Design and synthesis of thiobarbituric acid analogues as potent urease inhibitors

Article abstract of DOI:10.1016/j.molstruc.2021.129959

A series of thiobarbiturates 4a–4e and bis-thiobarbiturates analogues 5a–5o has been synthesized by condensing 1,3-diethylthiobarbituric acid 3 with a variety of aromatic aldehydes with varied structural features and substitution at active methylene position of thiobarbituric acid. Afterward, chemical structures of newly synthesized analogues of thiobarbituric acid were characterized through FT-IR, NMR spectroscopy and mass spectrometry. Subsequently, the inhibitory potential of thiobarbiturates 4a–4e and bis-thiobarbiturates analogues 5a–5o against urease enzyme was evaluated. The inhibitory potential of all synthesized analogues in terms of IC₅₀ value was observed in the range of 8.42 ± 0.32 to 79.34 ± 0.52 μM by comparing with thiourea (IC₅₀ 21.25 ± 0.15 μM) as a standard urease inhibitor. Out of these twenty analogues, most of the analogues exhibited potent inhibitory activity against urease. After interesting findings, structure activity relationship (SAR) has been established for all analogues. Docking studies revealed that synthesized analogues interacted with active site residues of bimetallic nickel center of the urease enzyme through, thiolate, π-π stacking and hydrogen bonding interactions.

Full text of DOI:10.1016/j.molstruc.2021.129959

Journal of Molecular Structure 1231 (2021) 129959
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design and synthesis of thiobarbituric acid analogues as potent urease
inhibitors
Matee Ullah Khana, Misbah Aslamb, Sohail Anjum Shahzada,∗, Zulfiqar Ali Khanc,∗∗
,
Nazeer Ahmad Khana, Muhammad Alid,∗∗∗, Sadia Naza,e, Jameel Rahmanb, Umar Farooqa
a Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan
b Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
c Department of Chemistry, Government College University, Faisalabad 38000, Pakistan
d Natural and Medical Sciences Research Center, University of Nizwa, Nizwa 611, Sultanate of Oman
e Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thiobarbiturates 4a–4e and bis-thiobarbiturates analogues 5a–5o has been synthesized by
condensing 1,3-diethylthiobarbituric acid 3 with a variety of aromatic aldehydes with varied structural
features and substitution at active methylene position of thiobarbituric acid. Afterward, chemical struc-
tures of newly synthesized analogues of thiobarbituric acid were characterized through FT-IR, NMR
spectroscopy and mass spectrometry. Subsequently, the inhibitory potential of thiobarbiturates 4a–4e
and bis-thiobarbiturates analogues 5a–5o against urease enzyme was evaluated. The inhibitory poten-
Received 1 November 2020
Revised 8 January 2021
Accepted 13 January 2021
Available online 19 January 2021
Keywords:
Synthesis of 1,3–diethyl-2-thiobarbituric
acid
tial of all synthesized analogues in terms of IC50 value was observed in the range of 8.42
0.32 to
79.34 0.52 μM by comparing with thiourea (IC50 21.25 0.15 μM) as a standard urease inhibitor. Out
Urease inhibition
of these twenty analogues, most of the analogues exhibited potent inhibitory activity against urease. After
interesting findings, structure activity relationship (SAR) has been established for all analogues. Docking
studies revealed that synthesized analogues interacted with active site residues of bimetallic nickel center
of the urease enzyme through, thiolate, π-π stacking and hydrogen bonding interactions.
Structure activity relationship (SAR)
Molecular docking studies
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
found that various pathological problems trigger the formation of
urinary stones. It also involves the progression of other diseases
The nickel (Ni) dependent hyperactive urease enzyme
(urea_amidohydrolase; E.C. 3.5.1.5) belongs to a family of phospho-
triesterases and amidohydrolases [1]. It is produced in a different
varieties of microorganisms including innumerable bacteria, algae,
plants, fungi and invertebrates as well as in soil [2]. Urease en-
zyme catalyzes the fast conversion of urea into ammonia (NH3)
and carbamic acid, which further disproportionate into carbon
dioxide (CO2) and another molecule of ammonia [3]. Uncontrolled
production of ammonia by overexpressed urease is lethal for
human cells. The overexpression of urease causes various patho-
logical conditions and damages the cells of living system. It is also
like pyelonephritis, hepatic coma, formation of ammonia, hepatic
encephalopathy, urolithiasis as well as urinary catheter encrus-
tation [4]. Urease of Helicobacter pylori potentially leads toward
the development of various diseases including gastric and peptic
ulcers which allows the bacteria to persevere in stomach even at
low pH range of stomach during the process of colonization [5,6].
Literature studies reveal that the gastric cancer has been ranked
as a fourth most occurring cancer and the second major source of
deaths related to cancer in the worldwide [7,8]. In agriculture, the
excessive activity of soil urease leads to significant economic and
environmental problems through anomalous production of large
quantity of ammonia into the surrounding environment during the
fertilization process of urea. Subsequently, plants are damaged due
to lack of their important nutrients as well as ammonia toxicity
and high level of soil pH [9].
∗
Corresponding author at: Department of Chemistry, COMSATS University Islam-
abad, Abbottabad Campus, Abbottabad 22060, Pakistan.
∗∗
CO-corresponding author at: Department of Chemistry, Government College
Infections caused by over-activity of urease can be treated by
inhibiting its activity. Various nitrogen containing heterocycles like
bis-thiobarbiturates [10a] are known to exhibit urease inhibitory
activity (Fig. 1). These synthetic compounds reduce the enzymatic
University Faisalabad, Faisalabad 38000, Pakistan.
∗∗∗
Co-corresponding author at: Natural and Medical Sciences Research Center, Uni-
versity of Nizwa, Nizwa 611, Sultanate of Oman.
E-mail
addresses:
sashahzad@cuiatd.edu.pk
(S.A.
Shahzad),
zulfiqarchem@gmail.com (Z.A. Khan), m.ali@unizwa.edu.om (M. Ali).
https://doi.org/10.1016/j.molstruc.2021.129959
0022-2860/© 2021 Elsevier B.V. All rights reserved.

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
Fig. 1. (A) Clinically used drugs for anti-ulcer (B) Current representative structures of urease inhibitors.
activity of urease by blocking the active site of the enzyme [10].
Such deactivation of urease enzyme leads to reduce its catalytic
efficiency for the conversion of urea into ammonia and carba-
mate or carbon dioxide. A wide variety of urease inhibitors have
been examined over the past few years for instance phospho-
rodiamidates, polyphenols, hydroxamic acid derivatives (Lithostat)
and imidazoles like lansoprazole and omeprazole (Fig. 1). However,
various side effects including teratogenicity of existing urease in-
hibitors are reported in literature. Further, in vivo usage of many
urease inhibitors was prevented owing to their toxicity or struc-
tural instability and degradation of phosphoramidates at low pH
[11]. Therefore, analogues with high inhibition potential and suit-
able hydrolytic stability are highly desirable for the possible treat-
ment of urease-based bacterial infections. For this purpose, current
research efforts are looking for potent urease inhibitors with high
inhibitory activity as well as good hydrolytic stability.
zole has been replaced with a phenyl substituted 1,3-thiobarbituric
acid moiety to provide more flexibility to accommodate within
the urease pocket site [28]. The versatile therapeutic application
of 1,3-thiobarbituric acid has been advanced by different substi-
tution pattern at aryl ring and functionalization of thiobarbituric
acid ring system [29]. We believe that nitrogen containing moiety
with two carbonyl group and additional presence of thiol group
would generate hydrogen bonding site as well as other stabilizing
electrostatic interaction within urease pocket [30]. Therefore, a set
of thiobarbiturates 4a–4e and bis-thiobarbiturates analogues 5a–
5o were synthesized by condensing 1,3-diethylthiobarbituric acid 3
with a variety of aromatic aldehydes by using ethanol as a solvent
(Scheme 1 & 2). Compounds 4a–4e were synthesized by treating
1.0 equivalent of 1,3-diethylthiobarbituric acid 3 with various aro-
matic aldehydes (Scheme 1).
