New dihydropyridine derivatives: Anti-inflammatory, analgesic and docking studies

Article abstract of DOI:10.1007/s00044-012-0156-1

The present article describes synthesis of new diethyl 2,6-dimethyl-4-(4- (2-substituted amino-2-oxoethoxy) phenyl)-1,4-dihydropyridine-3,5-dicarboxylates (6a-10b) following multistep synthetic route. Structures of newly synthesized intermediates and title compounds were established by spectral and elemental analyses. The final compounds were screened for their in vivo anti-inflammatory and analgesic activities by carrageenan-induced paw oedema and tail immersion methods, respectively. Moreover, molecular docking studies were carried out for active compounds 6c, 6d, 7d, 8 and 10b to study their mode of action, meanwhile in vivo results indicated that these compounds displayed rapid onset of anti-inflammatory action and exhibited prominent activity when compared with the standard drug. Compounds 6d and 7d carrying amide functionality showed the highest anti-inflammatory as well as analgesic activities. The molecular docking results emphasised the in vivo data and all docked molecules were found to display very low binding constant values in nanomolar scale.

Full text of DOI:10.1007/s00044-012-0156-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0156-1
ORIGINAL RESEARCH
New dihydropyridine derivatives: anti-inflammatory, analgesic
and docking studies
•
Shrikanth Ulloora Santhosh Kumar
•
•
Ramakrishna Shabaraya Airody Vasudeva Adhikari
Received: 30 March 2012 / Accepted: 12 June 2012
Ó Springer Science+Business Media, LLC 2012
Abstract The present article describes synthesis of new
diethyl 2,6-dimethyl-4-(4-(2-substituted amino-2-oxoeth-
oxy) phenyl)-1,4-dihydropyridine-3,5-dicarboxylates (6a–
10b) following multistep synthetic route. Structures of
newly synthesized intermediates and title compounds were
established by spectral and elemental analyses. The final
compounds were screened for their in vivo anti-inflam-
matory and analgesic activities by carrageenan-induced
paw oedema and tail immersion methods, respectively.
Moreover, molecular docking studies were carried out for
active compounds 6c, 6d, 7d, 8 and 10b to study their
mode of action, meanwhile in vivo results indicated that
these compounds displayed rapid onset of anti-inflamma-
tory action and exhibited prominent activity when com-
pared with the standard drug. Compounds 6d and 7d
carrying amide functionality showed the highest anti-
inflammatory as well as analgesic activities. The molecular
docking results emphasised the in vivo data and all docked
molecules were found to display very low binding constant
values in nanomolar scale.
Introduction
White blood cells are the main soldiers in our body and
they protect us from infection and foreign substances and
unwanted materials. This primary response of the body
to the pathogens and injury is referred as inflammation
(Kawai and Akira, 2006). Sometimes, however, the white
blood cells and their inflammatory chemicals cause damage
to the body’s tissues. In some diseases, the body’s defence
system inappropriately triggers an inflammatory response
when there are no foreign substances to fight off. Gener-
ally, non-steroidal anti-inflammatory drugs (NSAIDs) are
extensively used against such inflammatory responses. An
ideal anti-inflammatory drug should obstruct any type of
uncontrolled inflammatory stimulations and should not
interfere with normal defence action. In fact, the NSAIDs
inhibit the action of cyclooxygenase (COX-1 and COX-2)
enzymes (Chowdhury et al., 2009 and Puig et al., 2000),
which catalyses biosynthesis of prostaglandins (PGs).
However, the use of these NSAIDs may lead to several side
effects including gastric injury and that might later cause
gastric ulceration and renal injury (Navidpour et al., 2006
and Dannhardt and Kiefer, 2001). Therefore, there is an
immense need for new anti-inflammatory agents which do
not show such side effects. Further, most of the reported
NSAIDs act as anti-inflammatory as well as analgesic
agents. These agents produce their therapeutic effects by
inhibiting various prostaglandin substances involved in
development of pain, inflammation as well as regulation of
body temperature (Modi et al., 2012). Therefore, it is
desirable that new anti-inflammatory agents rather possess
analgesic property also.
Keywords Dihydropyridines ꢀ Amide ꢀ
Anti-inflammatory ꢀ Analgesic ꢀ Docking studies
S. Ulloora ꢀ A. V. Adhikari (&)
Department of Chemistry, National Institute of Technology
Karnataka, Surathkal, Mangalore 575 025, Karnataka, India
e-mail: avadhikari123@yahoo.co.in; avchem@nitk.ac.in;
avachem@gmail.com
S. Kumar
Department of Bio-informatics, Kuvempu University,
Shankaraghatta, Karnataka, India
Dihydropyridine (DHP) derivatives are an important
class of compounds in medicinal chemistry owing to their
wide range of biological applications. They are well-known
R. Shabaraya
Srinivas College of Pharmacy, Valachil, Mangalore,
Karnataka, India
123

Med Chem Res
calcium channel blockers. In addition, they display various
activities (Coburn et al., 1988), viz. antihypertensive (Jiang
et al., 1999 and Schleifer, 1999) hypnotic (Godfraid et al.,
1986), anticoagulant (Kumar et al., 2011), antihypoxic,
antiischemic (Khadilkar and Borkar, 1998), antitubercular
(Kharkar et al., 2002) and antimicrobial (Sirisha et al.,
2011) activities. In particular, several DHP derivatives
were also reported as potent anti-inflammatory agents
(Mishra and Mishra, 2007; Sadanandam et al., 1994 and
Komoda et al., 2010). Interestingly, DHPs were found to
bind adenosine receptors (A1, A2, A3) effectively in the
brain and bring about therapeutic effects successfully
(Ismail et al., 1995). In fact, in most of the cases, A3
adenosine receptor antagonists are being sought as poten-
tial anti-inflammatory agents (Beaven et al., 1994).
Amide is an important pharmacophoric linkage that is
shown to possess wide spectrum of biological activity.
Several amide derivatives were reported in the literature as
prominent anti-inflammatory agents (Raghavendra et al.,
2012; Galanakis et al., 2004; Takahashi and Miyazawa,
2012; Onkol et al., 2010 and Papadopoulou et al., 2005).
Moreover, they were shown to be powerful antimicrobial
(Padmavathi et al., 2011), antitubercular (Thomas et al.,
2011) as well as anticancer (Lu et al., 2010) agents. More
interestingly, amide group was found to increase selectivity
of anti-inflammatory agents towards COX-2 enzyme
(Kalgutkar et al., 2000). It was reported that conversion of
indomethacin, meclofenamic acid and ketoprofen to their
corresponding amides made them as selective COX-2
inhibitors.
explore their in vivo anti-inflammatory and analgesic
properties following standard methods, with the expecta-
tion that resulting molecules would display good pharma-
cological action. Accordingly, title compounds were
synthesized and characterized by spectral data. Later on,
they were subjected to in vivo anti-inflammatory and
analgesic activities by carrageenan-induced paw oedema
and tail immersion methods, respectively. Most of the
tested compounds exhibited prominent activity. Finally,
theoretical bioassay study was done using Molinspiration
server and found that PPAR-c is the most suitable receptor
for docking studies compared to other possible targets.
