Synthesis, in vitro and in silico studies of novel potent urease inhibitors: N-[4-({5-[(3-Un/substituted-anilino-3-oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides

Article abstract of DOI:10.1016/j.bmc.2018.06.005

The present article describes the synthesis, in vitro urease inhibition and in silico molecular docking studies of a novel series of bi-heterocyclic bi-amides. The synthesis of title compounds was initiated by benzoylation, with benzoyl chloride (1), of the key starter ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (2) in weak basic aqueous medium followed by hydrazide formation, 4, and cyclization with CS₂ to reach the parent bi-heterocyclic nucleophile, N-{4-[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-1,3-thiazol-2-yl}benzamide (5). Various electrophiles, 8a–l, were synthesized by a two-step process and these were finally coupled with 5 to yield the targeted bi-heterocyclic bi-amide molecules, 9a–l. The structures of the newly synthesized products were corroborated by IR, ¹H NMR, ¹³C NMR, EI-MS and elemental analysis. The in vitro screening of these molecules against urease explored that most of the compounds exhibit potent inhibitory potential against this enzyme. The compound 9j, with IC₅₀ value of 2.58 ± 0.02 μM, exhibited most promising inhibitory activity among the series, relative to standard thiourea having IC₅₀ value of 21.11 ± 0.12 μM. In silico studies fully augmented the experimental enzyme inhibition results. Chemo-informatics analysis showed that synthesized compounds (9a–l) mostly obeyed the Lipinski's rule. Molecular docking study suggested that ligand 9j exhibited good binding energy value (?7.10 kcal/mol) and binds within the active region of target protein. So, on the basis of present investigation, it was inferred that 9j may serve as a novel scaffold for designing more potent urease inhibitors.

Full text of DOI:10.1016/j.bmc.2018.06.005

Accepted Manuscript
Synthesis, in vitro and in silico studies of novel potent urease inhibitors: N-[4-
({5-[(3-Un/substituted-anilino-3-oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-
yl}methyl)-1,3-thiazol-2-yl]benzamides
Muhammad Athar Abbasi, Mubashir Hassan, Aziz-ur-Rehman, Sabahat Zahra
Siddiqui, Hussain Raza, Syed Adnan Ali Shah, Sung-Yum Seo
PII:
S0968-0896(18)31004-6
https://doi.org/10.1016/j.bmc.2018.06.005
BMC 14397
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 May 2018
3 June 2018
6 June 2018
Please cite this article as: Abbasi, M.A., Hassan, M., Aziz-ur-Rehman, Zahra Siddiqui, S., Raza, H., Shah, S.A.A.,
Seo, S-Y., Synthesis, in vitro and in silico studies of novel potent urease inhibitors: N-[4-({5-[(3-Un/substituted-
anilino-3-oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides,
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.06.005
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Synthesis, in vitro and in silico studies of novel potent urease inhibitors:
N-[4-({5-[(3-Un/substituted-anilino-3-oxopropyl)sulfanyl]-1,3,4-
oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides
Muhammad Athar Abbasia,b,*, Mubashir Hassana, Aziz-ur-Rehmanb, Sabahat Zahra Siddiquib,
Hussain Razaa, Syed Adnan Ali Shahc,d and Sung-Yum Seoa,*
a
College of Natural Science, Department of Biological Sciences, Kongju National University,
Gongju, 32588, South Korea
b Department of Chemistry, Government College University, Lahore-54000, Pakistan
d
c Faculty of Pharmacy & Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns),
Level 9, FF3, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia
a
*Corresponding Authors: Prof. Dr. Sung-Yum Seo, E-mail: dnalove@kongju.ac.kr Tel:
b
(+82)-416-8508503 and Dr. Muhammad Athar Abbasi, E-mail: abbasi@gcu.edu.pk Tel:
(+92)-42-111000010 Ext. 266.
1

ABSTRACT
The present article describes the synthesis, in vitro urease inhibition and in silico molecular
docking studies of a novel series of bi-heterocyclic bi-amides. The synthesis of title compounds
was initiated by benzoylation, with benzoyl chloride (1), of the key starter ethyl 2-(2-amino-1,3-
thiazol-4-yl)acetate (2) in weak basic aqueous medium followed by hydrazide formation, 4, and
cyclization with CS2 to reach the parent bi-heterocyclic nucleophile, N-{4-[(5-sulfanyl-1,3,4-
oxadiazol-2-yl)methyl]-1,3-thiazol-2-yl}benzamide (5). Various electrophiles, 8a-l, were
synthesized by a two-step process and these were finally coupled with 5 to yield the targeted bi-
heterocyclic bi-amide molecules, 9a-l. The structures of the newly synthesized products were
1
13
corroborated by IR, H-NMR, C-NMR, EI-MS and elemental analysis. The in vitro screening
of these molecules against urease explored that most of the compounds exhibit potent inhibitory
potential against this enzyme. The compound 9j, with IC50 value of 2.58 ± 0.02 µM, exhibited
most promising inhibitory activity among the series, relative to standard thiourea having IC50
value of 21.11 ± 0.12 µM. In silico studies fully augmented the experimental enzyme inhibition
results. Chemo-informatics analysis showed that synthesized compounds (9a-l) mostly obeyed
the Lipinski's rule. Molecular docking study suggested that ligand 9j exhibited good binding
energy value (-7.10 kcal/mol)) and binds within the active region of target protein. So, on the
basis of present investigation, it was inferred that 9j may serve as a novel scaffold for designing
more potent urease inhibitors.
Keywords: Bi-heterocycles, Thiazole, Oxadiazol, Bi-amides, Urease, cytotoxicity. Molecular
docking
2

1. Introduction
Thiazole is an important scaffold in heterocyclic chemistry and 1,3-thiazole ring is present
in many pharmacological active substances. Compounds containing the thiazole ring have been
reported as being histamine H3 antagonists, with herbicidal, anti-inflammatory, anti-tumoral and
selective cardio-depressant activities.1 Antimicrobial activities of some substituted thiazoles are
well established because it possess (S-C=N) toxophoric unit. Thiazoles have enhanced lipid
solubility with hydrophilicity. Also, thiazoles are easily metabolized by routine biochemical
reactions and are non-carcinogenic in nature.2 Aminothiazoles and related heterocycles represent
a novel class of potent and selective antitumor agents which exhibit nanomolar inhibitory
activity against a range of human breast, leukemia, lung, colon, CNS, melanoma, ovarian, renal
and prostate cell lines in vitro.3,4 Aminothiazoles, especially 2-aminothiazole derivatives have
been reported as antiviral, antimicrobial, anti-inflammatory and anti-tubercular agents.5,6 2-
aminothiazole analogues have also been reported as potential neuroprotective agents for the
treatment of neurological diseases and modulators of transcriptional repression for treatment of
Huntington’s disease.7 1,3,4-Oxadiazole is an important heterocycle and its different derivatives
possess an extensive spectrum of pharmacological activities such as antiviral, antibacterial,
antitumor, antituberculosis, analgesic, anti-inflammatory, anticonvulsant, anti-HIV, and anti-
Alzheimer activities.8-16
Urease (urea amidohydrolase, EC: 3.5.1.5) occurs throughout the animal and plant
kingdom, many microorganisms use this reaction to provide a source of nitrogen for growth, and
the enzyme plays an important role in plant nitrogen metabolism during the germination
process.17,18 The presence of urease activity in soils is exploited in the widespread agricultural
practice of urea-based fertilizer application for enhancing crop yields. Unfortunately, excessive
levels of soil urease can degrade fertilizer urea too rapidly and result in phytopathic effects and
loss of volatilized ammonia.19 Of medical and veterinary interest, urease is a virulence factor in
certain human and animal pathogens; it participates in the development of kidney stones,
pyelonephritis, peptic ulcers, and other disease states.20 The obvious remedy for treating bacterial
infection with antimicrobials, however, has often proven futile,21 and only a few combination
regiments has reached clinical practice. Recently, some 3-arylpropionylhydroxamic acid
derivatives22, reductive derivatives of flavonoids23, and bisindolylmethane thiosemicarbazides24
were reported as potential urease inhibitors, but still the need for alternative or novel treatment is
3

