Synthesis, physicochemical and anticonvulsant properties of new N-(pyridine-2-yl) derivatives of 2-azaspiro[4.4]nonane and [4.5]decane-1,3- dione. Part II

Article abstract of DOI:10.1002/ardp.200500219

A series of N-(pyridine-2-yl) derivatives of 2-azaspiro[4.4]nonane- (1a-e), 2-azaspiro[4.5]decane-(2a-e) and 6-methyl-2-azaspiro[4.5]decane-1,3-dione (3a-e) were synthesized and tested for their anticonvulsant activity in the maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure threshold tests. To explain the possible mechanism of action, the most active compounds N-(3-methylpyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-dione (1b), N-(3-methylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-dione (2b), N-(4-methylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-dione (2c), and N-(3-methylpyridine-2-yl)-6-methyl-2-azaspiro[4.5]decane-1,3-dione (3b) were tested in vitro for their influence on voltage-sensitive calcium channel receptors, however, they revealed low affinities. For all synthesized compounds the lipophilicity was determined by use of RP-TLC method. The correlation between the lipophilicity and anticonvulsant activity was obtained - the higher the lipophilicity the stronger the anticonvulsant efficacy.

Full text of DOI:10.1002/ardp.200500219

Arch. Pharm. Chem. Life Sci. 2006, 339, 255–261
K. Kaminski et al.
255
Full Paper
Synthesis, Physicochemical and Anticonvulsant Properties of
New N-(Pyridine-2-yl) Derivatives of 2-Azaspiro[4.4]nonane and
[4.5]decane-1,3-dione. Part II
Krzysztof Kaminski1, Jolanta Obniska1, Agnieszka Zagorska1, Dorota Maciag2
1 Department of Pharmaceutical Chemistry, Jagiellonian University Medical College, Krakow, Poland
2 Department of Pharmacobiology, Jagiellonian University Medical College, Krakow, Poland
A series of N-(pyridine-2-yl) derivatives of 2-azaspiro[4.4]nonane- (1a–e), 2-azaspiro[4.5]decane-
(2a–e) and 6-methyl-2-azaspiro[4.5]decane-1,3-dione (3a–e) were synthesized and tested for their
anticonvulsant activity in the maximum electroshock (MES) and subcutaneous pentylenetetra-
zole (scPTZ) seizure threshold tests. To explain the possible mechanism of action, the most active
compounds N-(3-methylpyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-dione (1b), N-(3-methylpyridine-2-
yl)-2-azaspiro[4.5]decane-1,3-dione (2b), N-(4-methylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-dione
(2c), and N-(3-methylpyridine-2-yl)-6-methyl-2-azaspiro[4.5]decane-1,3-dione (3b) were tested in
vitro for their influence on voltage-sensitive calcium channel receptors, however, they revealed
low affinities. For all synthesized compounds the lipophilicity was determined by use of RP-TLC
method. The correlation between the lipophilicity and anticonvulsant activity was obtained –
the higher the lipophilicity the stronger the anticonvulsant efficacy.
Keywords: 2-Azaspiro[4.4]nonane-1,3-dione / 2-Azaspiro[4.5]decane-1,3-dione / Anticonvulsant activity / Calcium
channel receptors affinity / Lipophilicity /
Received: October 5, 2005; accepted: November 14, 2005
DOI 10.1002/ardp.200500219
In our earlier studies, we described anticonvulsant
activity of many N-(pyridine-2-yl)-3-phenyl-pyrrolidine-
2,5-diones which displayed protection against both MES
and scPTZ tests [6–8]. In this series the most active com-
pound was N-(3-methylpyridine-2-yl)-3-(3-bromophenyl)-
pyrrolidine-2,5-dione with ED50 = 54.2 mg/kg (MES) and
ED50 = 110.3 mg/kg (scPTZ). The structure-activity relation-
ship analysis indicated that their anticonvulsant activity
was closely connected with the conformational and elec-
tronic properties of those molecules and highly
depended on the position of the methyl group at the pyr-
idine moiety [9, 10].
