Design, synthesis, docking study and urease inhibitory activity evaluation of novel 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-arylacetamide derivatives

Article abstract of DOI:10.1007/s00044-020-02683-5

In this paper, novel 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-arylacetamide derivatives (7a–l) are designed, synthesized, and evaluated in vitro for their urease inhibitor activities. The compounds are synthesized efficiently in three steps in high isolated yields from amines, 2-chloroacetyl chloride, hydrazinecarbothioamide, and carbon disulfide. The molecular docking simulation were performed using AutoDock4 by docking all synthesized compound and standard inhibitors into the crystal structure of Jack bean urease. Comparison between the urease inhibitory activity of compounds 7a–l with the IC₅₀ of (2.85–5.83 μM) and thiourea and hydroxyurea as standards inhibitors with the IC₅₀ of (22.00 and 100.00 μM, respectively) proved the high activity of the synthesized compounds against the mentioned enzyme. Docking results were in good agreement with experimental results and indicate that synthesized compounds could interact well with the active site of the urease enzyme and among all; compound 7j shows more favorable interactions with the active site which confirm its great inhibitory activity with IC₅₀ of 2.85 μM. Therefore, compound 7j might be a promising candidate for further evaluation.

Full text of DOI:10.1007/s00044-020-02683-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:729–742
https://doi.org/10.1007/s00044-020-02683-5
ORIGINAL RESEARCH
Design, synthesis, docking study and urease inhibitory activity
evaluation of novel 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-
arylacetamide derivatives
1 ●
1 ●
2 ●
1 ●
3 ●
Mohammad Nazari Montazer Mehdi Asadi Saeed Bahadorikhalili Faezeh Sadat Hosseini Arash Amanlou
4 ●
1,5
Mahmood Biglar Massoud Amanlou
Received: 11 October 2020 / Accepted: 2 December 2020 / Published online: 12 January 2021
The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
©
Abstract
In this paper, novel 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-arylacetamide derivatives (7a–l) are designed, synthesized,
and evaluated in vitro for their urease inhibitor activities. The compounds are synthesized efficiently in three steps in high
isolated yields from amines, 2-chloroacetyl chloride, hydrazinecarbothioamide, and carbon disulfide. The molecular
docking simulation were performed using AutoDock4 by docking all synthesized compound and standard inhibitors into
the crystal structure of Jack bean urease. Comparison between the urease inhibitory activity of compounds 7a–l with the
IC50 of (2.85–5.83 µM) and thiourea and hydroxyurea as standards inhibitors with the IC50 of (22.00 and 100.00 µM,
respectively) proved the high activity of the synthesized compounds against the mentioned enzyme. Docking results were
in good agreement with experimental results and indicate that synthesized compounds could interact well with the active
site of the urease enzyme and among all; compound 7j shows more favorable interactions with the active site which
confirm its great inhibitory activity with IC50 of 2.85 µM. Therefore, compound 7j might be a promising candidate for
further evaluation.
●
●
●
●
Keywords Urease inhibitor Anti-Helicobacter pylori Thiadiazole Enzyme inhibition Docking
Introduction
Urease is a 550 kDa nickel-containing enzyme, which
belongs to amidohydrolases and phosphotriesterase enzyme
family. Urease is composed of two subunits, named UreA
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02683-5) contains supplementary
material, which is available to authorized users.
(26.5 kDa) and UreB (60.3–61.6 kDa), and catalyzes the
conversion of urea to ammonia and carbon dioxide [1–3].
This enzyme is a cytoplasmic enzyme and could be found in
several cells, including bacteria, fungi, algae, and some
plant cells [4]. The conversion of urea to ammonia leads to
an increase in pH, which is essential for the survival of
Helicobacter pylori (H. pylori) [5, 6]. H. pylori is a well-
known bacterium, which is colonized in the human stomach
and causes gastrointestinal infection. It is proven that the
urease enzyme is essential for colonization of H. pylori in
gastric mucosa, and the role of this enzyme is critical for
colonization of the mentioned bacterium [7]. Regarding the
critical role of urease in the colonization of H. pylori, this
enzyme’s inhibition is an efficient way for the treatment of
infections caused by this bacterium. Phosphorodiamidates,
hydroxamic acid derivatives, nickel chelators, and thiol
*
Massoud Amanlou
amanlou@tums.ac.ir
1
2
3
4
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
School of Chemistry, College of Science, University of Tehran,
Tehran, Iran
Faculty of Specialized Veterinary Sciences, Science and Research
Branch, Islamic Azad University, Tehran, Iran
Drug Design and Development Research Center, Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical
Sciences, Tehran, Iran
5
Experimental Medicine Research Center, Tehran University of
Medical Sciences, Tehran, Iran

