127.74;
2H, Ar); 8.67 (s, 1H, NH2); 9.70 (s, 1H, NH2); 13C NMR (150 MHz,
128.94; 132.23; 133.08; 134.19; 135.17; 171.32; 181.93.
N-(4-Nitrophenylaceto)urea (b22). White powder, 58%, mp:
199.1–200.9 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.91 (s, 2H,
CH2); 7.57 (d, J ¼ 8.7 Hz, 2H, Ar); 8.20 (d, J ¼ 8.7 Hz, 2H, Ar); 9.46 (s,
1H, NH2); 9.50 (s, 1H, NH2); 11.40 (s, 1H, NH); 13C NMR (150 MHz,
DMSO-d6): 42.43; 123.86; 131.38; 142.81; 147.04; 171.72; 181.99.
N-(4-Fluorophenylaceto)urea (b23). White powder, 57%, mp:
165.4–166.5 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.71 (s, 2H,
CH2); 7.15 (t, J ¼ 8.9 Hz, 2H, Ar); 7.32 (dd, J ¼ 8.5 Hz, J ¼ 5.7 Hz, 2H,
Ar); 9.42 (s, 1H, NH2); 9.55 (s, 1H, NH2); 11.30 (s, 1H, NH); 13C NMR
(150 MHz, DMSO-d6): 41.87; 115.57 (d, J ¼ 21.1 Hz); 131.04 (d,
J ¼ 3.2 Hz); 131.78 (d, J ¼ 8.0 Hz); 161.74 (d, J ¼ 242.7 Hz);
172.75; 182.12.
CDCl3): 43.41; 122.37; 131.04; 131.14; 132.42; 170.59; 181.93.
N-(2-Chlorophenylaceto)urea (b13). White powder, 55%, mp:
148.8–151.2 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.91 (s, 2H,
CH2); 7.29–7.32 (m, 2H, Ar); 7.37–7.40 (m, 1H, Ar); 7.42–7.45 (m,
1H, Ar); 9.42 (s, 1H, NH2); 9.52 (s, 1H, NH2); 11.38 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 40.84; 127.60; 129.44; 129.47; 132.92;
133.04; 134.17; 171.73; 182.01; MS (ESI) m/z 229 (M þ H)þ; Anal.
Calcd for C9H9ClN2OS: C, 47.27; H, 3.97; Cl, 15.50; N, 12.25; S,
14.02; found: C, 47.21; H, 3.97; Cl, 15.52; N, 12.25; S, 14.03.
N-(3-Fluorophenylaceto)urea (b14). White powder, 58%, mp:
126.0–127.4 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.75 (s, 2H,
CH2); 6.95–7.23 (m, 3H, Ar); 7.30–7.52 (m, 1H, Ar); 9.43 (s, 1H, NH2);
9.54 (s, 1H, NH2); 11.32 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6):
42.37 (d, J ¼ 1.8 Hz); 114.20 (d, J ¼ 20.8 Hz); 116.74 (d, J ¼ 21.4 Hz);
126.07 (d, J ¼ 2.7 Hz); 130.67 (d, J ¼ 8.3 Hz); 137.52 (d, J ¼ 8.0 Hz);
162.45 (d, J ¼ 243.2 Hz); 172.28; 182.07.
N-(2-Naphthylaceto)urea (b24). White powder, 52%, mp:
1
202.6–204.4 ꢂC, H NMR (600 MHz, DMSO-d6) d (ppm): 3.90 (s, 1H);
7.45 (dd, J ¼ 8.4 Hz, J ¼ 1.7 Hz, 1H, Ar); 7.49 (dd, J ¼ 6.8 Hz,
J ¼ 1.6 Hz, 1H, Ar); 7.51 (dd, J ¼ 7.0 Hz, J ¼ 1.8 Hz, 1H, Ar); 7.81 (d,
J ¼ 1.8 Hz, 1H, Ar); 7.88 (d, J ¼ 8.1 Hz, 2H, Ar); 7.89 (d, J ¼ 8.7 Hz,
1H, Ar); 9.45 (s, 1H, NH2); 9.60 (s, 1H, NH2); 11.40 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 42.96; 126.28; 126.68; 127.94; 127.98;
128.18; 128.29; 128.34; 132.41; 132.59; 133.38; 172.85; 182.17.
