Novel (thio)barbituric-phenoxy-N-phenylacetamide derivatives as potent urease inhibitors: synthesis, in vitro urease inhibition, and in silico evaluations

Article abstract of DOI:10.1007/s11224-020-01617-6

A novel series of (thio)barbituric-phenoxy-N-phenylacetamide derivatives 7a-l was synthesized and evaluated against Helicobacter pylori urease. The latter assay revealed that all the synthesized compounds 7a-l (IC₅₀ = 0.69 ± 0.33–2.47 ± 0.23?μM

Full text of DOI:10.1007/s11224-020-01617-6

Structural Chemistry
https://doi.org/10.1007/s11224-020-01617-6
ORIGINAL RESEARCH
Novel (thio)barbituric-phenoxy-N-phenylacetamide derivatives
as potent urease inhibitors: synthesis, in vitro urease inhibition,
and in silico evaluations
Saeb Sedaghati1 & Homa Azizian2 & Mohammad Nazari Montazer1 & Maryam Mohammadi-Khanaposhtani3
&
Mehdi Asadi1 & Fatemeh Moradkhani1 & Mehdi Shafiee Ardestani4 & Mohammad Sadegh Asgari5 & Azadeh Yahya-
Meymandi6 & Mahmood Biglar7 & Bagher Larijani7 & Seyed Esmaeil Sadat-Ebrahimi1 & Alireza Foroumadi1
Massoud Amanlou1 & Mohammad Mahdavi7
&
Received: 2 June 2020 /Accepted: 11 August 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A novel series of (thio)barbituric-phenoxy-N-phenylacetamide derivatives 7a-l was synthesized and evaluated against
Helicobacter pylori urease. The latter assay revealed that all the synthesized compounds 7a-l (IC50 = 0.69 0.33–2.47
0.23 μM) were significantly more potent than two used standard inhibitors, thiourea (IC50 = 23 0.73 μM) and hydroxyurea
(IC50 = 100 1.7 μM). Docking study of the synthesized compounds demonstrated that these compounds as well fitted in the
urease active site. Moreover, molecular dynamic study of the most potent compound 7d showed that this compound created
important interactions with the active site flap residues, Cys592 and His593. Furthermore, in silico pharmacokinetic study
predicted that all the synthesized compounds are drug-like.
.
.
.
.
.
Keywords Urease Synthesis Barbituric acid Docking In silico N-phenylacetamide
Introduction
urease has been discovered as the key virulence factor of
many microorganisms such as Helicobacter pylori (H. pylori)
[3]. Indeed, H. pylori infection leads to peptic and duodenal
ulcer that could further result to gastric cancer that is the sec-
ond cause of death in worldwide [4]. One of the most impor-
tant ways to stop H. pylori activity is urease inhibitors [5]. On
the other hand, there are evidences that showed microbial
ureases are involved in the pathogenesis of diseases such as
Urease (EC 3.5.1.5) is a metalloenzyme of amidohydrolase
superfamily that catalyzes conversion of urea to ammonia
and carbon dioxide and therefore plays pivotal role in nitrogen
cycle [1]. In agriculture, the urea performance as efficient soil
fertilizer is restricted due to environmental concerns and corps
damage which is attributed to urease activity [2]. In addition,
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11224-020-01617-6) contains supplementary
material, which is available to authorized users.
3
* Mahmood Biglar
Cellular and Molecular Biology Research Center, Health Research
mbiglar@tums.ac.ir
Institute, Babol University of Medical Sciences, Babol, Iran
4
* Mohammad Mahdavi
Department of Radiopharmacy and Medicinal Chemistry, Faculty of
momahdavi@tums.ac.ir
Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
5
School of Chemistry, College of Science, University of Tehran,
1
Department of Medicinal Chemistry, Faculty of Pharmacy and
Tehran, Iran
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran, Iran
6
Department of Chemistry, Faculty of Science, University of Birjand,
Birjand, Iran
2
7
Department of Medicinal Chemistry, School of
Endocrinology and Metabolism Research Center, Endocrinology and
Pharmacy-International Campus, Iran University of Medical
Sciences, Tehran, Iran
Metabolism Clinical Sciences Institute, Tehran University of
Medical Sciences, Tehran, Iran

Struct Chem
kidney stones and pyelonephritis [6]. Therefore, discovery
and development of potent and safe urease inhibitors is an
important target for pharmaceutical researchers.
apparatus and are uncorrected. The reaction progress was
monitored by thin layer chromatography (TLC); silica gel
60F254 coated aluminum sheets (Merck, Germany) were visu-
alized by ultraviolet light at wavelength of 254 and 365 nm.
The IR spectra of compounds 7a-l was recorded on Nicolet
Magna FT-IR 550 spectrophotometer using KBr disks. All
synthetized compounds 7a-l were dissolved in DMSO-d6 to
Barbituric acid derivatives exhibited a wide range of bio-
logical activities such as sedative, hypotensive, tranquilizing,
and antibacterial [7–9]. Furthermore, several derivatives of
barbituric acid with high inhibitory activity against urease
had been reported [10, 11]. For example, 5-arylidene barbitu-
rates A exhibited significant anti-urease activity in compari-
son with thiourea as standard inhibitor (Fig. 1) [12].
Moreover, compounds B with phenoxy moiety and com-
pounds C with phenylacetamide moiety are effective inhibi-
tors against urease (Fig. 1) [13, 14].
According to mentioned points and in continuation of our
attempts for design of new and potent urease inhibitors, here-
in, we designed (thio)barbituric-phenoxy-N-phenylacetamide
scaffold (Fig. 1) [15–17]. Twelve derivatives of this scaffold
were synthesized via Knoevenagel condensation and were
evaluated against H. pylori urease. In silico induced fit
docking and molecular dynamic studies of these compounds
were performed to further investigate the interaction, orienta-
tion, and conformation in the active site of urease.
Furthermore, in silico drug-likeness and oral absorption pre-
diction of new compounds 7a-l were also performed.
1
obtain H and 13C NMR spectra on a Bruker FT-500 using
TMS as an internal standard. Elementar Analysen System
GmbH VarioEL CHN mode was used for elemental analysis
of title compounds.
In vitro urease inhibitory assay
The urease inhibitory activity of the synthesized compounds
7a-l was determined by Berthelot spectrophotometric method
[17]. In this assay, the anti-urease activity is calculated based
on blue colored indophenol produced by the reaction of phe-
nol with monochloramine, which is formed in situ through
reaction of ammonia (NH3) and hypochlorite (HOCl). The
absorbance of obtained indophenol was determined by a
Synergy H1 Hybrid multimode microplate reader (BioTek,
Winooski, VT, USA) at 625 nm.
The assay mixture included urea (850 μL, 30 mM, in phos-
phate buffer 100 mM, pH 7.4), compounds 7a-l (100 μL, at
the concentration of 0–10 mg/ml), and phosphate buffer
(100 mM, pH 7.4) with the total volume of 985 μL. Then,
15 μL of urease solution (JBU, EC 3.5.1.5, 3 mg/mL, the
phosphate buffer 100 mM, pH 7.4) was added to the assay
mixture, and the obtained mixture was incubated at 37 °C for
60 min. After that, the concentration of ammonia produced by
the activity of uninhibited urease enzyme was determined
through addition of solution A (500 μL, consist of 1% w/v
phenol (5.0 g) and 0.005% w/v sodium nitroprusside
Experimental details
Material and methods
All reagents and solvents were purchased from Sigma-Aldrich
(USA) and were used without further purifications. Melting
points of (thio)barbituric acid-phenoxy-N-phenylacetamide
derivatives 7a-l were determined on Kofler hot stage
Fig. 1 Design strategy for new urease inhibitors 7a-l

