Experimental and theoretical studies of 1,3,5-tris (bromomethyl)-2,4,6-trimethylbenzene with 2-pyridone

Article abstract of DOI:10.1002/poc.4188

In this present work, a new compound 1-({2,4,6-trimethyl-3,5-bis[(2-oxo-2H-pyridin-1-yl)methyl]phenyl}methyl)-2H-pyridin-2-one hydrate (M3P) has been synthesized and characterized by IR, ¹H, and ¹³C NMR and single-crystal X-ray diffraction analysis. The molecular geometry, frontier molecular orbital energies, and thermodynamic properties of the title molecule were explored using density functional theory (DFT) calculation via B3LYP method with 631++G (d,p) basis set. The compound (M3P) crystallizes in the space group of P-1 with unit cell parameter a = 9.8224(12) ?, b = 11.6383(14) ?, c = 11.8865(14) ?, α = 64.6410(10)°, β = 81.156(2)°, γ = 69.5910(10)°, V = 1150.8(2) ?3, and Z = 2. Furthermore, Hirshfeld surface analysis was performed to observe better intermolecular interactions in the crystal packing. The nature of the interaction of M3P with DNA and BSA protein was studied using molecular docking analysis. DNA binding studies were also done using UV spectrophotometric method. The compound moderately binds with CT-DNA which is comparable with the docking results. in vitro anti-microbial activities against some bacterial and fungal strains were investigated. M3P shows good response against bacteria Pseudomonas aeruginosa and Klebsiella pneumonia and fungi Candida albicans and Aspergillus niger.

Full text of DOI:10.1002/poc.4188

Received: 21 October 2020
DOI: 10.1002/poc.4188
Revised: 9 December 2020
Accepted: 26 December 2020
R E S E A R C H A R T I C L E
Experimental and theoretical studies of 1,3,5-tris
(bromomethyl)-2,4,6-trimethylbenzene with 2-pyridone
R. Nandini Asha1,2
| M. Usha Rahini1,2 | Caroline Daisy1,2
|
B. Ravindran Durai Nayagam1,2
| N. Bhuvanesh3
1Department of Chemistry and Research
Abstract
Centre, Popes' College (Autonomous),
Sawyerpuram, Tuticorin, 628251, India
In this present work, a new compound 1-({2,4,6-trimethyl-3,5-bis[(2-oxo-2H-
pyridin-1-yl)methyl]phenyl}methyl)-2H-pyridin-2-one hydrate (M3P) has been
synthesized and characterized by IR, 1H, and 13C NMR and single-crystal
X-ray diffraction analysis. The molecular geometry, frontier molecular orbital
energies, and thermodynamic properties of the title molecule were explored
using density functional theory (DFT) calculation via B3LYP method with 631
++G (d,p) basis set. The compound (M3P) crystallizes in the space group of
P-1 with unit cell parameter a = 9.8224(12) Å, b = 11.6383(14) Å, c = 11.8865
(14) Å, α = 64.6410(10)ꢀ, β = 81.156(2)ꢀ, γ = 69.5910(10)ꢀ, V = 1150.8(2) Å3,
and Z = 2. Furthermore, Hirshfeld surface analysis was performed to observe
better intermolecular interactions in the crystal packing. The nature of the
interaction of M3P with DNA and BSA protein was studied using molecular
docking analysis. DNA binding studies were also done using UV spectrophoto-
metric method. The compound moderately binds with CT-DNA which is
comparable with the docking results. in vitro anti-microbial activities against
some bacterial and fungal strains were investigated. M3P shows good response
against bacteria Pseudomonas aeruginosa and Klebsiella pneumonia and fungi
Candida albicans and Aspergillus niger.
2Manonmaniam Sundaranar University,
Tirunelveli, India
3Department of Chemistry, Texas A&M
University, College Station, TX, USA
Correspondence
B. Ravindran Durai Nayagam,
Department of Chemistry and Research
Centre, Popes' College (Autonomous),
Sawyerpuram-628251, Affiliated to
Manonmaniam Sundaranar University,
Tirunelveli-627012, Tamil Nadu,
Tuticorin, India.
Email: b_ravidurai@yahoo.com
K E Y W O R D S
2-pyridone, DFT, DNA binding, Hirshfeld surface analysis, molecular docking
1 | INTRODUCTION
of pyridone chemistry due to their vast biological
applications. The 2-pyridone compounds display numerous
Heterocyclic compounds are one of the important catego-
ries of organic compounds. A huge number of organic
compounds used in the medicinal field as drugs for vari-
ous diseases contain heterocyclic skeletons inclusive of
antibacterial, antifungal, antioxidants, and anticancer,
and so on.[1] Pyridones are a unique class of six-
membered aza-heterocycles and are present in several
natural and biologically active scaffolds.[2,3] Many
researchers have shown great interest in the synthesis
pharmacological activities like antibacterial,[4] antifungal,[5]
anti-inflammatory,[6] antiviral,[7] antitumor,[8] and anti-
Alzheimer's properties.[9] Due to a peptidomimetic functional-
ity of 1H-pyridin-2-one tautomer, it plays an essential role as
a scaffold in drug design.[10,11] The 2-pyridone core and its
tautomer, 2-hydroxypyridine, have been reported as a feature
of several new NSAIDs.[12–14] In recent years, 2-pyridones
have captivated much importance as these compounds have
been found to exhibit several biological activities, such as
J Phys Org Chem. 2021;34:e4188.
wileyonlinelibrary.com/journal/poc
© 2021 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/poc.4188

