Synthesis and spectral characterization of a new class of N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones: Antimicrobial, analgesic and antipyretic studies

Article abstract of DOI:10.1016/j.ejmech.2008.03.031

A series of N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones (13c-21c) were synthesized by the base catalyzed nucleophilic substitution of N-chloroacetyl-2,6-diarylpiperidin-4-ones obtained from their corresponding 2,6-diarylpiperidin-4-ones with N-methylpiperazine. These newly synthesized compounds were characterized by one- and two-dimensional NMR spectral studies. In all the cases, the piperazine ring adopted normal chair conformation with equatorial orientation of methyl group irrespective of the non-chair conformations of the piperidin-4-one moiety. All the compounds were screened for their possible antibacterial and antifungal activities against a spectrum of microbial agents besides analgesic and antipyretic activities. These biological studies proved that compounds 17c/18c against bacterial and 18c/20c against fungal strains exhibited promising antimicrobial activities whereas 17c/19c and 18c/19c showed beneficial analgesic and antipyretic profiles, respectively, at a concentration of 60 mg/kg and were also found to be more potent than the reference drug.

Full text of DOI:10.1016/j.ejmech.2008.03.031

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European Journal of Medicinal Chemistry 44 (2009) 577e592
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and spectral characterization of a new class
of N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones:
Antimicrobial, analgesic and antipyretic studies
G. Aridoss a,1, P. Parthiban a, R. Ramachandran a, M. Prakash b,
S. Kabilan a, , Yeon Tae Jeong
*
c,**
a Department of Chemistry, Annamalai University, Annamalainagar e 608 002, Tamil Nadu, India
b Department of Zoology, Annamalai University, Annamalainagar e 608 002, Tamil Nadu, India
c Division of Image and Information Engineering, Pukyong National University, San 100,
Yongdang-Dong, Nam-Gu, Busan 608-739, Republic of Korea
Received 29 August 2007; received in revised form 15 December 2007; accepted 21 March 2008
Available online 7 April 2008
Abstract
A series of N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones (13ce21c) were synthesized by the base catalyzed nucleophilic substi-
tution of N-chloroacetyl-2,6-diarylpiperidin-4-ones obtained from their corresponding 2,6-diarylpiperidin-4-ones with N-methylpiperazine.
These newly synthesized compounds were characterized by one- and two-dimensional NMR spectral studies. In all the cases, the piperazine
ring adopted normal chair conformation with equatorial orientation of methyl group irrespective of the non-chair conformations of the piper-
idin-4-one moiety. All the compounds were screened for their possible antibacterial and antifungal activities against a spectrum of microbial
agents besides analgesic and antipyretic activities. These biological studies proved that compounds 17c/18c against bacterial and 18c/20c against
fungal strains exhibited promising antimicrobial activities whereas 17c/19c and 18c/19c showed beneficial analgesic and antipyretic profiles,
respectively, at a concentration of 60 mg/kg and were also found to be more potent than the reference drug.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Piperidin-4-one; N-Methylpiperazine; Conformation; Antimicrobial; Analgesic; Antipyretic activities
1. Introduction
a number of pathological conditions made them the most use-
ful therapeutic agents in the world [2]. However, the routine
use of these agents was reported to be limited because of their
associated side effects mainly on gastrointestinal (GI) tracts.
Pain is an unpleasant sensory and emotional experience asso-
ciated with actual or potential organ and tissue damage [3].
Body inflammation is a unique pain inducer which the human
kind faces more often as an outcome of tissue damage devel-
oped by a series of microbial infections such as anorexia, pain
and fever which in turn shoots up the body temperature. In
order to combat these diseases caused by pathogens, it is usual
that chemotherapeutics, analgesic and antipyretic agents are
prescribed separately in clinical practices. However, multidrug
treatments for microbial diseases create a significant problem
among the patients with impaired organ functions [4]. This
laid the foundation for the search and design of new chemical
Despite the major breakthrough in many areas of modern
medicine during the past 10 decades, the successful treatment
towards multidrug-resistant pathogens (i.e., microbial isolates
such as fungi and bacteria) has become a serious problem and
remains a significant challenge over the last 10 years [1]. The
potentiality of non-steroidal anti-inflammatory drugs (NSAID)
to alleviate pain, inflammation and fever coupled with
* Corresponding author. Tel.: þ91 4144 238641.
** Corresponding author. Tel.: þ82 51 629 6411; fax: þ82 51 629 6408.
E-mail addresses: skabilen@rediffmail.com (S. Kabilan), ytjeong@pknu.
ac.kr (Y.T. Jeong).
1
Present address: Division of Image and Information Engineering, Pukyong
National University, San 100, Yongdang-Dong, Nam-Gu, Busan 608-739, Re-
public of Korea.
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.031

578
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
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 antimicrobial, analgesic and antipyretic activities together
by keeping inview the pharmaco-economic and frequent patient
compliance. While exploring for such a compound, we have
found that piperidone nucleus is an integral component of
countless alkaloids with marked biological properties [5e12].
Interest in 2,6-diarylpiperidin-4-ones 1 (Fig. 1) derives from
thevaried biological activities of this structural motif containing
compounds, that were evaluated for wide range of pharmaco-
logical activities [13,14] and in particular their potency towards
broad spectrum of microbial strains [15e22]. Generally, com-
pounds possessing an amide bond linkage have a wide range
of biological activities such as antimicrobial [23,24], anti-
inflammatory [25], antiviral, antimalarial and general anes-
thetics [26]. Furthermore, the amides derived from chloroacetyl
chloride also gain significant importance in medicinal field as
evidenced by their varied pharmacological activities [27e32].
Compounds with piperazine and its N-substituted deriva-
tives as an integral part are the most successfully employed
side chains as they exhibit a varied range of activity such as
antimicrobial [23,33], anticancer [34], anti-inflammatory
[35], antipsychotic [36], CNS agent [37], antagonists for
CCKB/gastrin receptor [38], melanocortin-4-receptor [39]
and in the treatment of Alzheimer’s disease [40].
Some clinically useful drugs such as Norfloxacin 2, Cipro-
floxacin 3, Enoxacin 4, Ofloxacin 5 and Levofloxacine 6
(Fig. 1) having piperazine or its 1-methyl derivative skeleton
as a vital part exhibit potential activity for respiratory, urinary,
gastrointestinal tracts, skin and soft tissue infections caused by
both classes of bacteria [41] while other drugs viz. Clothiapine
7, Loxapine 8 and Clozapine 9 (Fig. 1) with 1-methylpiperazine
unit were reported as efficient antipsychotic drugs. Ciprofloxacin
3 possesses modest activity against Gram-positive cocci whereas
its N-methylpiperazine derivative viz., Levofloxacine 6 has
enhanced potency against Gram-positive and atypical organisms
as it may be due to its better cell permeability and enhanced lip-
ophilicity [41,42]. This indicates that replacement of piperazine
moiety by its 1-methyl analogue has well pronounced impact on
the microbial potency. Moreover, recent literature [43] reveals
that linking of N-substituted piperazine to aryl or heteroaryl
moiety by two carbon chains having b-keto group 10 (Fig. 1)
has additional advantage in eliciting good biological response.
As a part of our ongoing research program to find out potent
broad spectrum antimicrobial agents for the past few years, we
have made certain modifications on the nitrogen site of 1 by
introducing various heterocyclic ring systems through two
carbon spacer units (either alkoxy [17,18] or acyl [15,16]), of
which, a few compounds were endowed with appreciable and
statistically significant antimicrobial activities. However, pre-
liminary in vitro antimicrobial studies of the core structure 1
O
O
F
COOH
R
R
1
2
N
R
N
Z
N
H
HN
R
R
1
2. Norfloxacin : R = ethyl; Z = H
3. Ciprofloxacin : R = cyclopropyl ; Z = H
4. Enoxacine
: R = ethyl; Z= N
O
O
F
COOH
F
COOH
N
N
N
N
N
O
N
O
H C
CH
3
HC
CH
3
3
3
5. Ofloxacin
6. Levofloxacin
CH
3
N
O
F
COOH
N
N
O
N
Z
N
R
X
Z
N
R
Y
1
10. R = cyclopropyl; ethyl
7. Clothiapine X = H; Y = S; Z = Cl
R1 = aryl; heteroaryl
8. Loxapine
9. Clozapine
X = H; Y = O; Z = Cl
X = Cl; Y = NH; Z = H
Fig. 1.