Hence, docking studies were undertaken for most active
agents to study their mode of interaction with PPAR-c and
thereby to get supportive coordination between in silico
and in vivo anti-inflammatory results. All compounds dis-
played good binding energy and low inhibition constants.
Compound 6d carrying substituted amino benzothiazole
emerged as the most active anti-inflammatory as well as
analgesic agent. The docking results are also compliment
the in vivo data.
Results and discussion
Chemistry
The required intermediates and final compounds 6a–10b
were synthesized according to Schemes 1 and 2, respec-
tively. The core ring, dihydropyridine was constructed
according to Hantzsch synthesis (Fassihi et al., 2009) by
refluxing 4-hydroxybenzaldehyde with two equivalents of
ethyl acetoacetate and ammonium acetate in ethanol. Then,
Keeping all the above points in view, it was contem-
plated to design and synthesize new series of dihydropyr-
idine derivatives (6a–10b) carrying amide functionality to
Scheme 1 Synthetic route of
intermediates
123

Med Chem Res
Scheme 2 Synthesis of target
compounds
O
O
Cl
CO2H
O
O
NHR
O
SOCl2, DCM
RT, 4 Hrs
COOEt
RNH2, DCM
Piperidine, RT, 8 hrs
EtOOC
EtOOC
COOEt
EtOOC
COOEt
N
H
N
H
N
H
[3]
[5]
[6a-6d]
SH
N
O
N
O
O
NH
O
O
NH
NH
NH2
R
O
O
RCOCl
CS2, KOH
DCM, RT, 8 Hrs
C2H5OH, Δ
EtOOC
COOEt
EtOOC
COOEt
EtOOC
COOEt
N
H
N
H
N
H
[8]
[7a-7e]
[4]
C2H5OH, H+
O
O
CH3COCH2COCH3
O
O
O
O
O
O
N
N
O
NH
O
O
O
NH
NH
N
O
CO2H
O
O
EtOOC
COOEt
COOEt
EtOOC
EtOOC
COOEt
N
H
N
H
N
H
[10b]
[10a]
[9]
the phenolic group was alkylated with ethyl chloroacetate
in DMF under nitrogen atmosphere to obtain the product 2.
Under this condition, only phenolic OH group, but not NH
group of DHP ring underwent alkylation. The alkaline
hydrolysis of ester 2 afforded the corresponding acid 3
which was later conveniently converted to acid chloride 5
and was coupled with various amines to obtain amides
6a–d in good yield. The ester 2 was transformed to its
hydrazide 4 by refluxing it with hydrazine hydrate in eth-
anol for about 4 h. At this reaction condition, the two ester
groups on DHP ring were found to be stable. Another set of
amides 7a–e were obtained by coupling hydrazide 4 with
different acid chlorides in the presence of piperidine base.
In another series, the intermediate 4 was cyclised to form
1,3,4-oxadiazole-2-thiol (8) by treating compound 4 with
carbon disulphide and potassium hydroxide followed by
subsequent neutralization. On the other hand, hydrazide 4
was condensed with acetyl acetone in alcoholic medium
containing trace of glacial acetic acid to obtain pyrazole
derivative 9. Finally, intermediate 4 was treated with suc-
cinic anhydride and phthalic anhydride under acidic med-
ium, to achieve 10a and 10b, respectively, in good yield.
Interestingly, reaction of 4 with succinic anhydride led to
ring opening reaction and resulted in an carboxylic acid
derivative 10a, while that with phthalic anhydride resulted
in substituted pthalimide derivative 10b.
Newly synthesized target compounds were character-
ized by FTIR, 1H NMR, 13C NMR and mass spectral
techniques followed by elemental analysis. Formation of
DHP ring was confirmed by FTIR spectrum of 1, where it
showed prominent peaks at 3337 and 1656 cm-1, due to
NH/OH and ester groups, respectively. This was also
1
confirmed by its H NMR spectrum, wherein it displayed
singlets at d 9.09 and d 8.77 ppm, which are attributed to
123

Med Chem Res
phenolic OH and NH protons of DHP ring, respectively. A
multiplet and a triplet at d 4.04 and d 1.15 ppm, respec-
tively correspond to two OCH2CH3 attached to DHP ring
were intern support the structure. Another characteristic
singlet at d 4.74 ppm was observed corresponding to C4
are summarized in the experimental section and their
characterization data are tabulated in Table 1.
Biological results
1
CH proton also support the proposed structure of 1. In H
The in vivo works have been performed in accordance with
the ethical standards on animal experimentations. From the
in vivo anti-inflammatory results, it is clear that newly syn-
thesized target compounds are reasonably active. It was
observed that the presence of electron donating groups on
aryl ring brought about enhancement in the activity. Also, the
presence of methyl group at para position of phenyl group in
compound 6c resulted in improved activity. Similar result
was observed for compound 7a which was more active than
un-substituted derivative 7b. Compound 6d carrying
2-amino benzothiazole group displayed the highest activity
when compared with standard drug, i.e. diclophenac. How-
ever, less activity was observed for compound 6b bearing
two methyl groups at 2nd and 6th positions of aryl ring. This
may be owing to steric hindrance offered by two methyl
groups adjacent to amide functionality of the molecule. As
the literature reveals, thiophene carboxamide derivatives are
good anti-inflammatory agents (Fakhr et al., 2009 and Hazra
et al., 2007). Similarly, in our study compounds 7c, 7d and 7e
bearing thiophene pharmacophore exhibited moderate-to-
good anti-inflammatory activity. In addition, un-substituted
thiophene derivative, 7d (43.95) displayed improved activity
when compared with those possessing halogen substituents
adjacent to carbonyl groups. Moreover, compound 8 con-
taining 1,3,4-oxadiazole-2-thiol showed good activity
(42.43) while compound 9 carrying substituted pyrazole
displayed moderate anti-inflammatory action (13.65). Fur-
thermore, compounds 6c (43.94), 6d (47.74) and 7d (43.95)
carrying substituted carboxamide groups, 8 (42.43), 10a
(34.85) and 10b (34.10) possessing 1,3,4-oxadiazole-2-thiol,
a carboxylic acid and phthalimide moieties, respectively
showed prominent activity and displayed rapid inhibition
just after injection of carrageenan irritant. Results of anal-
gesic activity screening indicated that almost all tested
compounds exhibited moderate activity. Particularly, sam-
ples 6d (7.23 0.02) and 7d (7.02 0.025) displayed the
highest activity in 60–90 min duration and their results were
comparable with standard drug diclophenac potassium. In
conclusion, compounds 6c, 6d, 7d, 8, 10a and 10b showed
enhanced activity both in anti-inflammatory and analgesic
studies with rapid onset of action when compared with the
standard. The anti-inflammatory results are tabulated in
Table 2. Analgesic data are given in Table 3.
