Study of the structure-bioactivity of fleximers: synthesis, crystal structure, Hirshfeld surface analysis, and anti-inflammatory assays

Article abstract of DOI:10.1016/j.molstruc.2021.130513

Synthesized and natural pyridones/pyridines derivatives exhibiting diverse biological activities. 2-pyridone has lactam-lactim tautomerization like thymine and uracil bases. In this study, COX-2 target based series of pyridone/pyridine linked fleximers were designed, synthesized and studied. All analogues binding affinity with COX-2 active site were studied through molecular docking, and anti-inflammatory activity studied by in vivo analysis. Weak interactions were studied to find binding sites among analogues through crystal packing, Hirshfeld surface analysis and in silico analysis. All the analogues exhibited anti-inflammatory activity, while compound (3) is the most active analogue among the series. In contrast, since compound (3) is a pyridine-phthalimide ring-containing analogue, the presence of a phthalimide group probably favors anti-inflammatory activity over other types of rings. The results suggested further investigations on compounds as anti-inflammatory prodrugs.

Full text of DOI:10.1016/j.molstruc.2021.130513

Journal of Molecular Structure 1239 (2021) 130513
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Study of the structure-bioactivity of fleximers: synthesis, crystal
structure, Hirshfeld surface analysis, and anti-inflammatory assays
a,d,∗
a
a
b
Ved Prakash Singh
Ashish Kumar Tewari
, Jayanta Dowarah , Brilliant N. Marak , Balkaran Singh Sran ,
c,∗
a
Department of Chemistry, School of Physical Sciences, Mizoram University, Aizawl-796004, Mizoram India
Department of Chemistry, Guru Nanak Dev University, Amritsar-143 005, India
b
c
Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi-221 005, India
Department of Industrial Chemistry, School of Physical Sciences, Mizoram University, Aizawl-796004
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesized and natural pyridones/pyridines derivatives exhibiting diverse biological activities. 2-pyridone
has lactam-lactim tautomerization like thymine and uracil bases. In this study, COX-2 target based se-
ries of pyridone/pyridine linked fleximers were designed, synthesized and studied. All analogues binding
affinity with COX-2 active site were studied through molecular docking, and anti-inflammatory activ-
ity studied by in vivo analysis. Weak interactions were studied to find binding sites among analogues
through crystal packing, Hirshfeld surface analysis and in silico analysis. All the analogues exhibited anti-
inflammatory activity, while compound (3) is the most active analogue among the series. In contrast,
since compound (3) is a pyridine-phthalimide ring-containing analogue, the presence of a phthalimide
group probably favors anti-inflammatory activity over other types of rings. The results suggested further
investigations on compounds as anti-inflammatory prodrugs.
Received 19 February 2021
Revised 10 April 2021
Accepted 12 April 2021
Available online 22 April 2021
Keywords:
Weak-interactions
NSAIDs
Pyridone
COX-2
Hirshfeld
SCXRD
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
receptor. It also contributes to enhancing binding affinity. These in-
teractions may be an essential factor in drug design strategy [7,8].
Inflammation is a defensive and healthy response of the body
to any noxious stimulus to a complex sustained response involving
the organism’s whole body, leading to an immunological response
of the transplanted tissues [1]. Non-steroidal anti-inflammatory
drugs (NSAIDs) are the most widely used anti-inflammatory drugs
2-pyridones represent a unique class of pharmacophores ob-
served in various antibiotics and therapeutic agents [9]. In recent
years, 2-pyridones have been found to exhibit several biological ac-
tivities, such as analgesic [10], anti-HIV [11], antitumoral [12], and
anti-inflammatory [13] properties. Pyridone derivatives exhibiting
various biological activities [14].
[
2]. Selective inhibition of the COX-2 isozyme provided a useful
concept that resulted in effective anti-inflammatory NSAIDs [3].
Celecoxib, Rofecoxib, are the most common selective COX-2 in-
hibitors.
Flexible pyridones have promising weak interaction as a model
system [15]. For good COX-2 inhibitory activity and selectiv-
ity, compounds should have a key pharmacophore [16]. Herein,
we used the COX-2-binding site as a reaction vessel to design
highly potent and selective inhibitors. We designed and synthe-
sized a range of pyridone/pyridine based building blocks and a
collection of corresponding polymethylene containing heterocyclic
compounds (1–8). After screening for their COX-2 inhibitory po-
tency, various building blocks were incubated in pairs with hu-
man recombinant COX-2 isozyme to test the capability of COX-
2 potent inhibitors. Compound 3 displayed a superior in vivo
anti-inflammatory activity profile compare to clinically used anti-
inflammatory drugs.
Structural biology and X-ray crystallography provide beneficial
information in developing drugs [4,5]. Non-covalent interactions
are of immense importance in biological systems. In biological sys-
tems, an H-bond competing process always exists because bulk
water interferes with all reversible biological processes and dif-
ferent types of energy compensation occurs during H-bond forma-
tion. The mechanism and contribution of each H-bonds to molec-
ular functions are still not clear [6]. Non-covalent interactions are
not only crucial in the orientation of an inhibitor binding in the
∗
Corresponding authors.
E-mail addresses: vpsingh@mzu.edu.in (V.P. Singh), tashish2007@gmail.com (A.K.
Tewari).
https://doi.org/10.1016/j.molstruc.2021.130513
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
2
. Materials and Methods
1682 (RCONR). MS (m/z): 283 (M+1). Element Analysis: (i). Calcu-
lated: C= 72.31%; H= 6.38%; N= 9.92%. (ii). Found: C= 72.19%; H=
6.28%; N= 9.61%. Crystallized in ethyl acetate and hexane (95:5).
2
.1. General procedure for the synthesis of compounds 1-8 (Scheme 1)
In
a
100 mL round-bottom flask, 4,6-dimethyl-2-oxo-1,2-
2.1.2. 4,6-dimethyl-2-(3-phenoxypropoxy)nicotinonitrile (2)
Yield= 0.22 g, 12%. Mp= 136-39°C. 1H NMR 300 MHz, 25°C,
dihydro-pyridine-3-carbonitrile (0.014 mol) was dissolved in DMF
and stirred for 15 min. Anhydrous potassium carbonate (0.028 mol)
or sodium hydroxide (0.014 mol) was then added into the flask
and the mixture was stirred for another 20 minutes. After that,
respective halides were added to the reaction mixture and con-
tinued stirring. The completion of the reaction was checked via
TLC (30% EtOAc & Hexane), in which two spots were visualized.
After completing the reaction, DMF was removed under reduced
pressure, and the reaction mixture was extracted with CHCl / H O
Si(CH ) (CDCl ) (δ): 2.18-2.25 (2H, p, CH CH CH , J= 6.0 Hz, 6.0
3
4,
3
2
2
2
Hz); 2.37 (3H, s, CH ); 2.44 (3H, s, CH ); 4.0-4.03 (2H, t, CH , J=
3
3
2
5.6 Hz, 5.6 Hz); 4.21-4.24 (2H, t, CH , J= 7.2 Hz, 7.2 Hz); 6.65 (1H,
2
s, Ar-H); 7.02-7.05 (2H, d, Ar-H, J= 8.1 Hz); 7.30-7.35 (1H, t, Ar-
13
H, J= 7.5 Hz, 7.5 Hz); 7.26-7.31 (2H, m, Ar-H). C NMR (75MHz,
CDCl ) (δ): 20.52, 20.78, 27.50, 36.30, 42.50, 64.49, 101.06, 109.45,
3
114.14, 115.36, 120.82, 129.32, 150.76, 157.88, 158.11, 160.85, 162.38.
IR (KBr, cm 1): 2251 (CN). MS (m/z): 283 (M+1). Element Anal-
ysis: (i). Calculated: C= 72.31%; H=6.38%; N= 9.92%. (ii). Found:
C=72.20%; H=6.24%; N=9.51%. Crystallized in ethyl acetate and
hexane (90:10).
−
3
2
(
3:1). The CHCl3 layer was dried with anhydrous Na SO . CHCl
2 4 3
was removed, and the product was purified by column chromatog-
raphy.
2
.1.1. 4,6-dimethyl-2-oxo-1-(3-phenoxypropyl)-1,2-dihydropyridine-3-
carbonitrile
1)
2.1.3. 1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-4,6-dimethyl-2-oxo-1,2-
dihydropyridine-3-carbonitrile
(
(3)
Yield = 1.12 g, 59%. Mp. 161-64°C. 1H NMR 300 MHz, 25°C,
Yield: 1.65 g, 37%. Mp. 159-63°C. 1H NMR 300 MHz, 25°C,
Si(CH ) (CDCl ) (δ): 2.25-2.31 (2H, p, CH CH CH , J= 6.0 Hz, 6.0
Si(CH3)4, (CDCl ) (δ): 2.05-2.10 (2H, q, CH CH CH ); 2.35 (3H, s,
3
4,
3
2
2
2
3
2
2
2
Hz); 2.42 (3H, s, CH ); 2.43 (3H, s, CH ); 4.15-4.18 (2H, t, CH , J=
CH ); 2.38 (3H, s, CH ); 3.80-3.84 (2H, t, NCH , J= 6.6 Hz, 6.6 Hz);
3
3
2
3
3
2
6
.0 Hz, 6.0 Hz); 4.56-4.59 (2H, t, CH , J= 6.0 Hz, 6.0 Hz); 6.66 (1H,
4.07-4.12 (2H, t, NCH , J= 7.8 Hz, 7.8 Hz); 6.00 (1H, s, Ar-H); 7.72-
2
2
s, Ar-H); 6.89-6.93 (2H, m, Ar-H); 6.94-6.95 (1H, t, Ar-H, J= 2 Hz, 2
7.74 (2H, d, Ar-H, J= 5.1 Hz); 7.84-7.87 (2H, t, Ar-H, J= 3.3 Hz, 4.8
Hz); 7.24-7.29 (2H, m, Ar-H). 13C NMR (75MHz, CDCl ) (δ): 19.88,
Hz). C NMR (75MHz, CDCl ) (δ): 20.57, 20.73, 27.37, 35.46, 42.81,
13
3
3
2
4.06, 28.81, 63.58, 64.06, 93.79, 114.39, 114.88, 117.39, 120.56,
101.46, 109.50, 115.27, 123.27, 131.74, 134.07, 150.01, 157.96, 160.67,
29.32, 154.17, 158.70, 160.55, 163.78. IR (KBr, cm 1): 2256 (CN),
−
168.13. IR (KBr, cm ): 2255 (CN), 1680 (RCONR), 1754 (RCONCOR).
−1
1
Scheme 1. Synthesis of compound 1-8.
2

