Synthesis, physicochemical and anticonvulsant properties of new N-phenylamino derivatives of 2-azaspiro[4.4]nonane- and [4.5]decane-1,3-diones: Part V

Article abstract of DOI:10.1016/j.ejmech.2007.02.024

The synthesis, physicochemical and pharmacological properties of new N-phenylamino derivatives of 2-azaspiro[4.4]nonane-1,3-dione (8-10), 2-azaspiro[4.5]decane-1,3-dione (11-18) and 3-cyclohexyl-pyrrolidine-2,5-dione (19, 20) derivatives were described. The anticonvulsant properties of those compounds were examined by a maximal electroshock (MES) and a pentylenetetrazole (scPTZ) tests, and their neurotoxicity was determined using a rota-rod test. The most active was N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.4]nonane-1,3-dione (9), which exhibited anti-seizure properties in the MES model at a dose of 100 mg/kg in mice and at a dose of 30 mg/kg in rats. To explain the possible mechanism of action, for chosen active derivatives N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.4]nonane-1,3-dione (9), N-[(4-bromophenyl)-amino]-2-azaspiro[4.4]nonane-1,3-dione (10), N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.5]decane-1,3-dione (12) and N-[(4-bromophenyl)-amino]-2-azaspiro[4.5]decane-1,3-dione (13) their influence on GABA_A receptors were tested in vitro. Moreover, for all compounds obtained the lipophilic properties were determined by use of RP-HPLC method.

Full text of DOI:10.1016/j.ejmech.2007.02.024

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 53e61
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, physicochemical and anticonvulsant properties of new
N-phenylamino derivatives of 2-azaspiro[4.4]nonane-
and [4.5]decane-1,3-diones: Part V
a
a,
b
*
´
Krzysztof Kaminski , Jolanta Obniska , Ma1gorzata Dyba1a
a Department of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland
b Department of Histochemistry and Pharmacobiology, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland
Received 19 October 2006; received in revised form 20 February 2007; accepted 27 February 2007
Available online 18 March 2007
Abstract
The synthesis, physicochemical and pharmacological properties of new N-phenylamino derivatives of 2-azaspiro[4.4]nonane-1,3-dione (8e
10), 2-azaspiro[4.5]decane-1,3-dione (11e18) and 3-cyclohexyl-pyrrolidine-2,5-dione (19, 20) derivatives were described. The anticonvulsant
properties of those compounds were examined by a maximal electroshock (MES) and a pentylenetetrazole (scPTZ) tests, and their neurotoxicity
was determined using a rota-rod test. The most active was N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.4]nonane-1,3-dione (9), which exhibited
anti-seizure properties in the MES model at a dose of 100 mg/kg in mice and at a dose of 30 mg/kg in rats. To explain the possible mechanism of
action, for chosen active derivatives N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.4]nonane-1,3-dione (9), N-[(4-bromophenyl)-amino]-
2-azaspiro[4.4]nonane-1,3-dione (10), N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.5]decane-1,3-dione (12) and N-[(4-bromophenyl)-amino]-
2-azaspiro[4.5]decane-1,3-dione (13) their influence on GABAA receptors were tested in vitro. Moreover, for all compounds obtained the
lipophilic properties were determined by use of RP-HPLC method.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 2-Azaspiro[4.4]nonane- and [4.5]decane-1,3-diones; 3-Cyclohexyl-pyrrolidine-2,5-diones; N-phenylamino derivatives; Anticonvulsant activity;
RP-HPLC
1. Introduction
necessity for novel, specific and low-toxic anticonvulsant
drugs [2e4].
Epilepsy, a common neurological disorder characterized by
recurrent spontaneous seizures arising from excessive electri-
cal activity in some portion of the brain, is a major, worldwide,
public health problem, which affects approximately 1% of the
population [1]. Despite the increasing understanding of the
pathogenesis of seizures and epilepsy, as well as a large num-
ber of new anticonvulsant drugs that have been marked during
recent years, at least 30% of epilepsies remain uncontrolled. In
addition, many of currently used anticonvulsants produce sig-
nificant undesirable harmful side effects, which may limit their
maximal usefulness. For these reasons, there is a growing
In recent years anticonvulsant properties of many 2-azaspiro
[4.4]nonane- and [4.5]decane-1,3-diones (spirosuccinimides)
with different substituents at the imide nitrogen atom have
been described [5e10]. The structure activity relationship stud-
ies conducted with these groups of compounds revealed that
their efficacy were highly dependent on the size of the cycloalkyl
system attached to the C3 spiro carbon atom and the kind of
aromatic group at the nitrogen atom of pyrrolidine-2,5-dione
ring. Moreover it was proved that the imine (eNHe) linker
joiningthe endocyclicnitrogenatomand aromaticmoietywases-
sential for anticonvulsant activity of those molecules. As a result
the most activewas N-phenylamino-2-azaspiro[4.4]nonane-1,3-
dione (1) (Fig. 1), with ED50 ¼ 72.61 mg/kg in the maximal
electroshock seizure test (MES) [11].
* Corresponding author. Tel.: þ48 12 658 82 16; fax: þ48 12 657 02 62.
E-mail address: mfobnisk@cyf-kr.edu.pl (J. Obniska).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.02.024

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K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
54
and 1-carboxy-1-(4-methyl-cyclohexane) (6) acetic acids were
prepared as previously reported [19]. The synthetic procedures
of 2-cyclohexyl succinic acid (7) have been described in a sep-
arate literature [20]. The final N-substituted spirosuccinimides
8e20 were obtained in a one-pot cyclization reaction of the
prepared dicarboxylic acids (2e7) and appropriately
substituted phenylhydrazines by heating them at ca. 190e
200 ꢀC for 1.5e2 h.
O
N
O
N
H
cmpd 1
The structures of the compounds synthesized were con-
firmed by the examination of their 1H NMR, MS and IR spectra.
