Novel indole based hybrid oxadiazole scaffolds with: N -(substituted-phenyl)butanamides: Synthesis, lineweaver-burk plot evaluation and binding analysis of potent urease inhibitors

Article abstract of DOI:10.1039/c8ra04987d

In the study presented herein, 4-(1H-indol-3-yl)butanoic acid (1) was sequentially transformed in the first phase into ethyl 4-(1H-indol-3-yl)butanoate (2), 4-(1H-indol-3-yl)butanohydrazide (3) and 5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazole-2-thiol (4)

Full text of DOI:10.1039/c8ra04987d

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Novel indole based hybrid oxadiazole scaffolds
with N-(substituted-phenyl)butanamides:
synthesis, lineweaver–burk plot evaluation and
binding analysis of potent urease inhibitors†
Cite this: RSC Adv., 2018, 8, 25920
ab
Majid Nazir,a Muhammad Athar Abbasi,
Aziz-ur-Rehman,a
*
Sabahat Zahra Siddiqui,a Hussain Raza,b Mubashir Hassan,b Syed Adnan Ali Shah,c
b
Muhammad Shahidd and Sung-Yum Seo
In the study presented herein, 4-(1H-indol-3-yl)butanoic acid (1) was sequentially transformed in the first phase
into ethyl 4-(1H-indol-3-yl)butanoate (2), 4-(1H-indol-3-yl)butanohydrazide (3) and 5-[3-(1H-indol-3-yl)propyl]-
1,3,4-oxadiazole-2-thiol (4) as a nucleophile. In the second phase, various electrophiles were synthesized by
reacting substituted-anilines, 5a–j, with 4-chlorobutanoyl chloride (6) to afford 4-chloro-N-(substituted-
phenyl)butanamides (7a–j). In the final phase, nucleophilic substitution reaction of 4 was carried out with
different electrophiles, 7a–j, to achieve novel indole based oxadiazole scaffolds with N-(substituted-phenyl)
butamides (8a–j). The structural confirmation of all the as-synthesized compounds was performed by spectral
and elemental analysis. These molecules were screened for their in vitro inhibitory potential against urease
enzyme and were found to be potent inhibitors. The results of enzyme inhibitory kinetics showed that
compound 8c inhibited the enzyme competitively with a Ki value 0.003 mM. The results of the in silico study
of these scaffolds were in full agreement with the experimental data and the ligands showed good binding
energy values. The hemolytic study revealed their mild cytotoxicity towards cell membranes and hence, these
molecules can be regarded as valuable therapeutic agents in drug designing programs.
Received 11th June 2018
Accepted 5th July 2018
DOI: 10.1039/c8ra04987d
rsc.li/rsc-advances
acts as a free radical scavenger and has a broad-spectrum of
antioxidant activity. It has a unique place in biological systems,
1. Introduction
playing crucial roles in the form of amino acid, growth hormone
and alkaloids. The indole core is also an important component
in many recent drugs for treatment of chemotherapy-induced
nausea and vomiting, cluster headache, or as antihyperten-
sive, antineoplastic and antimitotic agents.2–4 In addition, 1,3,4-
oxadiazole derivatives have played a crucial part in theoretical
development in heterocyclic chemistry and also used exten-
sively in organic synthesis.5,6 Considerable attention has been
focused on 2,5-disubstituted-1,3,4-oxadiazole containing
compounds due to their remarkable biological activity and also
their importance in the pharmaceutical eld. These compounds
are effectively being utilized as antibacterials. Some of these
compounds have also been recognized as anticancer, anti-
Parkinson, anti-HIV and anti-proliferative agents as well.7–9
Urease belongs to a family of highly conserved urea-
hydrolyzing enzymes. Urease is known to be one of the major
causes of disease induced by Helicobacter pylori, thus allowing
them to survive at low pH of the stomach and therefore, play an
important role in the pathogenesis of gastric and peptic ulcer.
Proteus mirabilis and Yersinia enterocolitica are responsible for
urolithiasis and are involved in the development of acute
pyelonephritis and infection-induced reactive arthritis,
Indole is an aromatic heterocyclic compound which exhibits
a distinct reactivity. Indole is composed of a benzo-pyrrole in
which the benzene and pyrrole rings are fused together through
the 2- and 3-positions of the pyrrole ring. Indole can be deproto-
nated at the nitrogen end of the pyrrole ring. The salts thus formed
prove to be good nucleophiles. The indole ring is found in many
natural products such as fungal metabolites, indole alkaloids, and
marine natural products. Highly ionic salts (e.g. Li+, K+) favor N-
substitutions. Soer counter ions favor C-3 substitution.1
Indoles are found in various natural products and have been
identied as compounds of chemical and biological impor-
tance. Indole is a highly conserved heterocyclic molecule that
aDepartment of Chemistry, Government College University, Lahore-54000, Pakistan.
