Design, synthesis and evaluation of anticonvulsant activity of pyridinyl-pyrrolidones: A pharmacophore hybrid approach

Article abstract of DOI:10.1002/ardp.201100140

Various 1-[6-(4-substituted phenyl)-3-cyano-4-(substituted phenyl)-pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acids (3a-t) were designed and synthesized by clubbing pyrrolidinones and pyridines, the two active anticonvulsant pharmacophores. All the synthesized compounds fulfilled the requirements of suggested pharmacophoric model for anticonvulsant activity. Their in vivo anticonvulsant evaluation was performed by maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) tests. The minimal motor impairment was assessed by rotorod test and the estimation of various liver enzymes was performed to check the magnitude of liver toxicity posed by the synthesized compounds. Compounds 3d and 3k displayed comparable anticonvulsant activity to the standard drugs with ED₅₀ values of 13.4 and 18.6 mg/kg in electroshock screen, repectively. The compounds 3d and 3k were also found to have encouraging anticonvulsant activity (ED₅₀ = 86.1 and 271.6 mg/kg, respectively) in scPTZ screen. Interestingly, they did not show any sign of motor impairment at the maximum dose administered and were not toxic to the liver.

Full text of DOI:10.1002/ardp.201100140

Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
185
Full Paper
Design, Synthesis and Evaluation of Anticonvulsant
Activity of Pyridinyl-Pyrrolidones: A Pharmacophore
Hybrid Approach
Nadeem Siddiqui1, Waquar Ahsan1, M. Shamsher Alam1, Ruhi Ali1, and Kamna Srivastava2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar,
New Delhi, India
2 Center for Biomedical Research, University of Delhi, Delhi, India
Various 1-[6-(4-substituted phenyl)-3-cyano-4-(substituted phenyl)-pyridin-2-yl]-5-oxopyrrolidine-3-
carboxylic acids (3a–t) were designed and synthesized by clubbing pyrrolidinones and pyridines,
the two active anticonvulsant pharmacophores. All the synthesized compounds fulfilled the
requirements of suggested pharmacophoric model for anticonvulsant activity. Their in vivo
anticonvulsant evaluation was performed by maximal electroshock seizure (MES) and
subcutaneous pentylenetetrazole (scPTZ) tests. The minimal motor impairment was assessed by
rotorod test and the estimation of various liver enzymes was performed to check the magnitude
of liver toxicity posed by the synthesized compounds. Compounds 3d and 3k displayed comparable
anticonvulsant activity to the standard drugs with ED50 values of 13.4 and 18.6 mg/kg in electroshock
screen, repectively. The compounds 3d and 3k were also found to have encouraging anticonvulsant
activity (ED50 ¼ 86.1 and 271.6 mg/kg, respectively) in scPTZ screen. Interestingly, they did not show
any sign of motor impairment at the maximum dose administered and were not toxic to the liver.
Keywords: Anticonvulsants / Pharmacophore / Pyridinyl / Pyrrolidones / Synthesis
Received: April 11, 2011; Revised: June 6, 2011; Accepted: June 20, 2011
DOI 10.1002/ardp.201100140
Introduction
lactam ring determines the activity and the only positions
that are available to explore are the first and the fourth
position [8]. A series of imidazo [1,2-a] pyrrolo [2,3-c] pyridines
was synthesized and evaluated for their anti-BVDV activities
in MDBK cells. The synthesized different analogues com-
pounds showed significant anti-BVDV activities [9].
The extensive structural comparison of different clinically
available antiepileptic drugs (AEDs) revealed few pharmaco-
phoric elements that are essential for anticonvulsant activity.
One of the important core templates is a nitrogen hetero-
atomic system, usually a cyclic imide, at least one carbonyl
group and aryl or alkyl groups attached to the heterocyclic
system [1–3]. One of such heteroatomic cyclic imide system is
pyrrolidone which constitutes the basic pharmacophore of
several newer AEDs as shown in Figure 1. Pyrrolidone moiety
has been exploited previously [4–7] to get various derivatives
that have shown encouraging anticonvulsant activity. The
SAR studies showed that the position of substitution on the
In the present study, the first position of the pyrrolidone
system was thought to be substituted with a hydrophobic
pyridine ring system because pyridine ring itself is an anti-
convulsant pharmacophore [10–12]. Clubbing the two phar-
macophores is expected to have synergistic action on the
anticonvulsant activity and it has also been reported earlier
[13]. Pyridine moiety was further substituted with two phenyl
rings possessing different functionalities in order to impart
added lipophilicity to the resulting molecule. The fourth
position of the pyrrolidone ring was substituted with small
carboxylic acid group as the pyrrolidone carboxylic acid has
been known to have anticonvulsant properties [7].
Correspondence: Nadeem Siddiqui, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Hamdard University, Hamdard Nagar,
New Delhi-110062, India
E-mail: nadeems_03@yahoo.co.in
Fax: þ91 1126059688 x 5307
Carboxylic acid group also acts as a hydrogen acceptor/
donor system, which along with the nitrogen atom of pyridine
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186
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
O
O
O
malononitrile and ammonium acetate in ethanol to yield the
substituted biphenyl nicotinonitriles (2a–t) which in turn
refluxed with itaconic acid in water to afford 1-[6-(4-substi-
tuted phenyl)-3-cyano-4-(substituted phenyl)-pyridin-2-yl]-5-
oxopyrrolidine-3-carboxylic acids (3a–t). The physicochemi-
cal data are presented in Table 1.