NMR spectrum of compound 2, disappearance of OH peak
at d 9.09 ppm clearly established that phenolic hydroxyl
group was alkylated but not NH group of DHP ring. In
FTIR spectrum of 3, appearance of new broad peak at
3343 cm-1 and a shift in carbonyl stretching frequency
from 1739 to 1723 cm-1 clearly confirmed hydrolysis of
ester 2. This was supported by 1H NMR spectrum, wherein,
a quartet and a triplet peaks that correspond to ester group
was disappeared. The intermediate acid chloride 5 was
directly used for next step without purification, as it is
highly reactive. The formation of amides, viz. 6a was
confirmed by its FTIR spectrum, wherein a new peak at
1672 cm-1 corresponding to amide group appeared. This
1
was also confirmed by its H NMR spectrum, wherein a
new peak at d 9.31 ppm was observed.
Conversion of ester 2 to a key intermediate 4 was ver-
ified by FTIR spectrum of hydrazide 4, wherein, shifting of
carbonyl stretching frequency from 1739 cm-1 to lower
frequency 1656 cm-1 was observed. Also, its 1H NMR
spectrum displayed two singlets at d 9.24 and d 4.28 ppm
confirming the presence of hydrazidic NH and NH2 groups.
Formation of various amides 7a–e from hydrazide 4 was
1
proved by their H NMR spectra. A shift in peak from d
1
9.24 to d 10.19 ppm in H NMR spectrum of 7a that cor-
responds to amide NH proton, confirmed its structure.
Similar pattern of peaks were observed for remaining
amide derivatives of the series also. Conversion of hydra-
zide 4 to 1,3,4-oxadiazole 8 was evidenced by its FTIR and
1H NMR spectra, wherein carbonyl and amine stretching
peaks disappeared and new characteristic peak at d
14.65 ppm that corresponds to tautomeric CSNH proton of
1,3,4-oxadiazole ring appeared. Similarly, conversion of
hydrazide to pyrazole derivative 9 was validated by its
FTIR spectrum where peaks that correspond to hydrazide
group disappeared. Moreover, its 1H NMR spectrum
showed two new peaks at d 2.45 and 2.19 ppm which are
attributed to two allylic methyl groups attached to pyrazole
ring. In FTIR spectrum of 10a, a new broad peak at
3390 cm-1 and shift of carbonyl stretching frequency to
1718 cm-1 were observed, confirming the presence of
1
carboxylic group. A new peak at d 12.08 ppm in H NMR
spectrum confirms the ring opening of succinic anhydride.
Disappearance of NH2 peak at d 4.28 ppm in 10b clearly
established the conversion of hydrazide 4 to phthalimide
derivative 10b. Finally, structures of all the target com-
pounds were confirmed by their 13C NMR and mass
spectral data. The spectral data of all the title compounds
Molecular docking studies
The observed in vivo anti-inflammatory and analgesic
potency for the new dihydropyridine derivatives prompted
123

Med Chem Res
Table 1 Characterisation data of final compounds 6a–d, 7a–e, 8, 9, 10a–b
COOEt
HN
O
R
COOEt
Compound
R
Mol. formula
C28H32N2O6
Mol. weight (g)
492.5
M.P. (°C)
Yield (%)
82
6a
165
O
NH
O
6b
C29H34N2O6
506.6
176
78
HN
CH3
H3C
H3C
6c
6d
7a
C28H32N2O6
C28H29N3O6S
C29H33N3O7
492.5
535.6
535.6
116
98
84
80
76
NH
O
N
S
NH
O
119
O
HN
NH
O
7b
7c
7d
C28H31N3O7
521.5
562.0
527.5
145
132
148
78
80
82
O
HN
NH
O
C26H28ClN3O7S
O
NH
NH
O
Cl
S
C26H29N3O7S
H3C
N
O
S
CH3
N
7e
C26H28ClN3O7S
562.0
138
79
O
Cl
HN
O
NH
N
8
C22H25N3O6S
459.5
173
72
SH
N
O
123

Med Chem Res
Table 1 continued
Compound
R
Mol. formula
C26H31N3O6
Mol. weight (g)
481.5
M.P. (°C)
Yield (%)
78
9
182
H3C
N
O
O
CH3
CO2H
N
10a
C25H31N3O9
517.5
547.5
184
140
82
80
NH
NH
O
10b
C29H29N3O8
O
N
HN
O
O
Table 2 Anti-inflammatory activity data of final compounds
Table 3 Analgesic activity data of final compounds
Compound Tail flick latency in secs
Compounds
Percentage inhibition
0 min
30 min
60 min
90 min
‘ hr
1 h
2 h
3 h
Control
Standard
6a
3.35 0.028 3.23 0.005 3.34 0.005 3.25 0.005
3.23 0.017 6.25 0.02 6.95 0.02 7.45 0.02
3.17 0.017 4.56 0.008 5.73 0.020 6.8 0.017
3.25 0.028 4.92 0.005 6.1 0.057 6.9 0.028
3.24 0.003 5.15 0.028 5.91 0.023 7.12 0.012
3.23 0.033 5.28 0.005 6.03 0.018 7.23 0.020
3.25 0.005 4.99 0.101 6.11 0.063 6.92 0.014
3.20 0.020 4.62 0.005 5.87 0.014 6.37 0.004
3.16 0.013 4.34 0.034 5.67 0.002 6.45 0.034
3.23 0.003 4.92 0.014 6.23 0.024 7.02 0.025
3.27 0.026 4.85 0.004 5.74 0.014 6.46 0.004
3.21 0.003 4.97 0.031 6.07 0.004 6.97 0.017
3.27 0.027 4.76 0.004 5.93 0.032 6.78 0.012
3.18 0.015 4.96 0.027 6.07 0.018 6.87 0.023
3.29 0.032 5.03 0.026 6.16 0.003 7.21 0.002
Standard
6a
16.67
08.33
15.92
43.94
47.74
16.67
09.10
10.61
43.95
12.89
42.43
13.65
34.85
34.10
41.03
16.05
26.28
55.13
54.50
35.92
29.50
39.73
55.12
42.30
54.50
32.70
42.95
42.30
61.11
08.90
47.79
62.22
64.45
46.67
44.45
57.24
62.20
52.78
61.70
46.11
57.78
57.22
65.26
31.14
53.89
61.07
65.86
47.31
44.91
56.28
61.07
53.30
61.08
48.50
56.88
59.28
6b
6b
6c
6c
6d
6d
7a
7a
7b
7b
7c
7c
7d
7d
7e
7e
8
8
9
9
10a
10b
10a
10b
us to carry out molecular docking studies of selected active
molecules. Currently, computer-aided docking studies are
being widely used to predict binding orientations of new
drug candidates with protein-receptor sites (Lengauer and
Rarey, 1996). The in silico studies were done using
AutoDock 4.0 (Morris et al., 2009) software and results
were found to be in good correlation with in vivo data,
which established the anti-inflammatory and analgesic
efficacy of new target molecules.
biochemical assay (Lewis et al., 2011). Based on these
observations, the predictive bio-assay study was carried out
for the final compounds using MOLINSPIRATION server,
and it was found that new ligands are most likely to be
Nuclear Receptor Modulators rather than being classical
enzyme inhibitors (COX) or GPCR ligands. Among
nuclear receptors, peroxisome proliferator-activated
receptor gamma (PPAR-c or PPARG) protein is most
suitable receptor for nuclear-receptor activators (Grone-
meyer et al., 2004; Barros et al., 2010) and is having
diverse ligand binding capacity for anti-inflammatory
agents as they were shown to act by inhibiting cyclooxy-
genase (COX) activity (Desvergne and Wahli, 1995).