E-mail: abbasi@gcu.edu.pk; Tel: +92-42-111000010 ext. 266
bCollege of Natural Science, Department of Biological Sciences, Kongju National
University, Gongju, 32588, South Korea
cFaculty of Pharmacy, Atta-ur-Rahman Institute for Natural Products Discovery
(AuRIns), Universiti Teknologi MARA, Level 9, FF3, Puncak Alam Campus, 42300
Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
dDepartment of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra04987d
25920 | RSC Adv., 2018, 8, 25920–25931
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
respectively. The typical remedy for treating bacterial infection round bottom ask. The reaction mixture was stirred for 14 h at
with antimicrobials has oen proven futile, and only a few room temperature (RT). Aer absolute conversion, the acid
combination regiments have reached clinical practice. Thus, hydrazide was obtained by distilling methanol off from the
the need for alternative or novel treatments is evident.10,11
reaction mixture. The precipitates were ltered, washed with
The bioactivity of indole and oxadiazole moieties prompted cold n-hexane and air-dried to get pure 4-(1H-indol-3-yl)
us to synthesize some new butanamide molecules bearing these butanohydrazide (3).
moieties altogether. Hence, in continuation of our previous
efforts on bioactivity study of indole containing compounds12,13
2.4 Synthesis of 5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazole-
and urease inhibitory activity of bi-heterocyclic bi-amide mole-
cules,14 the present investigation was designed to explore the
urease inhibitory potential and the kinetic mechanism of newly
synthesized bi-heterocyclic butanamides. The binding interac-
tions with the targeted enzyme were ascertained by molecular
docking, and the cytotoxicity of these scaffolds was evaluated by
hemolysis to identify their therapeutic utility as promising drug
candidates in drug discovery programs.
2-thiol (4)
4-(1H-indol-3-yl)butanohydrazide (0.13 mol; 3) in absolute
ethanol (30 mL) and KOH (0.13 mol) were placed in a round
bottom ask. Carbon disulde (0.26 mol) was added subse-
quently. The mixture was reuxed for 16 h. On completion of
the reaction, excess chilled distilled water and dil. HCl was
added to adjust to pH 5–6. The precipitates were ltered,
washed and dried to get the desired cyclized product, 4.
2. Materials and methods
2.1 General
2.5 Preparation of 4-chloro-N-(un/substituted-phenyl)
butanamides (7a–j)
Synthesis of various 4-chloro-N-(un/substituted-phenyl)
butanamides (7a–j) was carried out by reaction of various aryl
amines (5a–j) with 4-chlorobutanoyl chloride (6) in equimolar
quantities (0.001 moles) and shaking manually in 10% aqueous
Na2CO3 solution. Solid precipitates were formed aer 20–
30 min, and then ltered and washed with cold distilled water
to obtain the desired electrophiles, 7a–j.
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 systems.
Spots were detected by UV254. Gallonkamp apparatus was used
to detect melting points in capillary tubes. Infrared (IR) spectra
(n, cmꢀ1) were recorded by the pellet method using a Jasco-320-
A spectrophotometer. Elemental analyses were performed on
a Foss Heraeus CHN-O-Rapid instrument and were within
ꢁ0.4% of the theoretical values. Electron Impact Mass Spec-
trometry (EI-MS) spectra were measured on a JEOL JMS-600H
instrument with a data processing system. Proton Nuclear
Magnetic Resonance (1H-NMR) spectra (d, ppm) were recorded
at 600 MHz [Carbon Nuclear Magnetic Resonance (13C-NMR)
spectra, at 150 MHz] in DMSO-d6 using a Bruker Advance III
600 Ascend spectrometer with a BBO probe. The 1H-NMR
spectral peaks for interpretation are abbreviated 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. The structural characterization of all
compounds is given in the ESI.†
2.6 Synthesis of 4-({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)-N-(substituted-phenyl)butanamides
(8a–j)
5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazole-2-thiol (0.2 g, 4) was
added in DMF (5 mL) contained in a 250 mL round bottom ask
at room temperature, and a small amount of LiH was added,
followed by stirring for 30 min. Then, different 4-chloro-N-
(substituted-phenyl)butanamides (7a–j) were added separately
in equimolar amounts to each respective reaction mixture and
stirred for 60–70 hours. The completion of the reaction was
monitored by TLC; aer completion, the reaction mixture was
quenched with ice cold water (100 mL). The respective deriva-
tives, 8a–j, were collected through ltration or solvent extraction
based on the nature of the product.
2.2 Synthesis of ethyl 4-(1H-indol-3-yl)butanoate (2)
4-(1H-indol-3-yl)butanoic acid (0.2 mol; 1) dissolved in absolute
ethanol (70 mL) and a catalytic amount of concentrated sulfuric
acid (20 mL) were placed in a 500 mL round bottom (RB) ask
and reuxed for 8 h until the maximum completion of reaction,
as determined through TLC. At the end, the reaction mixture
was neutralized with 10% aqueous sodium carbonate (40 mL).
The product was isolated by solvent extraction with chloroform
(50 mL ꢂ 3). The solvent was distilled off and the required ester,
2, was obtained as a reddish brown liquid.
2.7 In vitro urease inhibitory activity
The Jack bean urease activity was determined by measuring the
amount of ammonia produced via the indophenols method, as
reported.15,16 The reaction mixtures, comprised of 20 mL of
enzyme (Jack bean urease, 5 U/mL) and 20 mL of compounds in
50 mL potassium phosphate buffer (100 mM urea, 10 mM
K2HPO4, 1 mM EDTA and 10 mM LiCl, pH 8.2), were incubated
ꢃ
for 30 min at 37 C in 96-well plate. Briey, 50 mL each of the
phenol reagents (1%, w/v phenol and 0.005%, w/v sodium
nitroprusside) and 50 mL of the alkali reagent (0.5%, w/v NaOH
and 0.1% sodium hypochlorite NaOCl) were added to each well.