F
N
N
N
H2N
H2N
H2N
F
O
O
O
Levetiracetam
Seletracetam
Brivaracetam
O
RC6H4
O
N
All the final compounds were characterized by different
1
spectral analytical techniques. The H NMR spectra showed
N
HN
OH
R'C6H4
CN
O
O
characteristic pattern of pyrrolidine moiety with two distinct
doublets of the two methylene protons in the range of 3.11–
3.27 and 4.40–4.49 ppm respectively. The methine proton of
the pyrrolidine nucleus was observed at 3.46–3.59 ppm. A
multiplet in the range of 6.76–8.11 ppm was assigned for the
aromatic protons. The hydroxyl group attached to the aro-
matic ring in respective compounds showed a singlet in the
range of 8.09–8.24 that was D2O exchangeable whereas the
hydroxyl group constituting the carboxylic acid functional-
ity was observed downfield in the range of 10.28–11.24 ppm.
The IR spectra further confirmed the structures of the com-
pounds and showed presence of sharp and intense peaks of
different functional groups present. The peak for phenolic
hydroxyl group was present in the range of 3534–3349 cmꢀ1
whereas that of carboxylic acid hydroxyl group was at 3412–
3143 cmꢀ1. The aromatic C–H stretching was observed in the
Compounds (3a-t)
Ethosuximide
Figure 1. Anticonvulsant agents having pyrrolidone system as
basic pharmacophore.
ring (electron donor) and phenyl rings (hydrophobic aryl ring)
fulfills the requirements of the suggested pharmacophore
model for anticonvulsant activity as shown in Figure 2.
Results and discussion
Synthesis
The synthesis of title compounds (3a–t) was carried out
according to Scheme 1. 4-Substituted acetophenones (1a–d)
were treated with substituted benzaldehydes in presence of
Figure 2. Figure showing anticonvulsant
agents possessing essential pharmacophoric
elements. HBA ¼ Hydrogen bond acceptor,
HBD ¼ hydrogen
bond
donor,
HAr ¼
hydrophobic aryl ring, PI ¼ positive ionizable
group, NI ¼ negative ionizable group.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
Synthesis and Pharmacology of Pyrrolidone Derivatives
187
R
R
R
(ii)
(i)
N
N
O
O
CH3
N
NH2
CN
CN
Scheme 1. Reagents and conditions: (i) Aryl
aldehydes, malononitrile, ammonium acetate,
ethanol; (ii) itaconic acid, 10% NaOH, water.
R'
R'
OH
(2a-t)
(3a-t)
O
(1a-d)
range of 3064–2979 cmꢀ1 and the C N stretching of the
nitrile group at 2298–2214 cmꢀ1. Two distinct peaks corre-
sponding to the two carbonyl functionalities were present in
the region 1726–1758 cmꢀ1 and 1687–1724 cmꢀ1 for the
ketonic and the carboxylic acid respectively.
was evaluated by rotorod method and the data are presented
in Table 2. Data for the reference compounds have been taken
from the literature.
In the phase I preliminary anticonvulsant screening, all
the compounds showed protection in MES screen which was
indicative of the good ability of these compounds to prevent
the seizure spread. Among these compounds, 3d, 3i, 3k and
3n showed protection from seizure at the lowest dose of
30 mg/kg after 0.5 h. Interestingly, 3d and 3k continued
the anticonvulsant activity after 4.0 h at the same dose
whereas compounds 3i and 3n also displayed protection after
4.0 h but at 100 mg/kg dose, which indicated the promising
nature of the compounds showing quick onset and long
duration of action at relatively lower doses. Compounds
Pharmacology
Anticonvulsant activity
The anticonvulsant activity was assessed by two most adopted
animal models electroshock (MES) and chemoshock (scPTZ)
methods. All the synthesized compounds were administered
intraperitoneally into mice using doses of 30, 100 and
300 mg/kg and the observations were taken at two different
time intervals (0.5 h and 4.0 h). Neurological impairment
Table 1. Physicochemical parameters of the title compounds (3a–t).
Compound
R
R0
Mol. Formulaa
(Mol. wt.)