N-(Diphenylaceto)urea (b25). White powder, 54%, mp:
169.5–172.7 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 5.40 (s, 1H,
CH); 7.23–7.32 (m, 6H, Ar); 7.33–7.38 (m, 4H, Ar); 9.52 (s, 1H, NH2);
9.61 (s, 1H, NH2); 11.49 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6):
56.77; 127.70; 129.03; 129.04; 139.00; 173.35; 182.12.
N-(2-Bromophenylaceto)urea (b15). White powder, 54%, mp:
179.0–179.5 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.92 (s, 2H,
CH2); 7.22 (td, J ¼ 7.7 Hz, J ¼ 1.7 Hz, 1H, Ar); 7.35 (td, J ¼ 7.4 Hz,
J ¼ 1.8 Hz, 1H, Ar); 7.38 (dd, J ¼ 7.6 Hz, J ¼ 1.9 Hz,1H, Ar); 7.60 (dd,
J ¼ 8.6 Hz, J ¼ 2.3 Hz, 1H, Ar); 9.41 (s, 1H, NH2); 9.52 (s, 1H, NH2);
11.38 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): 43.28; 125.06;
128.14; 129.63; 132.72; 133.00; 134.83; 171.69; 182.01.
N-(4-Methylphenylaceto)urea (b16). White powder, 55%, mp:
171.2–172.5 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 2.27 (s, 3H,
CH3); 3.65 (s, 2H, CH2); 7.12 (d, J ¼ 7.9 Hz, 2H, Ar); 7.17 (d,
J ¼ 7.9 Hz, 2H, Ar); 9.40 (s, 1H, NH2); 9.57 (s, 1H, NH2); 11.26 (s, 1H,
NH); 13C NMR (150 MHz, DMSO-d6): 21.13; 42.45; 129.41; 129.66;
131.84; 136.44; 173.04; 182.19.
N-(2-Methoxyphenylaceto)urea (b17). White powder, 61%, mp:
135.4–136.9 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.69 (s, 2H,
CH2); 3.75 (s, 3H, OCH3); 6.89 (td, J ¼ 7.4 Hz, J ¼ 1.0 Hz, 1H, Ar); 6.97
(dd, J ¼ 8.2 Hz, J ¼ 2.8 Hz, 1H, Ar); 7.17 (dd, J ¼ 7.5 Hz, J ¼ 1.7 Hz,
1H, Ar); 7.25 (td, J ¼ 7.8 Hz, J ¼ 1.5 Hz, 1H, Ar); 9.36 (s, 1H, NH2);
9.57 (s, 1H, NH2); 11.17 (s, 1H, NH); 13C NMR (150 MHz, DMSO-d6):
37.94; 55.90; 111.16; 120.66; 123.22; 128.94; 131.69; 157.66;
172.95; 182.10.
N-(2-Methylphenylaceto)urea (b18). White powder, 60%, mp:
135.6–137.1 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 2.23 (s, 3H,
CH3); 3.76 (s, 2H, CH2); 7.11–7.15 (m, 1H, Ar); 7.15–7.17 (m, 2H, Ar);
7.19 (dd, J ¼ 6.7 Hz, J ¼ 1.7 Hz, 1H, Ar); 9.41 (s, 1H, NH2); 9.57 (s,
1H, NH2); 11.29 (s, 1H, NH2); 13C NMR (150 MHz, DMSO-d6): 19.74;
40.69; 126.30; 127.52; 130.41; 130.68; 133.67; 137.20;
172.80; 182.11.
N-(4-Chlorophenylaceto)urea (b19). White powder, 57%, mp:
184.1–186.6 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.72 (s, 2H,
CH2); 7.31 (d, J ¼ 8.4 Hz, 2H, Ar); 7.39 (d, J ¼ 8.4 Hz, 2H, Ar); 9.43 (s,
1H, NH2); 9.54 (s, 1H, NH2); 11.31 (s, 1H, NH); 13C NMR (150 MHz,
DMSO-d6): 42.02; 128.77; 131.77; 132.12; 133.88; 172.48; 182.09.
