Synthesis, computational predictions of selective bioactive nicotinamide derivatives and in vitro biological evaluations

Article abstract of DOI:10.1016/j.molstruc.2019.06.048

A series of nicotinamide derivatives were designed and studied in silico molecular docking predictions against inflammatory responsible enzymes (cyclooxygenase-1(1PGG) and cyclooxygenase-2 (4-COX)). The well-predicted compounds were synthesized, characterized by the spectroscopic techniques namely, Proton Nuclear Magnetic Resonance, Carbon Nuclear Magnetic Resonance and the in vitro biological studies namely, anti-inflammatory activity and anti-diabetic activity. Furthermore, Density Functional Theory Calculations and Frontier Molecular Orbital by molecular electrostatic potential were investigated.

Full text of DOI:10.1016/j.molstruc.2019.06.048

Accepted Manuscript
Synthesis, computational predictions of selective bioactive nicotinamide derivatives
and in vitro biological evaluations
Jayaprakash Thirunavukarasu, Rosina Begam, A. Shajahan
PII:
S0022-2860(19)30772-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.06.048
MOLSTR 26690
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 April 2019
Revised Date: 11 June 2019
Accepted Date: 12 June 2019
Please cite this article as: J. Thirunavukarasu, R. Begam, A. Shajahan, Synthesis, computational
predictions of selective bioactive nicotinamide derivatives and in vitro biological evaluations, Journal of
Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.06.048.
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ACCEPTED MANUSCRIPT
Synthesis, Computational Predictions of Selective Bioactive Nicotinamide
Derivatives and in vitro Biological Evaluations
Jayaprakash Thirunavukarasua**, Rosina Begamb**, and A.Shajahanc
a,b,cDepartment of Chemistry, B S Abdur Rahman Crescent Institute of Science and Technology,
Chennai, Tamil Nadu, India.
ajp79.pharma@gmail.com
brosinashabari@gmail.com
cashajahan@crescent.education
**Both the authors have contributed equally for this research work.
Abstract
A series of nicotinamide derivatives were designed and studied in silico molecular docking
predictions against inflammatory responsible enzymes (cyclooxygenase-1(1PGG) and
cyclooxygenase-2 (4-COX)). The well-predicted compounds were synthesized, characterized by
the spectroscopic techniques namely, Proton Nuclear Magnetic Resonance, Carbon Nuclear
Magnetic Resonance and the in vitro biological studies namely, anti-inflammatory activity and
anti-diabetic activity. Furthermore, Density Functional Theory Calculations and Frontier
Molecular Orbital by molecular electrostatic potential were investigated.
Keywords: anti-inflammatory; anti-diabetic activity; cyclooxygenase; Molecular Docking
Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

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Synthesis, Computational Predictions of Selective Bioactive Nicotinamide
Derivatives and in vitro Biological Evaluations
Jayaprakash thirunavukarasu,a Rosina Begam,a and A.Shajahana*
aDepartment of Chemistry, B S Abdur Rahman Crescent Institute of Science and
Technology,chennai,tamilnadu India.
Corresponding Author: ashajahan@crescent.education
Graphical abstract

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Synthesis, Computational Predictions of Selective Bioactive Nicotinamide
Derivatives and in vitro Biological Evaluations
Abstract
A series of nicotinamide derivatives were designed and studied in silico molecular docking
predictions against inflammatory responsible enzymes (cyclooxygenase-1(1PGG) and
cyclooxygenase-2 (4-COX)). The well-predicted compounds were synthesized, characterized
by the spectroscopic techniques namely, Proton Nuclear Magnetic Resonance, Carbon
Nuclear Magnetic Resonance and the in vitro biological studies namely, anti-inflammatory
activity and antidiabetic activity. Furthermore, Density Functional Theory Calculations and
Frontier Molecular Orbital by molecular electrostatic potential were investigated.
Keywords: anti-inflammatory; antidiabetic activity; cyclooxygenase; Molecular Docking
Funding: This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.