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
MS (m/z): 336 (M+1). Element analysis: (i) Calculated: C=65.10%;
H=5.01%; N=12.51%. (ii). Found: C= 65.65%; H= 5.21%; N=12.39%.
Crystallized in ethyl acetate and hexane (95:5).
J= 5.7 Hz, 6.3 Hz); 6.00 (1H, s, Ar-H); 7.72-7.73 (1H, d, Ar-H, J=
3.3 Hz); 7.74-7.75 (1H, d, Ar-H, J= 3.0 Hz); 7.83-7.86 (2H, t, Ar-
13
H, J= 3.0 Hz, 3.6 Hz). C NMR (75 MHz, 25°C, Si(CH ) , CDCl ):
3
4
3
(
δ) 20.57, 20.73, 27.37, 35.46, 42.81, 101.46, 109.50, 115.27, 123.27,
31.74, 134.07, 150.01, 157.96, 160.67, 168.13. IR (KBr, cm ): 2256
2
2
.1.4.
−1
1
-(3-(1,3-dioxoisoindolin-2-yl)propoxy)-4,6-dimethylnicotinonitrile (4)
Yield: 0.71 g, 15%. Mp. 138-40°C. 1H NMR 300 MHz, 25°C,
(
CN), 1682 (RCONR), 1756 (RCONCOR). MS (m/z): 322 (M+1). El-
ement analysis: (i). Calculated: C= 67.28%; H= 4.68%; N= 13.09%
(ii). Found: C= 67.19%; H= 4.43%; N= 12.91%. Not crystallised.
Si(CH3) (CDCl ) (δ): 2.19-2.23 (2H, q, CH CH CH , J= 6.0 Hz);
4,
3
2
2
2
2
6
.37 (3H, s, CH ); 2.41 (3H, s, CH ); 3.88-3.93 (2H, t, NCH , J=
3
3
2
.9 Hz, 6.6 Hz); 4.44-4.48 (2H, t, OCH , J= 5.7 Hz, 6.3 Hz); 6.65
2
2.2. Crystal structure determination X-ray Crystallography
(
1H,s, Ar-H); 7.69-7.71 (2H, d, Ar-H, J= 3.9 Hz); 7.82-7.84 (2H, t, Ar-
13
H, J= 3.0 Hz, 2.4 Hz). C NMR (75MHz, CDCl ) (δ): 19.88, 24.34,
3
Single-crystal X-ray data for compounds 1-4, 6 & 7 were col-
27.84, 35.03, 64.23, 93.82, 114.62, 117.43, 123.07, 123.19, 132.00,
lected with an Oxford Diffraction Xcalibur CCD and Bruker Apex
33.78, 133.97, 154.16, 160.36, 163.51, 168.14. IR (KBr, cm 1): 2253
−
1
2
diffractometer. Crystallographic details of compounds 1-4, 6 & 7
(
CN), 1751 (RCONCOR). MS (m/z): 336 (M+1). Element analy-
have been summarized in Table 1 & 2.
sis: (i). Calculated: C=65.10%; H=5.01%; N=12.51% (ii). Found:
C=64.61%; H=4.96%; N=12.41%. Crystallized in ethyl acetate and
hexane (95:5).
2
.3. Hirshfeld Surface, finger-plot and weak interaction energy
analysis
2
.1.5. 1-(2-(4-formylphenoxy)ethyl)-4,6-dimethyl-2-oxo-1,2-
dihydropyridine-3-carbonitrile
5)
Inter-molecular interaction analyses for all compounds were
carried out using PLATON [17]. CrystalExplorer17 was used to gen-
erate Hirshfeld surfaces, fingerprint plots, and calculate interac-
tion energies [18]. The C-H bond lengths converted to normal-
ized values based on neutron diffraction results [19]. Interaction
energies were calculated employing the CE-B3LYP/6-31G(d,p) func-
tional/basis set combination and corrected for basis set superposi-
tion energy (BSSE) using the counterpoise (CP) method [20]. The
interaction energy is broken down as
(
Yield: 0.42 g, 21%.Mp. 154-156°C. 1H NMR 300 MHz, 25°C,
Si(CH3)4, (CDCl ) (δ): 2.39 (3H, s, CH ); 2.59 (3H, s, CH ); 4.42-
4
3
3
3
.47 (4H, p, 2CH ); 6.07 (1H, s, Ar-H); 6.93-6.94 (1H, d, Ar- H,
2
J= 1.8 Hz); 6.96 (1H, s, Ar-H); 7.81-7.82 (1H, d, Ar-H, J= 1.8
1
3
Hz); 7.83-7.84 (1H, d, Ar-H, J= 2.1 Hz); 9.88 (1H, s, -CHO).
C
NMR (75MHz, CDCl ) (δ): 20.88, 21.51, 44.85, 65.50, 101.66, 109.79,
3
1
14.57, 115.14, 130.51, 131.98, 151.30, 158.63, 160.99, 162.80, 190.59.
ꢀ
ꢀ
ꢀ
dis
ꢀ
IR (KBr, cm 1): 2251 (CN), 1678 (RCONR), 1710 (C=O). MS (m/z):
−
Etot = keleE + kpol E + kdisE + krepErep
ele
pol
2
5
96.9 (M+1). Element Analysis: (i). Calculated: C= 68.92%; H=
.41%; N= 9.46%. (ii). Found: C= 68.21%; H= 5.42%; N= 9.22%. Not
where the k values are scale factors (kele = 1.057; k
ele
= 0.740;
pol
ꢀ
kdis = 0.871; krep = 0.618), E
pol
represents the electrostatic com-
crystallised.
ꢀ
ꢀ
ponent, E
the polarization energy, E
the dispersion energy,
dis
ꢀ
and E rep the exchange-repulsion energy [21,22]. A contact sphere
2
.1.6. 2-(2-(4-formylphenoxy)ethoxy)-4,6-dimethylnicotinonitrile (6)
Yield: 0.93 g, 47%.Mp. 121-23°C. 1H NMR 300 MHz, 25°C,
˚
of 3.8 A was used to determine primary Inter-molecular contacts
for interaction energy calculations.
Si(CH3)4, (CDCl ) (δ): 2.45 (6H, s, 2CH ); 4.43-4.46 (2H, t, CH ,
3
3
2
J= 4.8 Hz, 5.4 Hz); 4.77-4.81 (2H, t, CH , J= 5.4 Hz, 4.8 Hz); 6.72
2
2
.4. In silico analysis of 1-8
(
1H, s, Ar-H); 7.06-7.09 (2H, d, Ar-H, J= 9.0 Hz); 7.83-7.86 (2H, d,
13
Ar-H, J= 9.0 Hz); 9.90 (1H, s, -CHO). C NMR (75MHz, CDCl ):
3
The ability of compounds to interact with the COX-2 was as-
(
δ): 20.04, 24.48, 64.68, 66.25, 94.14, 114.70, 114.99, 117.96, 130.22,
31.98, 154.66, 160.53, 163.364, 163.62, 190.78. IR (KBr, cm 1):
−
sessed by in silico studies. The crystal structure of COX-2 protein
was retrieved from the RSCB protein data bank (PDB id: 4RRW).
The protein preparation was done in chimera [23] by removing
co-crystallized ligands, cofactors, and embedded water molecules.
It was further processed by adding polar hydrogens and Kollman
charges in Autodock tools. The ligands were prepared in Chem-
Bio3D and optimized using the MMF94 force field. The active site’s
grid parameters were assigned based on the native ligand cover-
ing all the residues of the binding cavity, and subsequently, dock-
ing of compounds was carried out in the Autodock 4.2 program
1
2
258 (CN), 1717 (C=O). MS (m/z): 297 (M+1). Element Analysis:
i). Calculated: C= 68.92%; H= 5.41%; N= 9.46%. (ii). Found: C=
8.54%; H= 5.13%; N= 9.44%. Crystallized in ethyl acetate and hex-
ane (95:5).
(
6
2
2
.1.7.
-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)-4,6-dimethylnicotinonitrile (7)
Yield: 1.32 g, 31 %.Mp. 141-142°C. 1H NMR 300 MHz, 25°C,
Si(CH3)4, (CDCl ) (δ): 2.31 (3H, s, CH ); 2.42 (3H, s, CH ), 4.10-
3
3
3
[
24] using the Lamarckian Genetic Algorithm. The re-docking of
4
6
.14 (2H, t, NCH , J= 3.6 Hz); 4.67-4.71 (2H, t, OCH , J= 3.6 Hz);
2
2
compounds confirmed the validation of docking results. Further,
validation was done by re-docking the crystal structure of lumira-
coxib, extracted from COX-2 protein (PDB: 4RRW), and then su-
.65 (1H, s, Ar-H); 7.70-7.73 (2H, q, Ar-H, J= 3.0 Hz, 2.4 Hz, 3.0
13
Hz); 7.84-7.86 (2H, q, Ar-H, J= 3.0 Hz, 2.4 Hz, 3.0 Hz). C NMR
3 4 3
(75 MHz, 25°C, Si(CH ) , CDCl ): (δ) 20.03, 24.29, 37.07, 63.49,
perimposing it with the native lumiracoxib in COX-2 protein. The
docked lumiracoxib presented the RMSD value of 0.330 A˚ concern-
9
4.28, 114.55, 117.86, 123.33, 132.12, 133.94, 154.47,160.30, 163.27,
68.08. IR (KBr, cm−1): 2252 (CN), 1750 (RCONCOR). MS (m/z): 322
1
ing the native lumiracoxib. The analysis of docking results was car-
ried out using the pymol and Discovery studio visualizer.
(
M+1). Element analysis: (i). Calculated: C= 67.28%; H= 4.68%;
N= 13.09%. (ii). Found: C= 67.23%; H= 4.59%; N= 12.54%. Crystal-
lized in ethyl acetate and hexane (95:5).
2
.5. In vivo evaluation of the anti-inflammatory effects of compounds
2
.1.8. 1-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-4,6-dimethyl-2-oxo-1,2-
dihydropyridine-3-carbonitrile
8)
All compounds and Nimesulide, as a reference drug, were sub-
(
jected to in vivo anti-inflammatory analysis.
Yield: 0.66 g, 15.5 %. Mp. 135-137°C. 1H NMR 300 MHz, 25°C,
Experimental model of inflammation: carrageenan-induced
foot paw edema model used throughout the experiment for anti-
inflammatory analysis [25,26].
Si(CH3)4, (CDCl ) (δ): 2.37 (3H, s, CH ); δ 2.42 (3H, s, CH ); 4.03-
3
3
3
4
.07 (2H, t, NCH , J= 6.0 Hz, 6.0 Hz); 4.30-4.34 (2H, t, NCH ,
2
2
3

