A new class of anticonvulsants possessing 6 Hz activity: 3,4-Dialkyloxy thiophene bishydrazones

Article abstract of DOI:10.1016/j.ejmech.2009.05.026

Thirty nine new 3,4-di(substituted)oxy-N²,N⁵-bis(substituted)thiophene-2,5-dicarbohydrazides were synthesized starting from ethyl thiodiglycolate through multi-step reactions. In the synthetic sequence, 3,4-dihydroxythiophene-2,5-diester (1) was obtained by condensing the ethyl thiodiglycolate with diethyl oxalate. It was derivatized using different alkyl halides to give disubstituted thiophene esters (2-5), which were then converted to corresponding hydrazides (6-9) following usual methods. Finally, these hydrazides, on treatment with various substituted carbonyl compounds underwent smooth condensation to yield target hydrazones (10-13). The new compounds were characterized using FT-IR, ¹H NMR and ¹³C NMR, mass spectral and elemental analyses. The anticonvulsant activity of the title compounds was established after intraperitoneal (ip) administration in three seizure models, which include maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and 6 Hz screens and their neurotoxicity was also evaluated. Compound 11f has emerged as an active compound with no neurotoxicity in this series. Also, the structure-activity relationship of the tested compounds was discussed.

Full text of DOI:10.1016/j.ejmech.2009.05.026

European Journal of Medicinal Chemistry 44 (2009) 4376–4384
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new class of anticonvulsants possessing 6 Hz activity: 3,4-Dialkyloxy
thiophene bishydrazones
,
b
Ravi Kulandasamy a, Airody Vasudeva Adhikari a , James P. Stables
*
a Department of Chemistry, National Institute of Technology Karnataka, Surathkal, Srinivasnagar, Mangalore 575025. Karnataka, 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
Thirty nine new 3,4-di(substituted)oxy-N2,N5-bis(substituted)thiophene-2,5-dicarbohydrazides were
synthesized starting from ethyl thiodiglycolate through multi-step reactions. In the synthetic sequence,
3,4-dihydroxythiophene-2,5-diester (1) was obtained by condensing the ethyl thiodiglycolate with
diethyl oxalate. It was derivatized using different alkyl halides to give disubstituted thiophene esters (2–
5), which were then converted to corresponding hydrazides (6–9) following usual methods. Finally, these
hydrazides, on treatment with various substituted carbonyl compounds underwent smooth condensa-
tion to yield target hydrazones (10–13). The new compounds were characterized using FT-IR, 1H NMR
and 13C NMR, mass spectral and elemental analyses. The anticonvulsant activity of the title compounds
was established after intraperitoneal (ip) administration in three seizure models, which include maximal
electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and 6 Hz screens and their neurotoxicity
was also evaluated. Compound 11f has emerged as an active compound with no neurotoxicity in this
series. Also, the structure–activity relationship of the tested compounds was discussed.
Article history:
Received 18 March 2009
Received in revised form
13 May 2009
Accepted 20 May 2009
Available online 28 May 2009
Keywords:
Thiophene bishydrazones
Synthesis
Anticonvulsants
6 Hz test
Neurotoxicity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
(labeled B in Fig. 1) attached to azomethine side chains of thio-
phene, in order to study the effect of molecular dynamics on the
Thiophene derivatives have been reported to possess broad
spectrum of biological properties including anti-inflammatory [1],
analgesic [2], antidepressant [3], antimicrobial [4] and anticon-
vulsant activities [5–8]. Presently available active antiepileptic
drugs (AEDs) like tiagabine [5], etizolam [7], brotizolam [8] are
containing thiophene moiety in their structures as active phar-
macophore. Also, it has been established that the higher activity of
sodium phethenylate [6] is due to the presence of thiophene ring in
its structure. Further, compounds containing hydrazones and thi-
osemicarbazones with different types of substitution are well
documented for their anticonvulsant activity [9–14].
activity.
In continuation of our research program on design and synthesis
of new anticonvulsant agents, we herein report the multi-step
synthesis of hitherto unknown 3,4-di(substitute)oxy-N2,N5-bis
(substituted)thiophene-2,5-dicarbohydrazides, carrying active
groups and evaluation of their anticonvulsant activity by MES,
scMET and 6 Hz models. Also, we report their neurotoxicity by
rotorod method.
2. Chemistry
In our earlier work [15], we explored the anticonvulsant prop-
erty of many hydrazones containing 3,4-dipropyloxythiophene.
Amongst them, the compound 3,4-dipropyloxy-N2,N5-bis[1-(2-
thienyl)ethylidene]thiophene-2,5-dicarbohydrazide (Fig. 1) was
found to be lead. In search of potential anticonvulsant agents, it has
been thought of designing new hydrazones containing different
electron releasing groups at positions 3, 4 of thiophene ring
(labeled A in Fig. 1) and various substituted auxiliary thiophenes
The reaction sequence leading to the synthesis of title
compounds, viz. 3,4-di(substituted)oxy-N2,N5-bis(substituted)
thiophene-2,5-dicarbohydrazides (10–13) is shown in Scheme 1.
The dihydroxythiophene diester (1), obtained by condensing
diethyl oxalate with ethyl thiodiglycolate in alcoholic sodium eth-
oxide, was treated with different alkyl halides in the presence of
anhydrous potassium carbonate to yield 3,4-disubstituted esters
(2–5). Further, they were condensed with hydrazine hydrate in
alcoholic medium to give key intermediates, viz., 3,4-di
(substituted)oxythiophene-2,5-dicarbohydrazides (6–9). These
hydrazides were then conveniently converted to hydrazones by
refluxing them with appropriate aryl/alkyl aldehydes or ketones in
* Corresponding author. Tel.: þ91 0824 2474000x3203; fax: þ91 0824 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.2009.05.026

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
4377
–OCH2– protons resonated at
d
5.1 ppm. This indicated the forma-
CH3 H3C
A
tion of the compound 4. Further, its LC–mass spectrum confirmed
high purity of 4 and showed its molecular weight that corresponds
to C24H24O6S.
S
S
B
The conversion of the diester (4) to the corresponding hydrazide
(8) was evidenced by its spectral data. IR spectrum of compound 8
showed bands at 3294, 2982 and 1703 cmꢀ1 indicating the
presence of –NH2, benzyloxy and pC]O groups respectively. In its
1H NMR spectrum –NH2 group appeared as broad singlet at
O
O
B
H3C
CH3
NH
NH
N
N
S
d
4.5 ppm, while –OCH2– group resonated as a sharp singlet at
5.2 ppm. Further, aromatic protons appeared as multiplet at 7.3–
8.8 ppm.
showed peak at
76 ppm due to –OCH2– group. The signals appeared at 124 and
146 ppm correspond to thiophene carbons. The aromatic carbons
showed in the region of 128 and 134 ppm and the carbonyl group
of the hydrazide resonated at 160 ppm. In the mass spectrum, it
O
O
d
d
Fig. 1. 3,4-Dipropyloxy-N2,N5-bis[1-(2-thienyl)ethylidene]thiophene-2,5-dicarbohy-
drazide where A and B are the positions of derivatization.