However, synthesis of bis-thiobarbiturates analogues 5a–5o was
accomplished by treating 2.0 equivalent of 1,3-diethylthiobarbituric
acid 3 with a range of aromatic aldehydes (Scheme 2). Use of
ethanol as a medium of reaction makes this protocol greener and
more attractive synthetic method. Electron withdrawing substi-
tuted aromatic aldehydes enhance the rate of formation of bis-
thiobarbiturates analogues with higher yields. Further, additional
advantages are associated with a pseudo-three component proto-
col for preparation of 5a–5o. These advantages include completion
of reaction with reduced time, no need of any base as a catalyst,
easy handling of products with high yields and their purification
without using column chromatography.
Recently, 1,3-diethylthiobarbituric acid and their analogues
demonstrated a wide range of pharmacological and biological ac-
tivities, like use in anesthesia [12], antimicrobial [13], anticon-
vulsant [14], antifungal [15], antiviral [16], sedative and hyp-
notics [17], antitumor activities [18], as well as anticancer and
anti-inflammatory activities [19,20]. Moreover, thiobarbiturate ana-
logues displayed various biological activities like potential mush-
room tyrosinase inhibition [21], α-glucosidase inhibition [22], anti-
urease [23], antituberculosis [24], anti-proliferative activity [25],
radio-sensitization [26], as well as inhibition for diaminopimelate
aminotransferase [27]. Discovery of potent urease inhibitors with
reduced toxicity and increased structural stability is highly desir-
able for the treatment of urease mediated infections. Therefore,
structurally modified library of 1,3-diethylthiobarbituric acid com-
pounds were synthesized and further evaluated for urease inhibi-
tion activity.
The chemical structures of synthesized analogues of thiobarbi-
turic acid were characterized through various spectroscopic meth-
ods such as FT-IR, 1H NMR, 13C NMR and mass spectrometry. In 1H
NMR spectra, appearance of characteristic singlet peak in the range
8.40–8.78 ppm was attributed to CH olefinic moiety and aromatic
protons were observed in the range 6.46–9.48 ppm. Presence of
these characteristics signals clearly confirm the formation of de-
sired thiobarbiturate analogues 4a–4e (Scheme 1). Similarly, ob-
servation of singlet peak attributed to CH bridged proton at 5.60–
5.84 ppm and aromatic signals at 6.33–8.19 ppm confirms the for-
mation of bis-thiobarbiturates analogues 5a–5o (Scheme 2). Ring
hydrogens in compounds 5a–5o were not appeared due to pres-
ence of keto-enol tautomerism as shown in Scheme 2. Formation
of synthesized compounds was further confirmed by mass spec-
trometry analysis.
2. Results and discussion
2.1. Chemistry
The recent research for the development of unique urease in-
hibitors and distinctive biological potential of barbiturates family
encouraged us to design new analogues of 1,3-thiobarbituric acid
for the hope of finding new urease inhibitors with good inhibi-
tion potential. A benzimidazole ring in lansoprazole and omepra-
2

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
Scheme 1. Synthetic protocol for thiobarbituric acid analogues (4a–4e).
Scheme 2. Synthetic protocol for bis-thiobarbituric acid analogues (5a–5o).
2.2. Urease inhibition studies
ited potent inhibitory potential (entry 3–4, Table 1). This inhibi-
tion potential of both analogues is attributed to lone pair inter-
action of respective oxygen and sulfur with nickel atom of ure-
ase. The smaller difference in inhibitory activity between analogue
4a and 4b was observed due to similar electronic and steric na-
ture of thiophenyl and methylfuryl substituted compounds. In-
troduction of electron-donating groups in the para position of
phenyl ring for instance 4-ethoxyphenyl substituted compound 4c
and N,N-dimethylaminophenyl substituted compound 4d showed
less inhibitory activity (higher IC50 values) as compared to elec-
tron withdrawing substituted analogues (Table 1). The urease in-
hibitory activity decreased in the order of 4b > 4a > 4d >
4c > 4e.
Thiobarbituric acid analogues 4a–4e and bis-thiobarbituric acid
analogues 5a–5o were screened against urease enzyme. All the
analogues exhibited various degree of urease inhibition potential
with IC50 ranging from 8.42
0.32 to 79.34
0.52 μM by com-
paring with thiourea (IC50 21.25
0.15 μM) as a standard ure-
ase inhibitor (Tables 1 and 2). The majority of the newly syn-
thesized 1,3-diethylthiobarbituric acid derivatives exhibited potent
antiurease activity. The structural core 1,3-diethylthiobarbituric
acid remains same in all synthesized compounds as shown in
Schemes 1 and 2 and the only changes were made to prepare
simple thiobarbituric acid analogues (4a-4e) and bis-thiobarbituric
acid analogues (5a-5o) using different aromatic aldehydes. Struc-
ture activity relationship (SAR) revealed urease inhibitory poten-
tial mainly based upon various substitutions pattern at phenyl
ring (Figs. 2 and 3). The thiophene substituted compound 4a
Compounds 5a–5c and 5e–5k also exhibited potent urease
inhibition having IC50 values in the range of 17.31
8.42 0.32 μM that has demonstrated higher inhibitory activity
as compared to standard inhibitor thiourea (Table 2). The phenyl
0.25 to
(IC50 23.75
(IC50 22.53
0.42), 2-methylfuryl substituted compound 4b
0.76), 4-ethoxy substituted compound 4c (IC50
analogue 5a (IC50 11.56
12.74
0.32 μM), naphthyl analogue 5b (IC50
0.25 μM), m-
0.21 μM), biphenyl analogue 5c (IC50 17.31
47.54
0.74), N,N-dimethylaminophenyl substituted compound 4d
cyanophenyl analogue 5e (IC50 15.11
substituted analogue 5f (IC50 10.71
0.35 μM), 3-chlorophenyl
(IC50 42.52
(IC50 79.34
0.24) and 9-anthracenyl substituted compound 4e
0.35 μM), p-bromophenyl
0.52 μM) demonstrated promising inhibitory po-
analogue 5 g (IC50 11.85
(IC50 11.34 0.45 μM), 3,4,5-trimethoxyphenyl analogue 5i (IC50
12.32 0.63 μM), o-fluorophenyl 5j (IC50 8.42 0.32 μM) and
0.32 μM), p-nitropheyl analogue 5 h
tential (Table 1). Among these analogues, thiophenyl substituted
compound 4a and 2-methylfuryl substituted compound 4b exhib-
3

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
Table 1
Inhibitory activity of urease by thiobarbiturates analogues (4a–4e).