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
579
with chloroacetyl unit at ‘N’ (compound 11) has little effect to-
wards certain selected microbial strains [16] whereas nucleo-
philic substitution of morpholine [15] in 11 (i.e., compound
12; Fig. 2) gave encouraging results with improved activity
profile. Hence, by considering the potent pharmacological ac-
tivities of 1, 12 and piperazine, we have now aimed to widen
our knowledge of structureeactivity relationships (SAR) in
this structural framework by synthesizing a new class of novel
target compounds 13ce21c (Fig. 2) and investigated their pos-
sible antimicrobial, analgesic and antipyretic activities.
2,6-diarylpiperidin-4-ones significantly influences the chemi-
cal shift and coupling constant values of 13be21b which in
turn affect the normal chair conformation of the parent com-
pounds 13ae21a. By one- and two-dimensional NMR spectral
studies of chloroacetyl derivatives 13be21b, we have arrived
at the following conclusions:
(a) Existence of two rotomers A and B (Fig. 3) is due to the
restricted rotation about NeCO bond.
(b) The obtained average NMR spectra for these two rotomers
are mainly due to their faster interconversion on NMR
time scale in CDCl3 at room temperature.
2. Results and discussion
(c) Observation of broad singlet for the benzylic protons in
the most deshielded region suggested the co-planarity of
chloroacetyl moiety with the reference plane of the piper-
idone ring. Due to this, severe allylic strain may exist
between amide carbonyl group and phenyl moieties at
C-2 and C-6 positions which consequently alters the chair
conformation of piperidone framework into non-chair
conformations of its preference.
(d) In these flexible conformations, the ring may become flat-
tened at the nitrogen end. Therefore, the benzylic protons
fall in the planar region of the chloroacetyl group thereby
deshielded well whereas the methylene protons at C-5
resonated at two different chemical shifts with significant
splitting patterns.
2.1. Chemistry
The obvious synthetic pathway that leads to the title com-
pounds (13ce21c) is represented in Scheme 1. By adopting
the literature precedent [44], 2,6-diarylpiperidin-4-ones were
prepared in one pot multi-component Mannich reaction by con-
densing suitably substituted aromatic aldehydes, ketone and
ammonium acetate in 1:2:1 ratio using ethanol as solvent. By
keeping in view the better basicity and HCl scavenging power
of triethylamine, we have used this as a desirable catalyst for
the nucleophilic reaction of 13ae21a with chloroacetyl chlo-
ride than the other catalyst. The yields of N-chloroacetyl-2,6-
diarylpiperidin-4-ones were also significantly good while using
this catalyst. Further, nucleophilic substitution of N-methylpi-
perazine with these chloroacetyl derivatives was performed in
different solvents such as benzene, ethanol and toluene using
NEt3 again as base. Here, toluene was chosen as the best sol-
vent as it improves the yield of the title compounds (13ce
21c) appreciably compared to the remaining solvents used.
Preliminary examination of the formation of compounds by
FT-IR analysis confirmed the absence of NH stretching in piper-
azine and changes in the magnitude of amide carbonylstretching
By keeping these observations in mind, we have analyzed
the chemical shifts and conformations of the final compounds
13ce21c.
1
2.2.1. H NMR spectral analysis of 13ce21c
1H NMR assignment of compounds 13ce21c is made based
on their one- and two-dimensional NMR spectral studies. In all
the cases, the benzylic protons resonated as a broad singlet with
very low intensity in the most downfield region 5.44e
6.54 ppm. Akin to 13be21b, for symmetrically substituted de-
rivatives 16c, 18c, 20c, 21c, C-2 and C-6 benzyclic protons of
piperidone ring showed a common resonance whereas for the
unsymmetrically substituted compounds 13ce15c, 17c and
19c, they resonated individually. The observed broadening of
the benzylic proton signal suggests the existence of restricted
rotation about NeCO bond and also the in plane nature of
the substituent at heterocyclic nitrogen of piperidone moiety
with dynamically averaged plane of the piperidone system.
One-dimensional 1H NMR spectra of these compounds
showed a medium intense unresolved broad singlet and
a very sharp singlet in the aliphatic region 2.49e2.55 ppm
and 2.31e2.43 ppm with eight and three protons integral, re-
spectively. In 13ce21c, as the nitrogen of piperazine moiety
is having almost similar chemical environment on either
side, its ring methylene protons become equivalent and there-
fore the signals in the former region are assigned to four meth-
ylene protons while the signal in the latter region is
characteristic for the methyl protons of N-methylpiperazine
system. Moreover, irrespective of the restricted rotation of
the NeCO bond, the rotation about NeC bond of the
of 13be21b (for 13ce21c, it appeared at around 1660 cmꢀ1
besides the appearance of additional aliphatic stretching
frequencies in the region 2981e2694 cmꢀ1. Analytical data of
all the synthesized compounds are furnished in Table 1.
)
2.2. NMR spectral analysis and stereochemistry
It is very clear from our earlier reports [45] that the substi-
tution of chloroacetyl moiety at the heterocyclic ‘N’ of
O
O
R
R
1
2
R
R
1
2
4
5
6
3
2
1
6'
2'
N
N
2''''
R
R
O
R
O
R
X
N
b
b
11. X = Cl
12. X =
R = H, Me, OMe,Cl
R1 = H, Me, Et, i-Pr,
R2 = H, Me
a
a
O
N
N
CH
3
Target Compounds13c-21c
Fig. 2.

580
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
O
O
R
R
1
2
R
R
1
2
1. EtOH/Heat
HCl
O
O
2. NH
3
H
N
H
H
NH OAc
4
13a-21a
R
R
R
R
ClCH COCl
NEt
2
3
stirring
30-35 °C
O
O
R
R
1
R
R
1
2
2
4
3
5
6
N-Methylpiperazine/NEt
Tolune/reflux
3
2
1
2'
6'
N
N
2''''
R
O
R
R
O
R
N
Cl
13b-21b
b
b
a
a
N
CH
3
13c-21c
Entry
R1
R2
H
R
13 (a-c)
14 (a-c)
15 (a-c)
16 (a-c)
17 (a-c)
18 (a-c)
19 (a-c)
20 (a-c)
21 (a-c)
Me
Et
H
H
H
i-Pr
Me
Me
Me
Me
Me
Me
H
H
Me
H
H
Cl
Me
H
Cl
OMe
OMe
Me
Me
Me
Scheme 1.
piperazinoacetyl moiety is very fast and prevents the spine
spin coupling of all the methylene protons of piperazine
moiety. Owing to this, their resonances were observed as an
unresolved broad singlet. All these observations strongly
confirm the nucleophilic substitution of piperazine moiety in
place of chlorine. Moreover, disappearance of acetyl methy-
lene proton signal of 13be21b from its original position
(i.e., chemical shift) also strongly confirms this nucleophilic
substitution. Our earlier report [45] described clearly that pro-
tons of C-3, C-5 and acetyl methylene group of 13b appeared
as well-resolved signals with good splitting pattern at 3.06 (t),
2.83/3.17 (dd) and 3.88/3.93 (d) ppm, respectively. But, for its
corresponding piperazinoacetyl derivative (13c unsymmetri-
cally substituted), well-resolved signals were not observed
except a double doublet centered at 2.81 ppm. By comparing
chemical shift and coupling constant values of double doublet
at 2.81 ppm (2J5a,5e ¼ 17.99 Hz/3J5e,6a ¼ 5.91 Hz), it is conve-
niently assigned to H-5e. This is further confirmed by its one
proton integral value. For the precise assignment of acetyl
methylene, C-3 and H-5a protons of 13c, HOMOCOSY
(Fig. 4) experiment has been performed and the observed
correlations are furnished in Table 2. From the 1He1H

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
581
Table 1
Analytical data of bioactive compounds 13ce21ca
Compound
Molecular
formula
Molecular
weight
Yield
(%)
Reaction
time (h)
M.p
(ꢁC)
Elemental analysis
Observed (%)
Calculated (%)
C
H
N
C
H
N
13cb
14c
15c
16cb
17c
18c
19c
20c
21c
a
C25H31N3O2
C26H33N3O2
C27H35N3O2
C26H33N3O2
C25H29N3O2Cl2
C26H31N3O2Cl2
C27H35N3O4
C28H37N3O4
C28H37N3O2
405.53
419.56
433.59
419.56
474.42
488.45
465.59
479.61
447.61
88
82
81
85
87
80
82
79
74
8.0
9.5
61
128e129
105
74.04
74.45
74.50
74.41
63.27
63.93
69.67
70.14
75.13
7.71
7.92
8.15
7.94
6.15
6.42
7.59
7.79
8.34
10.37
10.02
9.70
10.03
8.86
8.58
9.01
8.77
9.39
74.04
74.43
74.79
74.43
63.29
63.93
69.65
70.12
75.13
7.70
7.93
8.14
7.93
6.16
6.40
7.58
7.78
8.33
10.36
10.02
9.69
10.02
8.86
8.60
9.03
8.76
9.39
8.5
9.0
Semisolid
Semisolid
82
10.0
9.5
8.0
Semisolid
Semisolid
Semisolid
10.0
9.5
All the compounds were purified by column chromatography using n-hexane:ethyl acetate (4:1) as eluent.
Molecular mass noted for 13c and 16c at 406 and 420 (M þ H)þ respectively are consistent with the proposed molecular formula.
b
correlations, it is found that the partially overlapped multiplet
in the region 3.02e3.28 ppm has good HOMO correlation
with H-2 [5.64 ppm (s)], H-5e [2.81 ppm (dd)] and methyl
protons at C-3 [1.12 ppm (d)]. By considering these correla-
tions, the partially overlapped multiplet with four proton inte-
gral is assigned beyond doubt to H-3a, H-5a and acetyl
methylene protons. As the signals for the remaining unsym-
metrically substituted compounds (14c, 15c, 17c and 19c)
did not vary appreciably, they were assigned in a similar fash-
ion. Likewise, for unambiguous assignment of symmetrically
substituted compounds (16c, 18c, 20c, and 21c), HOMO-
COSY spectrum was recorded for the representative com-
pound 16c and the noted correlations are illustrated in Table
2. In symmetrically substituted compounds, except shielding
of acetyl methylene protons by about 1 ppm, no appreciable
change in the chemical shift values of piperidone ring protons
was observed.
C-a carbons because the former carbons are present at g-posi-
tion with respect to the amide carbonyl group, thereby experi-
encing its electronic effect (about 1.5 ppm). The methyl
carbon of N-methylpiperazine moiety resonated in the region
of 45.4e45.9 ppm. All the signals corresponding to the
N-methylpiperazine moiety are further confirmed from the
HSQC (heteronuclear single quantum coherence) spectrum
recorded for the representative compounds 13c (Fig. 5) and
16c and the observed correlations are reproduced in Table 3.
As the proton NMR chemical shift values of the piperidone
ring system and the acetyl methylene group in 13c and 16c
are assigned perfectly, the respective carbon signals are also
established on the basis of the observed HSQC correlations
given in Table 3. A striking observation from the HSQC
correlations is that the resonances of acetyl methylene carbon
in 13ce21c were deshielded to about 30 ppm due to the
replacement of chlorine by N-methylpiperazine moiety and
observed in the region of 61.8e62.4 ppm. The known elec-
tronic and substituent effects explain the significant changes
in the carbon resonances. Likewise, NMR assignments of
the rest of the compounds were also made.
Therefore, from one- and two-dimensional NMR spectra,
the complete and unambiguous assignment of the individual
protons in piperidone and piperazine ring system of N-(N-
methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones was made.
2.2.2. 13C NMR spectral analysis of 13ce21c
In plane nature of piperazine ring with respect to the amide
carbonyl group is evidenced from the two intense signals
observed (54.4e55.0/52.9e53.5 ppm) for the methylene car-
bons of piperazine viz. C-a and C-b unlike their corresponding
protons. On the basis of the earlier studies [43,46], the upfield
signal is assigned to C-b carbons while the downfield signal to
O
O
R
R
1
R
R
1
2
2
N
N
R
R
R
R
O
O
Cl
Cl
A
B
Fig. 3.
Fig. 4. HOMOCOSY spectrum of 13c.

