Discovery of 4-aminobutyric acid derivatives possessing anticonvulsant and antinociceptive activities: A hybrid pharmacophore approach

Article abstract of DOI:10.1021/jm061431g

Antiepileptic drugs are often utilized in the treatment of neuropathic pain. The present study aims at the design and synthesis of newer γ-aminobutyric acid (GABA) derivatives with the combination of aryl semicarbazone and the GABA pharmacophores in order

Full text of DOI:10.1021/jm061431g

J. Med. Chem. 2007, 50, 2459-2467
2459
Discovery of 4-Aminobutyric Acid Derivatives Possessing Anticonvulsant and Antinociceptive
Activities: A Hybrid Pharmacophore Approach
Perumal Yogeeswari,*,† Jegadeesan Vaigunda Ragavendran,† Dharmarajan Sriram,† Yarravarapu Nageswari,† Ramkumar Kavya,†
Narayanan Sreevatsan,† Kaliappan Vanitha,† and James Stables‡
Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology & Science, Pilani-333031, India, and Preclinical
Pharmacology Section, Epilepsy Branch, National Institutes of Health, Bethesda, Maryland 20892-9020
ReceiVed December 14, 2006
Antiepileptic drugs are often utilized in the treatment of neuropathic pain. The present study aims at the
design and synthesis of newer γ-aminobutyric acid (GABA) derivatives with the combination of aryl
semicarbazone and the GABA pharmacophores in order to develop a multifunctional drug useful in the
treatment of neurological disorders like epilepsy and neuropathic pain. Various GABA semicarbazones were
synthesized and screened for anticonvulsant, peripheral analgesic, antiallodynic, and antihyperalgesic activities.
The structures of the synthesized compounds were confirmed by the use of their spectral data in addition
to elemental analysis. The synthesized derivatives of the inhibitory neurotransmitter GABA produced
anticonvulsant and antinociceptive actions in the acetic acid induced writhing test and peripheral nerve
injury (chronic constriction injury and L5 spinal nerve ligation) models of neuropathic pain. The underlying
mechanisms are expected to be enhancement of peripheral GABAergic neurotransmission owing to their
activity in the scPIC screen and due to various reports on the involvement of GABAergic pathway in peripheral
models of neuropathic pain.
Introduction
of anticonvulsant agents with lesser central nervous system side
effects and hepatotoxicity.12-19 Given the promising biological
profile of GABA derivatives and aryl semicarbazones, we
initiated a drug discovery program focusing on newer GABA
derivatives as the lead. In the present paper, effort has been
taken to design GABA semicarbazones by combining the GABA
and semicarbazone pharmacophores. Rather than following the
old pattern of first completing the development of novel
compounds as AEDs and then testing them as analgesics, we
proceeded to assess the new pharmacophoric hybrids for
anticonvulsant, analgesic, antiallodynic, and antihyperalgesic
activity in various rat models.
Neuropathic pain responds to many antiepileptic drugs
(AEDs), suggesting that it shares with epilepsy underlying
neurophysiological mechanisms. Specifically, most of these
drugs either stabilize membrane hyperexcitability or enhance
inhibitory γ-aminobutyric acid (GABA) and/or glycine neu-
rotransmission.1,2 Most antiepileptics have multiple potential
modes of action that may be beneficial for neuropathic pain.
Anticonvulsants, such as carbamazepine or phenytoin, have been
traditionally used for the management of neuropathic pain.
However, the efficacy of this class of drugs has not been
unequivocally established, and their use has often been associ-
ated with numerous side effects.3,4 More recently, some of the
newer anticonvulsants, in particular gabapentin and to a lesser
extent topiramate and lamotrigine, received increased attention
as analgesics for treating neuropathic pain.4 GABA is the major
inhibitory neurotransmitter in the mammalian brain. It has been
well documented that the reduction of GABAergic neuronal
activity plays an important role in a number of neurological
disorders, including epilepsy, anxiety, and pain. Gabapentin, a
structural analogue of GABA, has been found to exert significant
analgesic effects.5-7 However, despite the progress made with
these compounds, neuropathic pain remains under treated, and
in many patients gabapentin does not provide adequate pain
relief. In behavioral studies, it has been shown that various
chemical analogues of GABA attenuate allodynic and hyper-
algesic responses in chronic constriction injury rats.8-11 Hence
there is a current focus on screening the potential of anticon-
vulsants as therapeutic agents and also in the development of
newer lead molecules for neuropathic pain treatment. In the
recent literature, various aryl semicarbazones with established
pharmacophore requirements have been reported as a novel class
Chemistry
The synthesis of GABA semicarbazones was achieved as
presented in Figure 1. The method was based on an earlier
reported procedure.13,20 The starting material, 4-aminobutyric
acid, was treated with phenyl chloroformate in aqueous sodium
hydroxide at a range of 0-5 °C to yield 4-(phenoxycarbony-
lamino)butanoic acid with a yield of 70% after crystallization
with 95% ethanol. The carbamate, on condensation with
hydrazine hydrate in ethanol, gave the 4-(hydrazinecarbony-
lamino)butanoic acid. Finally, the required semicarbazone
derivatives (1-15, Table 1) were obtained by reaction of the
appropriate aryl/alkyl aldehydes, ketone, or isatin derivatives
with the 4-(hydrazinecarbonylamino)butanoic acid in ethanol
to give 4-(alkylidene/arylidene hydrazine carbonylamino)-
butanoic acids in yields ranging between 47% and 81%. The
purity was assessed by TLC using chloroform:methanol in the
ratio 9:1 as the eluant system. The structures were characterized
by both spectral and elemental analysis, and the data were within
( 0.4% of the theoretical values. In general, the IR spectra
showed the CdN peak at 1620-1590 cm-1 and the NH
stretching vibrations at 3220 cm-1. The synthesized compounds
exhibited characteristic amide bonds at 1650-1620 cm-1. The
1H NMR spectrum revealed that the hydrazino proton (dN-
NH) showed a singlet at δ 10.0-10.56 and the alkyl NH at
* To whom correspondence should be directed. Tel.: +91-
9414402890. Fax: +91-1596-244183. E-mail: pyogee@bits-pilani.ac.in.
† Birla Institute of Technology & Science.
‡ National Institutes of Health.
10.1021/jm061431g CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/24/2007

2460 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Yogeeswari et al.
Figure 1. Synthetic protocol of 4-(alkylidene/arylidene hydrazine carbonylamino)butanoic acids.
Table 1. Physical Data of the Synthesized Compounds
molecular
formula
molecular
weight
yield
(%)
mp
(°C)
a
no.
