Lipophilic 4-imidazoly-1,4-dihydropyridines: Synthesis, calcium channel antagonist activity and protection against pentylenetetrazole-induced seizure

Article abstract of DOI:10.1016/j.farmac.2003.11.013

A group of alkyl, cycloalkyl and aryl ester analogs of nifedipine, in which the o-nitrophenyl group at position 4 is replaced by a 2-phenyl-4(5)-imidazolyl substituent, were synthesized and evaluated as calcium channel antagonist using the high K⁺ contraction of guinea-pig ileal longitudinal smooth muscle, and the activity of 5a-d, 8b and 8f against pentylenetetrazole (PTZ)-induced seizure was assessed. The results for symmetrical esters showed that lengthening of the methylene chain in C₃ and C₅ ester substituents increased activity. When increasing of the length is accompanied by increasing the hindrance, the activity decreased. In contrast to symmetrical derivatives, comparison of the activities of asymmetrical esters showed that increasing the length of the methylene chain was accompanied by a decrease in their activity. The results demonstrate that 8a was more active, and 5c and 8f were similar in effect to that of the reference drug nifedipine. The time-course of anticonvulsant effect on PTZ-induced seizure threshold of said compounds was assessed and showed that increasing the lipophilicity decreases the time needed for maximum effect. Mice treated with intraperitoneal injection of 25 mg/kg of these derivatives all exhibited increase seizure threshold as compared with control.

Full text of DOI:10.1016/j.farmac.2003.11.013

IL FARMACO 59 (2004) 261–269
www.elsevier.com/locate/farmac
Lipophilic 4-imidazoly-1,4-dihydropyridines: synthesis,
calcium channel antagonist activity and protection
against pentylenetetrazole-induced seizure
Latifeh Navidpour a, Hamed Shafaroodi b, Ramin Miri c,
Ahmad Reza Dehpour b, Abbas Shafiee a,*
a Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences,
P.O. Box 14155-6451, Tehran 14174, Iran
b Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran 14174, Iran
c Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, P.O. Box 71345-1718, 71345 Shiraz, Iran
Received 31 July 2003; accepted 13 November 2003
Abstract
A group of alkyl, cycloalkyl and aryl ester analogs of nifedipine, in which the o-nitrophenyl group at position 4 is replaced by a
2-phenyl-4(5)-imidazolyl substituent, were synthesized and evaluated as calcium channel antagonist using the high K+ contraction of
guinea-pig ileal longitudinal smooth muscle, and the activity of 5a–d, 8b and 8f against pentylenetetrazole (PTZ)-induced seizure was
assessed. The results for symmetrical esters showed that lengthening of the methylene chain in C3 and C5 ester substituents increased activity.
When increasing of the length is accompanied by increasing the hindrance, the activity decreased. In contrast to symmetrical derivatives,
comparison of the activities of asymmetrical esters showed that increasing the length of the methylene chain was accompanied by a decrease
in their activity. The results demonstrate that 8a was more active, and 5c and 8f were similar in effect to that of the reference drug nifedipine.
The time-course of anticonvulsant effect on PTZ-induced seizure threshold of said compounds was assessed and showed that increasing the
lipophilicity decreases the time needed for maximum effect. Mice treated with intraperitoneal injection of 25 mg/kg of these derivatives all
exhibited increase seizure threshold as compared with control.
© 2003 Elsevier SAS. All rights reserved.
Keywords: 1,4-Dihydropyridine; Calcium channel antagonist; 2-Phenyl-imidazole; Pentylenetetrazole; Anticonvulsant
1. Introduction
entry [6]. Both burst-firing [7] and the PDS [8] can be
inhibited by calcium antagonists or exacerbated by a calcium
agonist [9]. Depression of epileptic discharges by calcium
antagonists has been shown in vivo in single neurons and
neuronal populations [10].
Animal models of epilepsy have helped to demonstrate
anticonvulsant effects with flunarizine [11–13], nimodipine
[4,14] and verapamil [15]. Another study [16] showed that
many calcium antagonists were effective against audiogenic
seizures in mice.