CLog Pb
Rfc value
Elemental analyses % found (calculated)
C
H
N
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
NO2
NO2
NO2
NO2
NO2
H
H
C23H16ClN3O3 (417.84)
C23H16ClN3O4 (433.84)
C23H16ClN3O4 (433.84)
C23H15Cl2N3O3 (452.29)
C24H18ClN3O3 (431.87)
C23H16BrN3O3 (462.30)
C23H16BrN3O4 (478.29)
C23H16BrN3O4 (478.29)
C23H15BrClN3O3 (496.74)
C24H18BrN3O3 (476.32)
C23H16N4O5 (428.40)
C23H16N4O6 (444.40)
C23H16N4O6 (444.40)
C23H15ClN4O5 (462.84)
C24H18N4O5 (442.42)
C23H17N3O3 (383.40)
C23H17N3O4 (399.40)
C23H17N3O4 (399.40)
C23H16ClN3O3 (417.84)
C24H19N3O3 (397.43)
4.98
4.01
4.51
5.69
5.48
5.13
4.16
4.66
5.84
5.63
4.01
3.04
3.54
4.73
4.51
4.26
3.29
3.79
4.98
4.76
0.46
0.41
0.43
0.53
0.56
0.61
0.44
0.40
0.54
0.59
0.61
0.47
0.43
0.57
0.59
0.63
0.45
0.44
0.61
0.63
66.09 (66.11)
63.64 (63.67)
63.66 (63.67)
61.10 (61.08)
66.76 (66.75)
59.73 (59.76)
57.78 (57.76)
57.74 (57.76)
55.64 (55.61)
60.53 (60.52)
64.44 (64.48)
62.14 (62.16)
62.13 (62.16)
59.72 (59.68)
65.12 (65.15)
72.07 (72.05)
69.15 (69.17)
69.19 (69.17)
66.13 (66.11)
72.51 (72.53)
3.85 (3.86)
3.74 (3.72)
3.74 (3.72)
3.36 (3.34)
4.21 (4.20)
3.48 (3.49)
3.39 (3.37)
3.35 (3.37)
3.06 (3.04)
3.78 (3.81)
3.79 (3.76)
3.64 (3.63)
3.65 (3.63)
3.25 (3.27)
4.08 (4.10)
4.44 (4.47)
4.32 (4.29)
4.28 (4.29)
3.87 (3.86)
4.86 (4.82)
10.11 (10.06)
9.67 (9.69)
9.71 (9.69)
9.25 (9.29)
9.70 (9.73)
9.06 (9.09)
8.76 (8.79)
8.75 (8.79)
8.42 (8.46)
8.85 (8.82)
13.10 (13.08)
12.63 (12.61)
12.63 (12.61)
12.14 (12.10)
12.69 (12.66)
10.94 (10.96)
10.55 (10.52)
10.50 (10.52)
10.09 (10.06)
10.56 (10.57)
2-OH
4-OH
4-Cl
4-CH3
H
2-OH
4-OH
4-Cl
4-CH3
H
2-OH
4-OH
4-Cl
4-CH3
H
2-OH
4-OH
4-Cl
H
H
H
H
3t
4-CH3
a
Solvent of crystallization: ethanol.
Log P was calculated using software ACD Labs Software 2.0.
Solvent system: toluene/ethyl acetate/formic acid (5:4:1).
b
c
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Table 2. Anticonvulsant activity and minimal motor impairment data of the synthesized compounds (3a–t).
Compound
Intraperitoneal injection into micea
scPTZ screen
MES screen
Neurotoxicity screen
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
300
100
100
30
100
300
100
30
300
300
–
–
100
–
–
–
100
100
–
–
300
300
100
–
–
–
300
300
–
300
–
–
–
–
–
–
300
300
300
300
–
300
100
–
300
–
–
300
–
300
–
–
300
100
–
300
300
–
–
–
–
–
300
300
100
30
300
30
300
100
30
300
100
–
–
300
300
–
–
30
300
300
100
100
–
–
–
300
100
–
–
100
100
100
300
–
300
100
100
–
300
300
100
300
–
300
300
300
–
–
–
–
–
3p
3q
3r
3s
–
100
300
30
–
100
–
–
–
100
–
300
3t
300
100
–
Phenytoin
Ethosuximide
Phenobarbital
30
–
30
–
100
–
–
300
–
100
a
Number of animals used ¼ 6; solvent used: polyethylene glycol; doses of 30, 100 and 300 mg/kg were administered i.p. The figures in
the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of the mice. The animals were examined
at 0.5 h and 4 h after injections were administered.
b
The dash (—) indicates an absence of activity at maximum dose administered (300 mg/kg)
3b, 3c, 3h, 3m, 3p and 3s were found to be active in the MES
screen after 0.5 h at a dose of 100 mg/kg body weight. Among
these compounds 3c, 3m and 3s continued to protect from
seizures at the same dose after 4.0 h. Rest of all the com-
pounds showed the activity at the higher dose of 300 mg/kg.
In the chemoshock (scPTZ) screen, compounds that were
found to be active included 3b, 3c, 3d, 3g, 3h, 3i, 3k, 3l,
3m, 3n, 3o, 3q, 3r and 3s. Compounds that showed significant
anticonvulsant activity in the scPTZ screen being active at
100 mg/kg dose were 3c, 3d, 3m, 3n and 3s after 0.5 h.
Compounds 3d and 3n showed the promising nature by
continuing the protection from the seizures at the same dose
after 4.0 h. These compounds being active in both the screens
proved to be having broad spectrum of action in dealing
with the convulsions. Compounds 3c, 3m and 3s were
also found to be active after 4.0 h but at a higher dose of
300 mg/kg.
In the phase II anticonvulsant screening, the most active
compounds 3d and 3k were quantitatively evaluated for their
anticonvulsant activity (ED50) and the results are presented in
Table 3. Both the compounds showed comparable anticon-
vulsant activity to the standard drugs. Compound 3d dis-
played significant anti-MES activity with ED50 of 13.4 mg/
kg which was comparable to the currently used antiepileptic
drugs phenytoin and carbamazepine and much better than
Table 3. Phase II quantitative anticonvulsant evaluation in mice.
a
Compound
ED50
MES
scPTZ
3d
3k
13.4 (10.1–17.1)
18.6 (14.4–24.4)
9.5 (8.1–10.4)
8.8 (5.5–14.1)
21.8 (21.8–25.5)
86.1 (73.3–0.101.6)
271.6 (244.3–0.312.4)
>300
Phenytoin
Carbamazepine
Phenobarbital
>100
13.2 (5.8–15.9)
In the neurotoxicity screening, compounds that were
devoid of minimal motor impairment at any dose were 3b,
3c, 3k, 3m, 3n, 3p, 3r and 3s. Rest all the compounds showed
some sign of neurotoxicity but were less toxic than the stand-
ard drug phenytoin.
Number of animals used ¼ 10; solvent used: polyethylene glycol
(0.1 mL, i.p.).
Dose in milligrams per kilogram body mass.