582
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
Table 2
Correlations in the HOMOCOSY spectrum of compounds 13c and 16c [d
(ppm)]
Table 3
Correlations in the HSQC spectrum of compounds 13c and 16c [d (ppm)]
Compound
13C signal
Correlations in the HSQC
spectrum (with 1H NMR)
Compound
Signal
Observed correlations
13c
1.12 (d, 3H, CH3 at C-3)
2.81 (dd, 1H, H-5e)
5.64 (br s, 1H, H-2)
3.02e3.28
3.02e3.28
3.02e3.28
5.64, 2.81, 1.12
13c
126.79e128.88
(aromatic carbons)
62.45 (NeCOCH2)
7.09e7.41
(aromatic protons)
3.02e3.28
3.02e3.28 (m, 4H, H-3a,
H-5a and NeCOCH2)
(H-3a, H-5a, NeCOCH2)
5.64 (H-2a)
6.05 (H-6a)
60.89 (C-2)
53.45 (C-6)
45.99 (C-3)
16c
1.06 (d, CH3 at C-3 and C-5)
3.12 (t, 2H, H-3a and H-5a)
5.52 (br s, 2H, H-2 and H-6)
3.12
5.52, 1.06
3.12
3.02e3.28
(H-3a, H-5a, NeCOCH2)
3.02e3.28
43.28 (C-5)
(H-3a, H-5a, NeCOCH2),
2.81 (H-5e)
2.49
2.2.3. Conformational analysis of 13ce21c
55.00[(CH2)2-a
of piperazine]
[(CH2)2-a and b of piperazine]
2.49
Broadening of benzylic protons supports the existence of
restricted rotation about NeCO bond and also the in plane
nature of the amide group with the dynamically averaged
plane of the piperidone ring [45,47]. Depending upon the sub-
stituents on the piperidone ring system, the possible non-chair
conformations for N-chloroacetyl-2,6-diarylpiperidin-4-one
system have been established earlier by our group [45].
Replacement of chlorine atom by N-methylpiperazine moiety
did not change the resonances of carbon and the associated
protons appreciably in 13be21b. This suggests that the piper-
idone ring system in N-(N-methylpiperazinoacetyl)-2,6-diary-
lpiperidin-4-ones also retains the same flexible non-chair
conformations and the substituent at its nitrogen is also in
plane with the reference plane of the piperidone.
53.45 [(CH2)2-b
of piperazine]
[(CH2)2-a and b of piperazine]
2.33 (NeCH3)
1.12 (CH3 at C-3)
45.99 (NeCH3)
14.09 (CH3 at C-3)
16c
127.25e128.66
7.01e7.30 (aromatic protons)
(aromatic carbons)
61.88 (NeCOCH2)
60.85 (C-2 and C-6)
45.48 (C-3 and C-5)
54.54 [(CH2)2-a
2.99 (NeCOCH2)
5.52 (H-2a and H-6a)
3.12 (H-3a and H-5a)
2.50
of piperazine]
52.99 [(CH2)2-b
of piperazine]
[(CH2)2-a and b of piperazine]
2.50 [(CH2)2-a
and b of piperazine]
2.32 (NeCH3)
1.06 (CH3 at C-3 and C-5)
45.48 (NeCH3)
14.14 (CH3 at C-3 and C-5)
Though the restricted rotation exists about NeCO bond, it
will not affect the chair conformation of the piperazine ring
system as evidenced from the unique resonance of the ring
methylene protons. However, difference in the chemical shift
of C-a and C-b carbons of the same reveals the co-planarity
of piperazine moiety with the amide system (here C-b carbons
are well within the planar region of the amide carbonyl group)
which in turn is in plane with the piperidone ring system.
1
Further, H and 13C resonances of N-methyl function have
almost the same magnitude as that of equatorially oriented
N-methyl substituted piperazine systems [48]. From this, it
is clearly conceived that the methyl group is most preferen-
tially in the equatorial orientation. On the basis of the above
made investigations, the compounds 13ce21c adopt the
following three different pairs (which are in equilibrium
with each other) of flexible non-chair conformations A, B
and C (Fig. 6) in which N-methylpiperazine ring adopts
normal chair conformation with equatorial orientation of
methyl group irrespective of the substituents at C-3 and/or
C-5 position of piperidin-4-one ring system.
2.3. Antimicrobial activity (structureeactivity
relationship)
The new class of compounds described in this paper have
been preliminarily evaluated for their antibacterial and anti-
fungal activities against a band of certain selected bacterial
(Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, Salmonella typhi and Klebsiella pneumoniae)
and fungal (Candida albicans, Rhizopus sp., Aspergillus niger
and Aspergillus flavus) pathogens in vitro by conventional
twofold serial dilution method [49]. The obtained results
were compared to Ciprofloxacin and Amphotericin B which
were used as the standard drugs for bacterial and fungal
strains, respectively. The antibacterial and antifungal potencies
Fig. 5. HSQC spectrum of 13c.

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
583
R2
which compound 16c recorded MIC at 50 mg/mL) while
against rest of the strains, the activity did not eliminate com-
pletely. This confirms the importance of methyl functionality
at C-5 position besides another methyl group at C-3 for the an-
tibacterial profile of 2,6-diarylpiperidin-4-one derivatives. Fur-
thermore, substitution of bulkier alkyl groups viz. ethyl
(compound 14c) or isopropyl (compound 15c) group in place
of methyl in 13c exerted moderate activity. Compared to 13c,
14c and 15c showed twofold added activity against K. pneu-
moniae and S. typhi, respectively, whereas against E. coli,
the activity was retained as such.
O
H
R
H
H
N
H3C
N
R1
N
R
H
O
R2
O
O
H
R
N
N
N
H3C
R1
H
Compounds 17c and 18c obtained due to the replacement of
phenyl groups in 13c and 16c, respectively, by para-
chlorophenyl group are significant in inhibiting the growth
of all the tested bacterial strains, particularly, 17c against P.
aeruginosa and 18c against S. aureus and K. pneumoniae are
most superior (6.25 mg/mL) while towards remaining bacterial
strains, their potencies fall in between 12.5 and 50 mg/mL.
Moreover, impotency of 13c and 14c even at a maximum con-
centration of 200 mg/mL towards certain selected bacterial
strains can remarkably be alleviated by the simple structural
modification carried out in compounds 17c and 18c.
H
R
H
Conformation A for compounds 16c,18c,20c and 21c
H
R
H
H
O
N
H3C
N
CH3
N
H
R
H
O
O
H
R
Substitution of para-methoxyphenyl group instead of phe-
nyl ring in 13c (compound 19c) showed two- to threefold
increased activity against all the tested bacterial strains except
against S. typhi for which no change in activity was noted. The
same kind of phenyl group substitution in 16c (compound 20c)
registered 200% improved inhibition potency for S. aureus and
E. coli whereas 100e200% decreased activity was noticed in
S. typhi and K. pneumoniae. Introduction of para-methyl-
phenyl groups at C-2 and C-6 positions of piperidone ring
system in 16c (compound 21c) registered onefold increased
activity towards P. aeruginosa while for rest of the organisms,
the same activity was retained.
N
O
N
N
R
H3C
H
CH3
H
H
H
Conformation B for compounds 13c,17cand 19c
Ph
H
H
H
H
R1
N
H3C
N
O
N
O
Ph
H
Among the compounds tested for their inhibition potency
towards the chosen bacterial strains, 18c, 20c against S.
aureus, 18c against E. coli, 17c against P. aeruginosa, S. typhi,
16c, 18c and 21c against K. pneumoniae recorded elevated
activity compared to the standard Ciprofloxacin drug whereas
the MIC values of 17c, 20c against E. coli, 18c against P.
aeruginosa, 15c, 18c against S. typhi, 17c and 20c against K.
pneumoniae were at par with the activity of the standard drug.
Ph
O
H
H
R1
N
N
N
H3C
O
H
H
Ph
Conformation C for compounds 14c and 15c
H
Fig. 6. Different conformations of compounds 13ce21c.
2.3.2. Antifungal activity
All the compounds were assessed to elicit their antifungal
potency against the selected fungal strains and their MIC
values fall in the range of 6.25e200 mg/mL. Compound 13c
with methyl group at C-3 position failed to exhibit inhibition
potency even at a maximum concentration of 200 mg/mL
against C. albicans and Rhizopus sp. whereas a modest inhibi-
tion (100 mg/mL) was noticed against A. niger and A. flavus.
Replacement of methyl by ethyl group (compound 14c)
showed a positive response towards C. albicans (100 mg/mL)
only but it was inactive against Rhizopus sp. Besides, a com-
plete elimination of inhibitory potency against A. flavus and
onefold decreased activity against A. niger was noted in 13c
if methyl group is replaced by ethyl group (i.e., compound
of the synthesized compounds are reproduced in Tables 4 and
5, respectively.
2.3.1. Antibacterial activity
Among the compounds 13ce16c with unsubstituted phenyl
groups (at C-2 and C-6), compound 16c only recorded better
activity against S. typhi and K. pneumoniae at a MIC of 25
and 12.5 mg/mL, respectively, whereas the remaining com-
pounds showed very less activity. Removal of one of the
methyl groups at C-5 from 16c (compound 13c) completely
eradicates the antibacterial activity even at a high concentra-
tion of 200 mg/mL against S. aureus and P. aeruginosa (for