Among the classes of compounds with well documen-
ted anticonvulsant activity are spirosuccinimide deriv-
atives [11, 12]. Based on these facts in our recent studies,
we replaced the phenyl moiety at the 3-position of pyrro-
lidine-2,5-dione ring with a cycloalkyl fragment to obtain
a new series of N-benzyl and N-aminophenyl 2-azaspiro-
[4.4]nonane- and 2-azaspiro[4.5]decane-1,3-dione deriv-
atives, among which N-aminophenyl-2-azaspiro[4.4]-
Introduction
Epilepsy is one of the most frequent neurological disor-
ders characterized by spontaneous recurrent seizures
arising from excessive electrical activity in a portion of
the brain. Clinically available anticonvulsant drugs exert
satisfactory control in 60–70% of patients, however
many of them also produce undesirable harmful side-
effects. Therefore, there is a growing necessity for novel
specific and low-toxicity anticonvulsant molecules [1, 2].
In the course of developing new potentially anticon-
vulsant active agents, our attention has been focused on
the group of pyrrolidine-2,5-dione derivatives (succini-
mides) variously substituted at the nitrogen atom as well
as at the 3-position of the imide ring [3–5].
Correspondence: Jolanta Obniska, Department of Pharmaceutical
Chemistry, Jagiellonian University Medical College, Medyczna 9, 30-688
Krakow, Poland.
E-mail: mfobnisk@cyf-kr.edu.pl
Fax: + 48 12 657-0262
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256
K. Kaminski et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 255–261
nonane-1,3-dione was the most active with ED50 = 72.61 for 1.5 h. Compounds 1a and 2a have been previously
mg/kg in the MES test [13].
described by El-Talbany et al. [16, 17] (no 1H-NMR dataavail-
In view of these data, we designed and synthesized in able), but none of them were tested for their anticonvul-
this work a new series of 2-azaspiro[4.4]nonane-, 2-aza- sant activity. All other compounds arenew ones.
spiro[4.5]decane- and 6-methyl-2-azaspiro[4.5]decane-1,3-
dione derivatives with pyridine-2-yl moiety at the nitro- Anticonvulsant activity
gen atom. The compounds were evaluated for their anti- The maximal electroshock (MES) and subcutaneous pen-
convulsant activity and neurotoxic properties within the tylenetetrazole (scPTZ) tests are claimed to detect com-
antiepileptic drug development (ADD) program (Epilepsy pounds affording protection against generalized tonic-
Branch, Neurological Disorders Program, National Insti- clonic seizures and generalized absence seizures, respec-
tute of the Neurological and Communicative Disorders tively. Thus the MES and scPTZ screens have become the
and Stroke (NINCDS), Bethesda).
most widely employed seizure models for early identifi-
It is well known that several antiepileptic drugs are cation of candidate anticonvulsants.
thought to interact with voltage-gated Ca2+ [14]. Taking
The investigated compounds 1a–e, 2a–e, and 3a–e
this fact into account, to explain the possible mechanism showed diversified anticonvulsant properties from active
of action for selected active compounds their influence at a dose 100 mg/kg or less (class 1) (2b, 2c, 2d, 3d, and 3e)
on voltage-gated calcium channels were investigated.
to active at a dose of 300 mg/kg (class 2) (1b–e, 2e, 3b, 3c)
Lipophilicity, the inherent property of the chemical and inactive ones (class 3) (1a, 2a, 3a). The results of anti-
compound is widely used as structural descriptor in convulsant identification studies in mice are shown in
rational drug design. Therefore, in this study we decided Table 1.
to evaluate the influence of the lipophilicity on anticon-
vulsant activity of the investigated derivatives.
Except for compounds 2c and 2e, effective only in the
scPTZ test, at doses of 100 and 300 mg/kg, respectively,
all other derivatives revealed protection in both MES and
scPTZ screens. The most active N-(3-methylpyridine-2-yl)-
2-azaspiro[4.5]decane-1,3-dione (2b) exhibited anti-scPTZ
activity at a dose of 30 mg/kg and anti-MES protection at
a dose of 100 mg/kg. The 6-methyl isomer 2d was less
Results and discussion
Chemistry
Compounds 1a–e, 2a–e, and 3a–e were synthesized active and inhibited the seizures at doses of 100 and
according to Scheme 1. The starting dicarboxylic acids 300 mg/kg in the MES and scPTZ test, respectively. Simi-
were prepared as reported previously [15]. First, in a reac- lar results were obtained for the N-(4,6-dimethypyridine-
tion of cyclopentanone, cyclohexanone, or 2-methylcyclo- 2-yl)-6-methyl-2-azaspiro[4.5]decane-1,3-dione (3e), where-
hexanone with ethyl cyanoacetate, the corresponding as 3d with one methyl group at 6-position of the pyri-
cycloalkylidene cyanoethyl esters were obtained. Treat- dine-2-yl moiety was more potent in the scPTZ screen
ment of the intermediate products with potassium cya- (100 mg/kg) as compared to its anti-MES activity (300 mg/
nide, followed by a hydrolysis, furnished the respective kg). Compounds 3b, 3c as well as methyl analogues of N-
acids. The final N-substituted spirosuccinimides (1a–e, (pyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-dione (1b–1e),
2a–e, and 3a–e) were obtained in a one-pot cyclization regardless on the number and position of the methyl sub-
reaction of the prepared acids and appropriately substi- stituents at the pyridine-2-yl moiety, revealed protection
tuted 2-aminopyridines by heating them at ca. 190–2008C against both screens at doses of 300 mg/kg. The unsubsti-
tuted compounds 1a, 2a, and 3a were found to be inac-
tive.