R1
R2
2-OH
4-NO2
4-Cl
3-NO2
4-N(CH3)2
H
4-CH3
3-NH2
4-NO2
H
Rf
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
H
H
H
H
C12H15N3O4
C12H14N4O5
C12H14N3O3Cl
C12H14N4O5
C14H20N4O3
C13H17N3O3
C14H19N3O3
C13H18N4O3
C13H16N4O5
C18H19N3O3
C18H18N3O3Br
C20H23N3O3
C11H19N3O3
C10H17N3O3
C13H14N4O4
264
294
283
294
292
263
277
278
308
325
404
353
241
227
290
55.0
56.0
69.0
47.0
65.0
59.0
49.0
61.0
59.0
66.0
79.0
55.0
55.0
58.0
81.0
212
216b
203
182
259
119
120
260
197
47
73
89
122
121
114b
0.71
0.65
0.63
0.71
0.73
0.77
0.78
0.81
0.72
0.82
0.86
0.87
0.71
0.65
0.77
H
CH3
CH3
CH3
CH3
C6H5
C6H5
4-Br
CH2-C6H5
CH2-C6H5
cyclohexylene
cyclopentylene
isatinyl
a The solvent system used was chloroform:methanol (9:1). b Melting with decomposition.
6.0-6.03, both of which were D2O exchangeable. All com-
pounds showed a characteristic D2O exchangeable singlet due
to OH proton of the acid function at δ 12.0-12.2. The aryl
ring protons resonated at δ ∼ 7.2-7.66 ppm.
anticonvulsant activity by following the standard anticonvulsant
drug development (ADD) program protocols.21-22 Table 2 lists
the results obtained from the initial anticonvulsant evaluation
of the synthesized compounds compared to the clinically proven
antiepileptics such as phenytoin, and ethosuximide also tested
at the same dose levels. The tests included one electrical and
three chemoshock tests, i.e., maximal electroshock seizure test
(MES), subcutaneous pentylenetetrazole (scPTZ) seizure thresh-
old test, subcutaneous strychnine (scSTY) pattern test, and
subcutaneous picrotoxin (scPIC) seizure threshold test. The acute
neurological toxicity was determined by the rotorod test. Five
Results and Discussion
The aim of this drug discovery program was to prepare newer
GABA derivatives with multiple pharmacological actions ef-
fective in the treatment of epilepsy and neuropathic pain. The
synthesized compounds (1-15) were evaluated at dose levels
of 30, 100, and 300 mg/kg intraperitoneally in mice for

4-Aminobutyric Acid DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2461
Table 2. Analgesic and Anticonvulsant Activity of the Synthesized Compounds
intraperitoneal administration to micea
MES
scPTZ
scSTY
scPIC
neurotoxicity
0.5 h 4 h
acetic acid induced writhingb
number of writhes
(per 30 min)
%
compd
control
1
2
3
4
5
6
7
8
9
10
11
12
13
14
0.5 h
4 h
0.5 h
0.5 h
4 h
0.5 h
4 h
inhibition
-
-
-
-
-
-
-
30
-
-
30
-
-
30
-
100
300
30
300
300
-
-
300
-
100
300
-
300
-
-
-
-
30
-
-
-
-
300
-
183.3 ( 3.8
19.0 ( 2.1
18.7 ( 0.9
59.3 ( 4.6
17.7 ( 5.5
13.0 ( 2.6
33.7 ( 1.4
32.5 ( 2.4
19.7 ( 0.9
61.0 ( 0.9
13.3 ( 1.2
21.0 ( 2.1
10.3 ( 2.6
32.0 ( 2.5
18.0 ( 1.5
47.3 ( 6.2
-
-
89
90
68
90
93
82
82
89
67
93
89
94
82
90
74
-
-
-
-
300
-
-
-
-
300
-
300
-
100
300
100
300
-
-
-
-
300
-
-
-
-
-
-
-
-
-
-
-
-
100
-
300
-
-
-
-
-
300
-
300
300
-
-
300
-
300
-
-
-
-
-
-
-
300
-
-
300
300
-
-
-
300
300
300
-
300
300
300
-
300
-
300
-
-
-
-
-
-
-
-
-
100
-
-
-
-
-
300
-
-
300
300
30
-
-
-
-
300
100
100
-
300
300
100
-
15
300
30
-
300
-
-
-
300
-
phenytoin
ethosuximide
aspirin
-
-
300c
-
-
-
-
-
-
-
-
-
-
-
-
-
5.7 ( 3.5
97
a Doses of 30, 100, and 300 mg/kg were administered. The figures in the table indicate the minimum dose whereby bioactivity was demonstrated in half
or more of the mice (three in each group). The animals were examined at 0.5 and 4 h. The line (-) indicates an absence of anticonvulsant activity or
neurotoxicity at the maximum dose tested. b The compounds were administered intraperitoneally at a dose level of 100 mg/kg. Control animals were administered
30% v/v PEG 400 in water. Each value represents the mean ( SEM of six mice significantly different from the control at P < 0.01 (Student’s t test).
c Ethosuximide at 100 mg/kg exhibited protection in one out of three mice.
compounds (8, 10, 11, 14, and 15) showed activity in the MES
screen indicative of their ability to prevent seizure spread.
Compounds 8 and 15 exhibited longer duration of action
(i.e., activity until 4 h interval) whereas compounds 10 and 11
showed shorter duration of action (0.5 h). In the scPTZ
screen, a test used to identify compounds that elevate seizure
threshold, four compounds (4, 7, 13, and 15) showed marginal
protection. These compounds showed protection at 300 mg/kg
at 0.5 h time period only. Six compounds (1, 3, 6, and 10-12)
showed protection in the subcutaneous strychnine-induced
seizure model. Compound 1 was the most effective in this
model exhibiting protection at 30 mg/kg for a longer duration
(until 4 h). Compound 6 showed activity at 100 mg/kg, and
other compounds showed activity at 300 mg/kg. All of the
active compounds exhibited a longer duration (0.5 and 4 h) of
action. In the scPIC-induced seizure threshold test, most
of the compounds exhibited activity indicative of the
possible involvement of GABA-mediation in the anticonvulsant
action. All of the compounds except 2, 8, 9, 11, and 14
showed protection in the scPIC test. Compounds 1 and 5 showed
pronounced activity at 30 mg/kg and compounds 3 and 12
showed activity at 100 mg/kg. In general, it appears that
except compounds 2, 5, 8, 9, and 14, all other compounds were
found to be effective in at least two models of seizure with
compounds 10 and 15 effective in three animal models that
include MES, scSTY or scPTZ, and scPIC screens. Compounds
8 and 14 showed activity only in the MES model, whereas
compound 5 showed activity in the scPIC model, and com-
pounds 2 and 9 were completely inactive in the anticonvulsant
screening.
To examine the potential therapeutic value of the newer
GABA derivatives (1-15) in the treatment of neuropathic pain,
the first part of this study examined the ability of the synthesized
derivatives of GABA to inhibit writhing responses in the acetic
acid induced writhing test, a chemical pain test used to evaluate
acute antinociceptive function. All of the tested compounds
suppressed the acetic acid induced writhing response signifi-
cantly (P < 0.01) in comparison to the control (Table 2). The
standard drug, aspirin, exhibited the highest percentage inhibition
(97.0%). Compounds 2, 4, 5, 10, 12, and 14 were the most active
compounds with percentage inhibition 90% or above. Of these
compounds, 12 was observed to be most active with 94%
inhibition.