evident. The discovery of potent and safe urease inhibitors has been a very important area of
pharmaceutical research due to involvement of ureases in different pathological conditions.
One of the key objectives of organic and medicinal chemists is to design and synthesize
the molecules having potent therapeutic values. The rapid development of resistance to existing
drugs generates a serious challenge to the scientific community. Consequently, there is a vital
need for the development of new drugs having potent activity. It has been revealed that minor
modification in the structure of such heterocycles can lead to quantitative as well as qualitative
changes in the biological activity.25 It prompted us to synthesize some novel bi-heterocyclic bi-
amides and explore their therapeutic potentials against the target enzyme supported by molecular
docking.
2. Results and discussion
2.1. Chemistry
The planned bi-heterocyclic bi-amides were synthesized through a multi-step protocol
depicted in Scheme-1 and the varying groups are listed in Table-1. The first step in the synthesis
involved benzoylation of ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (2) with benzoyl chloride (1)
in aqueous weak basic medium to give ethyl 2-[2-(benzoylamino)-1,3-thiazol-4-yl]acetate (3).
This ester was subjected to nucleophilic substitution with hydrazine hydrate in methanol to
acquire the corresponding hydrazide, N-[4-(2-hydrazino-2-oxoethyl)-1,3-thiazol-2-yl]benzamide
(4). This substitution reaction was completed by refluxing for two hours. The hydrazide, 4, was
made to react carbon disulfide in the presence of KOH, an activator for cyclization, to yield a
thiol containing nucleophile, N-{4-[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-1,3-thiazol-2-
yl}benzamide (5). Various electrophiles, were synthesized by reacting un/substituted anilines
(6a-l) with a connector 3-bromopropanoyl chloride (7) in a weak basic aqueous medium to give
a series of electrophiles, 3-bromo-N-(un/substituted-phenyl)propanamides (8a-l). The final step
in synthesis involved coupling of 5 with newly synthesized electrophiles, 8a-l, in DMF using
LiH as a base and an activator to obtain the targeted bi-heterocyclic bi-amides, N-[4-({5-[(3-
un/substituted-anilino-3-oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-
yl]benzamides (9a-l). The molecular structures of these derivatives were well established by IR,
EI-MS, 1H-NMR and 13C-NMR spectral data along with elemental analysis.
4

O
CH3
C
O
O
C
O
C
N
O
CH3
C
O
N
N
Cl
+
I
3
S
H
H2N
1
2
S
II
O
NH
SH
O
C
C
O
C
NH2
N
N
N
O
N
III
N
H
N
H
S
S
4
5
H
N
R1
R1
O
C
Br
NH2
C
O
IV
V
+
Br
Cl
7
8a-l
6a-l
R2
R2
O
1"
2"
H
R1
5"
3"'
S
N
1"'
6'
O
N
2"'
3""
C
3' 4'
3" 4"
1""
N
C
7
N
2'
5'
1'
O
9a-l
N
H
5
1
S
R2
5""
N-[4-({5-[(3-Un/substituted-anilino-3-oxopropyl)sulfanyl]-
3
1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides
Scheme-1:
oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides. Reagents
Conditions: (I) Aq. 2% Na2CO3 soln./stirring and heating for hrs. (II)
Outline
for
the
synthesis
of
N-[4-({5-[(3-un/substituted-anilino-3-
&
5
MeOH/N2H4·H2O/refluxing for 2 hrs. (III) EtOH/CS2/KOH/refluxing for 10 hrs. (IV) Aq. 10%
Na2CO3 soln./pH 9-10/vigorous manual shaking and stirring at RT for 2-3 hrs. (V)
DMF/LiH/stirring for 3-5 hrs.
Table-1. Different groups (-R1 & -R2) in scheme-1.
Compd.
6a, 8a, 9a
6b, 8b, 9b
6c, 8c, 9c
6d, 8d, 9d
6e, 8e, 9e
6f, 8f, 9f
-R1
-R2
Compd.
6g, 8g, 9g
6h, 8h, 9h
6i, 8i, 9i
-R1
2-CH3
-R2
6-CH3
4-CH3
5-CH3
-H
-H
-H
2-CH3
3-CH3
-H
-H
3-CH3
-H
3-CH3
4-CH3
2-CH2-CH3
4-CH2-CH3
4-O-CH2-CH3
6j, 8j, 9j
6k, 8k, 9k
6l, 8l, 9l
2-CH3 3-CH3
2-CH3 5-CH3
-H
-H
5

The structural characterization of one of lead compounds is discussed hereby in detail for
the expediency of the readers. The compound 9k was acquired as light brown solid in 81% yield
having melting point 179-180 °C. Its molecular formula, C24H23N5O3S2, was recognized by CHN
analysis data and molecular mass was justified by the molecular ion peak in its EI-MS at m/z
493. The mass fragmentation pattern also supported to erect the said molecular formula.
Moreover, counting the number of protons in its 1H-NMR spectrum and the carbon resonances in
its 13C-NMR spectrum, also supplemented the assignment of molecular formula. The vibrational
studies of the molecules were examined using FT-IR spectroscopy to affirm various
functionalities. The prominent absorption bands in IR spectrum appeared at 3340 (N-H
stretching), 3060 (C-H of aromatic ring), 2920 (-CH2- stretching), 1665 (C=O stretching), 1653
(C=N stretching) and 1575 (C=C stretching of aromatic ring). The most downfield signals in its
1H-NMR spectrum δ 12.67 (br.s, 1H, -NH-CO-7), and 9.94 (s, 1H, -NH-CO-3''') were assignable
to two amidic hetero-atom protons (Figure-1a). A benzoyl group in the molecule was obvious by
its discrete signals at δ 8.08 (dd, J = 1.2, 8.4 Hz, 2H, H-2 & H-6), 7.62 (br.t, J = 7.4 Hz, 1H, H-
4), and 7.52 (br.t, J = 7.5 Hz, 2H, H-3 & H-5) (Fig. 1b). The 4-ethylphenyl moiety was identified
by a typical A2B2 spin system in the aromatic region, represented by two ortho-coupled doublets
at δ 7.47 (br.d, J = 8.4 Hz, 2H, H-2'''' & H-6''''), and 7.12 (br.d, J = 8.4 Hz, 2H, H-3'''' & H-5''''),
along with peculiar signals of an ethyl group in aliphatic region at δ 2.54 (q, 2H, 4''''-CH2-CH3),
and 1.14 (t, 3H, 4''''-CH2-CH3) (Fig. 1b and Fig. 1c). A singlet at δ 7.18 (s, 1H, H-5') was rational
for a 1,3-thiazol-2-yl moiety while a methylene connecting the two heterocycles (2-amino-1,3-
thiazol-4-yl and 1,3,4-oxadiazol-2-yl) resonated at δ 4.33 (br.s, 2H, CH2-6'). The remaining two
triplets appearing at δ 3.46 (br.t, 2H, J = 6.7 Hz, CH2-1'''), and 2.86 (br.t, 2H, J = 6.7 Hz, CH2-
2''') were an attribute of S-substituted propanamoyl unit in the molecule.
6

Figure 1a. 1H-NMR spectrum of 9k.
Figure 1b. Expanded aromatic region of 1H-NMR spectrum of 9k.
7

Figure 1c. Expanded aliphatic region of 1H-NMR spectrum of 9k.
The 13C-NMR spectrum (Fig. 2) thoroughly corroborated the carbon skeleton of the
molecule. 5-Sulfanyl-1,3,4-oxadiazol-2yl heterocyclic core was identified through two
quaternary signals at δ 165.04 (C-2''), and 158.78 (C-5''). The 1,3-thiazol-2-yl-4-(substituted-
methyl) moiety was rational by two quaternary carbon resonances at δ 163.65 (C-2'), and 143.45
(C-4'), a methine signal at δ 111.10 (C-5'), while the methylene group connecting to two
heterocyles appeared at δ 35.58 (C-6'). The benzoyl group was clearly indicated by its peculiar
signals at δ 165.48 (C-7), 132.56 (C-4), 131.84 (C-1), 128.51 (C-2 & C-6), and 128.09 (C-3 & C-
5). The 4-ethylphenyl moiety was characterized by the signals for a 1,4-di-substituted phenyl
ring at δ 138.60 (C-1''''), 136.56 (C-4''''), 127.85 (C-3'''' & C-5''''), and 119.15 (C-2'''' & C-6'''')
while an ethyl group at 4-position was emblematic by the signals of δ 27.53 (4''''-CH2-CH3), and
15.63 (4''''-CH2-CH3). The S-substituted propanamide unit in the molecule was symbolic by three
carbon resonances 168.38 (C-3'''), 27.77 (C-2'''), and 27.41 (C-1'''). The mass fragmentation
pattern of this compound was also fully justifying the deduced units and the data of prominent
peaks is given in the experimental section. Thus, on the basis of collective spectral evidences, the
8

structure of 9k was confirmed and it was named N-{4-[(5-{[3-(4-ethylanilino)-3-
oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-thiazol-2-yl}benzamide. A similar pattern
was adopted for the structural characterization of other derivatives in the synthesized series.
Figure 2. 13C-NMR spectrum of 9k.
2.2. Urease inhibition and structure-activity relationship
The synthesized bi-heterocyclic bi-amides were evaluated for their urease inhibitory
potential. These derivatives displayed varying degree of inhibition in the range of 2.58 ± 0.02 to
52.12 ± 0.15 µM when compared with the standard inhibitor thiourea having value IC50 of 21.11
± 0.12 µM (Table 2). Some of the derivatives, for example, 9j, 9g and 9a, showed outstanding
urease inhibitory potential with IC50 values of 2.58 ± 0.02, 2.79 ± 0.04, and 2.95 ± 0.17 µM
respectively, which is many folds better than the standard thiourea.
Although the observed activity is the resultant of a whole molecule, but a limited
structure-activity relationship (SAR) was rationalized by analyzing the effect of different groups
9