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ASHA ET AL.
HIV-1 non-nucleoside reverse transcriptase inhibitors
(NNRTIs) and cardiotonic agents.[15,16] Amrinone is a
therapeutic drug candidate containing pyridine structural
unit which is used in the treatment of heart failure dis-
eases.[17] 1H-pyridin-2-ones are also promising compounds
for the preparation of modified nucleotides and
oligonucleotides.[18–21] The 2-pyridone skeleton with the
cis-amide substructure provides an ideal structure for
bifunctional catalysis and also supramolecular structures.
Moreover, 2-pyridones are of particular interest to
researchers due to their in vitro and in vivo
antibacterial potencies against the bacterial type II
DNA topoisomerases, which includes two highly
homologous enzymes DNA gyrase and topoisomerase
IV.[22] A series of novel compounds like 6-amino-1-
((1,3-diphenyl-1H-pyrazole-4-yl)methyleneamino)-4-(aryl)-2-
antimicrobial activities, and DNA binding studies of the
newly prepared compound have been investigated.
2 | EXPERIMENTAL METHODS
2.1 | General methods
All chemicals and solvents were acquired from commer-
cial sources with high purity and analar grade and used
as received unless otherwise noted. The melting point of
the title compound was measured in an open capillary
tube. The infrared spectrum was recorded on a JASCO
4600 (Japan) FT-IR spectrometer (in KBr pellet) with a
1
scan range from 400 to 4,000 cm−1. H- and 13C-NMR
spectra were recorded with a Bruker Avance III,
400 MHz, 9.4 Tesla superconducting magnet using CDCl3
as a solvent. Chemical shifts were reported as δ (ppm)
with tetramethylsilane as an internal standard. DNA
binding experiments were done using Jasco V-650 UV
spectrophotometer.
oxo-1,2-dihydropyridine-3,5
dicarbonitriles
were
synthesized and screened for their in vitro antibacterial
activity against Staphylococcus aureus, Streptococcus
pyogenes (Gram positive), Escherichia coli, Pseudomonas
aeruginosa (Gram negative) by broth dilution and cyto-
toxic activity by MTT assay. Pyridone derivatives have
been used as enzymes inhibitors.[23,24] The results indi-
cated that the compounds exhibited potent antibacterial
activity against bacterial strains at non-cytotoxic concen-
trations.[25] A 2-pyridone derivative namely perchlorate
salt of 1-(2-aminoethyl)-6-hydroxy-2-oxo-4-(3-pyridinyl)-1,-
2-dihydropyridine-3,5-dicarbonitrile was synthesized by a
group of researchers, and its interaction with CT-DNA
was explored. It was observed that the compound can
2.2 | Synthesis of 1,3,5-tris
(bromomethyl)-2,4,6-trimethylbenzene
Mesitylene (12.0 g, 0.100 mol), paraformaldehyde (10.0 g,
0.330 mol), and 50 ml of glacial acetic acid were mixed
with 70 ml of 31% HBr/acetic acid solution. The mixture
was kept for 12 h at 120ꢀC and then poured into 100 ml
of water. The product 1,3,5-tris (bromomethyl)-2,-
4,6-trimethylbenzene was filtered and dried in vacuum at
room temperature.[29]
bind to the DNA helix in
manner.[26]
a partial intercalative
Density functional theory (DFT) calculations in
conjugation with molecular docking analysis are inevita-
ble in exposing the nature of interactions between
compounds and protein in recent years.[27,28] It is impor-
tant to note that various forces including hydrogen
bonding, π– π stacking, and electrostatic interactions
along with other non-covalent interactions help the
molecules to be intact inside the binding pocket of
proteins. Geometry optimization of compounds using
DFT calculations followed by frontier molecular orbital
analysis can be done to understand the nature of binding
of these compounds into the BSA from molecular
docking analysis.
In view of the above facts, we have synthesized a
novel 2-pyridone derivative M3P from 1,3,5-tris
(bromomethyl)-2,4,6-trimethylbenzene and sodium salt
of 2-pyridone. The synthesized compound has been
confirmed by spectroscopic reports. Furthermore, DFT
calculations, Hirshfield surface analysis, biological
studies like docking with DNA and BSA protein,
2.3 | Synthesis of ({2,4,6-trimethyl-3,5-bis
[(2-oxo-2H-pyridin-1-yl)methyl]phenyl}
methyl)-2H-pyridin-2-one hydrate (M3P)
A mixture of 2,4,6-tris (bromomethyl)mesitylene (0.399 g,
0.001 mol) and 2-hydroxypyridine sodium salt (0.285 g,
0.003 mol) in water (30 ml) and methanol (30 ml) was
heated at 80ꢀC with stirring for 60 min. The compound
formed was filtered and dried (Scheme 1). It was then dis-
solved in chloroform and methanol (1:2 v/v) and allowed
to undergo slow evaporation process for a week. Fine
crystals were obtained. The purity of the compound was
checked by performing TLC, and the yield was noted as
85%. M.PtoC: 263.6. FT-IR (KBr, cm–1): 3731.58,
2920.16, 2361.41, 1735.62, 1665.59, 1598.7, 1472.38,
1377.89, 1235.18, 1019.19, 1022.09, 991.232, 867.81,
648.929, 509.115, 409.709 ( Figure S1). 1H NMR (CDCl3)