7.4 ppm and –NH– proton resonated as broad singlet at
13C NMR spectrum of the compound
d
8
d
d
the presence of catalytic amount of conc. HCl. The physiochemical
properties and characterization data of the title compounds are
summarized in Table 1. 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 3,4-dibenzyloxy-2,5-thiophene diester (4)
from 3,4-dihydroxy-2,5-thiophene diester (1) was confirmed by its
IR, 1H NMR and mass spectral studies. Its IR spectrum showed the
carbonyl stretching at 1704 cmꢀ1 while the stretching of –CH2– of
benzyl group appeared at 2982 cmꢀ1. In its 1H NMR spectrum,
d
d
showed the molecular ion peak at m/z 413 (M þ 1, 100%). The peak
matches with its molecular formula C20H20N4O4S. The peaks at m/z
381 and 355 were due to the fragmentation of molecular ion.
The structure of hydrazone 10n was established by its spectral
data. In its IR spectrum, peaks at 1731 and 1679 cmꢀ1 clearly
showed the presence of ester and amide linkages respectively in
the molecule. The structure of hydrazone 10n was further
confirmed by its COSY-1HNMR, which is shown in Fig. 2. Here, the
HO
OH
EtOOC
S
COOEt
A
+
O
O
EtOOC
COOEt
S
1
EtO
OEt
B
R
R
R
R
O
O
O
O
C
NH
O
H2N
EtOOC
COOEt
S
S
2 - 5
O
NH
6 - 9
NH2
D
R
R
where,
O
O
2, 6, 10 R: Propyl, R: Propyl,
3, 7, 11 R: Heptyl, R: Heptyl,
4, 8, 12 R: Benzyl, R: Benzyl,
5, 9, 13 R,R: -CH2CH2-
R2
R1
NH
O
N
S
O
NH
N
10 (a-r), 11 (a-j)
12 (a-e), 13 (a-f)
R1, R2 : aralkyl / aryl / heteroaryl groups
R1
R2
Scheme 1. Synthesis of 3,4-dialkyloxy bishydrazones. A: NaOEt, ethanol, Hþ; B: R–Br, K2CO3, DMF; C: NH2NH2$H2O, ethanol; D: aldehyde/ketone, ethanol, Hþ
.

4378
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
Table 1
Physicochemical and characterization data of compounds 10a–r.
C3H7 H7C3
O
O
R2
R2
R1
R1
NH
NH
N
N
S
O
O
10a-r
Compound
R1
R2
Mp (ꢁC)/Y (%)
Recry. Solvena
Elemental analysis (%): Found (cal.)
C
H
N
S
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
CH3
CH3
CH3
CH3
CH3
3-isatinyl
H
H
H
H
H
H
H
CH3
H
H
H
3-C4H3S
256–257/60
294–295/72
296–297/70
214–215/68
241 Decb/75
>300/65
299–300/68
293–294/65
259–260/77
295 Decb/72
249–250/67
251–252/65
261–262/63
153–254/68
275–276/68
270–271/70
274–275/63
210–211/60
MDC
DMF
DMF
MDC
DMF
DMF
DMF
DMF
MDC
DMF
DMF
MDC
MDC
CHCl3
DMF
DMF
DMF
MDC
54.02 (54.11)
41.75 (41.68)
47.98 (47.92)
41.84 (41.75)
42.99 (43.08)
58.59 (58.53)
57.58 (57.52)
53.70 (53.60)
62.35 (62.26)
57.87 (57.92)
59.50 (59.53)
60.95 (60.85)
55.69 (55.62)
53.25 (53.32)
62.15 (62.05)
54.85 (54.95)
42.27 (42.18)
73.99 (73.92)
5.30 (5.19)
3.85 (3.80)
4.31 (4.36)
3.87 (3.80)
3.69 (3.61)
4.59 (4.56)
5.58 (5.52)
4.58 (4.50)
6.58 (6.62)
4.95 (4.86)
5.48 (5.38)
5.90 (5.84)
4.75 (4.67)
6.66 (6.71)
6.99 (6.94)
5.31 (5.38)
3.35 (3.27)
8.11 (8.20)
10.59 (10.52)
8.19 (8.11)
9.39 (9.31)
8.18 (8.11)
8.39 (8.36)
14.58 (14.63)
9.51 (9.58)
14.45 (14.43)
14.59 (14.52)
9.58 (9.65)
10.75 (10.68)
10.20 (10.14)
9.78 (9.98)
10.45 (10.36)
8.11 (8.04)
18.15 (18.06)
13.99 (13.93)
16.08 (15.99)
13.85 (13.93)
14.39 (14.35)
5.55 (5.58)
5.55 (5.48)
5.45 (5.50)
5.51 (5.54)
5.45 (5.52)
6.15 (6.11)
5.85 (5.80)
5.64 (5.71)
5.99 (5.93)
4.69 (4.60)
6.19 (6.11)
4.39 (4.33)
3.11 (3.18)
5-Br, 2-C4H2S
5-Cl, 2-C4H2S
2-Br, 3-C4H2S
2,5-Cl, 3-C4H2S
3-OCH3, 4-OH, phenyl
4-Nitrophenyl
4-N(CH3)2 phenyl
4-COOH phenyl
2-OH phenyl
2-OCH3 phenyl
4-Cl phenyl
–CH2 COOC2H5
4-Diacetal phenyl
2-NH2, 3-pyridyl
2-Br, 5-NO2 phenyl
N-dodecyl-3-carbazolyl
21.42 (21.36)
11.42 (11.35)
8.39 (8.34)
H
Physicochemical and characterization data of compounds 11a–j.
H
15C7
O
C7H15
O
R2
R2
R1
R1
NH
NH
N
N
S
O
O
11a-j
Compound
R1
R2
Mp (ꢁC)/Y (%)
238–239/69
Recry. Solvena
Elemental analysis (%): Found (cal.)
C
H
N
S
11a
6-CH3-5, 6-dihydro-4H-thieno
[2,3-b] thiopyran-4-yl
3-Isatinyl
H
H
H
H
H
H
CHCl3
56.74 (56.81)
6.41 (6.36)
7.30 (7.36)
21.12 (21.06)
11b
11c
11d
11e
11f
11g
11h
11i
>300/72
194–195/70
202–203/72
159–160/65
213–214/74
152–153/75
223–224/65
193–194/65
193–194/75
DMF
62.83 (62.95)
62.13 (62.05)
71.49 (71.57)
67.59 (67.52)
58.69 (58.77)
61.29 (61.20)
73.89 (73.82)
75.01 (75.09)
59.53 (59.60)
6.10 (6.16)
6.97 (6.94)
7.07 (6.97)
7.41 (7.33)
6.14 (6.09)
7.25 (7.19)
6.78 (6.71)
8.89 (8.82)
6.81 (6.88)
12.30 (12.24)
8.09 (8.04)
9.95 (10.02)
9.10 (9.26)
12.05 (12.10)
7.19 (7.14)
7.12 (7.17)
7.59 (7.51)
8.76 (8.69)
4.78 (4.67)
4.51 (4.60)
3.90 (3.82)
5.21 (5.30)
4.64 (4.62)
4.01 (4.08)
4.15 (4.11)
2.81 (2.86)
14.91 (14.92)
4-OH, 3-OCH3 phenyl
N-ethyl-3-carbazolyl
Phenyl
4-NO2 phenyl
3,4,5-OCH3 phenyl
2-Fluorenyl
MeOH
CHCl3
MDC
MDC
MDC
DMF
H
CH3
N-dodec-yl-3-carbazolyl
2-Thienyl
MDC
CHCl3
11j
Physicochemical and characterization data of compounds 12a–e.
