Synthesis, in vitro urease inhibitory activity and molecular docking of 3,5-disubstituted thiadiazine-2-thiones

Article abstract of DOI:10.1002/jhet.3705

A series of 3,5-disubstituted-tetrahydro-thiadiazine-2-thione (1-16) have been synthesized, characterized by elemental analysis, infrared (IR), UV-visible, ¹H NMR, ¹³C NMR, and MS spectroscopic techniques, and screened against jack bean urease. Among 16 compounds, compounds (1), (2), (3), (4), (6), (7), and (9) demonstrated excellent urease inhibitory activity with IC₅₀ values (9.8?±?0.5, 11.0?±?0.6, 16.0?±?1.5, 17.2?±?0.5, 15.4?±?0.5, 19.7?±?0.4, and 15.8?±?0.2μM), respectively, even better than the standard thiourea (IC₅₀?=?21?±?0.01μM). However, compound (8) shows an almost same level of inhibition (IC₅₀?=?22.9?±?0.3μM), as like standard. In this work, we reported for the first time urease inhibitory activity of thiadiazine thiones and its molecular docking studies.

Full text of DOI:10.1002/jhet.3705

Received: 11 June 2017
DOI: 10.1002/jhet.3705
Revised: 24 April 2019
Accepted: 8 August 2019
A R T I C L E
Synthesis, in vitro urease inhibitory activity and molecular
docking of 3,5‐disubstituted thiadiazine‐2‐thiones
Muhammad Ishaq Ali Shah1,2
| Rasool Khan1
| Mohammad Arfan3 |
Abdul Wadood4 | Mehreen Ghufran4
1 Institute of Chemical Sciences,
Abstract
University of Peshawar, Peshawar,
A series of 3,5‐disubstituted‐tetrahydro‐thiadiazine‐2‐thione (1‐16) have been
Pakistan
1
2 Department of Chemistry, Abdul Wali
Khan University, Mardan, Pakistan
3 International Center for Chemical and
Biological Science, H.E.J. Research
Institute of Chemistry University of
Karachi, Karachi, Pakistan
synthesized, characterized by elemental analysis, infrared (IR), UV‐visible, H
NMR, 13C NMR, and MS spectroscopic techniques, and screened against jack
bean urease. Among 16 compounds, compounds (1), (2), (3), (4), (6), (7), and
(9) demonstrated excellent urease inhibitory activity with IC50 values
(9.8
0.5, 11.0
0.6, 16.0
1.5, 17.2
0.5, 15.4
0.5, 19.7
0.4, and
4 Department of Biochemicstry, Abdul
Wali Khan University, Mardan, Pakistan
15.8
0.2μM), respectively, even better than the standard thiourea
(IC50 = 21 0.01μM). However, compound (8) shows an almost same level
of inhibition (IC50 = 22.9 0.3μM), as like standard. In this work, we reported
for the first time urease inhibitory activity of thiadiazine thiones and its molec-
ular docking studies.
Correspondence
Muhammad Ishaq Ali Shah, Institute of
Chemical Sciences, University of
Peshawar, Peshawar 25120, Pakistan.
Email: smishaq_81@yahoo.com
1 | INTRODUCTION
biological activities, most notably antibacterial,[13] anti-
fungal,[14] antitubercular,[15] anthelmintic,[16] antiproto-
Urease is a nickel metalloenzyme, isolated from the vari-
ety of bacteria, fungi, and plant.[1,2] Urease isolated from
different sources have similar amino acid sequences and
a common active site structure that contains two nickel
ions; consequently, different ureases have the same
mechanism of enzymatic activity.[3,4] It has been reported
that urease has adverse effects on agriculture, human,
and stockbreeding.[5,6] Urease is considered as a lethal
factor in the infection of the urinary and gastrointestinal
tract of human and animals and causes the development
of kidney stones, chronic gastritis, duodenal ulcer, and
gastric cancer.[7] Consequently, anti‐ulcer drugs oriented
around urease inhibitor remains the center of interest.
Most recently, different types of compounds have been
reported as urease inhibitor including quinines,[8] imidaz-
oles,[9] hydroxamic acids,[10] barbiturates,[11] and thio‐
barbiturates.[12] Hence, in view of the above, there is high
need to develop new urease inhibitors.
zoal,[17] leishmanicidal,[18] antimycobacterial,[19] and
antiviral.[20]
In addition to its renowned biological potential, this
versatile heterocyclic nucleus has been reported as a
biolabile prodrug,[21] in the drug delivery system (DDS)
because of its characteristic features like high
liphophilicity and enzymatic hydrolysis. Subsequently,
different types of amino acids,[22] peptides,[23] and drug
based on primary amine have been reported to success-
fully attached to the THTT moiety,[24] which further
enhance their cellular uptake by improving lipophilicity
in the area where various physiological and enzyme activ-
ities causes the release of the drug molecule.
In spite of the tremendous progress over the last
decades, in the synthesis and evaluation of the pharmaceu-
tical and biological properties of the THTH moiety, there is
still no example of the screening of these compounds
against urease. Herein, we report for the first time the
screening of thiadiazine thione derivatives for their urease
inhibitory activities and their molecular docking studies.
The thiadiazine thione nucleus and its derivatives have
received particular attention due to its diverse range
J Heterocyclic Chem. 2019;1–8.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

2
SHAH ET AL.
absorption band in the range of 3300 to 3450 cm−1, while
carbonyl (C═O) and carbon sulfide (C═S) in the range of
1760 to 1690 cm−1 and 1510 to 1450 cm−1, respectively.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
1
Similarly, H NMR spectra show two separate singlets
The desired 3,5‐disubstituted‐tetrahydro‐thiadiazine‐2‐
thiones (1‐16) were synthesized in good to excellent yields
(65%‐95%), according to Scheme 1. The preparation of
compounds (1‐16) was achieved from the reaction of the
primary amine including aniline, benzylamine,
butylamine, o‐toluidine, and 2,4,6‐trimethylaniline with
carbon disulfide, which leads to respective potassium salt
of dithiocarbamate. Subsequently, its treatments with
formaldehyde and different types of amino acid and
amine including glycine, alanine, 6‐amino‐caproic acid,
2‐aminopyrimidine, and ethanolamine in the presence
of phosphate buffer (pH = 7.7) form respective 3,5‐
disubstituted thiadiazine‐2‐thiones (1‐16).
for C‐4 and C‐6 methylene of tetrahydro‐2H‐1,3,5‐
thiadiazine‐2‐thione nucleus around 5.27 to 3.64 ppm.