In the next phase of screening two well-known peripheral
neuropathic pain models were used that included the chronic
constriction injury (CCI) and L5 spinal nerve ligation (SNL)
models in rats. In the CCI model, the left sciatic nerve proximal
to the trifurcation point was constricted with four loose ligatures
using 3-0 braided silk thread, while in the SNL model, a tight
ligation was tied around the L5 spinal nerve using 3-0 braided
silk thread. Four nociceptive assays aimed at determining the
severity of behavioral neuropathic responses, namely allodynia
and hyperalgesia, were performed. The assays involved mea-
surement of the degree of spontaneous (ongoing) pain and tests
of hind limb withdrawal to cold and mechanical stimuli
(dynamic mechanical allodynia, cold allodynia, and mechanical
hyperalgesia). A minimum of 10 min separated the testing
procedures to reduce the influence of prior nociceptive testing.
The order of testing was as follows: spontaneous pain, dynamic
allodynia, cold allodynia, and lastly mechanical hyperalgesia.
Baseline sensory response values were measured for each group
of animals (n ) 4) preoperatively and 9 days postoperatively.
Animals displaying allodynic and hyperalgesic responses in both
the models were then administered the relevant drug according
to a predetermined randomization table, and testing was
reperformed at 0.5, 1, 1.5, 2.0, and 2.5 h postdrug administration.
All of the behavioral responses were timed with a stopwatch.
All of the compounds were tested at a single dose of 100 mg/
In the acute neurological toxicity screen, the compounds 1,
3-6, and 11-14 emerged as promising anticonvulsants with
less or no neurotoxicity. There was no separation between the
anticonvulsant dose and the neurotoxic dose (300 mg/kg) for
compounds 7, 8, 10, and 15, in which 15 showed greater
neurotoxicity at 0.5 h at 100 mg/kg. Compound 2 which was
ineffective in the seizure model showed neurotoxicity at 300
mg/kg at 0.5 h.

2462 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Table 3. Effects of Compounds on Spontaneous Pain in the CCI Model
Yogeeswari et al.
spontaneous pain (PWD) (s)a
study
group
predrug
0.5 h
1.0 h
1.5 h
2.0 h
2.5 h
control
1
2
5
6
7
8
12
40.6 ( 12.3
31.3 ( 3.4
21.2 ( 2.3
52.7 ( 8.7
42.9 ( 3.7
27.7 ( 2.7
12.7 ( 1.4
22.8 ( 1.3
48.6 ( 4.3
69.6 ( 4.7
66.8 ( 7.9
74.9 ( 4.6
45.9 ( 17.3NS
10.5 ( 2.0
10.2 ( 1.1
47.2 ( 1.2NS
13.2 ( 0.8
23.9 ( 2.4NS
1.7 ( 0.1
42.1 ( 5.6NS
19.3 ( 2.7NS
9.2 ( 2.1
35.3 ( 15.3NS
23.7 ( 1.3NS
13.6 ( 1.9NS
38.2 ( 1.5NS
18.3 ( 1.6
41.4 ( 7.7NS
11.2 ( 1.7
11.3 ( 0.6NS
11.9 ( 5.2
12.9 ( 2.6
10.9 ( 1.4
1.4 ( 0.1
39.9 ( 8.1NS
7.3 ( 1.4
17.2 ( 2.3NS
5.2 ( 2.1
48.8 ( 3.5NS
18.8 ( 3.0
21.8 ( 2.7NS
1.2 ( 1.0
10.6 ( 1.1
10.7 ( 0.9
2.8 ( 1.0
18.3 ( 2.1NS
0.9 ( 0.2
9.2 ( 1.2
9.7 ( 1.8
1.3 ( 0.6
3.2 ( 0.9
8.5 ( 0.2
15
38.6 ( 5.2NS
69.2 ( 3.2NS
24.6 ( 2.0
62.2 ( 3.8NS
34.9 ( 1.3NS
79.0 ( 2.6NS
17.8 ( 2.9
15.5 ( 2.5
21.6 ( 2.3
20.6 ( 1.9
71.2 ( 2.3NS
67.4 ( 5.8NS
11.0 ( 2.3
20.2 ( 2.7
67.8 ( 2.7NS
76.9 ( 6.9NS
18.1 ( 2.3
lamotrigine
carbamazepine
gabapentin
76.9 ( 1.0NS
32.8 ( 2.7NS
13.1 ( 3.4
a All compounds were administered intraperitoneally at a dose level of 100 mg/kg. Control animals were administered 30% v/v PEG 400 in water. Each
value represents the mean ( SEM of four rats, significantly different from predrug at P < 0.05; NS denotes not significant at P < 0.05 (one-way ANOVA,
followed by post-hoc Bonferroni test).
Table 4. Antihyperalgesic Effects of Compounds in the CCI Model
mechanical hyperalgesia (PWD) (s)a
study
group
predrug
0.5 h
1.0 h
1.5 h
2.0 h
2.5 h
control
1
3
5
7
8
11
13
12.1 ( 3.5
25.7 ( 2.4
14.8 ( 2.5
41.9 ( 29.0
28.8 ( 1.3
24.7 ( 0.1
23.6 ( 1.1
21.2 ( 1.3
72.1 ( 21.6
11.3 ( 0.3
34.8 ( 6.0
21.8 ( 1.9
45.6 ( 1.5
14.7 ( 5.9NS
7.5 ( 2.7
13.6 ( 2.4NS
7.7 ( 2.0
14.3 ( 4.1NS
4.4 ( 2.0
12.8 ( 5.6NS
4.1 ( 1.0
4.2 ( 1.1
5.8 ( 2.6
5.3 ( 0.2
6.6 ( 1.9
7.3 ( 1.2
5.2 ( 1.2
22.8 ( 8.1
5.0 ( 0.1
33.7 ( 1.9NS
21.7 ( 1.7NS
9.2 ( 0.4
14.5 ( 4.1NS
7.3 ( 1.9
7.9 ( 1.8NS
17.4 ( 12.8
27.3 ( 1.8NS
9.3 ( 0.1
7.8 ( 1.7NS
13.1 ( 10.9
13.2 ( 0.8
9.6 ( 0.1
4.4 ( 1.1
7.2 ( 1.4
9.7 ( 8.2
4.8 ( 1.2
9.6 ( 0.6
4.2 ( 1.1
9.9 ( 0.1
4.8 ( 0.6
20.5 ( 2.3NS
20.3 ( 1.1NS
32.1 ( 8.2
8.5 ( 2.0
5.1 ( 1.0
5.6 ( 1.5
24.8 ( 1.0NS
30.7 ( 0.9
4.9 ( 0.5
4.8 ( 1.0
7.0 ( 1.1
14
15
17.7 ( 7.9
8.8 ( 3.4NS
35.7 ( 1.0NS
22.9 ( 0.7NS
10.7 ( 0.4
25.3 ( 8.2
27.8 ( 9.3*
29.8 ( 2.9NS
24.0 ( 2.2 NS
8.4 ( 0.1
10.8 ( 3.9NS
36.2 ( 5.0NS
13.3 ( 3.0NS
36.8 ( 0.2NS
lamotrigine
carbamazepine
gabapentin
36.7 ( 0.9NS
15.7 ( 2.3NS
12.3 ( 0.5
a All compounds were administered intraperitoneally at a dose level of 100 mg/kg. Control animals were administered 30% v/v PEG 400 in water. Each
value represents the mean ( SEM of four rats, significantly different from predrug at P < 0.05; asterisk (*) represents the mean ( SEM of a pharmacologically
inactive response significantly different from predrug at P < 0.05, and NS denotes not significant at P < 0.05 (one-way ANOVA, followed by post-hoc
Bonferroni test).