ASHA ET AL.
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SCHEME 1 Synthesis of M3P
δ ppm: Py-H 7.33 (t, 1H), 6.74 (d, 1H), 6.65(d, 1H), 6.10
(t, 1H), N-CH2-5.22 (s,2H) and -CH3 2.21 (s,3H)
(Figure S2). 13C NMR (300 MHz CDCl3) δ ppm: 15.26,
44.99, 105.25, 119.40, 130.16, 132.16, 138.04,
139.40,161.60(C=O) (Figure S3).
functional together with the Lee-Yang-Parr (LYP) non-
local correlation functional, symbolized B3LYP using
6-311G++ basis set. Parameters such as ionization poten-
tial (I), electron affinity (A), electronegativity (χ), electro-
chemical potential (μ), hardness (η), softness (σ), and
electrophilicity index (ω) were calculated using previous
reports.[41–43]
2.4 | X-ray structure determination
Single crystals obtained were characterized using a
BRUKER Quest X-ray (fixed-Chi geometry) diffractometer.
The goniometer was controlled using the APEX3 software
suite.[30] The absorption correction program SADABS[31]
was employed to correct the data for absorption effects.
Systematic reflection conditions and statistical tests of the
data suggested that the space group P-1. A solution was
obtained readily using XT/XS in APEX3. The absence of
additional symmetry and voids was confirmed using
PLATON (ADDSYM). The structure was refined (weighted
least squares refinement on F2) to convergence.[32–35] Olex
2 was employed for the final data presentation and
structure plots.[36] Mercury 3.0 was used for generating
structures and molecular interaction drawings.
2.7 | Molecular docking studies
Molecular docking analysis was performed using Auto
Dock Vina software where an optimized structure of M3P
has been used.[44] DNA docking analysis of M3P has been
done by using the crystal structure of DNA duplex of
sequence
d
(CGCGAATTCGCG)2 dodecamer (PDB
ID:1BNA). Molecular docking studies have also been car-
ried out using BSA protein structure (PDB ID: 4F5S) which
was downloaded from a protein data bank (PDB).[45] The
interactions of M3P with DNA/BSA, including hydrogen
bonds and hydrophobic interactions, were analyzed using
Discovery studio 4.0 client[46] and PyMol.[47]
2.8 | Antimicrobial studies
2.5 | Hirshfeld surface analysis
Antimicrobial studies have been carried out using Agar
Well diffusion method against the Gram positive bacteria
assay namely Staphylococcus aureus and Streptococcus
pneumonia and Gram negative bacteria Klebsiella pneu-
monia, Pseudomonas aeruginosa, and Escherichia coli.
For antifungal screening, the disc diffusion method[48]
was used against fungi Candida albicans, Aspergillus
flavus, and Aspergillus niger. Gentamicin was used as a
standard drug for antibacterial screening and
amphotericin B for antifungal screening.
Intermolecular interactions, as well as surface properties
of the pro-molecule of M3P, were defined by Hirshfeld
surface[37] and 2D fingerprint plots[38] which were
successfully acquired from CrystalExplorer-17.5[39] using
the cif files of SCXRD data.
2.6 | Computational methodology
The single-crystal structure file (cif file) of the title com-
pounds was converted to mol2 files using Mercury 3.0.
These files were used as input files in the GAMESS
(US) software package[40] for energy and other calcula-
tions. Theoretical calculation of M3P in the gas phase
was performed using the analytical gradient method of
DFT with Becke's three parameters (B3) exchange
2.9 | DNA binding studies
DNA binding studies were performed using a spectropho-
tometric titration method. Experiments involving the
interaction of compounds with CT-DNA were performed