Hence, in the present study, PPAR-c protein was taken as
In recent days, the computer-aided bioassay method is
widely used to identify suitable receptors and interaction of
drugs with those targets (Sousa et al., 2006; Suresh et al.,
2011). Also, it has been established that computer-aided
results were found to compliment that of in vitro
123

Med Chem Res
˚
2.081 A, respectively. Also, it exhibited good binding
0
0
5
10
15
20
25
30
energy of -10.17 kcal/mol and binding constant of 34.92
nM. Ligand 6d exhibited one hydrogen bonding interaction
˚
with GLU343 (2.145 A) in its most suitable orientation and
-0.2
-0.4
-0.6
-0.8
-1
ICM
KI
showed the highest binding energy of -10.70 kcal/mol and
the lowest binding constant value (14.37 nM), which
clearly indicated its strong affinity towards receptor.
Compound 7d involved in three hydrogen bonding inter-
PI
EI
GPCR
NRL
˚
actions with SER342 (2.196 A), SER289 (1.902 A) and
˚
˚
ARG 288 (2.412 A) and displayed very good binding
-1.2
1.4
energy (-10.18 kcal/mol) as well as low inhibition con-
stant (34.36 nM). Similar kinds of results were obtained for
ligands 8 and 10b also. In general, compounds with CONH
groups exhibited good binding interactions with the pro-
tein-receptor than substituted cyclic systems. Particularly,
compound 6d bearing substituted 2-amino benzothiazole
ring displayed the highest binding affinity towards receptor
which is in good correlation with in vivo results. The
results of docking studies are summarised in Table 4 and
the ligand-receptor interaction of active compounds are
shown pictorially in Figs. 2 and 3.
Fig. 1 Graphical representation of computer-aided theoretical bio-
assay results. ICM ion channel modulators, KI kinase inhibitors,
PI protease inhibitors, EI enzyme inhibitors, GPCR G-protein cou-
pled receptor, NRL nuclear receptor ligand
receptor rather than COX-2 enzyme. The results of theo-
retical bio-assay study were pictorially represented in Fig. 1.
The computational work was carried out on windows7
platform with Intel Corei3 processor and 4 GB DD3 mem-
ory system. The required PPAR-c structure was recovered
from protein data bank (PDB ID: 3ADS). The LIGPLOT and
CASTp (Dundas et al., 2006) analyses were undertaken to
recognize any residues present in the active site.
Conclusions
The active site comprises of about fourteen amino acid
residues, viz. LEU330, LEU333, MET348, MET364,
LEU353, ILE341, GLU343, SER342, ILE281, PHE282,
CYS285, SER289, ILE326 and ARG288. Compounds 6c,
6d, 7d, 8 and 10b with prominent anti-inflammatory
activity were selected for docking study and they were
found to involve in strong hydrogen bonding interactions
with any of these amino acid residues. All the five ligands
displayed physical contacts through hydrogen bonding with
the active pocket residues. Specifically, at least one of the
two ester groups present on DHP ring was engaged in
hydrogen bonding with protein-receptor site. Beside the
ester group, phenoxy ether and carboxamide groups were
also found to participate in interactions with various amino
acids present in the active site. The cumulative interactions
of these groups of new target molecules with protein-
receptor are perhaps responsible for their good bio-activity.
The ligands exhibited very good binding energies
ranging from -9.63 to -10.70 kcal/mol. In addition, they
showed low inhibition constants in nanomolar scale. These
values are significant when compared to that of standard
drug (diclofenac sodium), which exhibited binding energy
of -6.6 kcal/mol and very high binding constant value
(14,320 nM). Diclofenac sodium exhibited two hydrogen
bond interactions with ARG288 and SER289 with a bond
In the present work, new DHP derivatives were succes-
sively synthesised and characterised by spectral techniques.
All the target molecules were subjected to in vivo anti-
inflammatory and analgesic screening. Their results indi-
cated that compound 6c, 6d, 7d, 8, 10b displayed rapid
onset of anti-inflammatory action and their results were
excellent when compared with standard drug diclophenac.
Also, molecular docking studies were carried out for the
highly active molecules and their results were in agreement
with in vivo data. The predictive bioactivity study revealed
that new target molecules were most likely to be nuclear
receptor modulators. They showed very good binding
energies and low binding constants in the nanomolar scale.
Particularly, compounds 6d and 7d comprising amide
Table 4 Binding energy and binding constants of the target
compounds
Ligands Binding Intermolecular Total
No of
internal hydrogen constant
Inhibition
energy
(kcal/
mol)
energy (kcal/
mol)
energy bonds
(kcal/
mol)
(nM)
6c
6d
7d
8
-10.17 -13.16
-10.7 -13.68
-0.71
-0.97
-2.15
-0.22
-1.99
2
1
3
2
3
34.92
14.37
34.36
67.21
86.61
-10.18 -13.76
-9.79 -10.38
-9.63 -12.32
˚
length of 1.835 and 1.883 A, respectively. Compound 6c
displayed two hydrogen bond interactions with MET364
and ARG288 residues with bond distance of 2.181 and
10b
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Med Chem Res
Fig. 2 Pictorial representation of ligand-receptor interactions of 6c
and 7d
Fig. 3 Representation of molecular interactions of 10b and diclofe-
nac with receptor
functional group displayed the highest anti-inflammatory
activity and low binding constant values. Also, they
exhibited very good analgesic property and hence emerged
as lead compounds. All docked molecules displayed high
binding energy and about thousand time low binding
constants when compared with standard diclophenac
sodium, which clearly proves the potency of the designed
target molecules. Thus, suitable modification of this DHP
structure may further enhance its activity and can be a good
scaffold for the development of newer bio-active materials.
Stuart SMP3 (BIBBY STERLIN Ltd. UK) apparatus and
were uncorrected. Infrared spectra were recorded on a
Nicolet Avatar 5700 FTIR (Thermo Electron Corporation).
1H NMR and 13C NMR spectra were obtained with Bruker-
400 MHz and 100 MHz FT-NMR spectrometer using TMS
as internal reference and DMSO-d6 as solvent. Elemental
analyses were performed on a Flash EA1112 CHNS ana-
lyzer (Thermo Electron Corporation). Mass spectra were
recorded on LC-MSD-Trap-XCT_Plus Mass Spectrometer.
Experimental
Procedure for synthesis of diethyl 4-(4-hydroxyphenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (1)
Chemistry
A mixture of 4-hydroxybenzaldehyde (2 g, 16.3 mmol)
ethyl acetoacetate (2.1 mL, 32.7 mmol) and ammonium
acetate (1.9 g, 24.5 mmol) in 20 mL of ethanol was
refluxed for 12 h. The reaction mixture was cooled on ice
bath and solid separated was filtered, washed with ethanol
and dried under vacuum. The product was recrystallized
using ethanol/DMF mixture. Yield 60 %, m.p. 240 °C.