(-R1 & -R2) attached to phenyl ring (aryl part) on the inhibitory potential. Figure 3 displayed the
general structural features of the synthetic compounds.
Oxadiazole
Part
Propanamide Part
Thiazole
Part
Aryl Part
R1
Benzamide Part
O
H
N
S
O
N
C
O
N
C
N
N
S
H
R2
Figure 5: General structural features of compounds 9a-l.
Among the three methylated regio-isomers, 9b, 9c and 9d, the molecule 9b bearing a 2-
methyl group exhibited better inhibitory potential (IC50 = 7.47 ± 0.29 µM), as compared to 9c
(IC50 = 12.23 ± 0.09 µM) in which methyl group was present at 3-position. A further decreasing
trend was observed in 9d (IC50 = 29.18 ± 0.45 µM), whereby the methyl group was attached to 4-
position of phenyl ring. It means that more efficient interactions are made with enzyme, when
methyl group is present at ortho position instead of meta and para position (Figure 6).
CH3
O
O
H
H
CH3
S
N
S
N
O
C
O
C
N
N
C
O
C
O
N
N
N
N
N
N
9b
S
9c
IC50 = 12.23 ± 0.09 µM
S
H
H
IC50 = 7.47 ± 0.29 µM
O
H
S
N
O
N
C
O
N
C
N
N
9d
S
CH3
H
IC50 = 29.18 ± 0.45 µM
Figure 6: Structure-activity relationship of compounds 9b, 9c, and 9d.
A similar trend in inhibitory activity was observed in 9j and 9k, but in an intensified
manner, when a relatively bulky ethyl group was present on phenyl ring. Compound 9j with 2-
ethyl group exhibited much better potential (IC50 = 2.58 ± 0.02 µM), when compared with 9k
10

(IC50 = 43.18 ± 0.19 µM) in which an ethyl group was inherited at 4-position. Indeed, 9j was
identified as most active compound among all synthetic analogues. This may be due to an
appropriate interaction of 2-ethylphenyl ring with the active site of enzyme (Figure 7).
CH3
CH3
O
O
H
H
S
N
S
N
O
C
O
C
N
N
C
O
C
O
N
N
N
N
N
N
9j
S
9k
S
H
H
IC50 = 2.58 ± 0.02 µM
IC50 = 43.18 ± 0.19 µM
Figure 7: Structure-activity relationship of compounds 9j and 9k.
On comparison of 9b and 9j it was rational that although both compounds bear an ortho-
substituent, but the presence of a relatively bulky 2-ethyl group in 9j made it a more suitable
inhibitor of the urease enzyme. Interestingly, the activity of this compound (IC50 = 2.58 ± 0.02
µM) was increased approximately up to three folds when methyl group in former (IC50 = 7.47 ±
0.29 µM) was replaced with an ethyl group in latter (Figure 8). It means that an increased steric
factor in this case was imparting better activity to the molecule.
CH3
CH3
O
O
H
H
S
N
S
N
O
C
O
C
N
N
C
O
C
O
N
N
N
N
N
N
9j
9b
S
S
H
H
IC50 = 2.58 ± 0.02 µM
IC50 = 7.47 ± 0.29 µM
Figure 8: Structure-activity relationship of compounds 9b and 9j.
However, when the inhibitory potential of para-substituted molecules, 9d (IC50 = 29.18 ±
0.45 µM) and 9k (IC50 = 43.18 ± 0.19 µM) was compared, an opposite trend was observed
regarding the bulkiness of groups. In this case, the former molecule with smaller methyl group
behaved as better inhibitor relative to latter molecule in which bulky ethyl group was present at
same position. Amazingly, presence of a relatively more polar 4-ethoxy group in 9l (IC50 = 23.24
± 0.41µM) resulted in an enhancement in the inhibitory potential of this molecule as compared to
11

9k, which can be attributed to the appropriate interactions of 4-ethoxyphenyl ring (Figure 9) with
active site of the enzyme.
O
O
H
H
S
N
S
N
O
C
O
C
N
N
C
O
C
O
N
N
N
N
N
N
9d
9k
S
S
CH3
O
H
H
IC50 = 29.18 ± 0.45 µM
IC50 = 43.18 ± 0.19 µM
CH3
H
S
N
O
C
N
CH3
C
O
N
N
N
9l
S
O
H
IC50 = 23.24 ± 0.41 µM
Figure 9: Structure-activity relationship of compounds 9d, 9k and 9l.
Among the di-methylated molecules (Figure 10), the compound 9g (IC50 = 2.79 ± 0.04
µM) having symmetrical di-ortho substituents was again found to be a suitable inhibitor than
other analogues, 9e (IC50 = 48.16 ± 0.19 µM) and 9f (IC50 = 48.16 ± 0.19 µM). The compound
9g was identified as second most active compound of the series.
CH3
CH3
O
O
H
H
S
N
CH3
S
N
O
C
O
C
N
N
C
O
C
O
N
N
N
N
N
N
9e
S
9f
IC50 = 15.25 ± 0.55 µM
S
H
H
IC50 = 48.16 ± 0.19 µM
CH3
CH3
O
H
S
N
O
N
C
O
N
C
N
N
H
9g
H3C
S
IC50 = 2.79 ± 0.04 µM
Figure 10: Structure-activity relationship of compounds 9e, 9f and 9g.
The comparison of activity of two other di-methylated regio-isomers (Figure 11) revealed
that shifting of methyl groups to 3 and 4-position in 9h (IC50 = 34.19 ± 0.21 µM) and to 3 and 5-
position in 9i (IC50 = 52.12 ± 0.15 µM) was going to decrease their inhibitory potential relative
to afore-discussed di-ortho substituted analogue 9g.
12

O
O
H
H
CH3
CH3
CH3
S
N
S
N
O
C
O
C
N
N
C
O
C
O
N
N
N
N
N
N
9h
S
9i
IC50 = 52.12 ± 0.15 µM
S
H
H
IC50 = 34.19 ± 0.21 µM
CH3
Figure 11: Structure-activity relationship of compounds 9h and 9i.
So, it was rational from the structure-activity relationship that when small or medium
sized group or groups are present at ortho position of phenyl ring (aryl part) in such compounds,
overall a better inhibitory potential is imparted to these molecules. Synthesized compounds can
be arranged in the following row according to their inhibitory activity: 9j > 9g > 9a > 9b > 9c >
9f > 9l > 9d > 9h > 9k > 9e > 9i (see Table 2 for IC50 values).
Table 2. Percent inhibition of synthesized compounds at 0.5 mM and IC50 values for urease.
UreaseInhibition
Compound
Aryl Part
Inhibition (%) IC50 (µM)
98.47 ± 0.12 2.95 ± 0.17
9a
H3C
98.22 ± 0.06 7.47 ± 0.29
9b
CH3
84.82 ± 0.13 12.23 ± 0.09
76.41 ± 0.12 29.18 ± 0.45
69.85 ± 0.14 48.16 ± 0.19
9c
9d
9e
CH3
H3C
CH3
H3C
77.93 ± 0.13 15.25 ± 0.55
9f
CH3
13