CH2C6H5 CH2C6H5
O
O
R2
R2
R1
R1
NH
NH
N
N
S
O
O
12a-e
Compound
R1
R2
Mp (ꢁC)/Y (%)
Recry. Solvena
Elemental analysis (%): Found (cal.)
C
H
N
S
12a
12b
12c
12d
12e
CH3
H
H
H
H
2-Thienyl
3,4,5-OCH3 phenyl
Phenyl
N-ethyl-3-carbazolyl
4-NO2 phenyl
205–206/68
270–271/78
240–241/72
272Decb/65
263 Decb/75
CHCl3
CHCl3
DMF
DMF
MDC
61.19 (61.12)
62.52 (62.49)
69.31 (69.37)
72.88 (72.97)
60.11 (60.17)
4.58 (4.49)
5.29 (5.24)
4.85 (4.79)
5.08 (5.14)
3.81 (3.86)
8.96 (8.91)
7.38 (7.29)
9.59 (9.52)
10.17 (10.21)
12.32 (12.38)
15.39 (15.30)
4.20 (4.17)
5.52 (5.45)
3.95 (3.90)
4.79 (4.72)

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
4379
Table 1 (continued)
Compound R1
R2
Mp (ꢁC)/Y (%)
Recry. Solvena
Elemental analysis (%): Found (cal.)
C
H
N
S
Physicochemical and characterization data of compounds 13a–f.
O
O
R2
R2
R1
R1
NH
NH
N
N
S
O
O
13a-f
Compound
R1
R2
Mp (ꢁC)/Y (%)
Recry. Solvena
Elemental analysis (%): Found (cal.)
C
H
N
S
13a
13b
13c
13d
13e
13f
H
CH3
H
H
H
N-ethyl-3-carbazolyl
2-Thienyl
3,4,5-OCH3 phenyl
Phenyl
2-Fluorenyl
4-NO2 phenyl
>300/75
>300/70
>300/68
>300/68
>300/70
>300/72
DMF
DMF
DMF
DMF
DMF
DMF
68.33 (68.25)
50.51 (50.62)
54.79 (54.72)
60.89 (60.82)
70.72 (70.80)
50.21 (50.32)
4.89 (4.82)
3.89 (3.82)
4.99 (4.92)
4.25 (4.18)
4.21 (4.29)
3.15 (3.07)
12.65 (12.57)
11.85 (11.81)
9.01 (9.12)
12.98 (12.90)
9.11 (9.17)
4.72 (4.79)
20.21 (20.27)
5.31 (5.22)
7.29 (7.38)
5.29 (5.25)
6.01 (6.11)
H
16.15 (16.02)
a
Recrystallization solvent.
Decomposed.
b
presence of –OCH2– group of ester and –OCH2– group of propyloxy
linkage was observed as multiplet at 4.2 ppm. Further, the three
protons of methyl group of ester and two protons of –CH2CO–
group resonated at 1.2 and 3.4 ppm respectively. The appearance
of singlet peak at 9.9 ppm showed the presence of –NH– group in
the molecule. Its structure was further evidenced by its 13C NMR
spectrum. The peaks at 150 and 156 ppm showed the presence of
>C]N– and amide carbonyl groups respectively. In the mass
spectrum, it displayed the molecular ion peak at m/z 540 (M þ 1,
100%) that matches with its molecular formula, C24H36N4O8S. It
followed the similar fragmentation pattern as described in our
earlier paper [15].
in its 1H NMR spectrum, the appearance of a triplet at
confirmed the presence of –OCH2– group of the alkoxy substit-
uent. Also, appearance of a singlet at 10.0 ppm showed the
d 4.3 ppm
d
d
d
presence of –NH– group in the molecule. The formation of 11j
d
was further confirmed by its 13C NMR spectrum wherein >C]N–
appeared at
d 147 ppm while –CONH– resonated at 156 ppm. The
d
mass spectrum of 11j displayed the molecular ion peak at m/z
646 (M þ 1, 100%), which matches with its molecular formula
C32H44O4N4S3.
The conversion of the hydrazide 8 to hydrazone 12b was
confirmed by various spectral methods. In its IR spectrum, peaks at
3240 cmꢀ1 and 1637 cmꢀ1 were due to –NH– and pC]O absorp-
tions respectively. Further, the peaks at 1237 and 1052 cmꢀ1
showed the presence of aralkyl ether in the molecule. 1H NMR
The IR spectrum of compound 11j showed sharp peaks at
3310, 1679 and 1636 cmꢀ1 indicating the presence of –NH–,
pC]O and pC]N– groups respectively in its structure. Further,
spectrum of it displayed two singlets at
d 3.8 and 5.3 ppm
Fig. 2. COSY 1H NMR spectrum of compound 10n.

4380
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
Table 3
indicating the presence of –OCH3 and –OCH2– groups respectively.
The azomethine proton and –NH– proton resonated as singlets at
Results of anticonvulsant activity by 6 Hz model.
d
7.5 and 10 ppm respectively, whereas aromatic protons appeared
Compound
Dose (mg/kg)
0.25 h
0.5 h
1.0 h
2.0 h
4.0 h
as multiplet in the region of 6.8–7.5 ppm. The formation of 12b
d
11e
10a
100
100
100
100
100
100
100
100
100
100
50
100
60
100
100
30
2/4
0/4
1/4
0/4
2/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
2/4
0/4
0/4
2/4
2/4
2/4
3/4
0/4
1/4
1/4
3/4
1/4
0/4
0/4
1/4
0/4
4/4
0/4
1/4
1/4
0/4
0/4
2/4
2/4
1/4
1/4
1/4
1/4
0/4
2/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
1/4
1/4
1/4
1/4
0/4
0/4
0/4
0/4
0/4
1/4
1/4
0/4
2/4
0/4
1/4
1/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
1/4
1/4
0/4
1/4
0/4
0/4
was further confirmed by its 13C NMR spectrum. Here the azome-
thine and amide carbons resonated at 146 and 156 ppm respec-
tively. Finally, its mass spectrum showed the molecular ion peak at
m/z 769 (M þ 1, 100%). This confirmed its molecular formula
C40H40N4O10S.