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Table 1
Crystal data and structure refinement for compounds 1-3.
Identification code
Compound 1
Compound 2
Compound 3
Empirical formula
Formula weight
Temperature/K
Crystal system
C17H18N2O2
C17H18N2O2
C19H17N3O3
282.33
282.33
335.36
293(2)
296(2)
100(2)
monoclinic
monoclinic
monoclinic
Space group
a/A˚
P21/c
P21/c
P21/n
16.0221(13)
10.6234(17)
6.9431(14)
b/A˚
c/A˚
6.8052(5)
9.0164(14)
14.055(3)
14.3470(12)
16.687(3)
16.597(3)
α/°
90
90
90
β/°
100.993(8)
108.293(9)
95.46(3)
γ /°
90
90
90
Volume/ A˚ 3
1535.6(2)
1517.6(4)
1612.3(6)
Z
4
4
4
ρcalcg/cm3
μ/mm 1
F(000)
1.221
1.236
1.382
0.081
0.082
0.096
600
600
704
Crystal size/mm3
Radiation
0.30 × 0.27 × 0.12
MoKα (λ = 0.71073)
5.18 to 49.998
0.21 × 0.18 × 0.14
MoKα (λ = 0.71073)
4.038 to 56.682
0.32 × 0.17 × 0.17
MoKα (λ = 0.71073)
3.8 to 50.08
2
ꢀ range for data collection/°
Index ranges
-19 ≤ h ≤ 18, -8 ≤ k ≤ 7, -17 ≤ l ≤ 10
5388
-14 ≤ h ≤ 14, -11 ≤ k ≤ 12, -22 ≤ l ≤ 22
48778
-8 ≤ h ≤ 8, -16 ≤ k ≤ 15, -19 ≤ l ≤ 17
10906
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
2669 [Rint = 0.0165, Rsigma = 0.0274]
2669/0/262
3770 [Rint = 0.0635, Rsigma = 0.0293]
3770/0/192
2836 [Rint = 0.0310, Rsigma = 0.0300]
2836/0/277
1.005
1.024
1.05
R1 = 0.0555, wR2 = 0.1521
R1 = 0.0830, wR2 = 0.1650
0.22/-0.23-
R1 = 0.0521, wR2 = 0.1265
R1 = 0.0826, wR2 = 0.1468
0.24/-0.29
R1 = 0.0706, wR2 = 0.1336
R1 = 0.0758, wR2 = 0.1363
0.34/-0.24
Final R indexes [all data]
Largest diff. peak/hole / e A˚
−
3
CCDC
1906729
1906728
653561
Table 2
Crystal data and structure refinement for compounds 4, 6 & 7.
Identification code
Compound 4
Compound 6
Compound 7
Empirical formula
Formula weight
Temperature/K
Crystal system
C19H17N3O3
C17H16N2O3
C18H15N3O3
335.36
296.32
321.33
100(2)
100(2)
100(2)
monoclinic
orthorhombic
triclinic
Space group
a/A˚
P21/c
Pbcn
P-1
15.682(3)
11.529(2)
7.7033(15)
b/A˚
c/A˚
8.5349(17)
14.635(3)
7.8382(16)
12.294(3)
17.718(4)
13.017(3)
α/°
90
90
95.62(3)
β/°
99.22(3)
90
93.41(3)
γ /°
90
90
102.36(3)
Volume/ A˚ 3
1624.2(6)
2989.6(10)
761.4(3)
Z
4
8
2
ρcalcg/cm3
μ/mm 1
F(000)
1.371
1.317
1.402
0.095
0.092
0.098
704
1248
336
Crystal size/mm3
Radiation
0.34 × 0.25 × 0.18
MoKα (λ = 0.71073)
5.26 to 58.46
0.57 × 0.3 × 0.25
MoKα (λ = 0.71073)
4.5 to 49.98
0.38 × 0.23 × 0.14
MoKα (λ = 0.71073)
3.16 to 50.06
2
ꢀ range for data collection/°
Index ranges
-21 ≤ h ≤ 20, -11 ≤ k ≤ 11, -16 ≤ l ≤ 15
13329
-12 ≤ h ≤ 13, -17 ≤ k ≤ 17, -21 ≤ l ≤ 19
19012
-7 ≤ h ≤ 9, -8 ≤ k ≤ 9, -15 ≤ l ≤ 15
4933
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
4332 [Rint = 0.0320, Rsigma = 0.0288]
4332/0/228
2639 [Rint = 0.0352, Rsigma = 0.0240]
2639/0/201
2648 [Rint = 0.0178, Rsigma = 0.0307]
2648/0/219
1.076
1.054
1.048
R1 = 0.0534, wR2 = 0.1296
R1 = 0.0600, wR2 = 0.1343
0.34/-0.25
R1 = 0.0374, wR2 = 0.0841
R1 = 0.0423, wR2 = 0.0867
0.22/-0.18
R1 = 0.0440, wR2 = 0.1007
R1 = 0.0482, wR2 = 0.1034
0.18/-0.24
Final R indexes [all data]
Largest diff. peak/hole / e A˚
−
3
CCDC
710837
653560
653562
Animals: All experiments were conducted on adult Wistar
strain albino rats, weighing between 150-200 g. The albino rats
were taken from the Central Animal House of the IMS, B.H.U. Ani-
mals were acclimatized for a week before using in the experiment
at an ambient temperature of 25°C± 2 °C in colony cages and 45-55
The protocol begans after approval by the Ethical Committee of the
Banaras Hindu University for animal experimentation at IMS, BHU
(# 10-11/84). All studies are reported in accordance with the AR-
RIVE guidelines [27].
Carrageenan induced paw edema in albino rats: Target com-
pounds were suspended in the aqueous carboxymethyl cellulose
(CMC, 1% w/v). The dose level of 10 mg/kg of NSAI drugs nime-
sulide and their equivalent doses of the target derivatives (1-8)
were administered orally. Control animals were similarly treated
%
relative humidity with the light-dark cycle in the departmental
animal room. Experimental protocol was carried out in accordance
with the International Guidelines for Use and care of animals used
in laboratory and with Indian laws for the scientific use of animals.
4

