Theoretical and computational insight into the supramolecular assemblies of Schiff bases involving hydrogen bonding and C[sbnd]H…π interactions: Synthesis, X-ray characterization, Hirshfeld surface analysis, anticancer activity and molecular docking analysis

Article abstract of DOI:10.1016/j.molstruc.2021.130223

The present study examines the significance of various non-covalent interactions in the supramolecular assembly of (E)-1-(1-(4-nitrophenyl)ethylidene)-2-phenylhydrazine 1c and (E)-3?bromo-N'-(1-phenylethylidene)benzohydrazide 2d. The synthesized compounds were fully characterized by spectroscopic methods and single crystal X-ray diffraction analysis. The topology of the supramolecular assemblies was controlled by various non-covalent interactions including classical hydrogen bonding, C[sbnd]H…π and Br…Br interactions which were examined in detail using several theoretical methods and DFT calculations. The optimized geometric parameters of compounds 1c and 2d were calculated using density functional theory (DFT/B3LYP) quantum chemical method with the 6–311++G(d,p) basis set using the crystallographic coordinates. Additionally, fragments contributing to the HOMO and LUMO molecular orbitals were investigated at the same level of theory. The nature and various types of intermolecular interactions in the crystal structures was also investigated by Hirshfeld surface analysis. The synthesized Schiff bases were also studied for their potential as drugs and physicochemical properties. Bioevaluation against four cancer cell lines (NCI-H460, NCI-H460/Bcl-2, MDA-MB-231 and MCF-7) showed that compound 1c was a more potent inducer of toxicity compared to 2d. The putative binding modes of the bioactive Schiff bases were investigated using molecular docking tools and the results revealed that both the inhibitors were stabilized in the active pocket of the enzyme via the formation of various interactions with the key amino acid residues.

Full text of DOI:10.1016/j.molstruc.2021.130223

Journal of Molecular Structure 1235 (2021) 130223
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Theoretical and computational insight into the supramolecular
–
assemblies of Schiff bases involving hydrogen bonding and C H…π
interactions: Synthesis, X-ray characterization, Hirshfeld surface
analysis, anticancer activity and molecular docking analysis
Hina Andleeba,b,∗, Lubna Danishb, Shiza Munawarb, Muhammad Naeem Ahmedc,
Imtiaz Khand,∗, Hafiz Saqib Alid, Muhammad Nawaz Tahire, Jim Simpsonf,∗,
Shahid Hameeda,∗
a Department of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan
b Sulaiman Bin Abdullah Aba Al-Khail – Centre for Interdisciplinary Research in Basic Science (SA-CIRBS), Faculty of Basic and Applied Sciences,
International Islamic University, Islamabad, Pakistan
c Department of Chemistry, The University of Azad Jammu & Kashmir, Muzaffarabad 13100, Pakistan
d Department of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, United
Kingdom
e Department of Physics, University of Sargodha, Sargodha, Pakistan
f Department of Chemistry, University of Otago, P.O. Box 56, Dunedin, 9054, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study examines the significance of various non-covalent interactions in the supramolec-
ular assembly of (E)-1-(1-(4-nitrophenyl)ethylidene)-2-phenylhydrazine 1c and (E)-3–bromo-N’-(1-
phenylethylidene)benzohydrazide 2d. The synthesized compounds were fully characterized by spectro-
scopic methods and single crystal X-ray diffraction analysis. The topology of the supramolecular assem-
Received 29 November 2020
Revised 21 February 2021
Accepted 1 March 2021
Available online 6 March 2021
–
blies was controlled by various non-covalent interactions including classical hydrogen bonding, C H…π
and Br…Br interactions which were examined in detail using several theoretical methods and DFT cal-
culations. The optimized geometric parameters of compounds 1c and 2d were calculated using density
functional theory (DFT/B3LYP) quantum chemical method with the 6–311++G(d,p) basis set using the
crystallographic coordinates. Additionally, fragments contributing to the HOMO and LUMO molecular or-
bitals were investigated at the same level of theory. The nature and various types of intermolecular inter-
actions in the crystal structures was also investigated by Hirshfeld surface analysis. The synthesized Schiff
bases were also studied for their potential as drugs and physicochemical properties. Bioevaluation against
four cancer cell lines (NCI-H460, NCI-H460/Bcl-2, MDA-MB-231 and MCF-7) showed that compound 1c
was a more potent inducer of toxicity compared to 2d. The putative binding modes of the bioactive Schiff
bases were investigated using molecular docking tools and the results revealed that both the inhibitors
were stabilized in the active pocket of the enzyme via the formation of various interactions with the key
amino acid residues.
Keywords:
Non-covalent interactions
Schiff base
Supramolecular assembly
Hirshfeld analysis
Electronic transitions
Anticancer activity
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
tigation of such self-assembly processes has fueled much research
in this active area of research [1,2]. These systems mainly rely
The construction of solid-state architectures utilizing purely
organic molecules has witnessed tremendous growth in
upon non-covalent interactions (i.e., hydrogen bonding, π…π in-
teractions, C–H…π interactions, and van der Waals interactions)
and this underpins their interesting applications in the use of
molecular recognition and complex biological systems [3-6] show-
ing their diverse utility as a powerful tool for the construction of
supramolecular architectures [7,8]. Among them, nonconventional
C–H/X (X = O, N, S and π electrons) hydrogen bonding [9-13] has
gained immense attention due to their ability to build supramolec-
a
supramolecular chemistry and crystal engineering, and the inves-
∗
Corresponding author.
E-mail
addresses:
hina.andleeb@iiu.edu.pk
(H.
Andleeb),
imtiaz.khan@manchester.ac.uk (I. Khan), jsimpson@alkali.otago.ac.nz (J. Simpson),
shameed@qau.edu.pk (S. Hameed).
https://doi.org/10.1016/j.molstruc.2021.130223
0022-2860/© 2021 Elsevier B.V. All rights reserved.