2.3 Synthesis of 4-(1H-indol-3-yl)butanohydrazide (3)
4-(1H-indol-3-yl)butanoate (0.15 mol.; 2) in 60 mL methanol and The absorbance at 625 nm was measured aer 10 min, using
hydrazine monohydrate (80%; 25 mL) were placed in a 500 mL a microplate reader (OPTIMax, Tunable). All the reactions were
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 25920–25931 | 25921

View Article Online
RSC Advances
Paper
performed in triplicate. The urease inhibition activities were exhaustiveness value ¼ 8 to maximize the binding conforma-
calculated according to the following formula:
tional analysis. We adjusted the grid box size on biding pocket
residues to sufficiently allow the ligand to move freely in the
search space. All the as-synthesized ligands were docked sepa-
rately against the target protein. In all of the docked complexes,
the conformational poses of ligand were keenly observed to
obtain the best docking results. The generated docked
complexes were evaluated on the basis of the lowest binding
energy (kcal molꢀ1) values and structure activity relationship
(SAR) analyses. The three dimensional (3D) graphical depic-
tions of all the docked complexes were accomplished by using
the UCSF Chimera 1.10.1 and Discovery Studio (2.1.0)
programs.22
Urease inhibition activity (%) ¼ (ODcontrol ꢀ ODsample ꢂ 100)/
ODcontrol
(1)
where ODcontrol and ODsample represents the optical densities in
the absence and presence of sample, respectively. Thiourea was
used as the positive control for the urease activity.
2.8 Kinetic analysis
Kinetic analysis was carried out to determine the mode of
inhibition. Compound 8c was selected on the basis of the lowest
IC50 value. The kinetics was carried out by varying the concen-
tration of urea in the presence of different concentrations of 8c
(0.00, 0.017, and 0.034 mM). Briey the urea concentration was
changed from 100, 50, 25, 12.5 and 6.25 mM for the urease
kinetics studies and the remaining procedure was the same for
all kinetic studies, as described in the urease inhibition assay
protocol. Maximal initial velocities were determined from the
initial linear portion of absorbance up to 10 minutes aer
addition of the enzyme at per-minute intervals. The mode of
inhibition of the enzyme was assayed via the Lineweaver–Burk
plot of the inverse of the velocities (1/V) versus the inverse of the
2.10 Hemolytic activity
Bovine blood sample was collected in EDTA, which was diluted
with saline (0.9% NaCl), and centrifuge at 1000 ꢂ g for 10 min.
Erythrocytes were separated and diluted in phosphate buffer
saline (PBS) of pH 7.4 and a suspension was made. The test
compound solution (20 mL, 10 mg mLꢀ1) was mixed with 180 mL
of RBCs suspension and incubated for 30 min at room
temperature. PBS was used as a negative control and Triton 100-
X was taken as a positive control.23,24 The experiments were
carried out with the control in triplicate. Statistical analysis was
performed by Microso Excel 2010 for all the measurements
and the results are presented as mean ꢁ SEM. The % age of
hemolysis was obtained by using the formula:
substrate concentration 1/[S] mMꢀ1
. The EI dissociation
constant, Ki, was determined by secondary plot of 1/V versus the
inhibitor concentration. The urease activity was determined by
measuring ammonia production using the indophenol method,
as reported previously.17,18 The results (change in absorbance
per min) were processed by using the SoMaxPro soware.
ð%Þ of hemolysis ¼
Absorbance of sample ꢀ Absorbance of negative control
ꢂ 100
Absorbance of positive control
2.9 Computational methodology
(2)
2.9.1 Retrieval of Jack bean urease from Protein Data Bank.
The jack bean urease structure (C. ensiformis) at 1.52 resolution
was obtained from the Protein Data Bank (PDB) having PDBID
4H9M (https://www.rcsb.org/structure/4h9m). The accessed
receptor molecule was further minimized by employing UCSF
Chimera 1.10.1 tool.19 The Ramachandran plot and values were
3. Results and discussion
3.1 Chemistry
accessed from the PDB. The protein architecture and statistical The convergent syntheses of the targeted bi-heterocyclic buta-
percentage values of the receptor proteins, a-helices, b-sheets, mide derivatives was accomplished in superb yields through
coils and turns were predicted from the online server, VADAR several steps in three phases of reactions. In the rst phase, 4-
1.8.20
(1H-indol-3-yl)butanoic acid (1) was subjected to esterication
2.9.2 Molecular docking simulation. Before docking the by ethanol in the presence of concentrated sulfuric acid taken in
experiment, all the synthesized chemical structures were a catalytic amount. Ethanol was used as reactant and also as
sketched in ACD/ChemSketch tool and accessed in mol format. a solvent in order to push the equilibrium towards the product,
Furthermore, the UCSF Chimera 1.10.1 tool was employed for as it was a reversible reaction. The product was collected by
energy minimization of each ligand, separately, having default solvent extraction aer the addition of a weak base and excess
parameters as follows: steepest descent steps, 100 with step size water. The base was added to neutralize the catalytic sulfuric
˚
˚
of 0.02 (A); conjugate gradient steps, 100 with step size 0.02 (A); acid and the unreacted carboxylic acid. The salts of these acids
and update interval: 10. Finally, Gasteiger charges were added were transferred to the aqueous layer, while the resultant ester
using Dock Prep in the ligand structure to obtain good structure was present in the organic phase during solvent extraction.
