Synthesis and anticonvulsant activity of some new bishydrazones derived from 3,4-dipropyloxythiophene

Article abstract of DOI:10.1016/j.ejmech.2009.02.009

A series of new 3,4-dipropyloxy-N²,N⁵-bis(substituted)thiophene-2,5-dicarbohydrazides (4-30) were synthesized from ethyl thiodiglycolate and diethyloxalate through multistep reactions. Following Dieckmann-Komppa reaction, the required precursor 3,4-dihydroxythiophene-2,5-diester (1) was prepared. This was derivatized with propyl bromide and further converted to corresponding hydrazide (3), which was finally transformed to targeted hydrazones (4-30) by conventional methods. The newly synthesized compounds were characterized using FT-IR, ¹H and ¹³C NMR, EI-MS and elemental analyses. The anticonvulsant activity of all the title compounds was investigated against maximal electroshock induced seizures (MES) and subcutaneous pentylenetetrazole (scMET) models and their neurotoxicity was also evaluated. Some of the selected compounds were subjected to 6 Hz test in order to evaluate their uncover activities. Compound 3,4-dipropyloxy-N²,N⁵-bis[1-(2-thienyl)ethylidene]thiophene-2,5-dicarbohydrazide (15) has emerged as a lead in this series with less neurotoxicity.

Full text of DOI:10.1016/j.ejmech.2009.02.009

European Journal of Medicinal Chemistry 44 (2009) 3672–3679
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticonvulsant activity of some new bishydrazones derived from
3,4-dipropyloxythiophene
,
b
Ravi Kulandasamy a, Airody Vasudeva Adhikari a , James P. Stables
*
a Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Srinivasnagar, Mangalore, Karnataka 575025, India
b Preclinical Pharmacology Section, Epilepsy Branch, National Institute of Health, Bethesda, USA
a r t i c l e i n f o
a b s t r a c t
A series of new 3,4-dipropyloxy-N2,N5-bis(substituted)thiophene-2,5-dicarbohydrazides (4–30) were
synthesized from ethyl thiodiglycolate and diethyloxalate through multistep reactions. Following Die-
ckmann–Komppa reaction, the required precursor 3,4-dihydroxythiophene-2,5-diester (1) was prepared.
This was derivatized with propyl bromide and further converted to corresponding hydrazide (3), which
was finally transformed to targeted hydrazones (4–30) by conventional methods. The newly synthesized
compounds were characterized using FT-IR, 1H and 13C NMR, EI-MS and elemental analyses. The anti-
convulsant activity of all the title compounds was investigated against maximal electroshock induced
seizures (MES) and subcutaneous pentylenetetrazole (scMET) models and their neurotoxicity was also
evaluated. Some of the selected compounds were subjected to 6 Hz test in order to evaluate their
uncover activities. Compound 3,4-dipropyloxy-N2,N5-bis[1-(2-thienyl)ethylidene]thiophene-2,5-dicar-
bohydrazide (15) has emerged as a lead in this series with less neurotoxicity.
Article history:
Received 12 August 2008
Received in revised form
27 January 2009
Accepted 12 February 2009
Available online 21 February 2009
Keywords:
Thiophene
Bishydrazones
Epilepsy
Anticonvulsants
Toxicity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
containing thiophene moiety in their structure as active pharma-
cophore. Also, the higher activity of sodium phethenylate [9] is due
Epilepsy is one of the most common neurological disorders of
the brain, due to sudden burst of abnormal electrical discharges. It
has been reported that people suffering from epilepsy is increasing
day by day in the world [1]. More than 20% of the world epileptic
populations are not contented with the marketed antiepileptic
drugs (AEDs). However, all currently approved anticonvulsant
agents have dose-related toxicity and idiosyncratic side effects [2].
Some of the currently available active drugs have not been directly
linked with any specific binding site to receptor within the brain. It
is too difficult to identify common pharmacophore responsible for
prevention or arrest of seizure activity mainly because of the
chemical diversity of organic compounds and their multiple
mechanism of action in controlling the seizures [3]. Hence, the
search for antiepileptic compounds with more selective activity
and minimum toxicity continues to be an area of investigation in
medicinal chemistry.
to the presence of thiophene ring in its structure. On the other
hand, many carbazones and semicarbazones have been docu-
mented as potent anticonvulsants [11–16]. Their activity is mainly
attributed to the presence of aryl binding site with aryl/alkyl
hydrophobic group, hydrogen bonding domain and electron donor
group, which are the essential requirements [17] for the molecules
to show potential activity. Further, introduction of the one more
aryl ring in the structure brings about the enhanced van der Waals
bonding at the receptor site that leads to increased potency.
The literature survey reveals that very few reports appeared on
anticonvulsant activity of different hydrazones [11,12], but none on
thiophene carbohydrazides. Against this background, it has been
thought of synthesizing novel thiophene derivatives containing
bishydrazones at position 2 and 5 and propyloxy group at positions 3
and 4, hoping that the resulting molecules would show enhanced
anticonvulsant activity. In the present molecular design, the thio-
phene ring with propyloxygroup acts as electron releasing group and
bishydrazones containing aryl/alkyl groups provide effective
hydrogen bonding and van der Waals bonding with receptor binding
site. Accordingly, Fig. 1 shows the combination of various pharma-
cophoric features required for an effective anticonvulsant. In contin-
uation of our research program on the synthesis of novel thiophene
derivatives exhibiting potential anticonvulsant activity, we herein
report the multistep synthesis of hitherto unknown 3,4-dipropyloxy-
N2,N5-bis(substituted) thiophene-2,5-dicarbohydrazides (4–30) and
Thiophene derivatives have been known to possess a variety of
biological activities such as anti-inflammatory [4], analgesic [5],
antidepressant [6], antimicrobial [7] and anticonvulsants [8–10].
Presently available active AEDs like tiagabine [8], etizolam [10] are
* Corresponding author. Tel.: þ91 824 2474000x3203; fax: þ91 824 2474033.