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ASHA ET AL.
using double distilled water with tris (hydroxymethyl)
aminomethane (Tris, 5 mM) and sodium chloride
(50 mM). The pH was adjusted, and the absorption titra-
tion experiments were performed by maintaining
50 μg/ml of the concentration of the title compound as
constant but with variable CT-DNA concentration from
10 to 50 μM. While measuring the absorption spectra,
equal amounts of DNA were added to both complex and
reference solutions to eliminate the absorbance of DNA
itself. The data were then fit into the following equation,
and the intrinsic binding constant Kb was calculated.
mesityl moiety and pyridone ring of M3P is associated
with the adjacent molecules to make two sp3 C–H…π and
two sp3 C–H… C interactions. These sp3 C–H… π and sp3
C–H… C interactions developed a typical edge-to-edge
arrangements through mesityl moiety and pyridone ring
with two neighboring M3P molecules. M3P exhibited
π–π stacking interactions in the crystal due to their aro-
matic moieties with a short contact distance <3.7 Å.
Three π–π stacking interactions were observed between
mesityl moiety and pyridone ring of M3P (A) with M3P
(C). These π–π stacking interactions created the perpen-
dicular arrangement of mesityl moiety and pyridone ring
in M3P, and these intermolecular interactions generated
a slip stacking arrangement with neighboring molecules.
The π… π stacking modes formed in M3P(A) and M3P
(B) namely C2(A)-C17(B) mesityl… mesityl with a dis-
tance of 4.02 Å, C11(A)-C19(B) pyridone–pyridone with a
distance of 3.75 Å and mesityl-pyridone C12(A)-C20
(B) with 3.58 Å displayed the existence of π–π stacking
interactions in the crystal between the aromatic mesityl
and pyridone moieties of M3P (A) and M3P (B)
(Figure 2(c)). The stacking pairs are not completely paral-
lel to each other. Hence, these π–π stacking interactions
created face-to-face 3-D parallel-displaced arrangements
throughout the crystal.[50] The M3P crystal packing
forms a supramolecular sheet like arrangement (Figure 2
(d)) in which the water molecule in the lattice acts as a
bridge between the layers.
½DNAꢁ=ðεa-εf Þ = ½DNAꢁ=ðεb-εf Þ + 1=½Kbðεb-εf Þꢁ
ð1Þ
where [DNA] is the concentration of DNA in base pairs, εa
the apparent absorption coefficient, εf the extinction coeffi-
cient of the free compound, and εb the extinction coeffi-
cient of the compound when fully bound to DNA
respectively. In the plots of [DNA]/ (εa-εf) vs [DNA], the
ratio of a slope to the intercept gives the Kb value.[49]
3 | RESULTS AND DISCUSSION
3.1 | Crystal structure analysis using
SCXRD
Single crystals of the title compound were collected and
subjected to SCXRD analysis. Crystallographic data and
structure refinement details of M3P are shown in
Table 1. Figure 1 shows ORTEP view and thermal ellip-
soids. The single-crystal X-ray diffraction data show that
it crystallizes in a space group of P-1. The crystal belongs
to triclinic system with the following lattice parameters:
a = 9.8224(12) Å, b = 11.6383(14) Å, c = 11.8865(14) Å,
α = 64.6410(10)ꢀ, β = 81.156(2)ꢀ, and γ = 69.5910(10)ꢀ,
and its unit cell volume is 1150.8(2) Å3.
Crystal structural analysis of the compound revealed
the presence of abundant non-covalent (weak bonding)
interactions such as intermolecular H-bonds, π–π stac-
king, and Van der Waals forces. Figure 2(a) shows the
unit cell packing arrangement of M3P viewed through
a* axis. Intermolecular H-bonding (Figure 2(b)) was
observed with the hydrogen atom in the mesityl ring in
one M3P moiety as a donor with the oxygen atom in the
2-pyridone ring of another neighboring M3P moiety as
an acceptor. The inter-planar distance and angle between
pyridone (A) and pyridone (B) rings [1.021 Å, 120.6(2)ꢀ]
made the formation of the zig-zag-type of arrangement.
Intermolecular hydrogen bonds are also observed in the
crystal packing which are formed between the hydrogen
atom of the water molecule present in the crystal with
the oxygen atom of the pyridone ring. The sp3 C-H of
The molecular structure of the compound is composed
of three pyridone rings and one mesityl ring. In M3P, the
torsion angles of C(2)-C(1)-C(10)-N(1), C(4)-C(3)-C(16)-N
(2), C(6)-C(5)-C(22)-N(3) are 179.51(19), 97.90(2), and
102.00(2), respectively. The torsion angle 97.90(2) repre-
sents that one of the pyridone rings is perpendicular to the
mesityl ring. The triclinic crystals of M3P showed the
orientation of pyridone rings; that is, A N1 (C11-C15), B
N2 (C17-C21), and C N3 (C23-C37) are perpendicular with
respect to the central aromatic ring (C1-C6) having dihe-
dral angels of 86.20ꢀ, 70.20ꢀ, and 77.00ꢀ, respectively.[51]
3.2 | Hirshfeld surface and 2D
fingerprint plot analysis
The area and volume of three-dimensional Hirshfeld sur-
face mapped on dnorm, di, de, curvedness and shape index
properties are 566.43A2 and 483.73A3, respectively. The
Hirshfeld surface of M3P mapped over a dnorm is shown
in Figure 3. The Hirshfeld surface mapped with dnorm was
generated with the color scale of −0.5016 (a.u) for red and
1.6293 (a.u) for blue. The bright red color spot inside the
contour highlighted the intermolecular interactions in the
crystal structure. The hydrogen bond contact between