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
with aqueous carboxymethyl cellulose (CMC, 1% w/v) after 15 min.
One percent carrageenin suspension was prepared in distilled wa-
ter. Below the plantar aponeurosis of the left hind paw 0.1 ml of
carrageenan, the solution was injected through a 26 gauge nee-
dle into the plantar surface. The volume of the paw was mea-
sured at different time intervals for three h after injection of car-
rageenin. The difference in paw volume was measured by pedal
edema.
Statistical analysis: Analysis of Variance (ANOVA) analyzed
data. Overall, ANOVA was significant (p0.05), then data was further
subjected to statistical analysis by the student’s t-test.
3. Results and discussion
3
.1. COX-2 isozyme binding site based drug design of building blocks
Based on the structural features and binding sites of the COX-2
Measurement of paw volume: Plathysmographic method used
to measure the rat’s hind paw volume up to the ankle joint. The
ankle joint was marked with a skin marking pencil, and the paw
was dipped in the mercury to measure the volume change. A dif-
ference in the paw volume before and after injection of the phlo-
gistic agents at regular interval of time was taken as a measure-
ment of pedal edema. The change in paw volume was taken in
active site, we concluded that the designed molecule should have
some criteria: (i) at least one of the building blocks (in our case,
the pyridone/pyridine ring) should possess a hydrophilic pharma-
cophore to facilitate its binding into the secondary-binding pocket
of the COX-2 isozyme; (ii) the hydrophobic component should
have a proper size, orientation, and size of selective inhibitor large
enough to block the hydrophobic site of the COX-2 isozyme (Fig. 1).
"
ml" of mercury displaced.
Fig. 1. Design of building blocks for COX-2 inhibitors. Structure-based designed compounds (1–8) and illustration of in situ click chemistry binding reaction interactions
inside the COX-2-binding pocket.
5

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Fig. 2. (A) Showing C-N–π interactions in molecule (1), symmetry code 1-x,-1/2+y,1/2-z; (B) showing packing in the molecule (2). (a) -x,2-y,1-z (b) -1+x,1.5-y.
(
iii) Both hydrophobic and hydrophilic groups should be connected
group Pbcn with Z = 8 (60). Compound 7 [33] crystallized in the
monoclinic space group P1 with Z = 2.
with the linker or spacer to approach hydrophilic and hydrophobic
sites of COX-2 isozyme [28]
In the crystal structure of compound 1, two molecules are
arranged in close proximity so that inter-molecular C-N…π, C-
H…O, C-H…N interactions present between the neighboring moi-
eties (Fig. 2, Table 3), which results in ABAB packing architecture
due to two planes of pyridone ring (head) and phenyl ring (tail)
of the moiety. Intermolecular H2...O1 and H6A...O1 nontraditional
Hence, to optimize these criteria, we synthesized
a range
of pyridone/pyridine-linked heterocycle derivatives with var-
ious spacers containing hydrophilic and hydrophobic groups.
Di/trimethylene linkers selected a spacer and pyridone/pyridine
as the hydrophilic end for the new building blocks as
pyridone/pyridine-containing compounds are quite prevalent
in many anti-inflammatory drugs. The design of building blocks
and target compounds is illustrated in Fig. 1, where the central
methylene spacer was adapted from the COX-2 isozyme bind-
ing site structure. Hydrophilic pharmacophore is located at the
one end of the building block, and the spacer was introduced
to facilitate the binding of this ring into the COX-2 secondary-
binding pocket. In contrast, the phenyl/phthalimide ring is at the
other end of the building block to block the hydrophobic site of
COX-2 isozyme. Extensive SAR data is available in the literature,
which describes the importance of hydrophilic and hydrophobic
pharmacophore for selective binding to the COX-2 isozyme [29].
Substitution patterns on these building blocks selected for this
study had structural similarity to curcumin, which has good
COX-1/COX-2 binding affinity. Previous findings and new ideas
corroborated by in silico aided drug design [4]. It is thus taking
the biological profile as COX-2 inhibitors, the Lumiracoxib, and
SC-558 nucleus selected as the framework for designing new
pharmacophore-based hybrids (Fig. 1).
hydrogen bond result in the R 1 (6) motif shown in Fig. 2. C−N...π
2
interactions further assist interlayer connectivity. The overall inter-
molecular network is stabilized mainly through off-set displaced
aromatic interactions between the pyridone ring. Lone pair…π
stacking interactions found between cyano group (N…π) is 3.48
˚
and 3.65 A (Fig. 2). The results displayed in Table 3 showed that
C−H...O bond distances for bifurcated hydrogen bonding are 2.29
˚
and 2.44 A, and angles on hydrogen atoms are 168.77° and 156.80°.
˚
Three C−H...N interactions bond distances are 3.0, 3.16, 2.86 A and
angles 122.44°,118.7°, 125.66°, respectively.
Partial crystal packing of compound 2 is in Fig. 2. Packing is
stabilized by alternate π…π, C-H…π, and C-H…N interactions.
The acute angle (dihedral angle) between the pyridine ring plane
and phenyl ring is 54.9(1)°. The molecules form corrugated chains
running along (110) plane due to C5-H5…N2 interactions, sug-
gesting that these protons fall under the influence of the elec-
tronic environment of electronegative nitrogen atoms. These chains
are, in turn, joined to each other due to C-H…π interactions
˚
˚
˚
(H2…Cg = 3.60 A), (H6…Cg = 3.61 A), (H7B…Cg = 2.84 A),
˚
˚
Further, by in silico analysis, it was decided to swap both phar-
macophoric rings with different sizes and volumes. Effects of vol-
ume, flexibility, and the substituent were assessed on selective
COX-2 inhibition activity of all analogues. All analogues flexibil-
ity was increased via a methylene linker to impart flexibility to
the overall hybridized structure for compatible binding with COX-
(H13…Cg = 2.93 A), (H17A…Cg = 3.44 A), which are edge-to-
face, tilted, and off-set (Table 3 and Fig. 2), extending the net-
work and form an extended chain. The result is a stacked struc-
ture, down the ab plane’s diagonal, with a head-to-tail architecture.
The network supported by other non-covalent interactions in the
form of week π…π interactions shown between two adjacent cor-
rugated chains (Fig. 2). There is face-to-face and off-set aromatic
2
(Fig. 1). The outcome of the design approach is supported by in
˚
interaction between two pyridines (3.98 A) and two phenyl rings
vivo bioactivity analysis. Finally, the consensual binding mode to
the COX-2 active site was validated by the crystallographic report
and in silico docking studies.
˚
(3.65 A).
Compound 3 is an asymmetrical, nonplanar molecule. It con-
sists of phthalimide and pyridone ring linked through tri-
a
methylene linker. The overall structure of 3 exhibits a stacked ar-
rangement of molecules that exhibit a combination of parallel-
displaced π…π, C-H…π, C-H…O, and C-H…N interactions in
which π…π and C-H…π interactions predominate (Table 3). Both
rings are arranged in the AB pattern in the vertical direction to
the ABAB pattern in crystal packing (Fig. 3). The acute angle (dihe-
dral angle) between the pyridone ring plane and the phenyl ring
3
.2. Crystal analysis
All the molecules are unsymmetrical fleximers. The pyri-
done/pyridine ring is linked with a phenyl ring, a phthalimide
ring. Compounds 1, 2 & 4 [30] crystallized in the monoclinic space
group P2 /n with Z = 4, 5, and 4 (14), respectively. Compound 3
1
˚
is 59.43°. The stacking distance for π…π is 3.560 and 3.643 A
[
31] crystallizes in the monoclinic space group P2 /c with Z = 4
1
˚
(
Fig. 3), while for C-H…π is 3.753 and 3.648 A. Lone pair…π in-
(
14). Compound 6 [32] crystallized in the orthorhombic space
6

