Synthesis, biological valuation, and QSAR studies of novel pyrazole bearing pyridyl oxadiazole analogues as potential antimicrobial agents

Article abstract of DOI:10.1007/s00044-016-1511-4

A new series of 1-(2-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)-3-(aryl)prop-2-en-1-ones (5a-l) were synthesized by a simple and efficient synthetic protocol. The newly synthesized compounds were characterized

Full text of DOI:10.1007/s00044-016-1511-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2016) 25:712–727
DOI 10.1007/s00044-016-1511-4
ORIGINAL RESEARCH
Synthesis, biological valuation, and QSAR studies of novel
pyrazole bearing pyridyl oxadiazole analogues as potential
antimicrobial agents
N. C. Desai1 G. M. Kotadiya1 A. R. Trivedi1 V. M. Khedkar2,3 P. C. Jha4
•
•
•
•
Received: 2 May 2015 / Accepted: 12 January 2016 / Published online: 16 February 2016
Ó Springer Science+Business Media New York 2016
Abstract A new series of 1-(2-(3-(4-nitrophenyl)-1-phe-
nyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-
yl)-3-(aryl)prop-2-en-1-ones (5a–l) were synthesized by a
simple and efficient synthetic protocol. The newly syn-
discriminate the actives from inactives which can serve as
guide to design molecules with improved potency. In silico
ADME predictions were performed to gauge their pharma-
cokinetic, safety and drug likeness profile.
1
thesized compounds were characterized by IR, H NMR,
13C NMR and Mass spectroscopy. The resulting structural
diversity was screened for its antimicrobial activity the
following bacterial and fungal strains: two Gram-positive
bacteria [Staphylococcus aureus (MTCC-96), Streptococ-
cus pyogenes (MTCC-442)], two Gram-negative bacteria
[Escherichia coli (MTCC-443), Pseudomonas aeruginosa
(MTCC-1688)] and three fungal species (C. albicans, A.
niger and A. clavatus). Following this, in vitro cytotoxicity
activity against HeLa cell lines was measured by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]
assay. The observations derived from the diverse assays
were utilized for building classification models based on a
binary QSAR approach termed recursive partitioning (RP)
analysis to probe the physic-chemical properties influencing
the SAR for molecules. The decision tree derived from RP
analysis could highlight structural characteristics that
Keywords Antimicrobial activity Á Cytotoxicity Á
1,3,4-Oxadiazole Á Pyrazole Á Pyridine Á QSAR
Introduction
The rapid development of antibacterial resistance con-
tributes greatly in the challenges occurring in the discovery
of new therapies for combating life-threatening infectious
diseases (Chu et al., 1996; O’Connell et al., 2013). Owing
to this increased microbial resistance, development of
newer classes of antimicrobial agents with novel mode of
actions and high efficiencies are urgently needed to fight
against the multidrug resistant infections (Silver, 2011).
Pyrazole derivatives are reported to exhibit significant
cyclooxygenase (COX-2) inhibitory (Fioravanti et al.,
2010), antiinflammatory (Bekhit et al., 2008) and antioxi-
dant (Ahmad et al., 2010) activity. In addition, some of its
derivatives are among the most therapeutically important
class of heterocyclic compounds and have attracted con-
siderable attention from both synthetic and medicinal
chemists due to their biological activities such as antimi-
crobial (Aiello et al., 2000), antiviral (El-Sabbagh et al.,
2009), anticancer (Xia et al., 2008). 1,3,4-Oxadiazole
heterocycles are very good bioisosteres of amides and
esters, contributing substantially in enhancing pharmaco-
logical activity by participation in hydrogen bonding
interactions with the receptors (Guimaraes et al., 2005).
Potent pharmacological activity of 1,3,4-oxadiazoles can
be attributed to the presence of toxophoric –N=C–O–
& N. C. Desai
dnisheeth@rediffmail.com
1
Division of Medicinal Chemistry, Department of Chemistry,
(UGC NON-SAP and DST-FIST Sponsored), Maharaja
Krishnakumarsinhji Bhavnagar University, Mahatma Gandhi
Campus, Bhavnagar, Gujarat 364 002, India
2
CombiChem Bio Resource Center, National Chemical
Laboratory, Pune 411008, India
3
School of Health Sciences, Discipline of Pharmaceutical
Sciences, University of KwaZulu-Natal, Westville, Durban
4000, South Africa
4
School of Chemical Sciences, Central University of Gujarat,
Sector-30, Gandhinagar 382030, India
123

Med Chem Res (2016) 25:712–727
713
linkage which may react with the nucleophilic centers of
the microbial cell (Rigo and Couturier, 1985). In addition,
1,3,4-oxadiazole derivatives display quite a broad spectrum
of biological activities such as antimicrobial (El-Emam
et al., 2004), antiviral (Tan et al., 2006), antitubercular
(Kumar et al., 2010), antimalarial (Bakht et al., 2010),
analgesic (Akhter et al., 2009), anti-inflammatory (Bur-
buliene et al., 2004), anticonvulsant (Almasirad et al.,
2004) and biological properties such as genotoxic studies
(Maslat et al., 2002) and lipid peroxidation inhibitor
(Farghaly et al., 2000). The azole group of heterocyclic
compounds possesses significant pharmacokinetic prop-
erty, lipophilicity that influences the ability of a drug to
reach the target by transmembrane diffusion and shows
promising activity against resistant TB by inhibiting the
biosynthesis of lipids (Andreani et al., 2001; Kini et al.,
2009). Pyridine derivatives occupy a pivotal position in
modern heterocyclic chemistry, and consequently pyridine
substructure is one of the most important heterocycles
found in natural products, pharmaceuticals and functional
materials (Teague, 2008; Movassaghi et al., 2007). More-
over, substituted pyridines are reported as leukotriene B-4
antagonists (Zhou et al., 2008).
nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 1 was
synthesized by a reported method (Yadlapalli et al., 2012).
Compound 1 on condensation with equimolar quantity of
isoniazid 2 by refluxing in 1,4-dioxane for 8 h afforded a
single product, identified as, N’-((3-(4-nitrophenyl)-1-phe-
nyl-1H-pyrazol-4-yl)methylene)isonicotinohydrazide 3.
Compound 3 showed stretching vibration at 3286 and
1689 cm-1 corresponding to secondary amine as amide
1
linker and carbonyl group, respectively. In addition, H
NMR revealed the appearance of broad singlet at d
10.46 ppm due to one proton of the secondary amine
group. Compound 3 underwent a cyclization reaction with
acetic anhydride under reflux condition resulting in 1-(2-(3-
(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-
1,3,4-oxadiazol-3(2H)-yl)ethanone 4 which was character-
1
ized by disappearance of imine proton signal in H NMR
spectroscopy. Claisen–Schmidt condensation of intermedi-
ate 4 with appropriate aromatic aldehydes in ethanolic
potassium hydroxide well-appointed the respective targeted
pyrazole bearing pyridyloxadiazoles acknowledged as 1-(2-
(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-
4-yl)-1,3,4-oxadiazol-3(2H)-yl)-3-(aryl)prop-2-en-1-ones
5a–l.
Fascinated by multifarious bioactivity of these hybrid
heterocycles and the above-mentioned observations, in
continuation of our search for new, potent, selective and
less toxic antimicrobial agents (Desai et al., 2013a, b),
we planned to synthesize a compact system having
pyrazole, oxadiazole and pyridine entities hoping to
develop compounds possessing noteworthy pharmaco-
logical properties. Taking the above points in consider-
ation, we studied antimicrobial action of the resultant
molecules against a wide range of different human
pathogenic microorganisms in order to obtain compre-
hensive SAR indications. In addition, cytotoxicity stud-
ies were also conducted in HeLa cell lines to evaluate
the ability of these compounds to inhibit cell growth.
Further, a binary QSAR using recursive partitioning (RP)
analysis was performed on these molecules to develop a
classification model based on their activity and physic-
ochemical, structural, electronic and thermodynamic
properties.
The structures of the final derivatives 5a–l were estab-
lished by IR spectra which showed disappearance of –CH3
band in intermediate 4. A strong absorption band between
1685 and 1692 cm-1 was assigned to[C=O group and its
1H NMR spectra revealed two doublets at 6.48–6.60 and
7.96–8.08 ppm for alkene protons attached to carbonyl
group and aryl group, respectively, alongside vanishing of
methyl group singlet. Further, 13C NMR spectrum of
compound 5f as a representative example displayed,
besides the expected methyl and aromatic signals, four
characteristic signals at 167.1, 141.8, 118.6 and 78.8 ppm
due to the carbons of [C=O, alkene carbon attached with
aryl group (=CH–Ar), alkene carbon attached with car-
bonyl group and asymmetric carbon, respectively. The
mass spectrum of 5f showed molecular ion peak at
m/z = 556.17 (M? 71 %), which is in agreement with its
proposed structure. Similarly, the spectral values for all the
compounds and C, H, N analysis are presented in the
experimental part.
