Syntheses, characterization and evaluation of novel 2,6-diarylpiperidin-4- ones as potential analgesic-antipyretic agents

Article abstract of DOI:10.1016/j.ejmech.2014.05.080

A novel series of N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-one derivatives (1c-3c and 5c) were synthesized, via base catalyzed nucleophilic substitution of N-chloroacetyl-2,6-diarylpiperidin-4-ones (1b-6b) with N-methyl piperazine. The newly synthesized compounds were characterized by FTIR, Mass and NMR spectral studies. All the compounds were screened for their possible analgesic and antipyretic activities. The compound 2c exhibited promising antipyretic activity, comparable to that of paracetamol at 60 mg/kg dose. The compounds 2b and 2c showed significant analgesic profile at a dose of 60 mg/kg and were also found to be more potent than the reference drug, diclofenac sodium. Thus, it can be concluded that the synthesized 2,6-diarylpiperidin-4- ones exhibit promising antipyretic and analgesic activities and could be potential drug candidates.

Full text of DOI:10.1016/j.ejmech.2014.05.080

European Journal of Medicinal Chemistry 82 (2014) 439e448
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Syntheses, characterization and evaluation of novel 2,6-
diarylpiperidin-4-ones as potential analgesic-antipyretic agents
Purnima Tripathi, Avinash C. Tripathi, Viney Chawla, Shailendra K. Saraf*
Division of Pharmaceutical Chemistry, Faculty of Pharmacy, Babu Banarasi Das Northern India Institute of Technology, Lucknow 226028, Uttar Pradesh,
India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-one derivatives (1ce3c and 5c)
were synthesized, via base catalyzed nucleophilic substitution of N-chloroacetyl-2,6-diarylpiperidin-4-
ones (1be6b) with N-methyl piperazine. The newly synthesized compounds were characterized by
FTIR, Mass and NMR spectral studies. All the compounds were screened for their possible analgesic and
antipyretic activities. The compound 2c exhibited promising antipyretic activity, comparable to that of
paracetamol at 60 mg/kg dose. The compounds 2b and 2c showed significant analgesic profile at a dose
of 60 mg/kg and were also found to be more potent than the reference drug, diclofenac sodium. Thus, it
can be concluded that the synthesized 2,6-diarylpiperidin-4-ones exhibit promising antipyretic and
analgesic activities and could be potential drug candidates.
Received 8 June 2013
Received in revised form
30 May 2014
Accepted 31 May 2014
Available online 2 June 2014
Keywords:
2,6-Diarylpiperidin-4-one
Antipyretic
© 2014 Elsevier Masson SAS. All rights reserved.
Analgesic
Piperidin-4-one
Mannich reaction
N-methyl piperazine
1. Introduction
Bradykinin, released from plasma kininogen and cytokines, such as
TNF-a, IL-1 and IL-8 appear to be particularly important in eliciting
Inflammation is a normal and essential response to any noxious
stimulus that threatens the host and may vary from a localized
response to a generalized response [1]. Generally, this response acts
to protect the host but at times it may lead to a spectrum of in-
flammatory diseases, thus necessitating medication to dampen or
abolish the inflammatory response [2].
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely
used to treat the signs and symptoms of inflammation and pain.
NSAIDs exert their effect mainly through the inhibition of cyclo-
oxygenase (COX), the key enzyme in prostaglandin (PG) biosyn-
thesis from arachidonic acid (AA). The potentiality of NSAIDs to
alleviate pain, inflammation and fever, coupled with a number of
pathological conditions, makes them one of the most useful ther-
apeutic agents [3].
NSAIDs are particularly effective when inflammation has caused
sensitization of pain receptors to normally painless mechanical or
chemical stimuli. Pain, that accompanies inflammation and tissue
injury, probably results from local stimulation of pain fibers and
enhanced pain sensitivity (hyperalgesia), in part a consequence of
increased excitability of central neurons in the spinal cord.
the pain related to inflammation. These agents liberate prosta-
glandins and probably other mediators that promote hyperalgesia.
Neuropeptides, such as substance-P, calcitonin gene-related pep-
tide (CGRP) may also be involved in eliciting pain. Non-opoid an-
algesics act by inhibition of cyclooxygenase (COX) enzyme, and
specially COX-2 which plays a key role in the production of in-
flammatory mediators like prostaglandins and prostacyclins from
cyclic endoperoxides. Prostaglandin PGE2 and prostacyclin PGI2
sensitize the pain receptors in various organs. Thus, stimuli which
are normally painless are able to elicit pain. Some other mediators
like bradykinin and cytokines, i.e. tissue necrosis factor (TNF-a),
interleukins (IL-1, IL-8) stimulate the COX enzyme and thus pro-
duce prostaglandins which act in mediating hyperalgesia [4].
Fever is a complex physiologic response triggered by infectious
or aseptic stimuli, due to increased concentrations of prostaglandin
E(2) (PGE(2)) within certain areas of the brain. These elevations
alter the firing rate of neurons that control thermoregulation in the
hypothalamus. Antipyretics, such as aspirin, have been widely used
since the late 19th century but the mechanisms by which they
relieve fever have only been characterized in the last few decades. It
is now clear that most antipyretics work by inhibiting the enzyme
cyclooxygenase and reducing the levels of PGE(2) within the hy-
pothalamus. Recently, other mechanisms of action for antipyretic
* Corresponding author.
E-mail addresses: dirpharmniec@gmail.com, sarafs@sify.com (S.K. Saraf).
http://dx.doi.org/10.1016/j.ejmech.2014.05.080
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

440
P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
Table 1
drugs have been suggested, including their ability to reduce
proinflammatory mediators, enhance anti-inflammatory signals at
sites of injury, or boost antipyretic messages within the brain.
Analysis of recent data suggests that multiple pathways may be
involved in the induction of fever by cytokines, such as local cyto-
kine production leading to signaling through vagal fibers, release of
cytokine-induced circulating mediators at the tissue level, the use
of membrane-bound cytokines as mediators, or the local release of
cytokines in the hypothalamus by circulating activated monocytes.
A multi pathway mechanism for the induction of fever is therefore
suggested [5].
Structure of compounds (1e10).
S. No.
Compound code
Structure
1
1b
Traditional NSAIDs such as aspirin, diclofenac, flurbiprofen and
ibuprofen are non-selective in their action. Therefore, chronic use
of NSAIDs may elicit appreciable gastrointestinal (GI) irritation,
bleeding and ulceration. The incidence of clinically significant GI
side effects is high (over 30%) and causes some patients to abandon
NSAID therapy. Thus, the discovery of COX-2 provided the impetus
for the development of drugs devoid of GI disorders while retaining
clinical efficacy as anti-inflammatory agents. However, some re-
ports showed that selective COX-2 inhibitors (coxibs) could lead to
adverse cardiovascular effects. Therefore, development of novel
compounds having anti-inflammatory and analgesic activity with
improved safety profile is still a necessity. Synthetic approaches
based upon chemical modification of existing NSAIDs have been
undertaken with an aim to improve their safety profile [6]. Also,
agents with new mechanisms of action are in great demand.