In the neurotoxicity screen only compounds 2a, 2b, 2c,
and 3b were devoid of toxicity at maximum admini-
strated dose (300 mg/kg). The other derivatives were
found to be toxic at a dose of 100 mg/kg (1e, 2d, 2e, 3c–e)
or 300 mg/kg (1a–d, 3a).
Compounds 2a, 2d, and 3b, randomly selected from
derivatives active or inactive in i.p. screen in mice, were
examined for their activity and toxicity at a dose of
30 mg/kg after p.o. administration into rats. The
obtained results are shown in Table 2. As can be seen
Scheme 1. Synthesis and structures of the title compounds.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
from these data, only compound 3b was moderately
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Arch. Pharm. Chem. Life Sci. 2006, 339, 255–261
Anticonvulsant Activity of Certain Spirosuccinimides
257
Table 1. Anticonvulsant, neurotoxicity screening results and RM0
values of investigated compounds.
Table 2. The results in rats after oral administration at a dose of
30 mg/kg.
Intraperitoneal injection in micea)
TOXb)
ASPd)
class
Com-
pound
Oral administration to rats
Com-
pound
MESa)
0.25 h 0.5 h 1 h
TOXb)
c)
MES
scPTZ
RM0
2 h
4 h
0.25 h 0.5 h 1 h
2 h
4 h
0.5 h 4 h 0.5 h 4 h 0.5 h 4 h
2a
2d
3b
1/4
0/4
2/4
0/4
0/4
1/4
0/4
0/4
2/4
1/4
0/4
1/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
300
30014
30014
30014
10014
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.78
0.85
0.84
0.95
0.97
0.95
1.28
1.12
1.14
1.17
0.90
1.25
1.05
1.15
1.32
3
2
2
2
2
3
1
1
1
2
3
2
2
1
1
300
300
300
300
–
300
300
300
300
–
a)
Maximal electroshock test, number of animals protected/
number of animals tested.
Rotorod test for neurological toxicity, number of animals
exhibiting toxicity/number of animals tested.
b)
100
–
100
–
30
–
–
100
300
300
–
300
300
100
300
100
10014
300
–
Table 3. Results of calcium-channels binding studies.
–
Compound
IC50 [lM]
300
300
300
100
10014, 33
100
10014
1b
2b
2c
3b
181.2
199.4
168.0
101.3
a)
Doses of 30, 100, and 300 mg/kg were administrated. The
figures in the Table indicate the minimum dose (mg/kg),
whereby bioactivity was demonstrated. The dash (–) indi-
cates an absence of activity at maximum dose admini-
strated.
model for early identification of molecules affording pro-
tection against absence seizures. Therefore, to search for
possible mechanism of action, the chosen active com-
pounds, 1b, 2b, 2c, and 3b were in-vitro evaluated on vol-
tage-sensitive calcium channels affinity, by their ability
to displace [3H]nitrendipine in rat cortical tissue. The
radioligand binding experiments, however, revealed low
affinities; IC50 ranged from 101.3 to 199.4 lM (Table 3).
These preliminary results suggest that anticonvulsant
activity is rather not related with voltage-gated calcium
channels.
b)
c)
Toxicity screen: the minimum dose of compound whereby
14
toxicity was exhibited. Response comments:
grasp rotorod, 33 tremors.
unable to
The relative retention parameters, calculated according to
equation: RM = RM0 + ac, where c is the concentration of n-
propanol in a mobile phase and a is the slope of the straight
line.