Antimicrobial activity
Results and discussion
Chemistry
The outcome of antimicrobial studies of newly synthesized
compounds revealed that these compounds have significant
antibacterial and antifungal activity (Table 1). The inter-
In our attempt to synthesize bioactive scaffolds, pyrazole,
pyridine and oxadiazole were identified as better target,
easy and cheaper to synthesis. The synthetic route was
followed as reported in Scheme 1. Starting material 3-(4-
mediate
3
showed poor antimicrobial activity
(MIC = [1000 lg/mL against P. aeruginosa and C. albi-
cans, 1000 lg/mL against S. aureus, S. pyogenes, A. niger
and A. clavatus). Cyclization of intermediate 3 with acetic
123

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Med Chem Res (2016) 25:712–727
Scheme 1 Synthetic route for
the preparation of title
compounds 5a–l
O2N
O2N
N
H
N
CHO
CONHNH2
N
1,4-dioxane
Reflux, 8 h
N
N
O
N
N
N
2
3
1
(CH3CO)2O
Reflux, 5 h
R
HC CH
CH3
O2N
O2N
O
N
O
N
R
N
N
N N
O
O
CHO
N
N
N
N
Ethanol, KOH
Reflux, 6-7 h
4
5a-l
-R
-R
-R
5a
5b
5c
5d
-H
5e
5f
5g
5h
-3-CH3
-4-CH3
-3-NO2
-4-NO2
5i
5j
5k
5l
-3-OH
-4-OH
-3-OCH3
-4-OCH3
-3-Cl
-4-Cl
-4-F
anhydride afforded 4 which displayed mild antibacterial
activity (MIC = 1000 lg/mL against C. albicans, 500 lg/
mL against S. aureus, E. coli, A. niger and A. clavatus).
Subsequently, compound 4 was transmuted into target
compounds 5a–l by means of Claisen–Schmidt condensation
with aromatic aldehydes. From antibacterial activity data, it
was observed that compounds 5e (3-CH3), 5f (4-CH3), 5j (4-
OH), 5k (3-OCH3) and 5l (4-OCH3) were most potent
against all the bacterial strains in the range of 12.5–50 lg/
mL. Compounds 5e (3-CH3) and 5j (4-OH) possessed very
good activity against S. aureus, E. coli and P. aeruginosa.
Substitution of 4-CH3 group in 5f and 3-OCH3 in 5k
improved the activity against S. aureus. The presence of
4-OCH3 in 5l produced highest activity at MIC of 12.5 lg/
mL against S. aureus. Compounds 5k (3-OCH3) and 5j (4-
OH) demonstrated very good activity, while compounds 5e
(3-CH3), 5f (4-CH3) and 5l (4-OCH3) displayed highest
inhibition against S. pyogenes. When we introduced 4-CH3
in 5f, 3-OCH3 in 5k and 4-OCH3 in 5l, it possessed excellent
activity against E. coli, while 5f (4-CH3) and 5k (3-OCH3)
demonstrated excellent activity against P. aeruginosa.
Furthermore, compound 5l (4-OCH3) having methoxy group
showed highest activity at MIC of 12.5 lg/mL against P.
aeruginosa.
In case of antifungal activity, compounds 5e (3-CH3),
5f (4-CH3) and 5k (3-OCH3) exhibited very good activity
against C. albicans. Introduction of hydroxy or methoxy
group at 4th position resulted in an enhanced activity and
it possessed highest activity against C. albicans. Com-
pounds 5e (3-CH3) and 5j (4-OH) displayed very good
activity, while compounds 5f (4-CH3) and 5k (3-OCH3)
exhibited excellent activity against A. niger. In addition,
compound 5l (4-OCH3) having methoxy group at 4th
position emerged as the most effective antifungal agent at
MIC of 12.5 lg/mL against A. niger. Furthermore, when
we used methyl group as a substituent in 5e, it demon-
strated very good activity, whereas compounds 5f (4-CH3)
and 5l (4-OCH3) displayed highest inhibition against A.
clavatus. Here, we have discussed and compared
antibacterial and antifungal activities taking standard
drugs chloramphenicol and griseofulvin, respectively, as
reference.
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Med Chem Res (2016) 25:712–727
715
Table 1 Results of antibacterial and antifungal screening of compounds 5a–l
Entry
–R
Minimum inhibitory concentration (MIC) lg/mL
Gram-positive bacteriaa Gram-negative bacteriab
Fungic
Ca
Sa
Sp
Ec
Pa
An
Ac
3
–
1000
500
500
500
200
500
50
1000
250
500
250
125
250
25
500
500
250
200
100
250
50
[1000
250
500
250
250
500
50
[1000
1000
500
500
250
500
50
1000
500
1000
200
200
250
50
1000
500
500
250
200
500
50
4
–
5a
5b
5c
5d
5e
5f
5g
5h
5i
–H
-3-Cl
-4-Cl
-4-F
-3-CH3
-4-CH3
-3-NO2
-4-NO2
-3-OH
-4-OH
-3-OCH3
-4-OCH3
25
25
25
25
50
25
25
500
500
100
50
250
500
200
50
250
250
125
50
250
500
100
50
500
500
100
25
500
250
200
50
500
500
100
100
50
5j
5k
5l
25
50
25
25
50
25
12.5
50
25
25
12.5
50
25
12.5
25
Chloramphenicol
50
50
–
–
–
Griseofulvin
–
–
–
–
500
100
100
a
Sa Staphylococcus aureus MTCC 96, Sp Staphylococcus pyogenes MTCC 442
Ec Escherichia coli MTCC 443, Pa Pseudomonas aeruginosa MTCC 1688
b
c
Ca Candida albicans MTCC 227, An Aspergillus niger MTCC 282, Ac Aspergillus clavatus MTCC 1323
Cytotoxicity study
Table 2 Levels of cytotoxicity induced by compounds 5a–l on HeLa
cells
In vitro cytotoxicity of title compounds 5a–l was evaluated
against human cervical cancer cell line (HeLa) by the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro-
mide (MTT) colorimetric assay, which measures the
reduction in the tetrazolium bromide salt into a formazan
dye by mitochondrial dehydrogenases in treated versus
untreated cells. The IC50 values obtained for these com-
pounds are shown in Table 2. Cytotoxicity results revealed
that the derivatives 5e, 5f and 5i–5l accounted no toxicity
at concentration of 100 lM (IC50 [ 100 lM), while other
derivatives showed moderate toxicity against HeLa cell
lines. It was confirmed that none of the tested compounds
exhibited any significant cytotoxic effects on HeLa cell
lines, suggesting that compounds have potential for their
in vivo use as antimicrobial agents.
Entry
Cytotoxicity
(IC50 lM)a
HeLab
5a
65.42
57.32
84.24
68.30
[100
[100
71.10
82.22
[100
[100
[100
[100
3.24
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
Recursive partitioning analysis
Doxorubicin
a
IC50 is the concentration required to inhibit 50 % of cell growth
Recursive partitioning (RP) is a simple statistical data
analysis technique that seeks to decipher the elusive rela-
tionships in complex datasets involving thresholds, inter-
actions and nonlinearity (Strobl et al., 2009; Choi et al.,
2006; Young and Hawkins 2004; Rusinko et al., 2002;
Blower et al., 2002; Manvar et al., 2013; Samal et al.,
b
HeLa human cervical cancer cell line
2013). These issues in the data hinder an analysis based on
assumptions of linearity such as multiple linear regression,
principal component analysis (PCA), or partial least
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Med Chem Res (2016) 25:712–727
squares (PLS) regression. This ‘binary QSAR’ approach
uses the best chemical features (i.e., the physicochemical
properties) to classify the dataset into smaller and more
homogeneous subgroups of higher and lower response
through essential decisive elements (descriptors). The
decision tree derived from the RP analysis qualitatively
predicts the activities or activity classes of dataset without
considering complex statistical analysis. The algorithm
accounts for all possible binary splits for each descriptor
and selects the optimal one by a splitting criterion-a Boo-
lean function BN (M) of the molecular descriptors,
returning ‘true’ or ‘false’. The resulting subsets recursively
splits, with every new subset representing a node at which
the splitting criterion is associated, until no further splitting
is possible or a threshold value of the termination rule is
reached. The branches of the decision tree signify the truth
values taken by the splitting criteria where a true response
to any given splitting criteria follows a branch to the
downside while the false response to the upside. While
there can be several nodes consisting only ‘active’ mole-
cules each representing different structural classes of
known active, the objective is to produce the RP model
with decision tree in which the final terminal nodes contain
either only ‘active’ or only ‘inactive’ molecules. In other
words, to produce terminal subsets of maximal homo-
geneity with regard to the measured activities of their
members. The RP analysis is inherently faster than other
grouping techniques for instance, clustering and the output
provides an easily interpretable graphical tree, as well as a
statistics table that includes correctly classified percent-
ages, the number of false positives, and compound member
information for each node.
provides information on false negatives as well as false
positives, depending on the class to be examined. The term
‘Overall% Predict Correct’, so-called overall prediction is
the percent of ratio of a total number of compounds cor-
rectly classified to the number of compounds predicted in
that class. It provides information on the accuracy of the
prediction when the whole set is being predicted with the
RP model. Finally, ‘enrichment factor’ for a particular
class is the ratio of percentage of compounds correctly
predicted to belong to that class over the original per-
centage of compounds belonging to that class.
RP model for activity against Gram-positive
bacteria and fungi
Recursive partitioning models obtained for biological
activity carried out against Gram-positive bacteria (S.
aureus) and Fungi (C. albicans) resulted in a same classi-
fication pattern and are therefore discussed together. This is
because the classification of dataset into class 1 (inactives)
and class 2 (actives) based on the MIC threshold of 100 lg/
mL resulted in a similar division for both these categories.
The statistical results of the RP model and activity pre-
dictions from it are summarized in Tables 3 and 4,
respectively.
Analysis of recursive partitioning model shows that in
class 1, i.e., the class of ‘inactives’, 8 out of the 8 com-
pounds were correctly classified as belonging to class 1.