In epileptic patients, studies of flunarizine have disagreed
as to whether it has useful anticonvulsant potential [17,18] or
not [19,20]. Worrisome neurologic side effects with flunariz-
ine [21] and the pharmacologic interactions of other AEDs
with verapamil [22] and diltiazem [23] promote the dihydro-
pyridines as candidates for investigation as AEDs in clinical
practice. Their lack of sedative side effects adds to their
benefits.
Recent interest in calcium antagonists as possible antiepi-
leptic drugs (AEDs) stems from various observations. Syn-
aptosomal release of excitatory neurotransmitters is depen-
dent on calcium influx [1], and many establishedAEDs, such
as phenytoin and carbamazepine, inhibit this process by
inhibiting calmodulin activation of calmodulin kinase II
[2,3], although the relevance of this to their mode of action is
still speculative. Initiation of seizure activity is associated
with synchronization of intrinsic burst-firing which is syn-
apse mediated [4]. The abnormal action potential (paroxys-
mal depolarizing shift, PDS), which occurs when epileptoge-
nic activity begins [5] is also dependent on calcium cellular
* Corresponding author.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.11.013

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L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
Table 1
Physical and calcium channel modulation data for compounds 5a–d
Compound
R
Melting point (°C)
Yield (%)
Formula
Calcium channel antagonist activity
(IC50 S.E.M.) a
5a
5b
5c
5d
CH3
247–249
204–206
224–226
243–245
80
72
53
54
C
C
C
C
20H21N3O4
22H25N3O4
24H29N3O4
26H33N3O4
1.36 ( 0.88) × 10–9
CH2CH3
CH2CH2CH3
C(CH3)3
1.68 ( 0.35) × 10–9
3.11 ( 0.59) × 10–10
2.40 ( 0.31) × 10–9
Nifedipine
2.75 ( 0.36) × 10–10
a The molar concentration of antagonist test compound causing a 50% in the tonic contractile response (IC50 S.E.M.) in guinea-pig ileum smooth muscle by
KCl (80 mmol/l) was determined graphically from dose–response curve. The number of experiments was six for all compounds.
Studies suggest that substitution of C4 o-nitrophenyl with
heterocycles retains pharmacologic activity. We have found
that bioisosteric replacement of the 4-aryl moiety with a
imidazolyl group yields analogous 4-imidazolyl-1,4-
dihydropyridines which exhibit potent calcium antagonist
activity [24–28]. A major goal of our present research is to
2. Chemistry
The synthesis of the 1,4-dihydropyridine derivatives 5a–d
(Table 1) and 8a–i (Table 2) was achieved following the steps
outlined in Fig. 1. Reaction of alcohol 2 with 2,2,6-trimethyl-
4H-1,3-dioxin-4-one 3 afforded the corresponding acetoace-
tic esters 4 (76–92% yield) [29]. The symmetrical analogs
5a–d (Table 1) were prepared by the classical Hantzsch
condensation [30] in which 2-phenyl-1H-imidazole-4(5)-
carboxaldehyde 1 was reacted with acetoacetic esters 4 and
ammonium hydroxide. The asymmetrical analogs 8a–i
(Table 2) were synthesized by a modified Hantzsch reaction,
using a procedure reported by Meyer et al. [31]. The physical
properties of final compounds are summarized in
Tables 1 and 2.
prepare
lipophilic
substituted
4-imidazolyl-1,4-
dihydropyridine with improved ability to cross the blood–
brain barrier.
The present study assessed the efficacy of some alkyl,
cycloalkyl and aryl 1,4-dihydro-2,6-dimethyl-4-(2-phenyl-
4(5)-imidazolyl)-3,5-pyridine dicarboxylate derivatives in
terms of calcium channel blocking and anticonvulsive
activity.