Data in parentheses are the 95% confidence limits.
a
b
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Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
Synthesis and Pharmacology of Pyrrolidone Derivatives
189
that of phenobarbital. It also showed encouraging activity in
the scPTZ screen with ED50 value of 86.1 mg/kg indicating the
versatile nature of the compounds showing good activity in
more than one type of seizures. Nevertheless, compound 3k
was also found to have considerable anticonvulsant activity
in MES screen having ED50 value of 18.6 mg/kg and in scPTZ
screen having ED50 value of 271.6 mg/kg.
ineffective in the screen. In general, the bromo substituted
derivatives were found to be comparatively more neurotoxic
than the other compounds of the series and the effect of other
functional groups on the neurotoxicity was found to be not
significant. SARs were established according to Phase 1 tests.
It has been observed that the maximum potency of the
drugs that act on the central nervous system (CNS) is obtained
with congeners having an optimum lipophilicity (log Po). The
Clog P values were taken from ACD Labs Software 2.0. and
the results are shown in Table 1. Almost all the compounds
are found to be lipophilic and having potent anticonvulsant
activity in both MES as well as scPTZ test. Due to their higher
lipophilicity they are expected to have rapid onset and
shorter duration of action.
Designing of the compounds was made in a way to study
the structure-activity relationship of the series. Two phenyl
rings attached to the pyridyl moiety were substituted with
different functional groups to delineate the effect of the
resulting structure on the activity. The phenyl ring attached
to the sixth position of the pyridyl ring was either kept
unsubstituted or was substituted with electron withdrawing
groups of various sizes at the fourth position. The other
phenyl ring that was attached to the fourth position of the
pyridyl moiety was substituted with various electron releas-
ing functionalities at different positions or left unsubsti-
tuted. Substitutions at the phenyl ring attached to the
sixth position of the pyridyl moiety had a marked effect
on the anti-MES activity since the nitro substituted com-
pounds were the most active of the series followed by chloro,
bromo and the unsubstituted derivatives. On the other hand,
substitutions in the phenyl ring attached at fourth position
of the pyridyl moiety also seemed to be determining the
activity in the MES screen as the 4-chloro derivatives were
the most potent among all, irrespective of the substituent at
the other phenyl ring. In general the para substituted deriva-
tives were more effective than the meta ones since the com-
pounds with hydroxyl group at the fourth position were
more active than the compounds with hydroxyl group at
the second position. Substitutions by the methyl group at
fourth position markedly decreased the activity in all cases
followed by the unsubstituted derivatives. In the scPTZ screen
also, the nitro and chloro substituted derivatives were found
to be more effective than the bromo and the unsubstituted
derivatives. Substitutions at the other phenyl ring also
seemed to be important as the unsubstituted derivatives
and the 4-methyl substituted derivatives were almost
Acute toxicity studies
The acute toxicity studies were performed on the albino mice
in order to assess any mortality or toxic effects shown by the
synthesized compounds at increased doses and the data are
presented in Table 4. The most active compounds were dis-
solved in PEG-400 and different doses were administered
intraperitoneally to mice and the behavioral changes are
seen and compared with phenytoin, carbamazepine and
phenobarbital. Unlike the standard drugs, it was found that
both the tested compounds did not produce any mortality or
gross effect on the central nervous system at any of the dose
levels tested, i.e., 5, 25, 125, 250 and 400 mg/kg. However, the
animals have exhibited some of the toxic signs such as chew-
ing, licking, salivation followed by brief period of sedation.
There was not any toxicity seen in the vehicle alone as evident
in the control group of animals.
Liver enzymes estimation
Since some currently used antiepileptic drugs boast liver
toxicity as one of the restraint, the liver enzyme estimation
of the most active compounds 3d and 3k were also performed
to check the enormity of liver toxicity and the data are
presented in Table 5. The concentrations of alkaline phos-
phatase, SGOT and SGPT were determined in serum and the
Table 4. Acute toxicity study data of selected compounds.
Compounds
Number of animals dead/Total number of animals tested
Dose (mg/kg), i.p.
5
25
125
250
400
Control
3d
3k
Phenytoin
Carbamazepine
Phenobarbital
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
3/6
3/6
3/6
0/6
0/6
0/6
4/6
3/6
3/6
Vehicle used: polyethylene glycol (0.4 mL, i.p.)
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Table 5. Liver enzymes estimation of selected compounds.
Treatment Alkaline phosphatase (Mean W SEM)
SGOT level (Mean W SEM)
SGPT level (Mean W SEM)
Control
3d
3k
22.82 ꢁ 0.542
19.84 ꢁ 0.421
29.11 ꢁ 0.716ꢂ
142.34 ꢁ 2.147
149.14 ꢁ 2.132
137.14 ꢁ 2.114
45.47 ꢁ 1.214
49.34 ꢁ 1.341
41.31 ꢁ 1.014
ꢂ
P <0.05. The mean levels of enzymes were calculated using ANOVA followed by Dunnett multiple comparison test.
values are represented as mean ꢁ SEM. Compound 3d
showed no any significant change in enzymes level as com-
pared to the control whereas, compound 3k was observed to
increase the alkaline phosphatase level moderately (P <0.05)
but the other values were not significant indicating the non-
toxic nature of the tested compounds.