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Medicinal Chemistry Research (2021) 30:729–742
Scheme 1 Structure of 1, 2, 4-
thiadiazole derivatives as urease
inhibitor compounds
compounds are four general groups whose inhibition
activities are proven in urease inhibition [8–11].
Heterocycles with appropriate structures, such as thia-
diazoles, barbituric acids, quinalozinones, and other struc-
tures, have been shown to be irreversibly binding to the
active site of urease enzyme. As some examples, urease
inhibitory activity of 5-aminomethylene barbituric and
thiobarbituric acids [12, 13], bis-thiobarbiturates [14], and
organometallic complexes of barbituric acids [15] have
previously reported. Among various heterocycles with
urease inhibitory activities, 1,2,4-thiadiazole derivatives are
of high interest, due to their various advantageous proper-
ties. 1,2,4-thiadiazole is a five-membered ring containing
one sulfur and two nitrogen heteroatoms in its structure
[
16]. 1,2,4-thiadiazole derivatives (Scheme 1) have various
biological activities, including anticancer [17], neuropro-
tective [18], antibiotic [19, 20], antiviral [21], antifungal
[
22, 23], and β-glucuronidase inhibition activities [24].
Urease inhibitory activity of various analogous of this
compound has been observed [25, 26]. 1,3,4-thiadiazole-2
Scheme 2 General structures of four urease inhibitor
(
3H)-thiones [27], N-[5-(5-nitro-2-heteroaryl)-1,3,4-thia-
diazol-2-yl]thiomorpholines [28], and 2-[(chlorobenzyl)
thio]-5-(5-nitro-2-furyl)-1,3,4-thiadiazoles [29] are a num-
ber of thiadiazoles with urease inhibitory activities. In
addition, conjugation of this heterocycle to coumarin [30]
and quinazolinones [31] has been shown to be urease
inhibitor agents. In addition, the interaction of thiadiazole
and triazole derivatives with the active site of urease and
their enzyme inhibition activity is proven [32–35].
compounds could place in the active site of the urease
enzyme with proper interactions, and in vitro urease inhi-
bitory activities of synthesized compounds have been
evaluated and compared with thiourea and hydroxyurea as
positive standards by Berthelot colorimetric method.
Materials and methods
Regarding the significance of urease inhibitors on the one
hand, and the inhibitory activities of the thiadiazole deri-
vatives, on the other hand, in this paper novel thiadiazole
containing compounds is synthesized, and their urease
inhibitory activities are evaluated. The compounds are
designed based on the proven urease inhibition activity of
General remarks
All the chemicals were purchased from Merck, Sigma-
Aldrich, and Acros Chemicals. Melting points of the
products were measured on a Kofler hot stage apparatus.
1
13
1
,3,4-thiadiazol moiety, due to its interaction with the
urease enzyme’s active site [36, 37]. For this purpose, novel
-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-arylacetamide
H and C NMR spectra were recorded on a Bruker FT-
500, using TMS as an internal standard. A Nicolet Magna
FTIR 550 spectrophotometer was used to record FTIR
spectra. Mass spectra of the products were recorded on an
Agilent Technology (HP) mass spectrometer performing
at ionization potential = 70 eV. CHN elemental analysis
2
derivatives are synthesized with thiadiazole moiety. The
synthetic route is presented in Scheme 2. Docking study
was done, and the results indicate that all synthesized