ASHA ET AL.
5 of 14
TABLE 1 Crystal data and structure refinement
CCDC number
Empirical formula
Formula weight
Temperature
1,495,831
27 H29 N3 O4
C
459.53
110.0 K
Wavelength
0.71073 Å
Crystal system
Space group
Triclinic
P-1
Unit cell dimensions
a = 9.8224(12) Å
b = 11.6383(14) Å
c = 11.8865(14) Å
1150.8(2) Å3
2
α = 64.6410(10)ꢀ
β = 81.156(2)ꢀ
γ = 69.5910(10)ꢀ
Volume
Z
Density (calculated)
Absorption coefficient
F(000)
1.326 mg/m3
0.090 mm-1
488
Crystal size
0.259 × 0.156 × 0.058 mm3
1.896 to 27.620ꢀ.
Theta range for data collection
Index ranges
-12 < =h < =12, -13 < =k < =15,
0 < =l < =15
Reflections collected
9,538
Independent reflections
Completeness to theta
Absorption correction
Max. and min. transmission
Refinement method
9,538
25.242ꢀ 100.0 %
Semi-empirical from equivalents
0.7456 and 0.6817
Full-matrix least squares on F2
9538/0/314
Data/restraints/parameters
Goodness-of-fit on F2
1.035
Final R indices [I > 2sigma(I)]
R indices (all data)
R1 = 0.0558, wR2 = 0.1344
R1 = 0.0958, wR2 = 0.1561
n/a
Extinction coefficient
Largest diff. peak and hole
0.885 and −0.277 e.Å-3
FIGURE 1 An ORTEP view and thermal ellipsoid of M3P

6 of 14
ASHA ET AL.
FIGURE 2 (a) Unit cell packing arrangement viewed through crystallographic a*-axis, (b) hydrogen bonding, (c) stacking interactions,
and (d) sheet-like arrangement viewed through crystallographic b-axis
hydrogen in water and carbonyl oxygen could be seen
over the Hirshfeld surface as bright red spots. The red
spots over the surface arise due to weak C-Hꢂ ꢂ ꢂC-H and
C-Hꢂ ꢂ ꢂπ interactions, where the intermolecular distance
between neighboring molecules is less than Van der Waals
radius. In the blue region on the Hirshfeld surface, the
intermolecular distance between neighboring molecules is
more than Van der Waals radius. The Hirshfeld surface of
M3P mapped over shape index and curvedness is shown
in Figure 3. Hirshfeld surface mapped over shape index is
the most sensitive method to determine the changes in the
surface shape of the molecule and can be used to identify
the two molecular Hirshfeld surfaces which touch one
another. The red triangles on them represented by hollow
regions indicated atoms of the π-stacked molecule above
them, and the blue triangles represented by bumps indi-
cated the atoms of the molecule inside the surfaces. As it
can be seen from Figure 3 the shape index plot has com-
plementary red and blue spots which indicated the stac-
king of molecules related by center of symmetry. The
curvedness mapped over Hirshfeld surface provided useful
information about the shape of the surface of the
molecule. Curvedness maps typically showed green flat
areas of the surface that corresponds to low values of
curvedness, separated by dark blue curvature areas which
correspond to high values of curvedness and thus divide
the surface into patches, indicating interactions between
neighboring molecules.[52] The curvedness map of the
compound as shown in Figure 3 showed the presence of
green flat patches present over pyridine moiety indicating
the planar π–π stacking between neighboring molecules.
In di, the color scale in the conformations was maintained
as 0.8022 (a.u) for red and 2.9108 (a.u) for blue. The fin-
gerprint plot created by combining di and de described a
2D representation of 3D Hirshfeld surface and provides a
summary of intermolecular contacts in the same way as
the Hirshfeld surface. The fingerprint plots for different
intermolecular interactions and Hirshfeld surfaces
corresponding to these interactions are given in Figure 4.
The summary of these contacts including reciprocal con-
tacts and their contributions are shown in Table 2. It can
be observed that the major interaction between neighbor-
ing molecules is between hydrogen atoms, that is, Hꢂ ꢂ ꢂH
which covers 56.3% of all interactions. The strong