In silico studies
Distance mapping
Comparison of the structures of the synthesized compounds
and other molecules with anticonvulsant activity were per-
formed to find out the structural elements essential for
action. The compounds selected for this comparison have
at least one aryl (R) hydrophobic domain, one electron donor
(D) and a hydrogen acceptor/donor unit (HAD). In an initial
Figure 3. Ortep diagram (50% probability) of 1-(3-cyano-4,6-diphenyl-
study, calculations on the basis of molecular mechanics, with
pyridin-2-yl)-5-oxopyrrolidine-3-carboxylic acid (3p) showing two
the force field based on CHARMM parameterization were
performed to obtain an overview on their minimum confor-
mation for bioactivity. The synthesized compounds were
examined to check whether they reflect the conditions of
the derived pharmacophore model. Analyses of the distance
relationship showed that synthesized compounds (3a–t) ful-
fill the essential demands of pharmacophore when compared
with other known anticonvulsant drugs. In case of the title
hydrogen bonding interactions (N. . .H) as dotted line.
Conclusion
In conclusion, the combined pharmacophore of pyrrolidone
and pyridine can be regarded as a new structural class of
anticonvulsants that have shown comparable anticonvulsant
activity to some standard drugs, with markedly lower neuro-
toxicity and hepatotoxicity. These compounds were found to
have diverse mode of action as they were effective in both the
seizure models. These can act as lead molecules for future
investigations and need to be explored further to get better
agents.
˚
compounds the distances R–D (4.2 A), R–HAD (7.7 A) and
˚
˚
D–HAD (4.8 A) were in conformity with the distances of
active anticonvulsant drugs that should be in the range of
˚
R–D (3.2–5.1 A), R–HAD (4.2–8.5 A) and D–HAD (3.9–5.5 A).
˚
˚
Three-dimensional structural analyses
The ORTEP diagram (50% probability) was drawn using the
software Ortep3v2 and the characteristics were studied. As
noted in Fig. 3 of the compound 3p, strong hydrogen
bonding was observed between the nitrogen atoms of the
nitrile moiety and the surrounding vinyl protons at two
places. First found between N15 and H36 where the distance
between the donor and the acceptor atom d(N. . .H) was found
Experimental
Chemistry
The melting points were determined in open capillary tubes in a
Hicon melting point apparatus and are uncorrected. The homo-
gen elemental analyses (C, H, N) of all compounds were per-
formed on the CHNS Elimentar (Analysen Systeme GmbH,
Germany) Vario EL III. All the Fourier transform infra red
(FT-IR) spectra were recorded in KBr pellets on a Jasco FT/IR
410 spectrometer. The 1H NMR spectra were taken on a
Bruker 400 Ultra shieldTM (400 MHz) NMR spectrometer.
Chemical shifts (d) are expressed in ppm relative to tetramethyl-
silane (TMS) as an internal standard. The purity of the
˚
to be 2.896 A. Second was observed between N15 and H46
˚
where (N. . .H) distance was found to be 3.005 A. These hydro-
gen bondings ensure the rings are planar. It was reported that
the planarity of molecules is an essential factor in determi-
nation of biological activity.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
Synthesis and Pharmacology of Pyrrolidone Derivatives
191
compounds was checked by thin layer chromatography (TLC)
on silica gel G (Merck) coated plates by using toluene/ethyl
acetate/formic acid (5:4:1) as solvent system. Iodine chamber
and UV lamp were used for the visualization of TLC spots.
The calculated log P values were taken from the ACD Labs
Software 2.0. and the results are shown in Table 1.
(d, 2H, COCH2), 6.86–7.91 (m, 9H, ArH), 11.14 (s, 1H, OH,
D2O exchangeable).
1-[6-(4-Chlorophenyl)-3-cyano-4-(4-methylphenyl)-
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acid (3e)
Yield 64%; mp 2328C; IR (KBr) cmꢀ1: 3219 (COO–H), 3056
–
(Ar-CHstr), 2917 (C-Haliph), 2219 (C N), 1714 (C O), 1702
–
–
(C OOH), 759 (C–Cl); 1H NMR (CDCl3): d 2.26 (s, 3H, CH3), 3.15
(d, 2H, NCH2), 3.53 (m, 1H, CHpyrr), 4.42 (d, 2H, COCH2), 6.89–7.94
(m, 9H, ArH), 10.86 (s, 1H, OH, D2O exchangeable).
General procedure for the synthesis of 2-amino-6-
(4-substituted phenyl)-4-(substituted phenyl)
nicotinonitriles (2a–t)
–
A mixture of various substituted acetophenones (1a–d, 0.01 mole)
and the appropriate aromatic aldehydes (0.01 mole) in
ethanol (40 mL) was treated with malonotrile (0.01 mole) and
ammonium acetate (2g). The reaction mixture was refluxed for
10 h, where a crystalline precipitate separated. The formed pre-
cipitate was filtered, washed with ethanol, dried and recrystal-
lized from acetic acid and ethanol.
1-[6-(4-Bromophenyl)-3-cyano-4-phenyl-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3f)
Yield 68%; mp 2788C; IR (KBr) cmꢀ1: 3312 (COO–H), 3016
–
–
(Ar-CHstr), 2298 (C N), 1743 (C O), 1714 (C OOH), 645 (C–Br);
–
–
1H NMR (CDCl3): d 3.16 (d, 2H, NCH2), 3.53 (m, 1H, CHpyrr), 4.42
(d, 2H, COCH2), 6.92–7.86 (m, 10H, ArH), 10.79 (s, 1H, OH,
D2O exchangeable).
