Effect of o-difluoro and p-methyl substituents on the structure, optical properties and anti-inflammatory activity of phenoxy thiazole acetamide derivatives: Theoretical and experimental studies

Article abstract of DOI:10.1016/j.molstruc.2019.127024

Thiazole derivatives (6a and 6b) have been synthesized and characterised by ¹H –¹³C NMR, as well as LC-MS spectra. The three-dimensional structures have been confirmed by single crystal X-ray diffraction method. 6a and 6b compounds have been crystallized in the Triclinic and the Orthorhombic systems with P-1 and Pbca space groups, respectively. Supramolecular structures revealed the stability of molecules with different intermolecular interactions and different crystal packing environment. Theoretical study by Density functional theory (DFT) with B3LYP functional based on highest basis set 6–311++G(d,p) was employed to calculate the geometry and compared to the experimental data. The electronic structures and intramolecular charge transfers have been investigated by using natural population and natural bond orbital analysis (NBO). Further, DFT studies were performed to assess the frontier molecular orbitals (FMOs), energy gap, softness, hardness, and others chemical reactivity. Hirshfeld surface was investigated to distinguish the different interatomic contacts and understand the crystal packing of molecules with aid of energy frameworks through different intermolecular interaction energies based on the anisotropy of the topology. Nonlinear optical property (NLO) of the synthesized molecules were predicted by (DFT) and examined experimentally by using second harmonic generation (SHG) and revealed the importance of high NLO based on the nature of substituents and conformation. Thiazole derivatives were assessed for anti-inflammation activity by in silico molecular docking studies against COX-1 and COX-2 protein receptors revealed prominent interactions with active site and further molecular dynamics confirms the stability of the protein-ligand model. In vitro assay against cyclooxygenase (COX) enzyme gave IC₅₀ values of 6a and 6b molecules with ortho-difluoro and para-methyl positions on benzoyl group, showed better inhibitor for COX-1 and COX-2, respectively.

Full text of DOI:10.1016/j.molstruc.2019.127024

Journal Pre-proof
Effect of o-difluoro and p-methyl substituents on the structure, optical properties and
anti-inflammatory activity of phenoxy thiazole acetamide derivatives: Theoretical and
experimental studies
Hussien Ahmed Khamees, Yasser Hussein Eissa Mohammed, Ananda S, Fares
Hezam Al-Ostoot, Sangappa Y, Saad Alghamdi, Shaukath Ara Khanum, Mahendra
Madegowda
PII:
S0022-2860(19)31124-X
DOI:
https://doi.org/10.1016/j.molstruc.2019.127024
MOLSTR 127024
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 July 2019
Revised Date: 26 August 2019
Accepted Date: 2 September 2019
Please cite this article as: H.A. Khamees, Y.H.E. Mohammed, A. S, F.H. Al-Ostoot, S. Y, S. Alghamdi,
S.A. Khanum, M. Madegowda, Effect of o-difluoro and p-methyl substituents on the structure, optical
properties and anti-inflammatory activity of phenoxy thiazole acetamide derivatives: Theoretical
and experimental studies, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.127024.
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Effect of o-difluoro and p-methyl Substituents on the Structure,
Optical Properties and Anti-inflammatory Activity of Phenoxy
Thiazole acetamide derivatives: Theoretical and Experimental Studies
Authors:
Hussien Ahmed Khamees1, Yasser Hussein Eissa Mohammed2,3, Ananda S1, Fares Hezam
Al-Ostoot2,4, Sangappa Y5, Saad Alghamdi6, Shaukath Ara Khanum2 and Mahendra
Madegowda1*
Affiliations:
1Department of Studies in Physics, Manasagangotri, University of Mysore, Mysuru-570006,
Karnataka, India.
2Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysuru- 570005
Karnataka, India.
3Department of Biochemistry, Faculty of Applied Science College, University of Hajjah,
Yemen.
4Department of Biochemistry, Faculty of Education & Science, University of Albaydaa,
Yemen.
5Department of Physics, Mangalore University, Mangalore, Karnataka State, 574199, India.
6Laboratory Medicine Department, Faculty of Applied Medical Science, Umm Al-Qura
University, Makkah, Saudi Arabia.
*Correspondence Author:
Dr. Mahendra Madegowda.
Email: mahendra@physics.uni-mysore.ac.in
DOS in Physics, Manasagangotri, University of Mysore, Mysuru- 570006, Karnataka, India.
1

Abstract
Thiazole derivatives (6a and 6b) have been synthesized and characterised by 1H -13C NMR, as well as LC-MS
spectra. The three-dimensional structures have been confirmed by single crystal X-ray diffraction method. 6a
and 6b compounds have been crystallized in the Triclinic and the Orthorhombic systems with P-1 and Pbca
space groups, respectively. Supramolecular structures revealed the stability of molecules with different
intermolecular interactions and different crystal packing environment. Theoretical study by Density functional
theory (DFT) with B3LYP functional based on highest basis set 6-311++G(d,p) was employed to calculate the
geometry and compared to the experimental data. The electronic structures and intramolecular charge transfers
have been investigated by using natural population and natural bond orbital analysis (NBO). Further, DFT
studies were performed to assess the frontier molecular orbitals (FMOs), energy gap, softness, hardness, and
others chemical reactivity. Hirshfeld surface was investigated to distinguish the different interatomic contacts
and understand the crystal packing of molecules with aid of energy frameworks through different intermolecular
interaction energies based on the anisotropy of the topology. Nonlinear optical property (NLO) of the
synthesized molecules were predicted by (DFT) and examined experimentally by using second harmonic
generation (SHG) and revealed the importance of high NLO based on the nature of substituents and
conformation. Thiazole derivatives were assessed for anti-inflammation activity by in silico molecular docking
studies against COX-1 and COX-2 protein receptors revealed prominent interactions with active site and further
molecular dynamics confirms the stability of the protein-ligand model. In vitro assay against cyclooxygenase
(COX) enzyme gave IC50 values of 6a and 6b molecules with ortho-difluoro and para-methyl positions on
benzoyl group, showed better inhibitor for COX-1 and COX-2, respectively.
Keywords: DFT studies; Energy framework; In Silico Docking; In vitro COX; Molecular
Dynamics; Structural Analysis.
2

1. Introduction
Thiazole moiety is one of the organic heterocyclic rings that comprises nitrogen and sulphur atoms. Its
derivatives are the most bioactive important compounds in pharmacological field that plays a major role in
designing new pharmacologically active drugs and to develop the existed ones, which are extensively exploited
in the medicinal chemistry [1] with versatile activities like: anti-oxidant[2], antipsychotic[3], antibacterial[4],
antitumor [5] and anti-inflammatory[6].
The inflammation is well known multifactorial process that specifically defines as local responses of
mammalian tissues to the injury by any agent. In more details, it is a multiple sequence of a cellular and
molecular responses acquired throughout the evolution to eliminate external agents and then give promotion to
repair the damaged tissues. Fundamentally, the inflammation can be categorized into two types: acute and
chronic. Acute inflammation type is a fast response to the injurious agent. On the onset of such injury there
could be promulgation to deliver mediators of host defense-leukocytes and plasma proteins at the injury site. On
the other hand, the chronic inflammation response has prolonged inflammation duration (weeks to months to
years). This could proceed with active inflammation and injury simultaneously [7-8].
Among all strategies and methods available for treatment of inflammation, the two groups of
arachidonic acid metabolites: prostaglandins and leukotrienes produced by Cyclooxygenase (COX) and
Lipoxygenase (LOX) respectively, play significant role in inflammation, where the free steroidal anti-
inflammatory drugs usually inhibit the COX-1 isoenzyme or the combination isoenzymes of COX-1 and COX-
2[8].
Since more than two decades, the researchers and pharmacists have focused on the synthesis and
development of the thiazole derivatives as anti-inflammatory agents. Pawan K. Sharma et al, (1997) [9]
prepared a series of 1,2-benzisothiazole compounds with thiazolyl-acetic acid moiety and evaluated the anti-
inflammation activity; the molecule with para-chlorophenyl substituents registered as the most potent anti-
inflammatory molecule.
B. Shivarama Holla et al, (2003) [10] synthesised and screened the anti-inflammatory and antibacterial
properties of a series of 2,4-disubstituted thiazole. The authors have reported the compounds with 3-Cl, 4-F
phenyl and 4-Cl substituents are the best potent anti-inflammatory activity having 94% and 93% of oedema
inhibition in comparison to that of standard ibuprofen. Also, Pran Kishore Deb et al, (2013) [11] have reported
on the synthesis and anti-inflammatory activity of some benzothiazole acetamide derivatives and suggested that
the compounds with chloro and fluorophenyl substituents exhibited good anti-inflammatory activities with 95%
and 88% of the standard indomethacin. Other researchers also revealed the significance of amino group at the
para position of the thiazole ring, which has an increase in anti-inflammatory activity [12].
In addition, thiazole derivatives have been proved the enhancement of nonlinear optical property
(NLO), exceptionally with unsymmetrical thaizole rings and lengthy π-conjugated system, which typically
possess a worth value of second harmonic generation (SHG) [13-14], which is suitable for technological and
industrial applications of electronic and optical materials. The importance of thiazole ring as NLO materials has
been studied and confirmed widely in various experimental [15] and theocratical [13] methods. Such property
3