For class 2 which is the class of ‘actives’, all 6 compounds
were also correctly classified as actives representing 100 %
intra-class predictivity. The overall prediction represented
by ‘Overall% Predict Correct’ was also found to be 100 %
signifying the accuracy of prediction when entire data set
was being predicted with the RP model. The enrichment
factor (1.75 for class 1 and 2.33 for class 2) also indicates
that the RP model is statistically significant and can be used
to classify new compound library. To evaluate the pre-
dictability and to avoid over fitting of data, the model was
subjected to fivefold cross-validation. This procedure
leaves out 5 % of the dataset and builds the model using
remaining molecules which are then utilized to predict the
The classification model was developed using 2D as
well as 3D physicochemical descriptors by varying the
statistical parameters discussed in the ‘Computational
studies’ section seeking to improve the following
parameters:
‘Class % Observed Correct’, a measure of intra-class
prediction accounting for the compounds predicted cor-
rectly to be in respective classes as a percentage of a total
number of compounds observed to be in each class. It
Table 3 Statistical results of RP model for activity against Gram-positive bacteria (S. aureus) and fungi (C. albicans)
Class
Value
Number of
molecules
%
Class % observed
correct
Overall % predicted
correct
Enrichment factor
RP model
1
2
0
1
8
6
57.14
42.16
100
100
100
100
1.75
2.33
Cross-validation
1
2
0
1
8
6
57.14
42.16
75
100
75
1.75
1.75
100
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Med Chem Res (2016) 25:712–727
717
Table 4 Activity prediction in terms of binary codes by recursive partitioning model
Structure Gram-positive (S. aureus) Fungi (C. albicans)
Gram-negative (E. coli) Physico-chemical properties
Activity RP predicted
activity
Activity RP predicted
activity
Activity RP predicted
activity
Apol
Dipole
moment
Radius of
gyration
Density
3
0
0
0
0
0
0
1
1
0
0
1
1
1
1
0
0
0
0
0
0
1
1
0
0
1
1
1
1
0
0
0
0
0
0
1
1
0
0
1
1
1
1
0
0
0
0
0
0
1
1
0
0
1
1
1
1
0
0
0
0
1
0
1
1
0
0
0
1
1
1
0
0
0
0
1
0
1
1
0
0
0
1
1
1
16,983.400 13.237
18,213.960 5.483
22,683.780 6.769
24,223.560 8.018
24,223.560 8.419
22,796.160 8.167
23,245.200 6.594
23,245.200 6.564
23,758.860 9.261
23,758.860 10.909
23,076.740 8.281
23,076.740 7.444
23,590.000 7.741
23,590.000 7.171
5.228
4.637
5.278
5.271
5.347
5.343
5.381
5.501
5.207
5.537
5.268
5.285
5.360
5.485
1.172
1.185
1.162
1.200
1.199
1.187
1.151
1.151
1.199
1.198
1.177
1.178
1.165
1.165
4
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
activity of eliminated subset. The statistics of cross-vali-
dation are depicted in Table 3. The acceptable percentage
of classification, 75 and 100 % obtained for class 1 and
class 2 with enrichment factor of 1.75 for each class
indicate that the classification model would be able to
classify and predict the activity (class) of new derivatives.
Results of the RP analysis are displayed as a decision
tree derived from the RP process splitting the whole dataset
into smaller subsets (nodes). The RP progresses sequen-
tially by examining all the descriptor variables to identify
the best criterion for splitting the dataset into ‘active’ or
‘inactive’ class.
A 3-leaf RP decision tree was obtained for the substi-
tuted pyridyl oxadiazoles, as depicted in Fig. 1, formed
with 3 terminal and 2 non-terminal nodes in which Class 1
(0) is plotted using Red, while Class 2 (1) using Green. At
each node, the molecules are split into groups, higher and
lower responses, according to their physicochemical char-
acteristics. The terminal nodes 1 and 3 represented by red
lines are class 1 (inactive) while the terminal node 2 rep-
resents class 2 (active) plotted as green. The final decision-
tree-based model correctly classified all the 14 molecules,
respectively, in class 1 and 2 using density and sum of
atomic polarizabilities (Apol) as decision points. It is
noteworthy that a true response to any particular split fol-
lows the branch to the downside, while a false response to
any split follows the branch to the upside in the decision
tree. The first primary split was observed on the basis of
density at a cut-off value of 1.18. It is a spatial descriptor
reflecting the type of atoms and how tightly they are
packed in a molecule and also signify transport and melt
Fig. 1 A 3-leaf recursive partitioning tree generated for prediction of
activity classes against Gram-positive bacteria (S. aureus) and fungi
(C. albicans). Those marked 1:0, 2:1 and 3:0, correspond to terminal
nodes 1–3 and each terminal node corresponds to the value of 1
(active) or 0 (inactive)
behavior. The dataset was split into two branches-those
molecules with density value more than 1.18 (4, 5b, 5c, 5d,
5g and 5h-all inactives) followed a branch to upside
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Med Chem Res (2016) 25:712–727
forming terminal node 1 and are correctly classified while
the remaining 8 molecules with density below cut-off fol-
lowed a branch to downside for further classification.
These molecules were then split on the basis of another
physicochemical parameter-sum of atomic polarizability. It
is an electronic descriptor reflecting the relative tendency
of charge distribution caused by the presence of nearby ion
or dipole. It is also associated with hydrophobicity of a
molecule and thus related to its biological activity. The
terminal node 2 contains 6 molecules (5e, 5f, 5i, 5j, 5k and
5l) with Apol value [22,739.97 which are categorized in
class 2 (active), while the remaining two molecules (3 and
5a) having the Apol less than 22,739.97 were correctly
classified as class 1 (inactive) and also formed the terminal
node 3. Therefore in order to arrive at an active molecules
with pyridyl oxadiazole scaffolds, it must be substituted
with the functional groups resulting in a molecule with
density less than 1.18 and Apol more than 22,739.97 units.
RP model for activity against Gram-negative
bacteria
In a manner similar to the RP model generated based on the
activity carried out against Gram-negative bacteria (S.
aureus) and Fungi (C. albicans), a recursive partitioning
model was obtained based on the activity carried out
against Gram-negative bacteria (S. aureus) for these
molecules as well. Considering the limitation of the size of
the dataset, a statistically significant classification model
was obtained. The statistical outcomes of the model are
summarized in Table 5, while the derived structure activity
relationship discussed in the next section.
Fig. 2 A 4-leaf recursive partitioning tree generated for prediction of
activity classes against Gram-negative bacteria (E. coli)
terminal node 4 contains 6 molecules (3, 4, 5a, 5b, 5g and
5i) with radius of gyration value less than 5.28 and have
been correctly predicted to be in class 1, i.e., inactives. The
remaining 8 molecules formed the non-terminal node and
were further split on the basis of another physicochemical
property dipole moment. It is a molecular electronic
property signifying the strength and orientation behavior of
the molecule in an electrostatic field which can be related
to the long-range ligand–receptor recognition and subse-
quent binding. Terminal node 3 contains 5 molecules (5e,
5f, 5j, 5k and 5l) with dipole moment less than 7.88 units
and is correctly assigned as class 2 (actives), while
remaining three molecules with the dipole moment above
this threshold formed the non-terminal node for further
splitting. These were split on the basis of their sum of
A 4-leaf RP decision tree was obtained for the substi-
tuted pyridyl oxadiazoles (5a–l) based on their physico-
chemical properties and biological activity carried out
against Gram-negative bacteria (Fig. 2). The structure of
the decision tree is made up of 4 terminal and 3 non-
terminal nodes. The terminal nodes 1 and 3 are class 2
(active), while the terminal node 2 and 4 represents class 1
(inactive). The first primary split is observed on radius of
gyration which is a spatial descriptor accounting for the
size of a molecule and its cut-off value is 5.28. The
Table 5 Statistical results for RP model for activity against Gram-negative bacteria
Class
Value
Number of molecules
%
Class % observed correct
Overall % predicted correct
Enrichment factor
RP model
1
2
0
1
8
6
57.14
42.16
100
100
100
100
1.75
2.33
Cross-validation
1
2
0
1
8
6
57.14
42.16
62.50
66.67
71.43
57.14
1.25
1.33
123

Med Chem Res (2016) 25:712–727
719
atomic polarizability at a cut-off value of 23,991.21. Two
molecules (5d and 5h) with Apol values less than
23,991.21 formed the terminal node 2, while molecule 5c
with Apol above the cut-off value formed the terminal node
1 and have been correctly classified as inactive and active,
respectively.
compounds that are doomed to fail and concentrate on lead
optimization efforts to enhance the desired ADME prop-
erties. Also considering the cost involved in the experi-
ments to generate the ADME data, the in silico approach
provides a cost-effective alternative to filter and optimize
the leads in early phase of drug discovery. We carried out
in silico ADME predictions for these substituted pyridy-
loxadiazole analogues with the objective to gain an insight
A close appraisal of this classification models suggests
that radius of gyration, dipole moment and sum of atomic
polarizabilities (Apol) are the physicochemical properties
that contribute to the activity against Gram-negative bac-
teria (E. coli). In order to arrive at a better molecule, it
must have radius of gyration more than 5.28 and dipole
moment less than 7.88. If the radius of gyration is more
than 7.88, then it should have Apol above 23,991.21 with
radius of gyration more than 5.28. Thus by judiciously
modifying these physicochemical parameters one can
arrive at better and more specific antibacterial molecules.
¨
into their drug-like characteristics. QikProp (Schrodinger,
LLC, New York) is an in silico adsorption, distribution,
metabolism and excretion (ADME) prediction program
incorporated in Schrodinger molecular modeling suite. The
in silico ADME prediction data are shown in Table 6.