Table 2
Physical and calcium channel modulation data for compounds 8a–i
Compound
R
n
Melting point (°C)
Yield (%)
Formula
Calcium channel antagonist activity
(IC50 S.E.M.) a
8a
8b
8c
8d
8e
8f
8g
8h
C6H11(cyclohexyl)
C6H11(cyclohexyl)
C6H11(cyclohexyl)
C6H11(cyclohexyl)
C6H11(cyclohexyl)
C6H5
0
1
2
3
4
1
2
3
4
232–233
211–212
176–178
217–218
170–172
194–195
180–182
175–178
163–165
49
54
48
40
58
48
67
58
52
C
C
C
C
C
C
C
C
C
27H33N3O4
28H35N3O4
29H37N3O4
30H39N3O4
31H41N3O4
28H29N3O4
29H31N3O4
30H33N3O4
31H35N3O4
5.85 ( 0.39) × 10–11
2.12 ( 0.28) × 10–9
9.71 ( 0.82) × 10–9
7.80 ( 0.38) × 10–9
3.98 ( 0.41) × 10–8
2.77 ( 0.46) × 10–10
1.85 ( 0.29) × 10–9
3.52 ( 0.60) × 10–9
4.42 ( 0.30) × 10–9
2.75 ( 0.36) × 10–10
C6H5
C6H5
8i
C6H5
Nifedipine
a Refer to footnote ‘a’ of Table 1.

L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
263
Fig. 1. Synthesis of 1,4-dihydropyridines having 2-phenyl-4(5)-imidazolyl substituents.
3. Experimental
imidazole), 7.32 (m, 3H, aromatic), 7.77 (m, 2H, aromatic);
MS, m/z (%): 367 (100), 351 (44), 335 (26), 307 (82), 275
(63), 248 (12), 223 (38), 165 (18), 145 (40), 104 (12), 77 (5).
3.1. Chemistry
3.1.1.2. Diethyl 1,4-dihydro-2,6-dimethyl-4-(2-phenyl-4(5)-
imidazolyl)-3,5-pyridine dicarboxylate (5b). Melting point:
204–206 °C; IR (KBr): m (cm–1) 3295 (NH), 1696 (CO); 1H
NMR (CDCl3): d 1.26 (t, 6H, CH3-ethyl), 2.28 (s, 6H, C2 and
C6-CH3), 4.20 (q, 4H, COOCH2), 5.02 (s, 1H, H4), 6.56 (s,
1H, H4-imidazole), 7.35 (m, 3H, aromatic), 7.69 (m, 2H,
aromatic).
Melting points were determined with a Reichert-Jung
hot-stage microscope (A Cambridge Instrument Company,
Vienna, Austria) and are uncorrected. 1H NMR spectra were
obtained with a Bruker 80 MHz spectrometer (Bruker Ana-
lytische Messetechnik, Rheinstetten, Germany) in d1-
chloroform or d6-DMSO and tetramethylsilane (TMS) was
used as an internal standard. Mass spectra were recorded
with a Finnigan Mat TSQ-70 spectrometer (Finnigan Mat,
Breman, Germany). Infrared spectra were acquired on a
Nicolet Magna 550-FT spectrometer (Nicolet, Madison,
USA). Elemental analyses were carried out with a Perkin-
Elmer Model 240-C apparatus (Perkin-Elmer, Norwalk, CT,
USA). The results of elemental analyses (C, H, N) were
within 0.4% of the calculated values. Silica gel HT-254 (E.
Merck, Darmstadt, Germany) was used for thin layer chro-
matography.
3.1.1.3. Dipropyl 1,4-dihydro-2,6-dimethyl-4-(2-phenyl-
4(5)-imidazolyl)-3,5-pyridine dicarboxylate (5c). Melting
point: 224–226 °C; IR (KBr): m (cm–1) 3303 (NH), 1683
1
(CO); H NMR (CDCl3): d 0.90 (t, 6H, CH3-propyl), 1.65
(m, 4H, CH2-propyl), 2.29 (s, 6H, C2 and C6-CH3), 4.10 (t,
4H, COOCH2), 5.08 (s, 1H, H4), 6.54 (bs, 1H, NH-
imidazole), 6.64 (s, 1H, H4-imidazole), 7.36 (m, 3H, aro-
matic), 7.71 (m, 2H, aromatic).