H3C
98.47 ± 0.07 2.79 ± 0.04
76.52 ± 0.13 34.19 ± 0.21
64.83 ± 0.11 52.12 ± 0.15
9g
9h
9i
H3C
CH3
CH3
CH3
CH3
H3C
CH2
98.69 ± 0.12 2.58 ± 0.02
74.24 ± 0.06 43.18 ± 0.19
9j
CH2 CH3
9k
O
CH2 CH3
88.82 ± 0.43 23.24 ± 0.41
9l
Thiourea
-
98.12 ± 0.18 21.11 ± 0.12
Note: IC50 values (concentration at which there is 50% enzyme inhibition) of compounds were
calculated from the inhibition data obtained after doing assays at high dilutions of the
compounds as given in assay method and data was computed using EZ–Fit Enzyme kinetics
software (Perrella Scientific Inc. Amherst, USA). Data is mean of three values (Mean ± S.E.M.,
n = 3).
2.3. In silico analysis
2.3.1. Chemo-informatics properties and Lipinski’s rule (RO5) of synthesized compounds
The predicted chemo-informatic properties were evaluated by computational tools.
Results exposed that compounds 9a-l have better predicted value of molecular weight (g/mol),
hydrogen bond acceptor and donor, logP, polar surface area (A2) and molar volume (A3). The
generated values of ligands, 9a-l, show good comparable behavior with standard values of all
selected properties (Table 3). Moreover, the Lipinski’s rule of five (RO5) results showed that
compounds, 9a-l, possess good molecular weight (g/mol), HBA and HBD values which are
significantly justified their drug likeness behavior.26 Results showed that molecular weights
14

(g/mol) of all compounds were within the range value (< 500 g/mol) except 9l which was a
higher molecular weight compound than the borderline value. The HBA, HBD and logP values
of synthesized compounds, 9a-l, were justifiable with the standard values. The RO5 deviation
(Mol. Wt = > 500 g/mol; HBD = > 5; HBA > 10; and logP = > 5) results in poor absorption of
compounds. Furthermore, the polar surface area (PSA) of a molecule is defined as the surface
sum over all polar atoms, primarily oxygen and nitrogen, also including their attached hydrogen
atoms. The PSA parameter is commonly used for drug's optimization ability to permeate cells.
Prior research data showed the standard value of PSA (< 89 A2).27 Our predicted results showed
that compounds, 9a-k, showed less than standard values PSA results except 9l which is 93 (A2).
The drug-likeness or drug score is amalgam of complex balance of various molecular
properties and structure features which determine the particular behavior of molecule as drug.
The basic parameters are hydrophobicity, electronic distribution, hydrogen bonding
characteristics, molecule size and flexibility and of course presence of various pharmacophoric
features influence the behavior of molecule in a living organism, including bioavailability,
transport properties, affinity to proteins, reactivity, toxicity, metabolic stability. Our results
showed that all the compounds, 9a-l, showed good drug score values, which depicted their good
drug like behavior and these molecules might be considered as suitable drug candidates against
urease. The overall predicted result values of all compounds are mentioned in Table 3.
Table 3. Chemo-informatic properties of ligands.
Properties
Mol.Wt
HBA
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
465 479 479 479 493 493 493 493 493 493 493 509
8
2
8
2
8
2
8
2
8
2
8
2
8
2
8
2
8
2
8
2
8
2
9
2
HBD
LogP
3.55 3.83 3.95 3.95 4.25 4.24 3.55 4.24 4.36 4.33 4.45 4.12
PSA (A2)
86 85 86 86 85 85 86 86 86 85 86 93
Mol Vol.(A3) 424 445 445 445 466 466 424 464 466 464 463 474
Drug Score 0.29 0.47 0.11 0.35 0.01 0.15 0.29 0.17 0.31 0.52 0.69 0.56
*Mol. Wt= Molecular Weight (g/mol), HBA= Hydrogen Bond Acceptor, HBD= Hydrogen Bond
Donor, PSA= Polar Surface Area.
15

2.3.2. Molecular docking analysis
2.3.2.1. Docking energy evaluation of synthesized compounds
To predict the best conformational position within the active region of urease, all the
synthesized ligands, 9a-l, were docked against receptor molecule. The generated docked
complexes were examined on the basis of minimum energy values (kcal/mol) and bonding
interaction pattern such as hydrogen and hydrophobic bonds, respectively. Docking results
justified that compounds 9d and 9e both exhibited same highest energy value -8.60 (kcal/mol),
compared to other analogues. However, all these compounds have no big docking energy value
difference more than standard error value. The standard error for Autodock is reported as 2.5
kcal/mol (http://autodock.scripps.edu/).28 Although, the basic nucleus of all the synthesized
compounds were similar, therefore most of ligands possess good efficient energy values with no
big energy value fluctuations (Table 4).
The docking energy calculation is done by equation 1.
∆Gbinding = ∆Ggauss + ∆Grepulsion + ∆Ghbond + ∆Ghydrophobic + ∆Gtors (1)
∆G gauss Attractive term for dispersion, two gaussian functions, ∆Grepulsion Square of the
distance if closer than a threshold value, ∆G hbond Ramp function - also used for interactions
with metal ions, ∆G hydrophobic Ramp function, ∆G tors Proportional to the number of rotatable
bonds
16

Table 4. The docking energy values.
Docking complexes Energy values (kcal/mol)
-8.30
-6.30
-6.80
-8.60
-8.60
-7.20
-8.10
-7.80
-7.10
-7.10
-7.60
-6.60
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
2.3.2.2. Binding pocket and structure activity relationship (SAR) analyses
The binding pocket is present in domain 4 where the nickel metal is present29. All the
docked structures were superimposed to check the binding configuration of all ligands in the
active region of target protein against urease. All the synthesized ligands were bound at same
position residues with little fluctuations and different conformational poses. The binding of all
ligands at same position also justified our docking reliability and predicted results (Fig. 12).
17

Figure 12. Docking structures of synthesized ligands
Based on in vitro and in silico results 9j was selected to understand the binding behavior
of ligand against target protein. However, the best conformational pose docked complex of 9j is
mentioned in Fig. 13. The docking result showed that two hydrogen bonds and two hydrophobic
interactions were observed in 9j-docked complex. Both oxygen molecules of amide groups in 9j
formed strong hydrogen bonds with Gln635 and Ala636 with bond lengths of 2.33 and 2.97 Å,
respectively. Similarly, a hydrophobic interaction was observed between 2-ethylphenyl group of
9j and Arg639 having bond distance of 4.05 Å. Another π- π interaction was observed between
oxadiazole ring and Ala440 having bond length of 4.61 Å. Hydrogen bonds play an important
role in molecular docking because they help to stabilize and strengthen the docked complexes.
The shorter distances (< 3.0-3.5 Å) give more stability to ligand-protein docking complexes
compared to larger ones having distance > 4.0 Å. However, for hydrophobic interactions,
distances vary up to 5 Å. Literature data also ensured the importance of these binding-pocket
residues with other urease inhibitors which strengthen our docking results.30-33 The comparative
18

binding energy and binding interaction analysis showed the significance of 9j which might be
considered as potent inhibitor by targeting jack bean urease. The docking complexes of all other
compounds are mentioned in supplementary data (Figs S2-S12).
Figure 13. Docking complex of 9j A) A general overview of docking depiction. The protein
structure is represented in dark blue color in surface format while ligand is highlighted in green
color. B) The closer view of binding-pocket interaction with best conformation position of ligand
against target protein. The ligand molecule is depicted in green color while their functional
groups, such as oxygen, amino and sulfur are showed in red, blue and yellow colors,
respectively. C) The docking complex is represented with ligand conformation. Amino acids are
highlighted in light orange color D) The closer view of docking complex. The residues involved
in hydrogen bonds are justified in red while hydrophobic interacted residues are labeled in purple
19