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Table 3
Non-covalent interactions ( A˚ ) for 1-4, 6, 7 in crystals. Cg is the centroid of the rings.
D-H…A
D-H
H…A
D….A
D-H...A
Symmetry Operation
Compound 1
C(2)-H(2)•••O(1)
0.952
1.028
0.976
0.843
0.985
-
2.298
2.441
3.026
3.164
2.858
3.484
3.653
3.459
3.549
3.644
3.644
3.526
–
168.77
156.80
122.44
118.7
125.66
-
x,1.5-y,-1/2+z
x,1.5-y,-1/2+z
x,1.5-y,-1/2+z
x,1.5-y,-1/2+z
1-x, -1/2+y,1/2-z
1-x,-1/2+y,1/2-z
1-x,-1/2+y,1/2-z
C(6)-H(6A)••O(1)
C(7)-H(7A)...N(2)
C(7)-H(7B)...N(2)
C(10)-H(10A)...N(2)
N(2)…π Cg(C1-C5, N1) first neighbor
N(2)…π Cg(C1-C5, N1) second neighbor
Compound 2
-
-
-
C(5)–H(5) ...N(2)
0.930
2.704
3.609
3.619
2.835
2.935
3.440
3.658
3.98
3.556
152
x+1,+y+1,+z
-1+x,1/2-y,-1/2+z
1-x,-y,-z
C(2)-H(2)…. π Cg(C1-C6)
C(6)-H(6)…. π Cg(C11-C14, N1)
C(7)-H(7B)…. π Cg(C11-C14, N1)
C(13)-H(13)…. π Cg(C1-C6)
C(17)-H(17A)…. π Cg(C1-C6)
Cg(C1-C6) π …. π C2(C1-C6)
Cg(C1-C5, N1)π …πCg(C1-C5, N1)
Compound 3
1-x,-y,-z
-1+x,1/2-y,-1/2+z
-1+x,1/2-y,-1/2+z
-
C(2)-H(2)…N(3)
0.982
0.935
0.934
0.943
0.953
1.004
0.929
2.642
2.648
2.664
2.595
2.466
2.439
3.753
3.648
3.560
3.560
3.643
3.298
3.423
3.196
3.384
3.307
3.605
3.504
-
124.38
140.69
116.78
141.36
147.07
156.50
67.56
–
x,-1+y,z
C(4)-H(4)…O(3)
1.5-x,-1/2+y,1/2-z
-1+x,y,z
C(5)-H(5)…O(1)
C(9)-H(9B)..O(1)
-x,1-y,-z
C(11)-H(11A)..O(1)
-x,1-y,-z
C(19)-H(20A)…O(2)
-1+x,y,z
C(4)-H(4)… π Cg(N2, C12-C16)
1/2-x,-1/2+y,1/2-z
1/2-x,-1/2+y,1/2-z
1/2-x,-1/2+y,1/2-z
1/2-x,-1/2+y,1/2-z
1/2-x,-1/2+y,1/2-z
C(3)-H(3)… π Cg(N2, C12-C16)
Cg(N1,C1,C6,C7,C8) π ••• N(3)
Cg (C2- C8)… π Cg(N2, C12-C16)
Cg(N2, C12-C16) … π Cg(N2, C12-C16)
Compound 4
–
–
–
-
-
-
C(6)-H(6)…O(1)
0.950
0.990
0.990
0.980
0.950
0.950
0.990
0.990
–
2.628
2.608
2.565
2.707
3.467
3.289
3.689
3.228
3.088
3.606
3.879
3.403
3.320
3.48
3.410
4.080
3.991
4.554
3.945
4.215
–
139.00
128.89
153.77
129.08
124.47
132.31
147.33
130.65
154.65
–
1-x,-y,1-z
C(10)-H(10A)…O(2)
x,1/2-y,-1/2+z
1-x,-1/2+y,1/2-z
2-x,-1/2+y,1.5-z
1-x,-y,1-z
C(11)..H(11A)…O(1)
C(18)-H(18B)…N(3)
C(4)-H(4)…π (N2, C12-C16)
C(5)- H(5)…π (N2, C12-C16)
C(9)- H(9B)… π Cg (C2-C7)
C(9)- H(9B)… π Cg (N2, C12-C16)
O(1)… π Cg (N1, C1, C2, C7, C8)
Cg (N1, C1, C2, C7, C8) π … π Cg (N1, C1, C2, C7, C8)
Cg (C2-C7) π …π Cg (N1, C1, C2, C7, C8)
Intramolecular
1-x,-y,1-z
1-x,-1/2+y,1/2-z
1-x,-1/2+y,1/2-z
1-x,-1/2+y,1/2-z
–
–
–
–
C9-H9A… O2
0.990
0.990
2.659
2.793
2.936
3.298
96.24
C11-H11A… O2
112.14
Compound 6
C4-H4…N2
0.951
0.980
0.990
0.989
0.990
0.950
0.950
0.990
0.950
2.711
2.543
2.690
2.712
2.709
2.574
2.594
3.345
3.547
3.728
3.727
3.685
3.720
3.626
3.455
3.139
3.367
3.367
3.385
3.536
3.989
3.422
161.63
154.89
107.88
123.99
124.29
148.53
171.74
124.35
89.11
1/2-x, -1/2+y, z
1/2-x, ½-y, ½+z
-1/2+x, -1/2+y, ½-z
-1/2+x, -1/2+y, ½-z
-1/2+x, ½-y, 1-z
1/2-x, -1/2+y, z
-1/2+x, ½-y, 1-z
-x,-y,1-z
C7-H7B…O4
C9-H9A…O4
C10-H10A…O4
C10-H10B…N2
C12-H12…O4
C16-H16…N2
C9-H9A…π Cg(C1-C5,N1)
C16-H16…π Cg(C11-C16)
Cg(C1-C5,N1) π …O2
Cg(C1-C5, N1) π …N1
Cg(C1-C5, N1) π …. π Cg(C11-C16)
Cg(C11-C16) π … π Cg(C11-C16)
Compound 7
1/2-x,-1/2+y,z
-x,-y,1-z
-x,-y,1-z
1/2-x,-1/2+y,z
-x,y,1/2-z
C4-H4…O2
0.951
0.980
0.980
0.950
0.950
0.950
0.950
0.951
2.611
2.730
2.476
2.480
2.592
2.459
2.601
3.560
3.477
3.311
3.359
3.547
3.432
3.525
3.392
3.091
3.158
3.358
3.437
3.547
144.81
138.53
155.32
122.09
118.53
157.70
146.93
81.50
-x, 2-y, 1-z
1-x, 2-y, 1-z
-x,2-y, 1-z
x, -1+y, z
x, -1+y, z
1-x, 2-y, -z
1-x, 2-y, -z
-x,2-y,1-z
1-x,2-y,1-z
-x,2-y,-z
C8-H8A…N2
C8-H8C….O2
C12-H12…O3
C13-H13…O3
C14-H14…O1
C15-H15…N2
C4-H4… π Cg(C1-C5,N1)
Cg(C1-C5,N1) π … π C2(C1-C5,N1)
O2… π Cg(C12-C15,C17,C18)
C12-H12… π Cg(C11, C16-C18,N3)
Cg(C1-C5,N1) π … π C4(C1-C5,N1)
Intramolecular
1.209
0.950
3.383
3.371
82.99
82.61
-x,2-y,-z
-x,2-y,1-z
C10-H10B… π Cg(C1-C5,N1)
Cg(C11, C16, C17, C18, N3) π …O1
0.990
3.820
3.501
4.258
7