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1.
Introduction
In recent years, computational studies play as a vital tool in drug development industries as
well as research communities. The new drug discovery takes very long time in years and the
synthesis roots also need an excess effort in terms of cost of chemicals particularly the time
consumption and it creates huge amount of environmental pollution by wet lab trials. Silico
approach is great tool to rectify and reduce the above problems significantly. Molecular
docking is a biological computational tool in drug design which helps in screening the drug to
be synthesized according to their docking score, affinity or binding mode and binding sites of
proteins and enzymes with drug as ligands.
Nicotinamide has a potent immunomodulatory effect in vitro, and have great potential for
treatment of human inflammatory disease[1]. Vitamin B-3 consists of nicotinic acid and
nicotinamide can bind with more than 200 enzymes. It produces NAD and NADP which
supports in cell metabolism. Nicotinamide has also an amide derivative of niacin or vitamin
B3, with an amide linked to an aromatic ring. It has also shown the properties such as
antioxidant, anti-inflammatory and anti-cell death/apoptosis[2]. A nicotinamide, a vitamin
B(3) derivative, has evoked by FoxO3 in human gestational tissues and also react with
proteins were exhibiting anti-inflammatory and anti-oxidant effects[3]. Nicotinamide
derivatives have a property of phosphodiesterase Type 4 (PDE4) inhibitors which is
beneficial for the inflammatory disease treatment[4].Therapeutic effective of nicotinamide
adenine dinucleotide (NAD) has studied for detection and treatment of congenital
malformations[5][6]. Combination of Nicotinamide and streptozotocin has studied for in vivo
evaluation of antibacterial activity[7]and can be used as whitening anti-aging smoothing
toner[8] and anti-allergic mask[9]. Since nicotinamide and derivatives has multiple

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therapeutic properties, there is good scope in medical and pharmaceutical fields to synthesis
of new nicotinamide derivatives[10].
In this research work, various nicotinamide derivatives were designed and compared by
molecular docking interactions against cyclooxygenase inhibitors COX-1 and COX-2. The
promising predicted nicotinamide derivatives were synthesized and also their biological
activities such as anti-inflammatory and antidiabetic activities were evaluated. The HOMO,
LUMO and molecular electrostatic potential studies were done through DFT computational
method to investigate the structure, band gap energy and the electron density of the
synthesized nicotinamide derivatives.
2.
Materials and methods
2.1. Materials
All chemicals were procured from Sigma-Aldrich, Merck and used as it is without any
further purification. Analytical grade (AR) solvents and double distilled water were
used throughout the biological studies. The reactions were monitored consequently by
Thin Layer Chromatography (TLC) using Silica gel 60F254 and visualized under UV
at the wavelength range from 254 to 366nm.
1H NMR (300MHz) and 13C NMR (75MHz) were recorded on Bruker NMR
instrument in CDCl3, DMSO-d6 and TMS as an internal standard. Electrospray
ionization mass spectra (ESI-MS) were recorded on LCQ Fleet mass spectrometer
(Thermo Fisher Instruments Ltd., US). Shimadzu UV-1800 UV-vis spectrophotometer
was used to record UV absorption measurement (for biological studies).

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2.2
Methods
2.2.1. Molecular docking study
Molecular docking of designed compounds into the COX enzyme was carried out using
Auto-Dock tool (version 4.2). Accelrys discovery studio client 4.1 visualizer was used for
visualizing the protein-ligand complex. The 3D structure of compounds was energetically
minimized using Chemdraw 12 (MMFF94, 5000 interactions) before docking. The 3D crystal
structure of the COX enzymes (1PGG.pdb and 4COX.pdb) was taken from PDB (Protein
Data bank). All bound water and ligands were eliminated from the protein and polar
hydrogen was added. Moreover, a grid box size of 60 × 60 × 60 points is set for all docking
in X, Y and Z directions. A grid spacing of 0.375 Å and 15 runs was created by using
Lamarckian genetic algorithm searches.
2.2.2 General procedure for synthesis of 2a, 2c and 2l
The 6-chloronicotinic acid (2g, 12.73 mmol) was dissolved in DMF (20 ml). In the
prepared solution, TBTU (4.90 g, 15.28 mmol) and TEA (2.66mL, 19.10 mmol) was
added. The reaction mixture was stirred for 30 minutes at room temperature. An amine
(12.73 mmol) was added and stirred for 3 hours at room temperature. The progress of
the reaction was monitored by TLC. The reaction mixture was then quenched with
crushed ice (50g) and stirred for 30 min. The obtained precipitate was filtered and
dried. Finally the product was purified by column chromatography using ethyl acetate:
petroleum ether as eluent. Yield: (2a:87%, 2c:86%, 2l: 88%).
2.2.2.1 Synthesisof 3a, 3c and 3l
In the stirred solution of intermediate 2a (or 2c or 2l) (0.5g, 2.154 mmol), toluene (10
ml) and KOH (4.83g, 8.619 mmol) were added. In the prepared solution, benzyl