ASHA ET AL.
7 of 14
FIGURE 3 Hirshfeld surface of M3P
hydrogen bond interactions between Oꢂ ꢂ ꢂH/Hꢂ ꢂ ꢂO are
represented by two large spikes at the bottom of the fin-
gerprint plot and accounted for 20.5% of all interactions.
This interaction is also indicated by two bright red spots
on the Hirshfeld surface (Figure 4). The fingerprint plots
show that H-C/C-H contacts comprise 18% of the total
Hirshfeld surface area for the molecule. They correspond
to all O-HꢂꢂꢂO interactions of which strong C-Hꢂ ꢂ ꢂπ
interactions appear as four light red spots on the Hirshfeld
surface (Figure 4). The contact between Carbon and Car-
bon (C-C/C-C) is another interaction comprising 3.4% of
all other interactions. This interaction is characterized by
two small spikes on sides of the fingerprint plot as shown
in Figure 4. The other minor contacts include C-N (0.7%),
N-H (0.6%), and C-O (0.4%) which account for the
remaining 7.3% of all interactions over Hirshfeld surface.
structures.[53–57] Parameters like total energy, EHOMO
,
ELUMO, ionization potential, electron affinity, electronega-
tivity, electrochemical potential, hardness, softness,
and nucleophilicity are given in Table 3. According
to Molecular Orbital (MO) theory, the Highest
Occupied Molecular Orbital (HOMO) and the Lowest
Unoccupied Molecular Orbital (LUMO) are collectively
called frontier molecular orbitals (FMOs). FMOs are the
most important factors affecting the bioactivity which
specifies how the molecule interacts with species. The
interaction between the molecule and the receptor of bac-
teria is dominated by π–π stacking and hydrophobic inter-
action among these FMOs.[58,59] HOMO and LUMO of
compounds are demonstrated in Figure 5. The electron
clouds in M3P showed that HOMO was located in one
pyridone (ring-I) and the LUMO in another pyridone ring
and not in the mesityl moiety. This confirmed the pres-
ence of intermolecular π–π stacking interactions between
pyridone units. The energy of the HOMO is linked to the
ionization potential, while LUMO energy is linked to
the electron affinity. The results reveal that the energy of
the HOMO orbital is equal to −5.7470 eV, and the energy
of the LUMO orbital is equal to −1.2030 eV, which gives
Theoretical studies
The energies of compounds were calculated at the
B3LYP/6-311G++ level of theory by the DFT method in
the gas phase, carried out from the experimental

8 of 14
ASHA ET AL.
an energy gap of about −4.5440 eV. This energy gap
describes well the molecular chemical reactivity.[60,61]
From Table 3, the parameters such as Ionization Potential
(I), Electron Affinity (A), and Electronic Chemical Poten-
tial (μ) are equal to −5.7470, 1.2030, and −3.4750 eV,
respectively. The chemical hardness (η) is found to be
2.2720 eV. Also, the molecule has a chemical softness
equal to 0.4401 eV−1. The electronegativity is computed
based on the HOMO and LUMO energies and is found to
be 3.4750 eV. The nucleophilicity value is found to be
3.6342. Finally, the maximum charge transfer index has
been calculated and found to be 1.529. Mulliken atomic
charge calculation plays a major role in the quantum
chemical calculations of the molecular system. As the
FIGURE 4 Fingerprint plot with contribution of various interactions for M3P