ED50 = 72.61 mg/kg
(MES test)
1
The H NMR spectra of the investigated compounds re-
Fig. 1. The structure of lead compound 1.
vealed that the characteristic chemical shifts agreed with their
proposed structures. The chemical shifts of the cyclopentane
and cyclohexane rings were observed as multiplets within
the range of d 1.74e2.37 ppm (8e10) and d 0.99e2.17 ppm
(11e18). The signals due to pyrrolidine-2,5-dione ring protons
appeared at about d 2.67e2.74 ppm, as singlets (8e13, 17,
18); d 2.48e2.73 as doublets (J ca. 18 Hz) (14e16) and
d 2.62e2.94 as multiplets (19 and 20). Appearance of broad
singlets within the range d 6.07e6.59 ppm corresponds to
the one proton of eNHe linker and confirms the formation
of imides 8e20. The methyl groups at positions 6, 7 and 8
of 2-azaspiro[4.5]decane-1,3-dione ring appeared at about
d 0.80e0.96 as doublets. All the aromatic protons were well
separated and observed at expected region.
The structures of selected compounds (9, 13 and 19) were
further verified by EI-MS spectra (70 eV). The m/z values of
molecular ion peaks were in complete agreement with cal-
culated molecular weight for individual compounds. The
fragmentation of the 2-azaspiro[4.4]nonane- (9) and 2-
azaspiro[4.5]decane-1,3-dione (10) rings was confirmed by
the characteristic ions at m/z 81, m/z 95, m/z 109 and m/z
123, which were within the range of 10e29% intensity. The
ions at m/z 83, m/z 99 and m/z 168 correspond to the fragmen-
tation of 3-cyclohexyl-pyrrolidine-2,5-dione (19). The frag-
mentation of imide moiety was observed as ions at m/z 176,
m/z 202 and m/z 204 (9); m/z 186, m/z 212 and m/z
214 (13); m/z 142, m/z 144 (19). The cleavage of the imine
(eNHe) linker confirmed characteristic ions m/z 160 (9),
m/z 170 (13) and m/z 127 (19).
Regarding the latter as a lead structure, in effort to obtain
compounds with enhanced efficacy, our attention was focused
on a group of N-phenylamino 3-spirosuccinimides. However,
both introduction of methyl substituents at the aromatic ring
or enlargement of the cycloalkyl system from cyclopentane
to cyclohexane decreased anticonvulsant activity [12]. On
the other hand, it was also shown that introduction of the
electron-withdrawing substituents (Br or Cl) was essential
for the anticonvulsant efficacy for several classes of succini-
mides [11,13e15].
In line with above data, as a continuation of our systematic
research on N-phenylamino spirosuccinimides, in the present
study we obtained a new series of 2-azaspiro[4.4]nonane-
and 2-azaspiro[4.5]decane-1,3-diones as well as their ana-
logues with additional methyl group at position 6, 7, or 8 of
2-azaspiro[4.5]decane-1,3-dione ring, with highly electronega-
tive substituents (Cl or Br) at the phenyl moiety. The aim of
these modifications was to examine the influence of such sub-
stitution on anticonvulsant activity. On the other hand, to in-
vestigate the role of the cycloalkyl system, attached to the
imide through the C3 spiro carbon atom, as a structural frag-
ment essential for activity, two analogues with cyclohexyl
moiety as a flexible substructure at position-3 of pyrrolidine-
2,5-dione ring were synthesized.
The GABA-ergic system is an important component of the
pathology of epilepsy and constitutes a vital target in the
search of novel antiepileptic drugs. It is also known that
several anticonvulsants interact with the specific binding sites
of the GABAA receptor complex [16,17]. Therefore to explain
the possible mechanism of action for selected active com-
pounds their GABAA receptor affinities were investigated.
Lipophilicity, the inherent property of the chemical com-
pound is widely used as structural descriptor in rational drug
design [18]. Thus in this study the lipophilic properties of
all derivatives were determined by use of RP-HPLC method.
We attempted also to evaluate the influence of this parameter
on anticonvulsant activity.
For all compounds investigated the IR spectra were re-
corded. The C]O groups of imide rings were observed within
the range v 1733e1716 cmꢁ1, whereas the aromatic moieties
(C]C) appeared between v 1597 and 1452 cmꢁ1
.
The detailed spectral data of each molecule are presented in
Section 7.
3. Pharmacology
3.1. In vivo test
2. Chemistry
3.1.1. Anticonvulsant screening
The initial anticonvulsant evaluation was performed with
the Antiepileptic Drug Development (ADD) Program, Epilepsy
Branch, Neurological Disorders Program, National Institute of
the Neurological and Communicative Disorders and Stroke
(NINCDS), Bethesda. The profile of anticonvulsant activity
The synthesis of compounds 8e20 was accomplished as
shown in Scheme 1. The starting 1-carboxy-1-cyclopentane
(2), 1-carboxy-1-cyclohexane (3), 1-carboxy-1-(2-methyl-
cyclohexane) (4), 1-carboxy-1-(3-methyl-cyclohexane) (5),

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K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
55
O
O
A
OH
OH
OH
2- 6
OH
O
O
7
cmpd
A
2
3
4
5
6
cyclopentane
cyclohexane
H2N
R
190 - 200°C
1.5 h
N
2-CH3 cyclohexane
3-CH3 cyclohexane
4-CH3 cyclohexane
H
A
O
O
N
N
R
R
O
O
N
N
H
H
8- 18
19, 20
cmpd
cmpd
R
A
R
8
cyclopentane
cyclopentane
cyclopentane
cyclohexane
cyclohexane
cyclohexane
2-Cl
19
20
2-Cl
2,4-Cl
9
2,4-Cl
4-Br
10
11
12
13
14
15
16
17
18
2-Cl
2,4-Cl
4-Br
2-Cl
2-CH3-cyclohexane
2-CH3-cyclohexane
2-CH3-cyclohexane
3-CH3-cyclohexane
4-CH3-cyclohexane
2,4-Cl
4-Br
2,4-Cl
2,4-Cl
Scheme 1. Synthetic protocol of compounds 8e20.
of compounds 8e20 was established in the maximal electro-
shock (MES) and subcutaneous pentylenetetrazole (scPTZ)
tests, after intraperitoneal injection into mice, at doses of 30,
100 and 300 mg/kg. The neurotoxic properties were measured
by the minimal motor impairment e rota-rod screen (TOX).