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alcohol (2.24 mL, 2.154 mmol) was added. The mixture was heated for an hour at
100°C. The reaction mixture was cooled to room temperature and extracted with ethyl
acetate (200 ml), washed with water (2 x 200 ml) and brine solution (200 ml). The
organic layer was separated and dried over anhydrous sodium sulphate. The solvent
was evaporated under reduced pressure. Finally the products were purified by column
chromatography using ethyl acetate:petroleum ether as solvent. Yield: (3a:82%,
3c:84%, 3l:78%).
2.2.2.1.1.
Spectral data for synthesized compounds
6-chloro-N-phenyl-nicotinamide (2a)
2.2.2.1.1.1.
1H NMR (300 MHz, CDCl3/TMS): δ=7.19 (t, J = 14.2 Hz, 1H), 7.38 (t, J =15.6 Hz, 2H),
7.45 (d, J = 8.4 Hz, 1H) , 7.61 (d, J =7.8 Hz, 2H), 7.96 (S, 1H), 8.15 (m, 1H), 8.85 (d, J =2.1
Hz, 1H)
13C NMR(75 MHz, CDCl3/TMS): δ=120.8, 124.6, 125.4, 129.3, 129.8, 137.3, 138.1, 148.1,
154.7, 163.0.
2.2.2.1.1.2.
6-chloro-N-(3-chloro-phenyl)-nicotinamide (2c)
1H NMR (300 MHz, CDCl3/TMS): δ= 7.17 (d, J =7.8 Hz, 1H), 7.31 (t, J =16.2 Hz, 1H),
7.48 (d, J =8.2 Hz, 2H), 7.74 (s, 1H) , 7.962 (S, 1H), 8.16 (m, 1H), 8.85( d, J =2.4 Hz, 1H)
13C NMR (75 MHz, CDCl3+DMSO-D6/TMS): δ=118.4, 120.3, 123.6, 123.9, 129.4, 129.2,
133.7, 138.2, 139.4, 149.0, 153.6, 163.0.
2.2.2.1.1.3.
N-(2, 4-dimethoxybenzyl)-6-chloropyridine-3-carboxamide (2l)
1H NMR (300 MHz, CDCl3/TMS): δ= 3.80 (S, 3H), 3.85(S, 3H), 4.56 (d, J =5.7 Hz, 2H),
6.48 (m,2H), 6.56 (S,1H), 7.26 (S, 1H), 8.05 (m,1H), 8.69 (d,1H)

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13C NMR (75 MHz, CDCl3/TMS): δ=39.9, 55.5, 104.2, 118.1, 124.3, 129.5, 130.9, 138.0,
148.0, 154.0, 158.7, 160.9, 164.2.
2.2.2.1.1.4.
6-benzyloxy-N-phenyl-nicotinamide (3a)
1H NMR (300 MHz, CDCl3/TMS): δ= 5.5 (S, 2H), 6.85 (m, 1H), 7.15 (t, J =14.7 Hz, 1H),
7.35 (m, 5H), 7.42 (d, J =19.2 Hz, 2H), 7.6 (d, J 7.8 Hz, 2H), 7.85 (S, 1H), 8.07 (m, 1H),
8.68(d, J =2.1 Hz, 1H)
13C NMR (75 MHz, CDCl3/TMS): δ=68.4, 111.5, 120.4, 124.4, 128.2, 128.6, 129.2, 137.7,
138.1, 146.5, 165.7.
ESI-MS Calculated, m/z 304.34, found 305.3 (m++1).
IR (KBr)νmax: 3352, 3296, 1605, 1526, 1486, 1352 cm-1.
2.2.2.1.1.5. 6-benzyloxy-N-(3-chloro-phenyl)-nicotinamide (3c)
1H NMR (300 MHz, CDCl3/TMS): δ= 5.44 (S, 2H), 6.87 (d, J =8.7 Hz, 1H), 7.12 (d J =2.8
Hz,, 1H), 7.25 (t, J =8.1 Hz, 1H), 7.42 (m, 4H), 7.44 (m, 4H), 7.73 (t, J =3.9 Hz, 1H),7.86
(S, 1H), 8.06 (m, 1H), 8.67 (d, 1H)
13C NMR (75 MHz, CDCl3/TMS): δ=68.4, 111.5, 118.5, 120.6, 123.9, 124.9, 128.2, 128.6,
130.1, 134.8, 136.7, 138.0, 138.9, 146.7, 164.2, 165.8.
ESI-MS Calculated, m/z 338.79 found 339.3 (m++1)
IR (KBr)νmax: 3438, 3280, 1605, 1593, 1489, 1308 cm-1.
2.2.2.1.1.6.
ester (3l)
N-[2-(2, 4-dimethoxy-benzylcarbamoyl)-ethylidine]-acetimidic acid benzyl