Struct Chem
(25.0 mg) in 500 mL distilled water) and solution B (500 μL,
consist of 0.5% w/v sodium hydroxide (2.5 g) and 4.2 mL
sodium hypochlorite (5% chlorine) in 500 mL distilled water)
and further incubated at 37 °C for 30 min. The percentage of
inhibited enzyme was determined by measuring the absor-
bance of blue colored indophenol through the following equa-
tion: I (%) = [1 − (T/C)] × 100.
In the above equation, I (%) is assigned to percent of en-
zyme inhibition, T (test) is assigned to the absorbance of com-
pounds 7a-l, positive control, and C is assigned to control
which is the absorbance of the solvent in the presence of
enzyme. Thiourea and hydroxyurea were used as standard
urease inhibitor. The IC50 values were determined using
PRISM 4.0 software (GraphPad, San Diego, CA, USA), and
the results are shown in mean standard error.
2018–4 suite) [22]. The appropriate pose for MD simulation
procedure of the compounds was achieved by IFD method.
In order to build the system for MD simulation, the protein-
ligand complexes were solvated with SPC explicit water mol-
ecules and placed in the center of an orthorhombic box of
appropriate size in the periodic boundary condition.
Sufficient counter-ions and a 0.15 M solution of NaCl were
also utilized to neutralize the system and to simulate the real
cellular ionic concentrations, respectively. The MD protocol
involved minimization, pre-production, and finally production
MD simulation steps. In the minimization procedure, the en-
tire system was allowed to relax for 2500 steps by the steepest
descent approach. Then the temperature of the system was
raised from 0 to 300 K with a small force constant on the
enzyme in order to restrict any drastic changes. MD simula-
tions were performed via NPT (constant number of atoms;
constant pressure, i.e., 1.01325 bar; and constant temperature,
i.e., 300 K) ensemble. The Nose-Hoover chain method was
used as the default thermostat with 1.0 ps interval and
Martyna-Tobias-Klein as the default barostat with 2.0 ps in-
terval by applying isotropic coupling style. Long-range elec-
trostatic forces were calculated based on particle-mesh-based
Ewald approach with the he cutoff radius for columbic forces
set to 9.0 Å. Finally, the system subjected to produce MD
simulations for 30 ns for each protein-ligand complex.
During the simulation, every 1000 ps of the actual frame
was stored. The dynamic behavior and structural changes of
the systems were analyzed by the calculation of the root mean
square deviation (RMSD) and RMSF. Subsequently, the en-
ergy-minimized structure calculated from the equilibrated tra-
jectory system was evaluated for investigation of each ligand-
protein complex interaction.
Molecular docking study
In order to find out the interactions mode of designed mole-
cules over urease enzyme, Maestro Molecular Modeling plat-
form (version10.5) by Schrödinger, LLC, was performed [18].
The X-ray crystallographic structure of JBU (in complex with
acetohydroxamic acid, AHA) was downloaded from the
Protein Data Bank (PDB ID; 4h9m) (www.rcsb.org). As
urease is reported to be functionally active in monomeric
state, all the docking studies were performed on single
monomer. In addition, prosthetic group and co-factors are not
directly involved in urease inhibition, so they are totally
removed before docking investigation. Water molecules and
co-crystallized ligands were removed from the enzymes
crystallographic structures. The 2D structures of all
synthesized compounds were drawn in Marvin 15.10.12.0
program (http://www.chemaxon.com) and converted into pdb
file. The Protein Preparation Wizard and the LigPrep module
were used to prepare protein and ligand structure properly [19,
20]. The missing side chains of the proteins were filled using
the Prime tool, and missing residues were updated.
The accurate side-chain, backbone conformational changes
or both during ligand binding at the active site of urease en-
zyme were predicted by IFD method using Glide software
(Schrödinger LLC 2018, USA) [21]. The AHA binding site
was used to generate the grid for IFD calculation. The maxi-
mum 20 poses with receptor and ligand van der Waals radii of
0.7 and 0.5, respectively, were considered. Residues within
5 Å of the AHA at the active site were refined followed by
side-chain optimization. Structures whose Prime energy is
more than 30 Kcal/mol are eliminated based on extra precious
Glide docking.
In vitro cytotoxicity assay
In vitro cytotoxicity assay of the all synthesized compounds
was determined against human breast cancer cell line MCF-7
cell line by MTT assay according to the previous work [23].
In silico ADME properties of synthesized compounds
QikProp module of Schrodinger was used to calculate drug-
likeness and oral absorption prediction of the synthesized
compounds [24].
General procedure for synthesis of 2-chloro-N-
phenylacetamide derivatives 3a-g
A mixture of aniline derivatives 1 (1 mmol) and 2-
chloroacetyl chloride 2 (1 mmol) in DMF was stirred at room
temperature for 30 min. At the end of the reaction (checked by
TLC), the reaction mixture was diluted with cold water and
poured into ice, and the obtained precipitate was filtered off.
Molecular dynamic simulation
Molecular simulations of this study were performed using the
Desmond v5.3 using Maestro interface (from Schrödinger