10b
10d
10f
10k
10o
10p
11a
11b
11d 11e
11f
In the IR spectrum of the hydrazone 13d, the appearance of
peaks at 3306, 1675 and 1577 cmꢀ1 clearly indicated the presence
of –NH–, pC]O and pC]N- functional groups respectively. Its 1H
NMR spectrum displayed a sharp singlet peak at
the presence of ethylenedioxy group. Further, the azomethine
proton appeared at 8.4 ppm while –NH– proton resonated at
10.7 ppm as singlets. The mass spectrum of 13d showed the
d 4.5 ppm due to
11g
11j
d
d
molecular ion peak at m/z 435 (M þ 1, 100%), which matches with
12c
13b
30
100
30
its molecular formula C22H18N4O4S.
3. Pharmacology
All the new 39 hydrazones were subjected to anticonvulsant
screening by maximal electroshock test (MES) and subcutaneous
metrazol (scMET) test using doses of 30,100 and 300 mg/kg and the
observation was carried out at two different time intervals (0.5 and
4 h). Only compounds 6, 7, 10e, 10f, 10h–l, 10q, 11e, 12a, 12c, 12e,
13b, 13c and 13e showed moderate to good activity while the
remaining compounds did not show either activity or toxicity at
maximum dose and their results are presented in Table 2. Further,
some of the selected compounds were tested for their activity at
6 Hz model. The results of screening at five different time points are
summarized in Table 3. The detailed experimental procedures are
given in the experimental section.
noticed that the presence of isatin, phenyl and thiophene moieties
as a distal ring in their structures is responsible for the observed
activity.
In scMET model, compounds 10e, 10k and 12c showed positive
response towards induced seizures at a dose of 300 mg/kg and 11e
displayed same level of protection at 0.5 h of time period. Here, the
activity is mainly attributed to the presence thiophene and phenyl
entities as a distal ring in the designed molecules.
As seen from the results of neurotoxicity screening, compounds
10e, 10f, 10q and 13c exhibited toxicity at a dose of 300 mg/kg
whereas the compounds 10h–j, 10l and 12e showed at a dose of
100 mg/kg. It has been observed that the occurrence of dichlor-
othiophene, isatin and nitro, trimethoxy substituted phenyl rings in
the molecules is responsible for the lower level of toxicity and
presence of groups, viz., nitro, N,N-dimethyl, carboxyl and methoxy
substituted phenyl rings in the structures caused higher level of
neurotoxicity. It is interesting to note that the remaining
compounds did not show any neurotoxicity at the maximum
administered dose level of 300 mg/kg. These results clearly indi-
cated that toxicity is not due to the presence of the basic moiety, i.e.,
3,4-di(substituted)oxy thiophene in the tested compounds.
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 of various tested hydrazones,
compounds 10a, 10f, 10k, 10o, 11e, 11f, 11j and 13b displayed good
activity compared to other compounds. These active compounds
contain thiophene, isatin and hydroxy, diacetyl, trimethoxy
substituted phenyl rings attached to the 3,4-disubstituted thio-
phene system. The compound 11f, carrying 4-nitrophenyl
substituted heptyloxythiophene displayed 100% protection against
induced convulsions at 0.5 h at the dose of 100 mg/kg, whereas
compounds 10b, 10d, 11b and 11g exhibited moderate activity.
4. Results and discussion
The results of MES study revealed that compounds 12c and 13b
showed activity at a dose of 300 mg/kg at 0.5 h and compounds 10f
and 12a displayed resistance at 4 h at the same dose. It has been
Table 2
Anticonvulsant activity and neurotoxicity of title compounds.
Compound
MESa
0.5 h
scMETb
0.5 h
Toxicityc
0.5 h
4 h
4 h
4 h
6
7
–
–
–
–
–
–
–
–
–
–
–
–
300
–
300
–
–
100
–
300
–
–
–
–
–
–
–
300
–
–
–
–
–
–
–
–
–
–
–
–
–
300
–
–
–
–
–
300
–
–
300
–
–
–
–
300
–
–
–
–
300
–
–
100
–
–
–
10e
10f
10h
10i
10j
10k
10l
10q
11e
12a
12c
12e
13b
13c
13e
300
100
100
100
100
–
100
300
–
–
–
100
–
300
–
300
–
300
–
–
–
–
–
–
–
5. Structure–activity relationship (SAR)
–
–
–
–
–
–
–
–
In our previous article [15], we reported that 3,4-dipropyloxy-
N2,N5-bis[1-(2-thienyl)ethylidene]thiophene-2,5-dicarbohydrazide
–
emerged as
a
lead and 3,4-dipropyloxy-N2,N5-bis(phenyl-
The figures in the table indicate the minimum dose where bioactivity was observed
in at least one or more of the mice. The dash (–) indicates an absence of activity at
the maximum dose of 300 mg/kg. Note: The remaining compounds in the series did
not show either activity or toxicity at a maximum dose of 300 mg/kg.
methylene) thiophene-2,5-dicarbohydrazide, 3,4-dipropyloxy-
N2,N5-bis(3,4,5-trimethoxyphenylmethylene)thiophene-2,5-dicar-
bohydrazide as moderate antiepileptic agents. In the present work,
based on the reported results, the active molecules have
been further derivatized with different substituents, in order to
a
Maximal electroshock test.
Subcutaneous metrazol seizure threshold test.
Rotorod toxicity.
b
c

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
4381
CH3 H3C
activity and displayed toxicity as per our previous report [15], in our
synthetic design propyloxy group has been replaced by heptyloxy
(11d) in order to find the structural requirement for better activity.
Surprisingly, the activity has enhanced to 75% and toxicity has
reduced to the maximum extent. Further, replacement of the
heptyloxy group by benzyloxy (12d) and ethylenedioxy (13a)
moieties has showed neither activity nor toxicity.
O
O
NH
NH
N
N
S
R3
C
R3
C
O
O
Fig. 3. Positions of derivatization of active molecules.
6. Conclusion
investigate the pharmacophoric elements, responsible for better
activity. In this approach, some of the toxic compounds have been
also derivatized.
In the present work, thiophene-2,5-dicarbohydrazide contain-
ing different types of substituents at its positions 3,4 and various
groups attached to the azomethine functional moiety, were
synthesized and characterized by spectral and elemental analyses.
Based on the findings reported in our earlier work, the position and
type of substituents were selected. All the compounds were sub-
jected to antiepileptic screening by standard methods. Amongst the
tested models, the 6 Hz screening results were promising. The
results revealed that compounds 10a, 10f, 10k, 10o, 10p, 11d–f, 11j
and 13b exhibited moderate to good activity. The compound 11f
displayed 100% protection and emerged as a lead in this series.