In the same way, the 13C NMR spectra of the com-
pounds show the characteristics signals for C═S, C‐4,
and C‐6 at δ:194.5, 69.7, 59.7, 195.0, 67.7, 57.1, 192.8,
68.2, 59.2, 194, 68.8, 59.7, 193.0, 67.6, 58.4, 192.8, 70.9,
59.4, 193.1, 71.5, 60.9, 198.3, 49.0, 47.7, 193.0, 71.0, 58.8,
193.4, 71.1, 58.8, 195.1, 72.2, 59.9, 194.0, 73.5, 58.5,
194.7, 73.9, 59.4, 194.3, 73.4, 60.8, 193.2, 72.0, 58.4,
194.2, 72.7, 57.7, in compounds 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, and 16, respectively. All the remain-
ing data are mentioned in the experimental section.
Structures of all the synthesized compounds were con-
firmed by spectroscopic techniques and microanalysis.
UV‐visible analysis of the tetrahydrothiadiazine‐2‐thiones
shows an absorption band at 205 to 290 nm. Infrared (IR)
spectroscopy shows carboxylic and alcoholic O–H
2.2 | Urease inhibitory activity and
molecular docking study
Here, our main focus was to screen all our synthesized
compounds (1‐16), for urease inhibitory activity against
SCHEME 1 Synthetic route of 3,5‐
disubstituted thiadiazine thiones
TABLE 1 In vitro activity of thiadiazine‐2‐thiones (1‐16) against Jack bean
S. No
R1
R2
IC50 SEM, μM Percent Inhibition Yield, %
1
Benzyl–
HOOCCH2–
9.8 0.5
11.0 0.6
16.0 1.5
17.2 0.5
99.6
96.3
99.8
97
72
65
68
76
86
80
77
70
82
70
75
80
70
90
95
92
—
2
Benzyl–
(CH3)2CH–
3
Benzyl–
HOCH2CH2–
CH3CH2CH2CH2–
4
Benzyl–
5
Benzyl–
HOOCCH2(CH2)4– 25.08 0.2
97
6
CH3CH2CH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2–
HOOCCH2(CH2)4–
HOOCCH2–
HOOCCH2–
HOCH2CH2–
C4H3N2–
15.4 0.5
19.7 0.4
22.9 0.3
97.5
96.9
99.2
98.3
95
7
8
9
HOOCCH2(CH2)4– 15.8 0.2
HOOCCH2(CH2)4– 35.08 0.2
10
11
12
13
14
15
16
HOOCCH2–
CH3CH2CH2CH2–
HOOCCH2–
HOCH2CH2–
HOOCCH2–
HOOCCH2(CH2)4–
—
28.08 0.2
96
C6H5–
46.3 0.35
98.0
—
o‐CH3C6H4–
NI
2,4,6‐(CH3)3‐C6H3–
2,4,6‐(CH3)3‐C6H3–
2,4,6‐(CH3)3‐C6H3–
—
—
—
—
—
—
Standard urease inhibitor Thiourea (standard urease inhibitor)
Abbreviations: NI, no inhibitory activity. SEM, standard error of the mean.
21 0.01
—

SHAH ET AL.
3
Jack bean urease as shown in Table 1. Structure activity
relationship of the synthesized compounds revealed that
the thiadiazine thione nucleus having benzyl group at
N‐3 position and ethanoic acid group at N‐5 position
exhibited significant activity, for example, compound 1
series, the most active compound was investigated by
docking study to explain their binding approach with tar-
get enzyme. Based on our above finding, we are optimis-
tic that thiadiazine thione nucleus based moiety will be
used to treat urease related problems in future.
(IC50 = 9.8
0.5μM). The activities decreased when
ethanoic group was replaced by isopropyl, butyl, and
hexyl group at position N‐5; for example, compounds 2
(IC50 = 11.0
0.6μM), 4 (IC50 = 17.2
0.5μM), and 5
4 | EXPERIMENTAL
4.1 | Materials
(IC50 = 25.08 0.2μM) show low activities as compare
with compound 1.
Similarly, decreased in activities was observed, when
benzyl group at position N‐3 was replaced by butyl group,
All solvents and chemical reagents were commercial
grade quality and were used without further purifica-
tion. All the reactions were monitored by thin layer
chromatography, using precoated silica gel (aluminum
sheets, layer thickness 0.2 mm and 60 HF‐254). Melting
points were determined in glass capillary tubes using a
Gallen Kamp melting point apparatus and are uncor-
rected. Structures of all the synthesized compounds
were determined with the help of spectroscopic tech-
niques including: UV (Shimadzu), IR (Bio‐Rad Win
IR), 1H NMR (Bruker), 13C NMR (Bruker), MS (Jeol
Mat 312), and elemental analysis (Carlo Erba 1106).
for example, compounds 6 (IC50 = 15.4
0.5μM), 7
(IC50 = 19.7
(IC50 = 15.8
found for compound 12 (IC50 = 46.3
0.4μM), 8 (IC50 = 22.9
0.2μM). Surprisingly, low activity was
0.35μM), when
0.3μM), and 9
the substituent at position N‐3 was replaced by phenyl
group, while activities completely abolished when
monomethyl, dimethyl, and trimethyl substituted phenyl
were used at N‐3 position, such as compounds 10, 11, 12,
and 14 as shown in Table 1, which might be due to
methyl group attached to the phenyl group.
To investigate the binding mode of the most active
compound (compound 1), the molecular docking was
performed to determine the binding interaction between
the active compound and urease enzyme. The docking
result illustrated that the compound 1 was accommo-
dated very well in the active site pocket. From the
docking conformation of the most active compound 1, it
was observed that this compound formed three hydrogen
bonds with the Glu 493, His 593, and Arg 609 residues of
the enzyme (Figure 1).