Struct Chem
The residue was washed with cold water to obtain pure 2-
N-(2,4-difluorophenyl)-2-(4-((4,6-dioxo-2-
chloro-N-phenylacetamide derivatives 3a-g.
thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7c)
General procedure for synthesis of 2-(4-
formylphenoxy)-N-phenylacetamide derivatives 5a-g
Yield 86%; Cream powder; mp 201–207 °C. IR (KBr, cm−1):
ν = 3337, 3023, 2966, 1681, 1H NMR (500 MHz, DMSO-d6)
δ 9.48 (s, 1H, 2NH), 8.37 (s, 2H, NH-amide), 8.20 (s, 1H, CH-
vinyl), 7.89 (d, J = 8.4 Hz, 2H, Ar), 7.75 (q, J = 8.9 Hz, 1H,
Ar), 7.38–7.30 (m, 1H, Ar), 7.18 (d, J = 8.6 Hz, 2H, Ar), 7.08
(t, J = 7.9 Hz, 1H, Ar), 4.91 (s, 2H, CH2); 13C NMR
(126 MHz, DMSO-d6) δ 177.41, 174.48, 166.87, 163.09,
160.69, 158.88, 155.08, 150.65, 132.19, 130.61, 126.95,
126.88, 122.27, 122.15, 115.64, 111.79, 111.59, 104.95,
104.75, 104.54, 67.28. Anal. Calcd for C19H13F2N3O4: C,
54.67; H, 3.14; N, 10.07. Found: C, 54.61; H, 3.12; N, 10.09.
A mixture of 2-chloro-N-phenylacetamide derivatives 3a-g
(1 mmol), 4-hydroxybenzaldehyde 1 mmol 4, and K2CO3 in
DMF was heated at reflux for 3 h. After completion of reaction
(checked by TLC), the reaction mixture was poured into
crushed ice and filtered and washed with cold water to give
pure 2-(4-formylphenoxy)-N-phenylacetamide derivatives
5a-g.
General procedure for synthesis of (thio)barbituric
acid-phenoxy-N-phenylacetamide derivatives 7a-l
N-(2,3-dichlorophenyl)-2-(4-((2,4,6-
trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7d)
A solution of (thio)barbituric acid 6a-b (1 mmol) and 2-(4-
formylphenoxy)-N-phenylacetamide derivatives 5a-g in
MeOH/EtOH (1:1, 10 mL) in presence of p-toluenesulfonic
acid (1 mmol) as catalyst was refluxed for 8–12 h (checked by
TLC). Then, the reaction mixture was filtered off, and residue
was washed with cold methanol to give pure (thio)barbituric
acid-phenoxy-N-phenylacetamide derivatives 7a-l.
Yield 89%; Cream powder; mp 178–180 °C. IR (KBr, cm−1):
ν = 3323, 3018, 2967, 1733. 1H NMR (500 MHz, DMSO-d6)
δ 10.50 (s, 1H, NH-amide), 9.92 (s, 2H, 2NH), 9.27 (s, 1H,
CH-vinyl), 8.36 (d, J = 8.6 Hz, 1H, Ar), 7.91 (d, J = 8.6 Hz,
1H, Ar), 7.52–7.49 (m, 1H, Ar), 7.21 (d, J = 9.5 Hz, 1H, Ar),
7.14 (d, J = 8.4 Hz, 1H, Ar), 7.08 (d, J = 8.4 Hz, 1H, Ar), 6.98
(d, J = 6.8 Hz, 1H, Ar), 4.95 (s, 2H, CH2); 13C NMR
(126 MHz, DMSO- d6) δ 191.83, 166.95, 162.88, 137.58,
136.57, 132.39, 132.22, 130.72, 128.64, 127.62, 125.04,
115.72, 115.03, 67.38. Anal. Calcd for C19H13Cl2N3O5: C,
52.55; H, 3.02; N, 9.68. Found: C, 52.53; H, 3.07; N, 9.63.
N-phenyl-2-(4-((2,4,6-trioxotetrahydropyrimidin-
5(2H)-ylidene)methyl)phenoxy) acetamide (7a)
Yield 92%; Cream powder; mp 185–190 °C. IR (KBr, cm−1):
ν = 3338, 3026, 2961, 1728, 1H NMR (500 MHz, DMSO-d6)
δ 10.00 (s, 2H, 2NH), 9.58 (s, 1H, NH-amide), 8.17 (s, 1H,
CH-vinyl), 7.34–7.30 (m, 3H, Ar), 7.06 (dd, J = 13.5, 7.9 Hz,
3H, Ar), 4.58 (s, 2H, CH2). 13C NMR (126 MHz, DMSO-d6)
δ 157.92, 151.11, 138.84, 138.13, 130.27, 129.78, 129.19,
129.10, 128.05, 120.30, 120.16, 115.18, 114.14, 69.03.
Anal. Calcd for C19H15N3O5: C, 62.46; H, 4.14; N, 11.50.
Found: C, 62.49; H, 4.12; N, 11.55.
N-(3-chloro-2-methylphenyl)-2-(4-((2,4,6-
trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7e)
Yield 91%; Cream powder; mp 181–183 °C. IR (KBr, cm−1):
ν = 3329, 3020, 2961, 1722. 1H NMR (500 MHz, DMSO-d6)
δ 9.89 (s, 2H, 2NH), 8.99 (s, 1H, NH-amide), 8.26 (s, 1H, CH-
vinyl), 7.91 (d, J = 8.4 Hz, 2H, Ar), 7.35–7.32 (m, 2H, Ar),
7.22 (t, J = 8.2 Hz, 3H, Ar), 4.90 (s, 2H, CH2), 2.20 (s, 3H,
CH3); 13C NMR (126 MHz, DMSO- d6) δ 191.82, 166.75,
163.08, 137.49, 134.23, 132.20, 131.48, 130.63, 127.36,
127.02, 125.40, 115.70, 67.44, 15.51. Anal. Calcd for
C20H16ClN3O5: C, 58.05; H, 3.90; N, 10.15. Found: C,
58.01; H, 3.87; N, 10.18.
N-(4-fluorophenyl)-2-(4-((2,4,6-
trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7b)
Yield 87%; Cream powder; mp 208–213 °C. IR (KBr, cm−1):
ν = 3327, 3020, 2966, 1731. 1H NMR (500 MHz, DMSO-d6)
δ 9.88 (s, 2H, 2NH), 9.76 (s, 1H, NH-amide), 8.30 (s, 1H, CH-
vinyl)7.89 (d, J = 8.8 Hz, 2H, Ar), 7.64 (dd, J = 8.9, 5.0 Hz,
2H, Ar), 7.20–7.14 (m, 4H, Ar), 4.84 (s, 2H, CH2); 13C NMR
(126 MHz, DMSO- d6) δ 191.79, 166.27, 163.17, 135.13,
135.11, 132.20, 130.58, 122.04, 121.98, 115.89, 115.71,
115.65, 67.51. Anal. Calcd for C19H14FN3O5: C, 59.53; H,
3.68; N, 10.96. Found: C, 59.48; H, 3.65; N, 10.94.
2-(4-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)phenoxy)-N-phenylacetamide (7f)
Yield 94%; Cream powder; 212–214 °C. IR (KBr, cm−1): ν =
1
3326, 3019, 2961, 1687. H NMR (500 MHz, DMSO-d6) δ
10.17 (s, 2H, 2NH), 9.61 (s, 1H, NH-amide), 8.09 (s, 1H, CH-