Entry
Compound
R
Yield (%)
IC50
SEMa (μM)
1
2
3
4
5
4a
2-Thiophenyl
89
90
88
81
86
23.75
22.53
47.54
42.52
79.34
0.42
0.76
0.74
0.24
0.52
4b
5-Methyl-2-furyl
4-Ethoxyphenyl
4-(Dimethylamino)phenyl
9-Anthracyl
4c
4d
4e
Thioureab
21.25
0.15
a
Standard error of the mean (n = 3).
b
Standard inhibitor.
Table 2
Inhibitory activity of urease by bis-thiobarbituric acid analogues (5a–5o).
Entry
Compound
R
Yield (%)
IC50
SEMa (μM)
1
5a
Phenyl
82
85
78
88
78
79
80
81
78
84
88
81
83
85
80
11.56
12.74
17.31
23.24
15.11
10.71
11.85
11.34
12.32
8.42
0.32
0.21
0.25
0.45
0.35
0.35
0.32
0.45
0.63
0.32
0.32
0.45
0.34
0.77
0.73
2
5b
2-Naphthyl
3
5c
4-Biphenyl
4
5d
4-Isopropylphenyl
3-Cyanophenyl
5
5e
6
5f
3-Chlorophenyl
4-Bromophenyl
4-Nitrophenyl
7
5 g
5h
8
9
5i
3,4,5-Trimethoxyphenyl
2-Fluorophenyl
10
11
12
13
14
15
5j
5k
4-Fluorophenyl
9.81
5l
2,3-Difluorophenyl
3,5-Bis-(trifluoromethyl)phenyl
2,3,5-Trifluorophenyl
2,4,5-Trifluorophenyl
21.43
25.92
28.54
26.54
5m
5n
5o
Thioureab
21.25
0.15
a
Standard error of the mean (n = 3).
b
Standard inhibitor.
p-fluorophenyl analogue 5k (IC50 9.81
0.32 μM) showed potent
stituted analogue 5d (IC50 23.24
0.45 μM) perhaps due to favor-
urease inhibition against standard thiourea among twenty synthe-
sized compounds (Table 1–2, Fig. 2 & 3).
able hydrophobic and π–π stacking interactions (Fig. 2).
Analogue 5f having m-chlorophenyl group (IC50 10.71
0.35 μM) exhibited potent urease inhibition (Fig. 2). This favor-
able inhibition activity might possibly be due to either hydro-
gen bonding or any other electrostatic interaction of chloro group
with nickel atom of urease. Analogue 5e bearing m-cyanophenyl
At first, it was observed that difference in urease inhibitory ac-
tivities was found owing to different nature of substituents and
their pattern on the phenyl ring. The compound 5a bearing phenyl
group (IC50 11.56 0.32 μM), compound 5b having naphthyl group
(IC50 12.74 0.21 μM) and compound 5c bearing 4-biphenyl group
group (IC50 15.11
0.35 μM) showed potent inhibitory potential
(IC50 17.31
0.25 μM) demonstrated potent inhibition potential
but found less active as compared to 5f bearing m-chlorophenyl
than standard thiourea (Fig. 2). The small difference in inhibitory
activity might be due to the presence of similar electronic na-
ture of phenyl, naphthyl and biphenyl groups at the same position
(Table 2, Fig. 2). Despite the hydrophobic nature of both biphenyl
and 4-isopropylphenyl, the inhibitory activity of biphenyl substi-
tuted analogue 5c is relatively higher than 4-isopropylphenyl sub-
group owing to different nature of attached substituent at same
position. The p–bromo analogue 5 g (IC50 11.85
0.32 μM) and
0.45 μM
p-nitrophenyl analogue 5 h with IC50 value of 11.34
demonstrated better inhibitory potential as compared to stan-
dard thiourea (Table 2). The comparable inhibitory potential of p–
bromo substituted analogue 5 g as compared to p-nitro attached
4

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
Fig. 2. Nature and position of substituents affect urease inhibitory activity.
Table 3
nickel atom in urease enzyme. Despite the electron donating na-
ture of both 4-isopropylphenyl and 3,4,5-trimethoxy groups, the
inhibitory activity of 4-isopropylphenyl substituted analogue 5d
Binding interactions revealed by molecular docking studies.
S. No.
Compound
Binding interactions
(IC50 23.24
0.45 μM) is less than 3,4,5-trimethoxy substituted
1
2
4-series
5-series
4a
4b
Pi-Sulfur (His593), H-bond (Arg609, His519)
Pi-Sulfur (His593, Met637),H-bond (Arg609,
Asp494)
analogue 5i (Table 2). It is believed that presence of 4-isopropyl
group on phenyl ring ruled out the possibility of hydrogen bond-
ing or π–π stacking interactions with active site of urease enzyme
(Fig. 2). Analogues 5j and 5k having o-fluoro (IC50 8.42 0.32 μM)
3
4d
H-bond (Arg439, Met637), Alkyl-alkyl
(Asp494, His593)
4
5
5d
5f
H-bond (Ala636), Alkyl-alkyl (His593)
H-bond (Arg439, Ala440), Alkyl-alkyl
(Gln635)
and p-fluoro (IC50 9.81
0.32 μM) were found the most po-
tent urease inhibitors among synthesized analogues of thiobarbi-
turic acid 4a–4e and bis-thiobarbituric acid 5a–5o (Table 2, Fig. 3).
It was believed that small difference in inhibitory activity be-
tween these two analogues 5j bearing 2-fluorophenyl and 5k hav-
ing 4-fluorophenyl might be due to difference in position of flu-
oro substituent on the phenyl ring (Fig. 3). Furthermore, pres-
ence of fluoro group at phenyl ring may have possibility of ei-
ther hydrogen bonding or any other favorable electrostatic inter-
action with nickel atom of urease enzyme. It was also learned
that presence of fluoro group at ortho position is more effective
as compared to para position in phenyl ring (Fig. 3). Likewise,
6
7
5 g
5i
H-bond (Arg439, Ala436, Met637),
H-bond (His593, Arg439), Alkyl-alkyl
(Ala636, Gln635)
8
5j
Pi-Pi (Arg439), H-bond (Arg439, Ala440)
analogue 5 h might be due to the presence of different nature
of electron withdrawing groups at the same position on phenyl
ring (Fig. 2). The 3,4,5-trimethoxy analogue 5i showed potent in-
hibition potential with IC50 value of 12.32
0.63 μM (Table 2).
The outstanding inhibition might be due to combined impact of
hydrogen bonding of three methoxy groups at benzene ring with
2,3-difluoro substituted analogue 5l (IC50 21.43
0.45 μM) also
5

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
Fig. 3. Relationship of both position and number of fluorine substituents toward urease inhibitory activity.
Fig. 4. Binding interactions of most potent inhibitor 4a, 4b and 4d into active pocket of urease (3D-view).