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
ular structures [14–19], biological structures [20–24] and anion re-
ceptors [25–28]. Inter- and intra-molecular hydrogen bonding have
been employed extensively in the generation of complex organized
systems due to the reversibility, specificity, directionality and co-
operativity of such interactions [29]. Among the weak hydrogen
bonds, C–H…π interactions are of considerable significance dis-
playing several unique features that have impacts on chiral recog-
nition, polymer chemistry, coordination chemistry, biochemistry,
and the structures of DNA and proteins [30]. In view of these sig-
nificant applications, and as a continuation of our work in this area
[31], it is of much interest to explore the role of non-covalent inter-
actions in new classes of compounds forming supramolecular ar-
chitectures. Hence, in the present report, we showcase the synthe-
sis and X-ray characterization of two Schiff bases namely (E)−1-
(1-(4-nitrophenyl)ethylidene)−2-phenylhydrazine 1c and (E)−3–
bromo-N’-(1-phenylethylidene)benzohydrazide 2d. The detailed de-
scription of the crystal packing and supramolecular assemblies
s), 8.37 (2H, d, J = 8.4 Hz), 7.98 (2H, d, J = 8.4 Hz), 7.20 (2H, d,
J = 7.8 Hz), 6.88–6.84 (1H, m), 6.79–6.72 (2H, m), 3.56 (3H, s); 13C
NMR (75 MHz, DMSO–d6) (δ/ppm): 146.74, 144.70, 142.80, 133.82,
129.21, 126.09, 123.90, 120.28, 112.78, 15.91; HRMS (ESI +ve): ex-
act mass calculated for C14 H14 N3O2 [M + H]+: 256.0851; found,
256.0844.
2.2.2. Preparation of
(E)−3–bromo-N’-(1-phenylethylidene)benzohydrazide (2d)
Step 1. To
a stirred solution of 3-bromobenzoic acid 2a
(0.02 mol) in ethanol (25 mL) was added concentrated sulfuric acid
(few drops) and the reaction mixture was heated to reflux. The
progress of the reaction was monitored by TLC at regular intervals.
After completion of the reaction (4 h), the mixture was concen-
trated in vacuo. The crude mixture was dissolved in ethyl acetate
and washed with 10% aqueous sodium bicarbonate (2 × 25 mL).
The organic layer was dried (Na2SO4), filtered and concentrated in
vacuo to afford ester 2b which was used in the next step without
further purification.
–
driven by hydrogen bonding, C H…π and Br…Br interactions is re-
ported. Further insights into these intermolecular interactions were
obtained using Hirshfeld surface analysis. Moreover, the biologi-
cal applications of the synthesized compounds were tested against
four human cancerous cell lines, i.e.; NCI-H460, NCI-H460/Bcl-2,
MDA-MB-231 and MCF-7. In addition, molecular docking analy-
sis was performed to elucidate the putative binding modes of the
Schiff bases.
Step 2. To a stirred solution of ethyl 3-bromobenzoate 2b
(0.01 mol) in ethanol (15 mL) was added hydrazine hydrate (80%,
0.08 mol). The resulting mixture was heated at reflux and progress
was monitored by TLC at regular intervals. After completion of the
reaction (5 h), the mixture was concentrated in vacuo. The crude
solid was filtered off, washed with cold water, and recrystallized
from 80% EtOH/H2O to afford 3-bromobenzohydrazide 2c [33].
Step 3. To a stirred solution of 3-bromobenzohydrazide 2c
(10 mmol) in ethanol (15 mL) was added acetophenone (11 mmol)
followed by glacial acetic acid (0.5 mL). The resulting mixture
was heated at reflux and the progress was monitored by TLC at
regular intervals. After completion of the reaction (30 min), the
mixture was concentrated in vacuo. The crude solid was filtered
off, washed with cold water, and recrystallized from ethanol to
afford (E)−3–bromo-N’-(1-phenylethylidene)benzohydrazide 2d as
transparent crystals in 85% yield [33]. m.p. 215–219 °C; FTIR (ATR,
2. Experimental
2.1. Chemicals and instrumentation
Reagents and solvents used for the synthesis of compounds
were purchased from Sigma-Aldrich, Merck, Alfa Aesar, and were
used without any further purification. All the reagents used were
of analytical grade. All reactions were carried out using oven-dried
glassware. All the reactions were monitored using pre-coated silica
gel (60F254 0.2 mm) TLC plates from Merck (Germany). The prod-
uct spots were visualized under UV light at 254 nm. Melting points
were recorded on a Gallenkamp melting point apparatus (MPD) in
open capillaries and are uncorrected. Infra-red (IR) spectra were
recorded on a Schimadzu Fourier Transform Infra-Red Spectropho-
tometer model 270 using the ATR (Attenuated total reflectance) fa-
cility. NMR spectra were recorded on a Bruker AV300 spectrom-
eter at room temperature. 1H and 13C NMR spectra were refer-
enced to external tetramethylsilane via the residual protonated sol-
vent (1H) or the solvent itself (13C). Chemical shifts are quoted in
parts per million (ppm). For DMSO–d6, the shifts are referenced to
2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR spectroscopy.
Coupling constant (J) values are reported to the nearest 0.5 Hz.
High-resolution mass spectra were recorded on a Micromass LCT
electrospray ionization mass spectrometer operating at a resolution
of 5000 full width half height.
cm−1): 3300 (N H), 1597 (C = N), 1550 (N = O stretch); 1H NMR
–
(300 MHz, DMSO–d6) (δ/ppm): 10.94 (1H, s), 8.06 (1H, s), 7.87–
7.80 (2H, m), 7.77–7.51 (2H, m), 7.48–7.35 (4H, m), 2.38 (3H, s); 13C
NMR (75 MHz, DMSO–d6) (δ/ppm): 180.75, 162.99, 156.89, 138.46,
136.76, 134.66, 131.03, 130.06, 128.84, 127.55, 126.96, 122.04,
15.26; HRMS (ESI +ve): exact mass calculated for C15H14 BrN2O
[M + H]+: 317.0211; Found, 317.0202.
2.3. Crystal growth development
Single crystals of compounds 1c and 2d suitable for X-ray
diffraction analysis were grown at room temperature from ethanol
as the solvent.
2.4. X-ray structure determination
Diffraction data for the compounds were collected at 296(2)
2.2. Synthesis
K
for 1c and at 293(2) K for 2d using Mo-Kα radiation
˚
(λ = 0.71073 A) on a Bruker Kappa APEXII CCD diffractometer,
processed using APEX2 and SAINT [34] with multi-scan absorp-
tion corrections applied using SADABS [35]. The structures were
solved by direct methods (SHELXS-97) [36] and refined using full-
matrix least-squares procedures with SHELXL-2018 [37] and TI-
TAN2000 [38]. All non-hydrogen atoms were refined anisotrop-
ically and hydrogen atoms were placed in calculated positions
with their thermal parameters refined isotropically with Ueq(H)
1.2Ueq(N/C). Molecular plots and packing diagrams were drawn us-
ing Mercury [39] and additional metrical data were calculated us-
ing PLATON [40]. Tables were prepared using WINGX [41]. Details
of the X-ray measurements and crystal data for the compounds are
given in Table 1. Crystals of 2d were not of high quality and this is
2.2.1. Preparation of
(E)−1-(1-(4-nitrophenyl)ethylidene)−2-phenylhydrazine (1c)
To a stirred solution of phenylhydrazine (1 mmol) in ethanol
ꢀ
(2 mL) at 0 °C was added 4 -nitroacetophenone (1 mmol) followed
by glacial acetic acid (0.5 mL). The reaction mixture was stirred at
0–15 °C for 30 min. The reaction progress was monitored by thin
layer chromatography (TLC) at regular intervals. After completion
of the reaction, the mixture was concentrated in vacuo. The crude
solid was filtered off, washed with water and recrystallized from
80% EtOH/H2O to afford 1c as red crystals in 88% yield [32]. m.p.
145–148 °C; FTIR (ATR, cm−1): 3300 (N H), 1597 (C = N), 1550
–
(N = O stretch); 1H NMR (300 MHz, DMSO–d6) (δ/ppm): 9.16 (1H,
2