conformation. Molecular docking experiment was employed on Thus, ethyl 4-(1H-indol-3-yl)butanoate (2) was obtained as
all the ligands, 8a–j, against Jack bean urease by using virtual a brownish liquid (solid at refrigeration). The second step was
screening too PyRx with the VINA Wizard approach.21 The values performed to convert the ethyl ester to the respective carbohy-
of the grid box parameters in the VINA search space (X ¼ 10.22, drazide, 3, by the nucleophilic hydrazine in the presence of an
Y ¼ 24.56 and Z ¼ 46.18) were adjusted with default organic solvent such as methanol, and the mixture was set to
25922 | RSC Adv., 2018, 8, 25920–25931
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
reux for 14 hours. Thus, 4-(1H-indol-3-yl)butanohydrazide (3)
With the help of 1H-NMR spectrum of this molecule, the
was obtained as a light brown coloured solid. The third step was indole heterocyclic core was identied clearly by the charac-
a cyclization to form a heterocyclic ring through reaction with teristic signals at d 10.80 (s, 1H, NH-1), 7.51 (br.d, J ¼ 7.8 Hz, 1H,
CS2 in a basic alcoholic medium. The resulting product was 5- H-7), 7.34 (br.d, J ¼ 8.1 Hz, 1H, H-4), 7.14 (dist.d, J ¼ 1.8 Hz, 1H,
[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazole-2-thiol (4), which had H-2), 7.06 (br.t, J ¼ 7.2 Hz, 1H, H-6) and 6.97 (br.t, J ¼ 7.2 Hz,
a mercapto group at its second carbon. In the second phase, 1H, H-5) ppm.12 Similarly, the resonances at d 7.21 (br.d, J ¼
different electrophiles were synthesized by reacting substituted- 7.9 Hz, 1H, H-60000), 7.00 (br.s, 1H, H-30000), 6.94 (br.d, J ¼ 7.7 Hz,
anilines (5a–j) with 4-chlorobutanoyl chloride (6) to afford 4- 1H, H-50000), along with two methyl signals at d 2.23 (s, 3H, CH3-
chloro-N-(substituted-phenyl)butanamides (7a–j). In the nal 40000) and 2.12 (s, 3H, CH3-20000) ppm, were typical for a 2,4-
phase, the acidic proton of 4 was replaced with the aforemen- dimethylphenyl group in the molecule. The C and N-substituted
tioned electrophiles, 7a–j, in the presence of LiH using aprotic butanamide group was identied by four signals at d 9.25 (s, 1H,
polar medium to obtain the required molecules, 8a–j, as CONH-1), 3.27 (t, J ¼ 7.1 Hz, 2H, CH2-4000), 2.47 (t, J ¼ 7.2 Hz, 2H,
sketched in Scheme 1 and structures of different derivatives CH2-2000) and 2.06–2.01 (m, 4H, CH2-3000 & CH2-20). In the up-eld
have been drawn in Fig. 1.
region of the spectrum, the signals of three intervening meth-
The structural analysis of one of the compounds is discussed ylene groups at d 2.87 (t, J ¼ 7.4 Hz, 2H, CH2-30), 2.77 (t, J ¼
here in detail for the articulacy of the reader. The molecule 8g 7.3 Hz, 2H, CH2-10), and 2.06–2.01 (m, 4H, CH2-3000 & CH2-20),
was obtained as a brown sticky liquid. Its molecular formula, were helpful in ascertaining the connectivity of the indole
C
25H28N4SO2, was established by CHN analysis data and its moiety from its 3-position to the 5-position of the 1,3,4-oxa-
molecular ion peak in its EI-MS spectrum (m/z 448 (Fig. 2)). The diazole scaffold. The 1H-NMR spectrum of this compound is
1
number of proton and carbon resonances in its H-NMR and shown in Fig. 3(a, b and c).
13C-NMR spectra were also in agreement with its deduced
The carbon skeleton of this molecule was also fully corrob-
molecular formula. Different functionalities in this molecule orated by its 13C-NMR spectrum, shown in the Fig. 4. The
were depicted by absorption bands in its IR spectrum at n 3226 signals in its 13C-NMR spectrum were rational for all the twenty
(N–H str.), 2942 (C–H str. of aromatic ring), 1673 (C]O str.), ve carbons, where the most downeld quaternary carbons
1648 (C]N Str.), 1585 (C]C aromatic str.), 1520, 1477, 1415 signals at d 168.32 (C-500) and 163.36 (C-200) belonged to the
(Str. for oxadiazole), 1159 (C–O–C Str.), 644 (C–S str.) cmꢀ1
.
cyclized 1,3,4-oxadiazole ring, thus conrming the formation of
Scheme 1 Outline for the synthesis of 4-({5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-N-(substituted-phenyl)butanamides (8a–
j). For –R1 and –R2, see structures of derivatives (Fig. 1). Reagents & conditions: (I) EtOH/H2SO4/refluxing for 8 h (II) MeOH/N2H4$H2O/refluxing
for 14 h (III) EtOH/CS2/KOH/refluxing for 16 h (IV) Aq. Na2CO3 soln/pH 9–10/vigorous shaking at RT for 20–30 minutes. (V) DMF/LiH/stirring for
60–70 h.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 25920–25931 | 25923

View Article Online
RSC Advances
Paper
Fig. 1 Structures of different derivatives, 8a–j.
this heterocyclic core. The other three quaternary carbons resonances appearing at d 122.97 (C-2), 121.35 (C-6), 118.66 (C-5
appearing at d 136.79 (C-8), 127.50 (C-9), and 113.78 (C-3) ppm, & 7), 111.83 (C-4) were also coherent with the indole moiety.13
were attributed to the indole moiety. The methine carbon The C and N-substituted butanamido group was conrmed by
Fig. 2 EI-MS spectrum of 8g.