ASHA ET AL.
9 of 14
FIGURE 4 (Continued)
system of electronic structure, dipole moment, polarizabil-
ity, and other molecular properties is affected by atomic
charges, the distribution of charge over the atoms leads to
the creation of donor and acceptor pairs producing charge
transfer in the molecule. The Mulliken population analy-
sis in M3P molecule was computed using B3LYP with
6-311G++ (d,p) basis set and is listed in Table 4. In our
molecule, the O61 atom has the highest negative charge
(−0.616) compared to other oxygen atoms (O1 = −0.5796,
O2 = −0.5939, and O3 = −0.5684) present in the rings.
The carbon atom involved with the hydrogen atom is
negative, whereas the remaining carbon atoms
are positively charged in M3P. The oxygen and nitrogen
atoms have more negative charge, whereas all the
hydrogen atoms have positive charges. The more positive
charge of carbon is found in the atoms C28, C40, C48,
and C60. It is mainly due to the substitution of negative
charge of oxygen and nitrogen atoms. The presence of a
large negative charge on O2 and the net positive charge
on H62, H63 atom may confirm the formation of
C48-O27 … H63 intermolecular hydrogen bond interac-
tion in solid state.
3.3 | Molecular docking studies
Molecular docking techniques are used to recognize the
drug–DNA interactions in rational drug design, as well as
in the mechanistic study by placing a small molecule into
the binding site of the target DNA.
The mode of interaction of the compound with DNA
was theoretically calculated by molecular docking studies
using DNA base pairs d (CGCGAATTCGCG)2 dodecamer
(PDB ID: 355D) using the Auto Dock tools. Minimum

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ASHA ET AL.
TABLE 2 Summary of contacts (reciprocal contact included)
TABLE 3 FMO energy parameters and global reactivity
and their contributions to the Hirshfeld surface in M3P
descriptors of M3P
Types of contacts
Percentage contributions
Molecular properties
M3P
H-H
O-H
C-H
C-C
C-N
N-H
C-O
56.3
20.5
18.0
3.4
Total energy (kcal/Mol)
−1511.549
−5.7470
−1.2030
−4.5440
5.7470
E
E
HOMO (eV)
LUMO (eV)
ΔE (eV)
0.7
Ionization potential (eV)
Electron affinity (eV)
Electronegativity (eV)
Electrochemical potential (eV)
Absolute hardness (eV)
Softness (eV)
0.6
1.2030
0.4
3.4750
−3.4750
2.2720
0.4401
energy conformation was obtained from the docking of
the compound with CT-DNA (Figure 6). Further, the
resulting structures are stabilized by Van der Waals and
hydrogen bond interactions with DNA. Some important
binding between donor and acceptor atoms is also avail-
able in the interactions. It reveals that the hydrogen and
carbonyl oxygen atoms play an important role as donor
atoms in hydrogen bonding. The binding energy of M3P
is found to be −6.4 kcal/mol.
The action of the drug needs a comprehensive under-
standing of compounds–protein interactions which has a
great role in rational drug designing.[62–65] Molecular dock-
ing results revealed that the binding affinity of M3P into
the binding pocket of BSA protein is −8.1 kcal/mol. The
most favorable docking poses are given in Figure 7. The
hydrogen bonding interaction was observed between
Asn404 with the oxygen atom of the pyridine ring. An
interaction between Met547 and nitrogen atom of the M3P
compound was also observed. The π–π interaction was also
between Leu397 amino acid with the mesityl ring. Three
amino acids namely Val408, Leu543, and Lys544 are
involved in hydrophobic interactions with the compound.
Nucleophilicity (eV)
Dipole moment (Debye)
3.6342
5.4148
3.4 | Antimicrobial evaluation
The M3P crystal was screened against some microorgan-
isms to evaluate their antimicrobial activities. The crystal
was evaluated for their in vitro antibacterial activity
against some Gram positive bacteria assay namely Staph-
ylococcus aureus and Streptococcus pneumonia and Gram
negative bacteria Klebsiella pneumonia, Pseudomonas
aeruginosa, and Escherichia coli. They were also evalu-
ated for their in vitro antifungal potency against Candida
albicans, Aspergillus flavus, and Aspergillus niger fungal
strains. The agar-diffusion method was used in the deter-
mination of the preliminary antibacterial and antifungal
activity. Gentamicin and amphotericin B were used as
reference drugs for antibacterial and antifungal species
respectively. The results were recorded for each
FIGURE 5 Molecular orbitals of M3P