N-(2-Nitrophenylaceto)urea (b20). White powder, 59%, mp:
177.6–178.4 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 4.17 (s, 2H,
CH2); 7.55 (dd, J ¼ 7.6, J ¼ 1.4 Hz, 1H, Ar); 7.59 (td, J ¼ 7.8,
J ¼ 1.5 Hz, 1H, Ar); 7.73 (td, J ¼ 7.5, J ¼ 1.3 Hz, 1H, Ar); 8.10 (dd,
J ¼ 8.2, J ¼ 1.3 Hz, 1H, Ar); 9.41 (s, 2H, NH2); 11.41 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 41.21; 125.24; 129.32; 129.88; 134.41;
134.45; 148.92; 171.52; 181.93.
N-(Biphenyl-4-yl)acetourea (b26). White powder, 52%, mp:
1
187.3–189.6 ꢂC, H NMR (600 MHz, DMSO-d6) d (ppm): 3.76 (s, 2H);
7.36 (t, J ¼ 7.4 Hz, 1H, Ar); 7.39 (d, J ¼ 8.1 Hz, 2H, Ar); 7.46 (t,
J ¼ 7.7 Hz, 2H, Ar); 7.62 (d, J ¼ 8.1 Hz, 2H, Ar); 7.65 (d, J ¼ 6.8 Hz,
2H, Ar); 9.44 (s, 1H, NH2); 9.59 (s, 1H, NH2); 11.35 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 42.49; 127.07; 127.18; 127.85; 129.38;
130.41; 134.15; 139.31; 140.33; 172.83; 182.18.
N-(1-Naphthylaceto)urea (b27). White powder, 57%, mp:
195.5–197.9 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 4.24 (s, 2H,
CH2); 7.46–7.49 (m, 2H, Ar); 7.53 (td, J ¼ 7.4 Hz, J ¼ 1.2 Hz, 1H, Ar);
7.57 (td, J ¼ 8.3 Hz, J ¼ 1.5 Hz, 1H, Ar); 7.84–7.89 (m, 1H, Ar); 7.94
(dd, J ¼ 8.1, J ¼ 1.4 Hz, 1H); 8.02 (d, J ¼ 7.8 Hz, 1H); 9.43 (s, 1H,
NH2); 9.54 (s, 1H, NH2); 11.48 (s, 1H, NH); 13C NMR (150 MHz,
DMSO-d6): 40.20; 124.51; 125.99; 126.24; 126.74; 128.07; 128.74;
128.95; 131.49; 132.32; 133.81; 172.78; 182.12.
N-(2-(benzo[d][1,3]dioxol-5-yl)aceto)urea (b28). White powder,
56%, mp: 153.5–155.0 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm):
3.60 (s, 2H, CH2); 5.99 (s, 2H, OCH2O); 6.75 (dd, J ¼ 8.0 Hz,
J ¼ 1.7 Hz, 1H, Ar); 6.85 (d, J ¼ 7.7 Hz, 1H, Ar); 6.86 (d, J ¼ 1.5 Hz,
1H, Ar); 9.41 (s, 1H, NH2); 9.56 (s, 1H, NH2); 11.23 (s, 1H, NH); 13C
NMR (150 MHz, DMSO-d6): 42.43; 101.34; 108.60; 110.21; 122.95;
128.40; 146.64; 147.63; 172.95; 182.16.
N-(3,4-Dihydroxyphenylaceto)urea (b29). White powder, 56%,
1
mp: 153.5–155.0 ꢂC, H NMR (600 MHz, DMSO-d6) d (ppm): 3.48 (s,
2H, CH2); 6.53 (dd, J ¼ 8.1 Hz, J ¼ 2.1 Hz, 1H, Ar); 6.65 (d, J ¼ 8.0 Hz,
1H, Ar); 6.68 (d, J ¼ 2.1 Hz, 1H, Ar); 8.79 (s, 1H, OH); 8.88 (s, 1H,
OH); 9.39 (s, 1H, NH2); 9.60 (s, 1H, NH2); 11.17 (s, 1H, NH); 13C NMR
(150 MHz, DMSO-d6): 42.29; 115.90; 117.04; 120.52; 125.52; 144.77;
145.53; 173.40; 182.25.