General procedure for the synthesis of 1-
[6-(4-substituted phenyl)-3-cyano-4-(substituted phenyl)-
1-[6-(4-Bromophenyl)-3-cyano-4-(2-hydroxyphenyl)-
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acid (3g)
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acids (3a–t)
A mixture of substituted nicotinonitriles (2a–t, 0.5 mol) and
itaconic acid (0.6 mol) in 150 mL of water was heated at reflux
for 15 h, and then treated with 280 mL 10% NaOH. After cooling
to 208C the reaction mixture was filtered and the filtrate was
acidified with aq HCl to pH 1. Resulting precipitate formed was
filtered, washed with water, dried and recrystallized with etha-
nol to the get the final compounds (3a–t).
Yield 54%; mp 2498C; IR (KBr) cmꢀ1: 3457 (O–H), 3345 (COO–H),
–
3045 (Ar-CHstr), 2249 (C–N), 1734 (C–O), 1709 (C OOH), 657
–
(C–Br); 1H NMR (CDCl3): d 3.13 (d, 2H, NCH2), 3.57 (m, 1H,
CHpyrr), 4.40 (d, 2H, COCH2), 6.87–7.91 (m, 9H, ArH), 8.13 (s,
1H, OH, D2O exchangeable), 11.19 (s, 1H, OH, D2O exchangeable).
1-[6-(4-Bromophenyl)-3-cyano-4-(4-hydroxyphenyl)-
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acid (3h)
Yield 71%; mp 2588C; IR (KBr) cmꢀ1: 3416 (O–H), 3347 (COO–H),
1-[6-(4-Chlorophenyl)-3-cyano-4-phenyl-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3a)
–
3016 (Ar-CHstr), 2214 (C N), 1748 (C–O), 1721 (C OOH),
–
629 (C–Br); 1H NMR (CDCl3): d 3.15 (d, 2H, NCH2), 3.59 (m, 1H,
CHpyrr), 4.43 (d, 2H, COCH2), 6.91–7.96 (m, 9H, ArH),
8.17 (s, 1H, OH, D2O exchangeable), 11.21 (s, 1H, OH,
D2O exchangeable).
Yield 62%; mp 2748C; IR (KBr) cmꢀ1: 3412 (COO–H), 3045
–
–
(Ar-CHstr), 2236 (C N), 1734 (C O), 1712 (C OOH), 816 (C–Cl);
–
–
1H NMR (CDCl3): d 3.12 (d, 2H, NCH2), 3.48 (m, 1H, CHpyrr), 4.42
(d, 2H, COCH2), 6.87–7.83 (m, 10H, ArH), 10.28 (s, 1H, OH,
D2O exchangeable).
1-[6-(4-Bromophenyl)-4-(4-chlorophenyl)-3-cyano-pyridin-
2-yl]-5-oxopyrrolidine-3-carboxylic acid (3i)
1-[6-(4-Chlorophenyl)-3-cyano-4-(2-hydroxyphenyl)-
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acid (3b)
Yield 73%; mp 2678C; IR (KBr) cmꢀ1: 3289 (COO–H), 3014 (ArHstr),
2267 (C N), 1745 (C–O), 1724 (C–OOH), 867 (C–Cl), 649 (C–Br);
1H NMR (CDCl3): d 3.17 (d, 2H, NCH2), 3.52 (m, 1H, CHpyrr),
4.43 (d, 2H, COCH2), 6.91–7.99 (m, 9H, ArH), 10.86 (s, 1H, OH,
D2O exchangeable).
Yield 54%; mp 2458C; IR (KBr) cmꢀ1: 3534 (O–H), 3143 (COO–H),
–
–
3013 (Ar-CHstr), 2247 (C N), 1741 (C O), 1687 (C OOH), 798
–
–
(C–Cl); 1H NMR (CDCl3): d 3.15 (d, 2H, NCH2), 3.51 (m, 1H,
CHpyrr), 4.40 (d, 2H, COCH2), 6.84–7.94 (m, 9H, ArH), 8.12 (s,
1H, OH, D2O exchangeable), 11.12 (s, 1H, OH, D2O exchangeable).
1-[6-(4-Bromophenyl)-3-cyano-4-(4-methylphenyl)-
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acid (3j)
Yield 47%; mp 2378C; IR (KBr) cmꢀ1: 3324 (COO–H), 3045
(Ar-CHstr), 2897 (C-Haliph), 2237 (C N), 1729 (C–O), 1717
(C–OOH), 678 (C–Br); 1H NMR (CDCl3): d 2.37 (s, 3H, CH3), 3.20
(d, 2H, NCH2), 3.49 (m, 1H, CHpyrr), 4.38 (d, 2H, COCH2), 6.87–7.94
(m, 9H, ArH), 10.91 (s, 1H, OH, D2O exchangeable).
1-[6-(4-Chlorophenyl)-3-cyano-4-(4-hydroxyphenyl)-
pyridin-2-yl]-5-oxopyrrolidine-3-carboxylic acid (3c)
Yield 61%; mp 2578C; IR (KBr) cmꢀ1: 3516 (O–H), 3267 (COO–H),
–
–
3048 (Ar-CHstr), 2229 (C N), 1728 (C O), 1694 (C OOH), 757
–
–
(C–Cl); 1H NMR (CDCl3): d 3.16 (d, 2H, NCH2), 3.48 (m, 1H,
CHpyrr), 4.43 (d, 2H, COCH2), 6.96–7.98 (m, 9H, ArH), 8.09 (s,
1H, OH, D2O exchangeable), 11.16 (s, 1H, OH, D2O exchangeable).