25924 | RSC Adv., 2018, 8, 25920–25931
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 3 (a) 1H-NMR spectrum of 8g. (b) Expanded aromatic region of 1H-NMR spectrum of 8g. (c) Expanded aliphatic region of 1H-NMR spectrum
of 8g.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 25920–25931 | 25925

View Article Online
RSC Advances
Paper
Fig. 4 13C-NMR spectrum of 8g molecule.
two discrete resonances, one for a carbonyl group at d 170.51 (C- the inhibitory potential. Fig. 5 displays the general structural
1000) and other for three methylene groups at d 34.62 (C-2000), features of the synthetic compounds.
32.15 (C-4000) and 25.68 (C-2000). The 2,4-dimethylphenyl group
Compound 8a (IC50 ¼ 0.0523 ꢁ 0.0044 mM) having a 4-
attached to a nitrogen atom was thoroughly substantiated by ethyoxyphenyl ring showed a comparable activity with that of 8b
three quaternary signals at d 134.63 (C-10000), 134.18 (C-20000) and (IC50 ¼ 0.0542 ꢁ 0.0211 mM) which bears a 2-ethylphenyl group.
132.22 (C-40000), three methine signals at d 131.17 (C-50000), 126.80 This indicates that the phenyl ring either having a 4-ethoxy or 2-
(C-30000) and 125.71 (C-60000), and two methyl signals at d 20.91 ethyl group behaves almost similarly with the active site of the
(CH3-40000) and 18.20 (CH3-20000). In the up-eld region of the enzyme. However, compound 8c, bearing an ethyl group at the
spectrum, three signals at d 27.02 (C-10), 24.77 (C-30), and 24.31 4-position of the phenyl ring, showed excellent inhibitory
(C-20) were characteristic for three connected methylenes, which potential (IC50 ¼ 0.0175 ꢁ 0.0065 mM) and was identied as the
adjoined the indole moiety with the heterocyclic oxadiazole core most active among all the synthetic analogues. This may be due
in the molecule. So, on the basis of the aforesaid cumulative to an appropriate interaction of the 4-ethylphenyl ring with the
evidences, the structure of 8g was conrmed and it was active site of enzyme (Fig. 6).
named N-(2,4-dimethylphenyl)-4-({5-[3-(1H-indol-3-yl)propyl]-
Among the three regio-isomers, 8d, 8e and 8f, the molecule
1,3,4-oxadiazol-2-yl}sulfanyl)butanamide. A similar method- 8e, bearing a 3-methylphenyl group, exhibited better inhibitory
ology was implemented for the structural characterization of potential (IC50 ¼ 0.0323 ꢁ 0.0119 mM), although, 8d with a 2-
the other derivatives in the synthesized series.
methylphenyl moiety, also possessed very similar activity (IC50
¼ 0.0717 ꢁ 0.0152 mM). A minute reducing trend in activity was
observed in 8f which had the same methyl group at the 4-
position of the phenyl ring (IC50 ¼ 0.1017 ꢁ 0.0299 mM), which
means that more efficient interactions are made with the
enzyme when a methyl group is present at the meta position
instead of the ortho or para position (Fig. 7).
Among the di-substituted molecules, compound 8h and 8j,
having di-ortho substitutions, were found to be more suitable
than the other analogues, and exhibited similar IC50 values:
0.0251 ꢁ 0.0083 and 0.0375 ꢁ 0.0058 mM, respectively, although
a methyl group at the 2-position in 8h was replaced with an
ethyl group in 8j. The compound, 8h, was the second most
active compound of the series. In other two, di-methylated
regio-isomers, 8g and 8i, the presence of two methyl groups at
3 and 5-positions of the phenyl ring in 8i attributed this
3.2 Urease inhibition and structure–activity relationship
The synthesized bi-heterocyclic butanamides, 8a–j, were
screened against urease of Canavalia ensiformis (Jack bean) and
were found to have potent inhibitory potential against this
enzyme, as was evident from their low IC50 (mM) values (Table
1), relative to the standard, thiourea, which has an IC50 value of
4.7455 ꢁ 0.0546 mM. The jack bean urease was used only as
a surrogate because the human urease is highly expensive and is
not feasible for such in vitro investigations.16–18
Although the observed activity is the resultant of the whole
molecule, a limited structure–activity relationship (SAR) was
rationalized by analyzing the effect of different aryl moieties on
25926 | RSC Adv., 2018, 8, 25920–25931
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Table 1 Urease inhibitory and hemolytic activity of bi-heterocyclic butanamides, 8a–ja
Urease activity
Hemolysis (%)
Compounds
Aryl part
IC50 ꢁ SEM (mM)
(Mean ꢁ SEM)
8a
0.0523 ꢁ 0.0044
0.0542 ꢁ 0.0211
3.16 ꢁ 0.05
2.41 ꢁ 0.03
8b
8c
8d
0.0175 ꢁ 0.0065
0.0717 ꢁ 0.0152
0.62 ꢁ 0.02
5.18 ꢁ 0.03
8e
8f
0.0323 ꢁ 0.0119
0.1017 ꢁ 0.0299
0.2002 ꢁ 0.0122
5.92 ꢁ 0.02
3.51 ꢁ 0.04
1.41 ꢁ 0.01
8g
8h
8i
0.0251 ꢁ 0.0083
0.1244 ꢁ 0.0318
2.63 ꢁ 0.02
7.32 ꢁ 0.03
8j
0.0375 ꢁ 0.0058
9.63 ꢁ 0.05
Thiourea
Triton X
4.7455 ꢁ 0.0546
—
—
89.11 ꢁ 0.01
a
SEM ¼ Standard error of the mean; values are expressed in mean ꢁ SEM. PBS Hemolysis ¼ 2.93 ꢁ 0.01%.