6

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
Fig. 5. Binding interaction pattern of compounds (a) 5f, (b) 5 g, (c) 5d, (d) 5i and (e) 5j inside active pocket of urease (3D-view).
exhibited potent inhibitory activity (Fig. 3). The inhibitory activ-
ity of 2,3-difluoro substituted analogue 5l might be due to hydro-
gen bonding or any equivalent binding interaction of highly elec-
tronegative two fluoro groups on phenyl ring with nickel atom
of urease enzyme. Similarly, 3,5-bis(trifluoromethyl) substituted
logue 5o (IC50 26.54
0.73 μM) also exhibited potent inhibitory
potential (Fig. 3). However, strategic impact of number of fluorine
groups on phenyl ring was also realized that monofluoro substi-
tuted analogues demonstrated better inhibitory activity as com-
pared to presence of di or trifluoro groups on phenyl ring. The in-
hibitory potential also depends on the position of fluorine group
in monofluoro substituted analogues. It is concluded that further
analogue 5 m (IC50 25.92
logue 5n (IC50 28.54
0.34 μM), 2,3,5-trifluorophenyl ana-
0.77 μM) and 2,4,5-trifluorophenyl ana-
7

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
For bis-thiobarbituric acid analogues (5a–5o) the best bind-
ing scores were observed for 5f (–12.20 kcal/mol),
5 g (–
17.49 kcal/mol), 5d (–17.86 kcal/mol), 5i (–16.83 kcal/mol) and 5j
(–15.87 kcal/mol). Molecular docking studies revealed that almost
all compounds interacted with Arg439 through H-bonding along-
side other interactions like π-cation and π-alkyl interactions as
depicted in Fig. 5a–5e and Table 3.
3. Conclusion
In summary, twenty analogues of 1,3-diethylthiobarbituric acid
were accessed in good yields under convenient reaction condi-
tions. A new diverse range of 1,3-diethylthiobarbituric acid ana-
logues were discovered as potent urease inhibitors. Several newly
synthesized analogues of thiobarbiturate and bis-thiobarbiturate
demonstrated outstanding inhibitory potential (IC50 17.31
0.25
to 8.42 0.32 μM) as compared to standard thiourea. In addition
to excellent inhibitory activity of analogues 5a–5o, fluorophenyl
substituted thiobarbituric acid analogues 5j and 5k having o-fluoro
(IC50 8.42
0.32 μM) and p-fluoro (IC50 9.81
0.32 μM) showed
Fig. 6. 3D view of urease from Jack bean (PDB ID: 3LA4).
extraordinary inhibitory activity. The SAR could potentially provide
useful insights into the future design of potent urease inhibitors
with desired pharmacokinetic properties. Molecular docking stud-
ies of bis-thiobarbituric acid analogues 5a–5o presented the best
binding scores for 5f (–12.20 kcal/mol), 5 g (–17.49 kcal/mol), 5d
(–17.86 kcal/mol), 5i (–16.83 kcal/mol) and 5j (–15.87 kcal/mol).
Molecular docking studies revealed that almost all compounds in-
teracted with Arg439 through H-bonding alongside other inter-
actions like π-cation and π-alkyl interactions. These compounds
could be promising leads for further suitable modification to de-
sign drug like scaffolds as potent urease inhibitors.
addition of fluoro groups on phenyl ring will not increase in-
hibitory activity of urease enzyme.
This SAR clearly demonstrates that electronic and steric nature
of substituents are playing important role in urease inhibition. It is
also realized that bis-thiobarbiturates analogues show more inhibi-
tion as compared to thiobarbiturates analogues. The reason might
be due more hydrogen bonding site and electrostatic interaction
of two pyrimidine rings including unique influence of substitution
pattern at phenyl rings. Furthermore, balance between electronic
and steric bulk of attached substituents in synthesized analogues
of thiobarbituric acid 4a–4e and bis-thiobarbituric acid 5a–5o is
certainly a contributing factor to either increase or decrease their
inhibitory potential. For example, anthracyl substituted compound
4. Materials and methods
All commercially purchased reagents were used without further
purification. The starting reagents were weighed by digital bal-
ance and were used at room temperature in the presence of air.
1H NMR and 13C NMR spectra were performed at 300, 400 MHz
and 100 MHz respectively using Bruker Avance III HD 400 MHz
NMR spectrometer at COMSATS University Islamabad, Abbottabad
Campus. CDCl3 was used as deuterated solvent for all NMR anal-
ysis. The spectroscopic data was measured in δ (ppm). The NMR
peaks were recorded in chemical shifts and corresponding cou-
pling constant were calculated in Hz. Infra-Red spectra (KBr) were
performed on a Bruker FT-IR Spectrometer Model Tensor II. The
spectra were measured in term of wavenumber (cm−1) of re-
gion 4000–600 cm−1. The completion of reaction was monitor
by thin layer chromatography (TLC) precoated silica gel aluminum
plates (Kieselgel 60, F254, Merck, Germany). For the detection of
chromatograms, iodine vapors and UV lamp (λmax 254 nm) were
used.
4e with IC50 value of 79.34
0.52 μM exhibited the least in-
hibitory activity. The reason of this greatly reduced inhibitory ac-
tivity might be associated to steric bulk of anthracenyl moiety. The
SAR showed that the nature and number of substituents as well as
substitution pattern greatly affected the inhibitory potential of syn-
thesized compounds. To understand detailed binding interactions
of these compounds with urease enzyme, molecular docking was
carried out against bimetallic urease enzyme using the MOE soft-
ware.
2.3. Molecular docking studies
Molecular docking studies of potent inhibitors of both thiobar-
biturates analogues (4a–4e) alongside bis-thiobarbituric acid ana-
logues (5a–5o) of urease enzyme were performed for better un-
derstanding of their binding interaction pattern into active pocket.
The active site of urease has Ni-atoms that could be targeted
by competitive inhibitors. The test compounds showed mostly H-
bonding interaction, π-sulfur, π-cation and metal contact interac-
tion with active site residues having binding score in the range
of –22.05 to –2.73 kcal/mol for 4a–4e while –21.60 to –4.75 for
5a–5e series. The best binding scores in case of thiobarbiturates
analogues (4a–4e) were observed for 4b (–20.61 kcal/mol), 4a (–
18.95 kcal/mol) and 4d (–22.05 kcal/mol) involving H-bonding in-
teraction with Arg609 and π-sulfur interaction with His593 in the
active pocket as revealed in Fig. 4a–b. Similarly, 4d also showed
good binding interactions with active site residues i.e. Met637 (H-
4.1. General procedure for the synthesis of 1,3-diethylthiobarbituric
acid (3)
In the reaction mixture of diethylmalonate 1 (0.19 g, 1.2 mmol)
and 1, 3-diethylthiourea 2 (0.13 g, 1.0 mmol), sodium ethoxide
(0.06 g, 1.0 mmol) was added. The above resulting reaction mix-
ture was refluxed for 8 h and white precipitates were appeared.
The resulting reaction mixture was diluted with 15 mL hot wa-
ter and hydrochloric acid was added to make the solution acidic.
The clear solution was filtered and cooled for overnight. The re-
sulting white precipitates were filtered. The obtained precipitates
were washed with cold water and dried.
˚
˚
bond; 3.08 A) and Arg439 (H-bond; 2.44, 2.54 A) having binding
score –22.05 kcal/mol as depicted in Fig. 4c and Table 3.