has been impacted by mutual affecting factors which includes asymmetric polarization, conjugate length, degree
of linearity conformation, intramolecular charge transfer (ICT), types of donor and acceptor groups as well as
their position [16-18]. The registered patents of thiazole derivatives are a proof of their importance in both
industrial and pharmaceutical fields [19].
In view of the above facts and information observed from the literature, it is clear to reveal that the
combination of thiazole moiety with 4-chloro phenyl, acetamide and fluorine substituents are important for the
profound anti-inflammatory activity. Hence, motivated by the above literature, we have made pertinent
objectives to synthesize 2-(phenoxy)-N-(4-(4-chlorophenyl) thiazol-2-yl) acetamide (scheme 1), which
specifically target to study the effect of para methyl and 2,6 ortho fluorine substituents on the phenoxy ring of
the thiazole derivative; including structural characterization, supramolecular, electron population, HOMO-
LUMO, electrostatic and molecular electrostatic potential (ESP-MEP), nonlinear optical property (NLO),
Hirshfeld surface analysis and energy frameworks as well as anti-inflammatory activities of the compounds. in
addition, these compounds were tested theoretically to know the fitness and binding possessions of the
compounds in the active site of COX-1 (pdb ID:1eqg) and COX-2 (pdb ID:1pxx) receptors by using docking
and molecular dynamic simulation methods.
2. Procedures and instruments
2.1 Materials and Methods
All chemicals have been bought from the Sigma Aldrich Chemical Co. Thin layer chromatographic technique
were used to check the purity of the compounds under UV-lamp with λ = 254 nm. Boiling and melting points
have been recorded with a digital thermometer on the Chemiline, Microcontroller based melting point / boiling
point-Cl725 apparatus. 1H and 13C NMR spectra have been determined on VNMRS-400 “Agilent-NMR” with
using dimethyl sulphoxide solvent. Chemical shifts recorded in parts/million downfield of tetramethylsilane.
The ElementarVario EL III elemental analyzer has been used for elemental analysis (N, C, and H), where the
result exhibited within ± 0.4 % of the theoretical values. Mass spectra has been recorded on VG70-70H
spectrophotometer.
Scheme. 1 Synthesis of 2-(phenoxy)-N-(4-(4-chlorophenyl) thiazol-2-yl) acetamide derivatives
4

2.2 Synthesis
Synthesis of 2-(phenoxy)-N-(4-(4-chlorophenyl) thiazol-2-yl) acetamide derivatives were implemented as
shown in the scheme 1. Initially with the dry acetone as the solvent obtained ethyl 2-phenoxyacetate, which has
been hydrolyzed with the solution of sodium hydroxide to provide 2-phenoxyacetic acid, the esterification of
phenols with ethylchloroacetate are done with anhydrous potassium carbonate. The final compounds were
synthesized by joining 2-phenoxyacetic acid with 4-(4-chlorophenyl) thiazol-2-amine by using dichloromethane
as a solvent and 2,6-lutidine as a base as well as o-(benzotriazol-1-yl)-N,N,N′,N′- tetra methyl uranium tetra
fluoro borate as coupling agent. The reaction was monitored by thin layer chromatography. The spectral data for
the final compounds 6a and 6b are presented in the Supplementary Material (Figs. S1 S8).
2.3 X-ray intensity data collection strategy
The X-ray intensity data for both 6a and 6b molecules were recorded at temperature of 293(2) K on a Bruker
AXS kappa Apex2 CCD Diffractometer, with fine-focus sealed tube radiation source and 0.71073 Å wavelength
of MoKα radiation. The procedure and reduction of data set was accomplished completely by using SAINT
PLUS [20]. SHELXS and SHELXL programs [21] have been adopted to solve and refine the structure,
respectively. While the geometrical calculations, molecular figures and Crystal packing were generated and
visualized by PLATON and MERCURY software, respectively [22-23]. CCDC numbers: 1842848 and 1828753
contains full crystallographic data of 6a and 6b crystals respectively, and available online at Cambridge
crystallographic data centre.
2.4 Nonlinear optical property (NLO)
The experiment of NLO property was performed at the Indian Institute of Science (IISc), Bengaluru, according
to the method of classical powder that developed by Kurtz and Perry [24] to investigate SHG efficiency. In this
method, the samples were powdered in microcrystalline state and then tightly filled in a capillary glass tube.
These were exposed to Nd:YAG laser with a wavelength of 1064 nm, 1.2 mJ input energy and 10 Hz repetition
frequency. The references materials were Urea and KDP samples with of 60 mV and 17 mV output values,
respectively.
2.5 In vitro COX assay
By using an enzyme immunoassay (EIA) kit (catalog number 560101, Cayman Chemical, Ann Arbor, MI,
USA), the procedures have been described in the Supplementary Material (subsection 1.2)
2.6 Computational studies
2.6.1 Density functional theory calculation (DFT)
The most suitable function for organic compounds “B3LYP” (Becke’s three parameter (B3)) [25] has been
adopted in DFT model for whole theoretical calculations, by using Gaussian09 and Gaussview5.0 programs
under 6-311++G(d,p) bases set[26] .
5

2.6.2 Hirshfeld surface analysis and energy framework calculations
The intermolecular contacts, 2D fingerprint and energy frame work of the title compounds have been studied by
using CrystalExplorer17.5 program [27]. The molecular Hirshfeld surface is one of the best graphical
visualization for realising the intermolecular interactions in 3D simulation. Such surface is built on dnorm given
by equation (1).
vdw
dnorm = (di -rivdw/ rivdw) + (de -revdw/re
)
(1)
where di and de are the distances from the surface to the nearest inside and outside atoms, respectively, and rivdW
and revdW are the van der Waals radii of the contact.
Energy frameworks are considered as a powerful method to understand the crystal packing of molecules by
combining the different intermolecular interaction energies including electric (Eele), dispersion (Edis),
polarisation (Epol), repulsion (Erep) and total (Etot) energy based on the anisotropy of the topology of pairwise
intermolecular interaction energies. The cylinders are representation of interaction energies between the two
molecules and the radius are directly related to the energy magnitude of the interaction with the same scale
factor to expand or contract them [28].
2.6.3 Docking Simulation
Docking studies were carried out by Schrodinger-Maestro Version 10.7.014 [29] with extra precision (XP)
docking module in Glide 5.5 (Grid-based ligand docking with energetics). Receptor structures of COX-1(pdb
ID: 1eqg) and COX-2 (pdb ID: 1pxx) have been downloaded from RCSB website (http://www.rcsb.org/pdb/)
and subjected to receptors preparation including remove of crystallized ligands, water, cofactors as well as
charge fixing and polar hydrogens were achieved. 2D ligands model were constructed via Maestro 2D sketcher
application. The ligands 6a and 6b have been prepared by Ligprep application in Schrödinger that includes the
minimization of energy, choosing proper chirality, addition of hydrogens, ring conformations and
stereochemistry. The minimization has been achieved by OPLS (optimized potentials for liquid simulations)
Model [30-31]. 3D Glide grid boxes have been generated with a size of (34x, 34y,34z) Å3 and (32x, 32y, 32z)
Å3 for COX-1 and COX-2 receptors, respectively with grid maps centred at 26.7, 33.9, 200.3 and 27.0, 24.1,
14.8 for COX-1 and COX-2, respectively. All the dockings have been performed for all receptor-ligand in Glide
Extra Precision Mode active. The best ligand-receptor conformations are listed in Table 10, showing the glide
score, docking score, glide energy, hydrogen bonds and Pi interactions. The visualization of results has been
implemented by Maestro workspace, BIOVIA [32], LigPlot+ [33] and PyMol [34].
2.6.4 Molecular Dynamic Simulations
Molecular Dynamic stimulations (MDS) have been accomplished by using Desmond module (developed by D.
E. shaw research) [35] embedded in Schrodinger-Maestro. The best conformation of the 6a-1eqg complex has
been subjected to the molecular dynamics to investigate the stability of receptor-ligand complex system by
immersing it in SPC (simple point charge) water model using orthorhombic box and OPLS 2005 force field. The
system was neutralized by adding Cl- ions. The neutralised system was built with 0.15 M concentration of salt
(NaCl) in 10 Å buffer and the system was relaxed by implementing Steepest Descent and the limited-memory
6