Medicinal Chemistry Research (2021) 30:729–742
731
was carried out with an Elementar Analysensysteme
GmbH VarioEL.
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(4-methoxyphenyl)
acetamide (7b)
Chemistry
Yield: 77%; mp: 176–178 °C; IR (KBr): 3334, 3318, 3056,
−1
1
2986, 1688, 1618, 1610 cm ; H NMR (DMSO-d6,
Synthesis of 2-chloro-N-arylacetamide
500 MHz) δ (ppm): 10.05 (s, 1H, NH), 7.47 (d, J = 5, 2H, H2,
H ), 7.30 (s, 2H, NH ), 6.8 (d, J = 5 Hz, 2H, H , H ), 3.96 (s,
6
2
3
5
1
3
An aniline derivative (2 mmol) was added to a flask con-
taining DMF (10 ml) at 0 °C and then 1 equivalent of 2-
chloroacetyl chloride added dropwise to the reaction mix-
ture. After the addition of 2-chloroacetyl chloride, the
reaction mixture was stirred at room temperature for 2 h,
monitored by TLC. After the reaction completion, the
reaction mixture was added to ice water. The precipitate
was filtered and dried overnight at atmospheric condition
and used directly in the next step [38].
2H, CH ), 3.80 (s, 3H, OCH ) ppm; C NMR (DMSO-d6,
2 3
125 MHz) δ (ppm) 170.4, 165.7, 155.9, 149.8, 132.4, 121.2,
+
114.4, 55.7, 55.6, 39.1, 39.2 ppm; mass spectra (M ) at m/z
296.36; Anal. Calcd. for C H N O S : C, 44.58; H, 4.08; N,
1
1 12 4 2 2
18.91. Found: C, 44.26; H, 4.33; N, 19.11%.
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(2-fluoro-4-
nitrophenyl)acetamide (7c)
Yield: 83%; mp: 172–174 °C; IR (KBr): 3360, 3321, 3058,
−1
1
Synthesis of 5-amino-1,3,4-thiadiazole-2-thiol
2978, 1695, 1624, 1610, 1554, 1356 cm ; H NMR
DMSO-d , 500 MHz) δ (ppm): 10.50 (s, 1H, NH), 8.98 (dd,
(
6
Hydrazinecarbothioamide (2 mmol) was dissolved in DMF
J = 2.9, J = 6.75, 1H, H ), 8.05 (m, 1H, H ), 7.59 (t,
3 5
13
(
10 ml), and then carbon disulfide (2 mmol) was added. The
J = 9, 1H, H ), 7.32 (s, 2H, NH ), 4.13 (s, 2H, CH );
C
6
2
2
reaction mixture was stirred at room temperature for 2 h.
After the reaction completion, the reaction mixture was
added to ice water, and the product was separated by fil-
tration [39].
NMR (DMSO-d , 125 MHz) δ (ppm): 170.6, 167.9, 159.3,
6
157.6, 155.5, 149.6, 144.2, 127.6, 127.5, 121.1, 120.9, 118.0,
+
117.3, 117.1, 39.9, 38.8, 38.7 ppm; mass spectra (M ) at m/z
329.01; Anal. Calcd. for C H FN O S : C, 36.47; H, 2.45;
1
0
8
5
3 2
N, 21.27. Found: C, 36.18; H, 2.74; N, 20.89%.
Synthesis of 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-
arylacetamide
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(4-methylisoxazol-
3-yl)acetamide (7d)
2
-Chloro-N-methylacetamide (2 mmol) and 5-amino-1,3,4-
thiadiazole-2-thiol (2.4 mmol) were added to a flask con-
taining ethanol (10 ml). Then, a solution of sodium hydro-
xide (2.4 mmol) in water (8 ml) was added to the reaction
mixture and stirred at room temperature for 3 h. After the
reaction completion, the solvent was evaporated by using
Rotary evaporator and the product was purified by recrys-
tallization from ethanol [40].
Yield: 82%; mp: 171–173 °C; IR (KBr): 3364, 3325, 3045,
2988, 1685, 1615, 1604 cm ; H NMR (DMSO-d6,
−
1
1
500 MHz) δ (ppm): 11.18 (s, 1H, NH), 7.31 (s, 2H, NH ),
2
3
1
6.61 (s, 1H, H ), 4.00 (s, 2H, CH ), 2.34 (s, 3H, CH );
C
4
2
3
NMR (DMSO-d , 125 MHz) δ (ppm): 170.5, 170.2, 166.7,
6
159.6, 158.4, 149.5, 96.7, 39.9, 38.5, 38.4, 12.6, 43.0 ppm;
+
mass spectra (M ) at m/z 271.31; Anal. Calcd. for
C H N O S : C, 35.42; H, 3.34; N, 25.81. Found: C, 35.76;
8
9
5
2 2
Spectral data
H, 3.61; N, 26.12%.
2
-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(4-bromobenzyl)
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(2-methoxyphenyl)
acetamide (7a)
acetamide (7e)
Yield: 71%; mp: 188–190 °C; IR (KBr): 3356, 3312, 3048,
Yield: 72%; mp: 174–176 °C; IR (KBr): 3348, 3313, 3055,
−
1
1
−1
1
2
960, 1686, 1620, 1608 cm ; H NMR (DMSO-d6,
2980, 1688, 1624, 1608 cm ; H NMR (DMSO-d6,
500 MHz) δ (ppm): 9.5 (s, 1H, NH), 9.20 (d, J = 8 Hz, 1H,
H ), 7.31 (s, 2H, NH ), 7.06 (m, 2H, H4, 5), 6.91 (t, J =
5
00 MHz) δ (ppm): 8.69 (t, J = 6.0 Hz, 1H, NH), 7.36 (d,
J = 8.4 Hz, 2H, H3, 5), 7.31 (s, 2H, NH ), 7.24 (d, J =
2
6
2
8
2
1
4
.4 Hz, 2H, H ), 4.28 (d, J = 6 Hz, 2H, CH -phe), 3.33 (s,
7.5 Hz, 1H, H ), 4.06 (s, 2H, CH ), 3.83 (s, 3H, OCH )
2
, 6
2
3 2 3
1
3
13
H, CH2); C NMR (DMSO-d , 125 MHz) δ (ppm): 170.4,
ppm; C NMR (DMSO-d , 125 MHz) δ (ppm): 170.5,
6
6
67.5, 159.3, 159.2, 149.4, 138.5, 131.8, 129.5, 128.6,
166.6, 149.9, 149.6, 127.5, 124.9, 121.5, 120.7, 111.6,
+
+
2.3, 38.2, 38.1 ppm; mass spectra (M ) at m/z 359.26;
56.2, 56.1, 38.8 ppm; mass spectra (M ) at m/z 400.02.
Anal. Calcd. for C H BrN OS : C, 36.78; H, 3.09; N,
Anal. Calcd. for C H FN O S C, 36.47; H, 2.45; N,
11
11
4
2
10
8
5
3 2
1
5.60. Found: C, 36.98; H, 3.21; N, 15.81%.
21.27. Found: C, 36.22; H, 2.26; N, 21.17%.