colors, respectively. Red and purple dotted lines, with distances mentioned in angstrom (Å), are
justified for hydrogen and hydrophobic bond distances. Two nickel atoms are represented in grey
circle.
3. Conclusion
We have described the successful synthesis of bi-heterocyclic bi-amide molecules through
a multi-step protocol under facile conditions and these compounds were obtained in good yields.
Our present study proved fruitful as we succeeded in finding some potent inhibitors against
urease enzyme. It can be inferred from the results of bioactivity and computational studies that
these newly synthesized chemical ligands can serve as structural templates in designing of novel
drugs against jack bean urease enzyme.
4. Experimental
All the chemicals, along with analytical grade solvents, were purchased from Sigma
Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates
were used for TLC with ethyl acetate and n-hexane as solvent system (30:70). Spots were
detected by UV254. Gallonkamp apparatus was used to detect melting points in capillary tubes.
IR spectra (ν, cm–1) were recorded by KBr pellet method in the Jasco-320-A spectrophotometer.
1H-NMR spectra (δ, ppm) were recorded at 600 MHz (13C-NMR spectra, at 150 MHz) in
DMSO-d6 using the Bruker Advance III 600 As- cend spectrometer using BBO probe. EI-MS
spectra were measured on a JEOL JMS-600H instrument with data processing system. The
coupling constant (J) is given in Hz and chemical shift ( ) in ppm. The abbreviations used in
interpretation of 1H NMR spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t,
triplet; br.t, broad triplet; q, quartet; quint, quintet; sex, sextet; sep, septet; m, multiplet; dist.,
distorted.
4.1. Procedure for the synthesis of ethyl 2-[2-(benzoylamino)-1,3-thiazol-4-yl]acetate (3)
Ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (2; 0.05 mol) was taken in a 2% aqueous
Na2CO3 solution (200 mL) in a 500 mL RB flask and benzoyl chloride (1; 0.05 mol) was added
drop wise and mixture was stirred for 30 minutes. The reaction mixture became a solid adhering
20

paste. Crushed ice (ca. 10 g) is then added to the contents of the flask and stirred well with a
glass rod. The thick reaction mixture gradually became soft by the gradual dispersion of traces of
benzoyl chloride in the aqueous phase during stirring and finally the supernatant aqueous layer
becomes clear. The reaction progress was observed by TLC using n-hexane and ethyl acetate
solvent system (6:4). After 5 hours stirring and gradual heating, the product was precipitated,
filtered and washed with water to get it freed from any adhering unreacted material. After drying,
ethyl 2-[2-(benzoylamino)-1,3-thiazol-4-yl]acetate (3) was obtained as a crystalline solid.
4.2. Synthesis of N-[4-(2-hydrazino-2-oxoethyl)-1,3-thiazol-2-yl]benzamide (4)
Ethyl 2-[2-(benzoylamino)-1,3-thiazol-4-yl]acetate (3; 0.05 mol) was taken in methanol
(200 mL) in a 500 mL RB flask, hydrazine hydrate (0.05 mol) was added drop wise and mixture
was refluxed for 02 hours. The reaction progress was observed by TLC using n-hexane and ethyl
acetate solvent system (4:6). After completion of reaction, the mixture was allowed to cool at
room temperature to get white colored precipitates of the product, N-[4-(2-hydrazino-2-
oxoethyl)-1,3-thiazol-2-yl]benzamide (4). It was filtered and washed with methanol.
4.3. Synthesis of N-{4-[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-1,3-thiazol-2-yl}benzamide (5)
The hydrazide (4; 0.025 mol) was taken in C2H5OH (20 mL) in a 250 mL RB flask at 28
oC and then solid KOH (0.024 mol) was added. The contents were dissolved on reflux. Carbon
o
disulphide (0.05 mol) was poured into the reaction mixture drop wise at 28 C and the solution
was refluxed again for 10 hours. The reaction progress was noted with TLC using n-hexane and
ethyl acetate solvent system (7:3). After completion of reaction, excess of ethanol was
evaporated and sufficient ice cold distilled water was added followed by addition of dilute HCl
till constant pH of 4-5. Light peach colored precipitates of N-{4-[(5-sulfanyl-1,3,4-oxadiazol-2-
yl)methyl]-1,3-thiazol-2-yl}benzamide (5) were filtered and washed with distilled water.
4.4. Synthesis of 3-bromo-N-(un/substituted-phenyl)propanamides (8a-l)
The un/substituted anilines (6a-l; 0.01 mol) were first suspended in water (20 mL) and
the aqueous Na2CO3 (10%, 4-5 mL) was added. This mixture was prepared in an iodine flask
(250 mL). 3-Bromopropanoyl chloride (7) was added in small pinches along with vigorous
21

stirring by hand. The mixture was set to stir on magnetic stirrer for 2-3 hours. The synthesized
electrophiles, 8a-l, were collected through filtration followed by washing with water and drying
for next step.
4.5. General procedure for the synthesis of N-[4-({5-[(3-un/substituted-anilino-3-
oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-yl]benzamides (9a-l)
The compound 5 (0.47 mmol) was dissolved in N,N-dimethyl formamide (DMF, 5-10
mL) in a 100 mL RB flask. Solid LiH (0.005g) was added and mixture was stirred for half an
hour for activation of 5. Then, from newly synthesized electrophiles, 8a-l, one in each respective
reaction was added in equimolar ratio and the mixture was further stirred for 3-5 hours. The
reaction was monitored by TLC using n-hexane and ethyl acetate solvent system (8:2). After
completion, ice cold distilled water was added to the reaction flask. The respective products, 9a-
l, were collected by filtration or solvent extraction.
4.5.1.
N-[4-({5-[(3-Anilino-3-oxopropyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-
yl]benzamide (9a)
o
Off white solid; Yield: 84%; m.p.: 207-208 C; Mol. Formula: C22H19N5O3S2; Mol.
Mass: 465 gmol-1; IR: 3340 (N-H stretching), 3045 (C-H of aromatic ring), 2923 (-CH2-
stretching), 1670 (C=O stretching), 1658 (C=N stretching), 1580 (C=C stretching of aromatic
ring); 1H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.91 (s, 1H, -NH-CO-3'''), 8.08 (br.d, J = 7.8 Hz, 2H,
H-2 & H-6), 7.63 (br.t, J = 7.5 Hz, 1H, H-4), 7.53 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.38 (br.d,
2H, J = 7.9 Hz, H-2'''' & H-6''''), 7.33 (br.t, J = 7.6 Hz, 2H, H-3'''' & H-5''''), 7.16 (br.t, 1H, J = 7.3
Hz, H-4''''), 6.82 (s, 1H, H-5'), 4.33 (br.s, CH2, H-6'), 3.48 (br.t, 2H, J = 6.7 Hz, CH2-1'''), 2.90
13
(br.t, 2H, J = 6.7 Hz, CH2-2'''); C-NMR: 168.68 (C-3'''), 165.68 (C-7), 165.04 (C-2''), 163.40
(C-2'), 159.64 (C-5''), 143.94 (C-4'), 138.38 (C-1''''), 132.54 (C-4), 131.82 (C-1), 128.68 (C-3''''
& C-5''''), 128.50 (C-2 & C-6), 128.08 (C-3 & C-5), 123.22 (C-4''''), 119.05 (C-2'''' & C-6''''),
111.17 (C-5'), 35.66 (C-6'), 27.68 (C-2'''), 27.52 (C-1'''); Anal. Calc. for C22H19N5O3S2 (465.09):
C, 56.76; H, 4.11; N, 15.04. Found: C, 56.82; H, 4.16; N, 14.88; EI-MS: m/z 465 (M)+, 373
[C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O]+, 77 [C6H5] +.
4.5.2. N-{4-[(5-{[3-Oxo-3-(2-toluidino)propyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-
thiazol-2-yl}benzamide (9b)
22