E-mail addresses: avchem@nitk.ac.in, avadhikari123@yahoo.co.in (A.V. Adhikari).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.009

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
3673
S
S
CH3
A
E
O
E
S
N
CH3
A
Cl
N
Na
HN
HB
N
A
N
HB
N
H3C
E
S
O
HOOC
HB
N
H3C
Etizolam
Phethenylate sodium
Tiagabine
CH3 H3C
E
O
O
O
R
A
O
S
NH
NH
N
N
R1
HB
R1
R
HB - Hydrogen Bonding Domain; E - Electron Donor; A - Aryl binding site
Fig. 1. Molecular design of new thiophene based antiepileptic agents by pharmacophore combination.
screening of their anticonvulsant activity by MES, scMET, and 6 Hz
models and neurotoxicity.
3286 cmꢀ1 and –NH– group at 3191 cmꢀ1 of hydrazide (3) conformed
the product formation. The compounds 5–30 behave similarly. 1H
NMR spectrum of it showed two triplets at
and multiplet (–CH2–) at 1.8 confirming the presence of propyloxy
group. The azomethine proton resonated at 8.4 (–CH]N–) as
singlet and the aromatic protons appeared as multiplet at 7.4–7.7.
The lone pCONH– proton resonated as sharp singlet at 11.2, while
the absence of broad peak at 5.0 (–NH2) and sharp singlet at 8.3
d 0.9 (–CH3), 4.2 (–OCH2–)
2. Chemistry
d
d
The reaction sequences employed for synthesis of title
compounds, viz. 3,4-dipropyloxy-N2,N5-bis(substituted)thiophene-
2,5-dicarbohydrazides (4–30) are shown in Scheme 1.
d
d
d
(–NH–) (which correspond to the peaks of bishydrazide) further
confirmed the formation of condensationproduct 4. The spectral data
of other hydrazones are given in the Experimental section.
The structure of hydrazone 4 was further confirmed by its Mass
spectrum. It displayed the molecular ion peak at m/z 492 (100%),
which is in agreement with the molecular formula C26H28N4O4S.
The major peaks at m/z 389, 372, 347 and 289 were due to the
fragmentation of molecular ion leading to formation of important
species with complex structures, as shown in Scheme 2. It has been
also seen that similar fragmentation patterns were observed for
other hydrazone derivatives.
The starting material, thiodiglycolic ester was prepared by
refluxing the corresponding acidand absolute alcohol in the presence
of sulfuric acid. This ester was condensed with diethyloxalate in the
presence ofsodium ethoxideto obtain compound(1). Thiscompound
was substituted with propyl bromide in the presence of anhydrous
potassium carbonate to get compound (2). The key intermediate,
3,4-dipropoxythiophene-2,5-dicarbohydrazide (3) was synthesized
by condensing corresponding ester (2) with hydrazine hydrate in
methanol. It was then converted to hydrazones by condensing it with
appropriate aryl/alkyl aldehydes or ketones in the presence of Conc.
HCl as catalyst. The physiochemical properties and characterization
data of compounds 4–30 are summarized in Tables 1 and 2.
3. Pharmacology
The structural assignments to new compounds were based on
their elemental analysis and spectral (FT-IR, 1H NMR, 13C NMR and
Mass) data. The formation of hydrazide 3 from the corresponding
ester 2 was confirmed by its IR and 1H NMR spectral data. Its IR
spectrum showed bands at 3286, 3191 and 1641 cmꢀ1 indicating
the presence of –NH2, pNH and pC]O groups respectively. In its 1H
The newly synthesized compounds were evaluated for anti-
convulsant activity by the maximal electroshock (MES), subcuta-
neous metrazol (MET) and neurotoxicity screening by Rotorod
technique. Some of the selected compounds were evaluated in 6 Hz
model at five time intervals. The results of anticonvulsant activity of
newly synthesized compounds 4–30 are presented in Tables 3 and
4. The detailed test procedures are given the Experimental section.
NMR spectrum it displayed two triplet peaks at
d 0.9 (–CH3), 4.1
(–OCH2–) and multiplet at 1.8 (–CH2–). Further it showed broad
peaks at 5.0 (–NH2) and singlet at 8.3 (–NH–) conforming the
formation of hydrazide group in the molecule.
The condensation of carbonyl compounds with hydrazide 3 to
form hydrazone 4 was evidenced from its spectral data. Its IR spec-
trum showed absorption bands at 3211, 2969, 1642 and 1601 cmꢀ1
indicating the presence of pNH, propyl, pC]O and pC]N groups
respectively. The absence of absorption bands due to –NH2 group at
4. Results and discussion
The results of MES test reveal that compounds 14, 15, 20, 28, 29
and 30 showed activity at a dose of 300 mg/kg (1/1 of the animals
protected at 4 h). The presence of coumarin, phenyl and thiophene

3674
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
OH
HO
EtOOC
S
COOEt
A
+
COOEt
O
O
EtOOC
S
OEt
EtO
1
B
CH3 H3C
CH3 H3C
O
O
O
O
C
NH
O
O
H2N
S
EtOOC
COOEt
S
NH
NH2
3
2
D
D
CH3 H3C
CH3 H3C
O
O
R
O
O
NH
O
O
R2
S
R1
NH
O
O
NH
N
NH
R2
S
25-30
N
4 - 24
R1
R
where R2 :
N
N
N
N
N
NH2
S
N
O
S
CH
S
O
3
S
CH
S
3
25
26
27
28
29
30
A: NaOEt, H+; B: CH3CH2CH2Br, K2CO3, DMF; C: NH2NH2H2O, EtOH; D: aldehyde/ketone, EtOH, H+.
Scheme 1. Synthesis of substituted bishydrazones. A: NaOEt, Hþ; B: CH3CH2CH2Br, K2CO3, DMF; C: NH2NH2 H2O, EtOH; D: aldehyde/ketone, EtOH, Hþ
.
moieties along with methyl group attached to azomethine carbon
of hydrazones showed marginal activity at higher concentration.
However, at lower concentration no activity was observed probably
due to lack of hydrogen bonding with receptor site. The marginal
activity of compounds 28, 29 and 30 may be due to the presence of
alicyclic substituents. The size of the alicyclic substituent did not
affect the activity. The results showed that compounds 19 and 22
displayed protection at 100 mg/kg dose (1/7 animals protected at
0.5 h). But this effect was not found to be linear at higher dose. The
observed activity in MES screening may be due to the presence of
electron releasing substituents in hydrazones. In scMET model,
compounds 4, containing phenyl group and 15, containing thio-
phene and methyl groups showed marginal efficacy at 300 mg/kg
dose (1/5 of the animals protected at 4 h) and compound 22
showed protection at a dose 30 mg/kg (1/5 of the animals protected
at 4 h).
a dose level of 100 mg/kg, whereas compounds 8, 21 and 25
showed toxicity at 300 mg/kg. The remaining compounds did not
show neurotoxicity at the maximum administered dose level of
300 mg/kg. These results clearly indicated that toxicity is not
attributed to the basic moiety, i.e., 3,4-dipropropyloxy thiophene,
but it is due to the presence of other substituents such as pyridine,
carbazole, furan, indole, coumarin and certain alkyl groups
attached to it. The observed toxicity in the above compounds may
be due to the active involvements of these substituents in the
metabolism.