The ASP classification is as follows: 1 – anticonvulsant activ-
ity at doses of 100 mg/kg or less; 2 – anticonvulsant activity
at doses of 300 mg/kg; 3 – compound inactive at doses of
300 mg/kg.
d)
Lipophilicity
Lipophilicity is a fundamental physicochemical property
of bioactive compounds that play a pivotal role in the
transport of a molecule through cellular membranes and
influences the localization of compound in the therapeu-
active in rat MES oral screen and revealed the time of
onset of anticonvulsant activity at 0.25 h and two peaks
of 50% protection at time points 0.25 h and 1 h. In addi-
tion, the tested substance protected 25% of animals at tic site of action.
0.5, 2 and 4 h. The total duration of satisfactory action of
this compound appeared to be quite short within 0.25 to
1 h. The derivative 2a showed marginal protection,
The most widely used measurement of the lipophilic
properties of different chemical molecules is nowadays
the chromatographic techniques in reversed-phase sys-
whereas 2d was inactive in rats. No evidence of neurologi- tem. In consequence, the correlation between lipophili-
cal toxicity was observed at the dose of 30 mg/kg admini-
strated orally.
city and biological activity can be based on chromato-
graphic retention parameters [20–23]. In our previous
Several anticonvulsants i.e. valproate, ethosuximide, studies [24], the correlation between anticonvulsant
that are known as effective drugs in absence epilepsy
exert their activity by the interaction with voltage-gated
Ca2+ channels [18, 19]. As described above, the results of
activity and chromatographic RM0 parameters was
obtained. Based on these facts, in the presented work we
determined the lipophilicity of all compounds by use of
preliminary pharmacological screening revealed that the RP-TLC method.
the majority of investigated derivatives exhibited protec-
tion in the scPTZ screen, recognized as the experimental
The examination of the chromatographic behaviour in
the RP-TLC system showed a linear correlation between
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K. Kaminski et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 255–261
the anticonvulsant activity was proved, the higher the
lipophilicity, the stronger anticonvulsant efficacy.
The obtained results revealed that a number of novel
N-(pyridine-2-yl) derivatives of spirosuccinimides were
moderately effective in MES as well as scPTZ screens, how-
ever none of them was more potent than molecules with
an aryl moiety at the 3-position of pyrrolidine-2,5-dione
[6-8]. Thus, the presence of an aromatic area at the 3-posi-
tion of an imide ring seems to be the most important fac-
tor for anticonvulsant activity.
The authors wish to thank Dr James Stables for providing
them with pharmacological data through the Antiepileptic
Drug Development Program (Epilepsy Branch, National Insti-
tute of Neurological Disorders and Stroke, National Institute of
Health, Bethesda, MD, USA). We are pleased to acknowledge
the generous financial support of this work by the Committee
for Scientific Research, Poland (Grant No 3 PO5F 024 25).
Figure 1. Comparison of mean RM0 parameters for active and
inactive compounds.
RM values and n-propanol concentration (v/v) in a mobile
phase. The relative retention parameters RM0 were deter-
mined by the extrapolation method, and are listed in
Table 1.
As expected, the lowest RM0 values were observed for
unsubstituted derivatives of 1a, 2a, and 3a. The change of
the ring size from cyclopentane (1a–e) to cyclohexane
(2a–e) increased the lipophilicity. Surprisingly, the pre-
sence of the additional methyl group at the cyclohexane
fragment (3a–e) caused the increase of RM0 parameters
only for compounds 3d and 3e in comparison to the
unsubstituted analogues 2d and 2e. Moreover, the RM0
data proved that the introduction of the methyl groups
into pyridine ring enhanced the lipophilicity.
As it is shown in Figure 1, the mean RM0 parameter for
inactive compounds was lower in comparison to active
ones (30, 100, and 300 mg/kg). Furthermore, in general,
the higher the lipophilicity (RM0) the more potent anti-
convulsant activity was demonstrated. The applied statis-
tical analysis (Mann-Whitney test) revealed that the dif-
ferences between RM0 values of active and inactive deriv-
atives were statistically significant (p a 0.05).
In conclusion, the most active were compounds with
cyclohexane ring as spiro nucleus and methyl groups
especially in 3- or 6-position of pyridine-2-yl moiety. It
was in agreement with our earlier experiments [6, 9, 10,
13] indicating that the presence of methyl substituent at
the 3-position of pyridine-2-yl ring is important for the
anticonvulsant activity. The change of the size of a spiro-
cycloalkyl unit to cyclopentane as well as the introduc-
tion of a methyl substituent into 6-position of 2-azaspiro-
[4.5]decane-1,3-dione fragment decreased the activity and
also increased the toxicity. The role of the lipophilicity in
Experimental
Chemistry
All chemicals and solvents were purchased from Merck (Darm-
stadt, Germany) and were used without further purification.