Inspection of the partition coefficient values (logPo/w) and
prediction of binding to human serum albumin shows that
most of these compounds are hydrophobic in nature. The
molecules display fairly good oral bioavailability with very
low susceptibility to acid hydrolysis in the stomach as
reflected from the % human oral absorption data. The
predicted apparent Caco-2 cell permeability which
accounts for the gut-blood barrier further supports these
predictions. Aqueous solubility data (logS) suggested rea-
sonably good solubility profile for these molecules. Low
values observed for predicted brain/blood partition coeffi-
cient signify that these molecules will have very low ten-
dency to cross the brain/blood brain barrier eliminating the
chances of CNS related toxicity. This is further supported
by the predicted apparent MDCK cell permeability data
which is considered to be a good mimic for the blood brain
barrier. Therefore, the overall in silico ADME predictions
appear to be favorable for future lead optimization.
Structure activity relationship
Results of antimicrobial activity of final compounds and
precursors clearly suggested that compounds 5a–l demon-
strated better antimicrobial activity than its intermediates 3
and 4. The substitution pattern of the three bioactive
pyrazole, pyridine and 1,3,4-oxadiazoles was carefully
selected to impart different electronic environment on the
targeted compounds. The antimicrobial activity was
extensively affected by substitution pattern on the phenyl
ring and the most active compounds contained electron
donating substituent at para or meta positions of the phenyl
ring. In contrast, the presence of inductively electron
withdrawing groups on the phenyl ring witnessed a sub-
stantial decrease in antimicrobial activity for compounds
5b–5d, 5g and 5h. Compounds 5e, 5f and 5j–5l were found
to be most potent antimicrobial agents. Out of them,
compounds 5l, substituted with electron donating methoxy
group at para position, demonstrated the highest antibac-
terial and antifungal activity with fourfold higher MIC
(12.5 lg/mL) as compared to standard chloramphenicol
and griseofulvin, respectively. The reason of these results
may be due to increase in the electron density which makes
the compounds more active against microorganisms and
enhances the antimicrobial potency (Ozaky et al., 2010).
Experimental
All reactions, except those in aqueous media, were carried
out by standard techniques for the exclusion of moisture.
Melting points were determined on an electro thermal
melting point apparatus by open capillary method and were
reported uncorrected. TLC on silica gel plates (Merck, 60,
F254) was used for reaction monitoring. Column chro-
matography on silica gel (Merck, 70–230 mesh and 230–400
mesh ASTH for flash chromatography) was applied when
necessary to isolate and purify the reaction products. Yields
refer to purified products and are not optimized. Elemental
analysis (% C, H, N) was carried out by a Perkin-Elmer 2400
CHN analyzer. IR spectra of all compounds were recorded
on a Perkin-Elmer FT-IR spectrophotometer in KBr; fre-
quencies are reported in cm-1. 1H NMR spectra were run on
Varian Gemini 300 MHz and 13C NMR spectra on Varian
Mercury-400, 100 MHz in DMSO-d6 as a solvent and
tetramethylsilane (TMS) as an internal standard. Chemical
shifts are reported as units (ppm) values. Mass spectra were
scanned on a Shimadzu LCMS 2010 spectrometer.
In silico ADME
Approximately 40 % drug candidates fail in clinical trials
owing to poor ADME (absorption, distribution, metabo-
lism, and excretion) properties. These late-stage failures
contribute significantly to the cost of new drug discovery
campaign. Thus, ability to detect problematic candidates in
the early phase of drug discovery will considerably reduce
the amount of time and resources being wasted on
123

720
Med Chem Res (2016) 25:712–727
Table 6 In silico ADME prediction data for substituted pyridyl oxadiazole analogues
MolID
MW
Hbond acceptor
Hbond donor
logPo/w
logS
Caco
logBB
MDCK
logKhsa
%HOA
3
412.41
454.44
542.55
577.00
577.00
560.54
556.58
556.58
587.55
587.55
558.55
558.55
572.58
572.58
6
7
7
8
8
8
7
7
9
9
8
8
8
8
1
0
0
0
0
0
0
0
0
0
1
1
0
0
4.16
3.37
5.36
5.83
5.85
5.59
5.69
5.69
4.74
4.58
4.94
4.88
5.39
5.42
-6.58
-5.30
-6.98
-7.58
-7.70
-7.33
-7.30
-7.54
-6.52
-6.97
-7.28
-7.29
-6.74
-6.99
180.12
221.48
425.49
477.55
442.24
437.83
523.87
447.34
106.61
53.68
-1.76
-1.21
-1.21
-1.02
-1.05
-1.10
-1.11
-1.22
-1.91
-2.36
-1.78
-1.87
-1.16
-1.25
77.57
96.99
0.70
0.28
0.85
0.95
0.96
0.88
0.98
1.01
0.70
0.73
0.93
0.94
0.77
0.81
91.70
88.66
79.48
83.09
82.62
81.02
82.99
81.77
65.08
58.78
69.28
67.45
68.24
67.57
4
05a
05b
05c
05d
05e
05f
05g
05h
05i
05j
05k
05l
196.44
480.75
503.14
366.77
245.96
207.36
44.01
20.96
157.20
130.10
519.92
467.00
66.96
54.57
243.96
217.23
Here MW = Molecular Weight (130.0/725.0), Number Hydrogen Bonds Donor groups (0.0/6.0), Number of Hydrogen Bonds Acceptor
groups(2.0/20.0), LogP = octanol/water partition coefficient(-2.0/6.5), LogS is Predicted aqueous solubility (-6.5/0.5), LogKhsa is the Pre-
diction of binding to human serum albumin (-1.5/1.5), log BB is Predicted brain/blood partition coefficient (-3.0/1.2); Caco is Apparent Caco-2
Permeability (nm/s) (\25 poor,[500 great); MDCK is Apparent MDCK Permeability (nm/s) (\25 poor,[500 great); %HOA is the % Human
Oral Absorption in GI (\25 % is poor). The values in bracket signify range for 95 % of drugs
Anhydrous reactions were carried out in oven-dried glass-
ware in nitrogen atmosphere.
CH=N), 140.9 (C, C-4 pyridine ring), 139.2 (C, C-1 nitro-
phenyl ring), 139.8 (C, C-1 phenyl ring), 130.4 (CH, C-5
pyrazol ring), 129.2 (2CH, C-3, C-5 phenyl ring), 126.2
(2CH, C-2, C-6 nitrophenyl ring), 126.8 (CH, C-4 phenyl
ring), 124.5 (2CH, C-3, C-5, nitrophenyl ring), 121.8 (2CH,
C-3, C-5, pyridine ring), 119.7 (2CH, C-2, C-6, phenyl ring),
113.1 (C, C-4 pyrazol ring); LCMS m/z 412.15 [M]? ? (1)
(82). Anal.Calcd. for C22H16N6O3: C, 64.07; H, 3.91; N,
20.38; Found: C, 64.18; H, 3.85; N, 20.30.
General procedure for the synthesis of 3-(4-
nitrophenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (1)
Synthesis of 3-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-
carbaldehyde 1 was achieved by reported method (Yadla-
palli et al., 2012).
General procedure for the synthesis of 1-(2-(3-(4-
nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-
4-yl)-1, 3, 4-oxadiazol-3(2H)-yl)ethanone (4)
General procedure for the synthesis of N0-((3-(4-
nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)
methylene)isonicotinohydrazide (3)
Acetic anhydride (0.03 mol) was added to compound N-
((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)
isonicotinohydrazide 3 (0.01 mol) and refluxed at 90 °C
for 5 h. After cooling, the reaction mixture was poured into
ice cold water. The precipitates were filtered, washed with
water, dried and recrystallized from ethanol (95 %).
Creamiest powder; m.p. 182 °C; IR (KBr) vmax 3064,
Compound 3-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-car-
baldehyde 1 (0.01 mol) and isoniazid 2 (0.01 mol) were
dissolved in 1,4-dioxane (20 mL), and the reaction mixture
was refluxed for 8 h. After cooling, the resulting crystals
were filtered and recrystallized from absolute alcohol. Yel-
lowish powder; m.p. 162 °C; IR (KBr) vmax 3286, 3064,
1689, 1562, 1512, 1488 cm-1 1H NMR (DMSO-d6,
;
2938, 1688, 1570, 1514, 1492, 1218 cm-1 1H NMR
;
400 MHz): d = 7.45–7.88 (9H, m, Ar–H), 7.96 (2H, d,
J = 7.7 Hz, C3-H & C5-H pyridine ring), 8.57 (1H, s,
–CH=N), 8.72 (1H, s, pyrazole ring), 8.98 (2H, d,
J = 7.8 Hz, C2-H & C6-H pyridine ring), 10.46 (1H, brs,
–NH D2O exch.); 13C NMR (DMSO-d6, 100 MHz):
d = 163.4 (C, C=O), 150.5 (C, C-3 pyrazol ring), 149.7 (2C–
H, C-2, C-6 pyridine ring), 147.9 (C, C-NO2), 143.4 (CH,
(DMSO-d6, 400 MHz): d = 2.12 (3H, s, CH3), 6.82 (1H, s,
C2-H oxadiazole ring), 7.44–7.86 (9H, m, Ar–H), 7.97 (2H,
d, J = 7.7 Hz, C3-H & C5-H pyridine), 8.12 (1H, s, pyra-
zole ring), 8.84 (2H, d, J = 7.9 Hz, C2-H & C6-H pyr-
idine); 13C NMR (DMSO-d6, 100 MHz): d = 168.9 (C,
C=O), 157.2 (C, C-5 oxadiazole ring), 149.8 (C, C-3,
pyrazol ring), 149.5 (2CH, C-2, C-6 pyridine ring), 139.8
123

Med Chem Res (2016) 25:712–727
721
3-(3-chlorophenyl)-1-(2-(3-(4-nitrophenyl)-1-phenyl-
1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)-yl)prop-2-en-1-one (5b)
(C, C-1, phenyl ring), 139.1 (C, C-1 nitrophenyl ring),
138.5 (C, C-4 pyridine ring), 129.4 (2CH, C-3, C-5, phenyl
ring), 126.8 (CH, C-4 phenyl ring), 126.3 (2CH, C-2, C-6
nitrophenyl ring), 124.7 (2CH, C-3, C-5 nitrophenyl ring),
124.1 (2CH, C-3, C-5 pyridine ring), 123.2 (CH, C-5
pyrazol ring), 119.7 (2CH, C-2, C-6 phenyl ring), 117.3 (C,
C-4 pyrazol ring), 78.2 (CH, C-2 oxadiazol ring), 23.6
(CH3, -COCH3); LCMS m/z = 454.12 [M?] ? (1) (78).