Some of the compounds were selected for 6 Hz model to iden-
tify their activity at five different time points, i.e., 0.25, 0.5, 1.0, 2.0
and 4 h. As observed from the results, amongst various tested
hydrazones, compounds 4, 7 and 15 showed good response at
a dose 100 mg/kg and hence they emerged as lead compounds.
These compounds have been selected for second phase of screening
in order to explore their efficacy in detail. It is interesting to note
that presence of methyl and thiophene moieties as substituents
attached to azomethine group of title compound (15) has increased
As seen from the results of neurotoxicity screening, compounds
6 and 26 showed toxicity at a dose 30 mg/kg. It has been observed
that compounds 5, 9, 10, 11, 12, 20, 23 and 27 exhibited toxicity at

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
3675
Table 1
Physical characterization data of new compounds, 4–24.
Cd No
R
R1
Mol. for/Mol. mass
Recry. Sol
Elemental analysis (%): Found (cal.)
Mp (ꢁC)/yield %)
C
H
N
S
4
5
6
7
8
9
H
H
H
H
H
H
H
H
C6H5
3-Indolyl
5-Nitro-2-furyl
3,4,5-Meth-oxyphenyl
2-Fluorenyl
2-Thienyl
C26H28N4O4S/492
C30H30N6O4S/570
C22H22N6O10S/562
C32H40N4 O10S/672
C40H36N4O4S/668
C22H24N4O4S3/504
C20H32N4O4S/424
C42H42N6O4S/726
MDC
DMF
DMF
DMF
DMF
DMF
MDC
DMF
DMF
DMF
MDC
MDC
CHCl3
CHCl3
DMF
DMF
DMF
CHCl3
DMF
DMF
CHCl3
63.52 (63.40)
62.58 (63.14)
46.62 (46.97)
57.01 (57.13)
71.42 (71.83)
52.30 (52.36)
56.41 (56.58)
69.10 (69.40)
70.29 (70.78)
66.01 (66.24)
64.28 (64.59)
54.01 (54.11)
57.42 (57.71)
60.03 (59.75)
64.05 (64.19)
49.32 (49.42)
62.37 (62.18)
54.32 54.52)
66.65 (66.85)
50.03 (50.13)
70.58 (70.78)
5.67 (5.73)
5.19 (5.30)
3.85 (3.94)
5.85 (5.99)
5.31 (5.43)
4.68 (4.79)
7.52 (7.60)
5.72 (5.82)
5.60 (5.63)
5.48 (5.56)
6.28 (6.20)
5.22 (5.30)
5.99 (6.06)
5.78 (5.79)
5.62 (5.72)
4.81 (4.90)
4.93 (4.91)
6.98 (7.12)
5.20 (5.30)
3.71 (3.75)
5.53 (5.63)
11.43 (11.37)
14.88 (14.73)
15.01 (14.94)
8.45 (8.33)
6.42 (6.51)
229–230/45
294–295/55
280–281/51
297–298/57
304–205/45
259–260/57
192–193/74
278–279/76
268–269/65
291–292/72
229–230/79
253–254/71
232–233/51
242–243/54
293–294/47
288–289/59
251–252/72
219–220/48
255 d*/50
5.69 (5.62)
5.82 (5.70)
4.62 (4.77)
4.89 (4.79)
19.11 (19.06)
7.48 (7.55)
4.31 (4.41)
5.02 (4.97)
5.00 (4.91)
6.25 (6.16)
18.09 (18.06)
6.72 (6.63)
6.15 (6.14)
5.43 (5.36)
18.03 (17.99)
4.89 (4.88)
8.20 (8.09)
4.86 (4.96)
11.89 (11.81)
5.05 (4.97)
8.23 (8.12)
11.32 (11.10)
13.39 (13.20)
11.61 (11.56)
8.75 (8.69)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1-Butyl
N-Ethyl-3-carbazolyl
4-Biphenyl
6-Methoxy-2-phenyl
Phenyl
2-Thienyl
2-Pyrrolyl
2-Pyridyl
3-Indolyl
2-Thiazolyl
3-Couma-rinyl
CH3
H
H
C38H36N4O4S/644
C36H36N4O6S/652
C28H32N4O4S/520
C24H28N4O4S3/532
C24H30N6O4S/498
C26H30N6O4S/522
C32H34N6O4S/598
C22H26N6O4S3/534
C34H32N4O8S/656
C18H28N4O4S/396
C36H34N6O4S/646
C34H30Br2N4O4S3/814
C38H36N4O4S/644
8.68 (8.58)
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
10.71 (10.76)
10.62 (10.52)
16.90 (16.86)
16.21 (16.08)
14.11 (14.04)
15.80 (15.72)
8.65 (8.53)
14.18 (14.13)
13.08 (12.99)
6.81 (6.88)
4-Pyridyl
5-Br-2-thienyl
C6H5
297–298/54
254–255/79
8.79 (8.69)
d*: decomposed.
the activity considerably. Also, introduction of phenyl (4) and tri-
methoxy phenyl (7) groups to azomethine carbon caused moderate
activity. These active molecules have satisfied the structural
requirements such as aryl binding site with a hydrophobic group,
hydrogen bonding domain –NHCO– group, electron donor group
and another hydrophobic aryl ring for showing the activity.
least contribution towards the neurotoxicity, it can be used as basic
moiety in structural designing of new molecules for AEDs.
6. Experimental procedure
6.1. Chemistry
5. Conclusion
All the chemicals and the solvents, purchased from Aldrich and
Merck were used without further purification. The progress of the
reaction was monitored by thin layer chromatography, performed
on a Silica gel 60 F254 coated Aluminium sheet. Melting points were
determined on open capillaries using a Stuart SMP3 (BIBBY STERLIN
Ltd. UK) apparatus and they are uncorrected. FT-IR spectra were
recorded on Nicolet Avatar 330 FTIR spectrophotometer. The 1H
NMR and 13C NMR spectra were recorded on Varian 300 & 400 MHz
NMR spectrophotometers using TMS as an internal standard.