Table 5. Effects of Compounds on Spontaneous Pain in the SNL Model
spontaneous pain (PWD) (s)a
study
group
predrug
0.5 h
1.0 h
1.5 h
2.0 h
2.5 h
control
3
5
7
10
11
12
13
51.6 ( 10.9
37.5 ( 2.2
76.4 ( 4.6
26.3 ( 1.3
26.3 ( 2.1
29.2 ( 2.0
25.9 ( 2.6
30.8 ( 1.3
10.8 ( 2.3
23.1 ( 2.3
85.8 ( 6.8
35.5 ( 2.9
56.8 ( 17.0 NS
33.1 ( 1.3NS
12.4 ( 1.5
58.9 ( 8.1NS
14.0 ( 2.9
21.2 ( 9.5
11.1 ( 1.0
18.5 ( 1.2NS
28.5 ( 2.0NS
7.9 ( 1.0
48.1 ( 10.5NS
12.7 ( 2.1
16.4 ( 3.9
6.4 ( 2.9
53.5 ( 8.4NS
11.2 ( 1.2
29.2 ( 5.8
2.9 ( 1.0
40.3 ( 5.4NS
15.9 ( 1.1
43.0 ( 4.7NS
3.1 ( 0.1
22.2 ( 1.3NS
15.1 ( 1.1NS
25.1 ( 1.1NS
22.2 ( 2.NS
21.2 ( 0.2NS
1.2 ( 0.1
13.4 ( 1.8NS
23.1 ( 1.1NS
8.9 ( 2.2
10.9 ( 1.1
10.1 ( 1.0
11.8 ( 1.1
10.6 ( 1.2
0.7 ( 0.1
8.8 ( 1.2
8.7 ( 0.1
7.4 ( 1.1
10.8 ( 1.7
9.8 ( 1.8NS
24.4 ( 3.6NS
94.9 ( 7.6NS
10.1 ( 0.6
20.2 ( 1.2NS
0.9 ( 0.1
10.8 ( 1.2
0.9 ( 0.1
15
lamotrigine
carbamazepine
gabapentin
15.6 ( 3.4NS
14.6 ( 1.0
13.3 ( 2.7NS
7.9 ( 0.9
12.9 ( 1.0
2.6 ( 0.6
22.9 ( 3.4NS
85.4 ( 8.9NS
9.1 ( 1.0
22.3 ( 3.7
10.2 ( 1.0
29.7 ( 1.8NS
a All compounds were administered intraperitoneally at a dose level of 100 mg/kg. Control animals were administered 30% v/v PEG 400 in water. Each
value represents the mean ( SEM of four rats, significantly different from predrug at P < 0.05; NS denotes not significant at P < 0.05 (one-way ANOVA,
followed by post-hoc Bonferroni test).
kg. The results along with data on standard drugs lamotrigine,
carbamazepine, and gabapentin are represented in Tables 3-6.
In the CCI model, compounds 6, 8, and 12 completely
reversed the spontaneous pain response throughout the time
period of testing (0.5-2.5 h), more effective when compared
to carbamazepine (Table 3). The times to peak effect of these
compounds were 1.5 h for 8 and 12 and 2.5 h for 6. Lamotrigine
was ineffective in this test, while gabapentin showed activity
from 1 h until 2.5 h of testing. Compound 2 showed activity
up to 1 h. The onset of action of compounds 5 and 7 was 2 h
and for 15 1.5 h. Compound 1 showed an irregular activity
profile as presented in Table 3. Compounds 3, 4, 9, 10-11,
and 13-14 were ineffective in this test.
Six compounds (1, 5, 6, 11, 12, and 15) were active in
attenuating the dynamic allodynic response (results given as
Supporting Information) throughout the entire 2.5 h experiment
similar to gabapentin. Compound 3 showed an irregular
response, and all other compounds were inactive. In the cold

4-Aminobutyric Acid DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2463
Table 6. Antihyperalgesic Effects of Compounds in the SNL Model
mechanical hyperalgesia (PWD) (s)a
study
group
predrug
0.5 h
1.0 h
1.5 h
2.0 h
2.5 h
control
1
3
5
6
8
10
13
15.7 ( 3.7
23.9 ( 1.2
34.9 ( 5.8
19.1 ( 3.6
34.3 ( 6.8
17.1 ( 3.0
32.1 ( 7.2
29.0 ( 3.2
36.6 ( 3.1
39.5 ( 10.1
17.9 ( 3.4
19.1 ( 2.6
24.4 ( 0.5
13.7 ( 2.5NS
17.6 ( 1.5NS
24.6 ( 3.8NS
4.7 ( 0.7
15.0 ( 2.1NS
17.3 ( 2.8NS
17.3 ( 2.8NS
5.8 ( 2.3
14.7 ( 2.6NS
10.6 ( 1.9
12.3 ( 1.8
5.5 ( 2.0
15.5 ( 3.7NS
11.3 ( 2.1NS
5.6 ( 1.2
12.1 ( 4.5NS
17.3 ( 1.1NS
17.9 ( 1.2NS
5.8 ( 1.4
4.4 ( 1.4
23.2 ( 8.4NS
2.4 ( 0.7
27.6 ( 3.3NS
6.5 ( 0.8
18.7 ( 2.9NS
7.3 ( 0.8
16.7 ( 1.3
19.5 ( 3.3NS
13.7 ( 1.2
11.3 ( 2.3
9.6 ( 1.3
17.3 ( 1.7NS
17.3 ( 2.2NS
15.0 ( 1.9
12.8 ( 2.6
8.6 ( 1.2
18.4 ( 1.6NS
14.7 ( 2.2NS
5.3 ( 0.9
19.9 ( 3.8NS
19.7 ( 3.0NS
9.9 ( 1.2
18.5 ( 0.1NS
15.6 ( 2.9NS
5.1 ( 1.9
14
15
4.7 ( 0.2
9.6 ( 3.2
45.7 ( 10.6NS
10.1 ( 2.3NS
6.6 ( 0.7
19.7 ( 1.2NS
6.4 ( 0.4
18.7 ( 1.3
17.3 ( 1.8NS
5.8 ( 1.2
lamotrigine
carbamazepine
gabapentin
22.5 ( 1.9NS
11.1 ( 3.1NS
20.1 ( 0.2NS
8.2 ( 1.1
5.2 ( 2.3
5.5 ( 1.6
9.9 ( 0.2
10.2 ( 0.2
10.0 ( 0.3
8.8 ( 0.3
a All compounds were administered intraperitoneally at a dose level of 100 mg/kg. Control animals were administered 30% v/v PEG 400 in water. Each
value represents the mean ( SEM of four rats, significantly different from predrug at P < 0.05; NS denotes not significant at P < 0.05 (one-way ANOVA,
followed by post-hoc Bonferroni test).