8

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
4.2. General procedure for the synthesis of thiobarbiturates analogues
(4a–4e)
ArH), 8.58 (s, 1H, =CH of olefinic moiety), 8.08 (dd, 2H, J = 6.7,
1.9 Hz, ArH), 7.88 (dd, 2H, J = 7.3, 2.5 Hz, ArH), 7.50˗7.55 (m, 4H,
ArH), 4.70 (q, 2H, J = 6.9 Hz, N˗CH2), 4.34 (q, 2H, J = 7.0 Hz,
N˗CH2), 1.46 (t, 3H, J = 7.0 Hz, CH3), 1.13 (t, 3H, J = 7.0 Hz, CH3);
13C NMR (100 MHz, CDCl3): δ 179.0 (C), 159.8 (C), 157.7 (CH), 157.1
(C), 131.0 (2C), 130.2 (2C), 129.3 (2CH), 128.6 (CH), 128.4 (C), 126.9
(2CH), 125.6 (2CH), 125.0 (2CH), 123.4 (C), 44.2 (CH2), 43.4 (CH2),
12.6 (CH3), 12.4 (CH3); HRMS: m/z calcd for C23H20N2O2S (M+):
388.1245; found: 388.1256
The equivalent amounts of 1,3-diethylthiobarbituric acid
3
(0.20 g, 1.0 mmol) and different aromatic aldehydes (1.0 mmol)
were dissolved in ethanol (15 mL). The reaction mixture was re-
fluxed for 4–5 h. The progress of reaction was monitor by thin
layer chromatography. After completion of reaction, the precipi-
tates were filtered. The obtained precipitates were washed with
cold methanol or cold ethanol (3 × 5 mL) and dried. Finally, re-
crystallization was completed with mixture of ethanol and chloro-
form solvents.
4.3. General procedure for the synthesis of bis-thiobarbiturates
analogues (5a–5o)
4.2.1. 1,3-Diethyl-5-(thiophen-2-ylmethylene)
thioxodihydropyrimidine-4,6–dione (4a) [31]
The non-equivalent amounts of 1,3–diethyl-2-thiobarbituric acid
3 (0.40 g, 2.0 mmol) and different aromatic aldehydes (1.0 mmol)
were dissolved in ethanol (15 mL). The reaction mixture was re-
fluxed for 3–4 h. The progress of reaction was monitor by thin
layer chromatography. After completion of reaction as indicated by
TLC, the appeared precipitates were filtered and washed with cold
ethanol or cold methanol (3 × 5 mL). Finally, recrystallization was
completed with mixture of ethanol and chloroform solvents.
Yellow solid, 89%, M.P 170–172 °C; IR (KBr) ʋ
;
1568, 1394, 1105 cm−1 1H NMR: (CDCl3, 300 MHz): δ 8.78 (s,
2980, 1656,
max
1H, =CH of olefinic moiety), 8.06 (dd, 1H, J = 5.1, 1.2 Hz, ArH),
7.94 (dd, 1H, J = 4.0, 1.2 Hz, ArH), 7.33 (dd, 1H, J = 5.1, 4.0 Hz,
ArH), 4.56˗4.65 (m, 4H, 2 × N˗CH2), 1.26˗1.38 (m, 6H, 2 × CH3);
13C NMR (100 MHz, CDCl3): δ 179.9 (C), 160.9 (C), 159.7 (C), 150.1
(CH), 145.7 (CH), 142.6 (CH), 137.5 (C), 128.5 (CH), 111.4 (C), 44.0
(CH2), 43.2 (CH2), 12.4 (CH3), 12.3 (CH3); HRMS: m/z calcd for
C13H14 N2O2S2 (M+): 294.0497; found: 294.0519.
ꢀ
4.3.1. 5,5 -(Phenylmethylene)-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5a) [32]
Bright yellow crystals, 82%, M.P 188–190 °C; IR (KBr) ʋ
max
4.2.2. 1,3-Diethyl-5-(5-methylfuran-2-yl)
2978, 1610, 1518, 1378, 1107 cm−1 1H NMR: (CDCl3, 400 MHz):
;
methylenethioxodihydropyrimidine-4,6–dione (4b)
δ 7.29˗7.36 (m, 3H, ArH), 7.15 (d, 2H, J = 8.12 Hz, ArH), 5.69 (s,
1H, -CH-Ar), 4.57˗4.73 (m, 8H, 4 × N˗CH2), 1.40 (t, 6H, J = 7.0 Hz,
2 × CH3), 1.31 (t, 6H, J = 6.9 Hz, 2 × CH3); 13C NMR (100 MHz,
CDCl3): δ 174.7 (C), 163.9 (C), 162.4 (C), 135.6 (C), 128.6 (CH),
126.9 (CH), 126.5 (CH), 97.6 (enolized C), 45.3 (2xCH2), 44.7
(2xCH2), 35.1 (CH), 12.2 (2xCH3), 12.1 (2xCH3); HRMS: m/z calcd
for C23H28N4O4S2 (M+): 488.1552; found: 488.1563.
Yellow solid, 90%, M.P 159–161 °C; IR (KBr) ʋ
;
1575, 1386, 1104 cm−1 1H NMR: (CDCl3, 300 MHz): δ 8.74 (d,
2980, 1662,
max
1H, J = 3.6 Hz, ArH), 8.40 (s, 1H, =CH of olefinic moiety), 6.46
(d, 1H, J = 3.9 Hz, ArH), 4.55˗4.63 (m, 4H, 2 × N˗CH2), 2.51 (s, 3H,
Ar-CH3), 1.30˗1.36 (m, 6H, 2 × CH3); 13C NMR (100 MHz, CDCl3):
δ 179.9 (C), 163.9 (C), 161.1 (C), 159.0 (C), 151.0 (C), 141.3 (CH),
131.8 (CH), 113.5 (CH), 110.1 (C), 44.0 (CH2), 43.3 (CH2), 14.8 (CH3),
12.44 (CH3), 12.40 (CH3); HRMS: m/z calcd for C14 H16 N2O3S (M+):
292.0882; found: 292.0932.
ꢀ
4.3.2. 5,5 -(Naphthalen-2-ylmethylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5b)
Light yellow powder, 85%, M.P 175–177 °C; IR (KBr) ʋ
max
4.2.3. 5-(4-Ethoxybenzylidene)−1,3-
2979, 1613, 1505, 1379, 1108 cm−1 1H NMR: (CDCl3, 400 MHz);
;
diethylthioxodihydropyrimidine-4,6–dione (4c)
δ 7.80˗7.86 (m, 3H, ArH), 7.74˗7.77 (m, 1H, ArH), 7.55 (s, 1H, ArH),
7.48˗7.50 (m, 2H, ArH), 5.84 (s, 1H, -CH-Ar), 4.59˗4.77 (m, 8H,
4 × N˗CH2), 1.43 (t, 6H, J = 6.9 Hz, 2 × CH3), 1.33 (t, 6H, J = 7.2 Hz,
2 × CH3); 13C NMR (100 MHz, CDCl3): δ 174.7 (C), 164.0 (C),
162.3 (C), 142.2 (CH), 132.6 (C), 132.0 (C), 131.5 (C), 126.6 (CH),
124.9 (CH), 124.5 (CH), 123.8 (CH), 123.6 (CH), 122.2 (CH), 96.7
(enolized C), 45.4 (2xCH2), 44.8 (2xCH2), 35.1 (CH), 12.1 (2xCH3),
12.0 (2xCH3); HRMS: m/z calcd for C27H30N4O4S2 (M+): 538.1708;
found: 538.1718.
Yellow solid, 88%, M.P 152–154 °C; IR (KBr)
ʋ
2983,
max
1655,1532, 1379,1104 cm−1 1H NMR: (CDCl3, 400 MHz): δ 8.52 (s,
;
1H, =CH of olefinic moiety), 8.37 (d, 2H, J = 8.0 Hz, ArH), 6.98 (d,
2H, J = 8.0 Hz, ArH), 4.56˗4.62 (m, 4H, 2 × N˗CH2), 4.17 (q, 2H,
J = 7.0 Hz, OCH2), 1.48 (t, 3H, J = 7.0 Hz, CH3 of ethoxy moiety),
1.32˗1.36 (m, 6H, 2 × CH3); 13C NMR (100 MHz, CDCl3): δ 179.9
(C), 164.3 (C), 161.5 (C), 160.2 (CH), 159.0 (C), 138.4 (2CH), 125.7
(C), 114.8 (C), 114.6 (2CH), 64.1 (CH2), 44.2 (CH2), 43.6 (CH2), 14.6
(CH3), 12.5 (CH3), 12.4 (CH3); HRMS: m/z calcd for C17 H20N2O3S
(M+): 332.1195; found: 332.1232.