In the present work, twenty-seven newly designed bispropy-
loxyhydrazones were successfully synthesized. Their structures
were confirmed by spectral and elemental analyses. Further, their
anticonvulsant activity was evaluated by MES, scMET and 6 Hz
models. Also, they were screened for neurotoxicity. From the results
of antiepileptic activity, it can be concluded that compounds 4, 7 and
15 displayed good activity with less neurotoxicity. Here the activity
is attributed to the presence of favorable structural environment
such as aryl binding site with a hydrophobic group, hydrogen
bonding domain –NHCO– group, electron donor group and another
hydrophobic aryl ring in these hydrazones. It can be also concluded
that the 3,4-dipropyloxythiophene acts as an important pharma-
cophore for showing anticonvulsant activity. As this moiety showed
Chemical shifts were reported in ppm (d) and signals were described
as singlet (s), doublet (d), triplet (t), quartet (q), broad (br) and
multiplet (m). The coupling constant (J) values are expressed in Hz.
The FAB mass spectra were recorded on a JEOL SX 102/DA-6000
spectrophotometer/Data system using Argon/Xenon (6 kV, 10 mA)
FAB gas, at 70 eV. Elemental analysis was carried out using FLASH EA
Table 2
Physical and characterization data of new compounds, 25–30.
Cd No
R2
Mol. for/Mol. mass
Recry. Sol
Elemental analysis (%): Found (cal.)
Mp (ꢁC)/yield(%)
C
H
N
S
25
26
b
,
b
-Diphenyl propiophenyl
C54H52N4O4S/852
MDC
DMF
75.91 (76.03)
51.63 (51.83)
6.11 (6.14)
4.87 (4.97)
6.65 (6.57)
8.53 (8.63)
3.71 (3.76)
24.91 (24.71)
234–235/64
262–263/63
6-Methyl-5,6-dihydro-4-H-
thieno [2,3-b] thiopyran-4-yl
6-Methyl-5,6-di-hydro-4-H-
thieno [2,3-b] thiopyran(2-
sulfonamide)-4-yl
C28H32N4O4S5/648
27
C28H34N6O8S7/806
DMF
41.50 (41.67)
4.32 (4.25)
10.52 (10.41)
27.65 (27.81)
>300/70
28
29
30
Cyclopentenyl
Cyclohexenyl
Cycloheptenyl
C22H32N4O4S/448
C24H36N4O4S/476
C26H40N4O4S/504
MDC
CHCl3
CHCl3
58.61 (58.91)
60.42 (60.48)
61.68 (61.88)
7.10 (7.19)
7.69 (7.61)
7.92 (7.99)
12.59 (12.49)
11.82 (11.75)
11.20 (11.10)
7.21 (7.15)
6.79 (6.73)
6.40 (6.35)
203–204/66
182–183/66
174–175/75

3676
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
C3H7H C3
7
H7C3
O
O
C3H7H7C3
O
O
H7C3
O
HO
O
O
+
+
H2N
O
O
+
O
+
O
S
O
S
S
m/z 185
m/z 227
S
H2N
m/z 270
C3H7H7C3
m/z 169
O
O
+
O
NH
O
N
S
+
NH
HN
N
m/z 372/373
.
m/z 104
C3H7H7C3
+
O
O
O
O
S
.
NH
NH
N
C3H7H7C3
+
N
m\z 492/493
O
O
O
C3H7H7C3
+
S
O
O
O
NH2
NH
N
m/z 389
NH
O
N
S
+
NH2
N
m/z 347
.
m/z 147
C3H7
O
+
C3H7
OH
HO
OH
OH
O
+
O
O
O
+
O
O
O
S
S
S
NH
NH2
NH
N
N
NH
m/z 289
N
m/z 331
m/z 347
Scheme 2. Mass fragmentation pattern of compound 4.
1112 series, CHNSO Analyzer (Thermo). Compound 1 and 2 were
synthesized from the reported procedures [18–20].
washed with pet. ether (40ꢁ–60ꢁ) and dried to get 0.7 g of 3 with
yield, 78%. Mp 138–139 ꢁC; IR (KBr, cmꢀ1 : 3286 cmꢀ1 (–NH2),
3191 cmꢀ1 (–NH–), 2964 cmꢀ1 (propyl) and 1641 cmꢀ1 (pC]O); 1H
NMR (CDCl3, 300 MHz) in ppm: 0.9 (t, 6H, –CH3), 1.8 (m, 4H,
) y
6.1.1. Synthesis of 3,4-dipropyloxythiophene-2,5-carbodihydrazide
(3)
d
–CH2–), 4.1 (t, 4H, –OCH2–), 5.0 (br, 4H, –NH2) and 8.3 (s, 2H, –NH–).
One gram (0.003 mol) of diethyl 3,4-dipropyloxythiophene
2,5-dicarboxylate was added to a solution of 1.6 ml (0.03 mol) of
hydrazine hydrate in 30 ml of ethanol. The reaction mixture was
refluxed for 2 h. Upon cooling the reaction mixture in cold water,
white needle-like crystals were obtained. The product was filtered,
6.1.2. General procedure for synthesis of bishydrazones (4–30)
A clear solution of 1.58 g (0.005 mol) of propyloxy thiophene
carbohydrazide in 15 ml of absolute ethanol was mixed with
0.5 ml of conc. Hydrochloric acid. To this 0.010 mol of appropriate

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
3677
4 h
Table 3
Anticonvulsant activity and neurotoxicity of compounds 4–30.