584
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
Table 4
In vitro antibacterial activities of compounds 13ce21c against selected bacterial strains (MIC in mg/mL)
Compound
R1
R2
R
Minimum inhibitory concentration (MIC)a in mg/mL
S. aureus
E. coli
P. aeruginosa
S. typhi
K. pneumoniae
13c
14c
Me
Et
H
H
>200b
100
200
50
100
>200
100
100
25
>200
200
100
50
200
100
50
200
50
H
H
15c
16c
i-Pr
Me
Me
Me
Me
Me
Me
H
H
200
12.5
25
Me
H
H
Cl
25
17c
18c
50
6.25
12.5
50
12.5
50
Me
H
Cl
6.25
100
12.5
50
12.5
50
6.25
100
25
19c
20c
OMe
OMe
Me
200
100
25
Me
Me
25
50
25
21c
Ciprofloxacin
100
25
12.5
25
25
12.5
50
a
MIC is the lowest concentration of an antimicrobial agent that will significantly inhibit the visible growth of a microorganism after a period of incubation.
No inhibition even at 200 mg/mL.
b
14c). Due to the introduction of bulkier isopropyl group in
activity against C. albicans and Rhizopus sp. whereas only
onefold improvement was noted against A. niger and A. flavus.
However, compared to 17c, only 100% improvement was noted
in compound 18c against Rhizopus sp. whereas for rest of the
strains, an appreciable enhancement was not observed.
Replacement of chlorine by methoxy group in 17c (com-
pound 19c) retained the same inhibition potency towards A.
flavus while for the remaining strains, 200% decreased inhibi-
tion potency was noticed. However, the same type of inter-
change in 18c (compound 20c) augments its microbial
potency against C. albicans and A. flavus by reducing its
MIC value to half of its magnitude. But twofold decreased
activity was noted against Rhizopus sp. Compound 21c, which
resulted by means of substituting methyl functionality in place
of chlorine in 18c did not influence the control of the multipli-
cation of C. albicans and Rhizopus sp. whereas a distinguished
activity was noted against A. niger and A. flavus at an inhibi-
tion value of 12.5 mg/mL.
place of ethyl function (compound 15c), a striking decrease
in the inhibitory concentration (50 mg/mL) was noted in the
control of Rhizopus sp. against which compounds 13c and
14c were completely inactive. Similarly, it exhibited promis-
ing activity towards A. niger and A. flavus but onefold
decreased activity was noted against C. albicans. Furthermore,
insertion of methyl group at C-5 position in 13c (compound
16c) expressed prominent inhibitory activity for all the tested
organisms with an MIC ranging from 50 to 100 mg/mL. It is
worth mentioning here that among the phenyl (at C-2 and
C-6) substituted piperidone systems (compounds 13ce16c),
compound 16c only recorded moderate to better activity.
It is very surprising from Table 5 that incorporation of
chloro functionality at the para position of phenyl group in
13c (compound 17c) resulted in well-pronounced impact
over C. albicans and Rhizopus sp. by controlling their growth
at a very low MIC i.e., at 6.25 and 12.5 mg/mL, respectively.
Against A. niger and A. flavus, MIC value of 17c was de-
creased by about 1/4 and 1/2, respectively, compared to 13c.
The same kind of phenyl group modification on 16c (com-
pound 18c) also enhanced the biological activity remarkably
and MIC was noted in the range of 6.25e25 mg/mL. Com-
pared to 16c, compound 18c showed threefold elevated
Among the compounds explored for their antifungal poten-
cies, 17c, 20c against C. albicans, 18c against Rhizopus sp.,
21c against A. niger and 20c, 21c against A. flavus expressed
400% elevated potency compared to the standard drug used
whereas rest of the compounds showed moderate to poor
activity.
Table 5
In vitro antifungal activities of compounds 13ce21c against selected fungal strains (MIC in mg/mL)
Compound
R1
R2
R
Minimum inhibitory concentration (MIC)a in mg/mL
C. albicans
Rhizopus sp.
A. niger
A. flavus
13c
14c
Me
Et
H
H
>200b
100
200
100
6.25
12.5
25
>200
>200
50
100
200
100
50
100
>200
100
50
H
H
15c
16c
i-Pr
Me
Me
Me
Me
Me
Me
H
H
Me
H
H
Cl
50
17c
18c
12.5
6.25
50
25
25
50
25
Me
H
Cl
19c
20c
OMe
OMe
Me
100
25
50
Me
Me
6.25
50
25
25
25
12.5
12.5
50
21c
Amphotericin B
12.5
50
25
a
MIC is the lowest concentration of an antimicrobial agent that will significantly inhibit the visible growth of a microorganism after a period of incubation.
No inhibition even at 200 mg/mL.
b

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
585
2.4. Analgesic activity
activity at all the doses. Replacement of chlorine in 17c by
methoxy function (compound 19c) also resulted in diminished
analgesic potency at 30 (48.2%) and 90 mg/kg (48.0%)
whereas at 60 mg/kg, the same potency (70.3%) was retained.
However, when compared to aspirin, a notable enhancement in
inhibition of writhing was observed in 19c.
Compounds 20c and 21c which resulted out of incorpora-
tion of methoxy and methyl functions, respectively, instead
of chlorine in 18c registered a drastic improvement in activity
at 30 and 60 mg/kg, while at 90 mg/kg a decline in analgesic
property was observed. In all the cases, the para substituted
phenyl bearing compounds display improved inhibition
potency compared to their analogues, the unsubstituted phenyl
bearing compounds.
Therefore, from this study the importance of the structural
units such as chlorine, methoxy or methyl function at the para
position of the phenyl groups besides methyl group at C-3 is
confirmed for the observed antinociceptive profile associated
with 17c and 19c. Besides, though these compounds exhibited
an elevated activity in all the doses, the inhibition of writhing
response was proved to be superior at 60 mg/kg in 17c
(70.4%), 19c (70.3%), 20c (59.1%) and 21c (54.6%) compared
to aspirin (57.9%) and was considered as an optimum dose to
execute antinociceptive activity.
Analgesic activity of the compounds 13ce21c was deter-
mined in a dose-dependent manner by acetic acid induced
writhing test in mice. The visceral pain induced by acetic
acid is due to the release of arachidonic acid via cyclohexage-
nase and prostaglandin (PG). The test compounds adminis-
tered orally (p.o.) at 30, 60 and 90 mg/kg (body weight) 1 h
prior to the administration of acetic acid showed a significant
reduction in writhing movement. Analgesic profiles of the
target compounds are given in Table 6.
It is clearly inferred from Table 6 that analgesic potency
estimated by classical acetic acid induced constriction for
certain compounds is comparatively equal or highly related
to the reference compound at all the three different doses.
The compounds with phenyl groups at C-2 and C-6 positions
of piperidone ring (13ce16c) did not exert appreciable activity
in all the three doses. These four compounds showed only
1.2e24.1% analgesic effect.
To our great surprise, introduction of chloro functionality at
the para position of phenyl framework in 13c (compound 17c)
produced statistically significant antinociceptic activity among
the three doses. Further, this activity is also superior to the
standard drug used at the same concentration. However,
a strong beneficial effect over analgesic profile was noted at
60 mg/kg (70.4%) than 30 (49.2%) and 90 mg/kg (61.6%)
doses. This expressive enhancement of the analgesic activity
has indicated us the pharmacophoric character of para-
chlorophenyl framework for the analgesic profile in this new
series of compounds.
2.5. Antipyretic activity
In the present investigation, hyperthermia was induced by
subcutaneous administration of brewer’s yeast in normal
saline. Pretreatment with test compounds at two different
doses (30 and 60 mg/kg) shows a better antipyretic activity
in a dose-dependent manner at different time intervals relative
to the standard indomethacin drug. The obtained results are
furnished in Table 7. A close survey of Table 7 shows that
compounds with para substituted phenyl groups at C-2 and
C-6 positions of piperidone ring system were found to be
equi-potent to indomethacin and even possess significant
activity compared to the standard drug in the same pharmaco-
logical protocol. However, the activity of compounds 18c and
But the introduction of another methyl group at C-5 posi-
tion in 17c (compound 18c) showed relatively decreased
Table 6
Analgesic activity of compounds 13ce21c
S.
no
Compound
Writhing (mean ꢂ SEM)
30 mg/kg
60 mg/kg
90 mg/kg
1
13c
15.15 ꢂ 0.70**
(6.3)
16.92 ꢂ 0.01**
(15.6)
17.18 ꢂ 0.05**
(17.7)
2
14c
14.87 ꢂ 0.55**
(8.0)
19.14 ꢂ 0.28**
(4.5)
20.62 ꢂ 0.20**
(1.2)
19c with para-chloro (with methyl
at C-3 and C-5) and para-
methoxy (with methyl at C-3 only) phenyl groups, respec-
tively, exhibited an excellent antipyretic activity at both the
concentrations compared to the standard drug. Further, these
compounds showed similar pharmacokinetic profile relative
to indomethacin as evidenced by the onset of hypothermic ef-
fect immediately after 30 min and further it lasted over a period
of 150 min. Besides, although introduction of methyl group at
C-5 position in 19c (compound 20c) and replacement of me-
thoxy by methyl function at the para position of phenyl group
in 20c (compound 21c) showed a reduction in rectal tempera-
ture, their activity is less than that of indomethacin. From this
study, it is also clearly known that removal of substituents
from the phenyl groups significantly reduced the antipyretic
potency at both the concentrations throughout the period of
study. Among the compounds tested for their antipyretic activ-
ity, introduction of substituents in the phenyl group only reg-
istered enhanced activity and in particular compounds 18c and
3
15c
15.24 ꢂ 0.08**
(5.6)
16.41 ꢂ 0.80**
(18.1)
20.41 ꢂ 0.15**
(2.2)
4
16c
13.35 ꢂ 0.18**
(17.4)
15.21 ꢂ 0.56**
(24.1)
17.98 ꢂ 0.25**
(13.9)
5
17c
8.22 ꢂ 0.90**
(49.2)
5.94 ꢂ 0.76**
(70.4)
8.02 ꢂ 0.45**
(61.6)
6
18c
12.54 ꢂ 0.44**
(22.5)
12.72 ꢂ 0.52**
(36.5)
12.75 ꢂ 0.25**
(38.9)
7
19c
8.38 ꢂ 0.13**
(48.2)
5.96 ꢂ 0.79**
(70.3)
10.85 ꢂ 0.10**
(48.0)
8
20c
8.70 ꢂ 0.84**
(46.2)
8.19 ꢂ 0.79**
(59.1)
12.80 ꢂ 0.15**
(38.7)
9
21c
9.29 ꢂ 0.08**
(42.6)
9.09 ꢂ 0.56**
(54.6)
13.95 ꢂ 0.22**
(33.1)
10
11
Aspirin
Control
8.92 ꢂ 0.12*
(44.8)
8.43 ꢂ 0.18*
(57.9)
12.81 ꢂ 0.01*
(38.6)
16.17 ꢂ 0.70
20.04 ꢂ 0.06
20.87 ꢂ 0.25
Significant levels: ** P < 0.05, * P < 0.01 compared to control. The values in
the parentheses are the percentage of inhibition compared to control.