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Fig. 3. (A) partial packing diagram showing C-H.. π, π-π interactions in the molecule (3), Symmetry code: 1/2-x,-1/2+y,1/2-z; (B) Partial packing diagram showing non-
covalent interactions in molecule (4) symmetry code 1-x,-1/2+y,1/2-z.
˚
the one-another axis in crystal packing. Apart from these interac-
tions, tetrafurcated (five centers) hydrogen bonding at oxygen atom
is also observed, i.e., four C-H…O and trifurcated (four centers) hy-
drogen bonding nitrogen atom is also observed, i.e., three C-H…N.
This tetrafurcated and trifurcated hydrogen bonding assist the lin-
ear chain formation as well as interlayer connectivity. Intermolecu-
teraction distance is 3.560 A, further assisted the interlayer con-
nectivity. Apart from these interactions, trifurcated (four centers)
hydrogen bonding at the oxygen atom is also observed, i.e., three
C-H...O. This trifurcated hydrogen bonding assists the linear chain
formation as well as interlayer connectivity. Intermolecular C-H…O
non-traditional hydrogen bond result in the C22(10), R 2 (6), R 2
2
2
14) & R 2 (15) graph-set notation. The C-H…O bond distances for
2
1
(
lar C-H...O nontraditional hydrogen bond result in the C2 (11), R2
(5) & R 2 (26) graph-set notation and C-H...N nontraditional hydro-
2
˚
trifurcated hydrogen bonding are 2.664, 2.595 & 2.466 A, and an-
2
2
2
4
1
gles on hydrogen atoms are 116.78°, 141.36°, and 147.07°, while the
C-H…N bond distance and angle on hydrogen atom are 2.64 A and
24.38°, respectively (Table 3).
gen bond result in the C2 (16), C2 (14), R4 (21) & R2 (7) graph-
set notation. However, interlayer connectivity is further assisted by
C−H…π and π…π interactions. The C-H…O bond distances for
˚
1
˚
Compound 4 is an asymmetrical, nonplanar molecule consist-
tetrfurcated hydrogen bonding are 2.712, 2.574, 2.690, and 2.543 A,
ing of phthalimide and pyridine moieties are arranged in close
and angles on hydrogen atoms are 123.99°, 148.53°, 107.88°, and
proximity so that intermolecular hydrogen bonds of the C-H…O
˚
154.89°, while C-H…N bond distance in the trifurcated hydrogen
bonding is 2.709, 2.594, and 2.711 A˚ and angle on hydrogen atom
(
2.56 A) type are formed between the two neighboring moieties,
which seems to be typical of 2-pyridones with D22 (14) graph-
set notation. Both rings are arranged in the AB pattern in one
axis and AA and BB pattern in another axis in crystal packing
are 124.29°, 171.74° & 161.63° respectively (Fig. 4, Table 3).
The crystal structure of compound 7 has folded crystal packing
due to intra-molecular C-H...π, O(lone pair)...π, non-covalent inter-
actions (Table 3) shown in Fig. 4. The acute angle (dihedral angle)
between the pyridone ring plane and the phenyl ring is 13.37°. Two
molecules are arranged in close proximity so that intermolecular
hydrogen bonds of the C−H…O type are formed between the two
neighboring moieties, which seems to be typical of 2-pyridones
with R 2 (22) & R 2 (22) graph-set notation. Both rings are ar-
(
Fig. 3). The acute angle (dihedral angle) between the pyridone ring
and phenyl ring plane is 78.81°, sufficient to show intramolecu-
˚
lar interactions. Intramolecular hydrogen bonding C-H…O (2.65 A)
and lone pair…π interactions assisted the folding of the molecule.
The C-H…O bond distances for trifurcated intramolecular hydrogen
˚
bonding are 2.793 and 2.659 A, and the angles on hydrogen atoms
2
2
are 99.24° and 112.14°. The stacking distance for π…π is 3.606
ranged in two planes and forming an AB pattern in crystal packing.
˚
The C-H…O bond distances for O2 linked trifurcated intermolecu-
lar hydrogen bonding are 2.611 and 2.476 A˚ and angles on hydrogen
and 3.879 A, while for C-H…π is 3.467, 3.289, 3.689, and 3.228
˚
A (Table 3, Fig. 3). Apart from these interactions, bifurcated (three
centers) hydrogen bonding at the oxygen atom is also observed,
i.e., two C-H…O. This bifurcated hydrogen bonding assists the lin-
ear chain formation as well as interlayer connectivity. Intermolec-
atoms are 144.81° and 155.32°, another O3 trifurcated intermolec-
˚
ular hydrogen bonding are 2.480 and 2.592A, and angles on hy-
drogen atoms are 122.09° and 118.53° (Table 3). The stacking dis-
ular nontraditional hydrogen bond result in the C22(13), C 2(11) &
R22 (11) graph-set notation. However, interlayer connectivity is fur-
˚
˚
tance for π…π is 3.477 A, while for C-H…π is 3.560 and 3.359A.
Apart from these interactions, lone pair…π interaction at the oxy-
2
˚
ther assisted by C-H…π and π…π interactions. The C-H…O bond
gen atom is 3.311 A, also observed (Fig. 4, Table 3). Intermolecular
C-H…O & C-H…N non-traditional hydrogen bond results in the C22
(10), R21 (6), R22 (16) & R22 (9) graph-set notation.
˚
distances for bifurcated hydrogen bonding are 2.628 and 2.565 A,
and angles on hydrogen atoms are 139 and 153.77°, while the C-
˚
H…N and other C-H…O bond distance are 2.707, 2.608 A and angle
on hydrogen atom are 129.08, 128.89°, respectively (Table 3).
In the crystal structure of compound 6, molecules are ar-
ranged in close proximity, So, that intermolecular π..π, C-H...π,
lone pair...π, C-H...O, C-H...N non-covalent interactions exists be-
tween the two neighboring moieties (Fig. 4, Table 3). The acute
angle (dihedral angle) between the pyridone ring plane and the
3.3. Hirshfeld surface and interaction energy analysis
Hirshfeld surface analysis provides a view of intermolecular in-
teractions of the whole molecule, rather than assumed weak inter-
actions [34]. It encouraged a new way of exploring packing modes,
surface interactions, and intermolecular interactions in molecu-
lar crystals. The 2D fingerprint of the Hirshfeld surface analy-
sis summarizes the different combinations of de and di across
the molecule’s surface. It indicates that various interactions are
present, and the surface’s relative area corresponds to each inter-
phenyl ring is 72.20°. The π…π stacking interactions distances are
˚
3
3
.685 and 3.720 A, while for C−H…π interactions distances are
˚
˚
.547 and 3.345 A and lone pair....π are 3.728 and 3.727A (Table 3).
Both rings are arranged in the AB pattern and are perpendicular to
8

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Fig. 4. (A) Partial packing diagram showing alternate plane and non-covalent interactions in molecule (6), symmetry code 2-x,-1/2+y,1.5-z; (B) partial packing diagram
showing an alternate layer of the ring in the alternate pattern in molecule (7); (C) partial packing diagram showing non-covalent interactions in molecule (7), symmetry
code 2-x,-1/2+y,1.5-z.
Fig. 5. (A) Fingerprint plots (B) Hirshfeld surfaces (C) л..л interaction in interactions energy calculation of compound 1; (D) Fingerprint plots (E) Hirshfeld surfaces (F) л..л
interaction in interactions energy calculation of compound 2.
action. The 2D fingerprint plots and decompose fingerprints are
shown in Figs. 5-7, and percentage contributions of N…H, O…H,
H…H, C…C, and C…H interactions are shown in the bar graph
contribution of C–H…π interactions in the crystal lattice, which
˚
are prominent between di + de = 3.0–3.3 A. There is a sharp
peak in the fingerprint plots for O…H interactions in the crystal
˚
(
Fig. 8). Interaction energies for 1–4, 6 & 7 were calculated using
lattice, which are prominent in between di + de = 2.3–2.6 A in
CrystalExplorer [21,22] and weak interactions observed are shown
in Tables 4 & 5.
the 2D plot of compound 2 (Fig. 5D). The C-H…N hydrogen bond-
ing & π…π interactions that result in the R22(11/12/13) rings (-
− −1
33.7 kJ mol 1) are stronger than C-N…H interactions (-11 kJ mol
There is a peak in the fingerprint plots for O…H interactions in
the crystal lattice, which are prominent between di + de = 2.2–
each). The crystal structure also shows CH...π interactions, simi-
larly, as found in the Hirshfeld analysis. There are no red bins in
the fingerprints plot that indicates the absence of π…π stacking
in compound 3 (Fig. 6A). There is a light wing in the fingerprint
that indicates the small contribution of C-H…π interactions in the
˚
2
.6 A in the 2D plot of compound 1. There are light red bins, and
˚
di + de (<3.8 A) in the fingerprints that indicates the presence of
π…π stacking (Fig. 5A & 5C). A light wing in the fingerprint indi-
cates the small contribution of C–H…π interactions in the crystal
lattice (Fig. 5A), which are prominent between di + de = 2.7–3.1
˚
crystal lattice, which are prominent between di + de = 2.9–3.1 A
˚
2
−1
A. The C-H…O results in the R (6) rings (-32.4 kJ mol ), which
(Fig. 6A & 6C). There is a sharp peak in the fingerprint plots for
O…H interactions in the crystal lattice, which are prominent be-
2
−1
is stronger than C-N…H interactions (-8.2 kJ mol each). Aromatic
˚
interactions were observed in weak interaction energy calculations
˚
tween di + de = 2.7–2.9 A in the 2D plot. The C-H…O hydrogen
−1
(
Fig. 5C). There are red bins, and di + de (<3.8 A) in the finger-
bonding interactions results in the R22(11) rings (-39.1 kJ mol ) in
prints that indicates the presence of π…π stacking in compound
(Fig. 5D & 5F). A light wing in the fingerprint indicates the small
extended structure and CH…O/CH…N hydrogen bonding resulting
in R 2(10) rings (-38.3 kJ mol
−1
each) in extended structure [20].
2
2
9