1-[3-Cyano-6-(4-nitrophenyl)-4-phenyl-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3k)
Yield 69%; mp 2478C; IR (KBr) cmꢀ1: 3349 (COO–H), 3037
1-[4,6-Bis-(4-chlorophenyl)-3-cyano-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3d)
–
–
(Ar-CHstr), 2279 (C N), 1737 (C O), 1710 (C OOH), 1538
–
–
–
Yield 51%; mp 2168C; IR (KBr) cmꢀ1: 3248 (COO–H), 2979
(N O); 1H NMR (CDCl3): d 3.24 (d, 2H, NCH2), 3.46 (m, 1H,
CHpyrr), 4.41 (d, 2H, COCH2), 6.86–7.87 (m, 10H, ArH), 10.84
(s, 1H, OH, D2O exchangeable).
–
–
–
(Ar-CHstr), 2216 (C N), 1726 (C O), 1698 (C OOH), 749 (C–Cl);
–
–
1H NMR (CDCl3): d 3.11 (d, 2H, NCH2), 3.56 (m, 1H, CHpyrr), 4.47
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192
N. Siddiqui et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
1H NMR (CDCl3): d 3.26 (d, 2H, NCH2), 3.48 (m, 1H, CHpyrr), 4.45
(d, 2H, COCH2), 6.78–8.04 (m, 10H, ArH), 10.67 (s, 1H, OH,
D2O exchangeable).
1-[3-Cyano-4-(2-hydroxyphenyl)-6-(4-nitrophenyl)-pyridin-
2-yl]-5-oxopyrrolidine-3-carboxylic acid (3l)
Yield 76%; mp 2828C; IR (KBr) cmꢀ1: 3447 (O–H), 3367 (COO–H),
–
–
2997 (Ar-CHstr), 2243 (C N), 1736 (C O), 1718 (C OOH), 1538
–
–
–
(N O); 1H NMR (CDCl3): d 3.27 (d, 2H, NCH2), 3.51 (m, 1H,
CHpyrr), 4.46 (d, 2H, COCH2), 6.82–7.92 (m, 9H, ArH), 8.22 (s, 1H,
OH, D2O exchangeable), 11.19 (s, 1H, OH, D2O exchangeable).
–
1-(3-Cyano-6-phenyl-4-(4-methylphenyl)-pyridin-2-yl)-5-
oxopyrrolidine-3-carboxylic acid (3t)
Yield 49%; mp 2728C; IR (KBr) cmꢀ1: 3412 (COO–H), 3048
(Ar-CHstr), 2844 (C–Ha),liph 2277 (C N), 1751 (C O), 1712
–
–
(C OOH); 1H NMR (CDCl3): d 2.38 (s, 3H, CH3), 3.22 (d, 2H,
NCH2), 3.46 (m, 1H, CHpyrr), 4.41 (d, 2H, COCH2), 6.81–8.11
(m, 10H, ArH), 10.76 (s, 1H, OH, D2O exchangeable).
1-[3-Cyano-4-(4-hydroxyphenyl)-6-(4-nitrophenyl)-pyridin-
2-yl]-5-oxopyrrolidine-3-carboxylic acid (3m)
–
–
Yield 79%; mp 2768C; IR (KBr) cmꢀ1: 3367 (O–H), 3287 (COO–H),
–
–
–
3051 (Ar-CHstr), 2278 (C N), 1747 (C O), 1718 (C OOH), 1545
1
–
(N O); H NMR (CDCl ): d 3.24 (d, 2H, NCH ), 3.48 (m, 1H, CHpyrr),
–
–
–
–
4.43 (d, 2H, COCH2), 6.87–7.93 (m, 9H, ArH), 8.16 (s, 1H, OH,
3
2
Pharmacology
D2O exchangeable), 11.24 (s, 1H, OH, D2O exchangeable).
The investigations were conducted on albino mice of either
sex (25–30 g). Animals were kept under standard conditions
at an ambient temperature of 25 ꢁ 28C and allowed free
access to food and water except at the time they were brought
out of the cage. All the experimental protocols were carried
out with the permission from Institutional Animal Ethics
committee (IAEC), form no. 502. Animals were obtained from
Central Animal House Facility, Hamdard University, New
Delhi-62. Registration no. and date of registration is 173/
CPCSEA, 28 Jan., 2000.
1-[4-(4-Chlorophenyl)-3-cyano-6-(4-nitrophenyl)-pyridin-
2-yl]-5-oxopyrrolidine-3-carboxylic acid (3n)
Yield 48%; mp 2378C; IR (KBr) cmꢀ1: 3315 (COO–H), 3026
–
–
–
(Ar-CHstr), 2249 (C N), 1737 (C O), 1718 (C OOH), 1549 (N O),
–
–
–
849 (C–Cl); 1H NMR (CDCl3): d 3.21 (d, 2H, NCH2), 3.49 (m, 1H,
CHpyrr), 4.41 (d, 2H, COCH2), 6.92–7.93 (m, 9H, ArH), 10.91 (s, 1H,
OH, D2O exchangeable).
1-[3-Cyano-6-(4-nitrophenyl)-4-(4-methylphenyl)-pyridin-
2-yl]-5-oxopyrrolidine-3-carboxylic acid (3o)
Yield 57%; mp 2628C; IR (KBr) cmꢀ1: 3354 (COO–H), 3017
–
(Ar-CHstr), 2867 (C-Haliph), 2245 (C N), 1758 (C O), 1721
–
–
Anticonvulsant activity
Maximal electroshock test (MES)
(C OOH), 1571 (N O); 1H NMR (CDCl3): d 2.48 (s, 3H, CH3),
3.25 (d, 2H, NCH2), 3.54 (m, 1H, CHpyrr), 4.44 (d, 2H, COCH2),
6.87–7.91 (m, 9H, ArH), 10.88 (s, 1H, OH, D2O exchangeable).