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1H NMR (300 MHz, CDCl3TMS: δ= 3.80 (S, 3H), 3.85 (S, 3H), 4.55 (d, J =5.7 Hz, 2H), 5.40
(S, 2H), 6.45 (m, 2H), 6.56 (S, 1H), 6.81 (d, J =8.7 Hz, 1H) 7.38 (m,5 H), 8.00 (d, J = 2.4
Hz, 1H), 8.54 (d, J =2.1 Hz, 1H)
13C NMR (75 MHz, CDCl3/TMS): δ=39.7, 55.5, 68.2, 98.8, 104.1, 111.1, 118.6, 124.2,
128.1, 128.6, 130.8, 136.9, 138.0, 146.2, 158.7, 160.7, 165.2, 165.4 .ESI-MS Calculated, m/z
378.42 found 379.4 (m++1)
IR (KBr)ν max: 3436, 3277, 1635, 1544, 1489, 1354, 1289, 1263 cm-1.
2.2.4 Anti-inflammatory activity
2.2.4.1. BSA denaturation technique
The anti-inflammatory activity was performed for the synthesized drugs 3a, 3c and 3l by
adopting the experimental procedure given by Sribalan et al [11]. The synthesized
compounds were screened to anti-inflammatory activity by using inhibition of albumin
denaturation technique with minor modification. The standard drug diclofenac sodium at the
final concentration of 20.11, 26.41, 58.7, 73.2 and 84.81 µg/ml was used. 2.8 ml of phosphate
buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations of 3a, 3c and 3l were taken,
so that final concentrations become 25, 50, 100, 150 and 200 µg/ml.
2.2.4.2.Egg albumin denaturation technique
The Anti-inflammatory activity was performed by adopting the method specified by Chandra
et al[12]. The reaction mixture (5 mL) consisted of 0.2 ml of egg albumin (from fresh hen’s
egg), 2.8 ml of phosphate buffered saline (PBS, pH 6.4) and 2 ml of varying concentrations
of 3a, 3c and 3l to give the final concentrations of 25, 50, 100,150 and 200 µg/ml. Same
volume of double-distilled water served as control. Then the mixtures were incubated at
(37±2°C) in a BOD incubator (Labline Technologies) for 15 min and then heated at 70°C for

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5 minutes. After cooling, their absorbance was studied at 660 nm (SHIMADZU, UV 1800)
by using vehicle as blank and their viscosity was determined by using Ostwald viscometer.
Diclofenac sodium at the final concentration of (32.43, 48.91, 72.47, 85.22 and 97.85µg/ mL)
was used as reference drug and treated similarly for determination of absorbance and
viscosity.
2.2.5. Antidiabetic activity
The synthesized nicotinamide derivatives were studied for antidiabetic activity at five
different concentrations by following the procedure suggested by Sribalan et al. [11]
2.2.6. DFT studies
All the computational calculations including representation of HOMO and LUMO in the
checkpoint files were developed with Gaussain 09W program using density functional theory.
The chemical structure of 3a, 3c and 3l was optimized with B3LYP/6.311 G basis set. The
Gauss view software package was used to visualize the computed structures including
HOMO, LUMO and molecular electrostatic potential (MEP) representations.