586
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
Table 7
Antipyretic activity of compounds 13ce21c
S. no.
Compound
Dose (mg/kg)
Body temperature ꢂ SEMa
30 min
60 min
90 min
120 min
150 min
1
2
3
4
5
6
7
8
9
13c
14c
15c
16c
17c
18c
19c
20c
21c
30
60
30
60
30
60
30
60
30
60
30
60
30
60
30
60
30
60
e
38.25 ꢂ 0.03
38.20 ꢂ 0.30
38.27 ꢂ 0.10
38.25 ꢂ 0.11
38.15 ꢂ 0.15
38.05 ꢂ 0.11
38.11 ꢂ 0.12
38.10 ꢂ 0.25
37.05 ꢂ 0.25
37.95 ꢂ 0.10
37.95 ꢂ 0.25
37.92 ꢂ 0.15
37.90 ꢂ 0.25
37.85 ꢂ 0.15
38.10 ꢂ 0.12
38.01 ꢂ 0.21
38.11 ꢂ 0.22
38.01 ꢂ 0.25
38.34 ꢂ 0.12
38.10 ꢂ 0.15
38.20 ꢂ 0.15
38.10 ꢂ 0.25
38.22 ꢂ 0.15
38.20 ꢂ 0.10
38.10 ꢂ 0.20
38.01 ꢂ 0.10
38.00 ꢂ 0.20
37.95 ꢂ 0.15
37.96 ꢂ 0.20
37.92 ꢂ 0.50
37.90 ꢂ 0.15
37.85 ꢂ 0.25
37.80 ꢂ 0.15
37.70 ꢂ 0.25
38.01 ꢂ 0.15
38.95 ꢂ 0.10
38.02 ꢂ 0.15
38.95 ꢂ 0.50
38.30 ꢂ 0.21
37.90 ꢂ 0.10
38.10 ꢂ 0.20
38.02 ꢂ 0.03
38.15 ꢂ 0.03
38.10 ꢂ 0.12
38.01 ꢂ 0.15
38.95 ꢂ 0.25
38.95 ꢂ 0.10
37.90 ꢂ 0.23
37.89 ꢂ 0.20
37.85 ꢂ 0.25
37.80 ꢂ 0.15
37.75 ꢂ 0.11
37.60 ꢂ 0.25
37.50 ꢂ 0.15
37.95 ꢂ 0.25
37.90 ꢂ 0.22
37.95 ꢂ 0.25
37.90 ꢂ 0.15
38.90 ꢂ 0.03
37.80 ꢂ 0.15
38.03 ꢂ 0.01
38.01 ꢂ 0.06
38.01 ꢂ 0.02
38.95 ꢂ 0.15
38.96 ꢂ 0.20
38.90 ꢂ 0.15
38.85 ꢂ 0.15
37.70 ꢂ 0.25
37.70 ꢂ 0.15
37.65 ꢂ 0.25
37.60 ꢂ 0.15
37.55 ꢂ 0.20
37.50 ꢂ 0.10
37.30 ꢂ 0.20
37.80 ꢂ 0.15
37.85 ꢂ 0.25
37.90 ꢂ 0.15
37.80 ꢂ 0.25
38.20 ꢂ 0.01
37.70 ꢂ 0.20
38.01 ꢂ 0.05
38.95 ꢂ 0.02
38.98 ꢂ 0.10
38.90 ꢂ 0.11
38.90 ꢂ 0.50
38.75 ꢂ 0.10
38.70 ꢂ 0.25
37.55 ꢂ 0.15
37.65 ꢂ 0.15
37.50 ꢂ 0.15
37.40 ꢂ 0.25
37.30 ꢂ 0.50
37.20 ꢂ 0.20
37.01 ꢂ 0.20
37.70 ꢂ 0.15
37.60 ꢂ 0.25
37.70 ꢂ 0.25
37.60 ꢂ 0.15
38.20 ꢂ 0.25
37.50 ꢂ 0.25
10
11
a
Control
Indomethacin
5
Significant levels: P < 0.05, P < 0.01 compared to control, respectively, for test compounds and reference drug.
19c showed superior activity as they were expected to inhibit
the synthesis of prostaglandin thereby inducing the hypothal-
amus to regulate the body temperature.
In order to probe structural requirements for optimal anti-
microbial, analgesic and antipyretic activities in this series
of compounds, the size of the substituents attached at C-3
and/or C-5 positions besides introducing electron withdrawing
or donating functional groups at para position of the phenyl
framework was examined in detail.
3. Conclusion
From the close survey of the antibacterial and antifungal re-
sults against a panel of microbial strains, the following SAR
can be concluded and is also revealed from the bar graphs
(Figs. 7 and 8). The antibacterial efficacy of compounds 17c
and 18c with para-chlorophenyl groups (C-2 and C-6) along
with methyl at C-3 and/or C-5 towards P. aeruginosa and S.
aureus/K. pneumoniae, respectively, is greater when compared
to the structurally analogous non-chlorine substituted com-
pounds. Besides, elevated antifungal activities are noted in
18c and 20c bearing para-chloro and para-methoxy functions,
respectively, at the phenyl ring besides methyl at C-3 and C-5
positions against all the tested fungal strains. However, the
All the novel target molecules were synthesized by the
direct nucleophilic substitution of N-methylpiperazine with
the corresponding N-chloroacetyl amides derived from 2,6-di-
arylpiperidin-4-ones. The complete one- and two-dimensional
NMR spectral studies reveal that piperazine moiety in all the
target molecules adopts chair conformation with equatorial
orientation of methyl group irrespective of the various non-
chair conformations adopted by the piperidone framework.
Moreover, broadening of the benzylic protons (at C-2 and C-6)
confirms the existence of restricted rotation in the molecules
and also the in plane nature of piperazinoacetyl moiety with
the dynamically averaged plane of the piperidone ring.
450
Staphylococcus aureus
Escherichia coli
400
350
300
250
200
150
100
50
Pseudomonas aeruginosa Salmonella typhi
Kleb siella pneumoniae
0
13c
14c
15c
16c
17c
Compound
18c
19c
20c
21c
Fig. 7. Comparison of antibacterial potency of compounds 13ce21c with Ciprofloxacin.