Broyden-Fletcher-Goldfarb-Shanno algorithms in a hybrid manner to ensure that there is no steric clashes or
inappropriate geometry. The simulation was enhanced under NPT ensemble for 1.2 ns by implementing the
Berendsen thermostat and barostat methods. With 300 K of temperature that maintained throughout the
simulation using the Nose-Hoover thermostat algorithm [36] and 1.01325 bar of pressure through Martyna-
Tobias-Klein Barostat algorithm [37]. The final production run was carried out for 1.2 ns and the trajectory
sampling was done at an interval of 4.8 ps.
3 Results and Discussions
3.1 Structural analysis
A block shaped colourless single crystals having dimensions 0.15 × 0.20 × 0.25 mm of 6a and 0.10 × 0.15 ×
0.15 mm for 6b compound were chosen for Single crystal X-ray diffraction study, which revealed that the
molecular systems were crystallized in different crystal systems, 6a in the Triclinic with space group P-1, and
6b in the Orthorhombic system with space group Pbca. The details of structure refinement and descriptions of
crystal data are given in Table 1. Fig. 1 illustrates the experimental ORTEP diagrams and predicted structure for
the molecules. The dihedral angles between the planes of thiazole ring and ring A are 9.95° and 16.51° for 6a
and 6b, respectively, indicating the coplanarity conformation. The noticeable change in conformation of the
compounds based on the substituents can be observed between the mean planes of ring A with thiazole ring and
ring B in 6a, which indicated by mean planes angle values of 132.42°. The same angle has been shown in the 6b
with slightly Smaller value of 83.58° (Fig. 2). The computed geometries of the compounds were compared with
the experimental X-ray values. Some selected molecular parameters are listed in Tables 2-4. Full list of
geometries are given in Tables S1-S3 of Supplementary Material. The atoms numbering is according to ORTEP
diagram (Fig. 1).
In the present study, X-ray values of C-C bonds in rings A and B from 1.357(5) Å to 1.396(3) Å, showing
agreement with DFT predicted data (i.e., 1.38462 to 1.40278 Å) and also some reported thiazole compounds
[38-39] have similar values. The small variations in the C-C bond lengths can be attributed due to the effects of
the substituents present on different positions resulting with change in the charge distribution of the benzene
ring [40-41]. This is clearly observed in the C-C bond lengths of rings A and B of the two molecules. In ring A,
both the molecules are substituted by p-chloro showing no much deviation, while the different substituents and
position in ring B reflected maximum difference of 0.01372 Å (0.021 Å) as mentioned by both DFT values and
XRD in parenthesis. The substitution of methyl group (electron donating) at para position in ring B (6b) has
shown an increase of the electrons density on the ring (see Table 6) resulting in the increase of length C19-C20
= 1.40278 Å (1.387(4) Å), which has the longest C-C bond lengths found in the phenyl ring B. On the other
hand, the substitution of two fluorine atoms (electron acceptor) at ortho positions in ring B (6a) has decreased
the electron density at the ring carbon atom resulting in the reduction of lengths for C18-C17= 1.38481Å
(1.364(4)Å), as well as C20-C19 = 1.38337Å (1.357(5)Å), which has the shortest lengths of C-C bond as
observed in the phenyl rings.
The effect of methyl group in ring B (6b) shows distortion in the ring yielding angles smaller than the typical
angle (hexagonal) of 120o. at the position of substitution i.e. C18-C19-C20 =117.5626o (116.8(3)o), recording
7

the smallest C-C-C angle in the molecules [42]. This effect is in reverse with fluorine atoms (6a) that exhibited
larger (120o). at the position of substitution i.e. C18-C17-C16=122.5198o (122.2(3)o) and C20-C21-
C16=122.4745o (122.9o), respectively, recording the largest C-C-C angle for phenyl rings.
The variations in the bond length of phenyl rings A and B have been investigated by standard deviation equation
(2):
ꢀ = ꢁꢃꢂ
(2)
where µ represent the mean.
ꢃ
∑
ꢄ
ꢈꢉ
ꢅꢆ − ꢇ
ꢆꢊꢂ
The deviation in length of rings A and B for 6a are (0.01048) Å and (0.00641) Å, respectively, showing the
bond lengths of the ring A seems to be a double distorted than that of ring B.
While the deviation of rings A and B for 6b are 0.00963 Å and 0.00955 Å, respectively, denoting that the
distortions of bond lengths of the ring A and B are seem to be similar.
Over all standard deviation results indicated that the substituents of difluoro atoms at the ortho positions of ring
B have more steric effect rather than the other ring A.
The agreement between the predicted values and experimental data were verified by using linear regression
model with coefficient regression value of R2= 0.99 for both compounds, as shown in Fig. S9 of Supplementary
Material. These results reflect the degree of agreement between theoretical and experimental data.
3.2 Supramolecular
H-bonds dominate in the supramolecular architecture of the crystal packing of 6a molecule, where C19-
H19...O13 interactions have linked the molecules into C9-chains when viewed down b-axis (Fig. 3I). The
adjacent chains are interlinked via two centrosymmetric C14-H14A…O13 bonds (Table 5), showing ring motif
pattern R2 (8), resulting in a chair type two-dimensional sheets in the ac plane (Fig. 3II). These sheets are
2
stacked one over the other via weak interactions showing three-dimensional supramolecular feature (Fig. 3III).
On the other hand, the crystal packing of 6b molecule down b-axis has showed C4-chain by connecting two
molecules via N11-H11...O13 hydrogen bond with additional short contact (O13…N11) (Fig. 4I). This chain is
reinforced by weak C-H… π interactions, showing C14-H14A....Cg(3)= 2.74 Å and symmetry (1/2-X,1/2+Y,Z)),
where Cg(3) is the gravity centre of ring B (C16-C21). This C4-chain chain is constituted together with C6-
chain via C14-H14B...N8 hydrogen bond. Altogether, these adjacent chains showed R2 (8) ring motif. The
2
neighbouring two motifs are linked by C10 chain via C5-H5...O15, S2…S2 short contacts as well as weak π …π
stacking, between Cg(1) thiazole ring and Cg(2) chlorophenyl ring, thereby forming into a two dimensional
sheet when viewed along b axis (Fig. 4II). These sheets are further stabilized by C-H… π interaction (with
distance C20-H20…Cg(2)= 2.84 Å and symmetry operation (1/2-X,-Y,1/2+Z)), as seen in crystal packing (Fig.
4II). The geometries of potential hydrogen bonds are listed in Table 5.
3.3 Firsthyperpolarizability (β) and Nonlinear optical property (NLO)
The First hyperpolarizability is a 3rd rank tensor described by 3D matrix (3 × 3 × 3) resulting in 27 components,
and finally reduced to only 10 components [43], as computed by gaussian program according to formula (3) to
get (β):
8