Anal.Calcd. for C24H18N6O4: C, 63.43; H, 3.99; N, 18.49;
Found: C, 63.57; H, 3.91; N, 18.39.
Yield 67 %; m.p. 217 °C; IR (KBr) vmax 3055, 3022, 1687,
1
1574, 1520, 1498, 1210, 753 cm-1; H NMR (DMSO-d6,
400 MHz): d = 6.54 (1H, d, J = 15.6 Hz, –COCH=), 6.80
(1H, s, C2-H oxadiazole ring), 7.40–7.86 (13H, m, Ar–H),
7.97(1H, d, J = 15.9 Hz, =CH–Ar), 8.09(2H, d, J = 7.9 Hz,
C3-H & C5-H pyridine), 8.21 (1H, s, pyrazole ring), 8.81 (2H,
d, J = 8.0 Hz, C2-H & C6-H pyridine); 13C NMR (DMSO-d6,
100 MHz): d = 167.1 (C, C=O), 157.1 (C, C-5 oxadiazole
ring), 149.8 (C, C-3, pyrazol ring), 149.3 (2CH, C-2, C-6
pyridine ring), 147.8 (C, C-4 nitrophenyl ring), 141.7 (CH,
Ph–CH=), 139.9 (C, C-1, phenyl ring), 139.2 (C, C-1 nitro-
phenyl ring), 138.5 (C, C-4 pyridine ring), 136.7 (C, C-1
benzylidene ring), 134.2 (C, C–Cl), 130.1 (CH, C-5 benzyli-
dene ring), 129.4 (2CH, C-3, C-5, phenyl ring), 128.1 (CH,
C-4 benzylidene ring), 126.9 (CH, C-6 benzylidene ring),
126.7 (CH, C-2 benzylidene ring), 126.5 (CH, C-4 phenyl
ring), 126.1 (2CH, C-2, C-6 nitrophenyl ring), 124.7 (2CH,
C-3, C-5 nitrophenyl ring), 124.1 (2CH, C-3, C-5 pyridine
ring), 123.1 CH, C-5 pyrazol ring, 119.9 (2CH, C-2, C-6
phenyl ring), 118.7 (CH, =CH–CO), 117.1 (C, C-4 pyrazol
ring), 78.7 (CH, C-2 oxadiazol ring); LCMS m/z = 576.15
[M?] ? (1) (72). Anal.Calcd. for C31H21ClN6O4: C, 64.53;
H, 3.67; N, 14.57; Found: C, 64.40; H, 3.58; N, 14.47.
General procedure for the synthesis of 1-(2-(3-(4-
nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-
4-yl)-1, 3, 4-oxadiazol-3(2H)-yl)-3-(aryl) prop-2-en-
1-ones (5a–l)
A mixture of intermediate compound 1-(2-(3-(4-nitro-
phenyl)-1-phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1, 3,
4-oxadiazol-3(2H)-yl) ethanone 4 (0.01 mol) and different
aromatic aldehydes (0.01 mol) was stirred in ethanolic
potassium hydroxide (0.01 mol) for 20 min at room tem-
perature. After stirring, the reaction mixture was refluxed
for 6–7 h and excess of solvent was distilled out to get final
compounds 5a–l.
1-(2-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-
(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)-3-
phenylprop-2-en-1-one (5a)
3-(4-chlorophenyl)-1-(2-(3-(4-nitrophenyl)-1-phenyl-
1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)-yl)prop-2-en-1-one (5c)
White crystal; m.p. 202 °C; IR (KBr) vmax 3054, 3018,
1685, 1574, 1518, 1496, 1211 cm-1; 1H NMR (DMSO-d6,
400 MHz): d = 6.48 (1H, d, J = 15.5 Hz, –COCH=), 6.85
(1H, s, C2-H oxadiazole ring), 7.37–7.86 (14H, m, Ar–H),
7.98 (1H, d, J = 16.2 Hz, =CH–Ar), 8.11 (2H, d,
J = 7.8 Hz, C3-H & C5-H pyridine), 8.22 (1H, s, pyrazole
ring), 8.80 (2H, d, J = 8.0 Hz, C2-H & C6-H pyridine);
13C NMR (DMSO-d6, 100 MHz): d = 167.2 (C, C=O),
157.1 (C, C-5 oxadiazole ring), 149.9 (C, C-3, pyrazol
ring), 149.3 (2CH, C-2, C-6 pyridine ring), 147.8 (C, C-4
nitrophenyl ring), 141.8 (CH, Ph–CH=), 139.8 (C, C-1,
phenyl ring), 139.2 (C, C-1 nitrophenyl ring), 138.5 (C,
C-4 pyridine ring), 135.1 (C, C-1 benzylidene ring), 129.4
(2CH, C-3, C-5, phenyl ring), 128.8 (2CH, C-3, C-5
benzylidene ring), 128.2 (2CH, C-2, C-6 benzylidene
ring), 127.8 (CH, C-4 benzylidene ring), 126.6 (CH, C-4
phenyl ring), 126.1 (2CH, C-2, C-6 nitrophenyl ring),
124.7 (2CH, C-3, C-5 nitrophenyl ring), 124.1 (2CH, C-3,
C-5 pyridine ring), 123.1 (CH, C-5 pyrazol ring), 119.9
(2CH, C-2, C-6 phenyl ring), 118.7 (CH, =CH–CO), 117.1
(C, C-4 pyrazol ring), 78.8 (CH, C-2 oxadiazol ring);
LCMS m/z = 542.16 [M?] ? (1) (78). Anal.Calcd. for
C31H22N6O4: C, 68.63; H, 4.09; N, 15.49; Found: C,
68.76; H, 4.17; N, 15.39.
Yellowish powder; m.p. 231 °C; IR (KBr) vmax 3062,
1
3020, 1692, 1572, 1520, 1492, 1214, 756 cm-1; H NMR
(DMSO-d6, 400 MHz): d = 6.53 (1H, d, J = 15.7 Hz,
–COCH=), 6.82 (1H, s, C2-H oxadiazole ring), 7.40–7.88
(13H, m, Ar–H), 7.98 (1H, d, J = 16.1 Hz, =CH–Ar), 8.10
(2H, d, J = 7.9 Hz, C3-H & C5-H pyridine), 8.24 (1H, s,
pyrazole ring), 8.82 (2H, d, J = 8.1 Hz, C2-H & C6-H
pyridine); 13C NMR (DMSO-d6, 100 MHz): d = 167.0 (C,
C=O), 157.1 (C, C-5 oxadiazole ring), 149.8 (C, C-3,
pyrazol ring), 149.4 (2CH, C-2, C-6 pyridine ring), 147.8
(C, C-4 nitrophenyl ring), 141.9 (CH, Ph–CH=), 139.8 (C,
C-1, phenyl ring), 139.2 (C, C-1 nitrophenyl ring), 138.6
(C, C-4 pyridine ring), 133.8 (C, C–Cl), 133.3 (C, C-1
benzylidene ring), 129.7 (2CH, C-3, C-5, phenyl ring),
129.2 (2CH, C-2, C-6 benzylidene ring), 128.9 (2CH, C-3,
C-5 benzylidene ring), 126.8 (CH, C-4 phenyl ring), 126.3
(2CH, C-2, C-6 nitrophenyl ring), 124.7 (2CH, C-3, C-5
nitrophenyl ring), 124.1 (2CH, C-3, C-5 pyridine ring),
123.2 (CH, C-5 pyrazol ring), 119.7 (2CH, C-2, C-6 phenyl
ring), 118.7 (CH, =CH–CO), 117.3 (C, C-4 pyrazol ring),
78.9 (CH, C-2 oxadiazol ring); LCMS m/z = 576.14
123

722
Med Chem Res (2016) 25:712–727
[M?] ? (1) (70). Anal.Calcd. for C31H21ClN6O4: C, 64.53;
pyridine ring), 123.3 (CH, C-5 pyrazol ring), 119.7 (2CH,
C-2, C-6 phenyl ring), 118.6 (CH, =CH–CO), 117.4 (C,
C-4 pyrazol ring), 78.6 (CH, C-2 oxadiazol ring), 21.6
(CH3, C-CH3); LCMS m/z = 556.16 [M?] ? (1) (78).
Anal.Calcd. for C32H24N6O4: C, 69.06; H, 4.35; N, 15.10;
Found: C, 69.18; H, 4.45; N, 15.20.
H, 3.67; N, 14.57; Found: C, 64.67; H, 3.57; N, 14.46.