Isopropyl 3-aminocrotonate 7 were purchased from
Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany).
3.1.1.4. Ditertiarybutyl 1,4-dihydro-2,6-dimethyl-4-(2-
phenyl-4(5)-imidazolyl)-3,5-pyridine dicarboxylate (5d).
Melting point: 243–245 °C; IR (KBr): m (cm–1) 3302 (NH),
3.1.1. General procedure for the synthesis of dialkyl 1,4-
dihydro-2,6-dimethyl-4-(2-phenyl-4(5)-imidazolyl)-3,5-
pyridine dicarboxylates (5a–d)
A solution of ammonium hydroxide (25%, 0.5 ml) was
added to a stirring solution of compound 1 [32] (175 mg,
1 mmol) and corresponding alkyl 3-oxobutanoate (2 mmol)
in methanol (10 ml). The mixture was protected from light
and refluxed overnight. After cooling the precipitate was
filtered and crystallized from methanol to give pure com-
pounds 5a–d.
1
1700 (CO); H NMR (CDCl3): d 1.40 (s, 18H, C(CH3)3),
2.19 (s, 6H, C2 and C6-CH3), 4.90 (s, 1H, H4), 6.55 (s, 1H,
H4-imidazole), 7.34 (m, 3H, aromatic), 7.81 (m, 2H, aro-
matic).
3.1.2. General procedure for the synthesis of alkyl(aryl)
2-[(2-phenyl-4(5)-imidazolyl)-methylene]-3-oxobutanoate
derivatives (6a–i)
A solution of compound 1 (345 mg, 2 mmol), correspond-
ing alkyl(aryl) acetoacetate (2 mmol), glacial acetic acid
(0.2 ml), piperidine (0.08 ml) and dry benzene (20 ml) was
refluxed for 2 h, during which the resultant water was re-
moved via a Dean-Stark trap. After cooling the benzene was
removed and the residue was purified by chromatography on
silica gel with chloroform/methanol (20:1 v/v), to give pure
compounds 6a–i as semisolid.
3.1.1.1. Dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-phenyl-
4(5)-imidazolyl)-3,5-pyridine dicarboxylate (5a). Melting
point: 247–249 °C; IR (KBr): m (cm–1) 3256 (NH), 1689
(CO); 1H NMR (CDCl3): d 2.30 (s, 6H, C2 and C6-CH3), 3.73
(s, 6H, COOCH3), 5.05 (s, 1H, H4), 6.62 (s, 1H, H4-

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L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
3.1.3. General procedure for the synthesis of alkyl(aryl)
isopropyl 1,4-dihydro-2,6-dimethyl-4-(2-phenyl-4(5)imi-
dazolyl)-3,5-pyridine dicarboxylates (8a–i)
6H, CH3-isopropyl), 1.57 (m, 17H, CH2CH2CH2-
cyclohexyl), 2.23 (s, 6H, C2 and C6-CH3), 4.10 (t, 2H,
CO2CH2), 5.02 (m, 2H, H4 and CO2CH), 6.08 (bs, 1H,
NH-imidazole), 6.59 (s, 1H, H4-imidazole), 7.29 (m, 3H,
aromatic), 7.67 (m, 2H, aromatic).
A solution of compounds 6a–i (1 mmol) and isopropyl
3-aminocrotonate 7 (140 mg, 1 mmol) in methanol was
protected from light and refluxed overnight. After cooling,
the solution was concentrated under reduced pressure and
purified by chromatography on silica gel with
chloroform/methanol (20:1 v/v). The product was crystal-
lized from ethyl acetate/acetone to give pure compounds
8a–i.