FTIR (ATR, cm-1): 3337, 2980, 1656, 1221. 1H NMR
All the chemicals used in the present work procured from
Sigma Aldrich and Lanchaster (UK). All the solvents are of
analytical grade. They were purchased and used as such
without any further purification. The progress of the reac-
tion was monitored by thin layer chromatography, per-
formed on a Silica gel 60 F254 coated aluminium sheet.
Melting points were determined on open capillaries using a
123

Med Chem Res
(DMSO-d6, 400 MHz, d ppm): 9.09 (s, 1H), 8.77 (s, 1H),
6.94–6.91 (d, 2H, J = 12 Hz), 6.59–6.56 (d, 2H,
J = 12 Hz), 4.74 (s, 1H), 4.04–3.92 (m, 4H), 2.23 (s, 6H),
1.15–1.11 (t, 6H, 8.0 Hz). MS (m/z, %): 346.4. Anal.
Calcd. for C19H23NO5: C, 66.07; H, 6.71; N, 4.06. Found:
C, 66.01; H, 6.69; N, 4.05.
room temperature to get crude hydrazide 4. The resulting
solid was filtered, washed with ethanol and recrystallized
from ethanol/DMF mixture. Yield 90 %, m.p. 192 °C.
FTIR (ATR, cm-1): 3348, 3228, 3224, 3099, 2978, 1672,
1
1656, 1203. H NMR (DMSO-d6, 400 MHz, d ppm): 9.24
(s, 1H), 8.73 (s, 1H), 7.04–7.02 (d, 2H, J = 8.8 Hz),
6.77–6.75 (d, 2H, J = 8.8 Hz), 4.78 (s, 1H), 4.39 (s, 2H),
4.28 (s, 2H), 4.01–3.94 (m, 4H), 2.23 (s, 6H), 1.14–1.10 (t,
6H, J = 7.0 Hz). MS (m/z, %): 418.1. Anal. Calcd. for
C21H27N3O6. C, 60.42; H, 6.52; N, 10.07; O, 23.0. Found:
C, 60.34; H, 6.57; N, 10.02; O, 22.97.
Procedure for synthesis of diethyl 4-(4-(2-ethoxy-2-
oxoethoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (2)
To a clear solution of 1 (3 g, 8.7 mmol) in 30 mL of DMF,
ethyl chloroacetate (1.1 mL, 9.01 mmol) and K2CO3
(2.4 g, 17.3 mmol) were added with stirring. The reaction
mixture was heated at 80 °C for 20 h under nitrogen and
cooled to room temperature. It was then quenched to ice
cold water with stirring. Resulting solid was filtered,
washed with water and dried. This was recrystallized with
hot ethanol. Yield 80 %, m.p. 106 °C. FTIR (ATR, cm-1):
3351, 2979, 2905, 1739, 1686, 1221. 1H NMR (DMSO-d6,
400 MHz, d ppm): 8.73 (s, 1H), 7.04–7.02 (d, 2H,
J = 6.8 Hz), 6.74–6.72 (d, 2H, J = 6.8 Hz), 4.78 (s, 1H),
4.66 (s, 2H), 4.16–4.11 (m, 2H), 4.00–3.93 (m, 4H), 2.23
(s, 6H), 1.20–1.16 (t, 3H, J = 7.2 Hz), 1.13–1.10 (t, 3H,
J = 6.8 Hz). MS (m/z, %): 432.2. Anal. Calcd. for
C23H29NO7: C, 64.02; H, 6.77; N, 3.25; O, 25.96. Found:
C, 63.96; H, 6.74; N, 3.23; O, 25.91.
Procedure for synthesis of diethyl 4-(4-(2-chloro-2-
oxoethoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (5)
To a solution of compound 3 (1 g, 2.32 mmol) in methy-
lene dichloride (10 mL), thionyl chloride (0.35 mL,
4.64 mmol) was added followed by one drop of DMF. The
resulting solution was stirred at 50 °C for about 4 h.
The solvent was evaporated under reduced pressure, and
the resulting crude product was taken as such for next step
without any purification.
General procedure for synthesis of amides (6a–d)
The intermediate acid chloride 5 (0.1 g, 0.22 mmol) was
dissolved in 5 mL of methylene dichloride and reacted
with different amines (0.22 mmol) in the presence of
piperidine (0.02 mL, 0.22 mmol). This reaction mixture
was stirred at room temperature for 8 h. The precipitated
product was filtered, washed well with methylene dichlo-
ride and dried. The crude product was recrystalized from
ethanol-DMF mixture. Same procedure was followed for
the synthesis of 7a–e, wherein intermediate 4 was made use
instead of 5. Their characterization and spectral data are as
follows.
Procedure for the synthesis of 2-(4-(3,5-
bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-
4-yl)phenoxy)acetic acid (3)
To a solution of ester 2 (1 g, 2.32 mmol), in 10 mL of
ethanol, sodium hydroxide (0.1 g in 10 mL water) solution
was added. The resulting mixture was refluxed for one hr.
It was then cooled to room temperature and neutralized
with conc. HCl. The crude compound 3 was recrystallized
from ethanol-chloroform mixture. Yield 76 %, m.p.
177 °C. FTIR (ATR, cm-1): 3342(br), 2982, 2912, 1723,
1
1671, 1203. H NMR (DMSO-d6, 400 MHz, d ppm): 8.74
Diethyl 4-(4-(2-(benzylamino)-2-oxoethoxy)phenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (6a)
(s, 1H), 7.04–7.02 (d, 2H, J = 8 Hz), 6.73–6.71 (d, 2H,
J = 8 Hz), 4.78 (s, 1H), 4.58 (s, 2H), 4.00–3.97 (m, 4H),
2.24 (s, 6H), 1.15–1.11 (t, 3H, J = 7.2 Hz). MS (m/z, %):
404.4. Anal. Calcd. for C21H27NO7: C, 62.52; H, 6.25; N,
3.47; O, 27.76. Found: C, 62.46; H, 6.24; N, 3.42; O, 27.72.
Yield 82 %, m.p. 165 °C. FTIR (ATR, cm-1): 3392 (br),
2960, 2872, 1672, 1663, 1203. 1H NMR (DMSO-d6,
400 MHz, d ppm): 9.31 (s, 1H), 8.75 (s, 1H), 7.76–6.77 (m,
9H), 4.87 (s, 1H), 4.62 (s, 2H), 4.24 (s, 2H), 4.01–3.93 (m,
4H), 2.23 (s, 6H), 1.14–1.10 (t, 6H, J = 7.0 Hz). 13C NMR
(DMSO-d6, 100 MHz, d ppm): 169.2, 167.5, 157.3, 141.8,
142.1, 134.4, 126.8, 125,4, 116.0, 112.2, 68.7, 62.5, 43.3,
16.4, 14.3. MS (m/z, %): 493.6. Anal. Calcd. for
C28H32N2O6. C, 68.28; H, 6.55; N, 5.69; O, 19.49. Found:
C, 68.24; H, 6.53; N, 5.62; O, 19.46.
