Crystal, Hirshfeld, ADMET, drug-like and anticancer study of some newly synthesized imidazopyridine containing pyrazoline derivatives

Article abstract of DOI:10.1016/j.molstruc.2019.07.031

To steer the selection of a potent drug, computer models have been fostered as a valid alternative to reduce pharmacokinetics related failure. The present study mainly focuses on the relationship between molecular properties and anticancerous activity of some newly synthesized aza heterocycles. Twelve new imidazo[1,2-a]pyridine incorporated pyrazoline derivatives were synthesized and were well characterized by ¹HNMR, ¹³CNMR, LC-MS analysis. X-ray study resolved the structure of 4g, 4i and 4j as monoclinic crystal system. To quantify the electrostatic potential distribution and percentage intermolecular contacts in crystal packing, Hirshfeld surface study was performed. Moreover, virtual screening focused on ADMET and drug-like attributes to identify a promising derivative among the series. The anticancerous activity of the compounds was evaluated against A549 cell line. The study was further validated by subjecting best active compounds to induced hemolysis, which finally confirmed 4j as a potent molecule both in computational and in vitro study.

Full text of DOI:10.1016/j.molstruc.2019.07.031

Accepted Manuscript
Crystal, Hirshfeld, ADMET, drug-like and anticancer study of some newly synthesized
imidazopyridine containing pyrazoline derivatives
Sharanya Kuthyala, Manjuanatha Hanumanthappa, S. Madan Kumar, Sana Sheik,
Nagaraja Gundibasappa Karikannar, Ashwini Prabhu
PII:
S0022-2860(19)30865-8
DOI:
https://doi.org/10.1016/j.molstruc.2019.07.031
MOLSTR 26784
Reference:
To appear in: Journal of Molecular Structure
Received Date: 9 April 2019
Revised Date: 20 June 2019
Accepted Date: 5 July 2019
Please cite this article as: S. Kuthyala, M. Hanumanthappa, S. Madan Kumar, S. Sheik, N.
Gundibasappa Karikannar, A. Prabhu, Crystal, Hirshfeld, ADMET, drug-like and anticancer study
of some newly synthesized imidazopyridine containing pyrazoline derivatives, Journal of Molecular
Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.07.031.
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Graphicl Abstarct

ACCEPTED MANUSCRIPT
Crystal, Hirshfeld, ADMET, drug-like and anticancer study of some newly
synthesized imidazopyridine containing pyrazoline derivatives
Sharanya Kuthyala a , Manjuanatha Hanumanthappa b, Madan Kumar S c, Sana Sheik d,
Gundibasappa Karikannar Nagaraja *, Ashwini Prabhu e.
a Department of Chemistry, Mangalore University, Mangalagangothri, Karnataka, India,
b Department of Biotechnology Kuvempu University Shankaraghatta, Shimoga, Karnataka, India,
c DST-PURSE Lab, Mangalore University, Mangalagangothri, Karantaka, India.
d Department of Botany, St. Aloysius College, Mangalore, Karnataka, India,
e Yenepoya Research centre, Yenepoya (Deemed to be university), Deralakatte, Mangalore, Karnataka,
India
Abstract
To steer the selection of a potent drug, computer models have been fostered as a valid alternative
to reduce pharmacokinetics related failure. The present study mainly focuses on the relationship between
molecular properties and anticancerous activity of some newly synthesized aza heterocycles. Twelve new
imidazo[1,2-a]pyridine incorporated pyrazoline derivatives were synthesized and were well characterized
by 1HNMR, 13CNMR, LC-MS analysis. X-ray study resolved the structure of 4g, 4i and 4j as monoclinic
crystal system. To quantify the electrostatic potential distribution and percentage intermolecular contacts
in crystal packing, Hirshfeld surface study was performed. Moreover, virtual screening focused on
ADMET and drug-like attributes to identify a promising derivative among the series. The anticancerous
activity of the compounds was evaluated against A549 cell line. The study was further validated by
subjecting best active compounds to induced hemolysis, which finally confirmed 4j as a potent molecule
both in computational and in vitro study.
Keywords:
Imidazopyridine-pyrazoline;
X-Ray;
Hirshfeld;
ADMET;
Anticancer;
Hemocompatibility.
1. Introduction
Cancer burden is rapidly increasing across the globe. The world health organization is estimating
that globally almost one in six deaths are caused by this disease, which has led to around 9.6 million
deaths in 2018 [1]. It is one with highest levels of attrition due to the failure of potential anticancer drug
entering clinical development and challenge for a successful drug is still making its way over multiple