The compounds studied revealed diversified anticonvulsant
properties from active at a dose of 100 mg/kg (9, 12 and 13) to
active at a dose of 300 mg/kg (8, 10, 15 and 16) and inactive
ones (11, 14, 17e20). The results of anticonvulsant identifica-
tion studies in mice are shown in Table 2.
Some selected compounds (8, 9 and 12), active in mice
were evaluated orally in rats in the MES test at a dose of
30 mg/kg at five time periods ranging from one quarter to
4 h post substance administration (Table 3). Compound 9
was chosen for phase II evaluation for quantification of
ED50 and TD50 values.
3.2. In vitro test
3.2.1. GABAA binding assays
To search for possible mechanism of action, the affinities of
chosen derivatives 9, 10, 12 and 13 for GABAA receptors were
assessed in vitro, by their ability to displace [3H]muscimol in
rat cortical tissue. These compounds were active only in the
MES (9, 13) and both MES and scPTZ tests (10, 12). This se-
lection was based on different molecular mechanisms respon-
sible for the induction of seizures in MES and scPTZ screens.
Furthermore it seems that epileptic activity of pentylenetetra-
zole is probably due to blockade of Clꢁ channel of GABA re-
ceptor complex [21], thus from this point of view the most
interesting are molecules showing anti-scPTZ protection (10,
12). However, the radioligand binding experiments revealed
no affinities for all compounds investigated. These preliminary

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K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
56
data suggest that anticonvulsant activity of molecules tested is
not related with GABAA receptors. The results obtained re-
main in agreement with previous investigations indicating
that anticonvulsant activity of compounds with nitrogen five
or six member heteroatomic system (usually imide) as core
fragment and attached aromatic group involves the influence
on voltage-sensitive sodium channels [22,23]. Therefore, it
seems to be the most probable mechanism of action of the
molecules studied.
at a dose of 300 mg/kg at 0.5 h. The introduction of additional
methyl group into position-6 of the 2-azaspiro[4.5]decane-1,3-
dione moiety decreased activity, namely the 2,4-dichloro
substituted derivative 15 showed anti-MES and anti-scPTZ
protection at a dose of 300 mg/kg at 4 h and 0.5 h, respectively,
whereas 16 was effective only at MES screen at a dose of
100 mg/kg at 2 h and 300 mg/kg at 4 h. The 2-chloro substituted
compound 14 was ineffective in both tests applied. The 7-methyl
(17) and 8-methyl (18) analogues of active anticonvulsant deriva-
tive 12 did not show any protection in both screens. To evaluate the
role of the cycloalkyl fragment attached to the C3 spiro carbon
atom in the aspect of influence on anticonvulsant activity of spiro-
succinimides described above, two 3-cyclohexyl-pyrrolidine-2,5-
dione derivatives (19, 20) have been synthesized. These molecules
were designed as analogues of respective active (12) or inactive
(11) spirosuccinimides. The results obtained revealed that the
introduction of the cyclohexyl moiety as a flexible fragment
at position-3 of the imide ring made both the compounds
inactive.
In the neurotoxicity screen compounds 8, 11, 12, 15, 17, 19
and 20 were devoid of toxicity at the maximum dose adminis-
trated (300 mg/kg). The other derivatives were found to be toxic
at a dose of 30 mg/kg (13), 100 mg/kg (10, 14, 18) or 300 mg/kg
(9, 16). The mice were unable to grasp rota-rod after administra-
tion of compounds 9 and 16 at a dose of 300 mg/kg.
Compounds 8, 9 and 12, randomly selected from derivatives
active 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 results obtained are shown in Table 3. As can be
seen from these data, only compound 9 was moderately active
in rat MES oral screen and revealed the time of onset of anticon-
vulsant activity at 0.25 h and one peak of 75% protection at time
points 2 h. In addition, tested substance protected 50% of ani-
mals at 0.25 h, 0.5 h and 1 h. The total duration of satisfactory
action of this compound was quite short within 0.25 he2 h. The
derivative 8 showed marginal protection, whereas 12 was in-
active in rats. No evidence of neurological toxicity was
observed at the dose of 30 mg/kg administrated orally.
4. Lipophilicity
Lipophilicity is a fundamental physicochemical property of
bioactive compounds that plays a pivotal role in the transport of
a molecule through cellular membranes and influences the lo-
calization of compound in the therapeutic site of action. The
most widely used measurements of the lipophilic properties
of different chemical molecules are nowadays the chromato-
graphic techniques in reversed phase system [24e26]. In con-
sequence, the correlation between lipophilicity and biological
activity can be based on chromatographic retention parameters
[27e29]. Therefore, in present work we determined the lipo-
philicity of all compounds by use of RP-HPLC method.
The examination of the chromatographic behaviour in the
RP-HPLC system showed a linear correlation between log k
values and acetonitrile concentration (v/v) in a mobile phase.
The relative retention parameters log kw were determined by
the extrapolation method, and are listed in Table 2.
5. Results and discussion
The maximal electroshock (MES) and subcutaneous penty-
lenetetrazole (scPTZ) tests are claimed to detect compounds
affording protection against generalized tonic-clonic seizures
and generalized absence seizures, respectively. Thus the
MES and scPTZ screens have become the most widely em-
ployed seizure models for early identification of candidate
anticonvulsants.