Further, compounds 10a and 10k came out as potential candidates
for further investigations. It can be concluded that increase in
hydrophobicity in the molecules brings about some degree of
activity in the series. Further, the screening results clearly support
that 3,4-dioxy substituted thiophene moiety is a seat of broad
spectrum antiepileptic activity and with least neurotoxicity. Finally
it can be readily concluded that change in the distal ring influences
the activity as well as toxicity of substituted thiophene hydrazones.
In our new synthetic design, active groups, viz., heptyl, benzyl
and ethylenedioxy side chains have been introduced at positions
3,4 of the thiophene backbone structure and further, the active
distal ring has been derivatized with different substituents. It has
been observed that the introduction of the heptyl group has not
influenced the MES and scMET screening results as seen in 11j.
But, no change in activity was observed in the case of benzyl
substituted (12a) and ethylenedioxy substituted (13b) 2-thienyl
hydrazones. When 2-thienyl hydrazone was replaced by benz-
aldehyde hydrazones (11e, 12c, 13d), it retained the activity only
in the compounds 11e and 12c. Further, the introduction of 2-
fluorenyl hydrazone groups (11h, 13e) showed resistance at
a dose of 300 mg/kg against the induced convulsion, whereas
presence of propyloxy substitution in thiophene ring failed to
produce any activity. It has been observed that replacement of
propyl with heptyl group (11b) causes loss of activity in the isatin
hydrazones.
In order to study the influence of substituents attached to
different positions of secondary aryl group on the activity of various
hydrazones, many derivatives as shown in Figs. 3 and 4 have been
synthesized. In these figures, labeled parts C and D indicate
different secondary aryl groups attached to azomethine functional
group of active hydrazones.
7. Experimental
7.1. Chemistry
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. The FT-IR spectra were
recorded on Nicolet Avatar 330 FT-IR spectrophotometer. The 1H
and 13C NMR spectra were recorded on Varian 300, 400 and
500 MHz NMR spectrophotometers using TMS as an internal
From the results, it has been noticed that introduction of 2-
hydroxy phenyl group to hydrazone (10k) has caused moderate
activity while substitution by 4-hydroxy phenyl group (10g) has
resulted in poor activity. On the other hand, replacement of
hydroxyl group (10k) by methoxy group (10l) in position 2 of
phenyl ring has reduced activity (Fig. 3). This may be attributed to
the formation of increased extent of hydrogen bonding during its
action. Further, incorporation of 4-nitrophenyl (11f) ring in the
place of 4-hydroxy phenyl (10g) has caused the activity to the
maximum level (100%) with no neurotoxicity. This clearly indicates
the role of electronic factor on the activity. As far as position of
linkage is concerned, change in the linkage of azomethine group
from second to third position (10a) has retained the activity and
introduction of halogen groups (10d and 10b) into the thiophene
ring, as given in Fig. 4, has reduced the activity when compared to
that of unsubstituted 2-thienyl hydrazone.
standard. 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 1112 series, CHNSO Analyser (Thermo).
Compounds 2–5 were synthesized from diethyl-3,4-diyhdrox-
ythiophene-2,5-dicarboxylate following the reported procedure [16].
Keeping in view of the observed fact that N-ethyl carbazole
hydrazone of propyloxy substituted thiophene did not show any
7.1.1. Synthesis of 3,4-di(substituted)thiophene-2,5-
carbonyldihydrazides (6–9)
CH3 H3C
D
S
D
One gram (0.003 mol) of diethyl-3,4-disubstiutedoxythiophene-
S
2,5-dicarboxylates (2–5) was added to
a solution of 1.6 mL
O
O
(0.03 mol) of hydrazine hydrate in 30 mL of ethanol. The reaction
mixture was refluxed for 2 h. Upon cooling the reaction mixture,
white precipitate was obtained. The products (6–9) were then
filtered, washed with alcohol and dried to get title compounds with
good yield. The compounds 6–9 were crystallized using appro-
priate solvents. The spectral data of compound 8 is given below.
H3C
CH3
NH
NH
N
N
S
O
O
Fig. 4. Positions of derivatization of active molecules.

4382
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
7.1.2. 3,4-Bis(benzyloxy)thiophene-2,5-dicarbohydrazide (8)
J ¼ 8), 6.90 (d, 2H, C6– of phenyl, J ¼ 8), 7.00 (t, 2H, C4– of phenyl,
J ¼ 8), 7.38 (t, 2H, C5– of phenyl, J ¼ 8), 8.13 (d, 2H, C6– of phenyl,
J ¼ 8), 8.58 (s, 2H, –CH]N–), 10.28 (s, 2H, –NH–); 13C NMR (CDCl3,
Mp: 159–60 ꢁC; Crystallization Solvent: Ethanol; IR (KBr, cmꢀ1
)
y
:
3294 cmꢀ1 (br, –NH2), 2982 cmꢀ1 (benzyloxy), 1703 cmꢀ1
(pC]O); 1H NMR (CDCl3, 400 MHz)
5.20 (s, 4H, –OCH2–), 7.36–7.40 (m,10H, phenyl), 8.85 (s, 2H, –NH–);
13C NMR (CDCl3, 400 MHz)
in ppm: 76.4 (–OCH2–), 124.8 (C3,4
d
in ppm: 4.52 (br, 4H, –NH2),
400 MHz) d in ppm: 10.4 (–CH3), 23.2 (–CH2–), 55.5 (–OCH3), 76.3
(–OCH2–), 110.8 (C4–phenyl), 120.9 (C5–phenyl), 121.8 (C6–phenyl),
125.4 (C1–phenyl), 127.1 (C3–phenyl), 131.9 (C3,4–thiophene), 144.1
(C2,5–thiophene), 146.8 (C2–phenyl), 156.9 (–CH]N–), 158.0
(–CONH–).
d
–
thiophene), 128.8–129.4 (C2,3,5,6–phenyl), 134.5 (C1–phenyl), 146.3
(C2,5–thiophene), 161.5 (–CONH–). MS (m/z, %): 413 (M þ 1, 100),
381 (20), 355 (20), 279 (10), 265 (10).
The characterization data of compounds 6, 7 and 9 matched
with the reported data [15,17,18].
7.1.9. 3,4-Dipropyloxy-N2,N5-bis(4-chlorophenylmethylene)
thiophene-2,5-dicarbohydrazide (10m)
IR (KBr, cmꢀ1
) y:
3217 cmꢀ1 (-NH–), 2966 cmꢀ1 (propyl),
7.1.3. General procedure for synthesis of bishydrazones 10a–r,
11a–j, 12a–e and 13a–f
1643 cmꢀ1 (pC]O), 1600 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
d
in ppm: 1.13 (t, 6H, –CH3, J ¼ 7.2), 1.93 (m, 4H, –CH2–),
A
clear solution of 0.005 mol of 3,4-disubstituted-
4.26 (t, 4H, –OCH2–, J ¼ 6.9), 7.3 (d, 4H, C3 and C5– of phenyl), 7.7 (d,
4H, C2 and C6– of phenyl), 8.2 (s, 2H, –CH]N–), 10.2 (s, 2H, –NH–).
thiophenecarbohydrazides (6–9) in 15 mL of absolute ethanol was
mixed with 0.5 mL of conc. hydrochloric acid. To this 0.01 mol of
appropriate carbonyl compound, dissolved in 10 mL of absolute
ethanol was added slowly while stirring. The reaction mixture was
heated to reflux for 4 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 all
the newly synthesized compounds are presented in Table 1. Their
spectral data are given below.