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3.
Results and Discussion
Chemistry of the synthesized drugs 3a, 3c and 3l
3.1.
3.1.1. Molecular docking studies
In this research fourteen different nicotinamide derivatives were designed and studied for
molecular docking with the selected enzyme proteins namely COX-1 and COX-2 for the
interaction of ligand-enzyme sites selectivity.
Cyclooxygenase is an enzyme involved in the conversion of arachidonic acid into
inflammatory prostaglandins and it is target for the anti-inflammatory drugs[13] .The two
isozymes of COX involved in prostaglandin biosynthesis are COX-1 and COX-2 [14].The
cyclooxygenase enzyme inhibition will provide pain relief and cure inflammation[13]. On the
basis of this, the enzymes 1PGG and 4COX were chosen for docking. All the three selected
nicotinamide derivatives (3a, 3c and 3l) were docked with COX-1(1PGG) and COX-
2(4COX) enzymes.
The designed derivatives of nicotinamide structures are as shown in Figure 1 and best three
compounds which shows higher molecular docking score are indicated in square brackets.

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Fig. 1 The designed nicotinamide derivatives for molecular docking studies

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The molecular docking scores of the interaction of COX-1(1PPG) of the designed compounds
ranges from -6.13 to -9.07 kcal/mol and the inhibition constant ranges from 0.22 to 32.22µM
(Table 1).Among fourteen different compounds which are designed for this study, five
compounds, namely, 3a, 3c, 3e, 3f and 3l exhibits higher docking scores -9.07,-8.81, -8.96, -
8.94 and -8.69 kcal/mol and inhibition constant of 0.22, 0.35, 0.272, 0.278 & 0.42µM
respectively with COX-1 (Table 1).
From Table 2, it can be seen that the molecular docking score of 3a with COX-2 is -9.12
kcal/mol and inhibition constant is 0.205µM. Similarly, the docking scores of the compounds
3c & 3l with COX-2 are -9.37& -9.08 kcal/mol with two and one hydrogen bonding
respectively. Because of higher value of docking scores and inhibition constants, the
compounds 3a, 3c& 3l were selected and docked with COX-1(1PGG) and COX-2(4COX)
enzymes.
Table 1
Molecular docking studies of designed compounds of nicotinamide derivative, 3a-3n against
cyclooxygenase inhibitors (COX-1)
Compounds
1PPG
Inhibition
Constant
(µm)
Interacting
residue
Docking score
(kcal/mol)
Number of hydrogen
bonds
Name
3a
3b
3c
3d
-9.07
-8.32
-8.81
-8.3
-
-
0.223
-
0.793
ASN392
-
-
0.35
1
0.827
ASN382

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3e
3f
-8.96
-8.94
-8.58
-7.65
-8.25
-7.24
-6.13
-8.69
-6.86
-8.02
-
0.272
0.278
0.511
2.49
-
-
-
-
3g
3h
3i
-
1
-
ASN382
0.893
4.9
-
3j
-
ASN382
3k
3l
-
32.22
0.427
9.32
-
1
-
THR206
3m
3n
-
-
-
1.32
Table 2
Molecular docking studies of designed compounds of nicotinamide derivative, 3a-3n against
cyclooxygenase inhibitors (COX-2)
4COX
Compounds
Docking score
(kcal/mol)
-9.12
Number of
hydrogen bonds
2
Inhibition
Constant
0.205 µm
Interacting
residue
3a
3b
ASN382,
TRP387
-8.66
2
450.0nm
ASN382,
TRP387
3c
3d
3e
3f
-9.37
-8.69
-8.52
-8.44
2
2
1
2
0.135µm
426.85nm
0.568 µm
0.62 µm
THR206,ASN382
ASN382,TRP387
ASN382
ASN382,TRP387