Microsomal PGE synthase-1 (mPGES-1), an inducible enzyme for
the production of pro-inflammatory PGE2 from PGH2, is also
upregulated in rheumatoid arthritis and osteoarthritis. The mPGES-
1 is a critically important mediator of inflammation, pain, angio-
genesis, fever, bone metabolism, tumorigenesis, atherosclerosis,
and reproduction. It presents an attractive target to achieve more
specific inhibition of PGE2 production while preserving production
of other PGs and, as such, has attracted considerable attention as a
“next-generation” anti-inflammatory drug [7]. All this laid the
foundation for the search and design of new chemical agents,
which are devoid of all the limitations and side effects of the drugs
available in the market. Hence, there is an urgent need for mono
therapy, with a biologically potent candidate, endowed with anal-
gesic and antipyretic activities together by keeping in view the
pharmaco-economic and frequent patient compliance. While
exploring for such a compound, it was observed that piperidone
nucleus is an integral component of countless alkaloids with
marked biological properties [8e15] such as antiviral [16], anti-
tumor [17], anti-inflammatory [18], central nervous system active
[19e24], local anesthetic [25], anticancer [26] and antimicrobial
activity [27]. Moreover, derivatives of piperidines are also biologi-
cally important and act as neurokinin receptor antagonists [28],
analgesic and antihypertensive agents [29e31]. Also, substituted
piperidin-4-ones are important synthetic intermediates for the
preparation of various alkaloids and pharmaceuticals. The utility of
substituents at second, third and sixth positions, particularly aro-
matic substituent at second and/or sixth positions with regard to its
biological activity has been well documented [32].
2
1c
3
2b
4
2c
5
3b
1,3-Dimethyl-2,6-diphenyl-4-piperidone has been found to be a
versatile intermediate in different types of reactions since it has
two reactive sites, carbonyl and a keto methylene group. This paved
the way for the synthesis of some heterocyclic compounds such as
tetrahydropyridine, diazepanone, oxazepanone, piperidone, pyr-
idopyrimidone, pyridopyrimidinethione, thiazolopyridine, fur-
anylmethylene and pyridoindole [33].
Generally, all piperidin-4-ones are synthesized by condensing
1 mol of ketone and 2 mol of aldehydes with 1 mol of ammonium
acetate, in the presence of ethanol [34]. Noller et al. synthesized a
number of 4-piperidones with different substituents on the 1,2,3,5
6
3c

P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
441
Table 1 (continued )
S. No. Compound code
Mannich reaction by condensing suitably substituted aromatic al-
dehydes, ketone and ammonium acetate in equimolar ratio, using
ethanol as the solvent (Scheme 1).
Structure
In view of the better basicity and HCl scavenging power of
triethylamine (NEt3), it was used in the nucleophilic reaction of
1ae6a with chloroacetyl chloride. The yields of N-chloroacetyl-2,6-
diarylpiperidin-4-ones were significantly better while using NEt3.
Further, nucleophilic substitution of N-methyl piperazine with
these chloroacetyl derivatives was performed in different solvents,
such as ethanol and toluene, using NEt3 again as a base. Toluene
was chosen as the best solvent for most of the derivatives as it
improved the yield of the title compounds (1b, 1c, 2b, 2c, 3b, 3c, 5a
and 5c) appreciably when compared to ethanol. For hydroxy de-
rivatives, chloroform was used as the solvent instead of toluene.
Increasing the non-polar environment led to easy nucleophilic
substitution of these derivatives (4b and 6b). The possible mecha-
nism of the reaction is outlined in Scheme 2.
7
4b
8
5b
The FT-IR analysis of the compounds confirmed the absence of
NH stretching in piperazine and changes in the magnitude of amide
carbonyl stretching at around 1652e1660 cmꢀ1 of 1be6b (for 1c,
2c, 3c and 5c, it appeared at around 1643 cmꢀ1), besides the
appearance of additional aliphatic stretching frequencies in the
region 2981e2694 cmꢀ1
.
3. Results and discussion
9
5c
The synthesized compounds were subjected to in-vitro studies
to predict the log P value and hydrolytic profile of the compounds.
3.1. Partition coefficient
Lipophilicity is represented by the descriptors log P (also known
as Kow or Pow) and is used to predict in-vivo permeability of active
compounds in drug discovery.
10
6b
Partition coefficient of compound 2c, having 2-(p-chlorophenyl)
and 6-(p-fluorophenyl) functionalities with methyl group at C-3
and N-methyl piperazine ring- was found to be 2.09. Thus 2c was
found to be most lipophilic compound of the series, having Log P
value of 2.09, followed by compound 2b (having 2-(p-chlor-
ophenyl), 6-(p-fluorophenyl) functionality with methyl at C-3 and
N-chloroacetyl moiety), with log P value of 1.72. Compound 6b,
having 2-(p-hydroxyphenyl), 6-(p-fluorophenyl) functionalities
with a methyl group at C-3 and N-chloroacetyl moiety was the least
lipophilic compound with a log P value of 1.14. Partition coefficient
data are given in Table 2.
and 6 positions. Glacial acetic acid was used instead of ethanol in
their procedure [35].
Piperazine is an interesting heterocyclic moiety as it is a con-
stituent of several biologically active molecules. The polar nitrogen
atoms, opposing each other in the piperazine ring, confer bioac-
tivity to the molecules and elicit favorable interactions with mac-
romolecules [36,37]. Slight change in the substitution pattern in
piperazine nucleus causes distinguishable change in the pharma-
cological activities. Compounds with piperazine and its N-
substituted derivatives, i.e. N-methyl, N-ethyl piperazines, as an
integral part, are the most successfully employed side chains since
they exhibit a varied range of activities such as antimicrobial
[38,39], anticancer [40], anti-inflammatory [41], antipsychotic [42],
CNS active [43], antagonists for CCKB(Cholecystokinin B)/gastrin
receptor [44], melanocortin-4-receptor [45] and in the treatment of
Alzheimer's disease [46,47].
3.2. In-vitro hydrolysis
Stability in gastric juice is of prime importance for the drugs
intended for oral administration. It is observed that many drugs
have low bioavailability as they are degraded in the stomach, due to
low pH (1e2). Simulated gastric fluid (SGF) mimics the gastric fluid
in terms of acidity and molarity and simulated intestinal fluid (SIF)
mimics the intestinal fluid in terms of basicity. These fluids are the
perfect media to determine the stability of drug candidates in vitro.
In the present study, synthesized compounds were tested in vitro
using SGF and SIF.