o
Off white solid; Yield: 81%; m.p.: 245-246 C; Mol. Formula: C23H21N5O3S2; Mol.
Mass: 479 gmol-1; IR: 3365 (N-H stretching), 3050 (C-H of aromatic ring), 2940 (-CH2-
stretching), 1674 (C=O stretching), 1655 (C=N stretching), 1570 (C=C stretching of aromatic
1
ring); H-NMR: 12.66 (s, 1H, -NH-CO-7), 9.92 (br.s, 1H, -NH-CO-3'''), 8.07 (br.d, J = 7.5 Hz,
2H, H-2 & H-6), 7.62 (br.t, J = 7.5 Hz, 1H, H-4), 7.52 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.43
(br.d, J = 7.5 Hz, 1H, H-6''''), 7.40 (s, 1H, H-5'), 7.27 (br.d, J = 7.9 Hz, 1H, H-3''''), 7.09 (br.t, J =
7.3 Hz, 1H, H-5''''), 6.93 (br.t, J = 7.5 Hz, 1H, H-4''''), 4.32 (br.s, 2H, CH2-6'), 3.46 (br.t, J = 6.7
Hz, 2H, CH2-1'''), 2.87 (br.t, J = 6.7 Hz, 2H, CH2-2'''), 2.26 (br.s, 3H, 2''''-CH3); 13C-NMR:
168.65 (C-3'''), 165.48 (C-7), 165.07 (C-2''), 163.64 (C-2'), 158.82 (C-5''), 143.44 (C-4'), 138.48
(C-1''''), 137.22 (C-2''''), 132.54 (C-4), 131.86 (C-1), 130.16 (C-6''''), 128.50 (C-2 & C-6), 128.09
(C-3 & C-5), 127.82 (C-5''''), 125.79 (C-3''''), 125.13 (C-4''''), 111.07 (C-5'), 35.64 (C-6'), 27.75
(C-2'''), 27.41 (C-1'''), 21.14 (2''''-CH3); Anal. Calc. for C23H21N5O3S2 (479.11): C, 57.60; H,
4.41; N, 14.60. Found: C, 57.52; H, 4.48; N, 14.50; EI-MS: (m/z) 479 (M)+, 373 [C16H13N4O3S2],
318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O]+, 91 [C7H7] +, 77 [C6H5] +.
4.5.3. N-{4-[(5-{[3-Oxo-3-(3-toluidino)propyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-
thiazol-2-yl}benzamide (9c)
o
Light brown solid; Yield: 79%; m.p.: 177-178 C; Mol. Formula: C23H21N5O3S2; Mol.
Mass: 479 gmol-1; IR: 3350 (N-H stretching), 3052 (C-H of aromatic ring), 2923 (-CH2-
stretching), 1670 (C=O stretching), 1657 (C=N stretching), 1576 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.95 (br.s, 2H, -NH-CO-3'''), 8.08 (br.d, J = 7.5 Hz,
2H, H-2 & H-6), 7.62 (br.t, J = 7.4 Hz, 1H, H-4), 7.52 (br.t, J = 7.5 Hz, 2H, H-3 & H-5), 7.41 (s,
1H, H-5'), 7.34 (br.d, J = 7.9 Hz, 1H, H-6''''), 7.17 (br.s, merged, 1H, H-2''''), 7.16 (br.t, merged, J
= 7.8 Hz, 1H, H-5''''), 6.85 (br.d, J = 7.3 Hz, 1H, H-4''''), 4.33 (br.s, 2H, CH2-6'), 3.46 (br.t, 2H, J
= 6.5 Hz, CH2-1'''), 2.87 (br.t, 2H, J = 6.5 Hz, CH2-2'''), 2.26 (br.s, 3H, 3''''-CH3); 13C-NMR:
168.57 (C-3'''), 165.48 (C-7), 165.07 (C-2''), 163.64 (C-2'), 158.79 (C-5''), 143.44 (C-4'), 138.80
(C-1''''), 137.84 (C-3''''), 132.54 (C-4), 131.98 (C-1), 128.50 (merged, C-2, C-6 & C- 5''''), 128.09
(C-3 & C-5), 123.92 (C-4''''), 119.60 (C-6'''''), 116.26 (C-2''''), 111.07 (C-5'), 35.64 (C-6'), 27.75
(C-2'''), 27.41 (C-1'''), 21.14 (3''''-CH3); Anal. Calc. for C23H21N5O3S2 (479.11): C, 57.60; H,
4.41; N, 14.60. Found: C, 57.48; H, 4.53; N, 14.46; EI-MS: (m/z) 479 (M)+, 373 [C16H13N4O3S2],
318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O]+, 91 [C7H7] +, 77 [C6H5] +.
23

4.5.4. N-{4-[(5-{[3-Oxo-3-(4-toluidino)propyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-
thiazol-2-yl}benzamide (9d)
o
Off white solid; Yield: 89%; m.p.: 203-204 C; Mol. Formula: C23H21N5O3S2; Mol.
Mass: 479 gmol-1; IR: 3367 (N-H stretching), 3059 (C-H of aromatic ring), 2926 (-CH2-
stretching), 1672 (C=O stretching), 1651 (C=N stretching), 1588 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.04 (br.s, 1H, -NH-CO-3'''), 8.08 (br.d, J = 7.4 Hz,
2H, H-2 & H-6), 7.63 (br.t, J = 7.5 Hz, 1H, H-4), 7.53 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.46
(br.d, J = 6.4 Hz, 2H, H-2'''' & H-6''''), 7.11 (br.d, J = 6.4 Hz, 2H, H-3'''' & H-5''''), 7.40 (s, 1H, H-
5'), 4.33 (br.s, 2H, CH2-6'), 3.46 (br.t, 2H, J = 6.7 Hz, CH2-1'''), 2.86 (br.t, 2H, J = 6.7 Hz, CH2-
2'''), 2.25 (s, 3H, 4''''-CH3); 13C-NMR: 168.59 (C-3'''), 165.66 (C-7), 165.07 (C-2''), 163.61 (C-2'),
158.78 (C-5''), 143.46 (C-4'), 136.39 (C-1''''), 132.59 (C-4), 132.02 (C-1), 138.78 (C-1''''), 132.10
(C-4''''), 129.05 (C-3'''' & C-5''''), 128.50 (C-2 & C-6), 128.09 (C-3 & C-5), 119.06 (C-2'''' & C-
6''''), 111.05 (C-5'), 35.62 (C-6'), 27.74 (C-2'''), 27.42 (C-1'''), 20.39 (4''''-CH3); Anal. Calc. for
C23H21N5O3S2 (479.11): C, 57.60; H, 4.41; N, 14.60. Found: C, 57.69; H, 4.36; N, 14.55; EI-MS:
(m/z) 479 (M)+, 373 [C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O]+,
91 [C7H7] +, 77 [C6H5] +.
4.5.6. N-{4-[(5-{[3-(2,3-Dimethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-
1,3-thiazol-2-yl}benzamide (9e)
o
Light brown solid; Yield: 87%; m.p.: 187-188 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3365 (N-H stretching), 3055 (C-H of aromatic ring), 2930 (-CH2-
stretching), 1668 (C=O stretching), 1644 (C=N stretching), 1545 (C=C stretching of aromatic
1
ring); H-NMR: 12.68 (s, 1H, -NH-CO-7), 9.46 (br.s, 1H, -NH-CO-3'''), 8.08 (br.d, J = 7.8 Hz,
2H, H-2 & H-6), 7.62 (br.t, J = 7.3 Hz, 1H, H-4), 7.52 (br.t, J = 7.5 Hz, 2H, H-3 & H-5), 7.17 (s,
1H, H-5'), 7.10 (br.d, J = 7.6 Hz, 1H, H-6''''), 7.02 (br.t, J = 7.6 Hz, 1H H-5''''), 6.98 (br.d, J =
7.1 Hz, 1H, H-4''''), 4.34 (br.s, 2H, CH2-6'), 3.47 (br.t, 2H, J = 6.6 Hz, CH2-1'''), 2.88 (br.t, 2H, J
= 6.6 Hz, CH2-2'''), 2.22 (br.s, 3H, 2''''-CH3), 2.03 (br.s, 3H, 3''''-CH3); 13C-NMR: 168.60 (C-3'''),
165.41 (C-7), 165.08 (C-2''), 163.58 (C-2'), 158.77 (C-5''), 143.47 (C-4'), 136.80 (C-1''''), 135.74
(C-3'''') 132.44 (C-4), 132.01 (C-1), 131.96 (C-2'''') 128.42 (C-2 & C-6), 128.01 (C-3 & C-5),
24