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3g
3h
3i
-8.51
-8.30
-7.83
-7.65
-6.39
-9.08
-6.13
-7.17
2
2
1
1
-
0.58µm
ARG222,TRP387
0.830 µm
1.81µm
ASN382,TRP387
ASN382
TRP387
-
3j
2.49µm
3k
3l
20.85 µm
0.221 µm
32.22 µm
5.58 µM
1
-
TRP387
-
3m
3n
1
TRP387
3.2.
Synthesis of 3a, 3c and 3l
Three compounds (3a, 3c & 3l) which were selected by theoretical studies of molecular
docking were synthesized. According to Scheme l, two steps were followed as a synthesis
route. In step one, the compounds 2a, 2c & 2l were synthesized and used as precursors for
the compounds, 3a, 3c & 3l respectively. 2a, 2c & 2l were synthesized through the carboxylic
acid amine coupling. In second step, compounds of 3a, 3c and 3l were synthesized by
reacting 2a, 2c & 2l with benzyl alcohol to form ether linkage by optimizing parameters
namely, base, solvent, temperature and reaction time (Table 3). As the highest percentage of
yield of 82% was observed with KOH as a base and toluene as the solvent system at 100° C
for 1 hour which were chosen as optimized conditions for all the three nicotinamide
derivatives (3a, 3c & 3l).
Table 3
Optimization of reaction conditions for synthesis of 3a, 3c& 3l.
S. No.
Base
Solvent
Temperature
(°C)
Reaction time
(hour)
Yield
(%)

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1
2
3
4
5
6
7
Na2CO3
K2CO3
NaOH
NaOH
KOH
CAN
CAN
35
35
24
24
6
-
-
EtOH
80
21
15
35
27
82
EtOH/H2O
EtOH
80
6
80
6
KOH
EtOH/H2O
Toluene
80
6
KOH
100
1
O
NH2
O
R
TBTU,TEA,
OH
N
R
H
DMF, 3h,
RT
Cl
N
Cl
N
R = H, methoxy and chloro
O
O
O
ether
formation
acid amine
coupling
OH
N
N
R
R
H
H
Cl
N
O
N
Cl
N
R = H, methoxy and chloro
Scheme 1. Synthesis route of selected nicotinamide derivative compounds
3.3. Anti-inflammatory activity:
Anti-inflammatory study was performed by two different techniques namely, BSA
denaturation and Egg albumin denaturation techniques using Diclofenac sodium as a
reference drug .In BSA denaturation technique, irrespective of the concentration of drug
used, the compound 3c shows highest inhibition percentage followed by 3a and 3l.Similarly
Table 5 shows the inhibition rate of protein order which is 3l<3a<3c by Egg Albumin
Technique. Thus, the inhibition rate of protein order can be given as 3l<3a<3c (Table 4 and
5).The higher inflammatory effect can be attributed to higher electrophilicity index of 3c

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(Table 7 DFT calculation).The compound 3c contains chlorine substituted phenolic bulky
group which retains and exhibits intense anti-inflammatory activity to drug. Drugs which has
higher inhibition rate of protein denaturation would be worthwhile for anti-inflammatory
drug development[17].
Table 4
Anti-inflammatory activity of selective nicotinamide derivatives by BSA denaturation
Technique
Concentration
Inhibition (%)
(µm)
25
3a
3c
3l
Standard
20.11
26.41
58.7
19.56
24.49
50.48
71.55
78.87
21.12
28.78
56.42
75.59
80.24
15.24
19.87
42.23
65.59
73.37
50
100
150
200
73.2
84.81
Standard drug (DiclofenacSodium)
Table 5
Anti-inflammatory activity of selective nicotinamide derivatives by Egg Albumin Technique.
Concentration
Inhibition (%)
(µm)
25
3a
3c
3l
Standard
32.43
28.82
43.37
69.54
31.13
45.68
71.12
24.49
39.89
65.57
50
48.91
100
72.47

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150
200
75.59
82.28
78.84
85.33
70.24
78.98
85.22
97.85
Standard drug (DiclofenacSodium)
3.4. Antidiabetic Activity
Many researchers have attempted to show that the nicotinamide based drugs exhibit
antidiabetic activity [15], [16]. Synthesized compounds were studied for anti-diabetic activity
at five different concentrations namely 25, 50, 100,150 & 200 µM along with the standard
drug acarbose which is used for type 2 diabetics. Table 6 shows the percentage of inhibition
by the synthesized compounds. From Table 6, it can be seen that the percentage of inhibition
followed the order of 3l>3a>3c. As the concentration of compounds, 3a, 3c and 3l were
increased from 25 to 200 (µM), the percentage of inhibition also increased and follows the
trend of standard drug. The N-(2, 4-dimethoxybenzyl)-6-(benzyloxy) pyridine-3-carboxamide
(3l) has revealed highest anti-diabetic activity.
Table 6
Antidiabetic activity of synthesized compounds 3a, 3c & 3l
Concentration
Percentage of Inhibition
(µm)
25
3a
3c
3l
Standard
16.74
26.42
52.46
65.56
91.23
12.24
20.23
42.78
60.69
78.24
11.25
19.56
39.56
57.65
75.52
14.56
23.32
48.71
62.34
85.23
50
100
150
200
Standard – Acarbose drug