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Table 1
Crystal data and structure refinement for 1c and 2d
Compound
1c
2d
Empirical formula
Formula weight
Temperature (K)
C
14H13N3O2
C15H13BrN2O
317.18
255.27
296(2)
293(2)
˚
Wavelength (A)
0.71073
0.71073
Crystal system
Space group
Triclinic
Orthorhombic
P b c a
P −1
˚
a (A)
8.2765(4)
6.9087(14)
7.7682(13)
51.548(9)
˚
b (A)
10.7242(4)
16.4881(7)
72.489(2)
˚
c (A)
α (°)
β (°)
γ (°)
90
80.039(2)
90
68.411(2)
90
3
˚
Volume (A )
1294.53(10)
4
2766.5(9)
Z
8
Dcalc (Mg/m3)
1.310
1.523
Absorption coefficient (μ, mm–1
)
0.091
2.965
F(000)
536
1280
Crystal size(mm3)
0.420 × 0.340 × 0.280
2.176 to 27.530
−10<=h<=10,
−13<=k<=13,
−21<=l<=21
21,473
0.380 × 0.200 × 0.180
1.580 to 26.999
−8<=h<=8,
−9<=k<=7,
−64<=l<=65
19,909
Theta range for data collection (°)
Index ranges
Reflections collected
Independent reflections
Completeness to theta = 25.242°
Refinement method
5931 [R(int) = 0.0354]
99.9%
3012 [R(int) = 0.0705]
100.0%
Full-matrix
least-squares on F2
5931 / 0 / 378
0.946
Full-matrix
least-squares on F2
3012 / 0 / 174
1.198
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I >2σ(I)]
R1 = 0.0484,
wR2 = 0.1329
R1 = 0.0754,
wR2 = 0.1570
0.023(3)
R1 = 0.1142,
wR2 = 0.2181
R1 = 0.1609,
wR2 = 0.2338
0.0024(5)
R indices (all data)
Extinction coefficient
−3
˚
Largest difference peak and hole (e A
)
0.193 and –0.168
2,019,512
0.659 and –0.902
984,718
CCDC reference number
reflected in the final R index, R1 = 11.43%. Despite this the struc-
ture solved and refined without difficulty and the poorer quality of
the data is reflected in the residuals on all of the reported param-
eters.
2.6. Molecular docking and physicochemical properties
Molecular docking studies were performed using Autodock
Vina (ver. 1.5.6) [49]. For this purpose, the crystal structure of
EGFR (PDB: 2GS6) was retrieved from protein data bank. The co-
crystallized ligand and water molecules were removed, and the
protein was converted to pdbqt format using Autodock Tools [50].
The structures of ligands 1c and 2d were sketched using Chem-
draw and were converted to 3D format by Openbabel (ver. 2.3.1)
[51]. PDBQT files were prepared in MGL Tools [50]. The other pa-
rameters were left as default. Finally, the conformations with the
most favorable binding free energies were selected for the analysis
of the interactions between the target enzyme and the inhibitors.
PyMOL version 1.8.8.2 [52] and Chimera 1.6 software [53] were
used for 3D molecular graphics, structural alignments, and visu-
alizations. Physicochemical properties were calculated using Swis-
sADME [54].
2.5. Computational details
Density functional theory (DFT) designates the physicochemi-
cal properties of various compounds in a precise and concise way
while presenting a balance between accuracy and computing econ-
omy [42]. In the present project, all calculations were carried out
using the Gaussian 09 software package [43]. The geometric pa-
rameters and the frequencies were computed in the gas phase
by DFT with the Becke’s three-parameter and the Lee-Yang-Parr
(B3LYP) hybrid functional using the 6–311++G (d,p) basis sets in-
cluding the Grimme dispersion correction (D3) [44]. The thermo-
dynamic calculations were performed using the B3LYP/6–311++G
(d,p) level of DFT theory in the gas phase and the effect of tem-
perature on the results was calculated using the Moltran software
[45]. The frequency calculations have been performed for both
compounds using same level of DFT at the standard temperature
of 298.15 K and a standard pressure of 1 atm. The title compounds
have all positive normal mode frequencies which shows that both
compounds are at local minima. The frontier molecular orbital
(FMO’s) analysis, other reactivity descriptors and electrostatic po-
tential maps were computed at the same DFT level. Furthermore,
the description of non-covalent interactions with reduced density
gradient (RDG) analysis [46] is presented using Multiwfn software
[47] and visualized by a visual molecular dynamic program (VMD)
[48].
2.7. Maintenance of cell culture
Human cancerous cell lines NCI-H460 and NCI-H460/Bcl-2
(non-small cell lung cancer), MDA-MB-231 and MCF-7 (Breast can-
cer) were gifted from Peter Scheurich’s Lab in Stuttgart, Germany.
All cell lines were cultured in RPMI-1640 (Gibco) medium supple-
mented with 5% Fetal Bovine Serum (FBS) and 1% PenStrep (peni-
cillin and streptomycin). The culture was kept in humidified incu-
bator at 37 °C with 5% CO2. Cell cultures were checked regularly
for mycoplasma to ensure the absence of any contamination.
3