Results and discussion
N-(2,4-Dichlorophenylaceto)urea (b21). White powder, 59%, mp:
166.6–167.2 ꢂC, 1H NMR (600 MHz, DMSO-d6) d (ppm): 3.92 (s, 2H,
CH2); 7.41 (dd, J ¼ 8.3 Hz, J ¼ 2.0 Hz, 1H, Ar); 7.43 (d, J ¼ 8.2 Hz, 1H,
Chemistry
A typical synthetic route towards arylacetothioureas is described
Ar); 7.61 (d, J ¼ 2.2 Hz, 1H, Ar); 9.43 (s, 1H, NH2); 9.48 (s, 1H, NH2); in Scheme 1. The arylacetyl chloride intermediate was synthesised

408
W.-Y. LI ET AL.
potency in enzyme assays than AHA were selected to test the
potential growth inhibition against H. pylori, and the results are
shown in Table 1. The assayed compounds showed no or very
weak antibacterial activity against H. pylori at neutral pH.
However, at low pH (about 4.0), compounds with significant inhib-
ition against urease in intact H. pylori cell showed impressive anti-
bacterial effect against H. pylori. This is consistent with that
revealed by Eaton et al. and Karita et al. as above mentioned4,5. In
general, these data indicated a potential for in vivo efficacy of
compounds such as b19 and b16 in clearing H. pylori infection.
Scheme 1. Synthesis of arylacetothioureas.
from an appropriate arylacetic acid by reaction with SOCl2.
Amidation was followed with thiourea to afford the corresponding
compound b.
Ligand affinity
Inhibitory activity against cell-free urease
Compounds with IC50 lower than 20 mM against extracted H. pylori
urease were selected for ligand-enzyme binding interaction based
on SPR, which allows for the determination of affinities and kinetic
parameters in a single experiment25. In the present paper, the
pure Jack bean urease from commercial was used for this assay
because of non-availability of pure H. pylori urease. The binding
affinities are shown in Table 2 and the representative SPR plots
are shown in Figure 1. The most active compound b19 displayed
All synthesised arylacetothioureas were evaluated for their inhibi-
tory activity against extracted H. pylori urease. As shown in Table
1, a compound bearing naphthyl (b24 and b27), benzo-1,3-diox-
ole (b28) or biphenyl (b26) moiety exhibited very low potency,
even showed inactivity. In contrast, some compounds from phe-
nylacetothioureas showed excellent potency with IC50 values
lower than that of the positive control AHA. Out of these com-
pounds, b19 was the most active inhibitor with IC50 of
0.16 0.05 mM, showing 158-fold more potency than AHA.
Replacement of the chloro group of compound b19 by other sub-
stitutions such as fluoro, bromo, methyl, and methoxy attenuated
urease inhibition by 27- to 968-fold, which suggested that only
suitable steric volume is tolerated. Movement of the chlorine from
the para position (b19) to the meta position (b7) produced a 397-
fold decrease in potency. In the case of meta-substituted deriva-
tives, a different result was observed. Replacement of chloro
group with fluoro (b14), bromo (b2), or methoxy (b5) group gives
a comparable activity. However, hydroxyl-substituted analogue
(b4) showed an about fourfold increase in potency in comparison
with b7, which may be attributed to a possible hydrogen-bond
building ability of the hydroxyl group. In comparison with the
meta-substituted derivatives, the ortho-analogous resulted in no
significant change in potency. It is to be noted that compound
the highest binding affinity to urease with
a KD value of
4.50 0.16 nM. For efficient and tight ligand binding, the rate con-
stant koff is of particular interest, because koff has a potential to
differentiate indistinguishable compounds with similar affinities26.
The results revealed that b19 displayed very slow dissociation
from the catalytic domain of urease with a koff 1.73 ꢀ 10ꢁ3 sꢁ1
indicating that b19 could be prioritised for further optimisation.
,
Inhibition kinetics
Compounds with IC50 lower than 20 mM against extracted H. pylori
urease were selected to perform kinetic assay for further insight
of the inhibition mechanism. Mazzei et al. confirmed that urease
shared the mechanism of catalysis and inhibition regardless of the
biological sources27,28. For excluding the possible interference, a
pure urease (Jack bean urease) was therefore used for kinetic
assays. As an example, Figure 2 describes the preincubation-time
dependence of urease inhibition by b19. The suppressed urease
activity maintained at relatively constant values with the increas-
ing preincubation time under different concentrations of b19,
resulting in nearly equal IC50 values. The obtained results indicate
that b19 can rapidly bind to the active site and inhibit urease in a
time-independent manner.
containing
a strong electron-withdrawing nitro group (b20)
resulted in a significant decrease in potency with IC50 value over
180 mM. In the case of double substituent on the phenyl ring,
compound with 3,4-dihyroxyl group resulted in IC50 values of
18.4 mM, being the most active in this series and showing more
potent than the positive control AHA.