allodynia produced in CCI rats, the paw withdrawal durations
(PWDs) were significantly reduced by the administration of
compounds 1, 5, and 12, whose onset of action was at 1 h
(results given as Supporting Information). Lamotrigine did not
show any protection in this test, while carbamazepine was
effective only until 1 h. All other compounds were found to be
ineffective in this test. Hyperalgesia evoked by a mechanical
pin-prick stimulus was effectively attenuated at all time-points
of study by compounds 1, 5, 8, and 14. Compounds 7, 11, and
15 showed significant reductions in paw withdrawal duration
with an onset of action at 1 h similar to gabapentin, whereas
compounds 3 and 13 had shown onset of action from 1.5 h.
Compounds which were inactive in this test included 2, 4, 6, 9,
10, 12, and the standard drugs lamotrigine and carbamazepine
(Table 4). Overall, it appears that, in the CCI model of
neuropathic pain, compounds that showed promising results
include 1 and 5 effective in all the four tests, 12 and 15 in three
out of four tests, and 6, 7, 8, 11, and 15 in two tests. Compounds
that showed moderate activity include 3 in two tests.
In the SNL model, the paw withdrawal durations due to
spontaneous ongoing pain were significantly reduced by com-
pounds 3, 7, and 12 from 1 h until 2.5 h similar to gabapentin,
while compounds 5 and 15 showed activity from 0.5 to 2 h.
Carbamazepine showed significant activity until 1.5 h only.
Compounds 10 and 11 showed onset of action at 2 h, while
that for compound 13 occurred at 1.5 h. All other compounds
including lamotrigine were ineffective in this test. Compounds
6 and 8, which were active in completely reversing the pain
response in the CCI model, were inactive in the SNL model
(Table 5).
The dynamic mechanical allodynia produced by SNL was
effectively reversed by all compounds except 8, 9, 10, 12, and
15. In fact, all of the compounds tested in the dynamic allodynia
assay showed a reversal effect at least in two time-points. Five
compounds (1, 6, 7, 11, and 14) were found to be completely
effective until 2.5 h, hence more effective than carbamazepine
which showed activity until 1 h and lamotrigine which was
ineffective. Compounds 3 and 5 were effective until 1.5 h and
compounds effective until 1 h were 2 and 13. Gabapentin
reversed the dynamic allodynia from 1 to 2.5 h (results given
as Supporting Information).
Cold allodynia produced by SNL model was completely
reversed by compound 1 as seen with CCI model. Administra-
tion of compounds 2-5, 9, 10, 12, and 13 to animals with an
allodynic response to cold stimuli showed no antiallodynic
activity. Compounds 5 and 12 were active in CCI rats.
Compounds 8 and 15 were found to be effective but only until
1 and 2 h, respectively. Other active compounds had different
times of onset as follows: 7 and 14 (1.5 h), 6 (1 h), 11 (2 h),
and gabapentin (1 h). Lamotrigine and carbamazepine which
were effective in CCI were inactive in the SNL model (results
given as Supporting Information).
Mechanical hyperalgesia produced by SNL was completely
reversed significantly at all time-points by compounds 5 and
14. Compound 8, which showed complete reversal until 2.5 h
in the CCI model, showed activity until 1.5 h only in the SNL
model. Gabapentin was effective after 1 h of observation.
Compounds that showed moderate activity included 3, 10, 13,
and 15. Carbamazepine showed a significant antihyperalgesic
effect with an onset of action at 1 h while lamotrigine showed
an irregular activity profile. Compounds 1 and 6 were active at
only one time-point of observation (Table 6).
Overall, it appears that, in the SNL model, compounds that
showed promising results included 3, 5, 6, 7, 11, 13, 14, and
15 in three out of four tests, and 8 and 10 in two tests while
compounds 2 and 12 exhibited moderate activity. The most
active compounds that had been found to be effective in both
neuropathic pain models would be compounds 1 and 5.
The results of this study indicated that the newer GABA
derivatives, apart from exhibiting significant analgesic activity
in the acetic acid induced writhing model, also possessed
antiallodynic and antihyperalgesic actions in both the CCI and
SNL models of neuropathic pain. It is well-established that
GABAergic mechanisms are involved in antinociceptive pro-
cesses. As evidence to this, peripheral administration of
GABAergic agents increases the antinociceptive effect of
morphine, but central administration inhibits this effect, sug-
gesting that multiple interactions might occur.23 Second, the
abdominal constriction response induced by acetic acid is a
sensitive procedure to establish peripherally acting analgesics.
This response is thought to involve local peritoneal receptors.24
As in the case of acute nociceptive pain states, there is
considerable evidence supporting a palliative role for GABAer-
gic neurotransmission in neuropathic pain conditions as well.
In 1999, Eaton et al.25 provided evidence for the involvement
of GABA in spinal antinociception, when a single intrathecal
injection of GABA permanently reversed neuropathic pain after
nerve injury. Moreover, a reduced spinal GABAergic tone has
been suggested just after nerve injury.26 Intrathecal administra-
tion of GABA receptor antagonists dose-dependently produced
tactile allodynia,27 suggesting that inhibiting endogenous GABA
can lead to an excited sensory state.

2464 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Yogeeswari et al.
In conclusion, we have shown that the synthesized derivatives
of the inhibitory neurotransmitter GABA produce anticonvulsant
and antinociceptive actions in the acetic acid induced writhing
test and peripheral nerve injury (CCI and SNL) models of
neuropathic pain. The underlying mechanisms are expected to
be enhancement of peripheral GABAergic neurotransmission
owing to their activity in the scPIC screen and due to various
reports on the involvement of GABAergic pathway in peripheral
models of neuropathic pain. This study presents the first report
on the antiallodynic and antihyperalgesic activities of GABA
semicarbazones. Further research is required to confirm the
hypothesized molecular mechanisms of action of the reported
compounds.
anticonvulsant activity with less neurotoxicity. These derivatives
were also observed to be useful in the treatment of acute and
chronic pain conditions.