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hurdles. Virtually, the statistics are rather bleak in terms of potential drug to tackle the causes that are
fueling cancer [2].
The heterocycles are so ubiquitous in medicinal chemistry, their unique physicochemical property
and intrinsic versatility, have poised them as a key structural component of many anticancerous drugs
available in the market today [3]. In addition, defined with dual mode of action, the hybrid molecule
strategy can lead to powerful therapeutics in current advances [4], as they are capable of functioning as
one single molecule, thus minimizing the drug resistance and lowering the drug-drug interaction.
Recently, imidazopyridine, a fused heterocycle is featuring as Drug Prejudice prominently in the
field of medicinal chemistry [5]. Their prevalence in the field is, due to their various therapeutic
properties including anticancerous, anti-inflammatory, analagesics, anxiolytic, antiviral [6, 7] etc. It is
also a core structure of many of the established drugs such as zolpidem, nicopidem, alpidem, fadrazole,
olpinone, rifaximin, zolmidine, saripidine etc. [8]. Many studies have reported the anticancer activity of
certain imiazopyridines and their different mode of action but it is important to reduce the dose to the
least extent possible in order to minimize the undesirable toxic effects on the test system. Besides,
diversely substituted pyrazolines with variety of functional groups have contributed significantly in the
field of drug discovery. Many new compounds have been introduced as antimicrobial [9], anticancerous
[10] anti-inflammatory, analgesic [11, 12], antidepressant [13] etc and many patented [14]. It is precisely
reported that pyrazolines are not only useful in various types of cancer treatment but also have the ability
to act as cancer chemopreventive agents [15]. Given all of this, in the current work we targeted the
integration of imidazopyridine and pyrazoline in a single molecule as hybrid.
Molecular shapes or Hirshfeld surface analysis (HS) plays important role in understanding
chemistry and biochemistry. This study represents the boundaries of a molecule in the crystalline
environment and interprets electron density distributions and crystalline interactions [16, 17]. This HS is
associated with properties like di (distance to the nearest internal atom) and de (distance to the nearest

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external atom) and dnorm (distance between internal and external atoms normalized by Vander Waals radii,
thus visualizes the fidelity of the crystal structure.
To detect the safer and potential drug candidate ADMET profile has been fostered for the early
stage estimation of parameters in the discovery process. These computer models have constituted a valid
alternative in time and resource-consuming process of drug discovery and developments. Thus, in silico
study enables a key to access pharmacokinetic, physicochemical and other drug-like parameters for one or
more molecules with fast predictive models [18, 19].
2. Results and Discussion
2.1. Chemistry
Scheme 1. : i) 3-Chloro-2, 4-pentadione, DCM, reflux, 4h; ii) -2OH/-3Cl Benzaldehyde, ethanol,
40% NaOH, RT, 24 h; iii) NH2NH2.H2O, Acetic/Propionic/Butyric acid, reflux, 8 h.
Fig. 1. Mechanism of pyrazoline formation.
Synthetic route and mechanism for the objective compounds are shown in Scheme 1 and Fig. 1.
As depicted, the synthesis of intermediate (3a-3d) was achieved in two steps [4], further cyclization with
the hydrazine hydrate in the presence of excess acetic/propionic/butyric acid gave the targeted hybrid