3-(4-fluorophenyl)-1-(2-(3-(4-nitrophenyl)-1-phenyl-
1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)-yl)prop-2-en-1-one (5d)
Cream powder; m.p. 201 °C; IR (KBr) vmax 3064, 3020,
1690, 1571, 1520, 1497, 1216, 1142 cm-1; 1H NMR (DMSO-
d6, 400 MHz): d = 6.58 (1H, d, J = 15.5 Hz, –COCH=),
6.84 (1H, s, C2-H oxadiazole ring), 7.41–7.84 (13H, m, Ar–
H), 7.97 (1H, d, J = 15.7 Hz, =CH–Ar), 8.08 (2H, d,
J = 7.6 Hz, C3-H & C5-H pyridine), 8.23 (1H, s, pyrazole
1-(2-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-
5-(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)-3-p-
tolylprop-2-en-1-one (5f)
White powder; m.p. 257 °C; IR (KBr) vmax 3058, 3020,
2938, 1687, 1570, 1522, 1495, 1215 cm-1 1H NMR
;
ring), 8.85 (2H, d, J = 8.0 Hz, C2-H & C6-H pyridine); 13
C
(DMSO-d6, 400 MHz): d = 2.37 (3H, s, CH3), 6.56 (1H, d,
J = 15.6 Hz, –COCH=), 6.83 (1H, s, C2-H oxadiazole
ring), 7.35–7.86 (13H, m, Ar–H), 7.97 (1H, d,
J = 16.0 Hz, =CH–Ar), 8.07 (2H, d, J = 7.7 Hz, C3-H &
C5-H pyridine), 8.24 (1H, s, pyrazole ring), 8.81 (2H, d,
J = 8.1 Hz, C2-H & C6-H pyridine); 13C NMR (DMSO-d6,
100 MHz): d = 167.1 (C, C=O), 157.2 (C, C-5 oxadiazole
ring), 149.8 (C, C-3, pyrazol ring), 149.2 (2CH, C-2, C-6
pyridine ring), 147.8 (C, C-4 nitrophenyl ring), 141.8 (CH,
Ph–CH=), 139.7 (C, C-1, phenyl ring), 139.2 (C, C-1
nitrophenyl ring), 138.5 (C, C-4 pyridine ring), 137.7 (C,
C-CH3), 132.3 (C, C-1 benzylidene ring), 129.2 (2CH, C-3,
C-5, phenyl ring), 128.9 (2CH, C-3, C-5 benzylidene ring),
128.3 (2CH, C-2, C-6 benzylidene ring), 126.6 (CH, C-4
phenyl ring), 126.1 (2CH, C-2, C-6 nitrophenyl ring), 124.7
(2CH, C-3, C-5 nitrophenyl ring), 124.1 (2CH, C-3, C-5
pyridine ring), 123.2 (CH, C-5 pyrazol ring), 119.8 (2CH,
C-2, C-6 phenyl ring), 118.6 (CH, =CH–CO), 117.3 (C,
C-4 pyrazol ring), 78.8 (CH, C-2 oxadiazol ring), 21.4
(CH3, C-CH3); LCMS m/z = 556.17 [M?] ? (1) (71).
Anal.Calcd. for C32H24N6O4: C, 69.06; H, 4.35; N, 15.10;
Found: C, 69.19; H, 4.26; N, 15.20.
NMR (DMSO-d6, 100 MHz): d = 167.3 (C, C=O), 162.1 (C,
C-F), 157.2 (C, C-5 oxadiazole ring), 149.8 (C, C-3, pyrazol
ring), 149.3 (2CH, C-2, C-6 pyridine ring), 147.7 (C, C-4
nitrophenyl ring), 141.6(CH, Ph–CH=), 139.9(C, C-1,phenyl
ring), 139.2 (C, C-1 nitrophenyl ring), 138.6 (C, C-4 pyridine
ring), 130.9 (C, C-1 benzylidene ring), 130.3 (2CH, C-2, C-6
benzylidene ring), 129.4 (2CH, C-3, C-5, phenyl ring), 126.5
(CH, C-4 phenyl ring), 126.1 (2CH, C-2, C-6 nitrophenyl
ring), 124.8 (2CH, C-3, C-5 nitrophenyl ring), 124.2 (2CH,
C-3, C-5 pyridine ring), 123.0 (CH, C-5 pyrazol ring), 119.7
(2CH, C-2, C-6 phenyl ring), 118.9 (CH, =CH–CO), 117.4 (C,
C-4 pyrazol ring), 115.6 (2CH, C-3, C-5 benzylidene ring),
78.8 (CH, C-2 oxadiazol ring); LCMS m/z = 560.17
[M?] ? (1) (75). Anal.Calcd. for C31H21FN6O4: C, 66.42; H,
3.78; N, 14.99; Found: C, 66.55; H, 3.69; N, 14.89.
1-(2-(3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-
(pyridin-4-yl)-1,3,4-oxadiazol-3(2H)-yl)-3-(m-
tolyl)prop-2-en-1-one (5e)
Brown crystal; m.p. 241 °C; IR (KBr) vmax 3059, 3022,
2936, 1686, 1569, 1522, 1496, 1211 cm-1 1H NMR
;
(DMSO-d6, 400 MHz): d = 2.36 (3H, s, CH3), 6.57 (1H, d,
J = 15.8 Hz, –COCH=), 6.82 (1H, s, C2-H oxadiazole
ring), 7.36–7.82 (13H, m, Ar–H), 7.96 (1H, d,
J = 16.1 Hz, =CH–Ar), 8.06 (2H, d, J = 7.7 Hz, C3-H &
C5-H pyridine), 8.21 (1H, s, pyrazole ring), 8.80 (2H, d,
J = 8.1 Hz, C2-H & C6-H pyridine); 13C NMR (DMSO-d6,
100 MHz): d = 167.1 (C, C=O), 157.1 (C, C-5 oxadiazole
ring), 149.8 (C, C-3, pyrazol ring), 149.2 (2CH, C-2, C-6
pyridine ring), 147.7 (C, C-4 nitrophenyl ring), 141.6 (CH,
Ph–CH=), 139.6 (C, C-1, phenyl ring), 139.1 (C, C-1
nitrophenyl ring), 138.7 (C, C-4 pyridine ring), 138.2 (C,
C-CH3), 135.2 (C, C-1 benzylidene ring), 129.2 (2CH, C-3,
C-5, phenyl ring), 128.7 (CH, C-5 benzylidene ring), 128.2
(CH, C-4 benzylidene ring), 126.8 (CH, C-2 benzylidene
ring), 126.5 (CH, C-4 phenyl ring), 126.1 (2CH, C-2, C-6
nitrophenyl ring), 125.4 (CH, C-6 benzylidene ring), 124.7
(2CH, C-3, C-5 nitrophenyl ring), 124.1 (2CH, C-3, C-5
3-(3-nitrophenyl)-1-(2-(3-(4-nitrophenyl)-1-phenyl-
1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)-yl)prop-2-en-1-one (5g)
White powder; m.p. 227 °C; IR (KBr) vmax 3062, 3021,
1
1692, 1572, 1525, 1502, 1217 cm-1; H NMR (DMSO-d6,
400 MHz): d = 6.58 (1H, d, J = 15.7 Hz, –COCH=), 6.91
(1H, s, C2-H oxadiazole ring), 7.44–7.98 (13H, m, Ar–H),
8.08(1H, d, J = 15.4 Hz, =CH–Ar), 8.21(2H, d, J = 7.7 Hz,
C3-H & C5-H pyridine), 8.29 (1H, s, pyrazole ring), 8.90 (2H,
d, J = 8.2 Hz, C2-H & C6-H pyridine); 13C NMR (DMSO-d6,
100 MHz): d = 167.1 (C, C=O), 157.1 (C, C-5 oxadiazole
ring), 149.9 (C, C-3, pyrazol ring), 149.3 (2CH, C-2, C-6
pyridine ring), 147.9 (C, C-4 nitrophenyl ring), 147.5 (C, C-3
benzylidene ring), 141.9 (CH, Ph–CH=), 139.8 (C, C-1,
phenyl ring), 139.1 (C, C-1 nitrophenyl ring), 138.4 (C, C-4
pyridine ring), 137.8 (C, C-1 benzylidene ring), 134.6 (CH,
123

Med Chem Res (2016) 25:712–727
723
C-6 benzylidene ring), 129.8 (CH, C-5 benzylidene ring),
129.4 (2CH, C-3, C-5, phenyl ring), 126.7 (CH, C-4 phenyl
ring), 126.2 (2CH, C-2, C-6 nitrophenyl ring), 124.8 (2CH,
C-3, C-5 nitrophenyl ring), 124.1 (2CH, C-3, C-5 pyridine
ring), 123.7 (CH, C-4 benzylidene ring), 123.1 (CH, C-5
pyrazol ring), 122.8 (CH, C-2 benzylidene ring), 119.8 (2CH,
C-2, C-6 phenyl ring), 118.6 (CH, =CH–CO), 117.1 (C, C-4
pyrazol ring), 78.7 (CH, C-2 oxadiazol ring); LCMS
m/z = 587.16 [M?] ? (1) (76). Anal.Calcd. for
C31H21N7O6: C, 63.37; H, 3.60; N, 16.69; Found: C, 63.50; H,
3.52; N, 16.58.
oxadiazole ring), 149.8 (C, C-3, pyrazol ring), 149.4 (2CH,
C-2, C-6 pyridine ring), 147.7 (C, C-4 nitrophenyl ring), 141.8
(CH, Ph–CH=), 139.6 (C, C-1, phenyl ring), 139.0 (C, C-1
nitrophenyl ring), 138.5 (C, C-4 pyridine ring), 135.5 (C, C-1
benzylidene ring), 130.1 (CH, C-5 benzylidene ring), 129.3
(2CH, C-3, C-5, phenyl ring), 126.7 (CH, C-4 phenyl ring),
126.3 (2CH, C-2, C-6 nitrophenyl ring), 124.8 (2CH, C-3, C-5
nitrophenyl ring), 124.2 (2CH, C-3, C-5 pyridine ring), 123.0
(CH, C-5 pyrazol ring), 121.2 (CH, C-6 benzylidene ring),
119.8 (2CH, C-2, C-6 phenyl ring), 118.9 (CH, =CH–CO),
117.7(CH, C-2benzylidenering), 117.1(C, C-4pyrazolring),
115.2 (CH, C-4 benzylidene ring), 78.7 (CH, C-2 oxadiazol
ring); LCMS m/z = 558.16 [M?] ? (1) (72). Anal.Calcd. for
C31H22N6O5: C, 66.66; H, 3.97; N, 15.05; Found: C, 66.54; H,
3.87; N, 15.15.