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3.5.
Cyclooxygenase inhibition
The synthesized nicotinamide derivative compounds 3a, 3c and 3l exhibited good in vitro
anti-inflammatory activity. Cyclooxygenase is the enzyme responsible for prostaglandin
production[18]. Because of the above properties, the protein 1PGG and 4COX are selected
for docking. All the three synthesized compounds were docked with COX-1(1PGG) and
COX-2(4COX) enzymes. Figures 2 and 5 show the interaction of 1PGG COX-1 with
compound 3a which shows, Met391 (5.08Å), Leu390 (4.60Å), Ala199 (4.19Å), Ala202
(5.38Å) make pi-alkyl interaction with benzyl ring and His388 (5.83Å) makes pi-pi-T-shaped
interaction in benzyl ring. Pyridine ring and carbonyl group makes pi-pi-T-shaped interaction
with His386 (5.01Å), carbonyl group makes pi-alkyl interaction with both His386 (4.69 Å)
and Phe210 (3.64Å) amino acid residues. Amide NH makes pi-alkyl interaction with His207
(3.20Å).The compounds of 3c reveals, Benzyl ring makes pi-sulfur interaction, pi-sigma
interaction and two pi-alkyl interaction with the amino acid residues of Met391 (5.32Å),
Leu390 (3.88Å), Ala199 (5.11Å), Ala202 (4.62Å). Pyridine ring makes pi-donor hydrogen
bond interaction with Thr206 (3.14Å).Carbonyl group makes two pi-alkyl interaction with
the amino acid residues of His386 (2.87Å), Phe210 (4.83Å).m-substituted chloro ring make
pi-donor hydrogen bond interaction with the amino acids of Asn382 (3.53Å). The 3l
compounds exhibits (Figures 4 and 7), Benzyl and pyridyl ring makes pi-pi T-shaped
interaction with the amino acid residues of His120 (4.18Å). Carbonyl and amide NH groups
make pi-alkyl interaction with His388 (4.73Å) and His207 (3.42Å). Methoxy group
substituted benzene ring makes pi-alkyl interaction and pi-sulfur interaction with
Ala202(4.35Å) and Leu390 (5.96Å).

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Fig. 2 1PPG(COX-1) interaction image of compound3a
Fig. 3 1PPG(COX-1) interaction image of compound 3c

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Fig. 4 1PPG(COX-1) interaction image of compound 3l
The synthesized compound 3c of 4COX images (Figures 3 and 6) shows, Benzyl ring make
conventional hydrogen bond interactions with Phe210 (3.16Å) and His214 (3.33Å) make
carbon hydrogen bond interaction with benzyl ring. Benzyl CH2 makes pi-donor hydrogen
bond interaction with His207 (4.04Å). O-CH2 group makes conventional hydrogen bond
interaction with Asn (382 Å). Pyridine ring make pi-pi-T-shaped interaction with His207
(4.38 Å). Carbonyl group makes two hydrogen bond interactions with amino acid residues of
Trp387 (2.66Å), Tyr385 (Å). Amide attached benzene ring makes pi-alkyl interaction with
the amino acid residues of Leu390 (5.29Å), Ala199(5.299Å), Leu391 (5.01Å).The 3c
compound shows, Benzyl CH2 group make conventional hydrogen bond interaction with
Asn382 (3.01Å). Pyridine Nitrogen makes hydrogen bond interaction with Asn382 (2.78 Å).
Pyridyl ring makes pi-pi-T-shaped interaction with the amino acid residues of His207
(4.63Å), His386 (4.31Å). Carbonyl group and amide NH group makes conventional
hydrogen bond interaction with the amino acid residues of Thr206 (2.57Å) and His388
(3.40Å). Chloro substituted benzene ring makes pi-alkyl interaction with Leu530 (Å) and