Results of in vitro hydrolysis (Table 3) showed that compound
1c, having 6-(p-chlorophenyl), 2-(p-dimethylaminophenyl) func-
tionalities with a methyl group at C-3 and N-methyl piperazine ring
was the most hydrolyzed with a hydrolysis of 31.70%. Compound
5c, having 6-(p-chlorophenyl), 2-(p-methoxyphenyl) functional-
ities with a methyl group at C-3 and N-methyl piperazine ring was
the least hydrolyzed with a hydrolysis of 1.57% in simulated gastric
In the present work, incorporation of both the moieties, i.e.
piperidin-4-one and secondary amine i.e. N-methyl piperazine, was
carried out in a Mannich base in anticipation of enhanced analgesic
and antipyretic activities.
2. Chemistry
By adopting the literature precedent [34,47] 2,6-diarylpiperidin-
4-ones (Table 1) were prepared in a one pot multi-component

442
P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
Scheme 1. The synthetic pathway to the title compounds (1b-6b, 1ce3c and 5c).
fluid. This indicates their stability in stomach and subsequent
bioavailability.
Compound 1c, having 6-(p-chlorophenyl), 2-(p-dimethylami-
nophenyl) functionalities with a methyl group at C-3 and N-methyl
piperazine ring was the most hydrolyzed with a hydrolysis of
77.60%. Compound 2b, having 2-(p-chlorophenyl), 6-(p-fluo-
rophenyl) functionalities with a methyl group at C-3 and N-chlor-
oacetyl moiety was the least hydrolyzed with a hydrolysis of 1.17%
Scheme 2. The possible mechanism of the reaction.

P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
443
Table 2
Partition coefficient of the synthesized compounds.
Comp. lmax in octanol (nm) Plotting for standard curve in
mg/ml Abs. of octanol phase after 24 h (y) Conc. in octanol (x) (
mg/ml) Conc. in water (mg/ml) Log P
4
8
12
16
20
1b
1c
2b
2c
3b
3c
4b
5b
5c
6b
338.0
336.5
273.0
282.5
280.5
284.5
303.5
283.0
288.5
340.0
0.102 0.150 0.197 0.248 0.293 0.281
0.213 0.368 0.532 0.691 0.852 0.816
0.022 0.051 0.087 0.119 0.148 0.145
0.145 0.195 0.243 0.294 0.344 0.341
0.014 0.033 0.053 0.071 0.092 0.086
0.020 0.041 0.062 0.086 0.108 0.105
0.023 0.052 0.082 0.109 0.139 0.127
0.046 0.075 0.101 0.127 0.152 0.147
0.045 0.087 0.132 0.180 0.219 0.213
0.062 0.093 0.123 0.150 0.179 0.170
18.92
19.13
19.43
19.84
19.27
19.63
18.37
19.03
19.36
18.66
1.08
0.87
0.57
0.16
0.73
0.37
1.63
0.97
0.64
1.34
1.24
1.35
1.72
2.09
1.42
1.53
1.05
1.29
1.48
1.14
in simulated intestinal fluid. This demonstrates their stability and
bioavailability through the intestine.
Therefore, from this study the importance of the structural units
such as fluorine, chlorine, methoxy and hydroxy functions at the
para position of the phenyl groups has been established for the
antinociceptive profile associated with these compounds.
Furthermore, in the acetic acid-induced writhing test, the index
of pain is evaluated through the nociceptive behavior characterized
by a motor activity (writhing). The drugs that decrease or impair
the motor activity may reduce the acetic acid-induced writhing,
without producing antinociception [4]. Therefore, the effect of the
synthesized derivatives on motor coordination of the animals was
assessed, using the rota rod test. It was evident from the study that
none of the synthesized derivatives were involved in altering the
motor coordination, as the mean fall-off time of the animals were
found to be more than 180 s. The animals treated with Diazepam
(positive control), at a dose of 2 mg/kg body weight, failed to
maintain the motor coordination in 60 s. Therefore, it may be
concluded from the rota rod study that the antinociceptive effect of
the synthesized derivatives was not associated with motor
impairment [51].
3.3. Analgesic activity
The analgesic activity of the compounds was determined by
acetic acid-induced writhing in mice, using diclofenac sodium as
the standard drug. Painful reactions in animals may be produced by
chemicals also. Intraperitoneal injection of phenylquinone, brady-
kinin or acetic acid produces pain reaction which is characterized as
a writhing response. The visceral pain induced by acetic acid is due
to the release of prostaglandins (PG) via arachidonic acid pathway
in the presence of cyclooxygenase enzyme. Constriction of
abdomen, turning of trunk (twist) and extension of hind legs are
taken as the reactions to chemically induced pain [4]. The test
compounds, when administered orally (p.o.) at 60 mg/kg (body
weight) 1 h prior to the administration of acetic acid, showed a
significant reduction in the writhing movement. Analgesic profiles
of the compounds are given in Table 4.
It is evident from Table 4 that analgesic potency estimated by
classical acetic acid-induced constriction for certain compounds is
comparatively equal or highly related to the reference compound at
the test dose. Introduction of chloro/fluoro functionality at the para
position of phenyl framework in compound 2b and 2c produced
statistically significant antinociceptic activity. Compound 2b dis-
played almost equipotent analgesic activity, having a percent in-
3.4. Antipyretic activity
The antipyretic activity of the compounds was determined by
Brewer's yeast-induced hyperthermia in rats, using paracetamol as
the standard drug. Hyperthermia was induced in rats by subcu-
taneous administration of Brewer's yeast in normal saline. This
caused an increased production of prostaglandins in the hypo-
thalamus and thus the thermoregulation process was affected [5].
Pretreatment with test compounds at 60 mg/kg dose showed a
better antipyretic activity relative to the standard drug at different
time intervals. Antipyretic effect of the compounds may be due to
the inhibition of prostaglandin synthesis in the central nervous
system, thereby reducing the rectal temperature. The observations
are recorded in Table 5.
The compounds with para substituted phenyl groups at C-2 and
C-6 positions of the piperidone ring system were found to be
equipotent to paracetamol in antipyretic activity, in the same
pharmacological protocol. Compound 2c, with para-chloro/fluoro
phenyl functionality (with methyl group at C-3), displayed most
potent antipyretic activity similar to that of the standard drug
(paracetamol) in 1 h, 2 h and 3 h, with rectal temperatures of
37.900 0.072, 37.160 0.075 and 37.180 0.080 respectively, in
comparison to the standard drug paracetamol, showing rectal
temperatures of 37.880 0.073, 37.140 0.051 and 37.100 0.055
in 1 h, 2 h and 3 h respectively. Further, this compound showed
similar onset of hypothermic effect as that of paracetamol imme-
diately after 60 min, which lasted over a period of 150 min. Com-
pound 3c, with chlorophenyl functionality replaced with para-
methoxyphenyl (with methyl group at C-3), also exhibited
hibition of 57.637
1.885, in comparison to the standard drug
diclofenac, having a percent inhibition of 56.515 2.029. Further,
compound 2b was substituted with N-methyl piperazine to pro-
duce compound 2c. Significantly, introduction of N-methyl piper-
azine framework in compound 2b showed excellent analgesic
activity with a percent inhibition of 65.487 1.711. This expressive
enhancement of the analgesic activity indicates the pharmaco-
phoric character of para chloro/fluoro phenyl functionality and N-
methyl piperazine framework for the analgesic profile in this new
series of compounds.