Struct Chem
vinyl), 7.91 (d, J = 7.7 Hz, 2H, Ar), 7.64 (d, J = 6.8 Hz, 2H,
Ar), 7.37–7.29 (m, 2H, Ar), 7.20 (d, J = 7.6 Hz, 2H, Ar), 7.10
(s, 1H, Ar), 4.87 (s, 2H, CH2); 13C NMR (126 MHz, DMSO-
d6) δ 191.81, 166.30, 163.24, 138.79, 132.55, 132.21, 130.57,
129.24, 129.15, 124.23, 120.31, 120.14, 115.65, 67.56. Anal.
Calcd for C19H15N3O4S: C, 59.83; H, 3.96; N, 11.02. Found:
C, 59.84; H, 3.96; N, 11.08.
N-(2,3-dichlorophenyl)-2-(4-((4,6-dioxo-2-
thioxotetrahydropyrimidin-5(2H) ylidene)methyl)
phenoxy) acetamide (7j)
Yield 93%; Cream powder; mp 190–192 °C. IR (KBr, cm−1):
ν = 3334, 3021, 2967, 1674. 1H NMR (500 MHz, DMSO-d6)
δ 10.50 (s, 2H, 2NH), 9.43 (s, 1H, NH-amide), 8.14 (s, 2H,
CH-vinyl), 7.91 (d, J = 8.6 Hz, 2H, Ar), 7.28 (d, J = 8.3 Hz,
2H, Ar), 7.22 (d, J = 8.6 Hz, 2H, Ar), 7.08 (d, J = 8.5 Hz, 2H,
Ar), 4.95 (s, 2H, CH2); 13C NMR (126 MHz, DMSO- d6) δ
191.83, 167.25, 166.92, 136.56, 132.22, 129.48, 128.64,
127.33, 125.03, 124.39, 115.72, 114.25, 67.38. Anal. Calcd
for C19H13Cl2N3O4S: C, 50.68; H, 2.91; N, 9.33. Found: C,
50.74; H, 2.96; N, 9.34.
2-(4-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)phenoxy)-N-(4-fluorophenyl)
acetamide (7g)
Yield 87%; Cream powder; mp 174–176 °C. IR (KBr, cm−1):
ν = 3334, 3028, 2966, 1677. 1H NMR (500 MHz, DMSO-d6)
δ 10.22 (s, 2H, 2NH), 9.76 (s, 1H, NH-amide), 8.18 (s, 1H,
CH-vinyl), 7.89 (d, J = 8.7 Hz, 2H, Ar), 7.65 (dd, J = 8.9,
5.0 Hz, 2H, Ar), 7.23–7.11 (m, 4H, Ar), 4.85 (s, 2H, CH2);
13C NMR (126 MHz, DMSO- d6) δ 191.81, 166.27, 163.17,
135.15, 132.21, 130.60, 122.05, 121.99, 115.90, 115.72,
115.66, 67.52. Anal. Calcd for C19H14FN3O4S: C, 57.14; H,
3.53; N, 10.52. Found: C, 57.11; H, 3.57; N, 10.59.
N-(3-bromophenyl)-2-(4-((4,6-dioxo-2-
thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7k)
Yield 90%; Cream powder; mp 200–203 °C. IR (KBr, cm−1):
ν = 3335, 3021, 2965, 1680. 1H NMR (500 MHz, DMSO-d6)
δ 10.33 (s, 2H, 2NH), 9.71 (s, 1H, NH-amide), 8.20 (s, 1H,
CH-vinyl), 7.97 (s, 1H, Ar), 7.89 (d, J = 8.6 Hz, 2H, Ar), 7.57
(d, J = 7.3 Hz, 1H, Ar), 7.29 (d, J = 7.9 Hz, 2H, Ar), 7.19 (d,
J = 8.7 Hz, 2H, Ar), 4.87 (s, 2H, CH2); 13C NMR (126 MHz,
DMSO-d6) δ 191.80, 166.75, 163.12, 162.83, 140.35, 132.20,
132.05, 131.26, 130.61, 129.91, 126.84, 122.44, 121.97,
118.89, 115.66, 67.45. Anal. Calcd for C19H14BrN3O4S: C,
49.58; H, 3.07; N, 9.13. Found: C, 49.54; H, 3.08; N, 9.08.
N-(2,4-difluorophenyl)-2-(4-((4,6-dioxo-2-
thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7h)
Yield 91%; Cream powder; mp 211–213 °C. IR (KBr, cm−1):
ν = 3334, 3019, 2958, 1674. 1H NMR (500 MHz, DMSO-d6)
δ 10.19 (s, 2H, 2NH), 9.48 (s, 1H, NH-amide), 8.20 (s, 1H,
CH-vinyl), 7.89 (d, J = 8.4 Hz, 2H, Ar), 7.75 (q, J = 8.9 Hz,
1H, Ar), 7.38–7.30 (m, 1H, Ar), 7.18 (d, J = 8.6 Hz, 2H, Ar),
7.08 (t, J = 7.9 Hz, 1H, Ar), 4.91 (s, 2H); 13C NMR
(126 MHz, DMSO- d6) δ 191.80, 166.87, 163.09, 132.19,
130.61, 126.95, 126.88, 122.27, 122.15, 115.64, 111.79,
111.59, 104.95, 104.75, 104.54, 67.28. Anal. Calcd for
C19H13F2N3O5S: C, 54.67; H, 3.14; N, 10.07. Found: C,
54.63; H, 3.13; N, 10.06.
N-(3-chloro-2-methylphenyl)-2-(4-((4,6-dioxo-2-
thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7l)
Yield 87%; Cream powder; mp 196–198 °C. IR (KBr, cm−1):
ν = 3334, 3016, 2963, 1688. 1H NMR (500 MHz, DMSO-d6)
δ 10.23 (s, 2H, 2NH), 9.14 (s, 1H, NH-amide), 8.20 (s, 1H,
CH-vinyl), 7.94–7.87 (m, 2H, Ar), 7.35–7.28 (m, 2H, Ar),
7.25–7.16 (m, 3H, Ar), 4.89 (s, 2H, CH2), 2.20 (s, 3H,
CH3); 13C NMR (126 MHz, DMSO- d6) δ 190.83, 166.25,
165.92, 135.56, 131.83, 131.22, 128.48, 127.64, 126.33,
124.03, 123.39, 114.72, 113.25, 66.38, 10.59. Anal. Calcd
for C20H16ClN3O4S: C, 55.88; H, 3.75; N, 9.77. Found: C,
55.86; H, 3.71; N, 9.79.
N-(2-chlorophenyl)-2-(4-((4,6-dioxo-2-
thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)
phenoxy) acetamide (7i)
Yield 88%; Cream powder; mp 182–184 °C. IR (KBr, cm−1):
ν = 3335, 3023, 2964, 1682. 1H NMR (500 MHz, DMSO-d6)
δ 10.38 (s, 2H, 2NH), 9.42 (s, 1H, NH-amide), 8.65 (s, 1H,
CH-vinyl), 7.84 (dd, J = 14.9, 8.3 Hz, 3H, Ar), 7.53 (d, J =
8.2 Hz, 1H, Ar), 7.36 (t, J = 7.7 Hz, 1H, Ar), 7.23 (t, J =
7.6 Hz, 1H, Ar), 7.15 (d, J = 8.6 Hz, 2H, Ar), 4.87 (s, 2H,
CH2); 13C NMR (126 MHz, DMSO- d6) δ 190.79, 165.86,
162.09, 131.18, 129.61, 125.93, 121.17, 114.64, 110.59,
108.13, 103.73, 66.28. Anal. Calcd for C19H14ClN3O4S: C,
54.88; H, 3.39; N, 10.10. Found: C, 54.83; H, 3.34; N, 10.11.
Results and discussion
Chemistry
The synthetic pathway for (thio)barbituric acid-phenoxy-N-
phenylacetamide derivatives 7a-l was depicted in Scheme 1.
It started with the reaction between aniline derivatives 1a-g

