E. Naydeno6a et al. / Il Farmaco 57 (2002) 189–194
193
3.1. Preparation of cycloalkanespiro-5-hydantoins
(1a–5a) by Bucherer–Lieb synthesis
3.4.2. Electrocon6ulsi6e shock
An electroshock stimulation (monophase rectangular
pulses with a current intensity of 50 mA, single phase
duration 1 ms; stimulation frequency of 50 Hz and a
trial duration of 0.2 s) was applied by means of silver
corneal electrodes inducing clonic–tonic seizures. A
sham electroshock was applied. The percentage of the
tonic seizure was calculated.
The 3-aminocyclohexanespiro-5-hydantoin, 3-amino-
cycloheptanespiro-5-hydantoin, and 3-amino-cyclodo-
decanespiro-5-hydantoin were administered i.p. at the
following doses: 400 mg/kg, 600 mg/kg, 30 min before
pentylenetetrazole or electroconvulsive shock.
The data of both seizure intensity and latency to first
seizure were assessed by multifactor ANOVA. The data
of mortality and the percentage of the tonic seizures
were analyzed by Fisher’s Exact Probability Two Tailed
test.
To a solution of cycloalkanone (290 mmol) in etha-
nol (225 ml) and water (200 ml) were added sodium
cyanide (28.5 g, 438 mmol) and ammonium carbonate
(119 g, 1.13 mol). The mixture was refluxed for 6 h with
stirring. After dilution with water, the cooled mixture
was acidified with concentrated hydrochloric acid. The
crude
cycloalkanespiro-5-hydantoin
precipitated
overnight upon cooling at 5 °C. Pure compound was
crystallized from water as colorless crystals.
3.2. Synthesis of 3-aminocycloalkanespiro-5-hydantoins
(1b–4b)
The corresponding cycloalkanespiro-5-hydantoin
(0.03 mol) was refluxed with 10 ml (0.2 mol) of 98%
NH2NH2·H2O for 1 h, and with 80% NH2NH2·H2O for
3.5 h (the correction of the concentration was done by
adding the corresponding amount of H2O to the reac-
tion mixture without interrupting the heating). The
reaction mixture was cooled, then poured over a small
amount of crushed ice. Upon standing, the product
crystallized slowly and was filtered, washed with a
minimum of cold water, dried, and recrystallized from
water or aqueous ethanol. Results are recorded in
Table 1.
3.5. Biological acti6ity in the enteric ner6ous system
3.5.1. Smooth muscle preparations
Male guinea pigs (250–300 g) were used in the
experiments. A segment of the ileum (excluding the
portion 15 cm proximal to the ileocaecal junction)
10–15 cm long, was isolated and the intraluminal con-
tents were removed with aerated Krebs solution con-
taining (mM): NaCl 120, KCl 5.9, NaHCO3 15.4,
NaH2PO4 1.2, MgCl2 2.5 and glucose 11.5. In order to
retain the myenteric plexus intact, segments of the
ileum approximately 2.0 cm long were cut out.
3.3. Synthesis of
3-aminocyclododecanespiro-5-hydantoin (5b)
3.5.2. Spontaneous mechanical acti6ity
5 g (0.02 mol) of the cyclododecanespiro-5-hydantoin
(5a) were dissolved in hot ethanol (25 ml) then 10 ml
(0.2 mol) 98% NH2NH2·H2O were added. The optimal
duration of the reaction was 10 h. The compound was
obtained according to the above method as a white
solid: Anal. Calc. for C14H25N3O2: C, 62.92; H, 9.36; N,
15.73. Found: C, 62.95; H, 9.30; N, 15.71%.
The isolated segments were mounted along the axis
of the longitudinal layer in 3 ml organ baths, contain-
ing Krebs solution, continuously aerated with 95% O2
and 5% CO2 at 36.5 °C. The spontaneous mechanical
activity as well as the compound- or electrically-evoked
responses of the longitudinal layer were followed under
isometric conditions after standard calibration of a
mechanoelectrical transducer (Experimetria Ltd., Hun-
gary) connected to a recording device TZ 4620 (Labo-
ratorni Pristroje, Praha) at a tension equivalent to a
load of 0.5 g. There was a 60 min equilibration period
before application of the compounds. Concentration–
response curves were constructed for each of the com-
pounds. The EC50 values, presented as means9SEM
and significance of the differences (Student’s t-test at
PB0.05 and PB0.01) were calculated using computer
programs [10]. The concentration–response curves were
further followed in the presence of cholinoblocker at-
ropine (3 mM) which remained in the organ bath before
retesting the compounds for 10 min.
3.4. Biological acti6ity on the central ner6ous system
Experimental seizure models (pentylenetetrazole
seizures and electrocon6ulsi6e shock)
The experiments were carried out on male Wistar
mice (10 mice per group), weighing 18–20 g.
3.4.1. Pentylenetetrazole seizure model
Pentylenetetrazole was injected subcutaneously in a
dose of 85 mg/kg. This dose was chosen on the basis of
preliminary experiments in which moderate seizures
were produced in the majority of animals. The effect
was evaluated by the differences between the control
and the experimental group of mice in seizure intensity
determined by a six point scale [9]. The animals were
observed 1 h after pentylenetetrazole injection.
3.5.3. Electrical stimulation
Electrical field stimulation was applied by a pair of
ring wire platinum electrodes (0.45 mm thick), to