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Table 4
Non-covalent interactions ( A˚ ) for 1-4, 6 & 7 in Hirshfeld energy calculations
Donor-H...Acceptor
D – H, A˚
H...A, A˚
D...A, A˚
D - H...A, o
Symmetry Operation
Compound 1
C(2)-H(2)...O(1)
C(6)-H(6A)...O(1)
C(15)-H(15)...N(2)
Compound 2
1.083
1.083
1.083
2.170
2.390
2.812
3.237(2)
3.410(2)
3.815(2)
137.10
151.11
153.90
x, -y+1/2
x, -y+1/2
x, -y+1/2
C(5)-H(5)...N(2)
C(7)-H(7B)...N(2)
Compound 3
1.083
1.083
2.569
2.814
3.556(2)
3.713(2)
151.11
146.40
x, y, z
-x, y+1/2
C(3)-H(3)...O(2)
C(4)-H(4)...O(3)
C(8)-H(8)...N(2)
C(15)-H(16)...O(2)
C(19)-H(20A)...O(2)
C(20)-H(20A)...O(2)
Compound 4
1.083
1.083
1.083
1.083
1.083
1.083
2.663
2.535
2.886
2.395
2.366
2.600
3.605(2)
3.423(18)
3.439(2)
3.242(2)
3.383(2)
3.196(2)
145.07
138.56
120.41
131.36
155.73
113.85
-x, -y, -z
-x, -y, -z
x, y, z
x, y, z
x, y, z
x, y, z
C(6)-H(6)...O(1)
C(10)-H(10A)...O(2)
C(11)-H(11A)...O(1)
Compound 6
1.083
1.083
1.083
2.530
2.550
2.481
3.403(2)
3.320(2)
3.480(2)
137.03
127.26
152.81
x, -y+1/2, z+1/2
x, -y+1/2, z+1/2
-x, y+1/2, -z+1/2
C(7)-H(7A)...O(4)
C(12)-H(12)...O(4)
C(16)-H(16)...N(2)
Compound 7
0.98
0.95
0.95
2.543
2.574
2.594
3.455(2)
3.3855(18)
3.537(2)
155
1/2-x,1/2-y,1/2+z
1/2-x,-1/2+y,z
144
171.7
-1/2+x,1/2-y,1-z
C(8)-H(8C)...O(2)
C(12)-H(12)...O(3)
C(13)-H(13)...O(3)
C(14)-H(14)...O(1)
C(15)-H(15)...N(2)
0.98
0.95
0.95
0.95
0.95
2.48
2.48
2.59
2.46
2.60
3.392(2)
3.091(2)
3.158(2)
3.358(2)
3.437(2)
155
122
119
158
147
-x,2-y,1-z
x,1+y,z
x,1+y,z
1-x,2-y,-z
1-x,2-y,-z
Fig. 6. (A) Fingerprint plots (B) Hirshfeld surfaces (C) C-H..л interaction in interactions energy calculation of compound 3; (D) Fingerprint plots (E) Hirshfeld surfaces (F) л..л
interaction in interactions energy calculation of compound 4.
˚
peak in the fingerprint plots for O…H interactions in the crystal
There are light red bins, and di + de (<3.2 A) in the fingerprints
˚
that indicates the presence of strong π…π stacking interactions in
compound 4 (Fig. 6D & 6F). There is a light wing in the fingerprint
that indicates the small contribution of C–H…π interactions in the
crystal lattice, which are prominent between di + de = 2.8–3.1 A
Fig. 6D). There is a sharp peak in the fingerprint plots for strong
lattice, which are prominent between di + de = 2.4–3.4 A in the
2D plot. The C-H…O hydrogen-bonding interactions that result in
2
the C2 (21) chain have calculated interaction energies of 14.3 kJ
mol 1. There are light red bins, and di + de (<3.8 A) in the finger-
prints that indicates the presence of π…π stacking in compound
7 (Fig. 7D & 7F). There is a light wing in the fingerprint that indi-
cates the contribution of C-H…π interactions in the crystal lattice,
−
˚
˚
(
O…H interactions in the crystal lattice, which are prominent be-
˚
tween di + de = 1.9–2.5 A in the 2D plot. The C-H…O hydrogen-
bonding interactions that result in the R (6) and R22 (14) rings
2
˚
which are prominent between di + de = 2.7–3.1 A (Fig. 7D). There
2
−1
have calculated interaction energies of -46.4 kJ mol
and 43.0 kJ
˚
is a sharp peak in the fingerprint plots for O…H interactions in the
mol 1, respectively. There are light red bins, and di + de (<3.8 A)
−
crystal lattice, which are prominent in between di + de = 2.3–3.4
˚
indicates the presence of π…π stacking in compound 6 (Fig. 7A &
A in the 2D plot of compound 7. The C-H…O hydrogen-bonding
2
7
C). There is a light wing in the fingerprint that indicates the small
interactions that result in the R2 (10) rings have calculated inter-
action energies of 26.0 kJ mol 1
−
.
contribution of C-H…π interactions in the crystal lattice, which are
˚
prominent between di + de = 2.9–3.3 A (Fig. 7A). There is a sharp
10

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Fig. 7. (A) Fingerprint plots (B) Hirshfeld surfaces (C) л..л interaction in interactions energy calculation of compound 6; (D) Fingerprint plots (E) Hirshfeld surfaces (F) л..л
Interactions in energy calculation of 7.
Fig. 8. Percentage contributions in the fingerprint in compounds 1- 4, 6, and 7 are shown in the bar graph.
3
.4. In silico analysis of compound 1–8
of compound 3 with COX-2 afforded one hydrogen bonding inter-
action between the cyano group’s nitrogen in pyridone and the
amino acid residue Ser530. Moreover, the phthalimide ring of com-
pound 3 established π-sulfur interaction with the Met522 residue,
two π-π T-shaped interaction with Tyr385 and Trp387 residues,
and two amide-π stackings with the Gly526 residue. At the same
time, the pyridone ring exhibited π-σ interaction with the Ala527
residue. The other interactions involved are π-alkyl and alkyl inter-
actions with the residues Val16, Arg120, Val349, Tyr355, Leu384,
Val523, and Leu531 (Fig. 9A). These interactions might have con-
tributed substantially to the high binding energy of compound 3
in the active site of COX-2, due to which compound 3 has ex-
pressed better in vivo anti-inflammatory activity over other com-
pounds. Meanwhile, Compound 4 exhibited two hydrogen bond in-
teractions with the residues Ser530 and Tyr355. It also established
π-cation interaction with the residue Arg513, π-lone pair inter-
actions with Leu352, π-sulfur interaction with Met522, π-σ in-
teractions with Tyr385 and Val523, and π-alkyl interactions with
the residues Leu352, Phe381, Tyr385, and Ala516 (Fig. 9B). Hydro-
Docking analysis was performed for synthesized compounds 1-
8
at the COX-2 enzyme active site and compared with the stan-
dard drugs Lumiracoxib and Nimesulide. Table 6 shows the dock-
ing scores of Lumiracoxib, Nimesulide, compounds 1-8 within the
active sites of COX-2 (Fig. 9). From the structure-activity relation-
ship (SAR) perspective, a straight comparison of inhibitory potency
and selectivity index profiles of all compounds 1–8 revealed that
except compound 2, all shows strong binding affinity with COX-
2
compared to that of Lumiracoxib. From the in vivo perspective,
a straight comparison of inhibitory potency and selectivity index
profiles of all compounds docking study was performed compared
with Nimesulide. In contrast, compound 3 & 4 binds strongly, well
inside the pocket of COX-2. Compounds have non-covalent interac-
tions as ligands with COX-2 in silico analysis.
The docking study indicated that phthalimide containing com-
pounds 3 & 4 exhibited the highest binding affinity towards COX-
2
isoform (−9.29 & −9.27 kcal/mol, respectively). The interaction
11

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Fig. 9. Non-covalent interactions in the active site of COX-2. (A) Compound 3 (B) Compound 4 (C) Overlay of compound 3 (orange), 4 (green) & Lumiracoxib (pink) in the
active site.
Table 5
used with same head and tail group to provide flexibility in ana-
Aromatic interaction in Hirshfeld analysis of compound 1-4,
logue but it is not enough to provide better compatibility like 3 &
6
& 7. Cg is the centroid of the rings
4
to bind with COX-2 receptor.
Cg(I)…Cg(J)
Cg-Cg
Symmetry Identifiers
Compound 1–8 binds with COX-2 through C-H…O, C-H…π,
Compound 1
Cg(1)…Cg(1)
Cg(2)…Cg(2)
Compound 2
Cg(1)…Cg(1)
Cg(2)…Cg(2)
Compound 3
Cg(1)…Cg(1)
Cg(1)…Cg(2)
Compound 4
Cg(1)…Cg(1)
Cg(2)…Cg(3)
Compound 6
Cg(1)…Cg(1)
Cg(1)…Cg(2)
Cg(2)…Cg(2)
Compound 7
Cg(1)…Cg(3)
Cg(1)…Cg(3)
Cg(2)…Cg(2)
Cg(2)…Cg(2)
Cg(3)…Cg(3)
π…π, etc., non-covalent interactions. Both the analogue 3 & 4
has phthalimide ring, which enhance polarization in the molecule
to increase the electrostatic binding affinity with COX-2 receptor
in the same way as observed in crystal packing and in Hirshfeld
surface analysis. The docking pose showed that one ring of all
molecules present in the same binding site of COX-2 protein. These
results reveal that compounds bind selectively to the COX-2 active
site. All ligands show non-covalent interactions through the lig-
and’s donor-acceptor property similar to those found in the Hirsh-
feld and single-crystal analyses. Conformations of all the molecules
inside the COX-2 pocket are of higher energy than the single-
crystal structure. Because of the higher flexibility, all compounds
deformed their geometry to close interactions with the neighbour-
ing side-chain residues, while compounds 3, 4 & 7 showed rela-
tively more robust interactions with side chain residues.
3.528(12)
3.661(12)
-x, y+1/2, -z+1/2
x, y, z
3.612(12)
3.661(12)
-x, -y, -z
-x, -y, -z
3.632(12)
3.334(11)
-x, -y, -z
-x+1/2, y+1/2, -z+1/2
3.611(12)
3.534(11)
-x, y+1/2
-x, -y, -z
3.9977(12)
3.6850(11)
3.7201(12)
-x,-y,1-z
1/2-x, 1/2+y,z
1/2-x,1/2+y,z
3.8519(13)
3.9642(13)
3.9419(13)
3.9472(13)
3.6687(12)
-x,2-y,-z
1-x,2-y,-z
-x,2-y,1-z
1-x,2-y,1-z
1-x,2-y,-z
3
.5. In vivo anti-inflammatory studies of compound 1–8
As a reference, the drug Nimesulide and all drugs were sub-
jected to in vivo anti-inflammatory studies using the carrageenan-
induced foot paw edema model [25,26,35]. The results of anti-
inflammatory activities are shown in Tables 7 & 8.
gen bonding interaction of compound 4 with the receptor is very
similar to Lumiracoxib, whereas another compound 3 interactions
overlap with standard and interact with Val349, Leu384, Tyr385,
Trp387, Val523, and Ala527 amino acids. In both compound 3 &
The present study results show that compound 3 possesses the
maximum anti-inflammatory effect at 180 min, 9.1%, whereas stan-
dard drug inhibits up to 13.7% (Table 7 & 8, Fig. 10). Other com-
pounds, i.e., 1, 4, 5, 6 and 8 show intermediate effects on inhibitory
properties. Compounds 2 & 7 show a negligible effect. As per the
in vivo analysis, compound 3 is more productive, and compound
4
monocyclic and bicyclic rings are linked through tri-methylene
spacer to provide more flexibility to make effective binding with
the COX-2 receptor. In compound 7 & 8, di-methylene spacer is
12