Procedure for synthesis of 2-(4-(3,5-bis(ethoxycarbonyl)-
2,6-dimethyl-1,4-dihydropyridin-4-yl)phenoxy) acetic acid
hydrazide (4)
To a clear solution of 2 (2.5 g, 5.8 mmol) in ethanol
(25 mL), hydrazine hydrate (0.5 mL, 10 mmol) was added,
and the mixture was refluxed for 4 h. It was then cooled to
123

Med Chem Res
Diethyl 4-(4-(2-(2,6-dimethylphenylamino)-2-
oxoethoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6b)
(s, 1H), 4.56 (s, 2H), 4.01–3.95 (m, 4H), 2.35 (s, 3H), 2.24
(s, 6H), 1.15–1.11 (t, 6H, J = 7.4 Hz). 13C NMR (DMSO-
d6, 100 MHz, d ppm): 167.2, 166.9, 165.3, 155.9, 145.0,
141.7, 129.6, 128.9, 127.4, 114.0, 112.0, 66.1, 58.9, 39.7,
20.9, 18.1, 14.1. MS (m/z, %): 536.6. Anal. Calcd. for
C29H33N3O7. C, 65.03; H, 6.21; N, 7.85; O, 20.91. Found:
C, 64.98; H, 6.19; N, 7.83; O, 20.88.
Yield 78 %, m.p. 176 °C. FTIR (ATR, cm-1): 3350, 3091,
2981, 2912, 1672, 1666, 1195. 1H NMR (DMSO-d6,
400 MHz, d ppm): 10.04 (s, 1H), 8.74 (s, 1H), 7.82–6.80
(m, 7H), 4.86 (s, 1H), 4.62 (s, 2H), 4.01–3.96 (m, 4H), 2.32
(s, 6H), 2.23 (s, 6H), 1.14–1.10 (t, 6H, J = 7.0 Hz). 13C
NMR (DMSO-d6, 100 MHz, d ppm): 169.2, 167.6, 157.4,
141.2, 137.3, 134.2, 129.8, 126.7, 124.2, 114.5, 113.1,
66.8, 61.4, 43.2, 16.3, 14.7, 14.2. MS (m/z, %): 507.4.
Anal. Calcd. for C29H34N2O6. C, 68.76; H, 6.76; N, 5.53;
O, 18.95. Found: C, 68.64; H, 6.73; N, 5.52; O, 18.89.
Diethyl 4-(N0-(benzoyl)-2-phenoxyacetohydrazide)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (7b)
Yield 78 %, m.p. 145 °C. FTIR (ATR, cm-1): 3345, 3234,
3056, 2979, 1689, 1645, 1210. 1H NMR (DMSO-d6,
400 MHz, d ppm): 10.36 (s, 1H), 10.16 (s, 1H), 8.74 (s, 1H),
7.95–6.83 (m, 9H), 4.80 (s, 1H), 4.57 (s, 2H), 4.01–3.96 (m,
4H), 2.24 (s, 6H), 1.15–1.11 (t, 6H, J = 7.2 Hz). 13C NMR
(DMSO-d6, 100 MHz, d ppm): 167.2, 166.9, 165.4, 155.9,
145.1, 141.5, 131.2, 129.6, 128.7, 127.3, 114.1, 112.2, 66.1,
58.7, 39.7, 18.1, 14.1. MS (m/z, %): 522.3. Anal. Calcd. for
C28H31N3O7. C, 64.48; H, 5.99; N, 8.06; O, 21.47. Found:
C, 64.39; H, 5.92; N, 8.02; O, 21.48.
Diethyl 4-(4-(2-(p-toluidino)-2-oxoethoxy)phenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (6c)
Yield 84 %, m.p. 116 °C. FTIR (ATR, cm-1): 3315, 3091,
2981, 2923, 1686, 1667, 1235. 1H NMR (DMSO-d6,
400 MHz, d ppm): 9.83 (s, 1H), 8.74 (s, 1H), 7.78–6.76 (m,
8H), 4.88 (s, 1H), 4.63 (s, 2H), 4.01–3.95 (m, 4H), 2.32 (s,
3H), 2.23 (s, 6H), 1.14–1.11 (t, 6H, J = 6.4 Hz). 13C NMR
(DMSO-d6, 100 MHz, d ppm): 169.3, 167.4, 158.1, 143.4,
138.3, 135.7, 129.7, 121.5, 114.3, 112.5, 66.8, 61.7, 43.2,
24.3, 16.4, 14.2. MS (m/z, %): 493.4. Anal. Calcd. for
C28H32N2O6. C, 68.28; H, 6.55; N, 5.69; O, 19.49. Found:
C, 68.14; H, 6.53; N, 5.62; O, 19.49.
Diethyl 4-(N0-(3-chloro thiophene-2-carbonyl)-2-
phenoxyacetohydrazide)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (7c)
Yield 80 %, m.p. 132 °C. FTIR (ATR, cm-1): 3342, 3238,
3017, 2965, 1664, 1645, 1212. 1H NMR (DMSO-d6,
400 MHz, d ppm): 10.24 (s, 1H), 10.18 (s, 1H), 8.74 (s,
1H), 7.95–6.85 (m, 6H), 4.79 (s, 1H), 4.58 (s, 2H),
4.01–3.95 (m, 4H), 2.24 (s, 6H), 1.15–1.11 (t, 6H,
J = 7.0 Hz). 13C NMR (DMSO-d6, 100 MHz, d ppm):
167.2, 166.6, 161.2, 155.8, 140.9, 138.1, 135.0, 129.8,
129.2, 114.1, 112.2, 67.1, 58.8, 40.0, 16.3, 14.2. MS (m/z,
%): 562.9. Anal. Calcd. for C26H28ClN3O7S. C, 55.56; H,
5.02; N, 7.48; O, 19.93. Found: C, 55.49; H, 5.02; N, 7.42;
O, 19.89.
Diethyl 4-(4-(2-(benzothiazol-2-ylamino)-2-
oxoethoxy)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (6d)
Yield 80 %, m.p. 98 °C. FTIR (ATR, cm-1): 3315, 3150,
3066, 2981, 1672, 1215. 1H NMR (DMSO-d6, 400 MHz, d
ppm): 11.12 (s, 1H), 8.75 (s, 1H), 7.78–6.68 (m, 8H), 4.84
(s, 1H), 4.62 (s, 2H), 4.00–3.96 (m, 4H), 2.23 (s, 6H),
1.14–1.10 (t, 6H, J = 7.2 Hz). 13C NMR (DMSO-d6,
100 MHz, d ppm): 176.2, 169.3, 167.2, 155.6, 150.2,
148.7, 134.6, 130.2, 125.6, 124.3, 122.3, 114.5, 112.4,
66.7, 61.7, 42.9, 16.3, 14.2. MS (m/z, %): 536.4. Anal.