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
587
450
400
350
300
250
200
150
100
50
Rhizopus sp.
Candida albicans
Aspergillus niger
Aspergillus flavus
0
13c
14c
15c
16c
17c
Compound
18c
19c
20c
21c
Fig. 8. Comparison of antifungal potency of compounds 13ce21c with Amphotericin B.
activity of 18c and 20c against Rhizopus sp. and C. albicans,
respectively, is remarkable. Further, the above said compounds
possess significantly higher activity compared to the standards
used in the same experimental protocol. The physicochemical
properties like relative hydrophobicity, charge and molecular
mass are expected to be the important characteristics for the
penetration into the cell wall and execute different effects on
the microbial strains. Moreover, conformations of the com-
pounds may also play a crucial role in eliciting good biological
responses. Therefore, the poor potency of the compounds with
bulkier substituents at the C-3 position may be related to their
stereochemical influence which in turn may affect the better
penetration of the N-(N-methylpiperazinoacetyl)-2,6-diarylpi-
peridin-4-one framework into the cell walls of the respective
strains. The elevated antimicrobial profiles of 17c, 18c and
20c may perhaps be due to their appropriate size, lipophilicity,
electronic effects of the substituents in the piperidone core
structure and the conformations adopted by the respective
compounds.
Similarly, analgesic potency of the compounds 13ce21c is
clearly revealed from the pictorial representation in Fig. 9. The
compounds 17c and 19c exhibited a noticeable analgesic activ-
ity at all the three doses. However, the activity at 60 mg/kg
(z70%) was found to be superior among others. It is consid-
ered that these compounds effectively reduced the wave of
constriction and elongation passing caudally along the abdom-
inal wall with twisting of truck and extension of the hind limb
in mice due to nociceptive property of acetic acid. Besides, the
analgesic property appears to be peripherally mediated and
could result from the combined inhibition effects of
prostaglandin and acetylcholine. In the case of antipyretic ac-
tivity, compounds 18c and 19c were endowed with a significant
potency at both the doses during the period of study. Here also,
the better hypothermic activity of the compounds may be due
to the inhibition of prostaglandin synthesis in the central
nervous system thereby reducing the rectal temperature. The
antinociceptic and antipyretic activities of these compounds
are better than the standard drugs used. In these in vivo studies
also the compounds without substitution in the phenyl group
did not exert an appreciable pharmacological activity.
From the preliminary microbiological and pharmacological
studies, it is concluded that presence of para substituted
phenyl groups at C-2 and C-6 positions besides methyl group
at C-3 and/or C-5 positions are the key factors in eliciting ben-
eficial biological profiles. Hence, among the nine compounds
used for this study, 18c and 19c are considered to be potent
candidates with promising biological and pharmacological
properties. Moreover, development of this class of compounds
may lead to some interesting chemical entities with improved
biological and pharmacological profiles than the standard
drugs. Therefore, this class of compounds may be used as tem-
plates to generate better drugs to combat microbial infections
and to alleviate pain and fever.
4. Experimental
4.1. General
The course of the reactions and purity of the products were
assessed by performing TLC. All the reported melting points
80
30 mg 60 mg 90 mg
70
60
50
40
30
20
10
0
13c
14c
15c
16c
17c
18c
Compound
19c
20c
21
c
Aspirin
Fig. 9. Comparison of analgesic potency of compounds 13ce21c with the control at three different doses.

588
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
were taken in open capillaries and are uncorrected. IR spectra
were recorded in AVATAR-330 FT-IR spectrophotometer
(Thermo Nicolet) and only noteworthy absorption levels
(reciprocal centimeters) are listed. 1H NMR spectra were
recorded at 400 MHz on BRUKER AMX 400 MHz spectro-
photometer using CDCl3 as solvent and TMS as internal stan-
dard. 13C NMR spectra were recorded at 100 MHz on
BRUKER AMX 400 MHz spectrophotometer in CDCl3.
1He1H COSY and one-bond 1He13C correlation spectra
were recorded on BRUKER DRX 500 MHz NMR spectrome-
ter using standard parameters. The chemical shift values were
reported in ppm (parts per million) and the spin multiplicities
are indicated as follows: s (singlet), br s (broad singlet), d (dou-
blet), dd (double doublet), t (triplet) and m (multiplet). 0.05 M
solution of the sample in CDCl3 was used for recording two-
dimensional NMR spectra. The tubes used for recording NMR
spectra were of 5 mm diameter. Mass spectra were recorded on
Jeol SX-102 (EI) and microanalyses were performed on Her-
aeus Carlo Erba 1108 CHN analyzer. Unless otherwise stated,
all the reagents and solvents used were of high grade and pur-
chased from Fluka and Merck. They were used as received
without any further purification.
was purified by column chromatography using n-hexane:ethyl
acetate mixture (4:1) as eluent.
4.3.1. Synthesis of N-(N-methylpiperazinoacetyl)-
3-methyl-2,6-diphenylpiperidin-4-one (13c)
By adopting the above general method, compound 13c was
obtained as pale white solid from 13b (1 g, 2.47 mmol) and
NMP (0.247 g, 2.47 mmol). IR (KBr) (cmꢀ1): 3058, 3028,
2935, 2845, 2818, 2791, 2694 (CeH stretching), 1713
(C]O stretching), 1642 (NeC]O stretching), 1498, 1453,
1415, 1347, 1289, 1232, 1163, 1135, 1079, 1015, 945, 908,
829, 747, 697, 650, 531, 463, 417. Mass (m/z): 406
(M þ H)þ, 332, 303, 280, 233, 202, 171, 131, 100 (100%),
1
77. H NMR (d ppm): 2.49 (br s, 8H, H-a and H-b protons
of NMP), 2.33 (s, 3H, CH3 of NMP), 3.02e3.28 (m, 4H, H-3a,
H-5a and NeCOCH2), 2.81 (dd, 2J5a,5e ¼ 17.99 Hz,
3J5e,6a ¼ 5.91 Hz, 1H, H-5e), 5.64 (br s, 1H, H-2), 6.05 (br s,
1H, H-6), 7.09e7.41 (m, 10H, phenyl protons), 1.12 (d,
J ¼ 5.01 Hz, 3H, CH3 at C-3). 13C NMR (d ppm): 55.00
(C-a carbons of NMP), 53.45 (C-b carbons of NMP), 62.45
(NeCOCH2), 60.89 (C-2), 53.45 (C-6), 45.99 (C-3), 43.28
(C-5), 209.77 (C]O at C-4), 171.44 (N-C]O), 141.11
(C-20 ipso), 141.48 (C-60 ipso), 126.79, 127.59, 127.69,
127.82, 128.49, 128.88 (other phenyl carbons), 45.99 (CH3
of NMP), 14.09 (CH3 at C-3).
By adopting the literature precedent [44], 2,6-diarylpiperi-
din-4-ones 13ae21a were prepared by the condensation of
appropriate ketones, aldehydes and ammonium acetate in
1:2:1 ratio using ethanol as solvent.
4.3.2. Synthesis of N-(N-methylpiperazinoacetyl)-3-ethyl-2,
6-diphenylpiperidin-4-one (14c)
4.2. General procedure for the synthesis
By adopting the above general method, compound 14c was
obtained as pale white solid from 14b (1 g, 2.38 mmol) and
NMP (0.238 g, 2.38 mmol). IR (KBr) (cmꢀ1): 3062, 3028,
2945, 2845, 2791, 2695 (CeH stretching), 1706 (C]O
stretching), 1643 (NeC]O stretching), 1560, 1498, 1452,
1414, 1348, 1286, 1229, 1162, 1133, 1077, 1012, 914, 830,
759, 696, 651, 532, 485. 1H NMR (d ppm): 1.05 (t, 3H,
J ¼ 7.32 Hz, CH2CH3 at C-3), 1.61e1.74 (m, 2H, CH2CH3
at C-3), 2.35 (s, 3H, CH3 of NMP), 2.55 (br s, 8H, H-a and
H-b protons of NMP), 2.67 (2J5a,5e ¼ 17.35 Hz,
3J5a,6a ¼ 9.15 Hz, 1H, H-5a), 2.99e3.04 (m, 2H, NeCOCHH,
H-3a), 3.18 (d, J ¼ 14.06 Hz, 1H, NeCOCHH), 5.70 (br s, 1H,
H-6), 6.23 (br s, 1H, H-2), 7.03e7.26 (m, 10H, phenyl pro-
tons). 13C NMR (d ppm): 11.83 (CH2CH3 at C-3), 23.32
(CH2CH3 at C-3), 44.27 (C-5), 45.52 (CH3 of NMP), 52.57
(C-3), 53.08 (C-b carbons of NMP), 54.66 (C-a carbons of
NMP), 55.92 (C-2 and C-6), 62.05 (NeCOCH2), 126.41,
127.46, 127.74, 128.45, 128.75, 129.13 (other phenyl car-
bons), 141.24 (C-20 ipso), 141.69 (C-60 ipso), 171.51 (Ne
C]O), 209.51 (C]O at C-4).
of N-chloroacetyl-2,6-diarylpiperidin-4-ones (13be21b)
To a well-stirred solution of 3-methyl-2,6-diphenylpiperi-
din-4-one 13a (1 equiv.) and triethylamine (1 equiv.) in
30 mL of dry benzene, chloroacetyl chloride (1 equiv.) in
20 mL of benzene was added dropwise through the addition
funnel for about half an hour. Stirring was continued with
mild heating using a magnetic stirrer. After the completion
of reaction, it was poured into water and extracted with ether
in three 50 mL portions. The combined ether extract was then
washed well with 3% sodium bicarbonate solution and dried
over anhydrous sodium sulphate. This upon evaporation and
subsequent recrystallization in distilled ethanol furnished the
compound 13b in pure form with good yield. The compounds
14be21b were also synthesized similarly.
2
3J5e,6a ¼ 5.73 Hz, 1H, H-5e), 2.88 (dd, J5a,5e ¼ 16.96 Hz,
4.3. General procedure for the synthesis of
N-(N-methylpiperazinoacetyl)-2,6-diarylpiperidin-4-ones
(13ce21c)
A
mixture of N-chloroacetyl-2,6-diarylpiperidin-4-one
(1 equiv.), triethylamine (1 equiv.) and N-methylpiperazine
[NMP] (1 equiv.) in toluene was refluxed for about 8e10 h.
After the completion of reaction, excess of solvent was re-
moved 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 sulphate. The residue thus obtained
4.3.3. Synthesis of N-(N-methylpiperazinoacetyl)-
3-isopropyl-2,6-diphenylpiperidin-4-one (15c)
By adopting the above general method, compound 15c was
obtained as pale red solid from 15b (1 g, 2.31 mmol) and NMP
(0.231 g, 2.31 mmol). IR (KBr) (cmꢀ1): 3060, 3032, 2973,
2943, 2815, 2789, 2693 (CeH stretching), 1702 (C]O
stretching), 1642 (NeC]O stretching), 1498, 1453, 1412,