4.2 | General procedure for the synthesis
of
3,5‐disubstituted‐tetrahydrothiadiazine‐2‐
thiones (1‐16)
To
a
magnetically stirred solution of primary
The compound 1 has biological activity with the IC50
amine/amino acid (20 mmol) and potassium hydroxide
(20%, 20 mmol) in 30 mL of water, carbon disulfide
(20 mmol) was added portion‐wise. Further, the reaction
materials were stirred for 4 hours, followed by addition
of 37% formaldehyde (40 mmol) and stirred for 1 hour.
Subsequently, the reaction material was filtered, and
the filtrate was transferred dropwise to a suspension of
amino acid/amine in 20 mL of phosphate buffer solu-
tion (pH 7.7) and stirs it for 1 hour. The reaction mate-
rial was filtered, and the filtrate was extracted with
dichloromethane. The aqueous solution was acidified
with 15% HCl. The precipitate formed was filtered under
suction and thoroughly washed with water and dried. At
the end, the product was recrystallized from ethanol.
The above general procedure represents a slightly modi-
fied version of already reported method.[19] The struc-
tures of the all the synthesized compounds were
value of (9.8
0.5μM) and the corresponding binding
energy of −5.98 kcal/mol. His 492 was observed making
an arene cation linkage with the benzene moiety of the
compound. The Glu 493 and His 593 formed hydrogen
bonding with the carboxyl moiety of the compound,
while His 593 formed hydrogen bond with the nitrogen
atom of the 3,5‐dimethyl‐1,3,5‐thiadiazinane‐2‐thione
moiety of the compound.
3 | CONCLUSION
In conclusion, a series of thiadiazine thione nucleus
based compounds were prepared in good yield. The
in vitro urease inhibition activity of these compounds
revealed that the compounds (1, 2, 3, 4, 6, 7, and 9) were
found more active than the reference compound thio-
urea, while the compounds 5 and 8 show same level of
inhibitory activity as like standard drug. Among the
1
characterized by UV, IR, H NMR, 13C NMR, MS, and
elemental analysis.

4
SHAH ET AL.
FIGURE 1 Solid ribbon representation
of intermolecular interaction between
compound 1 and urease crystal structure
[Color figure can be viewed at
wileyonlinelibrary.com]
(64), 65 (24), and 56 (26); Anal. calcd for C13H16N2O2S2:
C, 52.70; H, 5.40; N, 9.45. Found: C, 52.69; H, 5.40; N, 9.44.
4.2.1 | (5‐Benzyl‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl)acetic acid (1)
Benzylamine (2.18 mL, 20 mmol) and glycine (1.5 g,
20 mmol): recrystallized from ethanol, colourless solid,[21]
Yield = 72%, m.p = 135‐137°C; UV (methanol) λmax nm:
205, 290; IR (KBr) υmax cm−1: 3430 (COOH), 3030
(Aromatic–C–H), 2883 (Aliphatic–C–H), 1724 (C═O),
1491 (C═S); 1H NMR (CDCl3 + CD3OD) δ: 7.29‐7.21
(m, 5H, Ar‐H), 5.22 (s, 2H, C6H5CH2), 4.39 (s, 2H, H‐4),
4.29 (s, 2H, H‐6), 3.29 (s, 2H, NCH2COOH); 13C NMR
(CD3OD) δ: 194.5 (C═S), 172.2 (C═O), 136.9 (Ar‐C),
129.8, 129.5, and 129.1 (Ar–CH), 69.7 (C‐4), 59.7 (C‐6),
54.5 (CH2C6H5), 51.9 (NCH2); EI‐MS; m/z (rel. int. %):
281 (M+), 149 (8), 133 (12), 119 (6), 101 (4), 91 (100),
and 65 (12); Anal. calcd for C12H14N2O2S2: C, 51.06; H,
4.96; N, 9.92. Found: C, 51.05; H, 4.92; N, 9.90.
4.2.3 | 3‐Benzyl‐5‐(2‐hydroxyethyl)‐1,3,5‐
thiadiazinane‐2‐thione (3)
Benzylamine (2.18 mL, 20 mmol) and ethanolamine
(1.20 mL, 20 mmol): recrystallized from ethanol,
colourless solid, Yield = 68%, m.p = 99‐101°C; UV (meth-
anol) λmax nm: 225, 290; IR (KBr) υmax cm−1: 3304 (OH),
3030 (Aromatic–C–H), 2958 (Aliphatic–C–H), 1491
1
(C═S); H NMR (CD3OD) δ: 7.40‐7.28 (m, 5H, Ar–H),
5.33 (s, 2H, CH2C6H5), 4.41 (s, 2H, H‐4), 4.32 (s, 2H,
H‐6), 3.43 (t, 2H, J = 5.4 Hz, HOCH2CH2), 2.77 (t, 2H,
J = 5.4 Hz, NCH2CH2); 13C NMR (CDCl3) δ: 192.8
(C═S), 135.1 (Ar–C), 128.9, 128.6, and 128.3 (Ar–CH),
68.2 (C‐4), 59.2 (C‐6), 58.5 (CH2CH2OH), 53.7 (CH2C6H5),
52.4 (NCH2CH2OH); EI‐MS; m/z (rel. int. %): 268 (M+),
235 (18), 196 (8), 149 (11), 118 (13), 90 (100), 87 (31), 76
(16), 65 (13), and 56 (9); Anal. calcd for C12H16N2OS2: C,
53.73; H, 5.97; N, 10.44. Found: C, 54.72; H, 5.97; N, 10.43.