732
Medicinal Chemistry Research (2021) 30:729–742
2
-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(3-chlorophenyl)
for C H BrN O S: C, 36.49; H, 2.76; N, 17.02. Found: C,
1
0
9
4
2
acetamide (7f)
36.53; H, 2.65; N, 17.23%.
Yield: 82%; mp: 177–179 °C; IR (KBr): 3358, 3324, 3045,
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(5-methylpyridin-
2-yl)acetamide (7j)
−
1
1
2
996, 1678, 1625, 1608 cm ; H NMR (DMSO-d6,
5
00 MHz) δ (ppm): 10.45 (s, 1H, NH), 7.79 (s, H, H ), 7.41
2
(
2
d, J = 8 Hz, 1H, H ), 7.35 (t, J = 8 Hz, 1H, H ), 7.31 (s,
Yield: 86%; mp: 200–202 °C; IR (KBr): 3345, 3318, 3031,
4
5
1
3
−1
1
H, NH ), 7.13 (d, J = 8, H ), 3.99 (s, 2H, CH ) ppm;
C
2995, 1678, 1624, 1606 cm ; H NMR (DMSO-d6,
500 MHz) δ (ppm): 10.62 (s, 1H, NH), 8.15 (d, J = 2.5 Hz,
1H, H ), 7.93 (d, J = 8.5 Hz, 1H, H ), 7.61 (dd, J = 8.5 Hz,
2
6
2
NMR (DMSO-d , 125 MHz) δ (ppm): 170.5, 166.7, 159.2,
1
mass spectra (M ) at m/z 300.78; Anal. Calcd. for
C H ClN OS C, 39.93; H, 3.02; N, 18.63. Found: C,
6
49.6, 140.6, 133.6, 131.0, 123.8, 119.0, 118.0, 39.1 ppm;
3
6
+
J = 2.5 Hz, 1H, H ), 7.31 (s, 2H, NH ), 4.03 (s, 2H, CH ),
5 2 2
1
3
2.24 (s, 3H, CH3); C NMR (DMSO-d , 125 MHz) δ
1
0
9
4
2
6
4
0.32; H, 3.45; N, 18.98%.
(ppm): 170.5, 166.9, 159.2, 149.9, 149.8, 148.2, 139.0,
+
129.1, 113.4, 39.0, 17.7 ppm; mass spectra (M ) at m/z
2
-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(3,4-
281.04; Anal. Calcd. for C H N OS : C, 42.69; H, 3.94;
10 11 5 2
dimethoxyphenyl)acetamide (7g)
N, 24.89. Found: C, 42.81; H, 2.93; N, 24.76%.
Yield: 82%; mp: 194–196 °C; IR (KBr): 3350, 3318, 3052,
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(5-chloropyridin-2-
yl)acetamide (7k)
−
1
1
2
5
7
994, 1689, 1618, 1604 cm ; H NMR (DMSO-d6,
00 MHz) δ (ppm): 10.11 (s, 1H, NH), 7.30 (s, 2H, NH2),
.26 (d, J = 8.7 Hz, 1H, H ), 7.06 (dd, J = 7.5, 1.0 Hz, 1H,
Yield: 88%; mp: 187–189 °C; IR (KBr): 3348, 3314, 3028,
2
−
1
1
H ), 6.89 (d, J = 8.7 Hz, 1H, H ), 3.95 (s, 2H, CH ), 3.72 (s,
2979, 1690, 1616, 1602 cm ; H NMR (DMSO-d6,
500 MHz) δ (ppm): 10.92 (s, 1H, NH), 8.39 (d, J = 2.6 Hz,
1H, H ), 8.08 (d, J = 8.9 Hz, 1H, H ), 7.92 (dd, J = 8.9,
6
5
2
13
3
1
1
3
H, OMe), 3.71 (s, 3H, OMe) ppm; C NMR (DMSO-d6,
25 MHz) δ (ppm): 170.6, 165.7, 159.4, 149.9, 149, 145.5,
32.8, 112.5, 11.1, 104.8, 104.7, 56.2, 56.1, 55.9, 55.8,
3
6
J = 2.6 Hz, 1H, H ), 7.31 (s, 2H, NH ), 4.06 (s, 2H, CH );
5
2
2
+
13
9.3, 39.2, 40.0 ppm; mass spectra (M ) at m/z 326.39;
C NMR (DMSO-d , 125 MHz) δ (ppm): 170.5, 167.4,
6
Anal. Calcd. for C H N O S : C, 44.16; H, 4.32; N,
150.8, 149.6, 146.9, 138.5, 125.8, 114.9, 39.9, 39.7,
38.9 ppm; mass spectra (M ) at m/z 301.77; Anal. Calcd.
12
14
4
3 2
+
1
7.17. Found: C, 44.36; H, 4.27; N, 17.32%.
for C H ClN OS : C, 35.82; H, 2.67; N, 23.21 Found: C,
9
8
5
2
2
-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(2,5-
36.24; H, 2.19; N, 22.89%.
dichlorophenyl)acetamide (7h)
2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(3,4-
Yield: 86%; mp: 194–196 °C; IR (KBr): 3354, 3322, 3028,
dimethoxyphenethyl)acetamide (7l)
−
1
1
2
5
1
975, 1687, 1628, 1611 cm ; H NMR (DMSO-d6,
00 MHz) δ (ppm): 9.94 (s, 1H, NH), 7.95 (d, J = 2.5 Hz,
H, H ), 7.54 (d, J = 8.5 Hz, H ), 7.34 (s, 2H, NH ), 7.27
Yield: 88%; mp: 187–189 °C; IR (KBr): 3348, 3314, 3028,
−
1
1
2979, 1694, 1626, 1612 cm ; H NMR (DMSO-d6,
500 MHz) δ (ppm): 8.37 (t, J = 5.5 Hz, 1H, NH), 7.36 (s,
2H, NH ), 6.83 (d, J = 8.15 Hz, 1H, H ), 6.80 (s, 1H, H ),
2
3
2
1
3
(
dd, J = 8.5 Hz, J = 2.5 Hz, 1H, H ), 4.09 (s, 1H, CH ); C
4 2
NMR (DMSO-d , 125 MHz) δ (ppm): 170.6, 167.5, 159.2,
6
2
5
2
1
3
49.6, 136.3, 132.1, 131.4, 126.2, 124.4, 124.3, 39.7, 38.6,
8.5 ppm; mass spectra (M ) at m/z 333.95; Anal. Calcd.
6.69 (d, J = 8.15, 1H, H ), 3.73 (s, 3H, OCH ), 3.70 (s, 3H,
6 3
+
OCH ), 3.64(s, 2H, CH -S), 3.25 (t, J = 6.75, 2H, CH2-
3 2
1
3
for C H Cl N OS : C, 35.83; H, 2.41; N, 16.71. Found: C,
NH), 2.63 (t, J = 6.75, 2H, CH -phe); C NMR (DMSO-
1
0
8
2
4
2
2
3
6.17; H, 2.63; N, 16.64%.
d6, 125 MHz) δ (ppm): 169.1, 167.2, 164.0, 150.1, 149.0,
1
47.6, 132.2, 120.9, 112.9, 112.3, 55.8, 41.3, 39.7,
+
2
-((5-amino-1,3,4-oxadiazol-2-yl)thio)-N-(4-bromophenyl)
35.0 ppm; mass spectra (M ) at m/z 354.44; Anal. Calcd.
acetamide (7i)
for C H N O S : C, 47.44; H, 5.12; N, 15.81 Found: C,
1
4
18
4
3 2
47.24; H, 5.33; N, 15.63%.
Yield: 78%; mp: 190–192 °C; IR (KBr): 3350, 3316, 3015,
−
1
1
2
5
2
3
988, 1685, 1619, 1600 cm ; H NMR (DMSO-d6,
00 MHz) δ (ppm): 10.39 (s, 1H, NH), 7.55 (d, J = 9 Hz,
H, H3, 5), 7.50 (d, J = 9 Hz, 2H, H1, 6), 7.29 (s, 2H, NH2),
Urease inhibitory activity evaluation
The urease inhibitory activity of the test and inhibitor
compounds was evaluated based on the Berthelot colori-
13
.99 (s, 2H, CH ) ppm; C NMR (DMSO-d6, 125 MHz) δ
2
(
3
ppm): 170.5, 166.5, 149.7, 138.6, 132.1, 121.6, 115.6,
9.2 ppm; mass spectra (M ) at m/z 329.17; Anal. Calcd.
metric method. In this method, the reaction of ammonia
+
−
(NH ), hypochlorite (OCl ), and phenol will make a
3