Calcd. for C28H29N3O6S. C, 62.79; H, 5.46; N, 7.85; O,
17.92. Found: C, 62.64; H, 5.43; N, 7.82; O, 17.90.
Diethyl 4-(N0-(thiophene-2-carbonyl)-2-
phenoxyacetohydrazide)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (7d)
Yield 82 %, m.p. 148 °C. FTIR (ATR, cm-1): 3348, 3242,
3009, 2965, 1669, 1647, 1209. 1H NMR (DMSO-d6,
400 MHz, d ppm): 10.19 (s, 1H), 10.11 (s, 1H), 8.74 (s,
1H), 7.82–6.81 (m, 7H), 4.81 (s, 1H), 4.54 (s, 2H),
4.01–3.96 (m, 4H), 2.24 (s, 6H), 1.15–1.11 (t, 6H,
J = 7.2 Hz). 13C NMR (DMSO-d6, 100 MHz, d ppm):
167.2, 166.6, 161.2, 155.8, 142.8, 138.1, 137.4, 129.8,
129.2, 114.2, 112.3, 67.1, 58.8, 39.8, 16.3, 14.2. MS (m/z,
%): 528.9. Anal. Calcd. for C26H29N3O7S. C, 59.19; H,
5.54; N, 7.96; O, 21.23. Found: C, 59.08; H, 5.52; N, 7.91;
O, 21.19.
Diethyl 4-(N0-(4-methyl benzoyl)-2-
phenoxyacetohydrazide)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (7a)
Yield 76 %, m.p. 119 °C. FTIR (ATR, cm-1): 3346, 3226,
3056, 2982, 1674, 1648, 1212. 1H NMR (DMSO-d6,
400 MHz, d ppm): 10.19 (s, 2H), 8.74 (s, 1H), 7.78–7.76
(d, 2H, J = 8 Hz), 7.29–7.27 (d, 2H, J = 8 Hz), 7.06–7.04
(d, 2H, J = 8.4 Hz), 6.84–6.82 (d, 2H, J = 8.4 Hz), 4.80
123

Med Chem Res
Diethyl 4-(N0-(2-chloro thiophene-3-carbonyl)-2-
phenoxyacetohydrazide)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (7e)
J = 8.8 Hz), 6.78–6.76 (d, 2H, J = 8.8 Hz), 6.22 (s, 1H),
5.39 (s, 1H), 4.79 (s, 2H), 4.02–3.96 (m, 4H), 2.45 (s, 3H),
2.23 (s, 6H), 2.19 (s, 3H), 1.14–1.11 (t, 6H, J = 7.0 Hz).).
13C NMR (DMSO-d6, 100 MHz, d ppm): 168.1, 166.9,
155.9, 152.3, 145.0, 143.5, 141.0, 128.2, 113.8, 101.9,
65.8, 58.9, 39.5, 18.2, 14.1, 13.5. MS (m/z, %): 482.5.
Anal. Calcd. for C26H31N3O6. C, 64.85; H, 6.49; N, 8.73;
O, 19.94. Found: C, 64.80; H, 6.47; N, 8.74; O, 19.91.
Yield 79 %, m.p. 138 °C. FTIR (ATR, cm-1): 3332, 3213,
2997, 2946, 1665, 1643, 1202. 1H NMR (DMSO-d6,
400 MHz, d ppm): 10.23 (s, 1H), 10.14 (s, 1H), 8.74 (s,
1H), 7.80–6.84 (m, 6H), 4.82 (s, 1H), 4.54 (s, 2H),
4.00–3.95 (m, 4H), 2.24 (s, 6H), 1.15–1.11 (t, 6H,
J = 7.0 Hz). 13C NMR (DMSO-d6, 100 MHz, d ppm):
167.2, 166.4, 164.8, 156.2, 141.3, 139.4, 137.6, 134.5,
129.8, 128.7, 118.9, 114.3, 112.3, 67.3, 58.8, 40.2, 16.3,
14.2. MS (m/z, %): 563.2. Anal. Calcd. for
C26H28ClN3O7S. C, 55.56; H, 5.02; N, 7.48; O, 19.93.
Found: C, 55.48; H, 4.98; N, 7.45; O, 19.91.
Procedure for synthesis of 4-(2-(2-(4-(3,5-
bis(ethoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridin-4-
yl)phenoxy)acetyl)hydrazinyl)-4-oxobutanoic acid (10a)
A mixture of 4 (0.5 g, 1.21 mmol), succinic anhydride
(0.11 g, 1.21 mmol) in 10 mL of ethanol was refluxed in
the presence of 0.1 mL of glacial acetic acid for 6 h. After
complete reaction, pH of the reaction mixture was made
alkaline by adding sodium bicarbonate solution. The
product was extracted with ethyl acetate and aqueous layer
was neutralized with dil. HCl. Resulting solid was filtered
and recrystalised from ethanol. Yield 82 %, m.p. 184 °C.
FTIR (ATR, cm-1): 3390 (br), 2978, 1718, 1658, 1221. 1H
NMR (DMSO-d6, 400 MHz, d ppm): 12.08 (s, 1H), 9.99 (s,
1H), 9.81 (s, 1H), 8.73 (s, 1H), 7.04–7.02 (d, 2H,
J = 8.4 Hz), 6.79–6.77 (d, 2H, J = 8.4 Hz), 4.78 (s, 1H),
4.48 (s, 2H), 4.01–3.95 (m, 4H), 2.43–2.42 (t, 2H,
J = 3.2 Hz), 2.38–2.37 (t, 2H, J = 3.2 Hz), 2.23 (s, 6H),
1.14–1.11 (t, 6H, J = 7.2 Hz). 13C NMR (DMSO-d6,
100 MHz, d ppm): 167.8, 166.8, 164.7, 156.1, 145.2,
141.1, 135.8, 113.9, 102.3, 65.9, 63.9, 58.9, 38.9, 18.1,
14.1. MS (m/z, %): 518.2. Anal. Calcd. for C25H31N3O9. C,
58.02; H, 6.04; N, 8.12; O, 27.82. Found: C, 57.92; H, 6.02;
N, 8.09; O, 27.79.
Procedure for synthesis of diethyl 4-(4-((5-mercapto-1,3,4-
oxadiazol-2-yl)methoxy)phenyl)-2,6-dimethyl-1,4-
dihydropyridine-3,5-dicarboxylate (8)
An ethanolic KOH (0.1 g in 10 mL C2H5OH) solution was
prepared and hydrazide 4 (0.5 g, 1.21 mmol) was added to
it. The resulting solution was cooled to 5 °C and to this
carbon disulphide (0.2 mL, 2.42 mmol) was added while
stirring. This mixture was stirred at room temperature for
about one hour and then heated under reflux condition for
6 h. The resulting potassium salt was filtered and washed
with ethanol. The salt was dissolved in water and acidified
with dil. HCl under stirring. The resulting solid was filtered
and recrystallised from ethanol–chloroform mixture. Yield
72 %, m.p. 173 °C. FTIR (ATR, cm-1): 3334, 3082, 2933,
1662, 1492, 1213. 1H NMR (DMSO-d6, 400 MHz, d ppm):
14.65 (br, 1H), 8.74 (s, 1H), 7.07–7.05 (d, 2H, J = 8.8 Hz),
6.87–6.85 (d, 2H, J = 8.8 Hz), 5.15 (s, 1H), 4.79 (s, 2H),
4.00–3.94 (m, 4H), 2.23 (s, 6H), 1.13–1.09 (t, 6H,
J = 7.6 Hz). 13C NMR (DMSO-d6, 100 MHz, d ppm):
178.0, 166.9, 159.6, 155.3, 145.1, 141.9, 128.4, 114.1,
101.9, 59.5, 58.1, 38.9, 18.1, 14.1. MS (m/z, %): 460.3.