4.2.2 | 2‐(5‐Benzyl‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl)propanoic acid (2)
Benzylamine (2.18 mL, 20 mmol) and alanine (1.78 g,
20 mmol): recrystallized from ethanol, colourless solid,
Yield = 65%, m.p = 143‐144°C; UV (methanol) λmax nm:
210, 290; IR (KBr) υmax cm−1: 3400 (COOH), 3010 (Aro-
matic–C–H), 2870 (Aliphatic–C–H), 1700 (C═O), 1456
4.2.4 | 3‐Benzyl‐5‐butyl‐1,3,5‐
thiadiazinane‐2‐thione (4)
Benzylamine (2.18 mL, 20 mmol) and n‐butylamine
(1.97 mL, 20 mmol): recrystallized from ethanol, colourless
solid,[25] Yield = 76%, m.p = 108‐109°C; UV (methanol)
λmax nm: 210, 290; IR (KBr) υmax cm−1: 3025 (Aromatic–
C–H), 2862 (Aliphatic–C–H), 1452 (C═S); 1H NMR
(CD3OD) δ: 7.46‐7.29 (m, 5H, Ar–H), 5.33 (s, 2H,
CH2C6H5), 4.82 (s, 2H, H‐4), 4.37 (s, 2H, H‐6), 2.53 (t, 2H,
J = 7.3 Hz, NCH2CH2), 1.17‐1.11 (m, 2H, CH2CH2CH2),
1.10‐1.07 (m, 2H, CH2CH2CH3), 0.77 (t, J = 6.9 Hz,
CH3CH2); 13C NMR (CD3OD) δ: 194 (C═S), 137.3 (Ar–C),
1
(C═S); H NMR (CD3OD) δ: 7.32‐7.03 (m, 5H, Ar–H),
5.22 (s, 2H, C6H5CH2), 4.28 (s, 2H, H‐4), 4.22 (s, 2H, H‐6),
3.57 (q, 1H, J = 7.0 Hz, CH3CH), 1.12 (d, 3H, J = 7.0 Hz,
CHCH3); 13C NMR (CD3OD + CDCl3) δ: 195.0 (C═S),
176.2 (C═O), 137.0 (Ar‐C), 129.8, 129.7, and 129.1 (Ar–
CH), 67.7 (C‐4), 57.7 (CHCH3), 57.1 (C‐6), 54.7
(C6H5CH2), 16.5 (CH3CH); EI‐MS; m/z (rel. int. %): 296
(M+), 281 (19), 148 (23), 133 (24), 119 (20), 90 (100), 76

SHAH ET AL.
5
129.8, 129.7, and 129.3 (Ar–CH), 68.8 (C‐4), 59.7 (C‐6),
54.29 (CH2C6H5), 50.9 (NCH2CH2), 30.2 (NCH2CH2CH2),
21.03 (CH2CH2CH3), 14.0 (CH2CH3); EI‐MS; m/z (rel. int.
%) 247 (M+), 189 (2), 148 (2), 103 (7), 91 (100), 72 (12), 65
(27), and 51 (7); Anal. calcd for C14H20N2S2: C, 60.09; H,
7.14; N, 10.80. Found: C, 60.09; H, 7.12; N, 10.79.
colourless solid, Yield = 77%, m.p = 58‐60°C; UV (meth-
anol) λmax nm: 245, 285; IR (KBr) υmax cm−1: 3300 (OH),
2860 (Aliphatic–C–H), 1480 (C═S); H NMR (CD3OD) δ:
1
4.84 (s, 2H, H‐4), 4.50 (s, 2H, H‐6), 3.90 (t, 2H, J = 7.9 Hz,
NCH2CH2), 3.72 (t, 2H, J = 5.4 Hz, HOCH2CH2), 2.91
(t, 2H, J = 5.4 Hz, NCH2CH2), 1.69‐1.63 (m, 2H,
CH2CH2CH2), 1.40‐1.34 (m, 2H, CH2CH2CH3), 0.96
(t, 3H, J = 7.4 Hz, CH3CH2); 13C NMR (CD3OD + CDCl3)
δ: 193.1(C═S), 71.5 (C‐4), 60.9 (C‐6), 59.5 (CH2OH), 53.5
(NCH2CH2), 53.0 (NCH2CH2OH), 29.6 (NCH2CH2), 20.9
(CH2CH3), 14.1 (CH3CH2); EI‐MS; m/z (rel. Int. %): 234
(M+), 201 (3), 161 (2), 110 (15), 84 (100), 72 (35), and 57
(15); Anal. calcd for C14H18N2O2S2: C, 46.12; H, 7.74; N,
11.95. Found: C, 46.10; H, 7.72; N, 11.94.
4.2.5 | 6‐(5‐Benzyl‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl)hexanoic acid (5)
Benzylamine (2.18 mL, 20 mmol) and 6‐aminocaproic acid
(2.62 g, 20 mmol): recrystallized from ethanol, colourless
solid, Yield = 86%, m.p = 108‐110°C; UV (methanol) λmax
nm: 240, 315; IR (KBr) υmax cm−1: 3400 (COOH), 3020 (Aro-
matic–C–H), 2850 (Aliphatic–C–H), 1729 (C═O), 1460
(C═S); 1H NMR (CD3OD) δ: 7.46‐7.31 (m, 5H, Ar–H), 5.33
(s, 2H, CH2C6H5), 4.47 (s, 2H, H‐4), 4.37 (s, 2H, H‐6), 2.53
(t, 2H, J = 7.3 Hz, CH2CH2N), 2.18 (t, 2H, J = 7.3 Hz,
CH2CH2COOH), 1.47‐1.40 (m, 4H, CH2(CH2)2CH2), 1.18‐
1.10 (m, 2H, CH2CH2CH2); 13C NMR (CD3OD + CDCl3)
δ: 193.0 (C═S), 175.8 (C═O), 135.0 (Ar–C), 128.5, 128.2,
and 127.9 (Ar–CH), 67.6 (C‐4), 58.4 (C‐6), 53.4 (C6H5CH2),
49.7 (NCH2CH2), 33.5 (CH2COOH), 26.4 (NCH2CH2), 25.9
(NCH2CH2CH2), 24.1 (CH2CH2COOH); MS (FAB) m/z:
339; Anal. calcd for C16H22N2O2S2: C, 56.77; H, 6.55; N,
8.28. Found: C, 56.76; H, 6.55; N,8.27.
4.2.8 | 3‐Butyl‐5‐pyrimidin‐2‐yl‐1,3,5‐
thiadiazinane‐2‐thione (8)
n‐Butylamine (1.97 mL, 20 mmol) and 2‐aminopyrimidine
(1.90 g, 20 mmol): recrystallized from ethanol, colourless
solid, Yield = 70%, m.p = 99‐101°C; UV (methanol) λmax
nm: 275; IR (KBr) υmax cm−1: 2873 (Aliphatic–C–H), 1465
1
(C═S); H NMR (CD3OD) δ: 8.33 (d, 2H, J = 3.7 Hz,
Ar–CH), 6.74 (t, 1H, J = 4.7 Hz, Ar–H), 5.27 (s, 2H, H‐4),
4.84 (s, 2H, H‐6), 3.65 (t, 2H, J = 7.2 Hz, NCH2CH2),
1.63‐1.56 (m, 2H, CH2CH2CH2), 1.38‐1.31 (m, 2H,
CH3CH2CH2), 0.92 (t, 3H, J = 7.3 Hz, CH3CH2);13C NMR
(CD3OD) δ: 198.3 (C═S), 162.4 (Ar‐C), 159.4 (Ar–C),
113.0 (Ar–CH), 49.0 (C‐4), 48.3 (NCH2CH2), 47.7 (C‐6),
31.1 (CH2CH2CH2), 21.1 (CH3CH2CH2), 14.0 (CH3CH2);
MS (FAB) m/z: 286; Anal. Calcd for C11H16N4S2: C, 49.20;
H, 5.97; N, 20.89. Found: C, 49.19; H, 5.95; N, 20.86.
4.2.6 | (5‐Butyl‐6‐thioxo‐1,3,5‐thiadiazinan‐
3‐yl)acetic acid (6)
n‐Butylamine (1.97 mL, 20 mmol) and glycine (1.5 g,
20 mmol): recrystallized from ethanol, colourless solid,[21]
Yield = 80%, m.p = 97‐99°C; UV (methanol) λmax nm:
210, 285: IR (KBr): 3420 (COOH), 2954 (Aliphatic–C–
4.2.9 | 6‐(5‐Butyl‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl)hexanoic acid (9)
1
H), 1733 (C═O), 1508 (C═S); H NMR (CD3OD) δ: 4.53
(s, 2H, H‐4), 4.52 (s, 2H, H‐6), 3.97 (t, 2H, J = 7.8 Hz,
NCH2CH2), 3.30 (s, 2H, CH2COOH), 1.67‐1.61 (m, 2H,
CH2CH2CH2), 1.38‐1.33 (m, 2H, CH2CH2CH3), 0.96
(t, J = 7.3 Hz, 3H, CH2CH3); 13C NMR (CD3OD) δ:
192.8 (C═S), 172.8 (C═O), 70.9 (C‐4), 59.4 (C‐6), 52.9
(CH2COOH), 52.0 (CH2CH2N), 29.5 (CH2CH2CH2), 20.9
(CH2CH2CH3), 14.1 (CH3CH2); EI‐MS; m/z (rel. int. %)
248 (M+), 115 (22), 84 (86), 72 (96), 60 (58), and 57
(100); Anal. calcd for C9H16N2O2S2: C, 43.54; H, 6.45; N,
11.29. Found: C, 43.52; H, 6.42; N, 11.28.
n‐Butylamine (1.97 mL, 20 mmol) and 6‐amino caproic
acid (2.62 g, 20 mmol): recrystallized from ethanol,
colourless solid, Yield = 82%, m.p = 88‐90°C; UV (metha-
nol) λmax nm: 250, 285; IR (KBr) υmax cm−1: 3400 (COOH),
2865 (Aliphatic–C–H), 1695 (C═O), 1503 (C═S); 1H NMR
(CD3OD) δ: 4.47 (s, 2H, H‐4), 4.46 (s, 2H, H‐6), 3.80
(t, 2H, J = 7.9 Hz, NCH2CH2), 2.78 (t, 2H, J = 7.1 Hz,
NCH2CH2), 2.29 (t, 2H, J = 7.3 Hz, CH2CH2COOH), 1.68‐
1.62 (m, 4H, CH2(CH2)2CH2), 1.61‐1.55 (m, 2H,
CH2CH2CH2), 1.44‐1.40 (m, 2H, CH3CH2CH2), 1.39‐1.34
(m, 2H, CH2CH2CH2), 0.96 (t, 3H, J = 7.3 Hz, CH3CH2);
13C NMR (CD3OD) δ: 193.0 (C═S), 177.4 (C═O), 71.0
(C‐4), 58.8 (C‐6), 52.9 (NCH2), 50.9 (NCH2), 34.8
(CH2COOH), 29.5 (CH2CH2CH2), 28.1 (CH2CH2CH2),
27.5 (CH2CH2COOH), 25.3 (CH2CH2CH2), 20.9
(CH2CH2CH3), 14.1 (CH3CH2); EI‐MS; m/z (rel. int. %):
4.2.7 | 3‐Butyl‐5‐(2‐hydroxyethyl)‐1,3,5‐
thiadiazinane‐2‐thione (7)
n‐Butylamine (1.97 mL, 20 mmol) and ethanolamine
(1.20 mL, 20 mmol): recrystallized from ethanol,

6
SHAH ET AL.
246 (M+), 156 (17), 115 (32), 98 (95), 84 (100), 76 (97), 57
(88), and 51 (7); Anal. calcd for C13H24N2O2S2: C, 51.31;
H, 7.89; N, 9.21. Found: C, 51.30; H, 7.87; N, 9.20.
0.95 (t, 3H, J = 7.3 Hz, CH3CH2);13C NMR (CDCl3) δ:
194.0 (C═), 144.5 (Ar–C), 129.8, 128.2, and 126.9 (Ar–
CH), 73.5 (C‐4), 58.5 (C‐6), 50.1 (NCH2), 29.3 (NCH2CH2),
20.0 (NCH2CH2CH2), 13.8 CH2CH3); EI‐MS; m/z (rel. int.