Struct Chem
and 2-chloroacetyl chloride 2 in DMF to give 2-chloro-N-
phenylacetamide derivatives 3a-g. The latter compounds
reacted with 4-hydroxybenzaldehyde 4 in the presence of
K2CO3 in DMF to produce 2-(4-formylphenoxy)-N-
phenylacetamide derivatives 5a-g. Finally, desired com-
pounds 7a-l were obtained from a Knoevenagel condensation
between formaldehyde derivatives 5a-g and barbituric acid
derivatives 6a-b.
The comparison of urease inhibitory activity of barbituric
acid derivatives with their corresponding thio-barbituric acid
analogs revealed that barbituric acid analogs 7a, 7c, and 7d
respectively with un-substituted, 2,4-difluoro, and 2,3-
dichloro-phenylacetamide moiety were more active than their
thio-barbituric acid analogs 7f, 7h, and 7j. In contrast, urease
inhibitory activity of 4-fluoro and 2-methyl-3-chloro deriva-
tives 7b and 7e of barbituric acid series was less than their
thio-barbituric acid analogs 7g and 7l.
In vitro urease inhibitory activity
Docking study
Urease inhibitory activity of all the synthesized (thio)barbitu-
ric acid-phenoxy-N-phenylacetamide derivatives 7a-l was de-
termined against H. pylori urease. As can be seen in Table 1,
compounds 7a-l with IC50 values = 0.69 0.33 to 2.47
0.23 μM exhibited excellent inhibitory activities against ure-
ase in comparison with two used standard inhibitors hydroxy-
urea and thiourea with IC50s 100 1.7 and 23 0.73,
respectively.
Structurally, synthesized compounds 7a-l can be divided to
two series: barbituric acid derivatives 7a-e and thio-barbituric
acid derivatives 7f-l. In each series, substituent on phenyl ring
of N-phenylacetamide moiety was altered to optimize the ure-
ase inhibitory activity.
Among the barbituric acid derivatives, the most potent
compound was compound 7d with 2,3-dichloro substituents
on N-phenylacetamide moiety. This compound was also the
most potent compound among all the synthesized compounds.
Replacement of 2-chloro substituent of this compound with 2-
methyl substituent, as in compound 7e, led to a significant
decrease in inhibitory activity. The second potent compound
in this series was 2,4-difluoro derivative 7c. Removing the 2-
fluoro substituent of this compound slightly diminished inhib-
itory activity as observed in compound 7b. Moreover, un-
substituted derivative 7a has a slightly reduced inhibitory ac-
tivity compared with 4-fluoro derivative 7b.
The most potent compound among the thio-barbituric acid
derivatives 7f-l, was 4-fluoro derivative 7g while the weakest
compound in this series was the un-substituted compound 7 f.
The introduction of second fluorine atom onto the 2-position
of compound 7g, as compound 7h, led to a moderate decrease
in inhibitory activity. Moreover, the introduction of 2-chloro,
2,3-dichloro, 3-bromo or 2-methyl-3-chloro on phenyl ring of
N-phenylacetamide moiety did not improve anti-urease poten-
cy as observed in compounds 7i-l.
In order to investigate the interaction modes of the synthesized
compounds 7a-l in the active site of urease, docking study was
carried out on Jack bean urease (JBU, pdb ID: 4h9m) [17].
Urease inhibitor acetohydroxamic acid (AHA) is bounded to
active site of JBU.
The applied docking procedure reliability was validated by
re-docking of AHA over JBU based on glide docking proce-
dure. The docked conformation corresponding to the lowest
glide energy was selected as the most possible binding modes.
The root mean square deviation (RMSD) was calculated for
each ligand to measure the docking prediction accuracy. The
pose was counted optimal if its RMSD was found to be less
than 2 Å. The RMSD of the re-docked conformations of AHA
over JBU was 1.02 Å (Fig. 2), which is considered success-
fully docked.
The performed docking procedure was then applied to
evaluate the interaction between newly synthetized com-
pounds, 7a-l over the JBU active site in comparison with
thiourea as a reference urease inhibitor. The top scoring
pose of all compounds was analyzed inside the binding
site of JBU. In the binding model, all the compounds are
successfully occupied in the bi-nickel active site cavity.
Figure 3a shows that the fitting-in mechanism of
barbituric acid and thio-barbiturate moiety in the active
site of the enzyme is quite similar. The (thio)barbituric
acid ring tends to orient toward the two nickel atoms
through the ionized N (N−) and the carbonyl group at C4
position of the (thio)barbiturate ring (Fig. 3b) which is
similar to the behavior of the carbonyl oxygens in the
AHA.
Additionally, Fig. 3 b shows that thio-barbituric acid ring
appears to make some distance deviation from Ni-Ni center
which cased as a result of steric effect of the sulfur atom.
Scheme 1 Synthesis procedure for desired compounds 7a-l: (a) DMF, room temperature, 30 min; (b) K2CO3, DMF, reflux, 2 h; (c) p-TsOH,
MeOH/EtOH, reflux, 8–12 h