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Scheme 1. Synthesis of (E)−1-(1-(4-nitrophenyl)ethylidene)−2-phenylhydrazine 1c.
Scheme 2. Synthesis of (E)−3–bromo-N’-(1-phenylethylidene)benzohydrazide 2d.
2.8. Cytotoxicity assays
contrast searching for (E)−1-benzylidene-2-phenylhydrazine with-
out substituents on either of the benzene rings yielded an ex-
tensive 52 hits. Limiting potential substituents to the N-bound
ring reduced the hits significantly to 4 unique molecules. These
included (E)−1-phenyl-2-(1-phenylethylidene)hydrazine itself [57].
Other substituents on the N-bound benzene ring were lim-
ited to NH2 [58] and three separate reports of molecules with
OH substituents [59-61], each in the 2-position of the aro-
matic ring. The final hit, (E)−1-(1-(naphthalen-2-yl)ethylidene)−2-
phenylhydrazine [62] was the naphthalene analogue of the phenol
derivatives.
A day prior to experiments, cells were counted and 15,000
cells/well were seeded in 96-well plates. Next day cells were stim-
ulated with serial dilutions of both compounds (0–100 μM). After
24 h, media were removed, cells were washed with PBS and ad-
herent (living) cells were stained with crystal violet for 15 min at
room temperature. Plates were washed gently to remove excessive
dye and dried overnight. 50 μL Methanol (50 μL per well) was
added and the absorbance was measured in a microplate reader
at 550 nm. All the values were normalized to the values from un-
stimulated wells.
Similarly,
a
search
for
(E)−3–bromo-N’-(1-
phenylethylidene)benzohydrazide 2d, gave no hits, whereas the
search for (E)−3–bromo-N’-(1-phenylethylidene)benzohydrazide
without substituents on either of the benzene rings yielded 53
hits. Further limitation of substituents to the benzene ring ad-
jacent to the C = O group again reduced the hits significantly,
in this case to only two unique molecules. (E)−3–bromo-N’-(1-
phenylethylidene)benzohydrazide itself [63] and two reports of
1-(2-aminobenzoylhydrazono)−1-phenylethane [64,65].
3. Results and discussion
3.1. Synthetic chemistry
Schiff bases were prepared via the condensation reac-
tion of phenylhydrazine or 3-bromobenzohydrazide with
corresponding acetophenones using glacial acetic acid as il-
lustrated in Schemes
1
and
2
[33,55]. (E)−3–bromo-N’-(1-
phenylethylidene)benzohydrazide (2d) was prepared in three
steps starting from commercially available 3-bromobenzoic acid
2a. Acid-catalyzed esterification produced ethyl 3-bromobenzoate
2b [33] which on hydrazination using hydrazine hydrate (80%)
in ethanol furnished the desired 3-bromobenzohydrazide 2c
[33]. The condensation reaction of 3-bromobenzohydrazide
2c with acetophenone gave the desired (E)−3–bromo-N’-(1-
phenylethylidene)benzohydrazide (2d) in 85% yield. Both the title
structures (1c and 2d) were fully characterized by spectroscopic
and X-ray crystallographic methods.
3.2.1. Molecular structures of 1c and 2d
The structures of the two title compounds 1c and 2d are shown
in Schemes 1 and 2 with their crystallographic ally determined
molecular structures shown in Fig. 1 and 2, respectively.
Compound 1c crystallizes with two unique molecules, M1 and
M2, in the triclinic unit cell, Fig. 1. In M1, the H-atoms on the
C18 methyl group are disordered over two sites with occupancies
of 52(7) and 48(7)%, respectively. In M2, the C atoms of the N21
bound phenyl ring and their associated atoms are also disordered
over two sites with an occupancy ratio 58(3):42(3). The two disor-
der components of these phenyl rings are inclined to one another
by 2(1)°. Neither of the unique molecules are entirely planar with
the outer phenyl and nitrobenzene rings inclined to one another
by 8.95(11)° in M1 and 14.5(6)° in M2. Also the nitro substituents
are inclined to their phenyl rings by 3.6(2)° for M1 and 4(1)° for
M2. The ethylidene-hydrazine segments of both molecules are also
3.2. X-ray crystallography
A search of the Cambridge structural database version 5.41
November 2019 with 1 update March 2020 [56] for (E)−1-(1-
(4-nitrophenyl)ethylidene)−2-phenylhydrazine 1c gave no hits. In
4