2
2
2
z
1/2
= (
β
β
x + β y + β
)
(3)
2
2
2
2
2
2
2
2
2
2
2
2
yyz
β
x = β xxx+β xyy+β
β
y = β yyy+β xxy+ β
β z = β zzz+ β xxz+ β
yzz) and (
where, (
xzz), (
)
The computed β of 6a and 6b are 2.843 x10-30 and 1.8671 x10-30respectively, in electrostatic units. In
comparison with the KDP, βKDP is 6.85×10-31 esu [44], but 6a and 6b are more than 4- and 2-times of KDP. The
predicted dipole moment of former and latter are 4.2671 D and 3.6848 D respectively. The high values of
computed β could be attributed to the non-zero dipole moment which indicates non-uniform distribution of the
charges on the molecular atoms as well as the difference in molecular phase, due to theoretical isolated gas
phase.
Experimentally, the nonlinear optical activity of both compounds is confirmed by using potassium dihydrogen
phosphate (KDP) as reference material with (I2ω: KDP = 17 mV). The results for 6a and 6b are 10 mV and 2
mV, respectively. These values give the SHG efficiency of 6a and 6b powdered samples of about 0.6 and 0.1
times of standard KDP. Accordingly, 6b is just active for the NLO having very small value of SHG efficiency
indicating that the para methyl group has less effect for NLO property, which may be due to non-coplanar
conformation that exist between chlorophenyl ring and with other rings depicting almost perpendicular between
them (see subsection 3.1) and also due to lack of the π-electron system (see subsection 3.4.2). In 6a, the difluoro
atoms in ortho position has shown enhanced NLO property with SHG efficiency exceeding the half value KDP
standard material, and this could be ascribed due to the slightly acute noncoplanar conformation that exist
between the rings (see subsection 3.1) showing abundance and redundancy of π-electron system. Hence, the
degree of planarity influences the mobility of the electrons and also enhances NLO activity, while any
deviations from planarity would decrease the mobility of electrons within the π-conjugated molecular system,
resulting in the reduction of NLO activity [45-46].
3.4 Electronic structure
3.5.1 Natural population analysis (NPA)
The analysis of electron configuration on the natural atomic orbitals (NAOs) provide a satisfactory description
of net natural atomic charge, with core (Cor) , valence (Val), and Rydberg (Ryd) populations on each atom,
which supports to understand the molecular properties[47]. The dissimilarities in energies and occupancies of 6a
and 6b compounds are strongly proven the effects of the substitutions on the delocalization of charges that
resulted in the variations of intermolecular contacts, angles and bond lengths (see section 2.2). The selected
charge accumulations and electron configurations for all atoms in diverse sub-shells have been grouped in Table
6. Additional data of charges accumulation are presented in Table S4 of Supplementary Material.
For 6a compounds, the highest electronegative charges of -0.64177e, -0.58469e, -0.54525e and -0.51939 are
appearing on N11, O13, O15 and N8 atoms, respectively. However, in the case of 6b, same atoms resemble the
most of electronegative charges accumulation, but with slightly variation in magnitude of -0.63438e, -0.59134e,
-0.56553e and -0.52126e, respectively. The change in magnitude in this case can be attributed to the hydrogen
bond formation from these atoms (see Table 5) as well as C22 atom of methyl group (-0.57836e). The maximum
change in electronegativity of unsubstituted atoms C19 (-0.18531e) in 6a and 6b of C1 (-0.02721e) are
observed, and this change can be attributed to hydrogen bond formation with 6a (see Table 5). On the other
hand, the most electropositive atoms in 6a and 6b are C12(0.67264e, 0.68183e), S2(0.48039e, 0.48051) and
9

H11(0.40436e, 0.40209), respectively. The maximum change in electro positivity of hydrogen atoms are H14A
(0.23686e) and H19(0.21498e) in 6a. These hydrogens are involved in hydrogen bonds for 6a (see Table 5).
3.4.1 Natural bond orbital analysis (NBO)
The analysis of NBO provides the fairly information to portray the delocalization effects between principal
Lewis and non-Lewis orbitals, and further which shows the occupancy of electron loss from the filled
Lewis orbital (donor) to the vacant non-Lewis orbital (acceptor). However, the significance and strengths of
such interaction can be valued through the second order perturbation theory by applying the Fock matrix
equation (4) [45 44]
.
E(2) = ∆E(2) F(i,j)2 /
=
q
ε
- ε
(4)
i
j
i
ij
where E(2) is the stabilization energy,
q
is ith donor orbital occupancy, ε and ε are diagonal elements and
i j
i
F(i,j) is the off-diagonal NBO Fock matrix (i is donor, j
is acceptor). The magnitude of E(2) specify the
strength degree of electron contributing from i → j orbitals, that demonstrates the intramolecular charge
transfer (ITC) inside the molecular system[48]. The important selected results are tabulated in Table 7. More
Lewis and non-Lewis interactions are given in Table S5 of Supplementary Material. The highest stabilization
energy resulting from overlapping σ →σ* in 6b is formed between σ(C1-CL1)→σ*(C2-H2) and σ(C10-
S2)→[σ*(C4-C7), σ*(C9-N11)] with E(2) values : 34.47616, 22.71912, 21.63128 kJ/mol, respectively . In 6a the
strongest σ→σ* interactions resulted from overlapping between various bonds system σ(C12-N11), σ(C9-N11),
σ(C12-O13), σ(C9-N8) and unique antibonding σ*(N11-H11) with E(2) values of 687.8496, 379.2378,
109.2861, 76.69272 kJ/mol respectively. In both the molecules, the largest electron density (ED) value
decreases for σ and increases in σ*, that occurred via the interaction σ(N11-H11)→σ*(C9-S2), thereby indicate
the importance of amide group in ICT and crystal packing (see subsection 3.2). The most significant π→π*
overlapping interactions in 6b resulted from π(C3-C4) →π*(C1-C2) and [π(C20-C21), π(C18-C19)] → π*
(C16-C17) with E(2) of 94.39104, 89.24472, 88.15688 kJ/mol, respectively. While in 6a, interactions resulted
from π(C19-C18) →π*(C16-C17), π(C3-C4) →[π*(C1-C2), π(C20-C21)] →π*(C16-C17) with E(2) of
94.72576, 94.64208, 87.27824 kJ/mol, respectively. The disparity of NBO of the compounds appear in the
overlapping between the hybridization of π and σ orbitals, which are less significant in 6b when compared to 6a,
thus such interaction in former compound reached maximum E(2) values of 77.06928, 23.97432 kJ/mol, owing
to π(C16-C17) →σ*(C18-C19) and σ(C14-H14B) →π*(C12-O13) respectively. But, hyperconjugative
interactions in 6a resulted with the enormous stabilization energies with delocalization E(2) values of 2034.972,
964.3702, 824.4154, 793.9977 kJ/mol, which belong to π(C9-N8) →[σ*(N11-H11), σ*(C17-F23), σ*(C3-C2)]
and π(C12-O13) →σ*(N11-H11), respectively. The ICT due to lone pairs in 6b and 6a are almost having similar
association to the molecular stabilization values (Table 7).
3.4.2 Frontier molecular orbitals analysis (FMOs)
FMOs or HOMOs-LUMOs orbital indicate the highest occupied and lowest- unoccupied molecular orbitals,
respectively [44]. The energy differences between these orbitals is named as energy gap (ꢀEgap), that provides
very useful datum about the excited situation, electrical transport, chemical softness /hardness, chemical
reactivity of molecular system [49]. The surfaces illustration of, ground state HOMO and first excited state
10

LUMO are demonstrated in Fig. 5. The calculated energies of different FMOs, ꢀEgap and the values of global
chemical parameters are summarised in Table 8.
The HOMO (ground state) orbitals are same for both compounds (Fig. 5), but with a few concentrations has
been observed over the acetamide bridge (i.e., thiazole ring side chain) (Fig. 5). These electronic states are
completely overturned in the first excited orbitals (LUMO) through A and B rings, where these can be
understood as HOMO→LUMO transition inferring the transfer of ED from chlorophenyl aromatic ring and
acetamide bridge of the π-conjugated to the ring B, except CH3 donor group (Fig. 5). Accordingly, the ꢀEgap
and chemical reactivity parameters of the compounds are mainly connected to the thiazole ring and acetamide
group reflecting their importance in electronic property and activity of molecules.
3.4.3 Electrostatic and Molecular electrostatic potential (ESP & MEP)
The molecular electrostatic potential (MEP) is the 3D plot of electrostatic potential relating to the electron
density mapped onto the iso-electron density surface.
Fig. 6I, delineates the ESP using the energy surface, where the negative electrostatic potential areas are convex
bulge around (Cl1, N8, N11 and O13) atoms for both the compounds and as well as fluorine atoms for 6a only.
The zero potentials are designated by flat surface, as portrayed in chlorophenyl ring plane for the both
compounds. Furthermore, the areas with electron lacking are displayed as completely concave surfaces
curvature around methylphenoxy ring in 6b, but for 6a due to difluoro atoms it seems to be relatively smaller
electron deficient area, which confirms the more electronegativity nature of 6a when compared to 6b.
3D MEP is represented in the colour scheme that scaled from deepest red maximum electron concentration to
deepest blue maximum electron deficient (Fig. 6II). The MEP of the both compounds are almost same with
maximum electron concentration around O13, nucleophilic site, and maximum electron deficient about the
(H11) atom, electrophilic site; this is justified by having N11-H11…O13 hydrogen bonds for both compounds)
(see subsection 3.2). While the slightly less electron concentration represented by yellow colour is around O15,
N8 and Cl1 atoms, respectively. But in addition to that, 6a also exhibited light yellow colour around the fluorine
atoms, which causes more electron deficient in para position of ring B (C19-H19) when compared to 6b. As a
result, the hydrogen bond: C19-H19…O13 is generated only for 6a (see subsection 3.2). There are no much
significant variations in zero potential regions, where light green colour is depicted on the chlorophenyl ring for
both the compounds.
3.5 Hirshfeld surface
Hirshfeld surfaces of 6a and 6b have been constructed with volumes 419.14 Å3 and 419.14 Å3, respectively,
(Fig. 7I). The color of dnorm is defined as -0.3861 au (blue) and 1.3711 (red) at distances longer and less than
sum vdw radii, respectively. While white colour represents the exactly the sum of vdw radii with dnorm = 0. Both
curvedness and shape index lie in the domain ranging from -4.00 to 4.00 and -1.00 to 1.00 respectively (Fig. 7
(II and III)) [50].
The different positions of bright-red spots on Hirshfield surfaces shown for compounds in Fig. 7I, which proves
the different substituents effect on the structure.
11