126.87 (C-4''''), 125.02 (C-5''''), 123.43 (C-6''''), 111.13 (C-5'), 34.92 (C-6'), 27.95 (C-2'''), 27.36
(C-1'''), 20.02 (3''''-CH3), 13.92 (2''''-CH3); Anal. Calc. for C24H23N5O3S2 (493.12): C, 58.40; H,
4.70; N, 14.19. Found: C, 58.51; H, 4.79; N, 14.02; EI-MS: (m/z) 493 (M)+, 373 [C16H13N4O3S2],
318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O/C8H9]+, 91 [C7H7] +, 77 [C6H5] +.
4.5.7. N-{4-[(5-{[3-(2,5-Dimethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-
1,3-thiazol-2-yl}benzamide (9f)
o
Dull white solid; Yield: 88%; m.p.: 177-178 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3355 (N-H stretching), 3060 (C-H of aromatic ring), 2925 (-CH2-
stretching), 1666 (C=O stretching), 1649 (C=N stretching), 1546 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.95 (br.s, 1H, -NH-CO-3'''), 8.08 (br.d, J = 7.7 Hz,
2H, H-2 & H-6), 7.63 (br.t, J = 7.7 Hz, 1H, H-4), 7.59 (dist. s, 1H, H-6''''), 7.53 (br.t, J = 7.6 Hz,
2H, H-3 & H-5), 7.30 (br.d, J = 7.1 Hz, 1H, H-3''''), 7.17 (s, 1H, H-5'), 7.06 (br.d, J = 7.3, 1H, H-
4''''), 4.34 (br.s, 2H, CH2-6'), 3.48 (br.t, 2H, J = 6.7 Hz, CH2-1'''), 2.89 (br.t, 2H, J = 6.7 Hz, CH2-
2'''), 2.18 (br.s, 3H, 5''''-CH3), 2.16 (br.s, 3H, 2''''-CH3); 13C-NMR: 168.71 (C-3'''), 165.41 (C-7),
165.15 (C-2''), 163.41 (C-2'), 158.78 (C-5''), 143.94 (C-4'), 137.99 (C-1''''), 132.59 (C-4), 132.02
(C-1), 131.62 (5''''), 130.60 (C-2''''), 128.50 (C-2 & C-6), 128.09 (C-3 & C-5), 126.63 (C-5''''),
123.22 (C-4''''), 119.05 (C-6''''), 111.17 (C-5'), 35.66 (C-6'), 27.68 (C-2'''), 27.52 (C-1'''), 19.59
(5''''-CH3), 18.79 (2''''-CH3); Anal. Calc. for C24H23N5O3S2 (493.12): C, 58.40; H, 4.70; N, 14.19.
Found: C, 58.55; H, 4.76; N, 14.08; EI-MS: (m/z) 493 (M)+, 373 [C16H13N4O3S2], 318
[C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O/C8H9]+, 91 [C7H7] +, 77 [C6H5] +.
4.5.8. N-{4-[(5-{[3-(2,6-Dimethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-
1,3-thiazol-2-yl}benzamide (9g)
o
Light brown solid; Yield: 81%; m.p.: 188-189 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3368 (N-H stretching), 3029 (C-H of aromatic ring), 2920 (-CH2-
stretching), 1660 (C=O stretching), 1642 (C=N stretching), 1588 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.01 (br.s, 1H, -NH-CO-3'''), 8.08 (br.d, J = 7.8 Hz,
2H, H-2 & H-6), 7.63 (br.t, J = 7.6 Hz, 1H, H-4), 7.53 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.30
(br.d, J = 8.1 Hz, 2H, H-3'''' & H-5''''), 7.17 (s, 1H, H-5'), 7.06 (br.t, J = 8.1, H-4''''), 4.35 (br.s,
25

2H, CH2-6'), 3.48 (br.t, 2H, J = 6.7 Hz, CH2-1'''), 2.89 (br.t, 2H, J = 6.7 Hz, CH2-2'''), 2.18 (br.s,
6H, 2''''-CH3 & 6''''-CH3); 13C-NMR: 168.68 (C-3'''), 165.46 (C-7), 165.04 (C-2''), 163.59 (C-2'),
158.78 (C-5''), 143.49 (C-4'), 137.88 (C-1''''), 135.71 (C-2'''' & C-6''''), 132.49 (C-4), 132.01 (C-
1), 128.63 (C-3'''' & C-5''''), 128.46 (C-2 & C-6), 128.03 (C-3 & C-5), 127.94 (C-3'''' & C-5''''),
126.05 (C-4'''' ), 111.15 (C-5'), 3.96 (C-6'), 27.98 (C-2'''), 27.35 (C-1'''), 18.79 (2''''-CH3 & 6''''-
CH3); Anal. Calc. for C24H23N5O3S2 (493.12): C, 58.40; H, 4.70; N, 14.19. Found: C, 58.31; H,
4.66; N, 14.03; EI-MS: (m/z) 493 (M)+, 373 [C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243
[C12H9N3OS]+, 105 [C8H5O/C8H9]+, 91 [C7H7] +, 77 [C6H5] +.
4.5.9. N-{4-[(5-{[3-(3,4-Dimethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-
1,3-thiazol-2-yl}benzamide (9h)
o
Light brown solid; Yield: 84%; m.p.: 188-189 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3350 (N-H stretching), 3052 (C-H of aromatic ring), 2923 (-CH2-
stretching), 1670 (C=O stretching), 1651 (C=N stretching), 1570 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.94 (br.s, 1H, -NH-CO-3'''), 7.99 (br.d, J = 7.8 Hz,
2H, H-2 & H-6), 7.59 (br.t, J = 7.6 Hz, 1H, H-4), 7.51 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.28
(br.d, 1H, J = 2.1 Hz, H-2''''), 7.23 (dd, 1H, J = 2.2, 8.1 Hz, H-5''''), 7.17 (s, 1H, H-5'), 7.05 (br.d,
1H, J = 8.1 Hz, H-6''''), 4.34 (br.s, 2H, CH2-6'), 3.46 (br.t, 2H, J = 6.7 Hz, CH2-1'''), 2.87 (br.t,
13
2H, J = 6.7 Hz, CH2-2'''), 2.21 (br.s, 3H, 4''''-CH3), 2.19 (br.s, 3H, 3''''-CH3); C-NMR: 168.71
(C-3'''), 165.56 (C-7), 165.06 (C-2''), 163.51 (C-2'), 158.77 (C-5''), 143.49 (C-4'), 136.62 (C-1''''),
136.22 (C-3''''), 132.49 (C-4), 132.02 (C-1), 130.91 (C-4''''), 128.44 (C-2 & C-6), 128.03 (C-3 &
C-5), 125.50 (C-5''''), 123.30 (C-2''''), 121.60 (C-6''''), 111.14 (C-5'), 34.94 (C-6'), 27.97 (C-2'''),
27.39 (C-1'''), 19.58 (3''''-CH3), 18.73 (4''''-CH3); Anal. Calc. for C24H23N5O3S2 (493.12): C,
58.40; H, 4.70; N, 14.19. Found: C, 58.37; H, 4.77; N, 14.23; EI-MS: (m/z) 493 (M)+, 373
[C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O/C8H9]+, 91 [C7H7] +, 77
[C6H5] +.
4.5.10. N-{4-[(5-{[3-(3,5-Dimethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-
1,3-thiazol-2-yl}benzamide (9i)
o
Light brown solid; Yield: 77%; m.p.: 201-202 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3350 (N-H stretching), 3050 (C-H of aromatic ring), 2920 (-CH2-
26

stretching), 1670 (C=O stretching), 1647 (C=N stretching), 1580 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.49 (br.s, 1H, -NH-CO-3'''), 8.08 (br.d, J = 7.8 Hz,
2H, H-2 & H-6), 7.63 (br.t, J = 7.6 Hz, 1H, H-4), 7.53 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.17 (s,
1H, H-5'), 6.89 (br.d, J = 1.3 Hz, 2H, H-2'''' & H-6''''), 6.66 (br.d, J = 1.3, H-4''''), 4.35 (br.s, 2H,
CH2-6'), 3.48 (br.t, J = 6.9 Hz, 2H, CH2-1'''), 2.89 (br.t, J = 6.9 Hz, CH2-2'''), 2.26 (br.s, 6H, 3''''-
13
CH3 & 5''''-CH3); C-NMR: 168.62 (C-3'''), 165.46 (C-7), 165.12 (C-2''), 163.57 (C-2'), 158.76
(C-5''), 143.48 (C-4'), 139.24 (C-1''''), 137.55 (C-3'''' & C-5''''), 132.46 (C-4), 132.02 (C-1),
128.44 (C-2 & C-6), 128.01 (C-3 & C-5), 127.62 (4''''), 124.34 (C-2'''' & C-6''''), 111.17 (C-5'),
34.96 (C-6'), 27.97 (C-2'''), 27.37 (C-1'''), 21.59 (3''''-CH3 & 5''''-CH3); Anal. Calc. for
C24H23N5O3S2 (493.12): C, 58.40; H, 4.70; N, 14.19. Found: C, 58.53; H, 4.77; N, 14.11; EI-MS:
(m/z) 493 (M)+, 373 [C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105
[C8H5O/C8H9]+, 91 [C7H7] +, 77 [C6H5] +.
4.5.11. N-{4-[(5-{[3-(2-Ethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-
thiazol-2-yl}benzamide (9j)
o
Light brown solid; Yield: 83%; m.p.: 239-240 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3355 (N-H stretching), 3070 (C-H of aromatic ring), 2935 (-CH2-
stretching), 1666 (C=O stretching), 1644 (C=N stretching), 1575 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.94 (br.s, 2H, -NH-CO-3'''), 8.08 (br.d, J = 7.8 Hz,
2H, H-2 & H-6),7.63 (br.t, J = 7.5 Hz, 1H, H-4), 7.53 (br.t, J = 7.4 Hz, 2H, H-3 & H-5), 7.27
(br.d, 1H, J = 7.3 Hz, H-6''''), 7.22 (br.d, J = 7.2 Hz, 2H, H-3''''), 7.18 (s, 1H, H-5'), 7.15-7.14 (m,
1H, H-5''''), 7.18 (s, 1H, H-5'), 7.12-7.10 (m, 1H, H-4''''), 4.34 (br.s, 2H, CH2-6'), 3.47 (br.t, J =
6.7 Hz, 2H, CH2-1'''), 2.86 (br.t, J = 6.7 Hz, CH2-2''), 2.55 (q, J = 7.5 Hz, 2H, 2''''-CH2-CH3),
13
1.09 (t, 3H, J = 7.5 Hz, 2''''-CH2-CH3); C-NMR: 168.37 (C-3'''), 165.47 (C-7), 165.05 (C-2''),
163.63 (C-2'), 158.77 (C-5''), 143.44 (C-4'), 137.91 (C-1''''), 135.30 (C-2''''), 132.55 (C-4), 131.85
(C-1), 128.50 (C-2 & C-6), 128.48 (C-3''''), 128.09 (C-3 & C-5), 126.03 (C-5''''), 125.84 (C-4''''),
125.71 (C-6''''), 111.11 (C-5'), 35.58 (C-6'), 27.78 (C-2'''), 27.54 (2''''-CH2-CH3), 27.42 (C-1'''),
15.61 (2''''-CH2-CH3); Anal. Calc. for C24H23N5O3S2 (493.12): C, 58.40; H, 4.70; N, 14.19.
Found: C, 58.49; H, 4.78; N, 14.11; EI-MS: m/z 493 (M)+, 373 [C16H13N4O3S2], 318
[C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O/C8H9]+, 77 [C6H5] +.
27