%): 266 (M+), 135 (32), 99 (67), 77 (99), 65(9), 57(30), and
51 (100); Anal. calcd for C13H18N2S2: C, 58.64; H, 6.76; N,
10.52. Found: C, 58.62; H, 6.74; N, 10.50.
4.2.10 | 6,6′‐(2‐Thioxo‐1,3,5‐thiadiazinane‐
3,5‐diyl) dihexanoic acid (10)
6‐Aminocaproic acid (2.62 g, 20 mmol) and 6‐
aminocaproic acid (2.62 g, 20 mmol): recrystallized from
ethanol, colourless solid,[26] Yield = 70%, m.p = 143‐
144°C; UV (methanol) λmax nm: 210, 285; IR (KBr) υmax
4.2.13 | [5‐(2‐Methylphenyl)‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl]acetic acid (13)
cm−1
:
3400 (COOH), 2862 (Aliphatic–C–H), 1704
o‐Toluidine (2.18 mL, 20 mmol) and glycine (1.5 g,
20 mmol): recrystallized from ethanol, colourless solid,
Yield = 70%, m.p = 127‐129°C; UV (methanol) λmax nm:
215, 240; IR (KBr) υmax cm−1: 3400 (COOH), 3030 (Aro-
matic–C–H), 1716 (C═O), 1485 (C═S); 1H NMR (CD3OD)
δ: 7.27‐7.04 (m, Ar–H), 3.69 (s, 2H, H‐4), 3.64 (s, 2H, H‐6),
3.30 (s, 2H, CH2COOH), 2.22 (s, 3H, Ar–CH3);13C NMR
(CD3OD) δ: 194.7 (C═S), 172.7 (C═O), 144, 136.6 (Ar–
C), 132.3, 128, 129.7, and 127.7 (Ar–CH), 73.9 (C‐4),
59.4 (C‐6), 52.7 (CH2COOH), 17.8 (Ar–CH3); EI‐MS;
m/z (rel. int. %): 282 (M+), 250 (15), 235 (12), 149 (62),
120 (100), 117 (53), 91 (94), 77 (58), 65 (66), and 51 (40);
Anal. Calcd for C12H14N2O2S2: C, 51.0; H, 4.96; N, 9.92.
Found: C, 50.98; H, 4.94; N, 9.91.
1
(C═O), 1492 (C═S); H NMR (CD3OD) δ: 4.47 (s, 4H,
H‐4, H‐6), 3.98 (t, 2H, (CH2)‐7, J = 7.8 Hz), 2.78 (t 2H,
(CH2)‐7′, J = 7.1), 2.38 (t, 4H, (CH2)‐11 and (CH2)‐11′),
1.59‐1.70 (m, 8H, (CH2)‐8 and 8′, (CH2)‐10 and 10′),
1.36‐1.43 (m, 4H, (CH2)‐9 and 9′); 13C NMR
(CD3OD + CDCl3) δ: 193.4 (C═S), 177.5 (C═O), 71.1 (C‐
4), 58.8 (C‐6), 52.95 (C‐7), 50.95 (C‐7′), 34.83 (C‐11&11′),
28.10 (C‐8), 27.57 (C‐8′), 25.82 (C‐9′), 25.6 (C‐9), 27.2
(C‐10), 27.2 (C‐10′); EI‐MS; m/z (rel. int. %): 362(M+),
156 (20), 155 (18), 142 (13), 128(8), 111 (40), 98(18), and
55(66); Anal. Calcd for C15H28N2O4S2: C, 49.72; H, 7.18;
N, 7.73. Found: C, 49.70; H, 7.16; N, 7.71.
4.2.11 | 2,2′‐(2‐Thioxo‐1,3,5‐thiadiazinane‐
3,5‐diyl) diacetic acid (11)
4.2.14 | 5‐(2‐Hydroxyethyl)‐3‐mesityl‐1,3,5‐
thiadiazinane‐2‐thione (14)
Glycine (1.5 g, 20 mmol) and glycine (1.5 g, 20 mmol):
recrystallized from ethanol, colourless solid, Yield = 75%,
m.p = 208‐209°C; UV (methanol) λmax nm: 250, 285; IR
(KBr) υmax cm−1: 3430 (COOH), 2850 (Aliphatic–C–H),
1700 (C═O), 1475 (C═S); 1H NMR (CD3OD) δ: 4.74
(s, 2H, CH2), 4.60 (s, 2H, H‐4), 4.58 (s, 2H, H‐6),
3.81(s, 2H, CH2); 13C NMR (CD3OD) δ: 195.1 (C═S),
173.2 (C═O), 170.8 (C═O), 72.2 (C‐4), 59.9 (C‐6), 53.2
(CH2COOH), 52.4 (CH2COOH); Mass (FAB) m/z: 250;
Anal. Calcd for C7H10N2O4S2: C, 33.6; H, 4.0; N, 11.2.
Found: C, 33.59; H, 3.97; N,10.99.
2,4,6‐Trimethylaniline (2.18 mL, 20 mmol) and ethanol-
amine (1.20 mL, 20 mmol): recrystallized from ethanol,
colourless solid, Yield = 90% m.p = 126‐128°C; UV
(methanol) λmax nm: 240, 285; IR (KBr) υmax cm−1: 3315
(OH), 3020 (Aromatic–C–H), 2833 (Aliphatic–C–H),
1750 (C═O), 1475 (C═S); 1H NMR (CD3OD) δ: 6.90
(s, 2H, Ar–H), 4.65 (s, 2H, H‐4), 4.51 (s, 2H, H‐6), 3.75
(t, 2H, J = 6.9 Hz, CH2CH2OH), 3.22 (t, 2H, J = 6.9 Hz,
NCH2CH2OH), 2.29 (s, 3H, Ar–CH3), 2.14 (s, 6H, Ar–
CH3); 13C NMR (CD3OD + CDCl3) δ: 194.3 (C═S),
139.5, 138.5 (Ar‐C), 129.7 (Ar–CH), 73.4 (C‐4), 60.8
(C‐6), 58.4 (CH2OH), 54.6 (NCH2), 21.0, 18.4 (Ar–CH3);
EI‐MS; m/z (rel. int. %): 296 (M+), 250 (3), 177 (100),
119 (89), 104 (4), 91 (18), 87 (4), 76 (45), and 65 (6); Anal.
calcd for C14H20N2OS2: C, 56.72; H, 6.80; N, 9.45. Found:
C, 56.70; H, 6.79; N, 9.43.