The compounds investigated 8e20 showed diversified anti-
convulsant properties in the mouse i.p. MES and scPTZ
screens. Among all derivatives under study, the most active
were 2,4-dichloro substituted compounds 9 and 12, which re-
vealed protection in the maximal electroshock test (MES) at
a dose of 100 mg/kg at 0.5 h and 4 h, respectively. These mole-
cules were also effective at a dose of 300 mg/kg in the same
screen at time points 0.5 h (9) and 4 h (9, 12). Additionally,
compound 12, exhibited anti-scPTZ activity at a dose of
300 mg/kg at 4 h. The removal of the chloro atom at posi-
tion-4 of the aryl ring yielded less active derivative 8 which
showed protection in both MES and scPTZ screens at a dose
of 300 mg/kg at 0.5 h and inactive molecule 11. The 4-bromo
analogues 10 and 13 exhibited anti-MES activity at a dose of
100 mg/kg at time point 2 h (10) and 4 h (13) and also 300 mg/kg
both at 4 h. Compound 13 revealed, however, neurotoxicity
at a dose of 30 mg/kg and regardless of its activity according
to the procedures of ADD Program was ascribed to 4 ASP
class. Moreover derivative 10 showed anti-scPTZ protection
Compound 9 was chosen for phase II evaluation for quan-
tification of anticonvulsant activities (ED50 and TD50), against
MES and scPTZ e induced seizures after oral administration
into rats at time point 2 h. The measured median effective
dose was greater than 180 mg/kg in the MES test and greater
than 125 mg/kg in the scPTZ screen. The tested molecule re-
vealed TD50 value to be higher than 250 mg/kg.
The chromatographically determined lipophilicity (log kw)
provided a good basis for evaluation of structureelipophilicity
relationships. As expected, the highest lipophilicities were ob-
served for the 2,4-dichloro derivatives 9, 12, 15, 17, 18 and 20,
whereas the lowest was shown by the 2-chloro substituted
compounds 8, 11, 14 and 19. The change of the ring size
from cyclopentane (8e10) to cyclohexane (11e13) as well
as introduction of the additional methyl group into cycloalkyl
system (14e18) increased the lipophilicity. Furthermore the
log kw values were higher for 3-cyclohexyl-pyrrolidine-2,5-
diones (19, 20) in comparison to respective spirosuccinimide
derivatives (11, 12).

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K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
57
In our previous study we determined the lipophilicity of
a series of structurally related N-phenylamino and N-benzyl-
2-azaspiro[4.4]nonane- and [4.5]decane-1,3-dione derivatives.
The role of lipophilicity in the anticonvulsant activity was
proved; the active anticonvulsants were less lipophilic than
the inactive ones [27,28]. Thus, the next step of our study
was an attempt to evaluate the correlation between the anti-
convulsant activity and lipophilicity. In subsequent analysis
we used individual and average lipophilicities (i.e. mean
log kw values) separately for plots of compounds active at
doses of 100 and 300 mg/kg, in contrast with inactive deriva-
tives. Comparison of lipophilicity for active and inactive mole-
cules is depicted in Fig. 2. As it is shown the ranges for active
and inactive molecules overlap, however, mean lipophilicity
value for active anticonvulsants is lower compared to inactive
ones. The applied statistical analysis (ManneWhitney test)
revealed that the difference between log kw parameters be-
tween these groups is not statistically significant (U ¼ 11.0,
p ¼ 0.153).
spiro carbon atom in the aspect of anticonvulsant activity of
that type of compounds.
6. Conclusion
The results obtained revealed that number of novel N-phe-
nylamino-2-azaspiro[4.4]nonane- and [4.5]decane-1,3-dione
derivatives were effective in the MES and scPTZ screens,
the most widely employed seizure models for early identifica-
tion of candidate anticonvulsants. The most active was
N-[(2,4-dichlorophenyl)-amino]-2-azaspiro[4.4]nonane-1,3-
dione, which was chosen for quantification of ED50 and TD50
values. The anticonvulsant activity profile depended on kind
and position of substituents at the aryl moiety as well as the
size and manner of attachment of the cyclolkyl system at the
position-3 of the pyrrolidine-2,5-dione ring. The GABAA
binding assays suggest that anticonvulsant activity is not
related with GABAA receptors. The influence on voltage-
sensitive sodium channels seems to be the most probable
mechanism of action of molecules investigated. There was
no distinct correlation between the lipophilicity and anticon-
vulsant efficacy.
In summary, the most active were compounds with cyclo-
pentane and cyclohexane ring as spiro nucleus and two chloro
substituents at positions-2 and 4 or bromo atom at position-4
of the aryl ring. Surprisingly, it was not consistent with the
data obtained for N-pyridine-2-yl-, N-phenyl- and especially
structurally related N-benzyl-spirosuccinimides, where the
highest activity was observed for ortho-substituted compounds
[9e11]. It proves the unique role of the imine (eNHe) linker
joining the endocyclic nitrogen atom and aromatic moiety in
respect to anticonvulsant properties of such type of derivatives.
The introduction of additional methyl group at position 6, 7 or
8 of the 2-azaspiro[4.5]decane-1,3-dione moiety decreased the
activity. Furthermore, comparison of results obtained for the
spirosuccinimides and compounds with cyclohexyl moiety as
a flexible fragment at position-3 of the imide ring, proved an
essential role of cycloalkyl system attached through the C3
7. Experimental protocols
7.1. Chemistry
All the chemicals and solvents were purchased from Merck
(Darmstadt, Germany) and were used without further purifica-
tion. Melting points (m.p.) were determined in open capillaries
on a Buchi 353 melting point apparatus (Buchi Labortechnik,
¨
¨
Flawil, Switzerland) and are uncorrected. The purity of the
compounds was confirmed by the thin-layer chromatography
(TLC) performed on Merck silica gel 60 F254 aluminium
sheets (Merck; Darmstadt, Germany), using subsequent deve-
loping system: chloroform/acetone (9:1). Spots were detected
by their absorption under UV light (l ¼ 254 nm) and by visu-
alization with 0.05 mol I2 in 10% HCl. Elemental analysis for
C, H, and N was carried out by a micro method using the
elemental Vario EI III Elemental analyser (Hanau, Germany).
The results of elemental analyses were within ꢂ0.4% of the
theoretical values (Table 1).