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
589
1346, 1285, 1228, 1163, 1131, 1081, 1014, 914, 829, 754, 697,
1
129.17 (other aryl carbons), 133.61 (C-20000 ipso), 133.78
(C-60000 ipso), 139.29 (C-20 ipso), 139.51 (C-60 ipso), 171.11
(NeC]O), 208.56 (C]O at C-4).
651, 531, 498. H NMR (d ppm): 1.06 {d, J ¼ 6.72 Hz, 3H,
0
0
00
00
[CH(CH3 )(CH3 )] at C-3}, 1.11 {d, J ¼ 6.44 Hz, 3H,
[CH(CH3 )(CH3 )] at C-3}, 2.02e2.06 [m, 1H, CH(CH3)2 at
C-3], 2.43 (s, 3H, CH3 of NMP), 2.67 (br s, 8H, H-a and H-
4.3.6. Synthesis of N-(N-methylpiperazinoacetyl)-3,5-
b
protons of NMP), 2.71 (dd, 2J5a,5e ¼ 16.89 Hz,
dimethyl-2,6-bis( p-chlorophenyl) piperidin-4-one (18c)
By adopting the above general method, compound 18c was
obtained as pale reddish solid from 18b (1 g, 2.05 mmol) and
NMP (0.205 g, 2.05 mmol). IR (KBr) (cmꢀ1): 2979, 2937,
2833, 2798, 2738, 2694 (CeH stretching), 1716 (C]O
stretching), 1646 (NeC]O stretching), 1491, 1456, 1386,
1290, 1211, 1134, 1090, 1011, 980, 930, 828, 798, 727, 727,
3J5e,6a ¼ 6.14 Hz, 1H, H-5e), 2.83 (dd, J5a,5e ¼ 16.40 Hz,
3J5a,6a ¼ 8.56 Hz, 2H, H-3a and H-5a), 2.98 (br s, 1H,
NeCOCHH ), 3.27 (d, J ¼ 14.56 Hz, 1H, NeCOCHH), 5.54
2
(br s, 1H, H-6), 6.54 (br s, 1H, H-2), 6.95e7.19 (m, 10H, phe-
13
0
00
nyl protons). C NMR (d ppm): 20.31 [CH(CH3 )(CH3 ) at
0
00
C-3], 21.73 [CH(CH3 )(CH3 ) at C-3], 28.84 (CH(CH3)2 at
C-3), 44.23 (C-5), 45.88 (CH3 of NMP), 53.50 (C-b carbons
of NMP), 54.96 (C-a carbons of NMP), 54.96 (C-3), 56.42
(C-6), 58.39 (C-2), 62.25 (NeCOCH2), 126.41, 127.22,
127.81, 128.26, 128.54 (other phenyl carbons), 141.29 (C-20
and C-60 ipso), 171.32 (NeC]O), 209.56 (C]O at C-4).
1
650, 542, 516, 464. H NMR (d ppm): 1.06 (d, J ¼ 6.91 Hz,
6H, CH3 at C-3 and C-5), 2.36 (s, CH3 of NMP), 2.55 (br s,
8H, H-a and H-b protons of NMP), 3.02e3.13 (m, 4H,
H-3a, H-5a and NeCOCH2), 5.44 (br s, 2H, H-2 and H-6),
7.11 (d, 4H, aryl protons meta to chlorine), 7.30 (d, 4H, aryl
protons ortho to chlorine). 13C NMR (d ppm): 14.11 (CH3 at
C-3 and C-5), 45.47 (C-3 and C-5), 45.47 (CH3 of NMP),
52.95 (C-b carbons of NMP), 54.48 (C-a carbons of NMP),
60.41 (C-2 and C-6), 61.92 (NeCOCH2), 128.93, 129.09
(other aryl carbons), 133.89 (C-20000 and C-60000 ipso), 139.68
(C-20 and C-60 ipso), 171.59 (NeC]O), 210.25 (C]O at
C-4).
4.3.4. Synthesis of N-(Nemethylpiperazinoacetyl)-3,5
-dimethyl-2,6-diphenylpiperidin-4-one (16c)
By adopting the above general method, compound 16c was
obtained as grayish white semisolid from 16b (1 g,
2.38 mmol) and NMP (0.238 g, 2.38 mmol). IR (KBr)
(cmꢀ1): 3059, 3028, 2968, 2938, 2875, 2809 (CeH stretch-
ing), 1716 (C]O stretching), 1647 (NeC]O stretching),
1494, 1455, 1387, 1351, 1289, 1214, 1131, 1088, 1012, 918,
826, 762, 736, 704, 663, 624, 534, 422. Mass (m/z): 420
(M þ H)þ, 378, 353, 301, 280 (100%), 224, 202, 148, 133,
4.3.7. Synthesis of N-(N-methylpiperazinoacetyl)-3-methyl-
2,6-bis( p-methoxyphenyl)-piperidin-4-one (19c)
By adopting the above general method, compound 19c was
obtained as dark reddish semisolid from 19b (1 g, 2.15 mmol)
and NMP (0.215 g, 2.15 mmol). IR (KBr) (cmꢀ1): 3066, 2940,
2838, 2798 (C-H stretching), 1717 (C]O stretching), 1641
(NeC]O stretching), 1613, 1514, 1459, 1415, 1299, 1253,
1
106, 87, 77, 57. H NMR (d ppm): 2.50 (br s, 8H, H-a and
H-b protons of NMP), 2.32 (s, 3H, CH3 of NMP), 3.12 (t,
2H, H-3a and H-5a), 2.99 (s, 2H, NeCOCH2), 5.52 (br s,
2H, H-2 and H-6), 7.01e7.30 (m, 10H, phenyl protons),
1.06 (d, J ¼ 6.99 Hz, 6H, CH3 at C-3 and C-5). 13C NMR (d
ppm): 54.54 (C-a carbons of NMP), 52.99 (C-b carbons of
NMP), 61.88 (NeCOCH2), 60.85 (C-2 and C-6), 45.48 (C-3
and C-5), 211.35 (C]O at C-4), 171.77 (NeC]O), 141.39
(C-20 and C-60), 127.25, 127.74, 128.66 (other phenyl car-
bons), 45.48 (CH3 of NMP), 14.14 (CH3 at C-3 and C-5).
1
1181, 1137, 1032, 1082, 934, 835, 761, 557, 460. H NMR
(d ppm): 1.05 (d, J ¼ 5.18 Hz, CH3 at C-3), 2.34 (s, 3H,
CH3 of NMP), 2.55 (br s, 8H, H-a and H-b protons of
2
3
NMP), 2.74 (dd, J5a,5e ¼ 18.05 Hz, J5e,6a ¼ 6.03 Hz, 1H, H-
5e), 3.04e3.26 (m, 4H, H-3a, H-5a and NeCOCH2), 3.77,
3.79 (s, 6H, OCH3 at C-20000 and C-60000), 5.46 (br s, 1H, H-
2), 5.93 (br s, 1H, H-6), 6.75, 6.81 (2d, 4H, aryl protons ortho
to methoxy), 7.02, 7.10 (br s, 4H, aryl protons meta to me-
thoxy). 13C NMR (d ppm): 13.99 (CH3 at C-3), 43.25 (C-5),
45.59 (CH3 of NMP), 46.18 (C-3), 53.07 (C-b carbons of
NMP), 53.78 (C-6), 54.76 (C-a carbons of NMP), 55.24
(OCH3 at C-20000 and C-60000), 60.48 (C-2), 62.19 (Ne
COCH2), 113.86, 114.23, 128.06, 128.99 (other aryl carbons),
133.26 (C-20 ipso), 133.52 (C-60 ipso), 158.94, 159.06 (C-20000
and C-60000 ipso), 171.19 (NeC]O), 209.79 (C]O at C-4).
4.3.5. Synthesis of N-(N-methylpiperazinoacetyl)-
3-methyl-2,6-bis( p-chlorophenyl) piperidin-4-one (17c)
By adopting the above general method, compound 17c was
obtained as reddish semisolid from 17b (1 g, 2.11 mmol) and
NMP (0.211 g, 2.11 mmol). IR (KBr) (cmꢀ1): 2984, 2941,
2847, 2798 (CeH stretching), 1718 (C]O stretching), 1648
(NeC]O stretching), 1569, 1492, 1457, 1411, 1345, 1289,
1227, 1136, 1092, 1014, 940, 831, 753, 664, 545, 497, 472.
1H NMR (d ppm): 1.08 (d, J ¼ 6.82 Hz, CH3 at C-3), 2.37
(s, CH3 of NMP), 2.58 (br s, 8H, H-a and H-b protons of
4.3.8. Synthesis of N-(N-methylpiperazinoacetyl)-3,5
2
3
NMP), 2.82 (dd, J5a,5e ¼ 17.94 Hz, J5e,6a ¼ 6.19 Hz, 1H, H-
5e), 2.92e3.16 (m, 4H, H-3a, H-5a and NeCOCH2), 5.49
(br s, 1H, H-2), 5.96 (br s, 1H, H-6), 7.02, 7.11 (d, 4H, aryl
protons meta to chlorine), 7.20, 7.27 (2d, 4H, aryl protons or-
tho to chlorine). 13C NMR (d ppm): 14.23 (CH3 at C-3), 42.49
(C-5), 45.71 (CH3 of NMP), 45.95 (C-3), 53.23 (C-b carbons
of NMP), 54.05 (C-6), 54.79 and 54.97 (C-a carbons of NMP),
60.39 (C-2), 62.34 (NeCOCH2), 128.21, 128.57, 128.94,
-dimethyl-2,6-bis( p-methoxyphenyl) piperidin-4-one (20c)
By adopting the above general method, compound 20c was
obtained as reddish semisolid from 20b (1 g, 2.09 mmol) and
NMP (0.209 g, 2.09 mmol). IR (KBr) (cmꢀ1): 2938, 2837,
2798 (CeH stretching), 1713 (C]O stretching), 1635
(NeC]O stretching), 1607, 1513, 1458, 1397, 1295, 1250,
1179, 1135, 1031, 929, 834, 879, 654, 591, 539. H NMR (d
ppm): 1.05 (d, 6H, J ¼ 5.24 Hz, CH3 at C-3 and C-5), 2.31
1