4.2.12 | 5‐Butyl‐3‐phenyl‐1,3,5‐
thiadiazinane‐2‐thione (12)
Aniline (2.18 mL, 20 mmol) and n‐butyl amine (1.97 mL,
20 mmol): recrystallized from ethanol, colourless solid,
Yield = 80%, m.p = 102‐104°C; UV (methanol) λmax nm:
215, 280; IR (KBr) υmax cm−1: 3030 (Aromatic–C–H),
4.2.15 | 5‐Mesityl‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl)acetic acid (15)
1
2825 (Aliphatic–C–H), 1455 (C═S); H NMR (CD3OD) δ:
7.46‐7.18 (m, 5H, Ar‐H), 4.84 (s, 2H, H‐4), 4.69 (s, 2H,
H‐6), 3.0 (t, 2H, J = 7.2 Hz, NCH2CH2), 1.61‐1.55
(m, 2H, CH2CH2CH2), 1.46‐1.40 (m, 2H, CH3CH2CH2),
2,4,6‐Trimethylaniline (2.18 mL, 20 mmol) and glycine
(1.5 g, 20 mmol): recrystallized from ethanol, colourless

SHAH ET AL.
7
solid,[27] Yield = 95%, m.p = 148‐150°C; UV (methanol)
λmax nm: 215, 245; IR (KBr) υmax cm−1: 3460 (COOH),
3010 (Aromatic–C–H), 2885 (Aliphatic–C–H), 1733
6 | MOLECULAR MODELING
METHOD
1
The crystallographic structure of Jack bean urease (PDB
ID: 4H9M) was obtained from the RSCB Protein Data
Bank.[29] The bound ligand acetohydroxamic acid and
crystallographic water molecules were removed from the
structure, and hypothetical hydrogen atoms were added
to the X‐ray structure in standard geometries with the
Molecular Operating Environment (MOE) 2016.08. The
point charge of the two catalytic nickel cations was set
to +2. Gasteiger charges were calculated using AutoDock
Tools, and nonpolar hydrogens were merged. Docking
was performed with AutoDock[30] 4.2 with rigid protein
structure and fully flexible ligands. The 3D structure of
the compound of the series was saved as pdb files, which
were then transformed to pdbqt files after the charges of
the nonpolar hydrogen atoms were assigned with the aid
of ADT. The grid calculation was performed using the
Autogrid4 program; grid spacing of 0.375 Å parameters
was set. A grid spacing based on the default setting was
0.375 Å. And the gpf file was generated to run AutoGrid
for the calculation of the energetic map. After successful
calculations, the docking parameter file (dpf file) was pre-
pared to run AutoDock. Default settings were used with
an initial population size of 150 randomly placed individ-
uals, a maximum number of 2.5 × 106 (medium) energy
evaluations, and a maximum number of 2.7 × 104 gener-
ations. A mutation rate of 0.02, a crossover rate of 0.8, and
GA crossover mode of two points were chosen. One hun-
dred runs were generated by using Lamarckian genetic
algorithm searches. On successful completion of docking,
the resultant complex structures were selected based on
the most favorable free energy of binding.
(C═O), 1471 (C═S); H NMR (CD3OD + CDCl3) δ: 6.90
(s, 2H, Ar–H), 4.63 (s, 2H, H‐4), 4.52 (s, 2H, H‐6), 3.90
(s, CH2COOH), 2.24 (s, 3H, Ar–CH3), 2.14 (s, 6H, Ar–
CH3); 13C NMR (CD3OD + CDCl3) δ: 193.2 (C═S), 172.2
(C═O), 139.2, 138.5, and 134.9 (Ar–C), 130.5 (Ar–CH),
72.0 (C‐4), 58.4 (C‐6), 52.0 (CH2COOH), 21.1, 18.2 (Ar–
CH3); EI‐MS; m/z (rel. int. %), 310 (M+), 177 (100), 162
(40), 147 (39), 119 (37), 100 (13), 104 (7), and 91 (31),
and 65 (12); Anal. calcd for C14H18N2O2S2: C, 54.19; H,
5.80; N, 9.03. Found: C, 54.17; H, 5.78; N, 9.01.
4.2.16 | 6‐(5‐Mesityl‐6‐thioxo‐1,3,5‐
thiadiazinan‐3‐yl)hexanoic acid (16)
2,4,6‐Trimethylaniline and 6‐aminocaproic acid (2.62 g,
20 mmol): recrystallized from ethanol, colourless solid,
Yield = 95%, m.p = 122‐124°C; UV (methanol) λmax
nm: 215, 285; IR (KBr) υmax cm−1: 3420 (COOH), 3030
(Aromatic–C–H), 2867 (Aliphatic–C–H), 1726 (C═O),
1
1490 (C═S); H NMR (CD3OD) δ: 6.91 (s, 2H, Ar–H),
4.63 (s, 2H, H‐4), 4.50 (s, 2H, H‐6), 3.09 (t, 2H,
J = 8.9 Hz, NCH2CH2), 2.29 (t, 2H, J = 9.1 Hz,
CH2CH2COOH), 2.26 (s, 3H, Ar–CH3), 2.15 (s, 6H, Ar‐
CH3), 1.66‐1.61 (m, 4H, CH2(CH2)2CH2), 1.45‐1.41
(m, 2H, CH2CH2CH2); 13C NMR (CD3OD + CDCl3) δ:
194.2 (C═S), 176.0 (C═O), 139.4, 139.0, and 135.4
(Ar–C), 129.7 (Ar–CH), 72.7 (C‐4), 57.7 (C‐6), 51.9
(NCH2CH2), 34.9 (CH2COOH), 27.3 (NCH2CH2), 21.0
(NCH2CH2CH2), 17.9 (CH2CH2COOH); MS (FAB) m/z:
366; Anal. calcd for C18H26N2O2S2: C, 59.01; H, 7.10; N,
7.65. Found: C, 59.01; H, 7.08; N, 7.62.
ACKNOWLEDGMENTS
The authors highly acknowledge funding from HEC for
access to spectroscopic instruments and bioassays. The
authors are indebted to International Center for Chemical
and Biological Sciences, HEJ‐RIC, University of Karachi,
Pakistan, for providing access to spectroscopic facilities
and bioassay laboratory for the whole studies.