Table 3 (continued)
Cd No
Dose (mg/kg)
MESa
05 h
scMETb
0.5 h
Toxicityc
0.5 h
Cd No
Dose (mg/kg)
MESa
05 h
scMETb
0.5 h
Toxicityc
0.5 h
4 h
4 h
4 h
4 h
4 h
28
30
100
300
30
100
300
30
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
4
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
30
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
1/7
0/5
0/1
0/3
0/1
0/1
0/3
0/1
0/1
1/7
0/5
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
1/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/3
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
1/5
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/4
0/8
0/4
0/4
1/8
1/4
1/4
8/8
3/4
0/4
0/8
0/4
0/4
0/8
1/4
0/4
3/8
4/4
0/4
2/8
3/4
0/4
2/8
2/4
0/4
1/8
1/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
0/8
0/4
0/4
3/8
2/4
0/4
0/8
1/4
0/4
0/8
0/4
0/4
4/8
1/4
0/4
1/8
0/4
0/4
0/8
1/4
1/4
4/8
2/4
0/4
2/8
2/4
0/2
0/4
0/2
0/2
0/4
0/2
0/2
2/4
1/2
0/2
0/4
0/2
0/2
0/4
1/2
0/2
0/4
1/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
1/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
1/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
0/2
0/2
0/4
1/2
29
30
5
6
100
300
a
Maximal electroshock test (number of animals protected/number of animal
7
tested).
b
Subcutaneous metrazol seizure threshold test (number of animals protected/
number of animal tested).
8
c
Rotorod toxicity (number of animals exhibiting toxicity/number of animal
tested).
9
carbonyl compound, dissolved in 10 ml of absolute ethanol was
added slowly while stirring. The reaction mixture was heated to
reflux for 4–5 h and cooled to 10 ꢁC. The precipitated product was
separated by filtration and recrystallized from an appropriate
solvent. The physical and characterization data of compounds,
4–30 are presented in Tables 1 and 2. Their spectral data are given
below.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
6.1.3. 3,4-Dipropyloxy-N2, N5-bis(phenylmethylene)thiophene-2,5-
dicarbohydrazide (4)
IR (KBr, cmꢀ1
) y:
3211 cmꢀ1 (pNH), 2969 cmꢀ1 (propyl),
1642 cmꢀ1 (pC]O) and 1601 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
400 MHz)
d
in ppm: 0.9 (t, 6H, –CH3, J ¼ 7.2), 1.8 (m, 4H, –CH2–,
J ¼ 7.2), 4.2 (t, 4H, –OCH2–, J ¼ 7.2), 7.4 (m, 6H, C3, C4 and
C5–phenyl), 7.7 (m, 4H, C2 and C6–phenyl), 8.4 (s, 2H, N]CH–), 11.2
(s, 2H, –CONH–); MS (FAB) (m/z, %): 492 (Mþ, 100), 372 (80), 347
(20), 289 (70), 121 (50).
6.1.4. 3,4-Dipropyloxy-N2,N5-bis(1H-indole-2-
ylmethylene)thiophene-2,5-dicarbohydrazide (5)
IR(KBr, cmꢀ1 :3278 cmꢀ1 (pNH), 2964 cmꢀ1 (propyl),1662 cmꢀ1
)y
(pC]O) and 1603 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6, 400 MHz)
d in
ppm: 0.9 (t, 6H, –CH3, J ¼ 7.2), 1.8 (m, 4H, –CH2–, J ¼ 6.6), 4.2 (t, 4H,
–OCH2–, J ¼ 6.2), 7.2 (m, 4H, C5 and C6–indole), 7.4 (d, 2H, C3–Indole,
J ¼ 7.8), 7.8 (d, 2H, C7–indole, J ¼ 2.1), 8.2 (d, 2H, C4–indole, J ¼ 7.5), 8.5
(s, 2H, –N]CH–), 10.9 (s, 2H, –CONH–), 11.6 (s, 2H, indole–NH); MS
(FAB) (m/z, %): 570 (Mþ, 40), 492 (50), 459 (10), 372 (10).
6.1.5. 3,4-Dipropyloxy-N2,N5-bis[(5-nitro-2-
furyl)methylene]thiophene-2,5-dicarbohydrazide (6)
IR (KBr, cmꢀ1
) y:
3209 cmꢀ1 (pNH), 2966 cmꢀ1 (propyl),
1646 cmꢀ1 (pC]O) and 1601 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
Table 4
Results of anticonvulsant activity by 6 Hz model.
Cd No
Dose (mg/kg)
0.25 h
0.5 h
1.0 h
2.0 h
4.0 h
4
5
7
8
15
23
24
25
29
30
100
100
100
100
100
100
100
100
100
100
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
2/4
1/4
0/4
1/4
3/4
0/4
0/4
0/4
0/4
1/4
2/4
1/4
2/4
1/4
3/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
1/4
1/4
3/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
100
300
(continued on next page)

3678
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
400 MHz)
d
in ppm: 0.9 (t, 6H, –CH3, J ¼ 6.6), 1.7 (m, 4H, –CH2–), 4.2
–OCH2–), 7.3 (t, 2H, C4–pyridine), 7.7 (t, 2H, C5–pyridine), 8.3 (d, 2H,
C3–pyridine), 8.6 (d, 2H, C6–pyridine), 10.4 (s, 2H, –CONH–); MS
(FAB) (m/z, %): 525 (M þ 2, 100), 389 (40), 136 (20).
(t, 4H, –OCH2–), 7.2 (d, 1H, C3–furan), 7.8 (d, 1H, C4–furan, J ¼ 3.4),
8.3 (s, 2H, –N]CH–), 11.7 (s, 2H, –CONH–); MS (FAB) (m/z, %): 562
(Mþ, 70), 459 (100), 407 (30), 154 (100).
6.1.13. 3,4-Dipropyloxy-N2,N5-bis(1-methylethylidene)thiophene-
6.1.6. 3,4-Dipropyloxy-N2,N5-bis(9H-fluoren-2-
2,5-dicarbohydrazide (21)
ylmethylene)thiophene-2,5-dicarbohydrazide (8)
IR (KBr, cmꢀ1 : 3315 cmꢀ1 (pNH), 2971 cmꢀ1 (propyl),1678 cmꢀ1
) y
IR(KBr, cmꢀ1
)
y
:3298 cmꢀ1 (pNH), 2967 cmꢀ1 (propyl),1668 cmꢀ1
(pC]O) and 1630 cmꢀ1 (pC]N–); 1H NMR (CDCl3, 300 MHz)
d in
(pC]O) and 1599 cmꢀ1 (pC]N–); MS (FAB) (m/z, %): 668 (Mþ, 70),
ppm: 1.0 (t, 6H, –CH3 propyl, J ¼ 7.2), 1.9 (m, 4H, –CH2–), 2.0 (s, 6H,
492 (20), 460 (20), 434 (5), 236 (20), 208 (20), 165 (30), 154 (100).