3-(4-nitrophenyl)-1-(2-(3-(4-nitrophenyl)-1-phenyl-
1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-oxadiazol-
3(2H)-yl)prop-2-en-1-one (5h)
Creamiest powder; m.p. 247 °C; IR (KBr) vmax 3063, 3022,
3-(4-hydroxyphenyl)-1-(2-(3-(4-nitrophenyl)-1-
phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-
oxadiazol-3(2H)-yl)prop-2-en-1-one (5j)
1
1691, 1571, 1523, 1504, 1214 cm-1; H NMR (DMSO-d6,
400 MHz): d = 6.57 (1H, d, J = 15.7 Hz, –COCH=), 6.90
(1H, s, C2-H oxadiazole ring), 7.45–7.99 (13H, m, Ar–H),
8.06 (1H, d, J = 16.1 Hz, =CH–Ar), 8.20 (2H, d,
J = 7.7 Hz, C3-H & C5-H pyridine), 8.27 (1H, s, pyrazole
White crystal; m.p. 271 °C; IR (KBr) vmax 3447, 3060,
3020, 1689, 1568, 1523, 1497, 1212 cm-1 1H NMR
;
ring), 8.89 (2H, d, J = 8.2 Hz, C2-H & C6-H pyridine); 13
C
(DMSO-d6, 400 MHz): d = 6.60 (1H, d, J = 15.4 Hz,
–COCH=), 6.82 (1H, s, C2-H oxadiazole ring), 7.42–7.84
(13H, m, Ar–H), 7.97 (1H, d, J = 15.7 Hz, =CH–Ar), 8.09
(2H, d, J = 7.8 Hz, C3-H & C5-H pyridine), 8.21 (1H, s,
pyrazole ring), 8.87 (2H, d, J = 8.1 Hz, C2-H & C6-H
pyridine), 9.11 (1H, s, OH D2O exch.); 13C NMR (DMSO-
d6, 100 MHz): d = 167.1 (C, C=O), 157.8 (C, C–OH),
157.1 (C, C-5 oxadiazole ring), 149.9 (C, C-3, pyrazol
ring), 149.3 (2CH, C-2, C-6 pyridine ring), 147.8 (C, C-4
nitrophenyl ring), 141.7 (CH, Ph–CH=), 139.7 (C, C-1,
phenyl ring), 139.1 (C, C-1 nitrophenyl ring), 138.4 (C, C-4
pyridine ring), 130.7 (2CH, C-2, C-6 benzylidene ring),
129.4 (2CH, C-3, C-5, phenyl ring), 127.7 (C, C-1 ben-
zylidene ring), 126.7 (CH, C-4 phenyl ring), 126.2 (2CH,
C-2, C-6 nitrophenyl ring), 124.9 (2CH, C-3, C-5 nitro-
phenyl ring), 124.2 (2CH, C-3, C-5 pyridine ring), 123.1
(CH, C-5 pyrazol ring), 119.9 (2CH, C-2, C-6 phenyl ring),
118.7 (CH, =CH–CO), 117.3 (C, C-4 pyrazol ring), 115.9
(2CH, C-3, C-5 benzylidene ring), 78.8 (CH, C-2 oxadiazol
ring); LCMS m/z = 558.15 [M?] ? (1) (80). Anal.Calcd.
for C31H22N6O5: C, 66.66; H, 3.97; N, 15.05; Found: C,
66.53; H, 3.87; N, 15.16.
NMR (DMSO-d6, 100 MHz): d = 167.3 (C, C=O), 157.2
(C, C-5 oxadiazole ring), 149.7 (C, C-3, pyrazol ring), 149.3
(2CH, C-2, C-6 pyridine ring), 147.8 (C, C-4 nitrophenyl
ring), 147.1 (C, C-4 benzylidene ring), 141.9 (CH, Ph–
CH=), 141.2 (C, C-1 benzylidene ring), 139.7 (C, C-1,
phenyl ring), 139.1 (C, C-1 nitrophenyl ring), 138.3 (C, C-4
pyridine ring), 129.7 (2CH, C-3, C-5, phenyl ring), 129.1
(2CH, C-2, C-6 benzylidene ring), 126.7 (CH, C-4 phenyl
ring), 126.2 (2CH, C-2, C-6 nitrophenyl ring), 124.8 (2CH,
C-3, C-5 nitrophenyl ring), 124.1 (2CH, C-3, C-5 pyridine
ring), 123.9 (2CH, C-3, C-5 benzylidene ring), 123.1 (CH,
C-5 pyrazol ring), 119.7 (2CH, C-2, C-6 phenyl ring), 118.7
(CH, =CH–CO), 117.2 (C, C-4 pyrazol ring), 78.6 (CH, C-2
oxadiazol ring); LCMS m/z = 587.14 [M?] ? (1) (78).
Anal.Calcd. for C31H21N7O6: C, 63.37; H, 3.60; N, 16.69;
Found: C, 63.50; H, 3.52; N, 16.80.
3-(3-hydroxyphenyl)-1-(2-(3-(4-nitrophenyl)-1-
phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-
oxadiazol-3(2H)-yl)prop-2-en-1-one (5i)
Yellowish powder; m.p. 253 °C; IR (KBr) vmax 3451, 3058,
3020, 1689, 1571, 1523, 1498, 1214 cm-1; 1H NMR (DMSO-
d6, 400 MHz): d = 6.58 (1H, d, J = 15.6 Hz, –COCH=),
6.84 (1H, s, C2-H oxadiazole ring), 7.39–7.83 (13H, m, Ar–
H), 7.99 (1H, d, J = 15.6 Hz, =CH–Ar), 8.07 (2H, d,
J = 7.6 Hz, C3-H & C5-H pyridine), 8.22 (1H, s, pyrazole
ring), 8.87 (2H, d, J = 8.0 Hz, C2-H & C6-H pyridine), 9.16
(1H, s, OH D2O exch.); 13C NMR (DMSO-d6, 100 MHz):
d = 167.1 (C, C=O), 158.5 (C, C–OH), 157.1 (C, C-5
3-(3-methoxyphenyl)-1-(2-(3-(4-nitrophenyl)-1-
phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-
oxadiazol-3(2H)-yl)prop-2-en-1-one (5k)
White powder; m.p. 211 °C; IR (KBr) vmax 3056, 3018,
2830, 1688, 1568, 1516, 1494, 1216 cm-1 1H NMR
;
(DMSO-d6, 400 MHz): d = 3.86 (3H, s, OCH3), 6.59 (1H,
123

724
Med Chem Res (2016) 25:712–727
Biological assay
d, J = 15.5 Hz, –COCH=), 6.84 (1H, s, C2-H oxadiazole
ring), 7.38–7.82 (13H, m, Ar–H), 7.98 (1H, d,
J = 16.0 Hz, =CH–Ar), 8.06 (2H, d, J = 7.9 Hz, C3-H &
C5-H pyridine), 8.20 (1H, s, pyrazole ring), 8.82 (2H, d,
J = 8.0 Hz, C2-H & C6-H pyridine); 13C NMR (DMSO-d6,
100 MHz): d = 167.2 (C, C=O), 160.7 (C, C-OCH3),
157.2 (C, C-5 oxadiazole ring), 149.7 (C, C-3, pyrazol
ring), 149.4 (2CH, C-2, C-6 pyridine ring), 147.7 (C, C-4
nitrophenyl ring), 141.6 (CH, Ph–CH=), 139.6 (C, C-1,
phenyl ring), 139.0 (C, C-1 nitrophenyl ring), 138.3 (C, C-4
pyridine ring), 135.1 (C, C-1 benzylidene ring), 129.7 (CH,
C-5 benzylidene ring), 129.1 (2CH, C-3, C-5, phenyl ring),
126.8 (CH, C-4 phenyl ring), 126.2 (2CH, C-2, C-6 nitro-
phenyl ring), 124.7 (2CH, C-3, C-5 nitrophenyl ring), 124.1
(2CH, C-3, C-5 pyridine ring), 123.0 (CH, C-4 benzylidene
ring), 120.7 (CH, C-6 benzylidene ring), 119.8 (2CH, C-2,
C-6 phenyl ring), 118.7 (CH, =CH–CO), 117.1 (C, C-4
pyrazol ring), 113.8 (CH, C-4 benzylidene ring), 113.2
(CH, C-2 benzylidene ring), 78.7 (CH, C-2 oxadiazol ring),
55.9 (CH3, OCH3); LCMS m/z = 572.17 [M?] ? (1) (74).
Anal.Calcd. for C32H24N6O5: C, 67.13; H, 4.22; N, 14.68;
Found: C, 67.26; H, 4.14; N, 14.58.