Struct Chem
Table 1 Urease inhibitory activities of (thio)barbituric acid-phenoxy-N-phenylacetamide derivatives 7a-l
Compound
X
O
O
O
O
O
S
R
IC50 (μM)a Compound
1.62 0.14 7h
X
S
S
S
S
S
-
R
IC50 (μM)a
1.72 0.41
1.61 0.18
2.21 0.52
2.33 0.28
1.59 0.25
100 1.7
23 0.73
7a
7b
7c
7d
7e
7f
H
2,4-Difluoro
4-F
1.48 0.09 7i
2-Cl
2,4-Difluoro
2,3-Dichloro
2-CH3-3-Cl
H
1.29 0.18 7j
2,3-Dichloro
0.69 0.33 7k
3-Br
1.75 0.17 7l
2-CH3-3-Cl
2.47 0.23 Hydroxyurea
1.19 0.14 Thiourea
-
-
7g
S
4-F
-
On the other hand, the phenoxy-N-phenylacetamide moiety
adapts by flexible conformation in the large hydrophobic
opening of the active site flap pocket (Fig. 3c).
The molecular interactions of the best conformational pose
and energy-valued docked complex of compounds 7d, 7b, 7g,
and 7c are illustrated in Fig. 4.
Figure 4 a shows barbituric acid ring of compound 7d tightly
coordinated along the metal bi-nickel center and further stabi-
lized by H-bond interaction with His492 and Asp633. At the
middle part of the molecule, the benzylidene group formed
hydrophobic interaction through Cys592. Finally, the amide
group at the tail part of the compound formed H-bond with
His593, which consequently oriented the attached 2,3-dichloro
phenyl group in front of the active site flap entrance.
Compounds 7b, 7g, and 7c depict similar orientation and
interaction to 7d (metal coordination forming through (thio)-
barbituric acid ring, H-bond with Cys592 and His593) (Fig. 4
b, c, and d).
Fig. 2 The location of JBU active site over C-terminal (αβ)8 TIM barrel domain, close-up representation of active site, the AHA co-crystallized, and the
corresponding re-docked form are represented in green and cyan color, respectively

Struct Chem
Fig. 3 Representation of the compounds docking poses over the active
site: side view representation (a), close-up illustration of (thio)barbiturate
ring relative to the bi nuclear center (b), and upside view of the active site
which shows the orientation of the phenoxy-N-phenylacetamide relative
to the active site flap (colored in green color) (c)
It is noteworthy that Cys592 and His593 are of the key
residues in the urease active site. Ligand interacting with these
residues seems to be the most important for urease inhibition
because of decreasing the flexibility of mobile flap covering
the active site entrance followed by inhibiting the ureolytic
activity.
Based on crystal structures of the ureases, besides the con-
served residues in the active site, most of ureases share
conserved residues that make up the mobile flap [25],
which covers the active site. In JBU, the residues compris-
ing the mobile flap are 590–606 on α subunit part of the
enzyme. Comparing RMSF values of urease-compound
complexes shows that the residues of the flap region cov-
ering the active site, 590–606, would have significantly
lower RMSF value in urease-compound 7d rather than
urease-thiourea complex (Fig. 5). Consequently, com-
pound 7d tightly anchoring the helix-turn-helix motif over
the active-site cavity reduces the flexibility of flap residue
(590–609) by interacting with key amino acid residues and
results in the inhibition of urease activity.
Molecular dynamic study
In addition, to understand dynamics behavior and the de-
tailed interactions of the most potent compound 7d and
compare it with thiourea as urease standard inhibitor, mo-
lecular dynamic (MD) simulation was performed. RMSF,
which refers to the fluctuation of the Cα atom coordinates
from its average position throughout the simulation, indi-
cates the flexibility of protein backbone structure. Loops
with loosely organized structure have high RMSF value,
while helixes and sheets represent lower RMSF value.
In order to study the behavior of the active site covering
flap over the course of the MD simulation, the distances be-
tween Ile599 at the tip of the flap and Ala440 at the entrance of
active site channel in the urease-compound complexes are