Melting points (m.p.) were determined in open capillaries on a
Bꢀchi 353 melting point apparatus (Bꢀchi Labortechnik, Flawil,
Switzerland) and are uncorrected. The purity of the compounds
was confirmed by the thin-layer chromatography (TLC) perform-
ed on Merck silica gel 60 F254 aluminium sheets (Merck; Darm-
stadt, Germany), using the developing system chloroform:ace-
tone (9:1). Spots were detected by their absorption under UV
light (k = 254 nm) and by visualization with 0.05 mol I2 in 10%
HCl. The chemical structures were confirmed by elemental and
spectral analyses (1H-NMR). 1H-NMR spectra were obtained in a
Varian Mercury 300 MHz spectrometer (Varian Inc., Palo Alto,
CA, USA), in CDCl3, with TMS as an internal standard. Chemical
shifts are reported in d values (ppm) and J values in Hertz (Hz).
Signal multiplicities are represented by the following abbrevia-
tions: s (singlet), d (doublet), t (triplet), td (triplet of doublets), dt
(doublet of triplets), m (multiplet). Elemental analyses for C, H, N
were carried out with an Elementar Vario EL III (Hanau, Ger-
many).
General procedure for the preparation of the 1a–e, 2a–e,
and 3a–e
A total of 0.02 mol of the appropriate substituted 2-aminopyri-
dines were dissolved in 20 mL of water and 0.02 mol of cyclopen-
tane- acid, cyclohexane- acid or 1-(2-methylcyclohexane-1-carb-
oxy-1-acetic acid was gradually added. The mixture was heated
in an oil bath with simultaneous distillation of water. After the
water was completely removed, the temperature of the reaction
mixture was raised up to 1808C and maintained for 1.5 h. The
crude products were recrystallized from methanol.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 255–261
Anticonvulsant Activity of Certain Spirosuccinimides
259
N-Pyridine-2-yl-2-azaspiro[4.4]nonane-1,3-dione 1a
Compound 1a was obtained as white solid (yield 70%), m.p. 143–
1458C (1308C [16]). 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.76–2.30
(m, 8H, cyclopentane), 2.78 (s, 2H, imide), 7.29–7.37 (m, 2H, H-4,
H-5, pyridine), 7.85 (td, 1H, H-3, J = 7.70 Hz, pyridine), 8.66 (dt,
1H, H-6, J = 4.86 Hz, pyridine). Anal. calcd. for C13H14N2O2: C,
67.81; H, 6.13; N, 12.17. Found: C, 67.62; H, 6.02; N, 12.10.
N-(4-Methylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-
dione 2c
Compound 2c was obtained as white solid (yield 64%), m.p. 138–
1
1408C. H-NMR (CDCl3, 300 MHz) d (ppm): 1.33–1.97 (m, 10H,
cyclohexane), 2.41 (s, 3H, CH3 ), 2.74 (s, 2H, imide), 7.09–7.17 (m,
2H, H-3, H-5, pyridine), 8.49 (d, 1H, H-6, J = 5.22 Hz, pyridine).
Anal. calcd. for C15H18N2O2: C, 69.74; H, 7.02; N, 10.84. Found: C,
69.71; H, 7.01; N, 10.80.
N-(3-Methylpyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-
dione 1b
N-(6-Methylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-
Compound 1b was obtained as white solid (yield 55%), m.p.
135–1378C. 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.78–2.34 (m, 8H,
cyclopentane), 2.38 (s, 3H, CH3), 2.79 (s, 2H, imide), 7.28–7.32 (m,
1H, H-5, pyridine), 7.65–7.69 (m, 1H, H-4, pyridine), 8.47 (dt, 1H,
H-6, J = 4.77 Hz, pyridine). Anal. calcd. for C14H16N2O2: C, 68.83; H,
6.60; N, 11.47. Found: C, 68.72; H, 6.41; N, 11.27.
dione 2d
Compound 2d was obtained as white solid (yield 60%), m.p.
151–1538C. 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.38–1.98 (m,
10H, cyclohexane), 2.59 (s, 3H, CH3), 2.75 (s, 2H, imide), 7.07 (d,
1H, H-5, J = 7.69 Hz, pyridine), 7.22 (d, 1H, H-4, J = 7.70 Hz, pyri-
dine), 7.75 (t, 1H, H-3, J = 7.84 Hz, pyridine). Anal. calcd. for
C15H18N2O2: C, 69.74; H, 7.02; N, 10.84. Found: C, 69.60; H, 6.92;
N, 10.72.