molecule with slightly better inhibitory potential (IC50 ¼ 0.1244
3.3 Kinetic analysis
ꢁ 0.0318 mM) as compared to 8g (IC50 ¼ 0.2002 ꢁ 0.0122 mM) in
Based on our results, we selected the most potent compound 8c
to determine their inhibition type and inhibition constant on
jack bean urease. The potential of these compounds to inhibit
which the two methyl groups were present at the 2- and 4-
positions of the phenyl ring (Fig. 8).
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 25920–25931 | 25927

View Article Online
RSC Advances
Paper
compounds (8a–j) displayed good docking energy values (kcal
molꢀ1) and bind in the binding pocket of urease (Table 3). In all
the docking complexes the predicted energy values were similar
and no marked energy value difference was observed amongst
all the docking structures. Prior research show that the stan-
dard error for Autodock is 2.5 kcal molꢀ1 (http://
autodock.scripps.edu/). Therefore, to prioritize one ligand
over others, it must have higher than a 2.5 kcal molꢀ1 energy
value difference compared to other ligands. In all the docking
complexes, the predicted energy values difference was less than
the standard error energy value. This result was obtained
because the basic nucleus of all the synthesized compounds was
similar and most of the ligands possess similar and efficient
binding affinity values with no signicant energy uctuations.
The comparative docking analysis and inhibition potential
justied that 8c has good therapeutic potential compared to all
other compounds.
3.4.2 Binding pocket analysis. The binding pocket analysis
showed that all the synthesized ligands (8a–j) were narrowed in
the binding pocket of the target protein close to the nickel ions.
All the docked structures were superimposed to check the
binding congurations of all the ligands within the active
region of the target protein. The results show that all the
synthesized ligands were bound at same positioned residues
with different conformational poses in binding pocket. The
binding of all the ligands at same position also justify our
docking reliability and predicted results (Fig. 10A). Based on in
vitro analysis, 8c showed good enzyme inhibition potential
therefore, ligand 8c was selected to keenly observe the binding
interaction pattern. The 8c-docking complex showed that the
indole moiety showed its penetration toward nickel metal. The
substituted functional moiety showed good conformation
position inside the active region of the target protein (Fig. 10B).
3.4.3 Binding analyses of synthesized compounds against
jack bean urease. The ligands-protein binding analyses showed
that 8c was conned in the active binding pocket of the target
protein. The 8c-docked complex revealed a good conforma-
tional state with hydrogen bond and hydrophobic interactions
within the receptor binding pocket. The docking results of 8c–
Fig. 5 General structural features of compounds 8a–j.
the free enzyme and enzyme substrate complex was determined
in terms of the EI and ESI constants respectively. The kinetic
studies of the enzyme by the Lineweaver–Burk plot of 1/V versus
1/[S] in the presence of different compound concentrations gave
a series of straight lines (Fig. 9A). The results of compound 8c
showed that the compound intersected within the second
quadrant. The analysis showed that Vmax decreased with the
increase in inhibitor dosage, while Km remained the same. This
behavior indicated that compound 8c inhibited the urease non-
competitively to form an enzyme inhibitor complex. The
secondary plot of slope against the concentration of inhibitors
showed the enzyme-inhibitor dissociation constant (Ki)
(Fig. 9B).
The kinetic results are presented in the Table 2.
3.4 Structural and physiochemical assessment of jack bean
urease
Jack bean urease is a class of hydrolase protein containing
couple of nickel atoms in the active region of target protein. The
VADAR analysis justied that the Jack bean urease architecture
consists of 27% a-helices, 31% b-sheets and 41% coils. More-
over, Ramachandran plots also indicated that 97.5% of the
residues were present in the favored regions, which shows good
precision of phi (4) and psi (j) angles among the coordinates of
the jack bean urease structure (Fig. S1†).
3.4.1 Molecular docking binding energy analysis. The
ligand-protein docked complexes were analyzed on the basis of
the minimum energy values and ligand conformational inter-
actions pattern. The generated results showed that all the
Fig. 6 Structure–activity relationship of compounds 8a, 8b, and 8c.
Fig. 7 Structure–activity relationship of compounds 8d, 8e, and 8f.
25928 | RSC Adv., 2018, 8, 25920–25931
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 8 Structure–activity relationship of compounds 8g, 8h, 8i, and 8j.
Fig. 9 Lineweaver–Burk plots for inhibition of urease in the presence of compound 8c. (A) Concentrations of 8c were 0.00, 0.017 and 0.034 mM.