–CH3),2.2(s,6H, –CH3), 4.2(t, 4H, –OCH2–, J ¼ 7.2),9.9(s,2H,–CONH–).
6.1.7. 3,4-Dipropyloxy-N2,N5-bis(2-thienylmethylene)thiophene-
6.1.14. 3,4-Dipropyloxy-N2,N5-bis[1-phenyl(pyridin-4-
2,5-dicarbohydrazide (9)
yl)methylidene]thiophene-2,5-dicarbohydrazide (22)
IR (KBr, cmꢀ1
)
y
:
3222 cmꢀ1 (pNH), 2966 cmꢀ1 (propyl),
IR (KBr, cmꢀ1 3301 cmꢀ1 (pNH), 2966 cmꢀ1 (propyl),
) y:
1641 cmꢀ1 (pC]O) and 1592 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
400 MHz)
1670 cmꢀ1 (pC]O) and 1584 cmꢀ1 (pC]N–); MS (FAB) (m/z, %):
648 (M þ 2, 100), 450 (40), 408 (10), 198 (40).
d
in ppm: 0.9 (t, 6H, –CH3, J ¼ 6.9), 1.7 (m, 4H,
–CH2–, J ¼ 6.7), 4.2 (t, 4H, –OCH2–), 7.1 (t, 2H, C4–thiophene), 7.4 (d,
2H, C5–thiophene) and 7.6 (d, 2H, C3–thiophene, J ¼ 4), 8.6 (s, 2H,
–N]CH–), 11.2 (s, 2H, –CONH–); MS (FAB) (m/z, %): 504 (Mþ, 100),
378 (50), 352 (20), 294 (40), 227 (20), 169 (30), 154 (40).
6.1.15. 3,4-Dipropyloxy-N2,N5-bis[(5-bromo-2-thienyl)(1-
phenyl)methylidene]thiophene-2,5-dicarbohydrazide (23)
IR (KBr, cmꢀ1
) y:
3290 cmꢀ1 (pNH), 2968 cmꢀ1 (propyl),
1678 cmꢀ1 (pC]O) and 1636 cmꢀ1 (pC]N–); MS (FAB) (m/z, %):
814 (M þ 2, 100), 535 (50), 449 (20), 266 (60).
6.1.8. 3,4-Dipropyloxy-N2,N5-bis(butylidene)thiophene-2,5-
dicarbohydrazide (10)
IR (KBr, cmꢀ1
)
y:
3212 cmꢀ1 (pNH), 2965 cmꢀ1 (propyl),
6.1.16. 3,4-Dipropyloxy-N2,N5-bis(diphenylmethlidene)thiophene-
1645 cmꢀ1 (pC]O) and 1600 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
2,5-dicarohydrazide (24)
d
in ppm: 1.0 (t, 6H, –CH3 of propyl, J ¼ 7.2),1.1 (t, 6H, –CH3
IR (KBr, cmꢀ1 3293 cmꢀ1 (pNH), 2969 cmꢀ1 (propyl),
) y:
of butyl), 1.6 (m, 4H, –CH2–CH3 of butyl, J ¼ 7.5), 1.8 (m, 4H, –CH2– of
propyl, J ¼ 7.2), 2.4 (m, 4H, –CH2–CH2– of butyl, J ¼ 6.9), 4.2 (t, 4H,
–OCH2–, J ¼ 6.6), 7.6 (t, 2H, –N]CH–CH2, J ¼ 5.4), 9.9 (s, 2H, –CONH–).
1670 cmꢀ1 (pC]O) and 1625 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
in ppm: 0.7 (t, 6H, –CH3, J ¼ 7.2), 1.1 (m, 4H, –CH2–,
J ¼ 7.5), 3.6 (t, 4H, –OCH2–, J ¼ 7.2), 7.3 (m, 10H, phenyl), 7.6 (m,10H,
phenyl), 10.2 (s, 2H, –CONH–); 13C NMR (CDCl3, 400 MHz)
in ppm:
d
d
6.1.9. 3,4-Dipropyloxy-N2,N5-bis(1-phenylethylidene)thiophene-
2,5-dicarbohydrazide (14)
9.17 (–CH3), 22.0 (–CH2–), 75.1 (–OCH2–), 128–129 (phenyl), 136.9
(C2 and C5–thiophene), 146.1 (C3 and C4–thiophene), 154.5 (pC]N),
157.1 (pC]O); MS (FAB) (m/z, %): 647 (M þ 2, 100), 450 (20), 224
(20).
IR (KBr, cmꢀ1
) y:
3310 cmꢀ1 (pNH), 2963 cmꢀ1 (propyl),
1674 cmꢀ1 (pC]O) and 1630 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
d
in ppm: 1.0 (t, 6H, –CH3 propyl, J ¼ 7.2), 1.9 (m, 4H,
–CH2–, J ¼ 7.2), 2.3 (s, 6H, –CH3), 4.3 (t, 4H, –OCH2–, J ¼ 7.2), 7.4 (m,
6H, phenyl), 7.9 (m, 4H, phenyl); MS (FAB) (m/z, %): 523 (M þ 2,
100), 388 (40), 304 (20), 161 (30), 135 (20), 118 (60).
6.1.17. 3,4-Dipropyloxy-N2,N5-bis(6-methyl-5,6-dihydro-4-H-
thieno[2,3-b]thiopyran-4-ylidene)thiophene-2,5-dicarbohydrazide
(26)
IR (KBr, cmꢀ1
) y:
3322 cmꢀ1 (pNH), 2965 cmꢀ1 (propyl),
6.1.10. 3,4-Dipropyloxy-N2,N5-bis[1-(2-
1661 cmꢀ1 (pC]O) and 1592 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
400 MHz) in ppm: 0.9 (t, 6H, –CH3 propyl), 1.4 (d, 6H, –CH3), 1.9 (s,
thienyl)ethylidene]thiophene-2,5-dicarbohydrazide (15)
d
IR (KBr, cmꢀ1
)
y:
3304 cmꢀ1 (pNH), 2962 cmꢀ1 (propyl),
4H, C5–alicyclic), 2.6 (m, 2H, C6–alicyclic), 3.7 (s, 2H, –CH2–), 4.3 (t,
4H, –OCH2–), 7.4 (d, 2H, C4–thiophene, J ¼ 5.24), 7.5 (d, 2H, C5–
thiophene, J ¼ 7.5) 10.4 (s, 2H, –CONH–); MS (FAB) (m/z, %): 650
(M þ 2, 100), 451 (60), 425 (10), 409 (10), 367 (10), 199 (10), 182
(20).