The Figs. of 2-D fingerprint allow us to identify the participation of all atoms to the Hirshfeld surfaces (Fig. 8a).
Furthermore, the breakdown of entire contribution to atom pairs enable us to predict the significant contribution
to the crystal packing formation (Fig. 8(b-d)).
The interactions O…H that appear as a pair of characteristic spikes in both 6a and 6b (Fig. 8b), have the highest
contribution towards the packing of crystal, since: di+de ≈ 2.5 Å and 2.25 Å, respectively. The short O…H bond
in the latter refers to the presences of two donor atoms, namely, N and C atoms. These interactions are covered
with 10.4% and 10.8% of Hirshfeld surface for 6a and 6b, respectively.
In 6b, N…H interaction (Fig. 8c) is also contributing strongly toward the supramolecular architecture of crystal
packing, where the atoms show contact distance at ≈ 2.60 Å, with 5.2% contribution to the Hirshfeld surface.
The interatomic contacts F…H (Fig. 8c) and F…F (Fig. 8d) has 9.5% and 2.3% contribution to the Hirshfield
surface of 6a, with insignificant contribution to the crystal packing is only from the latter, as short contacts with
distance di+de ≈ 2.86 Å <2.94Å. For 6b, the minor contribution from C…C contacts (4.8%) (Fig. 8d) also
confirms the presence of π… π stacking in the crystal packing formation. This also could be shown on the shape
index surface with pattern of blue and red triangles (Fig. 7b) as well as the flat regions upon the curvedness
surface (Fig. 7c). The contribution of C…H and H…H to Hirshfeld surface for 6a and 6b comprising ranging
from 34.3 to 19.1 1% with no directly contribution to the crystal packing as depicted in Fig. S10 of
Supplementary Material.
3.6 Energy frameworks
The result analysis of energy frameworks for different types of interactions are listed in Table 9. The molecular
environments of both 6a and 6b have been built by fixing one molecule in its centre and about with determined
distance of 3.8 Å (Fig. 9). The benchmarked energies in this study have been scaled based on Mackenzie et al
[28]. While the scale factors for repulsion, polarization, electrostatic and dispersion interactions energies are
0.618, 0.871, 1.057 and 0.740, respectively [51]. In Fig. 9, the strengths of the energy framework are indicated
by the cylinders. In general, the predicted interaction energies reveal the significance of the acetamide group in
supramolecular architectures for both 6a and 6b as indicated in the energy framework (Fig. 9), showing with all
strong cylinder energies in the region around it. The coulomb interactions in 6a looks to be weaker in strength
than in 6b which could be justified by distance between molecular centroids (R) (see Table 9). This situation
reverses for dispersion interaction (Fig. 9 (I)). However, in both molecules the dispersion interactions are
predominant over electrostatics with values of energy reached -235.6 / -74.69 kJ/mol and -175.71 / -59.59
kJ/mol for 6a and 6a, respectively. The others including polarization and repulsion interaction energies are listed
in Table 9, with total energies computed are -224.08 kJ/mol and159.34/mol for 6a and 6b, respectively.
3.7 Docking Simulation
The current in silico docking has been intended to check the inhibitor activity of 6a and 6b ligands against
COX-1 and COX-2 protein receptors. Prior to docking simulations, the classical modes of inhibitor binding
poses were observed experimentally in the COX active site, which contain inhibitors with Arg-120 of COX-1
and COX-2 [52], respectively. Instead, there could be also some apparent modes of NSAID binding that may
result from differences in the speed and efficiency with which inhibitors can perturb the hydrogen bonding
network around Arg-120 and Tyr-355 [53].
12

The results of Docking simulations revealed the prominent interactions of 6a and 6b ligands with the active site
residues of COX-1 and COX-2 (Fig. 10). The best glide score has been registered for 6a ligand with COX-1 that
binds at the active site with -8.92 kcal/mol and forms hydrogen bonds with ARG120:O13 and TYR355:O15 at
distances of 2.16 and 1.87 Å, respectively. These bonds are similar to the co-crystal ligand bonding network and
appears stronger (Fig. 11) [53]. Furthermore, there are pi-pi stacking interactions with TYR355 and Cg(2), as
well as pi-cation interaction with ARG120 and Cg(3). In addition to latter, some hydrophobic interactions in the
active site have been observed and found to be similar to the co-crystal ligand interactions as observed in COX1
(Fig. 12).
Fig. 13 represents three-dimensional surface plot mode showing the 6a ligand enfoldment inside the active site
cleft of COX-1 protein receptor with zoom view and ribbon model representations.
Table 10 lists the glide scores, hydrogen bonds, pi interactions and others parameters for all docking simulations
that indicate the inhibitor activity of 6a and 6b compounds. We infer from the latter results that 6a and 6bare
found to be potential COX-1 and COX-2 inhibitors, respectively, and these virtual results matches well with in
vitro assay results (see Table 11). The other details of plots and conformation representations of all others
docking simulations can be obtained from Supplementary Material (See Figs. S11-S15).
3.8 Molecular Dynamics Simulation
The results of MDS has ensured the stability of protein-Ligand complex (viz., 6a-1eqg). The event trajectory
was observed to remain stable throughout the simulation process. The stability of the system was tested by the
root mean square deviation (RMSD) graph of the system during the simulation. Protein-ligand RMSD plot
revealed that the system has been reached the stable form at 0.3 ns on the trajectory and later it loosed its
stability after 1ns during the production run (Fig. 14A) Furthermore, the radius of gyration during the MDS was
found to be within the range of 1 Å, indicating the compactness of the models predicted (Fig. 14A). This result
has been supported by the Root Mean Square Fluctuation (RMSF) plot (Fig. 14B) in which the residues
fluctuations are found to be preferably more in the region where there is no much interactions involved with the
ligands. Hence, these validation studies indicate the higher plausibility of the predicted models. On the other
hand, the residues interactions with MDS results was also found to be almost similar with the docking results.
The oxygen atom of carbonyl group displaying water bridged interaction with ARG120 has 82% presence, and
also confirming the original hydrogen bonds with ARG120:O15 and TYR355:NH11, respectively. The pi-cation
and pi-pi stacking interactions were also observed via thiazole ring towards ARG120 and TYR355, respectively
(See Fig. 15).
3.9 Anti-inflammatory activity (Cyclooxygenase (COX) inhibition)
One of the main biological activity of thiazole derivative is their ability to act as anti-inflammatory agent. A
number of synthetic modifications, viz., with electron withdrawing/donating groups, heterocyclic infused,
biphenyl based or other substitutions, have been reported to affect the anti-inflammation activities of thiazole
derivatives [6, 9, 10,11, 12]. The current study involves synthesis of heterocyclic compounds containing thiazole
moiety and acetamide bridge (see Scheme 1). The efficacy of synthesized compounds 6a and 6b to inhibit
ovine COX-1 and COX-2 enzymes was determined through measuring their peroxidase activity using
13