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Table 2
Hydrogen bond distances (A) and angles (°) for 1c
˚
D-H…A
d(D-H)
d(H…A)
d(D…A)
<(DHA)
N(11)-H(11 N)…O(11)#1
C(16)-H(16)…O(11)#1
C(25A)-H(25A)…O(12)#1
C(18)-H(18A)… Cg5#2
C(28)-H(28A)… Cg2#3
C(211)-H(211)… Cg1#3
0.86
0.93
0.93
0.96
0.96
0.93
2.42
3.2142(19)
3.321(3)
3.212(5)
3.937(2)
3.914(2)
4.214(3)
153
148
134
126
142
130
2.50
2.50
3.301
3.114
3.552
Symmetry transformations used to generate equivalent atoms:.
#1 x-1, y + 1, z, #2 1-x, 1-y, -z, #3 1-x, -y, 1-z. Cg5, Cg1 and Cg2 are the
centroids of the C19—C114, C21A—C26A and C29—C214 rings, respectively.
Table 3
˚
Hydrogen bond distances (A) and angles (°) for 2d
D-H…A
d(D-H)
d(H…A)
d(D…A)
<(DHA)
Fig. 1. The asymmetric unit of 1c with ellipsoids drawn at the 30% probability level
and the M1 and M2 molecules identified by leading 1 and 2 characters in the num-
bering schemes. For clarity only the major disorder components of the H-atoms on
the C18 methyl group of M1 and the N21 bound benzene ring of M2 are shown.
N(1)-H(1)…O(1)#1
C(15)-H(15)…N(2)#2
C(11)-H11…Cg2#3
0.86
0.93
0.93
2.19
2.73
2.93
2.984(10)
3.539(13)
3.807(12)
153
146
158
Symmetry transformations used to generate equivalent atoms:.
#1 -x + 1/2, y-1/2, z #2 -x + 1/2, y + 1/2, z, #3 −1/2-x, −1/2 + y, z. Cg2
˚
planar with rms deviation of fitted atoms of 0.0022 A for M1 and
is the centroid of the C10…C15 ring.
˚
0.0018 A for M2.
In contrast, compound 2d crystallizes with a single molecule in
the orthorhombic unit cell. Again the molecule is not planar with
an angle of 54.8(3)° between the peripheral bromo–benzene and
Cg1 and Cg2 are the centroids of the C19—C114, C21A—C26A and
C29—C214 rings, respectively. Overall, these contacts combine to
stack the molecules along the a axis direction, Fig. 7. Hydrogen
bonds for 1c are shown in Table 2.
˚
phenyl rings with the bromo–substituent 0.110 A (13) from the ring
plane. However, the (Z)-N’-ethylideneformohydrazide section of the
molecule is reasonably planar with an rms deviation of the 6 fitted
˚
atoms of 0.0713 A.
3.2.3. Crystal packing of 2d
In the crystal of 2d classical N1-H1…O1 hydrogen bonds, sup-
ported by weaker C15-H15…N2 contacts, form chains of molecules
along the b axis direction, Fig. 8.
3.2.2. Crystal packing of 1c
In the crystal of 1c O21 acts as a bifurcated acceptor with N21-
H21N…O21 and C26A-H26A…O21 enclosing R1 ring motifs that
Chains also form along b as a result of C11-H11…π contacts to
the centroid of the C10—C15 phenyl ring, Fig. 9.
2
form chains of M1 molecules along the b axis direction. These
chains are linked to M1 molecules by C25A-H25A…O12 hydrogen
bonds forming sheets of M1 and M2 molecules in the bc plane,
Fig. 3.
Close Br…Br contacts form molecular dimers and, in the over-
all packing arrangement, link adjacent pairs of stacked molecules
along the c axis direction, Fig. 10. The molecules of 2d stack along
the a axis. Hydrogen bonds for 2d are shown in Table 3.
Atom O11 of M1 also acts as a bifurcated acceptor forming N1-
H11…O11 and C16-H16…O11 hydrogen bonds enclosing R1 ring
2
motifs. These contacts form chains of M1 molecules along the ab
diagonal, Fig. 4.
3.3. Hirshfeld surface analysis
An unusual feature of the packing of 1c is the presence of
Further details of the intermolecular interactions in 1c and 2d
were obtained using Hirshfeld surface analysis [66] with Hirsh-
feld surfaces and two-dimensional fingerprint plots generated with
Crystal Explorer [67]. Figs. 11 and 12 show the Hirshfeld surfaces
for opposite faces of the asymmetric units of 1c and 2d. For 1c
–
three inversion related C H…π contacts. Pairs of M1 molecules
form dimers along a through C18-H18A…Cg5 contacts (1-x, 1-y, -
z), Fig. 5. Dimers of M2 molecules also form along a, but in this
instance the dimers result from pairs of inversion related C211-
H211…Cg1 and C28-H28A…Cg2 contacts (1-x, -y, 1-z), Fig. 6. Cg5,
–
–
the bold red circles correspond to N H…O and C H…O hydrogen
Fig. 2. The molecular structure of 2d with ellipsoids drawn at the 30% probability level.
5

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 3. Sheets of M1 and M2 molecules of 1c formed in the bc plane.
–
Fig. 5. Inversion dimers of M1 molecules formed by C H…π contacts.
Fig. 4. Chains of M1 molecules of 1c along the ab diagonal.
–
Fig. 6. Inversion dimers of M2 molecules formed by pairs of C H…π contacts.
–
bonds while the weaker C H…π contacts appears as faint red cir-
–
cles. For 2d the N H…O hydrogen bond also features strongly with
–
weaker C H…N and H…H contacts showing less strongly.
Fingerprint plots for 1c and 2d, Figs. 13 and 14 show, as ex-
pected, that H…H contacts are the most prolific in both cases. A
significant feature of the surfaces of both of these compounds is
the considerable number of contacts that contribute to the over-
all surface with the included surface areas of the majority of these
being over 1%. For 1c, the strongest additional contributions come
from H…C/C…H and H…O/O…H contacts. Weaker H…N/N…H,
C…O, C…N, C…C and O…N interactions are also found, see also
Table 4. For 2d, in addition to the H…H contacts, H…C/C…H,
H…O/O…H and H…Br/Br…H contacts predominate with lesser
contributions from H…N/N…H, Br…C, Br…Br, C…C and C…N con-
tacts all above 1%; see also Table 4.
Fingerprint plots (Fig. 14) show that H…H contacts are the most
prolific with significant contributions also coming from H…C/C…H
and H…O/O…H contacts. Weaker H…N/N…H, C…O, C…N, C…C
and O…N interactions are also found, see also Table 5.
6

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 7. Overall packing of 1c viewed along the a axis direction.
–
Fig. 9. Chains of molecules of 2d along the b axis generated by C H…π contacts.
3.4. Theoretical (DFT) studies
3.4.1. HOMO-LUMO analysis
Frontier molecular orbitals (FMO’s), the highest occupied
molecular orbitals (HOMO) and lowest unoccupied molecular or-
bitals (LUMO) are important orbitals that determine the stability of
molecules. The HOMO acts as an electron donor while the LUMO
tends to be an acceptor orbital having the ability to accept elec-
trons. The difference in the HOMO-LUMO energy values charac-
terizes the molecular reactivity, optical polarizability, and kinetic
stability of the compound [68]. The HOMO-LUMO energy gaps for
compounds 1c and 2d are relatively close at 2.36 and 2.38 eV,
respectively, demonstrating that 2d is only slightly more stable
and less reactive than 1c. The shapes of higher occupied MO’s are
shown in Fig. 15 for both compounds which infer that the HOMO
orbitals are delocalized on the hydrazide sections of the molecules.
For 1c the LUMO lies on the substituted aromatic ring, while for 2d
the LUMO involves most of the molecule and the electronic transi-
tions occur from n→π∗ and from π→π∗.
Fig. 8. Chains of molecules of 2d along the b axis.
Fig. 10. Overall packing of 2d viewed along the a axis direction.
7