1678 cmꢀ1 (pC]O) and 1626 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
d
in ppm: 1.0 (t, 6H, –CH3 propyl, J ¼ 7.2), 1.9 (m, 4H,
–CH2–, J ¼ 7.2), 2.3 (s, 6H, –CH3), 4.3 (t, 4H, –OCH2–, J ¼ 6.9), 7.0 (t,
2H, C4–thiophene), 7.4 (m, 4H, C3 and C5–thiophene).
6.1.11. 3,4-Dipropyloxy-N2,N5-bis[1-(1H-pyrrol-2-
6.1.18. 3,4-Dipropyloxy-N2,N5-bis(dicyclohexylidene)thiophene-
yl)ethylidene]thiophene-2,5-dicarbohydrazide (16)
2,5-dicarbohydrazide (29)
IR (KBr, cmꢀ1
)
y
: 3311 cmꢀ1 (pNH), 2971 cmꢀ1 (propyl),1655 cmꢀ1
IR (KBr, cmꢀ1 3326 cmꢀ1 (pNH), 2931 cmꢀ1 (propyl),
) y:
(pC]O) and 1592 cmꢀ1 (pC]N–); 1H NMR (CDCl3, 300 MHz)
d
in
1675 cmꢀ1 (pC]O) and 1632 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz) in ppm: 1.0 (t, 6H, –CH3), 1.7–1.9 (m, 16H, cyclohexyl),
ppm: 1.0 (t, 6H, –CH3 propyl, J ¼ 7.2),1.9 (m, 4H, –CH2–, J ¼ 7.2), 2.3 (s,
6H, –CH3), 4.3 (t, 4H, –OCH2–, J ¼ 7.2), 6.9 (d, 2H, pyrrole–C5), 6.5 (d,
2H, pyrrole–C3), 6.2(d,2H, pyrrole–C4)10.1(s,2H, –CONH–),10.7(s,br,
d
2.3 (m, 4H, C4–cyclohexyl), 2.5 (m, 4H, –CH2–), 4.2 (t, 4H, –OCH2–),
10.0 (s, 2H, –CONH–); MS (FAB) (m/z, %): 478 (M þ 2, 100), 365 (50),
340 (10).
2H, –NH– pyrrole); 13C NMR (CDCl3, 400 MHz)
d (ppm): 10.00 (–CH3),
12.9 (–CH3), 23.49 (–CH2–), 76.2 (–OCH2–),109.3 (C3 and C4–pyrrole),
111.8 (C5–pyrrole), 122.4 (C2–pyrrole), 129.9 (C2 and C5–thiophene),
145.4 (C3 and C4–thiophene),146.6 (–C]N–),157.2 (pC]O); MS (FAB)
(m/z, %): 501 (M þ 2, 100), 377 (30), 335 (5), 293 (10).
6.2. Pharmacology
The anticonvulsant evaluations were carried out by the National
Institute of Health, National Institute of Neurological Disorders and
Strokes (NINDS), USA, following reported procedures.
6.1.12. 3,4-Dipropyloxy-N2,N5-bis[1-(pyridin-2-
yl)ethylidene]thiophene-2,5-dicarbohydrazide (17)
Male albino mice (CF-1 strain, 18–25 g) were used for experi-
mentation. The animals were housed in metabolic cages and
allowed free access to food and water. The synthesized compounds
were suspended in 0.5% methyl cellulose/water mixture or in
IR (KBr, cmꢀ1 : 3314 cmꢀ1 (pNH), 2969 cmꢀ1 (propyl) and
) y
1680 cmꢀ1 (pC]O); 1H NMR (CDCl3, 300 MHz)
–CH3 propyl), 1.9 (m, 4H, –CH2–), 2.5 (s, 6H, –CH3), 4.3 (t, 4H,
d in ppm: 1.1 (t, 6H,

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 3672–3679
3679
polyethylene glycol (PEG 200) and injected intraperitoneally into
mice and evaluated in the MES, scMET and neurotoxicity screens.
The substances were administered at doses of 30, 100 and 300 mg/
kg at two time intervals.
used a lower frequency (6 Hz) and longer duration of stimulation
(3 s).
Test compounds were pre-administered to mice via i.p. injec-
tion. At varying times, individual mice (four mice per time point)
were challenged with sufficient current delivered through corneal
electrodes to elicit a psychomotor seizure in 97% of animals (32 mA
for 3 s). Untreated mice would display seizures characterized by
a minimal clonic phase followed by stereotyped, automatistic
behaviors described originally as being similar to the aura of human
patients with partial seizures. Animals not displaying this behavior
were considered protected.
6.2.1. Maximal electroshock test (MES)
The MES [21–23] is a model for generalized tonic–clonic
seizures and it provides an indication of ability of a compound to
prevent seizure spread when all neuronal circuits in the brain are
maximally active. These seizures are highly reproducible and are
electrophysiological consistent with human seizures.
For all tests based on MES convulsions, 60 Hz of alternating
current (50 mA) was delivered for 2 s by corneal electrodes, which
were primed with an electrolyte solution containing an anesthetic
agent (0.5% tetracaine HCl). For Test 1, mice were tested at various
intervals following doses of 30, 100 and 300 mg/kg of test
compound given by i.p. injection of a volume of 0.01 ml/g. In Test 2,
mice were tested after a dose of 30 mg/kg (p.o) in a volume of
0.04 ml/g. Test 3 used varying doses administered via i.p. injection,
again in a volume of 0.04 ml/g. An animal was considered ‘‘pro-
tected’’ from MES-induced seizures upon abolition of the hind limb
tonic extensor component of the seizure.
Acknowledgements
The authors are thankful to the Head, SAIF, CDRI Lucknow and
the Chairman, NMRRC, IISc, Bangalore for providing mass, 1H NMR
and 13C NMR spectral data.
References
[1] P. Pevarello, A. Bonsignori, P. Dostert, F. Heidempergher, V. Pinciroli,
M. Colombo, R.A. McArthur, P. Salvati, C. Post, R.G. Fariello, M. Varasi, J. Med.
Chem. 41 (1998) 579–590.