colorimetric enzyme immune assay. Then the inhibitory activity of synthesized compounds and standard
compounds diclofenac were expressed with IC50 values. The results of COX inhibition assay indicated that the
tested compounds had a reasonable potent inhibitory activity against COX-2 with IC50 ≃ 9.11 µM & 3.1 µM
respectively. Further, these compounds (6a and 6b) exhibited COX-1 inhibition with IC50 values of 10.2 µM &
18.2 µM, respectively, as listed in Table 11. The results of COX-1/COX-2 inhibition assay indicated that the
compound 6b showed excellent COX-2 inhibition. The ortho-difluoro groups of 6a also played a significant role
in the COX-2 activity. IC50 values of the compound 6b with a para-methyl group has remarkably better COX-2
activity than COX-1. On overall, COX-2 activity for both the compounds show better inhibitory results
Conclusion
Thiazole derivatives (6a and 6b) have been synthesised and the final compounds were characterized via 1H and
13C NMR as well as LC-MS mass spectra. Two single crystals were obtained and crystal structures were
investigated by using X-ray diffraction method. Geometry parameters were calculated by using DFT method
with B3LYP/6-311++G(d,p) basis set, and the results were in close agreement with the experimental
parameters. NPA obtained by Second order Fock matrix analysis based on the charge accumulation revealed the
reason behind for crystal packing environment with different hydrogen bonds. NBO also revealed the
difference in stabilization and intramolecular charge transfer based on substituents. FMOs/ESP/MEP analyses
showed the importance of the acetamide and thiazole moieties in the chemical reactivity descriptors of the
molecules. The analysis of Hirschfeld surface with fingerprint plots revealed the types of interatomic contacts of
molecules. Energy frameworks were examined based on the different intermolecular interaction energies. The
activity of nonlinear optical property was determined by SHG experiment and substantiated by
hyperpolarizability (β) parameter calculation and revealed the importance of fluorene atoms in the property. In
silico molecular docking and dynamics simulations predicted potential COX inhibitor compounds 6a and 6b,
showing the prominent interactions in the active sites of protein receptors. Further, summarizing the current
investigations, we have efficiently synthesized some phenoxy thiazole acetamide derivatives 6a and 6b with
fluoro and methyl groups at different position of benzoyl ring and also evaluated for anti-inflammatory activity.
The activity data revealed that the compounds 6a and 6b showed better COX-1 and COX-2 inhibitory activity,
respectively.
Acknowledgements
The authors would like to thank Dr. G. Hemantha Kumar, Coordinator of HPC, DOS in High Performance
Computing and thankful to technical staff for providing Schrödinger application software and system in HPC
Lab. And also, thanks to Mis. Lakshmi. R. S for assistance in MDS analysis. We are grateful to SAIF, IIT
Madras, for providing single crystal and spectral data. Hussien Khamees wish to acknowledge the financial
assistance provided by Abs Community College, Yemen. Yasser Hussein Eissa Mohammed is thankful to the
University of Hajja, Yemen, for providing financial assistance. Fares Hezam Al-Ostoot gratefully acknowledges
the financial support provided by government of Yemen and Albaydaa University-Yemen. Special thanks to
Cytxon Bio solutions pvt. Ltd, Hubballi, India for biological assistance in this work. Shaukath Ara Khanum
thankfully acknowledges the financial support provided by VGST, Bangalore, under CISEE Programme
14

[Project sanction order: No. VGST/CISEE/282]. Dr. Mahendra M would like to thank University of Mysore for
awarding research grant under the head DV6/375/MRP/2017-18, Dated:12-02-2018.
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properties of an organic imidazolium diphenylacetate diphenylacetic acid single crystal. New J.
Chem, 42(4), 2439-2449.
49- Carey, F. A., & Sundberg, R. J. (2007) Advanced organic chemistry: part A: structure and mechanisms,
2nd ed., Springer Science & Business Media, New York.
50- McKinnon, J. J., Spackman, M. A., & Mitchell, A. S. (2004). Novel tools for visualizing and exploring
intermolecular interactions in molecular crystals. Acta Crystallogr. Sect. B: Struct. Sci, 60(6), 627-668.
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Intermolecular interactions in molecular crystals: what’s in a name?. Faraday Discuss, 203, 93-112.
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18

Table 1 Crystal data and structure refinement details.
Compound
6a
6b
Parameter
CCDC Number
Empirical formula
1842848
C17H11ClF2N2O2S
380.79
1828753
C18H15ClN2O2S
358.83
Formula weight
Temperature
293(2)
K
293(2) K
Wavelength
0.71073 Å
0.71073 Å
2.305° to 23.449°
Orthorhombic
Pbca
θ range for data collection
Crystal system
2.326° to 28.708°
Triclinic
Space group
P-1
a =8.9470(3) Å
b=10.0708(4)Å
c=10.5882(4)Å
=76.763(3)°
ß =88.242(2)°
ꢀ=63.626(2)°
829.26(6)Å3
a = 17.2242(18)Å
b=9.6307(12)Å
c=20.581(2)Å
=90°
Cell dimensions
Volume
ß = 90°
ꢀ= 90°
3413.9(7)Å3
Z
2
8
Density(calculated)
1.525 Mg m−3
1.396 Mg m−3
Absorption coe cient (µ)
0.391 mm−1
0.36 mm-1
F000
388
1488
Crystal size
0.15 × 0.20 × 0.25 mm
0.10 × 0.15 × 0.15 mm
Index ranges
-12 ≤ h ≤ 12
-13 ≤ k ≤ 13
-14 ≤ l ≤ 14
21870
-19≤ h ≤ 19
-10≤ k ≤10
-23≤ l ≤ 23
35709
Reflections collected
Independent reflections
Absorption correction
Refinement method
4285
2502
None
None
Full-matrix least-squares on F2
4285 / 0 / 227
Full-matrix least-squares on F2
2502 / 0 / 219
Data / restraints / parameters
Goodness-of-fit on F2
Final R value [I > 2σ(I)]
1.016
1.039
R1 = 0.0439, WR2 = 0.1061
R1= 0.0434, WR2= 0.0824
R indices (all data)
Largest di . peak and hole
R1 = 0.1096, WR2 = 0.1358
R1 = 0.0847, WR2 = 0.0993
0.217 and -0.265 e Å-3
0.186 and -0.200 e Å-3
Table 2 Selected Bond lengths(Å).
6a
6b
Length (Å)
Length (Å)
Atoms
S(2)-C(10)
Atoms
DFT B3LYP
6-311++G(d,p)
DFT B3LYP
6-311++G(d,p)
Experiment
Experiment
1.712(3)
1.719(2)
1.365(3)
1.221(3)
1.352(3)
1.385(3)
1.366(4)
1.357(5)
1.396(3)
1.364(4)
1.7368
S(2)-C(10)
S(2)-C(9)
1.707(3)
1.722(3)
1.387(3)
1.222(3)
1.352(4)
1.389(4)
1.364(4)
1.387(4)
1.388(4)
1.387(4)
1.73714
1.75187
1.38442
1.2177
S(2)-C(9)
1.75214
1.36468
1.21757
1.36558
1.38779
1.38462
1.38337
1.40269
1.38481
O(15)-C(16)
O(13)-C(12)
N(11)-C(12)
N(11)-C(9)
C(21)-C(20)
C(20)-C(19)
C(4)-C(5)
O(15)-C(16)
O(13)-C(12)
N(11)-C(12)
N(11)-C(9)
C(21)-C(20)
C(20)-C(19)
C(4)-C(5)
1.36814
1.38749
1.38738
1.40278
1.40263
1.39834
C(17)-C(18)
C(17)-C(18)