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Table 6
Docking Scores and interactions compared with Nimesulide and Lumiracoxib (Standard drug).
Binding affinity
(Kcal/mol)
Compound
Hydrogen Bonds
Residues involved in other interactions
Lumiracoxib (Standard drug)
−7.66
Arg120, Tyr355, Met522
Val349, Leu352, Phe381, Leu384, Tyr385,
Trp387, Val523, Ala527
(
halogen bond)
Nimesulide
1
−7.1
Arg120
Ser530
Val523, Leu352, Ser353
−8.05
Val349, Leu352, Leu384, Tyr385, Trp387,
Phe518, Met522, Val523, Ala527
Val349, Leu352, Tyr355, Leu384, Tyr385,
Trp387, Met522, Val523, Gly526, Ala527,
Val116, Arg120, Val349, Tyr355, Leu384,
Tyr385, Trp387, Met522, Val523, Gly526,
Ala527
2
3
−7.56
−9.29
Ser530
Ser530
4
5
6
7
8
−9.27
-7.6
Ser530, Tyr355
Ser530
–
Leu352, Phe381, Tyr385, Arg513, Ala516,
Met522, Val523,
Val116, Val349, Tyr355, Tyr385, Trp387,
Met522, Val523, Ala527, Leu531
Phe205, Val344, Tyr385, Trp387, Phe518,
Met522, Gly533, Leu534
−8.77
−8.91
-6.8
Val523
Ser530
Trp90, Leu352, Tyr 355, Leu384, Tyr385,
Phe518, Met522
Val116, Val349, Leu352, Tyr355, Leu359,
Tyr385, Trp387, Met522, Val523, Gly526,
Ala527, Leu531
Table 7
Percentage Edema Growth of compound 1-8.
Group
Percentage Edema Growth Relative to Control at Different Time Intervals (Mean± SEM)
0
(min)
30 (min)
90 (min)
180 (min)
Control
100± 0
100± 0
100± 0
100± 0
100± 0
100± 0
100± 0
100± 0
100± 0
100± 0
130.2± 6.45 (30.2)
113.7± 2.88 (13.7)
116.13± 2.77(16.13)
118.2± 2.96(18.2)
111.4± 5.11(11.4)
116.1± 3.78(16.1)
117.8± 3.47(17.8)
117.03± 2.77(17.03)
119.9± 2.90(19.9)
117.6± 3.78(17.6)
138.5± 4.45 (38.5)
122.3± 5.22 (22.3)
113.7± 4.25 (13.7)
117.2± 5.15(17.2)
118.2± 5.22(18.2)
109.1± 3.39(9.1)
∗
Nimesulide
123.5± 3.27 (23.5)
∗
∗
1
2
3
4
5
6
7
8
126.88± 3.35 (26.88)
130.88± 3.76 (30.88)
∗
∗
∗
120.4± 5.76 (20.4)
126.8± 3.53 (26.8)
117.2± 5.05 (17.2)
118.1± 5.89(16.8)
116.12± 5.25(16.12)
117.95± 5.12(17.95)
115.9± 4.25(15.9)
128.7± 5.31 (28.4)
∗
∗
126.88± 3.40 (26.88)
131.22± 3.76 (31.22)
∗
129.2± 6.21 (29.2)
N = 6 = Number of rats in each group. Results in parentheses indicate percentage change from respective
∗
∗∗
∗∗∗
∗∗∗
control group. p-value < 0.05 = ; < 0.01 = ; <0.001 =
∗
∗∗
p-value < 0.05 = ; < 0.01 = ; <0.001 =
Fig. 10. (A) Paw Edema Growth of the compound 1–8 at a different time interval (B) Percentage Edema Growth of compound 1–8.
2
is among the least active. Crystal analysis also gives informa-
anti-inflammatory effect in vivo analysis as found in docking anal-
ysis, but compounds 1, 4, 5 & 7 are not effective in vivo analysis as
per their binding interactions. Compound 3, 4, 7 and 8 are simi-
lar in term of flexibility and volume but anti-inflammatory activity
is not found in same order. All 3, 4, 7 and 8 are having differ-
ent electronic and structural properties. Compound 3 and 4 differs
in monocyclic ring of one arm of fleximer, whereas 7 and 8 are
tion about aromatic planes’ dihedral angle similarity between new
compounds and standard drugs. The surface area may also be one
of the factors for compound 3 to become more effective in binding
with COX-2. Structure 3 is very similar to Lumiracoxib (Standard
drug), which is very suitable for binding with the active site as per
docking results due to its flexibility. Compound 3 responds good
13

V.P. Singh, J. Dowarah, B.N. Marak et al.
Journal of Molecular Structure 1239 (2021) 130513
Table 8
Varanasi, India, is also acknowledged for providing the space and
facilities to perform biological analysis.
Paw Edema Growth of the compound 1–8 at a different time interval.
Paw edema at a different time interval (ml/rat) (Mean± SEM)
Group
0 (min)
30 (min)
90 (min)
180 (min)
Supplementary materials
∗
∗
∗
∗
Control
0.99± 0.05
1.26± 0.044
1.16± 0.065
1.08± 0.048
1.30± 0.058
0.98± 0.056
1.08± 0.048
1.12± 0.066
1.09± 0.035
1.32± 0.050
1.33± 0.048
1.35± 0.06
1.21± 0.072
1.15± 0.045
1.09± 0.077
1.30± 0.073
0.96± 0.063
1.09± 0.077
1.11± 0.070
1.10± 0.070
1.31± 0.063
1.31± 0.044
Nimu-silide
1.02± 0.054
0.93± 0.051
1.10± 0.064
0.88± 0.059
0.93± 0.051
0.95± 0.055
0.94± 0.050
1.11± 0.044
1.13± 0.046
1.26± 0.038
1.18± 0.065
1.44± 0.065
1.06± 0.062
1.18± 0.065
1.22± 0.052
1.19± 0.055
1.45± 0.060
1.46± 0.064
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130513.
1
2
3
4
5
6
7
8
∗
∗
∗
∗
∗
∗
References
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[
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;
∗∗
<
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&
9
[
[
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4
. Conclusion
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This study concluded that flexibility between the Pyri-
dine/pyridine ring and phenyl/phthalimide ring and number of
rings strengthens the parallel aromatic stacking interactions and
increased folding and hence close packing of the crystal. Further,
docking simulations revealed that fleximer 3, 4 & 7 experienced
the lowest binding energy at the COX-2 active site, but the con-
formation adopted was high energy. Compound 3 shows good in-
hibitory activity means it exists high energy conformation at the
active site inside the rat’s body. Hirshfeld surface analysis and
molecular conformation in crystal packing are showing good agree-
ment. Hirshfeld’s energy calculation revealed that dispersive and
electrostatic forces dominate over other forces in stabilizing the
crystal lattice through weak interactions. Compound 3 is the most
potent inhibitor of inflammation in vivo and in silico analysis. In
compounds 2 & 3, activity and volume are directly linked, but no
such relations are observed in compounds 1 and 2. It means that
multiple factors other than activity and volume are also involved
in the activity of the analog. Overall our study demonstrated that
compound 3 analog should be further investigated as an anti-
inflammatory prodrug candidate. The results reported herein might
be useful in drug design and understanding the robust state archi-
tecture of organic aromatic fleximers.
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Declaration of Competing Interest
The authors declare no conflicts of interest.
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