N-(4-Methylpyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-
dione 1c
Compound 1c was obtained as white solid (yield 61%), m.p. 97–
998C. 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.70–2.28 (m, 8H, cyclo-
pentane), 2.40 (s, 3H, CH3), 2.77 (s, 2H, imide), 7.10–7.17 (m, 2H,
H-3, H-5, pyridine), 8.49 (d, 1H, H-6, J = 5.22 Hz, pyridine). Anal.
calcd. for C14H16N2O2: C, 68.83; H, 6.60; N, 11.47. Found: C, 68.80;
H, 6.53; N, 11.38.
N-(4,6-Dimethylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-
dione 2e
Compound 2e was obtained as white solid (yield 55%), m.p. 99–
1
1018C. H-NMR (CDCl3, 300 MHz) d (ppm): 1.39–1.99 (m, 10H,
cyclohexane), 2.39 (s, 3H, CH3), 2.57 (s, 3H, CH3), 2.76 (s, 2H,
imide), 6.88 (s, 1H, H-5, pyridine), 7.07 (s, 1H, H-3, pyridine). Anal.
calcd. for C16H20N2O2: C, 70.56; H, 7.40; N, 10.29. Found: C, 70.49;
H, 7.25; N, 10.18.
N-(6-Methylpyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-
dione 1d
Compound 1d was obtained as white solid (yield 63%), m.p. 78–
808C. 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.80–2.32 (m, 8H, cyclo-
pentane), 2.63 (s, 3H, CH3), 2.81 (s, 2H, imide), 7.10 (d, 1H, H-5, J =
7.70 Hz, pyridine), 7.25 (d, 1H, H-4, J = 7.70 Hz, pyridine), 7.77 (t,
1H, H-3, J = 7.84 Hz, pyridine). Anal. calcd. for C14H16N2O2: C,
68.83; H, 6.60; N, 11.47. Found: C, 68.62; H, 6.49; N, 11.35.
N-(Pyridine-2-yl)-6-methyl-2-azaspiro[4.5]decane-1,3-
dione 3a
Compound 3a was obtained as white solid (yield 67%), m.p. 81–
838C. 1H-NMR (CDCl3, 300 MHz) dd (ppm): 1.06 (d, 3H, J = 6.88 Hz,
CH3), 1.82–2.41 (m, 9H, cyclohexane), 2.52 (d, 1H, J = 18.43 Hz,
imide), 2.91 (d, 1H, J = 18.43 Hz, imide), 7.31–7.41 (m, 2H, H-4, H-
5, pyridine), 7.89 (td, 1H, H-3, J = 7.70 Hz, pyridine), 8.68–8.71
(m, 1H, H-6, pyridine). Anal. calcd. for C15H18N2O2: C, 69.74; H,
7.02; N, 10.84. Found: C, 69.63; H, 6.93; N, 10.74.
N-(4,6-Dimethylpyridine-2-yl)-2-azaspiro[4.4]nonane-1,3-
dione 1e
Compound 1e was obtained as white solid (yield 59%), m.p. 100–
1028C. 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.80–2.32 (m, 8H,
cyclopentane), 2.39 (s, 3H, CH3), 2.58 (s, 3H, CH3), 2.80 (s, 2H,
imide), 6.92 (s, 1H, H-5, pyridine), 7.07 (s, 1H, H-3, pyridine). Anal.
calcd. for C15H18N2O2: C, 69.74; H, 7.02; N, 10.84. Found: C, 69.70;
H, 6.98; N, 10.73.
N-(3-Methylpyridine-2-yl)-6-methyl-2-azaspiro[4.5]
decane-1,3-dione 3b
Compound 3b was obtained as white solid (yield 57%), m.p.
1
175–1778C. H-NMR (CDCl3, 300 MHz) d (ppm): 0.96 (d, 3H, J =
6.92 Hz, CH3), 1.02–2.01 (m, 8H, cyclohexane), 2.04–2.15 (m, 1H,
cyclohexane), 2.22 (s, 3H, CH3), 2.58 (d, 1H, J = 18.46 Hz, imide),
2.78 (d, 1H, J = 18.43 Hz, imide), 7.28–7.32 (m, 1H, H-5, pyridine),
7.67 (t, 1H, H-4, J = 6.28 Hz, pyridine), 8.48 (d, 1H, H-6, J = 4.62 Hz,
pyridine). Anal. calcd. for C16H20N2O2: C, 70.56; H, 7.40; N, 10.29.