Struct Chem
Fig. 4 2D representation of ligand-residue interactions of compound 7d (a), compound 7b (b), compound 7g (c), and compound 7c (d) over urease
active site. Active site flap colored in green
analyzed. Figure 6 a displays representative Ile599-Ala440
separations for the urease-compound complex. In the thiourea
bound-state, the separation of these residues is about 32 Å
corresponding as open flap conformation, while in the case
of compound 7d, this distance varies only slightly oscillating
within 26 Å, respectively, which is related to the close flap
conformation (Fig. 6b). Based on the closed flap conforma-
tion, it is proposed that compound 7d inhibits urease activity
by stabilizing the reaction intermediate during the ureolytic
reaction [26].

Struct Chem
Fig. 5 RMSF of the urease Cα in
complexed with thiourea (colored
in blue) and compounds 7d
(colored in red) for over 30 ns
MD simulation time
In vitro cytotoxicity assay
Table 2 shows the descriptor values affecting the drug-
likeness according to the Lipinski rule of five. It is con-
sidered that all of the synthesized compounds follow of
Lipinski rule of five without any deviations and therefore
are drug-like.
Solubility and permeability are two main factors affecting
the oral absorption of a compound in the body [27]. Table 3
depicts the descriptor values which influence solubility, per-
meability, and finally the human oral absorption [28]. It shows
that all the synthesized compounds with the exception of com-
pounds 7k-l are oral active.
In vitro cytotoxicity of the synthesized compounds 7a-l was
evaluated against breast cancer cell line MCF-7 by MTT assay
[23]. This assay revealed that at 150 μM, all the studied com-
pounds were non-cytotoxic against MCF-7.
In silico pharmacokinetic study
Drug-likeness and oral absorption prediction of the synthe-
sized compounds 7a-l were calculated with QikProp module
of Schrodinger.
Fig. 6 MD simulated flap distance between Ala440-Ile599 in thiourea
(blue) and compound 7d (red) in urease bound-state (a). Representative
snapshots from MD simulations where the flap covering the active site
can adopt open conformations of the flap (blue) and closed conformation
(red) (b). The zoom of the active site residues shows catalytic residues in
green and nickel atoms in purple

Struct Chem
Table 2 The Lipinski rule of five properties of compounds 7a-l
Entry
Mw
HBDa
HBAb
LogP o/wc
RBCd
Rule of five violations
7a
7b
7c
7d
7e
7f
365.345
383.335
401.326
434.235
413.816
381.405
399.396
417.386
415.850
450.295
460.301
429.877
3
3
3
3
3
3
3
3
3
3
3
3
7.250
7.250
7.250
7.250
7.250
6.750
6.750
6.750
6.750
6.750
6.750
6.750
1.699
1.926
2.134
2.472
2.436
2.811
3.040
3.277
3.280
3.694
3.362
3.467
6
6
6
6
6
6
6
6
6
6
6
6
0
0
0
0
0
0
0
0
0
0
0
0
7g
7h
7i
7j
7k
7l
a Number of average hydrogen bond donor (recommended value 0.0–6.0); b number of average hydrogen bond acceptor (recommended value 2.0–20.0);
c predicted octanol/water partition coefficient (acceptable range from − 2 to 6.5); d number of rotatable bond (recommended value 2.0–20.0)
Conclusion
Furthermore, drug-likeness and oral absorption predictions
of the synthesized compounds 7a-l were performed. These
studies predicted that all the synthesized compounds are
drug-like and with the exception of compounds 7k-l are oral
active.
In conclusion, (thio)barbituric-phenoxy-N-phenylacetamide
derivatives 7a-l were synthesized and evaluated urease inhib-
itors. The obtained urease inhibitory activates showed that all
the synthesized compounds were more active than standard
urease inhibitors hydroxyurea and thiourea. Docking study of
the synthesized compounds demonstrated that these com-
pounds as well occupied active site cavity urease. Molecular
dynamic simulation of the most active compound 7d proposed
that this compound inhibited urease by stabilizing the pro-
duced intermediate during the ureolytic reaction.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
1. Sudkolai ST, Nourbakhsh F (2017) Urease activity as an index for
assessing the maturity of cow manure and wheat residue
vermicomposts. Waste Manag 64:63–66
2. Modolo LV, da Silva CJ, Brandão DS, Chaves IS (2018) A
minireview on what we have learned about urease inhibitors of
agricultural interest since mid-2000s. J Adv Res 13:29–37
3. Kosikowska P, Berlicki Ł (2011) Urease inhibitors as potential
drugs for gastric and urinary tract infections: a patent review.
Expert Opin Ther Pat 21:945–957
4. Konturek PC, Konturek SJ, Brzozowki T (2006) Gastric cancer and
helicobacter pylori infection. J Physiol Pharmacol 57:51
5. Dunn BE, Phadnis SH (1998) Structure, function and localization
of helicobacter pylori urease. Yale J Biol Med 71:63
6. Mobley HL, Island MD, Hausinger RP (1995) Molecular biology
of microbial ureases. Microbiol Mol Biol Rev 59:451–480
7. Ikotun AA, Ojo Y, Obafemi CA, Egharevba GO (2017) Synthesis
and antibacterial activity of metal complexes of barbituric acid. Afr
J Pure Appl Chem 5:97–103
Table 3 The calculated ADME properties of synthesized compounds
7a-l
a
Entry
Log Swat
PCacob
% HOAc
Rule of three violations
7a
7b
7c
7d
7e
7f
− 4.087
− 4.435
− 4.758
− 4.947
− 5.064
− 4.965
− 5.316
− 5.698
− 5.520
− 6.099
− 5.808
− 5.755
75.181
75.192
70.469
71.802
72.814
75.466
75.895
86.768
88.112
89.156
90.216
92.657
89.448
91.118
0
0
0
0
0
0
0
0
0
0
1
1
73.215
79.827
86.673
264.691
264.740
253.405
289.660
290.358
246.436
282.585
7g
7h
7i
7j
8. Vosooghi M, Farzipour S, Saeedi M, Shareh NB, Mahdavi M,
Mahernia S, Foroumadi A, Amanlou M, Shafiee A (2015)
Synthesis of novel 5-arylidene (thio) barbituric acid and evaluation
of their urease inhibitory activity. J Iran Chem Soc 12:1487–1491
9. Harris RA, Stokes JA (1982) Effects of a sedative and a convulsant
barbiturate on synaptosomal calcium transport. Brain Res 242:157–
163
7k
7l
a Predicted aqueous solubility in mol dm−3 (− 6.5–0.5); b predicted Caco-
2 cell permeability in nm/s; c percentage human oral absorption (< 25% is
poor and > 80% is high)