Figure 3 shows the nonlinear fitting curves (V vs. [S]) of the
most potential urease inhibitor b19 based on the general kinetics
equation (Equation (1), and the corresponding plots by linearly fit-
ting the fitting constants from V–[S] curves against [I], which pro-
Inhibitory activity against urease in intact cell
Encouraged by the results of the extracted urease assay, com-
pounds showing higher potency than AHA were selected to deter-
mine inhibitory activities against urease in intact H. pylori cell, and
the results are shown in Table 1. A 5- to 24-fold increase of IC50
values against urease in intact cell was observed in comparison
with those of extracted urease. Three compounds (b8, b16, and
b19) were found to be much more potent than AHA, showing
IC50 of 52.5 3.9, 35.7 1.7, and 3.86 0.10 mM. Not surprisingly,
the most potent compound b19 found in the extracted urease
assay was also exhibited the highest activity against urease in
intact H. pylori cell.
vided the kinetic parameters Ki and K0 of b19. Herein, Ki is the
i
dissociation constant for “urease-inhibitor ! urease and inhibitor”
and Ki0 is the dissociation constant for “urease-urea-
inhibitor ! urease-urea and inhibitor”1. The calculated Ki and Ki0
of b19 are 0.040 and 0.16 mM, respectively, suggesting: (1) b19 is
a reversible urease inhibitor (Ki ! 1 and Ki0 ! 1 indicate an irre-
versible inhibition); (2) b19 has a mixed competitive mechanism
(Ki and K0 are not ! 1, and Ki ¼ Ki0 ). Similarly, kinetic parame-
i
ters (Ki and K0 ) and inhibition types of other compounds were
i
also determined and are shown in Table 3. The values of Ki0 are
larger than the corresponding Ki for all tested compounds, sug-
gesting that the complex of urease-urea-inhibitor is less stable
Antibacterial activity
To get an insight of the possibility of the identified urease inhibi- than that of the urease-inhibitor and competitive inhibition has
tors for further drug developing, compounds showing higher relatively higher weight in the mixed competitive mechanism.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
409
Table 1. Structure, inhibitory activity (IC50), antibacterial activity (MIC50) against H. pylori urease of com-
pounds b1–b29.
IC50 (µM)
MIC (µM)
compound
Extracted
urease
97.9 6.8
Urease in
intact cella
ND
Ar1
Ar2
H
pH=7.0 pH=4.0
b1
b2
b3
ND
ND
ND
ND
ND
ND
H
H
ND
ND
58.4 4.3
63.0 4.6
b4
b5
b6
H
H
H
>64.0
ND
64.0
ND
ND
16.3 0.3
75.8 5.7
41.9 2.5
108.2 6.1
ND
ND
ND
b7
b8
H
H
ND
ND
8.0
ND
52.5 3.9
ND
63.6 5.1
64.0
9.93 0.90
b9
H
H
ND
ND
ND
ND
136
7
b10
24.2 0.6
16.3 1.2
22.9 0.4
54.2 4.8
207 16
b11
b12
b13
H
H
H
>64.0
ND
32.0
ND
98.3 5.9
189 18
ND
ND
ND
b14
b15
H
H
ND
ND
ND
ND
ND
ND
50.3 3.5
51.1 4.4
b16
b17
H
H
64.0
ND
2.0
ND
5.20 0.32
55.7 3.1
35.70 1.7
ND
b18
H
ND
ND
ND
46.2 1.8
b19
b20
H
H
32.0
ND
0.5
ND
0.16 0.05
183 12
3.86 0.10
ND
b21
b22
H
H
ND
ND
ND
ND
ND
ND
67.7 4.3
155 16
(continued)

410
W.-Y. LI ET AL.
b23
H
H
>64.0
8.0
14.6 1.0
93.6 5.5
b24
b25
b26
ND
ND
ND
ND
ND
ND
NA
ND
ND
ND
318 24
279 16
H
H
b27
ND
ND
NA
ND
ND
b28
b29
H
H
ND
ND
ND
ND
98
5
18.4 1.6
27.2 0.7
143
171
6
9
AHA
aND: no determination.
>64.0
16.0
Table 2. The binding affinity data for urease–thioureas interactions.