Experimental Section
Chemistry. Melting points were measured in open capillary tubes
on a Buchi 530 melting point apparatus and are uncorrected. Infrared
(IR) and proton nuclear magnetic resonance (1H NMR) spectra were
recorded for the compounds on Jasco IR Report 100 (KBr) and
Brucker Avance (300 MHz) instruments, respectively. Chemical
shifts are reported in parts per million (ppm) using tetramethyl silane
(TMS) as an internal standard. All exchangeable protons were
confirmed by addition of D2O. Mass spectra were measured with
a Shimadzu GC-MS-QP5000 spectrophotometer. Only molecular
ions (M+) and base peaks are given. Elemental analyses (C, H,
and N) were undertaken with a Perkin-Elmer model 240C analyzer,
and all analyses were consistent with theoretical values (within
(0.4%) unless indicated. The homogeneity of the compounds was
monitored by ascending thin layer chromatography (TLC) on
silicagel-G (Merck) coated aluminum plates, visualized by iodine
vapor and UV light. Developing solvents were chloroform-
methanol (9:1).
Structure-Activity Relationship
The bioevaluation led to an understanding of the importance
of the size of the group at the carbimino carbon atom. In the
preliminary anticonvulsant screening, replacement of the proton
on the carbimino carbon atom by methyl did not improve the
anticonvulsant activity profile when compared to compounds
with carbimino hydrogen. The benzylidene compounds were
less neurotoxic than their phenylethylidene derivatives. When
replaced with bulkier groups like phenyl (10), the spectrum of
activity improved with no separation from neurotoxicity, while
benzyl compounds (12) showed activity only in scPIC and
scSTY screens with no neurotoxicity. Generally at the 2- and
4-positions of the aryl ring, the electron donating group is
favored as seen with compounds 1, 3, 5, and 7 in which
compound 7 was found to exhibit neurotoxicity at the anticon-
vulsant dose. The electron withdrawing group at the 4-position
(2 and 9) was found to result in complete loss of activity. This
is in agreement with our earlier report on aryl semicarbazones.15
At the carbimino terminal, introduction of cycloalkylidene group
(13, 14) showed decreased activity and introduction of isatin-
imino function showed improved spectrum of activity in MES,
scPTZ, and scPIC but elevated the neurotoxicity. Analysis of
this data reveals that compounds 7, 8, 10, and 13-15 are poor
anticonvulsants as the neurotoxic dose is equal to the anticon-
vulsant dose. Overall it appears that compounds 1 and 5 emerged
as promising candidates with significant activity and not
neurotoxic at the anticonvulsive dose.
In the antiallodynic and antihyperalgesic assays in CCI and
SNL rats, among the benzylidene derivatives (1-5), compounds
1 and 5 with 2-hydroxy and 4-N,N-(dimethylamino) substituent,
respectively, showed more effectiveness in CCI rats in alleviat-
ing dynamic and cold allodynia and mechanical hyperalgesia.
While in SNL rats, compounds with an electron rich group at
4-position (especially 3 and 5) showed activity in three
behavioral assays compared to compounds with substitution at
the 2- (1) or 3-positions (4, 8) which were less active or totally
inactive. Among the phenylethylidene derivatives (6-9), com-
pounds with unsubstitution and substitution with electron
donating groups were active, while compound 9 with 4-nitro
group was found to be ineffective in both CCI and SNL rats.
Replacement of carbimino hydrogen with phenyl (10) showed
lesser effectiveness than dibenzyl (12) or 4-bromophenyl (11)
compound in CCI rats, while in the SNL rats, dibenzyl
compound (12) was less effective than 10 and 11. Replacement
of benzylidene group with cycloalkylidene group (13, 14)
showed moderate activity against spontaneous pain and me-
chanical hyperalgesia in CCI rats and pronounced activity in
SNL rats. Lastly, isatinimino derivative (15) showed similar
effectiveness in CCI and SNL rats.
Synthesis of 4-[(Phenoxycarbonyl)amino]butanoic Acid. 4-Ami-
nobutanoic acid (0.1 mol, 10.32 g) was dissolved in 10 mL of water
and was stirred at 0-5 °C vigorously. An equimolar amount of
phenyl chloroformate (0.1 mol, 15.65 mL) and 0.1 N sodium
hydroxide (10 mL) were added dropwise simultaneously. The
reaction mixture was maintained alkaline throughout the course of
the reaction. Stirring was continued for 2 h. The resulting precipitate
was filtered, dried, and recrystallized using ethanol. Mp 98 °C; IR
1
(KBr) νmax 3260, 3040, 1710, 1650-1590, 1240 cm-1; H NMR
(DMSO-d6, 300 MHz, δ ppm) 1.88 (m, 2H, CH2 â to COOH),
2.20 (t, 2H, CH2 R to COOH), 2.99 (t, 2H, CH2 γ to COOH), 6.77
(s, 1H, NH, D2O exchangeable), 7.2-7.34 (m, 5H, Ar-H), 12.32
(s, 1H, OH of COOH, D2O exchangeable); MS m/z 223 (M+, 100).
Anal. (C11H13NO4) C, H, N.
Synthesis of 4-(Hydrazinecarbonylamino)butanoic Acid.
4-[(Phenoxycarbonyl)amino]butanoic acid (0.05mol, 11.16 g) was
dissolved in 25 mL of ethanol. To it an equimolar amount of
hydrazine hydrate (99%) (0.05 mol, 2.425 mL) was added and
refluxed for 6 h after which the solvent was stripped off by
distillation in vacuo. The resultant product was dried and recrystal-
lized in 95% ethanol. Mp 202 °C; IR (KBr) νmax 3450, 3300, 1650-
1
1620, 1590-1540, 1310, 1210 cm-1; H NMR (DMSO-d6, 300
MHz, δ ppm) 1.82 (m, 2H, CH2 â to COOH), 2.20 (t, 2H, CH2 R
to COOH), 3.16 (t, 2H, CH2 γ to COOH), 5.34 (s, 2H, NH2-, D2O
exchangeable), 6.03 (s, 2H, NHCONH, D2O exchangeable), 12.24
(s, 1H, OH of COOH, D2O exchangeable); MS m/z 161 (M+, 100).
Anal. (C5H11N3O3) C, H, N.
General Procedure for the Synthesis of 4-(Alkylidene/
Arylidene Hydrazinecarbonylamino)butanoic Acids (1-15). To
a solution of 4-(hydrazinecarbonylamino)butanoic acid (0.005 mol,
0.806 g) in ethanol (20 mL) was added an equimolar quantity of
appropriate alkyl/aryl aldehydes or ketones (including isatin) in
ethanol (5-6 mL), and the mixture stirred for 1-3 h until the
completion of the reaction. The resultant precipitate was then
filtered, dried, and recrystallized from 95% ethanol. The physical
data of the compounds are presented in Table 1. The IR spectra of
the compounds were identical in the following aspects: 3320-
3200, 1650-1620, 1620-1590, 1350, 840 cm-1; 1H NMR (DMSO-
d6, 300 MHz, δ ppm) spectra and m/z of some of the representative
compounds are as follows.