Infrared spectra (IR) were recorded in FTIR Spectrometer
1600 (Perkin Elmer). 1H NMR spectra were obtained in a Varian
Mercury spectrometer (Varian Inc., Palo Alto, CA, USA), in
CDCl3, operating at 300 MHz. Chemical shifts are reported in
d values (ppm) relative to TMS d ¼ 0 (1H), as internal standard.
The J values are expressed in Hertz (Hz). Signal multiplicities
are represented by the following abbreviations: s (singlet),
d (doublet), dd (double doublet), m (multiplet). For chosen com-
pounds 9, 13 and 19 the mass spectra (MS) were recorded on
AMD-604 Mass Spectrometer operating at 70 eV.
7.1.1. General procedure for preparation of
compounds 8e20
To asuspension of 1-carboxy-1-cyclopentane (2), 1-carboxy-
1-cyclohexane (3), 1-carboxy-1-(2-methyl-cyclohexane) (4),
Fig. 2. Comparison of ranges and mean log kw parameters for active and inac-
tive compounds.

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K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
58
IR v (cmꢁ1): 1704, 1722 (C]O), 1554, 1520, 1492 (C]C).
C14H15Br1N2O2 (323.2).
Table 1
Analytical data for compounds 8e20
Cmpd Molecular formula, weight Analysis (calcd/found)
7.1.1.4. N-[(2-Chlorophenyl)-amino]-2-azaspiro[4.5]decane-
%C
%H
%N
1,3-dione (11). White powdery crystals. Yield: 69%; m.p.
8
C
14H15Cl1N2O2, 278.7
60.33, 60.31 5.42, 5.40 10.05, 10.06
53.69, 53.70 4.51, 4.49 8.94, 8.95
52.03, 52.02 4.68, 4.66 8.67, 8.66
61.54, 61.55 5.85, 5.83 9.57, 9.57
55.06, 55.07 4.93, 4.92 8.56, 8.57
53.43, 53.44 5.08, 5.06 8.31, 8.30
62.64, 62.62 6.24, 6.25 9.13, 9.12
56.32, 56.30 5.32, 5.33 8.21, 8.20
54.71, 54.73 5.45, 5.44 7.98, 7.99
56.32, 56.33 5.32, 5.30 8.21, 8.22
56.32, 56.30 5.32, 5.31 8.21, 8.20
62.64, 62.63 6.24, 6.22 9.13, 9.12
56.32, 56.31 5.32, 5.30 8.21, 8.22
1
195e197 ꢀC; H NMR (300 MHz, CDCl3): d 1.37e1.92 (m,
9
C14H14Cl2N2O2, 313.2
C14H15Br1N2O2, 323.2
C15H17Cl1N2O2, 292.8
10
11
12
13
14
15
16
17
18
19
20
10H, cyclohexane), 2.70 (s, 2H, imide), 6.50 (dd, J ¼ 8.20 Hz,
J ¼ 1.28 Hz, 1H, Haromat.), 6.57 (br s, 1H, NH), 6.87e6.93 (m,
C
15H16Cl2N2O2, 327.2
1H,
Haromat.), 7.09e7.12 (m, 1H, Haromat.), 7.32 (dd,
C15H17Br1N2O2, 337.2
C16H19Cl1N2O2, 306.8
C16H18Cl2N2O2, 341.2
J ¼ 7.97 Hz, J ¼ 1.54 Hz, 1H, Haromat.). IR v (cmꢁ1): 1727
(C]O), 1594, 1492 (C]C). C15H17Cl1N2O2 (292.8).
C
16H19Br1N2O2, 351.2
C16H18Cl2N2O2, 341.2
C16H18Cl2N2O2, 341.2
C16H19Cl1N2O2, 306.8
7.1.1.5.
N-[(2,4-Dichlorophenyl)-amino]-2-azaspiro[4.5]-
decane-1,3-dione (12). White powdery crystals. Yield: 71%;
m.p. 202e204 ꢀC; 1H NMR (300 MHz, CDCl3): d 1.37e
1.91 (m, 10H, cyclohexane), 2.70 (s, 2H, imide), 6.44 (d,
J ¼ 8.72 Hz, 1H, Haromat.), 6.50 (br s, 1H, NH), 7.10 (dd,
J ¼ 8.72 Hz, J ¼ 2.31 Hz, 1H, Haromat.), 7.34 (d, J ¼ 2.05 Hz,
1H, Haromat.). IR v (cmꢁ1): 1721 (C]O), 1488, 1452
(C]C). C15H16Cl2N2O2 (327.2).
C
16H18Cl2N2O2, 341.2
1-carboxy-1-(3-methyl-cyclohexane) (5), and 1-carboxy-1-
(4-methyl-cyclohexane) (6) acetic acids or 2-cyclohexyl-
succinic acid (7) (0.01 mol) in 10 ml of water, the appropriately
substituted phenylhydrazines (0.01 mol) were gradually added.
The mixture was heated in an oil bath with simultaneous
distillation of water. After complete removal of water the
temperature of the reaction mixture was raised up to 190e
200 ꢀC and maintained for 1.5 h. The crude products were
crystallized from isopropanol. The obtained solid residues
were purified by column chromatography on Silica gel 60
(Merck, Darmstadt, Germany) using chloroform/acetone mix-
ture (9:1) as a solvent. After evaporation of the solvents, the
oil products were recrystallized from isopropanol to afford
the desired compounds.
7.1.1.6. N-[(4-Bromophenyl)-amino]-2-azaspiro[4.5]decane-
1,3-dione (13). White powdery crystals. Yield: 67%; m.p.
1
186e188 ꢀC; H NMR (300 MHz, CDCl3): d 1.36e1.94 (m,
10H, cyclohexane), 2.67 (s, 2H, imide), 6.07 (br s, 1H, NH),
6.63e6.67 (m, 2H, Haromat.), 7.33e7.36 (m, 2H, Haromat.). IR v
(cmꢁ1): 1733, 1715 (C]O), 1593, 1492 (C]C). MS m/z (%):
338 (100) [M þ 2]þ, 336 (99) [M]þ, 214 (22), 212 (22), 186
(38), 170 (10), 123 (10), 95 (17). C15H17Br1N2O2 (337.2).