5 | UREASE INHIBITION
BIOASSAY (IN VITRO)
All urease inhibition assays were performed on 96‐well
microplate format using a 96‐well microplate reader
(Molecular devices, USA). The pure enzyme solution
and urea were incubated at 30°C for 15 minutes. The
buffer used in this assay was 10nM K2HPO4, 1mM LiCl2,
and 1mM EDTA buffer (pH 8.2). For ammonia determi-
nation using the indophenol method the absorbance at
630 nm was measured after every 2 minutes continuously
for 1 hour.[28] Results were compared with thiourea; the
standard inhibitor of urease. IC50 values were calculated
as a mean of three individual readings.
ORCID
Muhammad Ishaq Ali Shah
https://orcid.org/0000-0002-
9627-1627
Rasool Khan https://orcid.org/0000-0002-8556-458X
REFERENCES AND NOTES
[1] J. C. Polacco, M. A. Holland, Int. Rev. Cytol. 1993, 145, 65.

8
SHAH ET AL.
[2] H. L. Mobley, R. P. Hausinger, Microbiol. Rev. 1989, 53, 85.
[18] L. Monzote, A. M. Montalvo, L. Fonseca, R. Perez, M. Suarez,
H. Rodriguez, Arzneimittelforschung 2005, 55, 232.
[3] H. L. Mobley, M. D. Island, R. P. Hausinger, Microbiol. Rev.
1995, 59, 451.
[19] D. Katiyar, V. K. Tiwari, R. P. Tripathi, A. Srivastava, V.
Chaturvedi, R. Srivastava, B. S. Srivastava, Bioorg. Med. Chem.
2003, 11, 4369.
[4] P. A. Karplus, M. A. Pearson, R. P. Hausinger, Acc. Chem. Res.
1997, 30, 330.
[20] A. N. El‐Shorbagi, Eur. J. Med. Chem. 1994, 29, 11.
[5] Z. P. Xiao, T. W. Ma, W. C. Fu, X. C. Peng, A. H. Zhang, H. L.
Zhu, Eur. J. Med. Chem. 2010, 45, 5064.
[21] T. Aboul‐Fadl, A. El‐Shorbagi, Eur. J. Med. Chem. 1996, 31, 165.
[6] G. B. Thalita, S. W. John, O. A. C. Antunes, M. F. M. Estela,
[22] M. I. Abd‐Elrahman, M. O. Ahmed, S. M. Ahmed, T. Aboul‐Fadl,
A. El‐Shorbagi, Biophys. Chem. 2002, 97, 113.
Lett. Drug. Des. Dis. 2009, 6, 186.
[7] M. Houimel, J. P. Mach, I. Corthesy‐Theulaz, B. Corthesy, I.
Fisch, Eur. J. Biochem. 1999, 262, 774.
[23] A. N. El‐Shorbagi, Arch Pharm (Weinheim) 2000, 333, 281.
[24] M. Kot, W. Zaborska, J. Enzyme Inhib. Med. Chem. 2006, 21, 537.
[8] T. C. Kuhler, J. Fryklund, N. A. Bergman, J. Weilitz, A. Lee, H.
[25] M. Arfan, M. N. Tahir, M. I. A. Shah, M. S. Iqbal, Acta
Crystallogr. Sect. E. 2009, 65, o468.
Larsson, J. Med. Chem. 1995, 38, 4906.
[9] N. E. Dixon, T. C. Gazzola, R. L. Blakeley, B. Zermer, J. Am.
Chem. Soc. 1975, 97, 4131.
[26] R. Pérez, O. Reyes, M. Suarez, H. E. Garay, L. J. Cruz, H.
́
Rodrıguez, M. A. D. Molero‐Vilchez, C. Ochoa, Tetrahedron
[10] K. Kobashi, S. Takebe, N. Terashima, J. Hase, J. Biochem. 1975,
77, 837.
Lett. 2000, 41, 613.
[27] M. Arfan, M. N. Tahir, M. I. A. Shah, R. Khan, M. S. Iqbal, Acta
Crystallogr. Sect. E. 2009, 65, o902.
[11] A. Rauf, F. Ahmed, A. Qureshi, A. Khan, M. Qadir, M.
Choudhary, Z. Chohan, M. Youssoufid, T. B. Haddad, J. Chin.
Chem. Soc. 2011, 58, 528.
[28] M. W. Weatherburn, Anal. Chem. 1967, 39, 971.
[29] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat,
H. Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res.
2000, 28(1), 235.
[12] A. M. Qureshi, S. Mumtaz, A. Rauf, M. Ashraf, R. Nasar, Z. H.
Chohan, J. Enzyme Inhib. Med. Chem. 2015, 30, 119.
[13] S. Saraç, M. Ertan, A. Balkan, N. Yulug, Arch. Pharm. 1991,
324, 449.
[30] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew,
D. S. Goodsell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785.
[14] M. A. Hussein, A. N. El‐Shorbagi, A. R. Khallil, Arch. Pharm.
2001, 334, 305.
[15] S. A. A. El Bialy, A. M. Abdelal, A.‐N. El‐Shorbagi, S. M. M.
Kheira, Arch. Pharm. 2005, 338, 38.
How to cite this article: Shah MIA, Khan R,
Arfan M, Wadood A, Ghufran M. Synthesis, in vitro
urease inhibitory activity and molecular docking of
3,5‐disubstituted thiadiazine‐2‐thiones. J
Heterocyclic Chem. 2019;1–8. https://doi.org/
10.1002/jhet.3705
[16] M. Schorr, W. Dürckheimer, P. Klatt, G. Lämmler, G.
Nesemann, E. Schrinner, Arzmeim‐Forsch/Drug Res 1969, 19,
1807.
[17] J. Coro, R. Perez, H. Rodriguez, M. Suarez, C. Vega, M. Rolon,
D. Montero, J. J. Nogal, A. Gomez‐Barrio, Bioorg. Med. Chem.
2005, 13, 3413.