Medicinal Chemistry Research (2021) 30:729–742
733
blue-colored indophenol. The absorbance of formed blue
color will be measured at 635 nm. The comparison of
samples absorbance with the blank sample will provide the
data for the enzyme inhibition analysis.
Chem3D ultra version 8.0. and pdbqt format of all ligands
were made by ADT. Lamarckian genetic algorithm para-
meters were set as following, a population size of 150, and
6
2.5 × 10 maximum number of energy evaluations with ten
run jobs all other parameters were set to their default
values. AutoDock4.2 was used to calculate docking
simulations [42]. The most favorable docking poses were
used to display the result by PyMOL version 1 level [43]
and LIGPLOT version v.2.2 [44].
Experimental procedure The mixture of reaction for each
compound is consisted of 50 µl of urease enzyme solution
(
(
3 mg/ml) in phosphate buffer, pH 7.4, 850 µl urea solution
1.5 mg/ml) in phosphate buffer, pH 7.4, and 100 µl of each
compound for each concentration. After a 30 min incuba-
tion of the mixture at 37 °C, 100 µl from the incubated
solution added to a mixture of 500 µl solution A (0.5 g
phenol and 25 mg sodium nitroprusside in 500 ml distilled
water) and 500 µl solution B (2.5 g sodium hydroxide and
Results and discussion
Chemistry
4
.2 ml sodium hypochlorite (5%) in 500 ml distilled water)
and again incubated in 37 °C for 30 min.
The absorbance of formed blue colored measured at
In this paper, novel 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-
N-arylacetamide derivatives (7a–l) are synthesized in a
facile three-step reaction, as shown in Scheme 2. The first
step of the synthesis involves an aniline derivative reaction
with 2-chloroacetyl chloride to form the corresponding
2-chloro-N-arylacetamide (3). Separately, hydrazine-
carbothioamide and carbon disulfide reacted together to
form 5-amino-1,3,4-thiadiazole-2-thiol. Finally, the reaction
of 2-chloro-N-arylacetamide derivatives and 5-amino-1,3,4-
thiadiazole-2-thiol led to the synthesis of 2-((5-amino-1,3,4-
thiadiazol-2-yl)thio)-N-arylacetamide (7a–l). The products
were obtained in high isolated yields and characterized by
6
25 nm and the percentage of enzyme inhibition has been
determined according to this formula [1 − (T/C)] × 100,
where T is the test compound concentration and C is the
absorbance of solvent as the blank sample. The IC50
values for all compounds have been calculated by
GraphPad prism 8 software (GraphPad software Inc.,
San Diego, CA) [41].
Docking simulation
1
13
In order to prepare all input files, receptor and ligands,
AutoDockTools 1.5.6 (ADT) was used. Crystallographic
structure of Jack bean urease enzyme with PDB ID: 3LA4
was retrieved from protein data bank (www.rcsb.com) and
used as the receptor. The following has been done to
prepare the receptor. All water molecules were deleted.
Polar hydrogens were added, and nonpolar hydrogens
were merged. Kollman charges were added. The receptor
file saved as pdbqt format. Grid box of 50 × 50 × 50 Å (X,
Y, and Z) with grid-point spacing of 0.375 Å was located in
the active site of enzyme. AutoGrid 4.2 was used to
computing the grid maps of each atom type. The ligands
were sketched in 2D structure by MarvinSketch version
melting point, FTIR, H NMR, C NMR, MASS, and
elemental analysis (Scheme 3).
In vitro urease inhibitory activities
The aim of this study is to synthesize novel compounds
against H. pylori bacterium by the mechanism of urease
inhibition. Therefore, the synthesized 2-((5-amino-1,3,4-
thiadiazol-2-yl)thio)-N-arylacetamide derivatives (7a–l)
are evaluated for potent urease inhibitory activity. The
results are presented in Table 1. For better evaluation of
the urease inhibitory activity of the compounds 7a–l, the
activities of thiourea and hydroxyurea were evaluated, and
their activities were compared to compounds 7a–l.
1
5.2.2 and converted to 3D structure with pdb format by
Scheme 3 The synthesis of 2-((5-amino-1,3,4-thiadiazol-2-yl)thio)-N-arylacetamide derivatives (7a–l). Reagents and conditions: (i) DMF, 0 °C to
r.t.; (ii) DMF, r.t.; and (iii) ethanol, NaOH, r.t.