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molecules (4a-4l) in good yield (Table 1). The structures of all the synthesized compounds were analyzed
by FT-IR, 1HNMR, 13CNMR, LC-MS spectroscopy.
Sample
Code
R
R1
R2
Molecular
formula
Molecular
weight
%
Yield
Melting
point
(0C)
4-CH3-pyr
4-CH3-pyr
5-CH3-pyr
5-CH3-pyr
4-CH3-pyr
4-CH3-pyr
5-CH3-pyr
5-CH3-pyr
4-CH3-pyr
4-CH3-pyr
5-CH3-pyr
5-CH3-pyr
2-OH-C6H5
3-Cl-C6H5
2-OH-C6H5
3-Cl-C6H5
2-OH-C6H5
3-Cl-C6H5
2-OH-C6H5
3-Cl-C6H5
CH3CO-
CH3CO-
C20H20N4O2
C20H19ClN4O
C20H20N4O2
C20H19ClN4O
C21H22N4O2
C21H21ClN4O
C21H22N4O2
C21H21ClN4O
C22H24N4O2
C22H23ClN4O
C22H24N4O2
C22H23ClN4O
348.3984
366.8440
348.3984
366.8440
362.4642
380.8706
362.4642
380.8706
376.4515
394.8972
376.4515
394.8972
69
72
73
72
65
67
66
66
79
78
79
79
238-240
4a
4b
4c
4d
4e
4f
186-188
290-292
208-210
148-150
162-164
234-236
154-156
124-126
220-222
128-130
156-158
CH3CO-
CH3CO-
CH3CH2CO-
CH3CH2CO-
CH3CH2CO-
CH3CH2CO-
4g
4h
4i
2-OH-C6H5 CH3CH2CH2CO-
-3-Cl-C6H5 CH3CH2CH2CO-
2-OH-C6H5 CH3CH2CH2CO-
4j
4k
4l
3-Cl-C6H5
CH3CH2CH2CO-
Table 1. Physical data of imidazopyridine-pyrazoline hybrid molecules.
The FT-IR spectrum, manifested the presence of characteristic bands for O-H, C-H, C=O, C=C
and C=N group. The bands for 4a were observed at 3130, 2931, 1641, 1581 and 1456 cm-1 respectively. In
1HNMR spectrum (Fig.2) signal for acetyl-CH3, pyridine-CH3 and imidazole-CH3 protons resonated as a
singlet at 2.381, 2.417 and 2.472 ppm respectively. HA proton resonated at 3.106 ppm as a doublet of
doublet with coupling constant JAB = 17.2 Hz and JAX = 4.4 Hz. Similarly HB and HX appeared as doublet
of doublet at 4.017 and 5.581 ppm with the coupling constants JBA = 17.2 Hz & JBX = 12 Hz and JXA = 4.4
Hz & JXB = 11.6 Hz respectively. Among the aromatic protons, the signal for 2- hydroxyphenyl-5H
appeared at 6.704 ppm as a triplet with coupling constant J=7.6 Hz. The 2-hydroxyphenyl-6H proton
resonated at 6.834 ppm as a doublet with coupling constant 8 Hz. At 6.890 ppm, a doublet appeared for 2-
hydroxyphenyl-3H with J= 7.6 Hz. A singlet for imidazopyridine-9H was found at 7.424 ppm and a
doublet at 9.294 with J value 6.8 Hz was observed for imidazopyridine-6H. Phenolic-OH proton
resonated at 9.744 ppm as a singlet and remaining two aromatic protons resonated as multiplets at 7.031-
7.092 ppm respectively.. In 13CNMR, peaks for three methyl groups appeared at 15.7 ppm (acetyl-CH3),

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20.7 ppm (pyr-CH3) and 21.9 ppm (imid-CH3) respectively. All other characteristic peaks were within
43.1-166.9 ppm. LC-MS showed molecular ion peak at 349.80 (M+1) (Fig. 3).
Fig 2. 1HNMR of 4a.
Fig. 3. LC-MS of 4a.