Table 3 Selected Bond angles (°).
6a
6b
Angle
Angle
Atoms
Atoms
DFT B3LYP
6-311++G(d,p)
DFT B3LYP
6-311++G(d,p)
Experiment
Experiment
C(10)-S(2)-C(9)
87.78(12)
122.9(3)
117.3(2)
119.7(2)
109.1
87.6283
C(10)-S(2)-C(9)
87.86(15)
87.6233
C(16)-C(21)-C(20)
F(22)-C(21)-C(16)
N(8)-C(7)-C(4)
122.4745
117.8144
118.9470
110.8969
108.7511
122.5198
119.6672
119.6674
120.5929
C(20)-C(21)-C(16)
C(16)-C(21)-H(21)
N(8)-C(7)-C(4)
120.4(3)
119.8
119.8504
118.8984
118.9997
106.0733
107.9882
119.2799
119.6574
119.6887
117.5626
118.3(3)
108.5
O(15)-C(14)-H(14B)
H(14A)-C(14)-H(14B)
C(18)-C(17)-C(16)
C(2)-C(1)-CL1
O(15)-C(14)-H(14B)
H(14A)-C(14)-H(14B)
C(16)-C(17)-C(18)
C(2)-C(1)-CL1
107.8
107.5
122.2(3)
120.1(2)
124.1
118.8(3)
119.6(3)
124.0
S(2)-C(10)-H(10)
C(20)-C(19)-C(18)
S(2)-C(10)-H(10)
C(18)-C(19)-C(20)
121.5(3)
116.8(3)
Table 4 Selected Torsion angles (°).
6a
6b
Angle
Angle
DFT B3LYP
6-311++G(d,p)
0.0039
Atoms
Atoms
DFT B3LYP
Experiment
0.2(2)
Experiment
6-311++G(d,p)
C10-S2-C9-N8
-0.0007
C10-S2-C9-N8
0.3(2)
179.3(3)
180
C10 S2 C9 N11
C9 S2 C10 H10
H11 N11 C9 S2
F22 C21 C20 H20
F22 C21 C20 C19
N8 C7 C10 S2
178.9(2)
179.5
-179.9898
-179.7011
-179.9480
0.1615
C10 S2 C9 N11
C9 S2 C10 H10
H11 N11 C9 S2
H21 C21 C20 H20
H21 C21 C20 C19
N8 C7 C10 S2
-179.6545
-179.1010
179.9109
0.1226
178.7
-177.6
-0.1
-0.9
179.0(3)
0.7(3)
179.7731
-0.0653
179.9
-179.5633
-0.2294
-0.2(3)
-170.0(3)
2
O15 C14 C12 O13
F23 C17 C16 O15
C3 C2 C1 CL1
176.4(2)
-0.6(4)
179.6(2)
173.6256
-2.2192
O15 C14 C12 O13
O15 C16 C17 H17
C3 C2 C1 CL1
-166.5139
0.2333
179.9956
-179.5(3)
-179.9891
Table 5 Hydrogen bond geometry (Å,°) of 6a and 6b.
6a
6b
D-H…A
D-H
0.97
0.93
H…A D…A
D-H…A
160
D-H…A
D-H
H…A
D…A
D-H…A
162
C(14)-H(14A)...O(13)i
C(19)-H(19)…O(13)i i
2.49
2.52
3.4176(1)
3.4435(1)
N(11)-H(11)...O(13) i
0.86
2.24
3.0663(4)
169
C(5)-H(5)...O(15)ii
C(14)-H(14B)...N(8) iii
0.93
0.97
2.59
2.60
3.2478(4)
3.4030(4)
128
140
Symmetry transformations:
Symmetry transformations:
(i) 1/2-x,-1/2+y,z (ii) -1/2+x,1/2-y,-z (iii) 1/2-x,1/2+y,z
(iii) 1/2-x,1/2+y,z
(i) -x,1-y,-z (ii) 1+x,-1+y,z

Table 6 Selected charges accumulation and natural populations of 6a and 6b compounds.
Natural population (e) of 6a
Natural population (e) 6b
Atom
No.
Charge (e)
Total (e)
Charge (e)
Total (e)
Cor
Val
Ryd
Cor
Val
Ryd
N8
-0.51939
0.48039
-0.64177
0.40436
0.67264
-0.58469
0.23686
0.21343
-0.54525
0.33808
-0.18531
0.33027
0.21498
1.99926
9.99884
1.99930
0.00000
1.99945
1.99976
0.00000
0.00000
1.99978
1.99992
1.99921
1.99992
0.00000
5.50101
0.01912
0.04435
0.02564
0.00425
0.04356
0.01429
0.00240
0.00185
0.01740
0.01067
0.01637
0.00823
0.00164
7.51939
15.51961
7.64177
0.59564
5.32736
8.58469
0.76314
0.78657
8.54525
9.33808
6.18531
9.33027
0.78502
-0.52126
0.48051
1.99926
5.50289
0.01911
7.52126
15.51949
7.63438
0.59791
5.31817
8.59134
0.80285
0.78382
8.56553
0.79414
6.02721
0.78594
6.57836
S2
5.47642
5.61682
0.59139
3.28434
6.57063
0.76074
0.78473
6.52807
7.32749
4.16972
7.32212
0.78339
9.99884
1.99930
0.00000
1.99945
1.99976
0.00000
0.00000
1.99977
0.00000
1.99907
0.00000
1.99927
5.47634
5.60912
0.59370
3.27537
6.57744
0.80024
0.78193
6.54928
0.79141
4.01257
0.78332
4.57031
0.04430
0.02596
0.00421
0.04334
0.01414
0.00261
0.00189
0.01648
0.00273
0.01556
0.00262
0.00878
N11
H11
C12
O13
H14A
H14B
O15
F23
-0.63438
0.40209
0.68183
-0.59134
0.19715
0.21618
-0.56553
*0.20586
-0.02721
**0.21406
# -0.57836
C19
F22
H19
*H17, **H21 and #C22

Table 7 Selected Lewis and non-Lewis interactions from Second order perturbation theory analysis of Fock
matrix for 6a and 6b molecules.
6a
6b
Acceptor(j)
Donor(i)
EDa
Acceptor(j)
EDa
E(2)kJ/mol
∆Eb
F(i,j)c
Donor(i)
EDa
EDa
E(2)kJ/mol
∆Eb
F(i,j)c
π (C1-C2)
1.67848
π*(C4-C3)
π*(C7-C10)
π*(C5-C6)
0.35893
0.32303
0.31992
0.39375
0.01469
0.03704
0.03989
0.01469
0.01151
0.02830
0.01574
0.01273
0.01469
0.01367
0.07491
0.01469
0.01469
0.01469
0.21087
0.34736
0.01469
0.43615
0.34736
0.43615
73.0108
65.85616
86.23224
94.64208
379.2378
21.7568
0.30
0.26
0.27
0.26
5.21
0.93
1.03
5.36
0.26
0.04
2.30
2.45
4.35
1.16
0.84
5.15
5.57
4.76
0.52
0.31
5.57
0.24
0.29
0.27
0.065
0.057
0.067
0.069
0.614
0.062
0.067
0.280
0.125
0.087
0.619
0.408
1.338
0.057
0.054
0.822
0.341
0.849
0.052
0.066
0.286
0.068
0.068
0.068
σ(C1-CL1)
1.98927
σ*(C2-H2)
π*(C7-C10)
π*(C5-C6)
π*(C1-C2)
0.01274
0.32332
0.31982
0.39410
34.47616
64.05704
85.68832
94.39104
2.16
0.26
0.27
0.26
0.119
0.057
0.067
0.069
1.62907
1.97306
π(C3-C4)
1.62907
π(C3-C4)
π*(C1-C2)
σ(C9-N11)
σ(C10-S2)
σ(C9-N8)
1.98241
1.97303
1.98564
σ*(N11-H11)
σ*(C9-N11)
σ*(C4-C7)
σ*(C9-N11)
σ*(C4-C7)
σ*(C4-C5)
σ*(C4-C7)
σ*(C3-C4)
σ*(C1-C2)
σ*(C4-C7)
σ*(C1-CL1)
σ*(C9-S2)
0.03689
0.03989
0.02134
0.03989
0.02195
0.02780
0.03989
0.03186
0.07490
0.35900
0.39410
0.03689
0.01120
0.21121
0.33688
0.02268
0.39100
0.33688
0.39100
21.63128
22.71912
18.74432
15.85736
13.8072
0.94
1.03
1.06
1.08
1.24
1.24
1.08
0.83
0.84
0.29
0.27
1.10
1.13
0.53
0.30
1.09
0.26
0.29
0.27
0.062
0.067
0.062
0.057
0.057
0.058
0.058
0.057
0.055
0.068
0.066
0.067
0.054
0.052
0.067
0.059
0.067
0.069
0.069
σ(C10-S2)
σ(C2-C3)
22.8028
σ*(N11-H11)
σ*(C19-H19)
σ*(C17-F23)
σ*(C3-C2)
76.69272
302.545
1.97818
1.96779
964.3702
824.4154
336.017
π(C9-N8)
1.87026
13.93272
16.19208
20.83632
18.24224
83.21976
81.83904
21.3384
σ*(C2-H2)
σ(C5-C6)
σ(N11-H11)
π(C5-C6)
σ*(N11-H11)
σ*(C12-O13)
σ*(C9-S2)
2034.972
14.4348
1.96532
1.67877
σ(N11-H11)
1.96536
18.2004
π*(C3-C4)
σ(C12-N11)
σ(C12-O13)
π(C12-O13)
σ(C14-H14B)
π(C16-C17)
σ(C21-F22)
π(C19-C18)
1.98430
1.99516
1.98633
1.96596
1.68511
1.99502
1.68325
σ*(N11-H11)
σ*(N11-H11)
σ*(N11-H11)
π*(C12-O13)
π*(C19-C18)
σ*(N11-H11)
π*(C16-C17)
π*(C19-C20)
π*(C16-C17)
687.8496
109.2861
793.9977
24.81112
73.26184
76.52536
94.72576
83.80552
87.27824
π*(C1-C2)
σ(C7-N8)
σ(C9-S2)
1.97850
1.97976
1.96841
1.67485
1.97559
1.64995
σ*(C9-N8)
σ*(C10-H10)
π*(C12-O13)
σ*(C18-C19)
σ*(C16-C21)
π*(C16-C17)
π*(C18-C19)
π*(C16-C17)
13.34696
23.97432
77.06928
16.81968
88.15688
85.22808
89.24472
σ(C14-H14B)
π(C16-C17)
σ(C17-H17)
π(C18-C19)
π(C20-C21)
π(C20-C21)
1.67075
1.75100
1.68077
1.74837
π*(C12-O13)
π*(C9-N8)
0.21087
0.43102
0.03480
0.08317
0.43102
102.6335
73.80576
17.1544
0.34
0.34
1.12
0.59
0.22
0.083
0.073
0.060
0.104
0.073
π*(C12-O13)
π*(C9-N8)
0.21121
0.43144
0.02713
0.08222
0.43144
0.32332
101.5457
73.55472
18.99536
92.8848
0.34
0.35
1.15
0.59
0.22
0.26
0.082
0.073
0.064
0.104
0.073
0.062
LP (1)N11
LP(1)N11
LP (1)O15
LP (2)O13
1.97124
1.85545
σ*(C16-C17)
σ*(C12-N11)
π*(C9-N8)
LP(1)O15
LP(2)O13
1.96863
1.85610
1.59213
1.59213
σ*(C16-C17)
σ*(C12-N11)
π*(C9-N8)
93.88896
127.4446
126.9007
73.97312
LP (2)S2
1.59196
1.92407
LP (2)S2
π*(C7-C10)
0.32303
0.37501
73.55472
76.7764
0.26
0.43
0.062
0.086
π*(C7-C10)
LP (3)F22
LP (3)F22
a. Electron density.
b. Energy difference between donor orbital (i) and acceptor(j) orbital ∆E= E(i)-E(j) a.u.
c. F(i,j) is the Fock matrix element between i and j NBO orbitals in a.u unit.