734
Medicinal Chemistry Research (2021) 30:729–742
Table 1 The urease inhibitory activity and docking score of the synthesized compounds 7a–l
Entry Compound
ΔG binding (kcal/mol)
−5.85
IC50 (µM)a
4.35 ± 0.01
7
a
7
7
7
7
7
7
7
7
7
b
c
d
e
f
−5.40
−5.94
−5.65
−5.36
−5.79
−5.98
−5.81
−5.78
−6.12
5.29 ± 0.01
4.44 ± 0.01
4.84 ± 0.01
4.98 ± 0.01
4.02 ± 0.01
5.24 ± 0.01
3.99 ± 0.01
5.83 ± 0.01
2.85 ± 0.01
g
h
i
j

Medicinal Chemistry Research (2021) 30:729–742
Table 1 (continued)
735
Entry
Compound
ΔG binding (kcal/mol)
−6.12
IC50 (µM)a
7
k
3.57 ± 0.01
7
l
−5.08
−3.48
−5.45
3.99 ± 0.01
22.00 ± 0.03
100.00 ± 0.20
Thiourea
Hydroxyurea
aValues of IC50 are the mean ± SEM. All experiments were performed at least three times
It could be observed that The IC50 of thiourea and
hydroxyurea against urease have been 22.00 and
aromatic moieties are similar but 7l has ethylene spacer,
shows that the aliphatic spacer has raised the urease inhibi-
tory activity of 7l. IC50 of 7g and 7l are 5.239 and 3.986 µM,
which clearly proves the improvement of the inhibitory
activity of 7l after the introduction of the aliphatic saver part.
1
00.00 µM, respectively. The IC50 of compounds 7a–l
against the studied enzyme has been between
.85–5.832 µM (Table 1). All the compounds have shown
2
more inhibition activities of urease enzyme inhibitory than
thiourea and hydroxyurea. Compound 7j has been the most
active compound and showed the inhibition concentration
of 2.85 µM. Compounds 7k, 7l, and 7h have had excellent
urease inhibition activities in the concentrations of 3.56,
Docking study
Docking simulation was performed to survey the orienta-
tions, close interactions, binding modes, and binding energy
of all synthesized compounds in the active site of urease
enzyme. As could be seen in Fig. 1, similar to standard
compounds thiourea and hydroxyurea, all synthesized
compounds could be placed well in the active site of urease
enzyme by forming at least one hydrogen bond. However,
some compounds showed a more suitable orientation and
were located near Ni+ atoms.
2D representation and the detail interactions of all syn-
thesized compound and the standard compounds were listed
in Table 2. All compounds form at least one hydrogen bond
with the active site of urease enzyme. Modified residue
CME 592 plays a significant role in hydrogen bond for-
mation with compounds 7a, 7c, 7d, 7e, 7f, 7h, 7i, and 7l. In
addition, a variety of pi-interactions played an effective role
in the placement of compounds in the active site. However,
covalent bonds with nickel atoms, as seen in standard
compounds thiourea and hydroxyurea, were only found
with compounds 7g, 7j, and 7k.
3
.98, and 3.98 µM, respectively.
It could be observed in 7a with phenyl group as the
functional group in the structure of 2-((5-amino-1,3,4-thia-
diazol-2-yl)thio)-N-arylacetamides, the inhibition concentra-
tion has been 4.350 µM. Replacing the unsubstituted phenyl
by substituting ones with bulky groups in para position has
led to an increase in the compounds’ inhibition concentra-
tion. Compounds 7b and 7g with methoxy functionalities in
para position have increased the inhibition concentrations to
2
5
.285 and 5.239 µM, respectively. In addition, compound 7i,
which has bromo functionality in para position of phenyl
group has been less active than the unsubstituted compound
(
IC = 5.832 µM). The most active compounds were the
50
pyridine derivatives (compounds 7j and 7k with inhibition
activities of 2.853 and 3.567 µM, respectively). The intro-
duction of ethylene (–CH CH –) spacer between the phenyl
2
2
and amide group had a positive effect on the compound’s
activity (7I). A comparison between 7g and 7l, in which the