Replacement of one chloro/fluoro phenyl functionality in com-
pounds 3b, 3c, 4b, 5b, 5c and 6b by methoxy/hydroxy phenyl
functionality also resulted in diminished analgesic potency, with
percent inhibitions of 51.184 1.109, 55.199 1.528, 39.715 2.113,
44.126
1.365, 53.049
1.250 and 51.178
1.156 respectively.
However, replacement of one chloro/fluoro phenyl functionality in
compounds 1b and 1c by para dimethylaminophenyl group resul-
ted in poor analgesic potency, with percent inhibitions of
17.794 2.028 and 28.101 1.636. In all the cases, the para halogen
substituted phenyl bearing compounds displayed improved inhi-
bition potency as compared to their analogues, the para dimethy-
laminophenyl bearing compounds.

444
P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
Table 3
Data of In-vitro hydrolysis.
Compound
SGF
lmax
0 min
15 min
30 min
45 min
60 min
75 min
90 min
105 min
120 min
% Hydrolyzed
1b
1c
2b
2c
3b
3c
4b
5b
5c
6b
349.6
344.8
350.0
342.5
272.0
283.5
288.8
290.5
272.5
274.5
321.2
322.6
273.5
284.0
292.0
286.5
284.0
290.5
271.5
282.0
0.289
0.466
0.451
0.375
0.091
0.428
0.189
0.178
0.194
0.398
0.158
0.398
0.087
0.440
0.068
0.298
0.255
0.032
0.092
0.412
0.289
0.281
0.353
0.219
0.090
0.426
0.188
0.176
0.193
0.395
0.156
0.397
0.085
0.342
0.068
0.298
0.254
0.032
0.092
0.412
0.288
0.208
0.329
0.192
0.090
0.426
0.187
0.176
0.193
0.395
0.153
0.394
0.084
0.281
0.067
0.296
0.254
0.031
0.091
0.282
0.287
0.186
0.323
0.151
0.089
0.425
0.186
0.175
0.192
0.394
0.153
0.394
0.083
0.281
0.066
0.294
0.253
0.030
0.090
0.280
0.286
0.165
0.316
0.141
0.088
0.424
0.184
0.174
0.192
0.393
0.152
0.393
0.083
0.276
0.064
0.293
0.253
0.029
0.090
0.280
0.283
0.124
0.312
0.131
0.086
0.424
0.182
0.172
0.191
0.392
0.151
0.391
0.082
0.262
0.064
0.292
0.252
0.029
0.089
0.242
0.282
0.108
0.308
0.115
0.085
0.423
0.181
0.172
0.190
0.392
0.151
0.389
0.080
0.258
0.063
0.291
0.251
0.028
0.088
0.240
e
e
2.42%
76.82%
31.70%
77.60%
6.59%
1.17%
4.23%
4.49%
2.06%
2.01%
4.43%
2.51%
8.04%
42.73%
7.35%
2.68%
1.57%
18.75%
4.35%
43.20%
SIF
SGF
SIF
SGF
SIF
SGF
SIF
SGF
SIF
SGF
SIF
SGF
SIF
SGF
SIF
0.100
e
0.100
e
0.098
e
0.084
e
0.423
e
0.423
e
0.171
e
0.170
e
0.390
e
0.390
e
0.389
e
0.388
e
0.252
e
0.252
e
0.291
e
0.290
e
SGF
SIF
SGF
SIF
0.026
e
0.026
e
0.238
0.234
activity comparable to that of the standard in 1 h and 3 h, with
rectal temperatures of 38.180 0.059 and 37.100 0.045. Com-
pounds 2b, 3b, 4b, 5b, 5c and 6b, with one chloro/fluoro phenyl
functionality replaced with para-methoxyphenyl/para-
hydroxyphenyl (with methyl group at C-3), also displayed appre-
ciable antipyretic profile. Compound 1b, with one chlorophenyl
and one dimethylaminophenyl functionality (with methyl group at
C-3), displayed the least potent antipyretic activity followed by
compound 1c.
Among the compounds tested for their antipyretic activity,
introduction of substituents in the phenyl ring only registered
enhanced activity and, in particular, compounds 2c and 3c showed
superior activity.
Analgesic potency of the compounds 1be6b and 1ce5c has
been presented in Table 4. The compounds 2b and 2c exhibited
noticeable analgesic activity at the tested dose of 60 mg/kg. It is
probable that these compounds effectively reduced the wave of
constriction and elongation passing caudally along the abdominal
wall, with twisting of trunk and extension of the hind limb, in mice
due to nociceptive property of acetic acid. In the case of antipyretic
activity, compounds 2c and 3c exhibited significant potency at the
tested dose of 60 mg/kg during the period of study. Here also, the
antinociceptic and antipyretic activities of these compounds were
found to be better than the standard drugs used.
From the preliminary results of the study, it may be concluded
that the presence of para substituted phenyl groups at C-2 and C-6
positions, methyl group at C-3 position could result in high log P
value and less in-vitro hydrolysis, which in-turn are the key factors
in eliciting valuable biological profiles. The most potent compounds
for the analgesic activity (2b and 2c), in addition to the antipyretic
activity (2c and 3c), demonstrated modest in vitro hydrolysis pro-
files and enhanced Log P values. However, the least active com-
pound of the series (1c), exhibited a very high in vitro hydrolysis
and a low Log P value. Therefore, the high potency of these com-
pounds may be attributed to these factors also. Hence, among the
ten compounds used in this study, 2b, 2c and 3c were considered to
be potent candidates with promising pharmacological properties.
Moreover, development of this class of compounds may lead to
4. Conclusion
All the novel target molecules were synthesized by the direct
nucleophilic substitution of N-methyl piperazine, with the corre-
sponding N-chloroacetyl amides derived from substituted 2,6-
diarylpiperidin-4-ones. In order to probe structural requirements
for optimal analgesic and antipyretic activities in this series of
compounds, the methyl substituent attached at C-3 position be-
sides introducing electron withdrawing or electron donating
functional groups at para position of the phenyl framework was
examined.
Partition coefficient of compound 2c was found to be 2.09. Thus,
2c was found to be the most lipophilic compound of the series
followed by 2b, having a log P value of 1.72. Compound 6b was the
least lipophilic with a log P value of 1.14. In the series of synthesized
compounds, it was observed that the log P values of N-methyl
piperazine derivatives were higher than their corresponding N-
chloroacetyl derivatives.
Table 4
Analgesic activity of the substituted diaryl piperidin-4-ones.