Interaction
Kon (M–1ꢄs–1
)
Koff (s–1
)
KD (nM)
Urease–b4
Urease–b8
6.08 ꢀ 104 2.26 ꢀ 102 3.89 ꢀ 10–2 2.93 ꢀ 10–4 644.0 9.2
9.38 ꢀ 106 3.18 ꢀ 104 2.92 ꢀ 10–3 0.61 ꢀ 10–5 311.2 10.3
Urease–b11 5.72 ꢀ 105 4.35 ꢀ 103 3.81 ꢀ 10–2 7.60 ꢀ 10–4 666.7 13.7
Urease–b16 5.01 ꢀ 105 6.09 ꢀ 103 8.10 ꢀ 10–2 1.38 ꢀ 10–4 162.0 3.8
Urease–b19 3.82 ꢀ 106 5.79 ꢀ 104 1.73 ꢀ 10–3 2.57 ꢀ 10–5
4.50 0.16
Urease–b23 5.43 ꢀ 104 3.92 ꢀ 102 3.09 ꢀ 10–2 1.81 ꢀ 10–4 569.2 21.6
Urease–b29 1.98 ꢀ 104 1.75 ꢀ 102 1.55 ꢀ 10–2 0.44 ꢀ 10–4 782.5 21.3
Figure 2. Characterisation of urease inhibition by compound b19 for different
preincubation time.
of b19 establishes appropriate hydrophobic contacts with the
hydrophobic gap (Met317, Leu318, and Met366) under the mov-
able flap,
a
helix-turn-helix motif composed of residues
a313–a346. Moreover, benzene ring is further solid by an S–Hꢄꢄꢄp
with Cys321, and a C–HꢄꢄꢄN with Leu318. In addition, this model
also suggested that the aceto moiety as a donor as well as an
acceptor forms C–HꢄꢄꢄN and N–HꢄꢄꢄO hydrogen bonds with His322
and Arg338, respectively. The enzyme assay data and the molecu-
lar docking results indicated that compound b19 is a potential
inhibitor of urease.
Figure 1. Sensograms of interactions between b19 and urease.
Molecular docking
With the aim to explore the structural determinants of the guid-
ance for further SAR studies, molecular docking of the most
potent inhibitor b19 into urea binding site was performed, and
the binding model is depicted in Figure 4. This model revealed
that the monosubstituted thiourea moiety is nicely bound to urea
binding site (Figure 4(A)), and is of primary importance for its net-
work of interactions (Figure 4(B)): it coordinates the nickel ion and
establishes hydrogen bonds with N of His138, O of Asn168, and
Molecular dynamics
Molecular dynamic (MD) simulations were performed to under-
stand the dynamic properties of urease–b19 complexes, and to
provide some evidence for the suppression site identified by
molecular dockings. Figure 5 shows the evolutions of the root
mean square deviation (RMSD) values versus time in reference to
the energy minimised complex structure. RMSD change suggested
Me of Ala365, respectively. On the other hand, the benzene ring that simulation attains an equilibrium position within 1 ns, and

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
411
Figure 3. A velocity (V) was nonlinearly fitted against the concentrations of urea [S] in the presence of a specific concentration of compound b19. (B) (1) Ki: the fitted
ꢀ
ꢁ
ꢀ
ꢁ
constants (VK 1 þ K ) from the corresponding V–S plot were plotted against concentrations of b19 ([I]); (2) Ki0 : the fitted constants (
1 þ K0 ) from the corre-
½Iꢃ
½Iꢃ
i
1
Vmax
m
max
i
sponding V–S plot were plotted against concentrations of b19.
Table 3. Data of inhibition mechanism.
Compound
Type of inhibition
Ki (lM)
Ki0 (lM)
b4
b8
b11
b16
b19
b23
b29
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
Reversible
4.34
2.54
3.94
1.28
0.040
3.53
8.21
8.52
11.3
12.7
2.84
0.16
9.67
21.5
Figure 4. Predicted binding mode of ligand-urease (PDB code: 1e9y): (A) compound b19 shown as white sticks and the enzyme shown as surface. (B) Compound b19
shown as cyan sticks and enzyme shown as lines; Hydrogen bonds shown as red dashed lines.
the average RMSD fluctuation values of b19 were 1.1 Å after equi- profile against human normal hepatic cell line L-02 and normal
librium was reached. The very low deviation from docked position
indicated that molecular docking results of b19 are reliable, which
was also evidenced by the high binding affinity observed in
enzyme and SPR assays.