3.1.3.6. Benzyl isopropyl 1,4-dihydro-2,6-dimethyl-4-(2-
phenyl-4(5)imidazolyl)-3,5-pyridine dicarboxylate (8f).
Melting point: 194–195 °C; IR (KBr): m (cm–1) 3276 (NH),
1696 (CO); 1H NMR (CDCl3): d 1.27 (2d, 6H, CH3-
isopropyl), 2.30 (s, 6H, C2 and C6-CH3), 5.02 (m, 4H, H4 and
CO2CH and CO2CH2), 5.95 (bs, 1H, NH-imidazole), 6.62 (s,
1H, H4-imidazole), 7.28 (s, 5H, phenyl), 7.35 (m, 3H, aro-
matic), 7.70 (m, 2H, aromatic).
3.1.3.1. Cyclohexyl isopropyl 1,4-dihydro-2,6-dimethyl-4-
(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicarboxylate (8a).
Melting point: 232–233 °C; IR (KBr): m (cm–1) 3276 (NH),
1696 (CO); 1H NMR (CDCl3): d 1.24 (2d, 6H, CH3-
isopropyl), 1.70 (m, 10H, cyclohexyl), 2.28 (s, 6H, C2 and
C6-CH3), 4.95 (m, 2H, CO2CH), 5.02 (s, 1H, H4), 6.08 (bs,
1H, NH-imidazole), 6.59 (s, 1H, H4-imidazole), 7.35 (m, 3H,
aromatic), 7.71 (m, 2H, aromatic); MS, m/z (%): 463 (20),
420 (22), 380 (82), 338 (100), 294 (44), 278 (25), 249 (48),
236 (35), 196 (43), 149 (15), 123 (7).
3.1.3.7. Isopropyl phenylethyl 1,4-dihydro-2,6-dimethyl-4-
(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicarboxylate (8g).
Melting point: 180–182 °C; IR (KBr): m (cm–1) 3262 (NH),
1696 (CO); 1H NMR (CDCl3): d 1.25 (2d, 6H, CH3-
isopropyl), 2.25 (s, 6H, C2 and C6-CH3), 2.95 (t, 2H, CH2-
Ph), 4.38 (t, 2H, CO2CH2), 5.03 (s, 1H, H4), 5.13 (m, 1H,
CO2CH), 6.32 (bs, 1H, H-imidazole), 6.62 (s, 1H, H4-
imidazole), 7.01 (s, 5H, phenyl), 7.35 (m, 3H, aromatic), 7.69
(m, 2H, aromatic).
3.1.3.2. Cyclohexylmethyl isopropyl 1,4-dihydro-2,6-
dimethyl-4-(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicar-
boxylate (8b). Melting point: 211–212 °C; IR (KBr): m
(cm–1) 3276 (NH), 1696 (CO); 1H NMR (CDCl3): d 1.26 (2d,
6H, CH3-isopropyl), 1.30 (m, 11H, cyclohexyl), 2.30 (s, 6H,
C2 and C6-CH3), 3.72 (m, 3H, CO2CH2 and CO2CH), 5.05 (s,
1H, H4), 5.90 (bs, 1H, NH-imidazole), 6.65 (s, 1H, H4-
imidazole), 7.45 (m, 3H, aromatic), 7.79 (m, 2H, aromatic).
3.1.3.8. Isopropyl phenylpropyl 1,4-dihydro-2,6-dimethyl-4-
(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicarboxylate (8h).
Melting point: 175–178 °C; IR (KBr): m (cm–1) 3302 (NH),
1689 (CO); 1H NMR (CDCl3): d 1.26 (2d, 6H, CH3-
isopropyl), 1.92 (t, 2H, CH2-Ph), 2.30 (s, 6H, C2 and C6-
CH3), 2.62 (t, 2H, CH2), 4.20 (t, 2H, CO2CH2), 5.08 (m, 2H,
H4 and CO2CH), 6.12 (bs, 1H, NH-imidazole), 6.65 (s, 1H,
H4-imidazole), 7.28 (s, 5H, phenyl), 7.35 (m, 3H, aromatic),
7.70 (m, 2H, aromatic).