Group Treatment
No. of writhes
(mean SEM)**
% Inhibition
(mean SEM)**
I
II
Control CMC (0.5%)
Standard (diclofenac
sodium)
73.800 1.158
32.000 1.000
_
56.515 2.029
Results of in vitro hydrolysis showed that compound 1c was the
most hydrolyzed, with a percent hydrolysis of 31.70, and compound
5c was the least hydrolyzed, with a percent hydrolysis of 1.57, in
simulated gastric fluid. This indicated their stability in stomach and
subsequent bioavailability. Compound 1c was the most hydrolyzed
with a percent hydrolysis of 77.60, and compound 2b was the least
hydrolyzed, with a percent hydrolysis of 1.17 in simulated intestinal
fluid. This demonstrated their stability and bioavailability through
the intestine. The percent hydrolysis of the various synthesized
compounds in SGF and SIF are depicted in Table 3.
III
IV
V
VI
VII
VIII
IX
X
Compound 1 (1b)
Compound 2 (1c)
Compound 3 (2b)
Compound 4 (2c)
Compound 5 (3b)
Compound 6 (3c)
Compound 7 (4b)
Compound 8 (5b)
Compound 9 (5c)
Compound 10 (6a)
60.600 1.030
53.000 0.0707
31.200 1.068
25.400 0.927
36.000 0.707
33.000 0.707
44.400 0.927
41.200 0.860
34.600 0.509
36.000 0.707
17.794 2.028
28.101 1.636
57.637 1.885
65.487 1.711
51.184 1.109
55.199 1.528
39.715 2.113
44.126 1.365
53.049 1.250
51.178 1.156
XI
XII
** Indicates highly significant result with P < 0.01.

P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
445
Table 5
Antipyretic activity of the substituted diaryl piperidin-4-ones.
Gp Treatment
Initial rectal temp. (ꢁC) Rectal temp. after 18 h Rectal temp. 1 h after treatment Rectal temp. 2 h after treatment Rectal temp. 3 h after treatment
(Mean SEM)
(ꢁC) (Mean SEM)
(ꢁC) (Mean SEM)**
(ꢁC) (Mean SEM)**
(ꢁC) (Mean SEM)**
I
Control CMC 37.260 0.052
(0.5%)
39.200 0.089
39.300 0.071
39.320 0.102
39.400 0.071
II Standard
(Paracetamol)
37.240 0.051
39.280 0.087
37.880 0.073
37.140 0.051
37.100 0.055
III Cpd. 1 (1b)
IV Cpd. 2 (1c)
37.280 0.086
37.300 0.071
37.240 0.050
37.220 0.086
37.200 0.070
37.220 0.058
37.180 0.073
37.200 0.071
37.280 0.037
39.180 0.058
39.140 0.075
39.220 0.058
39.200 0.071
39.260 0.051
39.220 0.058
39.140 0.052
39.200 0.045
39.180 0.066
39.200 0.070
39.000 0.032
38.900 0.070
38.140 0.051
37.900 0.072
38.580 0.058
38.180 0.059
38.920 0.057
38.760 0.051
38.520 0.058
38.720 0.037
38.560 0.051
38.140 0.053
37.540 0.050
37.160 0.075
37.900 0.071
37.440 0.074
38.500 0.070
38.360 0.052
37.680 0.058
38.300 0.045
37.860 0.051
37.520 0.058
37.160 0.068
37.180 0.080
37.200 0.070
37.100 0.045
37.140 0.081
37.200 0.072
37.280 0.037
37.160 0.052
V
Cpd. 3 (2b)
VI Cpd. 4 (2c)
VII Cpd. 5 (3b)
VIII Cpd. 6 (3c)
IX Cpd. 7 (4b)
X
Cpd. 8 (5b)
XI Cpd. 9 (5c)
XII Cpd. 10 (6b) 37.260 0.068
** Indicates highly significant result with P < 0.01; Cpd. ¼ Compound; Gp ¼ Group.
some interesting chemical entities with improved biological and
pharmacological profiles than the standard drugs. Therefore, this
class of compounds may be used as templates to generate better
drugs to alleviate pain and fever.
5.2.1. N-chloroacetyl-2-(4-dimethylaminophenyl)-3-methyl-6-(4-
chlorophenyl)-piperidin-4-one (1b)
Stirring time 7.0 h; Dark brown colored crystalline solid; Yield
78.47% (Ethanol); Melting range (ꢁC): 115e118; IR (KBr) ( cmꢀ1):
(1654.81) NeCeO Stretching, (1718.46) C]O Stretching, (831.26)
AreH Bending; 1H NMR [(CDCl3)
in ppm: 1.052e1.075 (d,
n
d
5. Experimental protocols
J ¼ 6.9 Hz, 3H), 2.854e2.940 (q, J ¼ 25.8 Hz, 1H), 2.970e3.044 (q,
J ¼ 22.2 Hz, 1H), 3.087 (s, 6H), 3.131e3.150 (d, J ¼ 11.1 Hz, 1H), 3.948
(s, 2H), 5.359e5.383 (d, J ¼ 7.2 Hz, 1H), 5.863e5.901 (t, J ¼ 11.4 Hz,
1H), 6.818e6.843 (d, J ¼ 7.5 Hz, 2H), 6.690e6.720 (d, J ¼ 9.0 Hz, 2H),
7.304e7.332 (d, J ¼ 8.4 Hz, 2H), 7.164e7.182 (d, J ¼ 5.4 Hz, 2H)];
Anal. Calcd for C22H24Cl2N2O2: C ¼ 63.01%, H ¼ 5.77%, N ¼ 6.68%;
Found C ¼ 60.50%, H ¼ 5.26%, N ¼ 4.57%; MS: [Mþ] at m/z 419.34.
5.1. General
The chemicals and reagents were procured from S. D. Fine
Chemicals, Mumbai and were used as such. Reaction progress was
monitored by thin layer chromatography on silica gel G plates,
using iodine vapors and UV chamber as the visualizing agents. After
physical characterization, the compounds were subjected to spec-
tral analysis. UV spectra were recorded on a Double Beam spec-
trophotometer (Shimadzu-1700). IR spectra were recorded on an
FTIR Spectrophotometer (Shimadzu 8400S) and values are
expressed in cmꢀ1, and only noteworthy absorption levels (recip-
rocal centimeters) are listed. Mass spectra were recorded on JEOL-
Accu TOF JMS-T100LC Mass spectrometer and THERMO Finnigan
LCQ Advantage max ion trap Mass spectrometer. NMR spectra were
recorded on a Bruker DRX-300 spectrometer using CDCl3 as the
5.2.2. N-chloroacetyl-2-(4-chlorophenyl)-3-methyl-6-(4-
fluorophenyl)-piperidin-4-one (2b)
Stirring time 20.0 h; Light brown colored crystalline solid; Yield
80.59% (Ethanol); Melting range (ꢁC): 98e101; IR (KBr) ( cmꢀ1):
n
(1654.81) NeCeO Stretching, (1718.46) C]O Stretching, (835.12)
AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 1.032e1.072 (d,
J ¼ 12.0 Hz, 3H), 2.842e2.862 (d, J ¼ 6.0 Hz, 2H), 2.923e2.945 (d,
J ¼ 6.6 Hz, 1H), 3.953 (s, 2H), 4.040e4.056 (d, J ¼ 4.8 Hz, 1H),
4.072e4.181 (t, J ¼ 2.7 Hz, 1H), 6.973e6.991 (d, J ¼ 5.4 Hz, 2H),
7.140e7.169 (d, J ¼ 8.7 Hz, 2H), 7.234e7.242 (d, J ¼ 2.4 Hz, 2H),
7.306e7.334 (d, J ¼ 8.4 Hz, 2H)]; MS: [Mþ] at m/z 394.16.