7.1.1.7. N-[(2-Chlorophenyl)-amino]-6-methyl-2-azaspiro[4.5]-
decane-1,3-dione (14). White powdery crystals. Yield: 65%;
1
7.1.1.1. N-[(2-Chlorophenyl)-amino]-2-azaspiro[4.4]nonane-
m.p. 196e198 ꢀC; H NMR (300 MHz, CDCl3): d 0.84 (d,
1,3-dione (8). White powdery crystals. Yield: 72%; m.p.
J ¼ 6.67 Hz, 3H, CH3), 1.01e1.95 (m, 8H, cyclohexane),
2.04e2.13 (m, 1H, cyclohexane), 2.51 (d, J ¼ 18.72 Hz, 1H,
imide), 2.73 (d, J ¼ 18.72 Hz, 1H, imide), 6.55 (dd,
J ¼ 8.06 Hz, J ¼ 1.41 Hz, 1H, Haromat.), 6.59 (br s, 1H, NH),
6.87e6.92 (m, 1H, Haromat.), 7.09e7.15 (m, 1H, Haromat.), 7.33
(dd, J ¼ 7.95 Hz, J ¼ 1.28 Hz, 1H, Haromat.). IR v (cmꢁ1):
1726 (C]O), 1597, 1497 (C]C). C16H19Cl1N2O2 (306.8).
1
140e142 ꢀC; H NMR (300 MHz, CDCl3): d 1.74e2.37 (m,
8H, cyclopentane), 2.74 (s, 2H, imide), 6.53 (dd, J ¼ 8.20 Hz,
J ¼ 1.54 Hz, 1H, Haromat.), 6.59 (br s, 1H, NH), 6.87e6.93 (m,
1H,
Haromat.), 7.09e7.12 (m, 1H, Haromat.), 7.32 (dd,
J ¼ 8.20 Hz, J ¼ 1.41 Hz, 1H, Haromat.). IR v (cmꢁ1): 1730
(C]O), 1594, 1492 (C]C). C14H15Cl1N2O2 (278.7).
7.1.1.2.
N-[(2,4-Dichlorophenyl)-amino]-2-azaspiro[4.4]-
7.1.1.8. N-[(2,4-Dichlorophenyl)-amino]-6-methyl-2-azaspiro-
[4.5]decane-1,3-dione (15). White powdery crystals. Yield:
60%; m.p. 200e202 ꢀC; 1H NMR (300 MHz, CDCl3):
d 0.82 (d, J ¼ 6.92 Hz, 3H, CH3), 1.00e1.94 (m, 8H, cyclo-
hexane), 2.00e2.11 (m, 1H, cyclohexane), 2.50 (d,
J ¼ 18.46 Hz, 1H, imide), 2.72 (d, J ¼ 18.72 Hz, 1H, imide),
6.48 (d, J ¼ 8.72 Hz, 1H, Haromat.), 6.53 (br s, 1H, NH), 7.10
nonane-1,3-dione (9). White powdery crystals. Yield: 70%;
m.p. 154e156 ꢀC; 1H NMR (300 MHz, CDCl3): d 1.74e
2.23 (m, 8H, cyclopentane), 2.73 (s, 2H, imide), 6.46 (d,
J ¼ 8.46 Hz, 1H, Haromat.), 6.52 (br s, 1H, NH), 7.12 (dd,
J ¼ 8.72 Hz, J ¼ 2.31 Hz, 1H, Haromat.), 7.34 (d, J ¼ 2.05 Hz,
1H, Haromat.). IR v (cmꢁ1): 1723 (C]O), 1574, 1490
(C]C). MS m/z (%): 316 (10) [M þ 4]þ, 314 (64)
[M þ 2]þ, 312 (100.0) [M]þ, 204 (30), 202 (48), 176 (54),
160 (24), 109 (29), 81 (28). C14H14Cl2N2O2 (313.2).
(dd, J ¼ 8.72 Hz, J ¼ 2.31 Hz, 1H,
Haromat.), 7.34 (d,
J ¼ 2.31 Hz, 1H, Haromat.). IR v (cmꢁ1): 1721 (C]O), 1573,
1488 (C]C). C16H18Cl2N2O2 (341.2).
7.1.1.3. N-[(4-Bromophenyl)-amino]-2-azaspiro[4.4]nonane-
7.1.1.9. N-[(4-Bromophenyl)-amino]-6-methyl-2-azaspiro[4.5]-
1,3-dione (10). White powdery crystals. Yield: 75%; m.p.
decane-1,3-dione (16). White powdery crystals. Yield: 60%;
1
1
166e168 ꢀC; H NMR (300 MHz, CDCl3): d 1.73e2.20 (m,
8H, cyclopentane), 2.71 (s, 2H, imide), 6.42 (br s, 1H, NH),
6.63e6.67 (m, 2H, Haromat.), 7.33e7.36 (m, 2H, Haromat.).
m.p. 186e188 ꢀC; H NMR (300 MHz, CDCl3): d 0.80 (d,
J ¼ 6.92 Hz, 3H, CH3), 0.99e1.99 (m, 8H, cyclohexane),
2.01e2.11 (m, 1H, cyclohexane), 2.48 (d, J ¼ 18.72 Hz, 1H,

´
K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
59
imide), 2.70 (d, J ¼ 18.72 Hz, 1H, imide), 6.10 (br s, 1H, NH),
Haromat.). IR v (cmꢁ1): 1724 (C]O), 1594, 1491 (C]C).
C16H19Br1N2O2 (351.2).
methylcellulose at the dose levels of 30, 100, and 300 mg/kg
with anticonvulsant activity and neurotoxicity assessment at
0.5 h and 4 h intervals after administration. The results are pre-
sented in Table 2. Some selected derivatives from phase I (8, 9
and 12) underwent phase VIa in which they were adminis-
trated orally into rats using four animals at a fixed dose of
30 mg/kg for both the MES and the rota-rod toxicity tests.