4.5.12. N-{4-[(5-{[3-(4-Ethylanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-
thiazol-2-yl}benzamide (9k)
o
Light brown solid; Yield: 81%; m.p.: 179-180 C; Mol. Formula: C24H23N5O3S2; Mol.
Mass: 493 gmol-1; IR: 3340 (N-H stretching), 3060 (C-H of aromatic ring), 2920 (-CH2-
stretching), 1665 (C=O stretching), 1653 (C=N stretching), 1575 (C=C stretching of aromatic
1
ring); H-NMR: 12.67 (br.s, 1H, -NH-CO-7), 9.94 (s, 1H, -NH-CO-3'''), 8.08 (dd, J = 1.2, 8.4
Hz, 2H, H-2 & H-6), 7.62 (br.t, J = 7.4 Hz, 1H, H-4), 7.52 (br.t, J = 7.5 Hz, 2H, H-3 & H-5),
7.47 (br.d, J = 8.4 Hz, 2H, H-2'''' & H-6''''), 7.18 (s, 1H, H-5'), 7.12 (br.d, J = 8.4 Hz, 2H, H-3''''
& H-5''''), 4.33 (br.s, 2H, CH2-6'), 3.46 (br.t, 2H, J = 6.7 Hz, CH2-1'''), 2.86 (br.t, 2H, J = 6.7 Hz,
13
CH2-2'''), 2.54 (q, 2H, J = 7.5 Hz, 4''''-CH2-CH3), 1.14 (t, 3H, J = 7.5 Hz, 4''''-CH2-CH3); C-
NMR: 168.38 (C-3'''), 165.48 (C-7), 165.04 (C-2''), 163.65 (C-2'), 158.78 (C-5''), 143.45 (C-4'),
138.60 (C-1''''), 136.56 (C-4''''), 132.56 (C-4), 131.84 (C-1), 128.51 (C-2 & C-6), 128.09 (C-3 &
C-5), 127.85 (C-3'''' & C-5''''), 119.15 (C-2'''' & C-6''''), 111.10 (C-5'), 35.58 (C-6'), 27.77 (C-2'''),
27.53 (4''''-CH2-CH3), 27.41 (C-1'''), 15.63 (4''''-CH2-CH3); Anal. Calc. for C24H23N5O3S2
(493.12): C, 58.40; H, 4.70; N, 14.19. Found: C, 58.34; H, 4.76; N, 14.10; EI-MS: m/z 493 (M)+,
373 [C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 105 [C8H5O/C8H9]+, 77 [C6H5] +.
4.5.13. N-{4-[(5-{[3-(4-Ethoxyanilino)-3-oxopropyl]sulfanyl}-1,3,4-oxadiazol-2-yl)methyl]-1,3-
thiazol-2-yl}benzamide (9l)
o
Light brown solid; Yield: 83%; m.p.: 191-192 C; Mol. Formula: C24H23N5O4S2; Mol.
Mass: 509 gmol-1; IR: 3365 (N-H stretching), 3055 (C-H of aromatic ring), 2920 (-CH2-
stretching), 1666 (C=O stretching), 1651 (C=N stretching), 1585 (C=C stretching of aromatic
ring), 1520 (C=N stretching); 1H-NMR: 12.67 (s, 1H, -NH-CO-7), 9.92 (br.s, 2H, -NH-CO-3'''),
8.06 (br.d, J = 7.8 Hz, 2H, H-2 & H-6), 7.64 (br.t, J = 7.6 Hz, 1H, H-4), 7.53 (br.t, J = 7.6 Hz,
2H, H-3 & H-5), 7.46 (br.d, J = 8.0 Hz, 2H, H-2'''' & H-6''''), 7.18 (s, 1H, H-5'), 6.89 (br.d, J =
8.0 Hz, 2H, H-3'''' & H-5''''), 4.32 (br.s, 2H, CH2-6'), 3.98 (q, 2H, 4''''-O-CH2-CH3), 3.45 (br.t,
2H, J = 6.7 Hz, CH2-1'''), 2.88 (br.t, 2H, J = 6.7 Hz, CH2-2'''), 1.28 (t, 3H, 4''''-O-CH2-CH3); 13C-
NMR: 168.34 (C-3'''), 165.52 (C-7), 165.05 (C-2''), 163.43 (C-2'), 158.76 (C-5''), 155.42 (C-4''''),
143.47 (C-4'), 133.09 (C-1''''), 132.54 (C-4), 131.99 (C-1), 128.51 (C-2 & C-6), 128.09 (C-3 &
28

C-5), 121.62 (C-2'''' & C-6''''), 114.67 (C-3'''' & C-5''''), 111.07 (C-5'), 63.56 (4''''-O-CH2-CH3)
35.57 (C-6'), 27.78 (C-2'''), 27.54 (C-1'''), 14.92 (4''''-O-CH2-CH3); Anal. Calc. for C24H23N5O4S2
(509.12): C, 56.57; H, 4.55; N, 13.74. Found: C, 56.64; H, 4.64; N, 13.66; EI-MS: m/z 509 (M)+,
373 [C16H13N4O3S2], 318 [C13H10N4O2S2]+, 243 [C12H9N3OS]+, 121 [C8H9O] +, 105 [C8H5O]+, 77
[C6H5] +.
4.6. Urease inhibition assay
This enzyme assay is the customized form of the commonly known Berthelot assay.34 The
assay mixture of 85 µL is prepared containing 10 µL of phosphate buffer of pH 7.0 (in each well
in the 96-well plate), 10 µL of sample solution and 25 µL of enzyme solution (0.135 units).
Contents were pre-incubated at 37 ºC for 5 minutes. 40 µL of urea stock solution (20 mM) was
added to each well with incubation for 10 min at 37 ºC. It is followed by the addition of 115 µL
phenol hypochlorite reagents (freshly prepared by mixing 45 µL phenol with 70 µL of alkali) per
well. For color development, incubation was carried out for further 10 min at 37 ºC. Absorbance
was measured at 625 nm. The percentage enzyme inhibition and IC50 values were calculated
using the following formula:
Where, Control is the total enzyme activity without inhibitor and Test, the activity in the
presence of test compound. IC50 values were calculated using the EZ–Fit Enzyme kinetics
software (Perrella Scientific Inc. Amherst, US).
4.6.1. Statistical analysis
Statistical analysis was performed by Microsoft Excel 2010 for all the thrice measured
values and the results are presented as mean ± SEM.
4.7. Computational Methodology
4.7.1. Retrieval of Jack bean urease from Protein Data Bank
29