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2.2. Single crystal X-Ray crystallography study
The proposed molecular structure for the compound 4g, 4i and 4j were confirmed by the single
crystal XRD studies and are shown in Fig. 4. In the unit cell of the compound 4g, two molecules were
crystallized with solvent molecules (water and ethanol). The dihedral angles between pyrazole
(Cg6/Cg2:N3A/B-N4A/B-C10A/B-C12A/B) and phenyl rings (Cg8/Cg4:C13A/B-C18A/B) in molecule
A and molecule B were 77.76(15)0 and 87.56(17)0 respectively. The crystal structure was stabilized via
intermolecular hydrogen bonds (O---H...O, O---H...N, C---H...O) and intermolecular interactions (C---
H...π and π...π). The Cg1...Cg7: Cg3 N1B/C2B-C3B/C5B-C7B)...Cg7 and Cg6...Cg8 (C13A-C18A)
exists with a distance of 3.6758 (17), 3.6167 (16) and 3.7514(16) respectively.
The compound 4i crystallizes with butyric acid solvent. The dihedral angle between Cg1 (N1-
N2/C7-C9) and Cg4 (C1-C6) is 73.8(3)°. Intramolecular hydrogen bonds of the type C---H…N were
observed. The crystal structure was stabilized through intermolecular hydrogen bonds (O--- H…O),
intermolecular interactions (C---H…π) and π...π interactions between Cg2 (N3/C10/C11/N4/C13)…Cg2
(3.570 Å, symmetry 1-X,1-Y,1-Z).
In the crystal structure of 4j, the Cg2 (N3-N4/C8-C10) makes dihedral angles of 77.16(12)0 (C11-
C16) and 18.17(10)0 (N1/C1-C5/N2-C6-C7), with Cg4 and Cg5, respectively. The dihedral angle
between Cg4 and Cg5 is 80.25(10)0 and intramolecular hydrogen bonds C---H...N were observed. The
crystal structure was stabilized via intermolecular hydrogen bonds (C---H...O and C---H...N) and
intermolecular interactions (C---H...π and π...π) (Fig.4.). The Cg2...Cg4: exists with a distance of 3.7669
(14) Å.
Parameters
4g
4i
4j
CCDC number
Empirical formula
1568088
2(C21H22N4O2),
H2O, C2H6O
1889128
(C22H24N4O2)0.5,
(C4 H7O2)0.5
231.27
1587575
C22H23ClN4O
Formula weight
Temperature
Wavelength
788.93
293(2) K
0.71075 Å
394.89
293(2) K
0.71075 Å
293(2)
0.71075 Å

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Crystal system, space
group
monoclinic,
monoclinic,
monoclinic,
P21/n
P21/c
I2/a
Unit cell dimensions
a = 12.2407(6)
a = 15.3022(16)
b = 16.2017(14)
c = 20.3832(17)
β = 100.025(10)
a =8.5348(6) Å
b=29.9913(14)
Å
Å
b = 14.4681(8)
Å
c = 8.8956(7) Å
c = 23.9361(11)
β
= 115.865(9) º
Å
β
= 92.701(4) º
Volume
Z, Calculated density
Absorption coefficient
F(000)
4234.4(4) Å3
4, 1.238 Mg/m3
0.084 mm-1
1680
4976.3(8) Å3
16,1.237 Mg/m3
0.085 mm-1
1976
2048.9(3) Å3
4, 1.280 Mg/m3
0.206 mm-1
832
Crystal size
0.21x0.23x0.24
mm
0.17 x 0.23 x 0.27
mm
0.25
x0.32x+0.41
mm
Theta range for data
collection
1.7o to 25.7°
2.5o to 25.0o
2.6o to 25.0°
Limiting indices
-14
≤
≤
h
≤
14, -17 -14
17, -29
29
44905/8031
≤
h
≤
18, -19
≤ l ≤
≤
k
-10
≤
≤
h
≤
10, -35
35, -10
10
20789/3609
≤
k
≤
l
≤
k
≤ l
≤
19,-24 24
≤
≤
Reflections collected /
unique
25151/4388 [R(int)
= 0.105]
[R(int) = 0.068]
[R(int) = 0.048]
R - value
Refinement method
0.0694
0.0965 (0.2669)
Full-matrix least-
squares on
0.0492
Full-matrix
least-squares on
F2
Full-matrix
least-squares on
F2
Data / restraints /
parameters
4948 / 0 / 547
F24388 / 0 / 316
2875 / 0 / 256
Goodness-of-fit on F2
1.04
1.04
1.04
Largest diff. peak and
hole
0.23 and−0.22.
Å-3
0.24 and −0.25
0.29 and −0.32.
Å-3
Table 2. Crystal structure and refinement parameters.