Rats were tested at five time periods ranging from one quarter
to 4 h post substance administration. The results are shown in
Table 3.
Compound 9, active in mice and rats, was chosen for quan-
tification of the pharmacological parameters (ED50 and TD50).
The quantitative determination of the median effective dose
(ED50) and toxic dose (TD50) was performed at previously es-
timated time of peak effect after oral administration into rats.
Groups of eight rats received various doses of the compound
until at least two points were established between the limits
of 100% protection and 0% protection or minimal toxicity.
6.65e6.68 (m, 2H,
Haromat.), 7.32e7.36 (m, 2H,
7.1.1.10. N-[(2,4-Dichlorophenyl)-amino]-7-methyl-2-azaspiro-
[4.5]decane-1,3-dione (17). White powdery crystals. Yield:
1
68%; m.p. 204e206 ꢀC; H NMR (300 MHz, CDCl3): d 0.96
(d, J ¼ 6.16 Hz, 3H, CH3), 1.02e1.86 (m, 9H, cyclohexane),
2.69 (s, 1H, imide), 6.44 (d, J ¼ 8.72 Hz, 1H,
H
aromat.), 6.51 (br s, 1H, NH), 7.09e7.13 (m, 1H, Haromat.),
7.34 (d, J ¼ 2.31 Hz, 1H, Haromat.). IR v (cmꢁ1): 1723
(C]O), 1576, 1488 (C]C). C16H18Cl2N2O2 (341.2).
7.1.1.11. N-[(2,4-Dichlorophenyl)-amino]-8-methyl-2-azaspiro-
[4.5]decane-1,3-dione (18). White powdery crystals. Yield:
1
68%; m.p. 210e212 ꢀC; H NMR (300 MHz, CDCl3): d 0.95
(d, J ¼ 6.67 Hz, 3H, CH3), 1.00e2.17 (m, 9H, cyclohexane),
2.68 (s, 1H, imide), 6.44 (d, J ¼ 8.72 Hz, 1H,
H
aromat.), 6.51 (br s, 1H, NH), 7.08e7.13 (m, 1H, Haromat.),
7.3. GABAA receptor binding assay
7.34 (d, J ¼ 2.31 Hz, 1H, Haromat.). IR v (cmꢁ1): 1724 (C]O),
1590, 1489 (C]C). C16H18Cl2N2O2 (341.2).
The experiment was performed as described previously
[32]. Rats’ brains were homogenised in 20 volumes of ice-
cold 50 mM triseHCl buffer (pH 7.4) using a ULTRA
TURAX homogeniser. The homogenate is then centrifuged
at 20 000 ꢃ g for 20 min (0e4 ꢀC). The resulting supernatant
7.1.1.12. N-[(2-Chlorophenyl)-amino]-3-cyclohexyl-pyrroli-
dine-2,5-dione (19). White powdery crystals. Yield: 60%;
m.p. 150e152 ꢀC; 1H NMR (300 MHz, CDCl3): d 1.08e
1.82 (m, 10H, cyclohexane), 1.97e2.17 (m, 1H, cyclohexane),
2.62e2.93 (m, 3H, imide), 6.53 (dd, J ¼ 8.08 Hz, J ¼ 1.54 Hz,
1H, Haromat.), 6.57 (br s, 1H, NH), 6.87e6.92 (m, 1H, Haromat.),
Table 2
Anticonvulsant screening project (ASP) phase I results in mice
7.10e7.15 (m, 1H,
H
aromat.), 7.32 (dd, J ¼ 7.95 Hz,
d
J ¼ 1.28 Hz, 1H, Haromat.). IR v (cmꢁ1): 1720 (C]O), 1595,
1497, 1445 (C]C). MS m/z (%): 308 (32) [M þ 2]þ, 306
(100) [M]þ, 168 (8), 144 (10), 142 (34), 127 (43), 99 (11),
83 (11). C16H19Cl1N2O2 (306.8).
Cmpd Intraperitoneal injection in mice
log kw
ASPe class
MESa
scPTZb
TOXc
0.5 h 4 h
0.5 h 4 h
0.5 h 4 h
8
300
100
e
300
300
e
e
e
e
e
300
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
2.095
2.419
2.115
2.322
2.634
2.337
2.515
2.847
2
1
2
3
1
4
3
2
2
3
3
3
3
9
300f
100
e
10g
11
12
13
14
15
16g
17
18
19
20
300 300
7.1.1.13. N-[(2,4-Dichlorophenyl)-amino]-3-cyclohexyl-pyrro-
lidine-2,5-dione (20). White powdery crystals. Yield: 65%;
m.p. 160e162 ꢀC; 1H NMR (300 MHz, CDCl3): d 1.03e
1.82 (m, 10H, cyclohexane), 1.96e2.06 (m, 1H, cyclohexane),
2.62e2.94 (m, 3H, imide), 6.46 (d, J ¼ 8.72 Hz, 1H, Haromat.),
6.50 (br s, 1H, NH), 7.12 (dd, J ¼ 8.72 Hz, J ¼ 2.31 Hz, 1H,
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
100
100
e
e
30
100
e
300 300
300
e
e
e
e
e
e
e
e
300f 2.561
2.976
100 2.977
aromat.), 7.34 (d, J ¼ 2.31 Hz, 1H, Haromat.). IR v (cmꢁ1):
e
100
e
e
H
1716 (C]O), 1489, 1447 (C]C). C16H18Cl2N2O2 (341.2).
e
e
2.536
2.896
e
e
e
7.2. Anticonvulsant screening
The figures in the table indicate the minimum dose whereby bioactivity was
demonstrated.
a
Compounds 8e20 were pharmacologically pre-evaluated
within the Antiepileptic Drug Development (ADD) Program,
Epilepsy Branch, Neurological Disorders Program, National
Institute of the Neurological and Communicative Disorders
and Stroke (NINCDS), Bethesda, using procedures described
elsewhere [30,31].