Table 8 FMOs energy with global chemical parameters values of title compounds.
Property
Chemical parameters
6a
6b
-1.771
-6.0437
4.273
LUMO energy
HOMO energy
Energy gap
EL (eV)
-1.841
-6.065
4.224
6.065
1.841
2.112
0.2367
-3.953
3.953
3.699
EH (eV)
ꢀEgap = EL - EH (eV)
6.0437
1.771
Ionization potential ̓ = - EH
Electron affinity
hardness
̻ = - EL
2.1363
0.2340
-3.9073
3.9073
3.5732
ꢁ = (EL - EH)/2
ζ = 1/2ꢁ
Softness
Chemical potential ꢂ = (EL + EH)/2
Electronegativity
Electrophilicity
χ = - ꢂ
= ꢂ2/2ꢁ
Table 9 Different interaction energies (kJ/mol) of the molecules at B3LYP/6-31G(d,p).
6a
6b
N
2
1
2
1
1
1
2
1
1
Symop
x, y, z
R
E_ele
-2.86
E_pol
-0.444
-2.442
-0.444
-1.998
-0.888
-3.404
-1.406
-0.592
-0.074
-11.692
E_dis
-18.64
-70.81
-5.49
E_rep
5.01
E_tot
-16.49
-51.62
-6.69
N
2
1
2
2
2
2
2
2
Symop
R
E_ele
-7.08
-5.92
-1.46
E_pol
-0.814
-1.48
-0.15
-4.59
-0.59
-0.52
-0.22
-0.22
E_dis
-16.99
-52.35
-5.05
E_rep
9.02
E_tot
-15.86
-40.65
-4.19
10.07
6.12
16.2
4.50
7.40
9.12
10.07
11.21
15.14
x+1/2, -y+1/2, -z
-x, -y, -z
9.02
-x, -y, -z
x, y, z
-10.10
-3.49
29.29
2.29
5.90
19.10
2.47
x+1/2, -y+1/2, -z
-x+1/2, y+1/2, z
x, y, z
11.41
5.94
-x, -y, -z
-x, -y, -z
-x, -y, -z
x, y, z
-14.16
-10.89
-22.41
-10.04
-0.21
-64.28
-30.57
-21.60
-8.71
22.5
-55.94
-28.05
-29.61
-12.01
-32.87
-4.65
-3.81
-1.90
-1.90
-51.83
-8.19
33.19
4.57
-56.10
-8.86
13.41
14.40
6.74
9.63
-x+1/2, -y, z+1/2
x, -y+1/2, z+1/2
x, -y+1/2, z+1/2
11.16
12.65
10.54
-22.39
-5.84
8.40
-18.32
-5.61
2.35
-x, -y, -z
-x, -y, -z
-10.71
-0.074
-235.6
2.60
-8.32
-4.58
-13.07
5.44
-9.75
-0.53
0.74
Total
-74.69
96.98
-213.31
-59.59
-8.584
-175.71
84.54
-159.34
Symop: symmetry operation, R: Distance between molecular centroids (mean atomic position) in Å

Table 10 Schrödinger Docking Results of 6a and 6b ligands with COX-1 and COX-2 protein receptors.
Docking Glide Glide
score
Glide
energy
kcal/mol
H bonds
with
Residues
H Bond
length
(Å)
Pi interactions
Protein
Receptor Score
Glide
evdwa ecoulb
Ligands
6a
Pi-Pi
Pi-Cation
1.87
TYR355:NH11
ARG120:O13
1eqg
1pxx
-8.919
-6.834
-8.914
-6.829
-39.85
-13.91
-3.93
-7.8
-43.79
TYR355:Cg(2)
TYR355:Cg(3)
ARG120:Cg(3)
2.16
1.7
ARG120:Cg(3)
ARG120:Cg(1)
ARG120:Cg(3)
-21.67
TYR355:O13
1eqg
1pxx
-8.584
-8.805
-8.579
-8.800
-49.08
-26.98
0.71
-48.37
-28.29
ARG120:O13
ARG120:O15
1.93
2.15
6b
-1.31
a van der Waals and b Coulomb interaction energies
Table 11 In vitro COX-1/COX-2 enzyme inhibition assay of the synthesized compounds with IC50 (µM).
IC50 (ꢀM)a
COX-2SIb
Compounds
COX-1
COX-2
6a
6b
10.21
9.11
1.12
5.81
18.2
7.9
3.13
8.2
Diclofenac
0.96
a: IC50 value is the compound concentration required to produce 50% inhibition of COX-1or COX-2 for means of three
determinations using an ovine COX-1/COX-2 assay kit. The deviation from the mean is <10% of the mean value. b:
Selectivity index (COX-1 IC50/COX-2 IC50).

Fig. 1. Molecular structures of the molecules 6a and 6b: I) ORTEP diagrams drawn with 50% probability level
and II) Optimized structures at DFT/B3LYP/6-311++G(d,p) level.
Fig. 2. 6a showing with acute noncoplanar conformation (132.42o) and 6b with perpendicular noncoplanar
conformation (83.58) between the designated mean planes (Shown in red colour and green colour).
Fig. 3. Supramolecular architecture of the 6a compound: I) one dimensional, II) two-dimensional chair-type
sheets and III) three dimensional crystal packing viewed along b axis and showing two contacts C(19)-
H(19)...O(13) (blue) and C(14)-H(14A)...O(13) (orange).

Fig. 4. Supramolecular architecture of 6b compound: I) Crystal packing viewed down b axis. II) Close up view
of crystal packing diagram showing diverse contacts: i) N11-H11...O13 (Turquoise), ii) C-H …π: C14-
H14B....Cg(3) (light green), iii) C14-H14A...N8 (magenta), iv) C5-H5...O15 (blue), v) S…S (brown), vi) π …π:
Cg(1) … Cg(2) (red) and vii) C-H… π: C20-H20....Cg(2) (black).
Fig. 5. HOMO-LUMO orbitals with energy gap.

Fig. 6. I) ESP and II) MEP of 6a and 6b compounds obtained by DFT with B3LYP under 6-311++G(d.p) level.
Fig. 7. Comparison of Hirshfeld surfaces for 6a and 6b molecules mapped over: (I) dnorm for representing the
inter contacts and red spots denoting as hydrogen bonds, (II) Shape index and (III) Curvedness.

Fig. 8. 2D fingerprint plots of 6a and 6b showing: a) all contacts and (b-f) breakdown of contacts for specific
pairs of atoms.
Fig. 9. Comparison of the interaction energies of 6a and 6b molecules: I) Coulomb interactions (red), II)
Dispersion interactions (green) and III) Total interaction energies (blue) along a, b and c directions.