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 12. Hirshfeld surfaces for opposite faces (a) and (b) of the molecule of 2d,
mapped over dnorm in the range –0.4184 to 1.3501 a.u.
Fig. 11. Hirshfeld surfaces for opposite faces (a) and (b) of the asymmetric unit of
1c, mapped over dnorm in the range –0.2859 to 1.6689 a.u.
Table 4
3.4.2. Thermodynamic function analysis
Percentage contributions of interatomic con-
tacts to the Hirshfeld surface for the two
molecules that comprise the asymmetric unit
of 1c
The total energy of a molecule is the sum of the translational,
rotational, vibrational, and electronic energies i.e., E = Et + Er +
Ev + Ee. The statistical thermochemical analyses of the title com-
pounds are carried out considering the molecule to be at the stan-
dard temperature of 298.15 K and a standard pressure of 1 atm.
The thermodynamic properties such as the heat capacity (Cp), en-
thalpy (Hm), Gibb’s free energy (G) and entropy (S0) were calcu-
lated using the DFT/B3LPY program with the 6–311++G basis set
[69]. Thermodynamic data for the title compounds in the tempera-
ture range from 0 to 500 K were calculated using the Moltran soft-
ware [45] and are given in Fig. 16. From these results, it can be
predicted that, with the exception of the Gibb’s free energies, the
thermodynamics parameter increases with increasing temperature
as the vibrational intensities of the title compounds change with
temperature [70]. The Gibb’s free energies decreases with increase
in temperature which shows that the both compounds are stable
and do not decompose when temperature is increased in the range
investigated. The variation in the thermodynamic parameters with
temperature can be seen in Fig. 16. All the thermodynamic data
provides helpful information for further study of the compounds.
The correlation equations between the thermodynamic properties
and temperature satisfy a parabolic formula. The regression coeffi-
cients are also determined in the parabolic equations. A partition
function is one of the most important thermodynamic parameters
that links thermodynamics, spectroscopy and quantum theory and
is calculated by the first vibrational level or by the internuclear
potential energy values [71]. The partition function can be used
to calculate the statistical properties like entropy, free energy, rate
constants etc. of a system in thermodynamic equilibrium.
Contact
Included surface area (%)
H…H
42.2
22.6
19.4
5.2
H…C/C…H
H…O/O…H
H…N/N…H
C…O
4.3
C…N
3.3
C…C
1.5
O…N
1.3
O…O
0.2
N…N
0.1
Table 5
Percentage contributions of interatomic con-
tacts to the Hirshfeld surface for 2d
Contacts
Included surface area (%)
H…H
38.0
25.0
10.7
10.5
4.4
H…C/C…H
H…O/O…H
H…Br/Br…H
H…N/N…H
Br…C
3.8
Br…Br
3.0
C…C
2.1
C…N
1.9
N…N
0.5
C…O
0.2
8

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 13. Two-dimensional fingerprint plots for the asymmetric unit of 1c, (a), together with (b)-(g) separate contact types and included surface areas for the major individual
contacts. Minor contacts contributing less than 1% to the total surface area are not shown here but, for completeness, are shown in Table 4.
3.4.3. Molecular electrostatic potential
The most possible regions with blue colours are near the N − H
group which make a more reactive site for the nucleophilic attack
(strong attraction). Both these sites are also available for hydrogen
bonding due to high electronegative atoms i.e. nitrogen and oxy-
gen.
The molecular electrostatic potential (MEP) is the visual repre-
sentation of electronic density for the compounds of interest and is
a helpful parameter for the determination of electrophilic and nu-
cleophilic sites and hydrogen binding interactions [72]. MEP maps
are also useful for building a relationship of molecular structure
with its physicochemical properties [73]. The three-dimensional
MEP maps for compounds 1c and 2d are presented in Fig. 17 and
show the positive and negative sites of these compounds. The color
scheme represents the values of electrostatic potential which in-
creases from red to blue. Both MEP maps in Fig. 17 show that the
oxygen atoms with red color have negative electrostatic potential
and act as a possible site for electrophilic attack (strong repulsion).
3.5. Anticancer activity and computational studies
3.5.1. Drug-likeness
Before bioactivity screening, the compounds were analyzed for
their physicochemical properties, including size, shape, lipophilic-
ity, and polar surface area. Descriptors such as the number of ro-
tatable bonds, hydrogen bond donors, hydrogen bond acceptors,
9

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 14. Two-dimensional fingerprint plots for 2d, (a), together with (b)-(j) separate contact types and included surface areas for the major individual contacts. Minor contacts
contributing less than 1% to the total surface area are not shown here but, for completeness, are shown in Table 5.
Lipinski’s acceptors, Lipinski’s donors, Lipinski’s violations, Lipin-
ski’s drug-likeness, log P (logarithm of the octanol/water partition
coefficient), molecular weight and total polar surface area (TPSA)
were computed to access the drug-like properties of the synthe-
sized compounds. In silico calculation results revealed that both the
synthesized compounds fulfilled the Lipinski’s Ro5 [74] and Veber’s
Ro3 [75] cut-off limits, which suggested that they may have anti-
cancer potential
From the Lipinski Ro5 limit [74], to be considered as drug-like
the molecules must have molecular weights ≤ 500, log P ≤ 5, to-
˚
tal polar surface area (TPSA) < 140 A², number of hydrogen bond
donors (HBD) values ≤ 5 and hydrogen bond acceptor (HBA) values
≤ 10. Veber et al. suggested further modifications to Ro5 [75] with
number of rotatable bonds (NOR) of the molecule being ≤10 [74].
Molecules that violate more than one of these criteria may have
problems with their bioavailability. The detailed results of drug-
10

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 15. Frontier molecular orbitals for compounds 1c (left) and 2d (right).
Fig. 16. Variations in thermodynamic parameters with temperature and the correlation equations of 1c (left) and 2d (right).
Fig. 17. 3D molecular electrostatic potential map of 1c (left) and 2d (right).
likeness of the compounds 1c and 2d are tabulated in Table 6 and
3.5.2. Anticancer activity
from these results both compounds are seen fulfill the criteria to
be considered as drug-like molecules. Furthermore, the Log P val-
ues of compounds 1c and 2d indicate good absorption characteris-
tics.
The Schiff bases were also tested for their anticancer poten-
tial against breast and lung cancer cell lines. In both sexes, lung
cancer is the most commonly diagnosed cancer and the leading
cause of death closely followed by female breast cancer, prostate
cancer and colorectal cancer. Cancer is characterized by an accu-
11