solvent .Chemical shifts are reported in parts per million (
d
values),
using TMS as an internal standard (
d
-0 ppm for 1H NMR). The spin
multiplicities in 1H NMR are indicated as follows: s (singlet),
d (doublet), dd (double doublet), t (triplet) and m (multiplet).
Elemental analysis was performed on an Elemental Vario EL-III
analyzer.
5.2.3. N-chloroacetyl-2-(4-methoxyphenyl)-3-methyl-6-(4-
fluorophenyl)-piperidin-4-one (3b)
Stirring time 21.0 h; Brown colored crystalline solid; Yield
75.84% (Ethanol); Melting range (ꢁC): 121e123; IR (KBr) ( cmꢀ1):
n
5.2. General procedure for the synthesis of N-chloroacetyl-2,6-
(1654.81) NeCeO Stretching, (1718.46) C]O Stretching, (837.05)
diarylpiperidin-4-ones (1b, 2b, 3b and 5b)
AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 0.822e0.840 (d,
J ¼ 5.4 Hz, 3H), 3.159e3.170 (d, J ¼ 3.3 Hz, 2H), 3.782e3.808 (d,
J ¼ 7.8 Hz,1H), 3.895 (s, 3H), 3.956 (s, 2H), 4.511e4.517 (d, J ¼ 1.8 Hz,
1H), 6.781e6.810 (d, J ¼ 8.7 Hz, 2H), 6.872e6.904 (d, J ¼ 9.6 Hz, 2H),
6.952e6.961 (d, J ¼ 2.7 Hz, 2H), 6.996e7.056 (t, J ¼ 18.0 Hz, 1H),
7.827e7.856 (d, J ¼ 8.7 Hz, 2H)]; MS: [Mþ1]þ at m/z 390.17.
To a well-stirred solution of (1a, 2a, 3a and 5a) substituted 2,6-
diphenylpiperidin-4-one (1 equiv., 0.005 mol) and triethylamine
(1 equiv., 0.005 mol) in 30 ml toluene, chloroacetyl chloride
(1 equiv., 0.005 mol) in 30 ml toluene was added dropwise, for
about half an hour, through a dropping funnel. Stirring was
continued with mild heating (30e35 ꢁC) till the completion of the
reaction. After this, the mixture was poured into 10 ml water and
extracted with three 10 ml portions of ether. The collected ether
extracts were then washed well with 3% sodium bicarbonate so-
lution and dried over anhydrous sodium sulfate. The same upon
evaporation and subsequent recrystallization in distilled ethanol
afforded the compounds (1b, 2b, 3b and 5b) in pure form with good
yields. The completion of the reactions was monitored by TLC, using
the solvent system methanol: chloroform (2%).
5.2.4. N-chloroacetyl-2-(4-methoxyphenyl)-3-methyl-6-(4-
chlorophenyl)-piperidin-4-one (5b)
Stirring time 25.0 h; Brown colored crystalline solid; Yield 74.9%
(Ethanol); Melting range (ꢁC): 202e205; IR (KBr) (
n
cmꢀ1):
(1658.67) NeCeO Stretching, (1720.39) C]O Stretching, (833.19)
AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 1.051e1.093 (d,
J ¼ 12.6 Hz, 3H), 3.133e3.153 (d, J ¼ 6.0 Hz, 2H), 3.752 (s, 3H),
3.785e3.810 (d, J ¼ 7.5 Hz, 1H), 3.894 (s, 2H), 3.935e3.951 (d,

446
P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
J ¼ 4.8 Hz, 1H), 4.111e4.176 (t, J ¼ 19.5 Hz, 1H), 6.843e6.871 (d,
J ¼ 8.4 Hz, 2H), 6.785e6.809 (d, J ¼ 7.2 Hz, 2H), 7.167e7.182 (d,
J ¼ 4.5 Hz, 2H), 7.242e7.269 (d, J ¼ 5.1 Hz, 2H)]; Anal. Calcd for
5.4.1. N-(N-methylpiperazinoacetyl)-2-(4-dimethylaminophenyl)-
3-methyl-6-(4-chlorophenyl)-piperidin-4-one (1c)
Reflux time 13.0 h; Orange colored crystalline solid; Yield
C
21H21Cl2NO3: C ¼ 62.08%, H ¼ 5.21%, N ¼ 3.45%; Found C ¼ 61.64%,
50.62% (Ethanol); Melting range (ꢁC): 105e107; IR (KBr) ( cmꢀ1):
n
H ¼ 5.42%, N ¼ 2.10%; MS: [Mþ] at m/z 406.13.
(1643.24) NeCH2eC]O Stretching, (1714.69) C]O Stretching,
(833.19) AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 0.815e0.837 (d,
5.3. General procedure for the synthesis of N-chloroacetyl-2,6-
diarylpiperidin-4-ones (4b and 6b)
3H), 2.105 (s, 3H), 2.569e2.626 (q, 8H), 2.947 (s, 6H), 3.026e3.034
(d, 2H), 3.091 (s, 2H), 3.587e3.621 (d, 1H), 6.693e6.722 (d, 1H),
7.142e7.214 (t, 1H), 7.248e7.266 (d, 2H), 7.332e7.354 (d, 2H),
7.378e7.392 (d, 2H), 7.726e7.755 (d, 2H)]; Anal. Calcd for
To a well-stirred solution of (4a and 6a) substituted 2,6-
diphenylpiperidin-4-one (0.005 mol, 1 equiv.) and triethylamine
(0.005 mol, 1 equiv.) in 30 ml chloroform, chloroacetyl chloride
(0.005 mol, 1 equiv.) in 30 ml chloroform was added dropwise, for
about half an hour, through a dropping funnel. Stirring was
continued with mild heating at (30e35 ꢁC) till the completion of
the reaction. After this, the mixture was poured into 10 ml water
and extracted with three 10 ml portions of ether. The collected
ether extracts were then washed well with 3% sodium bicarbonate
solution and dried over anhydrous sodium sulfate. The same upon
evaporation and subsequent recrystallization in distilled ethanol
furnished the compound (4b and 6b) in pure form with good yields.
The completion of the reactions was monitored by TLC, using the
solvent system methanol: chloroform (4%).