[2] P.T. Flaherty, T.D. Greenwood, A.L. Manheim, J.F. Wolfe, J. Med. Chem. 39 (1996)
1509–1513.
[3] B. Malawska, K. Kulig, A.S. Piewaka, P. JamesStables, Bioorg. Med. Chem. 12
(2004) 625–632.
[4] K.I. Molvi, K.K. Vasu, S.G. Yerande, V. Sudarsanam, N. Haque, Eur. J. Med. Chem.
42 (2007) 1049–1058.
6.2.2. Subcutaneous metrazol seizure threshold test (scMET)
This is one of the commonly used tests to measure the ability of
a compound to control seizures produced from subcutaneous
injection of the Metrazol [24] in mice.
Animals were pretreated with various doses of the test
compound (in a similar manner to the MES test, although a dose of
50 mg/kg (p.o.) was the standard for Test 2 scMET). At the previ-
ously determined TPE of the test compound, the dose of Metrazol
which would induce convulsions in 97% of animals (CD97: 85 mg/kg
mice) was injected into a loose fold of skin in the midline of the
neck. The animals were placed in isolation cages to minimize stress
and observed for the next 30 min to see the absence of a seizure. An
episode of clonic spasms, approximately 3–5 s, of the fore and/or
hind limbs, jaws or vibrissae was taken as the endpoint. Animals,
which do not meet this criterion were considered protected.
[5] P. Broto, G. Moreau, C. Vandycke, Eur. J. Med. Chem. 9 (1984) 79–84.
[6] B.V. Asthalatha, B. Narayana, K.K. Vijaya Raj, N. Suchetha Kumari, Eur. J. Med.
Chem. 42 (2007) 719–728.
[7] N. Satheesha Rai, B. Kalluraya, B. Lingappa, S. Shenoy, V.G. Puranic, Eur. J. Med.
Chem. 43 (2008) 1715–1720.
[8] S. Arroyo, J. Salas-Puig, Rev. Neurol. 32 (2001) 1041–1043.
[9] P. James, Spurlock, Denton, Tex US 2366221, Jan 2 1945.
[10] Z. Polivka, J. Holubek, E. Svatek, J. Metys, M. Protiva, Collect. Czech. Chem.
Commun. 49 (1984) 621–623.
[11] H.J. Lankau, K. Unverferth, C. Grunwald, H. Hartenhauer, K. Heinecke,
K. Bernoster, R. Dost, U. Egerland, C. Rundfeldt, Eur. J. Med. Chem. 41 (2006)
1–6.
[12] J.R. Dimmock, R.N. Puthucode, J.M. Smith, M. Hetherington, J. Wilson Quail,
U. Pugazhenthi, T. Lechler, James P. Stables, J. Med. Chem. 39 (1996) 3984–3997.
[13] J.V. Ragavendran, D. Sriram, S. Patil, I.V. Reddy, N. Bharathwajan, J. Stables,
P. Yogeeswari, Eur. J. Med. Chem. 42 (2007) 146–151.
[14] P. Yogeeswari, R. Thirumurugan, R. Kavya, J.S. Samuel, J. Stables, D. Sriram, Eur.
J. Med. Chem. 39 (2004) 729–734.
[15] S. Gunizkuculguzel, A. Mazi, F. Sahin, S. Qzturk, J. Stables, Eur. J. Med. Chem. 38
(2003) 1005–1013.
[16] R. Thirumurugan, D. Sriram, A. Saxena, J. Stables, P. Yogeeswari, Bioorg. Med.
Chem. 14 (2006) 3106–3112.
[17] S. Pandeya, A.S. Raja, J. Pharm. Pharmaceut. Sci. 5 (3) (2002) 266–271.
[18] C.G. Overberger, J. Lal, J. Am. Chem. Soc. 73 (1951) 2956–2957.
[19] G. Daoust, M. Leclerc, Macromolecules 24 (1991) 455–459.
[20] Q.T. Zhong, J.M. Tour, J. Am. Chem. Soc. 120 (1998) 5355–5362.
[21] E.A. Swinyard, J.H. Woodhead, H.S. White, M.R. Franklin, General principles:
experimental selection, quantification and evaluation of anticonvulsants, in:
R.H. Levy, R.H. Mattson, B. Melrum, J.K. Penry, F.E. Dreifussed (Eds.), Antiepi-
leptic Drugs, 3rd ed., Raven Press, New York, 1989, pp. 85–102.
[22] H.S. White, M. Johnson, H.H. Wolf, H.F. Kupferberg, J. Ital, Neurol. Sci. 16 (1995)
73–77.
[23] H.S. White, J.H. Woodhead, M.R. Franklin, General principles: experimental
selection, quantification and evaluation of antiepileptic drugs, in: R.H. Levy,
R.H. Mattson, B.S. Meldrum (Eds.), Antiepileptic Drugs, 4th ed., Raven Press,
New York, 1995, pp. 99–110.
[24] E.A. Swinyard, L.D. Clark, J.T. Miyahara, H.H. Wolf, J. Physiol. 132 (1961) 97–102.
[25] M.S. Dunham, T.A. Miya, J. Am. Pharm. Assoc. Sci. Ed. 46 (1957) 208–209.
[26] M.E. Barton, B.D. Klein, H.H. Wolf, H.S. White, Epilepsy Res. 47 (2001) 217–227.
[27] J.E. Toman, G.M. Everett, R.K. Richards, Tex. Rep. Biol. Med. 10 (1952) 96–104.
6.2.3. Acute toxicity-minimal motor impairment (MMI)
Rotorod technique [25] is the most widely used method to
determine the acute toxicity of compounds in anticonvulsant
studies.
In experimental procedure, the mouse was placed on a rod that
rotates at a speed of 6 rpm, where the animal can maintain its
equilibrium for long periods of time. The animal was considered
toxic if it falls off this rotating rod three times during a 1-min
period. Similar procedure was followed for all the compounds to
evaluate neurotoxicity.
6.2.4. Minimal clonic seizure (6 Hz) test
Some clinically useful AEDs are ineffective in the standard
MES and scMET tests but still have anticonvulsant activities in
vivo. In order to identify potential AEDs with this profile, some
compounds were tested in the minimal clonic seizure (6 Hz or
psychomotor) test. Like the maximal electroshock (MES) test, the
minimal clonic seizure (6 Hz) test [26,27] was used to assess
compound’s efficacy against electrically induced seizures but