ASHA ET AL.
11 of 14
TABLE 4 Mulliken atomic charges of M3P
Atoms
O1
Charges
−0.5796
−0.5939
−0.5684
−0.5099
−0.5285
−0.5310
−0.0319
0.0371
Atoms
C33
H34
C35
H36
C37
H38
H39
C40
H41
C42
H43
C44
H45
C46
H47
C48
C49
H50
H51
C52
H53
C54
H55
C56
H57
C58
H59
C60
O61
H62
H63
Charges
−0.1266
0.0652
0.1943
0.0990
−0.0509
0.1379
0.1495
0.1985
0.1084
−0.1294
0.0660
0.0006
0.0760
−0.1215
0.0753
0.6078
−0.0273
0.1468
0.1255
0.1901
0.1057
−0.1251
0.0659
−0.0056
0.0728
−0.1242
0.0749
0.5856
−0.6160
0.2879
0.3032
O2
O3
N4
N5
N6
C7
C8
C9
−0.0744
0.0362
C10
C11
C12
C13
H14
H15
H16
C17
H18
H19
H20
C21
H22
H23
H24
C25
H26
H27
C28
C29
H30
C31
H32
−0.0764
0.0460
−0.3543
0.1288
0.1378
0.1409
−0.3576
0.1225
FIGURE 6 Molecular docking studies of M3P with DNA
0.1213
0.1432
−0.3528
0.1516
against Pseudomonas aeruginosa and Klebsiella pneumo-
nia when compared with the standard drug gentamicin
at a high concentration of 500 μg/ml. It shows moderate
activity against Staphylococcus aureus, Escherichia coli
and Streptococcus pneumonia. Antifungal screening of
M3P exhibited moderate activity against Aspergillus
flavus when compared with the standard drug
amphotericin B. Further, M3P shows good activity
against Candida albicans and Aspergillus niger. As evi-
dent from Table 5, inhibition against bacteria and fungi
increases with an increase in the concentration of the
compound.
0.1189
0.1426
−0.0855
0.1647
0.1222
0.5952
−0.1219
0.0787
−0.0080
0.0759
3.5 | DNA binding studies using the
spectrophotometric titration method
compound as the average diameter of inhibition zones
(IZ) of bacterial or fungal growth around the discs in mm
(Table 5).
Electronic absorption spectroscopy is an effective method
to determine the binding mode and intensity of organic
molecules with CT-DNA. The intrinsic binding constant
Kb for M3P was calculated as 9.18 × 104 M−1 indicating
that it can moderately bind with CT-DNA which is com-
parable with the docking studies. There was another typi-
cal observation from the UV absorption spectrum that
there was an increase in absorption for the compound
while increasing the DNA concentration, that is, hyper-
chromic shifts have been observed for the compounds
The results revealed that most of the tested com-
pounds displayed variable inhibitory effects on the
growth of the tested bacterial strains and also against
antifungal strains. From Table 5, it is clear that the anti-
microbial activity depends upon the concentration of the
compound used and a minimum inhibitory concentration
of 100 μg/ml and above is needed to kill the microorgan-
isms. It is noteworthy that M3P shows a good response

12 of 14
ASHA ET AL.
FIGURE 7 Molecular docking of M3P
with BSA
TABLE 5 Antibacterial activity and antifungal activity of M3P
Zone of inhibition (mm)
500 μg/ml 250 μg/ml
07–10 05–08
No
1
Name of the bacteria and fungi
Staphylococcus aureus (bacteria)
Streptococcus pneumonia (bacteria)
Klebsiella pneumonia (bacteria)
Pseudomonas aeruginosa (bacteria)
Escherichia coli (bacteria)
100 μg/ml
50 μg/ml
00–02
05–11
03–05
10–12
02–03
07–12
04–07
03–06
Std
02–05
18–21
04–07
17–20
01–03
16–19
06–09
07–10
12–15
20–23
10–13
23–26
08–12
21–24
12–15
11–14
2
19–21
11–14
24–28
05–08
24–27
08–11
12–15
21–24
06–09
20–23
02–05
19–23
05–08
08–11
3
4
5
6
Candida albicans (fungi)
7
Aspergillus flavus (fungi)
8
Aspergillus Niger (fungi)
Note. The bold emphasis denotes the highest activity of the title compound against the bacteria and fungi.
FIGURE 8 UV-Vis spectra for DNA binding
ability of M3P

ASHA ET AL.
13 of 14
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To sum up, a new crystal,1-({2,4,6-trimethyl-3,5-bis[(2-
oxo-2H-pyridin-1-yl)methyl]phenyl}methyl)-2H-pyridin-
2-one hydrate (M3P) has been synthesized and character-
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single-crystal X-ray diffraction analysis. Crystal structural
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ORCID
R. Nandini Asha https://orcid.org/0000-0002-8296-6380
B. Ravindran Durai Nayagam https://orcid.org/0000-
0001-7859-971X
[31] SADABS, BRUKER AXS Inc., 5465 East Cheryl Parkway,
Madison, WI 53711-5373 USA.
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How to cite this article: Asha RN, Rahini MU,
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