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 18. Cytotoxic effects of Schiff bases on cancer cell lines.
Table 6
and MDA-MB-231 and MCF-7 (breast cancer cell lines). The lung
cancer cell line NCI-H460 was quite sensitive to both compounds,
Physiochemical properties of compounds 1c and 2d
IC50 value of 1c is 35
5.5 μM which is close to that of the stan-
4.2 μM), whereas its counterpart with over-
Physicochemical property descriptors
Compound 1c
14H13N3O2
Compound 2d
dard drug used (30
Formula
C
C15H13BrN2O
expression of Bcl-2 showed resistance to compound 1c and 2d as
well as doxorubicin, which points to the importance of mitochon-
drial induced toxicity in these cell lines. Breast cancer cells were
also resistant to the toxicity induced by 1c and 2d at low concen-
trations as compared to doxorubicin. However, in both cell lines 1c
was a more potent inducer of cell death than 2d at higher concen-
trations.
Molecular weight
255.27 g/mol
317.18 g/mol
NoR
4
4
HBA
3
2
HBD
Log P
TPSA
1
1
2.09
2.48
˚
˚
70.21 A²
41.46 A²
Abbreviations used: NoR = Number of rotatable bonds; HBA = Hydrogen bond
acceptor; HBD = Hydrogen bond donor; Log P (Log P (o/w)) = logarithm of the
(octanol/water) partition coefficient; TPSA = Total polar surface area.
Cytotoxic effects of the two compounds were evaluated in lung
and breast cancer cell lines. All cells were seeded in 96 well plates
24 h prior to experiment. Stock solution (1 nM) of the compounds
was prepared in DMSO, and cells were stimulated with serial dilu-
tions of both compounds, DMSO was added as a control. 24 h post
stimulation A) morphological changes have been observed and im-
ages have been captured under microscope. B) Viable cells were
stained with crystal violet for 15 min and the absorbance was mea-
Table 7
IC50 (μM) value of compounds against lung and breast cancer
cell lines
Cell
1c
2d
Doxorubicin
lines
IC50 (μM
SD)
NCI-H460
35
90
65
82
5.5
5.5
5.4
6.5
68
4.6
5.5
30
60
26
42
4.2
5.5
8.5
5.5
sured at 550 nm. Values shown are mean
triplicates. GraphPad Prism 8 software was used to calculate the
mean SD and IC50
SD calculated from
NCI-H460/Bcl-2
MCF
N/A
90
MDA-MB-231
N/A
.
Epidermal growth factor receptors (EGFR) are frequently over-
expressed in breast and non-small cell lung cancer and is a po-
tential therapeutic target for this disease. EGFR is also known as
ErbB1 or HER1 and is a member of the receptor family tyrosine
kinase (RTK) of cell surface receptors [80]. EGFR regulates differ-
entiation, apoptosis, cell cycle progression, development, and tran-
scription [81]. Overexpression of the EGFR has been observed in
a variety of human cancers [82]. EGFR consists of an extracellu-
lar domain, a single hydrophobic transmembrane segment, an in-
tracellular portion with a juxtamembrane (JXM) segment, a pro-
tein kinase domain, and a carboxyterminal tail [83]. Eps8 is an
important active kinase substrate of EGFR that directly binds to
the juxtamembrane (JXM) domain of EGFR to form an EGFR/Eps8
complex. The EGFR/ Eps8 complex is involved in regulating can-
cer progression and might be an ideal target for antitumor ther-
apy [83]. Therefore, to study to putative binding modes, the com-
pounds 1c and 2d were analyzed by molecular docking studies to
target the EGFR/Eps8 complex in breast and non-small cell lung
cancer (NSCLC).
N/A where IC50 value is above 100 μM.
mulation of genetic variations and the loss of normal cellular reg-
ulatory processes. It is a main cause of death worldwide [76,77].
Although chemotherapy is the cornerstone of systemic cancer ther-
apy, it only has a modest effect on overall survival [78]. Thus, there
is a great need to develop novel therapeutic modalities to improve
survival rates [79].
In this work, the anticancer activity of compounds 1c and 2d
was evaluated, against lung (NCI-H460, NCI-H460/Bcl-2) and breast
cancer (MCF7, MDA-MB-231) cell lines. Cells were stimulated with
serial dilutions (0–100 μM) of the compounds and doxorubicin (a
positive control) for 24 h. The percentage viability after the 24 h
treatment is represented on graphs, Fig. 18, while IC50 values are
mentioned separately in Table 7.
Fig. 18 reveals that compound 1c is a more potent inducer of
toxicity in all four human cancer cell lines than 2d, including NCI-
H460 and NCI-H460/Bcl-2 (non-small cell lung cancer cell lines),
12

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
‘mouth-like’ structure (Fig. 19) and compete with the natural sub-
strate for the ATP binding site. Fig. 19 shows that the groove in the
‘mouth’ consists of five functional regions, which are the P-loop
(Ser720 to Gly724), the α-loop (Arg748 to Ser752), a hinge-region
(Gln791 to Leu798), a DFG motif (Thr854 to Arg858) and a Cα-
helix (Pro753 to Ser768).
The difference between inhibitory activities of analogues (1c
and 2d) studied in this work led us to make structural compar-
isons in terms of their intermolecular interactions that may me-
diate protein-ligand binding, their relative location in the active
site, their size, shape, and physicochemical properties. In order to
investigate the binding mode of the inhibitors and their interac-
tion with amino acid residues of EGFR (PDB ID: 2GS6), a molecular
docking study of the two synthesized compounds was performed.
The docked conformations further assisted in the identification of
the relative location of the co-crystallized inhibitors and the ref-
erence molecules in the protein architecture of EGFR. Doxorubicin
was used as a reference drug in biological screening; therefore, it
was also docked with the enzyme to compare its binding interac-
tion.
Fig. 19. A cartoon representation of the EGFR protein in a rainbow color pattern.
Some important regions of EGFR such as active site, hinge region, p-loop, activation
loop, α-loop, C-lobe and N-lobe are labelled.
3.5.3. Molecular docking studies
Computational approaches were employed to investigate the
possible basis for preferential binding of ligands to the active con-
formation of EGFR-TKD.
3.5.3.2. Molecular docking and binding analysis. For elucidation of
the molecular basis of the mechanism of inhibition, the com-
pounds were docked computationally to the active site of EGFR.
˚
In this study, first the protein structure (2GS6: 2.0 A) was retrieved
3.5.3.1. EGFR structure. EGFR,
a
transmembrane glycoprotein is
from Protein Data Bank (PDB). The protein was already bound with
ligands in its crystal structure. The bound co-crystallized ligands
were removed from the active pocket of the protein to study the
inhibition mechanism of the active compounds. All the ligands
were docked in 20 different conformations with variable energy
values. All the preferred docked conformations formed one cluster
inside the pockets as shown in Fig. 20.
composed of 1186 residues, including three parts: extracellular re-
ceptor region, transmembrane structure, and intracellular tyrosine
kinase region [80]. The structure and function of the intracellular
domain of EGFR (from Gly696 to Ile1018) has been a major topic
of research. There are seven α-helices and seven β-sheets in the
intracellular domain, and the inhibitor can be firmly locked in the
Fig. 20. Superimposition of the twenty different docked conformations of the inhibitors in the active site of EGFR (PDB: 2GS6); 1c is shown in red (A and B) forming two
clusters whereas 2d is shown in yellow (C and D), forming three clusters inside the pockets of the enzyme EGFR; a closer view. Receptor is shown in beige in surface A and
C and in ribbon form in B and D while the ligands are shown in sticks form. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
13