3.1.3.3. Cyclohexylethyl isopropyl 1,4-dihydro-2,6-
dimethyl-4-(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicar-
boxylate (8c). Melting point: 176–178 °C; IR (KBr): m
(cm–1) 3276 (NH), 1703 (CO); 1H NMR (CDCl3): d 1.24 (2d,
6H, CH3-isopropyl), 1.56 (m, 13H, CH2-cyclohexyl), 2.30 (s,
6H, C2 and C6-CH3), 4.15 (t, 2H, CO2CH2), 5.01 (m, 1H,
CO2CH), 5.04 (s, 1H, H4), 6.46 (s, 1H, H4-imidazole), 6.88
(bs, 1H, NH-imidazole), 7.34 (m, 3H, aromatic), 7.70 (m,
2H, aromatic).
3.1.3.9. Isopropyl phenylbutyl 1,4-dihydro-2,6-dimethyl-4-
(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicarboxylate (8i).
Melting point: 163–165 °C; IR (KBr): m (cm–1) 3276 (NH),
1703 (CO); 1H NMR (CDCl3): d 1.23 (2d, 6H, CH3-
isopropyl), 1.63 (m, 4H, CH2CH2-Ph), 2.28 (s, 6H, C2 and
C6-CH3), 2.57 (m, 2H, CH2), 4.14 (m, 2H, CO2CH2), 5.04
(m, 2H, H4 and CO2CH), 6.20 (bs, 1H, NH-imidazole), 6.62
(s, 1H, H4-imidazole), 7.13 (s, 5H, phenyl), 7.32 (m, 3H,
aromatic), 7.69 (m, 2H, aromatic).
3.1.3.4. Cyclohexylpropyl isopropyl 1,4-dihydro-2,6-
dimethyl-4-(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicar-
boxylate (8d). Melting point: 217–218 °C; IR (KBr): m
(cm–1) 3269 (NH), 1683 (CO); 1H NMR (CDCl3): d 1.25 (2d,
6H, CH3-isopropyl), 1.60 (m, 15H, CH2CH2-cyclohexyl),
2.29 (s, 6H, C2 and C6-CH3), 4.12 (t, 2H, CO2CH2), 5.02 (m,
1H, CO2CH), 5.10 (s, 1H, H4), 5.62 (bs, 1H, NH-imidazole),
6.65 (s, 1H, H4-imidazole), 7.42 (m, 3H, aromatic), 7.78 (m,
2H, aromatic).
3.2. Pharmacology
3.2.1. Determination of calcium channel antagonist
activity
Male albino guinea pigs (300–450 g) were purchased
from Pasteur Institute (Karaj, Iran). They had free access to
standard rodent chow (Dam-Pars Co., Tehran, Iran) and tap
water at all times. The animals were housed in a room
maintained at 23 2 °C temperature, 55 10% humidity and
on a 12 h light/dark cycle. The feeding was disrupted 1 d
before starting in vitro tests. The animals were sacrificed by a
3.1.3.5. Cyclohexylbutyl isopropyl 1,4-dihydro-2,6-
dimethyl-4-(2-phenyl-4(5)imidazolyl)-3,5-pyridine dicar-
boxylate (8e). Melting point: 170–172 °C; IR (KBr): m
(cm–1) 3335 (NH), 1670 (CO); 1H NMR (CDCl3): d 1.20 (2d,

L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
265
Fig. 5. The time-course of anticonvulsant effect of 5d (25 mg/kg, i.p.) on
PTZ-induced seizure threshold: 5d was administered 5, 10, 15 and 30 min
before PTZ and compared with CMC-treated mice. Data are expressed as
mean S.E.M. of eight mice. * P < 0.05 compared with CMC group.