Found: C, 70.42; H, 7.32; N, 10.18.
N-Pyridin-2-yl-2-azaspiro[4.5]decane-1,3-dione 2a
Compound 2a was obtained as white solid (yield 72%), m.p. 148–
1508C (1338C [17]). 1H-NMR (CDCl3, 300 MHz) d (ppm): 1.40–2.02
(m, 10H, cyclohexane), 2.79 (s, 2H, imide), 7.30–7.42 (m, 2H, H-4,
H-5, pyridine), 7.89 (td, 1H, H-3, J = 7.70 Hz, pyridine), 8.68–8.70
(m, 1H, H-6, pyridine). Anal. calcd. for C14H16N2O2: C, 68.83; H,
6.60; N, 11.47. Found: C, 68.75; H, 6.58; N, 11.40.
N-(4-Methylpyridine-2-yl)-6-methyl-2-azaspiro[4.5]
decane-1,3-dione 3c
N-(3-Methylpyridine-2-yl)-2-azaspiro[4.5]decane-1,3-
dione 2b
Compound 3c was obtained as white solid (yield 64%), m.p. 80–
1
828C. Yield: H-NMR (CDCl3, 300 MHz) d (ppm): 1.06 (d, 3H, J =
Compound 2b was obtained as white solid (yield 56%), m. p.
166–1688C. 1H-NMR (CDCl3, 300 MHz) dd (ppm): 1.36–1.95 (m,
10H, cyclohexane), 2.18 (s, 3H, CH3 ), 2.77 (s, 2H, imide), 7.30–
7.33 (m, 1H, H-5, pyridine), 7.66–7.69 (m, 1H, H-4, pyridine),
8.46–8.48 (m, 1H, H-6, pyridine). Anal. calcd. for C15H18N2O2: C,
69.74; H, 7.02; N, 10.84. Found: C, 69.62; H, 7.00; N, 10.79.
6.60 Hz, CH3), 1.36–2.40 (m, 9H, cyclohexane), 2.45 (s, 3H, CH3),
2.50 (d, 1H, J = 18.43 Hz, imide), 2.90 (d, 1H, J = 18.15 Hz, imide),
7.12–7.13 (m, 1H, H-5, pyridine), 7.19–7.21 (m, 1H, H-3, pyri-
dine), 8.54 (d, 1H, H-6, J = 5.22 Hz, pyridine). Anal. calcd. for
C16H20N2O2: C, 70.56; H, 7.40; N, 10.29. Found: C, 70.39; H, 7.39;
N, 10.10.
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260
K. Kaminski et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 255–261
a shaking water bath; at 258C for 30 min. Addition of a ligand
initiated the incubation, which was terminated by rapid filtra-
tion through GF/C Whatman fiberglass filters. The filters were
then rinsed twice with 5 mL portions of ice-cold incubation buf-
fer and placed in plastic scintillation minivials. A scintillation
fluid (aquascint) was added and the samples were counted for
radioactivity in a Beckman LS 3801 scintillation counter. Radi-
oligand binding data were analyzed using iterative curve fitting
routines (GraphPAD/Prism, Version 3.0 – San Diego, CA, USA).
The results are shown in Table 3.
N-(6-Methylpyridine-2-yl)-6-methyl-2-
azaspiro[4.5]decane-1,3-dione 3d
Compound 3d was obtained as white solid (yield 58%), m.p.
1
146–1488C. H-NMR (CDCl3, 300 MHz) d (ppm): 0.94 (d, 3H, J =
6.87 Hz, CH3), 1.36–1.98 (m, 8H, cyclohexane), 2.06–2.14 (m, 1H,
cyclohexane), 2.58 (d, 1H, J = 18.43 Hz, imide), 2.62 (s, 3H, CH3),
2.80 (d, 1H, J = 18.43 Hz, imide), 7.05 (d, 1H, H-5, J = 7.70 Hz, pyr-
idine), 7.24 (d, 1H, H-4, J = 7.70 Hz, pyridine), 7.75 (t, 1H, H-3, J =
7.84 Hz, pyridine). Anal. calcd. for C16H20N2O2: C, 70.56; H, 7.40;
N, 10.29. Found: C, 70.51; H, 7.31; N, 10.16.