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 21. A) An overlay of the docked orientations of the preferred conformations of compounds 1c and 2d and doxorubicin in the active pocket of EGFR (surface view-rainbow
color). B) Minimal energy conformations and relative positioning of all 3 variants. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
Fig. 22. Docking pose of compound 1c in 3D space; receptor is shown in cyan surface (left) and in ribbons (right). Ligand 1c is shown in red ball and stick model. Receptor
is shown in cyan colored ribbons while the key residues are shown in stick mode. Interacting residues are labeled in maroon. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
All the docked conformations for each compound were ana-
lyzed and it was found that the most favorable docked poses with
maximum number of interactions were those, which were ranked
the highest, based on the least binding energy. The binding en-
ergy was calculated as a negative value by the software. From the
molecular docking calculation results, we chose variants with the
minimal energy of the enzyme–inhibitor complex. The most favor-
able docking poses of the 20 docked conformations for each com-
pound were retained to investigate the interactions of the docked
conformations within the active site. The detailed docking results
are also discussed. It has been observed from conformational anal-
ysis and relative positioning that the compounds 1c and 2d are
positioned in the active pocket in the same way as doxorubicin
(Fig. 21).
and the carbonyl O of compound 2d In contrast doxorubicin forms
two hydrogen bonds with Thr766 and Thr830. It has also been ob-
served that the relative positioning of the two molecules (2d and
doxorubicin) is slightly different (Fig. 23). Compound 2d preferen-
tially occupies the space near the p-loop region while doxorubicin
prefers to reside between the p-loop and α-loop region. These re-
sults clearly conclude that good activities of these inhibitors are
attributed to quite low binding energy, effective positioning of the
compounds in the active site near activation loop and multiple in-
teractions with the key residues inside the active site of the target
structure.
3.5.4. Pharmacokinetics
The ADMET properties of the Schiff bases (1c and 2d) were cal-
culated using SwissADME (Table 8). It was found that both the
compounds could be absorbed by the human intestine. Never-
theless, the compounds were predicted as non-substrates for P-
glycoprotein (P-gp) which effluxes drugs and various compounds
to undergo further metabolism and clearance. Many of the hu-
man microsomal cytochrome P450s catalyze the metabolism of a
wide variety of compounds including xenobiotics and drugs. Drug
metabolism via the cytochrome P450 system has emerged as an
important determinant in the occurrence of several drug interac-
tions that can result in reduced pharmacological effects and ad-
verse drug reactions [84]. Most drugs can exhibit decreased effi-
cacy due to rapid metabolism, but drugs with active metabolites
can display increased drug effect and/or toxicity due to enzyme in-
It has been observed that compound 1c occupies the hydropho-
bic pocket of the binding site and makes hydrophobic interac-
tions in the active site of EGFR (Fig. 22). Both the phenyl rings
are positioned in a hydrophobic pocket formed by the side chains
of Leu694, Ala719, Val702 and Leu820. Most of the amino acid
residues form pi-alkyl interactions with aryl rings of compound 1c
(Fig. 22).
In compound 2d, the phenyl ring is adjacent to the carbonyl
moiety predominantly pi-alkyl hydrophobic interactions occur with
Ala719 and Leu820 (Fig. 23). However, the phenyl ring adjacent
to C = N linkage also forms pi-cation interactions with Lys721 at
˚
a distance of 3.44 A. Another important interaction of hydrazide
–
2d with the receptor is a hydrogen bond between O H of Thr766
14

H. Andleeb, L. Danish, S. Munawar et al.
Journal of Molecular Structure 1235 (2021) 130223
Fig. 23. Docking pose of ligands in 3D space; receptor is shown in ribbons and ligands are shown in ball and stick model. A) Compound 2d is shown as yellow sticks; B)
Reference compound doxorubicin is shown as green sticks; Receptor is shown in cyan colored ribbons while the key residues are shown in stick mode. Interacting residues
are labeled in maroon. Pink dotted lines indicate pi-pi interactions, orange dotted lines indicate pi-cation interactions while green dotted lines indicate hydrogen bond
interactions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 8
Acknowledgements
Pharmacokinetic properties of compounds 1c and 2d
Pharmacokinetic property descriptor
Compound 1c
Compound 2d
H.A. and L.D. are grateful to the Higher Education Commission
of Pakistan for financial support under SRGP program. J.S. thanks
the Chemistry Department, University of Otago for support of his
work. L.D. is thankful to Peter Scheurich Lab at institute of cell bi-
ology and immunology, university of Stuttgart, Germany for pro-
viding human cancerous cell lines NCI-H460 and NCI-H460/Bcl-2
(non-small cell lung cancer), MDA-MB-231 and MCF-7 (breast can-
cer).
GI absorption
High
Yes
No
High
Yes
No
BBB permeant
P-gp substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2d6 inhibitor
CYP3A4 inhibitor
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
No
Supplementary materials
duction [84]. Both the Schiff bases were predicted to have potential
against inhibition of CYP2C9, CYP2C19, and CYPA12 (Table 8). How-
ever, none of the imines are predicted to have potential to be the
inhibitor of CYP2d6 as shown in Table 8.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130223.
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
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