prostate cell line P69 using the colorimetric cell proliferation MTT
assay. As shown in Table 4, most of the assayed compounds
showed low cytotoxicity against these human normal cell lines
with viability over 90% at concentration of 25 mg/mL. It is to be
noted that the cell viability against the most potent compound
b19 of the two cell lines was more than 93%, suggesting the low
toxicity to mammalian cells. On the whole, the new identified ure-
ase inhibitor b19 showed lower cell toxicity than the positive con-
Cytotoxicity
The highly bioactive compounds with IC50 value against urease
lower than that of AHA were further evaluated for its toxicity trol AHA.

412
W.-Y. LI ET AL.
References
1. Liu Q, Ni W-W, Li Z, et al. Resolution and evaluation of 3-
chlorophenyl-3-hydroxy-propionylhydroxamic acid as antivir-
ulence agent with excellent eradication efficacy in
Helicobacter pylori infected mice. Eur J Pharm Sci 2018;121:
293–300.
2. Valenzuela-Valderrama M, Cerda-Opazo P, Backert S, et al.
The Helicobacter pylori urease virulence factor is required for
the induction of hypoxia-induced factor-1 in gastric cells.
Cancers 2019;11:799.
3. Liu Q, Shi W-K, Ren S-Z, et al. Arylamino containing hydroxa-
mic acids as potent urease inhibitors for the treatment of
Helicobacter pylori infection. Eur J Med Chem 2018;156:
126–36.
4. Eaton KA, Brooks CL, Morgan DR, et al. Essential role of ure-
ase in pathogenesis of gastritis induced by Helicobacter
pylori in gnotobiotic piglets. Infect Immun 1991;59:2470–5.
5. Karita M, Tsuda M, Nakazawa T. Essential role of urease
in vitro and in vivo Helicobacter pylori colonization study
using a wild-type and isogenic urease mutant strains. J Clin
Gastroenterol 1995;21:S160–S163.
Figure 5. The RMSD values obtained during 6 ns of molecular dynamic simula-
tion for urease and b19.
Table 4. Cell Viability of selected compounds on L-02 and P69 at concentration
of 25 lg/mL.
Viability (%)
Compound
L-02
93.7
P69
b4
b8
88.1
94.6
95.3
87.5
89.4
89.9
93.6
97.6
71.2
6. Rizvi F, Khan M, Jabeen A, et al. Studies on isoniazid deriva-
tives through a medicinal chemistry approach for the identi-
fication of new inhibitors of urease and inflammatory
markers. Sci Rep 2019;9:1–14.
92.1
108.1
97.0
91.5
113.8
96.8
96.1
80.2
b10
b11
b12
b16
b19
b23
AHA
7. Shi
W-K,
Deng
R-C,
Wang
P-F,
et
al.
3-
Arylpropionylhydroxamic acid derivatives as Helicobacter
pylori urease inhibitors: synthesis, molecular docking and
biological evaluation. Bioorg Med Chem 2016;24:4519–27.
8. Kanwal, Khan M, Arshia, et al. Syntheses, in vitro urease
inhibitory activities of urea and thiourea derivatives of
tryptamine, their molecular docking and cytotoxic studies.
Bioorg Chem 2019;83:595–610.
9. Rauf A, Shahzad S, Bajda M, et al. Design and synthesis of
new barbituric- and thiobarbituric acid derivatives as potent
urease inhibitors: structure activity relationship and molecu-
lar modeling studies. Bioorg Med Chem 2015;23:6049–58.
10. Yu H-Y, Guo S-H, Cheng J-Y, et al. Synthesis and crystal
structures of cobalt(III), copper(II), nickel(II) and zinc(II) com-
plexes derived from 4-methoxy-N0-(pyridin-2-ylmethylene)-
benzohydrazide with urease inhibitory activity. J Coord
Chem 2008;61:1212–20.