(B) The insets represent the plot of the slope or the vertical versus inhibitor 8c concentrations to determine inhibition constants. The lines were
drawn using linear least squares fit.
receptor complex showed that two hydrogen and a single bonding distances. Moreover, another p–p interaction was
hydrophobic interaction were observed at Asp494, Arg439 and observed between the indole ring and the aromatic His593
˚
His593 residues, respectively. The nitrogen of the oxadiazol ring residue with a bond distance of 5.01 A. The binding interaction
formed a strong hydrogen bond with Arg439 with a bond pattern within the binding pocket and against the interacted
˚
distance of 2.96 A. Similarly, the nitrogen of the indole ring also residues is mentioned in Fig. 11A and B. The 2D graphical
formed a hydrogen bond with Asp494 with a bond length of 2.96 depiction of all the other docking complexes is mentioned in
˚
A. Both amino acids are key residues in the active binding the ESI (Fig. S2–S10†). Literature reports also favor our docking
pocket site. Our ligand 8c showed good attachment inside the
active region of the target structure with good hydrogen
Table 3 Docking results of synthesized compounds
Docking complexes
Binding affinity (kcal molꢀ1
)
Table 2 Kinetic parameters of the jack bean urease for urea activity in
the presence of different concentrations of 8ca
8a
8b
8c
8d
8e
8f
8g
8h
8i
ꢀ8.3
ꢀ8.4
ꢀ8.4
ꢀ8.5
ꢀ8.6
ꢀ9.5
ꢀ8.6
ꢀ9.4
ꢀ8.5
ꢀ9.1
Concentration
(mM)
Vmax (DA/Min) Km (mM) Inhibition type
Ki (mM)
0.00
0.017
0.034
0.001724017
5.9
Non-competitive 0.003
6.11923 ꢂ 10ꢀ5 5.9
4.59941 ꢂ 10ꢀ5 5.9
a
Vmax is the reaction velocity, Km is the Michaelis–Menten constant, Ki
is the EI dissociation constant.
8j
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 25920–25931 | 25929

View Article Online
RSC Advances
Paper
Fig. 10 Binding pocket analysis of the ligands. (A) Superimposed docked structures (B) binding conformation of 8c within the binding pocket.
Fig. 11 Docking interaction 8c with receptor molecule. (A) The protein binding pocket in the surface format is represented in pink, while the
protein structure is highlighted in yellow in ribbon format. (B) The 8c-docking complex ligand structure is represented in green, while their
functional groups such as oxygen, nitrogen and sulphur are shown in red, blue and yellow, respectively. Amino acids are highlighted in light pink,
and red and purple dotted lines represent hydrogen and hydrophobic binding with the distance mentioned in angstrom (A˚). Nickel metal is
represented as grey circles.
results which strengthen our discussions on the ligand binding (2.41 ꢁ 0.03%) and 8h (2.63 ꢁ 0.02%) respectively, relative to
and conformational position within the active region of the that of Triton-X.
target protein.25–30
4. Conclusion
3.5 Hemolytic activity
All the as-synthesized bi-heterocyclic butamides, 8a–j, were also The targeted 2-({5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}
subjected to hemolytic assay to nd out their cytotoxicity sulfanyl)-N-(substituted-phenyl)butanamides
(8a–j)
were
prole. The results of the percentage hemolysis (%) are shown synthesized in good yields and these molecules, owing to their
in Table 1. Our results showed that all the compounds of this unique skeleton, possessed potent inhibitory potential against
series have moderate toxicity towards red blood cell membrane. urease. The molecular docking results were in complete agree-
Maximum membrane toxicity was shown by the compound 8j ment with enzyme inhibition data. These molecules were
(9.63 ꢁ 0.05%) which is feeble relative to the positive control, screened for their in vitro inhibitory potential against urease
Triton X having hemolysis of 89.11 ꢁ 0.01%. The minimum enzyme and were found to be potent inhibitors. The enzyme
toxicity was pragmatic in 8c (0.62 ꢁ 0.02%). Precisely, a very low inhibitory potential (0.0175 ꢁ 0.0065) and kinetics results
toxicity was also observed for molecules 8g (1.41 ꢁ 0.01%), 8b showed that compound 8c inhibited the enzyme competitively
25930 | RSC Adv., 2018, 8, 25920–25931
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
with Ki value 0.003 mM. The in silico study of these scaffolds was 13 K. Rubab, M. A. Abbasi, Aziz-ur-Rehman, S. Z. Siddiqui,
in full agreement with the experimental data and 8c showed
M. Ashraf and A. Shaukat, Trop. J. Pharm. Res., 2016b,
15(7), 1525–1533.
good interaction within the active region of the target protein,
having a good binding energy value (ꢀ8.4 kcal molꢀ1). Almost 14 M. A. Abbasi, M. Hassan, Aziz-ur-Rehman, S. Z. Siddiqui,
all the molecules also demonstrated low cytotoxicity. Therefore,
these studies conclude that the newly synthesized molecules
H. Raza, S. A. A. Shah and S.-Y. Seo, Bioorg. Med. Chem.,
2018, 26, 3791–3804.
might serve as promising drug candidates for further structural 15 M. W. Weatherburn, Anal. Chem., 1967, 39(8), 971–974.
optimizations and drug designing studies.