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Fig. 4. ORTEP of 4g, 4i and 4j.
2.3. Hirshfeld analysis
A graphical tool ‘Hirshfeld surfaces computational method’ was assessed, to explore and
visualize the intercontacts within the crystal lattice [20]. Conventional mapping of dnorm on molecular
Hirshfeld surfaces were highlighted and given in supplementary and Fig. 5 for 4g and 4j respectively.
Also the percentages of intercontacts were calculated and the 2D- finger print plots shown in the

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supplementary details and Fig.5. The compound 4g exhibits; C…C (2.8%), C…H (16.69 %), H…H (60.1
%), H…N (5.9 %), N…C (2.7 %), O…H (11.4 %) and other interactions in molecule A. In molecule B,
C…C (2.6%), C…H (16.3%), H…H (58.7%), H…N (6.2%), N…C (2.7%), O…H (12.8%) and other
interaction were the major interactions observed. In the associated ethanol molecule mainly H…N (7.8%),
H…H (78.8%) and O…H (12.7%) with other interaction were found and C…H (3.2%), H…N (10.8%),
O…H (40.1%) and H…H (45.7%) were observed in associated water molecule respectively. The
intercontacts C…C (1.3%), C…Cl (2.4%), C…H (17.9%), Cl…H (8.8%), Cl…N (1.4%), H…H (51.3%),
N…H (8.8%), O…H (74%) with other interactions were observed for 4j. Also the electrostatic potential
was mapped using wave function STO-3G basis set at Hatree-Fock theory over the range of ±0.030 au
and are given in supplementary details and Fig.5 [5]. The hydrogen donor potential is represented by blue
region, which indicates positive electrostatic potential, where as the negative electrostatic potential
(hydrogen bond acceptor) is visualized by red region.
Fig.5. a. Hirshfeld surfaces and their corresponding 2D finger print plots for the compound
4j; b. Electrostatic potential mapped on Hirshfeld surface (different orientations) with +-20 au. Red
regions correspond to negative electrostatic potential and blue region corresponds to positive
electrostatic potential.

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2.4. ADMET prediction
Predicted ADMET score has been tabulated in Table 3. Absorptivity efficacy of all 12 derivatives
were found to be very good (96.17-99.61%) via human intestinal tract. In addition they have shown
medium permeability in Caco-2 cell model, Madin-Darbg Canine Kidney (MDCK) model and Blood
Brain Barrier (BBB) Model. The pharmacodynamic behaviour of drug depends on uptake and
distribution, can possibly be ascertained by Plasma Protein Binding. Here the molecules have shown
medium binding affinity. Also, the toxicity prediction score, the carcinogenic and mutagenic effects of
pyrazoline derivatives were evaluated. All the compounds except 4i exhibited negative results to mouse
toxicity.
Comp.
HIA
(%)
BBB (%)
Caco2
(nm/sec) (nm/sec)
MDCK
PPB (%)
Ames
test
Mouse
toxicity
96.17
97.51
96.17
97.51
96.19
97.56
96.19
99.61
96.22
97.61
96.22
97.61
0.3604
1.296
28.30
38.35
29.11
38.36
32.43
41.22
33.06
55.43
35.22
43.83
35.87
43.83
21.96
123.38
41.76
79.92
2.287
27.39
30.51
7.827
0.9392
26.82
8.378
8.695
80.40
90.38
80.38
90.13
87.06
91.22
86.91
91.49
88.33
91.00
88.19
90.82
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Negative
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.2618
1.8250
0.3281
1.0342
0.2496
0.6967
0.2970
0.7791
0.2467
1.1975
4j
4k
4l
*Classification: (% HIA): 0-20 % (poorly absorbed) 20-70 % (moderately absorbed), 70-100 % (well
absorbed); BBB: 1(CNS active), < 1 (CNS inactive); Caco-2 cell permeability: < 4 (low
permeability), 4-70 (medium permeability), 70 (higher permeability); MDCK: < 25 (low
˃
˃
permeability), 25-500 (medium permeability), ˃ (higher permeability); plasma protein binding: > 90
(strongly bound), < 90 (weakly bound).
Table 3. ADMET Property Evaluation.
2.5 Drug-like prediction study
The concept of drug ability involves consideration of how chemical properties compare with
those approved drug to reduce the failure in clinical trials. Drug-like score has been tabulated in Table 4.
The number of rotatable bonds in 4i to 4j molecules is found to be 4 indicating more flexibility than 4a to
4h molecules (2 to 3). All molecules are having nitrogen bonding ability in the range from 5 to 6, while

