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TMS as an external standard. IR spectra were recorded on
Specord IR-75 spectrometer. Results of elemental analysis
for C, H, N by method of microanalysis performed in Depart-
ment of Organic Chemistry, Medical University Lublin were
in acceptable accordance with calculated values ( 0.7% for
C, 0.75% for N and 0.9% for H).
The purity of the obtained compounds was examined by
TLC method. Chromatography was performed on 10 × 10 cm
TLC plates precoated with silica gel RP-18 F254 and silica gel
Si 60 F254 (E. Merck, Darmstadt, Germany). Mobile phase
for normal system was prepared from mixtures of acetone,
toluene and acetic acid (8:2:1) and for reversed phase system
from methanol. Water and formic acid (5:5:0.5). Compounds
were dissolved in methanol (5 mg ml–1) and 10 µl samples of
these solutions were spotted on the plates.After development
in horizontal Teflon DS chambers (Chromdes, Lublin, Po-
land) and drying, the spots were visualized under UV light at
k = 254 nm.
10 animals each were selected by means of a randomized
schedule. The experiments were performed between 8 a.m.
and 3 p.m. The investigated compound 9 was administered
intraperitoneally (i.p.) in doses of 25, 50 and 100 mg/kg as
suspensions in a 3% Tween 80 in the constant volume of
10 ml/kg. Control animals received the equivalent volume of
solvent.
5.2.2. The influence of compound 9 on the motor
impairment in mice
Chimney test. The effect of compound 9 on motor impair-
ment was quantified with the chimney test [8]. Briefly, mice
had to climb up backwards in a plastic tube (3 cm in inner
diameter, 25 cm long). Mice unable to perform the task
within 60 s were considered to display motor impairment.
Motor impairment was quantified as the percentage of ani-
mals that failed to complete the test.
5.2.3. The influence of the tested compound 9 on the body
temperature
5.1.1. Synthesis (general procedure)
A 0.1 mol of the maleic acid anhydride in 20 ml of
anhydrous ethyl ether was added to the solution of 0.1 mol of
amidrazone (1–7) in 40 ml of anhydrous ethyl ether. Mixture
was left in the room temperature for 48 h. Precipitation was
collected and purified by crystallization from the water-
:methanol (1:1) mixture. The yields and physical and spectral
data of compounds (8–14) are given in Table 1.
The rectal body temperature in mice (measured with an
Ellab thermometer) was recorded 15, 30, 45, 60, 90 and
120 min after the administration of compound 9 in a dose
100 mg/kg i.p.
5.2.4. The influence of the investigated compound 9
on pentobarbital-induced sleep
Pentobarbital, at a dose of 100 mg/kg i.p., was given
30 min after administration of the tested compound in doses
of 50 and 100 mg/kg i.p. The period during which the ani-
mals lost righting reflex was regarded as a sleeping time.
5.1.2. X-ray crystallography
Crystal data for 11: space group P21/c, a = 15.446(3) Å,
b = 9.027(2) Å, c = 12.687(3) Å, b = 97.28(3)°, V = 1754.6(7)
Å3, Z = 4, dcalc = 1.277 g cm–3, µ = 0.804 mm–1.
X-ray diffraction data were measured on a KM4 diffrac-
tometer using variable scan speed (x – 2h scan mode) and
graphite-monochromatized CuKa radiation (k = 1.54178 Å).
Reflections were collected up to hmax = 79.93°; 3272 reflec-
tions were measured. Crystal structure was solved by direct
methods using the SHELXS97 [6] program and refined by
the full-matrix least squares on F2 using the SHELXL97 [7].
Non-hydrogen atoms were refined with anisotropic displace-
ment parameters. H-atom positions were located from the
geometry, and isotropic factors of 1.2 Ueq of the bonded
C/O-atoms were given, the ‘riding’ model was used in the
refinement. Final discrepancy factors are R1 = 0.0324,
wR2 = 0.1517 for I > 2r(I).
5.2.5. Pain reactivity in the “writhing syndrome test”
in mice for compound 9
Pain reactivity was measured in mice by the “writhing
syndrome” test of Witkin et al. [9]. Thirty minutes after the
administration of compound 9 in doses of 25, 50 and
100 mg/kg i.p., the animals were injected with 0.6% acetic
acid i.p. and the number of writhing episodes was counted for
30 min.
5.2.6. The influence of the investigated compound 9
on pentetrazole-induced convulsions
Pentetrazole, at a dose of 100 mg/kg s.c., was injected
30 min after the administration of the compound 9 in doses of
25, 50 and 100 mg/kg i.p. This dose produced the tonic
convulsions in 80% and mortality in 80% of non-pretreated
mice. The observation (during 30 min) of individual animals
to note occurrence of tonic convulsions as well as mortality
of mice started immediately after the injection of pentetra-
zole.
5.2. Pharmacology
5.2.1. Behavioural experiments
The study was conducted on Albino Swiss male mice
weighing 20–23 g purchased from a licensed dealer,
Górzkowska, Warsaw, Poland. The animals were housed in
colony cages with free access to tap water and food (standard
laboratory pellets, Bacutil, Motycz, Poland) and maintained
under 12/12 h light–dark cycle (light on from 7 a.m. to
7 p.m.). Experimental and control groups consisting of
5.2.7. Statistical analysis
Student’s t-test (for analyzing the data from the Ta-
bles 2 and 4) or Fisher’s two-tailed exact probability test (for
analyzing the data from the Table 3) were used to determine
Modzelewska-Banachiewicz, Bo?ena