Lipophilicity
N-(4,6-Dimethylpyridine-2-yl)-6-methyl-2-
The lipophilicity of all compounds was measured by reversed
phase thin layer chromatography (RP-TLC). TLC was performed
on 20620 cm RP-18F254S precoated silica plates (Merck). Solu-
tions (10 mg mL– 1) of the tested compounds were prepared in
chloroform, and 10 mL volumes were spotted onto the plates, 2
cm apart. Mixtures of the n-propanol content ranging between
30–65% (v/v) in a 5% increment and TRIS buffer (pH = 7.0) were
used as a mobile phases. The chromatography chamber was
saturated with the mobile phase for 30 min before use. After the
development of chromatograms (180 mm), the plates were dried
in the air at a room temperature and were inspected under UV
light (k = 254 nm). RF (retardation factor) values were estimated
as mean values of the results obtained from three determina-
tions. The retention parameters (RM) were calculated by means of
the equation:
azaspiro[4.5]decane-1,3-dione 3e
Compound 3d was obtained as white solid (yield 60%), m.p.
1
115–1178C. H-NMR (CDCl3, 300 MHz) d (ppm): 1.05 (d, 3H, J =
6.87 Hz, CH3), 1.21–1.98 (m, 8H, cyclohexane), 2.00–2.19 (m, 1H,
cyclohexane), 2.39 (s, 3H, CH3), 2.50 (d, 1H, J = 18.43 Hz, imide),
2.57 (s, 3H, CH3), 2.88 (d, 1H, J = 18.43 Hz, imide), 6.88 (s, 1H, H-5,
pyridine), 7.20 (s, 1H, H-3, pyridine). Anal. calcd. for C17H22N2O2:
C, 71.30; H, 7.74; N, 9.78. Found: C, 71.20; H, 7.62; N, 9.70.
Pharmacology
Anticonvulsant screening
The anticonvulsant evaluations were performed using proce-
dures described elsewhere [25, 26]. Phase I studies of the investi-
gated compounds involved three testes: maximal electroshock
(MES), subcutaneous pentylenetetrazole (scPTZ), and a rotorod
test for neurological toxicity (TOX). All the compounds were
injected intraperitoneally into mice, as a suspension in 0.5%
methylcellulose, at the dose levels of 30, 100, and 300 mg/kg at
0.5 and 4 h time periods. The results are presented in Table 1.
Selected compounds from phase I (2a, 2d, and 3b) underwent
phase VIa in which they were administrated orally into rats
using four animals at a fixed dose of 30 mg/kg for both the MES
and the rotorod toxicity tests. Rats were tested at five time peri-
ods ranging from one quarter to 4 h post-substance administra-
tion. The results are shown in Table 2.
RM = log (1/Rf –1)
The RM values were extrapolated to 0% of organic modifier
concentration to estimate the relative retention parameters
(RM0) by use of the equation:
RM = RM0 + ac
c is the concentration of n-propanol and a the slope of the
straight line [29].
The correlation coefficients were always A 0.97. The relative
retention parameters (RM0) are listed in Table 1.
Calcium channel binding
The experiment was performed as described previously [27]. The
rat brain was rapidly removed after decapitation, placed on an
ice-chilled porcelain plate, and the cerebral-cortices dissected
out. The tissues were homogenized using a Polyrton disintegra-
tor (setting 4, 10 s) at 0 8C in 20 volumes 50 mmol/L Tris-HCl buf-
fer (pH 7.6). The homogenate was centrifuged at 08C and
10006g for 10 min, the supernatant was decanted and recentri-
fuged at 08C and 25.0006g for 30 min, and the resulting pellet
was resuspended in the buffer and recentrifuged under the
same conditions. The pellet thus obtained (fraction P2) was
stored at –188C for no longer than 48 h. For incubation it was
reconstituted in the Tris-HCl buffer to give a final protein con-
centration (measured according to Lowry et al. [28]) of approxi-
mately 1.2 mg/mL. The radioligand [3H]nitrendypine (NEN, speci-
fic activity 87.0 Ci/mmol), for estimating voltage-dependent Ca2+
channels was used. The incubation mixture (final volume
550 mL) consisted of 450 mL membrane suspension, 50 mL of
radioligand solution (0.8 nM), and 50 mL buffer containing
seven concentrations (0.1 nM–100 mM) of tested compounds.
For measuring unspecific binding, nifedipine was used at a con-
centration of 10 mM. Incubation was carried out in duplicate, in
Statistical analysis
Statistical analyses were performed using the computer pro-
gram Statistica version 5, 1997 Edition, Copyrighti Statsoft, Inc.
1984–1997. Data with p a 0.05 value were considered to be sig-
nificant.
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