16 H. Raza, Q. Abbas, M. Hassan, S.-H. Eo, Z. Ashraf, D. Kim,
A. R. Phull, S. J. Kim, S. K. Kang and S.-Y. Seo, Pharm.
Biol., 2017, 55(1), 218–226.
Conflicts of interest
17 P. A. Channar, A. Saeed, F. Albericio, F. A. Larik, Q. Abbas,
M. Hassan, H. Raza and S.-Y. Seo, Molecules, 2017, 22(8),
1352–XXXC.
There are no conicts to declare.
18 A. Saeed, F. A. Larik, P. A. Channar, H. Mehfooz,
M. H. Ashraf, Q. Abbas, M. Hassan and S.-Y. Seo, Chem.
Biol. Drug Des., 2017, 90, 764–777.
Acknowledgements
19 E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch,
D. M. Greenblatt, E. C. Meng and T. E. Ferrin, J. Comput.
Chem., 2004, 25, 1605–1612.
The authors acknowledge the Higher Education Commission
(HEC) of Pakistan for nancial support regarding EI-MS
analysis.
20 L. Willard, A. Ranjan, H. Zhang, H. Monzavi, R. F. Boyko,
B. D. Sykes and D. S. Wishart, Nucleic Acids Res., 2003, 31,
3316–3319.
21 S. Dallakyan and A. J. Olson, Methods Mol. Biol., 2015, 1263,
243–250.
22 Studio D., Discovery,version 2.1. Accelrys,San Diego, CA,2008.
23 M. Shahid, S. A. Bukhari, Y. Gul, H. Munir, F. Anjum,
M. Zuber, T. Jamil and K. M. Zia, Int. J. Biol. Macromol.,
2013, 62, 172–179.
24 C. R. Yang, Y. Zhang, M. R. Jacob, S. I. Khan, Y. J. Yhang and
X. C. Li, Antimicrob. Agents Chemother., 2006, 50(5), 1710–
1714.
25 T. Abdul-Fattah, A. Saeed, P. A. Channar, Z. Ashraf, Q. Abbas,
M. Hassan and F. A. Larik, Chem. Biol. Drug Des., 2018, 91,
434–447.
26 P. A. Channar, A. Saeed, F. Albericio, F. A. Larik, Q. Abbas,
M. Hassan, H. Raza and S. Y. Seo, Molecules, 2017, 22, E1352.
27 A. Saeed, S. Ur-Rehman, P. A. Channar, F. A. Larik, Q. Abbas,
M. Hassan, H. Raza and S. Y. Seo, Drug Res., 2017, 67, 596–
605.
References
1 P. Hardik, D. Nilesh, P. Jagath and P. Bhagirath, Int. J. Drug
Res. Technol., 2012, 2(3), 225–230.
2 S. Agarwal, S. Cammerer, S. Filali, W. Frohner, J. Knoll and
M. P. Krahl, Curr. Org. Chem., 2005, 9, 1601–1614.
3 J. Hajicek, Czech. Chem. Commun., 2007, 72, 821–898.
4 M. A. Metwally, S. Shaaban, B. F. Abdel-Wahab and G. A. El-
Hiti, Curr. Org. Chem., 2009, 13, 1475–1496.
5 M. S. Chande, K. A. Puthamane, P. A. Brave, R. R. Khanwelkar
and D. S. Venkatraman, J. Braz. Chem. Soc., 2008, 19(1), 42–
52.
6 M. Yar, M. Bajda, R. A. Mehmood, L. R. Sidra, N. Ullah,
L. Shahzadi, M. Ashraf, T. Ismail, S. A. Shahzad,
Z. A. Khan, S. A. R. Naqvi and N. Mahmood, Lett. Drug Des.
Discovery, 2014, 11, 331–338.
7 M. Amir and S. Shahani, Indian J. Heterocycl. Chem., 1998, 8,
107–110.
8 J. Hazarika and J. C. S. Kataky, Indian J. Heterocycl. Chem.,
1998, 7, 83–92.
28 A. Saeed, P. A. Mahesar, P. A. Channar, F. A. Larik, Q. Abbas,
M. Hassan, H. Raza and S. Y. Seo, Chem. Biodiversity, 2017,
14, e1700035.
9 B. S. Holla, C. S. Prasanna, B. Poojary, K. S. Rao, K. Shridhara
and U. G. Bhat, Indian J. Chem., 2004, 43B, 864–868.
10 M. I. Choudhary, US pat. 20150368214 A1, 2015.
11 M. A. Lodhi, J. Hussain, M. A. Abbasi, A. R. Jassbi,
M. I. Choudhary and V. U. Ahmad, J. Enzyme Inhib. Med.
Chem., 2006, 21(5), 531–535.
29 A. Saeed, S. Rehman, P. A. Channar, F. A. Larik, Q. Abbas,
¨
M. Hassan, H. Raza, U. Florke and S. Y. Seo, J. Taiwan Inst.
Chem. Eng., 2017, 77, 54–63.
30 A. Saeed, F. A. Larik, P. A. Channar, H. Mehfooz,
M. H. Ashraf, Q. Abbas, M. Hassan and S. Y. Seo, Chem.
Biol. Drug Des., 2017, 90, 764–777.
12 K. Rubab, M. A. Abbasi, Aziz-ur-Rehman, S. Z. Siddiqui and
M. N. Akhtar, Trop. J. Pharm. Res., 2016a, 15(7), 1515–1524.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 25920–25931 | 25931