C
27H35ClN4O5: C ¼ 67.13%, H ¼ 7.30%, N ¼ 11.60%; Found C ¼ 79.18%,
H ¼ 5.80%, N ¼ 3.09%; MS: [Mþ] at m/z 483.35.
5.4.2. N-(N-methylpiperazinoacetyl)-2-(4-chlorophenyl)-3-methyl-
6-(4-fluorophenyl)-piperidin-4-one (2c)
Reflux time 18.0 h; Brown colored crystalline solid; Yield 63.02%
(Ethanol); Melting range (ꢁC): 178e180; IR (KBr)
(n
cmꢀ1):
(1643.24) NeCH2eC]O Stretching, (1720.39) C]O Stretching,
(833.19) AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 1.078e1.096 (d,
J ¼ 5.4 Hz, 3H), 2.291 (s, 3H), 2.793e2.813 (t, J ¼ 6.0 Hz, 4H),
2.860e2.873 (d, J ¼ 3.9 Hz, 2H), 3.09 (s, 2H), 3.294e3.340 (t,
J ¼ 13.8 Hz, 4H), 3.587e3.621 (d, J ¼ 10.2 Hz, 1H), 3.683e3.729 (t,
J ¼ 13.8 Hz, 1H), 6.880e6.896 (d, J ¼ 4.8 Hz, 2H), 7.026e7.033 (d,
J ¼ 2.1 Hz, 2H), 7.169e7.178 (d, J ¼ 2.7 Hz, 2H), 7.233e7.241 (d,
J ¼ 2.4 Hz, 2H)]; MS: [Mþ1]þ at m/z 458.24 .
5.3.1. N-chloroacetyl-2-(4-hydroxyphenyl)-3-methyl-6-(4-
chlorophenyl)-piperidin-4-one (4b)
Stirring time 21.0 h; Orange colored crystalline solid; Yield
5.4.3. N-(N-methylpiperazinoacetyl)-2-(4-methoxyphenyl)-3-
methyl-6-(4-fluorophenyl)-piperidin-4-one (3c)
45.32% (Ethanol); Melting range (ꢁC): 132e134; IR (KBr) ( cmꢀ1):
n
(1652.88) NeCeO Stretching, (1716.53) C]O Stretching, (833.19)
Reflux time 19.0 h; Dark brown colored crystalline solid; Yield
AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 0.830e0.852 (d,
73.34% (Ethanol); Melting range (ꢁC): 187e189; IR (KBr) ( cmꢀ1):
n
J ¼ 6.6 Hz, 3H), 3.659e3.695 (d, J ¼ 10.8 Hz, 2H), 3.947e3.951 (d,
J ¼ 1.2 Hz, 1H), 4.175 (s, 2H), 4.527 (s, 1H), 5.815e5.861 (d,
J ¼ 13.8 Hz, 1H), 6.567e6.592 (t, J ¼ 7.5 Hz, 1H), 6.823e6.837 (d,
J ¼ 4.2 Hz, 2H), 6.939e6.966 (d, J ¼ 8.1 Hz, 2H), 7.159e7.184 (d,
J ¼ 7.5 Hz, 2H), 7.306e7.328 (d, J ¼ 6.6 Hz, 2H)]; Anal. Calcd for
(1641.31) NeCH2eC]O Stretching, (1718.46) C]O Stretching,
(835.12) AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 0.811e0.833 (d,
J ¼ 6.6 Hz, 3H), 2.285 (s, 3H), 2.592e2.615 (t, J ¼ 6.9 Hz, 8H),
3.685e3.698 (d, J ¼ 3.9 Hz, 2H), 3.809 (s, 2H), 3.884e3.894 (d,
J ¼ 3.0 Hz, 1H), 4.007 (s, 3H), 4.085e4.097 (d, J ¼ 3.6 Hz, 1H),
6.552e6.588 (t, J ¼ 10.8 Hz, 1H), 6.795e6.824 (d, J ¼ 8.7 Hz, 2H),
6.907e6.914 (d, J ¼ 2.1 Hz, 2H), 7.024e7.032 (d, J ¼ 2.4 Hz, 2H),
7.157e7.182 (d, J ¼ 7.5 Hz, 2H)]; MS: [Mþ1]þ at m/z 454.30.
C
20H19Cl2NO3: C ¼ 61.24%, H ¼ 4.88%, N ¼ 3.57%; Found C ¼ 60.19%,
H ¼ 4.82%, N ¼ 3.82%; MS: [Mþ] at m/z 392.12.
5.3.2. N-chloroacetyl-2-(4-hydroxyphenyl)-3-methyl-6-(4-
fluorophenyl)-piperidin-4-one (6b)
5.4.4. N-(N-methylpiperazinoacetyl)-2-(4-methoxyphenyl)-3-
methyl-6-(4-chlorophenyl)-piperidin-4-one (5c)
Stirring time 23.5 h; Wine red colored crystalline solid; Yield
73.80% (Ethanol); Melting range (ꢁC): 192e195; IR (KBr) (
n
cmꢀ1):
Reflux time 20.5 h; Dark brown colored crystalline solid; Yield
(1658.67) NeCeO Stretching, (1720.39) C]O Stretching, (837.05)
71.16% (Ethanol); Melting range (ꢁC): 128e130; IR (KBr) ( cmꢀ1):
n
AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 0.835e0.856 (d,
(1641.31) NeCH2eC]O Stretching, (1718.46) C]O Stretching,
J ¼ 6.3 Hz, 3H), 3.028e3.051 (d, J ¼ 6.9 Hz, 2H), 3.096e3.115 (d,
J ¼ 5.7 Hz, 1H), 4.011 (s, 2H), 4.165 (s, 1H), 4.938e4.956 (d,
J ¼ 5.4 Hz, 1H), 5.846e5.941 (t, J ¼ 28.5 Hz, 1H), 7.028e7.056 (d,
J ¼ 8.4 Hz, 2H), 7.157e7.181 (d, J ¼ 7.2 Hz, 2H), 7.342e7.404 (d,
J ¼ 18.6 Hz, 2H), 7.743e7.771 (d, J ¼ 9.6 Hz, 2H)]; MS: [Mþ1]þ at m/z
376.14.
(831.26) AreH Bending; 1H NMR [(CDCl3)
d
in ppm: 0.857e0.880 (d,
J ¼ 6.9 Hz, 3H), 2.285 (s, 3H), 2.353e2.378 (t, J ¼ 7.5 Hz, 8H),
3.722e3.730 (d, J ¼ 2.4 Hz, 2H), 3.772e3.792 (d, J ¼ 6.0 Hz, 1H),
3.799 (s, 2H), 3.894 (s, 3H), 5.869e5.965 (d, J ¼ 1.8 Hz, 1H),
6.532e6.582 (t, J ¼ 15.0 Hz, 1H), 6.734e6.762 (d, J ¼ 8.4 Hz, 2H),
6.793e6.882 (d, J ¼ 26.7 Hz, 2H), 7.016e7.022 (d, J ¼ 1.8 Hz, 2H),
7.376e7.390 (d, J ¼ 4.2 Hz, 2H)]; Anal. Calcd for C26H32ClN3O3:
C ¼ 66.44%, H ¼ 6.86%, N ¼ 8.94%; Found C ¼ 64.98%, H ¼ 6.33%,
N ¼ 7.87%; MS: [Mþ1]þ at m/z 470.30.