Anal. Calcd. for C22H25N3O6S. C, 57.50; H, 5.48; N, 9.14;
O, 20.89. Found: C, 57.48; H, 5.47; N, 9.14; O, 20.85.
Procedure for the synthesis of diethyl 4-(4-(2-(1,3-
dioxoisoindolin-2-ylamino)-2-oxoethoxy)phenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (10b)
A solution of 4 (0.5 g, 1.21 mmol) and phthaleic anhydride
(0.2 g, 1.35 mmol) in 10 mL of ethanol containing 0.2 mL
of glacial acetic acid was refluxed for 6 h with stirring.
Upon completion of reaction, the reaction mixture was
cooled, filtered and washed with ethanol. The solid product
was recrystallised from ethanol–chloroform mixture. Yield
80 %, m.p. 140 °C. FTIR (ATR, cm-1): 3224, 2981, 1668,
Procedure for the synthesis of diethyl 4-(4-(2-(3,5-
dimethyl-1H-pyrazol-1-yl)-2-oxoethoxy)phenyl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (9)
To the ethanolic solution of 4 (0.5 g, 1.21 mmol), acetyl
acetone (0.43 mL, 1.21 mmol) was added followed by
addition of catalytic amount (0.2 mL) of glacial acetic acid
and the resulting solution was refluxed for 5 h. It was
cooled, filtered and recrystallised from ethanol–chloroform
mixture. Yield 78 %, m.p. 182 °C. FTIR (ATR, cm-1):
3326, 2996, 2934, 1665, 1214. 1H NMR (DMSO-d6,
400 MHz, d ppm): 8.73 (s, 1H), 7.04–7.02 (d, 2H,
1
1222. H NMR (DMSO-d6, 400 MHz, d ppm): 10.24 (s,
1H), 8.73 (s, 1H), 7.78–6.80 (s, 8H), 4.79 (s, 1H), 4.55 (s,
2H), 4.00–3.96 (m, 4H), 2.23 (s, 6H), 1.15–1.11 (t, 6H,
J = 7.0 Hz). 13C NMR (DMSO-d6, 100 MHz, d ppm):
167.8, 166.9, 164.9, 156.0, 145.0, 141.1, 135.6, 129.8,
123.8, 113.9, 102.3, 65.9, 63.9, 58.9, 38.9, 18.1, 14.1. MS
(m/z, %): 548.5. Anal. Calcd. for C29H29N3O8. C, 63.61; H,
123

Med Chem Res
Molecular docking studies
5.34; N, 7.67; O, 23.38. Found: C, 63.57; H, 5.32; N, 7.63;
O, 23.39.
The molecular docking studies were performed by using
AutoDock version 4.0 software (Huey et al., 2007). The
ligands were analysed for predictive bioactivity study by
using MOLINSPIRATION server and they were found to
be nuclear receptor modulator.
Anti-inflammatory study
Newly synthesized title compounds were screened for their
in vivo anti-inflammatory properties following Carra-
geenan-induced Paw Oedema method (Yoshimasa et al.,
1985) by taking Diclofenac sodium as standard drug. Male
or female Wistar strain rats with a body weight between
100 and 150 g were used. The animals were weighed and
divided into different groups (control, standard and the test
groups) of five rats each. The animals were starved over-
night. To ensure uniform hydration, the rats received 5 ml
of water by stomach tube (controls) or the test drug
(20 mg/kg) dissolved or suspended in the same volume.
Thirty minutes later, the rats were given a subcutaneous
injection of 0.05 ml of 1 % solution of Carrageenan into
the plantar side of the left hind paw. The paw was marked
with ink at the level of the lateral malleolus and immersed
in mercury up to this mark. The paw volume was measured
plethysmographically immediately after injection, after 30,
60, 120 and 180 min. Results were expressed as mean, by
one way ANOVA analysis followed by Dunnet’s-t test and
are summarised in Table 2. The in vivo work was per-
formed in accordance with the ethical standards on animal
experimentations.
Peroxisome Proliferator- Activated Receptor (PPAR-c)
was used as receptor for our docking studies. Ligand bound
crystallographic structure of protein receptors are available
in Protein Data Bank (Berman et al., 2000). Since indo-
methacin is a well-known agonist of PPAR-c receptor, in
our study, the crystal structure of indomethacin coupled
PPAR was taken, and structure of indomethacin was
replaced by synthesized ligands for docking studies. LIG-
PLOT analysis was done to identify the residues present in
the ligand-binding site. Additional residues were identified
by CASTp analysis.
The ligands were drawn in Chem Draw Ultra 6.0
assigned with proper 2D orientation. The correct atom
types and bond types were defined and hydrogen atoms
were added. The lowest energy conformer of the molecule
was selected by using PRODRG software (Schuttelkopf
and van Aalten, 2004). This structure was selected and
taken as input ligand structure in AutoDock, to carry out
simulation studies.
The receptor was cleaned from any possible ligands
before the docking studies. Receptor was modified for the
docking studies by incorporating united atom charges,
solvation parameters and polar hydrogens. Q site finder
server was used to predict ligand-binding site. In addition,
CASTp server was used to identify active site and to
measure its cavity size. The auto grid program available in
AutoDock server was used to generate grid maps. For each
ligand, a best conformer having higher binding energy,
minimum binding constant and maximum number of
hydrogen bonding interactions was chosen. Finally, dock-
ing runs were performed and schematic diagram of pro-
tein–ligand interactions was generated in LIGPLOT server.
Analgesic study
The analgesic study of active target compounds was done
by Tail immersion method (Stasi et al., 1988) by taking
Diclofenac potassium as standard drug. The animals were
weighed and divided into different groups (control, stan-
dard and the test groups) of five rats each. Young female
Wistar rats (170–210 g body weight) were used for the
study. They were placed into individual restraining cages
leaving the tail hanging out freely. The animals were
allowed to adapt to the cages for 30 min before testing. The
lower 5 cm portion of the tail was marked. This part of the
tail was immersed in a cup of freshly filled water of exactly
55 °C. Within a few seconds, the rat reacts by withdrawing
the tail. The reaction time was recorded in 0.5 s units by a
stopwatch. After each determination, the tail was carefully
dried. The reaction time was determined before and peri-
odically after either oral or subcutaneous administration of
the test substance, e.g. after 0, 30, 60 and 90 min. The cut
off time of the immersion was 15 s. The withdrawal time
of untreated animals was between 1 and 5.5 s. A with-
drawal time of more than 6 s therefore was regarded as a
positive response. The analgesic results are tabulated in
Table 3.
Acknowledgments The authors are thankful to NITK, Surathkal for
financial support and IISc, Bangalore for spectral facilities. Also, we
thank Syed Amir, Srinivas College of Pharmacy Mangalore for
helping to carry out biological screening studies.
Conflict of interest The authors declare that they have no conflict
of interest.
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