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Ala202 (4.54Å) amino acid residues and the compound shows the interaction of COX-2,
Again pyridine ring formed pi-donor hydrogen bond interaction with Asn382. Benzyl ring
and methoxy substituted ring was observed pi-pi-T-shaped interaction with His207 (4.35Å),
His388 (4.95Å).
Fig. 5 4COX-2 interaction image of compound 3a

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Fig. 6 4COX-2 interaction image of compound 3c
Fig. 7 4COX-2 interaction image of compound 3l
3.6.
Frontier Molecular Orbitals
From Density Functional Theory (DFT) computational studies, HOMO and LUMO energy
levels of frontier orbitals were constructed and analyzed. The DFT studies were performed to

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evaluate chemical properties (chemical potential, global hardness and softness) and electronic
properties (electrophilic index and energy band gap).
Among the synthesized compounds 3a, 3c & 3l, the compound 3c lying at the highest value
of HOMO -6.6650 and LUMO -1.6691 (Figure 9) with energy gap value of 4.9958 (eV) and
higher value of chemical potential -4.1671. The compounds 3a and 3l has the moderate value
of HOMO values of -6.4601 , -6.0674 and LUMO values of 4.8559, 4.9593 respectively
(Figures 8 and 10). When the HOMO and LUMO energy gap is higher, the kinetic energy
and the chemical interactions or the molecular interactions will be higher[19].The global
hardness and softness of the synthesized compounds of 3a, 3c and 3l were 2.4279, 2.4979,
2.4796 and 0.2059, 0.2001, 0.2016 respectively.
Table 7
DFT calculation of compound nicotinamide derivatives of 3a, 3c&3l.
S.
Compound
HOMO LUMO Band
gap(∆E)
Chemical
potential
Global
Global
Electrophilicity
index
No name
hardness
softness
1.
2
3a
-6.4601 -1.6041
-6.6650 -1.6691
-6.0674 -1.1080
4.8559 -4.0321
4.9958 -4.1671
4.9593 -3.877
2.4279
2.4979
2.4796
0.2059
0.2001
0.2016
3.3480
3.4758
2.5954
3c
3l
3

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Fig. 8 FMO diagram of nicotinamide derivative 3a

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Fig.9 FMO diagram of nicotinamide derivative 3c

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Fig. 10 FMO diagram of nicotinamide derivative 3l
3.7.
Molecular Electrostatic Potential
The molecular electrostatic potential (MEP) mapping shows the binding interaction capability
and the reactive sites for electrophilic and nucleophilic attacks of synthesized compounds.
MEP allows visualization of the charge distribution of molecule (electron density) on the
molecular surface[20].
Figures 11, 12 and 13 show differentiation of electron rich region, electron poor region and
zero electrostatic potential of the compounds 3a, 3c and 3l respectively. The red colour zone

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indicates negative region preferred for electrophilic attack and the blue colour zone express
the positive region which is preferred for nucleophilic attack. The negative region is mainly
used for hydrogen bond formation with enzymes and proteins.
From molecular docking studies,-OCH3group makes two hydrogen bond interaction with
His388 (3.34Å), Gln203Å. Carbonyl group make hydrogen bond interaction with His
385(2.41Å). Amide NH and pyridine makes carbon hydrogen bond interaction with the
amino acid residue of His207 (3.07Å) and His207 (2.46Å), His207 (3.14Å) respectively.
Fig.11 Molecular Electrostatic potential of compound 3a
Fig. 12 Molecular Electrostatic potential of compound 3c

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Fig. 13 Molecular Electrostatic potential of compound 3l
4.
Conclusions
In this study of molecular docking the selected three compounds of nicotinamides were
synthesized .These compounds have exhibited good anti-inflammatory and antidiabetic
properties along with better docking score with enzymes .The computational studies of DFT
& MEP revealed the details of electrophilicity and electron density which is needed for the
bioactive and chemical activation sites in drugs.
Acknowledgement
Researchers of this study acknowledges Biochemie Innovations Lab, Tindivanam for
utilizing facility meant for biological evaluations.
References
[1] J. S. Ungerstedt, M. Blombäck, and T. Söderström, “Nicotinamide is a potent inhibitor
of proinflammatory cytokines,” Clinical & Experimental Immunology, vol. 131, no. 1,
pp. 48–52, Jan. 2003.