736
Medicinal Chemistry Research (2021) 30:729–742
Fig. 1 3D representation of hydrogen bonds of all synthesized compounds in the active site of urease enzyme
Compound 7j and compound 7k, which contains pyridine
ring with methyl and chloro substituents, respectively, in
para position, were able to stay close to the nickel atom and
form covalent bond like standard compounds thiourea and
hydroxyurea and may exert their inhibitory effects by che-
571. It is integrating that covalent bond was occurred with
Ni 841 and Ni 842. Therefore, these favorable interactions
could well justify the inhibitory effects of compound 7j.
In silico ADME properties
+2
lating Ni group by disrupting the activity of the enzyme.
Due to the similarity of chlorine and methyl size, both
compounds showed a similar orientation in the active site
with similar lowest binding energy −6.12 kcal/mol. The
least IC50 value of these compounds may justify the effec-
tiveness of pyridine ring in creating the proper interaction
with the active site. Compound 7j with methylpyridin group
indicate lowest IC = 2.853 µM among all compounds. The
SwissADME web server (http://www.swissadme.ch/index.
php) [45] was used to determine the ADME properties of all
synthesized compounds. The calculated parameters were
listed in Table 3. Lipinski’s rule [46] was developed to set
an orally active drug or drugability of synthesized com-
pounds compared with the available drugs. The Lipinski’s
rule criteria are based on logP < 5, MW < 500, HBA ≤ 10,
and hydrogen bond donor (HBD) < 5 values. As is seen in
Table 3, all synthesized compounds passed the criteria of
the Lipenski’s rule. The total number of HBDs and the total
number of HBAs of all compounds are in the range.
Lipophilicity is estimated as the ratio of equilibrium of the
concentration of a compound between oil and a liquid phase
[47]. As seen all synthesized compounds demonstrate the
lipophilic properties except the compound 7d. It should be
mention that all compounds indicate the solubility class in
the water solubility parameter. The topological polar surface
5
0
molecular docking results showed that the compound 7j
could well place into the active site. As it is shown in
Fig. 2, following details interaction was observed between 2-
(
(5-amino-1,3,4-thiadiazol-2-yl)thio)-N-(5-methylpyridin-2-
yl)acetamide (7j) and urease active site: three hydrogen
bonds formed with His 593, Arg 609, and Asp 633.
Pi–sulfur interactions were seen with His 492 and Met 637.
In addition, a large number of Van der Waals interactions are
formed with KCX 490, His 407, His 409, Ala 440, Thr 441,
Glu 493, Asp 494, Tyr 544, His 545, Gly 550, and also Thr

Medicinal Chemistry Research (2021) 30:729–742
737
Table 2 2D interactions diagram of all synthesized compounds and descriptions
Entry
2D interactions diagram
Interactions description
7
a
Hydrogen bond: CME 592
Pi–cation: Arg 439
Pi–alkyl: Ile 411, Arg 639
Pi–sulfur: Met 588
Pi–lone pair: Gln 635
Van der Waals interactions: Ala 636, Met 637, Gly 638
7
b
Hydrogen bond: His 593
Carbon hydrogen bond: Asp 633, Ala 636
Pi–donor hydrogen bond: CME 592
Pi–alkyl: Ala 440
Pi–sulfur: Met 637
Pi–pi T shape: His 519
Van der Waals interactions: KCX 490, His 492, Asp 494, Gly
50, Arg 609, Ni 841, Ni 842
5
7
c
Hydrogen bond: CME 592
Pi–cation: Arg 439, Arg 639
Pi–sulfur: Met 588
Van der Waals interactions: Ile 411, Gln 635, Ala 636, Met 637,
Gly 638, Val 640
7
d
Hydrogen bond: CME 592, Gly 638
Pi–cation: Arg 439
Pi–alkyl: Ile 411, Arg 639
Pi–sulfur: Met 588
Van der Waals interactions: Ser 634, Gln 635, Ala 636, Met 637,
Val 640
7
e
Hydrogen bond: CME 592
Carbon hydrogen bond: Arg 439
Pi–alkyl: Ile 411, Met 588, Arg 639
Amide–pi stacked: Gly 638
Van der Waals interactions: Pro 573, Gln 635, Ala 636, Met 637