Fig. 2. The time-course of anticonvulsant effect of 5a (25 mg/kg, i.p.) on
PTZ-induced seizure threshold: 5a was administered 5, 10, 20, 30, 40 and
50 min before PTZ and compared with CMC-treated mice. Data are ex-
pressed as mean S.E.M. of eight mice. * P < 0.05, ** P < 0.01 compared
with CMC group.
Fig. 6. The time-course of anticonvulsant effect of 8b (25 mg/kg, i.p.) on
PTZ-induced seizure threshold: 8b was administered 2, 4 and 8 min before
PTZ and compared with CMC-treated mice. Data are expressed as
mean S.E.M. of eight mice. * P < 0.05, ** P < 0.01 compared with CMC
group.
Fig. 3. The time-course of anticonvulsant effect of 5b (25 mg/kg, i.p.) on
PTZ-induced seizure threshold: 5b was administered 10, 20, 30 and 40 min
before PTZ and compared with CMC-treated mice. Data are expressed as
mean S.E.M. of eight mice. * P < 0.05 compared with CMC group.
Fig. 4. The time-course of anticonvulsant effect of 5c (25 mg/kg, i.p.) on
PTZ-induced seizure threshold: 5c was administered 5, 10 and 20 min before
PTZ and compared with CMC-treated mice. Data are expressed as
mean S.E.M. of eight mice. ** P < 0.01 compared with CMC group.
Fig. 7. The time-course of anticonvulsant effect of 8f (25 mg/kg, i.p.) on
PTZ-induced seizure threshold: 8f was administered 2, 4 and 8 min before
PTZ and compared with CMC-treated mice. Data are expressed as
mean S.E.M.

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L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
blow to the head. The intestine was removed above the
ileocecal junction and a longitudinal smooth muscle seg-
ments of 2 cm length were mounted under a resting tension of
0.5 g. The segments were maintained at 37 °C in a 20 ml
jacketed organ bath containing oxygenated physiological
saline solution of the following (mmol) composition: NaCl,
137; CaCl2, 1.8; KCl, 2.7; MgSO4, 1.1; NaH2PO4, 0.4;
NaHCO3, 12 and glucose, 5. The muscles were equilibrated
for 1 h with a solution change every 15 min. The contractions
were recorded with a force displacement transducer (F-50)
on a Narco Physiograph (Narco Biosystems, Houston, TX,
USA). Test agents were prepared as 10–2 mol/l stock solu-
tions in DMSO and stored protected from light. Dilutions
were made with DMSO. The contractile response was taken
as the 100% value for the tonic (slow) component of the
response. The contraction was elicited with 80 mmol/l KCl.
Test compounds were cumulatively added and compound-
induced relaxation of contracted muscle was expressed as
percent of control. The IC50 values (concentration needed to
produce 50% relaxation on contracted ileal smooth muscle)
were graphically determined from the concentration–re-
sponse curves [33,34].
Fig. 8. Effect of different dosage of 5a (5, 10 and 25 mg/kg) on PTZ-induced
seizure threshold in mice: 5a was administered 30 min before PTZ. Data are
expressed as mean S.E.M. of eight mice. ** P < 0.01 compared with CMC
group.
The research protocol and experimental animals have
been approved by the ethics committee of Tehran University
of Medical Sciences.
3.2.2. Determination of anticonvulsant activity
3.2.2.1. Chemicals. Pentylenetetrazole (PTZ) was purchased
from Sigma (UK). It was dissolved in physiological saline
solution and all selected derivatives were dispersed in car-
boxy methyl cellulose (CMC, 2%) to such concentrations
that requisite doses were administered in a volume of
10 ml/kg. In all experiments PTZ was administered intrave-
nously (i.v.) and all other drugs were administered intraperi-
toneally (i.p.).
Fig. 9. Effect of different dosage of 5b (5, 10 and 25 mg/kg) on PTZ-
induced seizure threshold in mice: 5b was administered 30 min before PTZ.
Data are expressed as mean S.E.M. of eight mice. * P < 0.05 compared
with CMC group.