4-({[2-(2-Hydroxybenzylidene)hydrazino]carbonyl}amino)-
butanoic Acid (1). 1.80 (m, 2H, CH2 â to COOH), 2.20 (t, 2H,
CH2 R to COOH), 3.0 (t, 2H, CH2 γ to COOH), 6.01 (s, 1H, NHCO,
D2O exchangeable), 7.2-7.4 (m, 4H, Ar-H), 8.0 (s, 1H, carbimino
H), 9.9 (s, 1H, Ar-OH, D2O exchangeable), 10.56 (s, 1H, NHNd,
D2O exchangeable), 12.0 (s, 1H, OH of COOH, D2O exchangeable);
MS m/z 265 (M+, 100).
In conclusion, the present study revealed that some of the
newer GABA derivatives possessed a broad spectrum of
4-({[2-(4-Chlorobenzylidene)hydrazino]carbonyl}amino)-
butanoic Acid (3). 1.80 (m, 2H, CH2 â to COOH), 2.22 (t, 2H,

4-Aminobutyric Acid DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10 2465
CH2 R to COOH), 3.12 (t, 2H, CH2 γ to COOH), 6.0 (s, 1H, NHCO,
D2O exchangeable), 7.4-7.6 (m, 4H, Ar-H), 8.0 (s, 1H, carbimino
H), 10.36 (s, 1H, NHNd, D2O exchangeable), 12.0 (s, 1H, OH of
COOH, D2O exchangeable); MS m/z 283 (M+, 100).
4-[({2-[4-(Dimethylamino)benzylidene]hydrazino}carbonyl)-
amino]butanoic Acid (5). 1.80 (m, 2H, CH2 â to COOH), 2.22 (t,
2H, CH2 R to COOH), 3.0 (s, 6H, N(CH3)2), 3.12 (t, 2H, CH2 γ to
COOH), 6.0 (s, 1H, NHCO, D2O exchangeable), 7.2-7.4 (m, 4H,
Ar-H), 8.0 (s, 1H, carbimino H), 10.56 (s, 1H, NHNd, D2O
exchangeable), 12.0 (s, 1H, OH of COOH, D2O exchangeable);
MS m/z 292 (M+, 100).
animal to maintain equilibrium on the rod for at least 1 min in
each of the three trials.
3. Analgesic Activity. Acetic Acid Induced Writhing. Mice
were divided into groups of six each. Using the method of Siegmund
et al.,31 writhing was induced by an intraperitoneal injection of 0.1
mL of 3% v/v acetic acid. Test group mice received acetic acid 1
h after drug treatment. The number of writhings occurring for a 30
min time period was recorded. For scoring purposes, a writhe was
indicated by stretching of the abdomen with simultaneous stretching
of at least one hind limb. The percentage inhibition of the writhing
response was then calculated.
4-({[2-(1-Phenylethylidene)hydrazino]carbonyl}amino)-
butanoic Acid (6). 1.0 (s, 3H, carbimino CH3), 1.80 (m, 2H, CH2
â to COOH), 2.22 (t, 2H, CH2 R to COOH), 3.12 (t, 2H, CH2 γ to
COOH), 6.0 (s, 1H, NHCO, D2O exchangeable), 7.4-7.65 (m, 5H,
Ar-H), 10.0 (s, 1H, NHNd, D2O exchangeable), 12.22 (s, 1H,
OH of COOH, D2O exchangeable); MS m/z 263 (M+, 100).
4-[({2-[1-(3-Aminophenyl)ethylidene]hydrazino}carbonyl)-
amino]butanoic Acid (8). 0.98 (s, 3H, carbimino CH3), 1.80 (m,
2H, CH2 â to COOH), 2.22 (t, 2H, CH2 R to COOH), 3.12 (t, 2H,
CH2 γ to COOH), 5.86 (s, 2H, Ar-NH, D2O exchangeable), 6.0
(s, 1H, NHCO, D2O exchangeable), 7.0-7.22 (m, 4H, Ar-H),
10.56 (s, 1H, NHNd, D2O exchangeable), 12.0 (s, 1H, OH of
COOH, D2O exchangeable); MS m/z 278 (M+, 100).
4-[({2-[1-(4-Nitrophenyl)ethylidene]hydrazino}carbonyl)amino]-
butanoic Acid (9). 0.98 (s, 3H, carbimino CH3), 1.80 (m, 2H, CH2
â to COOH), 2.22 (t, 2H, CH2 R to COOH), 3.12 (t, 2H, CH2 γ to
COOH), 6.02 (s, 1H, NHCO, D2O exchangeable), 7.6-8.2 (m, 4H,
Ar-H), 10.56 (s, 1H, NHNd, D2O exchangeable), 12.0 (s, 1H,
OH of COOH, D2O exchangeable); MS m/z 308 (M+, 100).
4-({[2-(Diphenylmethylene)hydrazino]carbonyl}amino)-
butanoic Acid (10). 1.80 (m, 2H, CH2 â to COOH), 2.22 (t, 2H,
CH2 R to COOH), 3.12 (t, 2H, CH2 γ to COOH), 6.0 (s, 1H, NHCO,
D2O exchangeable), 7.2-7.8 (m, 10H, Ar-H), 10.56 (s, 1H,
NHNd, D2O exchangeable), 12.0 (s, 1H, OH of COOH, D2O
exchangeable); MS m/z 325 (M+, 100).
4-{[(2-Cyclohexylidenehydrazino)carbonyl]amino}butanoic
Acid (13). 1.32 (m, 4H, o-position of cyclohexane ring), 1.66 (m,
6H, m- and p-positions of cyclohexane ring), 1.80 (m, 2H, CH2 â
to COOH), 2.22 (t, 2H, CH2 R to COOH), 3.12 (t, 2H, CH2 γ to
COOH), 6.0 (s, 1H, NHCO, D2O exchangeable), 10.56 (s, 1H,
NHNd, D2O exchangeable), 12.0 (s, 1H, OH of COOH, D2O
exchangeable); MS m/z 241 (M+, 100).
4-({[2-(2-Oxo-1,2-dihydro-3H-indol-3-ylidene)hydrazino]-
carbonyl}amino)butanoic Acid (15). 1.82 (m, 2H, CH2 â to
COOH), 2.24 (t, 2H, CH2 R to COOH), 3.16 (t, 2H, CH2 γ to
COOH), 6.03 (s, 1H, NHCO, D2O exchangeable), 7.0-7.66 (m,
4H, Ar-H), 10.54 (s, 1H, NHNd, D2O exchangeable), 11.0 (s,
1H, NH of isatinyl, D2O exchangeable), 12.2 (s, 1H, OH of COOH,
D2O exchangeable); MS m/z 290 (M+, 100).