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the molecules 4a, 4c, 4e, 4g, 4i and 4k are showing single hydrogen bonding ability and the remaining
molecules are having no hydrogen bonding property. Hydrogen and nitrogen bonding ability of all
molecules has been directly related to TPSA property, where we found parallel increased value of 4a, 4c,
4e, 4g, 4i and 4k molecules (70.21) indicating higher TPSA property of those molecules, whose H and N
bonding ability is more. Lipophilicity (milog P) is important property, which influences oral availability
of the molecules. The miLogp of 4f, 4h, 4i, 4j, 4k and 4l molecules lies in the range 4.64 to 5.20,
indicating higher lipophilicity compared to 4a to 4e and 4g molecules (3.09 to 3.90). However, two
molecules (4j and 4l) are having highest 5.20 value (
≤ 5). Interestingly, milogp value proportionately
increased with increased volume of these molecules showing drug-like property.
Compound
MW miLogP TPSA nON nOHNH nrotb Volume
348.41
366.85
348.41
366.85
362.43
380.88
362.43
380.88
376.46
394.91
376.46
394.91
3.09
3.80
3.09
3.80
3.93
4.64
3.93
4.64
4.49
5.20
4.49
5.20
70.21
49.98
70.21
49.98
70.21
49.98
70.21
49.98
70.21
49.98
70.21
49.98
6
5
6
5
6
5
6
5
6
5
6
5
1
0
1
0
1
0
1
0
1
0
1
0
2
2
2
2
3
3
3
3
4
4
4
4
316.17
321.69
316.17
321.69
332.97
338.49
332.97
338.49
349.77
355.29
349.77
355.29
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
Table 4: Drug likeness score for compounds.
2.6. Anticancer study
Among the series of novel imidazopyridine hybrid derivatives tested, the compounds 4b, 4f, 4j
and 4d were found to possess significant anticancer activity with IC50 values of 43.56, 53.35, 44.49 and
48.52 µM respectively, against A549 lung adenocarcinoma cells. Increase in the cytotoxicity was found to
be concentration-dependent with these test compounds. IC50 values of the test compounds on A549 cells
are represented in Table 5. Cisplatin was used as a standard drug, which showed an IC50 value of 14.87
µM. Thus compounds 4b and 4j may be considered as a promising effective compound for further
validations.
Compound
Cytotoxicity
IC50 (µM)

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(100µM, mean %
± SD)
4a
4b
4c
4d
4e
4f
4g
4h
4i
2.08±0.31
80.30±0.61
0.79±0.19
77.09±0.69
3.33±0.40
71.17±0.71
58.82±1.14
34.89±0.87
13.64±0.57
85.31±0.71
8.63±1.73
41.26±0.23
-
43.56
-
48.52
-
53.35
-
-
-
4j
4k
4l
44.49
-
-
Table 5. Cytotoxicity of the compounds (4a-4l) on A549 cellline.
2.7. HRBC’s membrane stabilization
The prevention of hypotonicity induced human erythrocyte membrane lysis was assessed and
expressed as percentage protection. Thus the extent of inhibition against haemolysis was taken as a
measure of anti-inflammatory activity. The compounds at its 100 ꢀg/mL concentration showed 59-63 %
protection as compared with 66.9% protection produced by the Diclofenac at same concentration (Fig. 6),
where 4j was found to be most active with 63% protection.
Fig. 6. Percentage HRBC’s membrane stabilization of 4b, 4d, 4f and 4j.
3. Experiments
3.1 Materials and methods