–
–
–
Maximal electroshock seizure test was carried out according
to the standard protocol [14]. Albino mice were stimulated
through corneal electrodes to 50 mA current at a pulse of
60 Hz applied for 0.25 s. Animals were previously adminis-
tered with the test drug i.p. Abolition of hind limb tonic
extension spasm was recorded as the anticonvulsant activity.
Test compounds were dissolved in polyethylene glycol (PEG).
In the preliminary screening, each compound was adminis-
tered as an i.p. injection at three dose levels (30, 100 and
300 mg/kg body mass) and the anticonvulsant activity was
assessed after 0.5 h and 4.0 h intervals of administration.
1-(3-Cyano-4,6-diphenyl-pyridin-2-yl)-5-oxopyrrolidine-3-
carboxylic acid (3p)
Yield 62%; mp 2168C; IR (KBr) cmꢀ13378 (COO–H), 3064 (Ar-CHstr),
1
2215 (C N), 1737 (C O), 1719 (C OOH); H NMR (CDCl ): d 3.21 (d,
–
–
–
2H, NCH2), 3.51 (m, 1H, CHpyrr), 4.43 (d, 2H, COCH2), 6.91–7.94
–
3
(m, 11H, ArH), 10.93 (s, 1H, OH, D2O exchangeable).
1-[3-Cyano-4-(2-hydroxyphenyl)-6-phenyl-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3q)
Yield 64%; mp 2758C; IR (KBr) cmꢀ1: 3349 (O–H), 3267 (COO–H),
1
3049 (Ar-CHstr), 2229 (C N), 1737 (C O), 1717 (C OOH); H NMR
–
–
–
–
(CDCl3): d 3.22 (d, 2H, NCH2), 3.51 (m, 1H, CHpyrr), 4.46 (d, 2H,
COCH2), 6.92–7.97 (m, 10H, ArH), 8.21 (s, 1H, OH, D2O exchange-
able), 11.15 (s, 1H, OH, D2O exchangeable).
Subcutaneous pentylenetetrazole induced seizure test
(scPTZ)
The subcutaneous pentylenetetrazole test was performed
according to the known protocol [15]. This method utilizes
pentylenetetrazole (75 mg/kg) that produces seizures in
>95% of animals as a 0.5% solution subcutaneously in the
posterior midline. The animals were observed for 30 min.
Failure to observe even a threshold seizure (a single episode of
clonic spasms of at least 5 s duration) was defined as
protection.
1-[3-Cyano-4-(4-hydroxyphenyl)-6-phenyl-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3r)
Yield 49%; mp 2378C; IR (KBr) cmꢀ1: 3359 (O–H), 3247 (COO–H),
1
3027 (Ar-CHstr), 2259 (C N), 1746 (C O), 1712 (C OOH); H NMR
–
–
–
–
(CDCl3): d 3.16 (d, 2H, NCH2), 3.48 (m, 1H, CHpyrr), 4.49 (d, 2H,
COCH2), 6.76–7.94 (m, 10H, ArH), 8.24 (s, 1H, OH, D2O exchange-
able), 11.24 (s, 1H, OH, D2O exchangeable).
The pharmacological parameters estimated in phase I
screening was quantified in phase II screening.
Anticonvulsant activity was expressed in terms of the median
effective dose (ED50). For the determination of ED50 values,
1-[4-(4-Chlorophenyl)-3-cyano-6-phenyl-pyridin-2-yl]-
5-oxopyrrolidine-3-carboxylic acid (3s)
Yield 71%; mp 2678C; IR (KBr) cmꢀ1
: 3396 (COO–H), 3018
–
–
–
(Ar-CHstr), 2258 (C N), 1748 (C O), 1724 (C OOH), 848 (C–Cl);
–
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Arch. Pharm. Chem. Life Sci. 2012, 345, 185–194
Synthesis and Pharmacology of Pyrrolidone Derivatives
193
groups of 10 mice were given a range of intraperitoneal doses
of the test drug until at least three points were established in
the range of 10–90% seizure protection. From the plot of
these data, the respective ED50 values, 95% confidence inter-
vals, slope of the regression line, and the standard error of the
slope were calculated by means of the computer program by
Litchfield and Wilcoxon’s method [16].
Three-dimensional structural analyses
This was performed to delineate the role of three-dimen-
sional structures of synthesized compounds to their anticon-
vulsant activity. Compound 1-(3-cyano-4,6-diphenyl-pyridin-2-
yl)-5-oxopyrrolidine-3-carboxylic acid (3p) which represents
the prototype structure of the synthesized compounds was
chosen for the study. It was performed using the software
Ortep3v2 [21].
Neurotoxicity screening (NT)
One of the authors (W.A.) is thankful to Council of Scientific and Industrial
Research (CSIR), Government of India for the Senior Research Fellowship
(SRF) as financial assistance. We are also thankful to Shrenik Pharma Ltd.,
Mumbai, India for providing pentylenetetrazole as gift sample.
The minimal motor impairment was measured in mice by
the rotorod test. Animals were trained to stay on an accel-
erating rotorod of diameter 3.2 cm that rotates at 10 rpm.
Trained animals were given i.p. injection of the test com-
pounds 30, 100 and 300 mg/kg. Neurotoxicity was indicated
by the inability of the animal to maintain equilibrium on the
rod for at least 1 min in each of the trials [17].
The authors have declared no conflict of interests.
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www.archpharm.com