7.1.10. Ethyl 3-{[(3,4-dipropyloxy-5-{[2-(3-ethoxy-1-methyl-3-
oxopropylidene)hydrazino] carbonyl}-2-thienyl)carbonyl]
hydrazono}butanoate (10n)
IR (KBr, cmꢀ1
) y:
3313 cmꢀ1 (-NH–), 2966 cmꢀ1 (propyl),
1732 cmꢀ1 (ester pC]O), 1660 cmꢀ1 (amide pC]O), 1633 cmꢀ1
(pC]N–); 1H NMR (CDCl3, 500 MHz)
d
in ppm: 1.0 (t, 6H, –CH3– of
propyl, J ¼ 4.2), 1.30 (t, 6H, –CH3– of ester, J ¼ 4.2), 1.85 (m, 4H,
–CH2– of propyl), 2.06 (s, 6H, –CH3), 3.49 (s, 4H, –COCH2–), 4.20 (m,
8H, –OCH2– of ester and propyl), 9.97 (s, 2H, –NH–); 13C NMR
7.1.4. 3,4-Dipropyloxy-N2,N5-bis[1-(3-thienyl)ethylidene]
thiophene-2,5-dicarbohydrazide (10a)
(CDCl3, 500 MHz) d in ppm: 10.0 (–CH3 propyl), 14.1 (–CH3 ester),
IR (KBr, cmꢀ1
)
y
: 3308 cmꢀ1 (-NH–), 2969 cmꢀ1 (propyl),
16.1 (–CH3), 23.4 (–CH2–propyl), 44.2 (–COCH2–), 61.1 (–OCH2–),
76.2 (–OCH2–propyl), 125.5 (C3,4–thiophene), 146.7 (C2,5–thio-
phene), 150.5 (–CH]N–), 156.9 (–CONH), 169.6 (–C]O); MS (m/z,
%): 540 (Mþ, 100), 510 (5), 396 (100), 227 (10).
1675 cmꢀ1 (pC]O), 1535 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
d
in ppm: 1.06 (t, 6H, –CH3– of propyl, J ¼ 7.2), 1.88 (m,
4H, –CH2–), 2.33 (s, 6H, –CH3), 4.28 (t, 4H, –OCH2–), 7.31 (d, 2H, C4–
of thiophene, J ¼ 16.2), 7.62 (s, 2H, C2– of thiophene), 7.71 (d, 2H,
C5– of thiophene, J ¼ 4.5), 10.13 (s, 2H, –NH–).
7.1.11. 3,4-Dipropyloxy-N2,N5-bis[(N-dodecyl-9H-carbazole-3-yl)-
methylene]thiophene-2,5-dicarbohydrazide (10r)
7.1.5. 3,4-Dipropyloxy-N2,N5-bis[1-(2-bromo-3-thienyl)ethylidene]
IR (KBr, cmꢀ1 : 3263 cmꢀ1 (-NH–), 2924 cmꢀ1 (propyl and
) y
thiophene-2,5-dicarbohydrazide (10d)
dodecyl), 1661 cmꢀ1 (pC]O), 1630 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
400 MHz)
IR (KBr, cmꢀ1
)
y
:
3307 cmꢀ1 (-NH–), 2967 cmꢀ1 (propyl),
d
in ppm: 0.89 (t, 6H, –CH3– of dodecyl, J ¼ 8), 1.23
1677 cmꢀ1 (pC]O), 1548 cmꢀ1 (pC]N–); MS (m/z, %): 691 (M þ 1,
(t, 6H, –CH3– of propyl, J ¼ 8),1.31 (m, 36H, –CH2– of dodecyl),1.84 (m,
4H, –NCH2CH2– of dodecyl),1.98 (m, 4H, –CH2– of propyl), 4.30 (m, 8H,
–OCH2– and –NCH2–), 7.30 (t, 2H, C4–car), 7.40 (d, 4H, C8,9–car, J ¼ 8),
7.49 (t, 2H, C3–car, J ¼ 8), 7.90 (d, 2H, C5–car, J ¼ 8), 8.12 (d, 2H, C2–car,
J ¼ 8), 8.26 (s, 2H, C6–car), 8.47 (s, 2H, –CH]N–), 10.19 (s, 2H, –NH–);
100), 473 (30), 387 (20).
7.1.6. 3,4-Dipropyloxy-N2,N5-bis[4-(N,N-dimethyl)phenylmethylene]
thiophene-2,5-dicarbohydrazide (10i)
IR (KBr, cmꢀ1
)
y
: 3293 cmꢀ1 (-NH–), 2966 cmꢀ1 (propyl),
13C NMR (CDCl3, 400 MHz)
d in ppm: 10.6 (–CH3 propyl), 14.0
1666 cmꢀ1 (pC]O), 1594 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz)
(–CH3), 22.6 (–CH2CH3), 23.4 (–CH2–), 27.2–31.8 (–CH2–dodecyl), 43.1
(–NCH2–), 76.0 (–OCH2–),108.7 (C1–car),109.0 (C8–car),119.4 (C4–car),
120.0 (C3,5–car), 122.7 (C4a,b–car), 124.3 (C6–car), 125.0 (C3,4–thio-
d
in ppm: 1.11 (t, 6H, –CH3, J ¼ 7.5), 1.91 (m, 4H, –CH2–),
3.02 [s, 12H, –N(CH3)2], 4.22 (t, 4H, –OCH2–, J ¼ 6.9), 6.70 (d, 4H, C3
and C5– of phenyl), 7.63 (d, 4H, C2 and C6– of phenyl), 8.04 (s, 2H,
–CH]N–), 10.09 (s, 2H, –NH–).
phene), 126.0 (C2,7–car), 140.8 (C9a–car), 141.8 (C8a–car), 146.8 (C2,5
–
thiophene), 148.9 (–CH]N–), 156.7 (–CONH–); MS (m/z, %): 1008
(M þ 1, 100), 631 (30), 605 (15), 588 (15), 546 (10), 361 (80).
7.1.7. 3,4-Dipropyloxy-N2,N5-bis(2-hydroxyphenylmethylene)
thiophene-2,5-dicarbohydrazide (10k)
7.1.12. 3,4-Diheptyloxy-N2,N5-bis(2-oxo-1,2-dihydro-3H-indol-3-
IR (KBr, cmꢀ1
)
y
: 3296 cmꢀ1 (-NH–), 3000 cmꢀ1 (br –OH),
ylidene)thiophene-2,5-dicarbohydrazide (11b)
2973 cmꢀ1 (propyl), 1676 cmꢀ1 (pC]O), 1616 cmꢀ1 (pC]N–); 1H
NMR (DMSO-d6, 300 MHz)
IR (KBr, cmꢀ1 3257 cmꢀ1 (-NH–), 2925 cmꢀ1 (heptyl),
) y:
d
in ppm: 0.98 (t, 6H, –CH3, J ¼ 6.9), 1.76
1712 cmꢀ1 (pC]O), 1657 cmꢀ1 (pC]N–); MS (m/z, %): 687
(Mþ, 100), 656 (20), 604 (60), 527 (50), 383 (10), 355 (10), 339 (20),
312 (20).