4. Induction of Peripheral MononeuropathysCCI Model.
Unilateral mononeuropathy was produced in rats using the CCI
model performed essentially as described by Bennett and Xie.32
The rats were anesthetized with an intraperitoneal dose of pento-
barbital sodium (65 mg/kg) with additional doses of the anesthetic
given as needed. Under aseptic conditions, a 3 cm incision was
made on the lateral aspect of the left hindlimb (ipsilateral) at the
mid-thigh level with the right hindlimb serving as the control
(contralateral). The left paraspinal muscles were then separated from
the spinous processes, and the common left sciatic nerve was
exposed just above the trifurcation point. Four loose ligatures were
then made with a 4-0 braided silk suture around the sciatic nerve
with about 1 mm spacing. The wound was then closed by suturing
the muscle using chromic catgut with a continuous suture pattern.
Finally, the skin was closed using silk thread with horizontal-
mattress suture pattern. A sham surgery (n ) 4) was performed by
exposing the sciatic nerve, as described above, but not damaging
it. Povidone iodine ointment was applied topically on the wound,
and gentamicin antibiotic (4 mg/kg) was given intramuscularly for
5 days after surgery. The animals were then transferred to their
home cages and left for recovery.
5. Induction of Peripheral MononeuropathysSelective Seg-
mental L5 SNL Model. A left L5 spinal nerve ligation, as described
by Kim and Chung,33 was performed. The rats were anesthetized
with an intraperitoneal dose of pentobarbital sodium (65 mg/kg)
with additional doses of the anesthetic given as needed. Under
aseptic conditions, using the transverse processes of L6 as a guide,
the left paraspinal muscles were exposed and separated from the
spinous processes of L4 to S2 by blunt dissection. The L5 spinal
nerve was then exposed at the level of the dorsal root ganglion
and ligated tightly with a 4-0 braided silk suture. Only one tight
ligature was made in this model. After confirmation of hemostasis,
the wound was then closed by suturing at both muscle and skin
levels. A sham surgery (n ) 4) was performed by exposing the L5
spinal nerve, as described above, but not damaging it. Povidone
iodine ointment was applied topically on the wound, and gentamicin
antibiotic (4 mg/kg) was given intramuscularly for 5 days after
surgery. The animals were then transferred to their home cages
and left for recovery.
Pharmacological Methods. Albino mice (Swiss strain, 20-25
g) and albino rats (Sprague-Dawley, 200-320 g) of either sex
were used as experimental animals. All experiments were approved
by the Institutional Animal Ethics Committee. Animals were housed
six (mice) and four (rats) per cage at constant temperature under a
12 h light/dark cycle (lights on at 7:00 AM), with food and water
ad libitum. The synthesized compounds 1-15 were suspended in
30% v/v polyethylene glycol (PEG) 400 or 0.5% methyl cellulose/
water mixture.
1. Anticonvulsant Screening. The anticonvulsant evaluations
were undertaken partly by the National Institutes of Health, using
their reported procedures.21,28 Initially all compounds were admin-
istered i.p. at doses of 30, 100, and 300 mg/kg to one to four mice.
Activity was established using the MES, scPTZ, scSTY,29 and
scPIC30 tests.
6. Sensory Testing Using Nociceptive Assays. Spontaneous
Pain. Spontaneous pain was assessed for a total time period of 5
min as described previously by Choi et al..34 The operated rat was
placed inside an observation cage that was kept 5 cm from the
ground level. An initial acclimatization period of 10 min was given
to each of the rats. A total number of four rats (n ) 4) were assigned
to this group. The test consisted of noting the cumulative duration
that the rat holds its ipsilateral paw off the floor. The paw lifts
associated with locomotion or body repositioning were not counted.
It has been suggested that those paw lifts in the absence of any
overt external stimuli are associated with spontaneous pain and are
correlative of ongoing pain.
Dynamic Allodynia. All of the operated rats were assessed for
dynamic allodynic response according to the procedure described
by Field et al.35,36 The operated rat was placed inside an observation
cage that was kept 5 cm from the ground level. An initial
acclimatization period of 10 min was given to each of the rats. A
total number of four rats (n ) 4) were assigned to this group. A
positive dynamic allodynic response consisted of lifting the affected
paw for a finite period of time in response to mild stroking on the
plantar surface using a cotton-bud. This stimulus is non-noxious
2. Neurotoxicity Screening. Minimal motor impairment was
measured in mice by the rotorod test. The mice were trained to
stay on an accelerating rotorod that rotates at 10 revolutions per
minute. The rod diameter was 3.2 cm. Trained animals were given
an i.p. injection of the test compounds in doses of 30, 100, and
300 mg/kg. Neurotoxicity was indicated by the inability of the

2466 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 10
Yogeeswari et al.
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noted. If no paw withdrawal was shown within 15 s, the test was
terminated and animals were assigned this withdrawal time. Hence,
15 s effectively represented no withdrawal (results given as
Supporting Information).
Cold Allodynia. The rats demonstrating unilateral mononeur-
opathy were assessed for acute cold allodynia sensitivity using the
acetone drop application technique as described by Caudle et al.37
The operated rat was placed inside an observation cage that was
kept 5 cm from the ground level and was allowed to acclimatize
for 10 min or until exploratory behavior ceased. A total number of
four rats (n ) 4) were assigned to this group. Few drops (100-
200 µL) of freshly dispensed acetone were squirted as a fine mist
onto the midplantar region of the affected paw. A cold allodynic
response was assessed by noting down the duration of paw
withdrawal response. For each measurement, the paw was sampled
three times and a mean calculated. At least 3 min elapsed between
each test (results given as Supporting Information).
Mechanical Hyperalgesia. Mononeuropathic rats were assessed
for mechanical hyperalgesia sensitivity according to the procedure
described by Gonzalez et al.38 The operated rat was placed inside
an observation cage that was kept 5 cm from the ground level. An
initial acclimatization period of 10 min was given to each of the
rats. A total number of four rats (n ) 4) were assigned to this group.
Hindpaw withdrawal duration was measured after a mild pin-prick
stimulus to the midplantar surface of the ipsilateral (left) hindpaw.
A withdrawal was defined as being abnormally prolonged if it lasted
at least 2 s. The mean withdrawal duration was taken from a set of
three responses.
Statistical Analysis. All data are expressed as means ( standard
error of mean (SEM). The data were analyzed using Student’s t
test only when two means were compared (acute pain assay).
In the case of neuropathic pain studies, statistical significance
was determined for drug effects by one-way ANOVA, and
Bonferroni’s post hoc test was used for individual comparisons with
postoperative (predrug) values. In the first assay, significance was
assigned to a P value of less than 0.01, and in the chronic pain
assay, comparison results with a P value of less than 0.05 were
considered statistically significant. The statistical software package
PRISM (Graphpad Software Inc., San Diego, CA) was used for
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Acknowledgment. The authors thank the Council of Sci-
entific & Industrial Research, New Delhi, India, for funding
the project. J.V.R. deeply acknowledges the University Grants
Commission, India, for providing the Junior Research Fellow-
ship.
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Supporting Information Available: Behavioral assays of
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available free of charge via the Internet at http://pubs.acs.org.
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