3.2.2.2. Subjects. Male NMRI mice (24–30 g, Pasteur Insti-
tute of Iran) were used throughout this study. The animals
were housed in temperature-controlled room (24 1 °C) on a
12 h light/dark cycle with free access to food and water. All
procedures were carried out in accordance with institutional
guidelines for animal care and use. Each mouse was used
only once and each treatment group consisted of at least eight
animals.
3.2.2.3. Determination of seizure threshold. Threshold of
PTZ-induced seizures was determined by inserting a 30-
gauge butterfly needle into the tail vein of mice and the
infusion of PTZ (0.5%) at a constant rate of 0.5 ml/min to
unrestrained animals. Infusion was halted when forelimb
clonus followed by full clonus of the body was observed.
Minimal dose of PTZ (mg/kg of mice weight) needed to
induce clonic seizure was measured as an index of seizure
threshold.
Fig. 10. Effect of different dosage of 5c (5, 10 and 25 mg/kg) on PTZ-
induced seizure threshold in mice: 5c was administered 30 min before PTZ.
Data are expressed as mean S.E.M. of eight mice. * P < 0.05 compared
with CMC group.

L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
267
Fig. 14. Comparison of the highest dosage (25 mg/kg) of derivatives on
PTZ-induced seizure threshold.
Fig. 11. Effect of different dosage of 5d (5, 10 and 25 mg/kg) on PTZ-
induced seizure threshold in mice: 5d was administered 30 min before PTZ.
Data are expressed as mean S.E.M. of eight mice. * P < 0.05 compared
with CMC group.
Animals in experiment 1 received the highest doses of
selected derivatives (25 mg/kg), 2.5, 5, 10, 20, 30, 40 and
50 min prior to PTZ in distinct groups of mice in order to
determine the time of maximal effect.
In experiment 2, different doses of the said derivatives (5,
10 and 25 mg/kg) or vehicle were administered in appropri-
ate time thus obtained before determination of seizure thresh-
old [35].
3.2.2.4. Statistical analysis. Data are expressed as
mean S.E.M. The one-way analysis of variance (ANOVA)
followed by Turkey–Kramer multiple comparisons was used
to analyze the data. P < 0.05 was considered as the signifi-
cance level between the groups.
4. Results and discussion
Fig. 12. Effect of different dosage of 8b (5, 10 and 25 mg/kg) on PTZ-
induced seizure threshold in mice: 8b was administered 30 min before PTZ.
Data are expressed as mean S.E.M. of eight mice. ** P < 0.01 compared
with CMC group.
The calcium channel antagonist activity of 5a–d and 8a–i
determined as the concentration needed to produce 50%
inhibition of the guinea-pig ileal longitudinal smooth muscle
(GPILSM) contractility, are summarized in Table 1 (sym-
metrical compounds) and Table 2 (asymmetrical com-
pounds).
Comparison of the activities of symmetrical esters in alkyl
ester series (Table 1, 5a–d) indicates that lengthening of the
methylene chain in C3 and C5 ester substituents increases
activity (5a–c). When increasing of the length is accompa-
nied by increasing the hindrance (5d), the activity decreases.
Finally the results show that compound 5c is similar in effect
to the reference drug nifedipine.
In contrast to symmetrical derivatives, comparison of the
activities of asymmetrical esters shows that increasing the
length of the methylene chain is accompanied by a decrease
in their activity. The activity of compounds 8f is similar in
effect and 8a is more active than the reference drug nife-
dipine.
The structure–activity data indicate that the 4-(2-phenyl-
4(5)-imidazolyl) moiety is a bioisoester of o-nitrophenyl
group.
Fig. 13. Effect of different dosage of 8f (5, 10 and 25 mg/kg) on PTZ-
induced seizure threshold in mice: 8f was administered 30 min before PTZ.
Data are expressed as mean S.E.M. of eight mice. **P < 0.01 compared
with CMC group.

268
L. Navidpour et al. / IL FARMACO 59 (2004) 261–269
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