(m, 4H, –CH2–), 4.19 (t, 4H, –OCH2–), 6.95 (m, 4H, C4 and C3– of
phenyl), 7.30 (m, 2H, C2– of phenyl), 7.67 (d, 2H, C5– of phenyl), 8.63
(s, 2H, –N]CH–), 11.02 (s, 2H, –OH), 11.44 (s, 2H, –NH–); MS (m/z,
%): 525 (Mþ, 100), 389 (70), 363 (20).
7.1.13. 3,4-Diheptyloxy-N2,N5-[bis(4-hydroxy-3-
methoxyphenyl)methylene]thiophene-2,5-dicarbohydrazide (11c)
7.1.8. 3,4-Dipropyloxy-N2,N5-bis(2-methoxyphenylmethylene)
thiophene-2,5-dicarbohydrazide (10l)
IR (KBr, cmꢀ1 3290 cmꢀ1 (-NH–), 2925 cmꢀ1 (heptyl),
) y:
1654 cmꢀ1 (pC]O), 1591 cmꢀ1 (pC]N–); 1H NMR (MeOD,
300 MHz) in ppm: 0.94 (t, 6H, –CH3), 1.41–1.48 (m, 12H,
IR (KBr, cmꢀ1
)
y
: 3206 cmꢀ1 (-NH–), 2967 cmꢀ1 (propyl),
d
1640 cmꢀ1 (pC]O), 1599 cmꢀ1 (-N–); 1H NMR (CDCl3, 400 MHz)
in ppm: 1.13 (t, 6H, –CH3– of propyl, J ¼ 8), 1.91 (m, 4H, –CH2– of
propyl), 3.89 (s, 6H, –OCH3, J ¼ 8), 4.25 (t, 4H, –OCH2– of propyl,
–CH2CH2CH2CH3), 1.62 (m, 4H, –OCH2CH2CH2–), 1.96 (m, 4H,
–OCH2CH2–), 4.04 (s, 6H, –OCH3), 4.44 (t, 4H, –OCH2–), 4.70
(s, 2H, –OH D2O exchangeable), 6.92 (d, 2H, C5– of aromatic), 7.20
d

R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
4383
(d, 2H, C6– of aromatic), 7.78 (s, 2H, C2– of aromatic), 8.27 (s, 2H,
7.1.19. 3,4-Dibenzyloxy-N2,N5-bis[(N-ethyl-9H-carbazole-3-yl)-
–N]CH–).
methylene]thiophene-2,5-dicarbohydrazide (12d)
IR (KBr, cmꢀ1 : 3314 cmꢀ1 (-NH–), 2980 cmꢀ1 (benzyl and
) y
ethyl), 1680 cmꢀ1 (pC]O), 1596 cmꢀ1 (pC]N–); 1H NMR (DMSO-
d6, 300 MHz) in ppm: 1.34 (t, 6H, –NCH2CH3), 4.4 (d, 4H, –NCH2–,
7.1.14. 3,4-Diheptyloxy-N2,N5-bis(phenylmethylene)thiophene-2,5-
d
dicarbohydrazide (11e)
IR (KBr, cmꢀ1
) y:
3263 cmꢀ1 (-NH–), 2929 cmꢀ1 (heptyl),
J ¼ 6), 5.46 (s, 4H, –OCH2–), 7.2–8.4 (m, 24H, aromatic), 8.51 (s, 2H,
1656 cmꢀ1 (pC]O), 1603 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
400 MHz)
–CH]N–), 10.89 (s, 2H, –NH–).
d
in ppm: 0.88 (t, 6H, –CH3– of heptyl, J ¼ 6), 1.31 (m, 8H,
7.1.20. 3,4-Dibenzyloxy-N2,N5-bis(4-nitrophenylmethelene)
C5, C6– of heptyl), 1.40 (m, 4H, C4– of heptyl), 1.52 (m, 4H, C3– of
heptyl), 1.88 (m, 4H, C2– of heptyl), 4.28 (t, 4H, –OCH2–), 7.42 (t, 6H,
C3, C4, C5– of phenyl), 7.77 (q, 4H, C2, C6– of phenyl), 8.26 (s, 2H,
thiophene-2,5-dicarbohydrazide (12e)
IR (KBr, cmꢀ1
) y
: 3266 cmꢀ1 (-NH–), 2952 cmꢀ1 (benzyl),
1677 cmꢀ1 (pC]O), 1632 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
300 MHz) in ppm: 5.51 (s, 4H, –OCH2–), 7.42 (m, 4H, C2 and
–N]CH–), 10.27 (s, 2H, –NH–); 13C NMR (CDCl3, 400 MHz)
d in
d
ppm:13.9 (–CH3), 22.5 (–CH2CH3), 26.1–30.0 (–CH2–), 31.78
(–OCH2CH2–), 74.9 (–OCH2–), 125.0 (C3,4–thiophene), 127.7–128.6
(C2,3,5,6–phenyl), 130.5 (C4–phenyl), 133.4 (C1–phenyl), 147.0 (C2,5
C6– of benzyl), 7.82 (d, 4H, C2 and C6– of phenyl), 8.00 (m, 6H,
C3, C4 and C6– of benzyl), 8.30 (m, 4H, C3 and C4– of phenyl),
8.72 (s, 2H, –N]CH–), 10.55 (s, 2H, –NH–). MS (m/z, %): 680
(M þ 2, 15).
–
thiophene), 148.4 (–CH]N–), 157.0 (–CONH–).
7.1.15. 3,4-Diheptyloxy-N2,N5-bis(4-nitrophenylmethylene)
7.1.21. N5,N7-bis(3,4,5-trimethoxyphenylmethylene)-2,3-
thiophene-2,5-dicarbohydrazide (11f)
dihydrothieno[3,4-b][1,4]dioxine-5,7-dicarbohydrazide (13c)
IR (KBr, cmꢀ1
) y:
3279 cmꢀ1 (-NH–), 2926 cmꢀ1 (heptyl),
IR (KBr, cmꢀ1
) y:
3303 cmꢀ1 (-NH–), 2926 cmꢀ1 (EDOT),
1665 cmꢀ1 (pC]O), 1591 cmꢀ1 (pC]N–); 1H NMR (CHCl3,
300 MHz) in ppm: 0.89 (t, 6H, –CH3), 1.32–1.53 (m, 16H, C3, C4, C5
1670 cmꢀ1 (pC]O), 1577 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
300 MHz) in ppm: 3.62 (s, 6H, para-OCH3), 3.75 (s, 12H, meta-
d
d
and C6– of heptyl), 1.90 (m, 4H, –OCH2CH2–), 4.30 (t, 4H, –OCH2–,
J ¼ 6), 7.88 (d, 4H, C2 and C6– of aromatic), 8.22 (d, 4H, C3 and C5– of
aromatic), 8.42 (s, 2H, –N]CH–), 10.41 (s, 2H, –NH–).