ꢀ
ꢀ
4.3.3. 5,5 -([1,1 -Biphenyl]−4-ylmethylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5c)
4.2.4. 5-(4-dimethylaminobenzylidene)−1,3-
Light yellow solid, 78%, M.P 185–187 °C; IR (KBr) ʋ
max
;
2968, 1608, 1518, 1378, 1107 cm−1 1H NMR: (CDCl3, 400 MHz):
diethylthioxodihydropyrimidine-4,6–dione (4d) [32]
Red solid, 81%, M.P 208–210 °C; IR (KBr) ʋ
2983, 1655, 1581,
1H NMR: (CDCl3, 400 MHz): 8.39˗8.45 (m, 3H,
δ
7.61˗7.63 (m, 2H, ArH), 7.57 (dd, 2H,
J
=
6.62, 1.76 Hz,
max
1342, 1104 cm−1
;
ArH), 7.43˗7.47 (m, 2H, ArH), 7.36˗7.38 (m, 1H, ArH), 7.21 (d,
2H, J = 7.56 Hz, ArH), 5.71 (s, 1H, -CH-Ar), 4.59˗4.75 (m, 8H,
4 × N˗CH2), 1.41 (t, 6H, J = 7.0 Hz, 2 × CH3), 1.33 (t, 6H, J = 6.9 Hz,
2 × CH3); 13C NMR (100 MHz, CDCl3): δ 174.6 (C), 163.7 (C), 162.3
(C), 140.5 (C), 139.7 (C), 134.6 (C), 128.8 (CH), 127.3 (CH), 127.1
(CH), 127.0 (CH), 126.8 (CH), 97.4 (enolized C), 45.2 (2xCH2), 44.6
(2xCH2), 34.8 (CH), 12.1 (2xCH3), 12.0 (2xCH3); HRMS: m/z calcd
for C29H32N4O4S2 (M+): 564.1865; found: 564.1871.
ArH & =CH of olefinic moiety), 6.79˗6.86 (m, 2H, ArH), 4.57˗4.63
(m, 4H, 2 × N˗CH2), 3.19 (s, 6H, 2 × N˗CH3), 1.33˗1.36 (m, 6H,
2 × CH3); 13C NMR (100 MHz, CDCl3): δ 179.9 (C), 161.2 (C),
160.4 (C), 160.1 (CH), 153.9 (C), 138.4 (2CH), 125.7 (C), 114.8 (C),
110.9 (2CH), 44.18 (CH2), 43.17 (CH2), 41.6 (2xCH3), 12.4 (CH3), 12.2
(CH3); HRMS: m/z calcd for C17 H21N3O2S (M+): 331.1354; found:
331.1384.
ꢀ
4.2.5. 5-(Anthracen-9-ylmethylene)−1,3-
4.3.4. 5,5 -(4-Isopropylphenyl)methylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (4e)
diethylthioxodihydropyrimidine-4,6–dione (5d)
Dark blue solid, 86%, M.P 215–217 °C; IR (KBr) ʋmax 2927, 1671,
White powder, 88%, M.P 155–157 °C; IR (KBr) ʋ
2969, 1611,
max
1568, 1394, 1105 cm−1
;
1H NMR: (CDCl3, 400 MHz): δ 9.48 (s, 1H,
1514, 1368, 1106 cm−1 1H NMR: (CDCl3, 400 MHz): δ 7.18 (d,
;
9

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
J = 7.0 Hz, 2 × CH3), 1.33 (t, 6H, J = 6.9 Hz, 2 × CH3); 13C NMR
(100 MHz, CDCl3): δ 174.7 (C), 163.8 (C), 162.3 (C), 142.8 (C), 141.8
(C), 126.5 (C), 126.2 (C), 106.9 (CH), 106.3 (CH), 97.5 (enolized C),
53.3 (2xOCH3), 51.1 (OCH3), 45.3 (2xCH2), 44.7 (2xCH2), 34.5 (CH),
12.2 (2xCH3), 12.1 (2xCH3); HRMS: m/z calcd for C26H34N4O7S2
(M+): 578.1869; found: 578.1872.
2H, J = 8.2, ArH), 7.04 (d, 2H, J = 8.2, ArH), 5.65 (s, 1H, -CH-Ar),
4.56˗4.73 (m, 8H, 4 × N˗CH2), 2.90˗2.93 (m, 1H, CH of isopropyl
moiety), 1.39 (t, 6H, J = 7.0 Hz, 2 × CH3), 1.32 (t, 6H, J = 6.9 Hz,
2 × CH3), 1.26 (d, J = 6.9 Hz, 6H, 2 × CH3 of isopropyl moiety); 13C
NMR (100 MHz, CDCl3): δ 174.7 (C), 163.8 (C), 162.4 (C), 147.5 (C),
132.8 (C), 126.7 (CH), 126.4 (CH), 97.7 (enolized C), 45.3 (2xCH2),
44.7 (2xCH2), 34.8 (CH), 33.7 (CH), 24.1 (2xCH3), 12.23 (2xCH3),
12.17 (2xCH3); HRMS: m/z calcd for C26H34N4O4S2 (M+): 530.2021;
found: 530.2026.
ꢀ
4.3.10. 5,5 -(2-Fluorophenyl)methylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5j)
White powder, 84%, M.P 175–177 °C; IR (KBr) ʋ
2977,
max
1609, 1508, 1372, 1102 cm−1
;
1H NMR: (CDCl3, 400 MHz): δ
4.3.5. 3-Bis(1,3–diethyl-4,6-dioxothioxohexahydropyrimidin-5-
yl)methyl)benzonitrile (5e)
7.26˗7.31 (m, 1H, ArH), 7.12˗7.16 (m, 2H, ArH), 7.00˗7.05 (m, 1H,
ArH), 5.74 (s, 1H, -CH-Ar), 4.58˗4.74 (m, 8H, 4 × N˗CH2), 1.38 (t,
6H, J = 7.0 Hz, 2 × CH3), 1.31 (t, 6H, J = 7.0 Hz, 2 × CH3); 13C
NMR (100 MHz, CDCl3): δ 174.6 (C), 163.1 (C), 162.5 (C), 160.0
(d, 1xC, JC-F = 204 Hz), 129.3 (d, 1xCH, JC-F = 3.0 Hz), 129.0
(d, 1xCH, JC-F = 8.0 Hz), 124.0 (d, 1xCH, JC-F = 3.0 Hz), 122.9
(d, 1xC, JC-F = 10.0 Hz), 116.2 (d, 1xCH, JC-F = 23.0 Hz), 97.8
(enolized C), 45.3 (2xCH2), 44.7 (2xCH2), 31.8 (CH), 12.2 (2xCH3),
12.1 (2xCH3); HRMS: m/z calcd for C23H27FN4O4S2 (M+): 506.1448;
found: 506.1443.