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All the required chemicals were purchased from Spectrochem India and Sigma Aldrich. These
were used without any further purification. To check progress of the reaction Thin Layer Chromatography
plate-aluminium sheet was used which was coated with Silicagel 60F254. These were visualized by UV
1
light. Melting point was determined by open capillary method and it was uncorrected. HNMR and
13CNMR spectra were recorded on Bruker Avance III, 400MHz and Bruker Ascend, 400 MHz
respectively using DMSO as solvent. The IR spectrum was analysed on Bruker Alpha spectrometer, LC-
MS was carried out on Schimadzu LCMS-8030. The X-ray intensity data was collected at 296 K on a
Rigaku Saturn724 diffractometer using graphite monochromated Mo-Kα radiation.
3.3 General procedure for the synthesis of 4a-4l
The mixture of imidazopyridine-chalcone 3a-3d (0.01mol), hydrazine hydrate (0.01mol) in
excess of acetic/ propionic/butyric acid was heated under reflux for 6 hours. The reaction mixture was
cooled; the solid product formed was filtered, washed with cold water and recrystallized using suitable
solvent mixture.
3.2. Single crystal X-Ray crystallography study
Good quality crystals of 4g, 4i and 4j were grown in dimethylformamide solvent by slow
evaporation of the solvent at room temperature. The asymmetric unit of the title compounds is given in
Fig. 4. The crystal parameters and data refinement are given in the Table 2. The detailed specification
about software and steps followed are given in supplementary details. The structure was solved by direct
methods and refined by full-matrix least squares method on F2. Detailed account on structure refinement
of all the three crystals has been included in supplementary details.
3.3. Hirshfeld analysis
The intermolecular interactions within the crystal structure are quantified and visualized using
Hirshfeld surfaces computational method [21, 22, 23, 24]. The percentages of intercontacts are calculated

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and the 2D- finger print plots are shown in the figure. The Hirshfeld surfaces are highlighted by
conventional mapping of dnorm on molecular Hirshfeld surfaces.
3.4. ADMET prediction
The ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) is pre-requisite in
drug design and discovery studies since the attributes of the molecules play a critical role in pre-clinical
and clinical phase. PreADMET property is a web-based application software used to predict ADMET
(http://preadmet.bmdrc.kr/) of (4a to 4l) pyrazole derivatives. Further details are given in supplementary
details.
3.5. Drug-like Prediction study.
The concept of drug ability involves consideration of how chemical properties compare with
those approved drug to reduce the failure in clinical trials. In the present work, the molecular properties
and bioactivity of imidazopyridine derivatives (4a to 4l) was evaluated using Molinspiration
cheminformatics server (http:// www.molinspiration.com/). The server has wide range of tools for the
dispensation and manipulation of molecules including generation of molecule fragmentation,
tautomerization, normalization, calculation of various molecular properties needed in QSAR (Quantitative
Structure Activity Relationship) study such as molecular descriptors, selection of training test etc., also
supports fragment and crystal based virtual screening. In addition, the server calculates the molecular
properties based on Lipinski/ Pfizer's Rule of five (RO5) [25] to evaluate drug-like study that would make
it a likely orally active predicting bioactivity score for the most important therapeutic targets like GPCR
receptor kinase inhibitors, ion channel modulators, enzymes and nuclear receptors [26].
3.6. Anticancerous Study
Cytotoxicity of the compounds on A549 cells was evaluated using MTT assay [27], the details are
given in supplementary details.
3.7. HRBCs membrane stability assay

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The membrane stability assay was performed by slight modified method of Padmanabhan and
Jangle [28]. The detailed steps followed are discussed in supplementary details.
Conclusion
Herein we report a concise approach to synthesize a hybrid system containing imidazopyridine
and pyrazoline scaffolds. It was successfully characterized by FT-IR, 1HMR, 13CNMR, LC-MS and single
crystal XRD techniques. Hirshfeld surface analysis visualized the electrostatic potential distribution and
intermolecular close contacts in crystal packing. Through virtual screening 4b, 4d, 4f, 4h, 4j and 4l found
to be with good ADMET properties. Whereas, 4j and 4l showed good lipophilicity character. Further,
from MTT assay, 4b and 4j ascertained good anticancerous activity on A549 cell line. Additionally,
hemocompatibilty study validated the compound 4j with very good membrane stabilizing property. Thus
results of the present investigation reveals an insight into the possibility of developing 4j as potential
chemotherapeutic agents for lung cancer, after appropriate validation with pre-clinical and clinical
studies.
Acknowledgement
Authors are grateful to DST-PURSE-Mangalore University; STIC-Cochin, Kerala; MIT-
Manipal. Karnataka for providing spectroscopy analysis.
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Highlights
•
•
New imidazopyridine-pyrazolines were synthesized and well characterized.
Crystal structures of 4g, 4i and 4j were well defined and analyzed their Hirhsfeld
surfaces.
•
•
•
Virtual screening predicted ADMET property, Druglike and bioactivity scores.
Anticancerous activity against A549 was evaluated and validated by docking study.
HRBCs membrane stabilization property of most active compounds was evaluated.