738
Medicinal Chemistry Research (2021) 30:729–742
Table 2 (continued)
Entry 2D interactions diagram
Interactions description
7
f
Hydrogen bond: Arg 439, CME 592, Gln 635
Pi–alkyl: Ala 440, Met 588, Met 637
Pi–sulfur: His 594
Van der Waals interactions: Ile 411, His 593, Ala 636
7
g
Hydrogen bond: Asp 494, Arg 609, Ala 636
Carbon hydrogen bond: CME 592
Pi–alkyl: Ala 440, Met 637
Pi–sulfur: His 593
Salt bridge: Asp 633
Van der Waals interactions: KCX 490, His 407, His 409, His 492,
His 519, Gly 550, Gly 551, His 545, His 594
Covalent bond: Ni 841, Ni 842
7
h
Hydrogen bond: CME 592
Pi–alkyl: Ile 411
Pi–cation: Arg 439
Pi–sulfur: Met 588
Pi–donor hydrogen bond: Gly 638
Van der Waals interactions: Pro 573, Gln 635, Ala 636, Met 637
7
i
Hydrogen bond: CME 592, Asp 494
Pi–alkyl: Ala 440, His 545
Pi–cation: Arg 609
Pi–sulfur: Met 637
Halogen bond: Ala 636, Asp 633
Van der Waals interactions: Ala 436, His 492, His 519, Gly 550,
His 593, Ni 842, Ni 841

Medicinal Chemistry Research (2021) 30:729–742
Table 2 (continued)
739
Entry
2D interactions diagram
Interactions description
7
j
Hydrogen bond: His 593, Arg 609, Asp 633
Carbon hydrogen bond: CME 592, His 519
Pi–sulfur: His 492, Met 637
Van der Waals interactions: KCX 490, His 407, His 409, Ala 440,
Thr 441, Glu 493, Asp 494, Tyr 544, His 545, Gly 550, Thr 571
Covalent bond: Ni 841, Ni 842
7
k
Hydrogen bond: Asp 494, Asp 633
Carbon hydrogen bond: His 492, His 519
Pi–sulfur: His 593, Met 637
Van der Waals interactions: KCX 490, His 407, Ala 440, Glu 493,
His 545, Gly 550, Thr 571, CME 592, Arg 609, Ala 636
Covalent bond: Ni 841, Ni 842
7
l
Hydrogen bond: Arg 439, CME 592, Gln 635
Pi–sulfur: Met 588
Pi–alkyl: Ala 440
Pi–donor hydrogen bond: Met 637
Van der Waals interactions: Ile 411, Pro 573, His 593, Ala 636,
Gly 638, Arg 639
Thiourea
Hydrogen bond: His 492, Gly 550
Attractive charge: Asp 633
Van der Waals interactions: KCX 490, His 407, His 409, His 519,
Ala 440, Tyr 544, His 545, Gly 551, Thr 571, CME 592, Ala 636
Covalent bond: Ni 841, Ni 842

740
Medicinal Chemistry Research (2021) 30:729–742
Table 2 (continued)
Entry
2D interactions diagram
Interactions description
Hydroxyurea
Hydrogen bond: Ala 440, Gly 550, Ala 636
Salt bridge: Asp 633
Van der Waals interactions: KCX 490, His 409, Thr 441, His 492,
His 519, His 545, Gly 551, Met 639
Covalent bond: Ni 841, Ni 842
Fig. 2 Compound 7j in the active site of urease enzyme and its close interactions with residue and nickel atom in 3D and 2D display
Table 3 Calculated ADME
properties of all compounds
Entry MW (g/mol) H bond
acceptors
H bond donors TPSA
MLOGP
[49]
Lipinski ESOL class
[50]
2
(Å )
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
266.34
282.34
329.33
271.32
296.37
300.79
326.39
335.23
345.24
281.36
301.78
354.45
3
4
6
5
4
3
5
3
3
4
4
5
2
2
2
2
2
2
2
2
2
2
2
2
134.44
143.67
180.26
160.47
143.67
134.44
152.9
0.74
0.6
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
Soluble
0.15
−0.64
0.49
1.3
g
h
i
0.24
1.85
1.44
0.33
0.6
134.44
134.44
147.33
147.33
152.9
j
k
l
0.5
area (TPSA) [48] indicates the amount of molecular surface
arising from the polar fragment. The TPSA values higher
than 140 Å is related to and poor permeability to the cell
and 7i show good cell permeability. However, compounds
7b, 7e, 7j, and 7k indicate slight differences from the value
of criteria. Gather together of all parameters of the ADME,
all compounds indicate the acceptable degree of ADME
2
membrane. According to this value, compounds 7a, 7f, 7h,

Medicinal Chemistry Research (2021) 30:729–742
741
properties and may reflect the potential of drug-likeness
properties.
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