590
G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
(s, 3H, CH3 of NMP), 2.51 (br s, 8H, H-a and H-b protons of
NMP), 2.99 (s, 2H, NeCOCH2), 3.09 (t, 2H, H-3a and H-5a),
3.79 (s, 6H, OCH3 at C-20000 and C-60000), 5.45 (br s, 2H, H-2 and
H-6), 6.82 (d, 4H, aryl protons ortho to methoxy), 7.08 (s, 4H,
aryl protons meta to methoxy). 13C NMR (d ppm): 14.13 (CH3
at C-3 and C-5), 45.47 (C-3 and C-5), 45.71 (CH3 of NMP),
53.02 (C-b carbons of NMP), 54.58 (C-a carbons of NMP),
55.28 (OCH3 at C-20000 and C-60000), 60.35 (C-2 and C-6),
61.96 (NeCOCH2), 114.03, 128.94 (other aryl carbons),
133.54 (C-20 and C-60 ipso), 159.09 (C-20000 and C-60000 ipso),
171.68 (NeC]O), 211.63 (C]O at C-4).
fungi. The minimum inhibitory concentrations (MICs) were
recorded by visual observations after 24 h (for bacteria) and
72e96 h (for fungi) of incubation. Ciprofloxacin and Ampho-
tericin B were used as standards for bacterial and fungal stud-
ies, respectively. The percentage of antimicrobial potency of
the test compounds compared to reference standard was calcu-
lated by adopting the following equation
Antimicrobial potencyð%Þ
MICðmg=mLÞof reference compound
¼
ꢃ 100
MICðmg=mLÞof test compound
4.3.9. Synthesis of N-(N-methylpiperazinoacetyl)-3,5
4.5. Pharmacology
-dimethyl-2,6-bis( p-methylphenyl) piperidin-4-one (21c)
Byadoptingtheabovegeneralmethod, compound21cwasob-
tained as pale reddish semisolid from 21b (1 g, 2.09 mmol) and
NMP (0.209 g, 2.09 mmol). IR (KBr) (cmꢀ1): 3033, 2973,
2935, 2875, 2800 (CeH stretching), 1716 (C]O stretching),
1644 (NeC]O stretching), 1514, 1455, 1383, 1290, 1166,
Albino mice and Wister rats of both sexes (pregnant
females excluded) weighing 20e25 g and 200e250 g, respec-
tively (unless otherwise specified), were used to test the
following pharmacological studies. All these animals were
provided by Raja Muthia Medical College Hospital (RMMCH)
and experiments were performed after getting clearance from
IAEC (Institutional Animal Ethical Committee) and also by
strictly adhering to the ethical guidelines. The provided ani-
mals were allowed to adopt the lab condition for two days be-
fore being used for the study and were fed with standard pellet
diet 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 six animals each.
1
1138, 1086, 1014, 981, 927, 824, 726, 656, 521, 575, 444. H
NMR (d ppm): 1.04 (d, J ¼ 6.36 Hz, 6H, CH3 at C-3 and C-5),
2.33 (s, 6H, CH3 at C-20000 and C-60000), 2.41 (s, 3H, CH3 of
NMP), 2.59 (br s, 8H, H-a and H-b protons of NMP), 3.00 (s,
2H, NeCOCH2), 3.06e3.11 (m, 2H, H-3a and H-5a), 5.45 (br
s, 2H, H-2 and H-6), 7.06e7.36 (m, 8H, aryl protons). 13C
NMR (d ppm): 14.12 (CH3 at C-3 and C-5), 20.89 (CH3 at
C-20000 and C-60000), 45.61 (C-3 and C-5), 45.79 (CH3 of NMP),
53.35 (C-b carbons of NMP), 54.87 (C-a carbons of NMP),
60.61 (C-2 and C-6), 62.16 (NeCOCH2), 127.71, 129.24 (other
aryl carbons), 137.36 (C-20000 and C-60000 ipso), 138.57 (C-20 and
C-60 ipso), 171.76 (NeC]O), 211.65 (C]O at C-4).
4.5.1. Analgesic activity
Antinociceptive activity of the series of compounds was
determined by acetic acid induced writhing test [50] in Albino
mice. The test compounds were administered at three different
doses i.e., at 30, 60 and 90 mg/kg (body weight) and aspirin
was chosen as reference drug at the same concentration of
the test compounds. All the compounds and standard drug
were suspended in 1% sodium carboxymethyl cellulose
(CMC) and administrated orally (p.o.). The writhing syndrome
in test animals was elicited by injecting 1% acetic acid in
normal saline (10 mL/kg body weight) intraperitoneally 1 h
after the oral administration of the test compounds. Complete
extension of either hind limb was regarded as a writhing
response. After 5 min of injection of noxious agent, the
number of writhes was counted during the subsequent
30 min by keeping the animals in flat surface. A significant
reduction in the number of writhes by drug treatments as
compared to vehicle control animals was considered as a pos-
itive 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 test compound treated group and
C is the number of writhes in control group.
4.4. Microbiology: in vitro antimicrobial activity
In vitro activities of the compounds were tested in Sabour-
auds dextrose broth (SDB, Hi-media, Mumbai) for fungi and
in nutrient broth (NB, Hi-media, Mumbai) for bacteria by two-
fold serial dilution method [49]. The test compounds were dis-
solved in dimethylsulphoxide (DMSO) to obtain 1 mg/mL
stock solutions. Seeded broth (broth containing microorgan-
isms) was prepared in NB from 24 h old bacterial cultures
on nutrient agar (Hi-media, Mumbai) at 37 ꢂ 1 ꢁC while fun-
gal spores from 1e7 days old Sabourauds agar slant cultures
were suspended in SDB. The colony forming units (cfu) of
the seeded broth were determined by plating technique and ad-
justed in the range of 104e105 cfu/mL. The final inoculum
size was 105 cfu/mL for antibacterial assay and 1.1e
1.5 ꢃ 102 cfu/mL for antifungal assay. Testing was performed
at pH 7.4 ꢂ 0.2 for bacteria (NB) and at pH 5.6 for fungi
(SDB). Exactly, 0.4 mL of the solution of the test compound
was added to 1.6 mL of seeded broth to form the first dilution.
One milliliter of this was diluted with a further 1 mL of seeded
broth to give the second dilution and so on till six such dilu-
tions were obtained. A set of assay tubes containing only
seeded broth was kept as control and likewise solvent controls
were also run simultaneously. The tubes were incubated in
BOD incubators at 37 ꢂ 1 ꢁC for bacteria and 28 ꢂ 1 ꢁC for
Percentage inhibition of writhing ¼ ½1 ꢀ T=Cꢄ ꢃ 100
The reported values were the average of six determina-
tions ꢂ SEM of n (6) animals per group. The observed results

G. Aridoss et al. / European Journal of Medicinal Chemistry 44 (2009) 577e592
591
[14] N. Rameshkumar, A. Veena, R. Ilavarasan, M. Adiraj,
P. Shanmugapandiyan, S.K. Sridhar, Biol. Pharm. Bull. 26 (2) (2003) 188.
[15] G. Aridoss, S. Balasubramanian, P. Parthiban, S. Kabilan, Eur. J. Med.
Chem. 42 (2007) 851.
were statistically analyzed by Student’s t-test and ANOVA
(one way). The *P value of <0.05 or <0.01 was considered
as statistically significant or highly significant, respectively.
[16] G. Aridoss, S. Balasubramanian, P. Parthiban, R. Ramachandran,
S. Kabilan, Med. Chem. Res. 16 (4) (2007) 188.
4.5.2. Antipyretic activity
[17] G. Aridoss, S. Balasubramanian, P. Parthiban, S. Kabilan, Eur. J. Med.
Chem. 41 (2006) 268.
Antipyretic activity of the target compounds was deter-
mined by measuring the variation in the body temperature as
per the reported procedure [51]. Fever was induced to fasted
Wister rats by administering 2% brewer’s yeast in normal
saline (10 mL/kg body weight) subcutaneously. Initial rectal
temperature was recorded using a lubricated digital thermom-
eter with a precision of 0.1 ꢁC by inserting into the rectum of
the animal. The same thermometer was used for all the ani-
mals in each group in order to minimize the possible experi-
mental error. After 18 h, the animals that showed an increase
of 0.3e0.5 ꢁC in rectal temperature were selected for this
study. The test compounds were administered orally (at
a dose of 30 and 60 mg/kg body weight) as a suspension in
1% CMC and indomethacin (5 mg/kg) was used as a reference
drug. Control groups received the vehicle (1% CMC) only.
After the administration of the test compounds, rectal temper-
ature was measured successively after 30, 60, 90, 120 and
150 min. The reported values were the average of six determi-
nations ꢂ SEM of n (6) animals per group. The observed
results were statistically analyzed by Student’s t-test and
ANOVA (one way) for a significance level of *P < 0.05 or
*P < 0.01.
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One of the authors SK is grateful to University Grants
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Research, New Delhi, India, for financial support in the
form of Major Research Projects. We also thank NMR
Research Centre, IISc, Bangalore, for the facilities provided
to record NMR spectra. GA wishes to thank Pukyong National
University for the financial support through the 2007 Post-Doc
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