White solid, 78%, M.P 223–225 °C; IR (KBr) ʋ
2975, 1612,
max
1507, 1374, 1106 cm−1 1H NMR: (CDCl3, 400 MHz): δ 7.60 (d,
;
1H, J = 2.1 Hz, ArH), 7.39˗7.48 (m, 3H, ArH), 5.66 (s, 1H, -
CH-Ar), 4.53˗4.74 (m, 8H, 4 × N˗CH2), 1.41 (t, 6H, J = 6.9 Hz,
2 × CH3), 1.32 (t, 6H, J = 6.9 Hz, 2 × CH3); 13C NMR (100 MHz,
CDCl3): δ 174.7 (C), 164.0 (C), 162.4 (C), 137.7 (C), 131.1 (CH),
130.7 (CH), 130.4 (CH), 129.5 (CH), 118.9 (C), 112.9 (C), 96.6 (eno-
lized C), 45.4 (2xCH2), 44.8 (2xCH2), 35.0 (CH), 12.2 (2xCH3),
12.1 (2xCH3); HRMS: m/z calcd for C24H27N5O4S2 (M+): 513.1504;
found: 513.1511.
ꢀ
4.3.11. 5,5 -(4-Fluorophenyl)methylene-bis(1,3-
ꢀ
4.3.6. 5,5 -(3-Chlorophenyl)methylene-bis-(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5k) [22]
diethylthioxodihydropyrimidine-4,6–dione (5f)
White solid, 88%, M.P 192–194 °C; IR (KBr) ʋ
2973, 1615,
max
1505, 1381, 1109 cm−1 1H NMR: (CDCl3, 400 MHz): δ 7.11˗7.13 (m,
;
Light yellow solid, 79%, M.P 188–190 °C; IR (KBr) ʋ
;
1612, 1507, 1372, 1109 cm−1 1H NMR: (CDCl3, 400 MHz): 7.25˗7.27
2979,
max
2H, ArH), 7.00–7.05 (m, 2H, ArH), 5.64 (s, 1H, -CH-Ar), 4.56˗4.72
(m, 8H, 4 × N˗CH2), 1.39 (t, 6H, J = 7.0 Hz, 2 × CH3), 1.31 (t,
6H, J = 6.9 Hz, 2 × CH3); 13C NMR (100 MHz, CDCl3): δ 174.7
(C), 163.9 (C), 162.4 (C), 161.8 (d, 1xC, JC-F = 245 Hz), 131.3 (d,
1xC, JC-F = 3.0 Hz), 128.1 (d, 1xCH, JC-F = 7.0 Hz), 115.5 (d,
1xCH, JC-F = 22 Hz), 97.5 (enolized C), 45.3 (2xCH2), 44.7 (2xCH2),
34.6 (CH), 12.20 (2xCH3), 12.15 (2xCH3); HRMS: m/z calcd for
C23H27FN4O4S2 (M+): 506.1448; found: 506.1652.
(m, 2H, ArH), 7.11 (s, 1H, ArH), 7.02˗7.05 (m, 1H, ArH), 5.64 (s, 1H,
-CH-Ar), 4.49˗4.87 (m, 8H, 4 × N˗CH2), 1.38 (t, 6H, J = 7.0 Hz,
2 × CH3), 1.30 (t, 6H, J = 7.0 Hz, 2 × CH3); 13C NMR (100 MHz,
CDCl3): δ 174.1 (C), 163.9 (C), 162.1 (C), 136.9 (C), 132.1 (CH),
131.6 (CH), 131.4 (CH), 130.4 (CH), 111.8 (C), 96.7 (enolized C),
45.3 (2xCH2), 44.3 (2xCH2), 34.7 (CH), 12.2 (2xCH3), 11.9 (2xCH3);
HRMS: m/z calcd for C23H27ClN4O4S2 (M+): 522.1162; found:
522.1173.
ꢀ
4.3.12. 5,5 -((2,3-Difluorophenyl)methylene)-bis(1,3-
ꢀ
4.3.7. 5,5 -(4-Bromophenyl)methylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5l)
diethylthioxodihydropyrimidine-4,6–dione (5 g) [22]
White powder, 81%, M.P 192–194 °C; IR (KBr) ʋ
2979, 1612,
max
1507, 1372, 1109 cm−1 1H NMR: (CDCl3, 400 MHz): 7.05˗7.13 (m,
;
Light yellow solid, 80%, M.P 185–187 °C; IR (KBr) ʋ
;
1610, 1510, 1379, 1107 cm−1 1H NMR: (CDCl3, 400 MHz): δ 7.46 (d,
2975,
max
2H, ArH), 6.89˗6.93 (m, 1H, ArH), 5.76 (s, 1H, -CH-Ar), 4.57˗4.71
(m, 8H, 4 × N˗CH2), 1.38 (t, 6H, J = 7.0 Hz, 2 × CH3), 1.31 (t,
6H, J = 7.0 Hz, 2 × CH3); 13C NMR (100 MHz, CDCl3): δ 174.6 (C),
163.1 (C), 162.5 (C), 152.3 (dd, 1C, JC-F = 254, 14.0 Hz), 149.1 (dd,
1C, JC-F = 204, 14.0 Hz), 125.5 (d, 1C, JC-F = 7.0 Hz), 123.9 (br. s,
CH), 123.6 (q, 1xCH, JC-F = 7.0 Hz), 116.2 (d, 1xCH, JC-F = 18.0 Hz),
97.5 (enolized C), 45.3 (2xCH2), 44.8 (2xCH2), 31.97 (CH), 12.2
(2xCH3), 12.1 (2xCH3); HRMS: m/z calcd for C23H26F2N4O4S2 (M+):
524.1364; found: 524.1391.
2H, J = 8.2 Hz, ArH), 7.01 (d, 2H, J = 8.0 Hz, ArH), 5.60 (s, 1H, -CH-
Ar), 4.53˗4.66 (m, 8H, 4 × N˗CH2), 1.39 (t, 6H, J = 6.9 Hz, 2 × CH3),
1.31 (t, 6H, J = 6.9 Hz, 2 × CH3); 13C NMR (100 MHz, CDCl3): δ
174.1 (C), 163.7 (C), 162.1 (C), 147.6 (C), 137.3 (C), 133.8 (CH), 128.5
(CH), 123.1 (CH), 121.9 (CH), 96.3 (enolized C), 45.3 (2xCH2), 44.7
(2xCH2), 34.9 (CH), 12.3 (2xCH3), 12.1 (2xCH3); HRMS: m/z calcd
for C23H27BrN4O4S2 (M+): 566.0657; found: 566.0662
ꢀ
4.3.8. 5,5 -(4-Nitrophenyl)methylene-bis(1,3-
ꢀ
diethylthioxodihydropyrimidine-4,6–dione (5 h) [22]
4.3.13. 5,5 -(3,5-Bis(trifluoromethyl)phenyl)methylene-bis(1,3-
White solid, 81%, M.P 230–232 °C; IR (KBr) ʋ
2979, 1612,
1H NMR: (CDCl3, 400 MHz): δ 8.15˗8.19 (m,
diethylthioxodihydro-pyrimidine-4,6–dione (5 m)
max
1523, 1376, 1107 cm−1
;
White solid, 83%, M.P 205–207 °C; IR (KBr) ʋ
;
1518, 1365, 1110 cm−1 1H NMR: (CDCl3, 400 MHz): δ 7.82 (s,
2982, 1614,
max
1H, ArH), 8.02 (s, 1H, ArH), 7.50˗7.55 (m, 2H, ArH), 5.71 (s, 1H, -CH-
Ar), 4.56˗4.81 (m, 8H, 4 × N˗CH2), 1.41 (t, 6H, J = 6.9 Hz, 2 × CH3),
1.32 (t, 6H, J = 6.9 Hz, 2 × CH3); 13C NMR (100 MHz, CDCl3): δ
174.8 (C), 164.0 (C), 162.4 (C), 148.7 (C), 138.4 (C), 132.8 (CH), 129.6
(CH), 122.2 (CH), 122.0 (CH), 96.8 (enolized C), 45.4 (2xCH2), 44.8
(2xCH2), 35.1 (CH), 12.2 (2xCH3), 12.1 (2xCH3); HRMS: m/z calcd
for C23H27N5O6S2 (M+): 533.1403; found: 533.1410.