Conculsions
In summary, we have developed a series of mono-substituted thi-
oureas as potent H. pylori urease inhibitors. The most potent com-
pound b19 was identified as a reversible urease inhibitor, and
significantly inhibits extracted urease and urease in intact cell with
IC50 values of 0.16 0.05 and 3.86 0.10 mM, being 170- and 44-
fold more potent than clinically used drug AHA, respectively. The
SPR assay thus revealed that b19 exhibits very high urease affin-
ities in the low nM range, probably binding to the urea site and
showing very slow dissociation from the catalytic domain. Urease
inhibition assay, SPR assay, molecular docking studies and cell
proliferation assay suggested that the mono-substituted thioureas
are a new kind of urease inhibitors acting at the urea site, with
b19 having potential for further development as an agent to treat
H. pylori caused diseases.
11. Ni W-W, Liu Q, Ren S-Z, et al. The synthesis and evaluation
of phenoxyacylhydroxamic acids as potential agents for
Helicobacter pylori infections. Bioorg Med Chem 2018;26:
4145–52.
12. Xiao Z-P, Shi W-K, Wang P-F, et al. Synthesis and evaluation
of N-analogues of 1,2-diarylethane as Helicobacter pylori ure-
ase inhibitors. Bioorg Med Chem 2015;23:4508–13.
13. Wang X-D, Wei W, Wang P-F, et al. Synthesis, molecular
docking and biological evaluation of 3-arylfuran-2(5H)-ones
as anti-gastric ulcer agent. Bioorg Med Chem 2015;23:
4860–5.
14. Xiao Z-P, Peng Z-Y, Dong J-J, et al. Synthesis, structure–ac-
tivity relationship analysis and kinetics study of reductive
derivatives of flavonoids as Helicobacter pylori urease inhibi-
tors. Eur J Med Chem 2013;63:685–95.
Acknowledgements
Authors thank Dr Dong-Dong Li for molecular dynamics.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The work was financed by grants from National Natural Science
Foundation of China (grant No. 21867010), National
Demonstration Centre for Experimental Chemistry Education
15. Xiao Z-P, Ma T-W, Fu W-C, et al. The synthesis, structure and
activity evaluation of pyrogallol and catechol derivatives as
H. pylori urease inhibitors. Eur J Med Chem 2010;45:5064–70.
(grant No. 2018ZXCX17), and a Graduate Research Innovation 16. Sivapriya K, Suguna P, Banerjee A, et al. Facile one-pot syn-
Project supported by Jishou University (grant No. JGY201842). thesis of thio and selenourea derivatives: a new class of

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
413
potent urease inhibitors. Bioorg Med Chem Lett 2007;17: 23. SYBYL molecular modeling software, version SYBYL-X 2.1. St.
6387–91. Louis (MO): Tripos Inc.; 2015.
17. Taha M, Wadood A. Synthesis and molecular docking study 24. Ha NC, Oh ST, Sung JY, et al. Supramolecular assembly and
of piperazine derivatives as potent urease inhibitors. Bioorg
Chem 2018;78:411–7.
acid resistance of Helicobacter pylori urease. Nat Struct Biol
2001;8:505–9.
€
€
18. Bano B, Kanwal, Khan KM, et al. Synthesis, in vitro urease 25. Frostell A, Vinterback L, Sjobom H. Protein-ligand interac-
inhibitory activity, and molecular docking studies of thiourea
tions using SPR systems. Methods Mol Biol 2013;1008:
and urea derivatives. Bioorg Chem 2018;80:129–44.
139–65.
19. Amtul Z, Atta-ur-Rahman BSP, Siddiqui R, et al. Chemistry 26. Guo D, Hillger JM, IJzerman AP, Heitman LH. Drug-target
and mechanism of urease inhibition. Curr Med Chem 2002;9:
1323–48.
residence time—a case for G protein-coupled receptors.
Med Res Rev 2014;34:856–92.
20. Weatherburn MW. Phenol–hypochlorite reaction for deter- 27. Mazzei L, Cianci M, Musiani F, et al. Inactivation of urease by
mination of ammonia. Anal Chem 1967;39:971–4.
21. Methods for antimicrobial susceptibility testing of anaerobic
bacteria, M11. 7th ed. Wayne (PA): CLSI; 2007.
22. Patil S, Sistla S, Jadhav J. Screening of inhibitors for mush-
room tyrosinase using surface plasmon resonance. J Agric
Food Chem 2014;62:11594–601.
catechol: kinetics and structure. J Inorg Biochem 2017;166:
182–9.
28. Mazzei L, Cianci M, Contaldo U, et al. Insights into urease
inhibition by N-(n-butyl) phosphoric triamide through an
integrated structural and kinetic approach. J Agric Food
Chem 2019;67:2127–38.