5.4. General procedure for the synthesis of N-(N-
methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones (1c, 2c, 3c and
5c)
5.5. In-vitro studies
A mixture of N-chloroacetyl-2,6-diarylpiperidin-4-one deriva-
tive (1b, 2b, 3b and 5b e 0.005 mol, 1 equiv.), triethylamine
(0.005 mol, 1 equiv.) and N-methyl piperazine (0.005 mol, 1 equiv.)
in 30 ml of toluene was refluxed for about 13e20.5 h on water bath.
After the completion of the reaction, excess of solvent was removed
under reduced pressure. The final mass was poured into water to
remove the quaternary ammonium salt formed. This was then
extracted with ether three times and dried over anhydrous sodium
sulfate. The completion of the reaction was monitored by TLC, using
the solvent system n-hexane: ethyl acetate mixture (4:1).
The in-vitro studies included partition coefficient and hydrolysis
profile in SGF (simulated gastric fluid) and SIF (simulated gastric
fluid).
5.5.1. Determination of partition coefficient
Partition coefficient of the synthesized compounds was deter-
mined by “Shake Flask Method” [48]. Initially, 2 mg of the syn-
thesized compound was dissolved in 100 ml of the solvent (50 ml
water þ 50 ml octanol). It was then shaken for 30 min and kept

P. Tripathi et al. / European Journal of Medicinal Chemistry 82 (2014) 439e448
447
aside for 24 h to attain equilibrium. Two phases were separated and
the concentration of the drug was determined in octanol phase by
taking absorbance. Finally, found absorbance was extrapolated over
a standard curve so as to obtain the concentrations of the organic
phase. The log P values of the synthesized compounds were then
determined from the concentrations of the octanol phase obtained,
using the formula given below:
writhing syndrome in test animals was elicited by injecting 1% of
acetic acid in normal saline (10 ml/kg body weight) intraperito-
neally,1 h after the oral administration of the test compounds. After
5 min of injection of the noxious agent, the number of writhes was
counted during the subsequent 30 min by keeping the animals on a
flat surface. A significant reduction in the number of writhes by
drug treatments, as compared to vehicle control animals, was
considered as a positive analgesic response. The mean number of
writhes for each experimental group was compared with that of the
control group and the analgesic potency was expressed as percent
inhibition, determined using the following equation where T is the
number of writhes in the test compound treated group and C is the
number of writhes in control group.
Log P ¼ ½conc: of compound in octanolꢂ=
ꢃ ½conc: of compound in distilled waterꢂ
Similar procedure was adopted for all the synthesized com-
pounds [49].
Percentage inhibition of writhing ¼ ½1 ꢀ ðT=CÞꢂ*100:
5.5.2. Hydrolysis studies
For this, 10 mg of the synthesized compound was dispersed/
dissolved in 100 ml of simulated gastric fluid/simulated intestinal
fluid. The resulting mixture was shaken on an orbital shaker at a
5.6.2. Rota rod test
temperature of 37
2
ꢁC. Samples were withdrawn at regular in-
The test is used to evaluate the activity of drugs interfering with
motor coordination. The cardinal feature of the test is to ascertain
the impairment of motor performance, ataxia, loss of skeletal
muscle strength, and acute neurotoxicity produced by drugs in pre-
clinical studies. The animals were placed on a 1 in. diameter
knurled wooden rod, rotating at a speed of 6e10 rpm (Medicraft
Rota rod Apparatus). Diazepam at a dose of 2 mg/kg body weight
was taken as the positive control. The study was started 60 min
after the oral administration of the test compounds. Normal mice
remain on a rod rotating at this speed indefinitely. Neurologic
toxicity was defined as the failure of the animal to remain on the
rod for duration of 1 min or more [51].
tervals of 0, 15, 30, 45, 60, 75, 90, 105, 120 min. The samples were
suitably diluted, filtered and analyzed spectrophotometrically at
the designated absorption maxima of each compound [49]. Percent
hydrolysis was calculated using the following formulas:
% Hydrolysed in SGF ¼ ½ðabs: at 0 min ꢀ abs: at 90 minÞ
ꢃ =abs: at 0 minꢂ*100:
% Hydrolysed in SIF ¼ ½ðabs: at 0 min ꢀ abs: at 120 minÞ
ꢃ =abs: at 0 minꢂ*100:
5.6.3. Antipyretic activity
5.6. Pharmacology
Antipyretic activity of the target compounds was determined by
measuring the variation in the rectal temperature as per the re-
ported procedure [52]. Fever was induced in the fasted Wistar rats
by injecting 15% brewer's yeast in normal saline (10 ml/kg body
weight) subcutaneously. Initial rectal temperature was recorded
using a lubricated digital thermometer, by inserting it into the
rectum of the animal at a depth of about 7 mm. The same ther-
mometer was used for all the animals in each group in order to
minimize the possible experimental error. All the values are mean
of five independent measurements. After 18 h, the animals that
showed rectal temperature of at least 38 ꢁC were selected for this
study. Paracetamol (150 mg/kg) was used as the reference drug. The
test compounds and standard drug were administered orally as a
suspension in 0.5% CMC, at doses of 60 and 150 mg/kg body weight
respectively. Control groups received the vehicle (0.5% CMC) only.
After the administration of the standard/test compounds, the rectal
temperature was measured successively after 30, 60, 120 and
180 min.
Male Albino mice and Wister rats weighing 25e30 g and
200e300 g respectively (unless otherwise specified), were used for
the pharmacological studies. The animals were housed in poly-
propylene cages with steel net, in a temperature controlled room
under standard living conditions of temperature 25
5
ꢁC and
relative humidity of 55 5% with regular 12 h light and 12 h dark
cycles, and allowed free access to standard laboratory food and
water. However, the groups of animals chosen for the study were
fasted for 20e24 h before the treatment and were divided into
parallel groups containing five animals each. All the animals were
treated humanely in accordance with the guidelines laid down by
the Institutional Animal Ethics Committee (IAEC). Experiments
were performed after the protocols were approved by the IAEC and
also by strictly adhering to the ethical guidelines.
The reported values are the average of five determinations. The
observed results are expressed as mean SEM and were analyzed
by one-way analysis of variance (ANOVA, Dunnett's test) for the
possible significant identification between various groups. The *P
value of <0.05 or <0.01 was considered as statistically significant or
highly significant, respectively. Statistical analysis was carried out
using Graph pad InStat 3.0 (Graph pad software).
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