1H, ArH), 7.57 (s, 2H, ArH), 5.71 (s, 1H, -CH-Ar), 4.62˗4.76 (m, 6H,
3 × N˗CH2), 4.47˗4.52 (m, 2H, N˗CH2), 1.41 (t, 6H, J = 7.0 Hz,
2 × CH3), 1.30 (t, 6H, J = 7.0 Hz, 2 × CH3); 13C NMR (100 MHz,
CDCl3): δ 174.8 (C), 164.1 (C), 162.3 (C), 138.9 (C), 131.8 (q, 1C,
JC-F = 66.0 Hz), 127.2 (d, 1C, JC-F = 3.0 Hz), 124.8 (CH), 122.1 (CH),
120.9 (quintet, 1C), 119.3 (CH), 96.5 (enolized C), 45.4 (2xCH2), 44.8
(2xCH2), 35.2 (CH), 12.1 (2xCH3), 11.9 (2xCH3); HRMS: m/z calcd
for C25H26F6N4O4S2 (M+): 624.1300; found: 624.1205.
ꢀ
4.3.9. 5,5 -(3,4,5-Trimethoxyphenyl)methylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5i)
ꢀ
Light yellow powder, 78%, M.P 160–162 °C; IR (KBr) ʋ
2978,
4.3.14. 5,5 -(2,3,5-Trifluorophenyl)methylene-bis(1,3-
max
1611, 1516, 1375, 1104 cm−1
;
1H NMR: (CDCl3, 400 MHz): δ 6.33
diethylthioxodihydropyrimidine-4,6–dione (5n)
(d, 2H, J = 1.04 Hz, ArH), 5.64 (s, 1H, -CH-Ar), 4.55˗4.76 (m, 8H,
4 × N˗CH2), 3.87 (s, 3H, OCH3), 3.78 (s, 6H, 2xOCH3), 1.39 (t, 6H,
White powder, 85%, M.P 201–203 °C; IR (KBr) ʋ
2979, 1613,
max
1505, 1372, 1108 cm−1 1H NMR: (CDCl3, 400 MHz): δ 6.88˗6.90
;
10

M.U. Khan, M. Aslam, S.A. Shahzad et al.
Journal of Molecular Structure 1231 (2021) 129959
(m, 1H, ArH), 6.67˗6.69 (m, 1H, ArH), 5.73 (s, 1H, -CH-Ar), 4.57˗4.71
(m, 8H, 4 × N˗CH2), 1.38 (t, 6H, J = 7.0 Hz, 2 × CH3), 1.32 (t, 6H,
J = 7.0 Hz, 2 × CH3); 13C NMR (100 MHz, CDCl3): δ 174.6 (C),
163.2 (C), 162.5 (C), 126.8, 126.7, 111.1, 110.9, 104.5 (dd, JC-F = 22.0,
21.0 Hz, CH), 104.5 (CH), 97.0 (enolized C), 45.3 (2xCH2), 44.8
(2xCH2), 32.1 (CH), 12.1 (2xCH3), 12.0 (2xCH3); HRMS: m/z calcd
for C23H25F3N4O4S2 (M+): 542.1269; found: 542.1275.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
CRediT authorship contribution statement
Matee Ullah Khan: Conceptualization, Methodology, Writing -
original draft. Misbah Aslam: Investigation, Methodology, Visu-
alization. Sohail Anjum Shahzad: Funding acquisition, Investiga-
tion, Project administration, Supervision, Writing - review & edit-
ing. Zulfiqar Ali Khan: Data curation, Visualization, Writing - re-
view & editing. Nazeer Ahmad Khan: Investigation, Visualization.
Muhammad Ali: Funding acquisition, Project administration, Su-
pervision, Visualization. Sadia Naz: Formal analysis, Software, Val-
idation. Jameel Rahman: Investigation, Methodology, Validation,
Visualization.
ꢀ
4.3.15. 5,5 -(2,4,5-Trifluorophenyl)methylene-bis(1,3-
diethylthioxodihydropyrimidine-4,6–dione (5o)
White solid, 80%, M.P 180–182 °C; IR (KBr) ʋ
;
1513, 1379, 1108 cm−1 1H NMR: (CDCl3, 400 MHz): δ 6.87˗7.02
2977, 1610,
max
(m, 2H, ArH), 5.67 (s, 1H, -CH-Ar), 4.57˗4.69 (m, 8H, 4 × N˗CH2),
1.38 (t, 6H, J = 7.0 Hz, 2 × CH3), 1.32 (t, 6H, J = 7.0 Hz, 2 × CH3);
13C NMR (100 MHz, CDCl3): δ 174.1 (C), 163.7 (C), 162.1 (C), 136.7
(C), 121.1 (C), 120.8 (C), 107.7 (C), 106.5 (CH), 103.1 (CH), 96.6 (eno-
lized C), 45.4 (2xCH2), 44.9 (2xCH2), 32.4 (CH), 12.7 (2xCH3), 11.9
(2xCH3); HRMS: m/z calcd for C23H25F3N4O4S2 (M+): 542.1269;
found: 542.1276.
Acknowledgments
The authors are grateful to HEC Pakistan for providing
a
4.4. Urease inhibition assay
research grant vide 20-1933/NRPU/R&D/HEC/12/5017. Dr. S. A.
Shahzad would like to acknowledge HEC Pakistan for financial sup-
port under Project No. 5290/Federal/NRPU/R&D/HEC/2016.
The previously described Berthelot assay with slight modifica-
tions was carried out to evaluate urease inhibitory activity [33,34].
A total volume of 85 μL assay mixture contained 25 μL of 50 mM
phosphate buffer, pH 7, 10 μL of test compound, and 10 μL of ure-
ase enzyme solution (0.015 units, jack bean urease from Sigma).
The contents were preincubated at 37 °C for 10 min. Then, 40 μL
of 20 mM substrate (urea) was added per well and re-incubated for
further 10 min. After this, contents were pre-read at 625 nm us-
ing 96-well plate reader Synergy HT (BioTek Inc. USA). Then freshly
prepared mixture of phenol (45 μL) and alkali reagent (70 μL) was
added per well. For color development, incubation was continued
for further 10 min and then absorbance was noted at 625 nm. The
percentage inhibition was calculated by using this formula.
Inhibition (%) = 100 – [(Abs. of sample / Abs. of control) × 100]
Serial dilutions of active compounds were assayed, and their
percent inhibitions were determined, and data obtained was used
to calculate IC50 values using EZ-Fit Enzyme software (Perrella Inc.,
USA).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.129959.
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