Antibacterial assay
Antibacterial studies of newly synthesized compounds 5a–l
were carried out against the representative panel of Gram-
positive [Staphylococcus aureus (MTCC-96), Streptococ-
cus pyogenes (MTCC-442)] and Gram-negative [Escher-
ichia coli (MTCC-443), Pseudomonas aeruginosa (MTCC-
1688)] bacteria using conventional broth dilution method
(Desai et al., 2012; Hannan, 2000). All MTCC cultures
were collected from Institute of Microbial Technology,
Chandigarh. The activity of compounds was determined as
per National Committee for Clinical Laboratory Standards
(NCCLS) protocol using Mueller–Hinton Broth (Becton–
Dickinson, USA). Primary screening was done first for
antibacterial activity in six sets against E. coli, S. aureus, P.
aeruginosa and S. pyogenes at different concentrations of
1000, 500, 250 lg/mL. The compounds found to be active
in primary screening were similarly diluted to obtain 200,
125, 100, 62.5, 50, 25 and 12.5 lg/mL concentrations for
secondary screening to test in a second set of dilution
against all microorganisms. Inoculum size for test strain
was adjusted to 106 CFU/mL (Colony Forming Unit per
milliliter) by comparing the turbidity (turbidimetric
method). Mueller–Hinton Broth was used as a nutrient
medium to grow and dilute the compound suspension for
test organisms. 2 % DMSO was used as a diluent/vehicle to
obtain the desired concentration of synthesized compounds
and standard drugs to test upon standard microbial strains.
Synthesized compounds were diluted to 1000 lg/mL con-
centration, as stock solution. The control tube containing
no antibiotic was immediately subcultured [before inocu-
lation] by spreading a loopful evenly over quarter of a plate
of medium suitable for the growth of test organisms. The
culture tubes were then incubated for 24 h at 37 °C, and
the growth was monitored visually and spectrophotomet-
rically. Ten lg/mL suspensions were further inoculated on
an appropriate media, and growth was noted after 24 h and
48 h. The lowest concentration (highest dilution) required
to arrest the growth of bacteria was regarded as minimum
inhibitory concentration (MIC), i.e., the amount of growth
from the control tube before incubation (which represents
the original inoculum) was compared. Solvent had no
influence on strain growth. The result of this was greatly
affected by the size of inoculum. The test mixture con-
tained 106 CFU/mL organisms. DMSO and sterilized dis-
tilled water were used as negative control, while
chloramphenicol antibiotic (1 U strength) was used as
positive control. Standard drug used in the present study
was ‘chloramphenicol’ for evaluating antibacterial activity
3-(4-methoxyphenyl)-1-(2-(3-(4-nitrophenyl)-1-
phenyl-1H-pyrazol-4-yl)-5-(pyridin-4-yl)-1,3,4-
oxadiazol-3(2H)-yl)prop-2-en-1-one (5l)
White powder; m.p. 231 °C; IR (KBr) vmax 3058, 3019,
2831, 1688, 1569, 1518, 1490, 1215 cm-1 1H NMR
;
(DMSO-d6, 400 MHz): d = 3.87 (3H, s, OCH3), 6.58 (1H,
d, J = 15.7 Hz, –COCH=), 6.85 (1H, s, C2-H oxadiazole
ring), 7.40–7.84 (13H, m, Ar–H), 7.98 (1H, d,
J = 15.8 Hz, =CH–Ar), 8.06 (2H, d, J = 7.9 Hz, C3-H &
C5-H pyridine), 8.21 (1H, s, pyrazole ring), 8.84 (2H, d,
J = 8.0 Hz, C2-H & C6-H pyridine); 13C NMR (DMSO-d6,
100 MHz): d = 167.1 (C, C=O), 159.7 (C, C-OCH3),
157.1 (C, C-5 oxadiazole ring), 149.8 (C, C-3, pyrazol
ring), 149.3 (2CH, C-2, C-6 pyridine ring), 147.7 (C, C-4
nitrophenyl ring), 141.7 (CH, Ph–CH=), 139.8 (C, C-1,
phenyl ring), 139.0 (C, C-1 nitrophenyl ring), 138.5 (C, C-4
pyridine ring), 130.3 (2CH, C-2, C-6 benzylidene ring),
129.2 (2CH, C-3, C-5, phenyl ring), 127.6 (C, C-1 ben-
zylidene ring), 126.8 (CH, C-4 phenyl ring), 126.3 (2CH,
C-2, C-6 nitrophenyl ring), 124.7 (2CH, C-3, C-5 nitro-
phenyl ring), 124.2 (2CH, C-3, C-5 pyridine ring), 123.1
(CH, C-5 pyrazol ring), 119.7 (2CH, C-2, C-6 phenyl ring),
118.8 (CH, =CH–CO), 117.3 (C, C-4 pyrazol ring), 114.2
(2CH, C-3, C-5 benzylidene ring), 78.9 (CH, C-2 oxadiazol
ring), 55.8 (CH3, OCH3); LCMS m/z = 572.17
[M?] ? (1) (70). Anal.Calcd. for C32H24N6O5: C, 67.13;
H, 4.22; N, 14.68; Found: C, 67.01; H, 4.31; N, 14.58.
123

Med Chem Res (2016) 25:712–727
725
Table 7 Physico-chemical properties computed for the substituted pyridyloxadiazole analogues to be used for building recursive partitioning
based classification model
Physico-chemical properties
Description
Structural descriptor
Electronic descriptors
Topological descriptors
Spatial descriptors
Kappa indices
The number of H-bond donor and acceptor
Charge, Apol, HOMO, LUMO, Dipole moment
Wiener index (W), Zagreb index (Zagreb), Hosoya index (Z), Kier and Hall molecular connectivity index (v)
Radius of Gyration, Jurs descriptors, Molecular volume, Area, Density, Principal moment of inertia
The shape of molecule
E_state_keys
The electrotopological interaction for each atom
which showed 50 lg/mL MIC against E. coli, P. aerugi-
nosa, S. aureus and S. pyogenes.
mitochondrial succinate dehydrogenase. This product was
eluted from cells by addition of 150 mL of DMSO. The
absorbance at 570 nm was determined by an ELISA using
a ELX800 microplate spectrophotometer. The IC50 value
was defined as the concentration at which 50 % of the cells
could survive.
Antifungal assay
The newly prepared compounds 5a–l were screened for their
antifungal activity as primary screening in six sets against C.
albicans, A. niger and A. clavatus at various concentrations
of 1000, 500, 250 lg/mL. The primary active compounds
were similarly diluted to obtain 200, 125, 100, 62.5, 50, 25
and 12.5 lg/mL concentrations for secondary screening to
test in a second set of dilution against all fungi. The fungal
activity of each compound was compared with griseofulvin
as a standard drug, which showed 500, 100 and 100 lg/mL
MIC against C. albicans, A. niger and A. clavatus, respec-
tively. For fungal growth, in the present protocol, we have
used Sabourauds dextrose broth at 28 °C in aerobic condi-
tion for 48 h. DMSO and sterilized distilled water were used
as negative control, while griseofulvin (1 U strength) was
used as positive control.
Computational studies
For each of the biological activity carried out, i.e., against
Gram-positive (S. aureus), Gram-negative (E. coli) bac-
teria and fungi (C. albicans), an individual recursive
partitioning model was derived using Discovery studio
version 4.0 (Accelrys Inc. USA) software. The antibac-
terial and antifungal activities of the molecules under
study have been described in terms of their minimum
inhibitory concentration (MIC). The entire dataset was
split into two classes as inactive (0) and active (1) with
the class 1 containing molecules with MIC more than
100 lg/mL, while those having an MIC below this
threshold value are categorized as class 2. The numerical
descriptors that represent topological, geometric/struc-
tural, electronic and thermodynamic characteristics of a
molecule (Table 7) were computed for deriving the RP
model and decision tree was generated from the results
thereof. Based on the correlation matrices, the descriptors
with zero variance as well as those columns containing
95 % of zero values were eliminated. Also those
descriptors with cross-correlation coefficient more than
0.5 were eliminated since; they represent nearly the same
information. The resulting uncorrelated descriptors
formed the independent variables (X), while the biologi-
cal activity served as the dependent variable (Y) for the
RP analysis. The activity classes were given equal weight
(weighing by classes) where an active node is a node in
which the fraction of actives exceeds the fraction of
inactive molecules over the entire dataset (in case of
‘weight by observables’ a node with more than half of the
molecules being active will be considered ‘active’). Here,
the inactive categorized in an ‘active’ node and the vice
versa are accounted as ‘misclassified’. The splits were
Preliminary cytotoxicity activity (MTT assay)
In vitro cytotoxicity activity of compounds 5a–l was
measured by means of the IC50 using the MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay
method (Mosmann, 1983; Alley et al., 1988). The IC50
determination was performed according to the National
Committee for Clinical Laboratory Standards (NCCLS)
recommendations. All compounds were dissolved in 0.1 %
DMSO with the stock concentration of 10 g/L, and diluted
with medium freshly before drug administration. Cell lines
were seeded into 96-well plates at density of 8 9 103
cells/well. After 24 h seeding, each compound dilution
was added in duplicate and incubation continued at 37 °C
in a humidified atmosphere containing 10 % FBS, 1 %
glutamine and 50 lM/mL gentamicin sulfates in a 5 %
CO2 and 95 % air. After 24 h, 20 lL MTT reagents was
added at 5 mg/mL in PBS (filter sterilized, light protected,
and stored at 4 °C) per well, and after 4 h of incubation at
37 °C, MTT was converted to a blue formazan product by
123

726
Med Chem Res (2016) 25:712–727
‘Dr. D. S. Kothari Post-Doctoral Fellowship’ and start up grants,
respectively.
scored using Twoing rule scoring function to reduce the
misclassification costs; the pruning factor was varied
from 0 to 3. The values for maximum tree depth (lay-
ers\10) were also varied between 5 and 10, while default
values were set for maximum number of generic splits
(30) and the number of knots per variable (20). The RP
model thus obtained was validated using cross-validation,
with the number of cross-validation groups set to 5.
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