Struct Chem
10. Rahim F, Ali M, Ullah S, Rashid U, Ullah H, Taha M, Javed MT,
Rehman W, Khan AA, Abid OU, Bilal M (2016) Development of
bis-thiobarbiturates as successful urease inhibitors and their molec-
ular modeling studies. Chin Chem Lett 27:693–697
11. Khan KM, Rahim F, Khan A, Shabeer M, Hussain S, Rehman W,
Taha M, Khan M, Perveen S, Choudhary MI (2014) Synthesis and
structure–activity relationship of thiobarbituric acid derivatives as
potent inhibitors of urease. Bioorg Med Chem 22:4119–4123
12. Khan KM, Ali M, Wadood A, Khan M, Lodhi MA, Perveen S,
Choudhary MI, Voelter W (2011) Molecular modeling-based anti-
oxidant arylidene barbiturates as urease inhibitors. J Mol Graph
Model 30:153–156
13. Wahid S, Jahangir S, Versiani MA, Khan KM, Salar U, Ashraf M,
Farzand U, Wadood A, Taha M, Perveen S (2020) Atenolol thio-
urea hybrid as potent urease inhibitors: design, biology-oriented
drug synthesis, inhibitory activity screening, and molecular docking
studies. Bioorg Chem 94:103359
14. Abbasi MA, Hassan M, Siddiqui SZ, Raza H, Shah SA, Seo SY
(2018) Synthesis, in vitro and in silico studies of novel potent urease
inhibitors: N-[4-({5-[(3-un/substituted-anilino-3-oxopropyl)
sulfanyl]-1, 3, 4-oxadiazol-2-yl} methyl)-1, 3-thiazol-2-yl]
benzamides. Bioorg Med Chem 26:3791–3804
15. Asadi M, Mahdavi M, Mahernia S, Rezaei Z, Safavi M, Saeedi M,
Amanlou M (2018) Synthesis and urease inhibitory activity of some
5-aminomethylene Barbituric/Thiobarbituric acid derivatives. Lett
Drug Des Discov 15:428–436
16. Moghimi S, Goli-Garmroodi F, Allahyari-Devin M, Pilali H,
Hassanzadeh M, Mahernia S, Mahdavi M, Firoozpour L,
Amanlou M, Foroumadi A (2018) Synthesis, evaluation, and mo-
lecular docking studies of aryl urea-triazole-based derivatives as
anti-urease agents. Arch Pharm 351:1800005
18. Schrodinger LL (2012) Schrodinger suite 2012 induced fit docking
protocol; glide version 5.8. Schrodinger, LLC, New York
19. Schrödinger Release 2018–4: Schrödinger Release 2018–4 Protein
Preparation Wizard; Epik, Schrödinger, LLC, New York, NY,
2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime,
Schrödinger, LLC, New York, 2019
20. Schrödinger Release 2018–4: LigPrep, Schrödinger, LLC, New
York, 2018
21. Schrödinger Release 2018–4: Induced Fit Docking protocol; Glide,
Schrödinger, LLC, New York, NY, 2018; Prime, Schrödinger,
LLC, New York, 2018
22. Schrödinger Release 2018–4: Prime; MM-GBSA , Schrödinger,
LLC, New York, 2018
23. Mohammadi-Khanaposhtani M, Fahimi K, Karimpour-Razkenari
E, Safavi M, Mahdavi M, Saeedi M, Akbarzadeh T (2019)
Design, synthesis and cytotoxicity of novel coumarin-1, 2, 3-tri-
azole-1, 2, 4-oxadiazole hybrids as potent anti-breast cancer agents.
Lett Drug Des Discov 16:818–824
24. Schrödinger Release 2018–4: QikProp, Schrödinger, LLC, New
York, 2018
25. Ha NC, Oh ST, Sung JY, Cha KA, Lee MH, Oh BH (2001)
Supramolecular assembly and acid resistance of helicobacter pylori
urease. Nat Struct Mol Biol 8:505–509
26. Arqué X, Romero-Rivera A, Feixas F, Patiño T, Osuna S, Sánchez
S (2019) Intrinsic enzymatic properties modulate the self-propul-
sion of micromotors. Nat Commun 10:1–2
27. Van De Waterbeemd H, Gifford E (2003) ADMET in silico model-
ling: towards prediction paradise? Nat Rev Drug Discov 2:192–204
28. Jorgensen WL, Duffy EM (2002) Prediction of drug solubility from
structure. Adv Drug Deliv Rev 54:355–366
17. Azizian H, Nabati F, Sharifi A, Siavoshi F, Mahdavi M, Amanlou
M (2012) Large-scale virtual screening for the identification of new
helicobacter pylori urease inhibitor scaffolds. J Mol Model 18:
2917–2927
Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.