Maximal electroshock test.
Subcutaneous pentylenetetrazole test.
b
c
Rota-rod toxicity.
The relative retention parameters calculated according to equation:
d
log k ¼ log kw ꢁ S4, where S is the slope, 4 is the volume fraction of the or-
ganic modifier.
e
The ASP classification is as follows: 1 e anticonvulsant activity at doses of
100 mg/kg or less; 2 e anticonvulsant activity at doses of 300 mg/kg; 3 e
compound inactive at doses of 300 mg/kg, 4 e compound active, however,
toxic at a dose of 30 mg/kg.
Phase I studies of the compounds investigated involved
three tests: maximal electroshock seizure (MES), subcutane-
ous pentylenetetrazole seizure (scPTZ) and rota-rod test for
neurological toxicity (TOX). All the compounds were injected
f
Response comments: unable to grasp rota-rod.
Compounds 10 and 16 revealed additionally anti-MES activity at doses of
100 mg/kg at 2 h.
g
intraperitoneally into mice as
a
suspension in 0.5%

´
K. Kaminski et al. / European Journal of Medicinal Chemistry 43 (2008) 53e61
60
Table 3
The results in rats after oral administration at a dose of 30 mg/kg
The injection volume was 5 ml in all cases. Uracil (Sigmae
Aldrich) was used as the unretained compound to determine
the dead volume at the different acetonitrile concentrations
in the mobile phase. The retention times were measured at
room temperature by the UV detector at the lmax of the analy-
tes (214 nm). Three sets of measurements were conducted for
each compound and the mean value was used for further cal-
culations. The capacity factors log k were calculated by us
from equation: k ¼ (tr ꢁ t0)/t0, where tr and t0 are the retention
times for solute and unretained compound (uracil), respec-
tively. The log k values were then extrapolated to 100% water
content to estimate the relative retention parameters log kw,
according to equation: log k ¼ log kw ꢁ S4, where S is the
slope, 4 is the volume fraction of the organic modifier and
log kw is the chromatographic lipophilicity parameter that
characterizes the partition of the compound between a non-
polar hydrocarbon stationary phase and pure water [33]. The
log kw values better approximate the experimental conditions
of lipid phase/water partition system values and are recom-
mended for chromatographic measurement of lipophilicity in
QSAR studies [34].
Compd Oral administration to rats
MESa
TOXb
0.25 h 0.5 h 1.0 h 2.0 h 4.0 h 0.25 h 0.5 h 1.0 h 2.0 h 4.0 h
8
1/4
2/4
0/4
1/4 0/4 1/4 0/4 0/4
2/4 2/4 3/4 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
0/4 0/4 0/4 0/4
9
12
a
Maximal electroshock test, number of animals protected/number of ani-
mals tested.
b
Rota-rod test for neurological toxicity, number of animals exhibiting tox-
icity/number of animals tested.
was discarded and the pellet rehomogenised 20 volumes of
ice-cold 50 mM triseHCl buffer (pH 7.4) and centrifuged as
above. The pellet was resuspended and centrifuged for further
two times. The final pellet was stored at ꢁ20 ꢀC for at least
18 h. On the day of the assay, pellet was thawed at room tem-
perature, resuspended in 20 volumes of ice-cold 50 mM trise
HCl buffer (pH 7.4) and centrifuged at 20 000 ꢃ g for 28 min
(0e4 ꢀC).
Radioligand binding assays were performed in plates (Multi-
Screen/Millipore). The final incubation mixture (final volume
300 ml) consisted of 240 ml membrane suspension, 30 ml of
a 3 nM [3H]muscimol and 30 ml buffer containing eight concen-
trations (10ꢁ11e10ꢁ4 M) of tested compounds. For measuring
unspecific binding 100 mM GABA was applied. The sample
was incubated for 10 min at 0 ꢀC. The incubation was termi-
nated by rapid filtration over glass fiber filters (Whatman GF/
C) using a vacuum manifold (Millipore). The filters were then
washed 2 times with the buffer and placed in scintillation vials
with liquid scintillation cocktail. Radioactivity was measured
in a WALLAC 1409 DSA e liquid scintillation counter. All
assays were done in duplicates. Radioligand binding data were
analyzed using iterative curve fitting routines (GraphPAD/Prism,
Version 3.0 e San Diego, CA, USA).
7.5. Statistical analysis
Statistical analyses were performed using the computer
program Statistica version 5, 1997 Edition, CopyrightÓ Stat-
soft, Inc. 1984e1997.
Acknowledgements
The authors wish to thank Dr James Stables for providing
them pharmacological data through the Antiepileptic Drug
Development Program (Epilepsy Branch, National Institute
of Neurological Disorders and Stroke, National Institute of
Health, Bethesda, MD, USA).
We are pleased to acknowledge the generous financial sup-
port of this work by the Committee for Scientific Research,
Poland (Grant No 3 PO5F 024 25).
7.4. HPLC measurements
The analytical system consisted of a Waters (Milford, USA)
instrument, equipped with 515 HPLC pumps, a 600S flow
controller, 486 Tunable Absorbance Detector and injector:
Rheodyne 7161 valve (Cotati, CA, USA). The measurements
were performed on a LiChrospher C18 end-capped column,
250 ꢃ 4.6 mm (Merck, Darmstadt, Germany). Acetonitrile
for measurements was of gradient grade and was purchased
from Merck (Darmstadt, Germany). The water was filtrated
under vacuum a 0.45 mm HA Millipore filter (Millipore, Mil-
ford, MA, USA) before being mixed with acetonitrile. For
chromatographic analysis stock solutions of 1.0 mg/ml of the
samples in acetonitrile/water (1:1) were prepared and filtrated
through 0.2 mm Millipore filter (Millipore, Milford, MA,
USA). These solutions were kept in Eppendorf tubes at
room temperature. Isocratic runs were carried out with mix-
tures containing increasing acetonitrile volume fractions from
40% to 95% v/v in 5% increment. Flow rate was 1 ml/min.
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