OCH3), 4.41 (s, 4H, –OCH2CH2O–), 6.93 (s, 4H, C2 and C6– of phenyl),
8.26 (s, 2H, –CH]N–), 10.58 (s, 2H, –NH–).
7.1.22. N5,N7-bis(phenylmethylene)-2,3-dihydrothieno[3,4-b]
7.1.16. 3,4-Diheptyloxy-N2,N5-bis[1-(2-thienyl)ethylidene]
thiophene-2,5-dicarbohydrazide (11j)
[1,4]dioxine-5,7-dicarbohydrazide (13d)
IR (KBr, cmꢀ1
) y:
3306 cmꢀ1 (-NH–), 2926 cmꢀ1 (EDOT),
1675 cmꢀ1 (pC]O), 1577 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
300 MHz) in ppm: 4.50 (s, 4H, –OCH2CH2O–), 7.43 (s, 6H, C3, C4
IR (KBr, cmꢀ1
) y:
3310 cmꢀ1 (-NH–), 2926 cmꢀ1 (heptyl),
1679 cmꢀ1 (pC]O), 1636 cmꢀ1 (pC]N–); 1H NMR (CDCl3,
500 MHz)
d
and C5– of phenyl), 7.70 (d, 4H, C2 and C6– of phenyl), 8.43 (s, 2H,
–N]CH–), 10.73 (s, 2H, –NH–); MS (m/z, %): 435 (M þ 1, 100), 391
(20), 332 (10), 315 (10), 289 (10), 232 (20), 107 (5).
d
in ppm: 0.88 (t, 6H, –CH3– of heptyl, J ¼ 6.5), 1.30 (m,
8H, C5 and C6– of heptyl), 1.35 (m, 4H, C4– of heptyl), 1.45 (m, 4H,
C3– of heptyl), 1.85 (m, 4H, C2– of heptyl), 2.33 (s, 6H, –CH3), 4.30 (t,
4H, –OCH2–), 6.98 (s, 2H, C5– of thiophene), 7.32 (s, 4H, C3 and C4–
7.1.23. N2,N7-bis[(4-nitrophenyl)methylene]-2,3-
of thiophene), 10.00 (s, 2H, –NH–); 13C NMR (CDCl3, 500 MHz)
d
in
ppm: 13.6 (–CH3 hep), 22.0 (–CH3), 25.2 (–CH2CH3), 28.6–29.8
(–CH2CH2CH2–), 31.2 (–OCH2CH2), 74.4 (–OCH2–), 125.0 (C3,4
dihydrothieno[3,4-b][1,4]dioxine-5,7-dicarbohydrazide (13f)
IR (KBr, cmꢀ1
) y:
3290 cmꢀ1 (-NH–), 2940 cmꢀ1 (EDOT),
–
1672 cmꢀ1 (pC]O), 1580 cmꢀ1 (pC]N–); MS (m/z, %): 525 (M þ 1,
thiophene), 126.7 (C03,4–thiophene), 128.0 (C05–thiophene), 142.4
(C02–thiophene), 146.2 (C2,5–thiophene), 147.8 (>C]N–), 156.0
(–CONH–); MS (m/z, %): 646 (M þ 1, 100), 521 (5), 505 (50), 477 (5),
309 (50), 167 (40), 124 (50).
20), 482 (10), 378 (20), 361 (40), 232 (20).
7.2. Pharmacology
The evaluation of anticonvulsant activity was carried out by the
National Institute of Health, National Institute of Neurological
Disorders and Strokes (NINDS), USA, following reported
procedures.
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 polyethylene
glycol (PEG 200) and injected intraperitoneally into mice and eval-
uated by the MES, scMET and neurotoxicity screens. The substances
were administered at doses of 30, 100 and 300 mg/kg at two time
intervals.
7.1.17. 3,4-Dibenzyloxy-N2,N5-bis(3,4,5-trimethoxyphenylmethylene)
thiophene-2,5-dicarbohydrazide (12b)
IR (KBr, cmꢀ1
) y
: 3240 cmꢀ1 (-NH–), 2926 cmꢀ1 (benzyl),
1637 cmꢀ1 (pC]O), 1572 cmꢀ1 (-C]N–); 1H NMR (CDCl3,
400 MHz) in ppm: 3.89 (s, 18H, –OCH3), 5.36 (s, 4H, –OCH2–), 6.86
(s, 4H, phenyl), 7.48 (s, 12H, benzyl phenyl and –N]CH–), 10.04 (s,
2H, –NH–); 13C NMR (CDCl3, 400 MHz)
in ppm: 56.3 (–OCH3), 60.9
d
d
(–OCH2–), 104.9 (C2,6–trimethoxyphenyl), 126.0 (C3,4–thiophene),
128.7 (C4–benzyl), 129.4 (C2,3,5,6–benzyl), 129.4 (C1–trimethox-
yphenyl), 134.8 (C1–benzyl), 140.4 (C2,5–thiophene), 146.7
(–CH]N–), 148.3 (C4–trimethoxyphenyl), 153.4 (C3,5–trimethox-
yphenyl), 156.5 (–CONH–).
7.2.1. Maximal Electroshock (MES) Test
7.1.18. 3,4-Dibenzyloxy-N2,N5-bis(phenylmethylene)thiophene-2,5-
The MES [19–21] 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 maxi-
mally active. These seizures are highly reproducible and are elec-
trophysiological consistent with human seizures.
dicarbohydrazide (12c)
IR (KBr, cmꢀ1
) y
: 3263 cmꢀ1 (-NH–), 2926 cmꢀ1 (benzyl),
1673 cmꢀ1 (pC]O), 1605 cmꢀ1 (pC]N–); 1H NMR (DMSO-d6,
300 MHz) in ppm: 5.28 (s, 4H, –OCH2–), 7.30–7.61 (m, 20H,
d
aromatic), 7.95 (s, 2H, –CH]N–), 10.86 (s, 2H, –NH–); MS (m/z, %):
589 (M þ 1, 80), 498 (5), 443 (10), 413 (10), 385 (M þ Na, 10), 379
(10), 353 (5), 261 (40).
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

4384
R. Kulandasamy et al. / European Journal of Medicinal Chemistry 44 (2009) 4376–4384
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.
for 3 s). The 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 to be protected.
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.
7.2.2. Subcutaneous Metrazol Seizure Threshold (scMET) Test
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 [22] in mice.
References
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). The previously
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.
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7.2.4. 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 [24,25] was used to assess compound’s efficacy
against electrically induced seizures but 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